Autoimmunity and cytokines: pathogenesis and therapy

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52 Auto immunity and cytokines: pathogenesis and therapy Evangelos Th. Andreakos and Marc Feldmann Imperial College of Science, Technology and Medicine London, UK

Do not blame anybody for your mistakes and failures. Bernard M. Baruch

INTRODUCTION Although most self-reactive lymphocytes are eliminated by clonal deletion during their development in the thymus or in the periphery, some persist and can be detected in normal healthy individuals by various assays. Mechanisms of clonal anergy or clonal suppression operate to ensure that these cells do not usually attack self-tissues. However, in a number of common human diseases these mechanisms have been perturbed, resulting in the activation of selfreactive T and B cells and the generation of cellmediated and humoral immune responses against self-antigens. The response of the immune system against self-tissue is termed 'autoimmunity' and, when sustained, results in serious damage of organs that can sometimes be fatal. Autoimmune diseases affect 5-7% of the population, and the most common of them include rheumatoid arthritis (RA), systemic lupus erythematosus, Sjogren's syndrome,

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T. Lotze ISBN 0-12-689663-1, London

insulin-dependent diabetes mellitus, and Graves' and Hashimoto's thyroiditis. It is not known what combination of events triggers autoimmunity, but there is strong evidence for both genetic and environmental factors. Studies in autoimmune individuals and monozygotic twins have linked the susceptibility to autoimmune disease with the MHC genotype and particularly with the HLA class II antigens (Table 52.1). Thus, more than 80% of RA patients have the HLA-DR4 or HLA-DR1 genotype (Wordsworth and Bell, 1991) and more than 90% of insulin-dependent diabetics the HLA-DR3 or HLADR4 genotype (Bell et al., 1987). Other diseases such as ankylosing spondylitis are associated with HLA class I antigens (Nasution et al., 1997). The classical role of MHC molecules is in peptide binding and presentation to T cells (Townsend et al., 1989). Thus, MHC molecules may affect disease susceptibility by first 'shaping' the repertoire of naive T cells in the thymus, and then presenting antigenic

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TABLE 52.1 Susceptibility to autoimmunity is associated with the HLA genotype and the sex (adapted with permission from ]aneway and Travers, 1997) Disease

Prevalent HLA alleles

Relative risk

Sex ratio (female:male)

Ankylosing spondylitis Acute anterior uveitis Goodpasture's syndrome Graves' disease Hashimoto's thyroiditis Insulin-dependent diabetes mellitus Juvenile rheumatoid arthritis Multiple sclerosis Myasthenia gravis Pemphigus vulgaris Pernicious anemia Psoriatic arthritis Reiter's syndrome Rheumatoid arthritis Sjogren's syndrome Systemic lupus erythematosus Ulcerative colitis

B27 B27 DR2 DR3 DR5 DR4/DR3 and DR3/DQW8 B27/DR5 DR2 DR3 DR4 DR5 B7, B27 B27 Dw4/DR4 Dw3 DR3 B5

87.4 10.0 15.9 3.7 3.2 3.2 4 4.8 2.5 14.4 5.0 11.0 37.0 4.2 6 5.8 4.0

0.3 <0.5

determinants of proteins to the same T cells in the periphery to initiate antigen-specific immune responses. Association of disease susceptibility with MHC loci, however, is not necessarily due to inappropriate antigen presentation. In addition to MHC molecules, MHC loci encode a wide variety of other proteins such as peptide transporters (Bahram et al., 1991), proteasome enzymes (Kelly A. et al., 1991), hsp70 and tumor necrosis factors (LT-a, LT-~. TNF-a). These proteins are also involved in the initiation of antigen-specific immune responses and can influence disease susceptibility in an MHC moleculeindependent fashion. It should be stressed, however that MHC genotypes alone do not determine whether individuals will develop autoimmunity, as identical twins that share all their genes are more likely to develop disease than MHC-identical siblings. Genetic factors other than MHC are also essential for susceptibility as, for example, systemic lupus erythematosus that is strongly associated with inherited homozygous deficiency of the early proteins (C1, C4 or C2) of the classical pathway of complement (Kolble and Reid, 1993). Moreover, the hormonal status of the individual does also affect disease susceptibility as many autoimmune diseases show a strong sex bias (Figure 52.1). More than 70% of the patients of Sjogren's syndrome, SLE, autoimmune thyroid disease, rheumatoid arthritis, multiple sclerosis and myasthenia gravis are female,

4-5 4-5 -~1 2 3 3

1

3 19 9 -~1

implying that sex hormones are involved in disease pathogenesis (Whitacre, 2001). Sex hormones such as estrogen, progesterone and androgens are known to act directly to cells of the immune system, among their multiple functions, modulating antigen presentation, lymphocyte activation, cytokine production and/or cell homing. How exactly, however, these hormones influence the prevalence of autoimmune disease remains unclear. Females do develop stronger immune responses (Verthelyi, 2001) and so autoimmunity may be the price. Finally, the observation that autoimmune disease manifests in less than 50% of identical twins that share all their genes (e.g. the concordance rates of identical twins in rheumatoid arthritis is 15-35%) demonstrates that non-inherited factors are clearly of importance (Silman et al., 1993). Whether these are inherited agents or random events such as exposure to particular environmental conditions or pathogens is not known.

AUTOIMMUNE DISEASES CAUSE TISSUE DAMAGE INITIATED BY ANTIBODY OR T CELLS By definition, autoimmune diseases are due to sustained adaptive immune responses to self-antigens. As antigen is an intrinsic component of self-tissue,

C L I N I C A L A P P L I C A T I O N OF CYTOKINES A N D CYTOKINE I N H I B I T I O N

AUTOIMMUNE DISEASES CAUSE TISSUE DAMAGE INITIATED BY ANTIBODY OR T CELLS Genetic predisposition Hormonal status Environmental factors

Initiation

1 19 1

= Antigen presentation T cell activation

CYTOKINES

Perpetuation

Autoantigen reactive T cells

.,,

9

Antigen-presenting cells

CYTOKINES

Disease

1 EFFECTOR MECHANISMS

FIGURE 52.1 The development of autoimmunity consists of three stages, the initiation, the perpetuation and the disease stage.

autoimmunity results in extensive tissue damage. The nature of tissue damage, the pathology and the clinical expression of the disease depend on the antigen and tissue against which autoimmune responses are directed. Both antibody and cell-mediated immune responses can be involved. In autoimmune hemolytic anemia and autoimmune thrombocytopenic purpura, for example, the mechanisms of tissue damage are relatively understood and involve antibodies against autoantigen that induce cell destruction by direct lysis or most probably by accelerated clearance of autoantibodysensitized cells (Schreiber and Frank, 1972). Antibody interactions with complement and in particular with Fc receptors are well known to induce tissue damage (Ravetch and Clynes, 1998). In Hashimoto's thyroiditis or systemic lupus erythematosus (SLE) antibodies initiate a powerful inflammatory response that results in antibody-dependent and direct T celldependent cytotoxicity (Podleski, 1972; Calder et al., 1973). Powerful inflammatory responses are also observed in Goodpasture's syndrome where autoantibodies recognize extracellular antigens. Further, autoantibodies can cause disease when directed against cell-surface receptors by interfering with their function. Thus, in Graves' disease autoantibodies to the thyroid-stimulating hormone receptor stimulate the production of excessive thyroid

hormone (Drexhage et al., 1981), and in myasthenia gravis autoantibodies to the a chain of the nicotinic acetylcholine receptor block neuromuscular transmission (Almon et al., 1974; Lennon and Lambert, 1980). Sustained autoantibody responses require the activation of autoantigen-specific CD4 § T cells, cells that provide help to autoantigen-specific B cells. In addition, T cells can mediate tissue injury by themselves or by activating macrophages and inducing local inflammation. Such mechanisms seem to operate in rheumatoid arthritis and multiple sclerosis in which the disease tissues are heavily infiltrated by T lymphocytes and activated macrophages. Rheumatoid arthritis, in particular, seems to be initiated by CD4 § T cells specific for antigens expressed in the joint. T cells are also involved in the maintenance of local inflammation. Accumulation of polymorphonuclear leukocytes and macrophages may ultimately destroy the joint. During that process autoantibodies are produced and include the rheumatoid factor, an IgM anti-IgG antibody that may up-regulate cytokine production and account for part of the tissue damage observed. CD8 § T cells, on the other hand, are more important in other autoimmune diseases, such as insulindependent diabetes mellitus, where CD8 § T cells attack and destroy the insulin-producing [3cells of the pancreas.

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MODELS OF AUTOIMMUNITY A number of potential etiological mechanisms have been proposed to explain the induction of autoimmunity and it is possible that multiple mechanisms are involved, in different individuals. First, the 'release of anatomically sequestered antigens' model proposes that autoimmunity occurs as a result of the exposure of mature T cells to antigens that are sequestered from the circulation and are not thus seen by developing T cells in the thymus. Such antigens may be confined behind anatomical and immunosuppressive barriers such as the cornea, brain or testes, and include myelin basic protein (MBP) and thyroid peroxidase (TPO), two major autoantigens found in multiple sclerosis and thyroid autoimmunity respectively. Support for this theory comes from the clinical experience of the induction of autoimmune uveitis after eye injury (Gery and Streilein, 1994) or orchitis after vasectomy (Shahani and Hattikudur, 1981). The relevance of this concept, however, is unclear as there is increasing evidence that even 'hidden' antigens may at times be expressed in the thymus (Wekerle et al., 1996; Hanahan, 1998). The 'cryptic self' hypothesis is another model that has been proposed. It is based on the concept that T cell tolerance is generated against self-epitopes that are presented in the context of MHC in the thymus. However, self-reactive T cells may escape negative selection because they recognize cryptic epitopes of autoantigens (defined as epitopes not normally expressed when antigen is processed) rather than anatomically sequestered autoantigens. Cryptic epitopes may be due to ineffective antigen processing, dominance of a flanking epitope that competes for binding to the same MHC molecule, or aberrant expression of developmental or differentiation antigens. This model is comparable with the 'Bottazzo-Feldmann hypothesis' that autoimmunity occurs when MHC class II expression and antigenpresenting cell function is up-regulated and induced in cells not normally expressing class II molecules. Inducing factors may involve cytokines such as IFN7 released during local infection or trauma (Bottazzo et al., 1983) and may result in the presentation of previously sequestered antigens or cryptic epitopes. Support for this model comes from findings that local

IFN7 production in [3 islets is able to induce diabetes (Sarvetnick et al., 1990). Some thyroid-infiltrating TPO-specific T cell clones recognize cryptic epitopes which are presented by thyroid epithelial cells synthesizing TPO, but not by antigen-presenting cells taking up TPO as an exogenous antigen (Dayan et al., 1991; Quarantino et al., 1996). The relevance of this concept to other autoimmune diseases is not yet documented. A popular theory to explain autoimmunity is based on 'molecular mimicry'. A number of viruses and bacteria possess antigenic determinants that are identical or homologous, closely related to normal host cell components. During an infection, this may lead to cross-reactivity between immune responses to selfand foreign antigens, and activation and clonal expansion of autoreactive cells in the periphery (Oldstone, 1989). Many autoimmune diseases have been associated with the infection of particular pathogens, including ankylosing spondylitis with mycobaterium Klebsiella (Fielder et aL, 1995), Chagas disease with T r y p a n o s o m a cruzi (Van Voorhis and Eisen, 1989) and insulin-dependent diabetes mellitus with cocksackie virus (Ray et al., 1980; Baum et al., 1996). In addition, many proteins found in bacterial and parasitic pathogens have been shown to crossreact with mammalian proteins, such as the microbial heat-shock protein hsp65. Both antibody and T cell responses against hsp65 have been detected in rheumatoid arthritis (Thompson et al., 1991; De Graeff-Meeder et al., 1991) and insulin-dependent diabetes mellitus patients (Tun et al., 1994), and antihsp65 antibodies have been demonstrated to recognize glutamic acid decarboxylase, a pancreatic enzyme localized in the insulin-producing beta cells of the islets of Langerhans (]ones et al., 1993). However, definitive evidence of pathogenic molecular mimicry is lacking and bystander T cell activation that occurs during infection may actually be as important as molecular mimicry in inducing autoimmunity (Bottazzo et al., 1983; Horwitz et al., 1998; Benoist and Mathis, 2001). Numerous other theories and factors have also been proposed to help explain autoimmunity and include defective apoptosis (Vaux and Flavell, 2000; Eguchi, 2001; Beutler, 2001), TH1/TH2cytokine imbalance (O'Garra et al., 1997; Singh et al., 1999) or neuroendocrine hormones (Cutolo and Wilder, 2000) as potential etiological factors. Despite that, no mecha-

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

THE CYTOKINE SYSTEM IN RA

nism has been consistently shown to account for autoimmune disease. It may be that autoimmunity requires a combination of them rather than a single mechanism to occur.

STAGES OF AUTOIMMUNITY Autoimmunity is envisaged to contain three stages of the disease process (Figure 52.1). The first stage is 'initiation', during which autoimmunity is induced in genetically predisposed individuals and under the influence of specific presumably environmental factors. This stage is believed to involve a local inflammatory response with the release of cytokines and the activation of T cells, and is difficult to study as it is largely asymptomatic and usually goes unreported. Evidence for this is indirect and comes mainly from cancer patients where administration of IL-2 and IFN-cz often induces autoimmunity, especially of the thyroid (Burman et al., 1986; Atkins et al., 1988). Mthough autoimmunity resolves after cessation of the cytokine therapy, it can be severe and occurs with the presence of autoantibodies and the association with the disease susceptibility MHC genotype. The second stage of autoimmunity is 'perpetuation' and involves the continual interaction between tissue antigen-presenting cells and autoantigen-reactive T cells. This results in the persistence of autoreactive lymphocytes and the maintenance of inflammation. In thyroid disease, for example, there is evidence that thyroid epithelial cells can present antigen and thyrocyte-specific T cells can be isolated from the thyroid infiltrate (Londei et al., 1984, 1985; Dayan et al., 1991). In addition to the initiation and perpetuation stages of autoimmunity, there is the late or tissue-damaging stage of autoimmunity. During this stage, cytokines and other inflammatory mediators released by both lymphocytes and antigen-presenting cells induce the production of autoantibodies, the formation of immune complexes, the activation of complement and the extravasation and activation of cytotoxic T cells, natural killer cells, macrophages and polymorphonuclear leukocytes. This leads to extensive damage of the tissues, ultimately resulting in disability or even death.

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THE CYTOKINE SYSTEM IN RA The late or tissue-damaging stage of autoimmunity is the most relevant therapeutically and the most accessible for study. It is usually at this stage that patients seek medical attention and that disease is detected or reported. As in all stages of autoimmunity, this stage involves the production of cytokines that specifically regulate the inflammatory response and the tissue damage and repair mechanisms. Realizing the importance of cytokines as mediators of inflammation and immunity (reviewed in Oppenheim and Feldmann, 2001), we have undertaken for the last 16 years the task of studying the role of cytokines in rheumatoid arthritis. RA offers some major advantages for this type of study over other autoimmune diseases, such as multiple sclerosis and insulin-dependent diabetes mellitus, because biopsy tissue from the disease site at the height of the inflammatory process can be obtained (reviewed by Feldmann et al., 1996b). A number of methods have been used to analyse synovial cytokine expression which include immunohistology of flesh ex vivo tissue (Chu et al., 1991a), in situ hybridization of synovial fluid products (Wood et al., 1992), and short-term culture of synovial membrane cells in the absence of extrinsic stimulation (Brennan et al., 1989a). Immunohistology is particularly useful when determining which cells are the major cytokine producers, whereas short-term culture of synovial membrane cells permits the quantitative analysis of proteins released, and their regulation, and is the one we used most extensively. Soon, our studies and those of other groups in the field made us realize that the synovium of RA patients is rich in the expression of cytokines; virtually every cytokine known can be detected as summarized in Table 52.2. The abundance of most known cytokines in the rheumatoid synovium was not surprising as this site is inflamed and contains numerous activated T cells, macrophages, endothelium, fibroblasts and plasma cells, all of which are able to produce cytokines. The plethora of cytokines expressed highlighted the complexity of determining which cytokines might be important in the pathogenesis of the disease. The quantity of the cytokines detected is not necessarily a reflection of their importance, as different cytokines exert distinct functions and have various properties.

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TABLE 52.2 Cytokines expressed in r h e u m a t o i d synovial tissue ( a d a p t e d with p e r m i s s i o n f r o m F e l d m a n n et al. 1998) Cytokines

Expression mRNA protein

Reference

Pro-Inflammatory IL-la,13

+

+

TNFa

+

+

LT IL-6

+ +

+ +

GM-CSF

+

+

M-CSF LIF

+ +

+ +

Oncostatin M IL-2 IL-3 IL-7 IL-9 IL-12 IL-15 IFNa/[3 IFN7 IL-17 IL-18 Immunoregulatory IL-4 IL-10

+ + ? ? + + + + + +

+ +_ ? ? + + + + +

Brennan et al. (1989b); Buchan et al. (1988a); C h o m a r a t et al. 1995; Deleuran et al. (1992); Firestein et al. (1990) Br en n an et al. (1989b); Chu et al. (1991a); Di Giovine et al. (1988) Brennan et al. (1990b);'Saxne et al. (1988) Field et al. (1991); Firestein et al. (1990); Helle et al. (1991); Hirano et al. (1988); Houssiau et al. (1988) Alvaro-Gracia et al. (1989); Haworth et al. (1991); Xu et al. (1989) Firestein et al. (1990) Lotz et al. (1992); Okamoto et al. (1997); Waring et al. (1993) Okamoto et al. (1997) Buchan et al. (1988b); Firestein et al. (1990) Firestein et al. (1990)

_ +

+

IL-11

+

+

IL-13 TGF~

+ +

+ +

IL-8

+

+

Groa MIP-1 MCP-1

+ + +

+ + +

ENA-78 RANTES

+ +

+ +

FGF

+

+

PDGF

+

+

VEGF

+

+

Morita et al. (1998) McInnes et al. (1996) Hopkins and Meager (1988) Buchan et al. (1988b); Firestein et al. (1990) C h a b a u d et al. (1999); Kirkham et al. (1997) Yamamura et al. (1997) Miossec et al. (1990) Cohen et al. (1995); Cush et al. (1995); Katsikis et al. (1994); Llorente et al. (1994) H e r m a n n et al. (1998); Okamoto et al. (1997); Taki et al. (1998) Isomaki et al. (1996) Brennan et aL (1990a); Bucala et al. (1991); Chu et al. (1991b); Fava et al. (1991); G o d d ar d et aL (1992); Lafyatis et al. (1989); Lotz et al. (1990); Miossec et aL (1990)

Chemokines Brennan et al. (1990b); Hosaka et al. (1994); Koch et al. (1991) Hosaka et al. (1994) Hosaka et aL (1994); Koch et aL (1994b) Akahoshi et al. (1993); Hachicha et al. (1993); Hosaka et al. (1994); Koch et al. (1992); Villiger et al. (1992) Koch et al. (1994a) Rathanaswami et aL (1993)

Growth Factors Bucala et al. (1991); G o d d a r d et al. (1990); G o d d a r d et al. (1992); Sano et al. (1990); Sano et al. (1993) R e m m e r s et al. (1990); R e m m e r s et al. (1991); Sano et al. (1993) Fava (1994); Koch et al. (1994c)

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

THE CYTOKINE SYSTEM IN RA The fact, however, that most cytokines are expressed transiently and can be induced or inhibited by other cytokines suggested that a 'cytokine network' may exist in which cytokines regulate each other (Vilcek, 1997; Feldmann et al., 1996a). In the rheumatoid synovium, this network can be envisaged as a balance of pro-inflammatory (TNFcz, IL-1, IL-6) and antiinflammatory (IL-10, IL-11) cytokines shifted towards the pro-inflammatory side (Figure 52.2). To understand the regulation of cytokine production in the rheumatoid joint, we decided to use shortterm cultures of rheumatoid synovial membranes and study the effects of neutralizing antibodies against specific cytokines. Short term (5-6-day) cultures of all the cells extracted from rheumatoid synovial membranes provided a good model of certain aspects of the disease, as they contain 30% T cells, 30-40% macrophages, and fewer endothelial cells, fibroblasts, dendritic cells, plasma cells and B lymphocytes. These cells reaggregate in vitro, and spontaneously release the inflammatory mediators which are also produced in vivo (reviewed in Feldmann et al., 1996b). This type of culture is much more representative of the synovium than the passaged 'synoviocyte' which selects for the fibroblast-like cells that probably represent 10% of the total synovium. Our work with synovial cultures yielded some very interesting observations. First, addition of a neutralizing anti-TNFcz antibody, but not the closely related anti-LTa antibody reproducibly inhibited the production of IL-1 in these cultures (Brennan et al., 1989b). IL-1 was used as a readout because it had been previously shown to be important in cartilage and bone destruction (Gowen et al., 1983; Saklatvala et al., 1985). This result was unexpected, because many other signals present in

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the synovium, such as IL-1 itself, GM-CSE IFN7 or immune complexes were known to induce IL-1 in other systems. Second, addition of the anti-TNFcz antibody also inhibited the production of other proinflammatory mediators too, such as GM-CSF, IL-6 and IL-8 (Haworth et al., 1991; Butler et al., 1995). These observations were consistent with those of others (Alvaro-Gracia et al., 1991) and indicated that many of the major pro-inflammatory cytokines produced in the rheumatoid synovium are linked in a network or cascade with TNFcz at its apex (Figure 52.3), and hence suggested that inhibition of TNFcz could be a good therapeutic approach. Similar studies by us and others in murine models of arthritis such as collagen-induced arthritis have also confirmed the importance of TNFcz in the induction and maintenance of disease (Thorbecke et al., 1992; Williams et al., 1992; Piguet et al., 1992; Wooley et al., 1993). Interestingly, a 'spontaneous' model of arthritis was developed using a disregulated TNFcz transgene carrying a deletion in the 3' untranslated region of the TNFcz gene that is essential for its normal regulation (Keffer et al., 1991). The rheumatoid synovium was also found to have up-regulated a number of anti-inflammatory mediators that include IL-10 and IL-11. This prompted us to investigate their role in the regulation of proinflammatory cytokine production in rheumatoid synovial membrane cells. We found that neutralization of IL-10 by anti-IL-10 antibodies increased the spontaneous release of both TNFcz and IL-1, as well as IFNT, a cytokine barely detectable otherwise (Katsikis et al., 1994). In agreement with that, we found that addition of recombinant IL-10 to monocyte cultures down-regulated TNFa and IL-1 activity by inhibiting

Anti-inflammatory

IL-11

IL-10, IL-lra, sTRF-R

IL-10 IL-1 TNF~

GM-CSF

sTNF-R

IL-lra

IL-6

t ]Immune system I ~- TNF~

~- IL-1

Ik-6, 11_-8,GM-CSF Pro-inflammatory

Pro-inflammatory

Anti-inflammatory

FIGURE 52.2 Cytokine disequilibrium in rheumatoid arthritis (modified with permission from Feldmann et al., 1996a).

FIGURE 52.3 Cyto]dne cascade in r h e u m a t o i d arthritis (taken with permission from Feldmann et al.,

1996a).

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A U T O I M M U N I T Y AND CYTOKINES: PATHOGENESIS AND THERAPY

their expression and inducing the production of soluble TNF receptors and IL-1 receptor antagonist (de Waal Malefyt et al., 1991; Joyce et al., 1994). Similady, neutralization of IL- 11 by anti-IL- 11 antibodies increased the production of TNFa in rheumatoid synovial membrane cultures, whereas addition of recombinant IL-11 inhibited the production of TNFa and matrix mettaloproteinases 1 and 3 (Hermann et al., 1998). In summary, IL-10 and IL-11 seem to negatively regulate pro-inflammatory cytokine production in the rheumatoid synovium. Other potentially inhibitory cytokines, such as IL-4 and IL-13, are poorly expressed. Another group of anti-inflammatory mediators are soluble TNF and IL-1 receptors, and the IL-1 receptor antagonist that specifically inhibit the function of TNFa and IL-1 respectively. The soluble p55 and p75 TNF receptors are elevated in RA plasma and synovial fluid, and are also spontaneously released in rheumatoid synovial membrane cultures (Cope et al., 1992). Neutralization of the soluble TNF receptors with monoclonal antibodies increases the bioactivity of TNFa. At the same time, the soluble IL-1 receptor and the IL-1 receptor antagonist are also increased in RA synovial fluid and synovial membrane cells (RouxLombard et al., 1992; Firestein et al., 1992; Deleuran et al., 1992). However, despite the production of natural TNF and IL-1 inhibitors, TNFa and IL-1 bioactivity and signaling (such as NF-KB activation) is still detectable in rheumatoid synovial membrane cultures and in synovium by immunohistology (Marok et al., 1996; Sioud et al., 1998), suggesting that these mechanisms are insufficient in regulating the disease process. This supports the concept that rheumatoid arthritis is an imbalance between the production of pro-inflammatory and anti-inflammatory mediators (Figure 52.2).

THE CYTOKINE SYSTEM IN OTHER AIJTOIMMUNE DISEASES The role of cytoldnes has also been studied in a number of other autoimmune diseases and has been recently reviewed in a book by Brennan and Feldmann (1996). In Crohn's disease, cytokine production has been investigated by using immunohisto-

chemistry, whole biopsy cultures, and isolated lamina propria mononuclear cells (LPMC), and a large number of pro-inflammatory cytokines (TNFa, IL-1, IL-6) and chemokines (IL-8, MCP-1 and RANTES) were found to be elevated (Fiocchi, 1998). As in rheumatoid arthritis, soluble TNF receptors and the IL-1 receptor antagonist, the natural inhibitors of TNFa and IL-1, were also found to be increased (Noguchi et al., 1998) as was the immunoregulatory cytokine IL-10 (Autschbach et al., 1998). Addition of exogenous IL-10 has been shown to suppress the elevated proinflammatory cytokine production from Crohn's disease LPMC when delivered in the in vitro cultures or when administered rectally to patients with active disease (Schreiber et al., 1995). T cell-derived cytokines can also be detected in Crohn's disease tissue and are predominantly of a T m profile with elevated levels of IL-2 and IFNy (Mullin et al., 1992). Increased expression oflL-12 has also been demonstrated in LPMC from Crohn's disease mucosa (Monteleone et al., 1997). Although, however, Crohn's disease has many analogies to rheumatoid arthritis, the pro-inflammatory cascade with TNFa at its apex has not been described, although it is likely to operate in vivo as antiTNFR clinical trials have shown marked benefit (van Dullemen et al., 1995; Rutgeerts et al., 1999). The cytokine networks in other autoimmune diseases are less clearly defined. In Graves' disease and Hashimoto's thyroiditis, a large number of cytokines have been detected and include pro-inflammatory (TNFm LT, IL-1, IL-6) and immunoregulatory cytokines (IL-10), chemokines (IL-8) and T cell-derived cytokines (IL-2, IL-4, IFNy) (reviewed by GrubeckLoebenstein, 1996). Although thyroid epithelial cells can produce cytokines, most of the cytokines detected are due to the intrathyroidal lymphocytic infiltrate that is seen in thyroid diseases (GrubeckLoebenstein et al., 1989; Weetman and McGregor, 1994). In Sj6gren's syndrome salivary gland biopsies TNFR, LT, IL-1, IL-2, IL-4, IL-6, IL-10, TGF[~ and IFNy can be detected by polymerase chain reaction and in situ hybridization (reviewed by Skopouli and Moutsopoulos, 1996). Similarly, elevated levels of TNFa, IL-1, IL-2, IL-6, IL-10, IFN7 and soluble TNF receptors levels are increased in sera from patients with systemic lupus erythematosus (reviewed by Smolen et al., 1996) as are TNFa, LT, IL-1, IL-2 and

C L I N I C A L A P P L I C A T I O N OF CYTOKINES A N D CYTOKINE I N H I B I T I O N

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ANTI-TNFc~ THERAPY IN RHEUMATOID ARTHRITIS

IFN7 in multiple sclerosis lesions from patients with active disease (reviewed by Baker et al., 1996). In summary, it is becoming increasingly clear that cytokines are essential for the development, maintenance and pathology of autoimmunity. More detailed reviews of cytokines in autoimmunity can be found in two recent books, Cytokines in A u t o i m m u n i t y edited by Brennan and Feldmann (1996) and Cytokine Reference (also on the web) edited by Oppenheim and Feldmann (2001).

ANTI-TNFt~ THERAPY IN RHEUMATOID ARTHRITIS Based on various findings including the observation that TNFa is at the apex of the pro-inflammatory cascade in rheumatoid arthritis (Feldmann and Maini, 2001), clinical trials aimed at blocking TNFcz activity with infliximab, a neutralizing anti-TNFa antibody, were initiated in 1992. Infliximab (formerly known as cA2) is a chimeric mouse Fv, h u m a n IgG1 monoclonal antibody of high affinity and neutralizing capacity (Knight et al., 1993). The first phase I/II study was an open (non-placebo controlled) trial of infliximab in long-standing active RA patients who failed all prior therapy averaging four disease-modifying drugs. The results were very encouraging with patients reporting alleviation of symptoms such as pain, morning stiffness, tiredness and reduction of swollen and tender

joints within a week or two (Figure 52.4). Inflammation markers, such as C-reactive protein (CRP), were also reduced. This response, however, was temporary and lasted 8-22 weeks. Readministration of infliximab induced further benefit (Elliott et al., 1993, 1994a). A double-blind, randomized, placebo-controlled clinical trial then followed and finally established the efficacy of infliximab in controlling the signs and symptoms of RA. A 60-70% reduction in the measures of disease activity such as swollen or tender joint counts and CRP was observed (Elliott et al., 1994b). Although infliximab was effective as a monotherapy in 60-70% of the patients, it was important to determine its effect with established therapies and to evaluate if combined anti-TNFcz and anti-T cell therapy may provide additional therapy as it does in mouse models (Williams et al., 1999). Methotrexate (MTX) is one of the most effective and durable therapies for RA with effects on T cells too, so a phase II clinical trial was done to examine the effect of infliximab in patients with active disease despite therapy with MTX. Results from this study demonstrated a synergy between infliximab at low doses and MTX. The onset of the response was rapid, with a significant improvement in symptoms and signs by 2 weeks, and the majority of patients responding by 6 weeks (Figure

52.5). Over 60-70% change in individual parameters of disease activity was achieved, as well as joint protection, when hands and feet were examined by

Swollen joint count (0-28)

CRP (mg/I) ',r-'

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FIGURE 52.5 Efficacy of combination of infliximab and methotrexate (MTX) versus methotrexate and placebo. In (a), patients were treated with MTX (7.5 mg week -1) and either placebo, 3 or 10 mg kg -1 infliximab administered intravenously at time points indicated by the arrows, in a DMARD unresponsive patient group with active disease despite methotrexate therapy. Patients were examined for disease improvement at the indicated time points (with permission from Maini et al., 1998). In (b), patients were treated with MTX (15 mg week -I) and either placebo, 3 or 10 mg kg -1 infliximab every 4 or 8 weeks. After 30 weeks, the response of the patients was evaluated according to the ACR 20 criteria and the improvement in swollen joints (with permission from Maini et al., 1999). In (b), patients were treated with MTX (15 mg/week) and either placebo, 3 or 10 mg/kg infliximab every 4 or 8 weeks. After 30 weeks, the response of the patients was evaluated according to the ACR 20 criteria and the improvement in swollen joints (with permission from Maini et al. 1999). CLINICAL APPLICATION

OF C Y T O K I N E S A N D C Y T O K I N E I N H I B I T I O N

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autoradiographs after 54 weeks in a similarly designed phase III trial involving two doses of infliximab (3 mg and 10 mg) and high doses of MTX (Maini et al., 1999; van der Heijde, 1996). These observations formally confirmed the essential role of TNFa in RA and demonstrated that the combination of infliximab with MTX can prevent progressive joint damage over a 2-year period (Lipsky et al., 2000).

MECHANISM OF ACTION OF ANTI-TNFa THERAPY The efficacy of anti-TNF~ therapy in RA, prompted us to investigate the mechanisms of action of infliximab in the anticipation that this will lead to a better understanding of the disease process. Some important observations were made by studying clinical trial serum and synovial samples. First, anti-TNF~ down-regulated the expression of cytokines such as IL-1, IL-6, IL-8, MCP-1 and VEGF in the serum (Lorenz et al., 1996; Elliott et al., 1993; Paleolog et al., 1998; Taylor et al., 2000). The rapid kinetics of this reduction (e.g. IL-6 was normalized in less than 24 h) suggested that this is a direct consequence of TNF~ blockade in the cytokine network rather than a reduction in the n u m b e r of cytokine-producing cells (Charles et al., 1999) that might occur if anti-TNF~ leads to cell death. These results confirmed our early observations in RA synovial m e m b r a n e cultures (Brennan et al., 1989b). Second, anti-TNF~ reduced the trafficking of leukocytes into the joints. This is probably due to decreased chemokine production and ICAM-1, E-selectin and VCAM-1 adhesion molecule expression (Taylor et al., 2000; Paleolog et al., 1996; Tak, et al., 1996). Third, anti-TNF~ diminished angiogenesis in inflamed joints as assessed by computerized image analysis of the endothelium (Maini et al., 1999). Inhibition of VEGF production may partly account for that (Taylor et al., 2000). Finally, anti-TNF~ restored hematological abnormalities observed in RA patients such as anemia and elevated platelet counts, although the mechanism by which this occurs is not clear (Davis et al., 1997). One possibility is that this is a consequence of IL-6 reduction. The m e c h a n i s m of action of anti-TNF~ therapy is documented in more detail in Feldmann and Maini, 2001.

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ANTI-TNFa THERAPY OF OTHER DISEASES The success of anti-TNFR therapy in RA suggested that TNFR may also be a good therapeutic target in a number of other autoimmune diseases in which similar mechanisms may operate. Crohn's disease, a chronic inflammatory disease characterized by inflammation and granulomatous lesions of the intestinal mucosa, is one such example in which trials were initiated after disclosure of results with infliximab (Derkx et al., 1993). TNFR is elevated in mucosal biopsies as are a number of other cytokines, although it has never been shown that TNFR is at the apex of a pro-inflammatory cascade. Nevertheless, clinical trials were initiated using infliximab as the TNFR blocking agent. In an initial phase I/II open-label study, a single infusion of infliximab was sufficient to induce clinical and endoscopic improvement in eight out of ten patients with active steroid-resistant disease (van Dullemen et al., 1995). Persistent clinical response could be seen in 41% of the infliximab-treated patients compared with 12% of placebo control patients (Targan et al., 1997). Infliximab downregulated the endoscopic disease activity, as well as the inflammatory response in the mucosal layer (D'Haens et al., 1999; Baert et al., 1999). In the light of the studies in RA, the effect of multiple infusions of infliximab were also examined in patients that initially responded to the single infusion of infliximab (Rutgeerts et al., 1997). It was found that 51% of the infliximab patients remained in remission for the duration of the trial, compared with 21% placebo controls (Rutgeerts et al., 1999). Recently, infliximab obtained approval from the FDA and the European Authority for its use in humans for the treatment of Crohn's disease. Currently, a n u m b e r of clinical trials with antiTNFR blocking agents are being initiated in other a u t o i m m u n e conditions too. In two open-label pilot studies in spondyloarthropathy, infliximab was found to be effective in both axial and peripheral arthritis (Van Den Bosch et al., 2000; Brandt et al., 2000). In a phase I/II clinical trial in juvenile arthritis, etanercept diminished disease activity in 74% of the patients compared with placebo (Lovell et al., 2000). Administration of etanercept also benefited patients with psoriasis, as etanercept-treated

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patients showed a significantly higher response as judged by the proriasis activity and severity index than placebo-treated patients (Mease et al., 2000). Psoriatic arthritis disease activity was also reduced in these patients.

OTHER ANTI-CYTOKINE THERAPY Anti-cytokine therapy has not been limited to TNFa but has been extended to other cytokines. In RA, clinical trials with IL-1 receptor antagonist have been performed and results have been positive, but less dramatic. IL-lra administration revealed an improvement in clinical disease as judged by the ARC criteria (Bresnihan, 1998) and in joint destruction as determined by radiology (Jiang et al., 2000; Watt and Cobby, 2001). An alternative approach aiming to block IL-1 directly in the joints was recently used. It involved the transduction of synovial cells ex vivo with a retroviral vector expressing IL-lra, and transduced cells were re-implanted in the same patient 1 week before joint replacement surgery. Appropriate untransduced synovial cells were also implanted to the same patient in a separate joint to serve as a placebo control. However, when IL-lra expression was examined at the time of surgery, IL- Ira could be detected in both joints suggesting either that injected cells can migrate to neighbouring joints, or that that there is some communication between adjacent RA joints (Evans, 1999). Because of that, no further conclusions could be drawn. Anti-IL-6 therapy clinical trials have also been reported in RA. In one study, a murine neutralizing anti-human IL-6 antibody was used in a small number of patients and demonstrated a short-term improvement. In another open-label study, administration of a humanized monoclonal anti-IL-6 receptor antibody improved Clinical symptoms of RA and normalized acute-phase proteins within 2 weeks (Yoshizaki et al., 1998). Although onset of the benefit was slower than that of anti-TNFa, randomized trials are under way to provide more information about its efficacy.

ANTI-INFIAMMATORY CYTOKINE THERAPY IN AUTOIMMUNITY In addition to the anti-cytokine therapy, there is an alternative approach to inhibit pro-inflammatory cytokine production that involves the use of immunoregulatory cytokines, such as IL-10, IL-11 and IFN-[3. The efficacy of this approach has been tested in a number of autoimmune diseases with variable efficacy. In inflammatory bowel diseases, administration of IL-10 has demonstrated some modest clinical benefit in mild/moderate Crohn's disease patients, but no effect in ulcerative colitis patients (van Deventer et al., 1997; Fedorak et al., 1998). Following administration of IL-11 in patients with Crohn's disease, 42% of the treated patients demonstrated a clinical benefit compared with 7% of the placebo group (Sands et al., 1999). The dose of IL-11 used was well tolerated and was not associated with an increase in platelet counts (Sands et al., 1999). IL-10 and IL-11 have also been tested for their efficacy in RA. In two independent phase I clinical trials, RA patients treated with IL-10 or IL-11 showed no significant clinical improvement when compared with placebo (Maini et al., 1997; Moreland et al., 1999). More encouraging though, was the use of IL-10 in psoriasis. In an open-label study, nine out of ten patients demonstrated significant clinical benefit. In a double-blind, placebo-controlled study that followed, clinical improvement was mainly restricted in the skin but not the arthritic disease activity (psoriatic arthritis) (Asadullah et al., 1999; McInnes et al., 2001). IL-10-treated patients demonstrated suppressed monocyte function and TH1 but n o t TH2 cytokine production, as well as reduced T cell and macrophage infiltration and angiogenesis in the synovium (McInnes et al., 2001). The most successful story of immunoregulatory cytokine therapy is probably the use of IFN-[3 in multiple sclerosis. Administration of IFN-[3 decreases the relapse rates by 18-34% in patients with relapsing/remitting multiple sclerosis (Anonymous 1993, 1998a). This is of major importance in the management of multiple sclerosis, as the failure of patients to recover from relapses is a major cause of disability. IFN-[3 administration can also delay disease progression for 9-12 months in patients with secondary

CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

FUTURE THERAPY progressive multiple sclerosis (Anonymous, 1998b). IFN-[3 therapy is now considered to be a major advance in the treatment of multiple sclerosis, although its cost and moderate efficacy has led to few patients benefiting from its use.

FUTURE THERAPY Although anti-TNFa therapy works well as a monotherapy, optimal efficiency appears to be achieved when anti-TNFa blocking agents are administered in combination with methotrexate. This has been demonstrated for both infliximab (Maini et al., 1998; Maini et al., 1999; Lipsky et al., 2000) and etanercept (Weinblatt et al., 1999) and suggests that combination therapy is an approach that can improve on antiTNFa therapy as expectations from patients augment. Studies that we have undertaken on animal models of arthritis demonstrated that the benefit of anti-TNFa can be increased by its combination with anti-T cell therapy. Co-administration of neutralizing anti-TNFa antibodies with T cell depleting anti-CD4 antibodies (Williams et al., 1994), for example, synergize to ameliorate disease, as does the co-administration of antiTNFR with anti-CD3 antibodies or CTLA-4Ig fusion proteins (Williams, unpublished data). Thus, it is possible that in human rheumatoid arthritis too, additional anti-T cell therapy involving anti-CD4 or anti-CD3 antibodies or T cell-inhibitory agents, such as cyclosporin may lead to increased therapeutic benefit in the patients. Anti-cytokine or immunoregulatory cytokine-based therapies have proved to be very successful in the treatment of certain autoimmune diseases, such as RA, Crohn's disease and multiple sclerosis, but they have not been able to induce long-term disease remission in any case in the absence of continuous treatment. Their efficacy has been dependent on prolonged administration of the therapeutic agent. In KA, prolonged administration of anti-TNFa biologicals suffers from some major disadvantages. First, it requires injection of the anti-TNFa agent. Second and more importantly, chronic blockade of TNFa may seriously compromise normal immune functions, as TNFa is a multifunctional cytokine. TNFa-deficient mice, for example, demonstrate an increased risk of bacterial infections, such as Listeria m o n o c y t o g e n e s

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(Marino et al., 1997), although no increased risk of infection was observed in tLA patients treated with either infliximab or etanercept (Feldmann and Maini, 2001). In long term-treated patients the infection risk may be augmented. There is recent evidence that the incidence of tuberculosis is increased after TNFablockade in both tLA and Crohn's disease (Keane et al., 2001). However, by far the biggest practical drawback is the cost of the treatment. To design better therapeutic agents that may specifically block the 'pathological' TNFa expressed in the rheumatoid synovium without compromising the 'physiological' TNFa expressed as a result of normal immune function, we decided to investigate the intracellular signalling pathways that regulate TNFa production. We envisaged that the mechanisms involved in TNFa production in the rheumatoid disease would be different from those involved in normal immune function. This is made possible by the fact that the expression of the TNFa gene is under complex control: the 5' promoter region contains binding sites for multiple transcription factors that include NF-KB, AP-1, NF-IL-6 and NF-AT (Shakhov et al., 1990; Goldfeld et al., 1991; McCaffrey et al., 1994) and hence may be involved in transcriptional control, whereas the 3' untranslated region contains AU-rich elements (ARE) that may be involved in posttranscriptional or translational control through the action of p38 mitogen-activated protein kinase (MAPK) and mRNAbinding proteins (Lee et al., 1994). Evidence, however, about the relative contribution of these factors in TNFa has been conflicting. In the murine RAW 264 macrophage cell line and bone marrow-derived macrophages, for example, NF-KB was required for TNFR production (Shakhov et al., 1990; Han and Beutler, 1990). In contrast, in human lymphoid cell lines NF-AT rather than NF-KB was essential for TNFa production (Goldfeld et al., 1991; Tsai et al., 1996). The main TNFa-producing cells in the rheumatoid synovium are macrophages with a small contribution from T cells and endothelial cells (Feldmann et al., 1996b). We first decided to investigate the role of NF-KB in TNFR production in this cell-type. By developing an efficient adenoviral gene transfer technique to express wild-type or dominant negative forms of intracellular signalling molecules, we were able to examine the regulation of TNFa production in primary human macrophages (Foxwell

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et al., 1998). The results were very interesting: Overexpression of IKBa, the natural inhibitor of NF-~cB abrogated TNFa production in response to LPS, PMA or UV light stimulation, but not zymosan or antiCD45 stimulation (Bondeson et al., 1999b). These findings suggested that the involvement of NF-~cB in macrophage TNFa production is stimulus-specific, and further added to the complexity of the regulation of TNFa. In RA, the stimulus that induces TNFa production is not known and signalling observations made in primary macrophages cannot be extrapolated into RA disease tissue. To overcome this problem we examined the role of NF-~cB directly in rheumatoid synovial membrane cultures. Adenoviral gene transfer again constituted a powerful approach to genetically modify RA cells, with >90% of the cells expressing the transgene of interest after infection at a multiplicity of infection of 40 (Foxwell et al., 1998; Bondeson et al., 1999a). Using flow cytometry, we found that CD14 § CD3- synovial macrophages, CD14- CD3 § synovial T cells and CD14- CD3- synovial fibroblasts were readily infected (Plate 52.6a) (see Plate section) (Bondeson et al., 1999a). This allowed us to block NF-~cB activation in RA cells by overexpressing IKBa, and then examine the effect that had on TNFa production. We found that the levels of TNFa released in synovial membrane cell cultures was reduced by 70% (Plate 52.6b), suggesting that NF-KB is rate-limiting for the expression of TNFa in RA. We also examined the effect of NF-~cB inhibition in the expression of other pro-inflammatory mediators and found that IL-6 production was inhibited by 90%, IL-1 by 40%, and IL-8 by 30-40% although this was more variable (Bondeson et al., 1999a). Interestingly, however, overexpression of IKBa had only minimal effects on the production of the anti-inflammatory mediators IL-10 and IL-11, and only moderately inhibited the release of soluble TNF receptors. Even then, this was found to be an indirect effect arising from the inhibition of TNFa rather than a direct effect of NF-~cB in the regulation of their transcription. These results demonstrated that NF-KB selectively plays a major role in the regulation of proinflammatory cytokine gene expression in RA synovial tissue without affecting anti-inflammatory cytokine gene expression. The effect, thus, of blocking NF-~cB in RA may be very beneficial, as it may restore

the cytokine equilibrium in the joints by diminishing pro-inflammatory mediators without reducing the anti-inflammatory ones. However, the means for doing so safely are not yet available, but as many companies are working on NF-KB inhibitors, this may change in the near future. We investigated the role of NF-~cB in destructive processes that take place in the joint too. Matrix metalloproteinases (MMPs) are thought to be involved in the breakdown of human articular cartilage. Articular cartilage is primarily composed of collagen type II and proteoglycans, and MMP1, MMP13 and membranebound MMPs are capable of cleaving collagen type II (Knauper et al., 1997; Cowell et al., 1998). MMP3 lacks this capacity but is able to cleave other components of the extracellular matrix that include proteoglycans, fibronectin and laminin (Matrisian, 1990). The expression of MMPs has been previously proposed to be AP-l-dependent as studies in some systems have shown (Fini et al., 1998), although there was also evidence for AP-1-independent mechanisms also operating (Buttice et al., 1991; Quinones et al., 1994; Borghaei et al., 1998). When, however, we explored the effect of NF-KB inhibition in rheumatoid synovial membrane cells, we found that the production of MMP-1, MMP-3 and MMP-13 was markedly down-regulated. This was in contrast to the production ofTIMP-1, the inducible tissue inhibitor of MMPs enzymatic action, whose expression was up-regulated (Bondeson et al., 1999a). Whether this effect was due to a direct inhibitory effect of NF-KB in their gene expression or due to an indirect effect through cytokine inhibition is not clear, although NF-KB binding sites have been detected in the promoter element of MMP-1 (Angel et al., 1987; Tewari et al., 1996). Nevertheless, these findings demonstrate that NF-KB is essential not only for the inflammatory, but also for the destructive processes that operate in the rheumatoid joint. Another major pathway that controls inflammatory gene expression involves the action of mitogenactivated protein kinases (MAPIO. With respect to TNFcz expression, all three subtypes of MAPK have been reported to be involved, albeit to different extents. In monocytes/macrophages, p54 MAPK has been proposed to control TNFcz production at the translational level (Swantek et al., 1997), In contrast, p42/44 MAPK regulates TNFcz production at the tran-

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CONCLUSIONS

scriptional levels (Foey et al., 1998; Rutault et al., 2001), probably by phosphorylating Elk- 1, resulting in increased transcription of c-Fos and hence increased formation of AP-1 (Karin, 1995). Finally, p38 MAPK is the best studied of the MAPK and affects TNFR expression at multiple levels that involve transcriptional, post-transcriptional and translational mechanisms (Ridley et al., 1998; Dean et al., 1999; Rutault et al., 2001). Transcriptional regulation by p38 MAPK is mediated through the activation of the ATF2, Alk-1, Sap-l, MEF2A, MEF2C and CHOP/GADD153 transcription factors (Wang and Ron, 1996; Han et al., 1997; Wasylyk et al., 1998; Zhao et al., 1999), whereas translational control requires the phosphorylation of the MAPK-interacting kinases Mnkl and Mnk2 that in turn phosphorylate the eIF-4E component of the translation initiation complex (Waskiewicz et al., 1997), and the eIF-4E binding protein PHAS-1 (Lin et al., 1994). Post-transcriptional regulation of gene expression, on the other hand, involves the activation of MAPKAP kinase 2 that controls mRNA stability possibly by affecting the binding of ARE-binding proteins, such as TTP (Lai, 1999; Mahtani et al., 2001), AUF1 (Zhang et al., 1993), TIAR (Gueydan et al., 1999), Hel-N1 (Levine et al., 1993) and HuR (Myer et al., 1997; Dean et al., 2001). Transgenic mice, for example, that express TNFa transcripts lacking the ARE have an increased production of TNFa protein and develop chronic inflammatory polyarthritis and inflammatory bowel disease (Keffer et al., 1991; Kontoyiannis et al., 1999). With all these effects that p38 MAPK has on inflammatory gene expression, it is not surprising that inhibition of its activity markedly abrogates the production of TNFR, whereas inhibition of the other MAPK only partially does so. In addition, inhibition of p38 MAPK also reduces the production of IL-6 and IL-8 in primary human monocytes or macrophages (Ridley et al., 1998; Dean et al., 1999), establishing p38 MAPK as a primary target for the action of antiinflammatory drugs. To determine, however, whether the p38 MAPK pathway is also essential in the regulation of cytokine gene expression in rheumatoid arthritis synovium or other chronic inflammatory disease tissue the role of p38 MAPK needs to be addressed at the relevant disease tissues or models. Indeed, a study published only recently demonstrated that RPR200765A, a novel p38 MAPK inhibitor,

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reduces the incidence and progression of arthritis in the rat streptococcal cell wall arthritis model when administered in either prophylactic or therapeutic dosing regiments (McLay et al., 2001). In contrast, a separate study using SP600125, a novel p54 MAPK inhibitor, in the rat adjuvant-induced arthritis model found that the inhibition of p54 MAPK activity only modestly decreases paw swelling, but almost completely prevents joint destruction as measured by radiography (Han, 2001). It may be that combinations of inhibitors of different MAPK is required for optimal therapeutic efficiency. Clinical trials of p38 MAPK inhibitors in RA are under way and so this hypothesis will be evaluated soon. In summary, the production of TNFR and other inflammatory or destructive mediators in the rheumatoid joint seem to be NF-~:B- and MAPKdependent. Both NF-KB and p38 MAPK, in particular, are potential therapeutic targets for the treatment of rheumatoid arthritis. Further clinical trials involving inhibitors of I~:B kinases, the main kinases that activate NF-~:B, are expected to follow. As the understanding of the signalling pathways that regulate the inflammatory process in the rheumatoid joint increases, and as kinase inhibitors become more specific, less toxic, cheaper and easier to administer, this approach is likely to provide the future generation of anti-inflammatory drugs for the treatment not only of rheumatoid arthritis but of chronic inflammatory and autoimmune diseases in general. Although for many years kinase inhibitors were not selective enough to be used as safe and effective medicines, the recent success of the bcr-Abl tyrosine kinase inhibitor Gleevec (STI571) from Novartis for the treatment of chronic myeloid leukemia demonstrates that this is feasible and paves the way for other inhibitors too.

CONCLUSIONS The identification and cloning of numerous cytokines over the last 20 years has provided new challenges for the scientist of today of how to translate this large amount of knowledge into therapeutic benefit. Cytokines play a major role in a number of physiological and pathological processes including immunity, inflammation, repair, fibrosis and cancer. We and others realized that cytokines may also be important in

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autoimmunity, during disease development, but also disease p e r p e t u a t i o n and tissue damage. Thus, we and others u n d e r t o o k the major task of elucidating which cytokines m a y be key or rate-limiting to the pathogenesis of r h e u m a t o i d arthritis, which lead to the definition of TNFa as a major therapeutic target. This was confirmed in clinical trials that we initiated over a decade ago, and also d e m o n s t r a t e d a remarkable degree of benefit, of blocking a single molecule in a complex disease. The success of anti-TNF~ therapy in r h e u m a t o i d arthritis p r o m p t e d anti-TNF~ clinical trials in a n u m b e r of related diseases, including Crohn's disease, psoriatic arthritis and ankylosing spondylitis, which also proved to be highly beneficial. This seems to be the beginning of a new wave of target therapies for a n u m b e r of related inflammatory diseases that is going to be based on the increased u n d e r s t a n d i n g of disease pathogenesis and the role cytokines play in that. Initially, this m a y take the form of anti-cytokine blocking agents, such as neutralizing antibodies and soluble receptors, but subsequently more specific therapeutic agents targeting the intracellular m e c h a n i s m s involved in cytokine gene expression m a y predominate. Durability of the benefit, safety and p h a r m a c o e c o n o m i c issues will determine w h e t h e r this early promise will materialize and w h e t h e r it will prove to be a major breakthrough to the t r e a t m e n t of incurable and painful diseases.

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CLINICAL APPLICATION OF CYTOKINES AND CYTOKINE INHIBITION

(a)

1!4

Total cells (90%)

CD3+ CD14- (80%) FL

'!!'

1 -I--I

-

_'

CD14+ CD3- (98%)

CD3- CD14- (98%)

(b) 500

-

400

-

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100

-

, untreated

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FIGURE 52.6 (a) More than 90% of rheumatoid synovial cells can be infected with replication-deficient adenoviruses as shown by measuring f)-galactosidase activity by FACS. Rheumatoid T cells, macrophage-like and fibroblast-like cells are all efficiently infected (with permission from Bondeson et al. 1999a). (b) Infection of rheumatoid synovial cells with an adenovirus overexpressing I~:Ba (AdIKBa) but not a control adenovirus without insert (ADO) inhibits TNFa production (taken with permission from Foxwell et al. 1998).