Molecular insights into rheumatoid arthritis.

Molec.AspectsMed.Vol. 12, pp. 341-394,1991

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MOLECULAR INSIGHTS INTO RHEUMATOID ARTHRITIS D. L. Scott, D. A. W i l l o u g h b y a n d D. R. B l a k e

The Departments of Rheumatology and Experimental Pathology, St Bartholomew's Hospital Medical College, Charterhouse Square, London, U.K. The London Hospital Medical College, Whitechapel Road, London, U.K.

Contents

INTRODUCTION

342

THE PATHOGENESIS OF RHEUMATOID ARTHRITIS

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COMMUNICATIONS BETWEEN CELLS

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3.1 3.2 3.3

The Hypothalamic-Pituitary-AdrenalAxis Cytokines and growth factors Adhesion molecules

347 348 354

NEUROENDOCRINE MECHANISMS

359

THE EXTRACELLULAR MATRIX

362

PROTEOLYTIC ENZYMES

368

FREE RADICALS AND PROGRAMMED CELL DEATH

372

CONCLUSIONS

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1 INTRODUCTION

1.1

General Background

There is a paradox in the current understanding and treatment of inflammatory arthritis. On the one hand there has been an enormous increase in knowledge about the pathogenic mechanisms in arthritis and many new anti-rheumatic drugs have become available in the last 20 years. On the other hand the end results of arthritis and the long term effects of treatment remain poor, and the outcome for patients with inflammatory joint disease had not changed substantially in the last two or three decades. Hopefully increasing knowledge and new therapeutic approaches may substantially alter the course of arthritic disease, and it is likely that this will soon be the case for many patients with joint diseases. Our main objective in this review is to assess the importance of some of the changes in our understanding of arthritis and the potential value of new therapeutic approaches. Although there are many diverse types of arthritis, we will concentrate on the main form of inflammatory arthritis, rheumatoid arthritis, since understanding its pathogenesis will help elucidate the major mechanisms acting in other forms of peripheral arthritis, as there are considerable overlap between these disorders. Recent advances in our knowledge of rheumatic diseases converge from a variety of different disciplines; though they principally involve immunology and biochemistry. Attempts to place these into order of importance are inevitably eclectic, but they span the genetic basis of arthritis, ecosanoids and other inflammatory mediators, the cytokine network, free radicals, proteolytic enzymes, cell surface proteins and extracellular matrix proteins, T cells and the immune system, and the biochemistry of cell division and cell death. Studies in this area are a mixture of clinical investigations using samples from rheumatoid patients, in vitro studies using isolated lymphocytes and macrophages or cultured synovial cells, and in vivo studies using experimental models of arthritis. The genetic basis of rheumatoid arthritis is complex, and lies outside the scope of the present review. At the same time it may account for the development of the disease in some cases or explain its clinical course and progression in others. There is a clear cut association between the presence of the class II antigen HLA DR4 and the development of rheumatoid arthritis, although not all subjects with HLA DR4 have the disease; indeed only a minority have synovitis. Also not all patients with rheumatoid arthritis are HLA DR 4 positive, though those who are have more severe disease and are more likely to show progression of their arthritis. It is possible that there are specific subtypes of the DR4 antigen which are of especial importance in relation to rheumatoid arthritis, and this is the centre of current controversy.

Molecular Aspects of Rheumatoid Arthritis

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There are many mechanisms of potential pathogenic importance in rheumatoid arthritis, and it is impossible to summarise them all. Instead we have chosen to examine several of the most important mechanisms and to show how they inter-relate to each other. Thes~ include communications between cells, the roles of the extracellular matrix, proteolytic enzymes, neuroendocrine mechanisms, free radicals and of programmed cell death. These all relate to interactions between cells and their surrounding milieu in the rheumatoid synovium and thus relate to the molecular biology of rheumatoid disease.

1.2

Potential Novel Therapeutic Targets

These are several areas of theoretical interest as possible therapeutic targets to alter the inflammatory reaction or joint destruction in rheumatoid arthritis. The main novel areas for intervention at the molecular level are:

(a)

Local cell to cell communication, such as influencing cytokine and growth factor function, and cellular adhesion molecules.

Co)

More general cell to cell communication, either through the hypothalamic-pituitary-adrenalaxis, or through various neuro-endocrine mechanisms.

(c)

Altering the degradation of the local extracellular matrix by proteolytic enzymes.

(d)

Effecting reactive oxygen species and the associated mechanisms of programmed cell death and DNA damage.

Each of these areas are major sites of action for current anti-rheumatic drugs, with the exception of the effects of corticosteroids on the hypothalamic-pituitary-adrenal axis, though this is a secondary effect. The introduction of drugs acting in any of these areas could be a major innovation in the treatment of chronic arthritis. In recent years there has been a paucity of novel anti-rheumatic drugs, and there is a need for new therapies which will either improve the symptoms of active synovitis or have potential disease modifying roles.

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2 THE PATHOGENESIS

2.1

OF RHEUMATOID

SYNOVITIS

Main Pathological Changes

The pathology of the rheumatoid synovium has been described by several workers in considerable detail (Gardner, 1972; Fassbender, 1975). It is a chronic inflammatory synovitis characterised by an increased number of cells on the surface of the synovium, moderate numbers of lymphocytes in perivascular areas, together with aggregates of lymphocytes and plasma cells in the subintimal synovium, prominent new vessel formation in the synovium, and an increased connective tissue matrix into which the inflammatory cells have migrated. There are also considerable amounts of fibrin superficially within the synovium. The synovial lining cells are of macrophage and fibroblast lineage. They show a variable increased number in rheumatoid synovia resulting from local proliferation and recruitment of macrophage-like cells. Underlying the lining cell layer is a variable subintimal layer and beneath that a more dense fibrotic collagenous layer. The subintimal synovium contains many blood vessels, reticular connective tissue components, tissue macrophages and occasional giant cells, and a variable lymphocytic and plasma cell infiltrate. Lymphocytes often form follicular aggregates. There is some fibrosis, though this is inconsistent. At the margins of the synovium is the pannus which overlies the articular cartilage. It is often difficult to define a lining cell layer in the pannus and a continuous mass of cells is seen adjacent to the cartilage margins which extends into the deeper tissues. Most cells of the pannus are large and mononuclear. Many have fibroblastic appearance. There is debate as to whether these cells are chondrocytes released from their matrix or soft tissue fibroblasts which have "invaded" the cartilage. LymFhocytes, plasma cells and other components such as most cells are also seen in the pannus. The main features of rheumatoid synovitis are summarised in Table 1 and are shown in Figure 1. None of these are specific for rheumatoid arthritis. The characteristic feature of rheumatoid synovitis is its chronicity, and understanding the pathogenesis of the disease may require different approaches to unravelling its initiation and its persistence.

2.2

Relationship to Potential Therapeutic Targets

There are several reasons why cell-cell interactions, neuroendocrine mechanisms, the destruction of the synovial joint extracellular matrix by degradative mechanisms, and the effects of free radical species may have important pathogenic roles in rheumatoid synovium. These are described in detail


345

in the ensuing sections. But the general principles outlining their potential value as therapeutic targets merit some consideration. The main potential therapeutic targets can be summarised as follows: (a)

Inflammatory cells are localised within a joint and there is consequent hyperplasia of the synovium and local features of inflammation.

Co)

The inflammatory synovitis persists and is associated with systemic features of chronic inflammation.

(c)

There is tissue remodelling and destruction of the normal architecture of the joint.

These processes involve signalling between cells and their surrounding matrix and subsequent enzymic and free-radical mediated tissue damage. Although there are a whole range of different factors involved in signalling between cells, the areas of most current interest include cytokines, adhesion molecules, and steroids and neuro-endocrine factors, and these have formed the focus of this review.

TABLE 1

The main pathological features of rheumatoid arthritis

Synovial lining cell hyperplasia Superficial fibrin deposition Vascular proliferation Lymphocytic infiltration and variable lymphocytic aggregation Macrophages and plasma cells in subintimal synovium Variable synovial fibrosis Marginal pannus over articular cartilage Destruction of adjacent articular cartilage and bone

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Superficial fibrin deposit

Hyperplastic synovial lining layer

Increased vascularity often with 'cuffing' of lymphocytes

Follicular aggrigates of lymphocytes Deeper areas of fibrosis Figure 1 The pathology of the arthritic joint

q>%


3 COMMUNICATIONS

BETWEEN

347

CELLS

Cells communicate with each other in a variety of ways, and there have been major advances in our understanding of the different mechanisms involved in recent years. They can interact hormonally. Although the functions of the endocrine system have been known for many years, there has been a recent resurgence of interest in the roles of hormones in modulating the inflammatory response of arthritis, especially the part played by corticosteroids (Wilder and Sternberg, 1990). There are two other main mechanisms for cell communication relevant in arthritis: the first is the production of soluble cytokines such as interleukins, interferons, and tumour necrosis factor; the second is the interaction between the related receptor molecules on cell surfaces and their associated ligands on target cells, which are often referred to as adhesion molecules.

3.1 The Hypothalamic-Pituitary-Adrenal Axis There has been a resurgence of interest in the role of neuroendocrine factors in arthritis stems from studies of experimental arthritis in rats, in particular the streptococcal cell wall model (Wilder et al, 1989). In this model one inbred strain, Lewis female rats, are highly susceptible to developing severe T lymphocyte-dependant proliferative and erosive arthritis after intraperitoneal injection of streptococcal cell walls. Conversely another strain, Fischer female rats, develop only a minimal, transient arthritis following an identical injection of cell walls. A series of innovative studies using this model (Sternberg, Hill, et al, 1989; Sternberg, Young et al, 1989) have shown that Lewis rats have a very poor response from their hypothalamic-pituitary-adrenal axis to the stress of intraperitoneal injections of streptococcal cell walls. In comparison Fischer rats show a good stress response with rapid increases in serum ACTH and corticosteroid levels as well as increased hypothalamic release and synthesis of corticotrophin releasing hormone. Giving the Lewis rats physiologic levels of corticosteroids inhibits the development of arthritis. By contrast blocking the effects of corticosteroids in Fischer rats using a corticosteroid receptor antagonist leads to the development of severe arthritis in response to streptococcal cell walls. These results show the potential importance of the hypothalamic-pituitary-adrenal axis in the development of arthritis. Several years ago Munck et al (1984) suggested that the production of corticosteroids in the stress response of the hypothalamic-pituitary-adrenalaxis was counter-regulatory and suppressed inflammation mediated by the immune system. The experiments with Lewis rats supports this concept; Sternberg and her colleagues' findings imply that a relative deficiency of corticosteroids may allow the development of unchecked immune activation and the persistence of arthritis in the Lewis rats. But does this have any clinical significance? It is unlikely that corticosteroids can be used therapeutically at the initiation of rheumatoid arthritis because of the problems in defining when this is occurring. But the case for using corticosteroids therapeutically may be strengthened.

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Treating rheumatoid patients with prednisolone undoubtedly alters the course of their disease (Joint Committee of the Medical Research Council and the Nuffield Foundation, 1959; Weiss, 1989). However the high doses needed cause the unacceptable long-term problems of excessive cortico-steroid levels, the consequences of iatrogenic Cushing's syndrome. When therapy with cortico-steroids is undertaken in this way massive supra-physiologic doses are used. It is possible that more appropriate physiological doses may effect the pathological changes of rheumatoid disease without causing unwarranted adverse effects. These concepts of a new therapeutic role for steroids need evaluation.

3.2 Cytokines and Growth Factors Cytokines, cytokine inhibitors and other growth factors are recognised as important mediators of inflammation and joint destruction in rheumatoid arthritis (Arend and Dayer, 1990). The results of many in vitro and in vivo studies suggest cytokines may mediate cell-cell interactions that lead to the release of tissue damaging enzymes (Dayer and Demczuk, 1984). Much current research directed at finding new anti-rheumatic drugs is directed at agents which block the synthesis, release or effects of particular cytokines. Terminology is difficult in this area. Duff (1989) suggested an appropriate general term should be 'peptide regulatory factors', but this has not found widespread acceptance. Several classes of peptides are involved including interleukins, tumour necrosis factors, interferons, and a variety of peptide regulatory factors. These are summarised in Table 2. In rheumatoid arthritis the main site for the synthesis of these peptides in the CD14 positive tissue macrophages, including those in the synovial lining layer.

3.2.1 lnterleukin-1

Interleukin-1 (IL-1) is a 17-kdalton protein which is primarily a product of monocytes and macrophages (Dinarello, 1989). Two forms of IL-1 (o~ and f~) have been described with amino acid sequence homology of about 26%. IL-1I~ is the predominant form synthesised by human monocytes. Both forms bind to the same cell surface receptor. The systemic effects of IL-1 include fever, decreased appetite and the induction of metabolic changes. In rheumatoid arthritis the local effects of IL-1 may be more important. These local effects include: augmentation of T and B lymphocyte function; chemotaxis of neutrophils and other cells; proliferation of fibroblasts; and the production of prostaglandin Fa and collagenase by fibroblasts and chondrocytes. These latter effects have been studied in detail in cultured synovial cells and chondrocytes, and IL-1 could be a major pathogenic mediator of joint destruction in rheumatoid synovitis by stimulating the production and release of destructive enzymes (Dayer et al, 1986; Evrquoz et al, 1984). IL-1 may also play a role in the initiation of rheumatoid synovitis by assisting with the migration of other cells and by stimulating the responses of endothelial cells. IL-1


349

induction of PGF_a and collagenase production may also be responsible for some of the bone resorption and cartilage destruction of the latter stages of rheumatoid arthritis.

TABLE 2

Cytokines and growth factors involved in arthritis

Cytokines influencing synovial cells

Cytokines influencing neutrophils

Growth factor influencing synovial cells

Interleukin 1 Interleukin 6 TNFot

Interleukin 8

PDGF ILGF FGF TGFI~ CTAP III

In addition IL-1 may contribute to joint scarring and fibrosis by stimulating fibroblast proliferation either directly or indirectly through the induction of platelet derived growth factor and other peptide regulatory factors (Postlethwaite et al, 1988: Raines et al, 1989). It additionally affects the synthesis of extracellular matrix proteins, stimulating the production of fibronectin (Krane et al, 1985) and collagen type I (Postlethwaite et al, 1988) and reducing the synthesis of collagen type II by chondrocytes (Goldring et al, 1988). These effects of IL-1 on synovial fibroblasts represent an example of agonist and antagonist influences; both the mechanisms of tissue destruction and repair can be activated by the same cytokine. Studies on the levels and activities of IL-1 in rheumatoid disease have given conflicting results. Investigations using bioassays for IL-1 have given very variable results, possibly due to the presence of varying amounts ofinhibitors. Immunoassays have usually shown increased amounts of the cytokine in rheumatoid synovial fluid (Hopkins et al, 1988), while it is often barely detectable in the plasma. A comparison of immunoassay and bioassay results by Smith et al (1989) showed relatively poor concordance between different measurements of IL-1, suggesting variable amounts of IL-1 inhibitors were present. However there is evidence that synovial fluid IL-1 levels are related to general inflammatory activity indicated by the ESR and serum C-reactive protein level in rheumatoid patients (Mannami et al, 1989). Although synovial cells may not necessarily produce high levels of IL-1

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(Yamagata et al, 1988), rheumatoid synovial tissue obtained at arthroscopy synthesises large amounts of IL-1 in vitro.

3.2.2 lnterleukin-1 inhibitors

A variety of natural IL-1 inhibitors have been identified in cell supernatants and in human urine (l_arick, 1989). But the term 'IL-1 inhibitor' is a general one, and such substances could potentially act at many different levels in specific or non-specific ways; such as reducing IL-1 synthesis, binding to IL-1, blocking IL-1 receptors, and interfering with the post-receptor effects of IL-1. Arend et al (1989) characterised a 22-kdalton protein which acts as an IL-1 inhibitor by binding to the IL-1 receptor on immune and inflammatory cells and prevents IL-1 interacting with its target cells.

3.2.3 Tumour Necrosis Factor

Tumour necrosis factor c~ (TNFc0 is a 17-kdalton peptide produced by monocytes and macrophages. It is usually secreted together with IL-1, although production of these two proteins is apparently regulated and controlled independently. As monocytes mature into macrophages their ability to produce IL-1 decreases while TNFa production is relatively unaffected. TNFc~ binds to separate receptors on target cells, though it has similar biological functions to IL-1; for example it also stimulates PGEz and collagenase production by synovial cells cultured in vitro (Dayer et al, 1985). TNFa is present in rheumatoid synovial fluid and is synthesised by synovial tissue (Yocum et al, 1989). It is present in the majority of rheumatoid synovial fluid samples, especially in active disease (Saxne et al, 1988). In rheumatoid synovial biopsies it is predominantly in synovial lining cells and interstitial macrophages (Husby and Williams, 1988). Similar to IL-1 the local effects of TNFo~ may be counteracted by regulatory or inhibitory proteins. A specific inhibitor of TNFc~ is present in the urine of febrile patients and the supernatants of cells cultured from rheumatoid synovial fluids (Seckinger et al, 1988). This TNFa inhibitor is a 31-33 kdalton protein, which is different from the IL-1 inhibitor. It has been purified to homogeneity and carefully characterised and is unrelated to other inhibitory proteins (Seckinger et al, 1989). The TNFa inhibitor may be a soluble version of the cell surface receptor (Novick et al, 1989).

3.2.4 Interleukin 6

Interleukin 6 (IL-6) is a 26-kdalton cytokine that is produced by monocytes, T lymphocytes, and fibroblasts (Wong and Clark, 1988). Both IL-1 and TNFc~ induce the synthesis and secretion of IL-6.


351

Although IL-6 has similar actions to IL-1, it is a more potent inducer of hepatic synthesis of acute phase proteins and of immunoglobulin production by B lymphocytes. High levels of IL-6 are present in rheumatoid arthritic synovial fluids, and cultured synovial cells synthesise IL-6 (Guerne et al, 1989). IL-6 may both amplify some of the effects of IL-1 and TNFot and also induce the synthesis of acute phase proteins and rheumatoid factors. The interaction between IL-1 and IL-6 is not confined to synovial lining cells. Bender et al (1990) showed IL-1 induced IL-6 synthesis in human articular chondrocytes cultured in vitro, and these authors suggested the induction of IL-6 in this way could contribute to damage to the cartilage matrix in joint inflammation.

3.2.5 lnterleukin 8

Interleukin 8 (IL-8) is a novel cytokine which activates neutrophils. It is also known as neutrophilactivating peptide-1 (Baggiolini et al, 1989). IL-8 is generated as a 99 amino acid precursor with a characteristic leader sequence of 22 amino acids; several mature forms have been identified. It induces a range of responses in neutrophils including the expression of surface adhesion molecules and the production of reactive oxygen metabolites. IL-8 is a product of mononuclear phagocytes (Van Damme et al, 1988) and also fibroblasts and other cells. In rheumatoid arthritis IL-8 could bring about the accumulation of neutrophils which are considered a major source of cartilage degrading enzymes (Baggiolini et al, 1979). Preliminary studies (Seitz et al, 1990) have shown that IL-8 levels are high in synovial fluids from patients with rheumatoid arthritis.

3.2.6 Cytokine Regulation

How are the large number of different cytokines in the inflamed synovium regulated? In vitro studies of the synthesis of cytokine mRNA show there is only brief expression after stimulation. In contrast studies on rheumatoid synovial biopsies show, by in situ hybridisation, that there is persistent expression of mRNA to IL-1, TNFa, IL-6 and other cytokines (Brennan et al, 1991). This stability and persistence of cytokine production suggests it plays an important role in the pathogenesis of the chronicity of synovitis in rheumatoid disease. When synovial cells from rheumatoid joints are cultured in vitro the production of mRNA for IL-1 ~ and 1~persists for at least 5 days (Buchan et al, 1988). The interactions of different cytokines in the pathogenesis of synovitis is summarised in Figure 2. There is evidence that TNFot is the dominant signal regulating IL-1 production (Brennan et al, 1991), although other non-cytokine signals such as immune complexes may also be of significance (Chantry et al, 1989). There is similar evidence from experimental models of arthritis that production of TNFa precedes the production of IL-1 and IL-6 (Feldmann et al, 1990). There is therefore an apparent dominant position of TNFa in the cytokine response in rheumatoid disease.

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3.2. 7 Fibroblast Growth Factors

Fibroblast growth factors (FGF) are a family of structurally related proteins whose name does not do justice to their biological properties. At least 7 proteins have been isolated and the family includes acidic and basic FGFs, and a number of proto-oncogene products. They have low homology (19-25 %) with IL-1 tx and 1), which are quite different from members of the FGF group of proteins (Anonymous, 1990a). Basic FGF was the first to be isolated and biologically characterised and serves as example for proteins of this group. It is a 17 kdalton single chain polypeptide which is widely distributed in tissues. There is a high-affinity receptor for basic FGF (Lee et al, 1989). Basic FGF increases DNA synthesis and cell division and has a number of other effects on cells including a role in angiogenesis and the potential to act within cells as an intracrine growth factor (Logan, 1990). The ability of basic FGF to stimulate vascularisation and the formation of granulation tissue is important in wound healing (Buntrock et al, 1990) and this may also be relevant in rheumatoid arthritis.

TNF-e inhi

i,or

Tissue Destruction

IL-1 ----~ m t _ _ ~ . t ~

I

Tissue Remodelling

Figure 2 The interactionof cytokinesand cytokineinhibitorsin inflammatorysynevitis


353

Melnyk et at (1990) have shown that cultured rheumatoid synovial cells express the gene for basic FGF, secrete the protein, and also proliferate in response to basic FGF. The implication of their findings is that synovial cells themselves may play a role in stimulating their proliferation in an autocrine manner through modulators such as basic FGF. There is also evidence from studies by Sano et al (1990) that rheumatoid joints produce relatively large amounts of acidic FGF.

3.2.8 Transforming Growth Factor fl Transforming growth factor fl (TGF-g) was originally isolated as a 25 kdalton protein from platelets (Assoian et al, 1983). It is present in various inflammatory cells such as lymphocytes and macrophages (Kehrl et al, 1986; Assoian et al, 1987). It has potent immunomodulatory effects (Wahl et al, 1989). Although there are five different forms of TGF-g, the evidence suggests only TGF-gl and TGF-B2 are important in rheumatoid arthritis (Allen et al, 1990). TGF-I] has several functions which are potentially important in the pathogenesis of rheumatoid synovitis. These include: promotion and inhibition of flbroblastic cell growth (Sporn et al, 1987) and facilitation of extracellular matrix remodelling (Keski-Oja et al, 1988). In studies of rheumatoid synovial fluid Fava et al (1989) showed evidence of TGF-g-like activity by several criteria including: TGF-g receptor competition; immunological neutralisation of biological activity; and an increase in the number of cells growing in soft agar (a complex measure of cell transformation, which is stimulated by growth factors such as TGF-g). They found that TGF-I] in synovial fluid is often present in a latent form. High levels of synovial fluid TGF-g have also been reported by Brennan et al (1990), though these workers did not find especially elevated levels in rheumatoid arthritis; levels were similarly high in other arthropathies. Analysis of the isoforms of TGF-g in rheumatoid synovial fluid has shown that the majority is TGF-g2 (Lotz et al, 1990). There is evidence from Wahl et al (1990) TGF-g is a key cytokine in rheumatoid arthritis: it both inhibits IL-1 induced lymphocytic proliferation, but also promotes synovial fibroblast hyperplasia and pathology. The same workers have demonstrated the effectiveness of TGF-g in inducing synovial hyperplasia by local intra-articular injection in experimental models of arthritis (Allen et al, 1990).

3.2.9 Platelet Derived Growth Factor Platelet derived growth factor (PDGF) is released from the t~ granules of platelets during blood clotting (Deuel et al, 1985). PDGF-like polypeptides are released from a variety of mesenchymal cells such as macrophages (Martinet et al, 1986). Biologically active PDGF consists of two subunits, A and B, that form disulphide-linked homodimers and heterodimers. PDGF is mitogenic for fibroblasts and chondrocytes (Olashaw and Pledger, 1988). To elicit a mitogenic response PDGF must interact with

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a specific receptor on the cell surface; a glycoprotein of 180 kdalton (Pope-Brown et al, 1985) with tyrosine kinase activity in its cytoplasmic domain (Yarden et al, 1986). PDGF is the major mitogen in serum for connective tissue cells (Ross et al, 1986).

3.3 Adhesion Molecules Adhesion molecules have several functions in inflammatory diseases such as rheumatoid arthritis (Anonymous, 1990b). Not only they are involved in cell-cell adhesion but they also take part in antigen recognition (Dustin and Springer, 1989), act as co-stimulatory signals in T-cell activation (Shimizu et al, 1990), and stimulate effector mechanisms of activated lymphocytes such as cytotoxicity (Krensky et al, 1984; Kohl et al, 1984). To make the situation even more complicated some adhesion molecules interact with extra-cellular matrix components such as fibronectin and laminin; also some molecules principally involved in antigen recognition, such as the major histocompatibility complex antigens can also act as adhesion molecules in certain circumstances (Doyle and Strominger, 1987; Norment et al, 1988). The adhesion molecules act as specific cell surface receptors for a variety of different ligands. There are three main groups of adhesion molecules each binding to different ligands (Larson and Springer, 1990; Springer 1990). These are: molecules of the immunoglobulin supergene family; the integrins; and the selectins. Each of these has different characteristics and potential pathogenic roles in inflammatory synovitis. The classification and nomenclature of these proteins is complicated and still evolving; a simplifcation is shown in Table 3. Immunohistological studies have shown that non-inflamed synovial tissue has no staining for the PDGFreceptor (Rubin et al, 1988); in cases of active synovitis there is considerable amounts of immunoreactive PDGF-receptor. It is found around proliferating blood vessels, on strand connective tissue cells, and is especially marked on the fibroblast-like synovial lining cells. Immunoreactive PDGF-receptors are also prominent in marginal pannus tissue close to cartilage and bone. In vitro studies of cultured quiescent human fibroblasts have shown that PDGF stimulates DNA

synthesis. But unlike IL-1 and TNF-ot, PDGF does not stimulate PGE 2 synthesis; this is important because it does not thus generate a negative feedback loop on cellular proliferation. Another implication of this finding is that different cytokines may act on human synovial cells through different pathways. However, there is some controversy about the effects of PDGF on prostaglandin synthesis by synovial cells, because van Kempis et al (1989) have reported that PDGF does increase the production of PGFa by cultured synovial cells.


TABLE 3

Simple classification of adhesion molecules

Families

Subfamilies

Immunoglobulin Supergene

T cell receptor/CD3 CD2 receptor/LFA3

Integrins

gl integrins

VLA 1-6

f12 integrins

LFA1 Mac-1 p150,95

g3 integrins

Vitronectin receptor Platelet glycoprotein II6/IIIa

Selectins

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Examples

GLAM-1 LAM-1 PADG-EM

Adhesion molecules from the immunoglobulin supergene family include receptors which react with antigens and antigen-independent receptors. Examples of the former group are the T-cell receptor/CD3 and surface immunoglobulin. An example of the latter group is the CD2 receptor which reacts with the ligand lymphocyte function associated molecule-3 (LFA-3). The integrins are a different family of adhesion molecules. They are transmembrane glycoproteins with an t~/B heterodimer structure. They are further subdivided into 3 groups on the basis of different structures of their B-chains; these are g~ - 1~3 integrins. The 13~integrins are also called the VLA (very late activation) subfamily. It has 6 members termed VLA 1-6. These molecules are widely distributed throughout the tissues and principally interact with extracellular matrix components such as fibronectin, laminin and collagen. They are important in wound healing and cell migration in tissue remodelling and repair. The g2 subfamily is also known as the leucocyte integrins and has 3 members. These are:

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LFA-1, which binds to the ligands ICAM-1 (intercellular adhesion molecule-l) and ICAM-2; Mac-1 which binds to the ligand iC3b; and p150,95 which also binds to the ligand iC3b. Leucocyte integrins are exclusively expressed on leucocytes and are predominantly involved in immune adherence; they all belong to the CD11/CD18 heterodimer complex. The 1~3 integrins are known as the cytoadhesion subfamily. There are 2 members: the vitronectin receptor (VNR) and platelet glycoprotein lib/Ilia. These are expressed on endothelial cells and platelets respectively. The 1~3 integrins have similar ligands and functions to the VLA molecules. An example of the interaction of integrins with the extracellular matrix is shown in Figure 3.

cytoskeletal connections

/\ IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIl lllllllllllllllll[llllll cell membrane

integrin

131

5

C J

~

fibronectin in matrix

Figure 3 An example of integrin structure showing an interaction with fibronectin


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The integrins are an especially important group of adhesion molecules in chronic inflammatory disorders, and the last few years have seen a massive increase in the amount of work in this field. Several factors have contributed to fast progress. One important aspect was that the discovery of integrins brought together a large number of separate observations when it was recognised that a group of chicken adhesion proteins, the platelet protein gp Ilb/IIIa, a group of lymphocyte adhesion proteins (the VLA family of cell surface receptors), and receptors for fibronectin and vitronectin all had related structures and activities. Another factor was that work on integrins was preceded by many years of detailed evaluation of the extracellular matrix proteins for which integrins are the cell surface receptors. The name integrin was introduced to signify the role of these proteins in integrating the intracellular cytoskeleton with the extracellular matrix (Ruoslahti, 1991). The final group of adhesion proteins are the selectins. They are expressed on leucocytes and endothelial cells and are involved in leucocyte adhesion to endothelium in acute inflammation. There are 3 members of this group: endothelial cell adhesion molecule- 1 (ELAM- 1); leucocyte adhesion molecule- 1 (LAM-1); and platelet-activation-dependant granule-external membrane protein (PADG-EM, CD26). A wide variety of extracellular and intracellular factors can modify the expression of adhesion molecules, and this can be a quantitative or qualitative change (Arnaout, 1990). Factors which are known to upregulate the expression and/or adhesiveness of these molecules include: exposure to cytokines such as interleukin-1 and tumour necrosis factor (Rothlein et al, 1988); adhesion to extracellular matrix proteins such as fibronectin (Dougherty, Murdoch and Hogg, 1988); T-cell activation (Shimizu et al 1990); and viral infection (Wang et al, 1988). The distribution of cell adhesion molecules within the rheumatoid synovium has been studied immunohistologically by Hale et al (1989). In studies of frozen sections of the synovium they found that antibodies directed against LFA-3 and ICAM-1 both reacted with macrophage-like type A synovial cells and synovial fibroblasts, as well as with tissue macrophages and vessel endothelium. Using flow cytometry they found that anti-LFA-1 and anti-ICAM-1 reacted with synovial fibroblasts cultured in vitro; but antibodies to their ligands, CD2 and LFA-I did not react. These fndings showed that ligands for lymphocyte LFA-1 molecules and for T cell CD2 molecules (LFA-3) are widely distributed among cell types of the synovial microenvironment and provide numerous cell types with which lymphocytes can interact via these two adhesion pathways as part of the pathogenesis of rheumatoid arthritis. Hale et al also found that ICAM-1 expression on synovial fibroblasts is induced by both gamma interferon and tumour necrosis factor. There are several ways that adhesion molecules may take part in the pathological changes of rheumatoid synovitis. They are implicated in both the acute and chronic inflammatory responses, and also in the recirculation of lymphocytes and their localisation. A model of their role in acute and chronic inflammation is given by the various forms of hepatitis, including acute and chronic viral hepatitis. Expression of ICAM-1 on hepatocytes is increased in these forms of liver disease (Volpes

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et al, 1990). Similar patterns of change occur in the distribution of ICAM-1 on keratinocytes in the inflammatory dermatoses (Nickloff et al, 1990), on cerebral vascular endothelium in viral encephalitis and multiple sclerosis plaques (Sobel et al, 1990), and on thyroid epithelial cells in autoimmune thyroiditis (Weetman et al, 1990). The roles of adhesion molecules in lymphocyte recirculation and localisation are somewhat different, but they are also potentially relevant in rheumatoid synovitis. Lymphocytes from adults, when collected from specific lymph nodes, show a preference for recirculation to the type of node from which they came (Yednock and Rosen, 1989). This suggests priming by specific antigen may alter surface phenotype to enable selective recirct, lation of lymphocytes. As lymphocytes from the blood enter lymph nodes by binding to specialised 'high' endothelial cells an interaction between adhesion molecules and their ligands at these sites will be especially important in recirculation. Using blocking experiments with monoclonal antibodies a variety of cell adhesion molecules have been implicated in the 'homing' of lymphocytes (Butcher, 1986; Stoolman, 1989); these include LFA-1, VLA-4 and LAM-1 which are all found on lymphocytes. 'High' endothelial cells are found in the rheumatoid synovit, m and these interactions may be important in the 'homing' of lymphocytes to the synovium. The presence of LFA-3 on synovial cells of both macrophage and fibroblast lineage may also be significant in the arrival of lymphocytes in the synovium. Experiments by Haynes et al (1988) showed that LFA-3 positive synovial fibroblasts bind thymocytes. This binding is inhibited by antibodies to LFA-3. There may also be interactions between circulating inflammatory cells and the extracellular connective tissue matrix of the synovium. For example the B~ integrin VLA-4 is expressed on resting lymphocytes and monocytes and functions as both a cell receptor and a matrix receptor with fibronectin acting as a ligand (Hemler, 1990). There is extensive evidence that fibronectin is a major extracellular matrix protein in the rheumatoid synovium. Modulating the expression of adhesion molecules in inflammatory diseases experimentally by the use of specific antibodies, such as anti- CD11/CD18 antibodies, prevents the migration of leucocytes in animal models of acute inflammation and reperfusion injury (Arnout, 1990; Carlos and Harlan, 1990). But it is unwise to extrapolate these findings to human diseases, especially as the use of antibodies in an acute inflammatory situation is unlikely to be beneficial. Despite such reservations it may be possible to use alternative approaches to alter the interaction between receptor and ligand in the function of adhesion molecules and thereby favourably influence adhesion molecule function.


4 NEUROPEPTIDES

359

AND SYNOVITIS

The so-called "diffuse neuroendocrine system" is characterised by the synthesis and release of bioactive peptides at many sites throughout the body. Some of these peptides act as classical circulating hormones, while others have a more restricted effect, acting only in the vicinity of their release from endocrine, epithelial or neuronal cells at potent modulators, neurotransmitters, or trophic cells (Polak, 1989). More than 40 such regulatory peptides have now been identified and they form an ever expanding family of substances. Peptides produced within the nervous system - the neuropeptides are part of the inflammatory cascade. Until recently the innervation of articular tissues and the influence of nerves on the pathophysiology of joint disease had received little attention. But evidence is now emerging to show they have a far more important role than was previously reposed.

4.1 Innervation of Joints For many years it has been assumed that the nerve supply of the joint is minimal and relatively inconsequential (Freeman and Wyke, 1967). But detailed histological and immunohistological studies has in fact shown there is significant innervation of synovial joints. Classical histological studies using stains, such as methylene blue and silver stains, together with sensitive immunocytochemical methods using antisera raised against specific components of nerves, have shown a large number of small diameter nerves in the synovium.

4.2 Neuropeptides in the Joint Many nerves in the normal synovium contain neuropeptides (Granblad et al, 1988; Mapp et al, 1988; Konttinen et al, 1990). The main peptides (summarised in Table 4) are substance P, Calcitonin generelated peptide, and neuropeptide Y, and these will be described in detail. Substance P is a small polypeptide of 11 amino acids which belongs to a family of peptides, the tachykinins; these share a sequence of six amino acids at their carboxyteminal. Substance P occurs within small primary sensory neurones and has an extensive distribution throughout the body (Lembeck and Gause, 1982). Intradermal injection of substance P causes vasodilation, plasma extravasation, itching and hyperalgesia (Foreman, 1987). It also activates neutrophils (Serr et al, 1988), effect lymphocytes (Stomisz et al, 1987), causes interleukin release from macrophages (Kimball et al, 1988, and release of prostaglandin E~ and collagenase from synoviocytes (Lotz et al, 1987).

360

TABLE 4

D.L. Scott et aL

Neuropeptides found in synovial joints

Substance P Calcitonin Neuropeptide Y Gallamine Vasoactive intestinal polypeptide Enkephalin

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide produced by alternative processing of the primary transcript of the calcitonin gene. It is abundant in the sensory nervous system and is present in about half of primary sensory neuroses (Gibson et al, 1984). CGRP is a potent vasodilator and causes a prolonged increase in microvascular blood flow when injected extravascularly (Brain et al, 1989). It potentiates oedema induced by other mediators such as substance P (Brain et al, 1985). Neuropeptide Y is a 36 amino acid peptide. It is associated with catecholaminergic nerves and is implicated in the potent vasoconstrictor effects on the vasculature associated with these nerves (Lundberg et al, 1982; Allen and Bloom, 1986). Neuropeptide Y is localised in nerve fibres around blood vessels in the synovium (Mapp et al, 1990), unlike the peptides substance P and CGRP which also occurs in free nerve fibres.

4.3 Neurotransmitters in Synovial Fluid These studies of neurotransmitters in synovial fluid have predominantly examined levels of substance P. Devillier et al (1986) found that inflammatory synovial fluids contained more tachykinin - like immunoreactivity than non-inflammatory fluids. More recently Marshall et al (1990) studied synovial fluid levels of substance P and found they were higher than those of paired plasma samples, suggesting local production of substance P.


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4.4 Neurotransmitters and the Development of Synovitis Sparing of completely paralysed links in patients with rheumatoid arthritis has been reported in hemiplegic cases (Thompson and Bywaters, 1962) and poliomyelitis (Glick, 1967); this suggests a neurological mechanism is involved, possibly modulated by neurotransmitters. Intra-articular injection of substance P into experimental animals produces plasma extravasation and vasodilation (Brain and Williams, 1988). This is inhibited by specific substance P antagonists, which also inhibit the early inflammatory responses to intra-articular carrageenin. Konttinen et al (1990) have studied the distribution of neuropeptides in adjuvant arthritis in the rat. Using antisera reactive to substance P and CGRP they found differences between the distribution of these neuropeptides in normal synovia. In the synovium from animals with adjuvant arthritis there was a specific reduction in the lining zone and subintimal nerves of the synovium.

4.5 Neurotransmitters in the Rheumatoid Synovium Immunohistological studies have provided evidence for a neurogenic influence on the chronic synovitis of rheumatoid arthritis, comparing the distribution of neuropeptides from normal and rheumatoid joints (Mapp et al 1990; Granblad et al, 1988; Pereira da Silva and Carmo-Fonseca, 1990). In normal joints nerve fibres are present throughout the articular tissues; free fibres are found in the synovial intimal and subliming layers with nerve tissues and perivascular fibres present in deeper tissue. Substance P and CGRP immunoreactive fibres were found as free fibres in the synovial membrane and around blood vessels in the subliming layer and deeper tissues. Neuropeptide Y immunoreactive fibres are localised exclusively in blood vessels. By contrast nerve fibres and peptide immunoreactive fibres are markedly depleted in the intimal and the subintimal layers of rheumatoid synovia. Pereira da Silva and Carmo-Fonseca (1990) developed a quantitative comparison of immunostaining for substance P, CGRP and neuropeptide Y and found all of those were significantly reduced in rheumatoid synovia. There are several possible explanation for the reduced nerves and neuropeptides in rheumatoid synovia. The loss of neuropeptide immunoreactivity could be due to their local release. Alternatively there could be a concomitant loss of all nerve fibres due to synovial tissue hyperplasia without proliferation of new fibres or to degeneration of pre-existing nerves. Further studies are needed to elucidate which of these mechanisms are involved.

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5 THE EXTRACELLULAR

MATRIX

The cells of the synovium interact closely with their surrounding extracellular matrix and this defines many of their interactions. Thus the extracellular matrix acts as a scaffolding and also controls a number of cellular activities including: cell shape, cell migration, cell division and differentiation (Hay, 1981). Components of the matrix can also trap autoantigens and thus too may contribute towards disease pathology (Lake et al, 1985). The main components of the extracellular matrix are: collagens; proteoglycans and glycosaminoglycans; structural glycoproteins; and elastins. They are summarised in Table 5. Although they are all involved in the pathogenesis of arthritis, elastins have a relatively minor role.

TABLE 5

Main components of the extracellular matrix

Type of Component

Examples

Distribution

Collagen

Collagen Collagen Collagen Collagen

Non-collagenous structural glycoproteins

Fibronectin Laminin Vitronectin

Widely distributed in connective tissue Basement membrane component More limited pericellular distribution

Proteoglycans and glycosaminoglycans

Hyaluronic acid Heparan sulphate Keratan sulphate

Widely distributed Basement membranes proteoglycan Cartilage

I and III II IV V

Interstitial connective tissues Cartilage Basement membranes Pericellular extracellular matrix


5.1

363

C o l l a g e n s o f t h e Joint

Collagen is a ubiquitous protein found in a variety of forms in all body tissues. The main forms of collagen in connective tissues are collagens I, II and Ill. They are fibre forming collagens with a triple helical structure. Collagens I and III are found in the synovium and associated fibrous structures. Collagen II is the main collagen of cartilage. There are a number of other minor collagens and these are summarised in Table 6. The basic collagen molecule consists of three polypeptide (c0 chains, arranged in a triple helical configuration. Different types of collagen have different c~ chains and helical structures.

TABLE 6

Type

I II III IV V VI VII VIII IX X XI

The types of collagen

Tissue Distribution in the Joint

Synovial membrane, joint capsule and ligaments Articular cartilage Synovial lining and vascular tissues Basement membranes Interstitial tissues Interstitial tissues Anchoring fibrils Endothelial cells Articular cartilage Hypertrophic and mineralising cartilage Articular cartilage

Collagen molecules can be classified into three groups: group 1 have a continuous uninterrupted helical domains (types I, II, III, V and XI); group 2 have interrupted helical domains (types IV and VII); and group 3 have short a chains (types VI, IX and X) (Miller, 1985).

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Collagen and its breakdown products are implicated in the process of inflammation; they are directly chemotactic for monocytes and fibroblasts to sites of injury and tissue damage in the early phases of the inflammatory process. Biochemical studies (Eyre and Muir, 1976; Lovell et al, 1978) of synovial membrane collagen by assay of hydroxyproline and cyanogen bromide peptide analysis show the collagen content varies from 25 % to 50% of dry weight; similar amounts of type I and III collagens were present in normal and arthritic synovia. Immunohistochemical studies have localised the various collagens in normal and inflamed synovia from rheumatoid arthritis and other arthropathies (Linck et al, 1983; Okada et al, 1990). Collagens III and VI are present in small amounts; and collagen IV is present in the basement membrane of blood vessels in the synovium. Collagen III in the subintimal layer is associated with their collagen fibrils and is related to the silver staining reticulin fibres of the synovium (Scott et al, 1984a). There is some debate as to whether collagen IV is present in relation to the synovial lining layer and Pollock et al (1990) have shown that type IV collagen is present around synovial lining cells. Collagen II is present only in the articular cartilage of the joint. But degradation of the collagen is a major factor in cartilage damage caused by rheumatoid synovitis. Moveland et al (1989) showed collagen type II was present in synovial phagocytes from rheumatoid patients in a study of synovial fluids. This is a potential sensitive immunohistochemical marker of cartilage erosion in rheumatoid disease and shows the potential importance of phagocytic cells in joint damage. In rheumatoid disease, there is a balance between the synthesis and degradation of collagens. The proliferation of the synovium leads to an increase in the amount of collagen within the joint and also to a change in its distribution, with increasing amounts of collagen III in the synovial lining cell layer and subintimal layer. Degradation of collagen and its associated components of the extracellular matrix is accomplished by proteolytic enzymes, which are considered in detail in section 6. The degradation products of collagen may have complex effects on cells and may play a role in signalling between cells, showing the variety of interactions between the extracellular matrix and synovial cells in rheumatoid disease.

5.2 Glycosaminoglycans and P r o t e o g l y c a n s These include hyaluronic acid; chondroitin sulphate, dermatan sulphate, keratan sulphate and associated molecules; and heparan sulphate proteoglycan. Hyaluronic acid, the only glycosaminoglycan not covalently bound to protein is a linear repeating disaccharide of high molecular weight (over 10,000 kdalton) and it is present in most connective tissues (Balazs et al, 1986; Laurent and Fraser, 1986). It also forms the central axis of the proteoglycan aggregates of articular cartilage (Hardingham and


365

Muir, 1972). Hyaluronate is responsible for many of the viscous properties of synovial fluid, but is also involved in many reactions in the tissues and is involved with a specific cell adhesion molecule (Miyake et al, 1990). The interaction between hyaluronate, its cell surface receptor, and the cell cytoskeleton may have important pathogenic roles in determining the chronic inflammatory response (Lacy and Underhill, 1987; Alho and Underhill, 1989). Several studies have suggested that high molecular weight hyaluronan inhibits cellular proliferation of endothelial cells (Goldberg and Toole, 1987), other connective tissue cells and lymphocytes (Annastassiades and Roberton, 1984). Immunolocalisation studies have shown there is a marked increase in hyaluronate with free binding sites 'for its associated link protein in the rheumatoid synovium (Worral et al, 1989), though further studies are needed to define its exact distribution. Hyaluronic acid is also present in the serum and its levels are high in rheumatoid disease. Finally in rheumatoid synovial fluid there is an increase in the total amount of hyaluronan but a decrease in its concentration and a decrease in its molecular size. The small size probably represents degradation after synthesis rather than defective synthesis (Prehm, 1990) and this may be due to oxidative damage by free radicals. Proteoglycans such as chondroitin sulphate and keratan sulphate are major cartilage constituents. There is evidence they are involved in the rheumatoid synovium. Worral et al (1990) showed, in immunohistochemical studies, that there is an abnormal zonal distribution of these sulphated proteoglycans in the rheumatoid synovium. They considered that this may indicate unsuspected heterogeneity of the rheumatoid synovial extracellular matrix. Heparan sulphate proteoglycans are the other major proteoglycan of the extracellular matrix. They are present as integral cell surface proteins and as constituents of basement membranes (Fujiwara et al, 1984). They have important roles in embryogenesis when they interact with laminin and collagen type IV. Although they have not been examined in detail in the rheumatoid synovium, it is likely they have a similar distribution to laminin and collagen IV in the synovium. There is evidence (Bradbury and Parish, 1989) that heparin sulphate proteoglycan interacts with lymphocytes via specifc cell surface receptors, and this represents another example of the interaction between the extracellular matrix and inflammatory cells.

5.3 The Structural Glycoproteins This is a heterogenous group of proteins which includes fibronectin, laminin, chondronectin, vitronectin and possibly associated proteins such as tissue P component. The different proteins are summarised in Table 7. An additional, poorly characterised component, is a non-collagenous reticulin component which is present in silver staining reticulin fibres in the synovium.

366

TABLE 7

D.L. Scott et aL

The structural glycoproteins of the joint

Protein

Size

Distribution in the Joint

Fibronectin

450 kd

Lining cell layer and synovium

Chondronectin

180 kd

Cartilage

Laminin

800 kd

Basement membranes

Vitronectin

Lining cell layer

P component

Elastic tissue and fibrosing areas

Fibronectin is the structural glycoprotein which has been investigated in most detail. It is a dimer of 450 to 500 kdalton. It has a complex domain structure (Carsons, 1989) with binding domains for collagen, fibrin, DNA and cells. The cell-binding domain is especially important functionally. The cell attachment activity is localised to a 15 kdalton fragment and mediated by a tetrapeptide (Arg-Gly-AspSer). It is remarkable that the enteric cell binding activity of a 250 kdalton subunit is expressed by a tetrapeptide; the same sequence may also be involved in the cell-binding activity of other matrix adhesive proteins such as vitronectin (Ruoslahti and Pierschbacher, 1987). There are cellular and plasma forms of fibronectin; which differ in their solubility, with the cellular form being relatively insoluble. These forms of fibronectin can be differentiated by monoclonal antibodies. Fibronectin has a variety of roles in the biology of the extracellular matrix of potential relevance in rheumatoid arthritis including: its interaction with cells which can influence their phenotype and function; its binding to complement, probably mediated through the collagen-like fragment of C lq; and its binding to collagen which consists of matrix-matrix and cell-matrix interactions (Yamada et al, 1985). The distribution of fibronectin in the synovium has been extensively studied by immunohistochemistry (Scott et al, 1981; Shiozawa and Ziff, 1983). It is present with the synovial lining ceils and in their immediate extracellular matrix; it is a component of superficial fibrin deposits; it is seen in subintimal reticular connective tissue; and it is related to vascular basement membranes. Using monoclonal antibodies which differentiate cellular fibronectin from its plasma from Walle et al (1990) showed cellular fibronectin was present at sites of hyperplasia of the rheumatoid synovial membrane and in the


367

walls of small blood vessels. They also provided evidence for an interaction between T lymphocytes and fibronectin and they suggested cellular fibronectin may be a factor contributing to the infiltration of mononuclear ceils into the rheumatoid synovium. Fibronectin is sensitive to proteases, which cleave the protein at flexible regions connecting proteaseresistant domains. Fragments of fibronectin can modulate its function (Brown, 1983), and enhance morphological cell transformation and stimulate DNA synthesis. Scott et al (1985) both showed synovial fluid flbronectin is different from the plasma form of the molecule. Subsequently Griffiths et ai (1989) showed synovial fluid fibronectin was frequently fragmented, and it is likely that these fragments modulate the biological effects of fibronectin. The site of flbronectin synthesis within the synovium in rheumatoid arthritis has been the subject of debate, especially as the plasma form of the protein accounts for a considerable amount of the protein in the synovium. In situ hybridisation, studies have recently shown that fibronectin mRNA is predominantly found in the synovial lining cell layer (Waller et al, 1990). The other structural glycoproteins of the synovium have been studied in less detail in rheumatoid arthritis. Laminin is a large (800 kdalton) structural glycoprotein prominent in all basement membranes (Rohde et al, 1979) and as a receptor-bound component of the cell surface (Ekblom et al, 1986; Aumailley et al, 1987). Laminin is composed of three polypeptide chains, each a product of different genes (Barlow et al, 1987). The three chains are intertwined to form a cross-shaped structure. Laminin interacts strongly with other glycoproteins, including heparan sulphate proteoglycan, type IV collagen, and itself (Engel and Furthmayer, 1987). There is a close relationship between the distribution of Laminin and collagen type IV is the inflamed synovium and both are present in the basal laminae of the synovial blood vessels (Scott et al, 1984b). Pollock et ai (1990) have also identifed laminin in the intimal layer of the rheumatoid synovium, again in association with collagen type IV; they questioned whether this distribution meant the synovial cells formed an incomplete basementmembrane like structure, though it most likely represents the distribution of laminin as a receptorbound cell surface component. Vitronectin and P-component are other structural glycoproteins with potential involvement in the synovium. Recent studies (Wailer and Scott; unpublished observations) suggest vitronectin is a major component of the synovium with a similar distribution of fibronectin, though it is also related to elastic tissue. P-component is a minor constituent of the rheumatoid synovium and again is closely associated with elastic tissue (Butler et al, 1988). An important association of collagen type III and structural glycoproteins is seen in reticulin fibres. These are fine fibres which stain with silver stains; they consist of collagen type III, fibronectin and a non-collagenous reticulin component (Unsworth et al, 1982). In the inflamed synovium these reticulin fibres are predominantly found beneath the hyperplastic synovial lining cell layer (Scott et al,

368

D.L. Scott et aL

1984a). The non-collagenous reticulin component is also present around the small synovial blood vessels, though it is not a component of their basement membranes.

6 PROTEOLYTIC

ENZYMES

The resorption of the connective tissues of the joint seen in joint destruction in rheumatoid arthritis is a process similar to that at other sites of the body in normal events such as cell migration and tissue morphogenesis. Cell-cell and cell-matrix interactions are important in controlling the process, but the main factors involved are the production of proteolytic enzymes, activators and inhibitors. The current view of the most important enzymic mechanisms has been summarised by Murphy et al (1991): the initial step is the extracellular degradation of connective tissues involving secreted matrix metalloproteinases; in certain special environments acid pH cysteine proteinases are also active extracellularly; and in inflammatory systems serine proteases are released by invading cells. Mechanical disruption and free radicals can augment the enzymic processes. Matrix fragments are subsequently phagocytosed for intracellular processing within the lysosomal system (Murphy and Reynolds, 1985).

6.1 Metalloproteinases These enzymes have ubiquitous roles in matrix turnover both in normal remodelling and in destructive diseases such as rheumatoid arthritis. Most connective tissue cells including fibroblasts and endothelial cells secrete metalloproteinases: there are four types of these enzymes and together they have the combined ability to degrade all the components of the extracellular matrix (Table 8). The enzymes are Zn 2+ or Ca 2+ requiring and are secreted in a latent proform requiring activation; this activation involves the loss of a propeptide of about 10 kdalton and is induced by proteinases such as trypsin or plasmin (Sellars et al, 1978). The active enzymes are specifically inhibited by members of the family of tissue inhibitors of the metalloproteinases (TIMPs). The domain structure of the metalloproteinases is shown in Figure 4.


TABLE 8

369

Metalloproteinase enzymes of human connective tissue

Enzyme

Size (Latent)

Size (Active)

Substrates

Collagenase

55 kd

43 kd

Collagens I, II and III

Stromelysin

57 kd

48 kd

Proteoglycan core protein Collagen IV Fibronectin Laminin Elastin Denatured collagens

Gelatinase

72 kd

66 kd

Denatured collagens Collagens IV, V, VII

Domain 1

Domain 2

Domain 3 .) C terminus

N terminus ( Latent Part

Active Site

Figure 4 Domain structureof metalloproteinase

Matrix Binding

370

D.L. Scott et al.

Collagenase is the most specific metalloproteinase. It cleaves a Gly-Leu or Gly-Ile bond in the native helix of interstitial collagens type I, II and III at a position determined by the tertiary structure of the collagen molecule (Fields et al, 1987). Human fibroblast collagenase in its latent form is a 55 kdalton protein with a minor glycoslyated form of 59 kdalton; on activation these sizes are reduced to 43 kdalton and 48 kdalton respectively (Doherty and Murphy, 1990). The enzyme's activity for the different types of collagen is in the order III > I > II. Stromelysin is a metalloproteinase which degrades the protein core of proteoglycans causing the release of soluble glycosaminoglycans. It also degrades laminin, fibronectin, and type IV collagen (Okada et al, 1986). Latent human stromelysin is a 57 kdalton protein with a glycosylated form of 60 kdalton. After activation these become 48 kdalton and 50 kdalton species respectively. There is also a small truncated active fragment of 24 kdalton (Chin et al, 1985). Recent studies have identified two additional stromelysin-like proteins (Muller et al, 1988). One of these, stromelysin-2 is of similar size and activity of the main form of stromelysin (Nicholson et al, 1989). The other is a shortened version missing the C-terminal domain; it is termed putative metalloproteinase (PUMP-l) and its activities are under investigation (Quantin et al, 1989). The final enzyme in this family is gelatinase. It has a latent molecular weight of 72 kdalton (Sellers et al, 1978). On activation it degrades gelatin, which is itself degraded collagen, and also collagens type IV and V (Murphy et al, 1985). The active form has a molecular weight of 66 kdalton; the molecule is unglycosylated. The properties of gelatinase suggest two roles in vivo. Firstly, it further degrades small fragments of collagen released by the action of collagenase. Secondly, its ability to degrade native type IV collagen, which is a major constituent of basement membranes, indicates it may have a role during the invasion of this membrane by proliferating cells.

6.2 Tissue Inhibitor of Metalloproteinase The connective tissue metalloproteinases are all specifically inhibited by a tissue inhibitor of metalloproteinase (TIMP). This is a heavily glycosylated protein of molecular weight 29 kdalton (Cawston, 1986). TIMP is present in connective tissues, and has also be identified in the culture media of many connective tissue cells and in synovial fluid. Recent studies have shown that there are several forms of TIMP; with a second protein, TIMP-2, a largely unglycosylated protein of 23 kdalton (Murphy et al, 1991). Metalloproteinases are inhibited by various proteins in addition to TIMP; a2 macroglobulin is one of these and is especially important. Binding studies have shown that TIMP is unable to displace ~2 macroglobulin once a metalloproteinase is bound (Cawston and Mercer, 1986). In the inflammatory response of rheumatoid arthritis o~2 macroglobulin concentrations rise in plasma and there is greater


371

capillary permeability, allowing it to reach sites such as synovial tissues. These circumstances may make c~2 macroglobulin a more effective metalloproteinase inhibitor.

6.3 Molecular Studies of Metalloproteinases and Inhibitors The identification of human cDNAs encoding fibroblast collagenase, stromelysin and gelatinases has led to the elucidation of their amino acid sequences (Goldberg et al, 1986; Saus et al, 1988; Wilheim et al, 1989). From these studies it is possible to divide the molecules into three functional domains. The first domain of about N-terminal amino acids is like a propeptide which is removed on activation. The second domain is the active site domain. It contains an amino acid sequence that is conserved in all enzymes and which as marked homology with bacterial enzymes such as thermolysin. The third C-terminal domain of about 200 amino acids may play a part in matrix binding (Hunt et al, 1987).

6.4 Regulation of Metalloproteinases and TIMPs The metalloproteinases are controlled in several ways: by the synthesis of the latent enzyme; by the need for an activation mechanism; and by the presence of inhibitory TIMPs. Expression of metalloproteinase and TIMP by cultured connective tissue cells is regulated by a number of cytokines, growth factors, and hormones. MacNaul et al (1990) have compared the effects of IL-1 and TNFa on metalloproteinases and TIMP production by cultured human rheumatoid synoviocytes. They found that immunochemical analyses of the levels of metalloproteinases and TIMP proteins reflected the steady-state mRNA levels for their gene products. By examining mRNA production, MacNaul et al found stromelysin and collagenase were not always co-ordinately expressed; IL-1 was more potent than TNF-a in the induction of stromylisin expression; neither IL-1 nor IL-1 and TNF-ot had a synergetic effect on stromelysin expression. The importance of IL-1 in controlling the expression of synovial fibroblast collagenase mRNA has also been shown by McCachren et al (1989) in less detailed investigations. The activity of metalloproteinases can also be influenced by their extracellular activation. The prometaUoproteinases undergo a conformational change with the loss of about 80 N-terminal amino acids (Grant et al, 1987; Nagase et al, 1990). Proteolytic cleavage with trypsin, plasmin or similar enzymes causes activation, though a final self-cleavage event occurs. The presence of stromelysin during collagenase activation causes a marked potentiation of over 10 fold in collagenase activity (Murphy et al, 1987).

372

6.5

D.L. Scott et aL

Other Enzymes

Several pieces of evidence suggest that other proteolytic enzymes may have a role in the pathogenesis of joint destruction. For example studies in our own Unit (Moore AR, Iwamura H, Scott DL, Willoughby DA, submitted for publication) show a role for enzymes released by polymorphonuclear lymphocytes and cartilage damage in rheumatoid disease. Similarly, Weinberg et al (1991) have shown that the rheumatoid synovium has increased numbers of macrophages containing urokinase type plasminogen activator. There is considerable information supporting a role for the plasminogen activator/plasmin system in the inflammation within rheumatoid joints (Hamilton et al, 1991). Human articular chondrocytes in culture produce plasminogen activator, and this is stimulated by cytokines such as TNF-a (Campbell et al, 1990). The balance of inhibitors such as plasmin may be important in the control of rheumatoid joint damage. This will be influenced by other processes; for example, these inhibitors are especially influenced by free radicals which are often generated by rheumatoid inflammation, and this may render them ineffective.

7 FREE RADICALS,

DNA DAMAGE

AND CELL DEATH

Chronic inflammatory disorders such as rheumatoid arthritis are characterised by populations of cells with altered regulation and function. Several different free-radical-mediated processes may be involved. Oxygen radicals may be selectively toxic to certain susceptible cell types and certain subpopulations of cells. This could influence the energy charge state of the cell, and oxidative DNA damage is likely to play a critical role (Schraufstatter et al, 1987). Another potential mechanism is alteration of cell surface receptors by oxidants (Lunec and Blake, 1987). A final mechanism is that oxidative DNA damage to target cells might give rise to mutations (Harris, 1983).

7.1

Free Radical Chemistry

A free radical is an atom or molecule with one or more unpaired electrons, capable of independent existence. They may have a positive or negative charge or be neutral. Examples include the oxygen molecule, the hydrogen atom and most transition metals. The unpaired electron(s) characteristic of an oxygen-derived free radical, may confer a high level of instability and thus chemical reactivity of the molecule, although this is not the case for molecular oxygen. Consequently, oxygen free radicals are often tissue damaging and usually only exist at low steady state concentrations in vivo (Blake et al, 1987). The initiating event in the production of such reactive species is usually the partial reduction of oxygen to the superoxide anion radical (02-°) or H202. This may occur as part of a normal metabolic process or through a 'leakage' of electrons; for example, from the mitochondrial respiratory


chain.

373

The reaction sequences of molecular oxygen and its associated radicals and other reactive

species together with the involvement of transition metal catalysts is summarised in Table 9.

TABLE 9

Reactions of free radicals and other reactive species related to oxygen

Classification

Type of Reaction

Dismutation"

202-° + 2H ÷ ~ H202 + 02

Catalase °

H202 --, 21-120 + 02

(a)

Reduction of iron (III) to iron (II)

Fe 3+ + 0[ ° ~ (Fe 3+ - 0 [ ° / F e ~+ -02) --- Fe 2+ + 02

(b)

Fenton Reaction

Fe 2+ + H202 ~ Fe s+ + °0H + 0H-

Haber-Weiss Reaction

02.0 + H202--" 02 + °0H + OH

Myeloperoxidase

H202 + C1- + H + --, HOC1 + H20

(--- a + b)

Free radicals are denoted by a superscripted dot R ° * These are protective, the rest generate toxic species: 0H ° or HOC1

When polymorphonuclear leucocytes engulf microbes they rapidly consume oxygen. This is termed the respiratory burst. The oxygen consumption is utilised to produce reactive, oxygen derived species which are responsible for killing microbial pathogens.

At least two enzyme systems are responsible

374

D.L. Scott et aL

for generating microbicidal oxidants: a plasma membrane bound NADPH oxidase system incorporating cytochrome-b-245; and the myeloperoxidase system.

7.2 Free Radicals and Tissue Damage A consequence of uncontrolled production of free radicals is damage to biomolecules leading to altered function and disease. There is much direct and indirect evidence implicating radicals in the pathogenesis of rheumatoid synovitis. Many cells involved in the inflamed synovium, such as macrophages, neutrophils, lymphocytes, and endothelial cells, have the capacity when isolated and stimulated, to produce free radicals. When stimulated in the environment of critical biomolecules such as lipids, proteins, DNA, and glycosaminoglycans, they promote oxidative damage. Interest in the tissue destroying power of oxygen radicals was stimulated by the observation that they degrade hyaluronic acid, leading to a loss in its viscosity similar to that seen in rheumatoid synovitis (McCord, 1974). This appeared to explain the paradox of low viscosity of rheumatoid synovial fluid in the absence of detectable hyaluronidase; however, it is now known that rheumatoid synovial lining cells secrete short chain hyaluronic acid polymers (Vuorio et al, 1982), so it is not certain the contribution of oxygen radicals towards the depolymerisation process.

7.3 The Production of Radicals within the Inflamed Joint Although there is ample evidence to implicate radicals in the pathogenesis of inflammatory synovitis, this requires an adequate supply of oxygen and the oxygen tension in inflamed synovial fluid is low (Lund-Oleson, 1970). There is a need to explain how, in the relatively hypoxic environment of the inflamed joint, free radicals are generated. This led Woodruff et al (1986) to propose radicals are formed in synovitis by a hypoxic-reperfusion mechanism. Ischaemia induced damage occurs in many disorders, such as coronary artery disease. Although ischaemia itself can ultimately produce tissue death, in many clinical situations a substantial part of the injury is more properly termed reperfusion injury (McCord, 1985). Much of the injury occurs when oxygen is re-introduced to the tissue by the restoration of the blood supply. When this happens free radicals are formed in abundance due to the uncoupling of a variety of intracellular redox systems, causing substantial microvascular and parenchymal damage. Several mechanisms can lead to hypoxic-reperfusion injury in rheumatoid joints. In the normal knee the intra-articular pressure is at or slightly below atmospheric pressure (Dixon and Hawkins, 1990). In the normal knees contracting the quadriceps produces a subatmospheric pressure (Jayson and Dixon, 1970) whilst in rheumatoid patients, where there is a synovial effusion, quadriceps contraction produces


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high intra-articular pressures. This high pressure on exercise occludes the synovial capillary bed in arthritic patients with reperfusion injury (Blake et al, 1989). The mechanisms involved are summarised in Table 10.

TABLE 10

Factors leading to reperfusion injury in inflamed joints

Resting pressure increased with exercise Capillary shutdown with exercise leading to hypoxia Inflamed synovium containing xanthine oxidase in endothelium produces radicals Capillary hyperaemia catalyses radical production Iron decompartmentised catalyses radical production Radical production leading to tissue damage

7.4 Lipid Peroxidation The polyunsaturated lipids of cell membranes can be damaged by radicals in the process of lipid peroxidation, which causes cell membrane damage (Dormandy, 1989). Polyunsaturated lipids undergoing peroxidation give rise to lipid breakdown products such as diene conjugates and thiobarbituric acid reactive substances. These are useful markers of oxidative damage to lipids. Thiobarbituric acid reactive material is present in the synovial fluid from rheumatoid patients, consistent with lipid peroxidation occurring locally and its amount correlates with clinical indices of inflammation (Rowley et al, 1984). Exercise of the inflamed knee promotes lipid peroxidation within the joint (Merry et al, 1991).

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7.5 Protein Oxidation Free radical generating systems will damage many proteins involved in the inflammatory processes of rheumatoid synovitis in vitro. This has been shown for a variety of proteins including collagen (Greenwald et al, 1976), caeruloplasmin (Winyard et al, 1989), and immunoglobulin G (Lunec et al, 1985). Free radical damage leads to denaturation, loss of function, cross-linking, aggregation, and fragmentation. In view of the importance of rheumatoid factors in rheumatoid synovitis, the damage to immunoglobulin G is especially interesting; radical damaged immunoglobulin G develops a characteristic autofluorescence and forms both monomeric and aggregated complexes. Immunoglobulin G with these characteristics is present in rheumatoid synovial fluid.

7.6 DNA Oxidation Single and double strand scission of DNA, together with hydroxylation of its constituent bases, are characteristic of oxygen radical damage. Site specific hydroxyl radical degeneration catalysed by iron bound to cellular DNA is an important mechanism (Loeb et al, 1988). An example is the reaction of hydroxyl radical with the DNA nucleoside deoxyguanosine which leads to the formation of 8hydroxydeoxyguanosine (Kasai et al, 1986); this causes misreading of DNA templates (Kuchino et al, 1987). The production of oxygen radicals in ischaemic tissues involves changes in purine metabolism within ischaemic cells (McCord, 1985). The low oxygen concentrations during temporary ischaemia causes a decline in mitochondrial oxidative phosphorylation, which in turn increases the dependence of the cell on ATP production via anaerobic glycolysis. This latter process is inefficient and consequently leads to raised concentrations of ATP catabolites such as adenosine and its breakdown products including hypoxanthine and xanthine. Cellular levels of ATP consequently fall with consequent fluxes of ionic calcium across the cellular membrane which will ultimately lead to cell death. Oxygen radicals also damage DNA directly, causing DNA single strand breaks (Schraufstatter et al, 1987). Repair of these DNA strand breaks requires the involvement of the enzyme poly-ADP ribose polymerase in an energy dependant process utilising NAD, and this is also influenced by cell "energy charge" and ATP levels. Carson et al (1986)5 have suggested a role for DNA strand breaks in the process of '"programmed cell death". They suggested this phenomenon represents a "suitable response" of cells with damaged DNA that cannot be repaired. It could result from radical induced damage to DNA or reduced cell ATP levels and "energy charge". Preliminary work by Bhusate et al (1989) has shown that circulating mononuclear cells in rheumatoid arthritis have increased numbers of DNA strand breaks compared to both healthy controls and patients with osteoarthritis. This suggests that "programmed cell death" is a potentially important pathogenic


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mechanism in rheumatoid disease, though further work is needed to evaluate the clinical significance. However, if loss of "cell energy charge" is occurring in rheumatoid synovitis, there should be an associated increase in the catabolic products of ATP. Herbert et al (1988) showed there are increased hypoxanthine levels in rheumatoid synovial fluid, presumably the result of ATP catabolism in senescent joint cells. DNA damage is an early event when cells are killed by oxygen radicals. The capacity of endogenous intra-cellular anti-oxidants or of repair mechanisms for oxidative DNA damage could determine the susceptibility of different cell populations to killing. Lawley et al (1988) showed lymphocytes from patients with rheumatoid arthritis and other autoimmune diseases show increased sensitivity to the toxic effects of the alkylating agent N-methyl-N-nitrosourea compared to normal subjects. Similarly there is a failure in the DNA repair of 06-methylguanosine. Finally lymphocytes from rheumatoid patients are more susceptible to X-irradiation (Harris et al, 1985). These findings imply DNA damage may be more severe in rheumatoid patients to oxygen radical attack.

8 CONCLUSIONS

The interactions between the different cells of the rheumatoid synovium and their matrix in the inflamed joint are complex. Although there has been considerable work on some of the mechanisms involved, for example the roles of the interleukins, the importance of any set of mediators or modes of communication between cells is not well established in terms of controlling the whole process. To develop new therapeutic approaches for rheumatoid arthritis requires the identification of appropriate targets for therapy. In arthritis conventional therapies such as non-steroidal antiinflammatory agents have a valuable and established role; it is unlikely that further developments in these areas will give rise to new treatment modalities. But cell-cell signalling, adhesion molecules, and enzymes leading to tissue damage are all potential new outcomes for treatment. Some of these areas, such as inhibitors of interleukins or metalloproteases are already the subject of intense investigations. At present it is difficult to know which of these will be the most rewarding and fruitful approach. Despite this there can be no doubt that affecting the molecular biology of the rheumatoid joint will hold the key to therapeutic advance in this area.

ACKNOWLEDGEMENTS We are grateful for the support of the Arthritis and Rheumatism Council and the Joint Research Board of St Bartholomew's Hospital. Dr Scott is Muir Hambro Fellow of the Royal College of Physicians.

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