interleukin-8 and Arthritis

Studies have suggested the role of cytokines in inflammation, as determined by results obtained in vitro, or with assessments of clinical samples. However, extrapolation of in vitro results to an in vivo situation must be made with caution, and findings obtained from clinical samples tend to lack a causal relation between cytokines and inflammatory responses. Animal models of inflammation can be useful in understanding roles of cytokines at sites of inflammation. We examined the production kinetics and cellular sources of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-8, and IL-1 receptor antagonist (IL-1Ra), and obtained evidence for the involvement of these cytokines in a rabbit model of arthritis induced by lipopolysaccharide (LPS). We also attempted to analyze the inflammatory cytokine network among TNFalpha, IL-1beta, IL-8, and IL-1Ra. Understanding the role of cytokines in animal models paves the way to a better understanding of disease in humans.

Topics: Animals; Arthritis; Cytokines; Disease Models, Animal; Female; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-8; Neutrophils; Rabbits; Sialoglycoproteins; Tumor Necrosis Factor-alpha

Neutrophil infiltration into inflammatory sites is one of the hallmarks of acute inflammation. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to the infiltration at inflammatory sites. Interleukin-8 (IL-8), a novel leukocyte chemotactic activating cytokine (chemokine), is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. However, no definitive evidence has been presented on its role in recruiting and activating neutrophils in the lesions of various types of inflammatory reactions. We administered a highly specific neutralizing antibody against IL-8 in several types of acute inflammatory reactions, including lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, lung reperfusion injury, and acute immune complex-type glomerulonephritis. Anti-IL-8 treatment prevented neutrophil-dependent tissue damage as well as neutrophil infiltration in these conditions. These results suggest that IL-8 plays a causative role in acute inflammation by recruiting and activating neutrophils.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Arthritis; Cross Reactions; Dermatitis; Glomerulonephritis; Inflammation; Inflammation Mediators; Interleukin-1; Interleukin-8; Lipopolysaccharides; Lung Diseases; Neutrophil Activation; Neutrophils; Rabbits; Reperfusion Injury

The goal of this study was to evaluate the systemic and peripheral effects of preoperative administration of cyclooxygenase-2 inhibitor on pain and inflammation occurring with total knee replacement (TKR). Patients undergoing elective TKR were prospectively and randomly given oral rofecoxib (25 mg) or placebo (control group) 1 hour before surgery. All patients received an epidural combined with isoflurane anesthesia during the operation and patient-controlled epidural analgesia postoperatively. The outcome measures included pain scores during rest and movement of knee joints and cumulative morphine consumption. Femoral blood and knee joint drainage fluids were examined for leucocyte numbers and concentrations of cytokines (including IL-6, IL-8, IL-10, and TNF-alpha). Periarticular circumferential increments at 48 hours served as an indication of inflammatory edema. Pain scores during rest and knee joint movement on postoperative days 1 and 2 were better in those given rofecoxib than in control subjects, and cumulative morphine consumption for the first 24 hours was significantly reduced. Both groups had higher concentrations of IL-6 and IL-8 in knee drainage fluid compared with serum levels. Rofecoxib significantly decreased regional IL-6 and TNF-alpha level after surgery. Moreover, the incidence of febris and degree of local edema were lower in the rofecoxib group (P < .05), and peripheral IL-6 level significantly correlated with pain score at 48 hours. Preoperative administration of rofecoxib increases patient satisfaction with analgesia, reduces opioid requirement, and decreases both systemic and local anti-inflammation after TKR.. This randomized, double-blinded trial shows that preoperative administration of rofecoxib can greatly ameliorate the pain occurring with total knee joint replacement surgery and its accompanying reduction of general and local inflammatory reactions.

Topics: Administration, Oral; Aged; Analgesics, Opioid; Arthritis; Arthroplasty, Replacement, Knee; Chemotaxis, Leukocyte; Cyclooxygenase 2 Inhibitors; Drug Interactions; Edema; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; Lactones; Male; Middle Aged; Pain Measurement; Pain, Postoperative; Patient Satisfaction; Prospective Studies; Sulfones; Treatment Outcome; Tumor Necrosis Factor-alpha

With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and -activating properties, occurs in different NH

Topics: Arthritis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Proteomics; Synovial Fluid; Tandem Mass Spectrometry

We describe a 38-year-old woman who presented with a history of inflammatory arthritis, rash, and daily fevers. She was noted to have chronic parvovirus infection with persistently detectable viral titers and a novel mutation in the ELANE gene. ELANE encodes neutrophil elastase, a neutrophil serine protease with important antimicrobial effects, and is found as part of neutrophil extracellular trap (NET) complexes. Pathogenic ELANE mutations have been identified in forms of hereditary neutropenia. However, our patient never had neutropenia. Because of the striking clinical scenario, we investigated this mutation functionally.. NET formation by neutrophils was assessed by scanning electron microscopy. Neutrophil activation and neutrophil elastase production were evaluated by flow cytometry and fluorescent substrate-based functional assay, respectively. A multiplex assay was used to quantitate neutrophil inflammatory cytokine production. PyMOL software was used to generate 3-dimensional models of mutant elastase.. Activated neutrophils from the patient demonstrated a significantly decreased ability to form NETs on scanning electron microscopy, as well as quantitative defects in neutrophil activation and neutrophil elastase activity. The patient's neutrophils showed altered levels of interleukin-12 (IL-12) and IL-8, which are key cytokines for antiviral immunity and neutrophil chemotaxis. Three-dimensional mapping revealed that the mutation could alter protein folding and surface charge distribution, potentially perturbing protein trafficking. Thus, the mutation could affect neutrophil function by decreasing NETosis and altering key antiviral activities of neutrophils.. This is the first report of a human pathogenic ELANE mutation associated with a defect in NETosis and a distinct syndrome of recurrent viral infection and chronic inflammation.

Topics: Adult; Arthritis; Chronic Disease; Extracellular Traps; Female; Humans; Interleukin-12; Interleukin-8; Leukocyte Elastase; Mutation; Neutrophils; Parvoviridae Infections; Recurrence

Canine idiopathic polyarthritis (IPA) is characterized by increased numbers of polymorphonuclear leukocytes (PMNs) in the synovial fluid (SF). In humans, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 8 (CXCL8) recruit monocytes and neutrophils, respectively, and are involved in various inflammatory disorders. The aim of this study was to assess the roles of these chemokines in driving PMNs infiltration into the joints of dogs with IPA. SF samples were collected from dogs with IPA (n=19) and healthy controls (n=8), and the concentrations of SF CCL2 and CXCL8 were determined by ELISA. Dogs with IPA had significantly higher concentrations of CCL2 (3316±2452pg/ml, mean±SD) and CXCL8 (3668±3879pg/ml) compared with the healthy controls (235±45pg/ml and <15.6pg/ml, respectively). Then, an in vitro chemotaxis assay was performed using a modified Boyden chamber (pore size: 3μm). SF from IPA dogs had a chemoattractant activity for PMNs that purified from the peripheral blood of a healthy dog. We subsequently found that combination treatment with MK-0812 (an antagonist of CCL2 receptor) and repertaxin (an antagonist of CXCL8 receptors) significantly inhibited the migration of PMNs to SF from IPA dogs. Thus, expression of the CCL2 receptor (chemokine (CC motif) receptor 2 (CCR2)) was examined using polymerase chain reaction and immunocytochemistry. Canine peripheral blood PMNs exhibited significantly higher CCR2 mRNA expression levels than those in monocytes. In addition, we observed strong CCR2 expression on PMNs obtained from healthy controls and IPA dogs, although mononuclear cells did not express CCR2. Taken together, the data suggest that CCL2 acts as a canine PMNs chemotactic factor as well as CXCL8 and both CCL2 and CXCL8 facilitate the infiltration of PMNs into the joints of dogs with IPA.

Topics: Animals; Arthritis; Case-Control Studies; Chemokine CCL2; Chemotactic Factors; Chemotaxis, Leukocyte; Dog Diseases; Dogs; Female; Humans; Interleukin-8; Male; Neutrophils; Receptors, CCR2; RNA, Messenger; Synovial Fluid

The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.

Topics: Adult; Arthritis; Biomarkers; Case-Control Studies; Cytokines; Extremities; Fathers; Female; Humans; In Vitro Techniques; Interferon-gamma; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Middle Aged; Mutation; Nod2 Signaling Adaptor Protein; Nuclear Family; Pedigree; Sarcoidosis; Synovitis; Tumor Necrosis Factor-alpha; Uveitis

Osteochondral fractures are often seen on magnetic resonance imaging (MRI) of acutely injured knees, but their existence has gained little interest because of a lack of knowledge of their relation to treatment options and outcome. It is not clear whether acute phase synovial fluid (SF) concentrations of cartilage and bone markers and proinflammatory cytokines are different between traumatically injured knees with or without osteochondral fracture.. Acutely injured knees with an osteochondral fracture, particularly fractures with disrupted cortical bone, have higher concentrations of bone markers and cytokines than do knees without an osteochondral fracture.. Cross-sectional study; Level of evidence, 3.. Synovial fluid (hemarthrosis) was aspirated (median 1 day after injury) and 1.5-T MRI was performed (median 8 days after injury) in the acutely injured knee of 98 individuals (26% women; mean age, 23 years). As visualized on MRI, 39% knees had an osteochondral fracture with disrupted cortical bone, 30% had an osteochondral fracture with intact cortical bone, and 32% did not have an osteochondral fracture. Concentrations of sulfated glycosaminoglycan, ARGS aggrecan, cartilage oligomeric matrix protein, osteocalcin, secreted protein acidic and rich in cysteine (SPARC), osteopontin and proinflammatory cytokines (interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor [TNF]-α) were analyzed.. After adjusting for days between injury and SF aspiration, age at injury, and sex, knees with any osteochondral fracture (with or without disrupted cortical bone) had significantly higher SF concentrations of TNF-α (median [interquartile range (IQR)] = 9 [7-12] pg/mL vs. 7 [5-14] pg/mL; P = .013), whereas knees with an osteochondral fracture with disrupted cortical bone had significantly higher SF concentrations (medians [IQRs]) of SPARC (492 [328-754] ng/mL vs. 407 [140-685] ng/mL; P = .030), IL-8 (278 [148-628] pg/mL vs. 138 [67-413] pg/mL; P = .028), and TNF-α (11 [7-15] pg/mL vs. 7 [5-14] pg/mL; P = .004) compared with knees without an osteochondral fracture.. In acutely injured knees with hemarthrosis, a concomitant osteochondral fracture with disrupted cortical bone is associated with a higher degree of joint inflammation.

Topics: Acute Disease; Adolescent; Adult; Arthritis; Biomarkers; Cartilage Oligomeric Matrix Protein; Cartilage, Articular; Cross-Sectional Studies; Female; Fractures, Bone; Fractures, Cartilage; Hemarthrosis; Humans; Interleukin-8; Knee Injuries; Magnetic Resonance Imaging; Male; Middle Aged; Osteonectin; Patellar Dislocation; Posterior Cruciate Ligament; Soft Tissue Injuries; Suction; Synovial Fluid; Tibial Meniscus Injuries; Time Factors; Tumor Necrosis Factor-alpha; Young Adult

PGE2 is a potent lipid mediator of pain and oedema found elevated in RA. Microsomal prostaglandin E synthase-1 (mPGES-1) is a terminal enzyme of the PGE2 pathway inducible by proinflammatory cytokines. mPGES-1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High-mobility group box chromosomal protein 1 (HMGB1) is known to induce inflammation both by direct interaction with TLR4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB1 within the inflamed synovium, implies its pro-arthritogenic role in RA. We aimed to investigate the effects of IL-1β/HMGB1 complexes on mPGES-1 and other enzymes of the PGE2 pathway in synovial fibroblasts (SFs) from patients with arthritis. Furthermore, we studied the effect of COX-2 inhibition and IL-1RI antagonism on prostanoid and cytokine production by SFs. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL-1β significantly increased mPGES-1 and COX-2 expressions as well as PGE2 production, as compared to treatment with HMGB1 or IL-1β alone. Furthermore, NS-398 reduced the production of IL-6 and IL-8, thus indicating that IL-1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL-1RA completely abolished the induced PGE2 and cytokine production, suggesting an effect mediated through IL-1RI. IL-1β/HMGB1 complexes promote the induction of mPGES-1, COX-2 and PGE2 in SF. The amplification of the PGE2 biosynthesis pathway by HMGB1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.

Topics: Arthritis; Biosynthetic Pathways; Blotting, Western; Cells, Cultured; Chemokines; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cytokines; Dinoprostone; Drug Synergism; Fibroblasts; HMGB1 Protein; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1beta; Interleukin-6; Interleukin-8; Intramolecular Oxidoreductases; Nitrobenzenes; Prostaglandin-E Synthases; Sulfonamides; Synovial Membrane

Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

Topics: Animals; Arthritis; Blood Proteins; Cell Line; Disease Models, Animal; HeLa Cells; Humans; Immunoblotting; Interleukin-1beta; Interleukin-8; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; Mice; Mice, Inbred BALB C; Mice, Knockout; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Signal Transduction; Toll-Like Receptors; Transforming Growth Factor beta; Tumor Necrosis Factors

We studied the influence of IL-6 on chemokine production from peripheral blood mononuclear cells (PBMC), fibroblastic synovial cells and human umbilical vessel endothelial cells (HUVEC). Moreover, we examined the effect of IL-6 on the adhesion of U937 cells to HUVEC. For chemokine production, PBMC, fibroblastic synovial cells and HUVEC were cultured with IL-6 or IL-6 + soluble IL-6R (sIL-6R) for 24 h and then the production of MCP-1 and IL-8 were measured in supernatants. IL-6 and IL-6 + sIL-6R induced production of both MCP-1 and IL-8 in PBMC and synovial cells, respectively. In HUVEC, IL-6 + sIL-6R induced MCP-1 production, but inhibited IL-8 production. For adhesion molecule expression, the production of soluble form of adhesion molecules in HUVEC culture supernatant were measured by ELISA and the expression of adhesion molecules on cell surface were examined by flow cytometry analysis. Soluble ICAM-1 was detectable in IL-6 + sIL-6R-treated HUVEC and IL-6 + sIL-6R-induced ICAM-1 expression on cell membrane of HUVEC. In addition, U937 cells were added to HUVEC, which were pre-treated with IL-6 + sIL-6R for 24 h, and 3 h later attached U937 cells were counted. The adhesion of U937 cells to HUVEC was augmented when HUVEC was pretreated by IL-6 + sIL-6R. This adhesion was suppressed by anti-ICAM-1 antibody and anti-IL-6R antibody, but not by antibodies against VCAM-1 or E-selectin. In conclusion, IL-6 signaling plays an important role in inflammatory cell migration by increasing the rate of cell adhesion and by inducing chemokine production in inflamed joints.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Arthritis; Cell Adhesion; Cell Line; Cell Movement; Cells, Cultured; Chemokine CCL2; Chemokines; Endothelial Cells; Fibroblasts; Humans; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; Synovial Membrane

Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.. Differentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 microg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.. HIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.. Expression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1alpha protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.

Topics: Arthritis; Biomarkers; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line; Cells, Cultured; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-8; Macrophages; Matrix Metalloproteinase 9; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; RNA, Messenger; Signal Transduction; Synovial Membrane; Vascular Endothelial Growth Factor A

Expression of the orphan nuclear receptor NR4A2 is controlled by pro-inflammatory mediators, suggesting that NR4A2 may contribute to pathological processes in the inflammatory lesion. This study identifies the chemoattractant protein, interleukin 8 (IL-8/CXCL8), as a molecular target of NR4A2 in human inflammatory arthritis and examines the mechanism through which NR4A2 modulates IL-8 expression. In TNF-alpha-activated human synoviocyte cells, enhanced expression of IL-8 mRNA and protein correspond to temporal changes in NR4A2 transcription and nuclear distribution. Ectopic expression of NR4A2 leads to robust changes in endogenous IL-8 mRNA levels and co-treatment with TNF-alpha results in significant (p<0.001) secretion of IL-8 protein. Transcriptional effects of NR4A2 on the human IL-8 promoter are enhanced in the presence of TNF-alpha, suggesting molecular crosstalk between TNF-alpha signalling and NR4A2. A dominant negative IkappaB kinase antagonizes the combined effects of NR4A2 and TNF-alpha on IL-8 promoter activity. Co-expression of NR4A2 and the p65 subunit of NF-kappaB enhances IL-8 transcription and functional studies indicate that transactivation occurs independently of NR4A2 binding to DNA or heterodimerization with additional nuclear receptors. The IL-8 minimal promoter region is sufficient to support NR4A2 and NF-kappaB/p65 co-operative activity and NR4A2 can interact with NF-kappaB/p65 on a 39bp sequence within this region. In patients treated with methotrexate for active inflammatory arthritis, a reduction in NR4A2 synovial tissue levels correlate significantly (n=10, r=0.73, p=0.002) with changes in IL-8 expression. Collectively, these data delineate an important role for NR4A2 in modulating IL-8 expression and reveal novel transcriptional responses to TNF-alpha in human inflammatory joint disease.

Topics: Active Transport, Cell Nucleus; Arthritis; Cell Nucleus; DNA-Binding Proteins; Female; Gene Expression Regulation; Humans; Interleukin-8; Male; Nuclear Receptor Subfamily 4, Group A, Member 2; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Synovial Membrane; Transcription Factor RelA; Transcription Factors; Tumor Necrosis Factor-alpha

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

The objective of this study was to verify that the [F-18]FDG PET synovial uptake is correlated with the synovial fluid (SF) TNF-alpha concentration. Two rabbit models of acute inflammatory arthritis induced by human interleukin-8 and lipopolysaccharide were used. Modified standard uptake values (MSUVs) obtained from PET images of the animals were compared with results of SF TNF-alpha measurements. Statistically significant correlations were found between the MSUVs and the SF TNF-alpha ratios. An equation to estimate the TNF-alpha ratio from a MSUV was also derived.

Topics: Animals; Arthritis; Disease Models, Animal; Fluorodeoxyglucose F18; Humans; Inflammation; Interleukin-8; Joints; Lipopolysaccharides; Positron-Emission Tomography; Rabbits; Synovial Fluid; Synovial Membrane; Tumor Necrosis Factor-alpha

Type II collagen (CII) is a major component of hyaline cartilage, and antibodies against CII are found in a subgroup of patients with rheumatoid arthritis. We undertook this study to investigate whether and how antibodies directed against CII can form solid-phase immune complexes (ICs) with cytokine-inducing properties in a model theoretically resembling the situation in the inflamed joint, in which CII is exposed for interaction with anti-CII antibodies during periods of inflammation.. Sixty-five arthritis patients with varying levels of anti-native CII antibodies and 10 healthy controls were evaluated concerning anti-CII and cytokines induced in a solid-phase IC model. Monocytes were either depleted or enriched to define responder cells. Antibodies blocking Fc gamma receptors (Fc gammaR) were used to define the responsible T cell surface receptors.. ICs containing anti-CII from arthritis patients induced the production of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-8. We found a close correlation between enzyme-linked immunosorbent assay optical density values and induction of TNFalpha (r = 0.862, P < 0.0001), IL-1beta (r = 0.839, P < 0.0001), and IL-8 (r = 0.547, P < 0.0001). The anti-CII-containing IC density threshold needed for cytokine induction differed among peripheral blood mononuclear cell donors. Anti-CII-containing IC-induced cytokine production was almost totally abolished (>99%) after monocyte depletion, and receptor blocking studies showed significant decreases in the production of TNFalpha, IL-1beta, and IL-8 after blocking Fc gammaRIIa, but not after blocking Fc gammaRIII.. These findings represent a possible mechanism for perpetuation of joint inflammation in the subgroup of arthritis patients with high levels of anti-CII. Blockade of Fc gammaRIIa and suppression of synovial macrophages are conceivable treatment options in such patients.

Topics: Adult; Aged; Aged, 80 and over; Antigen-Antibody Complex; Antigens, CD; Arthritis; Collagen Type II; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-1; Interleukin-8; Male; Middle Aged; Monocytes; Receptors, IgG; Tumor Necrosis Factor-alpha

Z39Ig is a transmembrane protein containing two Ig homology domains with unknown functions. Immunohistochemical analyses of human carotid atherosclerotic plaques detected Z39Ig staining in areas rich in foamy macrophages. Z39Ig staining was also observed in macrophages in the lining layers and sublining areas of rheumatoid arthritis synovium. Z39Ig staining in the osteoarthritis synovium was restricted to macrophages in the lining layers. To identify the role(s) of Z39Ig in the function of macrophages, we used human monocytic cell lines TF-1A (Z39Ig-negative) and THP-1 (Z39Ig-positive). The expression of Z39Ig was induced in TF-1A cells ,when they were differentiated into macrophages by treatment with PMA. The stimulation of PMA-treated TF-1A or THP-1 cells with immobilized anti-Z39Ig mAb induced the secretion of IL-8 and matrix metalloproteinase (MMP)-9, which was dependent on NF-kappaB activation. These data indicate that the macrophage Z39Ig is involved in the pathogenesis of inflammatory diseases through chemokine induction, which will promote the migration of inflammatory cells into the lesion area, and MMP-9 induction, which will contribute to cartilage destruction or extracellular matrix degradation.

Topics: Antibodies, Monoclonal; Antigen-Antibody Reactions; Arthritis; Atherosclerosis; Cell Line; Humans; Immunohistochemistry; Inflammation; Interleukin-8; Macrophages; Matrix Metalloproteinase 9; NF-kappa B; Receptors, Complement; Tetradecanoylphorbol Acetate

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

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

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.

Topics: Aged; Arthritis; Bone and Bones; Chemokine CCL2; Chemokine CCL5; Female; Humans; Immunohistochemistry; Interferon-gamma; Interleukin-6; Interleukin-8; Male; Middle Aged; Osteoblasts

To determine cytokines and MHC class II alleles in Japanese patients with adult Still's disease (ASD) and clarify the association between those profiles and chronic articular disease.. Of 35 patients with ASD (13 men, 22 women, mean age at onset 34.0 yr), 17 (49%) had chronic arthritis (>6 months, chronic articular ASD) and 18 (51%) lacked chronic arthritis (systemic ASD). Cytokines and cytokine receptors in sera were measured by ELISA. Correlations of each cytokine with disease activity or C-reactive protein (CRP) were determined. MHC class II alleles were examined by polymerase chain reaction methods.. In chronic articular ASD, female gender was more frequent and liver dysfunction and myalgia were rarer than in systemic ASD. In active disease, the white blood cell count was lower, but total IgG was greater in patients with chronic articular ASD than in those with systemic ASD. Tumour necrosis factor (TNF) alpha, soluble TNF receptor 2 and interleukin (IL)-18 were increased in both types of ASD, even in remission. Soluble IL-2 receptors, IL-4 and IL-18 levels were correlated with disease activity or CRP value only in chronic articular ASD. Interferon gamma and IL-8 remained increased only in chronic articular ASD, even when disease activity, including IL-6 and CRP, was low. DRB1*1501 (DR2) and DRB1*1201 (DR5) alleles were more frequent in chronic articular than in systemic ASD, whereas DQB1*0602 (DQ1) was frequently observed in both types of ASD.. The present study suggests that ASD with chronic articular disease has distinct clinical, cytokine and immunogenetic profiles.

Topics: Adult; Antigens, CD; Arthritis; Biomarkers; C-Reactive Protein; Chronic Disease; Cytokines; Female; HLA-DQ Antigens; HLA-DQ beta-Chains; HLA-DR Antigens; HLA-DRB1 Chains; Humans; Interferon-gamma; Interleukin-18; Interleukin-4; Interleukin-6; Interleukin-8; Japan; Male; Receptors, Interleukin-2; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type II; Still's Disease, Adult-Onset; Tumor Necrosis Factor-alpha

We investigated the functional role of a CXC chemokine, growth-related protein (GRO), in the recruitment of neutrophils in lipopolysaccharide (LPS)-induced rabbit arthritis. The amounts of GRO in the synovial fluids (SF) reached the first peak (major) at 2 hours and the second peak (minor) at 9 hours after injection of LPS into the knee joints. Administration of anti-GRO mouse monoclonal antibody inhibited 54% of the peak leukocyte accumulation at 9 hours (neutrophils greater than 95%), which was similar to the inhibition by anti-IL-8 IgG (48%). Co-administration of these inhibitors increased the inhibition up to 70% at 9 hours and also inhibited 65% of the initial phase of leukocyte infiltration at 2 hours (neutrophils greater than 99%), which was not affected by a single administration of each inhibitor. The amounts of GRO in SF at 2 hours were not altered by either anti-TNFalpha mAb or anti-IL-8 IgG, but reduced by rabbit recombinant IL-1 receptor antagonist (rrlL-1Ra) by 39%. The inhibition by rrlL-1 Ra was augmented further to 59% with coadministered anti-TNFalpha mAb. In contrast, the amounts of GRO at 9 hours were reduced by rrlL-1Ra by 67%. There was no additional reduction in the amounts of GRO at 9 hours by either combination of rrlL-1Ra with anti-TNFalpha mAb or anti-IL-8 IgG. Administration of anti-GRO mAb did not alter TNFalpha or IL-8 contents in SF at their peak (2 hours), but reduced the amounts of IL-1beta at 6 hours and IL-1Ra at 9 hours by 42% and 49%, respectively. These results provide evidence for the following: (a) GRO as well as IL-8 are important mediators involved in the recruitment of neutrophils both in the early and the late phase of LPS-induced arthritis, (b) IL-1 produced in the early phase stimulates GRO production, (c) GRO plays a role in the later induction of IL-1beta and IL-1Ra, and (d) induction of GRO is not regulated by IL-8.

Topics: Animals; Antibodies, Monoclonal; Arthritis; Cell Line; Chemokines, CXC; Fluoroimmunoassay; Growth Substances; Immunoglobulin G; Immunohistochemistry; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-8; Leukocyte Count; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Neutrophils; Rabbits; Recombinant Proteins; Sialoglycoproteins; Synovial Fluid; 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

In the present study, we analyzed the cytokine network among TNFalpha, IL-1beta, IL-8, and IL-1 receptor antagonist (IL-1Ra) in a rabbit experimental model of acute gout. The production of TNFalpha in synovial fluids reached the peak at 2 hours after the intra-articular injection of monosodium urate (MSU) crystals. The production of IL-1beta and IL-8 reached the first peak at 2 hours and the second peak at 9 and 12 hours, respectively. The production of endogenous IL-1Ra reached the peak at 9 hours. The source of TNFalpha and the first phase of IL-8 was synovial cells, whereas infiltrating leukocytes were the source of the second phase of IL-8 and also of IL-1beta and IL-1Ra. The production of TNFalpha was not altered by either anti-lL-8 IgG or IL-1Ra. The first IL-1beta peak was reduced only with a combination of anti-TNFalpha mAb and anti-lL-8 IgG, whereas the second peak was significantly reduced by either inhibitor. The first IL-8 peak was not altered with anti-TNFalpha mAb or IL-1 Ra, whereas the second IL-8 peak was reduced with IL-1Ra. Anti-TNFalpha mAb or anti-lL-8 IgG significantly reduced the peak level of endogenous IL-1Ra. These cytokine inhibitors also attenuated the maximal leukocyte accumulation at 9 hours, but not the initial phase, which occurred within 2 hours. These results provide evidence that IL-8 and TNFalpha were responsible for the production of IL-1beta and IL-1Ra, and that IL-1beta was responsible for the second phase of IL-1beta and IL-8 production. Our data also suggest that the initial and the maximal phases of leukocyte influx are differently regulated. Finally, the intravenous injection of colchicine inhibited neutrophil infiltration without affecting the production of TNFalpha or the first peak of IL-8, suggesting that colchicine inhibits MSU crystal-induced arthritis by directly inhibiting the migration of neutrophils.

Topics: Animals; Antibodies, Monoclonal; Arthritis; Cell Movement; Colchicine; Crystallization; Cytokines; Female; Gout Suppressants; Immunoglobulin G; Immunohistochemistry; Interleukin-1; Interleukin-8; Leukocyte Count; Leukocytes; Neutrophils; Rabbits; Receptors, Interleukin-1; Recombinant Proteins; Tumor Necrosis Factor-alpha; Uric Acid

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.

Topics: Animals; Antibodies, Monoclonal; Arthritis; Cell Movement; Chemokine CCL2; COS Cells; Crystallization; Female; Fluoroimmunoassay; Immune Sera; Injections, Intra-Articular; Injections, Intravenous; Interleukin-1; Interleukin-8; Knee Joint; Lipopolysaccharides; Monocytes; Rabbits; Recombinant Proteins; Synovial Fluid; Tumor Necrosis Factor-alpha; Uric Acid

To examine the involvement of cytokines in the mechanisms of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine, MDP-Lys(L18)-induced arthritis, we analyzed interleukin-1 (IL-1), tumor necrosis factor (TNF), colony-stimulating factor (CSF), and neutrophil chemotactic factor (NCF) by bioassays in the rat macrophage-conditioned medium (Mluminal diameter-CM) stimulated by MDP-Lys(L18) in vitro and the synovial fluid from dogs treated subcutaneously with MDP-Lys(L18) for 14 days in vivo. The dog showed arthritis characterized by swelling of the knee joint, increased synovial fluid and thickened synovial membrane, and a single subcutaneous injection of MDP-Lys(L18) was previously shown to induced synovitis in rat tarsal joint. IL-1, TNF, CSF, and NCF activities in Mluminal diameter-CM were increased by MDP-Lys(L18), while only NCF activity was detected in the dog synovial fluid. Partial purification procedures revealed that NCF in Mluminal diameter-CM was not leukotriene B4 but a protein having heparin-affinity, and, in addition, immuno-reactive IL-8 was evident to be in Mluminal diameter-CM. The NCF activity in the dog synovial fluid was not inhibited by dialysis, showing that NCF is a protein substance, possibly a chemokine. These results suggest that MDP-Lys(L18) produces a chemokine, such as IL-8, which recruits neutrophils to the synovial membrane for subsequent development of synovitis in rats and dogs.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Arthritis; Benzoquinones; Cells, Cultured; Chemotaxis, Leukocyte; Colony-Stimulating Factors; Culture Media, Conditioned; Cycloheximide; Cytokines; Dogs; Gene Expression Regulation; Interleukin-1; Interleukin-8; Lipoxygenase Inhibitors; Macrophages, Peritoneal; Male; Neutrophils; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Stimulation, Chemical; Synovial Fluid; Tumor Necrosis Factor-alpha

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

Commercially available ELISA kits now make it possible to measure cytokines in biological samples and cell culture supernatants. We have compared the levels of IL-1 beta, IL-6, IL-8 and TNF-alpha in various pathological plasma and synovial fluids, and in supernatants of human monocytes activated by lipopolysaccharide (LPS). Measurements were performed using ELISA kits from different companies. A wide variation in values was obtained when measurements were deduced from the standard curves formed with the standard provided by the manufacturers. We also performed calibration curves for all ELISA kits, using the international standards provided by the NIBSC (UK). The coefficients of variation were then significantly improved for IL-6 and IL-8 measurements but not for IL-1 beta and TNF alpha assays. However, despite this attempt to obtain uniform measurements, none of the kits gave similar values for individual samples. These results suggest that the nature of the different pairs of monoclonal antibodies employed in each ELISA does not permit comparable recognition of cytokines in samples. Further work with the various kits is required to establish whether (i) denaturation of the recognized epitope within the natural cytokine, (ii) fragmentation of the cytokine following enzymatic cleavage, (iii) depolymerization, (iv) binding of cytokines to undefined ligands, (v) variable glycosylation of the natural cytokines (vi) recognition of precursor forms, interferes with the measurements.

Topics: Antibodies, Monoclonal; Arthritis; Cells, Cultured; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Epitopes; False Negative Reactions; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Interleukins; International Agencies; Leukocytes, Mononuclear; Lipopolysaccharides; Protein Denaturation; Reagent Kits, Diagnostic; Reference Standards; Reproducibility of Results; Sepsis; Synovial Fluid; Tumor Necrosis Factor-alpha

To determine the roles of IL-8 in inflammatory synovitis, examination was made of the results of continuously injecting human recombinant IL-8 into the knee joints of New Zealand while rabbits. Recombinant human IL-8 was infused continuously into the joint cavity at 75 ng/h for 14 days by a polypropylene catheter connected to a mini-osmotic pump implanted in each rabbit. Infiltration of inflammatory cells into joint cavity and histopathological changes in synovial tissue were examined at 7 and 14 days following the start of infusion. The continuous infusion of IL-8 for 14 days led to severe arthritis characterized by apparent erythema and joint pain, the accumulation of leucocytes, infiltration of mononuclear cells in synovial tissue, and marked hypervascularization in the synovial lining layer. IL-8 may be a factor which can contribute to the inflammatory process of chronic arthritis by mediating leucocyte recruitment and hypervascularization in inflamed joints.

Topics: Animals; Arthritis; Chemotaxis, Leukocyte; Injections, Intra-Articular; Interleukin-8; Neovascularization, Pathologic; Rabbits; Recombinant Proteins; Synovial Membrane

Topics: Arthritis; Crystallization; Humans; Interleukin-8; Leukocyte Count; Neutrophils; Synovial Fluid

Rheumatoid arthritis and related inflammatory joint diseases are characterized by massive infiltration of polymorphonuclear cells (PMN) into inflamed joints. Interleukin-8 (IL-8) has recently been identified as a leukocyte chemotactic and activating factor produced by activated tissue cells as well as monocytes/macrophages. Examination was made of the involvement of IL-8 in acute arthritis induced by injecting lipopolysaccharide (LPS) or interleukin-1 alpha (IL-1 alpha) into the joints of rabbits. The neutralizing antibody to rabbit IL-8 blocked almost completely the infiltration of PMN into the joints and provided protection from damage to tissue in the early phase of inflammation induced by LPS or IL-1 alpha. Mononuclear cell infiltration observed later was not inhibited by this antibody. This is the first paper to clearly demonstrate that IL-8 is an essential and major mediator determining whether PMN infiltration will occur in the early phase of experimental acute arthritis.

Topics: Animals; Antibodies; Arthritis; Disease Models, Animal; Interleukin-1; Interleukin-8; Lipopolysaccharides; Male; Neutralization Tests; Neutrophils; Rabbits

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

Expression of the C3 receptors CR1 and CR3 was investigated on neutrophils from paired peripheral blood and synovial fluid samples from 34 patients with inflammatory joint disease (21 patients with rheumatoid arthritis (RA) and 13 patients with other articular diseases (OAD)). Using monoclonal antibodies (anti-CD35, anti-CD11b) and immunofluorescence flow cytometric analyses the percentages of positively labeled cells and the relative fluorescence intensities (as a measure of receptor number) were determined. CR1 and CR3 were found to be present on the majority (> 85%) of circulating neutrophils from normal subjects, RA and OAD patients, and on synovial fluid neutrophils from both patient groups. A strong correlation between neutrophil CR1 and CR3 expression was observed in peripheral blood samples from normal subjects (r = 0.81; P = 0.001), RA (r = 0.79; P = 0.001), and OAD patients (r = 0.83; P = 0.001); in each case the levels of CR3 expression were approximately twice those recorded for CR1. Both CR1 and CR3 expression was upregulated on synovial fluid neutrophils compared with that observed on the corresponding peripheral blood cells. Mean percentage increases observed were: RA patients: CR1, 16.5% (P < 0.001) and CR3, 28.7% (P < 0.001); and OAD patients: CR1, 4.1% and CR3, 26.9% (P = 0.001). Correlation of serum and synovial fluid IL-6, IL-8, and immune complex levels with neutrophil CR1 and CR3 expression failed to demonstrate any significant relationship between the concentrations of these soluble factors and receptor expression. Upregulation of CR1 and CR3 receptors, reflecting neutrophil activation within the inflamed joint, is a consistent finding in patients with inflammatory arthropathies.

Topics: Adult; Aged; Antibodies, Monoclonal; Antigen-Antibody Complex; Arthritis; Complement Activation; Complement C3b; Female; Flow Cytometry; Humans; Interleukin-6; Interleukin-8; Macrophage-1 Antigen; Male; Middle Aged; Neutrophils; Receptors, Complement 3b; Synovial Fluid; Up-Regulation

Injury to cartilage is a recognized sequela of neutrophil activation in arthritic joints. This study examined the possibility that chondrocytes may play a direct role in intraarticular neutrophil activation. We demonstrate that IL-1 beta-stimulated primary and subcultured human articular chondrocytes, express the gene for the potent neutrophil chemotactic and activating cytokine, IL-8. Expression of IL-8 mRNA is also inducible by TNF-alpha and LPS and, to a lesser degree, by the chondrocyte growth factor, transforming growth factor-beta, but not by platelet-derived growth factor, acidic and basic fibroblast growth factor, or epidermal growth factor. Analysis of IL-1 beta-stimulated cartilage organ cultures by in situ hybridization demonstrates that chondrocytes in all zones of cartilage are rapidly induced to express the IL-8 gene in high copy number. Metabolically labeled IL-1 beta-stimulated chondrocytes synthesize IL-8 de novo, which comigrates on SDS-PAGE with IL-8 produced by synovial fibroblasts. Furthermore, the conditioned media of IL-1 beta-stimulated chondrocytes and cartilage organ cultures contain neutrophil chemotactic activity which is completely neutralized by a specific antibody to IL-8, establishing that a bioactive form of IL-8 is the major secreted neutrophil chemotactic factor. By using a specific RIA, we demonstrate that not only IL-1 beta, but also TNF-alpha and LPS can induce abundant IL-8 secretion from chondrocytes. In conclusion, articular chondrocytes are readily inducible to express the IL-8 gene and secrete biologically active IL-8 which can promote neutrophil-mediated inflammation and cartilage destruction.

Topics: Arthritis; Base Sequence; Cartilage, Articular; Gene Expression Regulation; Humans; Interleukin-1; Interleukin-8; Lipopolysaccharides; Molecular Sequence Data; Neutrophils; Organ Culture Techniques; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Arthritis; Chemotactic Factors; Complement Activating Enzymes; Complement Activation; Complement C3-C5 Convertases; Complement C5; Gout; Humans; In Vitro Techniques; Interleukin-8; Uric Acid