1 Pathogenesis of Crohn's disease

1 Pathogenesis of Crohn’s disease HUMPHREY

J. F. HODGSON

DM, FRCP

Professor of Medicine Imperial College School of Medicine, Division Du Cane Road, London W12 ONN, UK

of Medicine,

Medicine

A, Hammersmith

Hospital,

In the absence of a single initiating aetiological factor, most workers envisage Crohn’s disease as the manifestation of poorly regulated immune and inflammatory processes within the gut wall. Initially these responses may arise as a response to common antigens associated with the gut-bacterial products being amongst the most obvious candidates. In genetically predisposed individuals there is overexpression both of local immune response mechanisms in the gut wall (T-cells, B-cells and macrophages) and of systemic inflammatory cells (predominantly polymorphonuclear leukocytes), which are attracted into the inflamed gut through activation of adhesion molecules on the vascular endothelium. As a consequence a large number of pro-inflammatory processes are expressed in the gut wall, inadequately checked by the normal counter-inflammatory processes that should serve to limit inflammation. Defining the relative importance of the individual processes, and identifying critical steps that could be inhibited or enhanced for therapeutic purposes, is a major challenge of Crohn’s disease research. Key words: cytokines; mucosal immunology;

inflammation;

IBD-aetiology.

INTRODUCTION To clinicians, the diseases that are simplest to ‘understand’ are those caused by external agents-bacteria, viruses, or toxins. More complex are those in which the structure of organs is disturbed without obvious cause. Our attempts to understand Crohn’s disease illustrate this feature. Some workers seek an aetiological explanation with a single exogenous agent; others seek to understand the processes of damage that are occurring in the gut, believing that understanding those mechanisms, however they are initiated, will help define potential approaches to treatment. Many different potentially damaging processes have been defined in the gut wall in Crohn’s disease. In addition to defining pro-inflammatory mechanisms, much is being learnt of the counter-inflammatory processes in the gut that should limit inflammation, and of the repair processes involved in healing and in complications such as strictming. Modem biology, which BailliPre’s

Clinical

Gastroenterology-

Vol. 12, No. 1, March 1998 ISBN O-7020-2482-1 0950-3528/98/010001+ 17 $12.00100

1

Copyright 0 1998, by Bailli&re Tindall All rights of reproduction in any form reserved

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defines molecular mechanisms and cell-to-cell signalling processes, is well suited to defining new therapies acting via inhibition or enhancement of single mechanisms. It is probably too early to judge whether such approaches will be more effective than the traditional pharmacological approach of using corticosteroids and aminosalicylates, drugs that are notable for having a range of actions over a number of discrete inflammatory processes. This chapter opens by briefly discussing some concepts of inflammation in the normal mucosa, as a background to considering the search for an aetiological agent, followed by a review of the multiple mechanisms that may contribute to pathogenesis. Where relevant, abnormalities in the closely related condition of ulcerative colitis will be considered. INFLAMMATION

AND

THE

NORMAL

GUT

Even in normal health, there are inflammatory cells present in the gut mucosa-lymphocytes between epithelial cells, and lymphocytes, macrophages and plasma cells in the lamina propria. They colonize the gut in response to antigens in the gut lumen; we know this because there is a gradual increase in cell number after birth, and because experimental animals reared in a germ-free environment demonstrate a much sparser population of these cells. This has been summarized in the description of the normal gut as being in a state of ‘physiological inflammation’. One common concept for the development of inflammatory bowel disease is that this represents an exaggeration of these normal inflammatory responses. Exaggerated inflammatory responses could be initiated in a number of ways: response to a specific pathogen (the as-yet-unidentified aetiological agent); response to a greater than normal antigenic challenge from the gut lumen (for example a greater mucosal permeability allowing greater ingress); or a failure to control responses normally. This last concept was first suggested as a disorder of immunoregulation (the defective Tsuppressor cell of the 197Os), but now embraces disordered regulation of inflammatory responses, for example overproduction of a pro-inflammatory cytokine or deficient production of a counter-inflammatory cytokine. These possibilities are not mutually exclusive and, for example, once the mucosa has been initially inflamed from any cause, enhanced mucosal permeability is likely to be a consequence, permitting greater antigen ingress and stimulating further inflammation. As inflammation becomes amplified, damage is mediated not only by the resident cell population, but by recruitment of inflammatory cells from the general circulation. THE

SEARCH

FOR AN EXOGENOUS

AGENT

No agreement has emerged on a single infectious disease.

agent initiating

Crohn’s

PATHOGENESIS

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The concept of a possible mycobacterial cause, first suggested over 80 years ago, has been refined in recent years to investigate a possible association of Crohn’s disease with Mycobacterium paratuberculosis, which is an established cause of chronic enteritis in cattle (Cocito et al, 1994). Mycobacterium paratuberculosis has been cultured from a few patients with Crohn’s disease, and fragments of its DNA have been identified by some, but not other, laboratories, strikingly more commonly in Crohn’s disease tissue than ulcerative colitis or control tissues (Sanderson et al, 1992). Widespread contamination of milk with M. paratuberculosis DNA has been documented in Europe. It certainly appears that the diseased mucosa of Crohn’s disease is more likely to harbour these organisms, which are common in the environment, but the association may well be no more than that. Trials of anti-mycobacterial drugs have shown some improvement, but it is not clear that this is significantly greater than that reported with other antibiotic regimes (Prantera et al, 1994). Measles virus has provided an alternative candidate, with one laboratory reporting evidence of persistence of the virus in Crohn’s tissue using a variety of techniques including electron microscopy, immunogold identification and molecular hybridization techniques (Wakefield et al, 1997). This has been tied in with a report of a cohort effect of a measles epidemic in childhood with subsequent Crohn’s disease (Wakefield et al, 1995), and a striking report of three cases of serious Crohn’s disease in the children of three out of four mothers affected during pregnancy (Ekbom et al, 1996). Some of the epidemiological evidence has been heavily criticized, and other laboratories have been unable to demonstrate similar findings (Haga et al, 1996; Iizuka et al, 1995). Interpretation is difficult because both natural measles and attenuated vaccine strains might potentially be implicated, and as with other viruses, tissue sequestering for decades after initial infection can occur. Other individual exogenous agents that have been considered include particulate antigens such as silicates, presenting an indigestible challenge to the cellular immune system. Rather than a single exogenous agent, the alternative possibility is that a combination of antigens in the gut-many of which are bacteria-derivedinitiate the response. An impressive series of studies demonstrated that various immunologically deficient ‘knockout’ mice, which lack genes for specific immune functions, are prone to develop chronic inflammation of the bowel (Kuhn et al, 1993; Sadlak et al, 1993; Strober and Erhardt 1993). A wide variety of different immunological defects predisposed to inflammation in these animals, but a common strand was that the inflammation could be prevented by rearing the animals in a bacteria-free environment. AUTOANTIGENS An early theory for inflammatory bowel disease was that these represented auto-immune conditions with self antigens such as the colonic epithelium acting as target. Over the years the evidence for this in ulcerative colitis has

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remained relatively strong--colon-bound immunoglobulin in a pattern suggestive of an epithelial auto-antibody (Halstensen et al, 1992), and a tendency to develop a specific circulating auto-antibody, anti-neutrophil cytoplasmic antibody (ANCA) (Snook et al, 1989; Duerr et al, 1991). For Crohn’s disease the evidence is weaker, though there is some overlap, and a few patients with Crohn’s have ANCA (Kossa et al, 1995). It may well be that some auto-immune sensitization occurs in both conditions. For all potential antigens-exogenous or endogenous-as initiators of Crohn’s disease, it is important to remember that only a subgroup of individuals may be susceptible, and indeed the initiator of the inflammation may be different in different individuals. Recent genetic studies on Crohn’s disease families are thus highly relevant, both to infectious and noninfectious theories of Crohn’s aetiology. GENETIC

STUDIES

The familial tendency in Crohn’s disease is strong, with a concordance of about 50% in genetically identical twins, and lesser degrees with nonidentical twins and more remote relationships (Tysk et al, 1988). The ability to perform genome-wide linkage studies, sib-pair analysis comparing affected and unaffected relatives, and careful analysis of large collections of families has recently provided enticing insights. The close relationship between the two major forms of inflammatory bowel disease (IBD) has been confirmed, with well documented individual families containing both diseases (Satsangi et al, 1996a), but a number of candidate gene loci have emerged, some associated with ulcerative colitis, some with Crohn’s disease, and some with both. These studies confirm the clinical suspicions that these diseases are polygenic, and indeed also suggest that more than one pathway may lead to an individual manifesting IBD (Satsangi et al, 1996b). For Crohn’s disease, the loci that appear to be associated with the condition are an area of chromosome 16 (Hugot et al, 1996) and (shared with ulcerative colitis: Satsangi et al, 1996c), on chromosomes 3, 7 and 12. Regularly found in ulcerative colitis but not Crohn’s disease are associations with HLA locia finding possibly associated with a tendency to auto-immune processes. The candidate genes in these areas are interesting and with further study should shed more light. They include genes controlling growth factors and responses to growth factors, the structure of mucin glycoproteins, adhesion molecules and cytokine receptors, etc. At the moment, they point the way to a number of different pathways by which genetic susceptibility might contribute to both forms of IBD: abnormalities in mucus leading to enhanced antigen penetration from the gut; a tendency to develop autoimmune responses to colonic epithelial antigens in ulcerative colitis; more severe disease associated with a different response to a given cytokine or abnormality of an anti-inflammatory cytokine. Certainly at this stage genetic studies appear to be defining a multiplicity of factors that predispose to IBD, rather than a single factor.

PATHOGENESIS

OF CROHN’S

INFLAMMATORY

DISEASE

PROCESSES

5

IN THE

GUT

MUCOSA

To discuss the inflammatory processes, and the relevant control mechanisms, in the gut wall in Crohn’s disease, this section first considers the luminal-mucosal interface: is there good evidence of enhanced permeability predisposing to Crohn’s disease? Secondly the inflammatory processes in the mucosa will be considered-both cellular and humoral, including both locally mediated inflammation and that dependent on recruitment of systemic immunity. Thirdly the anti-inflammatory and repair mechanisms will be discussed.

PERMEABILITY

OF THE

INTESTINE

IN CROHN’S

DISEASE

Enhanced penetration of antigen from the lumen would be expected to lead to upregulation of immune processes in the mucosa. Once disease is established, abnormal permeability would be anticipated so studies on relatives may be more telling than those on patients. The initial study identifying increased permeability in relatives (Hollander et al, 1986) has proved difficult to confirm, with later studies now arguing against this proposal (Marteau et al, 1990; Munkholm et al, 1994). Permeability studies are however notoriously difficult, and regional differences may be difficult to elucidate. One specific aspect germane to gut permeability is intriguing. Clinical observations had identified a tendency of patients with selective IgA deficiency to develop Crohn’s disease (Hodgson and Jewell, 1977), and a perfusion study has identified a lower than normal jejunal IgA secretion in apparently unaffected small intestine (Marteau et al, 1990), which would be expected to be associated with greater antigen penetration, and might thus lay the foundation of an upregulation of immune responses to luminal contents.

THE INFLAMMATORY CROHN’S MUCOSA Cellular

PROCESSES

IN THE

aspects

The normal inflammatory infiltrate of the ‘physiologically inflamed’ normal gut is enhanced in Crohn’s disease with an increase in T-cells and plasma cells in the lamina propria, a denser population of macrophages, and an increase in the number of acute inflammatory cells, predominantly polymorphonuclear leukocytes. In parallel there is an increase in the number of pro-inflammatory molecules present-and these can be identified within cells, in the interstitial fluid, and in the lumen of the adjacent gut. Where such measures are available, generally by identification of cell surface markers, cells tend to be in activated form. The overall effect during

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disease relapse is a greater density of cells in the tissue, increased blood supply, increased interstitial fluid, and recruitment of inflammatory cells from the circulation to the diseased tissue. For the individual cell populations and pro-inflammatory mechanisms the details are listed below. T-cells

T-cell numbers in the lamina propria are increased severalfold, and they show surface activation markers such as CD45RO+ markers (Roman et al, 1996), and increased T-cytokine production (discussed below). Genetic analysis of different T-cell clones cultured from the mucosa shows that the cells are polyclonal, but studies of their repertoire (by investigating T-cell receptor rearrangements) indicates that they are probably reacting to a fairly restricted number of antigens (Gulwani-Akolker et al, 1996), including bacterial antigens (Duchmann et al, 1996). The specialized T-cells in the epithelium (intra-epithelial lymphocytes) are characteristically CD8+ (suggesting suppressor-cytotoxic phenotype) and express, for the most part, the a-P type of T-cell receptor; it is unclear whether intra-epithelial lymphocytes are increased in number in Crohn’s disease, and no clear picture has yet emerged about variations in receptor usage. B cells

The normal preponderance of IgA plasma cell in the lamina propria is maintained, but whilst the increase above normal in cells expressing this isotope of immunoglobulin is only modest, there are striking increases in IgG and IgM expressing cells (Brandtzaeg, 1985). The significance of this is that the local release of these latter immunoglobulins is likely to cause inflammation, whilst IgA-antigen combination is markedly less proinflammatory. Additionally, the IgG expressed in Crohn’s disease is (in contrast to that in ulcerative colitis) skewed to IgGl and IgG2 subclasses, which are in general associated with anti-bacterial rather than auto-immune responses (Kett et al, 1987). Monocytedmacrophages

During active IBD there are increased numbers of activated monocytes in the circulation, and increased numbers of macrophages in the mucosa. Macrophages are present throughout the thickness of the lamina propria, in activated form (evidenced by enhanced lysozyme expression (Stamp et al, 1992), RFD9+ve status and interleukin-2 (IL-2)-receptor expression), often clustered together (the fuller form of which in association with T-cells is presumably the granuloma), and some recently derived from the circulation (Rugtveit et al, 1994). Wakefield et al (1991) have pointed out that granulomas may form in blood vessels, and others have indicated that the wall of lymphatic vessels may be another site of predilection, suggesting

PATHOGENESIS

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DISEASE

the initiation of an inflammatory response in vessel walls as a possible early step in the inflammatory process of Crohn’s disease. This concept has been extended, with the help of the study of vascular casts from surgical resection specimens, to suggest that Crohn’s disease is predominantly a vasculitic inflammatory process. Whether or not that is so, the activated macrophages are probably major contributors to the production of the cytokines (see below). Polymorphonuclear

leukocytes

During acute relapse there is a striking increase in polymorphonuclear leukocytes (PMN) in the lamina propria, these cells being recruited from the general circulation. The processes by which these cells accumulate in the gut have been recently elucidated, and the process is highly active. During disease exacerbation the chemoattractant stimulus is such that over 60% of the circulating PMN pool may be en route to the gut (Saverymuttu et al, 1986), where cells pass through the epithelium (giving the appearance of cryptitis) and thence enter the lumen of the gut. The critical steps in this process of transmigration of cells through the vascular epithelium are the upregulation of the adhesion molecules on the vascular endothelial surface (e.g. E-selectin, intercellular adhesion molecule (ICAM): Butcher, 1991) which interact with the ligands on the PMN surface (Figure 1). These molecules are upregulated during active inflammatory bowel disease (Koizumi et al, 1992; Ohtani et al, 1992). The entire process of moving cells into the interstitium includes a series of steps (slowing of cell velocity,

q-

E-selectin

-bd

ICAMI VCAM

4

lntegrins -P

b

\< ‘t.v\ \ *x Leake~plasrna proteins

Figure 1. Illustration of the process involved in leukocyte margination and migration into the tissues (with thanks to Dr M. Bhatti). ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; PGI, prostaglandin I,: TNF, tumour necrosis factor; IL, interleukin; NO, nitric oxide; LPS, lipopolysaccharide.

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H. J. F. HODGSON

rolling along the endothelium, adhesion and then transmigration between endothelial cells) each controlled by combinations of different adhesion molecules and ligands, which present an attractive target for novel therapies (Pober and Cotran, 1990). The chemoattractant in the tissues and the lumen has not been definitely defined, and candidates include small bacterial derived peptides (e.g. FMLP), chemokines and eicosanoids (e.g. IL-8 and LTB4 as discussed below) derived from local inflammatory cells, and bacterial lipopolysaccharide. The importance of this recruitment of acute inflammatory cells has been demonstrated in the naturally occurring model of cotton top tamarin colitis, in which an exogenously administered antibody to one of the critical molecular partners (a-4 integrin) involved in cell migration caused a striking reduction in inflammation (Podolsky et al, 1993). The rate and magnitude of neutrophil accumulation in Crohn’s disease and ulcerative colitis appear similar. Other cell types Both mast cells (tissue basophils) and eosinophils are increased in the tissues in Crohn’s disease, and there is evidence for their being in an activated form. Active products include histamine, prostaglandins, platelet activating factor, and the basic and cationic proteins of eosinophils. Platelet activation may not only further contribute to inflammation, but underly the clinical prothrombotic state associated with Crohn’s disease, and perhaps contribute further to microvascular damage (Schaufelberger et al, 1994). Functional

consequences

of enhanced

cellular

numbers

These are highly complex, and lend themselves to the design of complex network maps to illustrate possible pathogenetic mechanisms (Figure 2). The increased numbers of activated cells within the mucosa can initiate or perpetuate inflammation by direct cell-mediated processes (cytotoxicity for other cell types, phagocytosis of particulate matter), by release of chemical mediators or, in the case of plasma cells, following antigenantibody complex formation resulting in complement activation and the subsequent release of activated complement components. In respect of many chemical mediators, particularly the pro-inflammatory cytokines, there is considerable overlap between the cell types and the individual cytokines produced. With a rapidly moving field, in which ‘new’ inflammatory cytokines are being discovered, and assays are technically demanding (Fukushima et al, 1995) (such chemicals being present in picogram amounts), there is no clear consensus as to which mediators are likely to be of greatest importance. It remains uncertain whether there are ‘key players’, control of which will interrupt the process of inflammation, and determining this-perhaps most effectively by clinical experimentation using highly specific antagonists-is one of the most exciting areas of Crohn’s disease research.

I

i

I

1 IL-a)\ 1

.F~gfIjf!Y::

Figure 2. Potential pathogenic process activated during inflammation in Crohn’s disease (with thanks factors; T, T-cell; B, B-cell; LPS, lipopolysaccharide; TNF, tumour necrosis factor; MIF, macrophage polymorphonuclear cell; ICAM, intercellular adhesion molecule; VCAM, vascular adhesion molecule.

Local tissue Eicosanoids Endorphin Cytokines

Histamine

1

to Dr M. Bhatti). inhibitory factor;

-+I

CSFs

CSFs, colony stimulating E-sel., E-selectin; PMN,

Vasoconstriction

I

c$!%kocytes

IL, interleukin; IFN, interferon;

1

Stem

\D

10

H.J.F.

HODGSON

Specific mediators

Complement The combination of IgG and IgM antibody with antigen within the mucosa is likely to be a highly pro-inflammatory event, leading to enhanced vascular permeability, and further chemoattraction of non-specific inflammatory cells. Complement deposition has been demonstrated immunohistochemically in diseased mucosa (interestingly the distribution in Crohn’s disease differs from that in ulcerative colitis; in the latter the distribution is along the epithelium, suggestive of an anti-colon antibodymediated effect, whereas it is more scattered in Crohn’s disease: Halstensen et al, 1992, 1993). Proteolytic

enzymes

Release of intracellular proteases, such as elastase, cathepsin G and collagenase, from PMN has been demonstrated by the presence of free proteases in plasma and in the lumenal fluid (Adeyemi et al, 1985; Adeyemi and Hodgson, 1991, 1992). Other PMN derived substances (myeloperoxidase, lysozyme and lactoferrin) are also detectable. Activated oxygen radicals PMNs in particular can generate activated oxygen radicals (hydroxyl radicals, superoxide, hypochlorite) when they are stimulated, and these short-lived substances can damage cellular protein, lipid and DNA. There is evidence that some of the naturally occurring regulators that mop up such radicals (e.g. the enzyme superoxide dismutase) may be deficient in IBD (Verspaget et al, 1988), and agents that replace these, or prevent radical generation (e.g. allopurinol) may have some therapeutic effect (Emerit et al, 1991). Reactive nitrogen metabolites Nitric oxide has emerged as a ubiquitous agent in controlling vascular tone via endothelial relaxation, and can also cause secretion and activate neutrophils; the free radical is generated from arginine by the enzyme nitric oxide (NO)-synthase, which exists in two forms-one constitutively expressed and one upregulated in inflammation. Initial studies indicated that enhanced NO production was more characteristic of ulcerative colitis than of Crohn’s disease, but enhanced NO-synthase activity has also been reported in Crohn’s disease (Fu et al, 1996). Eicosanoids Short-lived products of arachidonic metabolism-the prostanoids, thromboxanes and leukotrienes-are generated in enhanced amounts

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during active inflammation. They are likely to be derived from a number of cell sources-mononuclear cells, PMNs and epithelial cells. When sampled in the gut by the technique of rectal dialysis, the concentrations of leukotriene LTI34-a major chemoattractant for PMNs, have been notably elevated, but a variety of other species are also increased (Lauritsen et al, 1988; Rampton and Collins, 1993). The consequences of activation of these substances include enhanced fluid secretion, increased motility, increased vascular permeability and chemoattraction of other cells. As with other cell products, some of the substances classified under this heading are probably beneficial and aid healing-notably the role of prostaglandins in reducing neutrophil adherence, enhancing mucus release and aiding regenerative epithelial repair. It is possible to modulate eicosanoid function in various ways, e.g. specific inhibitors of synthesis of prostaglandins or leukotrienes (Rask-Madsen et al, 1992), thromboxane synthesis inhibitors (Collins et al, 1996), or fish-oil administration that diminishes LTB4 production (Belluzzi et al, 1996). It was such studies that led to the conclusion that the overall balance of prostaglandin production was favourable in IBD, and inhibitors of leukotriene and thromboxane synthesis appear to be of marginal rather than dramatic effectiveness. Pro-inflammatory

cytokines

Cells of the immune system, and the non-memory-specific cells activated in inflammation can produce a series of peptide products, the cytokines, of which the interleukin series has been the focus of a tremendous research drive. Common to most interleukins is a peptide structure (typically lo-40 kDa in size), their release from an inactive precursor, and their ability to interact with a variety of other cell types by specific surface receptors. They have many functions in common, and can in general be produced from more than one cell type when activated. Activation of cells to produce interleukins can be initiated by a variety of means-including other cytokines-but also by stimuli such as bacterial lipopolysaccharide (LPS). Indeed the basic in vitro model for generating pro-inflammatory cytokines from cells is the use of LPS, and there is strong reason to suppose that gut-derived LPS may be a predominant stimulus in the mucosa in IBD. To categorize this bewildering list of cytokines, they may be considered as those predominantly generated from macrophages (IL-l, IL-6, tumour necrosis factor-a (TNF-a)), and those predominantly associated with Tcells. With respect to the latter, a concept derived from mouse immunology has been widely discussed in the context of inflammatory bowel disease. T-cell derived cytokines may be considered in two large families , Thl and Th2. The former are involved in generating classical cell-mediated immunity (IL-2, interferon); the second are involved in humoral responses (IL-4, IL-5, IL-IO, IL-1 3). There are those who argue that ulcerative colitis is predominantly an expression of Th2 type responses, and Crohn’s disease predominantly Thl (Niessner and Volk, 1995). However, as discussed below, quantification of cytokines (using a variety of different techniques,

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H. J.F. HODGSON

such as immunohistochemistry, immunoasssay, RT-PCR) is not yet giving consistent results, and the validity of the rather simple Thl/Th2 concept in human immunology may well not stand the test of time. TNF- a TNF-a was the first cytokine so designated, and as such avoided being renamed an interleukin. As with IL-l and IL-6, TNF-a expression can be stimulated by exposing monocytes and macrophages to LPS, and it induces systemic and local inflammatory responses (fever, induction of acute phase proteins from hepatocytes, inducing expression of adhesion molecules on vascular endothelium etc). Thus many of the cardinal clinical features of active inflammatory disease can be explained by the actions of TNF, IL-l and IL-6. Data on tissue levels in IBD is contentious (Reimund et al, 1996), but recent studies suggest a more marked TNF response in Crohn’s disease than ulcerative colitis (Mazlam and Hodgson, 1992; Breese et al, 1994; Bouma et al, 1995). The current interest in TNF in Crohn’s disease is derived from a line of research originally pursued in rheumatoid arthritis, where it could be demonstrated that in the synovium TNF-a appeared to stand at the head of a cascade of pro-inflammatory cytokines, and administration of monoclonal antibodies to TNF-a could prevent this cascade both in vitro and, strikingly, in vivo (Elliott et al, 1994). Recently both uncontrolled (Van-Dulleman et al, 1995) and controlled studies in Crohn’s disease using monoclonal anti-TNF indicate that inflammatory episodes can be terminated by single administrations-a striking and rather unexpected result because of the evidence presented throughout this chapter that there is a multitude of inflammatory processes in train in the Crohn’s disease mucosa. IL-l and 11-6 There is stronger direct evidence of enhanced local generation of these cytokines in Crohn’s disease than there is for TNF-a (Grottrup-Wolfers et al, 1996), and for IL-6 the response appears more marked in Crohn’s disease than in ulcerative colitis. Mononuclear cells appear to be the major source (Youngman et al, 1993). The overall effects of these are similar to the effects of TNF-a. IL-2, IL-12 and y-interferon These key T-cell cytokines are responsible for the amplification of specific cell-mediated immunity and the sensitization of cellular targets to cytotoxicity. Recent work in the main (Fuss et al, 1996; Parronchi et al, 1997), suggests enhanced expression in Crohn’s disease although this is contentious, and experimental systems in which these amplificatory steps are inhibited (for example by inhibiting the cytokine IL-12 which is involved in the cascade) show a downregulation of intestinal inflammation (Strober et al, 1997).

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IL-8 This is a strongly chemotactic cytokine, associated with activated and attracted neutrophils, which is elevated in tissues in IBD. Some evidence suggests that it may play a more prominent role in ulcerative colitis than in Crohn’s disease, but it is widely expressed in Crohn’s disease, in a variety of cell types (macrophages, PMNs and epithelial cells: Mazzucchelli et al, 1994). Anti-inflammatory

cytokines

A number of molecules generated in inflamed tissue are counter-regulatory, diminish tissue damage, and initiate repair. These include the predominantly T-cell derived cytokines IL-4, IL-1 0 and IL-l 3, and the macrophage derived substance ILl-receptor antagonist. For example, exposure of IBD tissue to IL-10 in culture leads to a diminution of production of a wide variety of pro-inflammatory cytokines and eicosanoids (Wardle, 1994). There is some evidence that their production may be deficient in Crohn’s disease tissue. Reduced levels of tissue IL-10 and IL-4 have been reported in Crohn’s disease (Nielsen et al, 1996), and this work is now impacting in clinical research with exogenous IL-10 since it is safe to administer in active IBD, and is associated with clinical improvement, although fuller results are awaited. The counter-regulatory molecule IL-1 receptor antagonist works by the specific mechanism of occupying but not activating the receptor on cells that IL-l a and p react with (Dinarello, 1991). In inflammation in the bowel there appears to be a reduction in the proportion of this regulator present compared to the agonist IL-1 (Cominelli et al, 1994), leading to the suggestion that exogenous IL-1 receptor antagonist might also be therapeutically effective. Although demonstrated in experimental colitis (Cominelli et al, 1990), the clinical data to support this have not become available.

REPAIR

PROCESSES

The clinical and pathological features of Crohn’s disease reflect not only the initial inflammatory process but also repair and healing-restitution and regeneration of epithelium, and fibrosis and thickening of the submucosal layers being major components. The molecular mechanisms underlying these involve the generation of cytokines that, in particular, can act as growth factors (Babyatsky et al, 1996) but whose actions include not only the enhancement of cell proliferation, but morphogenesis, neovascularization and the laying down of extracellular matrix. Whilst most repair processes are clearly beneficial, repair that is associated with distortion or excessive fibrosis can present later complications. Within the inflamed mucosa, mononuclear cells express transforming growth factor p (TGF-P: Chiarpotto et al, 1997); stromal cells express keratinocyte growth factor (Brachle et al, 1996), and epithelial cells in particular express a variety of

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growth factors (trefoil peptides, TGF a, epidennal growth factor (Poulson et al, 1993)) which can play a role in tissue repair. Experimental work on skin wound healing has demonstrated how, for example, modulating TGF-P action can modify scarring, and exogenous epidermal growth factor (EGF) can ameliorate experimental colitis (Procaccino et al, 1994), and this field offers exciting new therapeutic possibilities in Crohn’s disease. CONCLUSION Numerous inflammatory mechanisms are activated in Crohn’s disease, and, in combination with other mechanisms yet to be discovered, give rise to the chronic inflammatory condition affecting the gut and sometimes extraintestinal tissues. The relative importance of each, and the factors that lead one individual and not another to develop the condition are being slowly elucidated. Some are genetic, but others are environmental. Clear epidemiological factors such as smoking, which predisposes to Crohn’s disease or at least to clinical manifestations of the disease, are being investigated to define how they impact: intriguing work is beginning to define the effects of nicotine and other products on manifestations of inflammatory responses for example. To the clinician, the stimulus for such work is that it may define new therapeutic approaches. With such a plethora of potential targets, the only effective way to establish the validity of any given approach is by well-designed, scientifically valid ethical studies in the most important person in the equation-the patient. REFERENCES Adeyemi EO & Hodgson HJF (1991) Lactoferrin: a correlate of disease activity in inflammatory bowel disease. European Jour-nal qf Gastroenterology and Hepatology 3: 51-56. Adeyemi EO & Hodgson HJF (1992) Faecal elastase reflects disease activity in active ulcerative colitis. Scandinavian Journal of Gastroenterology 27: 139-142. Adeyemi EO, Neumann S, Chadwick VS et al (1985) Circulating human leucocyte elastase in patients with inflammatory bowel disease. Gut 26: 1306-13 11, Babyatsky MW, Rossiter G & Podolsky DK (1996) Expression of transforming growth factors alpha and beta in colonic mucosa in inflammatory bowel disease. Gastroenterology 110: 975-984. Belluzzi A, Brignola C, Campieri M et al (1996) Effect of an enteric-coated fish-oil preparation on relapses in Crohn’s disease. New England Journal qf Medicine 13: 1557-1560. Bouma G, Odukerk-Pool M, Scharenberg JG et al (1995) Differences in the intrinsic capacity of peripheral blood mononuclear cells to produce tumour necrosis factor alpha and beta in patients with inflammatory bowel disease and healthy controls. Scandinavian Journal qf Gastroenterology 30: 1095-l 100. Brachle M, Madlener M, Wagner AD et al (1996) Keratinocyte growth factor is highly over-expressed in inflammatory bowel disease. American Journal of Pathology 149: 521-529. Brandtzaeg P (1985) Immunopathology of Crohn’s disease. Annals of Gastroenteemlogy and Hepatology, Paris 21: 202-220. Breese EJ, Michie CA, Nicholls SW et al (1994) Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology 106: 14551466. Butcher EC (1991) Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67: 1033-1036.

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