Pro- and anti-inflammatory cytokines in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the accumulation of inflammatory cells into the synovium and the destruction of joints. Cytokines are important regulators of the synovial inflammation. Some cytokines, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1, function by promoting inflammatory responses and by inducing cartilage degradation. Other cytokines, such as IL-4, IL-10 and IL-13, function mainly as anti-inflammatory molecules. Although anti-inflammatory cytokines are present in rheumatoid joints, in progressive RA their levels obviously are too low to neutralize the deleterious effects of proinflammatory cytokines. Inhibiting the action of proinflammatory cytokines by using specific cytokine inhibitors or anti-inflammatory cytokines is the basis for new therapies currently tested in patients with RA. Promising results on the use of neutralizing anti-TNF-alpha monoclonal antibodies in the treatment of RA have been reported. The results from a trial using recombinant IL-10 in the treatment of patients with RA are available in the near future and will be important in determining the therapeutic potential of this cytokine.     

Clinical trials on biologics in rheumatoid arthritis

BACKGROUND: Cytokines and T cells play a major role in the pathogenesis of rheumatoid arthritis (RA). Biologic targeting is a novel therapeutic approach. Published trials in humans are discussed in this paper. METHODS: CD4-positive T cells, proinflammatory cytokines like TNF-alpha, IL-1, IL-6 and gamma-IFN were major targets for therapy. Biologics were constructed of monoclonal human and non-human antibodies, chimerics of both, antiinflammatory regulatory proteins like IL-1 receptor antagonist, IL-10, and fusion proteins consisting of receptors and immunoglobulins. RESULTS: More than 2000 humans with RA were exposed to biologics in the last decade. Both, toxic and safe, efficient and non-efficient drugs were tested. Phase II data could not confirm preliminary phase I data in several drugs tested. The pharmacokinetic profile of biologics is influenced by frequent induction of human anti drug antibodies. CONCLUSION: The major role of cytokines in RA has been confirmed. Due to the limited long-term experience immunomodulation does not replace conventional pharmacotherapy.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

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

Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines

Methotrexate (MTX) is one of the most widely prescribed drugs in the treatment of rheumatoid arthritis (RA). The mechanism by which MTX exerts its anti-rheumatic effect has not yet been defined. The aim of the present study was to investigate the effect of MTX treatment (7.5-15 mg/week) on synovial tissue in RA. For this purpose, synovial biopsies were taken from 11 RA patients before and 16 weeks after initiation of MTX therapy. Immunohistochemistry was performed using monoclonal antibodies (MAb) specific for CD3, CD4, CD8, CD22, CD25, CD38, CD68, MAb67, Ki67, interferon gamma (IFN-gamma), interleukin (IL)-1alpha, IL-1beta, tumour necrosis factor alpha (TNF-alpha), E-selectin, ICAM-1 and VCAM-1. All parameters for disease activity improved during the period of treatment. Immunohistochemical analysis revealed a statistically significant decrease in scores for CD3, CD8, CD38, CD68, Ki67, IL-1beta, TNF-alpha and the adhesion molecules E-selectin and VCAM-1. The observed decrease in synovial scores for inflammatory cells, monokines and adhesion molecules suggests that the anti-inflammatory effect of MTX is, in part, dependent on a reduction in monokine-inducible vascular adhesion molecules and subsequent reduction of cell traffic into joints.     

Variability in cytokine and cell adhesion molecule staining in arthroscopic synovial biopsies: quantification using color video image analysis

OBJECTIVE: To investigate the variability in immunostaining for cytokines and cell adhesion molecules using multiple arthroscopically directed synovial biopsies from within a rheumatoid knee joint, quantitated by color video image analysis. METHODS: Needle arthroscopic biopsies were taken from multiple sites (4-7 sites) around a knee joint in 8 patients with rheumatoid arthritis (RA). In 5 patients, immunoperoxidase staining for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin 8 (IL-8), and IL-1beta as well as the IL-1 receptor antagonist protein (IL-1ra) was performed. In 3 patients, immunoperoxidase staining for the cell adhesion molecules E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule 1 (ICAM-1, CD54), and platelet endothelial cell adhesion molecule (PECAM, CD31) was performed. Immunostaining was quantified using color video image analysis. RESULTS: The overall probability of paired biopsies from the same RA knee joint being significantly different from each other due to sampling variation was at most 22% for cytokine staining (usually less than 10%). There were no significant differences between intrabiopsy and interbiopsy variability for cell adhesion molecule staining of the sublining and vessels. CONCLUSION: The variability in cytokine and cell adhesion molecule staining within any single biopsy usually reflects the variability between biopsies taken from different sites in the same rheumatoid joint when the immunostaining is quantified using color video image analysis. Therefore, only a small number of synovial biopsies are required to accurately determine the cytokine and cell adhesion molecule expression in a single joint.     

Identification of two closely related genes, inducible and noninducible carbonyl reductases in the rat ovary

OBJECTIVE: To investigate the potential role of cytokines in psoriatic arthritis (PsA) by assessing the profiles of the proinflammatory cytokines in synovial fluid (SF) of PsA in comparison with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, and IL-8 were measured in SF using ELISA. RESULTS: Levels of TNF-alpha, IL-1beta, and IL-8 were significantly higher in PsA SF than in OA SF, although lower than in RA SF. No difference was detected in the IL-6 levels between PsA and RA SF, both of which were much higher than in OA SF. CONCLUSION: The pattern of expression of proinflammatory cytokines seen in PsA is similar to that in RA. Since PsA is also a destructive arthropathy, cytokines, in particular TNF-alpha and IL-1, may be principle factors in joint destruction.     

Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient

OBJECTIVE: To investigate the production of proinflammatory cytokines and expression of cell adhesion molecules in the rheumatoid nodule. METHODS: Cytokine content (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], and IL-1 receptor antagonist [IL-1Ra]), at the messenger RNA (mRNA) and protein levels, and cell adhesion molecule expression were studied in 16 rheumatoid nodules and 6 synovial membranes. RESULTS: Macrophages in the rheumatoid nodules contained TNFalpha, IL-1beta, and IL-1Ra mRNA and protein, particularly in perivascular cells of the stroma and in the palisading layer. All cell adhesion molecules studied were expressed in both the rheumatoid nodules and synovial membranes, with increased expression of E-selectin in the rheumatoid nodule compared with the synovial membrane, and with the absence of vascular cell adhesion molecule 1 expression on cells of the palisading layer in the rheumatoid nodule. CONCLUSION: The presence of similar proinflammatory cytokines and cell adhesion molecules in the rheumatoid nodule and synovial membrane suggests that similar pathogenic processes result in the chronic inflammation and tissue destruction in these lesions.     

Endothelial cell activation by sera containing HLA antibodies is mediated by interleukin-1

BACKGROUND: It has been suggested that antibodies which are associated with chronic pathological conditions such as chronic rejection and autoimmune diseases have the capacity to activate endothelial cells by induction and up-regulation of adhesion molecules. It has also been suggested that HLA antibodies formed by patients awaiting transplantation can activate endothelial cells. These antibodies include HLA and those that bind to endothelial cells. METHODS: We have further investigated this phenomenon using monoclonal antibodies against HLA class I determinants and sera from aortic valve graft recipients, containing strong HLA antibodies. The effect of 24-hr incubation of antibodies/serum with human umbilical vein endothelial cells (HUVECs) on adhesion molecule expression was measured by flow cytometry. RESULTS: HLA monoclonal antibodies had no effect on ICAM-1 expression on HUVECs. Five of 31 (16%) patients' sera caused strong up-regulation of adhesion molecules (ICAM-1, vascular cell adhesion molecule-1, and E-selectin) but this did not correlate with HLA specificity, IgG, or IgM binding to HUVECs. The activity, found in whole serum and IgG-depleted fractions was inhibited by neutralizing antibodies against interleukin (IL)-1beta and tumor necrosis factor-alpha. Examination of patient sera for presence of IL-1beta demonstrated high levels of IL-1beta in all five sera (range, 30 -500 U/ml) as well as in samples from an additional three patients. CONCLUSION: The ability to activate endothelial cells detected in our patient sera was caused by cytokines and not antibody. Our observation that addition of cytokines to sera before separation into large and low molecular weight fractions demonstrated retention of cytokines in both fractions may be a confounding issue when investigating endothelial cell activation by patients' sera.     

Circulating levels of matrix metalloproteinases MMP-3 and MMP-1, tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP-1/TIMP-1 complex in rheumatic disease. Correlation with clinical activity of rheumatoid arthritis versus other surrogate markers

OBJECTIVE: To investigate whether plasma levels of matrix metalloproteinases 3 (MMP-3, stromelysin), MMP-1 (collagenase), tissue inhibitor of metalloproteinases 1 (TIMP-1), and MMP1/TIMP-1 complex (MT complex) are specifically elevated in erosive joint diseases compared to nonerosive rheumatic diseases, and to assess how these markers reflect the clinical activity of rheumatoid arthritis (RA) compared to circulating cytokines and markers of connective tissue turnover as well as established variables [C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and rheumatoid factor titer]. METHODS: Plasma levels of MMP-3, MMP-1, TIMP- 1, and MT complex were determined by ELISA. One hundred fifteen patients with RA, 20 with osteoarthritis (OA), 28 with psoriasis arthritis (PsA), 24 with ankylosing spondylitis (AS), 3 groups with systemic autoimmune diseases, and 30 healthy controls were analyzed. In patients with RA routine laboratory variables, circulating inflammatory cytokines [interleukin 1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-6], collagen degradation products, and markers of bone formation were determined in parallel and were correlated to 4 variables of clinical activity. RESULTS: MMP-3 levels were markedly elevated in RA compared to controls and OA, but also in all other groups, including 26 patients with systemic lupus erythematosus (SLE). MMP-1 levels were significantly elevated in RA, but also in OA, PsA, SLE, and mixed connective tissue disease. In contrast, MT complex was elevated in RA only. TIMP-1 was not different from controls. CRP levels, MMP-3, and ESR correlated best with clinical activity of RA. In contrast, there was no correlation of IL-1 and TNF-alpha and only a weak correlation of IL-6 with clinical measures. Among variables of connective tissue turnover, only pyridinoline and deoxypyridinoline crosslinks were weakly correlated with disease activity. CONCLUSION: Elevated MMP-3 and MMP-1 levels are not specific for RA or for erosive joint diseases in general. In contrast, elevated MT complex levels were observed in patients with RA. However, the correlation of MT-1 with clinical data was weaker than that of MMP-3. Elevated MMP-3 levels reflected disease activity of RA better than cytokine levels or markers of connective tissue turnover. However, MMP-3 levels do not exceed the association of CRP with clinical activity.     

Enhancement of SPARC (osteonectin) synthesis in arthritic cartilage. Increased levels in synovial fluids from patients with rheumatoid arthritis and regulation by growth factors and cytokines in chondrocyte cultures

OBJECTIVE: To investigate the roles of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) in arthritis, using cartilage and synovium specimens and synovial fluids (SF) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), and to examine the effects of cytokines, growth factors, and hormones on SPARC synthesis by chondrocytes in culture. METHODS: SPARC in cartilage and synovium was immunostained with monoclonal antibodies. SPARC synthesis by cultured chondrocytes was measured by Northern blot analysis, immunoblotting, and sandwich enzyme-linked immunosorbent assay. RESULTS: SPARC was identified in numerous chondrocytes in the superficial and middle zones and in regenerating chondrocytes of RA and OA joints, whereas such staining was absent in these zones of normal cartilage, except for weak signals from a few chondrocytes in the deep zone. In addition, SPARC synthesis was enhanced in synovial cells of RA and OA joints. The average SPARC level in SF was 10-fold higher in the RA than in the OA population. In rabbit articular chondrocyte cultures, administration of transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 increased SPARC levels at 24-48 hours, whereas interleukin-lbeta (IL-1 beta), IL-1 alpha, tumor necrosis factor alpha, lipopolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone decreased SPARC levels at 24-72 hours. TGF beta increased SPARC messenger RNA (mRNA) levels at 24 hours, whereas IL-1 beta caused a marked decrease in SPARC mRNA levels at 24 hours. Furthermore, IL-1 decreased the glycosylation of SPARC. CONCLUSION: These findings suggest that various growth factors and cytokines, including TGF beta 1 and IL-1 beta, regulate the production of SPARC by chondrocytes at pre- and posttranslational levels, and that SPARC synthesis is markedly enhanced in arthritic joints.     

Cytokines in rheumatoid arthritis. Potential targets for pharmacological intervention

The ingress of inflammatory leucocytes into the synovium is a crucial step in the pathogenesis of rheumatoid arthritis (RA). Cytokines are mediators involved in the inflammatory events, adhesive mechanisms, angiogenesis and osteopenia associated with RA. Pro- and anti-inflammatory cytokines, growth factors and chemokines all have an important role in these processes. Because the efficacy of currently used antirheumatic therapy is often limited, there is a need for more specific intervention strategies. Anticytokine therapy may include the use of monoclonal antibodies, antagonistic cytokines, soluble cytokine receptors, cytokine receptor antagonists, somatic gene transfer or other approaches. Hopefully, the study of cytokines and their interactions will lead to the development of new immunomodulatory strategies that will benefit patients with RA.     

New immunologic treatment of rheumatoid arthritis

Rheumatoid arthritis is a heterogeneous disease for which no satisfactory therapy is available. Conventional treatments have nonselective immunological effects. Cyclosporin A ensures partially selective immunotherapy. Recently developed selective immunotherapy methods target CD4+ T-cells and their receptors, antigen-presenting cells including macrophages with surface expression of class II HLA antigens, antigen peptide(s) presented by macrophages, class II HLA antigens or T-cell receptors. T-cell suppression can be achieved using monoclonal antibodies or the still experimental T-cell vaccination. Monoclonal antibodies against activation antigens, including CD4, CD5, CD25 (interleukin 2 receptor), CD52, and adhesion molecules, have been investigated in animal studies and open trials in humans. Monoclonal antibodies directed against class II HLA antigens or antigen-presenting cells have been studied in animals. Peptide immunotherapy has yielded promising results in a variety of animal models. Other immunotherapy methods target cytokines, especially those which promote inflammation. Encouraging preliminary results have been obtained in rheumatoid arthritis patients with interleukin-1 receptor antagonists and monoclonal antibodies against tumor necrosis factor alpha. Problems raised by selective immunotherapy for rheumatoid arthritis and potential indications are discussed.     

Absence of correlation between interleukin 6 and C-reactive protein blood levels in systemic lupus erythematosus compared with rheumatoid arthritis

To investigate if the low C-reactive protein (CRP) response frequently observed in systemic lupus erythematosus (SLE) is related to an impaired expression of interleukin 6 (IL-6), considered its main inducer, we studied serum IL-6 and CRP levels in 37 patients with SLE and 22 with rheumatoid arthritis (RA). Results show that in contrast to CRP, IL-6 levels are significantly higher in SLE than in RA. A linear regression analysis shows a positive correlation between levels of these 2 molecules in RA but not in SLE. Similarly, levels of fibrinogen, another acute phase protein mainly induced by IL-6, did not correlate with IL-6 in SLE. Our results suggest an impairment of part of the acute phase response to IL-6 that might play a role in the pathogenesis of SLE.     

Plasma and synovial fluid interleukin-1, interleukin-6 and substance P concentrations in rheumatoid arthritis patients: effect of the nonsteroidal anti inflammatory drugs indomethacin, diclofenac and naproxen

We performed an open, between patients, placebo controlled study in order to evaluate the effect of the treatment with the non steroidal anti inflammatory drugs indomethacin, diclofenac and naproxen on the concentrations of the cytokines IL-1 beta and IL-6 and of the neuropeptide substance P in plasma and synovial fluid of 24 rheumatoid arthritis patients. All patients had high synovial fluid cytokine and substance P levels, and high plasma cytokine levels at the beginning of the study. The treatment with the non steroidal anti inflammatory drugs significantly decreased both plasma and synovial fluid IL-6 and synovial fluid substance P in comparison to placebo, but did not affect IL-1 beta concentrations. This effect can participate in the therapeutic effect of non steroidal anti inflammatory drugs in rheumatoid arthritis.     

Analysis of type 1 and type 2 T cells in synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: It has been reported that CD4+ helper T cells play an important role in the pathogenesis of rheumatoid arthritis (RA). We evaluated the presence of intracellular cytokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) produced by CD4+ and CD8+ T cells in the synovial fluid and peripheral blood of patients with RA at the single cell level. METHODS: We used 3 color flow cytometric analysis. Synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) were stimulated with phorbol myristate acetate (PMA) and calcium ionophore. The stimulated SFMC and PBMC were triple stained with conjugated mononuclear antibodies (Mab) against cytokines and surface antigens after fixation and permeabilization with a saponine buffer solution. The cells were analyzed for intracellular cytokines (IFN-gamma, IL-4) and surface antigens (CD3, CD4, CD8) using a flow cytometer. RESULTS: The CD4/CD8 ratio was significantly lower in SFMC than in PBMC. The positive rates of IFN-gamma producing cells among CD4+ T cells were significantly higher than those of IL-4 producing cells in both the SFMC and the PBMC of patients with active RA. In the SF of these patients, we also found CD8+ T cells that produce IL-4 alone, or both IL-4 and IFN-gamma. CONCLUSION: In the SF of patients with RA, CD4+ type 1 T cells, which may infiltrate into the synovium and cause pathogenic immune responses in the tissue, are predominant. We believe this cell type also induces migration and activation of CD8+ type 2 T cells into the active site of inflammation, which appears to downregulate the activity of CD4+ type 1 T cells, modulating the excess immune response.     

The significance of spinal cord compression as the initial manifestation of lymphoma

Sex hormones have profound effects on immune responses and may influence the outcome of autoimmune diseases such as rheumatoid arthritis (RA). We investigated the effect of gonadal steroids on the production of interleukin-1 (IL-1) and IL-6, cytokines believed to be important in the pathogenesis of RA. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy male donors and male patients with RA, and were stimulated with lipopolysaccharide (LPS) in the presence of different concentrations of 17-beta-estradiol, progesterone or testosterone. In studies of cells from normal male donors, 17-beta-estradiol at pharmacological concentrations (> or = 10(-6) M) enhanced IL-1 and IL-6 secretion as well as the production of cell-associated IL-1. Progesterone and testosterone at similar concentrations inhibited IL-1 secretion but had no significant effect on IL-6 secretion or on the production of cell-associated IL-1. In studies of male RA donors, 17-beta-estradiol failed to enhance IL-1 or IL-6 secretion and progesterone failed to inhibit IL-1 secretion. The inhibitory effects of testosterone, however, appeared to be similar to that in normal donors. It is suggested that 17-beta-estradiol may promote IL-1 and IL-6 production and release, while gestation hormone, progesterone, and testosterone may inhibit IL-1 release in vivo. These data may partly explain the gender and age differences in the incidence of RA and the development of the disease in men with low and androgen levels.