Nitric oxide production in rheumatoid arthritis

Abstract

Nitric oxide (NO), originally found as endothelium-derived relaxing factor (EDRF), is a free radical synthesized by NO synthases (NOS). Two isoforms exist in NOS, i.e. constitutive NOS (cNOS) and inducible NOS (iNOS). Inflammatory cytokines such as interleukin-1, interferon-γ, tumor necrosis factor-α induce iNOS expression in various cells including macrophages. Enhanced NO production is observed in arthritic conditions both in rodent models and human. The onset of arthritis in rodent models is significantly inhibited by the NOS inhibitor, N ^G-monomethyl-l-arginine. These data suggest a possible involvement of NO in the induction and/or maintenance of rheumatoid arthritis.     

Nitric oxide as s-nitrosoproteins in rheumatoid arthritis

Objective. Nitric oxide (NO) is a free radical involved in inflammation and immune reactions. The presence of NO is usually assessed by assaying its degradation products, nitrite and nitrate. NO binds to thiol-containing proteins to form S-nitrosoproteins (S-NP). The aim of this study was to investigate the presence of S-NP, together with nitrite and nitrate, in patients with rheumatoid arthritis (RA). Methods. Forty patients with RA were studied and compared with 24 patients with osteoarthritis (OA) and 21 control subjects. Fourteen patients were treated with 3 consecutive pulses of methylprednisolone for flares of RA. Nitrite was measured by the Griess reaction, and nitrate by a spectrophotometric assay using nitrate reductase. Spectrofluorometry coupled with the inner filter effect was used for the measurement of S-NP. Results. S-NP was detected in all RA samples, both in serum and synovial fluid (SF). Serum and articular S-NP concentrations were correlated ( P < 0.03). In RA, nitrite and S-NP levels were higher in SF than in serum; higher SF levels of the 3 compounds were observed in RA than in OA. S-NP levels in RA patients decreased significantly ( P < 0.03) after pulse methylprednisolone treatment, in parallel with the clinical improvement. Conclusion. S-NP, a biologically active form of NO, was consistently present in RA, with higher concentrations within the arthritic joint. S-NP assays should be added to nitrite and nitrate assays for the evaluation of NO metabolism. S-NP could be a stable storage form of active NO in RA, and its measurement could be useful in evaluating pharmacologic interventions that modulate NO generation.     

Nitric oxide production during adjuvant-induced and collagen-induced arthritis

Objective. To investigate the role of nitric oxide (NO) production and NO synthase (NOS) induction during adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in Dark Agouti rats. Methods. Urinary nitrate excretion and immune NOS (iNOS) messenger RNA (mRNA) expression were measured in the joint, lymph node, spleen, and liver tissues following the induction of either AIA or CIA. Results. Urinary nitrate excretion and iNOS mRNA expression increased substantially during joint inflammation in both models of arthritis. However, the increases in urinary nitrate excretion and iNOS mRNA expression observed in the joint, liver, and spleen tissues during AIA were greater than those observed during CIA, although iNOS induction in the lymph nodes was similar for both models. A prior injection with Mycobacterium bovis heat-shock protein resulted in suppression of arthritis and NO production in AIA, but not in CIA. Conclusion. Differences in NO production during AIA versus CIA are a reflection of the fundamental pathophysiologic differences between these 2 models of arthritis. Thus, NO production in these 2 models could not be merely a nonspecific reaction to the adjuvant injection, nor simply a byproduct of local inflammation in the joint.     

Nitric oxide synthase is expressed in the lymphomononuclear cells of synovial fluid in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the expression of inducible nitric oxide synthase (iNOS) in subpopulations of peripheral blood and synovial fluid (SF) leukocytes in patients with rheumatoid arthritis (RA). METHODS: iNOS was detected in peripheral blood and SF samples after cell permeabilization, by 2 color immunofluorescence flow cytometry. Samples from 14 patients with RA and 8 with osteoarthritis (OA) were studied. Nitrite concentration was determined by Griess reaction, interleukin 1beta and tumor necrosis factor alpha by an immunoenzymatic assay, and C-reactive protein (CRP) by an immunonephelometric method. RESULTS: In SF, iNOS was detected in 11 of 14 patients with RA and 2 of 8 with OA. In blood cells, iNOS was detected in 8 of 14 patients with RA and none of the OA group. iNOS was consistently detected in monocytes and was not detected in granular cells. In RA, there was no correlation between the number of iNOS positive mononuclear cells and cytokine concentrations. CRP concentration was correlated with the number of iNOS positive mononuclear cells in RA SF samples. CONCLUSION: SF mononuclear cells from patients with RA express iNOS and are involved in NO production in the joint. The number of positive cells is correlated with CRP concentration, suggesting the implication of NO production in the inflammatory process.     

Increased inflammatory activity parallels increased basal nitric oxide production and blunted response to nitric oxide in vivo in rheumatoid arthritis

BACKGROUND: Endothelial dysfunction, defined as loss of bioactivity of NO in the vessel wall, is thought to precede atherosclerosis. OBJECTIVE: To determine whether endothelial dysfunction characterises patients with RA and whether these patients have increased inducible nitric oxide synthase (iNOS) dependent NO production in vivo. METHODS: and results: Twenty patients with RA and 33 normal subjects received intrabrachial artery infusions of endothelium dependent (acetylcholine (ACh)) and independent (sodium nitroprusside (SNP)) vasodilators to determine arterial responsiveness to NO. Basal flow and its percentage decrease by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of both iNOS and endothelium dependent NOS (eNOS), was used to determine the contribution of iNOS and eNOS dependent NO to basal flow. Both SNP (p<0.01) and ACh (p<0.05) increased blood flow significantly less in patients with RA than normal subjects. Serum concentrations of TNFalpha were, within the RA group, inversely correlated with blood flow responses to both SNP (r=-0.67, p=0.002) and ACh (r=-0.64, p<0.005). Basal flow was significantly increased in RA and correlated within this group with serum CRP (r=0.48, p<0.05), TNFalpha (r=0.61, p<0.01) concentrations, and ESR (r=0.68, p<0.002). L-NMMA decreased basal flow significantly more (-34+/-2%) in the patients with RA than the normal subjects (-24+/-3%, p<0.02), suggesting in view of the blunted response to ACh, increased iNOS activity. CONCLUSIONS: Patients with RA have a dual abnormality in NO dependent vascular function. Basal blood flow is increased in proportion to inflammatory activity and more inhibited by L-NMMA, suggesting increased iNOS activity, and responsiveness to NO is reduced.     

Regulation of interleukin-2 production in rheumatoid arthritis

The regulation of interleukin-2 (IL-2) production was investigated using mononuclear cells from synovial fluid (SF) and peripheral blood of 12 patients with classical and active rheumatoid arthritis. Decreased phytohemagglutinin (PHA) stimulated IL-2 production by lymphocytes was observed in rheumatoid peripheral blood (5.3 +/- 10.9 units/ml) and SF (3.8 +/- 5.2 units/ml) compared to peripheral blood from 12 normal donors (18.1 +/- 15.4 units/ml) and SF from 5 patients with other rheumatic diseases (11.9 +/- 10.9 units/ml). Indomethacin, phorbol myristate acetate and irradiation of suppressor cells increased IL-2 values in rheumatoid SF and peripheral blood but did not restore normal IL-2 production. IL-2 production did not correlate with clinical activity in patients with RA.     

Nitric oxide is a mediator of apoptosis in the rheumatoid joint

OBJECTIVE: To study the role of nitric oxide (NO) derived from the inducible nitric oxide synthase (iNOS) pathway in the induction of apoptosis in the rheumatoid joint. METHODS: Joint tissue was obtained from four rheumatoid arthritis (RA) patients, three osteoarthritis patients and two patients with a fractured neck of the femur (NOF#), and apoptotic cells were identified in cryosections using the TUNEL (terminal dUTP nick end labelling) assay. Expression of iNOS was determined using immunohistochemistry. NO synthesis and the effect of NOS inhibitors on apoptosis levels were studied in explant cultures of RA cartilage and synovium. RESULTS: Numbers of apoptotic cells were greatly increased in rheumatoid synovium and articular cartilage compared with NOF# and osteoarthritic synovium. Immunohistochemistry showed co-localization of iNOS staining and apoptosis in the synovial lining layer and articular cartilage. The NOS inhibitor L-NMMA (L-N(G)-monomethylarginine) strongly inhibited apoptosis in explant cultures of synovium and cartilage, and this was reversed by the NO donor S-nitroso-acetyl-penicillamine. CONCLUSION: This study indicates that NO acts as a mediator of apoptosis in RA and suggests that NOS inhibitors reverse this process.     

Nitric oxide production by monocytes in alcoholic liver disease

Nitric oxide, initially described as an endothelial-derived relaxing factor, has recently been recognised as a mediator of macrophage function. We have studied the production of nitric oxide by peripheral blood monocytes from both normal volunteers and alcoholics. This was measured indirectly by assessing nitrite formation. Normal monocytes were found to produce a basal level of nitrite, which could be stimulated more than 6-fold using endotoxin. This effect was abrogated by the addition of nitric oxide synthesis inhibitor, L-n-monomethyl-arginine. A striking difference was observed in the monocytes obtained from alcoholics with and without evidence of alcoholic hepatitis. Whereas the latter behaved in a similar manner to the controls, the former had markedly increased basal levels. In the hepatitis group there was also substantial inhibition of production by L-n-monomethyl-arginine. We believe that these results indicate that nitric oxide derived from monocytes may play a role in the pathogenesis of alcoholic liver disease, especially alcoholic hepatitis.     

Nitric oxide production in arterial vessels of cirrhotic rats

Indirect evidence exists implicating vascular nitric oxide in the pathogenesis of arterial vasodilation in cirrhosis. In the current study, a coincubation assay to estimate the vascular nitric oxide production was developed and the nitric oxide production by arterial segments of cirrhotic and control rats was assessed. In the assay, measurement of reporter monolayer cell-associated cGMP levels allows the influence of nitric oxide released by arterial segments to be determined. RFL-6 cells served as reporter cells. Nitric oxide production was determined in thoracic aorta and mesenteric arteries of 22 control rats, 10 cirrhotic rats without ascites, and 12 cirrhotic rats with ascites. Basal and bradykinin-stimulated (10⁻⁶ mol/L) intracellular content of nitric oxide-dependent cGMP was significantly higher in RFL-6 cells coincubated with aortic segments of cirrhotic rats with (21.3 ± 3.6 pmol/10⁵ cells, P < .05 and 44.7 ± 7.0 pmol/10⁵cells, P < .025) and without ascites (15.3 ± 3.0 pmol/10⁵cells, P < .05 and 43.2 ± 7.6 pmol/10⁵cells, P < .05) than in those incubated with aortic segments of control rats (9.7 ±1.3 and 19.5 ± 2.5 pmol/10⁵cells). RFL-6 cells exposed to bradykinin-stimulated mesenteric arterial segments of cirrhotic rats also showed increased cGMP content (ascitic: 2.73 ± 0.31 pmol/10⁵cells, P < .005; nonascitic: 2.58 ± 0.51 pmol/10⁵cells, P < .025) compared with cells exposed to control mesenteric arterial segments (1.28 ± 0.15 pmol/10⁵cells). No differences between cirrhotic and control vessels were observed after endothelium denudation. These results indicate that basal and bradykinin-stimulated vascular nitric oxide production is higher in cirrhotic rats with and without ascites than in control rats in and that the endothelial lining is the site where vascular L-arginine nitric oxide pathway activation takes place in experimental cirrhosis.     

Elevated nitric oxide production in rheumatoid arthritis: Detection using the fasting urinary nitrate:creatinine ratio

Objective. To develop a simple method for assessing endogenous nitric oxide (NO) production applicable to routine clinical practice in rheumatology. Methods. NO production was assessed in patients with rheumatoid arthritis (RA) as serum nitrate levels and as the urinary nitrate:creatinine ratio in morning samples of urine following an overnight fast. The influence of dietary intake of nitrate on these measurements was investigated in healthy volunteers. The clinical value of the urinary nitrate:creatinine ratio was validated in patients with infectious gastroenteritis, in whom its production is known to be increased. Results. Urinary nitrate:creatinine ratios were significantly elevated in patients with RA (average 3-fold elevation over controls; P < 0.005) or infectious gastroenteritis (average 10-fold elevation, P < 0.001). Serum nitrate was significantly elevated only in patients with infectious gastroenteritis (P < 0.001). Dietary intake of nitrate had no significant influence on the fasting morning urinary nitrate:creatinine ratio in the healthy volunteers, showing that this parameter is a useful indicator of endogenous NO production. Conclusion. We have developed a simple procedure for evaluating endogenous NO production that is readily applicable to routine clinical practice. Assessment of NO production will help in our understanding of its role in inflammatory conditions such as RA.     

Lymphokine production in rheumatoid arthritis and systemic lupus erythematosus

The production of interferon (IFN) by peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and of IFN and interleukin 2 (IL-2) in systemic lupus erythematosus (SLE) was compared with that of healthy controls. Patients with SLE showed a significant reduction in IL-2 production compared to controls if the PBL were irradiated before mitogen stimulation. No patient with RA or SLE studied had impaired IFN production regardless of disease activity and the IFN produced was always IFN-gamma in type. We conclude that there is an abnormality in IL-2 production in SLE but there is no abnormality in IFN-gamma production in either RA or SLE.     

Local production of complement proteins in rheumatoid arthritis synovium

OBJECTIVE: Complement has been repeatedly implicated in the pathogenesis of rheumatoid arthritis (RA) based on studies showing reduced levels of native complement components and increased levels of complement metabolites in plasma, synovial fluid (SF), and synovial tissue (ST) of RA patients. However, there is limited information on local production and activation of key factors of the complement cascade in RA synovium and their potential modulation by novel anticytokine therapies. This study was undertaken to characterize the expression of complement proteins and receptors in RA SF and ST. METHODS: Using in situ hybridization, immunohistochemistry, and Western blot techniques, we assessed the presence of complement proteins C3, factor B (FB), and C5b-9, as well as the expression of complement receptors C3aR and C5aR in rheumatoid synovium. C3 and FB levels in SF were determined by enzyme-linked immunosorbent assay. Functional assessment was performed by examining the effects of soluble tumor necrosis factor receptor (sTNFR) p55 gene transfer in the SCID mouse model of RA. RESULTS: Complement proteins and receptors could be localized in all RA synovial specimens, whereas in osteoarthritis (OA) synovium, only a few, single cells expressed complement proteins and receptors. No differences were noted in the concentration of C3 between RA and OA in SF; however, FB levels were markedly reduced in RA versus OA SF. In RA synovium, in contrast to OA synovium, local expression of complement factor and complement receptor messenger RNA was found throughout the various ST compartments, suggesting that activation of the complement cascade occurs in all parts of the rheumatoid synovium. Moreover, C5aR expression was up-regulated following overexpression of sTNFR p55 by adenovirus-based gene transfer. CONCLUSION: In summary, local complement production and activation may play an important role in RA, and specific modulation and inhibition of local complement production could be an attractive therapeutic target for RA.     

Nitric oxide levels and the severity of juvenile idiopathic arthritis

In this study, we evaluate the distribution of nitric oxide (NO) in the serum of juvenile idiopathic arthritis (JIA) patients, correlating it with parameters of the severity of the disease. Ninety-seven patients with mean age 11.7 years and disease duration 4.8 years, showing active disease or not, grouped as oligoarticular (n = 34), polyarticular (n = 29) and systemic (n = 34) group, presenting uveitis and positive RF with erosive arthritis or active disease and erosions had significantly high levels of NO than the inactive ones. NO correlated with TNF-α in the oligoarticular subtype (P < 0.03), with pain in the polyarticular subtype with active disease (P < 0.04) and with ESR in the systemic subtype with active disease (P < 0.03). TNF-α concentration was high in all patients with active disease, accompanying NO production. The data confirm the production of NO in JIA patients, indicating a possible positive correlation between the production of NO and severity of the disease.     

Nitric oxide production and apoptosis by GP120.

Nitric oxide (NO) is increased by gp120 in astrocytes and in monocyte-derived macrophages. Of the gp120 fragments (F1: amino acid 254-274, F2: amino acid 315-329, F3: amino acid 421-438), F1 has been shown to increase NO in astrocytes and gp120 also primes CD4+ T cells for apoptosis. Peripheral blood mononuclear cells (PBMCs) at 10(6)/ml (N = 10) were incubated at 24 and 72 hours in RPMI, 10% CO2 with low doses (100 nM) gp120 and high doses (400 nM) of the smaller fragments. Supernatants were collected and assayed for the relative contribution of gp120 and its fragments on NO production at both time points. Apoptosis was detected by in situ hybridization with and without 1 microgram/ml LPS as superantigen at 72 hours. The major contribution to apoptosis and NO production was from F1. At 24 hours F1 had a 1.9-fold increase from control, whereas F2 and F3 had 1.25- and 1.35-fold increases. At 72 hours both F1 and F2 had a 1.5-fold increase and F3 had a 1.33 increase. Thus, F1 contributed significantly to NO production at 24 hours. Both F1 and F2 had significant contributions to NO production at 72 hours. F1 had the most contribution to apoptosis both with and without lipopolysaccharide (LPS). These findings may contribute to further understanding the mechanism of HIV-induced apoptosis.     

Diclofenac inhibits monocyte superoxide production ex vivo in rheumatoid arthritis

Summary Effects of the nonsteroidal anti-inflammatory drug, diclofenac, on stimulated monocyte superoxide production were assessed directly in vitro and following treatment of patients with rheumatoid arthritis ex vivo. Diclofenac inhibited superoxide generation provoked by serum treated zymosan (STZ) and fluoride anion (F) but not by phorbol myristate acetate (PMA) in vitro. Following patient therapy, inhibition of superoxide production occurred when STZ and PMA, but not F were used as stimuli. No changes were seen in control subjects. The contrasting profiles of inhibition seen in vitro and ex vivo suggest an indirect effect on superoxide production during clinical use of the agent. These data are consistent with the hypothesis that anti-inflammatory drugs may act in rheumatoid arthritis by inhibiting phagocyte super-oxide anion production.     

Cytokine production by synovial T cells in rheumatoid arthritis.

OBJECTIVE: To investigate the production of cytokines by T cells in patients with rheumatoid arthritis (RA), reactive arthritis (REA) and osteoarthritis (OA). METHODS: The lymphokines interleukin (IL)-2, IL-4, interferon gamma (IFN-gamma) and tumour necrosis factor beta (TNF-beta), as well as the monokines IL-1, IL-6 and TNF-alpha, were measured by immunoassays in sera and synovial fluid (SF) from patients with RA, REA and OA. In addition, cytokine expression was studied by immunohistochemistry in synovial membrane tissue sections from patients with RA and OA. RESULTS: Almost 60% of RA sera contained at least one of the cytokines investigated, though in low concentrations, whereas cytokines were generally not detectable in sera from REA and OA patients. In contrast, cytokines were found in virtually all SF; thus, the majority of SF from RA patients contained IFN-gamma (median level 17 pg/ml) in addition to the monokines IL-6 (4700 pg/ml) and TNF-alpha (157 pg/ml). IFN-gamma and IL-6 (but not TNF-alpha) were also frequently measured in SF from REA patients, whereas OA samples typically contained only IL-6. Immunohistochemical analysis of tissue sections from RA patients revealed lymphokine expression in 0.1-0.3% of T cells, particularly IL-2 and IFN-gamma, and to a lesser extent also IL-4. Interestingly, the expression of TNF-alpha and IL-6 by synovial T cells was also observed. The majority of cytokine-expressing T cells were CD4-positive T-helper cells typically found in perivascular areas, whereas cytokine-producing CD8-positive T cells were found distributed throughout the synovium. As expected, in specimens from OA patients, T cells were much less abundant and expression of cytokines could not be detected. CONCLUSION: These data clearly demonstrate production of cytokines by T cells in RA synovial tissue, indicating that activated T cells play a role in the pathophysiological events of RA.