Immunoregulatory role of interleukin 10 in rheumatoid arthritis.

The presence and the role of interleukin 10 (IL-10), a potent cytokine synthesis inhibitory factor and antiinflammatory cytokine, were investigated in rheumatoid arthritis (RA). The expression of both mRNA and protein for IL-10 could be demonstrated in RA and osteoarthritis (OA) joints. Human IL-10 mRNA could be demonstrated by polymerase chain reaction amplification of cDNA made by reverse transcription of total RNA extracted directly from synovial tissue in five out of five RA and four out of five OA patients. IL-10 protein was demonstrated by specific immunoassay and immunohistology. IL-10 protein was spontaneously produced in all 11 RA and 17 OA synovial membrane cultures investigated, and this production was sustained for up to 5 d in culture in the absence of any extrinsic stimulation. IL-10 protein could also be detected by immunohistology in all five RA and four OA synovial membrane biopsies investigated, but not three normal synovial membranes. Immunohistology revealed that the IL-10 was localized to the synovial membrane lining layer and mononuclear cell aggregates. Immunofluorescence double staining revealed that the sources of IL-10 were monocytes in the lining layer, and T cells in the mononuclear cell aggregates. We found evidence that the IL-10 expression was functionally relevant, as neutralization of endogenously produced IL-10 in the RA synovial membrane cultures resulted in a two- to threefold increase in the protein levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and IL-1 beta, although IL-6 and IL-8 levels were not affected. The addition of exogenous recombinant IL-10 to the RA synovial membrane cultures resulted in a two- to threefold decrease in the levels of TNF-alpha and IL-1 beta. IL-8 levels were reduced by day 5; however, IL-6 levels were not affected by exogenous IL-10. Neutralization of the endogenous IL-10 in two out of seven RA synovial membrane cultures resulted in the expression of detectable levels of interferon gamma (561-1,050 pg/ml). Taken together, the above findings suggest that IL-10 is spontaneously produced in RA and OA and is an important immunoregulatory component in the cytokine network of RA, regulating monocyte and in some cases T cell cytokine production.

Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717.

OBJECTIVES: To evaluate the clinical efficacy of a novel synthetic peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, CLX-090717, in several in vitro cell culture systems and murine CIA, an experimental model of RA. METHODS: Peripheral blood monocytes purified by elutriation, and rheumatoid synovial cells isolated from clinical tissue were cultured with CLX-090717 and TNF-alpha release was measured. Molecular mechanism of action was analysed by western blotting and electrophoretic mobility shift assay. Thioglycollate-elicited murine peritoneal macrophages were cultured with CLX-090717 and lipopolysaccharide (LPS)-induced TNF-alpha release was assayed. Therapeutic studies were done in mice with established arthritis by evaluating clinical parameters and histology. In addition, type II collagen response of lymphocytes from mice with CIA was examined. RESULTS: CLX-090717 significantly inhibited spontaneous TNF-alpha release by RA synovial membrane cells, as well as LPS-induced TNF-alpha release from human and murine monocytic cells. Inhibition of TNF-alpha in monocytes was mediated partially through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, as judged by sustained levels of IkappaBalpha in cytosolic extracts and a reduced level of LPS-induced NF-kappaB activity in nuclear extracts. CLX-090717 reduced clinical signs of arthritis and damage to joint architecture when administered therapeutically to arthritic mice. Mechanisms of action in CIA involved the reduction in proliferation of arthritic lymphocytes to antigen in vitro as well as reduced TNF-alpha release. CONCLUSIONS: Our data suggest that the synthetic compound CLX-090717 has potential as a small molecular weight anti-inflammatory therapeutic for chronic inflammatory conditions.

Neutralizing IL-21 and IL-15 inhibits pro-inflammatory cytokine production in rheumatoid arthritis.

Interleukin IL-21 and IL-15 belong to the common gamma-chain receptor family. IL-15 represents a novel therapeutic target in rheumatoid arthritis (RA), whereas less is known about the role of IL-21 in human inflammatory diseases. We have analysed the effects of blocking IL-21 and IL-15 on spontaneous production of pro-inflammatory cytokines in RA synovial cell cultures. RA synovial membrane cells were cultured in the presence of an IL-21R-Fc chimera or a neutralizing IL-15 antibody and production of tumour necrosis factor (TNF)alpha, IL-6 and IL-1beta was measured by enzyme-linked immunosorbent assay (ELISA). Expression of IL-21 and IL-15 in RA synovium was measured by RT-PCR and ELISA. mRNA for IL-21 and IL-21R was detected in the culture cell lysates. Protein for IL-15 was found at detectable levels in the cell lysates. Both the IL-21R-Fc chimera and anti-IL-15 antibody inhibited cytokine release, although substantially more IL-21R-Fc was needed. IL-21R-Fc at the highest dose (100 microg/ml) significantly reduced TNFalpha production by 50%, IL-6 by 57% and IL-1beta by 81%. Anti-IL-15 antibody (5 microg/ml) significantly inhibited TNFalpha release by 51%, IL-6 by 37% and IL-1beta by 82% in line with previous published observations. The data confirm that IL-15 plays a role in RA and suggests that IL-21 is also involved in driving the pro-inflammatory cytokine response in RA.

Paradoxical effects of a synthetic metalloproteinase inhibitor that blocks both p55 and p75 TNF receptor shedding and TNF alpha processing in RA synovial membrane cell cultures.

We have previously hypothesized that the pro-inflammatory cytokine TNF alpha has a pivotal role in the pathogenesis of rheumatoid arthritis (RA). It mediates its effects by cross-linking surface p55 TNF receptors (TNF-R), which can be proteolytically cleaved to yield soluble fragments. Upon binding TNF alpha soluble TNF-R (sTNF-R) can inhibit its function. We investigated the enzymatic nature of the proteases involved in TNF-R cleavage, and found that this process is blocked by a synthetic inhibitor of matrix metallo-proteinase activity (MMP), BB-2275. Inhibition of TNF-R cleavage was observed in a number of different cell types, as detected by retention of surface bound TNF receptor and by less sTNF-R released into the cell supernatant. The augmentation of surface TNF-R expression was of biological relevance as TNF alpha-mediated necrosis of human KYM.1D4 rhabdosarcoma cells was enhanced approximately 15-fold in the presence of BB-2275. The addition of BB-2275 to rheumatoid synovial membrane cell cultures totally inhibited MMP activity and also significantly reduced the levels of soluble TNF alpha (P < 0.006), p55 sTNF-R (P < 0.006), and p75 sTNF-R (P < 0.004). Paradoxically, despite the reduction in soluble TNF alpha levels, the production of IL-1 beta, IL-6, and IL-8, cytokines whose production was previously demonstrated to be inhibited by the addition of neutralizing anti-TNF alpha antibody were not down-regulated by BB-2275. These results raise the interesting possibility that a close relationship exits between the enzyme(s) which process membrane-bound TNF alpha, and those involved in surface TNF-R cleavage. Furthermore our observations suggest that hydroxamate inhibitors of MMP activity which block TNF alpha secretion and TNF-R cleavage may not modulate down-stream effects of TNA alpha, and as such suggest that the precise specificity of these compounds will be highly relevant to their clinical efficacy in inflammatory diseases.

Chronic exposure to tumor necrosis factor (TNF) in vitro impairs the activation of T cells through the T cell receptor/CD3 complex; reversal in vivo by anti-TNF antibodies in patients with rheumatoid arthritis.

Experiments were designed to test the hypothesis that chronic exposure to tumor necrosis factor alpha (TNF) alters the function of activated T lymphocytes. Pretreatment of tetanus toxoid-specific T cell clones with TNF for up to 16 d impaired rechallenge proliferative responses to antigen in a dose- and time-dependent fashion. IL-2 and PHA responses were preserved. Prolonged treatment with TNF impaired production of IL-2, IL-10, IFN gamma, TNF, and lymphotoxin (LT) following stimulation with immobilized OKT3, and resulted in suboptimal expression of the IL-2R alpha chain (Tac) but not CD3, CD4, or HLA-DR antigens, when compared to untreated control cells. By contrast, pretreatment of T cells for prolonged periods in vitro with neutralizing anti-TNF monoclonal antibodies (mAb) enhanced proliferative responses, increased lymphokine production, and upregulated Tac expression following stimulation with OKT3. To determine whether TNF exerts immunosuppressive effects on T cells in vivo, we studied cell-mediated immunity in patients with active rheumatoid arthritis (RA), before and after treatment with a chimeric anti-TNF mAb. Treatment with anti-TNF restored the diminished proliferative responses of PBMC to mitogens and recall antigens towards normal in all patients tested. These data demonstrate that persistent expression of TNF in vitro and in vivo impairs cell-mediated immune responses.

Cytokines in autoimmunity.

It is now generally accepted that many cytokines are involved in the pathogenesis of autoimmune disease, either directly by causing tissue destruction or indirectly through the activation of autoreactive and inflammatory cells. Thus, cytokines, such as tumor necrosis factor-alpha, are implicated in the pathogenesis of rheumatoid arthritis based on in vitro studies on synovial tissue from patients with rheumatoid arthritis, which suggest that the effects of tumor necrosis factor-alpha are amplified by its potential to induce other pro-inflammatory cytokines, such as interleukin-1 and granulocyte-macrophage colony-stimulating factor. Transgenic mouse technology has shown that mice expressing the human tumor necrosis factor-alpha gene develop a polyarthritis. Interleukin-2 has also been identified by transgenic technology as a cytokine involved in the pathogenesis of insulin-dependent diabetes mellitus through the activation and stimulation of growth of autoreactive T cells.

Cytokines in autoimmunity.

The past few years have witnessed exciting developments regarding the role of cytokines in autoimmune diseases, particularly in rheumatoid arthritis and Crohn's disease, with the demonstration that anti-TNF alpha therapy is clinically beneficial and provides reproducible results. Recent contributions to this field, derived from in vivo studies in animal models of autoimmunity, and increasingly from clinical trials, have greatly enhanced our understanding of this field.

The importance of T cell interactions with macrophages in rheumatoid cytokine production.

The analysis of suppression of cytokines in rheumatoid synovial tissue and fluid pioneered the studies of human cytokines in diseased tissue due to the relative ease of staining samples, even at the height of the inflammatory process. These studies led to the study of synovial cytokine regulation, and the identification of TNF as a therapeutic target, which has been amply validated in clinical trials and now routine therapy. The next key question was how is TNF disregulated in synovium. Are there differences between the mechanisms of synovial TNF production compared to the production of protective TNF during an immune response? Are there differences between the induction of the pro-inflammatory TNF and the anti inflammatory IL-10? The analysis of the interaction of the two most abundant synovial cells, T lymphocytes and macrophages has provided interesting clues to new therapeutic approaches based on disrupting T-macrophage interaction.

Preparation of covalent IgG complexes of defined size and their clearance from the circulation of mice.

Covalent complexes of various sizes were made by coupling the photosensitive hapten, NAP (4-azido-2-nitrophenyl), to rabbit IgG and reacting the conjugates with rabbit anti-NAP antibodies. The resultant oligomers were stable, being eluted as the same discrete peaks after chromatography on gels both before and after injection into mice. Tests of their biological properties showed they fixed guinea pig complement in vitro in a size-dependent manner. Normal mice and mice with chronic endogenous circulating complexes cleared the oligomers according to size in a manner best described by a two-component exponential curve. There was no difference in the clearance rates between the two groups of mice. The advantages of these model complexes for examining the effect of circulating complexes on the mononuclear phagocyte system are discussed.

Cytokines in autoimmune disorders.

Cytokines are important protein mediators of immunity, inflammation, cell proliferation, differentiation, fibrosis, etc. (Oppenheim and Saklatvala, 1993). As these are the major biological processes underlying autoimmunity, it is not surprising that there is now convincing evidence that cytokines have an important role in the pathogenesis of autoimmunity (Brennan and Feldmann, 1996; Feldmann et al., 1996). There has been much progress since we first highlighted the role of cytokines such as IFN gamma in autoimmunity in the early 1980s (Bottazzo et al., 1983). The number of cytokines molecularly cloned has increased greatly, and the biochemical and structural basis of their action are partly understood, as cytokine genes and that of their receptors have been cloned. Knowledge of cytokine signalling is rapidly expanding (see Chapter XIII). In medical terms, clear evidence of the importance of cytokines in autoimmunity is demonstrated by therapeutic advances. Thus it is possible to dramatically improve patients with rheumatoid arthritis and Crohn's disease by blocking TNF alpha, and a new target for therapy, TNF alpha, has thus been validated for both these diseases.

TNF blockade in rheumatoid arthritis: implications for therapy and pathogenesis.

The role of the immune response in rheumatoid arthritis (RA) is a subject of debate, although it is widely believed to be a T-cell-driven disease. Progress is being hindered by lack of convincing evidence of a defined specific antigen initiating or perpetuating the response. Clinical trials using monoclonal antibodies directed against T-cell surface molecules such as CD4. CD5, and CD7 have thus far not provided evidence of efficacy. The negative data may reflect inadequate dosing or could suggest that indiscriminate depletion of T cells is insufficient by itself as a therapeutic strategy. Blocking proinflammatory cytokines (e.g. TNF alpha, IL-1) or augmenting anti-inflammatory cytokines (e.g. IL-10) offers an alternative approach to therapy. Clinical trials using monoclonal anti-TNF alpha have been particularly successful in controlling inflammation and markedly reducing acute phase proteins and cellular ingress. However, because disease invariably relapses, repeated therapy is necessary. Preliminary experience suggests that this is possible. Anti-TNF therapy for RA has defined a molecular target and new approach for treating immuno-inflammatory disorders.

Cytokine expression and networks in rheumatoid arthritis: rationale for anti-TNF alpha antibody therapy and its mechanism of action.

The cloning of cytokine cDNAs has permitted the analysis of cytokine expression in diseased sites such as rheumatoid joints. A very wide range of cytokines were detected, mostly with proinflammatory activities. From the analysis of cytokine regulation in rheumatoid joint cell cultures using neutralizing anti-cytokine antibodies, it was found that blockade of TNF alpha reduced the production of other proinflammatory cytokines. Hence TNF alpha was a potential therapeutic target. This concept was tested successfully in collagen induced arthritis in mice and led to clinical trials of anti-TNF alpha antibody in rheumatoid arthritis (RA) in humans. The mechanism of action of anti-TNF alpha will be discussed.

TNF alpha is an effective therapeutic target for rheumatoid arthritis.

Rheumatoid arthritis is the most common of a number of diseases in which inflammation and tissue destruction is driven by an autoimmune process. Current therapy is inadequate, and this has prompted major research efforts, both in academia and industry, to understand more about the pathogenesis, and hence provide the rationale for new therapeutic strategies. Here we review our studies of cytokine expression and regulation in rheumatoid joints, which has culminated in demonstrating that TNF alpha blockade, using a chimeric (human IgG1/K, mouse Fv) anti-TNF alpha antibody, cA2, markedly ameliorates arthritis. This defines a therapeutic target for rheumatoid arthritis.

p55 and p75 tumor necrosis factor receptors are expressed and mediate common functions in synovial fibroblasts and other fibroblasts.

There is increasing evidence that TNF-alpha is a cytokine of major importance in the pathogenesis of rheumatoid arthritis. Since TNF-alpha mediates its effects via high affinity receptors, we were interested in investigating their expression and function in cells from rheumatoid tissue. Synovial fibroblasts derived from rheumatoid synovial tissue are stimulated by TNF-alpha to proliferate and release cytokines, prostaglandins, proteases and protease inhibitors. We have evaluated through which receptor stimulation of DNA synthesis and the release of the proinflammatory agents, IL-6, IL-8 and PGE2 are induced. It was found that rheumatoid synovial fibroblasts express both the p55 and p75 TNF receptor, in a ratio of 4:1. TNF-alpha-stimulated synovial fibroblast DNA synthesis and the release of IL-6, IL-8 and PGE2 was inhibited by antagonist monoclonal antibodies against either the p55 or the p75 TNF receptor, although the blockade of the p55 TNF receptor had a more potent effect than inhibition of the p75 TNF receptor alone. Similarly, specific monoclonal antibodies, agonistic for either the p55 or p75 TNF receptor stimulated synovial fibroblast DNA synthesis, as well as IL-6, IL-8 and PGE2 release. Both p55 and p75 TNF receptors on dermal and gingival fibroblasts were also involved in TNF-alpha-mediated DNA synthesis and IL-6, IL-8 and PGE2 release, although differences in the levels of DNA synthesis and release of inflammatory cytokines and PGE2 were observed between the three fibroblast types.

Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures. Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist.

While there is an extensive literature on cytokine regulation in vivo using human cell lines or peripheral blood monocytes, very little is known about cytokine regulation within the multicellular environment of inflammatory sites in vivo. We have previously shown that in rheumatoid synovial membrane cultures, a complex, but pathophysiologically relevant mixture of cells, the addition of a neutralizing anti TNF-alpha antibody inhibits the production of IL-1 and GM-CSF, indicating the presence of a cytokine 'cascade' in this inflammatory tissue. In this paper we demonstrate that the interactivities between cytokines in rheumatoid arthritis also extends to other cytokines, such as IL-6 and IL-8, and that within the IL-1 family it is IL-1 beta in particular which is downregulated by neutralizing TNF-alpha activity. The cytokine interactions are unidirectional, in that neutralization of TNF-alpha reduced IL-1 beta, IL-6 and IL-8 production, whereas treatment of the rheumatoid synovial membrane cells with a neutralizing concentration of the IL-1 receptor antagonist (IL-1ra) reduced IL-6 and IL-8 production but not TNF-alpha production. These results suggest a rationale for the profound anti-inflammatory effects and consequent clinical benefit noted in RA patients treated recently in clinical trials with a chimeric anti-TNF-alpha antibody in vivo.

Targeting TNF alpha for the therapy of rheumatoid arthritis.

Our pre-clinical studies have demonstrated a pathogenic role for TNF alpha in RA. Firstly, TNF alpha and its receptors are upregulated and co-expressed in the synovium and cartilage-pannus junction of RA joints. Secondly, mononuclear cells from RA joints maintained in culture produce many cytokines with pro-inflammatory activity, including TNF alpha. Neutralizing TNF alpha antibodies in vitro reduces the production of these pro-inflammatory cytokines, including IL-1, IL-8, and GM-CSF. Thirdly, when injected into arthritic DBA/l mice with collagen-induced arthritis, monoclonal anti-TNF antibodies decrease inflammatory damage of joints. Clinical trials employing cA2, a monoclonal chimeric anti-TNF alpha antibody, in open-label and randomized placebo-controlled studies have demonstrated a dose-dependent efficacy with impressive improvement in disease activity and acute phase responses lasting several weeks. We conclude that TNF alpha is a critical mediator of inflammation in RA and is an important therapeutic target in this disease.

TNF-alpha in rheumatoid arthritis and prospects of anti-TNF therapy.

Our work has shown that TNF alpha is produced by cultured mononuclear cells from rheumatoid arthritis joints and appears to regulate the production of IL-1. Immunohistochemical examination has shown the presence of TNF alpha in the synovium, e.g. in the lining layer, some endothelial cells and most importantly, in the cells in the cartilage pannus junction. TNF receptors (both p55 and p75) have a similar distribution, thereby suggesting that TNF has the potential for autocrine and paracrine activity in the joint. The concept that TNF alpha is pathogenic in inflammatory arthritis has been validated by showing that neutralizing monoclonal anti-TNF antibodies significantly attenuate collagen-induced arthritis in mice. In preliminary trials in rheumatoid patients anti-TNF appears to have an impressive effect on indices of disease activity including C-reactive production and serum amyloid-A production. TNF alpha appears to be a relevant therapeutic target in rheumatoid disease.

Cytokine blockade in rheumatoid arthritis.

As we enter the 2000's it is clear that cytokine blockade is an effective therapeutic strategy for rheumatoid arthritis. In this brief review, we will review the rationale for anti TNFalpha therapy, the current status of therapy and focus on the regulation of TNFalpha production in rheumatoid synovium. New approaches to studying TNF regulation in RA and of elucidating the controversial role of T cells in this complex disease will be described.