Naturally Occurring Cytokine Inhibitors in Rheumatoid Arthritis

13 Naturally Occurring Cytokine Inhibitors in Rheumatoid Arthritis G a r y S. Firestein

INTRODUCTION Cytokines play a central role in the perpetuation of rheumatoid arthritis (RA). Their ability to induce cellular proliferation, metalloproteinase and prostaglandin production, expression of adhesion molecules, and secretion of other cytokines contributes to synovial inflammation and tissue destruction. The cytokine profile of synovitis has been carefully mapped, resulting in a detailed understanding of complex paracrine and autocrine networks in arthritis (Firestein and Zvaifler, 1990; Arend and Dayer, 1990). Although the inflamed joint contains a broad range of cytokines, the profile is not random since most of the factors detected are derived from macrophages and fibroblasts, including IL-1, IL-6, TNFer colony-stimulating factors (e.g. GM-CSF), and chemokines (e.g. IL-8) (Firestein and Zvaifler, 1990). In contrast, T-cell derived products like IFN~/, IL-2, or TNFI3 are difficult or impossible to detect. Although the relative absence lymphokines from T cells might simply reflect our inability to measure minute amounts in the synovial microenvironment, this observation has engendered a lively debate on the role of T cells in RA (Panayi et al., 1992; also Chapters 2 and 4, this volume). Positive cytokine feedback loops that amplify cellular responses are normally opposed by negative circuits that attempt to re-establish homeostasis. For instance, the natural IL-1 antagonist, IL-lra, is produced in vivo in response to endotoxin exposure in humans and probably serves to modulate the devastating effects of septic shock (Ulich et al., 1992). These negative feedback systems also appear to operate in RA in an attempt to re-establish synovial homeostasis. Clearly, defective or inadequate production of these factors could contribute to the perpetuation of chronic synovitis. In this chapter, some of these naturally occurring cytokine inhibitors will be discussed, with an emphasis on cytokines known to be important in synovitis. (The role of cytokines in the pathology of RA is also discussed in Chapters 2, 9, 10, 11, 12, 26 and 27, this volume). INTERLEUKIN 1 ANTAGONISTS The interleukin 1 system is essential to homeostasis and normal immune responses. IL-le~ and-[3 comprise the agonist arm of this family (Dinarello, 1991). Both are synthesized as 31-kDa precursor peptides; the precursor for IL-le~ is biologically active, but unprocessed IL-1[3 is inactive. Precursor-IL-1[3 is cleaved to the active 17kDa protein and released into the extracellular space after the cell is stimulated (e.g. by endotoxin or other cytokines) or in other specialized situations, including apoptosis Mechanisms and Models in Rheumatoid Arthritis Copyright 9 1995 Academic Press Ltd ISBN 0-12-340440-1 All rights of reproduction in any form reserved 261

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(programmed cell death) (Mosley et al., 1987; Dinarello, 1991; Hogquist et al., 1991). At least two high affinity surface receptors for IL-1 exist and bind to IL-ler and-[3. The type I IL-1 receptor (IL-1R) is expressed on a variety of cells, including T cells, synoviocytes, chondrocytes, and fibroblasts, while the type II receptor is expressed on B cells and macrophages (Dinarello, 1991). The functional role of the type II receptor is not clear, since it possesses only a very short cytosolic domain (29 amino acids compared to 213 amino acids for the type I receptor). A competitive antagonist that binds to both IL-1Rs known as the IL-1 receptor antagonist has also recently been identified (see below). IL-1 is critically involved in the pathogenesis of arthritis (Firestein and Zvaifler, 1990; Arend and Dayer, 1990). In addition to its role in antigen presentation to T cells, it also has profound effects on synovial cells. For instance, IL-1 stimulates fibroblast-like synoviocytes to proliferate, secrete cytokines like GM-CSF and IL-8, and produce metalloproteinases that can digest the extracellular matrix (Balavoine et al., 1986; Alvaro-Gracia et al., 1990; see Chapters 8 and 9, this volume). Injection of IL-1 directly into joints causes synovitis and parenteral administration markedly exacerbates experimental antigen-induced arthritis (Henderson and Pettipher, 1989; Chandrasekhar et al., 1990). Immunoreactive and biologically active IL-1 are present in RA synovial effusions, although the actual concentrations that have been reported vary considerably depending on the type of assay used (Miossec et al., 1986; Symons et al., 1989; Rooney et al., 1990). About 10% of rheumatoid synovial cells contain detectable levels of IL-l[3 mRNA (Firestein et al., 1990). The vast majority of cells expressing the IL-113 gene are macrophages. IL-1 protein is also found in these synovial macrophages. IL-1 positive cells in the synovium are interspersed throughout the intimal lining and sublining perivascular mononuclear cell aggregates and appear to be particularly abundant near the cartilage-pannus junction (Miossec et al., 1986; Koch et al., 1992). In light of the purported importance of IL-1, recent descriptions of specific inhibitors in the joints of patients with RA are surprising. IL-1 RECEPTOR ANTAGONIST (IL-lra) Biology of IL-lra IL-lra is a naturally occurring IL-1 inhibitor that was described in the supernatants of human monocytes as well as in the urine of febrile patients (Arend et al., 1985, 1991). IL-lra binds directly to type I and II IL-1 receptors (IL-1R) and competes with IL-1 for the ligand binding site (Carter et al., 1990; Hannum et al., 1990; Dripps et al., 1991b). It is a pure antagonist; that is, no detectable signal is transduced and the receptor-ligand complex is not internalized after it binds to the IL-1R (Dripps et al., 1991a). This is distinctly different from the IL-1 : IL-1R complex, which enters the cell and migrates to the nucleus. Although IL-lra binds to the IL-1 receptor with the same affinity as IL-le~ and IL-l[3, it is a relatively weak inhibitor because IL-1 activates cells even if only a small percentage of receptors are occupied. Hence, a large excess of the inhibitor is needed to saturate the receptor and block IL-l-mediated stimulation (Arend et al., 1990; Smith et al., 1991). Typically, the concentration of IL-lra in vitro must be 10- to 100-fold higher than IL-1 to achieve significant inhibition (see Fig. 1). Recombinant IL-lra inhibits a variety of IL-l-mediated events in cultured cells

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derived from the joint, including the induction of metalloproteinase and prostaglandin production by chondrocytes and synoviocytes. Two structural variants of IL-lra have been described: (a) secretory IL-lra, or slLlra, which is synthesized with a signal peptide that allows it to be transported out of cells (Carter et al., 1990; Eisenberg et al., 1990); and (b) intracellular IL-lra, or iclLlra, which lacks a leader peptide due to alternative splicing of mRNA and therefore remains intracellular (Haskill et al., 1991). slL-lra is a major product of mononuclear phagocytes, particularly mature tissue macrophages, while icIL-lra is produced by keratinocytes and other epithelial cells (Haskill et al., 1991; Bigler et al., 1992). Both forms may be produced simultaneously by fibroblasts and alveolar macrophages (Chan et al., 1993; Quay et al., 1993). The role of iclL-lra in the immune response is not established. It might represent a homeostatic mechanism in the skin, where iclLlra in keratinocytes could be released after tissue injury or necrosis. Alternatively, localized apoptosis might release intracellular proteins like icIL-lra into the microenvironment. While IL-1 and IL-lra production are, in many circumstances, closely linked, they clearly have distinct regulatory controls (Janson et al., 1991). The relative balance between IL-1 and IL-lra expression in vitro depends on a variety of factors. Cell maturity is one important variable, and IL-1 predominates in immature monocytes

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while IL-lra is more prevalent in mature macrophages. Cytokines also regulate the production of IL-lra, since IL-4 and GM-CSF increase secretion of IL-lra by monocytes. In some cases, both the agonist and antagonist are simultaneously produced by the same cell (Roux-Lombard et al., 1989; Janson et al., 1991). I L - l r a in rheumatoid arthritis

Because of the importance of IL-1 in RA, the discovery of IL-lra led to the obvious hypothesis that IL-lra deficiency contributes to the disease. Yet, unexpectedly high concentrations of the protein (up to 50ng/ml) are present in rheumatoid synovial effusions as measured with a sensitive and specific immunoassay (Malyak et al., 1994). Furthermore, immunohistochemical studies of rheumatoid synovium reveal large amounts of IL-lra protein in rheumatoid synovial tissue, particularly in perivascular mononuclear cells and the synovial intimal lining (Deleuran et al., 1992a; Koch et al., 1992; Firestein et al., 1992). IL-lra protein is especially abundant in and about regions containing IL-1 protein. The presence of IL-lra in synovium is not specific to RA, since it is also present in osteoarthritis synovial tissue, albeit in lesser amounts. Normal synovium is completely devoid of immunoreactive IL-lra. Single and double label immunohistochemistry studies show that macrophages are the primary source of IL-lra in the sublining region (Firestein et al., 1992). Both macrophages and a population of non-macrophages (presumably fibroblast-like synoviocytes) in the intimal lining contain immunoreactive IL-lra. One potential limitation of immunohistochemistry is that it cannot determine whether a protein is produced by the labelled cell or whether the protein is secreted elsewhere and subsequently internalized or bound by the cell. To address this question, molecular biology techniques have been used to study synovial gene expression. Northern blot analysis of whole tissue RNA extracts demonstrate abundant IL-lra mRNA, confirming local gene transcription (Firestein et al., 1992). In situ hybridization studies reveal that the distribution of IL-lra m R N A correlates with IL-lra protein. Further evidence of synovial IL-lra production is provided by the demonstration of glycosylated IL-lra in the supernatants of cultured synovial tissue cells by western blot analysis (see Fig 2). Bioassays of these supernatants indicate that synovial IL-lra is biologically active. How can these data be reconciled with the notion that IL-lra deficiency might contribute to synovitis? The answer might lie in the fact that the biological importance of IL-1 as a pro-inflammatory mediator depends on a variety of influences, especially the balance between IL-1 and its various inhibitors. Hence, the absolute amount of IL-lra and other inhibitors is not as important as their concentrations relative to IL-1. While is is impossible to measure the amount of IL-1 and IL-lra directly in the synovial microenvironment, accurate assays have determined the relative amounts of each produced by cultured synovial tissue cells. These studies show that the ratio of IL-lra to IL-1 is relatively low (from 1 : 1 in cell lysates to 4:1 in culture supernatants) compared to the 10- to 100-fold excess of IL-lra needed to block IL-l-mediated metalloproteinase and prostaglandin synthesis by mesenchymal cells (Arend et al., 1990; Smith et al., 1991; Firestein et al., 1994). The reason for low synovial IL-lra production is complex and appears to involve at least two distinct processes. First, fibroblast-like synoviocytes, which account for much of the immunoreactive IL-lra in the synovial intimal lining, selectively produce

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Figure 2. IL-lra production by synovial cells. Western blot analysis demonstrated immunoreactive IL-lra (also called IRAP, or IL-1 receptor antagonists protein) secretion by freshly isolated synovial tissue cells. Samples of 2-day culture supernatants of rheumatoid arthritis (RA), osteoarthritis (OA), and avascular necrosis (AVN) were assayed. Recombinant hIL-lra and supernatants from phorbol ester activated THP-1 cells were included as positive control for non-glycosylated and glycosylated forms of the protein, respectively. The majority of IL-lra secreted by synovial cells appeared to be the higher molecular weight glycosylated form. Other studies showed that the IL-lra was biologically active. From Firestein et al. (1992) with permission.

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the intracellular form of the IL-lra. Evidence for this conclusion is derived from two observations: (a) supernatants of cultured fibroblast-like synoviocytes contain little IL-lra, while cell lysates contain large amounts; and (b) quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrate that the vast majority of IL-lra mRNA is the alternatively spliced iclL-lra form in the fibroblastlike cells (Firestein et al., 1994). Therefore, most, if not all, of the IL-lra produced by these cells remains intracellular and is not normally secreted into the extracellular space. However, iclL-lra might be released from synoviocytes in vivo by alternative routes, such as apoptosis. A variety of stimuli can induce apoptosis, such as metabolic poisons (e.g. actinomycin D) or oxygen radicals. One characteristic finding in apoptotic cells is degradation of DNA into discrete nucleosomes that can be visualized using agarose gel electrophoresis. Of interest, this pattern has been observed in DNA isolated directly from arthritic synovium as well as from cultured synovial cells (Firestein, unpublished data). It is possible, therefore, that low levels of programmed cell death occur in situ and contribute to IL-lra release. A second reason for low synovial IL-lra production is a relative defect in IL-lra production by synovial tissue macrophages. Macrophages isolated from other sites, such as alveolar macrophages or monocyte-derived macrophages, are highly efficient IL-lra producers (Moore et al., 1992). Although purified synovial macrophages produce IL-lra, they only secrete about 1% as much as macrophages isolated from other sites (Tsai et al., 1992; Firestein et al., 1994). Low IL-lra secretion by synovial macrophages might result, in part, from excess alternative splicing to the intracellular form. One rather puzzling aspect of these studies is the relatively high concentration of IL-lra reported in RA synovial fluid compared to the small amounts produced by synovial tissue. For most cytokines, the synovium is the primary site of gene expression, and fresh synovial fluid mononuclear cells usually contain only limited amounts of cytokine mRNA (Firestein et al., 1990). Although the evidence is indirect, IL-lra might be an exception to this paradigm since it appears to be synthesized by neutrophils, which selectively accumulate in the intra-articular cavity. The concentrations of IL-lra in rheumatoid effusions correlate with the number of neutrophils in the synovial fluid, and IL-lra is perhaps the most abundant new protein synthesized by recently activated neutrophils (McColl et al., 1992). Moreover, recent data show that synovial fluid neutrophils in RA contain IL-lra protein, and that these cells secrete increased amounts if they are treated with GM-CSF, which is known to be present in synovial fluid (Xu et al., 1989; Malyak et al., 1994). Therefore, neutrophils might be an important source of IL-lra in joint effusions. If true, this would provide an explanation for the apparent discrepancy between the relative amounts of IL-lra found in these two joint compartments (i.e. synovial fluid and synovium), since granulocytes usually do not reside in the synovial membrane (Firestein et al., 1987). The presence of an IL-lra defect in arthritis implies that administration of pharmacologic doses of IL-lra would be beneficial, and clinical studies to test this hypothesis are in progress. Preliminary data indicate that the recombinant protein is well tolerated in humans and has a pharmacokinetic profile that is amenable to long-term therapy (even though very large doses would be needed). Unfortunately, a beneficial effect of IL-lra in some animal models of arthritis has been somewhat difficult to demonstrate. This might reflect the fact that IL-lra functions primarily as an anti-

NATURALLY OCCURRING CYTOKINE INHIBITORS IN RA 267 inflammatory agent and does not inhibit cell-mediated immune responses that might be important in arthritis (Nicod et al., 1992). In some contrived models, such as the direct injection of IL-1 into the joint, benefit has been reported (Henderson et al., 1991). Also, a physiologic role of IL-lra in the resolution of synovitis is supported by the correlation between synovial fluid IL-lra concentrations and clinical improvement in the acute arthritis of Lyme disease (Miller et al., 1993). It is important to remember that the relevance of animal models or Lyme disease to RA is unclear, and the only way to test the efficacy is to perform a controlled clinical study. See Chapters 2 and 26, this volume, for descriptions of the inhibition of other cytokines in RA and models of RA. ANTAGONISTIC CYTOKINES A second mechanism of cytokine inhibition in RA is the local production of a second cytokine that antagonizes the function of the first. While many antagonistic cytokines have been described, perhaps the best characterized in RA is transforming growth factor-beta (TGFI3). TGFJ3 is a complex growth factor with multiple isoforms whose actions in vitro depend on culture conditions, the cells used, and the concentrations of the protein (see Chapter 12, this volume). Overall, TGFI3 is a potent inhibitor of IL-1 biological activity. For instance, incubation of chondrocytes with IL-113 leads to a marked decrease in proteoglycan and collagen biosynthesis, and much of this effect is abolished if cells are pretreated with TGFI3. While the mechanism is not fully established, down-regulation of IL-1R expression might account for some of the inhibition since TGFI3 decreases the number of surface receptors by nearly 50% (Harvey et al., 1991; R6dini et al., 1993). The affinity of the IL-1R for its ligand is unchanged, however. TGFI3 can also indirectly contribute to IL-1 inhibition by inducing IL-lra production. The role of TGFI3 in arthritis is also complex. It can serve as an autocrine growth factor for cultured fibroblast-like synoviocytes (Goddard et al., 1990, 1992) and inhibits metalloproteinase production by synoviocytes and fibroblasts from a variety of sources (Raghu et al., 1989; Overall et al., 1989). This effect appears to be mediated at the level of gene expression, since TGFJ3 decreases collagenase mRNA synthesis. TGFI3-mediated matrix preservation is also enhanced by an increase in production of tissue inhibitors of metalloproteinases (TIMPs), a family of proteins that bind to metalloproteinases and inhibit their action. Furthermore, synthesis of matrix proteins like collagen is increased. The combination of decreased proteolysis and increased collagen production has led to the suggestion that TGFI3 plays an important role in wound healing. Although large amounts of TGFI3 protein are present within the joints of rodents with experimental inflammatory arthritis, its specific role in the pathogenesis of synovitis is very confusing: depending on the model used and the timing of administration, exogenous TGFI3 can either exacerbate or ameliorate experimental models of arthritis (Brandes et al., 1991; Cooper et al., 1992; Elford et al, 1992; Wahl et al., 1993). For example, parenteral treatment with recombinant TGFJ31 decreases the severity of polyarthritis in streptococcal cell wall arthritis. The benefit is observed if therapy is initiated either before immunization or after the onset of acute arthritis. In contrast, antibodies that neutralize TGFJ3 decrease synovial inflammation in other models of

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arthritis. Also, direct intra-articular injections with TGFI3M or TGFI32 cause acute synovitis with synovial lining hyperplasia. Intra-articular TGF[3 also accelerates collagen-induced arthritis. As with streptococcal cell wall arthritis, immunoreactive TGF[3 is abundant in RA synovium. High concentrations of the peptide are also found in synovial effusions (Lafyatis et al., 1989; Fava et al., 1989; Miossec et al., 1990c; Brennan et al., 1990; Lotz et al., 1990; Chu et al., 1991). The majority of synovial fluid TGF[3 is present in an inactive latent form, and mild acid treatment can convert it to the biologically active protein in vitro. While only a fraction of TGFI3 is active in synovial fluid, levels of the active protein are certainly high enough to be biologically meaningful. In fact, TGF[3 probably accounts for much of the IL-1 inhibitory activity in synovial fluid as measured by a thymocyte proliferation assay (Wahl et al., 1990). Hence, TGF[3, like IL-lra, appears to be a particularly important inhibitor of IL-1 activity in synovial effusions. Its function in synovial tissue is not established, but the diverse effects in animal models suggest that a clear cut role of TGF[3 as either pro- or antiinflammatory in RA is far from understood. Moreover, the animal data imply that systemic TGF[3 might have a beneficial immunoregulatory effect, while the presence of the cytokine in the joint cavity might even be deleterious. The roles of TGF[3 in arthritis are also discussed in Chapters 9 and 12, this volume. SOLUBLE IL-1 BINDING PROTEINS Soluble cytokine receptors or soluble binding proteins can absorb free cytokines and prevent them from engaging functional receptors on cells. While these obviously could inhibit cytokine action, it should be kept in mind that they might also function as carrier proteins that protect cytokines from degradation and deliver them directly to cells. However, the role of the soluble IL-1R in normal immunity or as a negative feedback mechanism in inflammation has not been established. A therapeutic potential for soluble IL-1R is possible, since recombinant IL-1R protects against synovitis induced by direct injection of IL-1 into joints. A novel soluble IL-1 binding protein has been identified in supernatants of a human B cell line and in RA synovial fluid and has recently joined the panoply of IL-1 inhibitors present in the rheumatoid joint (Symons et al., 1990, 1991). This 47-kDa protein is proteolytically cleaved from the cell surface by serine proteases and binds specifically to IL-113, but not to IL-lra or IL-la. Both the biologically active 17-kDa form of IL-113 as well as the inactive 31-kDa precursor bind to the protein. The purified binding protein is able to absorb only IL-l[3 from culture supernatants and, hence, prevents IL-113 from engaging cell surface IL-1 receptors. Therefore, it exhibits several characteristics that distinguish it from the type I IL-1R. Recent studies suggest that the binding protein might be a soluble form of the type II IL-1R (although both IL-la and IL-lra should bind to the protein if this is true). ANTI-IL-Icx ANTIBODIES Specific antibodies that bind to and neutralize cytokines can also inhibit cytokine activity. Very little is known about natural anticytokine antibodies in vivo, although one might anticipate their presence in diseases with marked B cell hyperreactivity. In

NATURALLY OCCURRING CYTOKINE INHIBITORS IN RA 269 this regard, it is notable that neutralizing anti-IL-lot antibodies are found in the blood of about 16% of RA patients and only 5% of normal individuals (Suzuki et al., 1991). Similar studies have not been performed on synovial fluid, although it would be surprising if it were devoid of anti-IL-lot activity since plasma proteins generally are also present in synovial effusions, albeit at lower concentrations. Many pertinent questions arise concerning this autoantibody. For instance, is it truly directed against IL-lot, or does it result from molecular mimicry? Does the antibody simply block ILlot activity or does it serve other functions, like delivering the cytokine to Fc-receptor bearing cells? Where is the site of antibody production (synovium versus peripheral lymphoid organs)? Is this antibody unique, or are antibodies that bind cytokines a general feature of RA? The use of specific cytokine-neutralizing antibodies in the study of the pathology of experimental arthritis is reviewed by Otterness et al. (Chapter 26, this volume). The administration of anti-TNF antibodies to RA patients is described by Maini et al. (Chapter 2, this volume). TNF-Ot ANTAGONISTS TNF BIOLOGY TNFOt, like IL-1, has been implicated in the pathogenesis of RA (Firestein and Zvaifler, 1990, reviewed in Chapter 2, this volume). TNF (originally so-designated because of its ability to cause haemorrhagic necrosis of a murine sarcoma), is synthesized by a variety of cells (including macrophages, fibroblasts, and T lymphocytes) as a 26-kDa precursor peptide (Kunkel et al., 1989; Vilcek and Lee, 1991). It exists in solution as an active homotrimer with three 17-kDa subunits. The TNFOt gene (as well as the closely related lymphocyte protein, TNF[3) is located in the major histocompatibility region of human chromosome 6. Like many cytokines, its expression is regulated, in part, by posttranscriptional events, and the rate of mRNA degradation is tightly controlled by an endogenous ribonuclease(s) that recognizes an AU-rich tail in the 3' untranslated region. TNFOt shares many biological functions with IL-1, including the ability to stimulate metalloproteinase production, cytokine synthesis, and proliferation of synoviocytes (Alvaro-Gracia et al., 1990). IL-1 and TNFOt are usually additive or synergistic in biological assays. TNFOt is readily detected in most rheumatoid synovial fluids using immunoassays, although biological activity is difficult to demonstrate (perhaps due to the presence of inhibitors (see below)) (Saxne et al., 1988; Miossec et al., 1990c; Westacott et al., 1990). Immunoreactive TNFOt is found in RA synovial tissue, particularly in the lining and in perivascular lymphoid aggregates (Deleuran et al., 1992b), and TNFOt mRNA is primarily localized to synovial macrophages (Firestein et al., 1990). SOLUBLE RECEPTORS Two types of TNFOt receptors have been characterized (Hohmann et al., 1989; Brockhaw et al., 1990; Hohmann et al., 1990; Schoenfeld et al., 1991). A 55-kDa receptor (415 amino acids) has lower affinity for TNFOt and exhibits a high degree of homology with the nerve growth factor receptor. A 75-kDa TNF-R (461 amino acids)

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binds to TNFe~ with about 5- to 10-fold greater affinity than does the 55-kDa TNF-R. The relative levels of surface expression of each receptor vary depending on the cell type, and distinct functions appear to be subserved by each (Browning and Ribolini, 1989; Hohmann et al., 1990). TNFI3 also binds to both TNF-Rs. A naturally occurring inhibitor of TNF activity was first identified in the urine of febrile patients (Seckinger et al., 1988). Purification of this material revealed that it was a solubilized form of the TNF-Rs (Engelmann et al., 1989; Seckinger et al., 1990). Additional studies have since shown that the extracellular domain of both the 55-kDa and 75-kDa receptors are shed from cell surfaces (Nophar et al., 1990; Porteu et al., 1991) and that proteolytic cleavage is responsible for release of the soluble receptors. The soluble receptors can form stable complexes with TNFe~ and thereby inhibit biological activity. Significant amounts of soluble p55 and p75 TNF-R (> 1 ng/ml) are present in the plasma of normal individuals and likely represent an important homeostatic mechanism (Novick et al., 1989). Very high levels of both soluble TNF-Rs are released into the blood of patients with inflammatory diseases, including RA, and in rheumatoid synovial fluid where levels can exceed 50ng/ml (see Fig. 3). This is considerably higher than the concentration of TNFe~ in the blood or synovial fluid (10100 pg/ml) and represents a formidable inhibitor. The concentrations of both p55 and p75 in synovial effusions probably explain why biologically active TNF is difficult to detect in RA synovial fluid despite the presence of immunoreactive protein (RouxLombard and Dayer, 1991; Cope et al., 1992; Heilig et al., 1992; Roux-Lombard et al., 1993). Quantitative studies of sTNF-R levels in synovial fluid show a positive correlation between the concentrations of p75 and p55, although there is a modest preponderance of the latter. There is no correlation between the cell count or the number of neutrophils in synovial effusions. While little is known about the regulation of receptor shedding in the joint, synovial tissue mononuclear cells do have increased surface expression and mRNA levels for both TNF-Rs compared to osteoarthritis synovial cells or peripheral blood cells (Brennan et al., 1992). Cultured fibroblast-like synoviocytes express the lower affinity 55-kDa TNF-R and continuously shed this receptor into culture supernatants (AlvaroGracia et al., 1993) (see Table 1). IFN~/, which markedly enhances surface expression of HLA-DR on synoviocytes, modestly increases expression of the lower affinity TNF-R without changing the affinity or the rate of receptor shedding. The functional significance of soluble TNF-R obviously depends on the relative amounts of agonist and antagonist. As noted above, concentrations of the soluble TNF-Rs are quite high compared to TNFa levels in the blood and in synovial effusions, where TNFot levels are typically 10 to 100 pg/ml. As with IL-1 antagonists, the balance between agonist and antagonist appears to be shifted towards the antagonist in the synovial fluid compartment. Studies designed to examine critically this ratio in the synovial membrane have not yet been performed for TNF-R, but the presumed importance of TNFa in the rheumatoid cytokine network suggests that the agonist probably prevails within the synovial microenvironment. ANTAGONISTIC CYTOKINES Cytokines that antagonize TNFc~ biological activity have also been described. IFN-r appears to be an important TNFe~ antagonist, as it inhibits TNFe~-mediated synovio-

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RA CC CC RA Serum SF Serum SF Figure 3. Soluble TNF-R concentrations in serum and synovial fluid. Very high concentrations of both the p55 (A) and p75 (B) soluble TNF-Rs are present in RA synovial fluid, particularly when compared to matched serum samples. However, serum samples still contain much more TNF-R than TNFc~. Note that while chondrocalcinosis (CC) samples contain soluble TNF-Rs, RA samples are quite a bit higher. From Roux-Lombard et al. (1993). cyte proliferation, GM-CSF secretion, and collagenase production (Alvaro-Gracia et al., 1990). Early data suggested that the inhibition was specific for TNFoL, but additional studies indicate that IFN~/also blocks some IL-l-mediated activities (Johnson

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Table 1. TNFcx receptor expression on fibroblast-like synoviocytes Soluble 55 kDa TNF-R* Condition Receptors/cell kDa Medium 2800 7.4 X 10-1~ M 112 + 48 IFN',/(100U/ml) 3400 7.0 X 10-1~ 128 + 50 * pg/ml/10-~cells. IFN~/modestly increases TNF-R expression without changing either the affinity or the rate of receptor shedding. Cells were incubated in medium or 100 U/ml of IFN3, for 24 h prior to receptor analysis. TNF-R assays were performed by Scatchard analysis using I~2-%TNFc~.Soluble 55-kDa receptors were assayed by ELISA. Adapted from Alvaro-Gracia et al. (1993).

et al., 1989; Hamilton et al., 1992). The relationship between TNFer and IFN~/is of

particular interest because TNFot also antagonizes some actions of IFN~/on synoviocytes, such as induction of H L A - D R expression. The ability of these cytokines to inhibit each others' stimulating action on synoviocytes has been termed 'mutual antagonism' (Alvaro-Gracia et al., 1990). This relationship is fairly specific to synoviocytes, since these two cytokines are generally additive or synergistic on other cell types (Zuber et al., 1988; Elias et al., 1988; Scharffetter et al., 1989). Originally, mutual antagonism between TNFcx and IFN~/on synoviocytes was thought to be true for all actions of these cytokines. However, more recent data demonstrate that both induce adhesion molecules like ICAM-1 and VCAM-1 on synoviocytes and their effects are additive or synergistic (Chin et al., 1990; Morales-Ducret et al., 1992). Therefore, TNFedIFN~/ mutual antagonism is selective and depends on the specific function examined. Studies of TNFcx/IFN~, antagonism have not yet elucidated the mechanism. One potential explanation is that they down-regulate the expression of each others' surface receptors. However, Scatchard analyses using iodinated cytokines indicate IFN~/ actually increases TNF-R display (Alvaro-Gracia et al., 1993). TNFot also paradoxically increases IFN~/ receptor surface expression. The interaction between the two cytokines appears to have effects at the level of gene expression since IFN~/inhibits the accumulation of G-CSF and GM-CSF mRNA in TNFedIL-1 treated synoviocytes. Antagonism between IFN~/and TNFe~ (as well as IL-1) raises the interesting hypothesis that IFN~/ deficiency contributes to rheumatoid synovitis. This notion is actually antithetical to early paradigms of RA, which purported that IFN~/ was a critical pro-inflammatory factor that regulated H L A - D R expression and antigen presentation in the joint. In fact, RA peripheral blood T cells have a specific defect in IFN~/production (Hasler et al., 1983; Combe et al., 1985) and only very low levels of IFN~/have been detected in synovial fluid (Firestein and Zvaifler, 1987; Miossec et al., 1990c). Furthermore, GM-CSF, not IFN~/, has been identified as the major HLA-DRinducing factor in supernatants of RA synovial cells (Alvaro-Gracia et al., 1989). These data suggest that IFN~/might be an anti-inflammatory cytokine that normally helps maintain synovial homeostasis and that the absence of IFN~/in the joint contributes to unopposed TNFe~- and IL-l-mediated stimulation. This hypothesis implies that 'replacement' therapy with pharmacologic doses of IFN~/might correct this defect and decrease joint inflammation. In fact, clinical trials have demonstrated modest improvement in RA patients treated with IFN~/(Cannon et al., 1989). Perhaps the complexity and redundancy of cytokine networks prevents a single agent like IFN~/from being more effective. For example, the relative absence of

NATURALLY OCCURRING CYTOKINE INHIBITORS IN RA 273 other suppressive cytokines, like IL-4, might also contribute to disease. This notion is supported by in vitro studies of synovial tissue explants showing that exogenous IL-4 decreases production of metalloproteinases and immunoglobulins (Miossec et al., 1992). This raises the possibility that combinations of cytokines that antagonize proinflammatory factors might be more effective than single agents. INTERLEUKIN 2

INTERLEUKIN 2 IN ARTHRITIS In contrast to IL-1 and TNFe~, the role of IL-2 in chronic RA is less well established. Early studies suggested that significant amounts of this T-cell growth factor are in synovial effusions, but recent data indicate that little, if any, IL-2 is present (Firestein et al., 1988; Miossec et al., 1990c). However, IL-2 mRNA has been detected in synovial tissue (Buchan et al., 1988); this apparent paradox might be due to the induction of tolerance in synovial T cells, since a similar phenotype can be produced in tolerant T cells in vitro (i.e. high mRNA, low protein production) (Schall et al., 1992). Although small amounts of IL-2 could potentially be produced in the synovial microenvironment, its role in established disease is undefined. SOLUBLE RECEPTORS The soluble IL-2 receptor (IL-2R) is one of the best characterized and most extensively studied soluble cytokine receptors in RA. Surface IL-2Rs are displayed on a many cells types, including monocytes, B cells, and activated T cells. It is normally expressed by a small percentage of resting peripheral blood T cells (about 1-2%), and activation by antigen or mitogen rapidly increases both the percentage of cells expressing the IL-2R as well as the surface density on the positive cells. The receptor is comprised of two membrane polypeptides (p55 and p75); high affinity IL-2 binding occurs when both are present and in physical contact with each other (Hatakeyama et al., 1989). The low affinity binding protein, p55 (also called Tac), consists of a 251 amino acid polypeptide with extracellular, transmembrane, and short cytosolic domains encoded by a single structural gene on chromosome 10 in humans. The full length Tac protein undergoes extensive N- and O-linked glycosylation. Activated IL-2 bearing cells spontaneously shed the extracellular domain of the p55 IL-2R (about 191 amino acids) after proteolytic cleavage (Robb and Kutny, 1987). The resultant soluble receptor binds to IL-2, albeit with low affinity. The soluble IL-2R/IL-2 complex is unstable and has a short half-life. Soluble IL-2R cannot compete with high affinity receptors on cell membranes for IL-2 binding (Miossec et al., 1990b). Hence, soluble IL-2Rs appear to be inefficient inhibitors of IL-2 biological activity and might have limited capacity for absorbing excess IL-2 or serving as carrier proteins. However, other studies suggest that soluble IL-2Rs do have some immunoregulatory potential (Kondo et al., 1988). The percentage of cells in the rheumatoid synovium that express the IL-2R is similar to that of resting peripheral blood lymphocytes (Cush and Lipsky, 1988). However, the percentage of synovial fluid T cells that are IL-2R positive is much higher (Lafton et al., 1989). Elevated concentrations of soluble IL-2R are present in

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serum and synovial effusions of patients with R A , and the levels of IL-2R correlate with disease activity (Keystone et al., 1988; Symons et al., 1988; Miossec et al., 1990a) (see Fig. 4). While it is assumed that activated T cells are the source of soluble IL-2R,

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Figure 4. Elevated soluble IL-2 receptors in rheumatoid arthritis. Levels of soluble IL-2R were measured in sera and synovial fluids (SF) from patients with RA and in sera from healthy controls. High levels were present in most rheumatoid samples. Evaluation of paired samples indicated that soluble IL-2R concentrations in synovial fluid were higher in serum suggesting local shedding of receptors (not shown). From Keystone et al. (1988).

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this has not been confirmed, and many other cells that express the IL-2R, like monocytes and B cells, are also potential sources. Osteoarthritis synovial effusions also contain low concentrations of soluble IL-2R. Because RA synovial fluids inhibit IL-2-mediated cell proliferation in vitro, it is only natural to assume that this activity is due to soluble IL-2R. This appears to be supported by the observation that IL-2 inhibitory activity correlates with the concentration of soluble IL-2R in synovial effusions (Symons et al., 1988; Miossec et al., 1990b). Column chromatography of synovial fluid reveals a fraction that putatively corresponds to the receptor/IL-2 complex (Symons et al., 1988). However, the short half-life of the intact complex makes copurification very difficult to confirm, and this fraction might actually contain a different inhibitor. This, along with the observation that soluble IL-2Rs are inefficient inhibitors of IL-2-mediated activation of T cells, suggest that the correlation between the inhibitory activity and sIL-2R levels might be coincidental. H I G H M O L E C U L A R W E I G H T IL-2 INHIBITORY ACTIVITY Although soluble IL-2Rs in synovial fluid do not appear to function as effective inhibitors of IL-2 biological activity, other factor(s) in RA SF do exhibit this property. While non-specific toxic effects (e.g. from hyaluronic acid) can potentially contribute to inhibition of IL-2-mediated cell proliferation, Miossec and colleagues have also demonstrated a high molecular weight inhibitor (Kashiwado et al., 1987). The gel purified factor, which is not an antibody, inhibits IL-2-mediated proliferation of mitogen-stimulated human peripheral blood T cells. GM-CSF

A variety of GM-CSF-mediated actions are likely important in RA, including the activation of granulocytes and induction of class II major histocompatibility antigens on macrophages (Alvaro-Gracia et al., 1989). Studies of GM-CSF inhibitors in RA are scant, although there is some evidence from bioassays that synovial fluid contains a factor(s) that inhibits GM-CSF induction of colony-formation by stem cells in the peripheral blood or bone marrow (Xu et al., 1989). While the nature of the inhibitory activity is unknown, recent studies describing a soluble GM-CSF receptor suggest that this is a possible candidate (Raines et al., 1991; Sasaki et al., 1992). This topic is reviewed in detail in Chapter 12, this volume. EXTRACELLULAR MATRIX AND CYTOKINE INTERACTIONS Originally thought to be an inert tissue framework for cells, the extracellular matrix is now known to play an important role in the regulation of cellular adhesion and activation. For instance, fibronectin stimulates synoviocyte metalloproteinase production by crosslinking surface fibronectin receptors (Werb et al., 1989). Within hours after the receptors are crosslinked, stromelysin and collagenase account for as much as 5% of proteins secreted by cultured synovial fibroblasts. The response is specific, since gelatinase and inhibitors of metalloproteinases like TIMP are not affected by fibronectin receptor engagement. The matrix can also indirectly participate in syno-

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vitis by activating cytokines like fibroblast growth factor (FGF). FGF function is greatly enhanced when it associates with matrix molecules containing negatively charged proteoglycans like heparan sulphate (Yayon et al., 1991). In contrast, some macromolecules inhibit cytokine activity by absorbing them from the extracellular milieu and preventing receptor engagement. The interaction of TGF[3 with decorin is an example of this phenomenon (Border et al., 1992). Decorin associates with type I collagen fibrils and contains large numbers of leucine residues. It inhibits TGF[3 biological activity by reversibly binding to the growth factor. The biological importance of this type of interaction has been demonstrated in animal models of glomerulonephritis where TGF[3 plays a pathogenic role. Administration of decorin in these circumstances significantly retards renal damage. Other cytokines, including GMCSF, can also bind to matrix macromolecules. It is unclear whether this process primarily serves to inhibit local cytokine function or acts as a reservoir to maintain a supply of growth factor that can be released later into the extracellular milieu long after the cell that produced it has migrated away or been down-regulated. ROLE OF CYTOKINE ANTAGONISTS IN SYNOVITIS The rheumatoid synovial membrane and synovial fluid are teeming with pro- and antiinflammatory factors. One can conclude a priori that the net balance in RA must be towards the former or else the disease would be self-limited. However, this balance is probably tenuous, and subtle shifts in cytokine levels could potentially have major effects that contribute to the waxing and waning nature of the disease. Local differences in the agonist:antagonist ratios might vary depending on the cellular composition of each compartment. For example, synovial tissue and synovial fluid neutrophil contents are very different, and this might contribute to regional variations in IL-lra concentrations. The synovium also exhibits a significant preponderance of CD4 § memory T cells while CD8 § T cells are more prevalent in synovial effusions (reviewed in Chapter 11, this volume). Compartmentalization is apparent within the synovial membrane itself, with regional variations in metalloproteinase and complement protein gene expression within lining and sublining regions (Firestein et al., 1991). Hence, the joint is an organized structure with each separate compartment demonstrating a distinct cell mix and cytokine milieu. Most current data suggest that cytokine antagonists are dominant in synovial fluid: TNFo~ biological activity is difficult or impossible to detect in effusions and other inhibitors like TGFI3 and IL-lra are abundant. The net result is an environment in which cell proliferation and activation are very much blunted. This might account for defective synovial fluid T-cell function, including decreased proliferation and cytokine production. The situation might be reversed in the synovium, which is probably a more relevant compartment for understanding the pathogenesis of RA. In this region, the balance between IL-1 and IL-lra appears to favour IL-1. Also, the sequelae of TNF-ot biological activity can be demonstrated in synovium despite persistent shedding of inhibitors like TNF-R. For instance, addition of anti-TNFa antibodies to synovial cell cultures inhibits production of IL-le~ and GM-CSF suggesting that biologically active TNFet plays a pivotal role (Haworth et al., 1991). The reasons for the differences between the tissue and fluid phase of the joint are not clear; perhaps cytokine antagonists are less effective (or agonists more effective) within the three-

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dimensional framework of the synovium where the extracellular matrix can present cytokines or their antagonists in a tightly controlled evironment. This contrasts with synovial effusion, where cytokine gradients and high levels in a microenvironment are inherently much more difficult to achieve due to the nature of a fluid compartment. Overall, despite abundant evidence of very effective cytokine antagonists in the joint, it is clear that amounts of the inhibitors are insufficient to counteract proinflammatory factors. Perhaps the inhibitors are not present in high enough concentrations, or perhaps they are not synthesized in the right location or at the correct time. The functions of cytokines in the initiation and perpetuation of RA is well established, and the possibility that the relative amounts of cytokine antagonists are too low is now being tested in clinical studies using pharmacologic doses of inhibitors like IL-lra or antibodies to TNFo~ (Chapter 2, this volume). However, we must be cautious not to overinterpret the results of such trials, since the comlexity and redundancy of the cytokine networks suggest that combinations of inhibitors will be needed. For instance, inhibition of IL-1 without addressing the effects of TNFc~ might have minimal benefit. Another issue that will ultimately have to be faced is the difficulty and expense of using parenteral recombinant proteins to treat chronic diseases like RA. A small molecule approach (i.e. organic molecules that inhibit cytokine production or action) will have significant advantages, although this might lack the exquisite specificity characteristic of antibody or recombinant protein therapy. REFERENCES Aivaro-Gracia JM, Zvaifler NJ & Firestein GS (1989) Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis. J Exp Med 170: 865-875. Alvaro-Gracia JM, Zvaifler NJ & Fircstein GS (1990) Cytokincs in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophagc colony-stimulating factor production by rheumatoid arthritis synoviocytes. J Clin Invest 86: 179(I-1798. Alvaro-Gracia JM, Yu C, Zvaifler NJ & Firestein GS (1993) Mutual antagonism between IFN-gamma and TNF-alpha on fibroblast-likc synoviocytes: paradoxical induction of IFN-gamma and TNF-alpha receptor expression. J Clin Immunol 13: 212-218. Arend WP (1991) lnterleukin 1 receptor antagonist. A new member of the interleukin 1 family. J Clin Invest 88: 1445-1451. Arend WP & Dayer J-M (1990) Cytokine and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum 33: 305-315. Arend WP, Joslin JG & Massoni RJ (1985) Effects of immune complexes on production by human monocytes of interleukin 1 or an interleukin 1 inhibitor. J Immunol 134: 3868-3875. Arend WP, Welgus HG, Thompson RC & Eisenberg SP (1990) Biological properties of recombinant human monocyte-derived interleukin 1 receptor antagonist. J Clin Invest 85: 1694-1697. Balavoine J-F, deRochemontiex B, Williamson K, Seckinger P, Cruchaud A & Dayer J-M (1986) Prostaglandin E2 and collagenase production by fibroblasts and synovial cells is regulated by urine-derived human interleukin 1 and inhibitor(s). J Clin Invest 78:1120-1124. Bigler CF, Norris DA, Weston WL & Arend WP (1992) Interleukin-I receptor antagonist production by human keratinocytes. J Invest Dermato198: 38-44. Border WA, Noble NA, Yamamoto T, Harper JR, Yamaguchi Y, Pierschbacher MD et al. (1992) Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease. Nature 360:361-364. Brandes ME, Allen JB, Ogawa Y & Wahl SM (1991) Transforming growth factor beta 1 suppresses acute and chronic arthritis in experimental animals. J Clin Invest 87:1108-1113. Brennan FM, Chantry D, Turner M, Foxwell B, Maini R & Feldmann M (1990) Detection of transforming growth factor-beta in rheumatoid arthritis synovial tissue: lack of effect on spontaneous cytokine production in joint cell cultures. Clin Exp lmmuno181: 278-285. Brennan FM, Gibbons DL, Mitchell T, Cope AP, Maini RN & Feldmann M (1992) Enhanced expression of

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Dripps DJ, Verderber E, Ng RK, Thompson RC & Eisenberg SP (1991b) Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils. J Biol Chem 266:20311-20315. Eisenberg SP, Evans R J, Arend WP, Verderber E, Brewer MT, Hannum CH et al. (1990) Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 343: 341-346. Elford PR, Graeber M, Ohtsu H, Aeberhard M, Legendre B, Wishart WL et al. (1992) Induction of swelling, synovial hyperplasia and cartilage proteoglycan loss upon intra-articular injection of transforming growth factor beta-2 in the rabbit. Cytokine 4: 232-238. Elias JA, Krol RC, Freundlich B & Sampson PM (1988) Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor and lymphotoxin. J Clin Invest 81: 325-333. Engelmann H, Aderka D, Rubinstein M, Rotman D & Wallach D (1989) A tumor necrosis factor-binding

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(1988) Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (IL-2 and IL-3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis. J Exp Med 168: 1573-1586. Firestein GS, Alvaro-Gracia JM & Maki R (1990) Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J Immunol 144: 3347-3353. Firestein GS, Paine MM & Littman BH (1991) Gene expression (collagenase, tissue inhibitor of metalloproteinases, complement, and HLA-DR) in rheumatoid arthritis and osteoarthritis synovium: quantitative analysis and effect of intraarticular corticosteroids. Arthritis Rheum 34: 1094-1105. Firestein GS, Berger AE, Tracey DE, Chosay JG, Chapman DL, Paine MM et al. (1992) IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium. J Immunol 149: 1054-1062. 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(1990) lnterleukin-1 receptor antagonist activity of a human interleukin-I inhibitor. Nature 343: 336-340. Harvey AK, Hrubey PS & Chandrasekhar S (1991) Transforming growth factor-beta inhibition of interleukin-1 activity involves down-regulation of interleukin-I receptors on chondrocytes. Exp Cell Res 195: 376-385. Haskill S, Martin G, Van Le L, Morris J, Peace A, Biegler CF et al. (1991) cDNA cloning of an intracellular form of the human interleukin 1 receptor antagonist associated with epithelium. Proc Natl Acad Sci USA 88: 3681-3685. Hasler F, Bluestein HG, Zvaifler NJ & Epstein LB (1983) Analysis of the defects responsible for the impaired regulation of EBV-induced B cell proliferation by rheumatoid arthritis lymphocytes. J l m m u n o l 131: 768-772. Hatakeyama M, Tsudo M, Minamoto S, Kono T, Doi T, Miyata T et al. (1989) Interleukin-2 receptor beta chain gene: generation of three receptor forms by cloned human alpha and beta chain cDNAs. Science 244:551-556. Haworth C, Brennan FM, Chantry D, Turner M, Maini RN & Feldmann M (1991) Expression of granulocyte-macrophage colony-stimulating factor in rheumatoid arthritis: regulation by tumor necrosis factoralpha. Eur J Immuno121: 2575-2579. Heilig B, Wermann M, Gallati H, Brockhaus M, Berke B, Egen O et al. (1992) Elevated TNF receptor plasma concentrations in patients with rheumatoid arthritis. Clin Invest 70: 22-27. Henderson B & Pettipher ER (1989) Arthritogenic actions of recombinant IL-1 and tumour necrosis factor alpha in the rabbit evidence for synergistic interactions between cytokines in vivo. Clin Exp Immuno175: 306-310. Henderson B, Thompson RC, Hardingham T & Lewthwaite J (1991) Inhibition of interleukin-l-induced synovitis and articular cartilage proteoglycan loss in the rabbit knee by recombinant human interleukin-1 receptor antagonist. Cytokine 3: 246-249. Hogquist KA, Nett MA, Unanue ER & Chaplin DD (1991) Interleukin 1 is processed and released during apoptosis. 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