3-nitrotyrosine and Arthritis--Rheumatoid

Rheumatoid arthritis (RA) patients display high levels of oxidative stress. Transient exercise-induced increases in oxidative stress are thought to be adaptive in healthy populations. This study investigated the effect of exercise on markers of oxidative stress in RA, following acute exercise and a period of exercise training.. Acute exercise study: RA patients (N = 12, age: 56 ± 11) undertook a bout of exercise (30-40 min, 70 % VO2MAX), and blood samples were taken before and after exercise to assess markers of oxidative stress. Training study: RA patients (N = 19, age: 56 ± 10) were randomised into either a control or exercise group, who undertook 3 exercise sessions per week (30-40 min @70 % VO2MAX) for 3 months. Plasma markers of oxidative stress (protein carbonyls (PC), lipid hydroperoxides (LOOH), 3-nitrotyrosine (3-NT), total antioxidant capacity (TAC) and catalase (CAT) activity), inflammation (interleukin-8 (IL-8) and C-reactive protein (CRP)) and nitric oxide metabolites (NOx) were assessed before and after training.. Acute exercise study: Protein carbonyls (PC) (+18 %) and NOx (+27 %) were significantly increased following exercise. Training study: 3-nitrotyrosine (3-NT) decreased (2.18 ± 1.78 to 1.10 ± 0.93 μM) in the exercise group only, alongside increases in aerobic fitness (24.45 ± 4.98 to 27.10 ± 4.51 ml/kg/min(-1)) and reductions in disease activity score (DAS: 3.47 ± 1.17 to 2.88 ± 0.76). PC, LOOH, TAC, IL-8, CRP and NOx concentrations, and CAT activity were unchanged in both groups.. Aerobic exercise training did not increase markers of oxidative stress in RA patients. 3-Nitrotyrosine and disease activity were decreased following exercise training.

Topics: Aged; Arthritis, Rheumatoid; Biomarkers; Down-Regulation; England; Exercise Therapy; Female; Humans; Inflammation Mediators; Lipid Peroxidation; Male; Middle Aged; Oxidative Stress; Physical Fitness; Protein Carbonylation; Severity of Illness Index; Time Factors; Treatment Outcome; Tyrosine

Etanercept, a tumor necrosis factor inhibitor, is an effective drug for patients with active rheumatoid arthritis (RA). Monocyte chemoattractant protein-1 (MCP-1) and nitrotyrosine (NT) are pro-inflammatory biomolecules associated with satiety and increased body weight. We evaluated whether MCP-1 and NT are associated with decreased inflammation or increased body mass during etanercept therapy in active RA patients.. RA patients with moderate to high disease activity were enrolled to receive add-on etanercept (25 mg subcutaneous injection, biweekly) for at least one year, combined with sustained treatment with conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs).. Forty patients received add-on etanercept and 15 received DMARDs alone. At the end of one year, etanercept significantly reduced the disease activity score of 28 joints, C-reactive protein, and erythrocyte sedimentation rate. Moreover, etanercept significantly increased the body weight, body mass index (BMI), as well as MCP-1 and NT levels, compared to that in the csDMARD-only group.. Increased serum MCP-1 and NT levels in RA patients with moderate to high disease activity, who underwent one-year etanercept treatment, might be attributed to increase in body weight and BMI rather than induction of more severe autoimmune inflammation.

Topics: Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Chemokine CCL2; Etanercept; Female; Humans; Male; Middle Aged; Severity of Illness Index; Tyrosine; Weight Gain

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.

Topics: Actins; Animals; Arthritis, Rheumatoid; Disease Models, Animal; Female; Humans; Malondialdehyde; Mice; Mice, Inbred C57BL; Molecular Dynamics Simulation; Muscle Contraction; Muscle Weakness; Muscle, Skeletal; Myosins; Oxidative Stress; Polymerization; Protein Processing, Post-Translational; Tyrosine

Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.. 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1β, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4. A77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1β. The serum levels of TNF-α, IL-1β, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4. The inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4

Topics: Aniline Compounds; Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Differentiation; Crotonates; Forkhead Transcription Factors; Heme Oxygenase-1; Humans; Hydroxybutyrates; Inflammation; Isoxazoles; Jurkat Cells; Leflunomide; Mice, Inbred BALB C; Mice, Inbred C57BL; NF-E2-Related Factor 2; Nitric Oxide Synthase Type II; Nitriles; Oxidative Stress; Signal Transduction; Spleen; Th17 Cells; Toluidines; Tyrosine

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

Topics: Arthritis, Rheumatoid; Calorimetry, Differential Scanning; Circular Dichroism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin G; Inflammation; Light; Microscopy, Fluorescence; Oxygen; Peroxynitrous Acid; Protein Structure, Secondary; Scattering, Radiation; Sepharose; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Temperature; Tryptophan; Tyrosine

To characterize the utility of nitrotyrosine (NT) as a biomarker for arthritis and joint injury.. Synovial fluid, plasma, and urine from patients diagnosed with osteoarthritis (OA), rheumatoid arthritis (RA), anterior cruciate ligament (ACL) injury, meniscus injury and pseudogout, and knee-healthy volunteers were analyzed for concentrations of NT, nitrate and nitrite (NO(x)), matrix metalloproteinase (MMP)-3, MMP-1, MMP-9, more than 40 chemokines and cytokines.. In OA, plasma and synovial fluid NT were increased versus healthy volunteers. Synovial fluid to plasma NT ratios were elevated in OA patients. Synovial fluid from patients with ACL and meniscus injury and pseudogout had increased levels of NT (P < 0.001). In these samples, NT levels significantly correlated with ARGS-aggrecan neoepitope generated by aggrecanase cleavage of aggrecan (P ≤ 0.001), cross-linked C-telopeptides of type II collagen (P < 0.001), MMP-1 (P = 0.008), and MMP-3 (P ≤ 0.001). In RA, plasma NT decreased following 6 months of anti-tumor necrosis factor (TNF) treatment. For every 1.1% change in log(10) NT, there was a 1.0% change in the log(10) disease activity scores (DAS28-3 CRP). Both predicted and observed DAS28-3 CRP showed a robust linear relationship with NT. RA plasma NT positively correlated with CRP, MMP-3 and interferon γ-induced protein 10.. NT may serve as a useful biomarker for arthritis and joint injury. In RA, NT is highly correlated with several biomarkers and clinical correlates of disease activity and responds to anti-TNF therapy.

Topics: Anterior Cruciate Ligament; Anterior Cruciate Ligament Injuries; Arthritis, Rheumatoid; Case-Control Studies; Chemokines; Chondrocalcinosis; Cytokines; Female; Humans; Male; Matrix Metalloproteinases, Secreted; Menisci, Tibial; Nitrates; Osteoarthritis, Knee; Synovial Fluid; Tibial Meniscus Injuries; Tyrosine

Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD).. Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS.. RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD.. We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.

Topics: Adult; Aged; Apolipoprotein A-I; Arthritis, Rheumatoid; Cardiovascular Diseases; Case-Control Studies; Cholesterol; Female; Halogenation; Humans; Lipoproteins, HDL; Male; Middle Aged; Oxidation-Reduction; Peptide Mapping; Peroxidase; Tandem Mass Spectrometry; Tyrosine

Atherosclerosis is accelerated in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). We investigated a possible association of oxidized low-density lipoproteins (ox-LDLs), nitric oxide (NO), 3-nitrotyrosine, vitamin A, vitamin E, and β-carotene serum levels with subclinical atherosclerosis in RA and PsA. By the use of ELISA, we observed higher ox-LDL levels in patients with intima-media thickness (IMT) > 1 than in patients with IMT ≤ 1 and a negative correlation between NO levels and IMT values. By the use of high-performance liquid chromatography, we determined higher levels of vitamin A in patients with PsA and IMT ≤ 1 than in controls and lower levels of β-carotene in patients with RA and PsA than in controls. β-carotene concentrations were negatively correlated to the duration of disease in RA. Our study confirms that ox-LDLs and NO may be markers of accelerated atherosclerosis in RA and PsA whereas vitamins seem to be associated only to the presence of the autoimmune disorders.

Topics: Adult; Aged; Arthritis, Psoriatic; Arthritis, Rheumatoid; Atherosclerosis; beta Carotene; Carotid Artery, Common; Carotid Intima-Media Thickness; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lipoproteins, LDL; Male; Middle Aged; Nitric Oxide; Risk; Tyrosine; Vitamin A; Vitamin E

Rheumatoid arthritis (RA) is an autoimmune disease of complex aetiology and pathogenesis. In recent years it has become evident that apoptotically modified histones exert a central role in the induction of autoimmunity, for example in systemic lupus erythematosus (SLE) and RA. Because nitric oxide (NO)-related species have been found in inflamed joints of arthritis patients, we investigated whether nitrotyrosine is present in sera of RA patients, and whether peroxynitrite-modified H2A histone is likely to be involved in the induction and progression of RA.. Commercially available H2A histone was modified in vitro by peroxynitrite. The RA patients were divided into three groups on the basis of CRP, nitrite, total protein and IgG level. Sera of RA patients with high-titre rheumatoid factor (RF) were analysed for autoantibodies against native DNA and native and peroxynitrite-modified H2A histone. Binding characteristics and specificity of the autoantibodies were analysed by direct binding, inhibition enzyme-linked immunosorbent assay (ELISA), and band shift assay.. Sera from control subjects contained almost negligible amounts of 3-nitrotyrosine (3-NT); lower levels were found in group 1 RA patients in comparison to group 2 and group 3 patients, where the level of nitrotyrosine was progressively higher. Enzyme immunoassay data showed preferential binding of RA autoantibodies to peroxynitrite-modified H2A histone in comparison with native H2A histone or native DNA. The results suggest that peroxynitrite modification of self-antigen(s) can generate neoepitopes capable of inducing RA characteristic autoantibodies.. The preferential binding of peroxynitrite-modified histones by autoantibodies derived from RA sera indicates a role for oxidatively modified and nitrated proteins in the initiation/progression of RA.

Topics: Adult; Arthritis, Rheumatoid; Autoantibodies; C-Reactive Protein; Disease Progression; Histones; Humans; Immunoglobulin G; Peroxynitrous Acid; Rheumatoid Factor; Tyrosine

The contribution of inducible nitric oxide synthase (iNOS) to oxidative/nitrative stress is well-documented in inflammation, but difficult to quantify. Using a novel, recently developed assay for 3-nitrotyrosine (3-NT), we characterized iNOS activity and its inhibition in preclinical models of inflammation. In particular, we utilized the 3-NT assay to assess the role of iNOS in the disease pathology as well as for proof of pharmacology of iNOS inhibitors in an acute endotoxin challenge model, in models of rheumatoid arthritis (RA) such as rat adjuvant- and collagen-induced arthritis (AIA and CIA) and a model of osteoarthritis (OA) such as rat sodium monoiodoacetate-induced arthritis (MIA). Quantification of nitrotyrosine was performed using immuno-affinity 2-D LC-MS/MS assay. This assay is a very specific and reproducible and is amenable to a number of biological fluids. Plasma levels of 3-NT were significantly elevated in an acute model of inflammation (rat LPS) and in models of rheumatoid arthritis (adjuvant- and collagen-induced arthritis), and osteoarthritis (monoiodoacetate-induced arthritis). Plasma 3-NT correlated with the severity of the inflammatory response; thus, a 20-fold increase was observed in the rat LPS model, a 10-fold increase in AIA, and only a 2.5-fold elevation in CIA. Pharmacological intervention with iNOS inhibitors decreased 3-NT levels and associated pathology. 3-NT determination allowed for better elucidation of the role of iNOS in RA and OA disease pathology and provided proof of pharmacology for NOS inhibitors in animal models of RA and OA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Biomarkers; Disease Models, Animal; Enzyme Inhibitors; Inflammation; Nitric Oxide Synthase Type II; Osteoarthritis; Rats; Severity of Illness Index; Tyrosine

We recently reported that methionine-loaded human umbilical vein endothelial cells (HUVECs) exported homocysteine (Hcy) and were associated with hydroxyl radical generation and oxidation of lipids in LDL. Herein we have analysed the Hcy-induced posttranslational modifications (PTMs) of LDL protein. PTMs have been characterised using electrophoretic mobility shift, protein carbonyl ELISA, HPLC with electrochemical detection and Western blotting of 3-nitrotyrosine, and LDL uptake by scavenger receptors on monocyte/macrophages. We have also analysed PTMs in LDL isolated from rheumatoid (RA) and osteo-(OA) arthritis patients with cardiovascular disease (CVD). While reagent Hcy (< 50 microM) promoted copper-catalysed LDL protein oxidation, Hcy released from methionine-loaded HUVECs promoted LDL protein nitration. In addition, LDL nitration was associated with enhanced monocyte/macrophage uptake when compared with LDL oxidation. LDL protein nitration and uptake by monocytes, but not carbonyl formation, was elevated in both RA and OA patients with CVD compared with disease-matched patients that had no evidence of CVD. Moreover, a direct correlation between plasma total Hcy (tHcy) and LDL uptake was observed. The present studies suggest that elevated plasma tHcy may promote LDL nitration and increased scavenger receptor uptake, providing a molecular mechanism that may contribute to the clinical link between CVD and elevated plasma tHcy.

Topics: Adult; Aged; Arthritis, Rheumatoid; Blotting, Western; Cardiovascular Diseases; Case-Control Studies; Cells, Cultured; Chromatography, High Pressure Liquid; Copper; Electrophoretic Mobility Shift Assay; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Homocysteine; Humans; Lipoproteins, LDL; Male; Methionine; Middle Aged; Nitrites; Osteoarthritis; Receptors, Scavenger; Tyrosine; Umbilical Veins

Increased reactive nitrogen species (RNS) production has been suggested in the pathogenesis of rheumatoid arthritis (RA), osteoarthritis (OA) as well as in systemic lupus erythematosus (SLE). They are known to have direct toxicity to cells. High concentrations of serum nitrite/nitrate and elevated urinary nitrate:creatine ratio has been found in patients with RA, OA and SLE. Reactive nitrogen species play a role in the chronicity of inflammatory reaction such as cartilage and bone destruction seen in patients with RA and OA. Arthritis is also associated with increased intra-articular formation of 3-nitrotyrosine (3-NT), which may contribute to joint damage. There is growing evidence that nitrative injury plays an important role in oxidative stress in the etiology and pathogenesis of SLE. 3-nitrotyrosine is thought to be a relatively specific marker of nitrosative damage mediated by nitric oxide (NO) and its by-products.. Commercially available poly l-tyrosine was exposed to nitrating species resulting in the formation of 3-nitrotyrosine. Antibodies present in synovial fluid and sera of 30 patients with rheumatoid arthritis, 15 patients with osteoarthritis and 15 patients with SLE were studied for their recognition of 3-NT by direct binding ELISA.. IgG from the synovial fluid (SF) of RA and OA patients, purified on protein A-Sepharose matrix, exhibited increased recognition of 3-NT, than the IgG isolated from the sera of RA and OA patients in competitive ELISA, whereas IgG isolated from the sera of SLE patients exhibited increased recognition of 3-NT, than the IgG isolated from the synovial fluid. There was a higher prevalence of antibodies against 3-NT in the synovial fluid than in the sera of patients with RA and OA. Higher level of anti-3-NT antibodies were found in the synovial fluid in the later stages of SLE when compared to the early stages but was not more than that found in the sera.. The RNS may be produced within the inflamed joints of RA and OA patients but not in SLE patients. The 3-NT levels also correlated directly with disease activity.

Topics: Arthritis, Rheumatoid; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin G; Lupus Erythematosus, Systemic; Osteoarthritis; Spectrum Analysis; Synovial Fluid; Tyrosine

Recently, glycogen synthase kinase-3 (GSK-3) has being identified as an ubiquitous serine-threonine protein kinase that participates in a multitude of cellular processes and plays an important role in the pathophysiology of a number of diseases. The aim of this study was to investigate the effects of GSK-3beta inhibition on the degree of arthritis caused by type II collagen (CII) in the mouse (collagen-induced arthritis; CIA). Mice developed erosive hind paw arthritis when immunized with CII in an emulsion in complete Freund's adjuvant (CFA). The incidence of CIA was 100% by day 28 in the CII-challenged mice and the severity of CIA progressed over a 35-day period with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of the cartilage at the joint margins. Treatment of mice with the GSK-3beta inhibitor TDZD-8 (1 mg/kg/day i.p.) starting at the onset of arthritis (day 25) ameliorated the clinical signs at days 26-35 and improved histological status in the joint and paw. Immunohistochemical analysis for nitrotyrosine, poly(ADP-ribose) (PAR), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) revealed a positive staining in inflamed joints from mice subjected to CIA. The degree of staining for nitrotyrosine, PAR, iNOS, and COX-2 was significantly reduced in CII-challenged mice treated with the GSK-3beta inhibitor. Plasma levels of tumor necrosis factor (TNF)-alpha and the joint tissue levels of macrophage inflammatory protein (MIP)-1alpha and MIP-2 were also significantly reduced by GSK-3beta inhibition. These data demonstrate that GSK-3beta inhibition exerts an anti-inflammatory effect during chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Chemokine CCL3; Chemokine CCL4; Chemokine CXCL2; Collagen Type II; Cyclooxygenase 2; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hindlimb; Interleukin-6; Leukocyte Count; Macrophage Inflammatory Proteins; Mice; Mice, Inbred DBA; Monokines; Nitric Oxide Synthase Type II; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Radiography; Thiadiazoles; Tumor Necrosis Factor-alpha; Tyrosine

Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues.. Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied.. Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macrophages, endothelial cells, and lymphocytes. Numerous bands of nitrated proteins were detected by Western blot analysis of 15 synovial tissue extracts. Inducible nitric oxide synthase (iNOS) was detected immunohistochemically in endothelial cells, macrophages, vascular smooth muscle cells, and synoviocytes.. Nitrotyrosine-containing proteins were found in essentially all synovia from RA and OA patients. The most prominent site of nitration in all cases was the stroma. iNOS, the likely source of the nitrating species, was found in a variety of cell types.

Topics: Aged; Aged, 80 and over; Arthritis, Rheumatoid; Blotting, Western; Female; Humans; Immunohistochemistry; Male; Middle Aged; Osteoarthritis; Peroxynitrous Acid; Staining and Labeling; Synovial Membrane; Tyrosine

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, whose activation has been linked to several physiologic pathways including those related to the regulation of insulin sensitivity. Here, we investigate effects of PPARgamma specific ligands, rosiglitazone and pioglitazone, on formation of nitrotyrosine and increased expression of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and intercellular adhesion molecule-1 (ICAM-1) in adjuvant-induced murine arthritis. Administration of rosiglitazone or pioglitazone (30 mg/kg, p.o.) significantly inhibited the adjuvant-induced increase in formation of nitrotyrosine and expression of iNOS on both ankle and temporomandibular joints. Rosiglitazone also inhibited the adjuvant-induced expression of M30 positive cells, as a marker of apoptosis, in the joint tissues. In addition, treatment with rosiglitazone or pioglitazone (30 microM) inhibited lipopolysaccharide plus tumor necrosis factor (TNF)-alpha-induced protein expression of iNOS, cyclooxygenase-2, ICAM-1 and nitrotyrosine formation in RAW 264 cells, a murine macrophage-like cell line. Rosiglitazone or pioglitazone inhibited increase in phosphorylated I-kappaB (pI-kappaB) expression, as an index of activation of nuclear factor (NF)-kappaB, in both joint tissues and RAW264 cells. Furthermore, in PPARgamma-transfected HEK293 cells, rosiglitazone inhibited the TNF-alpha-stimulated response using NF-kappaB-mediated transcription reporter assay. These results indicate that PPARgamma ligands may possess anti-inflammatory activity against adjuvant-induced arthritis via the inhibition of NF-kappaB pathway.

Topics: Animals; Arthritis, Rheumatoid; Cell Line; Cyclooxygenase 2; Freund's Adjuvant; Inflammation Mediators; Isoenzymes; Ligands; Male; Mice; Mice, Inbred BALB C; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Signal Transduction; Temporomandibular Joint; Transcription Factors; Tyrosine

To determine the localization of 3-nitrotyrosine (3-NT), a footprint marker of peroxynitrite (ONOO-) and other reactive nitrogen species, to the inflamed human synovium and to compare this with normal synovial and nonsynovial tissue of human and animal origin.. Monoclonal and polyclonal antibodies were used to investigate for 3-NT, inducible nitric oxide synthase (iNOS), macrophage marker CD68, and the vascular smooth muscle marker alpha-actin by avidin-biotin immunocytochemistry.. In the inflamed synovium, 3-NT was found in the vascular smooth muscle and macrophages. In normal human synovium, 3-NT was present in the vascular smooth muscle and some lining cells and was not associated with immunoreactivity for iNOS. Similarly, 3-NT could be demonstrated in the vascular smooth muscle cells of normal rats and iNOS knockout mice. It was not present in the vascular smooth muscle of healthy, nonsynovial tissue.. The synovial vasculature in histologically normal human and naive rodent synovium was alone among the normal tissues studied in exhibiting iNOS-independent immunoreactivity for 3-NT. These findings suggest a physiologic role for ONOO- in normal synovial vascular function.

Topics: Actins; Aged; Aged, 80 and over; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Arthritis, Rheumatoid; Female; Humans; Immunohistochemistry; Macrophages; Male; Mice; Mice, Knockout; Middle Aged; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Synovial Membrane; Tyrosine

To investigate the effects of M40403, a synthetic mimetic of superoxide dismutase (SOD), on collagen-induced arthritis (CIA) in rats.. CIA was elicited in Lewis rats by intradermal injection of 100 microl of an emulsion of bovine type II collagen (CII) in Freund's incomplete adjuvant at the base of the tail. A second injection was given on day 21.. Immunization induced an erosive arthritis of the hind paws. Macroscopic evidence of CIA first appeared as periarticular erythema and edema in the hind paws by days 24-26 after the first injection, with a 100% incidence by days 27. Severity progressed over a 35-day period. Radiography revealed soft tissue swelling and focal resorption of bone, together with osteophyte formation in the tibiotarsal joint. Histopathologic features included erosion of the articular cartilage at the joint margins and subchondral bone resorption associated with bone-derived multinucleated cell-containing granulomatous lesions. Treatment with M40403 (2-10 mg/kg/day) starting at the onset of arthritis (day 25) ameliorated the clinical signs on days 26-35 and improved the histologic findings in the joint and paw. Immunohistochemical analysis for nitrotyrosine (a marker of peroxynitrite formation) and poly(ADP-ribose) polymerase (PARP; a nuclear enzyme activated by DNA single-strand damage) revealed positive staining in the inflamed joints of CII-treated rats, suggestive of the formation of peroxynitrite and DNA damage, both of which were markedly reduced by M40403 treatment. Radiographic evidence of protection from bone resorption, osteophyte formation, and soft tissue swelling was apparent in the tibiotarsal joints of M40403-treated rats. Arthritic rats treated with M40403 gained weight at the same rate and to the same extent as normal, nonarthritic rats.. This study shows that a low molecular weight mimetic of SOD, M40403, attenuates the degree of chronic inflammation, tissue damage, and bone damage associated with CIA in the rat, and supports the possible use of SOD mimetics as therapeutic agents for the management of chronic diseases such as rheumatoid arthritis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Arthritis, Experimental; Arthritis, Rheumatoid; Arthrography; Collagen; Collagen Type XI; Disease Models, Animal; Interleukin-1; Joints; Male; Manganese; Molecular Weight; Organometallic Compounds; Proteins; Rats; Rats, Inbred Lew; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine; Weight Gain

In inflammatory arthritis, there is evidence indicating that the affected tissues produce large amounts of oxygen-free radicals and NO. Herein, we examine the biologic effects of exposure of IgG to hypochlorous acid (HOCl) and peroxynitrite (ONOO). The concentrations of IgG modified by chlorination and nitrosation were measured in synovial fluids from inflammatory and noninflammatory arthritis. Human IgG was exposed to increasing concentrations of HOCl and ONOO, and the resulting products were tested for complement component binding; binding to FcgammaRI; activation of polymorphonuclear neutrophils; effect on the Ab-combining site of Abs; and in vivo inflammatory activity in a rabbit model of acute arthritis. Rheumatoid synovial fluids contained significantly greater concentrations of nitrosated and chlorinated IgG compared with ostearthritic specimens. In vitro exposure of human IgG to HOCl and ONOO resulted in a concentration-dependent decrease in C3 and C1q fixation. The decrease in Fc domain-dependent biologic functions was confirmed by competitive binding studies to the FcgammaRI of U937 cells. HOCl-treated IgG monomer was 10 times less effective in competing for binding compared with native IgG, and ONOO-treated IgG was 2.5 times less effective. The modified IgGs were also ineffective in inducing synthesis of H(2)O(2) by human PMN. The Ag-binding domains of IgG also showed a concentration-dependent decrease in binding to Ag. The ability of the modified IgGs to induce acute inflammation in rabbit knees decreased 20-fold as gauged by the intensity of the inflammatory cell exudates. These studies clarify the modulating role of biological oxidants in inflammatory processes in which Ag-autoantibody reactions and immune complex pathogenesis may play an important role.

Topics: Acute Disease; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Binding Sites, Antibody; Complement C1q; Complement C3; Female; Free Radicals; Gout; Humans; Hydrogen Peroxide; Hypochlorous Acid; Immunoglobulin G; Male; Neutrophils; Nitrates; Osteoarthritis; Oxidation-Reduction; Oxygen; Rabbits; Receptors, IgG; Serum Albumin, Bovine; Synovial Fluid; Tyrosine

Peroxynitrite, formed by reaction of superoxide and nitric oxide, appears to be an important tissue-damaging species generated at sites of inflammation. In this paper, we compare the abilities of several biological antioxidants to protect against peroxynitrite-dependent inactivation of alpha 1-antiproteinase, and to inhibit tyrosine nitration upon addition of peroxynitrite. GSH and ascorbate protected efficiently in both systems. Uric acid inhibited tyrosine nitration but not alpha 1-antiproteinase inactivation. The possibility that ascorbic acid is an important scavenger of reactive nitrogen species in vivo is discussed.

Topics: alpha 1-Antitrypsin; Antioxidants; Arthritis, Rheumatoid; Ascorbic Acid; Humans; Lipid Peroxidation; Nitrates; Oxidation-Reduction; Oxidative Stress; Pancreatic Elastase; Synovial Fluid; Tyrosine

Reaction of nitric oxide (NO.) with superoxide radical generates peroxy-nitrite, which can decompose to products that nitrate aromatic amino acids. Such nitro-aromatics may be 'markers' of NO.-dependent oxidative damage. Blood serum and synovial fluid from patients with the inflammatory joint disease rheumatoid arthritis contain 3-nitrotyrosine. By contrast, body fluids from normal subjects and patients with osteoarthritis contain no detectable 3-nitrotyrosine; much lower levels were found in serum from patients in the early stages of rheumatoid arthritis. This is evidence that NO. plays a role in joint damage in rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Chronic Disease; Humans; Nitric Oxide; Spectrophotometry, Ultraviolet; Synovial Fluid; Tyrosine