Cytokines and Disease

Cytokines and Disease Marc Feldmann* and Fionula M. Brennan Cytokine and Cellular Immunology Division, Kennedy Institute of Rheumatology, Imperial College School of Medicine, 1 Aspenlea Road, Hammersmith, London, W6 8LH, UK * corresponding author tel: +44(0)208 383 4406, fax: +44(0)208 563 0399, e-mail: [email protected] DOI: 10.1006/rwcy.2000.01003.

INTRODUCTION Whereas the health of an individual, organ or cell requires the precise maintenance of the function of the product of the entire  105 genes in the human genome, pathology is potentially simpler. It could be due to changes in a very small number of molecules, initially possibly one. Subsequently there is usually consequential damage to various cells and tissues, so that other genes become abnormally expressed as a consequence, and it can be difficult to assess what the critical early events were. With the involvement of cytokines in essentially all biological processes, such as in cell growth, differentiation, inflammation, immunity, repair, and fibrosis (see the chapter on Introduction to the Role of Cytokines in Innate Host Defense and Adaptive Immunity), it is apparent that cytokines will be involved in a multitude of, and probably all pathological processes. Their role in these processes will be diverse, in some instances cytokines being pathogenic, in others protective and probably most of the time, innocent bystanders. This means that unraveling the role of cytokines in diseases is not as simple as some might wish. The detection of a cytokine, upregulated at the site of disease or damage is merely the first step in that difficult path and in itself does not imply pathogenic relevance. It is very difficult to generalize about the role of cytokines in disease, not only because of the diversity (over 100 known cytokines), but also since diseases are enormously heterogeneous. Nevertheless, this chapter will attempt to provide an introduction to this very complex, and rapidly growing, medically and economically important field.

How can we establish whether a cytokine is relevant to a disease process? There are multiple strategies for evaluating the roles of cytokines in disease. A key distinction to make concerns the role of a particular cytokine in animal models or in human diseases. The former is considerably easier to establish, as an overexpression of that cytokine in transgenic mice (e.g. Keffer et al., 1991), total abrogation in knockouts (Marino et al., 1997), and infusion of large amounts of neutralizing antibodies (Thorbecke et al., 1992) are all possible, admittedly with various degrees of time and effort. This is not the case in humans, of course. As the closeness of animal models to the corresponding human disease is highly variable, with only some aspects of disease pathogenesis of complex human diseases reflected faithfully in any of the animal models, extrapolation from animal models is potentially perilous. So what is `important' in an animal model only provides a working hypothesis for what is happening in the related human disease. With authentic human diseases, other research strategies also yield clues concerning the role of cytokines. These include genetic susceptibility associated with cytokine polymorphisms (e.g. Wilson et al., 1997), differential expression at sites of disease compared to uninvolved tissue (e.g. Feldmann et al., 1996), increased levels in the blood of cytokines (e.g. Charles et al., 1999) or their soluble receptors (Cope et al., 1992). In vitro models of local human diseases, which involve culturing the tissue from the diseased site, can be very useful (Buchan et al.,

36

Marc Feldmann and Fionula M. Brennan

1988a, 1988b) if they continue to produce the same spectrum of `pathological' molecules considered or known to be produced in vivo. These in vitro models can be used for therapeutic tests with antibodies or other reagents to block cytokines that may influence measurable indices of disease activity (Brennan et al., 1989a). However, the only definitive proof of the importance of a cytokine in the pathogenesis of a human disease is in vivo, in a clinical trial in the affected population, using a cytokine inhibitor if the cytokine is considered detrimental or the cytokine itself if it is considered beneficial. Only a limited number of such studies have been performed to date, but it is inevitable that many more will be executed in the near future (Wendling et al., 1993; Paty et al., 1993; Cooper et al., 1993; Peest et al., 1995; Bresnihan et al., 1996; Keystone et al., 1998; Maini et al., 1998; van den Bosch et al., 1998).

Cytokines involved in a disease may be present at augmented, normal or low levels There is now definitive evidence that excess TNF is involved in the pathogenesis of two chronic inflammatory diseases, rheumatoid arthritis (RA) and Crohn's disease (Elliott et al., 1993; Van Dullemen et al., 1995; Present et al., 1999). The critical evidence comes from double-blind, randomized, placebo-controlled trials and has led to FDA approval of TNF inhibitors in these diseases (an anti-TNF antibody Remicade, in Crohn's and RA, a TNFR fusion protein Enbrel in RA). For real certainty of the role of a cytokine in disease more than one such trial and more than one anticytokine agent would be useful and this has been accomplished in RA. The role of TNF in these chronic inflammatory diseases is discussed in more detail in the chapter on TNF. This level of proof is difficult, expensive and time consuming to acquire, but there is no alternative. Negative clinical trial results, however, are more difficult to interpret and do not necessarily invalidate the hypothesis. Abundant animal experimentation was consistent with the importance of TNF in sepsis syndrome (Beutler et al., 1985; Van Zee et al., 1992). However, these studies clearly demonstrated benefit only if a TNF inhibitor was used at, before, or very soon after the injection of LPS or bacteria. However, human sepsis syndrome occurs relatively late after the onset of bacterial infection, and despite thousands of patients treated with TNF inhibitors, there have been no successes (Wherry et al., 1993; Fisher et al., 1996). What does this mean? There are at least two

possibilities. One is that TNF is not important in the later stages of sepsis as it presents clinically, or that the clinical trial design involving hundreds of trial centers makes it very difficult to discern the possible therapeutic effect. Whereas TNF is pathogenic in RA, there is evidence that the opposite may be true in systemic lupus erythematosus (SLE). The clues come from animal models of disease. (NZB  NZW) F1 mice spontaneously develop, in an age- and sex-related fashion, IgM and then IgG autoantibodies to doublestranded DNA (dsDNA), and subsequently other manifestations of SLE, especially nephritis. In these mice, treatment with anti-TNF antibody makes them worse (Jacob and McDevitt, 1988) as does treatment with IL-10 (Ishida et al., 1994), which would also reduce TNF synthesis. Analysis of these mice indicated that they were genetically low producers of TNF. What about human systemic lupus erythematosus? The results are not formally proven. There is evidence that a subset with lupus nephritis has a genetically low production of TNF (Jacob et al., 1990). As part of the anti-TNF therapy clinical trials in rheumatoid arthritis, two patients developed both clinical and serological manifestations of SLE, one including lupus nephritis (Sander and Rau, 1997). Thus it is very likely that low TNF production is one of the pathogenic steps in human SLE.

Cytokine equilibria As discussed in the chapter on the Introduction to the Role of Cytokines in Innate Host Defense and Adaptive Immunity, cells in vivo are never exposed to a single cytokine. They are exposed to whole battery, simultaneously and sequentially, and at various concentrations. So cells must integrate the multiple signals they receive, either individually or collectively, and translate them into biological effects. To help understand this, we have formulated over the years a number of helpful oversimplifications, which can be expressed in diagrams (Figure 1 and Figure 2). It may seem at first glance paradoxical that in inflammatory diseases both the pro- and anti-inflammatory cytokines may be upregulated. However, close investigation has revealed this to be the case, and one of the most important concepts is that of the cytokine equilibrium, illustrated in Figure 1. This applies, for example, to the ratios of proinflammatory to antiinflammatory cytokine mediators, with the net effect depending not on any one cytokine, but on the totality of expression, the equilibrium. Of course in any one tissue there is a complex mosaic of many such

Cytokines and Disease Figure 1 Cytokine disequilibrium in rheumatoid arthritis. The cytokine profile at sites of inflammation such as rheumatoid synovial tissue is characterized by increased production of both pro- and anti-inflammatory molecules. Modified and reproduced with permission from Feldmann et al. (1996). TGF β MMP-3

IL-1 TNF

IL-10

sTNF-R

TIMP-1 TIMP-2

method increasing in popularity (because of its simplicity) is PCR for cytokine mRNA. This is a relatively poor tool for understanding the role of cytokines in disease, since cytokines are also abundantly expressed in health, and it is the protein, not the mRNA which signals to the cytokine receptors.

IL-1ra

Receptor expression

MMP-1

Anti-inflammatory Proinflammatory

Figure 2 Cytokine cascade in rheumatoid arthritis. Proinflammatory cytokines in rheumatoid arthritic synovium interact in a `network' or `cascade'. Modified and reproduced with permission from Feldmann et al. (1996). TIMP-1, TIMP-2 IL-10, IL-1ra, sTNF-R Anti-inflammatory Immune system

37

TNF

IL-1

Because of their action on very high-affinity receptors cytokines are potent signaling molecules. This means that the biological effect of cytokines also depends on the levels of the corresponding receptors expressed on their neighboring target cells. While the levels of cytokine receptors are far less variable than those of cytokines, and most cytokine receptors are constitutively expressed, levels of cytokine receptors are regulatable. The capacity to receive cytokine signals, especially for multichain receptor complexes, depends critically on receptor density, and so evaluating the role of cytokines in disease also involves an understanding of receptor expression. The variable expression of the IL-2 receptor  chain (CD25) is the best known example of variable cytokine receptor expression. Without CD25 the multifunction IL-2 receptor complex has a much lower affinity (Smith, 1988).

Proinflammatory IL-6, IL-8, GM-CSF etc. MMP-1, MMP-3

equilibria, in different spots; each cell has to summate the effects of signals on its cell surface, integrate them and then, if appropriate, produce secondary mediators, move, commit suicide, etc. Of much interest is the subdivision of CD4+ T lymphocytes into TH1 and TH2 cells. These cells are polarized into producing some but not all of the repertoire of cytokines that T cells can produce (Romagnani, 1991). TH1 cells, considered proinflammatory, produce IFN and IL-2; these are not produced by TH2 cells, which typically produce IL-4 and IL-5. TH2 cells are involved in allergic diseases. In the same way that it is the balance between proinflammatory/anti-inflammatory mediators that dictates the `outcome', rather than levels of either, so it is the balance between TH1 and TH2 and their products that dictates the effects. The concept of equilibrium emphasizes the need for accurate quantitation of cytokines in vitro, in animal models and in human pathology, if greater insight is to be gained. Poorly quantitative and indirect methods for measuring cytokine levels are of limited value. For example, one

Cytokine inhibitors may reflect cytokine expression Like all powerful entities, cytokines have their inhibitors. Cytokines function physiologically as local mediators and their inhibitors are involved in helping to localize their actions. As cytokine synthesis is sporadic, depending on local cell activation, but induced rapidly, it is consistent with the function of cytokine inhibitors that, unlike cytokines, they are present constitutively (e.g. Arend and Dayer, 1990), and are also inducible by the same type of stimuli as induce the corresponding cytokine, and perhaps also by the relevant cytokine itself. Hence the definition of a cytokine activity `profile' needs to also take into account the presence and quantities of the inhibitors. This can be done functionally by using bioassays that `automatically' take endogenous inhibitors into account (rather than the easy and hence popular ELISA assays). In some circumstances there are a variety of inhibitors, for example for IL-1, soluble type I and type II IL-1 receptor, and IL-1 receptor antagonist, and two soluble TNF receptors for TNF and LT.

38

Marc Feldmann and Fionula M. Brennan

The role of cytokines in a variety of biological, pathological processes and diseases While it is clearly not possible in an introductory chapter of reasonable size to discuss the role of cytokines in disease in any depth, we will attempt to illustrate the diversity of effects of cytokines in disease processes. In only a small number of these diseases is there sufficient information to definitely argue that cytokines play a key role in the disease process. This is only true so far for rheumatoid arthritis and Crohn's disease, where the blockade of TNF has reproducibly led to clear clinical benefit. However, these are unlikely to remain the only examples in this category. Because of the greater depth of knowledge in these diseases, we will start the more detailed discussion of cytokines in disease with these examples.

THE ROLE OF CYTOKINES IN INFLAMMATORY AND IMMUNE DISEASES The evident role of cytokines in immunity and inflammation has led to a widespread exploration of the role of cytokines in this group of diseases. Over the years the understanding of the cytokine network in these diseases has evolved dramatically. In the late 1980s and early 1990s it was widely believed that because a number of proinflammatory cytokines with closely overlapping properties (e.g. IL-1, TNF, GM-CSF) were expressed in inflammatory sites, blocking a single cytokine was unlikely to be clinically useful as the proinflammatory features would still be maintained by the remaining cytokines. Studies with antiTNF antibody in rheumatoid joint cell cultures first demonstrated that the expression of proinflammatory cytokines were coordinated (Brennan et al., 1989b), with TNF blockade leading to marked diminution of IL-1 bioactivity. These studies led to the concept of the cytokine cascade, as described in detail in the section on Rheumatoid arthritis. A wide spectrum of cytokines is involved in both the normal immune and inflammatory responses. Hence it is anticipated that in diseases of these systems, alterations in the levels of cytokines may occur, and could be important in the abnormal outcomes. There are clues from cytokine `knockouts' that lowered levels of certain mediators may be important in certain diseases. However, in the human diseases, it is much more likely that there is a reduced cytokine

level, rather than the abrogation of cytokine synthesis, which occurs with a targeted mutation. In this context, total lack of important immune mediators such as IL-2 and IL-10 lead to major immune and inflammatory disorders (Schorle et al., 1991; Kuhn et al., 1993). An excess of TNF in transgenic mice also produces a spectrum of pathology, with a destructive rheumatoid type of arthritis and a Crohn's like inflammatory bowel disease the most common manifestations (Keffer et al., 1991). In contrast, a lack of proinflammatory mediators, e.g. TNF, produces a much more subtle phenotype, discernible upon stressing the animals (Marino et al., 1997). There is a diverse spectrum of immune and inflammatory diseases, with differences in the phenotypes displayed. The most salient features are described below.

Rheumatoid arthritis The role of cytokines in the pathogenesis of rheumatoid arthritis (RA) has been the most extensively investigated of the human diseases since the mid-1980s (reviewed by Feldmann et al., 1996). RA is a chronic inflammatory disease that affects the peripheral synovial joints, resulting in inflammation and swelling, and eventually leading to destruction of the underlying connective tissue. The synovial membrane lining the joint capsule in RA becomes infiltrated with cells from the blood, mainly activated monocytes, T and B cells, while the tissue-resident cells, such as fibroblasts and endothelium, become activated and increase in number. The eventual destruction of the underlying cartilage and bone is now thought to result from the activities of these cells in the synovium and the cytokine and enzyme products that they release. In particular the `pannus' tissue, which migrates from the synovium into the cartilage, is thought to represent the `moving edge' of inflammation and, as such, is an important source of proinflammatory cytokines and other mediators. Cytokine Expression in RA Synovial Tissue Using a combination of immunohistological, mRNA studies, and protein assays, studies from our own group and others have documented that many cytokines are produced in RA synovium. These investigations have indicated the abundance of proinflammatory cytokines (Fontana et al., 1982; Buchan et al., 1988a, 1988b; Di Giovine et al., 1988; Eastgate et al., 1988; Hirano et al., 1988; Hopkins et al., 1988; Hopkins and Meager, 1988; Houssiau et al., 1988; Saxne et al., 1988; Brennan et al., 1989b;

Cytokines and Disease Chu et al., 1991; Deleuran et al., 1992; Wood et al., 1992), hematopoietic growth factors (Firestein et al., 1988; Xu et al., 1989; Alvaro-Garcia et al., 1989; Haworth et al., 1991), chemokines (Brennan et al., 1990a; Koch et al., 1991, 1992, 1993, 1994a, 1994b; Akahoshi et al., 1993; Hosaka et al., 1994), and fibrotic growth factors. These are summarized in Table 1 and reviewed by Feldmann et al. (1996). In the main, activated macrophages, fibroblasts, and endothelium produce the most abundant cytokines. In contrast there is a paucity of T cell-derived cytokines (Firestein et al., 1988; Saxne et al., 1988; Buchan et al., 1988a; Brennan et al., 1989a; Xu et al., 1989), leading to an ongoing debate regarding the importance of T cells in RA, especially in its late stages (Firestein and Zvaifler, 1990; Panayi et al., 1992). Recently, however, using more sensitive techniques, the presence of T cell-derived cytokines, IFN , IL-2, and lymphotoxin, which are characteristic of TH1 cells, has been described in RA tissue (Ulfgren et al., 1995a, 1995b; Steiner et al., 1999).

39

Table 1 Cytokine expression in rheumatoid arthritis Cytokine

Expression mRNA

Protein

IL-1, IL-1

+

+

TNF

+

+

LT

+



IL-6

+

+

GM-CSF

+

+

M-CSF

+

+

LIF

+

+

Oncostatin M

+

+

IL-2

+

+

IL-3

ÿ

ÿ

IL-7





IL-9





Proinflammatory

IL-12

+

+

Cytokine Networks in RA Synovial Tissue

IL-15

+

+

While the studies described above document the spectrum of cytokines produced in an inflammatory tissue such as that in RA, these observations do not indicate which are of major importance with respect to disease pathology, nor do they address the complex interactions between the cytokines. These issues have been addressed using an ex vivo system in which dissociated rheumatoid synovial tissue cells are cultured for short periods without exogenous stimulation, and the spontaneous production of cytokines detected in the presence or absence of cytokine antagonists (reviewed by Feldmann et al., 1996). Prolonged cytokine expression was detected, at both the mRNA level which was not due to increased half-life (Buchan et al., 1988b) and the protein level, mimicking what was presumed to occur in vivo. As it had been reported that TNF was a strong inducer of IL-1 production (Nawroth et al., 1986) we investigated the effect of neutralizing TNF on IL-1 synthesis. It was found that blockade of TNF but not TNF (LT) in these RA synovial cultures significantly inhibited IL-1 bioactivity (Brennan, 1989). As both IL-1 and TNF induce the resorption of cartilage and bone (Gowen et al., 1983; Saklatvala et al., 1985; Dayer et al., 1985; Thomas et al., 1987) this suggested that TNF blockade could have potent effects upon the disease process (Figure 2). Since there are many other possible inducers of IL-1, such as GM-CSF, IFN , and immune complexes, these results demonstrated the predominance of TNF, and led us to investigate whether other

IFN,

+

+

IFN

+

IL-17

+

+

IL-18

+

+

IL-4



ÿ

IL-10

+

+

IL-11

+

+

IL-13

+

+

TGF

+

+

IL-8

+

+

GRO

+

+

Immunoregulatory

Chemokines

MIP-1

+

+

MCP-1

+

+

ENA-78

+

+

RANTES

+

+

FGF

+

+

PDGF

+

+

VEGF

+

+

Mitogens

Marc Feldmann and Fionula M. Brennan

cytokines produced in the RA synovial cultures may also be TNF? dependent. It was found that GM-CSF, IL-6, and IL-8 (Haworth et al., 1991; Butler et al., 1995) were also downregulated by anti-TNF in these ex vivo cultures. In order to evaluate the role of IL-1 in the synovial cytokine network, IL-1 receptor antagonist was used, and it was found that IL-6, IL-8 were downregulated, but not TNF (Butler et al., 1995). These results clearly pointed to the possibility that blocking TNF may be useful therapeutically and it also highlighted the central or `pivotal' role of TNF in this inflammatory disease (Brennan et al., 1992). Most of the studies described above have been performed upon tissue from late-stage disease. Thus it is unclear whether or what cytokines are involved in promoting the earlier events, such as the TH1/proinflammatory immune response, which may involve IL-12 or IL-18, the most potent inducers of IFN . The role of IL-12 has been investigated in murine collagen-induced arthritis in DBA/1 mice. Thus, IL-12 coadministered with collagen type II boosts arthritis in these animals, and disease is ameliorated if IL-12 action is blocked with anti-IL-12 antibody (Malfait et al., 1998), which is particularly effective in conjunction with anti-TNF (Butler et al., 1999). Little is yet known about IL-18 expression in rheumatoid arthritis, but it is expected to be present at the early stages (Yamamura et al., 1997). Regulation of TNF in Rheumatoid Arthritis TNF production is a highly regulated process which includes involvement of both the 50 promoter and the 30 untranslated region of the gene (Beutler and Cerami, 1989). The 30 UTR contains an AU-rich motif, which regulates translation of the mRNA and tissue expression. Indeed replacement of this AU region in the human TNF gene expressed in transgenic mice results in the spontaneous development of arthritis (Keffer et al., 1991). In rheumatoid joints, the TNF is produced predominantly from CD68+ macrophage-like cells. The factor(s) that induce TNF synthesis in RA synovial joints have obviously been of much interest. Blockade of one likely candidate, IL-1, with the IL-1 receptor antagonist had no effect on TNF production (Butler et al., 1995). Others have suggested that the IL-2-like cytokine IL-15, which is found in RA synovium, may be of importance (McInnes et al., 1997). Thus, IL-15 was observed to activate T cells, which then induced TNF synthesis in monocytes in a cognate-dependent manner, and in vivo, arthritis disease in rodents was prevented with a soluble IL-15 receptor protein (McInnes et al., 1996; Ruchatz et al., 1998). However, it remains

unclear whether a gene such as IL-15, which contains multiple start codons, is translated efficiently into protein (Tagaya et al., 1996) in rheumatoid tissue. We have focused upon the hypothesis that TNF synthesis in RA tissue is T cell dependent, based upon the observation that removal of T cells from the monocytic/macrophage population markedly reduces the capacity of the latter to make TNF. Surprisingly, the few monocytic cells left in the T cellenriched fraction, together with the T cells, release more TNF than T cell-depleted synovial macrophages (Brennan, unpublished; Figure 3). Direct cell contact between monocytes and T cells provides for a potent stimulus for TNF and IL-1 production (Isler et al., 1993; Sebbag et al., 1997). The nature of the signals from T cells to macrophages is not well understood but has been studied. Using normal T cells/cell lines, several groups have investigated and proposed some possibilities, e.g. CD69 and LFA-1 (Isler et al., 1993; Manie et al., 1993), VLA4 (Laffon et al., 1991), (Alderson et al., 1993; Wagner et al., 1994; Kiener et al., 1995; Shu et al., 1995) as well as unidentified proteins in the range 25±35 kDa (Isler et al., 1993). We have observed additionally, that the manner in which the T cell is stimulated influences which cytokine are produced by the monocyte. Thus T cells activated with anti-CD3 antibodies mimicking an antigen signal induce abundant TNF and IL-10 synthesis in monocytes when co-cultured (Parry et al., 1997). In contrast, T cells activated for 8 days with a cocktail of cytokines (IL-2, TNF, and IL-6) when fixed and co-cultured with monocytes induce TNF but not IL-10 synthesis (Sebbag et al., 1997) (Figure 4). This raises the interesting possibility that chronic inflammation can be maintained as a consequence of proinflammatory cytokine production Figure 3 TNF production in RA synovial joint cell cultures is T cell dependent. Rheumatoid synovial cells were cultured either as a total population or without CD3+ T cells (removed by magnetic bead enrichment). TNF production was measured by ELISA. 750

TNFα (g/ml)

40

500

250

0

Total

T cell depleted

Day 2

Total

T cell depleted

Day 5

Cytokines and Disease Figure 4 T cell activation modulates the cytokine profile produced by monocytes following T cell monocyte cognate interaction. Macrophage

TNFα + IL-10

TNFα

Cytokine stimulus ‘bystander activation’

Antigen stimulus ‘specific activation’

Figure 5 Blockade of IL-11 activity enhances TNF production: synergy with IL-10. Rheumatoid arthritis synovial membrane cells (pooled data from five experiments) were cultured in triplicate, treated for 48 hours with neutralizing anti-IL-11 mAb (10 g/mL), neutralizing anti-IL10 mAb (10 g/mL) or both antibodies together, and supernatants tested for TNF by ELISA and illustrated as % level of TNF control. TNF production was significantly increased in cell cultures treated with anti-IL-11 mAb (2-fold), anti-IL-10 mAb (3-fold) or both antibodies (22-fold) compared with basal production, anti-IL-11 mAb-, and anti-IL-10 mAb-treated synovial membrane cell cultures, respectively. Modified and reproduced with permission from Hermann et al. (1998).

from macrophages, induced by `bystander' T cells, which themselves are activated by the cytokine products of the macrophage. In the absence of insufficient immunoregulation by cytokines such as IL-10, chronic inflammation ensues.

αIL-10 (10 µg/ml)

Anti-inflammatory Cytokines

αIL-11 (10 µg/ml)

The evaluation of cytokine expression in rheumatoid synovium revealed that some cytokines with chiefly anti-inflammatory properties were also resent in abundant quantities in rheumatoid synovium. These include TGF and IL-10 (Brennan et al., 1990a; Fava et al., 1991; Katsikis et al., 1994). In contrast, IL-4 production has not usually been identified, and its lack may be one of the factors accounting for the relative TH1 T cell preponderance in rheumatoid joints. There are conflicting data concerning IL-13 (Ulfgren et al., 1995b; Isomaki et al., 1996) and the expression and role of IFN/ is not well defined (Hopkins and Meager, 1988). The type I interferons have both pro- and antiinflammatory activities and it is not clear which, if either, predominates in RA. However, the recent evidence that IFN induces STAT4 phosphorylation in TH1 CD4+T cells (Rogge et al., 1997) in common with IL-12, may suggest that increased IFN in RA may predispose to TH1 rather than TH2 predominance. Our own interest has focused on the inhibitory cytokines, which are present in abundance in RA synovial tissue, and include TGF , IL-10, and IL-11. However, while TGF displays several anti-inflammatory effects, the addition to RA joint cell cultures had little effect on the spontaneous production of proinflammatory cytokines (Brennan et al., 1990a), suggesting that the quantities already present were probably at maximal effective concentration. In contrast, the addition of IL-10 diminished the production of IL-1 and TNF furthermore, blockade of endogenous IL-10 enhanced TNF levels by 3±4-fold (Katsikis et al., 1994). This suggests that IL-10 is an important immunoregulator of inflammation, and

41

αIL-11 + αIL-10

* ** * p <0.05 **p <0.01

**

Control

0

500

1000

1500

2000

2500

3000

% inhibition of TNFα control

that in RA synovial tissue the system is not in homeostasis. Recently, we investigated whether IL-11, a member of the IL-6 gene superfamily displayed anti-inflammatory effects similar to those of IL-10. It had been described that IL-11 inhibited LPS-induced TNF in mouse macrophages (Trepicchio et al., 1996). However, IL-11 alone in RA cultures had no effect on TNF synthesis, unless the soluble IL-11 receptor was added together with IL-11 (Hermann et al., 1998). Blockade of IL-11 enhanced TNF levels by 2-fold. However, if IL-10 and IL-11 were both blocked, TNF levels increased 22-fold (Figure 5). This suggests that both IL-10 and IL-11 are important endogenous immunoregulators in inflammatory tissue. IL-11 also inhibited the release of MMP-1 and MMP-3 whilst increasing TIMP levels in these cultures, suggesting that there is a direct action of IL-11 on fibroblastic cells. Targeting TNF in Disease In Vivo The definition that TNF was a therapeutic target, based on the in vitro studies described above, was confirmed with anti-TNF antibody in animal models of arthritis (Williams et al., 1992, 1995; Piguet et al., 1992; Thorbecke et al., 1992), and helped provide the rationale for anti-TNF antibody

Marc Feldmann and Fionula M. Brennan

therapy in patients with rheumatoid arthritis (Elliott et al., 1993, 1994). The clinical trials of anti-TNF antibody, using cA2, a high-affinity neutralizing chimeric antibody produced by Centocor, Inc., began in May 1992, and were successful in rapidly diminishing many aspects of disease activity, including symptoms (e.g. pain, joint stiffness), signs (joint swelling and tenderness) as well as biochemical markers of disease (e.g. C-reactive protein, CRP, and erythrocyte sedimentation rate, ESR). It was striking that this happened even in longstanding active patients who were resistant to existing therapy (Elliott et al., 1993). These results were confirmed in a randomized double-blind, placebo-controlled trial of cA2 (Elliott et al., 1994), and by other groups with different anti-TNF antibodies (Rankin et al., 1994; Salfield et al., 1998), and more recently with the p75 TNF receptor fusion protein Enbrel (Moreland et al., 1997). The cA2 trial demonstrated that in addition to a rapid fall in the acute phase response, serum levels of IL-6 were reduced within the first 24 hours of treatment, and reached normal levels in the majority of patients (Charles et al., 1999). Not all cytokines are detectable in serum samples, so it is not yet known if all the cytokines shown to be TNF dependent in the rheumatoid joint tissue ex vivo are in fact reduced in vivo. So far it is clear that IL-1, IL-6, IL-8, MCP-1, and VEGF are diminished by anti-TNF therapy in vivo. Furthermore, the trial demonstrated that endothelium was deactivated following anti-TNF therapy, with a marked reduction in circulating soluble E-selectin and ICAM-1, reduced trafficking of leukocytes to the inflamed synovium, associated with reduced expression of adhesion molecules on the blood vessels in synovium. One of the possible limitations of the effectiveness of anti-TNF therapy given as a short course may be the observation that cytokine inhibitors are also reduced after this therapy. Thus, serum IL-1Ra levels are diminished by about 50% and enter the normal range (Figure 6). The rate of change is rapid, just as rapid as changes in acute phase proteins, and it has been reported recently that IL-1Ra is produced in the liver by hepatocytes (Gabay et al., 1997) just like other acute phase proteins. With respect to soluble TNF receptors (sTNFR), there is also a rapid diminution in serum levels, which lasts for many weeks. The effect is statistically significant for p75 sTNFR, but not for the less abundant p55 sTNFR (Charles et al., 1999). There are no data available yet concerning other cytokine inhibitors, e.g. soluble IL-1 receptor. In contrast there is an abstract indicating that IL-10 levels may be increased (Ohshima et al., 1996).

Figure 6 Effect of cA2 on circulating IL-1Ra. Rheumatoid arthritis patients were treated with either placebo (circles); 1 mg/kg cA2 (triangles); or 10 mg/kg cA2 (filled squares). *p < 0.05, **p < 0.01, ***p < 0.001 compared with placebo. Reproduced with permission from Charles et al. (1999). 50 IL-1ra (change from day 0; pg/ml)

42

0

Placebo

– 50 –100 –150

* ***

***

**

– 200

***

– 250 01

3

10 mg/kg cA2 * p <0.05 **p <0.01 ***p <0.001

* 7

1 mg/kg cA2

14

28

Day

Blockade of Other Cytokines in RA In view of the many actions of TNF mediated by IL-1, and the synergy between these two cytokines, it would be predicted that IL-1 blockade might be just as effective as that of TNF. In murine collageninduced arthritis (CIA) that possibility has been tested and is the case, provided that therapy is performed when these cytokines are present (Wooley et al., 1993). If therapy is delayed until TNF is no longer expressed in mouse joints, then it is not surprising that it is not effective (Joosten et al., 1996). IL-1 blockade in human RA has involved the use of subcutaneous IL-1Ra, which is cleared rapidly, and the doses used probably do not block IL-1 receptors efficiently. This may account for the marginal results reported so far, with little reduction in inflammatory symptoms and signs (Campion et al., 1996; Bresnihan et al., 1996). IL-6 blockade in RA has been reported to be effective for 2±3 weeks, using a murine anti-IL-6 antibody (Wendling et al., 1993; Wijdenes et al., 1994). Professor T. Kishimoto has reported at meetings that anti-IL-6 receptor antibody is also effective in RA. Both therapies would block IL-6 signaling. TNF blockade diminishes the production of natural cytokine inhibitors such as IL-1Ra and soluble TNFR (Charles et al., 1999). This may be an important part of why TNF blockade alone does not, in the late-stage RA patients used for clinical trials, lead to a cure, as might have been considered possible on the basis of the equilibrium concept (Figure 1). One consequence of the anti-TNF antibody-induced reduction of natural cytokine inhibitors is that it would make sense to consider restoring them. The one that

Cytokines and Disease drops the most is IL-1Ra, which is currently in clinical trials, with marginal efficacy if used alone (Bresnihan et al., 1998). It may be more useful to use it in combination with anti-TNF antibody. Similarly, IL-10 may be more useful in conjunction with anti-TNF, as a maintenance therapy. Combination therapies of multiple types are very effective in collagen type II-induced arthritis (reviewed by Williams et al., 1998). The reduction in serum cytokines provides a useful laboratory test of whether therapy is effective, and may provide a useful surrogate marker for clinical efficacy. Serum IL-6 changes after anti-TNF antibody therapy are rapid, and reach normal levels. This assay could be a useful tool for ascertaining whether other antirheumatic therapies act by influencing the cytokine system. Whether the IL-6 assay is better than the conventional assays of CRP and ESR used by rheumatologists remains to be ascertained, especially as CRP and ESR are chiefly regulated by IL-6 (Andus et al., 1988; Geiger et al., 1988; Morrone et al., 1988) ± CRP directly, and ESR by IL-6-induced elevations of fibrinogen.

Osteoarthritis (OA) Osteoarthritis is the commonest joint disease and, unlike rheumatoid arthritis, its pathogenesis is poorly understood. It is not clear which type of pathology a `degenerative' disease of this type belongs to, and so there is no clear reference framework, as compared to autoimmunity or cancer. It is considered to be a heterogeneous disease, with a variety of predisposing risk factors, including genetic predisposition, obesity, and trauma (Cooper, 1994; Bullough, 1994). It is believed that there are a number of stages in the pathogenesis of human OA, which are recapitulated in the STR/ort mouse which develops pathology with similar histology (Chambers et al., 1997). First there is a cartilage-specific stage, in which matrix turnover is increased and chondrocytes divide, then a stage with hypertrophy of bone with the formation of osteophytes at the edge of joints and last of all, a stage with marked synovial membrane enlargement, leukocyte infiltration, inflammation, cytokine production and joint destruction, often leading to joint replacement surgery. It is only from this last stage that there is considerable information about cytokine expression, as it is the time that joint samples are obtained at surgery. The cytokine profile in late OA is typical of what would be anticipated from an activated cell population composed chiefly of macrophages, fibroblasts, and endothelial cells, with considerably less T cells

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than in RA (reviewed by Westacott and Sharif, 1996). Cytokines detectable include TNF, IL-1, IL-6, IL-8, LIF, and oncostatin M (Smith et al., 1997). These have been detected in synovial fluid, and in the supernatant of osteoarthritis synovial cell cultures. Inhibitors of cytokine action such as IL-1Ra, soluble TNFR, and TGF are also present in appreciable quantities in synovial fluid (SF) and synovial membrane (SM) cultures. Anabolic cytokines involved in maintaining cartilage function, such as TGF , FGF, and IGF1, are also found (e.g. Brennan et al., 1990a; Martel-Pelletier et al., 1990; Denko et al., 1990; Westacott and Sharif, 1996). Which angiogenic cytokines or chemokines are of major importance in the angiogenesis and cell recruitment that take place is currently not clear, although a role for macrophage inflammatory protein has been proposed by Koch et al. (1995).

Cytokine expression in Crohn's disease While there is strong evidence for a genetic association in Crohn's disease, the important genes have not yet been identified. All the classic autoimmune diseases have associations with certain HLA antigens. For example, rheumatoid arthritis is associated with HLA-DR4 and DR1, insulin-dependent diabetes mellitus with HLA-DR3 and DR4 and more closely with HLA-DQ8. Systemic lupus erythematosus (SLE) is associated with HLA B2 DR3, but in fact the strongest association within the HLA gene cluster is with the complement component C4. However, in Crohn's disease there has been much controversy, and reported associations are variable. Sometimes the disease is associated with DR1 or D95, but in more recent studies it is not associated with HLA at all, even within afflicted families (Satsangi et al., 1996). This suggests that the immunological aspects of Crohn's will differ from those of other diseases considered autoimmune. There has been a lot of interest in the relevance of mycobacteria and other bacteria to Crohn's disease, but there is no convincing link. At the cytokine level, the abnormalities in Crohn's appear to be closely related to those of RA. The strongest evidence for this is that both diseases have responded very well to therapy with an anti-TNF antibody (Remicade) (Van Dullemen et al., 1995; Targan et al., 1997; Present et al., 1999). Consistent improvement is seen in the majority of patients, lasting 8 or more weeks from a single infusion. Biopsies of affected sites via an endoscope have added

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considerably to our knowledge of the pathogenesis of Crohn's disease. There is a considerable infiltrate in the lamina propia of T cells and macrophages. There is evidence that these cells are activated, as judged by their surface markers and the production of cytokines. Cytokines of the proinflammatory TH1 type, such as IFN and IL-2, have been detected, whereas those of the proallergic TH2 type, such as IL-4 and IL-5, are difficult to find. There is evidence that the antigen-presenting function is upregulated; for example, high levels of HLA class II, extending to epithelium have been observed. This is likely to be a reflection of bioactive levels of IFN in the tissues. Macrophage proinflammatory cytokines, such as TNF, IL-1, IL-6, are also found (Radford-Smith and Jewell, 1996; Fiocchi, 1998). Anti-inflammatory mediators such as IL-10 are present. IL-4 levels are reduced compared to those in normal gut or associated with ulcerative colitis. TGF is present at elevated levels (Beck and Podolsky, 1999). The signals that drive the presence of these cytokines are not known. In animal models the Ox40 pathway has been implicated in therapeutic studies (Higgins et al., 1999). Chemokines expressed include IL-8, GRO, MCP-1, and more work needs to be performed to define the others. The relatively scarce results obtained with human tissue have been corroborated in a variety of animal models that resemble Crohn's disease, such as IL-10 knockouts, or the transfer of CD4+CD45RB+ T cells.

Ulcerative colitis (UC) The etiology and pathogenesis of ulcerative colitis is, like that of most diseases, incompletely understood. There are genetic associations (Fiocchi, 1998), including TNF 308 allele 2. Antibodies to neutrophils (pANCA) are very prevalent. Unlike Crohn's disease, where the inflammation is transmural, in UC the inflammation is more superficial. The cytokine profile has been studied. Chemokines such as MCP-1, IL-8, RANTES, ENA-28, and proinflammatory cytokines such as IL-1, TNF, IL-6, IL-15 have been described (Radford-Smith and Jewell, 1996; Fiocchi, 1998). The T cell cytokine profile is varied, but there is not the TH1 preponderance seen in Crohn's disease. Thus lamina propria T cells do not make large amounts of IFN . TH2 cytokines such as IL-5 are present, but IL-4 is not elevated compared to levels in normal gut; IL-10 is present. Unlike Crohn's disease, the response to anti-TNF antibody was not dramatic, emphasizing that at the cytokine level, UC is different from Crohn's disease.

Atherosclerosis Current concepts in this disease are changing rapidly. While it is still clear that abnormalities in lipid metabolism are of importance, it is now acknowledged that this does not explain all the phenomenology. Several studies, initially that of Russell Ross, have highlighted that atherosclerosis has the features of an inflammatory disease (Ross, 1989, 1999). More recently, autoimmunity and infection have been proposed (e.g. Wick et al., 1997; Danesh et al., 1997, Caligiuri et al., 1999). The cellular composition is analogous to that in other inflammatory diseases, with macrophages (often lipid laden, hence called `foam cells') and smooth muscle being the most abundant cells, with T lymphocytes and even some mast cells present. Early lesions termed `fatty streaks' only contain macrophages and T cells (Hansson and Libby, 1996). Adhesion molecules, such as ICAM-1 and VCAM-1 are also upregulated. The expression of cytokines in atherosclerotic plaques resembles that of other inflammatory sites. Cytokines expressed in the local lesions, and hence believed to be important in the pathogenesis, are TNF, MCP-1, IL-1, GM-CSF, IL-8, VEGF, MCP-4, BMP-6, CD40/CD40L, IFN , IL-12, and IL-6 (Libby and Galis, 1995; Libby et al., 1995; Raines and Ross, 1996). This profile of cytokines is very similar to that seen at other chronic inflammatory sites, such as in RA, and it is of interest that there is accelerated onset of atherosclerosis in RA patients. PDGF is also expressed in atherosclerotic plaques, and has been implicated in the migration of smooth muscle cells into the plaques. Expression of some of the receptors for the proinflammatory cytokines have been noted, such as TNFR, and presumably the others are also present, as would be expected on activated cells. Also present in atherosclerotic plaques are some cytokines that are believed to be protective. These include IL-10, which reduces the production of a whole gamut of proinflammatory cytokines, including most of those listed above. TGF has been reported widely to be present, and because it has deactivating effects on endothelium, macrophages, and smooth muscle, and it is abundant in sites of the arterial tree most prone to atherosclerosis, it is probable protective (Borkowski et al., 1995). Confirmatory evidence of the protective role of TGF comes from findings of genomic instability (deletions) in replication-prone microsatellites in the type II TGF receptor with consequent loss of function in cells from plaques but not in surrounding tissue (McCaffrey et al., 1997). While the pathogenesis of atherosclerosis is becoming clearer, the current state of knowledge

Cytokines and Disease does not yet point to clear-cut therapeutic cytokine targets. Adhesion molecules and chemokines are likely to be of major importance in the initial entry of monocytes and T lymphocytes into the sclerotic plaques. Which chemokines are of major importance is not known, but MCP-1, MCP-4, and RANTES are all present and hence are among the candidate chemokines involved in the early stages. MCP-1 is upregulated by physical stresses and strain, and so could be induced by physical means early in the atherosclerotic process (Wang et al., 1995). What induces the upregulation of adhesion molecules is not clear: lipids may influence this process as may the proinflammatory cytokines such as TNF and IL-1 present in these lesions. There is evidence for the recognition of oxidized low-density lipoproteins (OXLDL) by T cells in atherosclerotic plaques (Stemme, 1995). Of major interest is the recent funding of upregulated CD40L expression in endothelium, monocytes, smooth muscle, making CD40L/CD40 signaling of major relevance in the atherosclerotic plaque (Mach et al., 1997). There is evidence that blocking this pathway reduces lesions in apolipoprotein E-deficient mice (Mach et al., 1998). The most dangerous event in the life history of an atherosclerotic plaque is plaque rupture. This leads to `acute coronary syndromes', namely unstable angina or acute myocardial infarction. Plaque rupture is probably caused by the action of the matrix metalloproteinase enzymes produced by macrophages, smooth muscle, endothelium, etc., and this process can be regulated by proinflammatory cytokines, including TNF, IL-1, CD40 ligand. Plaque rupture leads to platelet accumulation and aggregation can rapidly lead to thrombosis. Understanding the cytokine control of the enzymes involved in plaque rupture, and in the upregulation of tissue factor which is a potent initiator of thrombosis could be very important, as it should lead to new therapeutic targets.

Cytokine expression in allergic disease (asthma, allergic rhinitis) It is now widely accepted that asthma in its varied forms is an inflammatory disorder of the airways in which mediators released from activated mast cells and eosinophils plays a major role (reviewed by Howarth et al., 1994; Barnes et al., 1998; Lee, 1998). Histologically there are epithelial lesions, thickening of basement membrane, and inflammatory infiltration consisting of activated eosinophils, TH2 lymphocytes

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and degranulated mast cells. Eosinophils are believed to be the prime proinflammatory effector cell causing bronchial damage, although it is considered that the TH2 cells also play a central role, both through specific allergen activation, and by releasing IL-4 (Moller et al., 1996b) and IL-5 (Shi et al., 1997), factors that regulate IgE production and eosinophil function. Such eosinophils primed by IL-3, IL-5, or GM-CSF can then bind to microvasculature, and be selectively chemoattracted by chemokines, in particular the chemokines. The amplification and maintenance of airway inflammation and the clinical exacerbations and chronicity of bronchial asthma depend on complex mechanisms in which TNF and IL-1 play fundamental roles (Sousa et al., 1996), such as the upregulation of vascular adhesion molecules involved in the recruitment of eosinophils and other inflammatory cells from the circulation. The importance of CXC chemokines, which attract monocytes and T cells, and CC chemokines (Gonzalo et al., 1998), which attract eosinophils and other cells, as local chemoattractants and activating stimuli is also recognized (Humbert and Ying, 1997). Thus in contrast to other chronic inflammatory diseases, such as rheumatoid arthritis, where T cell responses to autoantigens have been hard to identify, a large number of putative allergens have been proposed in asthma. Secondly, unlike RA, which is characterized by a predominantly TH1 infiltrate into the inflamed tissue, in asthma TH2 cells are predominant, with concomitant production of IL-4 and IL-5. Many attempts have been made to use information about the cytokine environment for therapeutic means and one potential target is IL-5, a cytokine important in eosinophil growth and recruitment. Antibodies to IL-5 are in clinical trials. The major receptor for chemokines eosinophils is CCR3, the receptor for eotaxin, MCP-3, MCP-4, and RANTES. Attempts to block this receptor with antibodies and small molecules are in progress. Allergic rhinitis (reviewed by Bachert et al., 1998) is thought to occur as a response to artificial or natural allergen exposure, which results in an increase in the number of eosinophils and basophils, mast cells, IgEpositive cells, macrophages, monocyte-like cells, Langerhans cells, and activated T cells which can be observed within the mucosa, and on the mucosal surface of the nose and sinuses. After antigen challenge, the release of proinflammatory and regulatory cytokines can be demonstrated, and TH2-type cytokine mRNA upregulation in allergic mucosa has been shown. As with asthma, proinflammatory cytokines initiate an adhesion cascade and activated T cells that create an `atopic' cytokine environment within the tissue, which also may be linked to the long-term

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selective recruitment of eosinophils. However, the acute selective migration of eosinophils after allergen challenge is not fully understood, nor is the role of chemokines in allergic and viral rhinitis.

Cytokine expression in other inflammatory lung diseases (sarcoid and pulmonary lung disease) Sarcoid and idiopathic pulmonary lung disease are inflammatory diseases of the lung in which cytokines are also considered to play a central role. However, unlike asthma, the T cell component is of TH1 cells, and not TH2 cells, resulting in a more inflammatory condition which develops into granulomatous tissue. The production of chemokines such as IL-8 from alveolar macrophages is thought to be of importance for the neutrophil migration into the airspaces of the lung in idiopathic pulmonary fibrosis (IPF) and the subsequent alveolar damage and fibrosis (Carre et al., 1991; Ziegenhagen et al., 1998). The recruitment of monocytes is characteristic of both IPF and sarcoidosis and the chemokine MIP-1 is found elevated in the bronchoalveolar lavage (BAL) fluid of patients with sarcoidosis and IPF (Standiford et al., 1993; Smith et al., 1995). The source of these chemokines is alveolar macrophages, but also fibroblasts within the interstitium. Particularly characteristic of these lung diseases is the formation of granulomatous tissue, and the fibrotic cytokines TGF and TNF have been implicated in this process (Kapanci et al., 1995). Sarcoid is further characterized by the large infiltration and expansion of activated oligoclonal CD4+ T cells and macrophages at sites of disease. Recently, the T cell chemokine and growth factor IL15 has been suggested to be of importance in the growth of these T cells (Agostini et al., 1996) which produce cytokines characteristic of TH1 cells, with elevated mRNA and protein levels of IFN , but not IL-4. Furthermore, expression of IL-12, a growth factor for TH1 cells, is increased, and is found in BAL fluid (Moller et al., 1996a).

Fibrosis The excess accumulation of collagen in a tissue is termed fibrosis. This is the end result of a complex series of events, and is a form of tissue repair. In inappropriate places or in excess, fibrosis leads to various types of pathology. Thus, in the eye, fibrosis

of the cornea or other parts of the eye leads to impaired vision, in the lung it leads to changes in the mechanical properties and gas/vascular exchange, in the liver occurring after liver damage it leads to reduced liver function and increased blood pressure in the local vessels, predisposing to their rupture and massive bleeding. A wide spectrum of pathological events can end up as fibrosis. In many instances, there is a preceding inflammatory response, which fails to resolve, becomes chronic and leads to the fibrotic state. Type I collagen is the main component of fibrous tissue, this is synthesized by fibroblasts and so cytokines regulating fibroblast growth and synthesis are of major importance in regulating fibrosis. One of the key problems in this field is that many cytokines regulate fibrosis, at various stages. The cytokines regulating fibroblast growth include proinflammatory cytokines such as IL-10, TNF, plateletderived growth factor (PDGF), many members of the fibroblast growth factor (FGF) family, e.g. basic FGF, acid FGF, KGF, and insulin-like growth factors, IGF-1 and IGF-2. Regulation of synthesis of matrix is influenced by all of the above, but TGF is considered the strongest signal for collagen synthesis (reviewed by Border and Noble, 1994). Which members of the TGF family are most important depends on the site: TGF 2 is the most abundant in the CNS and eye, and TGF 1 in the rest of the body. The progressive fibrosis of the liver, lung, kidney, heart, skin, eye, and bone marrow can result in a wide variety of pathological problems, leading to a high morbidity and mortality. There is an increasing understanding of the molecules involved in driving fibrosis, and so it is likely that therapeutic progress will be made. There is extensive literature in both animal models and human disease that TGF 1 is involved in many fibrotic events. In the human studies, expression in the local tissues is the criterion for suspecting involvement. In one instance, a therapeutic trial of TGF used for its anti-inflammatory action has initiated kidney damage, tending to reinforce that suspicion. To date, therapeutic trials of anti-TGF 1 have not been performed in humans. Trials of anti-TGF 2 are in progress for the prevention of fibrosis after eye surgery. In view of the inflammatory state preceding fibrosis, there is evidence that interfering with inflammatory mediators can reduce fibrosis. For example in the bleomycin-induced fibrosis (of lung) model there is evidence that blocking TNF (Piguet et al., 1987) or MIP-1 (Smith et al., 1995) or anti-TGF 1 can ameliorate the fibrosis. Other important mediators present in lung fibrosis include MCP-1, IL-8, and RANTES (Martinet et al., 1996).

Cytokines and Disease

CONCLUSION There is abundant evidence that cytokines are involved in a wide spectrum of diseases. There is now also evidence that targeting certain cytokines has led to clinical benefit, and so cytokines can be good therapeutic targets. We expect that this field will expand enormously in the next few years. The ratelimiting step in developing anticytokine therapeutics still remains a limited understanding of the cytokine network and of the molecules that are rate limiting. However, the success of anti-TNF therapy with Remicade and Embrel has led to new entrants in the field and a recognition of the importance of cytokines in health and disease.

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