interleukin-8 and Arthritis--Rheumatoid

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

The production of interleukin (IL)-26 was initially attributed to T cells, and in particular to Th17 cells. However, more recent findings indicate IL-26 production in natural killer (NK) cells, macrophages and fibroblast-like cells as well. It is known that IL-26 binds to the IL-20R1/IL-10R2 receptor complex on certain target cells, where it causes specific intracellular signaling and the secretion of IL-1β, IL-8 and TNF-α. In line with this type of proinflammatory role, IL-26 also increases chemotaxis of human neutrophils. Interestingly, high levels of IL-26 are present even in normal human airways, and endotoxin exposure further enhances these levels; this indicates involvement in antibacterial host defense. Studies on acute inflammatory disorders are few but there are studies showing the involvement of IL-26 in rheumatoid arthritis and inflammatory bowel disease. In conclusion, IL-26 is emerging as a potentially important player in host defense and may also be a pathogenic factor in the chronic inflammatory disorders of humans.

Topics: Arthritis, Rheumatoid; Chemotaxis; Chronic Disease; Humans; Immunity, Innate; Inflammation; Inflammatory Bowel Diseases; Interleukin-10 Receptor beta Subunit; Interleukin-1beta; Interleukin-8; Interleukins; Killer Cells, Natural; Macrophages; Receptors, Interleukin; Signal Transduction; Th17 Cells; Tumor Necrosis Factor-alpha

Antibodies against citrullinated proteins (ACPAs) are specific for rheumatoid arthritis (RA). ACPA-positive RA is a chronic inflammatory disease resulting from the complex interaction between genetic (mainly HLA class II genes) and environmental factors (mainly smoking). Recent findings have offered new insights into where, when and how anti-citrulline immunity develops. Some studies have found that a mucosal site, such as the lungs, may function as the initiating site for the immune response against citrullinated proteins, in line with the known association between smoking and ACPA. Other studies, focusing rather on the HLA associations, have suggested that cross-reactivity between microbial sequences and citrullinated self-proteins may lead to ACPA formation. Once ACPAs have developed, they can circulate throughout the body and upon reaching the joints exert direct pathogenic effects themselves. ACPAs can target first the bone compartment of the joints to activate osteoclasts and release interleukin (IL)-8 that in turn will promote bone loss and pain-like behaviour. In the current review, we will present the current understanding of the genetic associations in RA contributing to ACPA occurrence and offer insight in the latest findings explaining how and why autoimmunity generated in the lungs of genetically susceptible hosts might lead to chronic inflammation in the joints.

Topics: Anti-Citrullinated Protein Antibodies; Arthritis, Rheumatoid; Autoantibodies; Autoimmunity; Genetic Predisposition to Disease; HLA Antigens; Humans; Inflammation; Interleukin-8; Smoking

Topics: Animals; Arthritis, Rheumatoid; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Parvovirus B19, Human; Transcription Factors; Tumor Necrosis Factor-alpha; Viral Nonstructural Proteins

Topics: Animals; Antibodies, Monoclonal; Arthritis, Rheumatoid; Disease Models, Animal; Humans; Inflammation Mediators; Interleukin-8; Neutrophil Activation; Neutrophil Infiltration; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Synovial Fluid

Topics: Arthritis, Rheumatoid; Behcet Syndrome; Biomarkers; Bronchitis; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-10; Interleukin-7; Interleukin-8; Interleukin-9; Lupus Erythematosus, Systemic; Psoriasis; Reagent Kits, Diagnostic; Sjogren's Syndrome

A variety of factors have been identified that regulate angiogenesis, including the CXC chemokine family. The CXC chemokines are a unique family of cytokines for their ability to behave in a disparate manner in the regulation of angiogenesis. CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine amino acid residues separated by one non-conserved amino acid residue (i.e., CXC). A second structural domain within this family determines their angiogenic potential. The NH2 terminus of the majority of the CXC chemokines contains three amino acid residues (Glu-Leu-Arg: the ELR motif), which precedes the first cysteine amino acid residue of the primary structure of these cytokines. Members that contain the ELR motif (ELR+) are potent promoters of angiogenesis. In contrast, members that are inducible by interferons and lack the ELR motif (ELR-) are potent inhibitors of angiogenesis. This difference in angiogenic activity may impact on the pathogenesis of a variety of disorders.

Topics: Amino Acid Motifs; Angiogenesis Inhibitors; Animals; Arthritis, Rheumatoid; Chemokine CXCL10; Chemokines, CXC; Chronic Disease; Fibrosis; Humans; Inflammation; Interleukin-8; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Pulmonary Fibrosis; Receptors, Chemokine; Structure-Activity Relationship

Topics: Animals; Anti-Inflammatory Agents; Antibodies; Antibody Specificity; Arthritis, Rheumatoid; Autoantibodies; Blood Coagulation Factors; Chemokine CXCL5; Chemokines; Chemokines, CC; Chemokines, CXC; Chemotaxis, Leukocyte; Humans; Interleukin-8; Peptides; Receptors, Chemokine; Synovial Fluid; Synovitis

Epithelial neutrophil-activating protein 78 (ENA-78) is a member of the CXC chemokines and acts as a potent chemoattractant and activator of neutrophil function. On stimulation in vitro, ENA-78 is highly expressed in many cell types. ENA-78 protein levels are strongly elevated in synovial fluid and blood of patients with rheumatoid arthritis. By in situ hybridization and immunofluorescence staining, ENA-78 has been recognized as a major CXC chemokine expressed in epithelial cells of the intestinal mucosa of patients with Crohn's disease, ulcerative colitis, and acute appendicitis. A high expression of ENA-78 and interleukin-8 (IL-8) was also observed in the exocrine tissue of patients with chronic pancreatitis (CP). It is interesting to note that expression of IP-10, MIP-1alpha, and MCP-1 is high in healthy pancreatic tissue but low in tissue of patients with CP, suggesting a mutually exclusive expression of the ELR-CXC vs. non-ELR-CXC/CC chemokines. High-resolution studies of intracellular chemokines has revealed specific immunoreactivity for ENA-78 associated with the endoplasmic reticulum of many cell types. In contrast, GROalpha immunoreactivity was exclusively localized in the nucleus. Despite their common effects on neutrophil functions, the differential intracellular localization of ENA-78 and GROalpha suggests additional roles for these two chemokines in normal cell biology.

Topics: Arthritis, Rheumatoid; Chemokine CXCL5; Chemokines, CXC; Chronic Disease; Humans; Inflammatory Bowel Diseases; Interleukin-8; Monocytes; Pancreatitis; Respiratory Distress Syndrome

Topics: Animals; Arthritis, Rheumatoid; Interleukin-8

Topics: Arteriosclerosis; Arthritis, Rheumatoid; Chemokine CCL2; Chemotactic Factors; Humans; Interleukin-8; Lung Diseases; Neutrophils; Reperfusion Injury

Middle East Respiratory Syndrome (MERS) is a novel respiratory illness firstly reported in Saudi Arabia in 2012. It is caused by a new corona virus, called MERS corona virus (MERS-CoV). Most people who have MERS-CoV infection developed severe acute respiratory illness.. This work is done to determine the clinical characteristics and the outcome of intensive care unit (ICU) admitted patients with confirmed MERS-CoV infection.. This study included 32 laboratory confirmed MERS corona virus infected patients who were admitted into ICU. It included 20 (62.50%) males and 12 (37.50%) females. The mean age was 43.99 ± 13.03 years. Diagnosis was done by real-time reverse transcription polymerase chain reaction (rRT-PCR) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. Clinical characteristics, co-morbidities and outcome were reported for all subjects.. Most MERS corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. The presence of abdominal symptoms may indicate bad prognosis. Prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. Also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, COPD, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of ICU admitted MERS corona virus infected patients.. Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (. SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease.. A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.. HFNC did not significantly modify work of breathing in healthy subjects. However, a significant reduction in the minute volume was achieved, capillary [Formula: see text] remaining constant, which suggests a reduction in dead-space ventilation with flows > 20 L/min. (ClinicalTrials.gov registration NCT02495675).. 3 组患者手术时间、术中显性失血量及术后 1 周血红蛋白下降量比较差异均无统计学意义（. 对于肥胖和超重的膝关节单间室骨关节炎患者，采用 UKA 术后可获满意短中期疗效，远期疗效尚需进一步随访观察。.. Decreased muscle strength was identified at both time points in patients with hEDS/HSD. The evolution of most muscle strength parameters over time did not significantly differ between groups. Future studies should focus on the effectiveness of different types of muscle training strategies in hEDS/HSD patients.. These findings support previous adverse findings of e-cigarette exposure on neurodevelopment in a mouse model and provide substantial evidence of persistent adverse behavioral and neuroimmunological consequences to adult offspring following maternal e-cigarette exposure during pregnancy. https://doi.org/10.1289/EHP6067.. This RCT directly compares a neoadjuvant chemotherapy regimen with a standard CROSS regimen in terms of overall survival for patients with locally advanced ESCC. The results of this RCT will provide an answer for the controversy regarding the survival benefits between the two treatment strategies.. NCT04138212, date of registration: October 24, 2019.. Results of current investigation indicated that milk type and post fermentation cooling patterns had a pronounced effect on antioxidant characteristics, fatty acid profile, lipid oxidation and textural characteristics of yoghurt. Buffalo milk based yoghurt had more fat, protein, higher antioxidant capacity and vitamin content. Antioxidant and sensory characteristics of T. If milk is exposed to excessive amounts of light, Vitamins B. The two concentration of ZnO nanoparticles in the ambient air produced two different outcomes. The lower concentration resulted in significant increases in Zn content of the liver while the higher concentration significantly increased Zn in the lungs (p < 0.05). Additionally, at the lower concentration, Zn content was found to be lower in brain tissue (p < 0.05). Using TEM/EDX we detected ZnO nanoparticles inside the cells in the lungs, kidney and liver. Inhaling ZnO NP at the higher concentration increased the levels of mRNA of the following genes in the lungs: Mt2 (2.56 fold), Slc30a1 (1.52 fold) and Slc30a5 (2.34 fold). At the lower ZnO nanoparticle concentration, only Slc30a7 mRNA levels in the lungs were up (1.74 fold). Thus the two air concentrations of ZnO nanoparticles produced distinct effects on the expression of the Zn-homeostasis related genes.. Until adverse health effects of ZnO nanoparticles deposited in organs such as lungs are further investigated and/or ruled out, the exposure to ZnO nanoparticles in aerosols should be avoided or minimised.

Topics: A549 Cells; Acetylmuramyl-Alanyl-Isoglutamine; Acinetobacter baumannii; Acute Lung Injury; Adaptor Proteins, Signal Transducing; Adenine; Adenocarcinoma; Adipogenesis; Administration, Cutaneous; Administration, Ophthalmic; Adolescent; Adsorption; Adult; Aeromonas hydrophila; Aerosols; Aged; Aged, 80 and over; Aging; Agriculture; Air Pollutants; Air Pollution; Airway Remodeling; Alanine Transaminase; Albuminuria; Aldehyde Dehydrogenase 1 Family; Algorithms; AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase; Alzheimer Disease; Amino Acid Sequence; Ammonia; Ammonium Compounds; Anaerobiosis; Anesthetics, Dissociative; Anesthetics, Inhalation; Animals; Anti-Bacterial Agents; Anti-HIV Agents; Anti-Infective Agents; Anti-Inflammatory Agents; Antibiotics, Antineoplastic; Antibodies, Antineutrophil Cytoplasmic; Antibodies, Monoclonal, Humanized; Antifungal Agents; Antigens, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Antioxidants; Antitubercular Agents; Antiviral Agents; Apolipoproteins E; Apoptosis; Arabidopsis; Arabidopsis Proteins; Arsenic; Arthritis, Rheumatoid; Asthma; Atherosclerosis; ATP-Dependent Proteases; Attitude of Health Personnel; Australia; Austria; Autophagy; Axitinib; Bacteria; Bacterial Outer Membrane Proteins; Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Bariatric Surgery; Base Composition; Bayes Theorem; Benzoxazoles; Benzylamines; beta Catenin; Betacoronavirus; Betula; Binding Sites; Biological Availability; Biological Oxygen Demand Analysis; Biomarkers; Biomarkers, Tumor; Biopsy; Bioreactors; Biosensing Techniques; Birth Weight; Blindness; Blood Chemical Analysis; Blood Gas Analysis; Blood Glucose; Blood Pressure; Blood Pressure Monitoring, Ambulatory; Blood-Brain Barrier; Blotting, Western; Body Mass Index; Body Weight; Bone and Bones; Bone Density; Bone Resorption; Borates; Brain; Brain Infarction; Brain Injuries, Traumatic; Brain Neoplasms; Breakfast; Breast Milk Expression; Breast Neoplasms; Bronchi; Bronchoalveolar Lavage Fluid; Buffaloes; Cadherins; Calcification, Physiologic; Calcium Compounds; Calcium, Dietary; Cannula; Caprolactam; Carbon; Carbon Dioxide; Carboplatin; Carcinogenesis; Carcinoma, Ductal; Carcinoma, Ehrlich Tumor; Carcinoma, Hepatocellular; Carcinoma, Non-Small-Cell Lung; Carcinoma, Pancreatic Ductal; Carcinoma, Renal Cell; Cardiovascular Diseases; Carps; Carrageenan; Case-Control Studies; Catalysis; Catalytic Domain; Cattle; CD8-Positive T-Lymphocytes; Cell Adhesion; Cell Cycle Proteins; Cell Death; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Phone Use; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Cellulose; Chemical Phenomena; Chemoradiotherapy; Child; Child Development; Child, Preschool; China; Chitosan; Chlorocebus aethiops; Cholecalciferol; Chromatography, Liquid; Circadian Clocks; Circadian Rhythm; Circular Dichroism; Cisplatin; Citric Acid; Clinical Competence; Clinical Laboratory Techniques; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Clostridioides difficile; Clostridium Infections; Coculture Techniques; Cohort Studies; Cold Temperature; Colitis; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type XI; Color; Connective Tissue Diseases; Copper; Coronary Angiography; Coronavirus 3C Proteases; Coronavirus Infections; Cost of Illness; Counselors; COVID-19; COVID-19 Testing; Creatine Kinase; Creatinine; Cross-Over Studies; Cross-Sectional Studies; Cryoelectron Microscopy; Cryosurgery; Crystallography, X-Ray; Cues; Cultural Competency; Cultural Diversity; Curriculum; Cyclic AMP Response Element-Binding Protein; Cyclin-Dependent Kinase Inhibitor p21; Cycloparaffins; Cysteine Endopeptidases; Cytokines; Cytoplasm; Cytoprotection; Databases, Factual; Denitrification; Deoxycytidine; Diabetes Complications; Diabetes Mellitus; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diagnosis, Differential; Diatoms; Diet; Diet, High-Fat; Dietary Exposure; Diffusion Magnetic Resonance Imaging; Diketopiperazines; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Disease Progression; Disease-Free Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; DNA, Bacterial; DNA, Viral; Docetaxel; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Down-Regulation; Doxorubicin; Drosophila; Drosophila melanogaster; Drug Carriers; Drug Delivery Systems; Drug Liberation; Drug Repositioning; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy, Combination; Edema; Edible Grain; Education, Graduate; Education, Medical, Graduate; Education, Pharmacy; Ehlers-Danlos Syndrome; Electron Transport Complex III; Electron Transport Complex IV; Electronic Nicotine Delivery Systems; Emergency Service, Hospital; Empathy; Emulsions; Endothelial Cells; Endurance Training; Energy Intake; Enterovirus A, Human; Environment; Environmental Monitoring; Enzyme Assays; Enzyme Inhibitors; Epithelial Cells; Epithelial-Mesenchymal Transition; Epoxide Hydrolases; Epoxy Compounds; Erythrocyte Count; Erythrocytes; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagectomy; Estrogens; Etanercept; Ethiopia; Ethnicity; Ethylenes; Exanthema; Exercise; Exercise Test; Exercise Tolerance; Extracellular Matrix; Extracorporeal Membrane Oxygenation; Eye Infections, Fungal; False Negative Reactions; Fatty Acids; Fecal Microbiota Transplantation; Feces; Female; Femur Neck; Fermentation; Ferritins; Fetal Development; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Fibroblasts; Fibroins; Fish Proteins; Flavanones; Flavonoids; Focus Groups; Follow-Up Studies; Food Handling; Food Supply; Food, Formulated; Forced Expiratory Volume; Forests; Fractures, Bone; Fruit and Vegetable Juices; Fusobacteria; G1 Phase Cell Cycle Checkpoints; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Gastrectomy; Gastrointestinal Microbiome; Gastrointestinal Stromal Tumors; Gefitinib; Gels; Gemcitabine; Gene Amplification; Gene Expression; Gene Expression Regulation; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Neoplastic; Gene Expression Regulation, Plant; Gene Knockdown Techniques; Gene-Environment Interaction; Genotype; Germany; Glioma; Glomerular Filtration Rate; Glucagon; Glucocorticoids; Glycemic Control; Glycerol; Glycogen Synthase Kinase 3 beta; Glycolipids; Glycolysis; Goblet Cells; Gram-Negative Bacterial Infections; Granulocyte Colony-Stimulating Factor; Graphite; Greenhouse Effect; Guanidines; Haemophilus influenzae; HCT116 Cells; Health Knowledge, Attitudes, Practice; Health Personnel; Health Services Accessibility; Health Services Needs and Demand; Health Status Disparities; Healthy Volunteers; Heart Failure; Heart Rate; Heart Transplantation; Heart-Assist Devices; HEK293 Cells; Heme; Heme Oxygenase-1; Hemolysis; Hemorrhage; Hepatitis B; Hepatitis B e Antigens; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatitis B, Chronic; Hepatocytes; Hexoses; High-Throughput Nucleotide Sequencing; Hippo Signaling Pathway; Histamine; Histamine Agonists; Histidine; Histone Deacetylase 2; HIV Infections; HIV Reverse Transcriptase; HIV-1; Homebound Persons; Homeodomain Proteins; Homosexuality, Male; Hospice and Palliative Care Nursing; HSP70 Heat-Shock Proteins; Humans; Hyaluronan Receptors; Hydrogen; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydrolysis; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypoglycemia; Hypoglycemic Agents; Hypoxia; Idiopathic Interstitial Pneumonias; Imaging, Three-Dimensional; Imatinib Mesylate; Immunotherapy; Implementation Science; Incidence; INDEL Mutation; Induced Pluripotent Stem Cells; Industrial Waste; Infant; Infant, Newborn; Inflammation; Inflammation Mediators; Infliximab; Infusions, Intravenous; Inhibitory Concentration 50; Injections; Insecticides; Insulin-Like Growth Factor Binding Protein 5; Insulin-Secreting Cells; Interleukin-1; Interleukin-17; Interleukin-8; Internship and Residency; Intestines; Intracellular Signaling Peptides and Proteins; Ion Transport; Iridaceae; Iridoid Glucosides; Islets of Langerhans Transplantation; Isodon; Isoflurane; Isotopes; Italy; Joint Instability; Ketamine; Kidney; Kidney Failure, Chronic; Kidney Function Tests; Kidney Neoplasms; Kinetics; Klebsiella pneumoniae; Knee Joint; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Lactate Dehydrogenase 5; Laparoscopy; Laser Therapy; Lasers, Semiconductor; Lasers, Solid-State; Laurates; Lead; Leukocyte L1 Antigen Complex; Leukocytes, Mononuclear; Light; Lipid Peroxidation; Lipopolysaccharides; Liposomes; Liver; Liver Cirrhosis; Liver Neoplasms; Liver Transplantation; Locomotion; Longitudinal Studies; Lopinavir; Lower Urinary Tract Symptoms; Lubricants; Lung; Lung Diseases, Interstitial; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Lymphoma, Mantle-Cell; Lysosomes; Macrophages; Male; Manganese Compounds; MAP Kinase Kinase 4; Mass Screening; Maternal Health; Medicine, Chinese Traditional; Melanoma, Experimental; Memantine; Membrane Glycoproteins; Membrane Proteins; Mesenchymal Stem Cell Transplantation; Metal Nanoparticles; Metalloendopeptidases; Metalloporphyrins; Methadone; Methane; Methicillin-Resistant Staphylococcus aureus; Mexico; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Mice, Nude; Mice, SCID; Mice, Transgenic; Microarray Analysis; Microbial Sensitivity Tests; Microbiota; Micronutrients; MicroRNAs; Microscopy, Confocal; Microsomes, Liver; Middle Aged; Milk; Milk, Human; Minority Groups; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Models, Animal; Models, Molecular; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Epidemiology; Molecular Structure; Molecular Weight; Multilocus Sequence Typing; Multimodal Imaging; Muscle Strength; Muscle, Skeletal; Muscular Diseases; Mutation; Mycobacterium tuberculosis; Myocardial Stunning; Myristates; NAD(P)H Dehydrogenase (Quinone); Nanocomposites; Nanogels; Nanoparticles; Nanotechnology; Naphthalenes; Nasal Cavity; National Health Programs; Necrosis; Needs Assessment; Neoadjuvant Therapy; Neonicotinoids; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasm Transplantation; Neoplasms; Neoplastic Stem Cells; Netherlands; Neuroblastoma; Neuroprotective Agents; Neutrophils; NF-kappa B; NFATC Transcription Factors; Nicotiana; Nicotine; Nitrates; Nitrification; Nitrites; Nitro Compounds; Nitrogen; Nitrogen Dioxide; North Carolina; Nuclear Magnetic Resonance, Biomolecular; Nuclear Proteins; Nucleic Acid Hybridization; Nucleosomes; Nutrients; Obesity; Obesity, Morbid; Oceans and Seas; Oncogene Protein v-akt; Oncogenes; Oocytes; Open Reading Frames; Osteoclasts; Osteogenesis; Osteoporosis; Osteoporosis, Postmenopausal; Outpatients; Ovarian Neoplasms; Ovariectomy; Overweight; Oxazines; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxidoreductases; Oxygen; Oxygen Inhalation Therapy; Oxygenators, Membrane; Ozone; Paclitaxel; Paenibacillus; Pain Measurement; Palliative Care; Pancreatic Neoplasms; Pandemics; Parasympathetic Nervous System; Particulate Matter; Pasteurization; Patient Preference; Patient Satisfaction; Pediatric Obesity; Permeability; Peroxiredoxins; Peroxynitrous Acid; Pharmaceutical Services; Pharmacists; Pharmacy; Phaseolus; Phenotype; Phoeniceae; Phosphates; Phosphatidylinositol 3-Kinases; Phospholipid Transfer Proteins; Phospholipids; Phosphorus; Phosphorylation; Photoperiod; Photosynthesis; Phylogeny; Physical Endurance; Physicians; Pilot Projects; Piperidines; Pituitary Adenylate Cyclase-Activating Polypeptide; Plant Extracts; Plant Leaves; Plant Proteins; Plant Roots; Plaque, Atherosclerotic; Pneumonia; Pneumonia, Viral; Point-of-Care Testing; Polyethylene Glycols; Polymers; Polysorbates; Pore Forming Cytotoxic Proteins; Positron Emission Tomography Computed Tomography; Positron-Emission Tomography; Postprandial Period; Poverty; Pre-Exposure Prophylaxis; Prediabetic State; Predictive Value of Tests; Pregnancy; Pregnancy Trimester, First; Pregnancy, High-Risk; Prenatal Exposure Delayed Effects; Pressure; Prevalence; Primary Graft Dysfunction; Primary Health Care; Professional Role; Professionalism; Prognosis; Progression-Free Survival; Prolactin; Promoter Regions, Genetic; Proof of Concept Study; Proportional Hazards Models; Propylene Glycol; Prospective Studies; Prostate; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proteoglycans; Proteome; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-ret; Proto-Oncogene Proteins p21(ras); Proton Pumps; Protons; Protoporphyrins; Pseudomonas aeruginosa; Pseudomonas fluorescens; Pulmonary Artery; Pulmonary Disease, Chronic Obstructive; Pulmonary Gas Exchange; Pulmonary Veins; Pyrazoles; Pyridines; Pyrimidines; Qualitative Research; Quinoxalines; Rabbits; Random Allocation; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Histamine H3; Receptors, Immunologic; Receptors, Transferrin; Recombinant Proteins; Recurrence; Reference Values; Referral and Consultation; Regional Blood Flow; Registries; Regulon; Renal Insufficiency, Chronic; Reperfusion Injury; Repressor Proteins; Reproducibility of Results; Republic of Korea; Research Design; Resistance Training; Respiration, Artificial; Respiratory Distress Syndrome; Respiratory Insufficiency; Resuscitation; Retinal Dehydrogenase; Retreatment; Retrospective Studies; Reverse Transcriptase Inhibitors; Rhinitis, Allergic; Ribosomal Proteins; Ribosomes; Risk Assessment; Risk Factors; Ritonavir; Rivers; RNA Interference; RNA-Seq; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Small Interfering; Rosuvastatin Calcium; Rural Population; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Salivary Ducts; Salivary Gland Neoplasms; San Francisco; SARS-CoV-2; Satiation; Satiety Response; Schools; Schools, Pharmacy; Seasons; Seawater; Selection, Genetic; Sequence Analysis, DNA; Serine-Threonine Kinase 3; Sewage; Sheep; Sheep, Domestic; Shock, Hemorrhagic; Signal Transduction; Silver; Silymarin; Single Photon Emission Computed Tomography Computed Tomography; Sirolimus; Sirtuin 1; Skin; Skin Neoplasms; Skin Physiological Phenomena; Sleep Initiation and Maintenance Disorders; Social Class; Social Participation; Social Support; Soil; Soil Microbiology; Solutions; Somatomedins; Soot; Specimen Handling; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Spinal Fractures; Spirometry; Staphylococcus aureus; STAT1 Transcription Factor; STAT3 Transcription Factor; Streptomyces coelicolor; Stress, Psychological; Stroke; Stroke Volume; Structure-Activity Relationship; Students, Medical; Students, Pharmacy; Substance Abuse Treatment Centers; Sulfur Dioxide; Surface Properties; Surface-Active Agents; Surveys and Questionnaires; Survival Analysis; Survival Rate; Survivin; Sweden; Swine; Swine, Miniature; Sympathetic Nervous System; T-Lymphocytes, Regulatory; Talaromyces; Tandem Mass Spectrometry; tau Proteins; Telemedicine; Telomerase; Telomere; Telomere Homeostasis; Temperature; Terminally Ill; Th1 Cells; Thiamethoxam; Thiazoles; Thiophenes; Thioredoxin Reductase 1; Thrombosis; Thulium; Thyroid Cancer, Papillary; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Time Factors; Titanium; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; TOR Serine-Threonine Kinases; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transcriptome; Transforming Growth Factor beta1; Transistors, Electronic; Translational Research, Biomedical; Transplantation Tolerance; Transplantation, Homologous; Transportation; Treatment Outcome; Tretinoin; Tuberculosis, Multidrug-Resistant; Tuberculosis, Pulmonary; Tubulin Modulators; Tumor Microenvironment; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha; Twins; Ultrasonic Therapy; Ultrasonography; Ultraviolet Rays; United States; Up-Regulation; Uranium; Urethra; Urinary Bladder; Urodynamics; Uromodulin; Uveitis; Vasoconstrictor Agents; Ventricular Function, Left; Vero Cells; Vesicular Transport Proteins; Viral Nonstructural Proteins; Visual Acuity; Vital Capacity; Vitamin D; Vitamin D Deficiency; Vitamin K 2; Vitamins; Volatilization; Voriconazole; Waiting Lists; Waste Disposal, Fluid; Wastewater; Water Pollutants, Chemical; Whole Genome Sequencing; Wine; Wnt Signaling Pathway; Wound Healing; Wounds and Injuries; WW Domains; X-linked Nuclear Protein; X-Ray Diffraction; Xanthines; Xenograft Model Antitumor Assays; YAP-Signaling Proteins; Yogurt; Young Adult; Zebrafish; Zebrafish Proteins; Ziziphus

Binding immunoglobulin protein (BiP) is a human endoplasmic reticulum-resident stress protein. In pre-clinical studies it has anti-inflammatory properties due to the induction of regulatory cells. This randomized placebo-controlled, dose ascending double blind phase I/IIA trial of BiP in patients with active RA, who had failed accepted therapies, had the primary objective of safety. Potential efficacy was measured by DAS28-ESR and changes in biomarkers.. Twenty-four patients with active RA who had failed one or more DMARDs were sequentially assigned to three groups each of eight patients randomly allocated to receive placebo (two patients) or BiP (six patients), 1, 5 or 15 mg. Patients received a single i.v. infusion over 1 h and were observed as inpatients overnight. A 12-week follow-up for clinical, rheumatological and laboratory assessments for safety, efficacy (DAS28-ESR) and biomarker analysis was performed.. No infusion reactions or serious adverse drug reactions were noted. Adverse events were evenly distributed between placebo and BiP groups with no BiP-related toxicities. Haematological, renal and metabolic parameters showed no drug-related toxicities. Remission was only achieved by patients in the 5 and 15 mg groups, and not patients who received placebo or 1 mg BiP. Good DAS28-ESR responses were achieved in all treatment groups. The BiP responding patients showed significantly lower serum concentrations of CRP, 2 weeks post-infusion compared with pre-infusion levels, and of VEGF and IL-8 from the placebo group.. BiP (⩽15 mg) is safe in patients with active RA. Some patients had clinical and biological improvements in RA activity. BiP merits further study.. ISRCTN registry, http://isrctn.com, ISRCTN22288225 and EudraCT, https://eudract.ema.europa.eu, 2011-005831-19.

Topics: Adolescent; Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Biological Products; Biomarkers; Double-Blind Method; Female; Humans; Infusions, Intravenous; Interleukin-8; Lymphokines; Male; Middle Aged; Recombinant Proteins; Remission Induction; Treatment Outcome; Vascular Endothelial Growth Factor A; Young Adult

Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.

Topics: Arthritis, Rheumatoid; Cell Movement; Chemokine CCL2; Cytokines; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; Nicotinamide Phosphoribosyltransferase; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Synovial Membrane

Helicobacter pylori (H. pylori) is associated with chronic gastritis and gastric carcinogenesis. The effects of nonsteroidal anti-inflammatory drugs (NSAIDs), which exert chemopreventive effects on several cancers, on H. pylori-induced gastritis remain unknown. We investigated the effects of NSAIDs on gastric inflammation and the kinetics of gastric epithelial cells in H. pylori-induced gastritis.. Patients with rheumatoid arthritis or osteoarthritis who took NSAIDs for more than 1 month and complained of dyspeptic symptoms were recruited for this study. Patients not on any NSAIDs were included as non-NSAID user controls. All patients underwent diagnostic testing for H. pylori infection, esophagogastroduodenoscopy, and gastric biopsies. Neutrophil infiltration into gastric mucosa, expression of inducible nitric oxide synthase (iNOS), and apoptosis and proliferation of gastric epithelial cells were evaluated by immunohistochemistry. In an in vitro study, the effects of NSAIDs on production of interleukin (IL)-8 induced by H. pylori in a gastric epithelial cell line (AGS) were determined.. Numbers of neutrophils infiltrating the gastric mucosa, iNOS-expressing inflammatory cells and apoptotic cells, and proliferating cells in gastric epithelium were higher in H. pylori-positive groups than H. pylori-negative groups. Among H. pyloripositive groups, these parameters were lower in NSAID users than in non-NSAID users. NSAIDs inhibited the production of IL-8 induced by H. pylori in AGS cells.. These findings suggest that long-term use of NSAIDs normalizes the kinetics of gastric epithelial cells in patients with H. pylori infection by attenuating gastric mucosal inflammation, which may result in prevention of the gastric carcinogenesis associated with H. pylori infection.

Topics: Adult; Aged; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cell Line, Tumor; Epithelial Cells; Female; Gastric Mucosa; Gene Expression Regulation, Enzymologic; Helicobacter Infections; Helicobacter pylori; Humans; Interleukin-8; Male; Middle Aged; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Osteoarthritis; Time Factors

Synovial biomarkers are increasingly important in the development of novel therapeutic agents for the treatment of rheumatoid arthritis (RA). To identify biomarkers correlating with changes in clinical disease activity, real-time quantitative PCR (Q-PCR) was used to evaluate changes in synovial gene expression after treatment with corticosteroids. Patients with active RA received either oral prednisolone (n=10, 60 mg daily for the first week and 40 mg daily for the second week) or placebo (n=11) for 14 days. Real-time Q-PCR was used to quantify gene expression of tumour necrosis factor (TNF)alpha, IL1beta, IL8 and matrix metalloproteinase (MMP) 1 in synovial tissue samples obtained through an arthroscopic procedure before and after treatment. mRNA levels were reported as relative expression units compared with a cell-based standard. Statistical analysis was performed using an analysis of covariance model. Prednisolone markedly decreased IL8 and MMP1 expression compared with placebo, and the CIs excluded the likelihood of no effect. A trend towards reduction was seen in IL1beta and TNFalpha mRNA expression in the prednisolone group, although CIs included the value for no effect. These data suggest that Q-PCR can be used to measure synovial mRNA expression of mediators implicated in the pathogenesis of RA in small proof-of-concept trials.

Topics: Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Biomarkers; Cytokines; Female; Gene Expression Regulation; Glucocorticoids; Humans; Interleukin-1beta; Interleukin-8; Male; Matrix Metalloproteinase 1; Middle Aged; Polymerase Chain Reaction; Prednisolone; RNA, Messenger; Severity of Illness Index; Synovial Membrane; Treatment Outcome; Tumor Necrosis Factor-alpha

Leflunomide is a novel immunomodulating drug that has recently been approved as a disease-modifying antirheumatic drug for the treatment of rheumatoid arthritis (RA). The aim of this study was to determine the relationship between the clinical effects of leflunomide and neutrophil migration.. The effects of leflunomide and methotrexate on neutrophil chemotaxis were studied in 15 RA patients who participated in a prospective, randomized, double-blind clinical trial. When possible, neutrophil numbers were counted in synovial fluid (SF) samples at baseline and after 14 days, 4 months, and 1 year of treatment. The chemotactic properties of peripheral blood neutrophils from RA patients treated with either leflunomide or methotrexate were studied by the Boyden chamber technique, using the activators formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). The in vitro effects of A77 1726, the active metabolite of leflunomide, and methotrexate on peripheral blood neutrophils from 7 healthy control subjects were also investigated.. Both therapy groups exhibited clinical improvement, including rapid reductions in SF neutrophil counts and reduced joint swelling and tenderness. On day 14, 3 of 7 patients who received leflunomide showed no detectable effusions. There was a significant effect on neutrophil chemotaxis (P < 0.001), which was similar for leflunomide and methotrexate. The direct effects on the neutrophils diminished over time. Incubation of peripheral blood neutrophils from healthy controls with A77 1726 confirmed the inhibitory effect on chemotaxis.. Leflunomide treatment is beneficial in RA patients. Different mechanisms are operative in various phases of treatment, leading to decreased recruitment of inflammatory cells in the joints.

Topics: Aged; Aniline Compounds; Antirheumatic Agents; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Crotonates; Dose-Response Relationship, Drug; Double-Blind Method; Humans; Hydroxybutyrates; Immunosuppressive Agents; In Vitro Techniques; Interleukin-8; Isoxazoles; Leflunomide; Methotrexate; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitriles; Prospective Studies; Toluidines

The chemokine interleukin-8 (IL-8) is frequently associated with inflammatory diseases, and autoantibodies against IL-8 are present in the periphery at elevated levels in such conditions as rheumatoid arthritis (RA). Circulating free anti-IL-8 IgG autoantibodies correlate with inflammatory parameters and disease severity in RA. In this study, correlations were sought between these disease parameters and other antibody subclasses. We assayed IgM, IgA and IgG anti-IL-8 antibodies and IL-8 immunoglobulin immune complexes in the serum of 29 healthy controls and 56 patients with defined RA, and compared the results with clinical and humoral disease parameters. IgG and IgM antibodies directed against IL-8 were present in all samples. In the disease groups, all isotypes of free anti-IL-8 antibodies correlated with increasing humoral disease parameters like CRP and CIC and their related anti-IL-8 immune complexes. Samples which contained high titers of anti-IL-8 antibody subclasses and complexes were RF subclass-positive, while IgM RF-negative sera showed low levels of anti-IL-8 and complexes. Detectable levels of IgG and IgA RF were found in all sera. Patients with extra-articular organ manifestation showed significantly increased free IgA and IgA/IL-8 complexes, with no correlation to the IgA RF titer or IgA hypergamma-globulinemia. The highest titers were seen in two RA cases with vasculitis and in one patient with colitis. Polyclonal activation of the humoral antibody system, which normally precedes the resolution of an inflammatory response, can itself lead to secondary stimulation of inflammatory processes via immune complex formation. In the immune pathology of RA, it degenerates into a persistent chronic inflammation accompanied by progressive joint destruction. The presence of elevated IgA subclass anti-IL-8 autoantibodies in RA patients with extra-articular manifestations suggests these autoantibodies as a clinically useful marker of disease severity and extra-articular manifestations.

Topics: Adult; Aged; Antibodies, Anti-Idiotypic; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Female; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Interleukin-8; Male; Middle Aged; Prognosis; Reference Values; Sensitivity and Specificity; Severity of Illness Index

5 Patients with definite RA and knee effusions under constant doses of DMARD therapy were treated with up to 6 intraarticular injections of 10 mg methotrexate (MTX) every 3 to 7 days. A matched randomized control group who received a single i.a. injection of 40 mg triamcinolone hexacetonide (TC) was monitored according to the same protocol. The intraarticular granulocyte counts and IL-8 levels decreased in all MTX treated patients on day 10-13 and stayed low in those patients who could be re-evaluated after 13 weeks. Compared to the IL-8 levels, the other tested cytokine levels showed only minor changes on day 10-13. There was no need for re-injection in the TC group during the 13 week study phase. We conclude that intraarticular MTX therapy results in a strong decrease of SF-granulocyte counts. This effect may be due to the impairment of IL-8 mediated chemotaxis by decreased IL-8 synthesis in synovial fluid mononuclear cells. Clinically, repeated intraarticular MTX therapy results in a worse 13 week outcome than i.a. steroid treatment measured in an intention-to-treat analysis.

Topics: Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Injections, Intra-Articular; Interleukin-8; Knee Joint; Methotrexate; Middle Aged; Synovial Fluid

The G-1 column is an extracorporeal type granulocytapheresis device packed with 220 g cellulose acetate beads to which granulocytes and monocytes specifically adhere. A total of 59 rheumatoid arthritis patients with elevated granulocyte counts from 4 hospitals in Japan received 2 apheresis sessions of 1 h duration/week for a total of 8 times over a period of 4 weeks. About 55% of the leukocytes which entered the G-1 column were adsorbed onto the beads: 95% were granulocytes, 3.5% monocytes, and 0.4% lymphocytes. Clinical and efficacy assessments showed improvements in swollen joints (p < 0.01), tender joints (p < 0.001), the active joint score (p < 0.001), duration of morning stiffness (p < 0.01), and grip strength (p < 0.001). In good responders, the improvements were observed for up to 12 weeks following the last apheresis. Exacerbation was noted in 2 patients. It is suggested that the efficacy of the G-1 column is attributable to the removal or suppression of hyperactive leukocytes and inflammatory cytokines, inducing a kind of immunomodulation.

Topics: Adsorption; Adult; Aged; Arthritis, Rheumatoid; Blood Proteins; Cellulose; Cytapheresis; Female; Granulocytes; Humans; Interleukin-6; Interleukin-8; Leukocyte Count; Male; Middle Aged; Monocytes; Treatment Outcome; Tumor Necrosis Factor-alpha

Hypoxia and increased levels of inflammatory cytokines in the joints are characteristics of rheumatoid arthritis (RA). However, the effects of hypoxia and tumor necrosis factor-α (TNF-α) on interleukin (IL)-6 and IL-8 production on fibroblast-like synoviocytes (FLSs) remain to be clarified. This study aimed to explore how hypoxia and TNF-α affect the expression of IL-6 and IL-8 in human FLSs isolated from RA patients. Hypoxia or TNF-α treatment alone significantly increased the expression and promoter activity of IL-6, IL-8, and hypoxia-inducible factor-1α (HIF-1α). Treatment of hypoxic FLSs with TNF-α further significantly elevated the expression of these cytokines and enhanced promoter activity of HIF-1α, which was abrogated by treatment with the HIF-1α inhibitor YC-1. Similarly, TNF-α alone elevated the phosphorylation and promoter activity of nuclear factor-κBp65 (NF-κBp65) in the FLSs. These effects were further enhanced by the combined treatment of hypoxia and TNFα but were attenuated by the NF-κB inhibitor BAY11-7082. NF-κB-p65 inhibition decreased the effect of TNF-α on HIF-1α upregulation in the FLSs in response to hypoxia. The combination of hypoxia and TNF-α also significantly upregulated transforming growth factor-β-activated kinase 1 (TAK1) expression, and silencing TAK1 dramatically decreased NF-κB-p65, HIF-1α, IL-6, and IL-8 expression under the same conditions. Our results indicate that hypoxia and TNF-α synergistically increase IL-6 and IL-8 expression in human FLSs via enhancing TAK1/NF-κB/HIF-1α signaling.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Fibroblasts; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-6; Interleukin-8; NF-kappa B; Synoviocytes; Tumor Necrosis Factor-alpha

Topics: Animals; Arthritis, Rheumatoid; Cattle; Cells, Cultured; Cyclooxygenase 2; Cytokines; Dinoprostone; Fibroblasts; Interleukin-6; Interleukin-8; Lameness, Animal; Malates; Phosphatidylinositol 3-Kinases; Synovial Membrane; Synoviocytes; Tumor Necrosis Factor-alpha

Synovial monocytes (expressing CD14+CD16+) affect pro-inflammatory responses in the synovium microenvironment of psoriatic arthritis (PsA) and rheumatoid arthritis (RA). The effect of various drugs on those cells was evaluated.. Synovial fluid mononuclear cells (SFMCs) from PsA (n=29) and RA (n=11) patients were cultured with biologics or glucocorticoids (GCs). CD14+CD16+ cells were analysed by flow cytometry. TNF secretion was assessed by ELISA and changes in cytokine and matrix metalloproteinase-9 (MMP-9) mRNA by qPCR.. TNF inhibitors (i) [adalimumab (ADA) and infliximab (IFX)] significantly reduced the %CD14+CD16+ cells (p<0.04 and p<0.02, respectively) compared to IL-17Ai, IL-12/23i, and GCs in PsA patients' SFMCs. Similarly, those TNFi reduced the %CD14+CD16+ cells (p<0.05 and p<0.02, respectively) compared to IL-6Ri, CD20i and GCs in RA patients' SFMCs. TNFi (ADA p<0.01, IFX p=0.0003), and GCs (p<0.05) reduced TNF levels in PsA patients SFMCs supernatants. IFX down-regulated IL-1β mRNA (p<0.005) while GCs betamethasone (BET) (p<0.01) and methylprednisolone acetate (MPA) (p<0.005) led to IL-1β up-regulation. IFX down-regulated IL-8 and MMP-9 (p<0.01) and up-regulated IL-10 (p<0.005), and GCs did so to a greater extent (for IL-8, BET p<0.0001 and MPA p<0.005, for MMP-9, BET and MPA p<0.0001 and for IL-10, BET and MPA p<0.0001).. TNFi but not GCs reduced the inflammatory monocytes. Both TNFi and GCs inhibited TNF secretion but differently modulated IL-1β, IL-8, MMP-9 and IL-10 gene expression. Our data point to TNFi as a modulator of synovial monocytes.

Topics: Adalimumab; Arthritis, Psoriatic; Arthritis, Rheumatoid; Glucocorticoids; Humans; Infliximab; Interleukin-10; Interleukin-8; Matrix Metalloproteinase 9; Monocytes; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor Inhibitors; Tumor Necrosis Factor-alpha

Interleukin (IL)-40 is a new cytokine related to immune system function and malignancies. Recently, an association of IL-40 with rheumatoid arthritis (RA) and externalisation of neutrophil extracellular traps (NETosis) was found. As neutrophils are implicated in RA development, we investigated IL-40 in early stages of RA (ERA).. IL-40 was determined in serum of treatment naïve patients with ERA at baseline (n=60) and 3 months after initiation of conventional therapy and in healthy controls (HC; n=60). Levels of IL-40, cytokines and NETosis markers were measured by ELISA. NETosis was visualised by immunofluorescence. In vitro experiments were performed on peripheral blood neutrophils from ERA patients (n=14). Cell-free DNA was analysed in serum and supernatants.. Serum IL-40 was elevated in ERA compared with HC (p<0.0001) and normalised after 3 months of therapy (p<0.0001). Baseline serum IL-40 correlated with rheumatoid factor (IgM) (p<0.01), anti-cyclic citrullinated peptide (p<0.01) autoantibodies and NETosis markers (proteinase 3; neutrophil elastase (NE); myeloperoxidase) (p<0.0001). Levels of NE significantly decreased after therapy (p<0.01) and correlated with the decrease of serum IL-40 (p<0.05). In vitro, neutrophils enhanced IL-40 secretion following NETosis induction (p<0.001) or after exposure to IL-1β, IL-8 (p<0.05), tumour necrosis factor or lipopolysaccharide (p<0.01). Recombinant IL-40 up-regulated IL-1β, IL-6 and IL-8 (p<0.05 for all) in vitro.. We showed that IL-40 is significantly up-regulated in seropositive ERA and decreases after conventional therapy. Moreover, neutrophils are an important source of IL-40 in RA, and its release is potentiated by cytokines and NETosis. Thus, IL-40 may play a role in ERA.

Topics: Arthritis, Rheumatoid; Autoantibodies; Cytokines; Humans; Interleukin-8; Interleukins; Neutrophils

Interleukin (IL-) 6 is a critical factor in inflammatory processes of rheumatoid arthritis (RA). This is of high interest as the progression of RA may lead to the implantation of joint endoprostheses, which is associated with a pro-inflammatory increase in IL-6 in the periprosthetic tissue. Biological agents such as sarilumab have been developed to inhibit IL-6-mediated signaling. However, IL-6 signaling blockade should consider the inhibition of inflammatory processes and the regenerative functions of IL-6. This in vitro study investigated whether inhibiting IL-6 receptors can affect the differentiation of osteoblasts isolated from patients with RA. Since wear particles can be generated at the articular surfaces of endoprostheses leading to osteolysis and implant loosening, the potential of sarilumab to inhibit wear particle-induced pro-inflammatory processes should be investigated. Both in monocultures and indirect co-cultures with osteoclast-like cells (OLCs), human osteoblasts were stimulated with 50 ng/mL each of IL-6 + sIL-6R and in combination with sarilumab (250 nM) to characterize cell viability and osteogenic differentiation capacity. Furthermore, the influence of IL-6 + sIL-6R or sarilumab on viability, differentiation, and inflammation was evaluated in osteoblasts exposed to particles. Stimulation with IL-6 + sIL-6R and sarilumab did not affect cell viability. Except for the significant induction of RUNX2 mRNA by IL-6 + sIL-6R and a significant reduction with sarilumab, no effects on cell differentiation and mineralization could be detected. Furthermore, the different stimulations did not affect the osteogenic and osteoclastic differentiation of co-cultured cells. Compared to the osteoblastic monocultures, a decreased release of IL-8 was triggered in the co-culture. Among these, treatment with sarilumab alone resulted in the greatest reduction of IL-8. The co-culture also showed clearly increased OPN concentrations than the respective monocultures, with OPN secretion apparently triggered by the OLCs. Particle exposure demonstrated decreased osteogenic differentiation using different treatment strategies. However, sarilumab administration caused a trend toward a decrease in IL-8 production after stimulation with IL-6 + sIL-6R. The blockade of IL-6 and its pathway have no significant effect on the osteogenic and osteoclastic differentiation of bone cells derived from patients with RA. Nonetheless, observed effects on the reduced IL-8 secretion n

Topics: Arthritis, Rheumatoid; Cells, Cultured; Humans; Interleukin-6; Interleukin-8; Osteoblasts; Osteogenesis; Signal Transduction

High-temperature requirement serine protease A 2 (HtrA2) is known to be involved in growth, unfolded protein response to stress, apoptosis, and autophagy. However, whether HtrA2 controls inflammation and immune response remains elusive.. Expression of HtrA2 in the synovial tissue of patients was examined using immunohistochemistry and immunofluorescence staining. Enzyme-linked immunosorbent assay was used to determine the concentrations of HtrA2, interleukin-6 (IL-6), interleukin-8 (IL-8), chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor α (TNFα). Synoviocyte survival was assessed by MTT assay. For the downregulation of HtrA2 transcripts, cells were transfected with HtrA2 siRNA.. We found that the concentration of HtrA2 was elevated in rheumatoid arthritis (RA) synovial fluid (SF) than in osteoarthritis (OA) SF, and its concentrations were correlated with the number of immune cells in the RA SF. Interestingly, HtrA2 levels in the SF of RA patients were elevated in proportion to synovitis severity and correlated with the expression of proinflammation cytokines and chemokines, such as IL-6, IL-8, and CCL2. In addition, HtrA2 was highly expressed in RA synovium and primary synoviocytes. RA synoviocytes released HtrA2 when stimulated with ER stress inducers. Knockdown of HtrA2 inhibited the IL1β-, TNFα-, and LPS-induced release of proinflammatory cytokines and chemokines by RA synoviocytes.. HtrA2 is a novel inflammatory mediator and a potential target for the development of an anti-inflammation therapy for RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokines; Cytokines; Fibroblasts; Humans; Inflammation; Interleukin-6; Interleukin-8; Serine Endopeptidases; Serine Proteases; Synovial Membrane; Synoviocytes; Temperature; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is a systemic autoimmune disease causing joint dysfunction. As disease-modifying anti-rheumatic drugs (DMARDs) have poor efficacy in 20% to 25% of RA patients, additional novel RA medications are urgently needed. Schisandrin (SCH) has multiple therapeutic effects. However, whether SCH is effective against RA remains unknown.. To investigate how SCH affects the abnormal behaviours of RA fibroblast-like synoviocytes (FLSs) and further elucidate the underlying mechanism of SCH in RA FLSs and collagen-induced arthritis (CIA) mice.. Cell Counting Kit-8 (CCK8) assays were used to characterize cell viability. EdU assays were performed to assess cell proliferation. Annexin V-APC/PI assays were used to determine apoptosis. Transwell chamber assays were used to measure cell migration and invasion in vitro. RT-qPCR was used to assess proinflammatory cytokine and MMP mRNA expression. Western blotting was used to detect protein expression. RNA sequencing was performed to explore the potential downstream targets of SCH. CIA model mice were used to assess the treatment efficacy of SCH in vivo.. Treatments with SCH (50, 100, and 200 μΜ) inhibited RA FLSs proliferation, migration, invasion, and TNF-α-induced IL-6, IL-8, and CCL2 expression in a dose-dependent manner but did not affect RA FLSs viability or apoptosis. RNA sequencing and Reactome enrichment analysis indicated that SREBF1 might be the downstream target in SCH treatment. Furthermore, knockdown of SREBF1 exerted effects similar to those of SCH in inhibiting RA FLSs proliferation, migration, invasion, and TNF-α-induced expression of IL-6, IL-8, and CCL2. Both SCH treatment and SREBF1 knockdown decreased activation of the PI3K/AKT and NF-κB signalling pathways. Moreover, SCH ameliorated joint inflammation and cartilage and bone destruction in CIA model mice.. SCH controls the pathogenic behaviours of RA FLSs by targeting SREBF1-mediated activation of the PI3K/AKT and NF-κB signalling pathways. Our data suggest that SCH inhibits FLS-mediated synovial inflammation and joint damage and that SCH might have therapeutic potential for RA.

Topics: Animals; Antirheumatic Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Movement; Cell Proliferation; Cells, Cultured; Fibroblasts; Inflammation; Interleukin-6; Interleukin-8; Mice; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Synoviocytes; Tumor Necrosis Factor-alpha

To explore the role and underlying mechanism of Complement Factor H (CFH) in the peripheral and joint inflammation of RA patients.. The levels of CFH in the serum and synovial fluid were determined by ELISA. The pyroptosis of monocytes was determined by western blotting and flow cytometry. The inflammation cytokine release was tested by ELISA. The cell migration and invasion ability of fibroblast-like synoviocytes (FLS) were tested by Wound healing Assay and transwell assay, respectively. The potential target of CFH was identified by RNA sequencing.. CFH levels were significantly elevated in the serum and synovial fluid from RA and associated with high sensitivity C-reactive protein (hs-CRP), erythrocyte sedimentation rate (ESR), and disease activity score 28 (DAS28). TNF-α could inhibit CFH expression, and CFH combined with TNF-α significantly decreased cell death, cleaved-caspase 3, gasdermin E N-terminal (GSDME-N), and inflammatory cytokines release (IL-1β and IL-6) of RA-derived monocytes. Stimulated with TNF-α increased CFH levels in RA FLS and CFH inhibits the migration, invasion, and TNF-α-induced production of inflammatory mediators, including proinflammatory cytokines (IL-6, IL-8) as well as matrix metalloproteinases (MMPs, MMP1 and MMP3) of RA FLSs. The RNA-seq results showed that CFH treatment induced upregulation of eukaryotic translation initiation factor 3 (EIF3C) in both RA monocytes and FLS. The migration of RA FLSs was promoted and the expressions of IL-6, IL-8, and MMP-3 were enhanced upon EIF3C knockdown under the stimulation of CFH combined with TNF-α.. In conclusion, we have unfolded the anti-inflammatory roles of CFH in the peripheral and joints of RA, which might provide a potential therapeutic target for RA patients.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Complement Factor H; Cytokines; Fibroblasts; Humans; Inflammation; Interleukin-6; Interleukin-8; Synovial Membrane; Tumor Necrosis Factor-alpha

To investigate the ex vivo effect of the JAK1/2 inhibitor baricitinib on expression of pro-inflammatory mediators in rheumatoid arthritis (RA) fibroblast like synoviocytes (FLS) stimulated with TNFα, IL-1β and oncostatin M (OSM), and in RA synovial membrane cells (SMCs).. RA and osteoarthritis (OA) SMCs, were isolated from arthroplasty specimens of RA (n=8) and OA (n=8) patients, respectively, using enzymatic digestion followed by cell propagation to obtain RA (n=5) and OA (n=3) FLS. Normal FLS and normal human foreskin fibroblasts (HSF) were purchased from commercial sources. Fibroblasts were stimulated with cytokines with or without baricitinib. RA SMCs were cultured in the presence of baricitinib without stimulation. JAK/STAT activation and levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, RANTES and IP-10) were determined by qPCR, ELISA and MSD.. Baricitinib inhibited OSM-induced JAK signalling in RA synovial fibroblasts and effectively suppressed subsequent expression of the proinflammatory mediators IL-6, MCP-1 and IP-10. However, baricitinib was not effective in altering levels of spontaneously released TNFα, IL-6 and IL-8 in RA SMC. Although both TNFα and IL-1β signal independently of the JAK/STAT pathway, in HSF, but not in RA FLS, baricitinib significantly inhibited TNFα- and IL-1β-induced MCP-1 and IP-10 protein levels in a dose dependent manner. Furthermore, baricitinib did not inhibit TNFα- and IL-1β-induced expression of IL-6, IL-8 and MCP-1 in RA FLS.. These findings are consistent with known signalling pathways employed by OSM, TNFα and IL-1β, but our data suggest that in HSF, baricitinib may have anti-inflammatory effects via downstream modulation of cytokines and chemokines produced in response to TNFα or IL-1β.

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Azetidines; Cells, Cultured; Chemokine CCL5; Chemokine CXCL10; Fibroblasts; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Janus Kinase Inhibitors; Janus Kinases; Oncostatin M; Purines; Pyrazoles; RNA, Messenger; Signal Transduction; STAT Transcription Factors; Sulfonamides; Synovial Membrane; Synoviocytes; Tumor Necrosis Factor-alpha

To explore the role of interleukin 34 (IL-34) in rheumatoid arthritis (RA) and its related signalling pathways as well as the expression levels of IL-34 in collagen-induced arthritis (CIA) modelling mice.. Recombination IL-34 was used to stimulate cultured RA fibroblast-like synoviocytes (RA-FLS). The expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA), and the levels of phosphorylation signalling molecules were detected by western blotting assay (WB). After the establishment of the CIA model, paw indexes and serum IL-34 expression levels of mice were evaluated.. IL-34 significantly increased the secretion of IL-8 and TNF-α but had no significant effect on IL-6, and this effect could be impaired by signal inhibitors. At the same time, IL-34 activated multiple signalling pathways, whereas treating with inhibitors could reduce phosphorylation intensity. In animal experiments, mice in the model group had lost weight, and their paws were obviously swollen, ulcerous, and even stiffened. The hyperplasia of synovial tissue, infiltration of many inflammatory cells, and destruction of bone and cartilage from the typical pannus formation were also apparently observed.. IL-34 can mediate the production and secretion of IL-8 and TNF-α in RA-FLS cells through MAPKs, PI3K/Akt, JAK and NF-κB signalling pathways, while the expression of serum IL-34 in collagen-induced arthritis mice is also upregulated.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Fibroblasts; Interleukin-6; Interleukin-8; Interleukins; Mice; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Synovial Membrane; Tumor Necrosis Factor-alpha

Circular RNAs (circRNAs) can play a critical role in rheumatoid arthritis (RA) pathogenesis by involving gene regulation by competing for shared microRNAs (miRNAs), a family of small noncoding RNAs. MiR-130a-3p is a disease-related miRNA and Kruppel-like factor 9 (KLF9) is a zinc finger transcription factor, which are involved in RA pathogenesis. Here, we identified the action of circRNA circ_0130438 in regulating fibroblast-like synoviocytes (FLSs) stimulated by tumor necrosis factor α (TNF-α).. The direct relationship between miR-130a-3p and circRNA circ_0130438 or KLF9 was predicted by bioinformatics analysis and examined by a dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. CircRNA circ_0130438, miR-130a-3p and KLF9 factor expression levels were gauged by a quantitative real-time PCR (qRT-PCR) or a western blot method. Cell proliferation ability was analyzed by a 5-Ethynyl-2'-Deoxyuridine (EdU) staining assay. The transwell assay was used to evaluate cell migration and invasion capacities. The production levels of interleukin-1β (IL)-1β, IL-6 and IL-8 were assessed by enzyme-linked immunosorbent assay (ELISA).. The level of circRNA circ_0130438 was reduced in RA tissues (P = 0.0001) and FLSs isolated from RA tissues (P = 0.0001) compared with corresponding normal controls. Exposure of human fibroblast-like MH7A synoviocytes to TNF-α suppressed circRNA circ_0130438 expression (P < 0.0001). In contrast, the elevated expression of circRNA circ_0130438 suppressed the TNF-α-induced proliferation (P = 0.0047) and migration (P = 0.0023) of MH7A cells, as well as their pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production (P < 0.0001, P < 0.0001 and P < 0.0001). The circRNA circ_0130438 contained a miR-130a-3p binding site. Furthermore, the increase of miR-130-3p in TNF-α-stimulated MH7A cells reversed the effects of circRNA circ_0130438 elevation on cell proliferation (P = 0.0006), migration (P = 0.0406) and pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production (P = 0.0036, P < 0.0001 and P = 0.0004), indicating that miR-130a-3p was a functional mediator of circRNA circ_0130438 regulation. We also documented that KLF9 was a direct target and downstream effector of miR-130a-3p. Importantly, circRNA circ_0130438 enhanced KLF9 expression (P < 0.0001) in TNF-α-stimulated MH7A cells by functioning as a competing endogenous RNA (ceRNA) for miR-130a-3p (P = 0.0004).. Our findings demonstrate that the elevated expression of circRNA circ_0130438 suppresses TNF-α-induced migration, proliferation and pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) production of human MH7A cells by enhancing KLF9 expression by operating as a ceRNA for miR-130a-3p.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Cytokines; Fibroblasts; Humans; Inflammation; Interleukin-6; Interleukin-8; Kruppel-Like Transcription Factors; MicroRNAs; RNA, Circular; Synoviocytes; Tumor Necrosis Factor-alpha

STING (stimulator of interferon genes) has been recognized as an important signaling molecule in the innate immune response to cytosolic nucleic acids. Although it has been proposed that STING signaling pathway may play a pathogenic role in developing autoimmune and autoinflammatory diseases, its involvement in rheumatic disease processes remains to be elucidated. Here, we evaluated STING protein levels, expression and relationship with inflammatory parameters in synovial fluid (SF) of patients with psoriatic arthritis (PsA), rheumatoid arthritis (RA), gout, calcium pyrophosphate crystal-induced arthritis (CPP-IA), osteoarthritis (OA), and OA with CPP crystals (OA + CPP). The correlation with its negative regulator, nuclear factor erythroid 2-related factor 2 (Nrf2), was also investigated. SFs from 72 patients were analyzed for white blood cell (WBC) count, polymorphonuclear cell percentage (PMN%), and IL-1β, IL-6, IL-8, extra- and intracellular STING levels. STING and Nrf2 expression was also determined. WBC count and PMN% were greater in SF from inflammatory arthritis, while they were lower in OA groups. RA and gouty SFs have the highest levels of IL-1β, IL-8, and IL-6; while OA and OA + CPP showed the lowest concentrations. Gout and RA had the highest intracellular STING levels, while extracellular STING was greater in CPP-IA and OA SFs. STING was not detectable in PsA. STING mRNA was lower in PsA than other arthritides. Nrf2 mRNA was not detectable in OA. This study determines the presence of STING in SF of different arthritides, except for PsA, and suggests that it may be involved in pathogenesis and progression of arthropathies.

Topics: Arthritis, Psoriatic; Arthritis, Rheumatoid; Gout; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Membrane Proteins; NF-E2-Related Factor 2; Osteoarthritis; Synovial Fluid

Pinolenic acid (PNLA), an omega-6 polyunsaturated fatty acid from pine nuts, has anti-inflammatory and anti-atherogenic effects. We aimed to investigate the direct anti-inflammatory effect and anti-atherogenic effects of PNLA on activated purified CD14 monocytes from peripheral blood of patients with rheumatoid arthritis (RA) in vitro. Flow cytometry was used to assess the proportions of CD14 monocytes expressing TNF-α, IL-6, IL-1β, and IL-8 in purified monocytes from patients with RA after lipopolysaccharide (LPS) stimulation with/without PNLA pre-treatment. The whole genomic transcriptome (WGT) profile of PNLA-treated, and LPS-activated monocytes from patients with active RA was investigated by RNA-sequencing. PNLA reduced percentage of monocytes expressing cytokines: TNF-α by 23% (p = 0.048), IL-6 by 25% (p = 0.011), IL-1β by 23% (p = 0.050), IL-8 by 20% (p = 0.066). Pathway analysis identified upstream activation of peroxisome proliferator-activated receptors (PPARs), sirtuin3, and let7 miRNA, and KLF15, which are anti-inflammatory and antioxidative. In contrast, DAP3, LIF and STAT3, which are involved in TNF-α, and IL-6 signal transduction, were inhibited. Canonical Pathway analysis showed that PNLA inhibited oxidative phosphorylation (p = 9.14E-09) and mitochondrial dysfunction (p = 4.18E-08), while the sirtuin (SIRTs) signalling pathway was activated (p = 8.89E-06) which interfere with the pathophysiological process of atherosclerosis. Many miRNAs were modulated by PNLA suggesting potential post-transcriptional regulation of metabolic and immune response that has not been described previously. Multiple miRNAs target pyruvate dehydrogenase kinase-4 (PDK4), single-immunoglobulin interleukin-1 receptor molecule (SIGIRR), mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6) and acetyl-CoA acyltranferase2 (ACAA2); genes implicated in regulation of lipid and cell metabolism, inflammation, and mitochondrial dysfunction. PNLA has potential anti-atherogenic and immune-metabolic effects on monocytes that are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation regulates key miRNAs that are involved in metabolic, mitochondrial, and inflammatory pathways.

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Atherosclerosis; Cells, Cultured; Cytokines; Humans; Interleukin-6; Interleukin-8; Linolenic Acids; Lipopolysaccharides; MicroRNAs; Monocytes; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder which mainly affects small joints, occurs most commonly in middle-aged adults, and can be fatal in severe cases. The exact etiology of RA remains unknown. However, uncontrolled expression of pro-inflammatory cytokines and chemokines can contribute to the pathogenesis of RA.. In the current study, we assessed the potential of serum concentrations of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-8, and C-C motif chemokine ligand (CCL)5 as early predictive markers for RA.. In addition to clinical examination, blood samples were collected from 100 Saudi patients recently diagnosed with early RA for basic and serological tests, including rheumatoid factor (RF), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Sera of 32 healthy individuals were used as controls. Specific enzyme-linked immunosorbent assay was used to quantify the serum IL-1β, IL-6, TNF-α, IL-8, and CCL5 levels in the samples.. Our results indicated that RF, CRP, and ESR levels were higher in RA patients compared to controls. Furthermore, serum levels of IL-1β, IL-6, IL-8, and CCL5, but not TNF-α, significantly increased in RA patients compared to controls.. Overall, the findings suggested that IL-1β, IL-6, IL-8, and CCL5 can be used as biomarkers in the early diagnosis of RA.

Topics: Adult; Arthritis, Rheumatoid; Biomarkers; C-Reactive Protein; Humans; Interleukin-6; Interleukin-8; Middle Aged; Rheumatoid Factor; Saudi Arabia; Tumor Necrosis Factor-alpha

IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1β or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1β-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.

Topics: Animals; Arthritis, Rheumatoid; Cells, Cultured; Disease Models, Animal; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Mice; Neutrophils; Synovial Membrane

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Fibroblasts; Glucose; Humans; Inflammation; Interleukin-6; Interleukin-8; Molecular Docking Simulation; Rats; Synovial Membrane

Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA.. The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo.. L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1β, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice.. CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Topics: Amino Acids; Animals; Arginine; Arthritis, Experimental; Arthritis, Rheumatoid; Cationic Amino Acid Transporter 1; Cell Movement; Cell Proliferation; Cells, Cultured; Chromatography, Liquid; Cytokines; Fibroblasts; Interleukin-6; Interleukin-8; Mice; Mice, Inbred CBA; RNA, Messenger; RNA, Small Interfering; Synovial Membrane; Synoviocytes; Tandem Mass Spectrometry

Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease that is still not well understood in terms of its pathogenesis and presents diagnostic and therapeutic challenges. Monocytes are key players in initiating and maintaining inflammation through the production of pro-inflammatory cytokines and S100 proteins in RA. This study aimed to test a specific DNA methylation inhibitor (RG108) and activator (budesonide) in the regulation of pro-inflammatory mediators-especially the S100 proteins. We also searched for new biomarkers of high disease activity in RA patients. RNA sequencing analysis of healthy controls (HCs) and RA monocytes was performed. Genes such as the S100 family, TNF, and IL-8 were validated by qRT-PCR following DNA-methylation-targeted drug treatment in a monocytic THP-1 cell line. The concentrations of the S100A8, S100A11, and S100A12 proteins in the sera and synovial fluids of RA patients were tested and correlated with clinical parameters. We demonstrated that RA monocytes had significantly increased levels of S100A8, S100A9, S100A11, S100A12, MYD88, JAK3, and IQGAP1 and decreased levels of IL10RA and TGIF1 transcripts. In addition, stimulation of THP-1 cells with budesonide statistically reduced the expression of the S100 family, IL-8, and TNF genes. In contrast, THP-1 cells treated with RG108 had increased levels of the S100 family and TNF genes. We also revealed a significant upregulation of S100A8, S100A11, and S100A12 in RA patients, especially in early RA compared to HC sera. In addition, protein levels of S100A8, S100A11, and S100A12 in RA synovial fluids compared to HC sera were significantly increased. Overall, our data suggest that the S100A8 and S100A12 proteins are strongly elevated during ongoing inflammation, so they could be used as a better biomarker of disease activity than CRP. Interestingly, epigenetic drugs can regulate these S100 proteins, suggesting their potential use in targeting RA inflammation.

Topics: Arthritis, Rheumatoid; Biomarkers; Budesonide; Calgranulin A; Calgranulin B; Epigenesis, Genetic; Homeodomain Proteins; Humans; Inflammation; Interleukin-8; Repressor Proteins; S100 Proteins; S100A12 Protein

Topics: Adult; Arthritis, Rheumatoid; Atherosclerosis; Cell Line, Tumor; Cholesterol, HDL; Chromatography, Liquid; Female; Foam Cells; Humans; Interleukin-1beta; Interleukin-8; Lipoprotein(a); Macrophages; Male; Mass Spectrometry; Middle Aged; Pilot Projects; RNA, Messenger; THP-1 Cells

Topics: Animals; Arthritis, Rheumatoid; Cartilage; Cells, Cultured; Cholestenes; Cytokines; Fibroblasts; Humans; Interleukin-1beta; Interleukin-8; Mice; Mice, Inbred C57BL; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Synovial Membrane; Tumor Necrosis Factor-alpha

We evaluated the associations of anti-citrullinated protein antibodies (ACPAs) and serological and cytological levels of neutrophil extracellular trap (NET) formation in rheumatoid arthritis (RA) patients.. Serum levels of myeloperoxidase-DNA and elastase-DNA complexes (NET remnants) were examined in 51 patients with RA and 40 healthy controls using a modified enzyme-linked immunosorbent assay. Neutrophils were isolated by density gradient centrifugation. IgG antibodies were purified by affinity chromatography. NET formation in RA and control neutrophils was assessed by microscopy in vitro. NETs were purified and co-incubated with fibroblast-like synoviocyte (FLS) cells. Interleukin (IL)-6 and IL-8 mRNA expression and protein levels in FLS cells were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.. In RA patients, NET remnants in the peripheral circulation were higher in extremely high ACPA titers when compared to in moderate ACPA titers. And IgG antibodies containing ACPA can stimulate neutrophils to form NETs in a concentration-dependent manner. Furthermore, significantly higher expression of the pro-inflammatory cytokines IL-6 and IL-8 is detected after FLS cells interacted with NETs which derived from neutrophils stimulated with ACPA-containing IgG antibodies.. Anti-citrullinated protein antibodies may enhance NET formation and contribute to inflammation development in RA by stimulating NET formation, such as by subsequent activation of FLS cells by NETs.

Topics: Aged; Anti-Citrullinated Protein Antibodies; Arthritis, Rheumatoid; Case-Control Studies; Female; Humans; Immunoglobulin G; Interleukin-6; Interleukin-8; Male; Middle Aged; Neutrophils; Synoviocytes; Tumor Necrosis Factor-alpha

This study elucidates the mechanism of CCL25 and CCR9 in rheumatoid arthritis (RA). RA synovial fluid (SF) expresses elevated levels of CCL25 compared to OA SF and plasma from RA and normal. CCL25 was released into RA SF by fibroblasts (FLS) and macrophages (MΦs) stimulated with IL-1β and IL-6. CCR9 is also presented on IL-1β and IL-6 activated RA FLS and differentiated MΦs. Conversely, in RA PBMCs neither CCL25 nor CCR9 are impacted by 3-month longitudinal TNF inhibitor therapy. CCL25 amplifies RA FLS and monocyte infiltration via p38 and ERK phosphorylation. CCL25-stimulated RA FLS secrete potentiated levels of IL-8 which is disrupted by p38 and ERK inhibitors. CCL25 polarizes RA monocytes into nontraditional M1 MΦs that produce IL-8 and CCL2. Activation of p38 and ERK cascades are also responsible for the CCL25-induced M1 MΦ development. Unexpectedly, CCL25 was unable to polarize RA PBMCs into effector Th1/Th17 cells. Consistently, lymphokine like RANKL was uninvolved in CCL25-induced osteoclastogenesis; however, this manifestation was regulated by osteoclastic factors such as RANK, cathepsin K (CTSK), and TNF-α. In short, we reveal that CCL25/CCR9 manipulates RA FLS and MΦ migration and inflammatory phenotype in addition to osteoclast formation via p38 and ERK activation.

Topics: Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Chemokine CCL2; Chemokines, CC; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Interleukin-8; Macrophages; Monocytes; Osteoclasts; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, CCR; Signal Transduction; Synovial Fluid

To determine the association of polyunsaturated fatty acid (PUFA)-derived lipid mediators with progression from rheumatoid arthritis (RA)-related autoimmunity to inflammatory arthritis (IA).. We conducted a prospective cohort study using data from the Studies of the Etiology of Rheumatoid Arthritis (SERA). SERA enrolled first-degree relatives (FDRs) of individuals with RA (FDR cohort) and individuals who screened positive for RA-related autoantibodies at health fairs (screened cohort). We followed up 133 anti-cyclic citrullinated peptide 3.1 (anti-CCP3.1)-positive participants, 29 of whom developed IA. Lipid mediators selected a priori were quantified from stored plasma samples using liquid chromatography tandem mass spectrometry. We fit multivariable Cox proportional hazards models for each lipid mediator as a time-varying variable. For lipid mediators found to be significantly associated with IA, we then examined interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor (TNF) as potential statistical mediators.. For every 1 natural log pg/ml increase in the circulating plasma levels of proinflammatory 5-HETE, the risk of developing IA increased by 241% (hazard ratio 2.41 [95% confidence interval 1.43-4.07]) after adjusting for age at baseline, cohort (FDR or screened), and shared epitope status. The models examining 15-HETE and 17-HDHA had the same trend but did not reach significance. We did not find evidence that the association between 5-HETE and IA risk was influenced by the proinflammatory cytokines tested.. In a prospective cohort of anti-CCP-positive individuals, higher levels of 5-HETE, an important precursor to proinflammatory leukotrienes, is associated with subsequent IA. Our findings highlight the potential significance of these PUFA metabolites in pre-RA populations.

Topics: Adult; Aged; Anti-Citrullinated Protein Antibodies; Arthritis, Rheumatoid; Autoimmunity; Cohort Studies; Disease Progression; Docosahexaenoic Acids; Family; Fatty Acids, Unsaturated; Female; Humans; Hydroxyeicosatetraenoic Acids; Incidence; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Mediation Analysis; Middle Aged; Multivariate Analysis; Proportional Hazards Models; Prospective Studies; Tumor Necrosis Factor-alpha

We previously reported that synovial mast cells (MCs) from patients with rheumatoid arthritis (RA) produced TNF-α in response to immune complexes via FcγRI and FcγRIIA. However, the specific functions of synovial MCs in RA remain unclear. This study aimed to elucidate those functions. Synovial tissues and fluid were obtained from RA and osteoarthritis (OA) patients undergoing joint replacement surgery. Synovium-derived, cultured MCs were generated by culturing dispersed synovial cells with stem cell factor. We performed microarray-based screening of mRNA and microRNA (miRNA), followed by quantitative RT-PCR-based verification. Synovial MCs from RA patients showed significantly higher prostaglandin systhetase (PTGS)1 and PTGS2 expression compared with OA patients' MCs, and they produced significantly more prostaglandin D

Topics: Aged; Aged, 80 and over; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Histamine Release; Humans; Immunity, Innate; Interleukin-8; Lymphocytes; Male; Mast Cells; MicroRNAs; Middle Aged; Osteoarthritis; Prostaglandin D2; Receptors, IgG; RNA, Messenger; Signal Transduction; Synovial Fluid; Synovial Membrane

Autoantibodies are detected in most patients with rheumatoid arthritis (RA) and can be of the IgM, IgG or IgA subclass. Correlations between IgA autoantibodies and more severe disease activity have been previously reported, but the functional role of IgA autoantibodies in the pathogenesis of RA is ill understood. In this study, we explored the effect of IgA immune complexes on osteoclast mediated bone resorption.. Anti-citrullinated peptide antibody (ACPA) and anti-carbamylated protein (anti-CarP) antibody levels of the IgA and IgG isotype and rheumatoid factor (RF) IgA were determined in synovial fluid (SF) of RA patients. Monocytes, neutrophils, and osteoclasts were stimulated with precipitated immune complexes from SF of RA patients or IgA- and IgG-coated beads. Activation was determined by neutrophil extracellular trap (NET) release, cytokine secretion, and bone resorption.. NET formation by neutrophils was enhanced by SF immune complexes compared to immune complexes from healthy or RA serum. Monocytes stimulated with isolated SF immune complexes released IL-6 and IL-8, which correlated with the levels of ACPA IgA levels in SF. Osteoclasts cultured in the presence of supernatant of IgA-activated monocytes resorbed significantly more bone compared to osteoclasts that were cultured in supernatant of IgG-activated monocytes (p=0.0233). Osteoclasts expressed the Fc receptor for IgA (FcαRI; CD89) and Fc gamma receptors. IgA-activated osteoclasts however produced significantly increased levels of IL-6 (p<0.0001) and IL-8 (p=0.0007) compared to IgG-activated osteoclasts. Both IL-6 (p=0.03) and IL-8 (p=0.0054) significantly enhanced bone resorption by osteoclasts.. IgA autoantibodies induce release of IL-6 and IL-8 by immune cells as well as osteoclasts, which enhances bone resorption by osteoclasts. We anticipate that this will result in more severe disease activity in RA patients. Targeting IgA-FcαRI interactions therefore represents a promising novel therapeutic strategy for RA patients with IgA autoantibodies.

Topics: Animals; Antigen-Antibody Complex; Arthritis, Rheumatoid; Autoantibodies; Bone Resorption; Cattle; Extracellular Traps; Humans; Immunoglobulin A; Interleukin-6; Interleukin-8; Knee Joint; Osteoclasts; Synovial Fluid

Benzoylaconitine (BAC), the main hydrolysate of aconitine, is a lower toxic monoester type alkaloid considered as the pharmacodynamic constituent in Aconitum species. In this study, the effects and mechanisms of BAC on production of inflammatory cytokines interleukin (IL)-6 and IL-8 were investigated in IL-1β-stimulated human synovial SW982 cells. The SW982 cells were incubated with BAC (0, 5 and 10 µM) before stimulating with IL-1β (10 ng/mL). The results revealed that BAC suppressed gene and protein expression of IL-6 and IL-8 induced by IL-1β. BAC decreased activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Akt. BAC also inhibited degradation of inhibitor of kappaB (IκB)-α, phosphorylation and nuclear transposition of p65 protein. The results demonstrate that BAC exerts an anti-inflammatory effect dependent on MAPK, Akt and nuclear factor-κB (NF-κB) pathways in human synovial cells stimulated with IL-1β, suggesting that BAC may be exploited as a potential therapeutic agent for rheumatoid arthritis (RA) treatment.

Topics: Aconitine; Arthritis, Rheumatoid; Cell Line; Cell Survival; eIF-2 Kinase; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphorylation; Sarcoma, Synovial; Signal Transduction

Fibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated.. siRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-β1, TGF-β2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and β-catenin in FLSs.. DKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, β-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased.. By inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

Topics: Adult; Arthritis, Rheumatoid; Blotting, Western; Cell Proliferation; Cells, Cultured; Female; Fibroblasts; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Middle Aged; RNA, Small Interfering; Synoviocytes; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Synovial fluids of rheumatoid arthritis (RA) patients commonly contain high concentrations of soluble CD14 (sCD14). To investigate its potential role in RA pathogenesis, we tested whether sCD14 binding transmits a signal to fibroblast-like synoviocytes from RA patients (RA-FLS).. The induction of pro-inflammatory cytokines, chemokines, and mediators by sCD14 stimulation of RA-FLS was quantified by real-time PCR and ELISA. Cell proliferation was assessed by the BrdU assay. LPS-RS, a Toll-like receptor 4 (TLR-4) antagonist, was used to block TLR-4 signaling.. Soluble CD14 induced the expression of IL-6 mRNA and secretion of the protein. The expression of other pro-inflammatory cytokines and mediators, such as TNF-α, IL-8, intercellular adhesion molecule-1 (ICAM-1), MMP-3, and RANK ligand (RANKL), was also induced by sCD14. In addition, sCD14 stimulation promoted RA-FLS proliferation. LPS-RS abolished IL-6, IL-8, and ICAM-1 mRNA induction by sCD14 in RA-FLS. On the other hand, TNF-α and IL-17A increased TLR-4 expression by RA-FLS and amplified their sCD14-induced IL-6 expression.. Soluble CD14 transmits inflammatory signals to RA-FLS via TLR-4. The effects of sCD14 may be augmented in inflammatory milieu. Our results suggest that sCD14 is involved in the pathogenesis of RA and may be a novel therapeutic target.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Chemokines; Cytokines; Fibroblasts; Humans; Inflammation Mediators; Intercellular Adhesion Molecule-1; Interleukin-17; Interleukin-6; Interleukin-8; Lipopolysaccharide Receptors; Solubility; Synoviocytes; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.

Topics: Arthritis, Rheumatoid; Biological Products; Blood Proteins; Chromatography, Gel; Etanercept; Extracellular Vesicles; Humans; Immunoglobulin G; Interleukin-8; Promoter Regions, Genetic; Transcriptional Activation; Tumor Necrosis Factor-alpha

BACKGROUND Rheumatoid arthritis (RA) is an inflammatory disorder that is present in approximately 1% of the world's population. This study was aimed to investigate the effect of retinoic acid-platinum (II) complex [RT-Pt(II)] on rheumatoid arthritis (RA) and to explore the mechanism involved. MATERIAL AND METHODS MH7A cell viability was determined by MTT assay and apoptosis was assessed using FACSCalibur flow cytometry. RT-PCR and Western blot assays were used for assessment of mRNA and proteins levels. RESULTS Treatment of rheumatoid arthritis with RT-Pt(II) significantly reduced the levels of IL‑1ß, IL-6, IL-8, MMP-1, and MMP-13 in synovial fluid of mice in a dose-dependent manner. The expression of iNOS and COX-2 mRNA and protein in rheumatoid arthritis rats was also significantly inhibited by treatment with RT-Pt(II). The TNF-alpha-induced proliferation of MH7A cells was alleviated by RT-Pt(II) treatment in a concentration-dependent manner. Moreover, RT-Pt(II) treatment induced apoptosis and caused arrest of cell cycle in MH7A cells. The activation of MEK/NF-kappaB pathway was downregulated by RT-Pt(II) treatment in MH7A cells. CONCLUSIONS In summary, the present study demonstrated that RT-Pt(II) inhibits TNF-alpha-induced inflammatory response, suppresses cell viability, and induces apoptosis in rheumatoid arthritis synovial cells. Moreover, RT-Pt(II) exhibited its effect through targeting the MEK/NF-kappaB pathway. Therefore, RT-Pt(II) can be used for the development of treatments for rheumatoid arthritis.

Topics: Animals; Antirheumatic Agents; Apoptosis; Arthritis, Rheumatoid; Cell Line; Coordination Complexes; Cyclooxygenase 2; Disease Models, Animal; Gene Expression Regulation; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 13; Mice; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Nitric Oxide Synthase Type II; Platinum Compounds; Rats; Rats, Sprague-Dawley; Signal Transduction; Synovial Fluid; Synoviocytes; Tretinoin; Tumor Necrosis Factor-alpha

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Topics: Aged; Arthritis, Rheumatoid; Calcium; Cannabidiol; Cell Proliferation; Cell Survival; Female; Fibroblasts; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; Middle Aged; Synovial Fluid; Synovial Membrane; TRPA1 Cation Channel; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) remains a cause of morbidity and mortality in many patients while new treatments have changed the face of the disease. Despite the emergence of these new drugs, cardiovascular (CV) diseases remain more frequent in RA patients compared with the general population. However, predictive biomarkers of RA severity and precise guidelines to manage the CV risk in these patients are still lacking. Pro-inflammatory cytokines contribute both to RA and CV pathogenesis. Focusing on IL-17A, high levels of bioactive IL-17A were associated with destruction in RA but also during myocardial infarction. The study aimed to assess the relationship between bioactive IL-17A, destruction and the occurrence of CV events (CVE) in RA patients with a very long follow-up. Thirty-six RA patients were followed between 1970 and 2012 in Lyon, France. They were tested for bioactive IL-17A and clinical and biological characteristics were recorded at baseline. Then, the occurrence of CVE was registered during the follow-up. To study the bioactive fraction of IL-17A, the bioassay used the ability of human umbilical vein endothelial cells to produce IL-8 in presence of RA plasma samples with or without an anti-IL-17A antibody. Bioactive IL-17A level at baseline was higher in RA patients who later experienced a CVE compared to those without (0.77 vs 0.21 ng/ml,

Topics: Adult; Arthritis, Rheumatoid; Biomarkers; Bone Resorption; Cardiovascular Diseases; Endothelium, Vascular; Female; Follow-Up Studies; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-17; Interleukin-8; Joints; Male; Young Adult

The clinical efficacy of specific interleukin-6 inhibitors has confirmed the central role of IL6 in rheumatoid arthritis (RA). However the local role of IL6, in particular in synovial fibroblasts (SF) as a direct cellular target to IL6/sIL6R signal is not well characterized. The purpose of the study was to characterize the crosstalk between TNFα and IL6/sIL6R signaling to the effector pro-inflammatory response of SF.. SF lines were stimulated with either TNFα, IL6/sIL6R, or both together, for the time and dose indicated for each experiment, and where indicated, cells were treated with inhibitors actinomycin D, adalimumab, ruxolitinib and cycloheximide. mRNA expression of cytokines, chemokines and matrix metalloproteases (MMPs) were analyzed by quantitative RT-PCR. Level of IL8/CXCL8 and CCL8 in culture supernatants was measured by ELISA. Mononuclear and polymorphonuclear cells migration assays were assessed by transwell using conditioned medium from SF cultures. Statistical analyses were performed as indicated in the corresponding figure legends and a p-value < 0.05 was considered statistically significant.. The stimulation of SF with IL6/sIL6R and TNFα, cooperatively promotes the expression of mono- and lymphocytic chemokines such as IL6, CCL8 and CCL2, as well as matrix degrading enzymes such as MMP1, while inhibiting the induction of central neutrophil chemokines such as IL8/CXCL8. These changes in the pattern of chemokines expression resulted in reduced polymorphonuclear (PMN) and increased mononuclear cells (MNC) chemoattraction by SF. Mechanistic analyses of the temporal expression of genes demonstrated that the cooperative regulation mediated by these two factors is mostly induced through de novo transcriptional mechanisms activated by IL6/sIL6R. Furthermore, we also demonstrate that TNFα and IL6/sIL6R cooperation is partially mediated by the expression of secondary factors signaling through JAK/STAT pathways.. These results point out to a highly orchestrated response to IL6 in TNFα-induced SF and provide additional insights into the role of IL6/sIL6R in the context of RA, highlighting the contribution of IL6/sIL6R to the interplay of SF with other inflammatory cells.

Topics: Adalimumab; Arthritis, Rheumatoid; Cell Line; Cell Movement; Chemokine CCL8; Chemokines; Cycloheximide; Cytokines; Dactinomycin; Fibroblasts; Gene Expression Regulation; Humans; Inflammation; Interleukin-6; Interleukin-8; Janus Kinases; Kinetics; Matrix Metalloproteinases; Nitriles; Pyrazoles; Pyrimidines; Receptors, Interleukin-6; Signal Transduction; STAT Transcription Factors; Synovial Membrane; Tumor Necrosis Factor-alpha

Adequate tissue engineered models are required to further understand the (patho)physiological mechanism involved in the destructive processes of cartilage and subchondral bone during rheumatoid arthritis (RA). Therefore, we developed a human in vitro 3D osteochondral tissue model (OTM), mimicking cytokine-induced cellular and matrix-related changes leading to cartilage degradation and bone destruction in order to ultimately provide a preclinical drug screening tool. To this end, the OTM was engineered by co-cultivation of mesenchymal stromal cell (MSC)-derived bone and cartilage components in a 3D environment. It was comprehensively characterized on cell, protein, and mRNA level. Stimulating the OTM with pro-inflammatory cytokines, relevant in RA (tumor necrosis factor α, interleukin-6, macrophage migration inhibitory factor), caused cell- and matrix-related changes, resulting in a significantly induced gene expression of lactate dehydrogenase A, interleukin-8 and tumor necrosis factor α in both, cartilage and bone, while the matrix metalloproteases 1 and 3 were only induced in cartilage. Finally, application of target-specific drugs prevented the induction of inflammation and matrix-degradation. Thus, we here provide evidence that our human in vitro 3D OTM mimics cytokine-induced cell- and matrix-related changes-key features of RA-and may serve as a preclinical tool for the evaluation of both new targets and potential drugs in a more translational setup.

Topics: Aged; Arthritis, Rheumatoid; Bone and Bones; Calcium Phosphates; Cartilage, Articular; Chondrocytes; Cytokines; Female; Fibroblasts; Humans; In Vitro Techniques; Interleukin-6; Interleukin-8; Lactate Dehydrogenase 5; Macrophage Migration-Inhibitory Factors; Male; Mesenchymal Stem Cells; Middle Aged; Synovial Membrane; Tissue Engineering; Translational Research, Biomedical; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is a debilitating joint disease characterized by chronic inflammation, pathologic alteration of fibroblast‑like synoviocytes (FLS), destruction of cartilage and bone, and the formation of an invasive pannus. RA‑FLS exhibit increased proliferation and resistance to apoptosis. The retinoid X receptor (RXR) has a role in regulating cell cycle, differentiation and apoptosis, and agonism of RXR has been investigated as a treatment strategy in several types of cancer. However, there is little research on the effects of RXR agonism in other diseases. Bexarotene is a novel selective RXR ligand used in the treatment of T‑cell lymphoma. In the present study, bexarotene was used to investigate the involvement of RXR in tumor necrosis factor‑α (TNF‑α)‑induced RA conditions in human FLS. To the best of our knowledge, this is the first time that RXR has been demonstrated to be expressed in FLS and to be downregulated in response to TNF‑α stimulation. The present study also demonstrated that bexarotene exerted an anti‑inflammatory effect by downregulating expression of interleukin (IL)‑6, IL‑8, monocyte chemoattractant protein‑1, and high mobility group box‑1. Notably, bexarotene also rescued the TNF‑α‑induced downregulation of the anti‑inflammatory cytokines IL‑4 and transforming growth factor‑β1. Bexarotene treatment exhibited a potential protective effect against cartilage degradation by downregulating the expression of matrix metalloproteinase (MMP)‑1, MMP‑3 and MMP‑13. In addition, the present results demonstrated that the effects of bexarotene were mediated through the p38 mitogen‑activated protein kinase/nuclear factor‑κB pathway, via inhibition of p38 protein and the inhibitor α of κB phosphorylation. Taken together, the present findings demonstrated the potential of RXR agonism using bexarotene as a treatment against the development and progression of RA.

Topics: Anti-Inflammatory Agents; Apoptosis; Arthritis, Rheumatoid; Bexarotene; Cell Proliferation; Cells, Cultured; Chemokine CCL2; Cytokines; Down-Regulation; Fibroblasts; Humans; Inflammation; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Retinoid X Receptors; Signal Transduction; Synoviocytes; Tumor Necrosis Factor-alpha

The purpose of this study was to explore the role and mechanism of transient receptor potential M(2) (TRPM(2)) in antigen-induced arthritis (AIA) mice. Twelve C57BL/6 mice and 12 TRPM(2) knockout mice were divided into 4 groups, includingwild type control group, wild type AIA group, TRPM(2) knockout control group and TRPM(2) knockout AIA group, with 6 mice in each group. Methylated bovine serum albumin (mBSA) was used to establish AIA mouse model. The degree of joint swelling and inflammatory cell infiltration were recorded, as well as synovial hyperplasia of the knee joints. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression of interleukin (IL)-6, IL-8, chemokine ligand 6 (CXCL-6) and tumor necrosis factor alpha (TNFα) mRNA in synovial cells of knee joints. The results showed that compared with the wild-type AIA group, the TRPM(2) knockout AIA group had more significant synovial proliferation and inflammatory cell infiltration in the synovial tissue.The neutrophil and macrophage counts rather than monocytes in the knee joints of TRPM(2) knockout AIA group were higher than those in wild-type AIA mice. The expression of IL-6, IL-8 and CXCL-6 mRNA were significantly increased in the knock out mice. In summary, TRPM(2) may inhibit inflammatory cytokines such as IL-6 and IL-8 in knee joints of AIA mice by reducing the infiltration of neutrophils and macrophages, the refore alleviates the manifestations of knee arthritis.. 本研究初步探索瞬时受体电位M(2)（TRPM(2)）在抗原诱导关节炎（AIA）小鼠中的作用及机制。取C57BL/6小鼠12只，TRPM(2)基因敲除小鼠12只，分为野生型对照组、野生型AIA组、TRPM(2)基因敲除对照组、TRPM(2)基因敲除AIA组，每组6只。采用甲基化牛血清白蛋白（mBSA）建立野生型小鼠和TRPM(2)基因敲除小鼠AIA模型，进行小鼠膝关节肿胀程度、炎性细胞浸润、滑膜增生评分：实时荧光定量聚合酶链反应检测膝关节滑膜细胞白细胞介素（IL）-6、IL-8、趋化因子配体6（CXCL-6）、肿瘤坏死因子α（TNFα）mRNA表达。结果显示，与野生型AIA组小鼠比，TRPM(2)基因敲除AIA组小鼠膝关节肿胀更明显；膝关节滑膜细胞增生，滑膜组织炎性细胞浸润明显增加，膝关节中中性粒细胞、巨噬细胞计数升高，单核细胞无明显差异；IL-6、IL-8、CXCL-6 mRNA表达水平明显增加，TNFα mRNA表达无明显变化。TRPM(2)可通过减少AIA小鼠膝关节中性粒细胞和巨噬细胞的浸润，抑制小鼠膝关节IL-6、IL-8等炎性细胞因子表达，减轻AIA小鼠膝关节炎症状。.

Topics: Animals; Antigens; Arthritis, Experimental; Arthritis, Rheumatoid; Chemokine CXCL6; Interleukin-6; Interleukin-8; Knee Joint; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction; Serum Albumin, Bovine; Synovial Membrane; Tumor Necrosis Factor-alpha

Obesity-in which free fatty acid (FFA) levels are chronically elevated-is a known risk factor for different rheumatic diseases, and obese patients are more likely to develop osteoarthritis (OA) also in non-weight-bearing joints. These findings suggest that FFA may also play a role in inflammation-related joint damage and bone loss in rheumatoid arthritis (RA) and OA. Therefore, the objective of this study was to analyze if and how FFA influence cells of bone metabolism in rheumatic diseases. When stimulated with FFA, osteoblasts from RA and OA patients secreted higher amounts of the proinflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, growth-related oncogene α, and monocyte chemotactic protein 1. Receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin, and osteoblast differentiation markers were not influenced by FFA. Mineralization activity of osteoblasts correlated inversely with the level of FFA-induced IL-6 secretion. Expression of the Wnt signaling molecules, axin-2 and β-catenin, was not changed by palmitic acid (PA) or linoleic acid (LA), suggesting no involvement of the Wnt signaling pathway in FFA signaling for osteoblasts. On the other hand, Toll-like receptor 4 blockade significantly reduced PA-induced IL-8 secretion by osteoblasts, while blocking Toll-like receptor 2 had no effect. In osteoclasts, IL-8 secretion was enhanced by PA and LA particularly at the earliest time point of differentiation. Differences were observed between the responses of RA and OA osteoclasts. FFA might therefore represent a new molecular factor by which adipose tissue contributes to subchondral bone damage in RA and OA. In this context, their mechanisms of action appear to be dependent on inflammation and innate immune system rather than Wnt-RANKL pathways.

Topics: Aged; Animals; Arthritis, Rheumatoid; Cells, Cultured; Female; Humans; Interleukin-8; Leukocytes, Mononuclear; Linoleic Acid; Male; Mice, Inbred C57BL; Middle Aged; Osteoarthritis; Osteoblasts; Osteoclasts; Palmitic Acid

Our previous study revealed that Yin Yang 1(YY1) played an important part in promoting interleukin (IL)-6 production in rheumatoid arthritis (RA). However, whether YY1 has any role in regulation of IL-8 in RA remains unclear. YY1 and IL-8 expression in RA patients were analyzed by real-time polymerase chain reaction (PCR). Ingenuity pathway analysis (IPA) was used to analyze the signaling pathway involved in YY1-induced IL-8 production. The expression of YY1 and proteins involved in the pathway were detected by Western blot and enzyme-linked immunosorbent assay (ELISA). Migration of neutrophils was performed by chemotaxis assay. In this study, we found that high expression of IL-8 was positively associated with YY1 expression in RA. Blocking YY1 expression by YY1-short hairpin (sh)RNA lentivirus reduced IL-8 production. Mechanistically, we showed YY1 activated IL-8 production via the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. Further, using a co-culture system consisting of peripheral blood mononuclear cells (PBMC) and neutrophils, we found that migration of neutrophils would be inhibited by YY1 RNA interference. Finally, using the collagen-induced arthritis animal model, we showed that treatment with the YY1-shRNA lentivirus led to reduction of IL-8 levels and attenuation of inflammation and neutrophil infiltration in vivo. Our results reveal a role of YY1 involved in neutrophil infiltration in RA via the PI3K/Akt/mTOR/IL-8 signaling pathway. YY1 may be a new therapeutic target for treatment of RA.

Topics: Arthritis, Rheumatoid; Chemotaxis; Humans; Interleukin-8; MAP Kinase Signaling System; Neutrophil Infiltration; Neutrophils; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; TOR Serine-Threonine Kinases; YY1 Transcription Factor

Histone deacetylases (HDACs) are important in chronic inflammation, and inflammatory responses affect synovium-derived mesenchymal stem cell (SMSC) function in temporomandibular joint repair. However, the effect of HDACs on SMSC inflammatory activation remains unclear. In this study, temporomandibular joint fibroblast-like synoviocytes obtained from osteoarthritis patients met the minimal mesenchymal stem cell criteria. Interleukin 1β (IL-1β) upregulated IL-6 and IL-8 expression in SMSCs through nuclear factor-κB (NF-κB) pathway activation. IL-6 and IL-8 upregulation were blocked by broad-acting HDAC inhibitors SAHA and LBH589. MC1568 alleviated IL-1β activation of SMSCs, whereas CI994 and FK228 produced a minimal or opposite effect in vitro. We also found HDAC10 was highly associated with localized IL-1β expression in vivo and in vitro. HDAC10 knockdown alleviated IL-1β-mediated SMSC activation and blocked NF-κB pathway activation. Conversely, HDAC10 overexpression promoted IL-6 and IL-8 expression and IL-1β-mediated NF-κB pathway activation. In conclusion, HDAC10 upregulation contributed to IL-1β-mediated inflammatory activation of SMSCs, indicating that HDAC10 may be a novel therapeutic target.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Histone Deacetylases; Humans; Hydroxamic Acids; Interleukin-1beta; Interleukin-8; Mesenchymal Stem Cells; NF-kappa B; Osteoarthritis; Pyrroles; Signal Transduction; Synovial Membrane; Synoviocytes; Temporomandibular Joint; Transcriptional Activation; Up-Regulation

To identify PBMC-expressed genes significant for RA, and to ascertain their upstream regulatory factors, as well as downstream functional effects relevant to RA pathogenesis. We performed peripheral blood mononuclear cells (PBMCs) transcriptome-wide mRNA expression profiling in a case-control discovery sample. Differentially expressed genes (DEGs) were identified and validated in PBMCs in independent samples. We also generated genome-wide SNP genotyping data, and collected miRNA expression data and DNA methylation data from PBMCs of the discovery sample. Pearson correlation analyses were conducted to identify miRNAs/DNA methylations influencing DEG expression. Association analyses were conducted to identify expression-regulating SNPs. The key DEG, SAMD9, which was reported to function as a tumor suppressor gene, was assessed for its effects on T cell proliferation, apoptosis, and inflammatory cytokine expression. A total of 181 DEGs (Fold Change ≥ 2.0, Bonferroni adjusted p ≤ 0.05) were discovered in PBMCs. Four DEGs (SAMD9, CKLF, PARP9, and GUSB), upregulated with RA, were validated independently in PBMCs. Specifically, SAMD9 mRNA expression level was significantly upregulated in PHA-activated Jurkat T cells in vitro, and correlated with 8 miRNAs and associated with 22 SNPs in PBMCs in vivo. Knockdown of SAMD9 could transiently promote Jurkat T cell proliferation within 48 h and significantly induce TNF-α and IL-8 expression in T cells. SAMD9 expression is (epi-) genetically regulated, and significantly upregulated in PBMCs in RA patients and in activated T cells in vitro. SAMD9 might serve as a T cell activation marker but act as an anti-inflammatory factor.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Epigenesis, Genetic; Female; Genome-Wide Association Study; Humans; Interleukin-8; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Male; Polymorphism, Single Nucleotide; Proteins; T-Lymphocytes; Tumor Necrosis Factor-alpha

The receptor activator of nuclear factor κB ligand (RANKL) is an important factor for osteoclastogenesis and contributes to the pathology of rheumatoid arthritis (RA); thus, the anti-RANKL antibody (Ab) has been expected to protect joint destruction in RA patients. IL-8 also has osteoclastogenic activity; however, the role of IL-8 in the bone pathology of RA as well as the relation between IL-8 and RANKL remain unclear. In the present study, clinical observation revealed serum IL-8 levels of 611 pg ml-1 in RA patients with anti-RANKL Ab and 266 pg ml-1 in the same patients without anti-RANKL Ab. In vitro assay showed that anti-RANKL Ab induced production of IL-8 from pre-osteoclast-like cells (OCLs), and IL-8 promoted the formation of OCLs from peripheral monocytes even without RANKL activity. We further showed that treatment with FK506 (tacrolimus) possibly inhibits the increase in IL-8 levels in RA patients with anti-RANKL Ab, and in vitro assay confirmed that FK506 suppressed IL-8 production in pre-OCLs. These results suggest that inhibition of RANKL induces the change in osteoclastogenesis-promoting factor from RANKL to IL-8, and FK506 may be a valuable combination drug to support the use of anti-RANKL Ab in treatment of RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Denosumab; Female; Humans; Interleukin-8; Male; Middle Aged; Osteogenesis; RANK Ligand

An increased repertoire of potential osteoclast (OC) precursors could accelerate the development of bone-erosive OCs and the consequent bone damage in rheumatoid arthritis (RA). Immature dendritic cells (DCs) can develop into OCs, however, the mechanisms underlying this differentiation switch are poorly understood. We investigated whether protein citrullination and RA-specific anti-citrullinated protein Abs (ACPAs) could regulate human blood-derived DC-OC transdifferentiation. We show that plasticity toward the OC lineage correlated with peptidyl arginine deiminase (PAD) activity and protein citrullination in DCs. Citrullinated actin and vimentin were present in DCs and DC-derived OCs, and both proteins were deposited on the cell surface, colocalizing with ACPAs binding to the cells. ACPAs enhanced OC differentiation from monocyte-derived or circulating CD1c

Topics: Anti-Citrullinated Protein Antibodies; Antigens, CD1; Arthritis, Rheumatoid; Cell Differentiation; Cell Lineage; Cell Plasticity; Cell Transdifferentiation; Cells, Cultured; Citrullination; Dendritic Cells; Humans; Interleukin-8; Monocytes; Osteoclasts; Protein-Arginine Deiminases

This study aims to explore the regulatory effect of microRNA-193a-3p on rheumatoid arthritis (RA) and its underlying mechanism.. Expression level of microRNA-193a-3p in synovial tissues extracted from 30 RA patients and healthy controls was detected by quantitative Real-time polymerase chain reaction (qRT-PCR). MH7A cells were subjected to TNF-α induction for constructing the in vitro RA model. After transfection of microRNA-193a-3p inhibitor in MH7A cells, proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine levels of interleukin 6 (IL-6) and IL-8 in MH7A cells. Subsequently, the dual-luciferase reporter gene assay was carried out to verify the binding condition between microRNA-193a-3p and IGFBP5. Rescue experiments were conducted to evaluate the proliferation and apoptosis of MH7A cells with knockdown of microRNA-193a-3p and IGFBP5.. MicroRNA-193a-3p was highly expressed in synovial tissues of RA patients and TNF-α-induced MH7A cells than those of controls. TNF-α induction significantly increased the proliferative rate of MH7A cells, reaching the peak at 96 h. After knockdown of microRNA-193a-3p, the promoted proliferation by TNF-α induction was significantly inhibited. In addition, TNF-α induction significantly inhibited the apoptosis of MH7A cells. After inhibition of microRNA-193a-3p expression, the inhibited apoptosis by TNF-α induction remarkably increased. TNF-α induction upregulated levels of IL-6 and IL-8 in MH7A cells, which were remarkably reduced after the microRNA-193a-3p knockdown. Dual-luciferase reporter gene assay confirmed that IGFBP5 could bind to microRNA-193a-3p, and its expression was negatively regulated by microRNA-193a-3p. The regulatory effects of microRNA-193a-3p on proliferation and apoptosis of MH7A cells were reversed by IGFBP5 knockdown.. MicroRNA-193a-3p is highly expressed in the synovial tissues and cells of rheumatoid arthritis. MicroRNA-193a-3p participates in the process of rheumatoid arthritis by regulating the proliferation, apoptosis and inflammatory response of MH7A cells through targeting IGFBP5.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Line; Cell Movement; Cell Proliferation; Disease Progression; Gene Knockdown Techniques; Humans; Insulin-Like Growth Factor Binding Protein 5; Interleukin-6; Interleukin-8; MicroRNAs; Synovial Membrane; Synoviocytes; Tumor Necrosis Factor-alpha; Up-Regulation

To investigate the influence of micro ribonucleic acid (miR)-21 on rats with rheumatoid arthritis (RA) through the Wnt signaling pathway.. A total of 30 rats were divided into three groups: control group (healthy rats, n=10), model group (rat model of RA, n=10), and MiR group (rat model of RA injected with miR-21 lentivirus, n=10). The paw volume, arthritis indexes, and protein expression level in each group were analyzed by means of paw volume and arthritis index measurement, reverse transcription-polymerase chain reaction (RT-PCR) assay, and fluorescent Western blotting.. The expression levels of inflammatory factors declined in MiR group compared with those in model group, while they were higher in model group than those in control group and MiR group (p<0.05). At 15 d after transfection with lentivirus, the paw volume in MiR group was smaller than that in model group, which was decreased markedly with the extended time of transfection (p<0.05). On the 30th d, MiR group had a remarkably smaller paw volume than model group. In comparison with that in control group, the paw volume in model group was increased notably from the 7th d and displayed a significant difference in the 30th d (p<0.05). The arthritis indexes in MiR group were lower than those in model group; however, there were no apparent inflammations at the joints at 15 d after drug administration. Moreover, the longer the time of drug administration was, the less apparent the inflammations at the joints will be. The inflammations at the joints were ameliorated evidently on the 30th d in MiR group (p<0.05). Compared with those in control group, the inflammations in model group were increased significantly from the 7th d, with significant differences in the 30th d (p<0.05). The messenger RNA (mRNA) expression levels of interleukin-6 (IL-6), IL-8, and Wnt in MiR group were higher than those in control group, but lower than those in model group (p<0.05), while they were higher in model group than those in control group (p<0.05). The expression level of Wnt protein was decreased in MiR group compared with that in model group (p<0.05), and model group had a prominently elevated expression level of Wnt protein in comparison with control group (p<0.05).. MiR-21 overexpression can repress the expressions of IL-6 and IL-8 and relieve the symptoms of RA by down-regulating the Wnt signal.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Dependovirus; Female; Gene Expression Regulation; Genetic Vectors; Interleukin-6; Interleukin-8; MicroRNAs; Rats; Wnt Signaling Pathway

Rheumatoid arthritis (RA) is associated with abnormal B cell-functions implicating antibody-dependent and -independent mechanisms. B cells have emerged as important cytokine-producing cells, and cytokines are well-known drivers of RA pathogenesis. To identify novel cytokine-mediated B-cell functions in RA, we comprehensively analysed the capacity of B cells from RA patients with an inadequate response to disease modifying anti-rheumatic drugs to produce cytokines in comparison with healthy donors (HD). RA B cells displayed a constitutively higher production of the pathogenic factors interleukin (IL)-8 and Gro-α, while their production of several cytokines upon activation via the B cell receptor for antigen (BCR) was broadly suppressed, including a loss of the expression of the protective factor TRAIL, compared to HD B cells. These defects were partly erased after treatment with the IL-6-signalling inhibitor tocilizumab, indicating that abnormal IL-6 signalling contributed to these abnormalities. Noteworthy, the clinical response of individual patients to tocilizumab therapy could be predicted using the amounts of MIP-1β and β-NGF produced by these patients' B cells before treatment. Taken together, our study highlights hitherto unknown abnormal B-cell functions in RA patients, which are related to the unbalanced cytokine network, and are potentially relevant for RA pathogenesis and treatment.

Topics: Antibodies, Monoclonal, Humanized; Arthritis, Rheumatoid; B-Lymphocytes; Chemokine CCL4; Chemokine CXCL1; Humans; Interleukin-6; Interleukin-8; Nerve Growth Factor; TNF-Related Apoptosis-Inducing Ligand

The fibroblast-like synoviocytes (FLSs) has the aggressive phenotype, which is very important for cartilage destruction in rheumatoid arthritis (RA). To the pathology of RA, the increased FLSs migration, activation and proliferation are essential factors. Oxymatrine is a traditional Chinese herb, which is the extraction from the root of Sophora flavescens and regarded as quinolizidine alkaloid compounds and has been shown to inhibit inflammation, proliferation and migration in vitro or vivo. However, whether oxymatrine effects in the treatment of RA FLSs is undefined. In our study, the inhibition of oxymatrine in RA FLSs inflammation, proliferation and migration in RA FLS are evaluated. We found that oxymatrine decreased the IL-6 and IL-8 expression and the proliferation, migration and invasion of RA FLSs. We also evaluated the molecular mechanisms and we found the effect of oxymatrine on NF-κB activation. The results showed that oxymatrine inhibited the activity of NF-κB. And the treatment activity of oxymatrine on collagen-induced arthritis (CIA) was further explored by us. Thus, we conclude that oxymatrine may protect joint destruction of RA by inhibiting synoviocyte activation, migration, invasion, and proliferation.

Topics: Adult; Alkaloids; Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Movement; Cell Proliferation; Cells, Cultured; Drugs, Chinese Herbal; Female; Fibroblasts; Humans; Inflammation; Interleukin-6; Interleukin-8; Male; Mice; Mice, Inbred DBA; Middle Aged; NF-kappa B; Quinolizines; Signal Transduction; Sophora; Synovial Membrane; Transcriptional Activation

IL-11 has been detected in inflamed joints; however, its role in the pathogenesis of arthritis is not yet clear. Studies were conducted to characterize the expression and functional significance of IL-11 and IL-11Rα in rheumatoid arthritis (RA). IL-11 levels were elevated in RA synovial fluid (SF) compared to osteoarthritis (OA) SF and plasma from RA, OA and normal individuals (NLs). Morphologic studies established that IL-11 was detected in lining fibroblasts and macrophages in addition to sublining endothelial cells and macrophages at higher levels in RA compared to NL synovial tissues. Since IL-11Rα was exclusively expressed in RA fibroblasts and endothelial cells, macrophages were not involved in IL-11 effector function. Ligation of IL-11 to IL-11Rα strongly provoked fibroblast infiltration into RA joint, while cell proliferation was unaffected by this process. Secretion of IL-8 and VEGF from IL-11 activated RA fibroblasts was responsible for the indirect effect of IL-11 on endothelial cell transmigration and tube formation. Moreover, IL-11 blockade impaired RA SF capacity to elicit endothelial cell transmigration and tube formation. We conclude that IL-11 binding to endothelial IL-11Rα can directly induce RA angiogenesis. In addition, secretion of proangiogenic factors from migrating fibroblasts potentiated by IL-11 can indirectly contribute to RA neovascularization.

Topics: Arthritis, Rheumatoid; Endothelial Cells; Female; Fibroblasts; Humans; Interleukin-11; Interleukin-11 Receptor alpha Subunit; Interleukin-8; Joints; Male; Neovascularization, Pathologic; Transendothelial and Transepithelial Migration; Vascular Endothelial Growth Factor A

This study examined the expression of the inhibitory receptor, leucocyte-associated immunoglobulin (Ig)-like receptor-1 (LAIR-1) in fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients to investigate its potential role in the modulation of inflammatory cytokines, matrix metalloproteinases (MMPs) and invasiveness of synoviocytes. LAIR-1 expression in synovial tissues from RA patients, osteoarthritis patients and healthy donors was analysed by immunohistochemistry. The membrane-bound form (mLAIR-1) was detected by flow cytometry. Factors involved in inflammation and MMP activity in FLS were analysed by quantitative polymerase chain reaction (qPCR). LAIR-1 expression was higher in the synovia of the RA patients than those of the osteoarthritis patients. Co-immunostaining of vimentin/LAIR-1 demonstrated that LAIR-1 was localized mainly in FLS in the RA patients. Surprisingly, primary FLS isolated from the RA patients had low levels of mLAIR-1 expression, with cytoplasmic distribution. The extracellular domain of LAIR-1 was shed from the cell surface in response to tumour necrosis factor (TNF)-α, and this process could be blocked by serine protease inhibitors. Additional experiments indicated that LAIR-1 over-expression reduced FLS invasion considerably, which reduced simultaneously the mRNA levels of interleukin (IL)-6, IL-8 and MMP-13 in the presence of TNF-α. Our study demonstrated that LAIR-1 is an anti-inflammatory molecule, and was up-regulated in FLS in the RA patients; however, cell-surface LAIR-1 could be shed from cells in the inflammatory microenvironment in RA. This may weaken the interaction of LAIR-1 with its ligand, thus reducing the anti-inflammatory effects of LAIR-1. These findings suggested that LAIR-1 may be an important factor involved in the mediation of the progressive joint destruction in RA.

Topics: Arthritis, Rheumatoid; Biopsy; Cell Proliferation; Cells, Cultured; Fibroblasts; Flow Cytometry; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 13; Polymerase Chain Reaction; Receptors, Immunologic; Synovial Membrane; Synoviocytes; Tumor Necrosis Factor-alpha

Longer pre-centrifugation times alter the quality of serum and plasma samples. Markers for such delays in sample processing and hence for the sample quality, have been identified.. Twenty cytokines in serum, EDTA plasma and citrate plasma samples were screened for changes in concentration induced by extended blood pre-centrifugation delays at room temperature. The two cytokines that showed the largest changes were further validated for their "diagnostic performance" in identifying serum or plasma samples with extended pre-centrifugation times.. In this study, using R&D Systems ELISA kits, EDTA plasma samples and serum samples with a pre-centrifugation delay longer than 24 h had an IL16 concentration higher than 313 pg/mL, and an IL8 concentration higher than 125 pg/mL, respectively. EDTA plasma samples with a pre-centrifugation delay longer than 48 h had an IL16 concentration higher than 897 pg/mL, citrate plasma samples had an IL8 concentration higher than 21.5 pg/mL and serum samples had an IL8 concentration higher than 528 pg/mL.. These robust and accurate tools, based on simple and commercially available ELISA assays can greatly facilitate qualification of serum and plasma legacy collections with undocumented pre-analytics.

Topics: Adult; Arthritis, Rheumatoid; Biomarkers; Blood Chemical Analysis; Centrifugation; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-16; Interleukin-8; Male; Middle Aged; Pancreatitis; ROC Curve; Specimen Handling; Temperature; Time Factors; Young Adult

Inflammation is characterized by the infiltration of leukocytes from the circulation and into the inflamed area. Leukocytes are guided throughout this process by chemokines. These are basic proteins that interact with leukocytes to initiate their activation and extravasation via chemokine receptors. This is enabled through chemokine immobilization by glycosaminoglycans (GAGs) at the luminal endothelial surface of blood vessels. A specific stretch of basic amino acids on the chemokine, often at the C terminus, interacts with the negatively charged GAGs, which is considered an essential interaction for the chemokine function. Short-chain peptides based on this GAG-binding region of the chemokines CCL5, CXCL8, and CXCL12γ were synthesized using standard Fmoc chemistry. These peptides were found to bind to GAGs with high affinity, which translated into a reduction of leukocyte migration across a cultured human endothelial monolayer in response to chemokines. The leukocyte migration was inhibited upon removal of heparan sulfate from the endothelial surface and was found to reduce the ability of the chemokine and peptide to bind to endothelial cells in binding assays and to human rheumatoid arthritis tissue. The data suggest that the peptide competes with the wild-type chemokine for binding to GAGs such as HS and thereby reduces chemokine presentation and subsequent leukocyte migration. Furthermore, the lead peptide based on CXCL8 could reduce the disease severity and serum levels of the proinflammatory cytokine TNF-α in a murine Ag-induced arthritis model. Taken together, evidence is provided for interfering with the chemokine-GAG interaction as a relevant therapeutic approach.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Chemokines; Chemotaxis, Leukocyte; Glycosaminoglycans; Humans; Interleukin-8; Mice; Peptides

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblast-like synoviocytes (FLS) play a central role in RA initiation, progression, and perpetuation. Prior studies showed that sirtuin 1 (SIRT1), a deacetylase participating in a broad range of transcriptional and metabolic regulations, may impact cell proliferation and inflammatory responses. However, the role of SIRT1 in RA-FLS was unclear. Here, we explored the effects of SIRT1 on the aggressiveness and inflammatory responses of cultured RA-FLS. SIRT1 expression was significantly lower in synovial tissues and FLS from RA patients than from healthy controls. Overexpression of SIRT1 significantly inhibited RA-FLS proliferation, migration, and invasion. SIRT1 overexpression also significantly increased RA-FLS apoptosis and caspase-3 and -8 activity. Focusing on inflammatory phenotypes, we found SIRT1 significantly reduced RA-FLS secretion of TNF-α, IL-6, IL-8, and IL-1β. Mechanistic studies further revealed SIRT1 suppressed NF-κB pathway by reducing p65 protein expression, phosphorylation, and acetylation in RA-FLS. Our results suggest SIRT1 is a key regulator in RA pathogenesis by suppressing aggressive phenotypes and inflammatory response of FLS. Enhancing SIRT1 expression or function in FLS could be therapeutic beneficial for RA by inhibiting synovial hyperplasia and inflammation.

Topics: Adult; Aged; Apoptosis; Arthritis, Rheumatoid; Arthroplasty, Replacement; Case-Control Studies; Caspase 3; Caspase 8; Cell Cycle; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Middle Aged; Primary Cell Culture; Signal Transduction; Sirtuin 1; Synovial Membrane; Synoviocytes; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated peptide antibodies (ACPAs) and neutrophils infiltrating the synovial fluid (SF) of the affected joints. The aim of this work was to analyze whether the presence of ACPAs in SF is associated with neutrophil infiltration and with their phenotype in the inflamed joints of RA patients. We found that in the presence of ACPAs, the number of synovial neutrophils correlated with severe disease activity. The SF were divided according to synovial ACPA levels in negative- (<25 U/mL), low- (25-200 U/mL) and high level (˃200 U/mL; ACPA

Topics: Adult; Aged; Anti-Citrullinated Protein Antibodies; Arthritis, Rheumatoid; Female; Humans; Intercellular Adhesion Molecule-1; Interleukin-17; Interleukin-6; Interleukin-8; Leukocyte Count; Male; Middle Aged; Neutrophils; Reactive Oxygen Species; Synovial Fluid

Rheumatoid arthritis (RA) is a high morbidity and disability disease with numerous inflammatory cells infiltrating in interstitial of articular cartilages and bones. As the most abundant inflammatory cells, neutrophil has been reported that their apoptosis changed gradually in the circumstance of RA. Apoptosis, one modality of programmed cell death (PCD), is closely associated with autophagy, which indicates neutrophil autophagy may also alter in RA. Flow cytometry, western blotting, immunohistochemistry, immunofluorescence, transmission electron microscope and multiplex antibody microarray were used to comparative investigate the status of neutrophil autophagy in patients with RA and in vitro. The results showed that the expression of autophagy related LC3 protein was up-regulated with lower lysosomal pH in neutrophils from synovial fluid of RA and changed under stimulation of CQ and small RNA interferences (siRNAs) Atg5 transfection, which proved in acute promyelocytic leukemia HL-60 cell lines, predominantly a neutrophilic promyelocyte, treated by plasma and synovial fluid from RA. We further found out the concentration of IL-6, IL-8, IL-10 and MCP-1 was higher in their synovial fluid which may mediate neutrophil autophagy in RA via cytokine-cytokine receptor interaction and IL-17 signaling pathway. Our results indicate that neutrophil autophagy may be a novel perspective to understand the pathology which may provide a new maker to diagnose RA and IL-8, IL-10, MCP-1 specific antagonists and neutrophil autophagy target inhibitors may improve the therapeutic effect of RA someday.

Topics: Adult; Aged; Arthritis, Rheumatoid; Autophagy; Chemokine CCL2; Female; HL-60 Cells; Humans; Interleukin-10; Interleukin-6; Interleukin-8; Male; Microtubule-Associated Proteins; Middle Aged; Neutrophils; Synovial Fluid; Up-Regulation

Rheumatoid arthritis (RA) is a chronic and refractory autoimmune joint disease. Fibroblast-like synoviocytes (FLS) produce inflammatory cytokines and are involved in the migration and invasion of panuus tissue, which leads to the destruction of joints in RA. Receptor for hyaluronan mediated motility (RHAMM), is known to be one of the important receptors for hyaluronic acid. It has the ability to regulate migration of fibrocytes and infiltration of inflammatory cells. Here,we explored the mechanisms of RHAMM in RAFs.. Quantitative PCR and western blot were performed to test the expression of RHAMM in synoviocytes of RA patients and osteoarthritis (OA) controls. Collagen antibody-induced arthritis (CAIA) was used to investigate the RHAMM expression in mouse synovial issues. RHAMM siRNA was used to detect the function of RHAMM in FLS.. RA-FLS has a significantly higher expression of RHAMM than OA-FLS. Expression of RHAMM in joint synovial tissue was markedly increased in the CAIA mice compared with the controls. RHAMM silencing using SiRNA was not only decreased the production of IL-6 and IL-8, but also inhibited the migration and invasion of RA-FLS.. RHAMM has an important role in the FLS induced modulation of inflammation and destruction of joints in RA.

Topics: Animals; Arthritis, Rheumatoid; Cell Migration Assays; Cells, Cultured; Disease Progression; Extracellular Matrix Proteins; Gene Silencing; Humans; Hyaluronan Receptors; Interleukin-6; Interleukin-8; Mice; RNA, Messenger; RNA, Small Interfering; Synovial Membrane; Synoviocytes

To study the diagnostic value of P2X7 receptor for rheumatoid arthritis (RA) and its role in the inflammatory response.. With the synovial tissues from 25 patients with bone and joint replacement as the control,the synovial tissues of 25 RA patients were examined for the relative expression of P2X7 receptor mRNA using qRT-PCR.In an immortalized RA synovial cell line (MH7A),the effect of P2X7 receptor knockdown via a small interfering RNA were examined on the productions of the inflammatory cytokines including interleukin-1β(IL-1β),IL-6,and IL-8 using ELISA.. The RA patients showed significantly higher levels of P2X7 receptor mRNA expression in the synovial tissue than the control patients.P2X7 receptor had a good diagnostic value for RA.The expression levels of IL-1β,IL-6,and IL-8 were positively correlated with the levels of P2X7 receptor in the synovial tissues of RA patients (. RA patients show elevated P2X7 receptor level in the synovial tissue, which has a good diagnostic value for RA.Blocking P2X7 receptor can inhibit inflammatory factor secretion and suppress inflammatory reactions.

Topics: Arthritis, Rheumatoid; Case-Control Studies; Cell Line; Gene Knockdown Techniques; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X7; RNA, Messenger; Synovial Membrane

Neutrophils play a crucial role in the pathophysiology of rheumatoid arthritis (RA) via the release of reactive oxygen species (ROS), proteases and cytokines. Orally active Janus kinase (JAK) inhibitors (JAKi), e.g. baricitinib and tofacitinib, have high clinical efficacy in RA but are linked with neutropenia and increased infections. Our aim was to determine the effect of JAK inhibition with baricitinib and tofacitinib on healthy control and RA neutrophil lifespan and function. RA (n = 7) and healthy control (n = 7) neutrophils were treated with baricitinib or tofacitinib for 30 min, prior to incubation in the absence or presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon (IFN)-γ. JAKi prevented GM-CSF- and IFN-γ-induced apoptosis delay in RA and healthy control neutrophils in a dose-dependent manner. Baricitinib decreased the rate of chemotaxis towards interleukin (IL)-8, but not f-Met-Leu-Phe (fMLP) in RA neutrophils. While healthy control neutrophils incubated with GM-CSF became primed to produce ROS in response to stimulation with fMLP and phorbol-12-myristate-12-acetate (PMA), RA neutrophils produced increased levels of ROS without the need for priming. JAKi prevented ROS release from primed healthy control neutrophils in response to fMLP, but had no effect on ROS production by RA neutrophils. Baricitinib reversed GM-CSF priming of ROS production in response to fMLP in healthy control, but not RA, neutrophils. We conclude that incubation with JAKi prevents chemotaxis of RA neutrophils towards IL-8, but does not prevent the production of ROS or increase the level of apoptosis. This may be due to the in-vivo exposure of RA neutrophils to priming agents other than those that activate JAK/signal transducer and activator of transcription (STAT) signalling.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Azetidines; Case-Control Studies; Cell Movement; Cells, Cultured; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Janus Kinases; Male; Middle Aged; Neutrophils; Piperidines; Protein Kinase Inhibitors; Purines; Pyrazoles; Pyrimidines; Pyrroles; Reactive Oxygen Species; Respiratory Burst; Sulfonamides; Tetradecanoylphorbol Acetate; United Kingdom

The increase in Rheumatoid arthritis (RA) associated mortality is predominantly due to accelerated coronary artery and cerebrovascular atherosclerosis with increased risk of ischemic heart disease about 50% in RA patients compared to controls.. To study the pathogenesis of ischemic heart disease in RA, role of inflammatory cytokine interplay, disease activity and rheumatoid factor positivity.. Eighty RA patients and 44 healthy controls were included. All subjects were younger than 45years for females and 55years for males with exclusion of all traditional risk factors for atherosclerosis. Interleukin (IL) 1, 6 and 18 were assessed in all subjects. RA patients fulfilled ACR/EULAR 2010 criteria and were subjected to Dobutaminestress-echocardiography, diseases activity assessed by DAS-28, X-ray hands for Larsen score and function assessment by HAQ.. RA patients had significantly higher serum IL 1, 6 and 18 than controls (p=0.00 in all). Thirty four (42.5%) patients had hypertensive reaction on Dobutamine-stress-echocardiography, four of them had ischemic change, and 46 (57.5%) had normal reaction. All patients with hypertensive reaction had positive RF (p=0.00), 10 had DAS-28>5.1, 20 had DAS-28 from 3.2 to5.1 and 4 were in remission (p=0.001). CRP was higher in patients with hypertensive reaction (p=0.003) while serum levels of IL1, 6 and 18 showed no significant difference. In all patients, serum levels of IL1, 6 and 18 showed significant positive correlation with VAS, HAQ and DAS-28 (p<0.001 in all). Only IL18 showed significant positive correlation with X-ray score in all patients.. Disease activity and RF positivity play an important risk factor for ischemic heart disease in RA. Serum levels of IL1, 6 and 18 did not help much in detecting patients at risk of ischemic heart disease. Better control of RA disease activity with early remission helps in preventing cardiac complications. More studies on larger number of patients are needed for better understanding of mechanism of ischemic heart disease in RA.

Topics: Adult; Arthritis, Rheumatoid; Atherosclerosis; Coronary Artery Disease; Cross-Sectional Studies; Cytokines; Female; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Middle Aged; Myocardial Ischemia; Risk Factors; Severity of Illness Index

In this study, we found that catechins found in green tea (EGCG, EGC, and EC) differentially interfere with the IL-1β signaling pathway which regulates the expression of pro-inflammatory mediators (IL-6 and IL-8) and Cox-2 in primary human rheumatoid arthritis synovial fibroblasts (RASFs). EGCG and EGC inhibited IL-6, IL-8, and MMP-2 production and selectively inhibited Cox-2 expression. EC did not exhibit any inhibitory effects. When we looked at the expression of key signaling proteins in the IL-1β signaling pathway, we found all the tested catechins could inhibit TAK-1 activity. Therefore, the consumption of green tea offers an overall anti-inflammatory effect. Molecular docking analysis confirms that EGCG, EGC, and EC all occupy the active site of the TAK1 kinase domain. However, EGCG occupies the majority of the TAK1 active site. In addition to TAK1 inhibition, EGCG can also inhibit P38 and nuclear NF-κB expression whereas EC and EGC were not effective inhibitors. Our findings suggest one of the main health benefits associated with the consumption of green tea are due to the activity of EGCG and EGC which are both present at higher amounts. Although EGCG is the most effective catechin at inhibiting downstream inflammatory signaling, its effectiveness could be hindered by the presence of EC. Therefore, varying EC content in green tea may reduce the anti-inflammatory effects of other potential catechins in green tea.

Topics: Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Catalytic Domain; Catechin; Cells, Cultured; Cyclooxygenase 2 Inhibitors; Fibroblasts; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 2; Molecular Docking Simulation; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phytotherapy; Plants, Medicinal; Protein Binding; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-jun; Signal Transduction; Synovial Membrane; Tea

High-resolution atomic force microscopy (AFM) was used for the in situ evaluation of the anti-inflammatory effects of triptolide on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to understand the anti-RA effects of triptolide, based on the morphological and biophysical changes observed in RA-FLS. RA-FLS have been reported to play a primary role in inflammatory bone destruction during the development of RA and thus are regarded as an important target for RA treatment. Triptolide pretreatment significantly inhibited tumor necrosis factor-α-induced expression of the interleukin (IL)-1β, IL-6, and IL-8 genes in MH7A cells. Using AFM, we showed that triptolide-induced morphological damage in MH7A cells by inducing significant ultrastructure changes in the membrane, which were closely related to triptolide-induced apoptosis in MH7A cells. Using force measurements determined with AFM, triptolide was shown to increase the stiffness of MH7A cells. These findings not only revealed the strong anti-inflammatory effects of triptolide on RA-FLS, highlighting triptolide as a potential anti-RA agent, but also revealed the possible use of AFM for studying anti-inflammatory responses in RA-FLS, which we expect to be developed into a potential tool for anti-RA drug studies in RA-FLS.

Topics: Anti-Inflammatory Agents; Apoptosis; Arthritis, Rheumatoid; Cell Line; Cell Membrane; Cell Proliferation; Diterpenes; Epoxy Compounds; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Microscopy, Atomic Force; Phenanthrenes; Synoviocytes; Tumor Necrosis Factor-alpha

To investigate the effects of silencing transforming growth factor-β activating kinase 1 (TAK1)on the expressions of IL-6 and IL-8 induced by TNF-α in fibroblast-like synoviocytes, and to explore the role of TAK1 in rheumatoid arthritis (RA).. The synthesized TAK1 siRNA and scrambled siRNA (ScRNA) were transferred into cultured RA fibroblast-like synoviocyte line MH7A by lipofectamine. The expressions of the pro-inflammatory mediator IL-6 and IL-8 and the levels of phospho-P38(p-P38), phospho-C-Jun NH2-terminal kinase(p-JNK), phospho-extracellular signal-regulated kinase(p-ERK), phospho-p65(p-p65) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha(IκBa) were examined.. Silencing of TAK was demonstrated in synoviocytes transfected by TAK siRNA. TAK1 silencing markedly attenuated the expression of IL-6 and IL-8 in the presence of TNF-α. TAK1 silencing inhibited the activation of p38 and JNK MAPK. TAK1 silencing also inhibited activation of nuclear factor-κB (NF-κB).. TAK1 silencing attenuated the expression of IL-6 and IL-8 in synoviocytes induced by TNF-α

Topics: Arthritis, Rheumatoid; Cell Line; Fibroblasts; Gene Silencing; Humans; Interleukin-6; Interleukin-8; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; NF-kappa B; Synoviocytes; Tumor Necrosis Factor-alpha

Midkine (MK) is involved in cell proliferation, differentiation, migration, and survival. In this study, we measured serum MK levels in rheumatoid arthritis (RA) and investigated the correlation of serum MK with RA disease activity. Expression and effect of MK in RA synovial tissue were also examined.. Serum MK and production of inflammatory mediators by rheumatoid synovial fibroblasts (RSFs) were measured by enzyme-linked immunosorbent assay. MK expression in synovial tissue was examined by immunohistochemistry. MK receptor expression was analyzed by RT-PCR and Western blotting.. RA patients had a significantly higher serum MK level than healthy controls. In RA patients, the MK level was correlated with DAS28-ESR, disability index of the Health Assessment Questionnaire, and rheumatoid factor level. The serum MK level tended to be decreased by anti-TNF therapy. MK was expressed by synovial lining cells in RA synovial tissues and it enhanced the production of IL-6, IL-8, and CCL2 by RSFs. RSFs expressed LDL receptor-related protein 1, candidate receptor for MK.. The serum MK level could be a marker of disease activity in RA and an indicator of a poor prognosis. MK may have a role in the pathogenesis of RA via induction of inflammatory mediators.

Topics: Aged; Arthritis, Rheumatoid; Biomarkers; Cells, Cultured; Chemokine CCL2; Cytokines; Female; Fibroblasts; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Male; Middle Aged; Midkine; Patient Acuity; Synovial Membrane

Studies were performed to uncover the significance of obesity in rheumatoid arthritis (RA) and preclinical models.. Preclinical arthritis models were used to examine the impact of obesity on disease onset and remission. Conditioned media from RA adipose tissues were used to investigate the mechanism contributing to joint neutrophil influx and M1 macrophage differentiation observed in early and remission phases of arthritis.. We report that mice fed with high fat diet (HFD) have an earlier onset of collagen-induced arthritis (CIA) compared with mice on regular diet. However, the differences in CIA joint swelling between the two diet groups are lost once disease is established. We found that early arthritis triggered by obesity is due to elevated joint MIP2/interleukin-8 levels detected in CIA as well as in the RA and mouse adipose tissues and the effect of this chemokine on neutrophil recruitment. Although active disease progression is similarly affected in both diet groups, arthritis resolution is accelerated in lean mice while joint inflammation is sustained in obese mice. We document that HFD can prolong toll-like receptor (TLR)4-induced arthritis by increasing joint monocyte migration and further remodelling the recruited cells into M1 macrophages. Consistently, we show that adipose condition media can transform RA and wild-type naïve myeloid cells into M1 macrophages; however, this function is impaired by TLR4 blockade or deficiency.. We conclude that despite established disease being unaffected by obesity, the early and the resolution phases of RA are impacted by obesity through different mechanisms.

Topics: Adipose Tissue; Animals; Arthritis, Rheumatoid; Cell Movement; Chemokine CXCL2; Collagen; Cytokines; Dietary Fats; Disease Models, Animal; Interleukin-8; Joints; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neutrophils; Obesity; Signal Transduction; Toll-Like Receptor 4

Gastrodia elata (GE), which belongs to the Orchidaceae family, was found to possess anti-inflammatory activity. However, the effect of GE on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) remains largely unknown. Thus, the aim of this study was to investigate the effects of GE on tumor necrosis factor-α (TNF-α)-induced inflammatory response in RA-FLS and the underlying molecular mechanism was also explored. Our results demonstrated that GE significantly attenuated TNF-α-induced IL-6 and IL-8 production in RA-FLS. GE also inhibited TNF-α-induced MMP-3 and MMP-13 expression in RA-FLS. Furthermore, pretreatment with GE significantly attenuated TNF-α-induced the expression of p-p65 and IκBα degradation in RA-FLS. In conclusion, this study demonstrated for the first time that GE attenuated inflammatory response by inhibiting the NF-κB pathway signaling in RA-FLS. Thus, GE might have a therapeutic potential towards the treatment of RA.

Topics: Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Dose-Response Relationship, Drug; Fibroblasts; Gastrodia; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Phytotherapy; Plant Extracts; Plants, Medicinal; Proteolysis; Signal Transduction; Synovial Membrane; Time Factors; Transcription Factor RelA; Tumor Necrosis Factor-alpha

To explore disease-associated molecules in rheumatoid arthritis (RA), we comprehensively analyzed phosphoproteins purified from RA synoviocytes.. Synoviocytes were obtained from three patients with RA and three patients with osteoarthritis (OA). Profiles of phosphoproteins purified from the synoviocytes were compared by two-dimensional differential gel electrophoresis (2D-DIGE) between the RA and OA groups. Protein spots with significantly different phosphorylation levels were identified by mass spectrometry. Recombinant protein of annexin A4 (ANXA4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA4-transfected and non-transfected synoviocytes under a tumor necrosis factor-α (TNFα)-stimulated condition.. In 2D-DIGE, 318 phosphoprotein spots were detected, of which 94 spots showed significantly different intensities between the two groups (P < 0.05). Among the 94 spots, 22 spots showed two-fold or higher intensity and one spot showed less than 1/2-fold intensity in the RA group compared to the OA group. From the 22 spots, 11 phosphoproteins were identified, which included kinases, carrier and chaperone proteins, cytoskeletal proteins, proteases and calcium-binding proteins. One of the identified calcium-binding proteins was ANXA4, an exocytosis-regulating protein. The transfected rANXA4 was found to be phosphorylated intracellularly, and secretion of chemokine (C-X-C motif) ligand 1 and interleukin-8 induced by TNFα stimulation was significantly suppressed by the transfection (P < 0.01).. The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA4 may be one of therapeutic targets in RA.

Topics: Aged; Annexin A4; Arthritis, Rheumatoid; Biomarkers; Cells, Cultured; Chemokine CXCL1; Female; Humans; Interleukin-8; Middle Aged; Osteoarthritis; Phosphoproteins; Phosphorylation; Proteomics; Synoviocytes; Tandem Mass Spectrometry; Tumor Necrosis Factor-alpha; Two-Dimensional Difference Gel Electrophoresis

To investigate the effects of BET bromodomain protein inhibition on inflammatory activation and functional properties of rheumatoid arthritis synovial fibroblasts (RASF).. The expression of the BET bromodomain proteins BRD2, BRD3 and BRD4 was analysed in synovial tissue by immunohistochemistry. RASF were stimulated with tumour necrosis factor (TNF)-α, interleukin (IL)-1β and toll-like receptor (TLR) ligands (Pam3, pIC and lipopolysaccharide (LPS)) in the presence or absence of the BET inhibitor I-BET151, or siRNA targeting BRD2, BRD3 and BRD4. RASF expression of inflammatory mediators, including MMP1, MMP3, IL-6 and IL-8, was measured by q-PCR, q-PCR array and ELISA. Cellular viability, apoptosis, proliferation and chemoattractive properties of RASF were investigated using MTT, cell apoptosis ELISA, BrdU-based proliferation and transwell migration assays.. BRD2, BRD3 and BRD4 proteins were detected in rheumatoid arthritis (RA) synovial tissue, expressed in both RASF and macrophages. I-BET151 suppressed cytokine and TLR ligand-induced secretion of MMP1, MMP3, IL-6 and IL-8, and mRNA expression of more than 70% of genes induced by TNF-α and IL-1β. Combined silencing of BRD2, BRD3 and BRD4 significantly reduced cytokine and TLR ligand-induced expression of a subset of gene products targeted by I-BET151, including MMP1, CXCL10 and CXCL11. I-BET151 treatment of RASF reduced RASF proliferation, and the chemotactic potential for peripheral blood leucocytes of RASF conditioned medium.. Inhibition of BET family proteins suppresses the inflammatory, matrix-degrading, proliferative and chemoattractive properties of RASF and suggests a therapeutic potential in the targeting of epigenetic reader proteins in RA.

Topics: Arthritis, Rheumatoid; Cell Cycle Proteins; Fibroblasts; Gene Expression; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; Interleukin-1beta; Interleukin-6; Interleukin-8; Lipopolysaccharides; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Nuclear Proteins; Osteoarthritis; Protein Serine-Threonine Kinases; RNA-Binding Proteins; RNA, Messenger; Synovial Membrane; Toll-Like Receptors; Transcription Factors; Tumor Necrosis Factor-alpha

We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage.

Topics: Adult; Aged; Apoptosis; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Case-Control Studies; Cell Line; Chaperonin 60; Citrulline; Cloning, Molecular; Female; Humans; Interleukin-6; Interleukin-8; Joints; Male; Middle Aged; Mitochondrial Proteins; Osteoblasts; Protein Binding; Protein Processing, Post-Translational; Recombinant Proteins; Toll-Like Receptor 4

Transforming growth factor β-activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin-1β (IL-1β), tumor necrosis factor (TNF), and Toll-like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA).. The effect of TAK1, IL-1 receptor-associated kinase 1 (IRAK-1), and TNF receptor-associated factor 6 (TRAF6) inhibition was evaluated in IL-1β-stimulated human RA synovial fibroblasts (RASFs). Western blotting, immunoprecipitation, and 20S proteasome assay were used to study the ubiquitination process in RASFs. The efficacy of epigallocatechin-3-gallate (EGCG), a potent antiinflammatory molecule, in regulating these processes in RASFs was evaluated. Molecular docking was performed to examine the interaction of EGCG with human TAK1, IRAK-1, and TRAF6. These findings were confirmed using a rat model of adjuvant-induced arthritis (AIA).. Inhibition of TAK1, but not IRAK-1 or TRAF6, completely abrogated IL-1β-induced IL-6 and IL-8 synthesis in RASFs. EGCG inhibited TAK1 phosphorylation at Thr(184/187) and occupied the C(174) position, an ATP-binding site, to inhibit its kinase activity. EGCG pretreatment also inhibited K(63) -linked autoubiquitination of TRAF6, a posttranslational modification essential for TAK1 autophosphorylation, by forming a stable H bond at the K(124) position on TRAF6. Furthermore, EGCG enhanced proteasome-associated deubiquitinase expression to rescue proteins from proteasomal degradation. Western blot analyses of joint homogenates from rats with AIA showed a significant increase in K(48) -linked polyubiquitination, TAK1 phosphorylation, and TRAF6 expression when compared to naive rats. Administration of EGCG (50 mg/kg/day) for 10 days ameliorated AIA in rats by reducing TAK1 phosphorylation and K(48) -linked polyubiquitination.. Our findings provide a rationale for targeting TAK1 for the treatment of RA with EGCG.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Blotting, Western; Catechin; Disease Models, Animal; Female; Fibroblasts; Humans; Immunoprecipitation; In Vitro Techniques; Interleukin-1 Receptor-Associated Kinases; Interleukin-1beta; Interleukin-6; Interleukin-8; Lysine; MAP Kinase Kinase Kinases; Molecular Docking Simulation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rats; Rats, Inbred Lew; Synovial Membrane; TNF Receptor-Associated Factor 6; Ubiquitination

Synovial fibroblast hyperplasia, T-cell hyperactivity, B-cell overactivation, and the self-perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia-inducible factor-1α (HIF-1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF-1α regulates interactions between RASFs and T cells and B cells. We report here that HIF-1α promotes the expression of inflammatory cytokines IL-6, IL-8, TNF-α, and IL-1β, and cell-cell contact mediators IL-15, vascular cell adhesion molecule (VCAM)-1, thrombospondin (TSP)-1, and stromal cell-derived factor (SDF)-1 in RASFs. Furthermore, HIF-1α perpetuates RASF-mediated inflammatory Th1- and Th17-cell expansion while differentially inhibiting regulatory B10 and innate-like B cells, leading to increased IFN-γ, IL-17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF-1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF-1α may provide new therapeutic strategies for overcoming this persistent disease.

Topics: Arthritis, Rheumatoid; Autoantibodies; B-Lymphocytes; Cells, Cultured; Chemokine CXCL12; Cytokines; Fibroblasts; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-15; Interleukin-17; Interleukin-6; Interleukin-8; Synovial Membrane; T-Lymphocytes; Thrombospondin 1; Vascular Cell Adhesion Molecule-1

Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process.. Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin.. Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin.. We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Topics: Animals; Arthritis, Rheumatoid; Autoantibodies; B-Lymphocytes; Bone and Bones; Bone Resorption; Cell Culture Techniques; Chemokines; Citrulline; Female; Humans; Hydrolases; Immunohistochemistry; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Middle Aged; Osteoclasts; Protein-Arginine Deiminases; Receptors, Interleukin-8; Sulfonamides; Synovial Fluid; X-Ray Microtomography

Topics: Animals; Arthralgia; Arthritis, Rheumatoid; Autoantibodies; Bone and Bones; Bone Resorption; Chemokine CXCL1; Citrulline; Female; Humans; Hydrolases; Interleukin-8; Male; Nociception; Osteoclasts

Topics: Animals; Arthralgia; Arthritis, Rheumatoid; Autoantibodies; Bone and Bones; Bone Resorption; Chemokine CXCL1; Citrulline; Female; Humans; Hydrolases; Interleukin-8; Male; Nociception; Osteoclasts

Osteoclast-associated receptor (OSCAR) is an activating receptor expressed by human myeloid cells. Collagen type I (ColI) and collagen type II (ColII) serve as ligands for OSCAR. OSCAR-collagen interaction stimulates RANK-dependent osteoclastogenesis. We have recently reported that OSCAR promotes functional maturation of monocyte-derived dendritic cells. OSCAR is upregulated on monocytes from rheumatoid arthritis (RA) patients with active disease, and these monocytes show an increased proosteoclastogenic potential. In the current study, we have addressed a functional role for an OSCAR-collagen interaction on monocytes. We show that OSCAR-ColII signaling promoted the survival of monocytes. Moreover, ColII stimulated the release of proinflammatory cytokines by monocytes from healthy donors, which could be completely blocked by an anti-OSCAR monoclonal antibody. Mononuclear cells from the synovial fluid of RA patients plated on ColII secreted TNF-α and IL-8 in an OSCAR-dependent manner. Global RNA profiling showed that components of multiple signaling pathways relevant to RA pathogenesis are regulated at the transcriptional level by OSCAR in monocytes. Thus, OSCAR can play a proinflammatory role in monocyte-derived cells and may contribute crucially on multiple levels to RA pathogenesis.

Topics: Antibodies, Monoclonal; Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type II; Dendritic Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-8; Monocytes; Osteoclasts; Receptors, Cell Surface; Signal Transduction; Synovial Fluid; Tumor Necrosis Factor-alpha

To compare the effects of TNF-α and IL-17A on osteogenic differentiation of isolated fibroblast-like synoviocytes (FLS) from healthy donors, osteoarthritis (OA) and rheumatoid arthritis (RA) patients.. FLS were cultured in osteogenic medium, with and without TNF-α and/or IL-17A. Extracellular matrix mineralization was evaluated by alizarin red staining and alkaline phosphatase activity (ALP) measurement. mRNA expression was analyzed by qRT-PCR for Wnt5a, BMP2 and Runx2, genes associated with osteogenesis, for DKK1 and RANKL, genes associated with osteogenesis inhibition and Schnurri-3, a new critical gene in the cross talk with osteoclasts. IL-6 and IL-8 production was measured by ELISA.. In osteogenic medium, matrix mineralization and increased ALP activity indicated that FLS can undergo osteogenic differentiation, which was increased with TNF-α and IL-17A. The expression of osteogenesis activators (BMP2 and Wnt5a) was increased with cytokines and that of the osteogenesis inhibitor DKK1 was decreased. There was no difference between all three cell types. In contrast, RA FLS were particularly sensitive to the synergistic increase of Shn3 with TNF-α and IL-17A. Levels of IL-6 and IL-8 were also higher for RA-FLS, compared to healthy and OA FLS.. IL-17A and/or TNF-α treatment favor an osteogenesis induction in isolated FLS, independent of their origin. RA-FLS were more sensitive to the synergistic increase of Schnurri-3 expression. Combined with the higher levels of inflammation, this may in turn activate osteoclastogenesis, leading to increased bone destruction seen in destructive arthritis.

Topics: Alkaline Phosphatase; Arthritis, Rheumatoid; Bone Morphogenetic Protein 2; Calcification, Physiologic; Calcium; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; DNA-Binding Proteins; Extracellular Matrix; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-17; Interleukin-6; Interleukin-8; Osteoblasts; RANK Ligand; RNA, Messenger; Synoviocytes; Tumor Necrosis Factor-alpha; Wnt-5a Protein

G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gβγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.

Topics: Arthritis, Rheumatoid; Cell Line, Tumor; Chemotaxis, Leukocyte; Guanine Nucleotide Dissociation Inhibitors; Humans; Interleukin-8; Leukopoiesis; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Polymorphism, Single Nucleotide; Tretinoin

Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) lead to cartilage destruction, and the activation of NF‑κB is important in the proliferation of FLSs. Heparin is a glycosaminoglycan, which is widely used as an anticoagulant. In the present study, the effect of heparin on the tumor necrosis factor (TNF)‑α induced proliferation of FLSs was investigated. Western blot and polymerase chain reaction analyses were used to assess the expression levels of cytokines. The results revealed that TNF‑α induced the expression of interleukin (IL)‑6, IL‑8, TNF‑α and cyclin D1. Heparin inhibited the growth rate of the FLSs induced by TNF‑α. Heparin also decreased the TNF‑α‑induced mRNA and protein expression levels of IL‑6, IL‑8, TNF‑α and cyclin D1 in a dose‑dependent manner. Immunofluorescence analysis showed that the expression of cytoplasmic TNF‑α was significantly reduced by heparin treatment. Furthermore, the levels of p65 and inhibitor of nuclear factor (NF)‑κB phosphorylation were inhibited by heparin treatment, suggesting that heparin induced the inhibition of NF‑κB. In conclusion, the results of the present study revealed that heparin inhibited the TNF‑α‑induced proliferation, cytokine production, expression of cyclin D1 and activation of NF‑κB signaling in FLSs, indicating the therapeutic potential of heparin in the treatment of RA.

Topics: Anti-Inflammatory Agents; Anticoagulants; Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Fibroblasts; Heparin; Humans; Interleukin-6; Interleukin-8; NF-kappa B; Synoviocytes; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) patients with early elevations of antibodies against collagen type II (CII) have a distinct acute onset phenotype, associated with cytokine induction by surface-bound anti-CII-containing immune complexes (ICs) and high C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Polymorphonuclear granulocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) are abundant in the vicinity of CII in RA joints, and both PMN and PBMC reactivity against anti-CII IC individually relate to early joint destruction and early elevation of CRP and ESR in RA. We searched for CII-dependent mechanisms that might attract PMNs and PBMCs to RA joints. Human PBMCs and PMNs were stimulated with anti-CII ICs and control ICs, either individually or in cocultures. Cocultured PMNs and PBMCs stimulated with anti-CII ICs synergistically augmented production of the chemokines CXCL8, RANTES and MCP-1, whereas downregulation was seen with control IC. This upregulation was unique to chemokines, as TNF-α, IL-1β, and GM-CSF were downregulated in anti-CII IC-stimulated cocultures. The coculture-associated chemokine upregulation depended on endogenous TLR4 ligand(s) and functionally active PMN enzymes, and was partially mediated by GM-CSF. As anti-CII levels peak around the time of RA diagnosis, this mechanism can attract inflammatory cells to joints in early RA and intensify the anti-CII-associated acute onset RA phenotype.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Autoantibodies; Cells, Cultured; Chemokine CCL2; Chemokine CCL5; Coculture Techniques; Collagen Type II; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Humans; Interleukin-8; Joints; Leukocytes, Mononuclear; Toll-Like Receptor 4

Previously, we demonstrated that the urine proteome signature of patients with rheumatoid arthritis (RA) reflects inflammation-related cellular processes. Here, we measured interleukin (IL)-6, IL-8, and chemokine ligand 2 (CCL2) concentrations in the urine of RA patients and prospectively investigated their role in predicting RA activity and prognosis. One hundred seventy-three RA patients and 62 non-RA controls were recruited. Urinary IL-6, CCL2, and IL-8 levels were elevated in RA patients and correlated well with disease activity. Urinary IL-6 level at presentation was an independent risk factor of radiographic progression at 1 and 3 years. High urinary IL-6 level increased the risk ratio of radiographic progression by 2.9-fold, which was comparable to high serum CRP. Moreover, combination of urinary IL-6 and serum CRP measures synergistically increased the predictability of radiographic progression. In a subgroup with normal ESR, patients with the highest tertile of urinary IL-6 were at 6.4-fold greater risk of radiographic progression. Conclusively, high urinary IL-6 level at presentation is an independent risk factor for radiographic progression of RA, reflecting disease activity. Urinary IL-6 in combination with serum CRP may be a useful parameter for estimating RA prognosis.

Topics: Adult; Arthritis, Rheumatoid; Chemokine CCL2; Disease Progression; Female; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged

Due to their role in inflammatory metabolic diseases, we hypothesised that free fatty acids (FFA) are also involved in inflammatory joint diseases. To test this hypothesis, we analysed the effect of FFA on synovial fibroblasts (SF), human chondrocytes and endothelial cells. We also investigated whether the toll-like receptor 4 (TLR4), which can contribute to driving arthritis, is involved in FFA signalling.. Rheumatoid arthritis SF, osteoarthritis SF, psoriatic arthritis SF, human chondrocytes and endothelial cells were stimulated in vitro with different FFA. Immunoassays were used to quantify FFA-induced protein secretion. TLR4 signalling was inhibited extracellularly and intracellularly. Fatty acid translocase (CD36), responsible for transporting long-chain FFA into the cell, was also inhibited.. In rheumatoid arthritis synovial fibroblasts (RASF), FFA dose-dependently enhanced the secretion of the proinflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, as well as the matrix-degrading enzymes pro-MMP1 and MMP3. The intensity of the response was mainly dependent on the patient rather than on the type of disease. Both saturated and unsaturated FFA showed similar effects on RASF, while responses to the different FFA varied for human chondrocytes and endothelial cells. Extracellular and intracellular TLR4 inhibition as well as fatty acid transport inhibition blocked the palmitic acid-induced IL-6 secretion of RASF.. The data show that FFA are not only metabolic substrates but may also directly contribute to articular inflammation and degradation in inflammatory joint diseases. Moreover, the data suggest that, in RASF, FFA exert their effects via TLR4 and require extracellular and intracellular access to the TLR4 receptor complex.

Topics: Arthritis, Psoriatic; Arthritis, Rheumatoid; CD36 Antigens; Chemokine CCL2; Chondrocytes; Endothelial Cells; Fatty Acids, Nonesterified; Fibroblasts; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Osteoarthritis; Signal Transduction; Synovial Membrane; Toll-Like Receptor 4

To examine the effect of hypoxia on Signal Transducer and Activator of Transcription 3 (STAT3)-induced pro-inflammatory pathways in rheumatoid arthritis (RA).. Detection of phospho-STAT3 was assessed in RA synovial tissue and fibroblasts (RASFC) by immunohistology/immunofluorescence. Primary RASFCs and a normal synoviocyte cell line (K4IM) were cultured under hypoxic and normoxic conditions±Stat3-siRNA, HIF-siRNA or WP1066 (JAK2-inhibitor). HIF1α, p-STAT3, p-STAT1 and Notch-1IC protein expression were analysed by western blot. Functional mechanisms were quantified by invasion chamber, matrigel and migration assays. IL-6, IL-8, IL-10 and matrixmetalloproteinases (MMP)-3 were quantified by ELISA. Notch-1 receptor, its DLL-4 ligand and downstream target genes (hrt-1, hrt-2) were quantified by real-time PCR. The effect of WP1066 on spontaneous secretion of pro/anti-inflammatory cytokines and Notch signalling was examined in RA synovial explants ex vivo.. p-STAT3 was increased in RA synovium compared with control (p<0.05). Hypoxia induced p-STAT3, p-STAT1 and HIF1α expression, an effect blocked by Stat3-siRNA and WP1066. Hypoxia-induced cell invasion, migration and cytokine production were inhibited by Stat3-siRNA (p<0.05) and WP1066 (p<0.05). While HIF1α siRNA inhibited hypoxia-induced p-STAT3 detection, Stat3-siRNA also inhibited hypoxia-induced HIF1α. Furthermore, hypoxia-induced Notch-1IC, DLL4, hrt-1 and -2 expression were significantly inhibited by WP1066 (p<0.05). Finally, in RA synovial explant cultures ex vivo, WP1066 decreased spontaneous secretion of IL-6, IL-8 and MMP3 (p<0.05), Notch-1 mRNA (p<0.05) and induced IL-10 (p<0.05).. This is the first study to provide evidence of a functional link between HIF1α, STAT3 and Notch-1 signalling in the regulation of pro-inflammatory mechanisms in RA, and further supports a role for STAT blockade in the treatment of RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-10; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; Middle Aged; Receptor, Notch1; Signal Transduction; STAT3 Transcription Factor; Synovial Membrane

To study the effect of oral administration of dimethyl dimethoxy biphenyl dicarboxylate (DDB) on adjusting angiogeneic/inflammatory mediators and ameliorating the pathology of bones in rats with collagen-induced arthritis (CIA).. Wistar rat model of CIA was set up using bovine collagen type II. Fifty rats were divided into five groups randomly: normal, CIA model, DDB treatment, methotrexate (MTX) treatment, and combined DDB+MTX treatment. Ankle joints of rats were imaged with digital X-ray machine to show the destruction of joints. Fore and hind paw and knee joints were removed above the ankle joint then processed for haematoxylin and eosin staining. Plasma levels of vascular endothelial growth factor (VEGF), platelet derived growth factor, interleukin-8 (IL-8), IL-4, tumor necrosis factor α (TNF-α), and cyclooxygenase-2 (COX-2) were quantified by enzyme-linked immunosorbent assay. Nitric oxide levels were detected by Griess reagent.. Compared with the CIA model group, a remarkable reduction in various angiogenic (VEGF and IL-8) and inflammatory mediators (TNF-α, IL-4 and COX-2) after treatment with DDB either alone or combined with MTX P<0.05 or P<0.01). Histopathological and X-ray findings were confirmatory to the observed DDB anti-arthritic effect. The DDB-treated group showed amelioration in signs of arthritis which appeared essentially similar to normal.. Our data shed light on the therapeutic efficacy of DDB in experimental rheumatoid arthritis (RA) compared with a choice drug (MTX) and it may be offered as a second-line drug in the treatment of RA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Collagen; Cyclooxygenase 2; Dioxoles; Enzyme-Linked Immunosorbent Assay; Female; Interleukin-4; Interleukin-8; Methotrexate; Nitric Oxide; Platelet-Derived Growth Factor; Radiography; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo.. Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested.. TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice.. Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.

Topics: Animals; Antibodies, Bispecific; Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Disease Models, Animal; Drug Synergism; Fibroblasts; Granulocyte Colony-Stimulating Factor; Humans; In Vitro Techniques; Interleukin-17; Interleukin-8; Metalloproteases; Mice; Mice, Transgenic; Synovial Membrane; Tumor Necrosis Factor-alpha

Lunasin, a peptide with 43 amino acid residues and initially isolated and identified in soybean cotyledon, has gained extensive attention due to its anti-inflammatory and anticancer properties. However, its treatment efficacy on rheumatoid arthritis (RA) and corresponding mechanisms have not been reported. Herein, the synovial fibroblasts harvested and isolated from patients with RA were treated with lunasin at various concentrations to examine the proliferation, apoptosis status, and corresponding cell cycle of cultured RA synovial fibroblasts. Meanwhile, the underlying mechanisms of lunasin for RA treatment are explored through Western blot, real-time PCR, ELISA, and luciferase reporter assays. Lunasin significantly inhibited the proliferation and induced the apoptosis of cultured RA synovial fibroblasts. In addition, lunasin reduced the production of interleukin-6 (IL-6), IL-8, and matrix metalloproteinase-3 (MMP-3) and suppressed the activation of NF-κB in cultured RA synovial fibroblasts but did not reveal obvious modulation on the secretion and gene expression of MMP-1. Therefore, lunasin will have promising potential as a novel nutritional supplement or drug candidate for RA due to its potency of suppressing synovial cell proliferation and decreasing the production of proinflammatory cytokines and MMPs in synovial cells.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Proliferation; Dose-Response Relationship, Drug; Female; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; Soybean Proteins; Synovial Membrane

The purpose of this study was to relate central inflammation to autonomic activity (heart rate variability (HRV)) in patients with rheumatoid arthritis (RA) and fibromyalgia (FM). RA patients had reduced parasympathetic activity and FM patients had increased sympathetic activity compared to healthy controls. Comparisons between RA and FM showed higher cerebrospinal fluid (CSF) interleukin (IL)-1β inversely correlated to parasympathetic activity in RA. The FM patients had higher concentrations of CSF IL-8, IL-1Ra, IL-4 and IL-10, but none of these cytokines correlated with HRV. In conclusion, we found different profiles of central cytokines, i.e., elevated IL-1β in inflammatory pain (RA) and elevated IL-8 in dysfunctional pain (FM).

Topics: Adult; Arthritis, Rheumatoid; Autonomic Nervous System; Cytokines; Female; Fibromyalgia; Heart Rate; Humans; Interleukin-1beta; Interleukin-8; Middle Aged; Pain; Pain Measurement; Radioimmunoassay; Spinal Puncture; Statistics, Nonparametric

Dysregulated apoptosis of monocytes is a pathogenic feature of rheumatoid arthritis (RA). The aim of this study was to investigate the role of TRAIL and TRAIL-induced apoptosis in patients with RA.. Cell surface expression and serum concentrations of TRAIL were determined in 63 patients with RA, and TRAIL-induced monocyte apoptosis was quantified. Surface expression of TRAILR-1, TRAILR-2, TRAILR-3, TRAILR-4, CXCR1, and CXCR2 was determined, and intracellular signal transduction was investigated. In 8 patients with RA, clinical and laboratory parameters of disease activity were investigated longitudinally, before and after initiation of treatment with tumor necrosis factor (TNF) inhibitors.. Serum concentrations of both TRAIL and interleukin-8 (IL-8) were increased in patients with RA, while cell surface expression of the TRAIL receptors TRAILR-1, TRAILR-2, TRAILR-3, and TRAILR-4 was diminished. TRAIL-induced monocyte apoptosis was significantly decreased in RA due to increased TRAIL-induced IL-8 secretion by RA monocytes. The combined effect of TRAIL and IL-8 on monocytes resulted in activation of antiapoptotic pathways, including p42/44 MAPK and p38. Susceptibility to TRAIL-induced apoptosis was restored in RA monocytes after 3 months of TNF inhibition.. In RA, circulating monocytes with the potential to produce proinflammatory cytokines appear to have defects in several pathways of apoptosis induction, among which is a deficiency in TRAIL-induced apoptosis. Although this resistance to apoptosis might contribute to perpetuation of the disease, it remains to be determined whether specific induction of apoptosis could be therapeutically beneficial.

Topics: Apoptosis; Arthritis, Rheumatoid; Autocrine Communication; Case-Control Studies; Cells, Cultured; Humans; Interleukin-8; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Monocytes; p38 Mitogen-Activated Protein Kinases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

To investigate whether a narrow spectrum kinase inhibitor RV1088, which simultaneously targets specific MAPKs, Src and spleen tyrosine kinase (Syk), is more effective at inhibiting inflammatory signalling in rheumatoid arthritis (RA) than single kinase inhibitors (SKIs).. elisas were used to determine the efficacy of RV1088, clinically relevant SKIs and the pharmaceutical Humira on pro-inflammatory cytokine production by activated RA synovial fibroblasts, primary human monocytes and macrophages, as well as spontaneous cytokine synthesis by synovial membrane cells from RA patients. In human macrophages, RNAi knockdown of individual kinases was used to reveal the effect of inhibition of kinase expression on cytokine synthesis.. RV1088 reduced TNF-α, IL-6 and IL-8 production in all individual activated cell types with low, nM, IC50 s. SKIs, and combinations of SKIs, were significantly less effective than RV1088. RNAi of specific kinases in macrophages also caused only modest inhibition of pro-inflammatory cytokine production. RV1088 was also significantly more effective at inhibiting IL-6 and IL-8 production by monocytes and RA synovial fibroblasts compared with Humira. Finally, RV1088 was the only inhibitor that was effective in reducing TNF-α, IL-6 and IL-8 synthesis in RA synovial membrane cells with low nM IC50 s.. This study demonstrates potent anti-inflammatory effect of RV1088, highlighting that distinct signalling pathways drive TNF-α, IL-6 and IL-8 production in the different cell types found in RA joints. As such, targeting numerous signalling pathways simultaneously using RV1088 could offer a more powerful method of reducing inflammation in RA than targeting individual kinases.

Topics: Acetamides; Adalimumab; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Dasatinib; Dose-Response Relationship, Drug; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Macrophages; Monocytes; Naphthalenes; Oxazines; Piperidines; Primary Cell Culture; Protein Kinase Inhibitors; Pyrazoles; Pyridines; Pyrimidines; Pyrroles; RNA, Small Interfering; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha; Urea

To evaluate the effects of physiologically relevant concentrations of multimeric adiponectin isoforms and leptin on the function of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).. FLS, isolated from the synovial tissue of 21 RA patients, were stimulated for 24 h with interleukin (IL)-1β (1 ng/mL) and adiponectin isoforms [fraction enriched with high-molecular-weight (HMW) oligomers and middle-molecular-weight (MMW) hexamers or low-molecular-weight (LMW) trimers, 10 μg/mL each], or leptin (10 ng/mL), either separately or in a combination of IL-1β and the respective adipokine. Moreover, cells were pre-treated for 24 h with adipokines, then stimulated for 8 h with IL-1β. The concentrations of IL-6, IL-8, matrix metalloproteinase (MMP)-3, and dickkopf (DKK)-1, an inhibitor of osteoblastogenesis, in culture supernatants, as well as the concentrations of leptin, HMW, MMW, and LMW adiponectin in sera and synovial fluid (SF) samples, were measured by specific enzyme-linked immunosorbent assays (ELISAs).. In comparison with sera, SF samples contained similar amounts of leptin, lower amounts of total adiponectin but a higher proportion of the LMW isoform. Separately added IL-1β and HMW/MMW adiponectin, but not LMW adiponectin or leptin, up-regulated the release of IL-6, IL-8, and MMP-3 from FLS but no synergy was observed in co-stimulation experiments. However, pre-treatment of FLS with HMW/MMW or LMW significantly raised the IL-1β-triggered secretion of MMP-3 and IL-6 or MMP-3, respectively.. Adiponectin not only triggers pro-inflammatory and pro-destructive activities of rheumatoid FLS but also pre-disposes these cells to a stronger response to IL-1β. Thus, it is likely that adiponectin is more important in the initiation phase than in the chronic phase of RA.

Topics: Adiponectin; Arthritis, Rheumatoid; Cells, Cultured; Female; Fibroblasts; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1beta; Interleukin-6; Interleukin-8; Leptin; Male; Matrix Metalloproteinase 3; Middle Aged; Molecular Weight; Protein Isoforms; Synovial Membrane

Mast cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). In particular, their activation by interleukin-33 (IL-33) has been linked to the development of arthritis in animal models. The aim of this study was to evaluate the functional responses of human mast cells to IL-33 in the context of RA.. Human mast cells were stimulated with IL-33 combined with plate-bound IgG or IgG anti-citrullinated protein antibodies (ACPAs), and their effects on monocyte activation were evaluated. Cellular interactions of mast cells in RA synovium were assessed by immunofluorescence analysis, and the expression of messenger RNA (mRNA) for mast cell-specific genes was evaluated in synovial biopsy tissue from patients with early RA who were naive to treatment with disease-modifying antirheumatic drugs.. IL-33 induced the up-regulation of Fcγ receptor type IIa and enhanced the activation of mast cells by IgG, including IgG ACPAs, as indicated by the production of CXCL8/IL-8. Intriguingly, mast cell activation triggered with IL-33 and IgG led to the release of mediators such as histamine and IL-10, which inhibited monocyte activation. Synovial mast cells were found in contact with CD14+ monocyte/macrophages. Finally, mRNA levels of mast cell-specific genes were inversely associated with disease severity, and IL-33 mRNA levels showed an inverse correlation with the levels of proinflammatory markers.. When human mast cells are activated by IL-33, an immunomodulatory phenotype develops, with human mast cells gaining the ability to suppress monocyte activation via the release of IL-10 and histamine. These findings, together with the presence of synovial mast cell-monocyte interactions and the inverse association between the expression of mast cell genes at the synovial level and disease activity, suggest that these newly described mast cell-mediated inhibitory pathways might have a functional relevance in the pathogenesis of RA.

Topics: Adult; Aged; Antigen-Antibody Complex; Arthritis, Rheumatoid; Autoantibodies; Down-Regulation; Female; Gene Expression; Histamine Release; Humans; Interleukin-10; Interleukin-33; Interleukin-8; Interleukins; Macrophages; Male; Mast Cells; Middle Aged; Monocytes; Peptides, Cyclic; Receptors, IgG; RNA, Messenger; Synovial Membrane; Up-Regulation

This study aimed to investigate the clinical, immunological, and radiologic predictors of response to tumor necrosis factor (TNF)-α antagonist therapy in Chinese rheumatoid arthritis (RA). Ninety RA patients were divided into two groups according to their responsiveness to TNF-α antagonist therapy at 1 month: group A (responders) and group B (non-responders). After 3 months of therapy, all the 90 patients were re-assessed and re-divided into another two groups: group C (responders) and group D (non-responders). Serum samples and clinical characteristics as well as radiographic features were collected at baseline, first month, and third month post-initial administration of TNF-α antagonist. Serum TNF-α, interleukin (IL)-6, IL-8, IL-34, and matrix metalloproteinase (MMP)-3 were measured by enzyme-linked immunosorbent assay (ELISA). Disease activity and Sharp score were evaluated. (1) Comparisons between groups A and B: subjects in group A showed a lower level of erythrocyte sedimentation rate (ESR) and a higher level of albumin (ALB) at baseline than that of group B (p < 0.05). The cutoff value of ALB for prediction was ≥34.9 g/l and that of ESR was ≤55.5 mm/h. (2) Comparisons between groups C and D: group C showed lower levels of ESR, health assessment questionnaire (HAQ), and IL-34 at baseline (p < 0.05). The threshold for prediction were as follows: ESR ≤60 mm/h, HAQ ≤1.3125, and IL-34 ≤194.12 pg/ml. (3) The serum cytokines were positively correlated with C-reactive protein (CRP) and disease activity index, while ALB was negatively correlated with CRP and disease activity. Baseline ALB ≥34.9 g/l or ESR ≤55.5 mm/h might predict a good response at 1-month treatment of TNF-α antagonist, while baseline ESR ≤60 mm/h, HAQ ≤1.3125, and IL-34 ≤194.12 pg/ml might predict a good response at 3-month treatment.

Topics: Adult; Aged; Albumins; Antirheumatic Agents; Arthritis, Rheumatoid; Blood Sedimentation; C-Reactive Protein; China; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-6; Interleukin-8; Interleukins; Male; Matrix Metalloproteinase 3; Middle Aged; Surveys and Questionnaires; Treatment Outcome; Tumor Necrosis Factor-alpha; Young Adult

Rheumatoid arthritis (RA) is a symmetrical polyarticular autoimmune disease of unknown etiology. In this present study, we observed that the adenomatous polyposis coli (APC) expression is down-regulated and the expression of microRNA (miR)-663 increased significantly in synovium from RA patients compared with control. Target gene prediction for miR-663 revealed that the mRNA of APC gene, which is a member of the canonical Wnt signaling pathway, has a miR-663 binding site in its 3'-untranslated region (3'UTR). The result showed that increased miR-663 suppressed the APC expression significantly, and this down-regulation of APC expression triggered the activation of canonical Wnt signaling through accumulation of β-catenin in fibroblast-like synoviocytes (FLS). In addition, increased miR-663 induced the FLS proliferation and the expression MMP3 and fibronectin during disease development. Therefore, miR-663 can be considered as a critical regulator of RA pathogenesis and can be utilized for developing miRNA-based therapeutic agents for RA patients.

Topics: 3' Untranslated Regions; Adenomatous Polyposis Coli Protein; Adult; Arthritis, Rheumatoid; beta Catenin; Binding Sites; Cell Line; Cell Proliferation; Female; Fibronectins; Humans; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; MicroRNAs; Middle Aged; Synovial Membrane; Wnt Signaling Pathway

The CD30/CD30L signalling system has been implicated in the pathogenesis of several autoimmune and inflammatory conditions. In rheumatoid arthritis (RA), soluble CD30 (sCD30) levels reflect the recruitment of CD30(+) T cells into the inflamed joints and correlate with a positive response to immunosuppressive therapy. The aim of our report was to clarify the role of CD30/CD30L signalling system in the pathogenesis of RA. Our analysis of the CD30L(+) T cell subsets in peripheral blood (PB) and synovial fluid (SF) of RA patients and of the related cytokine profiles suggests the involvement of CD30/CD30L signalling in polarization of T cells towards a Th17 phenotype with proinflammatory features. Moreover, in RA SF nearly 50% of Treg cells express CD30, probably as an attempt to downmodulate the ongoing inflammation. We also show here that the engagement of CD30L on neutrophils stimulated with CD30/Fc chimera may play a crucial role in RA inflammation since activated neutrophils release IL-8, thus potentially amplifying the local inflammatory damage. In conclusion, the results obtained suggest that the complex CD30/CD30L signalling pathway is implicated in the pathogenesis and progression of RA synovitis through a concerted action on several immune effector cells.

Topics: Arthritis, Rheumatoid; CD30 Ligand; Female; Humans; Inflammation; Interleukin-8; Ki-1 Antigen; Male; Middle Aged; Neutrophils; Signal Transduction; Synovial Fluid; T-Lymphocytes, Regulatory; Th17 Cells

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2-200 pg/ml) or CCL20 (5-500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5-2.7-fold), ALP (1.6-1.7-fold), and IL-6 protein production (1.3-1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3-5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3-5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.

Topics: Aged; Arthritis, Rheumatoid; Bone Remodeling; Bone Resorption; Cell Differentiation; Cell Division; Cells, Cultured; Chemokine CCL20; Culture Media, Conditioned; Cytokines; Extracellular Matrix Proteins; Female; Gene Expression Regulation; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Osteoblasts; Osteoclasts; Osteoporosis; Receptors, CCR6; Receptors, Interleukin-8A; Tumor Necrosis Factor-alpha

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine produced by monocytes/macrophage that plays a pathological role in rheumatoid arthritis (RA). In this study, we investigate the effect of thymoquinone (TQ), a phytochemical found in Nigella sativa, in regulating TNF-α-induced RA synovial fibroblast (RA-FLS) activation. Treatment with TQ (1-5μM) had no marked effect on the viability of human RA-FLS. Pre-treatment of TQ inhibited TNF-α-induced interleukin-6 (IL-6) and IL-8 production and ICAM-1, VCAM-1, and cadherin-11 (Cad-11) expression in RA-FLS (p<0.01). Evaluation of the signaling events showed that TQ inhibited TNF-α-induced phospho-p38 and phospho-JNK expression, but had no inhibitory effect on NF-κB pathway, in RA-FLS (p<0.05; n=4). Interestingly, we observed that selective down-regulation of TNF-α-induced phospho-p38 and phospho-JNK activation by TQ is elicited through inhibition of apoptosis-regulated signaling kinase 1 (ASK1). Furthermore, TNF-α selectively induced phosphorylation of ASK1 at Thr845 residue in RA-FLS, which was inhibited by TQ pretreatment in a dose dependent manner (p<0.01). Pre-treatment of RA-FLS with ASK1 inhibitor (TC ASK10), blocked TNF-α induced expression of ICAM-1, VCAM-1, and Cad-11. Our results suggest that TNF-α-induced ASK1-p38/JNK pathway is an important mediator of cytokine synthesis and enhanced expression of adhesion molecule in RA-FLS and TQ, by selectively inhibiting this pathway, may have a potential therapeutic value in regulating tissue destruction observed in RA.

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; MAP Kinase Kinase Kinase 5; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha

Interleukin-29 (IL-29), a critical member of type III interferons (IFNs) family, has been implicated in protecting against viral infection and modulating autoimmune inflammation. Toll-like receptor 4 (TLR4) plays a crucial role in synovial inflammation and may contribute to the pathogenesis of rheumatology arthritis (RA). However, little is known about the modifying effect of IL-29 on TLR4-mediated inflammation in RA. We aim to investigate the potential association between IL-29 and TLR4 in RA.. Peripheral blood mononuclear cells (PBMCs) and serum from 77 patients with RA and 70 controls were collected to determine levels of IL-29 and TLR4 mRNA by real-time polymerase chain reaction (PCR). Levels of IL-29 and TLR4 in synovial tissues and fluid from 25 RA patients and 24 controls were detected by enzyme-linked immunosorbent assay (ELISA) or western blot assay, respectively. RAW264.7 cells were stimulated by lipopolysaccharide (LPS) and/or IL-29. The production of inflammatory cytokines including IL-6, IL-8 as well as TNF-α and the activation of nuclear factor-κB (NF-κB) signaling were determined.. In comparison with controls, increased IL-29 was observed in PBMCs, synovial tissue, serum and synovial fluid of patients with RA. Besides, TLR4 was significantly elevated in PBMCs and synovium of RA patients. Moreover, IL-29 was positively associated with TLR4 in RA, suggested by Pearson's correlation analysis. When RAW264.7 cells were stimulated by LPS with or without IL-29 in vitro, IL-29 could enhance LPS-mediated TLR4 expression and the production of IL-6, IL-8 and TNF-α in RAW264.7 cells via the activation of NF-κB signaling.. The present study suggests, for the first time, that IL-29 can aggravate LPS/TLR4-mediated inflammation in RA depending on NF-κB signaling activation.

Topics: Animals; Arthritis, Rheumatoid; Cell Line; Female; Humans; Inflammation; Interferons; Interleukin-6; Interleukin-8; Interleukins; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Mice; Middle Aged; NF-kappa B; Signal Transduction; Synovial Fluid; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

To observe the anti-inflammatory effects of honokiol in primary cultures of peripheral blood mononuclear cells of rheumatoid arthritis patients, the pro-inflammatory cytokines and potential targets were investigated.. The levels of GM-CSF, IL-1β, TNF-α and IL-8 were determined by ELISA assay. The genes and proteins expression were analyzed by real-time PCR and Western blotting respectively.. The serum IL-1β, TNF-α and GM-CSF levels were 1.76-, 2.16- and 3.57-fold increased in patients with RA as compared to those of control group. Honokiol inhibited the expression levels of IL-1β, TNF-α, GM-CSF and IL-8 in PBMCs with a dose-dependent manner. Measurements obtained from supernatants were positively correlated between TNF-α and IL-1β, moreover, similar results found TNF-α levels positively correlated with GM-CSF and IL-8 activity in the supernatants of PBMCs isolated from RA patients. Furthermore, the mRNA and protein expression of IL-1β, GM-CSF and IL-8 were up-regulated when the PBMCs exposure to TNF-α, however, honokiol treatment significantly reversed the expression of IL-1β, TNF-α and GM-CSF in response to TNF-α with a dose-dependent manner.. This study demonstrates that honokiol could possess potential anti-inflammatory effects and inhibits TNF-α-induced IL-1β, GM-CSF and IL-8 production in PBMCs from rheumatoid arthritis patients.

Topics: Adult; Aged; Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Biphenyl Compounds; Case-Control Studies; Cells, Cultured; Dose-Response Relationship, Drug; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; Lignans; Male; Middle Aged; Primary Cell Culture; Signal Transduction; Tumor Necrosis Factor-alpha

Loss of the regulatory mechanisms that avoid excessive or constitutive activation of NF-κB may be associated with chronic inflammatory disorders, including rheumatoid arthritis (RA). After massive sequencing of 158 regulators of the NF-κB pathway in RA patients, we focused on a scarcely known gene, ASCC1, and showed that it potently inhibits the expression of NF-κB target genes (TRAIL, TNF-α, cIAP-1, IL8) and blocks activation of a NF-κB-luciferase reporter construct in five different human cell lines. Therefore, ASCC1 may contribute to avoiding a pathologic activation of this transcription factor. A truncated variant of ASCC1 (p.S78*) was found in RA patients and control individuals. Functional in vitro studies revealed that truncation abrogated the NF-κB inhibition capacity of ASCC1. In contrast with full-length protein, truncated ASCC1 did not reduce the transcriptional activation of NF-κB and the secretion of TNF-α in response to inflammatory stimuli. We analyzed the clinical impact of p.S78* variant in 433 patients with RA and found that heterozygous carriers of this variant needed more disease-modifying antirheumatic drugs, and more patients with this genotype needed treatment with corticoids and biologic agents. Moreover, the truncated allele-carrier group had lower rates of remission compared with the full-length variant carriers. Overall, our findings show for the first time, to our knowledge, that ASCC1 inhibits NF-κB activation and that a truncated and inactive variant of ASCC1 is associated with a more severe disease, which could have clinical value for assessing the progression and prognosis of RA.

Topics: Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Base Sequence; Carrier Proteins; Cell Line, Tumor; Enzyme Activation; Female; Gene Expression Regulation; HEK293 Cells; HeLa Cells; Humans; Inhibitor of Apoptosis Proteins; Interleukin-8; Male; MCF-7 Cells; NF-kappa B; Sequence Analysis, DNA; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Transcriptional Activation; Tumor Necrosis Factor-alpha

This study was performed to evaluate the contribution of adiponectin to the production of interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-13 in human endothelial cells and osteoblasts in arthritic joints. Cultured human umbilical vascular endothelial cells (HUVECs) and osteoblasts were stimulated with adiponectin (1 or 10 μg ml(-1)) or IL-1β (0.1 ng ml(-1)) in the presence or absence of hypoxia for 24 h. The protein expression patterns were examined by analyzing culture supernatants using the enzyme-linked immunosorbent assay (ELISA). Adiponectin significantly stimulated the production of VEGF, MMP-1 and MMP-13 in osteoblasts but not in endothelial cells, whereas it significantly stimulated the production of IL-6 and IL-8 in both endothelial cells and osteoblasts. The increase in VEGF production induced by adiponectin was significantly greater than that induced by IL-1β. The production of IL-6 and IL-8 in adiponectin-stimulated endothelial cells was approximately 10-fold higher than that in IL-1β-stimulated endothelial cells; in osteoblasts, adiponectin-induced IL-6 and IL-8 secretion was approximately twofold higher than that induced by IL-1β. In addition, IL-8 production in endothelial cells was approximately sevenfold higher than in osteoblasts. However, IL-6 levels were similar between the two cell types, suggesting that adiponectin may be involved in the production of IL-8 in endothelial cells, which may have an important role in neutrophil recruitment to arthritic joints. Furthermore, the increases in protein expression induced by adiponectin were differentially regulated by hypoxia. In conclusion, adiponectin has a more important role than does IL-1β in the production of mediators that drive synovitis and joint destruction in endothelial cells and osteoblasts at physiological concentrations.

Topics: Adiponectin; Arthritis, Rheumatoid; Cell Hypoxia; Cell Line; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 1; Osteoblasts; Vascular Endothelial Growth Factor A

Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), interleukin (IL)-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated.. Patients (n = 105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. Disease activity score (DAS-28) was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine (Cl-Tyr), homocitrulline (Hcit), IL-8, and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum.. DAS-28 and CRP levels were not significantly different between groups. MPO activity, and MPO, Cl-Tyr, and Hcit levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients.. MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and Hcit in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations.

Topics: Arthritis, Rheumatoid; Cytokines; Female; Humans; Interleukin-8; Male; Peroxidase; Synovial Fluid

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) contribute to the destruction of cartilage and bone by production of metalloproteinases (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM). Bufalin, a major component of Venenum Bufonis, can attenuate the invasion of various cancer cells. Here, we investigated the effects of bufalin on tumor necrosis factor-alpha (TNF-α)-induced invasion of RAFLSs. Western blot analysis and electrophoretic mobility shift assay were conducted to analyze the nuclear translocation of p65/nuclear factor-kappa B (NF-κB) and NF-κB DNA-binding activity. Semiquantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were performed to assess the expression of cytokines. Our results revealed that TNF-α significantly increased p65 translocation into nucleus (P < 0.01) and enhanced NF-κB DNA-binding activity, which were dose-dependently inhibited by bufalin. Furthermore, bufalin attenuated the TNF-α-induced interleukin-1beta (IL-1β), IL-6, and IL-8 production in RAFLSs in a concentration-dependent manner. Interestingly, TNF-α-induced invasion of RAFLSs was dampened by the pretreatment of bufalin. Additionally, bufalin decreased the mRNA abundance and secretion of MMP-9 in TNF-α-treated RAFLSs. Our results reveal that bufalin can inhibit TNF-α-induced NF-κB activation, cytokine production, invasion, and MMP-9 expression in RAFLSs, indicating a therapeutic potential of bufalin on RA.

Topics: Arthritis, Rheumatoid; Bufanolides; Cartilage; Cell Nucleus; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 9; Medicine, Chinese Traditional; NF-kappa B; Synovial Fluid; Tumor Necrosis Factor-alpha

To evaluate the therapeutic efficacy of dexamethasone (DM) and methotrexate (MTX) entrapped within polysialic acid (PSA)-trimethyl chitosan (TMC) nanoparticles using an in vitro model of rheumatoid arthritis (RA).. The loading capacity of the PSA-TMC nanoparticles was determined. An RA in vitro model was developed by stimulating a synovial cell line with a proinflammatory mediator. Multiplex immunoassay was used to determine changes in the secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the in vitro model following administration of the DM- and MTX-loaded nanoparticles.. The loading capacity of the PSA-TMC nanoparticles was approximately 0.1 mg of drug/mg of nanoparticle. When applied to our in vitro model of RA, there were no significant differences in the concentrations of IL-6 and IL-8 when comparing the free drugs and drug-loaded nanoparticles, administered at concentration of 0.1 mg/ml and 1.0 mg/ml, respectively.. The present study verified that MTX and DM are able to retain bioactivity when loaded into PSA-TMC nanoparticles. Although in vitro efficacy was not increased, the in vivo efficacy will likely be enhanced by the site-specific targeting conferred by nanoparticle entrapment.

Topics: Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Cell Line; Chitosan; Dexamethasone; Drug Carriers; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Methotrexate; Nanoparticles; Sialic Acids; Synovial Membrane

In RA, synoviocytes cause increased oxidative stress, leading to mitochondrial alterations that may participate in the pathogenesis of RA. Here we investigated whether mitochondrial dysfunction induces inflammatory responses in cultured normal human synoviocytes, a hallmark of RA.. Mitochondrial dysfunction was induced with the inhibitor oligomycin. The effects of mitochondrial dysfunction on cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and IL-8 expression; cellular and mitochondrial reactive oxygen species (ROS) production; nuclear factor-κB (NF-κB) activation and p65 translocation were studied. ROS scavengers (N-acetylcysteine and mitoTEMPO) and an NF-κB inhibitor (BAY-117085) were used to investigate the pathways involved. The natural anti-inflammatory antioxidant resveratrol was also tested.. Mitochondrial dysfunction per se significantly stimulated mitochondrial ROS production as well as low-grade expressions of COX-2, PGE2 and IL-8. Interestingly, mitochondrial dysfunction induced by pretreatment of synoviocytes with oligomycin synergized with IL-1β to increase the expression of these inflammatory mediators. The inflammatory effects of mitochondrial damage appeared to be dependent on ROS production and NF-κB activation since the inflammatory response was counteracted by both N-acetylcysteine and mitoTEMPO and it was also reduced by BAY-117085. Antimycin A and paraquat (inhibitors of mitochondrial function) also induced inflammatory responses. Furthermore, resveratrol significantly reduced the inflammatory response by decreasing ROS production and NF-κB activation.. These data suggest that mitochondrial dysfunction could induce an inflammatory response in normal human synoviocytes and sensitize these cells, causing a significant amplification of the inflammatory response induced by IL-1β. Resveratrol may represent a promising strategy in controlling the synovial inflammatory response.

Topics: Aged; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Middle Aged; Mitochondria; NF-kappa B; Oligomycins; Oxidative Stress; Reactive Oxygen Species; Resveratrol; Stilbenes; Synovial Membrane

Sour cherry seed extract (SCE) was evaluated for its capacity to inhibit lipopolysaccharide-treated human peripheral blood T cells expressing tumor necrosis factor-alpha, and the chemokine interleukin-8. Both proteins are diagnostic biomarkers for inflammatory pathologies. Peripheral blood leukocytes from 11 rheumatoid arthritis (RA) patients and 8 healthy control subjects were co-cultured for 24h in lipopolysaccharide and the extract, then evaluated by flow cytometry for T cell activation and by enzyme-linked immunoassay for lymphocyte-associated heme oxygenase-1 (HO-1) expression. There was a dose-dependent decrease in expression of the immunophenotypes: CD3+TNF-α+, and CD3+IL8+ in cultures from RA patients to a greater extent than in cells from healthy participants. These results suggest that the extract may have a modulatory roll in RA and other inflammatory disorders via the induction of HO-1, thus abating oxidative stress and strengthening regulation of pro-inflammatory signaling pathways.

Topics: Adult; Arthritis, Rheumatoid; CD3 Complex; Cells, Cultured; Female; Heme Oxygenase-1; Humans; Interleukin-8; Leukocytes, Mononuclear; Lipopolysaccharides; Middle Aged; Plant Extracts; Prunus; Seeds; T-Lymphocytes; Tumor Necrosis Factor-alpha

Cigarette smoking is a recognized environmental risk factor for the development and progression of rheumatoid arthritis (RA). RA synovial fibroblasts (RASF) actively contribute to inflammation and joint destruction in this chronic inflammatory autoimmune disease. In the current study, we investigated the influence of cigarette smoke on the inflammatory and matrix-destructive properties of RASF. Furthermore, the functional role of Sirtuin 6 (SIRT6) in the regulation of the signalling induced by cigarette smoke or by tumor necrosis factor alpha (TNFα) was elucidated. We demonstrated that stimulation with cigarette smoke extract (CSE) enhances the pro-inflammatory and matrix-destructive potential of RASF by inducing the production of pro-inflammatory cytokine interleukin 8 (IL8) and the matrix-destructive enzyme matrix metalloproteinase 1 (MMP1), but not of IL6 and MMP3. Moreover, we could show that the expression of MMP1 is specifically regulated by SIRT6. Treatment of RASF with CSE or TNFα increased the levels of SIRT6. The expression of SIRT6 was also enhanced in vivo in synovial tissues of RA smokers and in joints of mice exposed to cigarette smoke. Silencing of SIRT6 specifically increased basal as well as CSE- and TNFα-induced production of MMP1, demonstrating that SIRT6 plays an important role in restricting MMP1 expression. In conclusion, the upregulation of SIRT6 in RASF under CSE or TNFα stimulation functions as a counterregulatory mechanism attenuating the production of the matrix-destructive enzyme MMP1. This is the first study revealing the protective function of SIRT6 in the cigarette smoke-induced signalling.. Cigarette smoke induces pro-inflammatory and matrix-destructive responses in RASF. Cigarette smoke enhances the expression of SIRT6 in vitro and in vivo. TNFα increases the levels of SIRT6. SIRT6 diminishes MMP1 production under cigarette smoke extract and TNFα stimulation.

Topics: Adult; Aged; Animals; Arthritis, Rheumatoid; Cells, Cultured; Complex Mixtures; Female; Fibroblasts; Gene Expression Regulation; Gene Silencing; Humans; Interleukin-8; Male; Matrix Metalloproteinase 1; Mice, Inbred C57BL; Middle Aged; Nicotiana; RNA, Messenger; Signal Transduction; Sirtuins; Smoke; Synovial Membrane; Tumor Necrosis Factor-alpha

Lysophosphatidic acid (LPA) is a pleiotropic lipid mediator that promotes motility, survival, and the synthesis of chemokines/cytokines such as interleukin-8 (IL-8) and interleukin-6 by human fibroblast-like synoviocytes from patients with rheumatoid arthritis (RAFLS). In those cells LPA was reported to induce IL-8 secretion through activation of various signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK), p42/44 MAPK, and Rho kinase. In addition to those pathways we report that mitogen- and stress-activated protein kinases (MSKs) known to be activated downstream of the ERK1/2 and p38 MAPK cascades and CREB are phosphorylated in response to LPA. The silencing of MSKs with small-interfering RNAs and the pharmacological inhibitor of MSKs SB747651A shows a role for both MSK1 and MSK2 in LPA-mediated phosphorylation of CREB at Ser-133 and secretion of IL-8 and MCP-1. Whereas CREB inhibitors have off target effects and increased LPA-mediated IL-8 secretion, the silencing of CREB1 with short hairpin RNA significantly reduced LPA-induced chemokine production in RAFLS. Taken together the data clearly suggest that MSK1 and MSK2 are the major CREB kinases in RAFLS stimulated with LPA and that phosphorylation of CREB1 at Ser-133 downstream of MSKs plays a significant role in chemokine production.

Topics: Arthritis, Rheumatoid; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Chemokine CCL2; Cyclic AMP Response Element-Binding Protein; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Interleukin-8; Lysophospholipids; Oxadiazoles; Phosphorylation; Ribosomal Protein S6 Kinases, 90-kDa; RNA Interference; Serine; Synovial Membrane

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.. RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.. Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman's ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman's ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman's ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman's ρ = 0.50), BME score (P = 0.04, approximate Spearman's ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman's ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman's ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman's ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.. The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Chemokine CCL2; Chemokine CCL4; Cross-Sectional Studies; Female; Hand Joints; Humans; Immunoassay; Inflammation Mediators; Interleukin-6; Interleukin-8; Magnetic Resonance Imaging; Male; Middle Aged; Radiography; Reproducibility of Results; Sensitivity and Specificity; Synovial Membrane; Synovitis; Tissue Culture Techniques; Ultrasonography, Doppler, Color

Observations in both animal models of arthritis and patients with rheumatoid arthritis (RA) suggest a role for dopamine and its receptors in RA. Because synovial fibroblasts (SFs) contribute to inflammation and joint destruction in RA, the aim of this study was to investigate dopaminergic pathways in SFs obtained from patients with RA and, for comparison, in SFs from patients with osteoarthritis (OA) undergoing knee joint replacement surgery.. The expression of all dopamine receptors (D1 -D5 ) and dopamine transporter was assessed by immunofluorescence and immunohistochemical staining. The levels of dopamine receptor and tyrosine hydroxylase messenger RNA were measured by real-time polymerase chain reaction. The intracellular content of dopamine, its precursor, and its main metabolites was assayed by high-performance liquid chromatography. The influence of dopamine on proinflammatory interleukin-6 (IL-6) and IL-8, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-2 was studied in SFs.. SFs possess an intrinsic dopaminergic system, including dopamine receptors, dopamine transporter, and tyrosine hydroxylase, and contain dopamine, its precursor, and its main metabolites. SFs from patients with RA, in comparison with those from patients with OA, showed increased expression of dopamine receptors D1 and D5 , and exogenous dopamine strongly inhibited the production of IL-8 in patients with RA.. SFs from patients with RA and patients with OA show a dopaminergic phenotype. The expression of D1-like dopamine receptors was higher in RASFs, and this increased expression may lead to antiinflammatory effects, as demonstrated by the expression of IL-8. Studies in animal models and patients with RA are needed to assess the therapeutic potential of endogenous, local production of dopamine in synoviocytes.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dopamine; Dopamine Plasma Membrane Transport Proteins; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Osteoarthritis; Receptors, Dopamine; Synovial Membrane; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5.

Topics: Acetylation; Arthritis, Rheumatoid; Chemokine CCL20; Chemokine CXCL12; Chromatin; Computational Biology; Epigenesis, Genetic; Fibroblasts; Humans; Interleukin-8; Methylation; Promoter Regions, Genetic; Signal Transduction; Synovial Membrane; T-Box Domain Proteins; Transcription, Genetic

The relevance of regulatory B cells in the maintenance of tolerance in healthy individuals or in patients with immune disorders remains understudied. In healthy individuals, CD19(+)CD24(hi)CD38(hi) B cells suppress CD4(+)CD25(-) T cell proliferation as well as the release of interferon-γ and tumor necrosis factor-α by these cells; this suppression is partially mediated through the production of interleukin-10 (IL-10). We further elucidate the mechanisms of suppression by CD19(+)CD24(hi)CD38(hi) B cells. Healthy CD19(+)CD24(hi)CD38(hi) B cells inhibited naïve T cell differentiation into T helper 1 (T(H)1) and T(H)17 cells and converted CD4(+)CD25(-) T cells into regulatory T cells (T(regs)), in part through the production of IL-10. In contrast, CD19(+)CD24(hi)CD38(hi) B cells from patients with rheumatoid arthritis (RA) failed to convert CD4(+)CD25(-) T cells into functionally suppressive T(regs) or to curb T(H)17 development; however, they maintained the capacity to inhibit T(H)1 cell differentiation. Moreover, RA patients with active disease have reduced numbers of CD19(+)CD24(hi)CD38(hi) B cells in peripheral blood compared with either patients with inactive disease or healthy individuals. These results suggest that in patients with active RA, CD19(+)CD24(hi)CD38(hi) B cells with regulatory function may fail to prevent the development of autoreactive responses and inflammation, leading to autoimmunity.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Antigens, CD19; Arthritis, Rheumatoid; B-Lymphocytes; CD24 Antigen; Cell Differentiation; Female; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Male; Middle Aged; T-Lymphocytes, Regulatory; Tumor Necrosis Factor-alpha; Young Adult

Topics: Arthritis, Rheumatoid; Calcitonin Gene-Related Peptide; Capsaicin; Case-Control Studies; Humans; Interleukin-6; Interleukin-8; Substance P; Synovial Membrane; TRPV Cation Channels

Recently, phosphatidylserine (PS) has received attention for its anti-inflammatory effect; however, the molecular mechanisms of its action have not been fully understood. Thus, we hypothesized that PS might have antiarthritic and anti-inflammatory effects. To test this hypothesis, the in vitro anti-inflammatory effect of soybean-derived PS was tested on interleukin (IL)-1β-stimulated fibroblast-like synoviocytes from rheumatoid arthritis patients (RA-FLS) by measuring the levels of IL-6, IL-8, prostaglandin E(2), and vascular endothelial growth factor by enzyme-linked immunosorbent assay. The analgesic and antiarthritic activities of PS were investigated in rat models of carrageenan-induced acute paw pain and arthritis. The former was evaluated with a paw pressure test; the latter, by measuring paw volume and weight distribution ratio. In addition, the participation of mitogen-activated protein kinase signaling in the anti-inflammatory and antiarthritic effects of PS was investigated in RA-FLS. Phosphatidylserine inhibited the production of inflammatory mediators IL-6; IL-8; vascular endothelial growth factor; and, in particular, prostaglandin E(2) in IL-1β-stimulated RA-FLS. These effects were associated with abrogation of inhibitor of nuclear factor-κBα phosphorylation and suppression of p38 and c-jun amino terminal kinase but not extracellular signal-regulated kinase 1/2 phosphorylation. In rats, PS also showed a significant inhibitory effect on arthritic and nociceptive symptoms induced by carrageenan. These findings suggest that PS has anti-inflammatory and antiarthritic effects in vitro and in in vivo animal models; thus, PS should be further studied to determine its potential use as either a pharmaceutical or dietary supplement for alleviating arthritic symptoms.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Carrageenan; Enzyme Activation; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylserines; Prostaglandins E; Rats; Rats, Sprague-Dawley; Synovial Membrane; Vascular Endothelial Growth Factor A

The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti-citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

Topics: Arthritis, Rheumatoid; Autoantibodies; Autoantigens; Citrulline; Humans; Interleukin-17; Interleukin-6; Interleukin-8; Microscopy, Fluorescence; Neutrophils; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha

To investigate the effect of α-defensin-1 on the expression of IL-6, IL-8 and MMPs as well as the signal transduction mechanisms responsible for their expression in RA fibroblast-like synoviocytes (FLS).. The concentrations of α-defensin-1 in SF were measured by ELISA. In RA FLS, mRNA expression of IL-6, IL-8 and MMPs and activation of signalling molecules were examined by real-time PCR, western blotting and electrophoretic mobility shift assay.. Concentrations of SF α-defensin-1 were significantly increased in RA patients compared with OA patients. The levels of mRNA expression of IL-6, IL-8, MMP-1 and MMP-3 were significantly increased in RA FLS treated with α-defensin-1 compared with controls. Furthermore, α-defensin-1 activated JNK and ERK in RA FLS, respectively. Treatment of RA FLS with ERK or JNK inhibitors prior to α-defensin-1 treatment resulted in reduced expression of IL-6, IL-8, MMP-1, and MMP-3 compared with controls. Remarkably, treatment of RA FLS with an ERK inhibitor prior to α-defensin-1 stimulation significantly reduced production of IL-6 and MMP-1 by approximately 71% and 98% compared with controls, respectively. The JNK inhibitor significantly suppressed α-defensin-1-induced MMP-1 production by approximately 73% compared with controls. Finally, there was a significant induction of NF-κB DNA binding activity in response to α-defensin-1.. Our results suggest that α-defensin-1 may play a role in RA pathogenesis by regulating the production of MMPs as well as IL-6 and IL-8. These processes were dependent on the regulation of the JNK and/or ERK and NF-κB pathways.

Topics: alpha-Defensins; Arthritis, Rheumatoid; Blotting, Western; Case-Control Studies; Cells, Cultured; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinases; Middle Aged; Osteoarthritis; Protein Kinase Inhibitors; Real-Time Polymerase Chain Reaction; Signal Transduction; Statistics, Nonparametric; Synovial Membrane

The aim of this study was to evaluate the effects of host modulation therapy on periodontal and biochemical parameters. Sixteen rheumatoid arthritis patients newly scheduled for anti-tumour necrosis factor (TNF) therapy were screened for 30 days. Periodontal parameters (clinical attachment level, probing pocket depth, bleeding on probing, plaque index and gingival index) as well as salivary and gingival crevicular fluid (GCF), interleukin (IL)-1β, IL-8 and monocyte chemoattractant protein-1 (MCP-1) levels of the patients were evaluated at baseline and on the 30th day of therapy. GCF volume, IL-1β and IL-8 levels (p = 0.007, p = 0.017 and p = 0.009, respectively) of the periodontitis patients significantly decreased. Although there was a decrease in all these parameters in healthy patients, it was below statistical significance. Salivary IL-8 and MCP-1 levels significantly decreased in periodontitis patients (p = 0.028 and p = 0.013, respectively), but IL-1β levels remained unchanged. These results suggest that TNF blockers may significantly modify host response in terms of biochemical parameters of the periodontium and may mask significant associations such as those reported between periodontitis and rheumatoid arthritis.

Topics: Adult; Arthritis, Rheumatoid; Chemokine CCL2; Dental Plaque Index; Female; Gingival Crevicular Fluid; Humans; Inflammation; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Periodontal Index; Periodontitis; Periodontium; Saliva; Tumor Necrosis Factor-alpha; Young Adult

Th17 cells are critically involved in autoimmune disease induction and severity. Recently, we showed that Th17 cells from patients with rheumatoid arthritis (RA) directly induced a proinflammatory loop upon interaction with RA synovial fibroblasts (RASF), including increased autocrine IL-17A production. To unravel the mechanism driving this IL-17A production, we obtained primary CD4(+)CD45RO(+)CCR6(+) (Th17) cells and CD4(+)CD45RO(+)CCR6(-) (CCR6(-)) T cells from RA patients or healthy individuals and cocultured these with RASF. IL-1β, IL-6, IL-23p19, and cyclooxygenase (COX)-2 expression and PGE2 production in Th17-RASF cultures were higher than in CCR6(-) T cell-RASF cultures. Cytokine neutralization showed that IL-1β and IL-6, but not IL-23, contributed to autocrine IL-17A induction. Importantly, treatment with celecoxib, a COX-2 inhibitor, resulted in significantly lower PGE2 and IL-17A, but not IFN-γ, production. Combined celecoxib and TNF-α blockade more effectively suppressed the proinflammatory loop than did single treatment, as shown by lower IL-6, IL-8, matrix metalloproteinase-1 and matrix metalloproteinase-3 production. These findings show a critical role for the COX-2/PGE2 pathway in driving Th17-mediated synovial inflammation in an IL-23- and monocyte-independent manner. Therefore, it would be important to control PGE2 in chronic inflammation in RA and potentially other Th17-mediated autoimmune disorders.

Topics: Arthritis, Rheumatoid; CD4 Antigens; Celecoxib; Cells, Cultured; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Female; Fibroblasts; Humans; Inflammation; Interferon-gamma; Interleukin-17; Interleukin-1beta; Interleukin-23 Subunit p19; Interleukin-6; Interleukin-8; Leukocyte Common Antigens; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Middle Aged; Pyrazoles; Receptors, CCR6; Sulfonamides; Synovial Membrane; Th17 Cells; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membrane of affected joints. It has been shown that several kinds of cytokine were increased in synovial fluid, while the underlying mechanism remains poorly understood.. NF-κB activator 1 (Act1) is a recently identified protein binding to the IκB kinase complex. Our study aimed to investigate the expression of Act1 induced by cytokine IL-17 stimulation in SW982 cells.. The human synovial sarcoma cell line SW982 and primary cultured RA fibroblast-like synovial cells were used. RT-PCR and Western blot assays were selected to investigate the genetic and protein expression of Act1. Additionally, four independent Act1 small interfering RNA (siRNA) oligonucleotides were designed and obtained according to the GenBank cDNA, the sequence of Act1 (Traf3ip2). Finally, enzyme-linked immunosorbent assay (ELISA) double antibody sandwich was used to assay supernatant IL-6 and IL-8 concentrations.. The Act1 mRNA expression level increased significantly after stimulation with IL-17 (5-100 ng/ml) in SW982 cells. Additionally, the level of Act1 mRNA expression correlated positively with the concentration of IL-17 (p < 0.01). IL-17 induced IL-6 and IL-8 in SW982 cells was in a concentration- and time-dependent way. Furthermore, ELISA assay revealed that IL-17 (20 ng/ml) significantly increased IL-6 (1927.4 ± 288.77 versus 786.5 ± 172.42 ng/ml, p < 0.01) and IL-8 levels (984.8 ± 95.09 ng/ml versus 307.1 ± 90.83 ng/ml, p < 0.01) compared with control group after stimulation for 24 h. However, transfection of Traf3ip2 siRNA markedly decreased IL-6 (995.9 ± 115.30 ng/ml versus 1816.1 ± 273.27 ng/ml, p < 0.01) and IL-8 levels (575.6 ± 65.96 ng/ml versus 929.4 ± 124.39 ng/ml, p < 0.01) compared to transfection negative control. These findings suggested that IL-6 and IL-8 level induced by IL-17 in SW982 cells could be reversed by down-regulation of Act1 expression level with Traf3ip2 siRNA.. Our results suggested that Act1 might play a key role in the pathophysiology and the treatment of RA.

Topics: Adaptor Proteins, Signal Transducing; Arthritis, Rheumatoid; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Interleukin-17; Interleukin-6; Interleukin-8; RNA Interference; RNA, Messenger; Signal Transduction; Synovial Membrane; Time Factors; Transfection; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; Up-Regulation

This study was conducted to evaluate phagocyte function in patients with age-related chronic inflammatory conditions. It included 95 patients with PMR, 17 with GCA, 40 with EORA, and 25 age-matched HCs. Serum IL-8 was determined with a bead array. The chemotactic capacity, phagocytic ability, and oxidative burst activity of circulating leukocytes were determined with flow cytometry kits. Patients with active chronic inflammatory diseases showed a significant increase in circulating levels of IL-8 that remained elevated in patients with PMR or EORA, despite treatment. No correlation was found between circulating IL-8 and the migratory capacity of neutrophils. Neutrophils from patients with active EORA without stimulus and after fMLP stimuli showed a higher capacity to migrate than those of the HCs (P=0.033). The phagocytic activity of granulocytes in the patients with GCA was significantly higher than in the HCs and the patients with PMR or EORA (P<0.05). The percentage and MFI of phagocytes that produce ROIs when stimulated with Escherichia coli was significantly reduced in neutrophils and monocytes from the patients with age-restricted inflammatory conditions. We concluded that the effector functions of phagocytes, determined to be chemotaxis, phagocytosis, and oxidative burst, are deregulated in age-restricted inflammatory disorders and may have a pathogenic role.

Topics: Acute-Phase Reaction; Age Factors; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Chronic Disease; Female; Giant Cell Arteritis; Humans; Interleukin-8; Male; Middle Aged; Neutrophils; Phagocytes; Polymyalgia Rheumatica; Respiratory Burst

IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.

Topics: Amino Acid Substitution; Animals; Anti-Inflammatory Agents; Arthritis, Rheumatoid; Binding Sites; Cattle; Drug Design; Drug Evaluation, Preclinical; Guanidine; Heparitin Sulfate; Humans; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Protein Binding; Protein Denaturation; Receptors, Interleukin-8A

We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.. The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.. The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.. We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.

Topics: Arthritis, Rheumatoid; Cell Line; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Flow Cytometry; Gene Expression; Humans; Interferons; Interleukin-6; Interleukin-8; Interleukins; Lipopolysaccharides; Peptidoglycan; Poly I-C; Receptors, Cytokine; Receptors, Interleukin-10; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptor 4

To investigate the effect of selective phosphodiesterase (PDE) 4 inhibitors on nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNFα) and interleukin-8 (IL-8) secreted by peripheral blood mononuclear cells (PBMCs) in patients diagnosed as rheumatoid arthritis with interstitial lung disease (RA-ILD).. PBMCs isolated from 15 healthy volunteers (group A) and 20 patients with untreated active RA-ILD (group B) were cultured in vitro. PBMCs from healthy subjects were considered as normal control. PBMCs from RA-ILD patients were divided into four groups with different treatment: blank group (B1), theophylline group (B2), selective PDE4 inhibitor rolipram group (B3), and glucocorticoid group (B4) with dexamethasone. The expression of NF-κB was determined by immunocytochemical staining, and the levels of TNFα and IL-8 in the culture supernatant were detected by enzyme linked immunosorbent assay (ELISA).. (1) The activity of NF-κB and the levels of TNFα and IL-8 in group B1 were significant higher than that in group A (P < 0.01). Compared with group B1, three parameters above were similar to those in group B2 (P > 0.05), while group B3 and group B4 had significant decreased levels of three parameters (P < 0.01); IL-8 level in group B4 was significantly lower than that in group B3 (P < 0.05). (2) TNFα and IL-8 levels were positively correlated with NF-κB activity in group B (r = 0.902 and 0.735, P < 0.01 respectively). (3) The reduction of TNFα and IL-8 levels were positively correlated with reduction of NF-κB activity after intervention of rolipram in group B3 (r = 0.874, P < 0.01; r = 0.561, P < 0.05 respectively).. NF-κB activation and proinflammatory cytokines were involved in the pathogenesis of RA-ILD. selective PDE4 inhibitors may inhibit the production of inflammatory cytokines by inhibiting the activity of the transcription factor NF-κB in PBMC, thus inhibiting the inflammatory reaction of RA-ILD.

Topics: Adult; Aged; Arthritis, Rheumatoid; Case-Control Studies; Cells, Cultured; Female; Humans; Interleukin-8; Leukocytes, Mononuclear; Lung Diseases, Interstitial; Male; Middle Aged; NF-kappa B p50 Subunit; Phosphodiesterase 4 Inhibitors; Tumor Necrosis Factor-alpha

It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.. RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.. We found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.. Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA.

Topics: Adult; Aged; Aged, 80 and over; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Blotting, Western; Chromatin Immunoprecipitation; Cysteine-Rich Protein 61; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Flow Cytometry; Humans; Immune System Diseases; Interleukin-8; Leukocyte Disorders; Male; Mice; Mice, Inbred DBA; Microscopy, Confocal; Middle Aged; Neutrophil Infiltration; Real-Time Polymerase Chain Reaction; Synovial Membrane

Notch signalling pathways are critical for angiogenesis and endothelial cell (EC) fate; however the mechanisms regulating these processes in the inflamed joint remain to be elucidated. Here, we examine whether Notch signalling mediates vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang2)-induced vascular function.. Notch-1 intracellular domain (Notch-1 IC), Notch-4 IC, Delta-like-ligand 4, Hes-related transcriptional repressors-1 and 2 (Hrt-1, Hrt-2) mRNA and/or protein expression was measured by Real-time PCR and/or western blot. VEGF/Ang2 induced EC function was assessed using transwell invasion chambers, matrigel tube formation assays and wound repair scratch assays±Notch-1 siRNA or an γ-secretase inhibitor N-(N-(3,5-Difluorophenacetyl-L-alanly))-S-phenylglycine-t-Butyl Ester (DAPT) in RA synovial explants or human microvascular EC. Interleukin (IL)-6 and IL-8 were measured by ELISA and MMP2 and 9 by gelatine zymography.. Notch-1 IC and Notch-4 IC protein expressions were demonstrated in RA and psoriatic arthritis synovial biopsies, with minimal expression observed in Osteoarthritis (OA). VEGF and Ang2 induced Notch-1 IC/ Notch-4 IC protein expression in synovial explant cultures and human microvascular EC levels were further potentiated by VEGF/Ang2 stimulation in combination. Notch-1, Delta-like-ligand 4, and Hrt-2 mRNA expression were significantly induced by VEGF and Ang2 alone and in combination. Furthermore VEGF/Ang2-induced EC invasion, angiogenesis and migration were inhibited by Notch-1 siRNA or DAPT. Conditioned media from VEGF/Ang2 stimulated RA synovial explants induced EC tube formation, an effect that was inhibited by DAPT. Finally, DAPT significantly decreased VEGF/Ang2 induced IL-6, IL-8, MMP2 and 9 expressions in RA synovial explants.. Notch-1 mediates VEGF/Ang2-induced angiogenesis and EC invasion in inflammatory arthritis.

Topics: Adult; Aged; Aged, 80 and over; Angiopoietin-2; Arthritis, Psoriatic; Arthritis, Rheumatoid; Cell Proliferation; Endothelial Cells; Female; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Neovascularization, Pathologic; Osteoarthritis; Proto-Oncogene Proteins; Receptor, Notch1; Receptor, Notch4; Receptors, Notch; Signal Transduction; Synovial Membrane; Vascular Endothelial Growth Factor A

Two of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro.. Primary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array.. Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed.. Significantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Chondrocytes; Cytokines; Granulocyte Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-6; Interleukin-8; Osteoarthritis; Statistics, Nonparametric; Synovial Fluid; Vascular Endothelial Growth Factor A

Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of glycoprotein VI (GPVI), a surface glycoprotein receptor for collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve Spleen Tyrosine Kinase (SYK), best known as an upstream activator of Bruton's Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by convulxin or collagen, specific ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector, phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor, RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets, convulxin stimulation resulted in elevated production of pro-inflammatory cytokines, IL-6 and IL-8, an effect which was dose-dependently blocked by RN486. The effects are specific as RN486 abrogated platelet aggregation induced by GPVI ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of inflammation driven by pMP-induced FLS cytokines production.

Topics: Agammaglobulinaemia Tyrosine Kinase; Arthritis, Rheumatoid; B-Lymphocytes; Blood Platelets; Catalysis; Cell-Derived Microparticles; Coculture Techniques; Humans; Interleukin-6; Interleukin-8; Lectins, C-Type; Lymphocyte Activation; Phospholipase C gamma; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Membrane Glycoproteins; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptors, Antigen, B-Cell; Synovitis

Interleukin (IL)-17A and Th17 cells are critically involved in T cell-mediated synovial inflammation. Besides IL-17A, Th17 cells produce IL-22. Recently, Th22 cells were discovered, which produce IL-22 in the absence of IL-17. However, it remains unclear whether IL-22 and Th22 cells contribute to T cell-mediated synovial inflammation. Therefore, we examined the potential of IL-22 and Th22 cells to induce synovial inflammation and whether IL-22 is required for T cell-mediated experimental arthritis.. Peripheral and synovial Th17 and Th22 cells were identified and sorted from patients with rheumatoid arthritis (RA). Co-culture experiments of these primary T cell populations with RA synovial fibroblasts (RASF) were performed. The in vivo IL-22 contribution to synovial inflammation was investigated by inducing T cell-mediated arthritis in IL-22 deficient mice and wild-type mice.. Peripheral Th17 and Th22 cell populations were increased in patients with RA and present in RA synovial fluid. In T cell-RASF co-cultures, IL-22 in the presence of IL-17A had limited effects on IL-6, IL-8, matrix metalloproteinase-1 (MMP-1) and MMP-3 production. Furthermore, primary peripheral blood and synovial Th17 cells were more potent in the induction of these factors by RASF compared with Th22 cells. In line with this, similar synovial inflammation and disease severity was found between IL-22 deficient and wild-type mice in T cell-mediated experimental arthritis.. These findings show that IL-17A/Th17 cell-mediated synovial inflammation is independent of IL-22 and Th22 cells. This implies that targeting IL-17A/Th17 cells, rather than IL-22/Th22 cells, should be the focus for treatment of T cell-mediated synovial inflammation.

Topics: Adult; Aged; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Coculture Techniques; Female; Humans; Interleukin-17; Interleukin-22; Interleukin-6; Interleukin-8; Interleukins; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Receptors, CCR6; Synovitis; Th17 Cells; Up-Regulation; Young Adult

It was recently demonstrated that the cholinergic anti-inflammatory pathway can modulate host inflammatory responses via cholinergic mediators or via electrical stimulation of the vagus nerve. Here, we investigated whether nicotine, a selective cholinergic agonist, plays any anti-inflammatory role in rheumatoid arthritis fibroblast-like synoviocytes (FLS). We observed that low concentrations (0.1-100 μM) of nicotine did not affect FLS viability in lactate dehydrogenase release test or the MTT assay. Nicotine at concentrations of 0.1-10 μM dose reduced the protein and mRNA expression of IL-6 and IL-8 induced by tumor necrosis factor-α (TNFα). Nicotine also inhibited nuclear factor (NF)-κB (p65) translocation from the cytoplasm to the nucleus, based on Western blotting and immunocytochemical analysis. In conclusion, nicotine can inhibit the TNFα dependant inflammatory pathway in synoviocytes by suppressing the activation of the NF-κB pathway.

Topics: Arthritis, Rheumatoid; Cell Nucleus; Cell Survival; Cells, Cultured; Cytoplasm; Dose-Response Relationship, Drug; Humans; Interleukin-6; Interleukin-8; NF-kappa B; Nicotine; RNA, Messenger; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha

Primary synovial cells with high passage number demonstrate increased production of proinflammatory mediators in response to inflammatory stimuli compared with cells with low passage number. This study used synovial cells to learn how different numbers of serial subculture passages affect the production of proinflammatory mediators in response to interleukin (IL)-1β. Synovial cells were serially subcultured in flasks until passage 7. During cell passage, synovial cells were treated with IL-1β for 24 h. Levels of proinflammatory mediators were analyzed by ELISA, real-time PCR, and Western blot. Synovial cells at passage 7 had elongated morphology and higher activity of β-galactosidase, a marker of senescence, than those at passage 3. Production of IL-6, IL-8, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE(2)) in response to IL-1β was significantly increased in cells at passage 7 compared with passage 3. To evaluate the mechanism of this different response, IL-1 receptor expression was studied in cells stimulated with IL-1β. Compared with cells at passage 3, cells at passage 7 had stronger expression of IL-1 receptors 1 and 2, as well as significantly increased expression of both receptors, in response to IL-1β. This finding suggests that differential production in response to inflammatory stimuli associated with cell passages should be considered when in vitro experiments are used to evaluate the production levels of proinflammatory mediators.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; beta-Galactosidase; Cells, Cultured; Cellular Senescence; Dinoprostone; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Receptors, Interleukin-1; Synovial Membrane; Vascular Endothelial Growth Factor A

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-κB pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-α-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-α-induced ERK and NF-κB activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-α-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Humans; Interleukin-6; Interleukin-8; MAP Kinase Kinase Kinases; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; NF-kappa B; Phosphatidylethanolamine Binding Protein; raf Kinases; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha

Human six-transmembrane epithelial antigen of prostate4 (STEAP4), an ortholog of mouse tumor necrosis factor-α-induced adipose-related protein (TIARP), plays a role in tumor necrosis factor (TNF)-dependent arthritis models. However, its role in rheumatoid arthritis (RA) is still obscure. This study explored such a role for STEAP4. The expressions of STEAP4, TNFα, and IL-6 were compared in synovia of RA and osteoarthritis patients. STEAP4 induction was examined in TNFα-stimulated fibroblast-like synoviocytes (FLS) in vitro. FLS (with/without TNFα stimulation) were also analyzed for IL-6 expression after STEAP4 knockdown, using siRNA or transfection with STEAP4-plasmid DNA. IL-8, cell proliferation, and apoptosis were also evaluated in STEAP4-overexpressing FLS. The expression of STEAP4 in joints correlated with TNFα expression, specifically in RA synovium. In the cultured FLS, STEAP4 protein expression was augmented by TNFα activation, and localized in endosomal/lysosomal compartments. STEAP4 downregulation by siRNA enhanced the expression of IL-6 mRNA, while STEAP4 overexpression suppressed IL-6 and IL-8 expression, inhibited cell proliferation, and induced apoptosis via caspase-3. The results indicated that human STEAP4 is regulated by TNFα in synovium, where it controls IL-6 secretion and proliferation of FLS, suggesting that STEAP4 might potentially suppress the pathogenesis of TNFα-induced arthritis such as RA.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Fibroblasts; Gene Expression; Gene Silencing; Humans; Interleukin-6; Interleukin-8; Jurkat Cells; Membrane Proteins; Osteoarthritis, Knee; Oxidoreductases; RNA, Small Interfering; Synovial Membrane; Transfection; Tumor Necrosis Factor-alpha

To delineate the functional significance of IL-17 Receptor (IL-17RA) and characterize the IL-17 producing T cell (Th17) subpopulation in psoriatic arthritis (PsA). Mononuclear cells from blood and synovial fluid (SF) were obtained from PsA (n=20), rheumatoid arthritis (RA, n=20) and osteoarthritis (OA, n=20) patients. Synoviocytes (FLS) were isolated from the synovium of RA (n=5), PsA (n=5) and OA (n=5) patients. IL-17RA expression in FLS was identified by western blotting (WB) and flowcytometry. T lymphocytes derived from the SF of these patients were studied to identify and phenotype the Th17 cells. The functional significance of IL-17RA was determined by evaluating its regulatory role on the production of proinflammatory cytokines and endopeptidase. IL-17RA expression was found to be significantly higher in FLS of RA (15.7%±4.9) and PsA (4.5%±0.9) in comparison to OA (1.14%±0.9). Western blot analyses showed that the relative intensity (RI) of IL-17RA protein was higher in RA and PsA compared to OA (Fisher exact, P<0.01). A significant enrichment of IL-17-producing CD4+ T cells (7.9%±2.8) was observed in the SF of PsA patients compared to that of OA patients (P<.001). Compared to OA-FLS, recombinant IL-17 induced higher levels of IL-6, IL-8, and MMP-3 production in PsA-FLS. Blockage of IL-17RA with an anti-IL-17RA antibody inhibited the production of IL-6, IL-8, and MMP-3. This is the first report to demonstrate the functional significance of IL-17RA in PsA. Results of this study support the hypothesis that IL-17RA blocking antibodies have the potential to be a therapeutic option for psoriatic arthritis.

Topics: Antibodies, Blocking; Arthritis, Psoriatic; Arthritis, Rheumatoid; Case-Control Studies; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Receptors, Interleukin-17; Synovial Fluid; Th17 Cells

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder, primarily affecting the articular structures and synovial membranes of multiple joints. Beside pharmacologically based treatments, sulphur bath therapy has long been used as a therapy for patients suffering from different rheumatic disorders. But scientific reports about the beneficial effects of H(2)S as well as about the underlying molecular mechanisms are controversial and rare.. Fibroblast-like synoviocytes (FLS) derived from RA and OA-patients were treated with the H(2)S-donor sodium hydrogen sulphide (NaHS). IL-6 release was quantified by enzyme-linked immunosorbent assay (ELISA). Gene expression of IL-6, IL-8 and COX-2 as well as of the matrix metalloproteinases (MMPs) MMP-2, MMP-3 and MMP-14 was monitored by quantitative real-time PCR (qRT-PCR). Modulation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2 was analysed by Western blotting.. High concentrations of H(2)S (above 0.5mM) elevated the expression of pro-inflammatory genes in RA- and OA-FLS. This was accompanied by activation of p38 and ERK1/2 MAPK. H(2)S-induced expression of IL-6, IL-8 and COX-2 was completely blocked by specific inhibitors of p38 and ERK1/2 MAPK and NF-κB.. H(2)S is a potent gaseous molecule that can upregulate the expression of a series of pro-inflammatory genes in RA and OA-FLS. Therefore, caution is advised in patients with active RA when taking sulphur bath therapy.

Topics: Arthritis, Rheumatoid; Balneology; Cells, Cultured; Cyclooxygenase 2; Fibroblasts; Gene Expression Regulation; Humans; Hydrogen Sulfide; Inflammation Mediators; Interleukin-6; Interleukin-8; MAP Kinase Signaling System; Matrix Metalloproteinases; Mineral Waters; Osteoarthritis; Sulfides; Synovial Membrane

TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.. RAFLS were obtained from surgical synovial specimens and used at passage 5-10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.. TWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.. TWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.

Topics: Apoptosis; Arthritis, Rheumatoid; Cytokine TWEAK; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Receptors, Tumor Necrosis Factor; Recombinant Proteins; RNA, Messenger; Synovial Membrane; Transcription Factor RelB; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; TWEAK Receptor

The recently described IL-33 acts as a pro-inflammatory cytokine, inducing the expression of multiple responses in the target cells. Although a nuclear localization of IL-33 has been described, its exact functional relevance is presently unknown. The present study was conducted to analyze the effects of IL-33 on the TNF-α induced synthesis of the pro-inflammatory mediators IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the pro-destructive molecules matrix metalloproteinase-1 (MMP-1), MMP-3, and TIMP-1 of rheumatoid arthritis synovial fibroblast (RA-SFs) using RNA overexpression and silencing. TNF-α significantly induced IL-33 mRNA expression and protein synthesis in RA-SFs. TNF-α-induced IL-33 protein expression was mediated via p38 signaling. Immunohistochemistry for IL-33 clearly showed that nuclear translocation of IL-33 was induced in TNF-α stimulated RA-SFs. IL-33 overexpression enhanced TNF-α-induced pro-inflammatory and pro-destructive functions in RA-SFs. IL-33 silencing significantly downregulated TNF-α-induced pro-inflammatory functions, whereas TNF-α-induced pro-destructive functions were less influenced by IL-33 silencing. This study identifies IL-33 as a critical regulator/enhancer of TNF-α-induced functions in RA-SFs, pointing to a central role of this cytokine in the perpetuation of pro-inflammatory and pro-destructive processes in rheumatoid arthritis (RA) and other inflammatory and degenerative diseases.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Down-Regulation; Fibroblasts; Humans; Interleukin-33; Interleukin-6; Interleukin-8; Interleukins; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; p38 Mitogen-Activated Protein Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Synovial Membrane; Tissue Inhibitor of Metalloproteinase-1; Tumor Necrosis Factor-alpha

Th17 cells have been implicated in rheumatoid arthritis (RA). We hypothesized that the interaction of T cells with bone marrow-derived mesenchymal stem cells (BM-MSCs) or with fibroblast- like synoviocytes (FLS) might, with the help of T cell-secreted inflammatory cytokines (i.e., interleukin-17A [IL-17A], tumor necrosis factor α [TNFα], and/or interferon-γ [IFNγ]), promote Th17 cell expansion and activation.. Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were cocultured with BM-MSCs or FLS from RA patients or osteoarthritis (OA) patients. Cocultures were exposed to phytohemagglutinin with or without IL-17A, TNFα, or IFNγ. Quantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and cytofluorometry were used to measure IL-17A production.. Interaction of PBMCs with BM-MSCs inhibited Th1 and Th2 responses, but promoted Th17 cell expansion, as early as 24 hours, as demonstrated by increases in retinoic acid receptor-related orphan nuclear receptor γ or IL-17A messenger RNA (mRNA) levels, IL-17A secretion levels, and IL-17A-secreting cell frequency, as well as by T cell switching to the Th17 pathway after 2 rounds of stimulation with MSCs. IL-17A production was also increased in PBMCs stimulated with anti-CD3 plus anti-CD28 or in isolated CD3+ or CD45RO+ T cells, thus demonstrating the role of T cell activation. Levels of mRNA for IL-6, IL-8, and IL-1β were further amplified when T cell-secreted inflammatory cytokines were added. Interestingly, OA FLS or RA FLS also enhanced IL-17A and IL-6 production, but only RA FLS enhanced IFNγ and IL-1β production. We further demonstrated that MSC-mediated Th17 promotion requires caspase 1 activation by using an inhibitory peptide and measuring its activity.. We found that the interaction of MSCs or FLS with T cells promotes the activation and expansion of Th17 cells through caspase 1 activation. Since proinflammatory and T cell-secreted inflammatory cytokines are also amplified, this mechanism may participate in the chronicity of RA.

Topics: Arthritis, Rheumatoid; Bone Marrow Cells; Caspase 1; Cells, Cultured; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; Mesenchymal Stem Cells; Osteoarthritis; Synovial Membrane; Th17 Cells; Up-Regulation

To determine whether adiponectin may have synergistic effects in combination with the proinflammatory cytokine interleukin (IL)-1β regarding the production of proinflammatory mediators during arthritic joint inflammation, synovial cells from rheumatoid arthritis (RA) patients were treated with adiponectin, IL-1β, and their combination for 24 h. Culture supernatant was collected and analyzed by enzyme-linked immunosorbent assay for levels of IL-6, IL-8, prostaglandin E(2) (PGE(2)), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Adiponectin-mediated intracellular signaling pathways were investigated to elucidate the molecular mechanisms underlying their synergy. The association of proinflammatory mediators with adiponectin was investigated in the synovial fluid of arthritis patients. Adiponectin functioned synergistically with IL-1β to activate IL-6, IL-8, and PGE2 expression in RA fibroblast-like synoviocytes; Levels of VEGF, MMP-1, and MMP-13 were not synergistically stimulated. Adiponectin and IL-1β each increased the expression of both adiponectin receptor 1 and IL-1 receptor 1. However, adiponectin and IL-1β did not synergistically support the degradation of IκB-α or the nuclear translocation of NF-κB. Synergistically increased gene expression was significantly inhibited by MG132, an NF-κB inhibitor. Supporting the in vitro results, IL-6 and IL-8 levels were positively associated with adiponectin in synovial joint fluid from patients with RA, but not osteoarthritis (OA). In conclusion, adiponectin and IL-1β may synergistically stimulate the production of proinflammatory mediators through unknown signaling pathways during arthritic joint inflammation. Adiponectin may be more important to the pathogenesis of RA than previously thought.

Topics: Adiponectin; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 2; Gene Expression Regulation; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Joints; Matrix Metalloproteinases; NF-kappa B; Obesity; Osteoarthritis; Receptors, Adiponectin; Receptors, Interleukin-1; Synovial Fluid

Type I interferons (IFNs) have emerged as potential activators of the IFN signature and elevated STAT-1 expression in rheumatoid arthritis (RA) synovium, but mechanisms that induce synovial IFN expression are unknown. Recently, tumor necrosis factor α (TNFα) was shown to induce a delayed IFN response in macrophages. We undertook this study to test whether TNFα, classically thought to activate inflammatory NF-κB target genes in RA, also contributes to the "IFN signature" in RA synovial macrophages.. Synovial fluid (SF) macrophages purified from 24 patients with RA and 18 patients with spondylarthritides (SpA) were lysed immediately after isolation or were cultured ex vivo in the absence or presence of blockade of endogenous type I IFN or TNFα. Expression of IFN-inducible target genes was measured by quantitative reverse transcription-polymerase chain reaction, and expression of their corresponding proteins was measured by enzyme-linked immunosorbent assay.. Expression of an IFN signature and STAT1 in RA synovial macrophages was suppressed when type I IFNs or TNFα were blocked, whereas TNFα blockade did not affect expression of IFN response genes or STAT1 in SpA synovial macrophages. RA SF suppressed the IFN signature in RA synovial macrophages and in TNFα-, IFNα-, and IFNβ-stimulated control macrophages. Type I IFNs suppressed expression of IL8 and MMP9 in RA synovial macrophages and in TNFα-stimulated control macrophages.. Our findings identify a new function of TNFα in RA synovitis by implicating TNFα as a major inducer of the RA synovial IFN response. The results suggest that the expression of IFN response genes in RA synovium is regulated by interplay between TNFα and opposing homeostatic factors expressed in the synovial microenvironment.

Topics: Adult; Arthritis, Rheumatoid; Humans; Interferon Type I; Interleukin-8; Macrophages; Matrix Metalloproteinase 9; Spondylarthritis; STAT1 Transcription Factor; Synovial Fluid; Tumor Necrosis Factor-alpha

To investigate the effects of heat shock protein 72 (Hsp72) on the expression of IL-6 and IL-8 and activation of NF-kappaB in synoviocytes from patients suffered from rheumatoid arthritis (RA).. IL6 and IL8 concentrations in culture supernatants were measured using enzyme-linked immunosorbent assays (ELISA). Nuclear translocation of NF-kappaB and degradation of the inhibitory protein IkappaBalpha were examined using immunohistochemistry and Western blot.. Hsp72 down-regulated IL-6 and IL-8 production in RA synoviocytes induced by tumor necrosis factor-alpha (TNF-alpha). Hsp72 inhibited nuclear translocation of NF-kappaB and degradation of IkappaBalpha induced by TNF-alpha.. Hsp72 has an anti-inflammatory effect on RA by down-regulation of IL-6 and IL-8 in synoviocytes, which is mediated through inhibiting the activation of NF-KalphaB signal pathways.

Topics: Arthritis, Rheumatoid; Cells, Cultured; HSP72 Heat-Shock Proteins; Humans; I-kappa B Proteins; Interleukin-6; Interleukin-8; NF-kappa B; NF-KappaB Inhibitor alpha; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.

Topics: Adult; Antimalarials; Artemisinins; Artesunate; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Female; Flow Cytometry; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Middle Aged; Phosphorylation; Signal Transduction; Statistics, Nonparametric; Synovial Membrane; Vascular Endothelial Growth Factor A

Chemokines promote leucocyte traffic into the synovium, leading to the initiation and progression of the rheumatoid arthritis (RA). The aim of the study was to determine the effects of etanercept, a soluble tumour necrosis factor receptor (sTNFr), on the serum chemokines levels in patients with active RA. Patients were treated with 50 mg of subcutaneous injection of etanercept per week and methotrexate (10-25 mg/week). Serum levels of interleukin-8 (IL-8), RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 (MCP-1) were assessed by ELISA at months 0, 3, 6, 9 and 12, prior to injection. 3-month treatment with etanercept diminished serum concentrations of IL-8, RANTES and MCP-1 (P < 0.05, P < 0.01 and P < 0.001, respectively). Subsequent etanercept administrations prolonged decrease in serum chemokines levels and in the case of IL-8 even intensified the reduction of its concentration in serum. These changes were accompanied by significant decrease of disease activity score (DAS28) (in all cases P < 0.001). Prior to the first etanercept administration, serum concentrations of studied chemokines correlated with markers of RA activity such as the erythrocyte sedimentation rate (ESR) and DAS28. Following next drug injection such associations were less or not significant. Therapy with etanercept and MTX not only caused a clinical improvement but also diminished serum chemokines levels in RA patients. Further treatment with etanercept sustained chemokines suppression.

Topics: Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Blood Sedimentation; Chemokine CCL2; Chemokine CCL5; Chemokines; Etanercept; Female; Humans; Immunoglobulin G; Interleukin-8; Male; Middle Aged; Receptors, Tumor Necrosis Factor

To investigate the interplay between IL-32 and tumour necrosis factor alpha (TNFα) during the chronic inflammation of rheumatoid arthritis (RA) and to assess whether anti-TNFα treatment of RA patients modulates synovial IL-32 expression.. Induction of IL-32γ by Pam3Cys, lipopolysaccharide, IL-1β or TNFα was investigated in human fibroblast-like synoviocytes (FLS). Stimulation of TNFα production by IL-32γ was studied by adenoviral overexpression of IL-32γ (AdIL-32γ) and lipopolysaccharide stimulation of THP1 cells. Silencing of endogenous IL-32 was employed to study cytokine regulation in FLS. AdIL-32γ followed by TNFα stimulation was performed in FLS to investigate cytokine induction. Immunohistochemistry was applied to study IL-32 expression in synovial biopsies from RA patients.. TNFα potently induced IL-32γ expression in FLS. Increased TNFα, IL-1β, IL-6 and CXCL8 production was observed after IL-32γ overexpression and lipopolysaccharide stimulation of THP1 cells. TNFα stimulation of FLS after silencing IL-32γ resulted in diminished IL-6 and CXCL8 production, whereas IL-32γ overexpression resulted in enhanced IL-6 and CXCL8 levels. Remarkably, the mechanism through which IL-32γ overexpression induced TNFα, IL-1β and CXCL8 was by counteracting messenger RNA decay. Importantly, treatment of RA patients with anti-TNFα resulted in significant reduction of IL-32 protein in synovial tissue.. TNFα is a potent inducer of endogenous IL-32 expression and IL-32 itself contributes to prolonged TNFα production, thus inducing an important auto-inflammatory loop. Treatment of RA patients with anti-TNFα antibodies diminished IL-32 expression in synovial tissue. The potent anti-inflammatory effect of TNFα blockade in RA patients may be partly due to the reduction of synovial IL-32 expression.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Gene Silencing; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-6; Interleukin-8; Interleukins; Lipopolysaccharides; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha

Neutrophils participate in the initiation and progression of rheumatoid arthritis (RA) although the exact mechanisms responsible for neutrophil accumulation in rheumatoid joints are not understood. This study compared the adhesive and chemotactic functions of neutrophils from RA patients in activity (DAS28 > 3.2) and not in activity (DAS28 < 2.6) and observed the effects of different treatment approaches on these functions. Neutrophils were isolated from healthy controls (CON), and patients with active or inactive RA in use of therapy not specific for RA (NSAIDs), in use of DMARDs and in use of anti-TNF-α therapy. Adhesive and chemotactic properties were evaluated using in vitro assays; adhesion molecule expression was assessed by flow cytometry and real-time PCR and circulating chemokines were determined by ELISA. No significant alterations in the adhesive and chemotactic properties of neutrophils from active RA were observed when compared to CON neutrophils, independently of treatment regimen. In contrast, neutrophils from RA patients in disease remission presented reduced adhesive properties and a lower spontaneous chemotactic capacity, in association with decreased adhesion molecule expression, although profiles of alterations differed for those patients on DMARDs and those on anti-TNF-α therapy. Circulating levels of the major neutrophilic chemokines, IL-8 and epithelial neutrophil activating peptide-78, were also significantly decreased in those patients demonstrating a clinical response. Remission of RA appears to be associated with ameliorations in aspects important for neutrophil adhesion and chemotaxis; whether these alterations contribute to decrease neutrophil migration to the synovial fluid, with consequent improvements in the clinical manifestations of RA, remains to be determined.

Topics: Adult; Aged; Antibodies, Monoclonal; Antirheumatic Agents; Arthritis, Rheumatoid; Case-Control Studies; Cell Adhesion; Cell Adhesion Molecules; Chemokines; Chemotaxis, Leukocyte; Female; Gene Expression; Humans; Infliximab; Interleukin-8; L-Selectin; Male; Middle Aged; Neutrophils; Remission Induction; Tumor Necrosis Factor-alpha; Young Adult

Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA).. In the present study, we compared RA fibroblast-like synoviocytes (FLS) that were resistant or sensitive to TRAIL-induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA).. Sensitivity to TRAIL-induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL-induced apoptosis. TRAIL-sensitive cells expressed significantly lower levels of TRAILR-1, and silencing of TRAILR-1 increased TRAIL-induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG-to-TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage.. These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL-induced apoptosis is associated with a disease-promoting activity of TRAIL in RA.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Apoptosis; Arthritis, Rheumatoid; Female; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Osteoarthritis; Osteoprotegerin; Severity of Illness Index; Spondylarthritis; Synovial Membrane; TNF-Related Apoptosis-Inducing Ligand; Young Adult

The aim of this study was to analyze both the constitutive and induced expression and function of double-stranded RNA (dsRNA; Toll-like receptor 3 [TLR-3], retinoic acid-inducible gene I [RIG-I], and melanoma differentiation-associated gene 5 [MDA5]) and single-stranded RNA (ssRNA; TLR-7) receptors in osteoarthritis (OA) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), by studying the transcription factors involved and the subsequent effects on antiviral interferon-β (IFNβ), the proinflammatory CXCL8 chemokine, and matrix metalloproteinase 3 (MMP-3). An additional goal was to study the effect of vasoactive intestinal peptide (VIP).. The expression of TLR-3, TLR-7, RIG-I, and MDA5 in cultured FLS was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and Western blotting. Transcription factors were studied using the ELISA-based TransAM transcription factor kit. The expression of IFNβ, CXCL8 (interleukin-8), and MMP-3 was analyzed by RT-PCR and ELISA.. FLS expressed TLR-3, TLR-7, RIG-I, and MDA5. The expression of TLR-3 and RIG-I was higher in RA FLS, while the expression of TLR-7 and MDA5 was higher in OA FLS. Stimulation with poly(I-C) induced the activation of IFN regulatory factor 3 (IRF-3), NF-κB, and activator protein 1 (AP-1) c-Jun as well as the subsequent production of IFNβ, CXCL8, and MMP-3. VIP reduced the activation of IRF-3 and the production of IFNβ in both OA and RA FLS. Imiquimod induced the activation of NF-κB, AP-1 c-Fos, and AP-1 c-Jun and the synthesis of CXCL8 and MMP-3. VIP significantly diminished MMP-3 production only in imiquimod-treated RA FLS.. The results of this study revealed a prominent function of FLS in the recognition of both dsRNA and ssRNA, which may be present in the joint microenvironment. This study also advances the healing function of the endogenous neuroimmune peptide VIP, which inhibited TLR-3-, RIG-I-, MDA5-, and TLR-7-mediated stimulation of antiviral, proinflammatory, and joint destruction mediators.

Topics: Aminoquinolines; Arthritis, Rheumatoid; Cells, Cultured; DEAD-box RNA Helicases; Fibroblasts; Humans; Imiquimod; Interferon Inducers; Interferon Regulatory Factor-3; Interferon-beta; Interferon-Induced Helicase, IFIH1; Interleukin-8; Matrix Metalloproteinase 3; Osteoarthritis; Receptors, Retinoic Acid; RNA, Double-Stranded; Synovial Fluid; Toll-Like Receptor 3; Toll-Like Receptor 7; Transcription Factors; Vasoactive Intestinal Peptide

Various cytokines and inflammatory mediators are known to be involved in the pathogenesis of rheumatoid arthritis (RA). We hypothesized that polymorphisms in selected inflammatory response and tissue repair genes contribute to the susceptibility to and severity of RA.. Polymorphisms in TNFA, IL1B, IL4, IL6, IL8, IL10, PAI1, NOS2a, C1INH, PARP, TLR2 and TLR4 were genotyped in 376 Caucasian RA patients and 463 healthy Caucasian controls using single base extension. Genotype distributions in patients were compared with those in controls. In addition, the association of polymorphisms with the need for anti-TNF-α treatment as a marker of RA severity was assessed.. The IL8 781 CC genotype was associated with early onset of disease. The TNFA -238 G/A polymorphism was differentially distributed between RA patients and controls, but only when not corrected for age and gender. None of the polymorphisms was associated with disease severity.. We here report an association between IL8 781 C/T polymorphism and age of onset of RA. Our findings indicate that there might be a role for variations in genes involved in the immune response and in tissue repair in RA pathogenesis. Nevertheless, additional larger genomic and functional studies are required to further define their role in RA.

Topics: Adult; Age of Onset; Aged; Arthritis, Rheumatoid; Case-Control Studies; Cohort Studies; Genotype; Humans; Inflammation Mediators; Interleukin-8; Male; Middle Aged; Polymorphism, Genetic; Severity of Illness Index; Tumor Necrosis Factor-alpha

The underlying mechanisms for the subsets of self-limiting, intermittent or chronic and deforming arthritis in systemic lupus erythematosus (SLE) are not well understood. We performed a cross-sectional analysis of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α) and joint status in 47 SLE patients (79% females, age 42 years, disease duration 8.6 years). All cytokines levels were significantly elevated in SLE patients compared with controls, but only IL-2 and IL-8 levels were higher than in patients with rheumatoid arthritis. SLE patients with ongoing synovitis (19%) and joint deformities (11%) had increased erythrocyte sedimentation rate (ESR), IL-6 and anti-dsDNA Ab levels. IL-6 levels correlated with ESR, anti-dsDNA Ab and haemoglobin, but not with C-reactive protein levels. Arthritis constitutes a considerable burden of disease in SLE over time, and joint deformations are associated with longstanding disease and arthritis flare rates. IL-6 is a potential biomarker and therapeutic target in the prevention of joint damage in SLE arthritis.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Blood Sedimentation; Case-Control Studies; Cross-Sectional Studies; Cytokines; Female; Humans; Interleukin-2; Interleukin-6; Interleukin-8; Joint Deformities, Acquired; Lupus Erythematosus, Systemic; Male; Middle Aged; Registries; Synovitis

To investigate the mechanism in vivo for the regulation of inflammation of patients with RA by infliximab, we measured serum levels of chemokine ligand (CCL) 2, CCL3, CXCL8, and expression of CCL2 receptor chemokine receptor (CCR) 2 on CD4 T cells from patients with rheumatoid arthritis (RA).. Forty-four patients with were enrolled in our study. Twenty-four patients received infliximab combined with methotrexate. Twenty patients received methotrexate alone. Serum levels of the chemokines CCL2, CCL3, and CXCL8 were quantified using commercial enzyme-linked immunosorbent assay kits. Flow cytometry was used to analyze the expression of CCR2 on CD4 T cells.. The mean CCL2 levels in the infliximab-treated patients decreased significantly from 885.20 ± 323.52 pg/mL at pretreatment to 454.65 ± 185.03 pg/mL (P < 0.05) at 30 weeks after the initial treatment. Fluorescence density of CCR2 expression on CD4 T cells were significantly reduced after infliximab treatment.. CCL2/CCR2 system in patients with active RA may be sensitive to anti-tumor necrosis factor-α therapy and suggest that CCL2 plays a crucial role in the pathogenesis of RA. CCR2 may be an important target for therapy in RA.

Topics: Adult; Anti-Inflammatory Agents; Antibodies, Monoclonal; Arthritis, Rheumatoid; CD4-Positive T-Lymphocytes; Chemokine CCL2; Chemokine CCL3; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Humans; Infliximab; Interleukin-8; Male; Middle Aged; Models, Biological; Receptors, CCR2; Tumor Necrosis Factor-alpha

To identify novel proteins involved in the pathogenesis of rheumatoid arthritis (RA) and to characterise the identified proteins based on pathogenic and therapeutic aspects.. The authors applied differential phosphoproteomic analysis to articular synoviocytes between RA and osteoarthritis (OA) to identify proteins differently phosphorylated between RA and OA. Focusing on annexin VII (Anx7), one of the highly phosphorylated proteins in RA, the authors prepared Anx7-transgenic C57BL/6 (Anx7-Tg-B6) mice to evaluate their susceptibility to collagen-induced arthritis (CIA). In addition, the authors examined the effect of anti-Anx7 antibodies (Abs) on CIA and serum levels of cytokines in wild-type DBA/1J mice, which are known to be susceptible to CIA, and in Anx7-Tg-B6 mice. In vitro, the authors examined the effect of the Anx7 knockdown by small interfering RNA on the secretion of cytokines in rheumatoid synoviocytes and the human synovial sarcoma cell line SW982.. The Anx7 transgene altered the CIA-resistant B6 mice to CIA-susceptible ones. The Abs treatment suppressed CIA even in the wild-type DBA/1J mice. The serum levels of cytokines including interleukin 6 (IL-6) and TNFα were not altered by the Abs treatment in vivo. On the other hand, the knockdown of Anx7 by small interfering RNA caused downregulation of IL-8 secretion in vitro.. These results indicate that Anx7 participates in the pathogenesis of RA partly through the secretion of IL-8. The study data have demonstrated the pathogenic roles and therapeutic significance of Anx7 in RA for the first time.

Topics: Adult; Aged; Animals; Annexin A7; Antibodies, Neutralizing; Arthritis, Experimental; Arthritis, Rheumatoid; Cytokines; Disease Susceptibility; Female; Gene Knockdown Techniques; Humans; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Phosphorylation; Proteomics; Synovial Membrane

To evaluate the modulation of RhoA/Rho kinase (ROCK) signaling pathway, a small Rho GTPase that is considered as an important modulator in inflammatory responses, on Toll-like receptor-2 mediated chemokine secretion in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients.. The RhoA activity was measured by a pull-down assay. And the ROCK activity was assessed by Western blot. The secretion of chemokines was measured by ELISA (enzyme-linked immunosorbent assay). MTT test was used to detect the cellular viability.. The stimulation of peptidoglycan (PG, 5 mg/L) increased the levels of IL-8 (interleukin-8), RANTES (regulated upon activation normal T cell expressed & secreted) and MCP-2 (monocyte chemotactic protein-2) and boosted the activities of RhoA and ROCK versus the unstimulated RA FLS. And these effects of PG were suppressed by anti-TLR-2 monoclonal antibody. Inhibition of RhoA and ROCK with a specific inhibitor inhibited the secretion of IL-8, RANTES and MCP-2 in PG-induced RA FLS.. The present study provides novel evidence that the RhoA/ROCK signal pathway modulates the TLR-2-mediated secretion of chemokines in RA FLS. It suggests that the inhibition of RhoA/ROCK may be a new therapeutic approach for RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL5; Chemokine CCL8; Female; Fibroblasts; Humans; Interleukin-8; Male; rhoA GTP-Binding Protein; Signal Transduction; Synovial Fluid; Synovial Membrane; Toll-Like Receptor 2

Sinomenine, an alkaloid isolated from the root of Sinomenium acutum, has been used to alleviate the symptoms of rheumatic diseases. Liang Miao San (LMS), composed of the herbs Rhizoma Atractylodis (Cangzhu) and Cotex Phellodendri (Huangbai), is another traditional Chinese medicine formula for rheumatoid arthritis (RA) treatment. Although numerous studies have demonstrated the potential anti-inflammatory activities of sinomenine and LMS, the underlying intracellular mechanisms regulating the anti-inflammatory activities of sinomenine and LMS on human primary fibroblast-like synoviocytes (FLS) from RA patients and normal control subjects have not been elucidated.. We investigated the in vitro anti-inflammatory activity of sinomenine and LMS on inflammatory cytokine tumor necrosis factor (TNF)-α-mediated activation of human normal and RA-FLS. The underlying intracellular signaling molecules were analyzed quantitatively using flow cytometry.. Sinomenine was found to significantly inhibit TNF-α induced cell surface expression of vascular cell adhesion molecule (VCAM)-1 and release of inflammatory cytokine and chemokine IL-6, CCL2 and CXCL8 from both normal and RA-FLS (all p<0.05). Moreover, the suppression of sinomenine on TNF-α induced VCAM-1 expression and IL-6 release of RA-FLS was significantly higher than that of normal FLS (p<0.05). LMS significantly inhibited TNF-α-induced inflammatory chemokines CXCL10 and CCL5 release from both normal and RA-FLS, with significantly higher suppression on CXCL10 secretion in RA-FLS than that of normal FLS (all p<0.05). Further investigations showed that sinomenine and LMS could significantly suppress TNF-α-induced phosphorylation of inhibitor κBα and extracellular signal-regulated protein kinase, the central signaling molecules mediating TNF-α-induced VCAM-1 expression and chemokine production.. Our results therefore provide a new insight into the differential anti-inflammatory activities of sinomenine and LMS through the suppression of TNF-α-activated FLS by modulating distinct intracellular signaling pathways in RA.

Topics: Anti-Inflammatory Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Case-Control Studies; Cells, Cultured; Chemokine CCL2; Chemokine CCL5; Chemokine CXCL10; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flow Cytometry; Humans; I-kappa B Proteins; Inflammation Mediators; Interleukin-6; Interleukin-8; Medicine, Chinese Traditional; Morphinans; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

To analyse the expression of SIRT1 in synovial tissues and cells of patients with rheumatoid arthritis (RA) and to study the function of SIRT1 in inflammation and apoptosis in RA.. Levels of SIRT1 expression were analysed in synovial tissues and cells from patients with RA by real-time PCR and western blotting before and after stimulation with toll-like receptor ligands, tumour necrosis factor α (TNFα) and interleukin 1β (IL-1β). Immunohistochemistry was used to study the localisation of SIRT1. Fluorescence activated cell sorting analysis was performed to investigate the effect of SIRT1 on apoptosis. Peripheral blood monocytes and rheumatoid arthritis synovial fibroblasts (RASFs) were transfected with wild-type or enzymatically inactive SIRT1 expression vectors or with siRNA targeting SIRT1. Cytokine analysis of IL-6, IL-8 and TNFα were performed by ELISA to study the role of SIRT1 on proinflammatory mediators of RA.. SIRT1 was found to be constitutively upregulated in synovial tissues and cells from patients with RA compared to osteoarthritis. TNFα stimulation of RASFs and monocytes resulted in further induced expression levels of SIRT1. Silencing of SIRT1 promoted apoptosis in RASFs, whereas SIRT1 overexpression protected cells from apoptosis. Inhibition of SIRT1 enzymatic activity by inhibitors, siRNA and overexpression of an enzymatically inactive form of SIRT1 reduced lipopolysaccharide-induced levels of TNFα in monocytes. Similarly, knockdown of SIRT1 resulted in a reduction of proinflammatory IL-6 and IL-8 in RASFs.. The TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Arthritis, Rheumatoid; Biopsy; Carbazoles; Cells, Cultured; Cytokines; Female; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Middle Aged; Monocytes; NF-kappa B; Osteoarthritis; RNA, Messenger; Signal Transduction; Sirtuin 1; Synovial Membrane; Tumor Necrosis Factor-alpha

Hyperuricemia-mediated uric acid crystal formation may cause joint inflammation and provoke the destruction of joints through the activation of inflammasome-mediated innate immune responses. However, the immunopathological effects and underlying intracellular regulatory mechanisms of uric acid crystal-mediated activation of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) have not been elucidated. Therefore, we investigated the in vitro effects of monosodium urate crystals, alone or in combination with the inflammatory cytokines tumor-necrosis factor (TNF)-α or interleukin (IL)-1β, on the activation of human FLS from RA patients and normal control subjects and the underlying intracellular signaling mechanisms of treatment with these crystals. Monosodium urate crystals were able to significantly increase the release of the inflammatory cytokine IL-6, the chemokine CXCL8 and the matrix metalloproteinase (MMP)-1 from both normal and RA-FLS (all P<0.05). Moreover, the additive or synergistic effect on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS was observed following the combined treatment with monosodium urate crystals and TNF-α or IL-1β. Further experiments showed that the release of the measured inflammatory cytokine, chemokine and MMP-1 stimulated by monosodium urate crystals were differentially regulated by the intracellular activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways but not the p38 mitogen-activated protein kinase pathway. Our results therefore provide a new insight into the uric acid crystal-activated immunopathological mechanisms mediated by distinct intracellular signal transduction pathways leading to joint inflammation in RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Drug Synergism; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Humans; Hyperuricemia; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Matrix Metalloproteinase 1; Signal Transduction; Synovial Fluid; Tumor Necrosis Factor-alpha; Uric Acid

Rheumatoid arthritis (RA) is a disease with significant gender differences in its prevalence and clinical features. Interleukin (IL)-1 and tumor necrosis factor (TNF) α produced by synoviocytes are principle inflammatory and destructive mediators of RA. We found that a potent androgen, 5α-dihydrotestosterone (DHT) inhibits IL-1α-induced production and mRNA expression of IL-8, IL-6 and IL-1β from RA patient-derived fibroblast-like synovial cell line MH7A. Promoter analysis of the IL-8 gene revealed that nuclear factor (NF)-κB activation is critical for its transcriptional activation by IL-1α, and DHT inhibited the IL-1α-induced NF-κB activation in a manner dependent on the androgen receptor (AR). DHT also inhibited the effects of TNFα on the cells overexpressed with AR, indicating that sufficient expression level of functional AR was necessary for the inhibitory effect of DHT on TNFα. These results suggest that androgen contributes to the prevention against RA and its gender difference by inhibiting IL-1α or TNFα-induced proinflammatory cytokine production from synovial fibroblast-like cells by inhibiting NF-κB activation in a manner depending on AR.

Topics: Androgens; Arthritis, Rheumatoid; Cell Line; Dihydrotestosterone; Female; Fibroblasts; Humans; Inflammation Mediators; Interleukin-1alpha; Interleukin-8; Interleukins; Male; NF-kappa B; Receptors, Androgen; RNA, Messenger; Synovial Fluid; Transcriptional Activation; Tumor Necrosis Factor-alpha

Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17), which are able to indirectly induce the recruitment of neutrophils. Recently, human Th17 cells have been phenotypically characterized and shown to express discrete chemokine receptors, including CCR2 and CCR6. Herein, we show that highly purified neutrophils cultured with interferon-gamma plus lipopolysaccharide produce the CCL2 and CCL20 chemokines, the known ligands of CCR2 and CCR6, respectively. Accordingly, supernatants from activated neutrophils induced chemotaxis of Th17 cells, which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment, neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally, we report that, although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and interferon-gamma release, these latter cells cannot be activated by IL-17A and IL-17F, because of their lack of IL-17RC expression. Collectively, our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells, which may represent a useful target for the treatment of chronic inflammatory diseases.

Topics: Arthritis, Rheumatoid; Cell Communication; Chemokine CCL2; Chemokine CCL20; Crohn Disease; Female; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-17; Interleukin-8; Male; Neutrophil Infiltration; Neutrophils; Receptors, CCR2; Receptors, CCR6; Receptors, Interleukin; Synovial Fluid; T-Lymphocytes, Helper-Inducer; Tumor Necrosis Factor-alpha

To test whether there are differences in the levels and ratios of 6 pro- and 3 anti-inflammatory cytokines produced by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) subjects compared to controls.. 79 participants (42 seropositive RA patients and 37 healthy controls) were enrolled in this study. The production levels in mitogen-stimulated PBMCs of the 6 proinflammatory cytokines (IFN-gamma, TNF-alpha, TNF-beta, IL-8, IL-17, IL-18) and 3 anti-inflammatory cytokines (IL-4, IL-10, IL-13) were assayed by ELISA using kits obtained from Immunotech SA. The ratios of pro- to anti-inflammatory cytokines were calculated for all participants.. There were significantly elevated levels of IL-8 and IL-10, and reduced levels of IFN-gamma, IL-4, and IL-17 in mitogen-stimulated PBMC culture supernatants of RA subjects compared to controls. Of the 18 pro-/anti-inflammatory cytokine ratios, 3 ratios (TNF-alpha/IL13, IL-8/IL-4 and IL-8/IL-13) were significantly higher in RA patients compared to controls; and 6 were higher in controls (IFN-gamma/IL-4; IFN-gamma/IL-10; IFN-gamma/IL-13; TNF-beta/IL10; IL-17/IL-10; IL-18/IL-10).. Activated PBMCs of RA patients, regardless of disease activity, showed higher-level production of IL-8 and IL-10 compared to controls; lower-level production of IFN-gamma, IL-4, and IL-17; and elevated ratios of TNF-alpha/IL-13, IL-8/IL-4 and IL-8/IL-13.

Topics: Adult; Arthritis, Rheumatoid; Case-Control Studies; Cells, Cultured; Female; Humans; Interleukin-10; Interleukin-13; Interleukin-4; Interleukin-8; Leukocytes, Mononuclear; Male; Middle Aged; Severity of Illness Index; Tumor Necrosis Factor-alpha

To assess whether serum levels of CC and CXC chemokines correlate with disease activity in patients with rheumatoid arthritis (RA), and to determine whether these effects predict clinical response.. Serum levels of the chemokines CC (CCL2, CCL5) and CXC (CXCL8, CXCL9, CXCL10) were quantified at baseline and after 12 weeks of treatment with disease-modifying antirheumatic drugs or biologic agents in 28 patients using flow cytometry. Serum from 40 healthy individuals was collected for comparison at baseline. Response to treatment was classified according to the European League Against Rheumatism (EULAR) response criteria. Remission of disease was defined as a Disease Activity Score < 2.6.. The baseline serum concentrations of CC and CXC chemokines were significantly elevated in patients with active RA compared to healthy controls (p < 0.05) except for CCL2. Significant improvement in all disease activity measurements was observed after 12 weeks of treatment. Seventeen (60.7%) patients achieved good to moderate response based on the EULAR response criteria, and 5 (17.9%) patients achieved remission. The improvement in clinical activity in patients with RA was accompanied by a significant reduction in the serum concentration of CXCL9 and CXCL10 (p < 0.001). A significant reduction in the serum level of CXCL10 was also observed in the group that achieved EULAR response. Serum concentration of CCL5 remained significantly elevated in patients with RA (n = 5) who achieved remission compared to the healthy controls (p < 0.05).. Serum concentration of CXCL9 and CXCL10 may serve as sensitive biomarkers for disease activity in patients with RA.

Topics: Adult; Aged; Analysis of Variance; Antirheumatic Agents; Arthritis, Rheumatoid; Biomarkers; Chemokine CCL2; Chemokine CCL5; Chemokine CXCL10; Chemokine CXCL9; Female; Flow Cytometry; Humans; Interleukin-8; Male; Middle Aged; Patient Selection; Prognosis; Remission Induction; Severity of Illness Index; Surveys and Questionnaires; Treatment Outcome

Nuclear factor-kappaB (NF-kappaB) is involved in the pathophysiology of rheumatoid arthritis (RA) and is considered to be a feasible molecular target in treating patients. In the RA joint tissues, activation of NF-kappaB is often observed together with high amounts of the proinflammatory cytokines tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta. TNFalpha and IL-1beta are known to stimulate NF-kappaB signaling and are produced as the effect of NF-kappaB signaling, thus forming a vicious cycle leading to a self-perpetuating nature of rheumatoid inflammation and expansion of such inflammatory response to other joints. Because a kinase called IkappaB kinase complex (IKK) is involved in the NF-kappaB activation cascade, we examined the effect of a novel IKK inhibitor, (7-[2-(cyclopropyl-methoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride; CHPD), on the production of inflammatory cytokines from rheumatoid synovial fibroblasts (RSF). TNFalpha stimulation induced production of inflammatory cytokines such as IL-6 and IL-8 in RSF, and the extent of IL-6 and IL-8 induction was dramatically reduced by CHPD under noncytotoxic concentrations. Likewise, expression of il-6 and il-8 genes was significantly reduced by CHPD. In addition, chromatin immunoprecipitation assays revealed that the DNA binding of NF-kappaB (p65) to il-8 promoter in RSF was induced after TNFalpha stimulation and that, upon CHPD treatment to RSF for 1 h, the NF-kappaB binding to il-8 promoter was significantly decreased. Here, we have demonstrated that an IKKbeta inhibitor, CHPD, acts as an effective inhibitor for the production of inflammatory cytokines in response to proinflammatory cytokines. These findings indicate that such a IKKbeta inhibitor could be a feasible candidate for an antirheumatic drug.

Topics: Adult; Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Female; Fibroblasts; Humans; I-kappa B Kinase; Interleukin-6; Interleukin-8; Male; Middle Aged; NF-kappa B; Oxazines; Phosphorylation; Pyridines; RNA, Messenger; Signal Transduction; Synovial Membrane

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H2S. Therefore, this topic encouraged us to investigate the influence of H2S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast-like synoviocytes) were treated with different concentrations of an exogenous H2S donor (NaHS). At defined time points, secretion of IL-6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen-activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL-6 and IL-8. Data provided prove that, in FLSs, constitutive as well as IL-1beta-induced expression of IL-6 is transiently and partially down-regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H2S deactivates p44/42 MAPK (ERK1/2). Long-term exposure of FLSs to H2S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL-6 expression. Presented data seem of importance for studying (patho-) physiological functions of H2S and also for re-evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.

Topics: Air Pollutants; Arthritis, Rheumatoid; Cells, Cultured; Enzyme Activation; Fibroblasts; Humans; Hydrogen Sulfide; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Synovial Membrane; Transcription Factor RelA

In the present study, we examined the effect of the pro-inflammatory cytokine IL-32gamma, the most biologically active isoform, and its related molecules in fibroblast-like synoviocytes (FLS).. FLS were isolated from synovial tissues of rheumatoid arthritis (RA) patients. The secretion and expression of IL-6 and IL-8 were examined by ELISA and real-time PCR, and the activation of signaling molecules was evaluated by Western blot, electrophoretic mobility shift assay (EMSA), real-time PCR, and siRNA transfection.. By IL-32gamma stimulation in RA FLS, the expressions of IL-6 and IL-8 were increased significantly, and the phosphorylated Erk1/2 and AP-1 were expressed prominently in Western blot and EMSA. In the Erk1/2 inhibited cells, IL-32gamma stimulation did not increase the mRNA expression of IL-6 and IL-8.. Our results suggest that IL-32gamma stimulation can induce the production of IL-6 and IL-8 from RA FLS via Erk1/2 activation.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Humans; Interleukin-33; Interleukin-6; Interleukin-8; Interleukins; Mitogen-Activated Protein Kinase 3; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Synovial Membrane; Transcription Factor AP-1

Acute phase apoprotein Serum Amyloid A (A-SAA), which is strongly expressed in rheumatoid arthritis synovial membrane (RA SM), induces angiogenesis, adhesion molecule expression, and matrix metalloproteinase production through the G-coupled receptor FPRL-1. Here we report alternative signaling through the high-density lipoprotein receptor scavenger receptor-class B type 1 (SR-B1). Quantitative expression/localization of SR-B1 in RA SM, RA fibroblast-like cells (FLCs), and microvascular endothelial cells (ECs) was assessed by Western blotting and immunohistology/fluorescence. A-SAA-mediated effects were examined using a specific antibody against SR-B1 or amphipathic alpha-Helical Peptides (the SR-B1 antagonists L-37pA and D-37pA), in RA FLCs and ECs. Adhesion molecule expression and cytokine production were quantified using flow cytometry and ELISA. SR-B1 was strongly expressed in the RA SM lining layer and endothelial/perivascular regions compared with osteoarthritis SM or normal control synovium. Differential SR-B1 expression in RA FLC lines (n = 5) and ECs correlated closely with A-SAA, but not tumor necrosis factor alpha-induced intercellular adhesion molecule-1 upregulation. A-SAA-induced interleukin-6 and -8 production was inhibited in the presence of anti-SR-B1 in human microvascular endothelial cells and RA FLCs. Moreover, D-37pA and L-37pA inhibited A-SAA-induced vascular cell adhesion molecule-1 and intercellular adhesion molecule expression from ECs in a dose-dependent manner. As SR-B1 is expressed in RA synovial tissue and mediates A-SAA-induced pro-inflammatory pathways, a better understanding of A-SAA-mediated inflammatory pathways may lead to novel treatment strategies for RA.

Topics: Arthritis, Rheumatoid; Arthroscopy; Biopsy; CD36 Antigens; Dose-Response Relationship, Drug; Endothelium, Vascular; Gene Expression Regulation; Humans; Inflammation; Interleukin-6; Interleukin-8; Peptides; Phenotype; Serum Amyloid A Protein; Synovial Membrane

Angiogenesis, which is a critical step in the initiation and progression of rheumatoid arthritis (RA), involves pro-angiogenic factors, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). We investigated the role of Toll-like receptor 3 (TLR3) in the regulation of pro-angiogenic factors in RA fibroblast-like synoviocytes (FLS).. FLS were isolated from RA synovial tissues and stimulated with the TLR3 ligand, poly (I:C). The levels of VEGF and IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assays, and the mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction. The expression patterns of VEGF and IL-8 in the RA synovium and osteoarthritis (OA) synovium were compared using immunohistochemistry.. The expression levels of TLR3, VEGF, and IL-8 were significantly higher in the RA synovium than in the OA synovium. VEGF and IL-8 production were increased in the culture supernatants of RA FLS stimulated with poly (I:C), and the genes for these proteins were up-regulated at the transcriptional level after poly (I:C) treatment. Treatment with inhibitors of nuclear factor-kappaB (NF-κB), i.e., pyrrolidine dithiocarbamate and parthenolide, abrogated the stimulatory effect of poly (I:C) on the production of VEGF and IL-8 in RA FLS.. Our results suggest that the activation of TLR3 in RA FLS promotes the production of proangiogenic factors, in a process that is mediated by the NF-κB signaling pathway. Therefore, targeting the TLR3 pathway may be a promising approach to preventing pathologic angiogenesis in RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Humans; Interleukin-8; Neovascularization, Pathologic; NF-kappa B; RNA, Messenger; Synovial Membrane; Toll-Like Receptor 3; Vascular Endothelial Growth Factor A

The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.

Topics: Adiponectin; Arthritis, Rheumatoid; Cells, Cultured; Chemotaxis; Fibroblasts; Humans; Interleukin-8; Neutrophils; Protein Kinase Inhibitors; Receptors, Adiponectin; Signal Transduction; Synovial Fluid

Although recent clinical studies have shown that laser therapy acts as an anti-inflammatory effector in the treatment of some diseases, little is known about the mechanism by which it acts in rheumatoid arthritis (RA) patients. The purpose of our work was to examine how irradiation with linear polarized infrared light (LPIL) suppresses inflammatory responses in the MH7A rheumatoid fibroblast-like synoviocyte cell line. We initially confirmed the effects of two disease-modifying anti-rheumatic treatments, LPIL irradiation and dexamethasone (Dex) administration, under experimental inflammatory conditions using gene chip technology. We found that LPIL exerted a smaller effect on gene transcription than Dex; however, IL-1beta-inducible target genes such as the CXCL type chemokines IL-8, IL-1beta and IL-6 were all clearly suppressed by LPIL to the same degree as by Dex. We also found that IL-1beta-induced release of IL-8 from MH7A cells was completely blocked by pretreatment with the (IL-8) inhibitor Bay11-7085, indicating that activation of NF-kappaB signaling plays an important role in the secretion of IL-8. Although the levels of NFKB1 and RELA transcription were unaffected by IL-1beta stimulation, phosphorylation of RelA S276 was suppressed by both LPIL and Dex. Thus LPIL likely exerts its anti-inflammatory effects by inhibiting the release of the inflammatory chemokine IL-8. A fuller understanding of the anti-inflammatory mechanism of LPIL in rheumatoid synoviocytes could serve as the basis for improved treatment of RA patients in the future.

Topics: Arthritis, Rheumatoid; Cell Line; Dexamethasone; Gene Expression Regulation; Humans; Inflammation; Infrared Rays; Interleukin-1beta; Interleukin-8; Laser Therapy; Phosphorylation; Synovial Membrane; Transcription Factor RelA; Transcription, Genetic

Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA.. Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-kappaB was measured by electrophoretic mobility shift assay.. NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-kappaB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13.. Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Gene Expression; Gene Silencing; Humans; Interleukin-6; Interleukin-8; Lipopolysaccharides; Matrix Metalloproteinases; NF-kappa B; Nod2 Signaling Adaptor Protein; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Synovial Membrane; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Up-Regulation

Angiogenesis in synovia is a characteristic of RA patients. We examined whether IL-6 or TNF-alpha induce tubule formation in a co-culture system of fibroblast-like synovial cells from RA patients (RA-FLS) and human umbilical vein endothelial cells (HUVEC). The effects of IL-6 and TNF-alpha on the expression of angiogenic factors in RA-FLS and HUVEC, and the proliferation of HUVEC were also studied. IL-6 + sIL-6R induced tubule formation, whereas IL-6 alone did not. IL-6/sIL-6R-induced tubule formation was completely suppressed by the addition of either anti-IL-6R or anti-VEGF antibody. TNF-alpha did not induce tubule formation. On the contrary, it decreased CD31-positive area compared with the control. IL-6 + sIL-6R augmented VEGF production in RA-FLS, whereas IL-6 alone did not. Anti-IL-6R antibody suppressed IL-6/sIL-6R-induced VEGF production, but not spontaneous VEGF production. In contrast, TNF-alpha did not induce VEGF production from RA-FLS and HUVEC. IL-6 + sIL-6R stimulation of RA-FLS strongly induced mRNA expression of VEGF, but not of other angiogenic factors, such as EGF, bFGF, TGF-beta, IL-1, TNF-alpha and IL-8. Neither IL-6 nor IL-6/sIL-6R promoted HUVEC proliferation, whereas TNF-alpha significantly inhibited VEGF-induced HUVEC proliferation. In conclusion, IL-6/sIL-6R complex showed angiogenic activity via the production of VEGF from RA-FLS, but TNF-alpha was anti-angiogenic in our experimental system.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Coculture Techniques; Endothelium, Vascular; Fibroblast Growth Factors; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Neovascularization, Physiologic; Receptors, Interleukin-6; RNA, Messenger; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Endothelial Growth Factor A

In the present study, we investigated the ability of microparticles isolated from synovial fluids from patients with rheumatoid arthritis or osteoarthritis to induce the synthesis and release of key cytokines of B-lymphocyte modulation such as B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor by rheumatoid fibroblast-like synoviocytes.. Microparticles were analyzed in synovial fluids from patients with rheumatoid arthritis, osteoarthritis, microcristalline arthritis, and reactive arthritis. In addition, microparticle release after activation from various cell lines (CEM lymphocyte and THP-1 cells) was assessed. Microparticles were isolated by differential centrifugation, and quantitative determinations were carried out by prothrombinase assay after capture on immobilized annexin V. B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release was evaluated by enzyme-linked immunosorbent assay.. Microparticles isolated from synovial fluids obtained from rheumatoid arthritis and osteoarthritis patients or microparticles derived from activated THP-1 cells were able to induce B cell-activating factor, thymic stroma lymphopoietin, and secretory leukocyte protease inhibitor release by rheumatoid arthritis fibroblast-like synoviocytes. Conversely, CEM-lymphocytes-derived microparticles generated by treatment with a combination of PHA, PMA and Adt-D did not promote the release of B cell-activating factor but favored the secretion of thymic stroma lymphopoietin and secretory leukocyte protease inhibitor by rheumatoid arthritis fibrobast-like synoviocytes. However, microparticles isolated from actinomycin D-treated CEM lymphocytes were not able to induce B cell-activating factor, thymic stroma lymphopoietin, or secretory leukocyte protease inhibitor release, indicating that microparticles derived from apoptotic T cells do not function as effectors in B-cell activation.. These results demonstrate that microparticles are signalling structures that may act as specific conveyors in the triggered induction and amplification of autoimmunity. This study also indicates that microparticles have differential effects in the crosstalk between B lymphocytes and target cells of autoimmunity regarding the parental cells from which they derive.

Topics: Aged; Aged, 80 and over; Arthritis, Rheumatoid; B-Cell Activating Factor; B-Lymphocytes; Cell-Derived Microparticles; Cytokines; Female; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Lymphocyte Activation; Male; Middle Aged; Receptor Cross-Talk; Secretory Leukocyte Peptidase Inhibitor; Synovial Fluid; Synovial Membrane; Thymic Stromal Lymphopoietin

LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTbetaR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells.. Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12- Myristat-13-Acetat)+ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of approximately 60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-g pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93+/-9.41 vs. 129.53+/-49.14 and 172.13+/-77.64; p<0.0005).. These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma levels of soluble LIGHT in patients with chronic infections suggest a role of LIGHT in systemic inflammatory activation.

Topics: Adolescent; Adult; Aged; Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Drug Combinations; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Hepatitis C, Chronic; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Ionomycin; Leukocytes, Mononuclear; Macrophages; Middle Aged; Molecular Weight; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thromboplastin; Tumor Necrosis Factor Ligand Superfamily Member 14; Umbilical Veins; Up-Regulation; Young Adult

Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the alpha7 subunit of the nicotinic acetylcholine receptor (alpha7nAChR) and its function in rheumatoid arthritis (RA).. The potential role of the alpha7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An alpha7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate-labeled alpha-bungarotoxin, which binds specifically to the alpha7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the alpha7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dupalpha7, a variant alpha7 transcript. Next, we studied the functional role of the alpha7nAChR in RA FLS by examining the effects of alpha7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS.. A screen using an Ad.shRNA library against 807 transcripts revealed that a specific alpha7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The alpha7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found alpha7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dupalpha7 mRNA. Specific alpha7nAChR agonists reduced tumor necrosis factor alpha-induced IL-6 and IL-8 production by FLS.. The alpha7nAChR and its dupalpha7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the alpha7nAChR could provide a novel antiinflammatory approach to the treatment of RA.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Neurotransmitter Agents; Polymerase Chain Reaction; Receptors, Nicotinic; RNA, Messenger; Synovial Membrane

To ascertain the effect of CyPA and IL-8 in chemotaxis of neutrophil and the level of IL-8 from the CyPA effecting of peripheral blood from RA patients.. 12 RA patients matched 4 healthy people were studied. Chemotaxis of IL-8 was measured by Boyden chamber on neutrophil of RA patients and that of 4 normal healthy people controls were studied; the level of IL-8 on neutrophil of RA patients peripheral blood after the effecting of CyPA was assessed by ELISA. Correlations between CyPA and IL-8 were observed in RA.. The chemotaxis of neutrophil which IL-8 mixed with CyPA antibody was lower than IL-8(P<0.05); The secretion of RA peripheral blood in IL-8 was higher than normal people, as well as the secretion of RA peripheral blood in IL-8 after effecting of CyPA was higher than before (P<0.05), but the level of IL-8 after blocking CyPA was not changed.. CyPA could affect the chemotaxis of IL-8 in the neutrophil of RA patients' peripheral blood. The secretion of IL-8 is accelerated by CyPA on neutrophil of RA patients'peripheral blood.

Topics: Adult; Aged; Antibodies, Monoclonal; Arthritis, Rheumatoid; Chemotaxis; Culture Media, Conditioned; Cyclophilin A; Enzyme-Linked Immunosorbent Assay; Female; HL-60 Cells; Humans; Interleukin-8; Male; Middle Aged; Neutrophils

High expression of galectin 3 at sites of joint destruction in rheumatoid arthritis (RA) suggests that galectin 3 plays a role in RA pathogenesis. Previous studies have demonstrated the effects of galectins on immune cells, such as lymphocytes and macrophages. This study was undertaken to investigate the hypothesis that galectin 3 induces proinflammatory effects in RA by modulating the pattern of cytokine and chemokine production in synovial fibroblasts.. Matched samples of RA synovial and skin fibroblasts were pretreated with galectin 3 or tumor necrosis factor alpha (TNFalpha), and the levels of a panel of cytokines, chemokines, and matrix metalloproteinases (MMPs) were determined using enzyme-linked immunosorbent assays and multiplex assays. Specific inhibitors were used to dissect signaling pathways, which were confirmed by Western blotting and NF-kappaB activation assay.. Galectin 3 induced secretion of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor, CXCL8, and MMP-3 in both synovial and skin fibroblasts. By contrast, galectin 3-induced secretion of TNFalpha, CCL2, CCL3, and CCL5 was significantly greater in synovial fibroblasts than in skin fibroblasts. TNFalpha blockade ruled out autocrine TNFalpha-stimulated induction of chemokines. The MAPKs p38, JNK, and ERK were necessary for IL-6 production, but phosphatidylinositol 3-kinase (PI 3-kinase) was required for selective CCL5 induction. NF-kappaB activation was required for production of both IL-6 and CCL5.. Our findings indicate that galectin 3 promotes proinflammatory cytokine secretion by tissue fibroblasts. However, galectin 3 induces the production of mononuclear cell-recruiting chemokines uniquely from synovial fibroblasts, but not matched skin fibroblasts, via a PI 3-kinase signaling pathway. These data provide further evidence of the role of synovial fibroblasts in regulating the pattern and persistence of the inflammatory infiltrate in RA and suggest a new and important functional consequence of the observed high expression of galectin 3 in the rheumatoid synovium.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Chemokine CCL3; Chemokine CCL5; Chemokines; Cytokines; Fibroblasts; Galectin 3; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Phosphatidylinositol 3-Kinases; Signal Transduction; Skin; Synovial Membrane; Tumor Necrosis Factor-alpha

Cytokines and chemokines are key regulatory molecules involved in rheumatoid arthritis (RA). B-cell depletion therapy improves RA clinically but its mechanism is not completely understood. One possible mechanism for this therapy is the modification of the proinflammatory cytokine homeostasis of RA. We show here that the levels of the proinflammatory chemokine IL-8 in serum samples from RA patients unexpectedly increased by up to 100-fold 8 weeks after the administration of rituximab, despite clinical improvement. We also show that RA patients produced anti-IL-8 autoantibodies and that their levels dropped after RTX treatment. Moreover, we identified antibody-IL-8 immune complexes in the synovial fluid and serum of RA patients, and found that the amount of these complexes decreased after the administration of RTX. Our results indicate that B-cell depletion therapy modifies the cytokine-autoantibody network by reducing the levels of anti-cytokine autoantibodies and, consequentially, the formation of antibody-cytokine immune complexes.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigen-Antibody Complex; Antirheumatic Agents; Arthritis, Rheumatoid; Autoantibodies; B-Lymphocytes; Cytokines; Female; Humans; Infliximab; Interleukin-8; Lymphocyte Depletion; Male; Middle Aged; Rituximab; Synovial Fluid

Patients with rheumatoid arthritis often conduct bathing in hot mineral water with a high concentrations of sulfate compounds in the water and ambient air. We investigated the effect of hyperthermia and sulfur as possible stress factors at transcriptional level in several proinflammatory genes in fibroblast like synoviocytes. We mimicked the classical balneological treatment. Cells were exposed to 30 minutes of hyperthermia (41-42 degrees C) or sulfur (2 mM NaHS). Indeed, both factors were acting as stressors, inducing a profound expression of heat shock protein 70 (HSP70). Stimulation of the cells with IL1beta induced a series of proinflammatory genes (IL1alpha, IL1beta, TNFalpha, IL8, monocyte chemoattractant peptide-1 and COX-2), but if the cells were treated with hyperthermia prior to IL1beta expression, gene expressions were significantly decreased up to 8 h. Treatment with sulfur alone induced expression of observed genes up to 12 h. We may conclude that hyperthermia as a balneological mean has indeed a protective effect on cells, but sulfur, which at first we considered as an antiinflammatory mean, had actually an opposite effect and induced expression of proinflammatory genes. Our data confirmed that the effect of hyperthermia as balneological mean treatment is beneficial, but sulfur treatment must be taken in reconsideration.

Topics: Arthritis, Rheumatoid; Balneology; Baths; Chemokine CCL2; Cyclooxygenase 2; Fever; Fibroblasts; Humans; Inflammation; Interleukin-1alpha; Interleukin-1beta; Interleukin-8; Sulfur; Synovial Membrane; Tumor Necrosis Factor-alpha

Environmental factors are involved in RA pathogenesis and epidemiological studies have suggested that smoking is an environmental risk factor for RA. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the major toxic components in cigarettes. To clarify the biological effects of smoking in RA, we investigated the role of TCDD in RA pathogenesis.. Human synovial tissue was obtained from RA and OA patients and aryl hydrocarbon receptor (AhR) expression in these tissues was evaluated using immunohistochemistry and real-time PCR. Expression of various cytokines was measured by real-time PCR following stimulation of RA synoviocytes with different concentrations of TCDD. To study the role of AhR, we treated RA synoviocytes with alpha-naphthoflavone, a known AhR antagonist. To evaluate which signal transduction pathways were stimulated by the TCDD-AhR interaction, we used inhibitors of nuclear factor-kappaB (NF-kappaB) and extra-cellular stimulus-activated kinase (ERK).. Higher AhR mRNA and protein levels were observed in RA synovial tissue than in OA tissue. TCDD up-regulated the expression of IL-1beta, IL-6 and IL-8 through binding to AhR, and this effect was transmitted via the NF-kappaB and ERK signalling cascades. AhR expression in synovial cells was up-regulated by TNF-alpha.. TNF-alpha activates AhR expression in RA synovial tissue, and that cigarette smoking and exposure to TCDD enhances RA inflammatory processes. TCDD induces inflammatory cytokines via its association with AhR, resulting in stimulation of the NF-kappaB and ERK signalling cascades. Thus TCDD exposure, such as smoking exacerbates RA pathophysiology.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dose-Response Relationship, Drug; Humans; Interleukin-1beta; Interleukin-6; Interleukin-8; NF-kappa B; Osteoarthritis, Knee; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Synovial Membrane; Tumor Necrosis Factor-alpha; Up-Regulation

To investigate the effect of poly(ADP-ribose) polymerase (PARP) inhibition on the production of inflammatory mediators and proliferation in tumour necrosis factor (TNF)-stimulated fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).. Cultured FLS from patients with RA were treated with two PARP inhibitors, 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinona (DPQ) or 4-amino-1,8-naphthalimida (ANI) before TNF stimulation. PARP-1 expression was also suppressed in RA FLS by small interfering RNA (siRNA) transfection. Expression and secretion of inflammatory mediators were analysed by quantitative polymerase chain reaction and by enzyme-linked immunosorbent assay, respectively. Proliferation of RA FLS was also determined. Mitogen-activated protein kinase (MAPK) activity was analysed by western blot assay and activator protein (AP)-1 and nuclear factor (NF)kappaB binding by electrophoretic mobility shift assay.. We show, for the first time, that PARP inhibition either with specific inhibitors or by siRNA transfection significantly reduced TNF-induced cytokine and chemokine expression in FLS from patients with RA. PARP inhibitors also decreased TNF-induced RA FLS proliferation. PARP inhibition reduced TNF-induced JNK phosphorylation and AP-1 and NF kappaB binding activities were partially impaired by treatment with PARP inhibitors or by PARP-1 knockdown.. PARP inhibition reduces the production of inflammatory mediators and the proliferation of RA FLS (in response to TNF), suggesting that PARP inhibitors could have therapeutic benefits in RA.

Topics: 1-Naphthylamine; Apoptosis; Arthritis, Rheumatoid; Blotting, Western; Cell Proliferation; Cells, Cultured; Depression, Chemical; Electrophoretic Mobility Shift Assay; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Isoquinolines; Mitogen-Activated Protein Kinases; Naphthalimides; NF-kappa B; Piperidines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Quinolones; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Synovial Membrane; Transcription Factor AP-1; Tumor Necrosis Factors

Surgery and trauma is accompanied by changes in blood levels of certain cytokines and chemokines including interleukin-6 (IL-6) and interleukin-8 (IL-8). However, there is little data on correlations between local and systemic levels of these mediators during orthopedic surgeries in rheumatoid arthritis (RA) patients who already show increased levels of proinflammatory cytokines due to their disease. We aimed to measure dynamics of blood and drainage fluid levels of IL-6 and IL-8 in postoperative period in rheumatoid arthritis patients undergoing knee replacement surgery and correlate these changes with blood levels of C-reactive protein (CRP), body temperature and pain. We report that blood and drainage fluid levels of IL-6 and IL-8 showed significantly increasing trend during the 36-h period after the surgery. Drainage fluid levels of both cytokines were significantly higher in comparison with blood, indicating their local production in the operated joint. In contrast, levels of CRP were higher in blood than in drainage fluid. Despite the fact that the levels of tested cytokines had already been high in RA patients before surgery, we conclude that after surgery their levels were being much significantly enough high in drainage fluid to reflect dominated local inflammatory reaction to surgical stress and trauma.

Topics: Aged; Arthritis, Rheumatoid; Arthroplasty, Replacement, Knee; C-Reactive Protein; Cytokines; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Male; Middle Aged

Cytokines are the major mediators of joint damage in chronic arthritis. Data on synovial fluid (SF) concentration of Th17 cell-derived cytokine interleukin 17 (IL-17) in patients with juvenile idiopathic arthritis (JIA) are sparse. We measured levels of IL-17 in SF specimens from children with enthesitis-related arthritis (ERA) and polyarticular JIA (poly-JIA), and studied the ability of IL-17 to produce matrix metalloproteinases (MMP) and cytokines by fibroblast-like synoviocytes (FLS) from patients with ERA.. IL-17 levels were measured in SF of patients with ERA (n = 43), poly-JIA (n = 17), rheumatoid arthritis (RA; n = 35), and osteoarthritis (OA; n = 10) by ELISA. In patients with JIA, 10 paired serum samples were also assayed. FLS were cultured from SF of patients with ERA and subsequently stimulated for 48 h by IL-17 or tumor necrosis factor-alpha. Later the production of IL-6, IL-8, MMP-1, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1 was measured in the culture supernatants by ELISA.. Median IL-17 levels in SF were higher in patients with JIA [28 pg/ml (range 0-200)] compared to OA [0 pg/ml (range 0-84); p < 0.001] and RA (p < 0.05). The levels were comparable between poly-JIA patients and the ERA group. The median SF IL-17 levels were significantly higher compared to serum levels in children with JIA (p < 0.005). In ERA, SF IL-17 correlated with number of swollen joints (r = 0.35; p < 0.05), number of joints with limited mobility (r = 0.55; p < 0.001), and number of tender joints (r = 0.46; p < 0.01); however, no correlation was seen with erythrocyte sedimentation rate. IL-17 induced FLS to produce IL-6, IL-8, MMP-3, and MMP-1. However, there was no effect on the production of TIMP.. Increased IL-17 levels in ERA SF correlate with disease activity and this may be due to increased production of MMP and cytokines by IL-17.

Topics: Adolescent; Adult; Arthritis, Juvenile; Arthritis, Rheumatoid; Case-Control Studies; Cells, Cultured; Child; Child, Preschool; Cytokines; Female; Fibroblasts; Humans; Interleukin-17; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Matrix Metalloproteinases; Osteoarthritis; Synovial Fluid

We previously reported that 10 mg/day of simvastatin significantly reduced clinical scores of rheumatoid arthritis (RA) in patients with active RA with hypercholesterolemia. We have also reported that a certain pharmacological concentration of simvastatin, i.e., 0.05-0.1 microM, inhibits the production of interleukin 6 (IL-6) and IL-8 and the cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) derived from patients with RA in vitro. We investigated other effects of simvastatin on FLS from the standpoint of cell viability and apoptosis.. RA FLS were cultured with or without 0.05-50 microM simvastatin for 48 h. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured by flow cytometric analysis using propidium iodide and annexin-V. Caspase-3 and -9 activities were analyzed by colorimetric assays.. High concentrations of simvastatin, i.e., 1.0-50 microM, reduced cell viability and induced prominent apoptosis in FLS in a dose-dependent manner. The apoptosis induced by simvastatin was caspase-3- and caspase-9-dependent. These effects were completely reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not in the presence of farnesyl-pyrophosphate. Further, a geranylgeranyl transferase inhibitor and a RhoA kinase inhibitor mimicked the effect of simvastatin.. These data, together with our previous report, suggest that low (pharmacological range) and high concentrations of simvastatin affect FLS differently: (1) at a low concentration, it inhibits IL-6 and IL-8 production and the cell proliferation of FLS induced by TNF-alpha (2) at high concentrations, it induces apoptosis in FLS. Understanding this dose-dependent biphasic effect of simvastatin may prove important for its clinical applications in the treatment of RA.

Topics: Apoptosis; Arthritis, Rheumatoid; Caspase 3; Caspase 9; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblasts; Flow Cytometry; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Interleukin-8; Simvastatin; Synovial Membrane

Pain sensitization and the related secretion of neuropeptides from sensory nerve terminals are proinflammatory in osteoarthritis (OA), rheumatoid arthritis (RA), and adjuvant-induced polyarthritis. In contrast, endogenous opioids such as the recently discovered endomorphins (EMs) are antiinflammatory. However, the role of endogenous EMs such as EM-1 and EM-2 has never been investigated in OA and RA.. We established a highly sensitive radioimmunoassay to detect EM-1 and EM-2. In patients with RA and patients with OA, immunohistochemistry for EM-1 and EM-2 was performed, and double-staining was used to identify EM-positive cells. The effects of EM-1 and EM-2 on the secretion of interleukin-6 (IL-6) and IL-8 from human synovial tissue were studied by tissue superfusion, and the therapeutic effects of EM-1 were tested in a rat model of adjuvant-induced polyarthritis.. EM-positive cells were located in the sublining area and vessel walls but were particularly evident in the highly inflamed lining area. Human macrophages, T cells, and fibroblasts stained positive for EMs. The synovial density of EM-positive cells was higher in patients with OA than in those with RA. EM-1 inhibited synovial secretion of IL-6 in patients with RA and secretion of IL-8 in patients with RA and those with OA (maximum 10(-10)M). EM-2 inhibited IL-8 secretion only from RA tissue (maximum 10(-10)M). In rats with adjuvant-induced polyarthritis, thymus, spleen, and synovial tissue contained significantly more EM-1 than was observed in controls. Rats with adjuvant-induced polyarthritis benefited from EM-1 treatment.. EM-1 had antiinflammatory effects in patients with OA or RA and in a model of adjuvant-induced polyarthritis. Local enhancement of EM-1 might be an interesting therapeutic option in different forms of arthritis.

Topics: Aged; Arthritis; Arthritis, Experimental; Arthritis, Rheumatoid; Female; Humans; Immunohistochemistry; Interleukin-6; Interleukin-8; Male; Middle Aged; Oligopeptides; Osteoarthritis; Synovial Membrane

Fibroblast-like synoviocytes (FLS) are potentially directly involved in the propagation of inflammation. We have previously shown evidence of an expanded activated population of natural killer (NK) cells in spondylarthritis (SpA) patients. In the present study, we sought to determine whether the interaction between NK cells and FLS from SpA patients results in a proinflammatory response.. Autologous NK cells and FLS were obtained from 6 patients with SpA, 4 patients with rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to study the activation, proliferation, and survival of NK cells in contact with FLS. Cytokine and stromal factor production were measured by a multiple cytokine bead assay.. NK cells both adhered to and migrated beneath the FLS monolayer (pseudoemperipolesis). FLS from SpA and RA patients supported increased pseudoemperipolesis, activation, cytokine production, and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8, IL-1beta, and IL-15, was increased in cocultures of NK cells and FLS, particularly in those from RA and SpA patients. Production of interferon-gamma, RANTES, and matrix metalloproteinase 3 (MMP-3) by NK cell and FLS coculture was greatest in SpA patients. Surface expression of IL-15 on FLS was significantly increased in SpA and RA patients, but not OA patients. Blockade with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells.. FLS promote the migration, activation, and survival of NK cells. The interaction of NK cells with FLS results in increased IL-15 expression by FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to joint inflammation. This response was much more marked in SpA and RA patients as compared with OA patients.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Communication; Cells, Cultured; Chemokine CCL2; Chemokine CCL5; Cytokines; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-15; Interleukin-1beta; Interleukin-6; Interleukin-8; Killer Cells, Natural; Male; Matrix Metalloproteinase 3; Middle Aged; Osteoarthritis, Knee; Spondylarthritis; Synovial Membrane

Rheumatoid arthritis (RA) is a chronic, symmetric polyarticular joint disease and serum amyloid A (SAA) is an acute-phase protein that is upregulated during the course of RA. We investigated the role of SAA in the pathogenesis of RA.. Fibroblast-like synovial cells (FLS) were established from RA joints. SAA-stimulated expression of cytokines from FLS was evaluated by ELISA. Nuclear factor-kappaB (NF-kappaB) activation by SAA was evaluated by luciferase assay. NF-kappaB activation and IkappaBalpha degradation were evaluated by Western blotting and nuclear localization of p65 subunit of NF-kappaB in FLS. Expression of receptor for advanced glycation end-products (RAGE) in synovial tissue was evaluated by immunohistochemical study. Effects of preincubation of soluble RAGE on NF-kappaB activation by SAA was evaluated by Western blotting of IkappaBalpha.. SAA stimulated the transcriptional activation by NF-kappaB in a dose-dependent manner and induced expression of the proinflammatory cytokines interleukin 6 (IL-6) and IL-8. Higher expression of RAGE in synovial tissue from patients with RA was noted. SAA induced IkappaBalpha degradation, with the peak effect around 30 minutes. Preincubation of SAA with soluble recombinant RAGE protein prevented SAA-induced IkappaBalpha degradation. SAA stimulation promoted nuclear translocation of NF-kappaB, whereas preincubation of SAA with RAGE inhibited nuclear translocation.. Our data suggested that the SAA-RAGE-stimulated NF-kappaB signaling pathway has an important role in the pathogenesis of RA.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblasts; Humans; Interleukin-6; Interleukin-8; NF-kappa B; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Serum Amyloid A Protein; Signal Transduction; Synovial Membrane

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a master regulator in the cellular response to hypoxic conditions, and rheumatoid synovial tissue is known to exist under hypoxic conditions. Therefore, this study was conducted to determine the contribution of HIF-1alpha to hypoxia-induced MMP and cytokine production in fibroblast-like synoviocytes (FLS).. RA FLS were transfected with either a plasmid that expresses HIF-1alpha or an empty vector as a control, and then cultured under normoxia (21% O(2)). Also, FLS were transfected with either HIF-1alpha small interfering RNA (siRNA) or control siRNA, and cultured under hypoxic conditions (1% O(2)). Following transfection, the amounts of MMP and cytokine mRNAs and HIF-1alpha protein were examined using real-time RT-PCR and western blotting, respectively.. The expression of HIF-1alpha, MMP-1, MMP-3, IL-6 and IL-8 was markedly enhanced in FLS that were cultured under hypoxia. We confirmed that transient transfection of HIF-1alpha overexpressing vector or siRNA had occurred using western blotting, and in vitro studies conducted using FLS transfected with HIF-1alpha overexpression vector showed that they had significantly increased MMP-1, MMP-3 and IL-8 expression levels. Further, hypoxia-induced MMP-3 expression was significantly attenuated by knock-down of HIF-1alpha, whereas hypoxia-induced IL-8 or MMP-1 expression was not significantly repressed by HIF-1alpha siRNA.. Hypoxia-induced MMP-3 expression is exclusively regulated by HIF-1alpha, and hypoxia-induced MMP-1 or IL-8 expression appears to have salvage pathways other than the HIF-1alpha pathway. Together, these data provide new insight regarding the mechanism by which hypoxia participates in joint inflammation and destruction in RA.

Topics: Arthritis, Rheumatoid; Cell Hypoxia; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoenzyme Techniques; Interleukin-8; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Membrane

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-8 production caused by leptin in both rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). RASF and OASF expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-8 production. Leptin-mediated IL-8 production was attenuated by OBRl receptor antisense oligonucleotide, JAK2 inhibitor or STAT3 small interference RNA (siRNA). Transfection with insulin receptor substrate (IRS)-1 siRNA or dominant-negative mutant of p85 and Akt or pretreatment with phosphatidylinositol 3-kinase inhibitor (Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (PDTC) and NF-kappaB inhibitor peptide also inhibited the potentiating action of leptin. Stimulation of RASF with leptin activated IkappaB kinase alpha/beta (IKK alpha/beta), p65 phosphorylation at Ser(276), p65 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked IL-8 expression. The binding of p65 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 acetylation on the IL-8 promoter was enhanced by leptin, which was inhibited by wortmannin, Akt inhibitor or IRS-1 siRNA. These results suggest that leptin increased IL-8 production in synovial fibroblast via the OBRl/JAK2/STAT3 pathway, as well as the activation of IRS1/PI3K/Akt/NF-kappaB-dependent pathway and the subsequent recruitment of p300.

Topics: Adaptor Proteins, Signal Transducing; Arthritis, Rheumatoid; Binding Sites; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Histones; Humans; Insulin Receptor Substrate Proteins; Interleukin-8; Janus Kinase 2; Leptin; NF-kappa B; Osteoarthritis; p300-CBP Transcription Factors; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Receptors, Leptin; Signal Transduction; STAT3 Transcription Factor; Synovial Membrane

Steroid hormone receptors such as glucocorticoid receptors, androgen receptors, and oestrogen receptors alpha (ERalpha) and beta (ERbeta) have been identified in synovial cells of patients with rheumatoid arthritis and osteoarthritis.. To find a quantitative relationship between the number of receptor positive cells and markers of inflammation, and to compare the two groups of patients with rheumatoid arthritis and osteoarthritis.. A total of 36 patients with rheumatoid arthritis (n = 17) and osteoarthritis (n = 19) were included, and receptor positive cells and cellular markers of synovial inflammation were quantified by immunohistochemistry and ELISA (interleukin 6 (IL6) and IL8).. Patients with rheumatoid arthritis showed a higher degree of histologically determined inflammation compared with those with osteoarthritis. However, synovial density of gluco-corticoid receptor positive (GR+), androgen receptor positive (AR+), ERalpha+ and ERbeta+ cells were not different among patients with rheumatoid arthritis and osteoarthritis. In patients with osteoarthritis, the density of GR+ cells positively correlated with the density of AR+, ERalpha+ and ERbeta+ cells (p = 0.007), which was not observed in patients with rheumatoid arthritis. This indicates positively coupled steroid hormone receptor expression in patients with osteoarthritis but not in those with rheumatoid arthritis. In patients with rheumatoid arthritis, secretion of synovial IL6 and IL8 positively correlated with the density of ERalpha+ and ERbeta+ cells (not with gluco-corticoid receptor and androgen receptor), which was not found in the synovium of patients with osteoarthritis. This indicates that inflammatory factors might up regulate the expression of oestrogen receptors in patients with rheumatoid arthritis, or vice versa.. In patients with osteoarthritis, expression of different steroid receptors is positively coupled, which was not observed in the synovium of patients with rheumatoid arthritis. This uncoupling phenomenon in rheumatoid arthritis might lead to an imbalance of the normal synovial homeostasis.

Topics: Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biomarkers; Cell Count; Enzyme-Linked Immunosorbent Assay; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Immunohistochemistry; Inflammation; Interleukin-6; Interleukin-8; Male; Middle Aged; Osteoarthritis; Receptors, Androgen; Receptors, Glucocorticoid; Receptors, Steroid; Statistics, Nonparametric; Synovial Membrane

To investigate whether the cytokine-inducing properties of surface-bound collagen type II (CII)-containing immune complexes (IC), which were reported earlier, have any clinical impact.. Anti-CII serology was analysed in 274 patients with early rheumatoid arthritis (RA). Patients with increased levels of anti-CII were followed serially for 1-5 years with regard to anti-CII IC-induced levels of tumour necrosis factor (TNF)alpha, interleukin (IL)1beta and IL8. Levels of antibodies and IC-induced cytokines were compared with clinical indices over 5 years of follow-up.. 5/100 healthy controls and 24/274 (8.8%) patients with RA exhibited increased levels (>29 arbitrary units (AU)/ml) of anti-native CII antibodies, a non-significant difference. 9/274 (3.3%) patients with RA and no controls comprised a discrete group with high anti-CII levels>450 AU/ml. These high anti-CII level sera were associated with induction of pro-inflammatory cytokines by anti-CII-containing IC formed in vitro. 8/9 patients with high baseline anti-CII levels exhibited a parallel decline in antibody levels, IC-induced cytokines, C reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Anti-CII-positive patients had significantly increased levels of CRP and ESR at baseline, but not later during the follow-up.. Anti-native CII-positive patients with RA have a distinct clinical phenotype characterised by an early acute phase response that might be driven by anti-CII-containing IC in joint cartilage.

Topics: Acute Disease; Antigen-Antibody Complex; Antirheumatic Agents; Arthritis, Rheumatoid; Autoantibodies; Biomarkers; Blood Sedimentation; C-Reactive Protein; Cells, Cultured; Collagen Type II; Cytokines; Female; Humans; Inflammation Mediators; Interleukin-1beta; Interleukin-8; Male; Middle Aged; Phenotype; Prospective Studies; Severity of Illness Index; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is characterized by infiltrations of inflammatory cells accompanied by neovascularization in the joint. We hypothesized that cell activation via the toll-like receptor (TLR) may be involved in the induction of angiogenic molecules, which are relevant to the pathogenesis of RA. RA fibroblast like synoviocytes (FLS) were stimulated with TLR-2 ligand bacterial peptidoglycan (PGN), TLR-4 ligand lipopolysaccharide (LPS) and various cytokines. Vascular endothelial growth factor (VEGF) and IL-8 were measured by ELISA in culture supernatants; mRNA levels were assessed by RT-PCR and real time PCR. The levels of TLR-2, VEGF and IL-8 were analyzed by dual immunohistochemistry in RA synovium and compared with osteoarthritis (OA). Regulation of MyD88, IRAK4, IRAK1, IRAK-M and TRAF-6 mRNA expression levels by PGN were analyzed by RT-PCR. Phosphorylation of I kappa B alpha was evaluated by western blotting. Levels of VEGF and IL-8 were upregulated in culture supernatants of RA FLS stimulated with PGN, similar to the levels of IL-1beta and IL-17 stimulation. Neutralization of TLR-2 with a blocking monoclonal antibody significantly reduced both VEGF and IL-8 levels (P<0.05), which reflected the functional relevance of TLR-2 activation to the induction of VEGF and IL-8 production. Downstream intracellular signaling following TLR-2 stimulation involved MyD88-IRAK-4-TRAF-6 pathways, resulting in NF-kappaB activation. Thus, TLR-2 activation in RA FLS by microbial constituents could be involved in the induction of VEGF and IL-8 and thereby promote inflammation either directly or via angiogenesis. This possibly contributes to the perpetuation of synovitis in patients with RA.

Topics: Angiogenesis Inducing Agents; Antibodies; Arthritis, Rheumatoid; Cells, Cultured; Cytokines; Fibroblasts; Gene Expression; Humans; I-kappa B Proteins; Interleukin-1 Receptor-Associated Kinases; Interleukin-8; Lipopolysaccharides; Myeloid Differentiation Factor 88; NF-kappa B; Peptidoglycan; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; TNF Receptor-Associated Factor 6; Toll-Like Receptor 2; Up-Regulation; Vascular Endothelial Growth Factor A

To evaluate the potential of using prednisolone phosphate (PSLP)-containing 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) liposomes to treat rheumatoid arthritis (RA), we examined their ability to bind human fibroblast-like synovial (HFLS) cells and their effects in these cells. To test for binding, Lissamine rhodamine B-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine)-labelled PSLP-containing TRX-20 liposomes were added to HFLS cells, and the fluorescence intensity of the rhodamine bound to the cells was evaluated. Rhodamine-labelled PSLP-containing liposomes without TRX-20 were used as a negative control. To evaluate the uptake of liposomes by the HFLS cells, we used TRX-20 liposomes containing 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and p-xylene-bis-pyridinium bromide (DPX), and observed the cells by fluorescence microscopy. The effects of the PSLP in TRX-20 liposomes on HFLS cells were assessed by the inhibition of the production of two inflammatory cytokines (interleukin 6 and granulocyte macrophage colony-stimulating factor) and one inflammatory chemokine (interleukin 8). The interaction of the PSLP-containing TRX-20 liposomes with HFLS cells was approximately 40 times greater than that of PSLP-containing liposomes without TRX-20. PSLP-containing TRX-20 liposomes bound to HFLS cells primarily via chondroitin sulfate. TRX-20 liposomes taken up by the cell were localized to acidic compartments. Furthermore, the PSLP-containing TRX-20 liposomes inhibited the production of the inflammatory cytokines and the chemokine more effectively than did the PSLP-containing liposomes without TRX-20. These results indicate that PSLP-containing TRX-20 liposomes show promise as a novel drug delivery system that could enhance the clinical use of glucocorticoids for treating RA.

Topics: Adult; Arthritis, Rheumatoid; Benzamidines; Cells, Cultured; Fatty Acids; Female; Fibroblasts; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Liposomes; Middle Aged; Prednisolone; Synovial Membrane; Tumor Necrosis Factor-alpha

Nonbiological therapeutics are frequently used for the treatment of patients with rheumatoid arthritis (RA). Because the mechanisms of action of these therapeutics are unclear, the authors aimed to elucidate the molecular effects of typical antirheumatic drugs on the expression profile of RA-related genes expressed in activated synovial fibroblasts. For reasons of standardization and comparability, immortalized synovial fibroblasts derived from RA (RASF) and normal donors (NDSF) were treated with methotrexate, prednisolone, or diclofenac and used for gene expression profiling with oligonucleotide microarrays. The cytotoxicity of the antirheumatic drugs was tested in different concentrations by MTS tetrazolium assay. Genes that were differentially expressed in RASF compared to NDSF and reverted by treatment with antirheumatic drugs were verified by semiquantitative polymerase chain reaction and by chemiluminescent enzyme immunoassay. Treatment with methotrexate resulted in the reversion of the RA-related expression profile of genes associated with growth and apoptosis including insulin-like growth factor binding protein 3, retinoic acid induced 3, and caveolin 2 as well as in the re-expression of the cell adhesion molecule integrin alpha6. Prednisolone reverted the RA-related profile of genes that are known from inflammation and suppressed interleukins 1beta and 8. Low or high doses of diclofenac had no effect on the expression profile of genes related to RA in synovial fibroblasts. These data give the first insight into the mechanisms of action of common antirheumatic drugs used for the treatment of arthritides. Synovial fibroblasts reflect the disease-related pathophysiology and are useful tools for screening putative antirheumatic compounds.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Cell Death; Cell Line, Transformed; Fibroblasts; Gene Expression Profiling; Humans; Immunoassay; Interleukin-1beta; Interleukin-8; Methotrexate; Oligonucleotide Array Sequence Analysis; Prednisolone; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Synovial Fluid

This study was performed to investigate whether synovial proliferation (SP) differentially affects hypoxia in the joint cavities of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Thirty RA and 42 OA patients who underwent synovitis assessment were classified into two groups based on the presence or absence of SP, as revealed by musculoskeletal ultrasonography. Synovial fluids (SFs) from the knee joints were analyzed for interleukin (IL)-8, pO(2), and white blood cell counts and blood samples were analyzed for erythrocyte sedimentation rate (ESR). No difference was found between the OA patients with and without SP in terms of SF oxygen tension (SF pO(2)) or IL-8 level, whereas the RA patients had significantly lower SF pO(2) levels in their knee joints than did the OA patients with SP, and the RA patients had higher levels of IL-8 in their joints than did the OA patients. The counts of infiltrated immune cells in the SF and tissues were much higher for patients with RA and SP than for those with OA and SP. The ESRs were not found to be correlated with SP in OA patients but were negatively correlated with SF pO(2) levels in RA patients. We conclude that ultrasonographically detected SP in OA patients does not generate a more hypoxic SF than that found in OA patients without SP. The SFs from RA patients with SP are hypoxic, which indicates that SP may have different impacts on hypoxia in the joint cavities of RA and OA patients.

Topics: Arthritis, Rheumatoid; Blood Sedimentation; Cell Proliferation; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hypoxia; Immunohistochemistry; Interleukin-8; Knee Joint; Male; Osteoarthritis, Knee; Oxygen; Severity of Illness Index; Synovial Fluid; Synovial Membrane; Ultrasonography

To investigate the relationship between macrophage migration inhibitory factor (MIF) levels and clinical measures in rheumatoid arthritis (RA), and the potential for regulation of angiogenesis in RA.. Serum and synovial fluid (SF) levels of MIF and vascular endothelial growth factor (VEGF) in patients with RA were determined by sandwich ELISA, and the relationships among MIF, VEGF, and RA clinical measures were analyzed. RA synovial fibroblasts were cultured with recombinant human MIF (rhMIF) and the production of VEGF and interleukin 8 (IL-8) were measured in the conditioned media. The angiogenic effect of MIF was examined using established measures of angiogenesis in vitro.. Erythrocyte sedimentation rate, C-reactive protein, and the daily dosage of oral prednisolone were correlated with SF levels of MIF. The SF levels of MIF were found to be higher in patients with bony erosion than in those without (69.2 +/- 11.4 ng/ml vs 44.0 +/- 6.2 ng/ml; p = 0.045). MIF levels had good correlation with VEGF levels (r = 0.52, p < 0.001 in sera, and r = 0.6, p < 0.001 in SF). Production of the angiogenic factors VEGF and IL-8 was enhanced in cultured RA synovial fibroblasts stimulated by rhMIF. Endothelial tube formation was augmented when the endothelial cells were cultured with the conditioned media from rhMIF-pretreated SF mononuclear cells, and this phenomenon was reversed by anti-VEGF antibody.. SF MIF may reflect the clinical activity in patients with RA, and rhMIF induces the angiogenic factors in RA synovial fibroblasts. These results suggest that MIF may be an important cytokine in the perpetuation of the angiogenic and inflammatory processes in patients with RA.

Topics: Angiogenesis Inducing Agents; Arthritis, Rheumatoid; Biomarkers; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Interleukin-8; Leukocytes, Mononuclear; Macrophage Migration-Inhibitory Factors; Male; Middle Aged; Neovascularization, Physiologic; Osteoarthritis, Knee; Recombinant Proteins; Synovial Fluid; Synovial Membrane; Up-Regulation; Vascular Endothelial Growth Factor A

To explore the source of the p19 subunit of interleukin-23 (IL-23) in joints with rheumatoid arthritis (RA), the effects of IL-1beta and tumour necrosis factor (TNF)-alpha on IL-23 gene expression in RA fibroblast-like synoviocytes and the effect of IL-23 on proinflammatory cytokines.. Expression of IL-23 p19 in joints was examined by immunohistochemical analysis of patients with RA and osteoarthritis (OA). The effects of IL-1beta and TNF-alpha on the expression, of IL-23 p19 and IL-12 p35 subunits in human fibroblast-like synoviocytes from RA patients (HFLS-RA) were determined by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative PCR and western blotting assay. Blockade of nuclear factor kappaB (NF-kappaB) or AP-1 activation was used to verify the involvement of intracellular signal pathways of the induction of p19. IL-23-induced IL-8 and IL-6 productions were determined in HFLS-RA by RT-PCR and enzyme-linked immunosorbent assay.. IL-23 p19 was expressed in the synovium from RA, but not from OA patients. Similar to the protein expression, IL-23 p19 mRNA could be detected by RT-PCR in four of five RA synovial fluid mononuclear cells (SFMC). IL-1beta and TNF-alpha could induce RA fibroblast-like synoviocytes to produce the IL-23 p19 subunit. The effects of IL-1beta were much stronger than TNF-alpha. These responses were observed in both a dose-responsive and time-dependent manner. IL-1beta produced weakly enhanced gene expression of the p35 subunits of IL-12. IL-1beta also promotes the p35 expression, a subunit of IL-12, but weakly. In addition, the NF-kappaB and the AP-1 inhibitors down-regulated the expression of IL-23 p19 mRNA induced by IL-1beta. IL-23 receptor (IL-23R) was of constitutive expression in HFLS-RA. Moreover, IL-23 up-regulated the IL-8 and IL-6 mRNA and protein levels in a dose-dependent manner in HFLS-RA.. Our results demonstrate that IL-23, produced by mononuclear cells in synovial fluid with RA and HFLS-RA, promotes inflammatory responses in RA by inducing IL-8 and IL-6 production from HFLS. IL-1beta regulates IL-23 p19 expression via NF-kappaB and AP-1 pathways. This report also demonstrates that IL-23 could promote inflammatory responses in HFLS-RA by stimulating IL-8 and IL-6 production.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dose-Response Relationship, Immunologic; Gene Expression Regulation; Humans; Interleukin-17; Interleukin-1beta; Interleukin-23 Subunit p19; Interleukin-6; Interleukin-8; NF-kappa B; Receptors, Interleukin; RNA, Messenger; Synovial Fluid; Synovial Membrane; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.

Topics: Arthritis, Rheumatoid; Blood; Catalysis; Dinoprostone; HeLa Cells; Humans; Inflammation; Inhibitory Concentration 50; Interleukin-6; Interleukin-8; Lipopolysaccharides; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Monocytes; Proto-Oncogene Proteins; Synovial Fluid

Monoclonal antibody against TNF-alpha such as infliximab has shown clinical efficacy in controlling the inflammatory signs and symptoms of rheumatoid arthritis (RA), but the detailed immunotherapeutic mechanism is not fully understood. We investigated 19 patients with active RA who were treated with infliximab (3 mg/kg) at weeks 0, 2, 6 and 14. Peripheral blood was obtained from the patients at weeks 0 and 14 and cultured with mitogens phytohaemagglutinin (PHA) and lipopolysaccharide (LPS). The concentrations of cytokines and soluble adhesion molecules (sICAM-1, sICAM-3, sE-selectin, sP-selectin, sVCAM-1 and sPECAM-1) in supernatant fluids or plasma were measured by flow cytometry and ELISA. After infliximab treatment, the absolute and percentage increases in release of inflammatory cytokine TNF-alpha and potent neutrophil chemoattractant IL-8 upon PHA and LPS activation were significantly decreased when compared to those of before treatment (all P<0.01). The increased releases of IL-6, IL-1beta, IL-18 and IL-12 upon mitogen activation were similar before and after infliximab treatment (all P>0.05). Plasma concentrations of these cytokines and soluble adhesion molecules did not differ significantly before and after infliximab treatment. Our study suggests that the reduction in synovial inflammation may be due to the decreased production of TNF-alpha and IL-8, and hence the number of neutrophils and other pro-inflammatory leukocytes infiltrating into the inflamed sites.

Topics: Adult; Aged; Antibodies, Monoclonal; Arthritis, Rheumatoid; Cytokines; Female; Humans; Infliximab; Interleukin-8; Male; Methotrexate; Middle Aged; Tumor Necrosis Factor-alpha

Recently, we found that administration of glucosamine to adjuvant arthritis, a model for rheumatoid arthritis, suppressed the progression of arthritis in rats. To clarify its anti-inflammatory mechanism, we evaluated the actions of glucosamine on the activation of synoviocytes in vitro.. Synoviocytes isolated from human synovial tissues were stimulated with interleukin (IL)-1beta in the presence of 0.01-1 mM glucosamine. IL-8 and prostaglandin (PG) E(2) were measured by ELISA, and nitric oxide was quantitated by Griess assay. IL-8 mRNA was detected by RT-PCR. Furthermore, the effect of glucosamine on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the binding of [(125)I] IL-1beta to its receptors were examined using a primary human synovial cell line (CSABI- 479).. Glucosamine significantly suppressed the IL-1beta-induced IL-8 production as well as its mRNA expression (p < 0.05) at 1 mM. Furthermore, glucosamine (1 mM) inhibited the IL-1beta-induced nitric oxide and PGE(2) production (p < 0.05). Moreover, glucosamine suppressed the IL-1beta-induced phosphorylation of p38 MAPK (p < 0.05 at >0.1 mM) and the IL-1beta-binding to its receptors (p < 0.05 at 1 mM).. These observations suggest that glucosamine can suppress the IL-1beta-mediated activation of synoviocytes (such as IL-8-, nitric oxide- and PGE(2)-production, and phosphorylation of p38 MAPK), thereby possibly exhibiting antiinflammatory actions in arthritis.

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Cells, Cultured; Dinoprostone; Glucosamine; Humans; Interleukin-1beta; Interleukin-8; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Messenger; Synovial Membrane

To determine whether subgroups of rheumatoid arthritis (RA) patients classified according to their synovial vascular pattern have a different expression of angiogenic mediators or exhibit distinct clinical or biological characteristics.. Arthroscopies were performed in 27 patients with RA and synovial samples were obtained. Vascular morphology was classified in three patterns: straight (S), tortuous (T) and mixed (M). Immunostaining was performed with anti-vascular endothelial growth factor (anti-VEGF), anti-vascular endothelial growth factor receptor (VEGFR)-1, anti-VEGFR-2, anti-IL-8 and anti-TGF-beta, and measured by digital image analysis. Serum levels of VEGF, TGF-beta and IL-8, and clinical, radiographic and serological data were also analysed.. Eleven (41%) patients had the S pattern, nine (33%) the M pattern and seven (26%) the T pattern. The S and M groups had a higher prevalence of rheumatoid factor positivity and erosive disease, and higher levels of markers of systemic inflammation compared with the T group. Synovial expression of VEGF was higher in the S and T groups compared with the M group, whereas TGF-beta was higher in the T compared with the S and M groups. Distinct synovial distribution of VEGF and TGF-beta between groups was also observed.. This preliminary study suggests that RA patients with the S and M patterns share different clinical, biological and serological characteristics compared with those with the T pattern, which may constitute a group with less severe disease. Differences in the intensity and distribution of synovial expression of VEGF and TGF-beta observed between groups could have pathophysiological relevance. However, larger, prospective multicentre studies would be need to determine the clinical relevance of vascular patterns in RA.

Topics: Adult; Aged; Angiogenesis Inducing Agents; Antirheumatic Agents; Arthritis, Rheumatoid; Arthroscopy; Female; Humans; Immunoenzyme Techniques; Interleukin-8; Male; Middle Aged; Neovascularization, Pathologic; Prognosis; Severity of Illness Index; Synovial Membrane; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy.. RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay.. Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate.. Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Antagonism; Drug Combinations; Fibroblasts; Gene Expression; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Interleukin-8; Mevalonic Acid; Polyisoprenyl Phosphates; RNA, Messenger; Simvastatin; Synovial Membrane; Tumor Necrosis Factor-alpha

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

Topics: Arthritis, Rheumatoid; Cell Survival; Cells, Cultured; Clusterin; Fibroblasts; Humans; I-kappa B Kinase; Immunohistochemistry; In Situ Hybridization; Interleukin-6; Interleukin-8; NF-kappa B; Osteoarthritis; Phosphorylation; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Synovial Fluid; Synovial Membrane; Transgenes; Tumor Necrosis Factor-alpha

The aim of the present study was to determine synovial levels of ELR (+) CXC chemokines, known to attract mainly neutrophils to inflamed tissues by binding the neutrophil chemokine receptors CXCR1 and CXCR2 and promoting neovascularization in patients with various inflammatory disorders. The study group consisted of 14 patients with Behçet's disease and nine with familial Mediterranean fever. Fourteen patients with rheumatoid arthritis and 16 with osteoarthritis served as controls. Synovial chemokine levels were measured by two-step sandwich enzyme-linked immunosorbent assay, and significant differences were found in the various chemokines studied. In addition to its angiogenic properties, increased synovial levels of interleukin-8 by attraction of more neutrophils to synovial fluids might also be responsible for the acute synovitis in patients with Behçet's disease. However, the absence of chronic changes with the eventual development of pannus and erosions might result from relatively lower expression of interleukin-8 and the transient, short-lived nature of the arthritis observed in these patients.

Topics: Adult; Angiogenesis Inhibitors; Arthritis, Rheumatoid; Behcet Syndrome; Chemokine CXCL1; Chemokine CXCL5; Chemokines, CXC; Familial Mediterranean Fever; Female; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Male; Microtubule-Associated Proteins; Middle Aged; Neovascularization, Pathologic; Osteoarthritis, Knee; Synovial Fluid

To obtain fibroblast-like synovial cells (FLS) from synovial fluid (SF).. SF aspirated from joints of patients with rheumatoid arthritis (RA), other types of inflammatory arthritis, and osteoarthritis (OA) was centrifuged and the resulting cell pellet resuspended in growth medium. After 2 days, nonadherent cells were removed. FLS were also cultured from surgical specimens of synovial tissue (td-FLS). Phenotype characterization of fluid derived FLS (fd-FLS) was accomplished by flow cytometry and immunohistochemistry staining. Tumor necrosis factor-alpha (TNF-alpha) induced interleukin 6 (IL-6), IL-8, and cyclooxygenase 2 (COX-2) mRNA levels were assessed.. Second and later passage fd-FLS exhibited uniform fibroblast-like morphology. Fd-FLS and td-FLS expressed a similar profile of cell surface antigens including the fibroblast marker Thy-1. Less than 2% of either cell type expressed surface markers characteristic of dendritic cells, phagocytic cells, T cells, or leukocytes. Immunohistochemistry staining revealed the presence of fibroblast products prolyl-4 hydroxylase, procollagen I, and procollagen III in both culture types. TNF-a induced increases in IL-6, IL-8, and COX-2 mRNA were suppressed by dexamethasone in both fd-FLS and td-FLS.. FLS can be cultured from SF. The fibroblast phenotype was confirmed by analysis of surface antigens and intracellular proteins. Inflammatory mediators produced after stimulation of both fd-FLS and td-FLS were suppressed by dexamethasone. In addition to providing a more accessible source of FLS, fd-FLS may also facilitate study of synovial cells in early RA when tissue specimens are not readily available.

Topics: Arthritis; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 2; Dexamethasone; Fibroblasts; Flow Cytometry; Gene Expression; Humans; Immunoenzyme Techniques; Interleukin-6; Interleukin-8; Knee Joint; Membrane Proteins; Osteoarthritis, Knee; Phenotype; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Ubiquitination affects various immune processes and E3 ubiquitin ligases (E3) play an important role in determining substrate specificity. We identified 11 human E3 ligase genes of potential importance in pathogenesis of autoimmune diseases by search of public databases and screened them for candidacy of biological investigation with case-control linkage disequilibrium tests on multiple SNPs in the genes using rheumatoid arthritis (RA) as a model of autoimmune diseases. Significant association with RA was observed in an SNP in intron 3 of Cullin 1 (CUL1) that affected transcriptional efficiency of the promoter activity in lymphocytic cell lines. Quantitative expression analysis revealed that CUL1 mRNA was highly detected in lymphoid tissues including spleen and tonsil, and was specifically expressed in T and B lymphocytes in fractionated peripheral leukocytes. Histological evaluation of tonsils indicated that CUL1 protein expression was relatively specific for maturing germinal centers. Suppression of CUL1 expression had influence on the phenotype of T-cell line, that is, it inhibited IL-8 induction, which is known to play an important role in the migration of inflammatory cells into the affected area seen in RA. Our data suggest that the regulation of CUL1 expression in immunological tissues may affect the susceptibility of RA via altering lymphocyte signal transduction.

Topics: Arthritis, Rheumatoid; Cell Cycle Proteins; Cullin Proteins; Genetic Predisposition to Disease; Humans; Interleukin-8; Jurkat Cells; Lymphocytes; Polymorphism, Single Nucleotide; RNA, Small Interfering; Signal Transduction; Ubiquitin-Protein Ligases

To explore the role of several chemokines in rheumatoid arthritis (RA) and their clinical significance.. ELISA was used to detect the serum levels of interleukin (IL)-8, interferon-gamma inducible protein (IP)-10, and RANTES in 58 active RA patients, 29 clinically remissive RA patients, 21 osteoarthritis (OA) patients, and 30 healthy volunteers. Clinical data of these patients were compared between different groups.. The serum levels of IL-8, IP-10 and RANTES in the active RA patients were significantly higher than those of the healthy controls and OA patients. The serum level of IP-10 of the active patients was higher than that of the clinically remissive patients. The index IL-8/IP-10 x RANTES/IP-10 of the active patients was higher than that of the healthy controls (P = 0.01). The serum RANTES level was positively correlated with erythrocyte sedimentation rate, C-reactive protein, platelet count, and numbers of swollen joints, and was negatively correlated with the hemoglobin level. The serum level of IP-10 was negatively correlated with X-ray image grade of hand and wrist joints.. The serum levels of IL-8, IP-10 and RANTES significantly increase in active RA. The level of RANTES may be a useful additional marker for disease activity and the level of IP-10 can be used as an index of prognosis.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Chemokine CCL5; Chemokine CXCL10; Chemokines, CXC; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Middle Aged

To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia.. CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8.. The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1-4 and glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. (125)I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. (125)I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or glypican-4 antibodies.. Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.

Topics: Adult; Aged; Arthritis, Rheumatoid; Binding Sites; Case-Control Studies; Endothelial Cells; Female; Heparitin Sulfate; Humans; Immunohistochemistry; Interleukin-8; Male; Membrane Glycoproteins; Microcirculation; Middle Aged; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Syndecan-3; Synovial Membrane

Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-alpha soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 microM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1beta-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 microM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process.

Topics: Active Transport, Cell Nucleus; Aniline Compounds; Anti-Inflammatory Agents; Apoptosis; Arthritis, Rheumatoid; Cells, Cultured; Crotonates; Cyclooxygenase 2; Cytokines; Dinoprostone; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Hydroxybutyrates; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-11; Interleukin-8; Isoxazoles; Leflunomide; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Nitriles; p38 Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Receptors, Tumor Necrosis Factor, Type I; Sialoglycoproteins; Synovial Fluid; Time Factors; Toluidines; Trypsin

Studies indicate the genetic, biological, and clinical heterogeneity of rheumatoid arthritis (RA). Recently the histological diversity of RA has been postulated. We investigated whether serum concentrations of interleukin 8 (IL-8), RANTES (regulated upon activation normal T cell expressed and secreted), and monocyte chemoattractant protein-1 (MCP-1) are correlated with histological appearance of the rheumatoid synovitis.. Using ELISA we assessed IL-8, RANTES, and MCP-1 concentrations in serum of 47 patients with RA and 30 patients with osteoarthritis (OA).. Morphological analysis of synovial specimens distinguished 2 types of rheumatoid synovitis. Twenty-eight RA samples presented diffuse infiltrates of mononuclear cells with no specific microanatomical organization and were categorized as diffuse synovitis. In the remaining 19 specimens, classified as follicular synovitis, formation of lymphocytic follicles with germinal center-like structures was observed. Serum levels of studied chemokines were increased in patients with RA compared to the OA control group (p < 0.001 for all comparisons). Concentrations of IL-8, RANTES, and MCP-1 were highest in serum of RA patients with follicular synovitis in comparison with patients with diffuse synovitis (p < 0.01, p < 0.01, and p < 0.05, respectively) and could distinguish RA patients with these 2 histological disease patterns. Serum levels of chemokines correlated with markers of disease activity such as erythrocyte sedimentation rate, C-reactive protein concentrations, and Disease Activity Score.. Distinct histological variants of rheumatoid synovitis associated with different serum levels of IL-8, RANTES, and MCP-1 reflect clinical activity of the disease and confirm the concept of RA heterogeneity.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Biopsy, Needle; Case-Control Studies; Chemokine CCL2; Chemokine CCL5; Chemokines; Disease Progression; Female; Humans; Immunohistochemistry; Interleukin-8; Male; Middle Aged; Probability; Prognosis; Reference Values; Risk Assessment; Sensitivity and Specificity; Severity of Illness Index; Synovitis

Examination of expression of the chemokine macrophage inflammatory protein-3a (CCL20/Mip-3alpha) in blood polymorphonuclear neutrophils (PMN) and synovial fluid (SF) PMN of patients with rheumatoid arthritis (RA).. Paired samples of blood PMN and SF PMN were obtained from 11 patients with RA. In addition, SF was prepared from 9 patients with osteoarthritis (OA) and 10 patients with juvenile idiopathic arthritis (JIA). PMN were isolated via density centrifugation to a purity of 98%. Total RNA was isolated and the expression of CCL20 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In some experiments blood PMN obtained from healthy donors were stimulated with individual SF of patients with RA. For quantitative considerations, CXCL8, CCL20, interleukin 1, and tumor necrosis factor-alpha (TNF-alpha) levels were determined in SF by ELISA.. In SF of RA patients CCL20 and CXCL8 levels were elevated, up to 7.5 ng/ml and 23.6 ng/ml, respectively. No significant level of either chemokine was found in SF of patients with JIA and OA. CCL20 mRNA was undetectable in blood PMN of all patients with RA. In SF PMN, CCL20 mRNA was found in 6/11 RA patients. Expression of CCL20 mRNA in 5/6 SF PMN samples was observed in the absence of detectable TNF-alpha levels in SF. Cell culture experiments, however, confirmed the ability of TNF-alpha in SF to induce CCL20 mRNA expression in blood PMN. Notably, expression of CCL20 was also found in PMN after stimulation with SF lacking TNF-alpha.. Recruitment of PMN to the synovial microenvironment induces expression of CCL20 mRNA independent of the concentrations of TNF-alpha accumulating in SF of patients with RA.

Topics: Adult; Aged; Arthritis, Juvenile; Arthritis, Rheumatoid; Case-Control Studies; Cells, Cultured; Chemokine CCL20; Chemokines, CC; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Macrophage Inflammatory Proteins; Male; Middle Aged; Neutrophils; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Our recent work showed that fibroblast-like synovial cells (FLS) could differentiate into adipocyte-like cells in vitro in response to stimulation with peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand. The aim of the present study was to determine the role of cytokines in the regulation of FLS differentiation to adipocyte-like cells.. FLS isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and from normal synovial tissues were incubated with the synthetic PPAR gamma ligand troglitazone to induce adipocyte-like differentiation of the cells.. Production of interleukin (IL)-6, IL-8 and matrix metalloproteinase-3 was reduced in adipocyte-like cells compared with FLS. DNA binding activity of nuclear factor kappa B (NF-kappa B) was clearly inhibited in adipocyte-like cells. Cultivation of FLS with interferon gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) or IL-1 beta inhibited the expression of PPAR gamma as well as CCAAT/enhancer binding protein (C/EBP) nuclear activity, and thus suppressed adipocyte-like cell differentiation in vitro.. Our results indicate the importance of PPAR gamma and C/EBP in adipocyte-like cell differentiation of FLS and that the process is influenced by inflammatory cytokines, and suggest that the proinflammatory character of FLS in patients with RA is diminished during adipocyte-like cell differentiation.

Topics: Adipocytes; Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Chromans; Cytokines; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Osteoarthritis; Receptors, Cytoplasmic and Nuclear; Synovial Membrane; Thiazolidinediones; Transcription Factors; Troglitazone

Recent studies of the pathogenesis of rheumatoid arthritis (RA) have revealed that both synovial fibroblasts and T cells participate in the perpetuation of joint inflammation as dynamic partners in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. In this study, we investigated the role of IL-17, a major Th1 cytokine produced by activated T cells, in the activation of RA synovial fibroblasts. Transcripts of IL-17R (IL-17 receptor) and IL-17RB (IL-17 receptor B) were present in fibroblast-like synoviocytes (FLS) of RA patients. IL-17R responded with increased expression upon in vitro stimulation with IL-17, while the level of IL-17RB did not change. IL-17 enhanced the production of IL-6 and IL-8 in FLS, as previously shown, but did not affect the synthesis of IL-15. IL-17 appears to be a stronger inducer of IL-6 and IL-8 than IL-15, and even exerted activation comparable to that of IL-1beta in RA FLS. IL-17-mediated induction of IL-6 and IL-8 was transduced via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB, while CD40 ligation and p38 MAPK (mitogen-activated protein kinase) are not likely to partake in the process. Together these results suggest that IL-17 is capable of more than accessory roles in the activation of RA FLS and provide grounds for targeting IL-17-associated pathways in therapeutic modulation of arthritis inflammation.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Fibroblasts; Humans; Interleukin-15; Interleukin-17; Interleukin-6; Interleukin-8; Middle Aged; Mitogen-Activated Protein Kinases; NF-kappa B; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Interleukin; Receptors, Interleukin-17; Signal Transduction; Synovial Membrane; Transcription Factor RelA

To determine the impact of type II collagen (CII)-reactive T cells on the production of chemokines in the joints of patients with rheumatoid arthritis (RA).. T cell proliferative responses to bovine CII were assayed in synovial fluid (SF) mononuclear cells and peripheral blood mononuclear cells. CII-stimulated T cells were cocultured with fibroblast-like synoviocytes (FLS). The expression of interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1 alpha) in the sera, SF, and supernatant of the CII-stimulated T cells and FLS coculture was measured by enzyme-linked immunosorbent assays.. The levels of IL-8, MCP-1, and MIP-1 alpha in SF were significantly higher than those in paired sera of RA patients. IL-8, MCP-1, and MIP-1 alpha levels in SF were strongly correlated with T cell responses to CII. When FLS were cocultured with CII-stimulated T cells, the production of IL-8, MCP-1, and MIP-1 alpha was significantly increased. This increase correlated well with the T cell proliferative response to CII. Chemokine production by coculture of CII-stimulated T cells and FLS was mediated mainly by direct cell-cell contact through CD40 ligand-CD40 engagement.. Our data indicate that the presence of CII-reactive T cells in RA joints can increase the production of chemokines such as IL-8, MCP-1, and MIP-1 alpha through interaction with FLS. This chemokine production is mediated by cell-cell contact, including CD40 ligand-CD40 engagement. These results suggest that CII-reactive T cells play a crucial role in the amplification and perpetuation of the inflammatory process in the rheumatoid synovium.

Topics: Adult; Aged; Antibodies, Monoclonal; Arthritis, Rheumatoid; CD40 Antigens; Cell Communication; Cells, Cultured; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokines; Coculture Techniques; Collagen Type II; Female; Fibroblasts; Humans; Interleukin-8; Macrophage Inflammatory Proteins; Male; Middle Aged; Synovial Fluid; Synovial Membrane; T-Lymphocytes

Interleukin-8 (IL-8) plays an important role in the migration of inflammatory cells into the synovium and joint fluids in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-8 inductive activity of the macrophage migration inhibitory factor (MIF) in RA synovial fibroblasts. The regulatory mechanism of IL-8 was compared with that of IL-1beta.. MIF-induced IL-8 and IL-1beta transcriptional activation was studied in RA synovial fibroblasts by Northern blot analysis, enzyme-linked immunosorbent assay, and electromobility shift assay. The effect of anti-MIF antibody administration on murine passive collagen-induced arthritis (CIA) was also evaluated by histologic examination and reverse transcriptase-polymerase chain reaction.. MIF up-regulated the IL-8 messenger RNA (mRNA) and protein levels in a dose-dependent manner. The IL-8 mRNA up-regulation started 1 hour poststimulation by MIF, and reached a maximum level at 6 hours. IL-1beta mRNA was also up-regulated by MIF. The mRNA up-regulation of IL-8 and IL-1beta by MIF was inhibited by 2 tyrosine kinase inhibitors, a protein kinase C (PKC) inhibitor, an activator protein 1 (AP-1) inhibitor, and by an NF-kappaB inhibitor. A cAMP-dependent kinase inhibitor did not inhibit it. MIF enhanced AP-1 and NF-kappaB binding activities in a dose-dependent manner. Passive CIA enhanced mRNA levels of macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractants and, moreover, migration and proliferation of inflammatory cells within the synovium, which were suppressed by administration of an anti-MIF antibody.. MIF may play an important role in the migration of inflammatory cells into the synovium of rheumatoid joints via induction of IL-8. MIF up-regulates IL-8 and IL-1beta mRNA via tyrosine kinase-, PKC-, AP-1-, and NF-kappaB-dependent pathways.

Topics: Arthritis, Rheumatoid; Chemokine CXCL2; Chemokines; Cytokines; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fibroblasts; Gene Expression; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-8; Macrophage Migration-Inhibitory Factors; Monokines; NF-kappa B; Osteoarthritis, Knee; Protein Kinase Inhibitors; RNA, Messenger; Sialoglycoproteins; Signal Transduction; Synovial Membrane; Transcription Factor AP-1; Transcription, Genetic; Up-Regulation

Madimadi, a Korean folk medicine, has been applied to treat rheumatoid arthritis (RA). However, its mechanisms of action have not been examined. The involvement of inflammatory cytokines, particularly TNF-alpha, IL-1beta, and IL-8, resulting in local inflammation in the pathogenesis of RA is now widely accepted. Madimadi dose-dependently inhibited TNF-alpha, IL-1beta, and IL-8 production from activated human mast cells (HMC-1). RT-PCR revealed inhibition of TNF-alpha and IL-1beta transcription in activated HMC-1. In addition, we confirmed potent inhibition of TNF-alpha and IL-1beta production by Madimadi using purified human blood PBMC from an active RA group, but not from healthy or disease control groups. These novel insights into the immunosuppressive action of Madimadi are likely to impact the clinical use of this agent.

Topics: Arthritis, Rheumatoid; Case-Control Studies; Cell Line; Cell Survival; Gene Expression Regulation; Herbal Medicine; Humans; Interleukin-1; Interleukin-8; Korea; Mast Cells; Medicine, East Asian Traditional; Phytotherapy; Tumor Necrosis Factor-alpha

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine important in animal models of rheumatoid arthritis (RA). We investigated the utilization by MIF of mitogen activated protein (MAP) kinase signalling pathways in the stimulation of fibroblast-like synoviocytes (FLS), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin 6 (IL-6) and IL-8 expression.. Cultured human RA FLS were treated with recombinant MIF. Activation of MAPK was measured by Western blotting and blocked using specific inhibitors. The expression of COX-2, PGE(2), IL-6, and IL-8 were measured using flow cytometry, ELISA, and real-time polymerase chain reaction.. MIF induced the phosphorylation of FLS p38 and extracellular-signal regulated kinase (ERK) MAP kinase. MIF significantly induced COX-2 and IL-6 protein and mRNA expression as well as PGE(2) and IL-8 production. Antagonism of p38 MAP kinase inhibited MIF induction of COX-2, PGE(2), and IL-6. In contrast, antagonism of ERK had no effect on COX-2, PGE(2), or IL-6. Neither antagonist inhibited MIF-induced IL-8.. MIF activates RA FLS COX-2 and IL-6 expression via p38 MAP kinase activation and induces IL-8 via p38 and ERK MAP kinase-independent pathways.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Enzyme Activation; Humans; Interleukin-6; Interleukin-8; Isoenzymes; Macrophage Migration-Inhibitory Factors; Membrane Proteins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Synovial Membrane

In the present study, we have shown for the first time that a plant steroid, diosgenin, causes an inhibition of the growth of fibroblast-like synoviocytes from human rheumatoid arthritis, with apoptosis induction associated with cyclooxygenase-2 (COX-2) up-regulation. Celecoxib, a selective COX-2 inhibitor, provoked a large decrease in diosgenin-induced apoptosis even in the presence of exogenous prostaglandin E2, whereas interleukin-1beta, a COX-2 inducer, strongly increased diosgenin-induced apoptosis of these synoviocytes. These findings suggest that the proapoptotic effect of diosgenin is associated with overexpression of COX-2 correlated with overproduction of endogenous prostaglandin E2. We also observed a loss of mitochondrial membrane potential, caspase-3 activation, and DNA fragmentation after diosgenin treatment.

Topics: Aged; Apoptosis; Arthritis, Rheumatoid; Caspase 3; Caspases; Cell Proliferation; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Diosgenin; DNA Fragmentation; Fibroblasts; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Isoenzymes; Membrane Potentials; Membrane Proteins; Middle Aged; Mitochondria; Plant Extracts; Prostaglandin-Endoperoxide Synthases; Synovial Membrane; Up-Regulation

To investigate the pathophysiological significance of cathepsin B and thrombin in synovial fluid (SF) from patients with rheumatoid arthritis (RA).. Thrombin and cathepsin B activities of samples from patients with RA and osteoarthritis (OA) were measured using fluorogenic synthetic substrates. The concentration of interleukin 8 (IL-8) in SF was measured by ELISA. The effect of thrombin on the proliferation of synovial fibroblast-like cells (SFC) was examined by measuring 3H-thymidine incorporation. The effect of thrombin on the release of IL-8 and cathepsin B from SFC was investigated. The expression of IL-8 mRNA in SFC after stimulation with thrombin was evaluated using real-time quantitative RT-PCR. The effect of recombinant IL-8 on the activation of cathepsin B was examined using the knee joints of rabbits.. In SF supernatants, cathepsin B and thrombin-like activity was significantly higher in RA than in OA, and there was a significant correlation between them. Cathepsin B activity was also significantly higher in SF cells and synovial tissue extracts from RA patients than in those from OA patients. There was a significant correlation between cathepsin B activity and the concentration of IL-8 in RA SF. Thrombin enhanced the proliferation of SFC in a dose-dependent manner. Thrombin significantly enhanced the release of IL-8 from SFC as well as the expression of IL-8 mRNA in SFC. IL-8 induced activation of cathepsin B in the knee joints of rabbits. However, thrombin did not directly increase cathepsin B activity in SFC.. In RA, thrombin was found to be related to the enhanced growth of SFC and the release of IL-8 from these cells; thus thrombin is probably related to worsening of inflammation through the recruitment of leukocytes (neutrophils), which release cathepsin B into the SF. Thrombin can induce activation of cathepsin B in SFC via increased expression of IL-8.

Topics: Adult; Aged; Aged, 80 and over; Animals; Arthritis, Rheumatoid; Cathepsin B; Female; Fibroblasts; Humans; Interleukin-8; Male; Middle Aged; Osteoarthritis; Rabbits; Synovial Fluid; Synovial Membrane; Thrombin

The aim of this study was to assess the synovial fluid (SF) neurotransmitter excitatory amino acid (EAA) levels, including glutamate (Glu) and aspartate (Asp), in the context of SF levels of other amino acids, TNF-alpha and chemokines from patients with active arthropathies. The SF was collected from patients with active rheumatoid arthritis (RA), gout, or osteoarthritis (OA). The SF samples were analysed for levels of neurotransmitters glutamate and aspartate, tumour necrosis factor-alpha (TNF-alpha), Regulated upon Activation Normally T-cell Expressed and Secreted (RANTES), macrophage inhibitory factor-1 alpha (MIP-1alpha) and interleukin 8 (IL-8). SF WBC counts were also determined. Correlations between SF EAA, TNF-alpha and chemokines were determined by the Pearson product-moment correlation. Primary cultures derived from SF from active RA and gout patients were incubated with added l-glutamate, to assess if exposure to Glu could increase TNF-alpha levels. There were significant elevations in SF EAA, SF TNF-alpha and SF RANTES in RA patients compared to gout or OA patients. Significant correlations between SF EAA and SF RANTES, MIP-1alpha and IL-8 levels were seen, and SF EAA and SF TNF-alpha or SF WBC levels approached significance. Addition of exogenous neurotransmitter glutamate significantly increased TNF-alpha levels in primary cell cultures derived from RA and gout patients. The SF neurotransmitter EAA levels significantly correlated to selected SF chemokine levels, in clinically active RA, gout and OA patients, independent of disease. Added Glu resulted in significantly increased TNF-alpha levels in primary synovial cell cultures. These data expand the relationship of SF neurotransmitter EAA levels to SF cytokines and chemokines in patients with clinically active arthritis, and suggest that neurotransmitters Glu and Asp contribute to peripheral inflammatory processes.

Topics: Adult; Aged; Arthritis; Arthritis, Rheumatoid; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Chemokines; Chromatography, High Pressure Liquid; Excitatory Amino Acids; Female; Gout; Humans; Interleukin-8; Macrophage Inflammatory Proteins; Male; Middle Aged; Osteoarthritis; Synovial Fluid; Tumor Necrosis Factor-alpha

IL-1 is one of the key mediators involved in the pathogenesis of rheumatoid arthritis (RA) and is known to affect the level of gene expression in various settings. We investigated the effects of IL-1beta on the expression of 240 genes in rheumatoid synovial fibroblasts (RSFs) using a cDNA microarray. Total RNAs were prepared from RSFs stimulated with IL-1beta and hybridized to the microarray. The fluorescence intensity of each gene was compared between the control and IL-1beta-treated cells. To confirm the data obtained from the microarray analysis, the level of gene expression was also examined by ELISA, Northern blot, or Western blot depending on the genes to be analyzed. The genes whose levels were significantly changed by IL-1beta in the microarray analysis could be divided into three categories; inflammatory mediators, matrix-modifying enzymes, and apoptosis-associated molecules. The increase in the mRNA levels of IL-6, IL-8, MCP-1, and GRO-1 was confirmed by determining their protein levels from the cell culture supernatant using ELISA. The increase in the level of two matrix-degrading enzymes, MMP-1 and MMP-3, was reproducibly observed by an ELISA method, while the decrease in the level of TIMP-3, an inhibitor of MMPs, was confirmed by Northern blot analysis. The fluorescence intensity of two apoptosis-related genes, caspase-3 and Bcl-xL, was significantly lowered. The decreased protein level of caspase-3 was also found. Our data suggested that IL-1beta could provoke a series of responses in RSFs leading to the pathologic status of RA, including enhancement of inflammatory cytokines, imbalanced production of MMPs and TIMPs, and dysregulation of apoptosis.

Topics: Animals; Apoptosis; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Chemokine CXCL1; Chemokines, CXC; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinases; Oligonucleotide Array Sequence Analysis; RNA; Synovial Membrane

To investigate the effect of the p38 mitogen activated protein kinase (MAPK) inhibitor RWJ 67657 on inflammatory mediator production by rheumatoid synovial fibroblasts (RSF).. RSF were pretreated with RWJ 67657 and stimulated with TNF alpha and/or IL-1 beta. Protein levels and mRNA expression of MMP-1, MMP-3, TIMP-1, IL-6, and IL-8 were determined, as was mRNA expression of COX-2 and ADAMTS-4.. MMP-3 production was significantly inhibited at 1 microM RWJ 67657 and MMP-1 production at 10 microM, while TIMP-1 production was not inhibited. Inhibition of IL-6 and IL-8 protein production was seen at 0.1 microM RWJ 67657. Expression profiles of mRNA were in accordance with protein production. Inhibition of COX-2 mRNA expression occurred at 0.01 microM RWJ 67657.. RWJ 67657 inhibits major proinflammatory mediator production in stimulated RSF at pharmacologically relevant concentrations. These findings could have important relevance for the treatment of rheumatoid arthritis.

Topics: ADAM Proteins; ADAMTS4 Protein; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 2; Fibroblasts; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Imidazoles; Inflammation Mediators; Interleukin-1; Interleukin-6; Interleukin-8; Isoenzymes; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Membrane Proteins; Metalloendopeptidases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Procollagen N-Endopeptidase; Prostaglandin-Endoperoxide Synthases; Protein Kinase Inhibitors; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; Stimulation, Chemical; Synovial Membrane; Tissue Inhibitor of Metalloproteinase-1; Tumor Necrosis Factor-alpha

To investigate concentrations of proinflammatory cytokines in the sera and their mRNA expression in biopsy specimens of evanescent rash and synovitis from patients with active untreated adult onset Still's disease (AOSD).. We measured serum levels of interleukin 6 (IL-6), IL-8, and tumor necrosis factor (TNF-alpha) by immunochemiluminescence method and serum IL-18 levels by ELISA in 50 patients with active untreated AOSD, 20 patients with active rheumatoid arthritis (RA), and 20 healthy controls. Multivariate analysis was used to evaluate the correlation between serum cytokine levels and disease activity and clinical features of AOSD. We also evaluated the expression of cytokine transcripts by real-time quantitative polymerase chain reaction in biopsy specimens of evanescent rash and synovitis from 12 patients with active untreated AOSD.. Significantly higher levels of IL-6, IL-8, IL-18, and TNF-alpha in sera were found in patients with active untreated AOSD compared to healthy controls. Serum levels of IL-6 and IL-18 correlated well with clinical activity score of AOSD patients. Multiple logistic regression analysis showed that serum IL-6 level was a possible predictor for the occurrence of evanescent rash (p = 0.0593), serum IL-8 level was a significant predictor of persistent arthritis, and serum IL-18 level predicted occurrence of liver dysfunction. The levels of mRNA expression of IL-6, IL-18, and IL-8 were significantly higher in the biopsy tissue of Still's rash from AOSD patients compared with those in controls. Levels of mRNA expression of IL-18, IL-8, and TNF-alpha were significantly higher in the synovial membranes of AOSD patients compared with those in osteoarthritis controls. Significantly lower levels of TNF-alpha and IL-8 were found in the sera and in the synovial membranes of AOSD patients compared with those in RA patients. AOSD patients who had a chronic articular course had significantly higher levels of serum IL-8 compared with those who had a monocyclic systemic course.. Significantly higher levels of IL-6, IL-8, IL-18, and TNF-alpha were seen in both sera and pathological tissues of patients with active AOSD. The associations between levels of cytokine profile and distinct clinical manifestations and various patterns of disease course suggest the heterogeneity of pathogenesis in AOSD.

Topics: Adult; Arthritis, Rheumatoid; Cytokines; Exanthema; Female; Humans; Interleukin-18; Interleukin-6; Interleukin-8; Male; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Still's Disease, Adult-Onset; Synovitis; Tumor Necrosis Factor-alpha

There is evidence that autoimmunity plays a significant role in the pathogenesis of relapsing polychondritis (RP). This study was designed to investigate circulating levels of various cytokines in relation to the etiology of this rare disorder, and to compare the pattern of cytokine elevations in RP with that in another autoimmune disease, rheumatoid arthritis (RA).. Serum from 22 patients with active RP and an equal number of age- and sex-matched healthy controls and RA patients were available for analysis. The following cytokines were measured: interleukin-1beta (IL-1beta), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, interferon-gamma (IFNgamma), tumor necrosis factor alpha, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1beta (MIP-1beta). Results were analyzed by nonparametric Mann-Whitney test with Holm stepdown adjustment for multiple testing.. The levels of 3 of these cytokines showed significant differences between RP patients and controls. Compared with controls, mean serum levels of MCP-1, MIP-1beta, and IL-8 were all much higher in patients with active RP. In contrast, RA patients showed a more general increase in all cytokines measured, with much higher levels of IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IFNgamma, G-CSF, GM-CSF, MCP-1, and MIP-1beta compared with controls.. Levels of 3 serum cytokines were significantly higher in RP patients than in age- and sex-matched controls. One of these 3 cytokines, IL-8, was not significantly elevated in RA samples. Overall, in RP, a more discrete group of cytokines exhibited significantly increased levels than was found in RA. Each of the 3 cytokines that were elevated in RP is a proinflammatory chemokine, characteristic of activation of the monocyte and macrophage lineage, and in the case of IL-8, also of neutrophils. These data suggest a major role for a cell-mediated immune response in the pathophysiology of RP.

Topics: Adult; Aged; Arthritis, Rheumatoid; Autoimmunity; Case-Control Studies; Chemokine CCL2; Chemokine CCL4; Cytokines; Female; Humans; Interleukin-8; Macrophage Inflammatory Proteins; Macrophages; Male; Middle Aged; Monocytes; Polychondritis, Relapsing

The effect of platelet-derived growth factor (PDGF)-AA on the inflammation in rheumatoid arthritis (RA) and osteoarthritis (OA) was investigated using cultured fibroblast-like synoviocytes (FLS) obtained from RA and OA patients as well as control nonarthritic (NA) individuals. PDGF-AA increased the mRNA and protein expressions of proinflammatory cytokines, interleukin (IL)-1beta and IL-8 in RA FLS. Biological activity of IL-1 in the culture supernatant of RA FLS was also increased by PDGF-AA stimulation. Interestingly, PDGF-AA synergized with tumour necrosis factor (TNF)-alpha to upregulate the protein expressions of IL-1beta and IL-8. PDGF-induced enhancement of the IL-1beta and IL-8 mRNA expressions was also observed in OA FLS. However, the expression of these proinflammatory cytokines in NA FLS did not change by PDGF treatment, suggesting that the inflammatory condition might have modified the biological effects of PDGF. In accordance with the enhanced expression of inflammatory cytokines, the activity of nuclear factor kappaB was also induced in response to PDGF-AA in RA FLS. These results suggest that PDGF-AA plays an important role in the progression of RA inflammation, and inhibiting PDGF activity may be useful for the effective RA treatment.

Topics: Arthritis, Rheumatoid; Chemokine CCL4; Humans; Interleukin-1; Interleukin-8; Macrophage Inflammatory Proteins; NF-kappa B; Platelet-Derived Growth Factor; Synovial Membrane

Topics: Adult; Aged; Arthritis, Rheumatoid; Cytokines; Female; Humans; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Male; Middle Aged; Receptors, Interleukin-2

We determined serum metalloproteinase-3(MMP-3) and inflammatory cytokine(IL-6, IL-8) levels in patients with rheumatoid arthritis(RA). Sera were obtained from 307 healthy subjects(female 140, male 167), 54 RA patients, and 17 osteoarthritis (OA). The MMP-3 concentrations in healthy female and male were 43.3 +/- 15.3 ng/ml and 90.7 +/- 26.0 ng/ml, respectively. The serum MMP-3 levels in male were significantly higher than those in female (p < 0.0001). MMP-3 levels in RA patients(259.1 +/- 34.2 ng/ml) were significantly higher than OA(43.6 +/- 6.1 ng/ml) or healthy controls. There was a significant correlation between MMP-3 and CRP(r = 0.586), IL-6(r = 0.345) levels in serum. In contrast, no significant correlation was observed between MMP-3 and IL-8(r = 0.19), or CA-RF(r = 0.052) levels. However, there were some cases with high MMP-3 levels in CA-RF-negative patients definitely diagnosed as RA. These findings suggest that MMP-3 determination is useful for the early diagnosis and the follow-up during the treatment for RA patients.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biomarkers; Female; Follow-Up Studies; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Matrix Metalloproteinase 3; Middle Aged

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared with patients with osteoarthritis. This study examines differentially expressed genes after the stimulation of fibroblast-like synoviocytes of RA patients by IL-17. Among these genes we identified the following: tumor necrosis factor-stimulated gene-6 (TSG-6), IL-6, IL-8, GRO-beta, and bone morphogenetic protein-6 with an expression 3.6-10.6-fold that in the unstimulated control. IL-17 augmented the expression of TSG-6, a hyaluronan-binding protein, in a time- and dose-dependent manner. IL-17 showed additive effects with IL-1beta and tumour necrosis factor-alpha on the expression of TSG-6, IL-6 and IL-8. The mitogen-activated protein kinase p38 seems to be necessary for the regulation of TSG-6 expression by IL-17, as shown by inhibition with SB203580. Our results support the hypothesis that IL-17 is important in the pathogenesis of RA, contributing to an unbalanced production of cytokines as well as participating in connective tissue remodeling.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Adhesion Molecules; Cells, Cultured; Drug Synergism; Fibroblasts; Humans; Hyaluronic Acid; Interleukin-1; Interleukin-17; Interleukin-6; Interleukin-8; Middle Aged; Synovial Membrane; Transcriptional Activation; Tumor Necrosis Factor-alpha; Up-Regulation

To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA).. Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined.. IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13.. Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Collagenases; Cytokines; Endothelial Growth Factors; Endothelium, Vascular; Fibroblasts; Humans; I-kappa B Kinase; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-6; Interleukin-8; Lipopolysaccharides; Lymphokines; Macrophages; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinases; Monocytes; NF-kappa B; Protein Serine-Threonine Kinases; Skin; Synovial Membrane; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand dependent transcriptional factor known to be a regulator of adipogenesis. Recent studies have also shown that stimulation of PPARgamma inhibits the transcriptional activities of other nuclear factors and down-regulates proinflammatory cytokine synthesis in T cells and monocytes. We examined, in the present study, the functional significance of PPARgamma expressed in fibroblast-like synovial cells (FLS) isolated from patients with rheumatoid arthritis (RA). Incubation of FLS with a synthetic PPARgamma ligand, troglitazone, inhibited endogenous production of TNF-alpha, IL-6 and IL-8, as well as matrix metalloprotease-3 (MMP-3), without inducing apoptosis of the cells. The gelatinase activity of FLS culture media was also inhibited by troglitazone. Electrophoretic mobility shift assay (EMSA) showed a significant reduction in the DNA binding activity of NF-kappaB in troglitazone-treated FLS in response to TNF-alpha or IL-1beta. Moreover, long-term cultivation of FLS with troglitazone resulted in morphological changes with marked lipid accumulation in these cells. Our results show a negative regulatory function for PPARgamma on cytokine and MMP production together with inhibition of cytokine-mediated inflammatory responses in rheumatoid synovial cells. Our results also suggest that FLS could differentiate into adipocyte-like cells in the presence of proper stimulatory signals including PPARgamma.

Topics: Adipocytes; Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Chromans; DNA; Fibroblasts; Gelatinases; Humans; In Vitro Techniques; Interleukin-6; Interleukin-8; Ligands; Matrix Metalloproteinase Inhibitors; NF-kappa B; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Synovial Membrane; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is an aggressive inflammatory disease in which chemokines are thought to recruit leukocytes and induce angiogenesis. The aim of this study was to investigate the effects of sulfasalazine (SASP) and its metabolites, sulfapyridine (SP), and 5-aminosalicylic acid (5ASA) on chemokine production by RA synovial tissue explants and interleukin (IL)-1beta-stimulated RA synovial tissue fibroblasts using enzyme-linked immunosorbent assays and flow cytometry. Synovial tissue explants from RA patients secreted a decreased amount of the chemokines IL-8 and growth-related gene product alpha (GROalpha) when treated with SASP over a broad range of concentrations based on the typical clinical dosage of 2 g/day. SP had a significant effect in that it decreased RA synovial tissue explant secretion of IL-8 (22%), GROalpha (55%), and monocyte chemotactic protein-1 (MCP-1) (42%) (P < 0.05). 5ASA had no effect on RA synovial tissue explant production of IL-8 and MCP-1, while increasing GROalpha production. In IL-1beta-stimulated RA synovial tissue fibroblasts, SASP significantly increased chemokine secretion, while SP significantly decreased IL-8 (24%) and GROalpha (21%) secretion (P < 0.05). Flow cytometry showed that the number of IL-8 expressing RA synovial tissue fibroblasts did not significantly change following SP treatment. These data suggest that SASP may function to reduce inflammation in RA through the effects of its metabolite SP to reduce the secretion of the inflammatory chemokines IL-8, GROalpha, and MCP-1.

Topics: Adult; Aged; Anti-Inflammatory Agents, Non-Steroidal; Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Culture Media, Conditioned; Female; Fibroblasts; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-8; Male; Mesalamine; Middle Aged; Sulfapyridine; Sulfasalazine; Synovial Membrane

In synovial fluid (SF) from patients with rheumatoid arthritis (RA), neutrophils are exposed to proinflammatory mediators endowed with either anti-apoptotic or pro-apoptotic properties. We investigated neutrophil apoptosis in the presence of SF from 11 RA patients.. SF was obtained from affected knees of 11 patients with RA. Human neutrophil apoptosis was evaluated by light microscopic examination and flow-cytometric analysis of annexin V binding. Immune complex-induced neutrophil activation was evaluated as superoxide anion production. Adenosine levels in SF were detected by chromatographic analysis and cytokine levels were studied by enzyme-linked immunosorbent assay.. Spontaneous and immune complex-triggered neutrophil apoptosis was reduced by SF from eight out of 11 patients. Immune complex-induced neutrophil activation was unaffected by SF. The cytokines tested had no role in promoting the anti-apoptotic activity of SF. On the contrary, the anti-apoptotic activity of SF was found to depend on the presence of adenosine. Adenosine levels detected in the various samples of SF correlated significantly with the anti-apoptotic activity of the fluids and with the number of apoptotic neutrophils detected in the articular exudate.. The microenvironment of rheumatoid SF is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils. Adenosine plays a crucial role in this phenomenon, which is related to anti-apoptotic activity.

Topics: Adenosine; Adult; Apoptosis; Arthritis, Rheumatoid; Cells, Cultured; Culture Media; Cytokines; Female; Flow Cytometry; Fluorescent Antibody Technique; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-15; Interleukin-2; Interleukin-8; Knee Joint; Male; Middle Aged; Neutrophils; Sensitivity and Specificity; Synovial Fluid; Tumor Necrosis Factor-alpha

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits (125)I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC(50) = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC(50) = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-alpha, IL-8, PGE(2), leukotriene B(4), and leukotriene C(4) levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-alpha and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC(50) = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Chronic Disease; Female; Humans; In Vitro Techniques; Interleukin-8; Lipopolysaccharides; Neutrophils; Ovalbumin; Rabbits; Receptors, Interleukin-8B; Recombinant Proteins; Urea

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.

Topics: Antibodies, Monoclonal; Arthritis, Rheumatoid; B-Lymphocytes; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Humans; Integrin alpha4; Integrin alpha4beta1; Integrin beta1; Interleukin-6; Interleukin-8; Stromal Cells; Synovial Membrane

Bone turnover markers (osteocalcin, bone-ALP, beta-crosslaps--CTX) and cytokines (IL-1 alpha, IL-8 and IL-10) in hip joint fluid were analyzed in patients with aseptic loosening of prosthesis before revision surgery, in patients with rheumatoid arthritis and with idiopathic coxarthrosis for comparison.. Bone turnover markers were determined by electrochemiluminescence, colorimetric or ELISA method and cytokines by ELISA in joint fluid collected at the beginning of the surgery.. Patients with loose implants had lower concentration of the resorption marker than cases with rheumatoid arthritis and coxarthrosis (7771 +/- 3322 vs 25986 +/- 16059 p < 0.01 and 23047 +/- 32556 pmol/L, p < 0.003) and over tenfold lower concentration of osteocalcin, the bone formation marker (p < 0.04 i p < 0.01). Concentration of IL-8 was elevated and similar in patients with loosening and rheumatoid arthritis while in cases with osteoarthrosis the mean value was twice lower. The anti-inflammatory IL-10 was highly elevated only in cases with prosthesis loosening. Additionally, a negative correlation was observed between CTX and IL-10 was and positive between IL-10 and time to revision surgery.. We conclude that increased local production of inflammatory cytokines leading to uncoupling of bone turnover is a part of the loosening process.

Topics: Aged; Aged, 80 and over; Alkaline Phosphatase; Arthritis, Rheumatoid; Arthroplasty, Replacement, Hip; Biomarkers; Bone Resorption; Collagen; Colorimetry; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Hip Joint; Humans; Interleukin-1; Interleukin-10; Interleukin-8; Male; Middle Aged; Osteoarthritis, Hip; Osteocalcin; Peptide Fragments; Prosthesis Failure; Synovial Fluid

During acute inflammation, leukocytes release proteolytic enzymes including matrix metalloproteinases (MMPs), but the physiopathological mechanisms and consequences of this process are not yet fully understood. Neutrophils, the predominant leukocyte type, produce neutrophil collagenase (MMP-8) and gelatinase B (MMP-9) but not the tissue inhibitors of MMPs. After stimulation, these cells also activate MMPs chemically. In arthritic diseases, neutrophils undergo great chemoattraction to the synovium, are activated by interleukin-8, and are stimulated to release gelatinase B in vivo. Production levels and net activities of gelatinase B were found to be absent in degenerative osteoarthritis but significantly increased in rheumatoid arthritis. The cleavage sites in cartilage type II collagen by gelatinase B were determined by a combination of reverse phase high-performance liquid chromatography, Edman degradation, and mass spectrometry analysis. The analysis revealed the site specificity of proline and lysine hydroxylations and O-linked glycosylation, the cleavage specificities by gelatinase B, and the preferential absence and presence of post-translational modifications at P2' and P5', respectively. Furthermore, gelatinase B leaves the immunodominant peptides intact, which are known from studies with (autoreactive) T cells. Lysine hydroxylation was detected at a critical position for T-cell activation. These data lend support to the thesis that extracellular proteolysis and other post-translational modifications of antigenic peptides may be critical in the establishment and perpetuation of autoimmune processes.

Topics: Amino Acid Sequence; Animals; Arthritis, Rheumatoid; Binding Sites; Cattle; Collagen Type II; Humans; Immunodominant Epitopes; Interleukin-8; Matrix Metalloproteinase 9; Models, Immunological; Molecular Sequence Data; Neutrophils; Peptide Fragments; Protein Denaturation; Protein Processing, Post-Translational; Substrate Specificity; Synovial Fluid

An excess of the proinflammatory substance IL-18 is present in joints of patients with rheumatoid arthritis (RA), and expression of IL-18 receptor (IL-18R) regulates IL-18 bioactivity in various cell types. We examined the expression of IL-18R alpha-chain and beta-chain and the biologic effects of IL-18 in fibroblast-like synoviocytes (FLS) after long-term culture. The presence of both IL-18R chains was a prerequisite for IL-18 signal transduction in FLS. However, all FLS cultures studied were either resistant or barely responsive to IL-18 stimulation as regards cell proliferation, expression of adhesion molecules ICAM-1 and vascular cell adhesion molecule (VCAM)-1, and the release of interstitial collagenase and stromelysin, IL-6 and IL-8, prostaglandin E2, or nitric oxide. We conclude that the presence of macrophages or IL-18R+ T cells that can respond directly to IL-18 is essential for the proinflammatory effects of IL-18 in synovitis in RA.

Topics: Arthritis, Rheumatoid; Cell Division; Cells, Cultured; Collagenases; Culture Media, Conditioned; Dinoprostone; Fibroblasts; Humans; Intercellular Adhesion Molecule-1; Interleukin-18; Interleukin-18 Receptor alpha Subunit; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Nitric Oxide; Receptors, Interleukin; Receptors, Interleukin-18; RNA, Messenger; Signal Transduction; Synovial Membrane; U937 Cells; Vascular Cell Adhesion Molecule-1

Density of sympathetic nerve fibers in synovial tissue was lower in patients with rheumatoid arthritis (RA) compared to those with osteoarthritis (OA). This was accompanied by norepinephrine (NE) release from synovial tyrosine hydroxylase positive cells (TH+ cells). We investigated the role of TH+ cells and NE in synovial inflammation.. Synovial tissue of 34 patients with RA and 36 with OA who underwent knee joint replacement surgery was characterized using immunohistochemistry and a synovial tissue superfusion technique, respectively. In culture experiments with mixed synoviocytes, the effect of NE on secretion of interleukin 6 (IL-6), IL-8, tumor necrosis factor (TNF), and matrix metalloproteinase-3 (MMP-3) was investigated.. Tissue density of TH+ cells was higher in RA compared to OA (63.9 vs 34.2 cells/mm2; p = 0.017). Basal NE release from synovial tissue correlated highly significantly with density of TH+ cells in RA (Rrank = 0.573, p = 0.001) but not in OA (Rrank = 0.102, NS). Basal NE release correlated with the degree of inflammation in RA (Rrank = 0.420, p = 0.021) but not in OA (Rrank = 0.174, NS), and with spontaneous IL-8 secretion in RA (Rrank = 0.581, p = 0.001) but not in OA (Rrank = 0.160, NS). Only in RA, density of TH+ cells correlated positively with spontaneous secretion of IL-6, IL-8, and MMP-3. We confirmed the extensive loss of sympathetic nerve fibers in RA compared to OA (0.32 vs 3.1 nerve fiber/mm2; p < 0.001). The ratio of sympathetic to sensory nerve fibers was 1 to 5 in RA and 2 to 1 in OA. A ratio of 1.0 separates almost all patients into 2 diseases groups (RA vs OA). Prior prednisolone treatment of RA patients was related to decreased spontaneous cytokine secretion, a lower density of T cells, CD163+ macrophages and TH+ cells, a lower degree of inflammation, and reduced synovial NE secretion. NE was able to inhibit secretion of IL-6 (in OA), IL-8 (in RA), and TNF (in RA and OA) in culture experiments.. TH+ cells and release of NE are strongly linked to a higher degree of synovial inflammation. Culture experiments indicate that NE has antiinflammatory properties at higher concentrations (10(-5) M). NE secretion of TH+ cells may be an antiinflammatory mechanism to counteract local inflammation. Thus, TH+ cell derived NE can be an important local factor of immunomodulation in synovial inflammation.

Topics: Adrenergic Fibers; Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biomarkers; Cell Count; Cells, Cultured; Humans; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 3; Middle Aged; Norepinephrine; Osteoarthritis; Predictive Value of Tests; Substance P; Synovial Membrane; Tumor Necrosis Factor-alpha; Tyrosine 3-Monooxygenase

Interleukin-17 (IL-17) has been characterized as a proinflammatory cytokine produced by CD4+ CD45RO+ memory T cells. Overproduction of IL-17 was detected in the synovium of patients with rheumatoid arthritis (RA) compared to patients with osteoarthritis. In contrast to the restricted expression of IL-17, the IL-17 receptor (IL-17R/CDw217) is expressed ubiquitously. Using a real-time RT-PCR assay, we detected similar absolute levels of IL-17R mRNA expression in fibroblast-like synoviocytes (SFC) from patients with RA (mean 9 pg/microg total RNA; ranged from 0.1 pg to 96 pg IL-17R mRNA/microg total RNA) compared to synoviocytes of non-RA patients. Analysis of the IL-17R surface expression confirmed the results obtained for IL-17R mRNA expression. Exposure of SFC to IL-17 led to a mRNA induction of CXC chemokines IL-8, GRO-alpha and GRO-beta. An anti-IL-17 antibody blocked these effects of IL-17. The MAPK p38 appears necessary for the regulation of IL-8, GRO-alpha and GRO-beta expression as shown by inhibition with SB203580. The inhibitors genistein (tyrosine kinase inhibitor) and calphostin C (inhibitor of protein kinase C) reduced significantly the IL-17-stimulated mRNA expression of IL-8, GRO-alpha and GRO-beta in SFC, whereas PD98059 (inhibitor of MEK-1/2) was without effect. Pharmacological drugs used in therapy of RA, such as cyclosporin and methotrexate, induced a fourfold increase of IL-17R mRNA expression and augmented the IL-17-stimulated IL-8 expression. Our results support the hypothesis that IL-17/IL-17R may play a significant role in the pathogenesis of RA contributing to an unbalanced production of cytokines as well as participating in connective tissue remodelling.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Cyclosporine; Fibroblasts; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-17; Interleukin-8; Kinetics; MAP Kinase Signaling System; Methotrexate; NF-kappa B; Receptors, Interleukin; Receptors, Interleukin-17; Recombinant Proteins; RNA, Messenger; Signal Transduction; Synovial Membrane; Transcription, Genetic; U937 Cells

Transforming growth factor (TGF)-beta1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF-beta1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non-arthritic individuals. mRNA expressions of IL-1beta, tumour necrosis factor (TNF)-alpha, IL-8, macrophage inflammatory protein (MIP)-1alpha and metalloproteinase (MMP)-1 were increased in RA and OA FLS by TGF-beta1 treatment, but not in non-arthritic FLS. Enhanced protein expression of IL-1beta, IL-8 and MMP-1 was also observed in RA FLS. Moreover, TGF-beta1 showed a synergistic effect in increasing protein expression of IL-1beta and matrix metalloproteinase (MMP)-1 with TNFalpha and IL-1beta, respectively. Biological activity of IL-1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF-beta1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)-kappaB and activator protein (AP)-1 were shown to increase by TGF-beta1 as well. These results suggest that TGF-beta1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL3; Chemokine CCL4; Cytokines; Fibroblasts; Humans; Interleukin-1; Interleukin-8; Macrophage Inflammatory Proteins; Matrix Metalloproteinase 1; NF-kappa B; Osteoarthritis; RNA, Messenger; Synovial Membrane; Transcription Factor AP-1; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.

Topics: Angiogenesis Inhibitors; Animals; Arthritis, Rheumatoid; Chemokine CCL2; Collagen; Electrophoretic Mobility Shift Assay; Endostatins; Endothelial Growth Factors; Endothelium, Vascular; Gene Transfer Techniques; Genetic Therapy; Humans; Immunoenzyme Techniques; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Joints; Lymphokines; MAP Kinase Kinase 4; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase Kinases; Neovascularization, Pathologic; Peptide Fragments; RNA; Synovitis; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Macrophages that accumulate in the synovium of rheumatoid arthritis patients play an important role in the pathogenesis of this inflammatory disease. However, the mechanism by which macrophages are attracted into the inflamed synovium and accumulate there has not been completely delineated. The results of this study show that rheumatoid arthritis synovial stromal cells produce the chemokines monocyte chemotactic protein-1 and IL-8, and these have the capacity to attract peripheral monocytes. These results suggest that one of the mechanisms by which macrophages accumulate in the inflamed synovium is by responding to the chemokines produced locally.

Topics: Adjuvants, Immunologic; Arthritis, Rheumatoid; Cell Line; Cell Movement; Cell-Free System; Chemokine CCL2; Humans; Interleukin-8; Monocytes; Receptors, Chemokine; RNA, Messenger; Stromal Cells; Synovial Membrane; Tumor Necrosis Factor-alpha

Paired synovial tissue samples were obtained from both clinically uninvolved (CU) and clinically involved (CI) knee joints of eight rheumatoid arthritis (RA) patients. In addition, biopsies were taken from five control subjects. We observed the expression of the chemokines CXCL8, CXCL9, CXCL10, CCL2 and CCL4 in CI and CU joints of RA patients. In particular, CXCL8 protein levels were specifically increased in CI joints compared with CU joints, which was confirmed by immunohistochemistry and in situ hybridization.

Topics: Arthritis, Rheumatoid; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin-8; Knee Joint; Synovial Membrane

Angiogenesis, the growth of new blood vessels, is vital to the ingress of inflammatory leukocytes in rheumatoid arthritis (RA) synovial tissue and to the growth and proliferation of RA pannus. The factors that mediate the growth of new blood vessels have not been completely defined. This study examined the ability of Glu-Leu-Arg (ELR)-containing chemokines to induce angiogenesis in the RA joint.. To reflect angiogenic activity in vivo, we selected a model using whole human synovial tissue rather than isolated cells. Tissues were examined by immunohistochemistry and enzyme-linked immunosorbent assay, and tissue homogenates were immunoneutralized and assayed for their ability to induce endothelial cell chemotaxis and rat corneal neovascularization.. Cells expressing interleukin-8 (IL-8) and epithelial neutrophil activating peptide 78 (ENA-78) were located in proximity to factor VIII-related antigen-immunopositive endothelial cells. RA homogenates produced more IL-8 and ENA-78 compared with normal synovial tissue homogenates. Moreover, homogenates from RA synovial tissue produced significantly more chemotactic activity for endothelial cells in vitro and angiogenic activity in the rat cornea in vivo than did normal synovial tissue homogenates. The effects of IL-8 and ENA-78 accounted for a significant proportion of the chemotactic activity of endothelial cells and angiogenic activity found in RA synovial tissue homogenates.. These results indicate that the ELR-containing chemokines IL-8 and ENA-78 are important contributors to the angiogenic activity found in the inflamed RA joint. It is possible that efforts aimed at down-regulating these chemokines offer a novel targeted therapy for the treatment of RA.

Topics: Adult; Aged; Aged, 80 and over; Animals; Arthritis, Rheumatoid; Arthroplasty; Chemokine CXCL5; Chemokines, CXC; Chemotaxis, Leukocyte; Endothelium, Vascular; Female; Humans; Interleukin-8; Male; Middle Aged; Neovascularization, Pathologic; Neutrophil Activation; Synovial Membrane

The difference in cell proliferation and release of soluble factors in response to interleukin 1beta (IL-1beta) in fibroblasts obtained from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) and from normal skin has been investigated.. The cells were treated with recombinant IL-1beta in the presence or absence of pharmacological agents for 24 h or 48 h.. Cell proliferation was examined by WST-1 assay, and the amounts of interleukin-6 (IL-6), interleukin-8 (IL-8), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF), matrix metalloproteinase-1 (MMP-1), and prostaglandin E2 (PGE2) were measured by enzyme linked immunosorbent assay (ELISA).. IL-1beta dose-dependently enhanced the proliferation of all fibroblasts. The proliferative response to IL-1beta in RA synovial fibroblasts was greater than that in OA synovial and skin fibroblasts. However, there was no difference in spontaneous levels of soluble factors between OA and RA fibroblasts, though medium concentrations of IL-1beta-released VEGF, MMP-1, and PGE2, but not cytokines, in RA were slightly higher than those in OA. Ability to release soluble mediators was pronouncedly increased at 3 h to 9 h after stimulating fibroblasts with IL-1beta for 1 h. The proliferative response to IL-1beta in all fibroblasts was inhibited by dexamethasone and the NF-kappaB inhibitor hymenialdisine but not the cyclooxygenase 2 (COX-2) inhibitor NS-398. But PGE2 prevented proliferation of RA fibroblasts when added to medium up to 3 h after IL-1beta stimulation. Dexamethasone also inhibited the release of IL-6, IL-8, and PGE2 induced by IL-1beta in both OA and RA fibroblasts. NS-398 exhibited an inhibition of IL-1beta-induced IL-6 production as well as PGE2 production. Hymenialdisine inhibited IL-6 production and reduced IL-8 production dependent on synovial cell strains. Methotrexate had no effect on the response to IL-1beta in synovial fibroblasts.. The present results indicate that the activation of NF-kappaB plays an important role in the proliferative response to IL-1beta in human fibroblasts, and suggest that PGE2 acts as a modulator of cell proliferation in inflamed synovial tissue. It appears that the ability to produce soluble factors in RA synovial fibroblasts is not intrinsic. However, the response to IL-1beta in RA cells seems to be greater than that in OA cells.

Topics: Arthritis, Rheumatoid; Cell Division; Dexamethasone; Dinoprostone; Endothelial Growth Factors; Fibroblasts; Glucocorticoids; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Kinetics; Lymphokines; Macrophage Colony-Stimulating Factor; Matrix Metalloproteinase 1; Osteoarthritis; Solubility; Synovial Membrane; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p < 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 +/- 5% decrease in HMVEC migration (p < 0.05). IL-18 appears to act on HMVECs via alpha(v)beta(3) integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p < 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p < 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-alpha, as evidenced by adding neutralizing anti-TNF-alpha Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p < 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.

Topics: Angiogenesis Inducing Agents; Animals; Arthritis, Rheumatoid; Cell Division; Cell Line; Cell Migration Inhibition; Cell Movement; Chemokine CXCL5; Chemokines; Chemokines, CXC; Chemotactic Factors; Collagen; Drug Combinations; Drug Implants; Endothelium, Vascular; Granuloma; Humans; Immune Sera; Interleukin-18; Interleukin-8; Laminin; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Porifera; Proteoglycans; Receptors, Vitronectin; Recombinant Proteins; Synovial Fluid; Tumor Necrosis Factor-alpha

As in rheumatoid arthritis (RA), it was demonstrated recently that bacterial fragments of DNA or rRNA are present in the joint and therefore could play a role in inducing or perpetuating the disease, this work was initiated to define mechanisms that account for the stimulatory activities of the oral streptococcal modulin, protein I/II, on fibroblast-like synoviocytes (FLSs) from RA patients. FLSs from RA patients were stimulated with protein I/II, and expression of interleukin (IL)-6 and IL-8 mRNA was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Immunoblotting by antibodies specific for activated forms of MAPKs and electrophoretic mobility shift assays (EMSAs) were performed to study downstream signalling, which allowed the synthesis of IL-6 and IL-8. We reported that protein I/II interactions with FLSs from RA patients trigger the synthesis and release of IL-6 and IL-8. We also demonstrated that protein I/II enhances the phosphorylation of ERK 1/2, p38 and JNKs and that ERK 1/2 and JNK MAPKs seem to play a more important role than p38 in protein I/II-mediated synthesis of IL-6 and IL-8. Our experiments also indicated that stimulation of FLSs with protein I/II induces nuclear translocation of NF-kappaB, AP-1-binding activity and that NF-kappaB plays a major role in IL-6 and IL-8 secretion from activated cells.

Topics: Anti-Bacterial Agents; Apigenin; Arthritis, Rheumatoid; Bacterial Proteins; Cells, Cultured; Curcumin; Enzyme Inhibitors; Flavonoids; Humans; Imidazoles; Interleukin-6; Interleukin-8; MAP Kinase Signaling System; Membrane Glycoproteins; NF-kappa B; Pyridines; Signal Transduction; Streptococcus; Sulfasalazine; Synovial Membrane

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.

Topics: Arthritis, Rheumatoid; Chemokine CCL2; Chemokine CCL5; Chemokine CXCL12; Chemokines, CXC; Enzyme Activation; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Receptors, Chemokine; Synovial Membrane

Topics: Arthritis, Rheumatoid; Cell Line; Dose-Response Relationship, Drug; Fibroblasts; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Synovial Membrane; Tumor Necrosis Factor-alpha

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor alpha (TNF-alpha)- or interleukin-1beta (IL-1beta)-induced production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor kappaB (NF-kappaB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Cell Nucleus; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; Interleukin-1; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha

To verify the hypothesis that in rheumatoid arthritis (RA), tumor necrosis factor alpha (TNFalpha) plays a critical role in regulating leukocyte trafficking and chemokine levels.. Ten patients with longstanding RA received a single 10 mg/kg infusion of anti-TNFalpha monoclonal antibody (cA2). The articular localization of autologous granulocytes, separated in vitro and labeled with 111In, was studied by analysis of gamma-camera images both before and 2 weeks after treatment. At the same sequential time points, synovial biopsy samples were assessed for infiltrating CD3+ T cells, CD22+ B cells, and CD68+ macrophages. Synovial tissue expression of the chemokines interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, Groalpha, and RANTES was also determined. Serum IL-8 and MCP-1 concentrations were measured by enzyme-linked immunosorbent assay.. Anti-TNFalpha therapy in RA significantly reduced 111In-labeled granulocyte migration into affected joints. There was a simultaneous and significant reduction in the numbers of infiltrating synovial CD3+ T cells, CD22+ B cells, and CD68+ macrophages and in the expression of IL-8 and MCP-1, with a trend toward a reduction in serum concentrations of these chemokines.. TNFalpha blockade reduces synovial expression of the chemokines IL-8 and MCP-1 and diminishes inflammatory cell migration into RA joints.

Topics: Aged; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Arthritis, Rheumatoid; B-Lymphocytes; CD3 Complex; Cell Adhesion Molecules; Cell Movement; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Female; Growth Substances; Humans; Immunoglobulins, Intravenous; Indium Radioisotopes; Intercellular Signaling Peptides and Proteins; Interleukin-8; Joints; Lectins; Leukocytes; Macrophage Inflammatory Proteins; Male; Middle Aged; Neutrophils; Radionuclide Imaging; Sialic Acid Binding Ig-like Lectin 2; Synovial Fluid; Synovial Membrane; T-Lymphocytes; Tumor Necrosis Factor-alpha

Rheumatoid arthritis (RA) is accompanied by synovial inflammation, proliferation, and cartilage destruction. The reasons the activation of synovial fibroblasts often persists despite antiinflammatory therapy are not known. One possibility is that the synovial membrane becomes gradually repopulated with immature mesenchymal and bone marrow cells with altered properties. To explore this hypothesis, we have investigated the expression in RA synovial tissues of various embryonic growth factors from the wingless (wnt) and frizzled (fz) families, which have been implicated in cell-fate determination in both bone marrow progenitors and limb-bud mesenchyme. Reverse transcriptase-PCR analysis revealed expression of five wnt (wnt1, 5a, 10b, 11, and 13) and three fz (fz2, 5, and 7) isoforms in RA synovial tissues. Osteoarthritis synovial tissues expressed much less wnt5a and fz5. Northern blotting confirmed the overexpression of wnt5a and fz5 in RA synovial tissues, in comparison to a panel of normal adult tissues. Compared with normal synovial fibroblasts, cultured RA fibroblast-like synoviocytes expressed higher levels of IL-6, IL-8, and IL-15. Transfection of normal fibroblasts with a wnt5a expression vector reproduced this pattern of cytokine expression and stimulated IL-15 secretion. These results suggest that the unusual phenotypic properties of RA fibroblasts may be attributable partly to their replacement with primitive fibroblast-like synoviocytes with characteristics of immature bone marrow and mesenchymal cells. Clear delineation of the signaling pathway(s) initiated by the wnt5a/fz5 ligand-receptor pair in the RA synovium may yield new targets for therapeutic intervention.

Topics: Arthritis, Rheumatoid; Blotting, Western; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Frizzled Receptors; Humans; Interleukin-15; Interleukin-6; Interleukin-8; Ligands; Osteoarthritis; Protein Isoforms; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Receptors, Neurotransmitter; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Tissue Distribution; Transfection; Wnt Proteins; Wnt-5a Protein

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial beta-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1beta by 85% after 24 h. Likewise, TNF-alpha levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-alpha levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1alpha production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.

Topics: Adenoviridae; Adult; Arthritis, Rheumatoid; Chemokine CCL2; Chemokine CCL4; Chemokine CCL5; Chemokine CXCL1; Chemokine CXCL5; Chemokines, CXC; Chemotactic Factors; Culture Media, Conditioned; Cytokines; Dinoprostone; Female; Genetic Therapy; Genetic Vectors; Growth Substances; Humans; Hyaluronan Receptors; Intercellular Adhesion Molecule-1; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-13; Interleukin-8; Macrophage Inflammatory Proteins; Male; Middle Aged; Organ Culture Techniques; Recombinant Proteins; Solubility; Synovial Membrane; Tumor Necrosis Factor-alpha

Taurine chloramine (Tau-Cl) has been shown to inhibit the production of proinflammatory cytokines (interleukin-6 [IL-6] and IL-8) by fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. The present study was conducted to elucidate the mechanism of inhibitory action exerted by Tau-Cl.. The effects of Tau-Cl on 1) the transcription of genes coding for IL-6 and IL-8, and 2) the activity of nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors, which are crucial for the transcription of these cytokine genes, were investigated in FLS isolated from the synovial tissue of RA patients. FLS were cultured in vitro for 3-6 passages and stimulated with recombinant human IL-1beta (1 ng/ml) in the presence of either Tau or Tau-Cl, which were added simultaneously with the stimulus at concentrations of 250 microM or 500 microM. The relative expression of IL-6 and IL-8 messenger RNA (mRNA) was evaluated after 4 hours of stimulation, using competitive reverse transcriptase-polymerase chain reaction. The DNA binding activity of NF-kappaB and AP-1 was examined 30 minutes and 2 hours after cell stimulation, respectively, using electromobility gel shift assay.. IL-1beta triggered a significant rise in the activity of transcription factors NF-kappaB and AP-1, followed by an elevation of cytokine IL-6 and IL-8 mRNA expression. Tau-Cl, but not Tau, reduced IL-1beta-triggered cytokine mRNA expression, exerting stronger inhibitory activity on the levels of IL-6 than on those of IL-8. Importantly, Tau-Cl also diminished the activity of NF-kappaB and, to a lesser extent, that of AP-1 transcription factor. Neither IL-1beta nor Tau-Cl affected the activity of octamer transcription factor 1.. Tau-Cl inhibition of IL-6 and IL-8 synthesis in FLS from RA patients results from the ability of this compound to diminish the activity of the major transcriptional regulators (NF-kappaB and AP-1), which subsequently reduces the transcription of these cytokine genes.

Topics: Aged; Arthritis, Rheumatoid; DNA-Binding Proteins; Female; Fibroblasts; Host Cell Factor C1; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Male; Middle Aged; NF-kappa B; Octamer Transcription Factor-1; Synovial Membrane; Taurine; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic

The sensory nervous system with the 2 neurotransmitters substance P (SP) and calcitonin gene related peptide (CGRP) is proinflammatory in experimental models of arthritis. The role of the sympathetic nervous system with norepinephrine (NE), adenosine, beta-endorphin, and methionine enkephalin (MENK) is not clearly understood. We studied the influence of these neurotransmitters on secretion of interleukin 6 (IL-6) and IL-8 in primary cultures of synovial fibroblasts of patients with rheumatoid arthritis (RA) compared to osteoarthritis (OA).. Fibroblasts were isolated using fresh synovial tissue of 5 patients with RA and 5 with OA who underwent knee joint replacement surgery. Modulation of spontaneous secretion of IL-6 and IL-8 was investigated in vitro using the neurotransmitters noted above.. In RA fibroblasts, CGRP increased IL-6 and IL-8 secretion at 10(-10) to 10(-8) M (p at least < 0.01), which was not observed in OA fibroblasts. SP had no effect on either cytokine in RA fibroblasts but stimulated IL-8 secretion at 10(-8) M in OA fibroblasts (p < 0.01). In RA fibroblasts, adenosine and NE inhibited secretion of both cytokines at low concentrations (10(-8) M; p < 0.01). However, in OA fibroblasts there was a NE induced increase of IL-8 and IL-6 secretion at 10(-7) and 10(-6) M (p < 0.01), but no inhibition at lower concentrations (10(-8) M; p = NS). In RA fibroblasts, beta-endorphin and MENK inhibited IL-8 secretion at 10(-9) to 10(-7) M (p < 0.01), whereas in OA fibroblasts the dose response curve was shifted to lower concentrations (10(-12) M, 10(-11) M; p < 0.01).. In OA fibroblasts, the sympathetic neurotransmitters were stimulatory at higher concentrations. CGRP was the most potent stimulatory neurotransmitter in RA fibroblasts whereas the sympathetic adenosine, NE, beta-endorphin, and MENK were inhibitory. This indicates a dualism of action of sympathetic and sensory neurotransmitters, with inhibitory and stimulatory effects on cytokine secretion of RA fibroblasts.

Topics: Aged; Arthritis, Rheumatoid; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Middle Aged; Neurons, Afferent; Neurotransmitter Agents; Osteoarthritis; Sympathetic Nervous System; Synovial Membrane

Concentrations of interleukin (IL)-6 and IL-8 in serum and synovial fluid obtained from patients with osteoarthritis (OA) of the knee were determined by the chemiluminescence-ELISA (CL-ELISA) method, the sensitivity of which is 100-1,000 times greater than that of the conventional ELISA method. The results were compared with those obtained from patients with rheumatoid arthritis (RA) and from healthy subjects. The mean IL-6 and IL-8 levels in synovial fluid indicated higher concentrations in RA than in OA. The IL-6 and IL-8 levels in serum were significantly higher in RA and OA relative to controls. Among OA patients in whom remarkable improvement was noted in hydrarthrosis, the synovial fluid IL-6 and IL-8 levels at the initial examination were relatively higher, and were markedly decreased after treatment with sodium hyaluronate (NaHA). Among those in whom no improvement was noted in hydrarthrosis, the synovial fluid IL-6 and IL-8 levels at the time of initial examination were relatively lower, and hydrarthrosis was not significantly improved even after treatment with NaHA. In addition, there was a tendency for the synovial fluid IL-6 and IL-8 levels to decrease as HA levels increased. Evaluation of X-ray findings revealed that the IL-6 levels in synovial fluid at the initial examination in low-grade cases tended to be significantly higher than in high-grade cases. In low-grade cases, as determined by X-ray findings, there was a significant decrease in IL-6 levels in synovial fluid after treatment with NaHA.

Topics: Adjuvants, Immunologic; Arthritis, Rheumatoid; Biomarkers; Enzyme-Linked Immunosorbent Assay; Humans; Hyaluronic Acid; Interleukin-6; Interleukin-8; Knee Joint; Luminescent Measurements; Osteoarthritis; Reference Values; Regression Analysis; Synovial Fluid

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-kappaB, C/EBPbeta, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IkappaBalpha, or dominant-negative versions of C/EBPbeta or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-kappaB activation significantly reduced the spontaneous and IL-1beta-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1ss-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPbeta modestly reduced constitutive and IL-1beta-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-kappaB, C/EBPbeta, and c-Jun were constitutively activated in RA FLS, but only NF-kappaB and c-Jun activity increased after IL-1beta. The reduction of cytokines by IkappaBalpha was mediated through inhibition of NF-kappaB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-kappaB was essential for IL-6 expression, because fibroblasts in which both NF-kappaB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-kappaB for the regulation of the constitutive and IL-1beta-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-kappaB as a therapeutic goal in RA.

Topics: Adenoviruses, Human; Arthritis, Rheumatoid; CCAAT-Enhancer-Binding Proteins; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Immunologic; Fibroblasts; Genetic Vectors; Humans; I-kappa B Proteins; Interleukin-1; Interleukin-6; Interleukin-8; NF-kappa B; NF-KappaB Inhibitor alpha; Proto-Oncogene Proteins c-jun; Skin; Synovial Membrane; Transcription Factor AP-1; Transcriptional Activation

The analysis of cytokine profiles plays a central part in the characterization of disease-related inflammatory pathways and the identification of functional properties of immune cell subpopulations. Because tissue biopsy samples are too small to allow the detection of cytokine protein, the detection of mRNA by RT-PCR analysis is often used to investigate the cytokine milieu in inflammatory lesions. RT-PCR itself is a qualitative method, indicating the presence or absence of specific transcripts. With the use of internal or external standards it may also serve as a quantitative method. The most widely accepted method is quantitative competitive RT-PCR, based on internal shortened standards. Recently, online real-time PCR has been introduced (LightCycler), which allows quantitation in less than 30 min. Here, we have tested its use for the analysis of cytokine gene expression in different experimental in vitro and ex vivo settings. First, we compared quantitative competitive RT-PCR with real-time RT-PCR in the quantitation of transcription levels of the CD4(+) cell-specific chemoattractant Interleukin-16 during the maturation of monocyte-derived dendritic cells, and found a good correlation between both methods. Second, differences in the amounts of IL-16 mRNA in synovial tissue from patients with rheumatoid arthritis and osteoarthritis as assessed by real-time RT-PCR paralleled differences in the level of IL-16 protein in the synovial fluid. Finally, we employed real-time RT-PCR to study the cutaneous expression of several cytokines during experimental immunomodulatory therapy of psoriasis by Interleukin-10, and demonstrate that the technique is suitable for pharmacogenomic monitoring. In summary, real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression even with small quantities of tissue. The results obtained do not differ from those generated by quantitative competitive RT-PCR.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Dendritic Cells; Gene Expression; Humans; Interferon-gamma; Interleukin-10; Interleukin-16; Interleukin-8; Monocytes; Osteoarthritis; Psoriasis; Receptors, Interleukin-8B; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha

Methotrexate (MTX) is one of the most used medicines in rheumatoid arthritis [r. a.] treatment. There are many scientific reports which present influence of MTX on inflammatory process. In many centers influence of MTX on cytokines level have been investigated. Thanks to clinical studies it has been performed that MTX holds up activity and productions of Il-8, releasing of TNF-alpha and reduction of concentration Il-6. MTX holds up proliferation of monocytes, macrophages and synoviocytes. It has been indicated that MTX decreases synthesis of B-4 leucotrien in neutrophils, decreases level of neutral proteases, holds up cellular immunity and has antiproliferative influence on endothelial cells. There are reports, that MTX reduces expression of endothelial cells adhesive proteins and synthesis of chemotactic factors, stopping migration of leucocytes to tissues.

Topics: Animals; Arthritis, Rheumatoid; Cytokines; Humans; Immunity; Interleukin-1; Interleukin-6; Interleukin-8; Methotrexate

The effect of Sinomenine on IL-8, IL-6, IL-2 and mIL-2R produced by peripheral blood mononuclear cells was investigated by using cell culture, radioimmunoassay and flow cytometry. It was showed that production of IL-8 and mIL-2R was inhibited, but the levels of IL-6 were enhanced by Sinomenine. Our results also demonstrated that Sinomenine did not have any effect on the production of IL-2. The study demonstrated that Sinomenine was able to regulate the production of cytokines. This may be one of the mechanisms by which Sinomenine works on rheumatoid arthritis.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Humans; Interleukin-2; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Morphinans

To measure serum and synovial fluid (SF) levels of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in patients with juvenile rheumatoid arthritis (JRA) and to compare them with adult rheumatoid factor-positive rheumatoid arthritis (RA).. IL-8 and MCP-1 were measured by immunoassay (1) in sera obtained from 55 children with JRA and from 16 adults with RA, and (2) in SF obtained from 30 children with JRA and 11 adults with RA.. Patients with active systemic JRA had serum levels of IL-8 and MCP-1 higher than in controls (p<0.01) and in patients with active polyarticular or pauciarticular JRA (p<0.05). In patients with RA serum MCP-1 levels were higher than in patients with the 3 JRA onset types, while no difference was found for IL-8 levels. Patients with systemic JRA and with current systemic features had serum levels of IL-8 and MCP-1 higher (p = 0.03 and p = 0.04, respectively) than patients in which systemic features had subsided. No significant differences in SF IL-8 or MCP-1 levels were found among the 3 JRA onset types or adults with RA. In patients with JRA SF leukocyte counts were correlated with SF IL-8 levels (p = 0.002), but not with MCP-1 levels. Moreover, SF levels of both IL-8 and MCP-1 were correlated with those of IL-1beta (p<0.001) and IL-6 (p<0.01), but not with those of TNF-alpha.. Elevated serum levels of IL-8 and MCP-1 in patients with systemic JRA with current systemic features at sampling suggest systemic production of the 2 chemokines during systemic phases of the disease. Similar SF levels of IL-8 and MCP-1 among the 3 JRA onset-types and RA suggest comparable local production of the 2 chemokines.

Topics: Adolescent; Adult; Age of Onset; Aged; Arthritis, Juvenile; Arthritis, Rheumatoid; Blood Sedimentation; Chemokine CCL2; Child; Child, Preschool; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukocyte Count; Middle Aged; Receptors, Tumor Necrosis Factor; Synovial Fluid; Tumor Necrosis Factor-alpha

Gold compounds have long been used in the treatment of rheumatoid arthritis (RA). However, their actions in RA have not been clarified. In this study, we examined the effect of one of the monovalent gold compounds, aurothioglucose (AuTG), on the IL-1-induced production of IL-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) from rheumatoid synovial fibroblasts (RSF) isolated from three RA patients. IL-6 and IL-8 induction but not GM-CSF induction was inhibited in most of the RSF after pretreatment with AuTG. Since gene expression of these cytokines is known to be under the control of a common transcription factor, NF-kappaB, the effect of AuTG on the cellular localization of NF-kappaB (p65 subunit) and on NF-kappaB-DNA binding was examined. Although AuTG treatment did not prevent NF-kappaB nuclear translocation, AuTG blocked the DNA-binding activity of NF-kappaB when examined in vitro. Morphologically, both metal-specific cell staining using p-dimethylaminobenzylidene rhodamine and transmission electron microscopic examinations demonstrated the accumulation of metal gold in the cytoplama and some organella (mitochondria and lysosomes) of the AuTG-treated RSF. These results indicate that one of the anti-rheumatic actions of AuTG might be through its inhibitory action on NF-kappaB.

Topics: Antirheumatic Agents; Arthritis, Rheumatoid; Aurothioglucose; Cells, Cultured; Fibroblasts; Gold Compounds; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Microscopy, Electron; NF-kappa B; Rhodamines; Staining and Labeling; Synovial Membrane

Thioredoxin (TRX) is a cellular reducing catalyst induced by oxidative stress and is involved in the redox regulation of transcription factors such as NF-kappaB. We found that the serum TRX concentration was elevated in patients with rheumatoid arthritis (RA) as compared with values from healthy individuals and patients with osteoarthritis (33.6 +/- 35.1 vs 11.8 +/- 6.6 ng/ml, p < 0.01). Moreover, the TRX concentration in the synovial fluid (SF) was much more elevated in RA patients than in osteoarthritis patients (103.4 +/- 53.3 vs 24.6 +/- 17.4 ng/ml, p < 0.001). Multiple regression analysis revealed that the serum C-reactive protein value was better correlated with the linear combination of SF TNF-alpha and SF TRX values than with SF TNF-alpha alone, suggesting that TRX might play a subsidiary role in the rheumatoid inflammation. We thus examined the effect of TRX on the TNF-alpha-induced IL-6 and IL-8 production using rheumatoid synovial fibroblast cultures. The extents of IL-6 and IL-8 production in response to TNF-alpha were greatly augmented by TRX as compared with TNF-alpha alone. TRX alone did not have such effects. We also found that TRX appeared to accelerate the nuclear translocation of NF-kappaB, a major transcriptional regulator for production of IL-6 and IL-8 on stimulation with TNF-alpha. Consistent with these findings, the IkappaBalpha phosphorylation at Ser32 and its subsequent degradation in response to TNF-alpha was facilitated by TRX. These findings indicate that the elevated TRX concentration in SF of RA patients might be involved in the aggravation of rheumatoid inflammation by augmenting the NF-kappaB activation pathway.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Biological Transport; C-Reactive Protein; Cell Nucleus; Cells, Cultured; DNA-Binding Proteins; Female; Fibroblasts; Humans; I-kappa B Proteins; Interleukin-6; Interleukin-8; Male; Middle Aged; NF-kappa B; NF-KappaB Inhibitor alpha; Osteoarthritis; Regression Analysis; Synovial Fluid; Thioredoxins; Tumor Necrosis Factor-alpha

Topics: Adolescent; Adult; Aged; Arthritis, Rheumatoid; Female; Humans; Hydrocortisone; Interleukin-8; Male; Middle Aged; Monocytes; Synovial Fluid

Chemokines play a key role in modulating leukocyte functions at sites of inflammation. To assess chondrocyte contribution to the chemotactic environment of inflamed joints the intracellular content of CC and CXC chemokines was investigated. IL-8, GROalpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression was evaluated by flow cytometric analysis and RT-PCR in chondrocytes isolated from cartilage specimens obtained from patients with osteoarthritis and rheumatoid arthritis and multiorgan donors as normal controls. All the chemokines except RANTES were found in normal chondrocytes, with different degrees of staining intensity. In osteoarthritis and rheumatoid arthritis patients, an enhancement of IL-8, GROalpha, MIP-1alpha and MIP-1beta was observed.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Separation; Chemokine CCL2; Chemokine CCL3; Chemokine CCL4; Chemokine CCL5; Chemokine CXCL1; Chemokines; Chemokines, CXC; Chemotactic Factors; Chondrocytes; Flow Cytometry; Growth Substances; Humans; Immunohistochemistry; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Interleukin-8; Macrophage Inflammatory Proteins; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction

The aim of this work was to determine differences in pro- and anti-inflammatory cytokine and adhesion molecule expression in synovial tissue from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Synovial tissue samples were obtained from patients with RA and OA, and from healthy individuals. The expression of mRNA of interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10, IL-13, tumour necrosis factor-alpha (TNF-alpha) and transforming growth-factor-beta1 (TGF-beta1) was evaluated by the polymerase chain reaction (PCR). In addition, IL-8 and IL-10 transcripts were measured by quantitative PCR. The expression of IL-8 and IL-10 proteins was determined by immunoperoxidase staining. To evaluate the inflammatory stage of synovial tissue, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression was also determined. RA patients were found to display higher levels of adhesion molecules than patients with OA. PCR analysis showed a similar profile of cytokine transcripts between the OA and RA groups. Gene expression of IL-4 and IL-13 in synovium was undetectable. In contrast, IL-1beta, IL-6, IL-8, IL-10, TNF-alpha and TGF-beta1 transcripts were expressed by both groups. Increased levels of IL-8 and IL-10 transcripts and their proteins were observed in synovium from RA patients when compared to patients with OA and healthy controls. Thus, our data show that IL-8, IL-10, ICAM-1 and VCAM-1 expression levels are higher in synovial tissue from patients with RA than in similar tissue from patients with OA.

Topics: Adult; Arthritis, Rheumatoid; Cytokines; Gene Expression; Humans; Immunoenzyme Techniques; Intercellular Adhesion Molecule-1; Interleukin-10; Interleukin-8; Middle Aged; Osteoarthritis; Peroxidase; Polymerase Chain Reaction; Synovial Membrane; Vascular Cell Adhesion Molecule-1

The large T antigen of SV40 (LT) has been widely used to immortalize primary cells for various studies. In this study, synovial fibroblasts of a patient from rheumatoid arthritis (RA) were transformed with LT gene to analyze the effect of SV40-mediated transformation on the production of cytokines, such as IL-6, IL-8, and GM-CSF, that are under the control of interleukin-1 beta (IL-1 beta), a physiological inducer of nuclear factor kappa B (NF-kappa B). It was noted that the basal levels of GM-CSF and IL-8 were upregulated, whereas that of IL-6 was downregulated. Moreover, the extents of induction of these cytokines in response to IL-1 beta were markedly downregulated in synovial fibroblasts transformed by LT as compared from parental cells. Although IL-1 beta could translocate NF-kappa B to the nucleus in all cells, some of the transformed cells exhibited nuclear translocation of NF-kappa B even before the stimulation with IL-1 beta, suggesting that transformation of LT resulted in the constitutive activation of NF-kappa B, either directly or indirectly. In order to examine whether LT downregulate the kappa B-dependent gene expression, we performed the transient luciferase gene expression assay. We found that cotransfection of LT did not downregulate the kappa B-dependent gene expression that was stimulated with L-1 beta. These observations suggest that the apparent inhibitory effect of LT on the IL-1-induced expression of cytokines may not be through its direct action on the NF-kappa B transactivation.

Topics: Antigens, Polyomavirus Transforming; Arthritis, Rheumatoid; Cell Line, Transformed; Cell Transformation, Viral; Cells, Cultured; Cytokines; DNA, Recombinant; Endothelial Growth Factors; Female; Fibroblasts; Fluorescent Antibody Technique, Indirect; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Lymphokines; Middle Aged; Recombinant Proteins; Simian virus 40; Synovial Membrane; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Synovial inflammation in patients with rheumatoid arthritis (RA) is characterised by the presence of large numbers of highly activated monocytes and macrophages. The importance of these cells in the aethiopathogenesis and prognosis of RA is increasingly recognised. The object of this report is to determine whether monocytes and monocyte derived macrophages of RA patients produce increased cytokine mRNA levels.. Monocyte derived macrophages from RA patients and healthy controls were cultured either in the absence or presence of lipopolysaccharide. The expression levels of the mRNAs encoding GAPDH, interleukin 1beta (IL1beta), IL8, and alpha(2) macroglobulin in these cells were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR).. Activated monocyte derived macrophages from RA patients produce significantly higher IL8 mRNA levels than activated macrophages from healthy controls. By contrast, resting RA and control macrophages produce similar levels of IL8 mRNA. Culturing of activated macrophages in the presence of RA or control sera has no effect on the expression levels of IL8 mRNA. No significant differences between RA and control macrophages were observed in the expression levels of IL1beta and alpha(2) macroglobulin mRNAs.. These data indicate that the increased IL8 mRNA production capacity of RA macrophages upon activation is an intrinsic property of these cells, and is not attributable to factors present in the circulation. Based on these observations, it is postulated that this innate hyper-responsiveness of RA macrophages contributes to the high IL8 levels present in the synovial fluid of rheumatoid joints, and is implicated in the chemotactic gradient leading to the homing of leucocytes to the joints.

Topics: Adult; Arthritis, Rheumatoid; Case-Control Studies; Female; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Interleukin-1; Interleukin-8; Macrophage Activation; Macrophages; Middle Aged; Monocytes; RNA, Messenger; Synovial Membrane

To evaluate the role of chondrocytes in producing CXC chemokines [interleukin 8 (IL-8), growth related gene product (GRO-alpha)] and CC chemokines [monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1alpha), RANTES] in patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and subjects after traumatic injury (PT).. Articular cartilage specimens were obtained from 38 patients with OA and 18 with RA undergoing joint replacement surgery. Healthy human cartilage was obtained from femoral condyles removed after trauma in 11 subjects with no history of joint pathology (PT cases). Chondrocytes were isolated from articular cartilage by sequential enzymatic digestion and cultured in vitro. Chemokine production was investigated in unstimulated condition and after 72 h incubation with proinflammatory [IL-1beta, tumor necrosis factor-alpha (TNF-alpha)] and antiinflammatory [transforming growth factor-beta1 (TGF-beta1), IL-10] mediators. Chemokine concentrations in cell supernatants were evaluated by ELISA.. Chondrocytes produce all these chemokines to a different extent. IL-1beta was a more potent stimulus than TNF-alpha in inducing production of all chemokines except MCP-1. We found no statistical differences among chondrocytes isolated from OA, RA, and PT for chemokine production in either basal conditions or after cytokine stimulation. IL-1beta induced chemokine production can be modulated by TGF-beta1 in different ways according to the various chemokines, while IL-10 does not affect IL-1beta induced chemokine production.. Chondrocytes produce IL-8, GRO-alpha, MCP-1, MIP-1alpha, and RANTES. Proinflammatory factors (IL-1beta, TNF-alpha) effectively upregulate chemokine production, but production is scarcely modulated by the antiinflammatory mediators TGF-beta and IL-10. Chondrocyte derived chemokines may play a role in triggering the mechanisms involved in pathogenesis and persistence of joint diseases.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Arthritis, Rheumatoid; Base Sequence; Biomarkers; Cartilage, Articular; Cells, Cultured; Chemokine CCL5; Chemokines; Chemokines, CC; Female; Humans; Inflammation; Interleukin-8; Macrophage Inflammatory Proteins; Male; Middle Aged; Molecular Sequence Data; Monocyte Chemoattractant Proteins; Osteoarthritis; Polymerase Chain Reaction; Statistics, Nonparametric

Synovial tissues from Rheumatoid Arthritis (RA) were divided into three groups based on their histopathological findings and compared for their expression of IL-8 and monocyte chemotactic and activating factor (MCAF) by using immunohistochemistry and in situ hybridization. The levels of IL-8 as well as those of MCAF were markedly higher in the synovial fluid from RA joints. Synovial lining cells (SLC) and macrophages had an ability to produce IL-8 at an early phase of the disease. The presence of MCAF was restricted in macrophages at this stage. On the other hand, the production of IL-8 as well as MCAF were prominent in most components of the joints such as SLC, migrated monocytes, sublining fibroblastoid cells, endothelial cells or migrated neutrophils at an active phase. The expression of IL-8 or MCAF was low in fibrotic synovitis of RA. These data indicate that IL-8 generated from SLC and macrophages may participate to the inflammatory process in the early synovitis of RA.

Topics: Arthritis, Rheumatoid; Arthroplasty, Replacement, Knee; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin-8; Macrophages; Monocyte Chemoattractant Proteins; Osteoarthritis; Synovial Fluid; Synovial Membrane; Transcription, Genetic

Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression.. SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites.. HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78.. These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

Topics: Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Biocompatible Materials; Cell Division; Chemokine CCL2; Chemotaxis; Collagen; Drug Combinations; Endothelium, Vascular; Fibroblast Growth Factor 2; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-8; Laminin; Mesalamine; Proteoglycans; Solubility; Sulfapyridine; Sulfasalazine

To examine whether taurine (Tau) or its physiologic chlorinated derivative, taurine chloramine (Tau-CI), affects proliferation of, and proinflammatory cytokine (interleukin-6 [IL-6] and IL-8) production by, fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients.. FLS, isolated from the synovial tissue of 19 RA patients and cultured in vitro for 3-6 passages, were stimulated with the recombinant human cytokines IL-1beta (1 ng/ml), tumor necrosis factor alpha (TNFalpha; 10 ng/ml), or IL-17 (10 ng/ml) in the presence of either Tau or Tau-Cl, which were added at concentrations of 50-500 microM. Tau and Tau-Cl were added simultaneously with, 2 hours before, or 24 hours after the stimuli. The concentrations of IL-6 and IL-8 were determined in culture supernatants using specific enzyme-linked immunosorbent assays. Proliferation of FLS was estimated on the basis of 3H-thymidine incorporation into the cells, which were cultured for 72 hours in the presence of recombinant human basic fibroblast growth factor (bFGF) (1 ng/ml) and Tau or Tau-Cl, which were added simultaneously at the beginning of the culture.. Cultured in vitro, RA FLS spontaneously secreted low levels of IL-6 and IL-8, but when RA FLS were stimulated with IL-1beta, TNFalpha, or IL-17, significantly higher amounts of IL-6 and IL-8 were produced. Tau-Cl, but not Tau, inhibited cytokine-triggered synthesis of IL-6 (50% inhibitory concentration [IC50] approximately 225 microM) and IL-8 (IC50 approximately 450 microM) when added simultaneously with the stimuli. However, IL-17-induced production of IL-8 was not affected by Tau-Cl. In the cells prestimulated with IL-1beta for 24 hours, Tau-Cl still inhibited synthesis of IL-6, but did not affect IL-8 production. Moreover, Tau-Cl inhibited spontaneous and bFGF-triggered proliferation of FLS in a dose-dependent manner. Neither Tau nor Tau-Cl affected cell viability.. The results of these studies demonstrate that Tau-Cl inhibits production of proinflammatory cytokines by RA FLS, as well as proliferation of these cells. Thus, Tau-Cl may act as a physiologic modulator of FLS functions related to their pathogenic role in RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Division; Female; Fibroblasts; Humans; Inflammation Mediators; Interleukin-1; Interleukin-6; Interleukin-8; Male; Middle Aged; Synovial Membrane; Taurine; Time Factors

We simultaneously measured the concentrations of parathyroid hormone related peptide (PTHrP) and cytokines in synovial fluid (SF) to clarify the relationship between PTHrP and cytokine network in the SF of elderly patients with arthritis. SF was collected from knee joints of five RA patients aged 66+/-11 years old and nine osteoarthritis (OA) patients aged 80+/-9 years old. PTHrP in SF was measured by enzyme-linked immunosorbent assay (ELISA), whereas tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-8 (IL-8) in SF were all measured by ELISA. The PTHrP levels in the SF of RA patients (2.56+/-0.89 pmol/l) were significantly (p<0.05) higher than those of OA patients (1.66+/-0.17 pmol/l). TNF-alpha, IL-1beta, IL-2 and IL-6 concentrations in SF of RA were also significantly higher than those in SF of OA (TNF-alpha 22.5+/-14.8 vs 4.8+/-3.0 pg/ml, p<0.01; IL-1beta 11.8+/-11.4 vs 1.4+/-1.3, p<0.05; IL-2 59.9+/-46.6 vs 12.5+/-8.0 pg/ml, p<0.05; IL-6 18424+/-8901 vs 3547+/-2948 pg/ml, p<0.01). The concentrations of IL-4 and IL-8 in SF of RA were similar to those of OA. Immunohistochemical studies revealed the presence of immunoreactive PTHrP in synovial fibroblasts from RA and OA. Among cytokines, only IL-6 was positively correlated with PTHrP levels in SF (r=0.685, p<0.01). In the culture of synovial cells from RA and OA, PTHrP was produced in RA more than OA after phorbol 12-mysistate 13-acetate (TPA) stimulation. These results indicate that PTHrP and cytokines, especially IL-6, might be involved in the inflammatory processes of elderly RA and OA. This is the first study in which PTHrP and cytokine levels were simultaneously examined in synovial fluid of elderly RA and OA.

Topics: Aged; Aged, 80 and over; Aging; Arthritis, Rheumatoid; Cytokines; Female; Fibroblasts; Humans; Interleukin-1; Interleukin-2; Interleukin-4; Interleukin-6; Interleukin-8; Knee Joint; Male; Middle Aged; Osteoarthritis; Parathyroid Hormone-Related Protein; Proteins; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Synovial fluids (SF) from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), gout, and osteoarthritis (OA) were investigated for the levels of interleukin (IL)-1 beta, IL-6 and IL-8, tryptophan (Trp) and indoleamine 2,3-dioxygenase (IDO) activity. Significant differences exist in the levels of IL-1 beta between inflammatory arthritides RA, PsA and gout and non inflammatory arthritis, such as OA. The highest concentration of IL-1 beta was found in RA, that showed high levels also of IL-6 and IL-8. In the same disease we also found the highest IDO activity and the lowest Trp concentration. In addition, IDO activity seems to be related with the decrease in Trp, as demonstrated by the inverse correlation found between these two substances in the SF of all patients.

Topics: Adult; Arthritis; Arthritis, Psoriatic; Arthritis, Rheumatoid; Cytokines; Enzyme-Linked Immunosorbent Assay; Gout; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-1; Interleukin-6; Interleukin-8; Leukocytes; Middle Aged; Neutrophils; Osteoarthritis; Synovial Fluid; Tryptophan; Tryptophan Oxygenase

To elucidate the role of interleukin (IL)-8, a chemotactic factor for neutrophils, in dialysis-related arthritis (DRA) of patients on long-term hemodialysis, the concentration of IL-8 was measured in the synovial fluids of DRA patients with acute arthralgia and joint swelling, and was compared with those in patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). We noted a marked elevation of IL-8 in the joint fluids of patients with DRA and RA as compared with OA. Furthermore, to determine the role of IL-8 in synovitis, we examined the in vivo effect of intra-articular injection of human recombinant IL-8 on leukocyte infiltration into the joint space of rabbits. A single injection of IL-8 to the joints of rabbits induced rapid infiltration of neutrophils into the joint space and synovial tissues, which reached a maximum in four hours. The oral administration of indometacin farnesil (a prodrug that is converted to indomethacin after intestinal absorption) before the injection of IL-8 alleviated the infiltration of neutrophils. When human synovial cells were incubated with tumor necrosis factor (TNF)-alpha, the expression of IL-8 mRNA and IL-8 production in the cultured synovial cells were increased. The TNF-alpha-stimulated expression of IL-8 mRNA and IL-8 production in the cultured synovial cells were markedly inhibited by dexamethasone. In conclusion, IL-8 levels were markedly elevated in the joint fluids of patients with DRA. Interleukin-8 released from synovial cells may be an important factor to induce acute inflammation in DRA. Dexamethasone and indomethacin may be effective for DRA by inhibiting the production and chemotactic actions of IL-8, respectively.

Topics: Adult; Aged; Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cells, Cultured; Dexamethasone; Female; Gene Expression; Humans; Indomethacin; Interleukin-1; Interleukin-8; Kidney Failure, Chronic; Leukocytes; Male; Middle Aged; Osteoarthritis; Rabbits; Renal Dialysis; RNA, Messenger; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Chemokines, including interleukin-8 (IL-8), function as key mediators in diverse inflammatory disorders via promoting the recruitment, proliferation, and activation of vascular and immune cells. IL-8 levels are elevated in inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, and periodontal disease, that also exhibit progressive bone loss. Therefore, it is possible that IL-8 contributes to the osteopenia associated with these pathological conditions. Although macrophages, neutrophils, and endothelial cells are considered the primary sources of inflammation-induced IL-8 increases, we report here for the first time that human bone marrow-derived osteoclast-like cells (hOCL) as well as authentic bone-resorbing human osteoclasts (hOC) isolated from osteoporotic femoral heads express messenger RNA (mRNA) for IL-8 and secrete high levels of IL-8 during culture. Basal IL-8 release by cultured hOC or hOCL was orders of magnitude greater than the release of the proinflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor-alpha. At a cellular level, in situ hybridization analysis revealed that IL-8 mRNA was expressed in resorbing hOC of rheumatoid arthritic pannus and was substantially greater than that expressed in hOC of noninflammatory giant cell tumor of bone tissue. Therefore, the potential inflammation-mediated induction of IL-8 was directly assessed using cultured hOCL. IL-8 release was stimulated by proinflammatory signals (IL-1alpha, tumor necrosis factor-alpha, lipopolysaccharide, or phorbol 12-myristate 13-acetate), unaffected by various other osteotropic modulators (transforming growth factor-beta1 and -beta3, IL-6, 17beta-estradiol, or calcitonin) and was decreased by interferon-gamma, vitamin D3, and the antiinflammatory glucocorticoid dexamethasone. Changes in IL-8 secretion were paralleled by corresponding changes in IL-8 mRNA steady state levels. We conclude that hOC and hOCL synthesize and secrete high constitutive and inflammation-stimulated levels of the chemokine IL-8. Consequently, hOC-derived IL-8 could act as an important regulatory signal for bone, vascular, and immune cell recruitment and activation during normal and pathological bone remodeling.

Topics: Arthritis, Rheumatoid; Calcitriol; Cells, Cultured; Dexamethasone; Humans; Interleukin-8; Osteoclasts; RNA, Messenger

SA96 (generic name, bucillamine) is a disease-modifying anti-rheumatoid arthritis (RA) drug with immunological effects. This compounds has two sulfhydryl groups in its molecule, and the differences and similarities between this drug and D-penicillamine, which is also a sulfhydryl group-containing anti-rheumatic drug, have frequently been discussed. To clarify the pharmacological differences between these two drugs, we examined the concentrations of the compounds and its metabolites in serum and synovial fluid, paying special attention to the metabolites of SA96 produced in vivo. SA96 was metabolized in a very short time to SA981 which is a disulfide compound formed by intramolecular binding of two sulfhydryl groups, and transferred to synovial fluid. In addition SA981 had significant suppressive effects on IL-6 and IL-8 production by synovial cells in vitro. These results demonstrate that SA96, which has two sulfhydryl groups, exhibits anti-rheumatic effects via a pharmacological action clearly different from that of D-penicillamine.

Topics: Adjuvants, Immunologic; Adult; Aged; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antirheumatic Agents; Arthritis, Rheumatoid; Cells, Cultured; Cysteine; Disulfides; Female; Gas Chromatography-Mass Spectrometry; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Penicillamine; Rats; Structure-Activity Relationship; Synovial Fluid

Tetranectin (TN) was assessed in paired synovial fluid (SF) and serum (S) samples from 27 patients with rheumatoid arthritis (RA), 23 with seronegative spondylarthritis (SSA) and 22 with osteoarthritis (OA). RA patients had a stronger correlation between serum and SF TN and a higher SF/S TN ratio than did SSA and OA patients. Moreover, the SF/S TN ratio exceeded 1 in most RA patients but not in SSA and OA patients, indicating the possibility of intra-articular TN synthesis in RA. A strong correlation of serum and SF TN with known inflammatory markers was observed in RA. The TN/proteinase inhibitors (PIs: alpha1-antitrypsin, alpha2-macroglobulin) molar ratio in SF was lower in RA and SSA patients to a statistically significant degree than in OA patients. In RA, in contrast to SSA and OA, this ratio correlated positively with the SF interleukin-8 (IL-8), responsible for neutrophil recruitment and degranulation, and negatively with erythrocyte sedimentation rate, serum C-reactive protein and fibrinogen, known markers of disease activity. In conclusion, patients with RA showed lower serum TN levels, a higher SF/S TN ratio and a lower SF TN/PI molar ratio than did SSA and OA patients, suggesting the implication of TN in the impaired regulation of fibrinolysis associated with the inflammatory process.

Topics: Adult; Aged; alpha 1-Antitrypsin; alpha-Macroglobulins; Arthritis, Rheumatoid; Biomarkers; Blood Proteins; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Lectins; Lectins, C-Type; Male; Middle Aged; Osteoarthritis; Serologic Tests; Spondylitis, Ankylosing; Synovial Fluid

We have compared three monocyte isolation procedures for their suitability in the analysis of cytokine mRNA expression in circulating monocytes. Monocytes were isolated from peripheral blood (1) using antiCD14 coated magnetic beads, (2) by Ficoll centrifugation, or (3) by Ficoll centrifugation followed by plastic adherence. The effect of the isolation procedure on the cytokine mRNA expression levels of the isolated monocytes was evaluated by RT-PCR. Results show that the expression of cytokine mRNAs determined in monocytes isolated using antiCD14 coated magnetized beads reflects best the cytokine mRNA levels in circulating monocytes. We subsequently applied this method to the analysis of cytokine mRNA expression levels in rheumatoid arthritis and control monocytes, which revealed that RA and control monocytes isolated by antiCD14 beads produce similar, very low TNF alpha, IL-1beta, and IL-8 mRNA levels.

Topics: Arthritis, Rheumatoid; Female; Humans; Interleukin-1; Interleukin-8; Leukocytes, Mononuclear; Male; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Necrosis Factor-alpha

The objective was to assess the congruity of gliostatin/platelet-derived endothelial cell growth factor (GLS PD-ECGF) with other clinical markers of rheumatoid arthritis (RA) and to define its molecular mechanism of action in the complicated cytokine network during RA pathogenesis. Immunoassay systems were used to quantify GLS or cytokine levels in laboratory and clinical samples. Expression levels of GLS were determined by reverse transcription-polymerase chain reaction methods. The GLS levels in synovial fluid were correlated with interleukin-1 (IL-1) and IL-8. The serial data of serum GLS levels reflected well changes in the disease activity during the clinical course of four representative patients with RA. In cultured fibroblast-like synoviocytes, tumour necrosis factor-alpha (TNF-alpha), IL-1, IL-6 and IL-8 induced GLS expression. In conclusion, our results suggest that the serum GLS level, mostly derived from cytokine-stimulated synoviocytes, was a useful clinical marker of RA.

Topics: Arthritis, Rheumatoid; Biomarkers; Blood Platelets; Endothelial Growth Factors; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Nerve Tissue Proteins; Synovial Fluid; Therapeutic Equivalency; Thymidine Phosphorylase; Tumor Necrosis Factor-alpha

To study the role of integrin receptors in the invasion of cartilage by rheumatoid synovial fibroblasts (RSF).. RSF were cocultured with cartilage slices alone or in the presence of various potential activators or inhibitors. The penetration of the cartilage surface by RSF was determined by live-cell imaging of fluorescent-labeled cells.. Interleukin-1beta (IL-1beta) and IL-8 stimulated the RSF invasion of cartilage. Invasion was specific for RSF and required a concentration gradient of IL-1beta. The IL-1beta-activated invasion of cartilage was inhibited by anti-IL-1 antibodies, IL-1 receptor antagonist, and collagenase inhibitors. RSF invasion was also inhibited by antibodies to alpha4, alpha5, alphaV, and beta1 integrins.. In this study, an IL-1beta concentration gradient was required for RSF invasion into cartilage, raising the possibility that in vivo invasion may be induced by IL-1beta released by chondrocytes. The IL-1beta activation of RSF assayed in vitro may contribute to the RSF invasion of cartilage in vivo. Cartilage invasion requires the availability of beta1 and alpha4, alpha5, and alphaV integrins and the presence of collagenase activity.

Topics: Antibodies; Antigens, CD; Arthritis, Rheumatoid; Cartilage, Articular; Cells, Cultured; Fibroblasts; Humans; In Vitro Techniques; Integrin alpha4; Integrin alpha5; Integrin alphaV; Integrin beta1; Integrins; Interleukin-1; Interleukin-8; Matrix Metalloproteinase Inhibitors; Osteoarthritis; Receptors, Interleukin-1; Synovial Membrane

To study the mechanism by which hypoxia and inflammatory cytokines mediate angiogenesis in the rheumatoid pannus through their effects on the fibroblast-like type B synoviocyte, the major cell type of normal synovia.. Fibroblasts were prepared from synovial tissue of healthy and diseased individuals, and cultured in the presence of various stimuli. The expression of vascular endothelial growth factor (VEGF) was assessed by ELISA and reverse transcription polymerase chain reaction.. Unlike normal fibroblasts, synovial fibroblasts from rheumatoid arthritis (RA) and osteoarthritis constitutively secreted significant levels of VEGF, which is known to act directly on endothelial cells. VEGF secretion was further inducible by both hypoxia and interleukin 1beta (IL-1beta) and these increases were additive. In contrast, tumor necrosis factor alpha was unable to induce VEGF expression.. Under hypoxia or IL-1 stimulation, conditions common to the inflamed synovium, type B synoviocytes secrete increased levels of VEGF, which is likely to act on nearby endothelia, promoting angiogenesis. The constitutive expression of VEGF in rheumatoid synovial fibroblasts may reflect an altered phenotype involved in the pathology of RA.

Topics: Alternative Splicing; Arthritis, Rheumatoid; Cell Hypoxia; Cells, Cultured; Endothelial Growth Factors; Endothelium, Vascular; Fibroblasts; Gene Expression; Humans; Interleukin-1; Interleukin-8; Lymphokines; Neovascularization, Physiologic; Osteoarthritis; Synovial Membrane; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments.. Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1beta (IL-1beta), IL-8, and tumor necrosis factor alpha (TNFalpha) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E2 (PGE2) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFalpha, IL-8, IL-1beta, and the IL-1 receptor antagonist protein (IL-1Ra).. MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFalpha in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFalpha in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1beta and IL-1Ra or synovial fluid IL-1beta and PGE2 was found.. MP therapy rapidly reduces IL-8 and TNFalpha levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFalpha expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.

Topics: Aged; Aged, 80 and over; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Female; Humans; Injections, Intravenous; Interleukin-8; Male; Methylprednisolone; Middle Aged; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Tumor necrosis factor (TNF)-alpha is present in synovial fluid of patients with rheumatoid arthritis. It induces the expression of proinflammatory cytokines in synovial cells. Based on our recent finding that reactive oxygen intermediates play important roles in mediating TNF-alpha action, we examined the effect of an antioxidant bioflavonoid, quercetin, on TNF-alpha induced expression of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cultured human synovial cells.. The amounts of mRNA for IL-8 and MCP-1 were determined by Northern blot analysis. Electrophoretic mobility shift assays (EMSA) were performed for the detection of a transcription factor, nuclear factor-kappa B (NF-kappa B).. Addition of quercetin suppressed TNF-alpha induced increase in the mRNA for IL-8 and MCP-1 in a dose dependent manner. Quercetin did not affect the stability of these mRNA. H2O2 mediated induction of IL-8 and MCP-1 genes was also inhibited by quercetin. EMSA revealed that quercetin inhibited the activation of NF-kappa B by TNF-alpha.. Quercetin suppresses TNF-alpha mediated stimulation of IL-8 and MCP-1 expression, at least in part, by inhibiting the activation of NF-kappa B.

Topics: Arthritis, Rheumatoid; Blotting, Northern; Cells, Cultured; Chemokine CCL2; DNA Probes; Humans; Hydrogen Peroxide; Interleukin-8; NF-kappa B; Quercetin; Recombinant Proteins; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha

Exposure of human rheumatoid synovial fibroblasts (RSF) to interleukin 1beta (IL-1beta) results in the coordinate up-regulation of 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX II) and subsequent biosynthesis of prostaglandin E2 (PGE2). We have recently demonstrated, through the use of oligonucleotide decoys and antisense, the participation of the proinflammatory transcription factor, nuclear factor kappaB (NFkappaB), in the regulation of the prostanoid-metabolizing enzymes. Hymenialdisine, a marine natural product has recently been characterized as an inhibitor of NFkappaB activation and exposure of IL-1-stimulated RSF-inhibited PGE2 production in a concentration-dependent manner (IC50 approximately 1 microM). Alternatively, both an analog, aldisine, and the protein kinase C inhibitor, RO 32-0432, were without affect. Direct action of hymenialdisine on IL-1-induced NFkappaB activation was demonstrated by a significant reduction (approximately 80%) in NFkappaB binding to the classical kappaB consensus motif (as assessed by electrophoretic mobility shift assay) and inhibition of stimulated p65 migration from the cytosol of treated cells (as assessed by Western analysis). Consistent with the role of NFkappaB in the transcriptional regulation of COX II and 85-kDa PLA2, hymenialdisine-treated RSF did not transcribe the respective mRNAs in response to IL-1. This led to reductions in their respective protein levels and subsequent reductions in the ability to produce PGE2. Specificity of action is suggested as IL-1-stimulated interleukin-8 (IL-8) production, which is known to be an NFkappaB-regulated event, was also inhibited by hymenialdisine, whereas IL-1-induced production of vascular endothelial growth factor, a non-NFkappaB-regulated gene, was not affected by exposure to hymenialdisine. Taken together, hymenialdisine inhibits IL-1-stimulated-RSF PGE2 formation acting predominately through modulation of NFkappaB activation and offers an interesting novel tool to evaluate the role of NFkappaB in inflammatory disease.

Topics: Adult; Arthritis, Rheumatoid; Azepines; Cells, Cultured; Dinoprostone; Endothelial Growth Factors; Humans; Interleukin-1; Interleukin-8; Lymphokines; NF-kappa B; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Pyrroles; Synovial Membrane; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

Topics: Amino Acid Sequence; Animals; Arthritis, Rheumatoid; Base Sequence; Cytokines; Dinoprostone; Endothelium, Vascular; Fibroblasts; Granulocyte Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Herpesvirus 2, Saimiriine; Humans; Inflammation; Interferon-gamma; Interleukin-17; Interleukin-6; Interleukin-8; Interleukins; Lymphocytes; Macromolecular Substances; Mice; Molecular Sequence Data; Open Reading Frames; Recombinant Proteins; Reference Values; Sequence Homology, Amino Acid; Skin; Stromal Cells; Synovial Membrane; T-Lymphocytes; Transfection; Tumor Necrosis Factor-alpha; Viral Proteins

T-cell infiltration into synovium is a crucial process for rheumatoid arthritis (RA). To investigate the mechanism of T-cell infiltration, we studied T-cell attracting activity in synovial tissue extracts of RA or osteoarthritis (OA) whose synovium lacks T-cell accumulation. RA extracts attracted twofold more T cells than OA extracts. By gel filtration column chromatography the activity of RA extracts was separated into two peaks; one was eluted at the 67-kDa region and the other was at the 12-kDa region, while the latter was absent in OA extracts. The activity eluted at the 12-kDa region was absorbed mostly by an antibody against IL-8/NAP-1, a potent T-cell chemotactic factor. IL-8/NAP-1 concentrations in RA extracts were much higher than those in OA extracts and correlated to T-cell attracting activity eluted at the 12-kDa region. The checkerboard analysis revealed that the 67-kDa activity was chemokinetic but not chemotactic. These results suggest that IL-8/NAP-1 is the major T-cell chemoattractant in RA-synovium.

Topics: Aged; Arthritis, Rheumatoid; Chemotactic Factors; Chemotaxis, Leukocyte; Female; Humans; Immunosorbent Techniques; Interleukin-8; Male; Middle Aged; Molecular Weight; Synovial Membrane; T-Lymphocytes

To study whether the induction of mRNA for interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and collagenase by tumor necrosis factor-alpha (TNF-alpha) is suppressed by antioxidants in human synovial cells. TNF-alpha has been shown to exert some of its effects by stimulating production of reactive oxygen intermediates in some cell lines other than synovial cells.. Amounts of mRNA for IL-8, MCP-1, and collagenase were determined by Northern blot analysis. Electrophoretic mobility shift assays were performed for the detection of a transcription factor, nuclear factor-kappa B(NF-kappa B). The concentration of IL-8 in the medium was determined by ELISA.. TNF-alpha increased the expression of IL-8, MCP-1, and collagenase mRNA in human synovial cells. NF-KB known to induce IL-8 gene transcription was also increased in nuclear extracts from the synovial cells treated with TNF-alpha. Prior addition of antioxidant, N-acetyl-L-cysteine (NAC) or 2-oxothiazolidine-4-carboxylate (OTC), suppressed TNF-alpha stimulated expressions of IL-8, MCP-1, and collagenase mRNA in a dose dependent manner. Treatment with NAC also suppressed TNF-alpha induced increase in NF-kappa B. The changes of IL-8 in the medium reflected the mRNA levels. Hydrogen peroxide (H2O2) induced the expression of mRNA for the cytokines but not collagenase mRNA, and NAC suppressed the effect of H2O2.. Our data suggest that TNF-alpha induces expression of proinflammatory cytokines such as IL-8 and MCP-1 through generation of reactive oxygen intermediates and subsequent activation of NF-kappa B in human synovial cells, and the antioxidants may inhibit, at least in part, the activation of NF-kappa B by TNF-alpha. These results indicate that antioxidants such as NAC may be useful in treating rheumatoid arthritis.

Topics: Acetylcysteine; Antioxidants; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Collagenases; Free Radical Scavengers; Gene Expression Regulation, Enzymologic; Humans; Hydrogen Peroxide; Interleukin-8; NF-kappa B; Pyrrolidonecarboxylic Acid; RNA, Messenger; Synovial Membrane; Thiazoles; Thiazolidines; Transcription, Genetic; Tumor Necrosis Factor-alpha

To examine circulating levels of cytokines and cytokine inhibitors and their production by blood mononuclear cells (MNC) in patients with active rheumatoid arthritis (RA) before treatment with methotrexate (MTX) and inactive disease upon treatment as well as healthy control individuals.. Interleukin-1 receptor antagonist (IL-1ra), soluble tumor necrosis factor receptors p55 and p75 (sTNFr; p55 and p75), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and monocyte chemoattractant protein 1 (MCP-1) were assessed by immunoassays in sera and MNC culture supernatants of 27 patients with RA with active disease before and 14 patients with inactive disease during MTX treatment, and 10 healthy controls.. Levels of circulating IL-1ra, sTNFr p55 and p75 were higher in patients with active RA compared to those with inactive disease or controls. At the cellular level, resting MNC of patients with active RA released more IL-1 beta and IL-8, but less IL-1ra, and showed a lower ratio of IL-1ra:IL-1 beta than MNC of patients without inflammatory symptoms or healthy controls. In addition, unstimulated and in vitro lipopolysaccharide stimulated MNC cultures of patients with inactive RA released higher amounts of sTNFr p75 than MNC of patients with active RA.. Circulating levels of IL-1ra and sTNFr as well as IL-1 beta, IL-8, and sTNFr p75 release from MNC and the ratio of IL-1ra:IL-1 beta production by these cells serve as markers to assess complete disease remission in patients with RA during MTX treatment.

Topics: Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Female; Humans; Interleukin-1; Interleukin-8; Male; Middle Aged; Receptors, Interleukin-1; Receptors, Tumor Necrosis Factor; Remission Induction; Solubility

In this study, we investigated the IL-1 beta converting enzyme (ICE) family cysteine proteases responsible for the Fas-mediated apoptosis of rheumatoid arthritis (RA) synoviocytes and their involvement in proinflammatory cytokine production. CPP32 inhibitor, but not ICE inhibitor, was capable of inhibiting the Fas-mediated apoptosis of RA synovial cells. CPP32, but not ICE, was activated in response to anti-Fas stimulation. IL-8, but not IL-1 beta, was secreted from the anti-Fas-stimulated RA synoviocytes even in the presence of CPP32 inhibitor. These results demonstrated that CPP32, but not ICE, is the predominant cysteine protease that mediates the Fas-mediated apoptosis of RA synovial cells. We also demonstrated that anti-Fas stimulation of RA synoviocytes leads to IL-8 secretion independently of the CPP32-mediated apoptosis, which would accelerate inflammation.

Topics: Apoptosis; Arthritis, Rheumatoid; Caspase 1; Caspase 3; Caspases; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Interleukin-1; Interleukin-8; Oligopeptides; Synovial Membrane

There is persistent excessive production of a number of pro-inflammatory cytokines in patients with rheumatoid arthritis (RA). A number of experimental therapies have been found to be effective in the treatment of RA, although the effects of these therapies on cytokine production have not been evaluated. One such experimental therapy involves administration of a MoAb to intercellular adhesion molecule-1 (ICAM-1) that has been shown to be clinically beneficial in approximately 60% of the patients, with some patients responding for up to 11 months. The current studies were carried out to determine whether the success of this therapy was associated with changes in mRNA levels for pro-inflammatory and other monocyte-derived cytokines in peripheral blood mononuclear cells (PBMC). Cytokine mRNA levels were assessed in freshly isolated unstimulated PBMC by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) after minimal in vitro manipulation of the cells. Anti-ICAM-1 MoAb administration was followed by an increase in IL-1 beta, IL-8, and tumour-necrosis factor-alpha (TNF-alpha) mRNA detected at 2 or 24 h after the initial infusion in the clinical responders, but no persistent change in these cytokine mRNA levels were observed that correlated with clinical benefit. By contrast, IL-6 mRNA levels declined immediately after the first MoAb infusion and reached pretreatment values variably after the 5 day treatment course. IL-6 mRNA levels remained significantly reduced in patients responding to therapy 1 months after treatment. Fluctuations in monocyte numbers within the PBMC after treatment did not account for the observed changes in cytokine mRNA levels. The results suggest that a decline in IL-6 mRNA levels is a pharmacological action of the MoAb to ICAM-1. Moreover, persistently diminished IL-6 mRNA levels induced by anti-ICAM-1 MoAb might be associated with the long-term benefit of this therapy. In addition, monitoring the activation status of monocytes in circulating PBMC may be useful in predicting response to therapy and warrants further investigation in a larger study population.

Topics: Antibodies, Monoclonal; Arthritis, Rheumatoid; Humans; Infusions, Intravenous; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Interleukin-8; Monocytes; RNA, Messenger; Tumor Necrosis Factor-alpha

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

Topics: Arthritis, Rheumatoid; Blotting, Northern; Cyclooxygenase 2; Dinoprostone; Fibroblasts; Humans; In Vitro Techniques; Interleukin-1; Interleukin-8; Isoenzymes; Membrane Proteins; NF-kappa B; Oligonucleotides, Antisense; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Synovial Membrane; Transcription Factor RelA; Up-Regulation

To evaluate the role of interleukin-8 (IL-8) on the activation of neutrophils in rheumatoid arthritis (RA), we measured IL-8 and the adhesion molecules, L-selectin (CD62L), CD1 1b and CD18, on neutrophils in paired peripheral blood and synovial fluid of RA patients. Synovial fluid IL-8 levels were significantly increased compared to peripheral blood. L-selectin was split off and CD1 1b and CD 18 were upregulated on neutrophils in the synovial fluid. A positive correlation occurred between the IL-8 concentration and CD18 or CD1 1b densities on neutrophils in the synovial fluid (r = 0.75, P < 0.005 and r = 0.60, P < 0.05, respectively). Peripheral blood neutrophils of the patients were desensitised with IL-8 in vivo, as shown by the significantly lower L-selectin shedding after in vitro IL-8 stimulation: 1.6 times decrease for patients vs 3.2 for controls (P < 0.05). In conclusion, these results add further evidence for the role of IL-8 in the activation of neutrophils in RA.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; CD11 Antigens; CD18 Antigens; Humans; Interleukin-8; L-Selectin; Middle Aged; Neutrophil Activation; Neutrophils; Synovial Fluid

The relationships between synovial fluid (SF) interleukin-8 (IL-8) and neutrophil turnover as measured by cytidine deaminase (CD), and SF metabolites were studied in 28 patients, 16 with rheumatoid arthritis (RA; median age and disease duration 62 and 14 yr, respectively) and 12 with seronegative polyarthritis (SNP; median age and disease duration 32 and 5 yr, respectively). Knee SF samples were aspirated using indwelling cannulae following a period of rest for 1 h. SF IL-8 levels (measured by an ELISA) were significantly elevated in RA compared to SNP (median 2.35 vs 0.22 ng/ml, P < 0.001), as were median levels of CD (55.8 vs 8.11 U/ml, P < 0.01), lactic acid (29.6 vs 16.6 mg/dl, P < 0.001), glucose (57.9 vs 84.5 mg/dl, P < 0.05) and the lactate to glucose ratio (0.85 vs 0.19, P < 0.001). Measures of disease activity, C-reactive protein, plasma viscosity and articular index were also elevated in RA compared to SNP (P < 0.05). SF IL-8 levels correlated strongly with CD, lactate, glucose and the lactate to glucose ratio when both disease groups were considered together (P < 0.001). These parameters also showed some association with the measures of disease activity (P < 0.05). All these associations were less marked when the individual disease groups were analysed separately. These results suggest that factors responsible for neutrophil accumulation and priming (probably IL-8) are present in SF, and these coincide with products of their activation (CD). The degree of neutrophil turnover is linked to the anaerobic metabolism of the synovial cavity.

Topics: Adult; Aged; Antibodies; Arthritis; Arthritis, Rheumatoid; C-Reactive Protein; Cytidine Deaminase; DNA; Female; Glycolysis; Humans; Interleukin-8; Male; Middle Aged; Neutrophils; Synovial Fluid

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast-like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon-gamma (IFN-gamma)-treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and 3H-thymidine incorporation was counted. Intercellular adhesion molecule-1 (ICAM-1), LFA-3 and vascular cell adhesion molecule-1 (VCAM-1) were detected on the surface of both RA and OA synoviocytes. However, ICAM-2, B7-1 and B7-2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up-regulated expression of ICAM-1, LFA-3 and VCAM-1 by IFN-gamma, IL-1beta or 12-o-tetradecanoyl phorbol 13-acetate (TPA) were markedly suppressed by clarithromycin in a dose-dependent manner at concentrations between 0.1 and 10 microg/ml. The production of IL-1beta, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-1alpha and tumour necrosis factor-alpha (TNF-alpha) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL-10 production. Finally, autologous T cells were stimulated by IFN-gamma-treated synoviocytes in response to PPD. As clarithromycin suppressed HLA-DR and costimulatory molecule expression was enhanced by IFN-gamma, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen-specific T cell proliferation induced by synoviocytes.

Topics: Anti-Bacterial Agents; Antibodies, Monoclonal; Antigens, CD; Arthritis, Rheumatoid; B7-1 Antigen; CD58 Antigens; Cell Adhesion Molecules; Cells, Cultured; Clarithromycin; Cytokines; Dose-Response Relationship, Drug; Fibroblasts; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; HLA-DR Antigens; Humans; Immunoenzyme Techniques; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-1; Interleukin-10; Interleukin-6; Interleukin-8; Lymphocyte Activation; Osteoarthritis; Synovial Membrane; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tuberculin; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Topics: Arthritis, Rheumatoid; Chronic Disease; Humans; Interleukin-6; Interleukin-8; Synovitis; T-Lymphocytes

To determine whether concentrations of cytokines and matrix-degrading enzymes in synovial fluid of patients with rheumatoid arthritis or osteoarthritis are associated with the degree of bone-destruction in the same joint.. Determination of Interleukin-1 alpha, IL-1 beta, IL-1-receptor-antagonist, IL-6, IL-8, tumor necrosis factor alpha (by ELISA), collagenase-activity and caseinase-activity (by substrate-assays) in the SF (knee) of patients with RA (n42) or OA (n35). The degree of bone-destruction was assessed radiographically.. SF cytokine- and enzyme-levels were higher in patients with RA than in those with OA. In the RA group, SF-levels of TNF alpha were positively correlated with the degree of bone destruction of the respective joint. No correlation was found between radiographically assessed joint changes and SF-concentrations of other cytokines, enzyme activities, serum CRP, or duration of disease. In the OA-group, none of the examined parameters was associated with the degree of joint destruction.. Our data may support the assumption of TNF alpha playing an important role in joint destruction in RA. Possible alternative conclusions are discussed.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Bone Diseases; C-Reactive Protein; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-6; Interleukin-8; Knee Joint; Middle Aged; Osteoarthritis; Sialoglycoproteins; Synovial Fluid; Tumor Necrosis Factor-alpha

To evaluate the relation between synovial fluid (SF) concentrations of interleukin-6 (IL-6) and other mediators of inflammation which are responsible for joint degradation in rheumatoid arthritis (RA).. We measured IL-6, IL-1 beta, tumour necrosis factor alpha (TNF alpha), granulocyte macrophage colony stimulating factor, IL-8, and polymorphonuclear leucocyte (PMNL) chemotaxis and degranulation in SF from patients with RA (n = 30) in the early phase of the disease.. In a cross-sectional study IL-6 concentrations correlated with those of IL-1 beta, IL-8 and with PMNL activation as reflected by lactoferrin concentrations. In a longitudinal study, changes in IL-6 concentrations correlated with changes in TNF alpha, IL-8 and lactoferrin concentrations.. IL-6 in SF appears to reflect the local proinflammatory, potentially erosive activity in RA. This supports the use of acute phase proteins, which are mainly induced by IL-6, as variables to monitor the course of RA.

Topics: Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Cross-Sectional Studies; Follow-Up Studies; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Lactoferrin; Neutrophil Activation; Prospective Studies; Synovial Fluid; Tumor Necrosis Factor-alpha

Topics: Adult; Aged; Aged, 80 and over; Antibodies; Arthritis, Rheumatoid; Female; Follow-Up Studies; Humans; Interleukin-1; Interleukin-8; Male; Methotrexate; Middle Aged; Prognosis; Treatment Outcome

Normal human polymorphonuclear neutrophils (PMN) can spontaneously produce the third component of complement (C3) in in vitro culture as detected by ELISA. This C3-producing capacity of PMN can be augmented by TNF-alpha (20 ng/ml) and bacterial lipopolysaccharide (100 ng/ml), but not by IL-1 beta or IL-8. The C3 production by PMN was found to be temperature dependent and was suppressed by the addition of protein inhibitor. The C3 mRNA in PMN could be detected by reverse transcription assisted polymerase chain reaction (RT-PCR) after TNF-alpha or LPS stimulation for 6 hours. To further understand C3 production by peripheral blood PMN in rheumatoid arthritis (RA), spontaneous and TNF-alpha stimulated production of C3 by peripheral PMN were compared in 15 cases of active RA, 15 inactive RA and 15 normal individuals. We failed to find any significant difference among the three groups. We conclude that PMN plays a negligible role in C3 hypercomplementemia in patients with active RA.

Topics: Arthritis, Rheumatoid; Base Sequence; Cells, Cultured; Complement C3; Cycloheximide; DNA Primers; Gene Expression; Humans; Interleukin-1; Interleukin-8; Kinetics; Lipopolysaccharides; Molecular Sequence Data; Neutrophils; Polymerase Chain Reaction; Protein Synthesis Inhibitors; Reference Values; Tumor Necrosis Factor-alpha

Characteristic cellular changes have previously been reported in the bone marrow of patients with rheumatoid arthritis (RA). We investigated the levels of various cytokines in RA bone marrow.. We studied 25 patients with RA (22 women and 3 men) and 10 trauma patients (7 women and 3 men) as non-RA controls. Twelve kinds of cytokines [interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, tumor necrosis factor (TNF)-alpha, and TNF-beta] were assayed by ELISA in iliac bone marrow serum (BMS), tibial BMS, and peripheral blood serum.. Markedly elevated levels of IL-6 and IL-8 were detected in iliac BMS, and much lower levels were found in tibial bone marrow and peripheral blood serum. The levels of IL-6 and IL-8 in iliac BMS showed a close relationship to the extent of synovial proliferation.. Iliac bone marrow may be an important site for the production or accumulation of IL-6 and IL-8 in RA, and these cytokines may influence synovial proliferation in patients with polyarthritis.

Topics: Adult; Aged; Arthritis, Rheumatoid; Bone Marrow; Bone Marrow Cells; Female; Granulocyte Colony-Stimulating Factor; Humans; Ilium; Interleukin-6; Interleukin-8; Male; Middle Aged; Synovitis; Tibia

The accumulation of polymorphonuclear leukocytes (PMN) in synovial fluid is a common feature of rheumatoid arthritis (RA). We studied the chemotactic response of PMN obtained from the synovial fluid and from the peripheral blood of patients with RA using a modified Boyden's method, in which interleukin-8 (IL-8) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as a chemotactic agent. The IL-8-induced response of peripheral blood PMN from 15 patients with RA did not differ from that of 15 healthy controls. A decreased chemotactic response to IL-8 was, however, observed in PMN from the synovial fluid of 12 patients with RA compared with peripheral blood cells of the same individual. This defective chemotactic ability of PMN was inversely correlated with the number of infiltrating cells in the synovial fluid. We also obtained similar results with FMLP. These results indicate that the chemotactic ability of PMN may be reduced after migrating to the synovial fluid.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Female; Humans; Interleukin-8; Leukocyte Count; Male; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Synovial Fluid

To investigate the influence of corticosteroid pulse (CP) therapy on soluble interleukin 2 receptor (sIL-2R), interleukin 6 (IL-6) and IL-8 levels in patients with active rheumatoid arthritis (RA).. Twenty-five patients with active RA were studied before and after treatment with intravenous CP therapy. In 12 patients lymphocyte subsets were also assessed.. All patients showed a significant decrease in clinical disease activity variables after 4 to 8 weeks of CP therapy. Baseline levels of sIL-2R were higher among patients with active RA than healthy controls; after CP therapy, sIL-2R levels decreased significantly, but not as much as the erythrocyte sedimentation rate or C-reactive protein. In most cases baseline levels of both IL-6 (n = 23) and IL-8 (n = 17) could be measured and both decreased significantly after CP therapy. Cortisol levels were suppressed shortly after CP therapy but had returned to pretreatment values at 4 weeks. After CP therapy there was a temporary increase in the number of HLA-DR positive cells and a decrease in the number of CD3 positive cells, while the CD4/CD8 ratio and IL-2 receptor (CD25) positive cells remained unchanged.. High dose corticosteroids influence serum levels of sIL-2R, IL-6 and IL-8 in patients with active RA.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Dehydroepiandrosterone; Dehydroepiandrosterone Sulfate; Dexamethasone; Drug Administration Schedule; Drug Therapy, Combination; Female; Gold; HLA-DR Antigens; Humans; Hydrocortisone; Infusions, Intravenous; Interleukin-6; Interleukin-8; Lymphocyte Subsets; Male; Methotrexate; Middle Aged; Receptors, Interleukin-2; Rheumatoid Factor; Sex Hormone-Binding Globulin; Sulfasalazine

Synovial fluid aspirated from 34 patients with symptomatic rheumatoid arthritis (RA) was evaluated for the presence of human cytomegalovirus (CMV) genomic material using polymerase chain reaction (PCR), and for levels of interleukin 8 (IL-8) and IL-6 using enzyme-linked immunoadsorbence assay. IL-8 and IL-6 levels were significantly higher in CMV DNA-positive RA patients than CMV DNA-negative RA patients and at least 10-fold higher than in both corresponding control groups of patients with osteoarthritis (OA). These findings suggest an association between elevated IL-8 and IL-6 levels and the presence of the CMV genome in RA patients.

Topics: Arthritis, Rheumatoid; Cytokines; Cytomegalovirus; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-6; Interleukin-8; Osteoarthritis; Polymerase Chain Reaction; Synovial Fluid

Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA).. We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot.. SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils.. These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.

Topics: Arthritis, Rheumatoid; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-8; Neutrophils; RNA, Messenger; Sialoglycoproteins; Synovial Fluid

Synovial pannus represents a hypertrophic and locally invasive connective tissue response to chronic inflammation that accounts in large part for the periarticular destruction of rheumatoid arthritis. Synovial fibroblasts cultured from rheumatoid synovia have been found to display an increased rate of proliferation and the constitutive expression of collagenases, growth factors, and inflammatory cytokines. The existence in rheumatoid synovium of both a pro-inflammatory state and growth dysregulation led us to investigate the expression by synovial fibroblasts of the closely homologous cytokines GRO alpha (gro/MGSA), GRO beta (MIP-2 alpha), and GRO gamma (MIP-2 beta). These cytokines are released by a variety of cell types and display overlapping growth regulatory and pro-inflammatory activities. In contrast to expectations, the majority of synovial fibroblast cell lines derived from osteoarthritic or non-inflammatory synovia showed a relative increase in the constitutive expression of GRO alpha and GRO beta when compared to synovial fibroblasts obtained from rheumatoid synovia. Considered together with evidence that GRO alpha is a growth regulator that modulates the expression of metalloproteinase activity, these findings provide evidence for a differential pathway of cytokine activation that may downregulate the proliferative and erosive response to chronic arthritis.

Topics: Actins; Arthritis, Rheumatoid; Base Sequence; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Consensus Sequence; Cytokines; DNA Primers; Fibroblasts; Gene Expression; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Sulfur Radioisotopes; Synovial Membrane; Transcription, Genetic

This study analyses the mRNA and protein production and their regulation of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8 and IL-6 by synovial fibroblasts obtained from patients with RA and OA. M-CSF was found to be produced constitutively as opposed to other cytokines. Stimulation of the cells with IL-1 beta caused a marked increase of GM-CSF, IL-8, IL-6 and as well as of M-CSF mRNA levels. In parallel, a time-dependent increase of M-CSF, GM-CSF, IL-8 and IL-6 protein production was observed. Among the cytokine mRNAs examined only that of M-CSF exhibited a pronounced stability in unstimulated synovial fibroblasts, whereas the other cytokines displayed short mRNA half-lives of 1-2 h. Induction by IL-1 beta markedly prolonged IL-8, IL-6 and GM-CSF mRNA half-lives to > 8 h which indicates increased mRNA stability. These findings suggest that among the cytokines that are produced in the inflamed synovium M-CSF may be particularly important for sustaining long-term influx, activation and survival of mononuclear phagocytes. GM-CSF, IL-8 and IL-6, by contrast, may be more involved in more acute cellular responses.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Female; Fibroblasts; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-6; Interleukin-8; Macrophage Colony-Stimulating Factor; Male; Osteoarthritis; RNA, Messenger; Synovial Membrane

Activated synoviocytes are major effector cells in the pathogenesis of rheumatoid arthritis (RA) because of their capacity to secrete a variety of inflammatory mediators. Among these mediators, the chemotactic proteins monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-8) are likely to contribute to the recruitment of inflammatory cells into the arthritic joint. We examined the effects of anti-rheumatic drugs on the MCP-1 and IL-8 production by cultured RA synoviocytes exposed to pro-inflammatory agonists. Both chemotactic cytokines were quantified by specific enzyme-linked immunosorbent assays (ELISA), and found to accumulate in the culture supernatants. Although the time course of formation was similar, the yield of IL-8 was three to 10-fold higher than that of MCP-1. Non-steroidal anti-inflammatory drugs inhibited the synthesis of prostaglandins, but did not influence the production and release of both chemotactic cytokines. Of three disease-modifying drugs tested, dexamethasone and gold sodium thiomalate (GST) inhibited the production of IL-8 and MCP-1, while methotrexate (MTX) was inactive. Dexamethasone reduced the production of MCP-1 and IL-8 by 20-65% and 60-80%, respectively, whilst GST inhibited MCP-1 and IL-8 synthesis in suboptimally, but not in optimally stimulated synoviocytes. Taken together, these results show that the production of MCP-1 and IL-8 is similarly affected by anti-rheumatic drugs and that dexamethasone is the most potent inhibitor suggesting that part of the anti-rheumatic action of glucocorticoids is due to prevention of accumulation of chemotactic cytokines acting on neutrophils and monocytes.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Rheumatoid; Cells, Cultured; Chemokine CCL2; Chemotactic Factors; Cytokines; Dexamethasone; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gold Sodium Thiomalate; Humans; Interleukin-1; Interleukin-8; Kinetics; Methotrexate; Prostaglandins; Prostaglandins E; Sensitivity and Specificity; Synovial Membrane

High levels of many cytokines, including interleukin (IL)-1, IL-6 and IL-8, were found in various arthropathies suggesting that they play a role in the pathogenesis of disease, although their relationship with the type and activity of disease is still not clear. The synovial fluid (SF) of 24 patients with rheumatoid arthritis (RA), 19 with psoriatic arthritis (PA) and 33 with osteoarthritis (OA) was analyzed for IL-1 beta, IL-6 and IL-8. The highest concentration of the three cytokines was found in the SF of RA. IL-beta detectable levels (> or = 20 pg/ml) were observed in 8/24 (33.3%) patients with RA, in one patient with PA but in no patient with OA. IL-6 (mean +/- SD) (1610.37 +/- 1781.65 pg/ml) was higher in RA than in PA (672.47 +/- 867.40 pg/ml, p = 0.043) and OA (89.45 +/- 120.52 pg/ml, p = 0.0001). IL-8 (1042.72 +/- 698.64 pg/ml) was higher in RA than in PA (660.36 +/- 625.11 pg/ml, p = 0.03) and OA (89.9 +/- 45.88 pg/ml, p = 0.0001). A correlation between IL-1 beta, IL-6 and IL-8 was found in RA. In all patients a correlation between IL-6 and IL-8 levels was found; moreover, these two cytokines were associated with SF indices of inflammation, such as white blood cells (WBC) count and total protein (TP) concentration. Our findings suggest that these interrelationships play a role in the evolution of more severe erosive arthropathy such as RA.

Topics: Arthritis; Arthritis, Psoriatic; Arthritis, Rheumatoid; Female; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Knee Joint; Leukocyte Count; Male; Muramidase; Osteoarthritis; Synovial Fluid

We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.

Topics: Arthritis; Arthritis, Rheumatoid; Base Sequence; Biological Assay; Blotting, Northern; Chemokine CXCL5; Chemokines, CXC; Chemotaxis, Leukocyte; Humans; In Vitro Techniques; Interleukin-1; Interleukin-8; Macrophages; Molecular Sequence Data; Neutrophils; Oligonucleotide Probes; Osteoarthritis; Recombinant Proteins; Reference Values; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Interleukin-8 (IL-8) may play an important role in the development of synovitis in rheumatoid arthritis (RA), in that it is a powerful chemoattractant for neutrophils and T cells. The aim of this study was to examine the distribution of IL-8 in the synovial membrane and cartilage, from RA, osteoarthritis (OA) and normal joints. By immunohistochemical techniques, IL-8 was shown to be present in the lining layer cells in RA (87%) and in OA (62%). By contrast, only a few of the normal synovial lining layer cells (14%) contained IL-8. Deeper in the membrane the number of IL-8 positive cells decreased. Only vessels were highly positive for IL-8. At the RA cartilage-pannus junction 26% of the cells contained IL-8, whereas at the OA cartilage-pannus junction 8% of the cells were IL-8 positive (P < 0.05). Chondrocytes present in joint surface cartilage stained positive for IL-8 in an average of 20% of the cells of both RA and OA. These results provide histological evidence that IL-8 is present in the arthritic synovial tissue and cartilage, and is distributed in a manner that may form a chemotactic gradient, which favours localisation of neutrophils to the joint lumen.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cartilage, Articular; Female; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Interleukin-8; Joints; Male; Middle Aged; Osteoarthritis; Synovial Membrane; Tissue Distribution

We have examined the ability of interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-1 receptor antagonist protein (IL-1ra) to regulate spontaneous interleukin-8 (IL-8) production in cultured SF mononuclear cells (SFMC) from RA. Furthermore, we examined whether IL-4, IL-10, or IL-1ra could influence the production of the arachidonic acid products leukotriene B4 (LTB4), 12-hydroxy-eicosatetraenoic acid (12-HETE) and 15-hydroxy-eicosatetraenoic acid (15-HETE). IL-4 induced a maximal suppression of 75% in the IL-8 secretion in SFMC from 10.0 ng/ml down to 2.5 ng/ml after 24 h and from 17.2 ng/ml to 4.2 ng/ml after 72 h of culture. IL-10 induced a 55% inhibition of the IL-8 secretion at 24 h and a 40% inhibition at 72 h. IL-1ra did not change the spontaneous IL-8 secretion from rheumatoid SFMC. We also examined, whether addition of IL-4, IL-10 or IL-1ra was able to modulate formation of the arachidonic acid products LTB4, 12-HETE and 15-HETE in cultured SF cells, stimulated with the calcium ionophore A23187. 15-HETE was not detected in untreated cultures, nor in IL-10 or IL-1ra treated cultures. IL-4, however, stimulated the formation of the anti-inflammatory mediator; 15-HETE (23 ng/10(6) cells). These results suggest that IL-4 or IL-10, could have beneficial anti-inflammatory effects in RA.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arthritis, Rheumatoid; Humans; Hydroxyeicosatetraenoic Acids; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-4; Interleukin-8; Leukotriene B4; Sialoglycoproteins; Synovial Fluid

A chronic inflammatory disease may be characterized by an accumulation of activated leukocytes at the site of inflammation. Since the chemokine RANTES may play an active role in recruiting leukocytes into inflammatory sites, we investigated the ability of cultured human synovial fibroblasts isolated from patients suffering from rheumatoid arthritis to produce this chemokine and compared its regulation to that of the closely related chemokine gene, interleukin-8 (IL-8). In unstimulated synovial fibroblasts, the expression of mRNA for both chemokines was undetectable, but was increased in both a time- and dose-dependent manner upon stimulation with the monokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta). Preincubation of the cells with cycloheximide "superinduced" the level of IL-8 mRNA stimulated by TNF alpha and IL-1 beta and RANTES mRNA stimulated by IL-1 beta, but decreased the expression of RANTES mRNA in response to TNF alpha. In addition, differential regulation of these genes was noted when synovial fibroblasts were stimulated with a combination of cytokines. IL-4 down-regulated and IFN gamma enhanced the TNF alpha- and IL-1 beta-induced increase in RANTES mRNA, whereas the induction of IL-8 mRNA by TNF alpha or IL-1 beta was inhibited by IFN gamma and augmented by IL-4. Moreover, a combination of TNF alpha and IL-1 beta synergistically induced IL-8 mRNA expression, whereas under the same conditions, the level of expression of RANTES mRNA was less than that induced by TNF alpha alone. These observations were also reflected at the level of chemokine secretion. These studies demonstrate that by expressing the chemokines RANTES and IL-8, synovial fibroblasts may participate in the ongoing inflammatory process in rheumatoid arthritis. In addition, the observation that these chemokine genes are differentially regulated, depending upon the presence of different cytokines, indicates that the type of cellular infiltrate and the progress of the inflammatory disease is likely to depend on the relative levels of stimulatory and inhibitory cytokines.

Topics: Arthritis, Rheumatoid; Base Sequence; Cells, Cultured; Chemokine CCL5; Cycloheximide; Cytokines; Dactinomycin; Fibroblasts; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-8; Lymphokines; Molecular Sequence Data; RNA, Messenger; Synovial Membrane; Tumor Necrosis Factor-alpha; Up-Regulation

IL-8 was measured in knee joint synovial fluid of 60 patients with rheumatoid arthritis, 8 with gout, 6 with osteoarthritis and 4 with meniscus lesions. IL-8 could be demonstrated in most SF samples. The highest levels were observed in rheumatoid joint effusions, yet mean levels were not significantly different between the different subgroups (mean +/- SE; RA 1537 +/- 3049 pg/ml, gout 570 +/- 952 pg/ml, OA/ML 178 +/- 188 pg/ml). In RA patients, IL-8 levels could not be related to various serological, clinical or radiological parameters. However, a correlation was observed between SF levels of IL-8 with those of lactate, LDH, beta 2-microglobulin and glucose. These observations suggest that next to the laboratory parameters IL-8 will be a parameter of the activity of the local inflammatory process. The results also demonstrate that IL-8 is not a disease-specific marker of joint inflammation.

Topics: Arthritis, Rheumatoid; Gout; Humans; Interleukin-8; Osteoarthritis; Synovial Fluid

To investigate both the involvement of chemokines in general and the relative importance of specific chemokines in rheumatoid arthritis (RA), we characterized the effect of the monokines tumor necrosis factor alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) on the synthesis of neutrophil-activating factors by synovial fibroblasts isolated from the joints of patients with RA.. Neutrophil-stimulating activity was assessed by determining intracellular calcium mobilization. IL-8 synthesis and secretion was assessed by specific enzyme-linked immunosorbent assay, and IL-8 messenger RNA (mRNA) levels were determined by Northern blot.. Treatment of synovial fibroblasts with IL-1 beta and TNF alpha resulted in the production of an activity which induced intracellular calcium mobilization in peripheral blood neutrophils. The 2 monokines combined had a synergistic effect on the release of the neutrophil-stimulating activity. The effect of the 2 monokines required gene transcription and translation, and closely mimicked the pattern of IL-8 secretion induced in these cells by the monokines. We confirmed that the majority of the neutrophil-stimulating activity was IL-8 by 3 different approaches: cross-desensitization experiments with IL-8, melanoma growth-stimulatory activity, and neutrophil-activating peptide 2, stimulation of calcium mobilization in cells transfected with the IL-8 receptor complementary DNA, and inhibition of the activity following pretreatment of the supernatants with an anti-IL-8 antibody. TNF alpha and IL-1 beta induced a time- and dose-dependent release of immunoreactive IL-8. A synergistic effect of TNF alpha and IL-1 beta was also observed for both IL-8 production and accumulation of IL-8 mRNA.. These results indicate that the monokines TNF alpha and IL-1 beta synergistically activate IL-8 expression and protein secretion by synovial fibroblasts, and that under these conditions, IL-8 appears to be the major neutrophil-activating factor released.

Topics: Arthritis, Rheumatoid; Drug Synergism; Fibroblasts; Gene Expression; Humans; Interleukin-1; Interleukin-8; Neutrophils; Proteins; Synovial Membrane; Tumor Necrosis Factor-alpha

Melanoma growth-stimulatory activity (MGSA)/GRO is well characterized as a potent neutrophil chemoattractant. In the present study, we have demonstrated that MGSA induced a dose-dependent decrease in the expression of interstitial collagens by rheumatoid synovial fibroblasts. The decrease was observed over a dose range of 0.6-6.0 nM MGSA. This effect was specific, as MGSA had no demonstrable effect on the expression of collagen-degrading metalloproteinases, nor did it affect the collagenase inhibitor, tissue inhibitor of metalloproteinases. It also had no effect on the proliferation rate of these fibroblasts, unlike its mitogenic effect on melanoma cells. The ability to inhibit collagen expression was also demonstrated by another member of the C-X-C branch of the platelet factor 4 superfamily, interleukin-8 (IL-8), but not by RANTES, MIP-1 alpha, or MIP-1 beta, which belong to the C-C branch. Steady-state levels of expression of MGSA and IL-8 transcripts in normal adult tissues were dissimilar, suggesting that expression may be an important level at which the activity of these cytokines is regulated. Direct binding experiments with 125I-MGSA on synovial fibroblasts have allowed us to identify an MGSA receptor with a KD of 10.1 nM and approximately 75,000 binding sites/fibroblast. 125I-MGSA binding was specific and could not be displaced by unlabeled IL-8. These results suggest that MGSA, as well as IL-8, may play a role other than that of neutrophil chemo-attractant and more specifically, may be important in the regulation of collagen turnover.

Topics: Arthritis, Rheumatoid; Base Sequence; Blotting, Northern; Cell Division; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Collagen; Cytokines; Fibroblasts; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Kinetics; Melanoma; Multigene Family; Synovial Membrane; Transcription, Genetic; Tumor Cells, Cultured

In response to interleukin 1 or tumor necrosis factor, human synovial cells and fibroblasts expressed several genes encoding known chemotactic factors or related proteins. Transcripts for interleukin 8 (IL-8), gro/MGSA, pAT 464, IP-10, pAT 744 and Monocyte Chemotactic and Activating Factor (MCAF) accumulated rapidly in IL-1 and TNF-treated cells. The inhibition of protein synthesis led to the superinduction of IL-8 and gro/MGSA mRNAs in IL-1, but not in TNF-treated cells. Thus, IL-1 and TNF are likely to regulate the expression of these mRNAs by different mechanisms. Important cell-specific differences in mRNA accumulation characterized the expression of chemotactic factor genes. Moreover, only a subset of the same genes was activated in quiescent cells stimulated by serum. Therefore, genes encoding closely related proteins each had a distinct pattern of expression. continuous stimulation of fibroblasts and synovial cells with IL-1 resulted in high and prolonged expression of IL-8 and gro/MGSA mRNAs. These results extend the list of chemotactic factor genes expressed by mesenchymal cells in vitro and suggest a pivotal role for these cells in processes such as chronic inflammation.

Topics: Arthritis, Rheumatoid; Blotting, Northern; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Fibroblasts; Gene Expression Regulation; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1; Interleukin-8; Multigene Family; Osteoarthritis; Protein Biosynthesis; RNA, Messenger; Synovial Fluid; Tumor Necrosis Factor-alpha

Angiogenic factors produced by monocytes-macrophages are involved in the pathogenesis of chronic inflammatory disorders characterized by persistent angiogenesis. The possibility was tested that interleukin-8 (IL-8), which is a cytokine that is chemotactic for lymphocytes and neutrophils, is also angiogenic. Human recombinant IL-8 was potently angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells. Angiogenic activity present in the conditioned media of inflamed human rheumatoid synovial tissue macrophages or lipopolysaccharide-stimulated blood monocytes was equally blocked by antibodies to either IL-8 or tumor necrosis factor-alpha. An IL-8 antisense oligonucleotide specifically blocked the production of monocyte-induced angiogenic activity. These data suggest a function for macrophage-derived IL-8 in angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.

Topics: Animals; Arthritis, Rheumatoid; Base Sequence; Cell Division; Cells, Cultured; Chemotaxis; Cornea; Dose-Response Relationship, Drug; Endothelium, Vascular; Fibroblast Growth Factor 2; Humans; Interleukin-8; Macrophages; Mice; Molecular Sequence Data; Monocytes; Neovascularization, Pathologic; Oligonucleotides, Antisense; Rabbits; Rats; Recombinant Proteins; Synovial Fluid; Tumor Necrosis Factor-alpha; Umbilical Veins

Interactions between interleukin 8 (IL-8) and endothelial cells play an important role in the emigration of mononuclear cells from the blood into areas of inflammation. We examined the ability of specific second-line antirheumatic drugs to regulate (IL-8) gene expression and protein secretion in interleukin 1 (IL-1) stimulated human umbilical vein endothelial cells and peripheral blood mononuclear cells. The drugs sodium aurothiomalate, D-penicillamine and sulphasalazine were all able to modulate IL-8 mRNA synthesis in and protein secretion from endothelial cells. A bimodal effect was observed: at low concentrations IL-8 was suppressed, whereas higher concentrations resulted in an increased IL-8 production. In endothelial cells, treatment with hydrocortisone led to a linear suppression of IL-8 production in concentrations ranging from 0.5 micrograms/ml up to 500 micrograms/ml. Sulphapyridine, auranofin, hydroxychloroquine and methotrexate, had no effect on IL-8 secretion in endothelial cells. By contrast, 5-aminosalicylic acid induced a threefold increase in the IL-8 release. In peripheral blood mononuclear cells it was only possible to suppress the IL-8 production by hydrocortisone treatment. These results indicate that suppression of IL-8 production in endothelial cells could be an important factor in the mode of action for a number of second-line antirheumatic drugs.

Topics: Anti-Inflammatory Agents; Arthritis, Rheumatoid; Endothelium, Vascular; Gene Expression; Gold Sodium Thiomalate; Humans; Hydrocortisone; In Vitro Techniques; Interleukin-1; Interleukin-8; Penicillamine; RNA, Messenger; Sulfasalazine

The content of interleukin-8 (IL-8) in the synovial fluid and its production by blood and synovial fluid mononuclear cells (PBMC and SFMC) was compared in rheumatoid arthritis (RA) and various other inflammatory rheumatic disorders. The study included 125 patients and 20 healthy individuals. The highest concentrations of IL-8 were found in the synovial fluids and culture supernatants of PBMC and SFMC from patients with seropositive RA. Only PBMC from seropositive patients, and not from other rheumatic diseases, exhibited significant spontaneous release of IL-8 that correlated with serum IgM rheumatoid factor titers. Gold sodium thiomalate (GST) and methotrexate (MTX) inhibited the spontaneous and stimulated IL-8 production by PBMC by 55-86% at 50 and 10 micrograms/ml, respectively. Two main conclusions were drawn: (1) rheumatoid factors appeared to be a major cause of enhanced IL-8 production in seropositive RA, and (2) inhibition of IL-8-mediated neutrophil migration and activation could be part of the mechanism of action of GST and MTX.

Topics: Anti-Inflammatory Agents; Arthritis, Psoriatic; Arthritis, Rheumatoid; Gold Sodium Thiomalate; Humans; Immunoglobulin M; Interleukin-8; Methotrexate; Monocytes; Rheumatic Diseases; Rheumatoid Factor; Synovial Fluid

Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.

Topics: Arthritis, Rheumatoid; Base Sequence; Chemokine CCL2; Chemotactic Factors; Fibroblasts; Humans; Immunohistochemistry; Interleukin-8; Macrophages; Molecular Sequence Data; RNA, Messenger; Synovial Fluid

Human neutrophil activating peptide/interleukin 8 (NAP-1/IL-8) has been shown to activate neutrophils to degranulate in vitro and to be a potent chemotactic agonist for neutrophils and lymphocytes in vitro and in vivo. It may therefore be a mediator of inflammatory conditions such as rheumatoid arthritis (RA). Levels of NAP-1/IL-8 were low or undetectable in serum samples from 53 patients with RA. Circulating levels of antibodies to NAP-1/IL-8 showed a strong correlation with the level of quantified C reactive protein and with the number of arthritic joints. These autoantibodies, in a similar manner to quantified C reactive protein, correlated with disease activity and are associated with a lack of clinical improvement when the patient is treated with systemic steroids. This observation indicates an important role for interleukin 8 and its autoantibodies in the inflammatory processes of RA, and may provide a clinically useful marker for the diagnosis of disease severity.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Autoantibodies; Autoantigens; C-Reactive Protein; Female; Humans; Immunoglobulin G; Interleukin-8; Male; Middle Aged

Topics: Arthritis, Rheumatoid; Interleukin-8; Kinetics; Leukocytes, Mononuclear; Osteoarthritis; Synovial Fluid; Time Factors

A sensitive and specific radioimmunoassay for human interleukin-8 (IL-8) was developed using isotopically labeled homogenous natural protein. The detection limit (20% inhibition of 125I-IL-8 binding) was 30 pg/100 microliters; 50% displacement occurred at 140 pg/100 microliters. There was no cross-reactivity with the structurally and functionally related neutrophil-activating peptides 2 and 3 up to 500 ng/100 microliters. The intra- and inter-assay coefficients of variation were 4 and 7%, respectively. In vitro experiments showed that human fibroblasts triggered by interleukin-1, double-stranded RNA or virus release immunoreactive and biologically active IL-8 in a dose- and time-dependent manner. Monocytes produce immunoreactive IL-8 in the 100 ng/ml range when exposed to plant mitogen, bacterial endotoxin, virus or IL-1. Although the radioimmunoassay was more sensitive than the chemotaxis assay (detection limit 0.6 ng/ml versus 10 ng/ml) a correlation between concentrations of immunoreactive IL-8 and neutrophil chemotactic activity in the supernatants from stimulated monocytes and fibroblasts was observed. In synovial fluids from patients with inflammatory joint disease, IL-8 was clearly demonstrable, but there was no correlation between IL-8 levels and general parameters of disease activity (erythrocyte sedimentation rate and serum levels of C-reactive protein). Synovial fluids from patients with rheumatoid arthritis, seropositive for rheumatoid factor, contained significantly higher concentrations of IL-8 than synovial fluids from seronegative rheumatoid arthritis patients and patients with non-rheumatoid arthritis joint inflammation. There was a highly significant correlation between IL-8 levels and serum titers of rheumatoid factor. These findings suggest that the molecular mechanisms underlying joint inflammation may be distinct in different types of arthritis.

Topics: Adult; Aged; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Female; Fibroblasts; Humans; Interleukin-1; Interleukin-8; Male; Middle Aged; Monocytes; Radioimmunoassay; Rheumatoid Factor; RNA, Double-Stranded; RNA, Viral; Sensitivity and Specificity; Synovial Fluid

Topics: Adult; Aged; Aged, 80 and over; Antibodies; Arthritis, Rheumatoid; C-Reactive Protein; Female; Humans; Immunoglobulin G; Interleukin-8; Male; Middle Aged; Synovial Fluid

Topics: Arthritis, Rheumatoid; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Monocytes; Synovial Membrane

The synovial fluid in affected joints of rheumatoid arthritis (RA) patients contains many cells, in numbers strongly correlated with the severity of disease. As the disease worsens and the cell count increases, the polymorphonuclear leucocyte becomes the predominant cell type. Although the inflammatory cytokines interleukin 1 (IL-1) and tumour necrosis factor (TNF) have no direct neutrophil-attractant activity, they are both potent inducers of interleukin 8 (IL-8) in a variety of cell types. Chemotactic attraction of neutrophils is a major activity of IL-8. Examination of a number of synovial fluids showed that significant levels of IL-8 are present in a high proportion of RA cases (10 out of 17), at concentrations directly related to the number of cells in the joint, and to circulating C-reactive protein (CRP) levels. The cytokine is present only at background levels in other diseases accompanied by arthritic manifestations, including systemic lupus erythematosus (SLE) and induced arthritis. The progressive joint destruction seen in all cases where high IL-8 levels were measured, coupled with the neutrophil-rich cell count and the strong correlation between concentration of IL-8 and both serum CRP and cellular influx into the joint, is strongly suggestive of a pathogenic role for IL-8 in RA.

Topics: Arthritis, Rheumatoid; C-Reactive Protein; Cell Count; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-8; Knee Joint; Synovial Fluid; Triamcinolone

Production of the neutrophil-activating peptide (NAP)-1/IL-8 by mononuclear phagocytes from patients with RA and from control subjects was studied under various conditions. Mononuclear cells from bone marrow (BMMC), PBMC, and synovial fluid (SFMC) were cultured for up to 48 h in the absence or presence of Escherichia coli LPS, different interleukins, interferon-gamma, zymosan, or immune complexes, and the neutrophil-stimulating activity released into the culture medium was determined. As shown by neutralization with an antiserum raised against human recombinant NAP-1/IL-8, over 90% of this activity could be attributed to NAP-1/IL-8. In unstimulated mononuclear cells from control individuals and BMMC from RA patients, the production of NAP-1/IL-8 was very low and was enhanced moderately by stimulation with LPS. By contrast, the spontaneous production of NAP-1/IL-8 was 3- to 10-fold higher in PBMC and even much higher in SFMC from RA patients. In all instances, the yield of NAP-1/IL-8 could be enhanced by stimulation in culture. In addition to LPS, rheumatoid factor-containing immune complexes, zymosan, and IL-1 were highly effective in inducing NAP-1/IL-8 production, while IL-3, GM-CSF, tumor necrosis factor (TNF), and IL-2 were somewhat less potent. An inhibitory effect was obtained with IFN-gamma, which significantly decreased the spontaneous NAP-1/IL-8 release from SFMC and the IL-1- and LPS-induced NAP-1/IL-8 from RA and control PBMC. Inhibition was also observed with glucocorticoids. The production of NAP-1/IL-8 was markedly reduced by dexamethasone in phagocytosis-stimulated PBMC, and almost totally inhibited in SFMC obtained from joints after intraarticular administration of betamethasone. By contrast, the cyclooxygenase inhibitor, indomethacin, tended to increase the NAP-1/IL-8 yield from PBMC in culture.

Topics: Arthritis, Rheumatoid; Cells, Cultured; Chemotactic Factors; Glucocorticoids; Humans; Immunoglobulin G; Indomethacin; Interferon-gamma; Interleukin-8; Neutrophils

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.

Topics: Animals; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Fibroblasts; Humans; Immunohistochemistry; Interleukin-8; Macrophages; Rabbits; Synovial Fluid; Synovial Membrane

Increased amounts of interleukin-8 (IL-8) were detected in synovial fluids of patients with active rheumatoid arthritis (RA) by radioimmunoassay (RIA). The concentration of IL-8 correlated directly with the number of infiltrating neutrophils in synovial fluids. To elucidate the role of IL-8 in neutrophil accumulation at the site of synovitis, the in vivo effects of intraarticular injection of recombinant IL-8 (rIL-8) on leukocytes infiltration into the joint space and synovium were examined. Following a single injection of rIL-8 into the knee joint space of rabbits, redness of the joint and limp became apparent after 4 h and were associated with the rapid infiltration of neutrophilic leukocytes into the joint space and synovial tissues. These effects were time dependent, first becoming evident at 1 h and reaching a plateau in 4 h, and also dose dependent, with a minimal effect being elicited by 100 ng per joint. Although neutrophils were present in the greatest number at 4 h, subsequently mononuclear cells accumulated and became apparent in considerable number after 8 h. Synovial lining cells became ovoid, pleomorphic, and multilayered at 24 h. IL-8 had no effect on the breakdown of proteoglycan of articular cartilage. Based on these findings, IL-8 released from monocytes and synovial cells may be an important contributor to leukocyte accumulation and inflammatory events in the joints of RA.

Topics: Animals; Arthritis, Rheumatoid; Cell Movement; Glycosaminoglycans; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Joints; Leukocytes; Osteoarthritis; Rabbits; Recombinant Proteins; Synovial Fluid; Synovitis

Gelatinases (type IV collagenases) produced by normal peripheral blood leukocytes were studied by the use of a substrate conversion assay. When monocytes were stimulated with IL-1 beta discrete amounts of a 85-kDa gelatinase were detected. This type of gelatinase comigrated with a phorbol ester-inducible metalloproteinase from human tumor cells. The levels of induction of the monocytic enzyme after stimulation with IL-1, double-stranded RNA, LPS, and mitogens paralleled those of the secondary cytokine IL-6. When peripheral blood neutrophils were stimulated with IL-8 or PMA significant amounts of a 91-kDa neutrophil gelatinase were released, whereas with IL-1 beta no effect was observed. Both neutrophil and monocyte gelatinases cross-reacted in immunoprecipitation experiments with tumor cell-derived gelatinases. Further evidence for structural similarity between the IL-1-inducible monocytic (85 kDa) and the IL-8-regulated neutrophilic (91 kDa) gelatinases was obtained after purification of the proteins to homogeneity: both gelatinases possessed an identical amino terminal amino acid sequence and appeared as truncated forms of gelatinase from tumor cells. Synovial fluids of arthritic joints contained extremely high concentrations of the 91-kDa gelatinase. The concentrations of this type of gelatinase were correlated with the titers of the marker cytokine IL-6. The controlled production and activity of leukocyte-derived gelatinase may play an essential role in local proteolysis of the extracellular matrix and in leukocyte migration. In the arthritis patient this enzyme might contribute to the pathogenesis of joint destruction and might constitute a useful marker of disease status.

Topics: Amino Acid Sequence; Arthritis, Rheumatoid; Cytokines; Fibroblasts; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Leukocytes; Metalloendopeptidases; Molecular Sequence Data; Monocytes; Neutrophils; Synovial Fluid

The presence of neutrophils in the synovial joint of patients with rheumatoid arthritis (RA) is thought to be due to the activity of chemotactic factors released by activated cells in the joint. We have shown in this report, for the first time, the abundance of one such factor, interleukin 8 (IL 8), in the synovial fluid of patients both with RA and other non-RA joint diseases, and the spontaneous production of IL 8 mRNA by RA synovial cells in culture. There was no correlation between the levels of chemotactic activity and IL 8 protein, suggesting that other factors with similar neutrophil chemotactic activity are also present in the synovial fluid exudate. In support of this concept neither the level of chemotactic activity nor IL 8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex. Such migration is likely to be due to a number of chemotactic signals in addition to IL 8, which may either synergize with, or inhibit, the action of IL 8.

Topics: Adult; Aged; Arthritis, Rheumatoid; Chemotaxis, Leukocyte; Humans; In Vitro Techniques; Interleukin-1; Interleukin-8; Middle Aged; Neutrophils; RNA, Messenger; Synovial Fluid; Tumor Necrosis Factor-alpha

The production of a lymphokine, polymorphonuclear leukocyte (PMN) chemotactic factor (CF) was studied in lymphocytes obtained from patients with rheumatoid-factor positive rheumatoid arthritis (RA). Control lymphocytes and PMN were obtained from normal individuals. The stimulation of the lymphocytes was by phytohemagglutinin (PHA) and by mixed leukocyte culture. CF was produced to the same degree by the cells from normals and from patients with RA by either type of stimulation. There is significantly less 3H-Thymidine uptake in cells from RA patients as measured by counts per minute although the stimulation index was the same. This can be accounted for by decreased proliferation of RA cells.

Topics: Arthritis, Rheumatoid; Chemotactic Factors; Chemotaxis; Humans; Interleukin-8; Lymphocyte Activation; Lymphocytes; Neutrophils; Phytohemagglutinins