Recent Developments in the Immunology of Inflammatory Bowel Disease

FRONTLINES– TOPIC REVIEW

Recent Developments in the Immunology of Inflammatory BowelDisease

T. T. MACDONALD, G. MONTELEONE & S. L. F. PENDER

Department of Paediatric Gastroenterology, St Bartholomews and the Royal London School of Medicine and Dentistry, London, EC1A 7BE, UK

(Received 1 October 1999; Accepted 11 October 1999)

MacDonald TT, Monteleone G, Pender SLF. Recent Developments in the Immunology of InflammatoryBowel Disease. Scand J Immunol 2000;51:2–9

Crohn’s disease and ulcerative colitis are caused by excessive immune reactivity in the gut wall. Analysis ofthe type of immune responses ongoing in diseased gut has revealed important features which suggest thatthese conditions are different. In Crohn’s disease tissue there is considerable evidence for an ongoing T helpercell type 1 response, with excess interleukin-12, interferon-g and TNF-a. There is circumstantial evidence inpatients that this response is directed against the normal bacterial flora and definitive evidence in mousemodels that T cell responses to the flora cause gut disease. In ulcerative colitis, the role of tissue damagingT cell responses in the gut mucosa is much less clear and there is more evidence that the lesion is owing toantibody-mediated hypersensitivity. Although different types of immune reactions initiate tissue injury inboth Crohn’s disease and ulcerative colitis, the downstream events which actually damage the tissue are thesame in each condition. Elevated cytokine concentrations in the mucosa lead to the production of excessmatrix degrading enzymes by gut fibroblasts, loss of mucosal integrity and ulceration. The same processalso leads to an increased production of epithelial growth factors such as KGF Keratinocyte GrowthFactor by gut fibroblasts and produces the crypt cell hyperplasia characteristic of all gut inflammatoryconditions.

Professor Thomas T MacDonald, Department of Paediatric Gastroenterology, 59 Bartholomew’s Close, StBartholomew’s and the Royal London School of Medicine and Dentistry, London EC1A 7BE, UK

INTRODUCTION

Inflammatory bowel disease (IBD) is caused by excessive andtissue damaging chronic inflammatory responses in the gut wall.Down-regulating this immune response allows the mucosa toheal and gut function to return to normal. Corticosteroids areextremely good at inhibiting immune responses and in themajority of patients they are the mainstay of therapy. They donot alter the natural history of IBD. There is also some doubt asto the extent to which they actually downregulate local inflam-mation, as opposed to inhibiting the systemic effects of the localinflammation. However about 30% of all patients are steroidrefractory and it is in this subgroup that new therapeutic regimes,based on the knowledge of the local tissue-damaging immuneresponses are being targeted.

Since the development of immunology as a distinct disciplinein the 1950s and 1960s there have been many thousands ofpublications on the immunology of IBD, the majority of whichhave contributed little because they have been descriptive rather

than mechanistic. In recent years however, with increasinglysophisticated tools to look at gut immune responses as well as amuch better understanding of molecular immunology, consider-able progress has been made, and there is now some consensusover the types of immune reactions which are ongoing in Crohn’sdisease (CD) and ulcerative colitis (UC). It has to be admittedhowever, that more progress has been made with Crohn’s diseasethan with UC.

MOUSE MODELS HAVE TOLD US THATT-CELL RESPONSES TO THE RESIDENTBACTERIAL FLORA OF THE GUT CANPRODUCE CHRONIC INFLAMMATORYBOWEL DISEASE

Until recently, the only animal models for inflammatory boweldisease consisted of the induction of nonspecific damage to theintestinal mucosa using acetic acid or dextran sulfate. A verypopular and informative model uses an intracolonic application

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of the contact sensitizing agent trinitrobenzene sulfonic acid(TNBS) in mice or rats. This elicits a Th1 response in the colonicwall and tissue injury. In rats, disease is chronic and resemblesCD in man, but in mice, it is the experience of the majority ofinvestigators that the disease is mild and self limiting. In the lastfew years more specific models have become available that havegreatly augmented our understanding of the regulation of muco-sal immune activation. These are summarized in Table 1. Most ofthese models are a result of targeted deletions of immuneresponse genes in mice (interleukin-2 (IL-2), IL-10, T-cellreceptor, TGF-b, SMAD3, Gai2) or overexpression of immunor-egulatory molecules such as gp39 or IL-7 transgenics. Finally,the important progress has come from models in which naive T-lymphocytes from normal mice are transferred into T-celldeficient mice (for reviews of most of these models see [1]).The results from these experiments can be condensed into severalpivota1 conclusions. First, most immunoregulatory events in

mucosal inflammation (proinflammatory as well as regulatory)are controlled by CD4þ T-lymphocytes. Secondly, mucosalinflammation may result from both CD4 Th1-biased (summar-ized in 1), and Th2-biased T-lymphocyte differentiation [2, 3].Thirdly, mucosal T-lymphocyte activation is antigen-dependent,and the responsible antigens originate from intestinal bacteria[4, 5].

The models involving manipulation of the host immunity havenot only told us that many different types of immune alterationscan lead to IBD, but have also provided tools with which to testvarious therapeutic regimes, such as IL-10 or anti-tumour necro-sis factor-a(TNF-a) antibody therapy. However, IBD also occursin mouse models with a normal immune system. A substrain ofthe C3H/HeJ mouse strain develops a spontaneous IBD early inlife and this again appears to be mediated by a CD4 Th1 responseto the flora [6]. The most interesting mouse to develop IBDhowever, is a chimera in whom gut epithelial E-cadherin func-tion is disrupted by a dominant negative N-cadherin. When thesemice become mature their intestinal epithelium is a mosaic ofnormal epithelium and leaky epithelium, where the N-cadherinprevents E-cadherin from signalling to the cell to maintainpolarity. In those areas of leaky gut, a chronic inflammatorybowel disease develops [7]. Along the same lines, Viney andcolleagues have studied a mouse strain in whom the multi drugresistance 1a gene is disrupted. These mice also develop IBD[8]. Transfer of normal marrow into irradiatedmdr1aknockoutmice also results in IBD, but the reciprocal transfer does not.This indicates that some radioresistant nonimmune componentis responsible for the development of IBD, and the currentnotion is that the absence ofmdr1a from the gut epitheliumallows increased antigen uptake from the gut lumen.

CROHN’S DISEASE HAS THE IMMUNESTIGMATA OF A T HELPER TYPE 1 IMMUNERESPONSE

Direct evidence for a role of CD4þ T-lymphocytes in CD islacking, but circumstantial evidence supports the notion that theconclusions from mouse models may be extended to the humansituation. First, active CD is characterized by an increasednumber of activated mucosal T-lymphocytes secreting inter-feron-g (IFN-g), and by increased mucosal production of cyto-kines that activate Th1-lymphocytes (IL-12 and IL-18) [9–12].There is a single report that IL-4 transcripts are elevated in theearly ileal lesions of recurrent CD [13], but this may be a specialtransient situation. Secondly, CD may disappear during thedevelopment of AIDS, or after bone marrow transplantation[14, 15]. Thirdly, treatment with depleting anti-CD4 antibodiesmay induce remissions in CD (as suggested by a small anduncontrolled trial) and anti-TNF antibody treatment mayowe its beneficial effects to counteracting activated mucosal T-lymphocytes (see below) [16, 17].

Fourth, CD, but not UC is classically associated with non-caseating granulomata, the hallmark of cell mediated immunity.

There is also circumstantial evidence that the gut flora is also

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Table 1. Animal models of spontaneous inflammatory bowel disease

Models Reference

Knockout modelsTcR-a Cell 1993 75 : 275–82TcR-b Cell 1993 75 : 275–82MHC Class II Cell 1993 75 : 275–82IL-2 Cell 1993 75 : 253–61IL-10 Cell 1993 75 : 263–74SMAD3 EMBO 1999 18 : 1280–91TGF-b Proc Natl Acad Sci USA 90 : 770–74Gai2 Nature Genetics 199 5 10 : 143–50TNF-a ARE Immunity 1999 10 : 387–98Cathepsin D EMBO 1995 14 : 3599–3608

Transgenic modelsIL-7 J Exp Med 1998 187 : 389–402HLA-B27 (rat) Cell 1990 63 : 1099–1112Gp39 Int Immunol 1997 9 : 1111–22Ag specific TcR transgenic mice J Immunol 1999 162 : 7208–16STAT-4 J Immunol 1999 162 : 1884–88

Leaky gut epitheliumN-cadherin dominant negativechimeric mice Science 1995 270 : 1203–7mdr1aknockouts J Immunol 1998 161 : 5733–44

Transfer modelsBone marrow→tge26 mice Immunity 1995 3 : 27–35CD4þ CD45RBhi→SCID Int Immunol 1993 5 : 1461–71CD4þ →SCID Eur J Immunol 1994 24 : 2803–12

Spontaneously coliticC3H/HeJ Bir mice Gastro 1994 107 : 1726–35

Unknown originEnteric glia deleted mice Cell 1998 93 : 189–201

important in CD. Antibiotic therapy is a useful adjunct for distalCD and is of therapeutic benefit in extending remission [18]and there have been studies in patients showing that aftersurgery, disease does not occur if the faecal stream is diverted[19, 20]. There are now a number of studies ongoing to determineif altering the flora with probiotics can change the diseaseprogression in CD.

In view of this predominant role of T-lymphocytes in thepathogenesis of CD it is of importance to understand how theimmune system regulates antigen-dependent T-lymphocyte acti-vation (for review see [21, 22]). Briefly summarized, threemechanisms seem to be important: firstly, the engagement ofthe T-lymphocyte receptor in the absence of a second signal (i.e.costimulation through B7 and CD28) leads to anergy; secondly,most activated T-lymphocytes are destined to undergo pro-grammed cell death (apoptosis) through Fas/Fas ligand inter-actions; and thirdly, counterregulatory cytokines (i.e. IL-10) caninterfere with the T-lymphocyte activation. Interestingly, thelast two mechanisms (in part) explain the effects of anti-TNFantibody and IL-10 treatment in CD. Moreover, mucosal T-lymphocytes from patients with active CD are relatively resistantto Fas-mediated apoptosis, which may explain the uncontrolledactivation that is observed in this condition [23].

THE NATURE OF THE T-CELL RESPONSE INUC IS STILL UNCLEAR

UC in its classical form differs so much from CD in itshistopathologically and clinically presentation that it is likelyto be a different disease. The mucosa is replete with neutrophils,there are crypt abscesses, and severe epithelial damage. Inrabbits, a local immunoglobulin (Ig)G immune complex reactionelicited in already damaged mucosa produces a lesion which isvirtually indistinguishable from UC in patients [24].

The idea that UC might represent some kind of Th2 type lesionis attractive but is still largely unfounded. Fuss and colleaguesshowed that isolated lamina propria CD4 T cells from UCpatients made more IL-5 when activatedin vitro with CD3/CD28 ligation than similar cells from CD patients or normals[25]. However IL-4 was not increased and overall, the CD4 cellsfrom UC patients produced IFN-g in an order of magnitudegreater than that they made IL-5. Others have also shown that IL-4 is decreased in UC [26, 27]. Where the Th2 paradigm mighthave some merit is in the sense of helping antibody responses. InUC, the inflamed bowel is filled with IgG plasma cells and IgG1,colocalizing with complement component C3b can be seen onthe surface of epithelial cells [28]. This IgG response is pre-sumably T-cell dependent and generated in organized gut asso-ciated lymphoid tissue.

There are other features of UC suggestive of antibody-mediated autoimmunity. It is associated with another putativeautoimmune disease, primary sclerosing cholangitis [29],and patients also have autoantibodies to the perinuclearcomponent of neutrophils (p-ANCA) [30] and epithelialtropomysin [31]. The fact that the disease is organspecific

and can be cured by colectomy is also suggestive of auto-immunity.

IL-12 MAY BE THE CYTOKINE RESPONSIBLEFOR DRIVING TH1 RESPONSES IN CROHN’SDISEASE

Studies in either human or experimental models have shown thatduring chronic colitis the differentiation of T-helper cells towarddefined cytokine secretion patterns is driven by locally releasedmolecules. Indeed in CD, IFN-g production is strictly associatedwith the expression of IL-12, a heterodimeric cytokine composedof two covalently linked subunits (p40 and p35). Transcripts forboth IL-12 subunits have been detected in gastric and intestinalmucosa of patients with recent onset and established CD [11, 12,32]. In addition, lamina propria mononuclear cells isolated fromeither spared or inflamed mucosal areas of CD patients releasefunctionally active IL-12, indicating that the IL-12 synthesis maynot be an epiphenomenon of active inflammation. One of themost powerful and relevant activities of IL-12 is its ability toinduce Th1 cell differentiation and IFN-g production [33].Consistent with these data it has been proven that neutralizationof endogenous IL-12 results in a dramatic decrease in the numberof IFN-g-producing cells in CD mucosa [34]. Moreover, con-vincing data have also been provided in the murine-inducedcolitis, which mimics some characteristics of CD, that mucosalinflammation can be abrogated by neutralizing IL-12 antibodies[35].

In contrast to CD, IL-12 is rare or undetectable in colonicmucosa of patients with UC [12]. It has been recently shown thata protein encoded by the Epstein-Barr-induced gene 3 (EBI3),sharing 27% of the amino acid sequence identity with the p40component of IL-12, can form a heterodimer with the p35subunit. This new heterodimer might function as an IL-12antagonist and favour the down-regulation of Th1 cytokines[36]. Interestingly, enhanced EBI3 expression has been detectedin inflamed mucosa of UC patients but not in active CD [37],suggesting that defective production of counterbalancing mole-cules may be contributory to the development of Th1 typelymphocytes in CD.

IL-12 mediates its biological activities through a specificreceptor composed of two subunits (b1 and b2). Both subunitsare required to form a functional receptor althoughb2 expressionis crucial in controlling Th1 cell lineage commitment [38, 39].By analogy with other Th1-mediated diseases, CD T-LPLexpress high levels ofb2 compared to UC and normal mucosa[40]. Binding of IL-12 to its receptor results in a rapid activationof the JAK-2 and TYK-2 signalling proteins and finally dimer-ization of STAT molecules. Although IL-12 can activate bothSTAT-3 and STAT-4 proteins, the effect of IL-12 on Th1 celldifferentiation depends specifically on expression of STAT-4[41, 42]. Indeed T cells from STAT-4-deficient mice manifestimpaired IFN-g production in response to IL-12 and are unable toefficiently promote the development of colitis when transferredin immunodeficient mice [43]. On the other hand overexpression

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of STAT-4 proteins in transgenic mice results in the induction ofcolitis which is characterized by the presence of a diffuseinfiltration of IFN-g-and TNF-a-secreting cells in the intestinalwall [44]. Taken together these data support the view that theactivation of the IL-12/STAT-4 signalling pathway is importantin immune-mediated injury in the gut. Consistent with this, wehave recently shown that activation of T cells in the laminapropria of foetal human gut with IL-12 and anti-CD3 results insevere mucosal degradation associated with the production ofTh1-type cytokines and matrix metalloproteinases (personalunpublished observations).

The IL-12-induced synthesis of IFN-g by lamina propria Tlymphocytes may be enhanced by other cytokines produced inhuman intestine such as IL-7 and IL-15 [45]. In addition, recentstudies have shown that in CD mucosa there is an enhancedproduction of biologically active IL-18, another cytokine withparticular relevance to Th1 responses [46, 47]. Immunohisto-chemical analysis has localized IL-18 to both lamina propriamononuclear cells and intestinal epithelial cells. In these cells,the expression of IL-18 is invariably associated with activesubunits of IL-1b converting enzyme, a molecule capable ofcleaving the precursor form of IL-18 to the active protein [47].Neutralization of IL-18 synthesis results in a significant inhibi-tion but not abrogation of CD lamina propria mononuclear cellIFN-g synthesis, further supporting the concept that IL-18 servesas a strong costimulatory factor of IL-12-driven Th1 responses[48].

A critical question remains as to what induces IL-12 in CD gutand which mechanisms regulate IL-12 secretion. IL-12 is pro-duced by antigen presenting cells mostly in response to bacteriaor bacterial products [33]. Since the development of Th1-mediated colitis in mouse models requires the presence ofnonpathogenic luminal bacterial flora, the inflammation may bedriven by microbial stimulation of IL-12 production. However,in contrast to autologous monocytes, normal intestinal macro-phages fail to produce detectable levels of IL-12 when stimulatedin vitro with bacterial products [12]. But as in CD the vastmajority of mononuclear cells infiltrating inflamed mucosa, andsubmucosa are recruited from circulation [49], it is thus likelythat IL-12 may at least in part be produced by recently recruitedmonocytes exposed to bacterial products. This is also supportedby the finding that in CD mucosa, IL-12-producing cells arepositively stained for the CD14 marker [34].

Figure 1 illustrates the pathways by which excess IL-12 candrive Th1 responses in the mucosa.

THE DOWNSTREAM EVENTS, SUBSEQUENTTO ELEVATED LOCAL NUMBERS OFINFLAMMATORY CELLS AND CYTOKINES,WHICH MEDIATE MUCOSAL DAMAGE

There have been a number of studies where evidence has beenproduced to suggest that cytokines have direct effect on gut cells.Many of these have used epithelial cell linesin vitro, but there isevidence that direct injection of cytokines has a damaging effect

on the gut [50, 68]. It is also self-evident that free radicalproduction by inflammatory cells, especially neutrophils, mustplay a major role in injury in acute inflammation in the mucosa.In addition cytokine-induced overexpression of vascular adhe-sion molecules will draw inflammatory cells into the gut andperpetuate inflammation. Nonetheless, in the last few years,studies from the authors’ laboratory have suggested thatdirect effects of cytokines plays a less important role than theregulatory role of cytokines on growth factor and matrix metallo-proteinase production by resident gut stromal cells.

Matrix metalloproteinases

A crucial step in ulceration of the gut wall in IBD is theactivation of a protease cascade, in particular the rapid upregula-tion of transcripts for the matrix metalloproteinases (MMPs) byproinflammatory cytokines. MMPs are a group of proteaseswhich have a putative Zn2þ-binding site HEXXH, and all requireCa2þ for stability, they also exhibit a preferred cleavage speci-ficity for the N-terminal side of hydrophobic residues [51, 52].MMPs can be subdivided into four different groups according totheir substrate specificity, these are collagenases, stromelysins,gelatinases and membrane type metalloproteinases (MT-MMPs).They are secreted in pro-enzyme forms requiring extracellularactivation except MT-MMPs. MMPs can be activated extracel-lularly by various agents such as plasmin or free radicals but theycan also interact to activate each other [53, 54]. MMP activity istightly regulated by their natural inhibitors, the tissue inhibitorsof metalloproteinase (TIMPs) [55]. Four TIMPs have beenidentified so far. As the result of their extensive substratespecificity, activated MMPs are able to degrade all classes ofextra-cellular matrix.

There have only been a few studies of MMPs in IBD. The firststudies came from Baileyet al. who performed immunohisto-chemistry and showed high levels of extracellular MMPs in areasof tissue injury in IBD [56]. The most important and convincing



Fig. 1. Diagrammatic illustration of how enteric bacteria entering thelamina propria can induce IL-12 production by monocytes which thendrives Th1 responses, leading to chronic cell-mediated inflammation.

evidence of MMPs in IBD was provided by Saarialho-Kereet al.[57] and Vaalamoet al. [58] who performedin situhybridizationshowing high degrees of expression of MMPs and TIMPs aroundulcer beds. They also observed that the cellular source of MMPsin IBD was fibroblast-like stromal cells. We have also observedan upregulation of stromelysin-1 in IBD compared to normalintestine whereas TIMP expression remain consistently high inboth groups of individuals (R. Heuschkel unpublished data).Similar results in ulcerative colitis patients have been made byothers [59].

Functionally we have been able to demonstrate an importantrole for MMPs in degradation of the gut mucosa during immunereactions. Activation of lamina propria T cells in explant culturesof foetal human small intestine by the lectin pokeweed mito-gen (PWM) for 72 h results in a strong Th1 biased responseand severe mucosal destruction [60]. This response is T-celldependent as it is inhibitable by cyclosporin A and FK506 [60].

The first clue that proteases might be important in this modelcame from the fact that PWM-stimulated explants showedevidence of the degradation of sulfated glycosaminoglycans inthe lamina propria. This phenomenon was inhibitable bya2-macroglobulin, suggesting that proteases were important [61].Further observations showed that this T-cell mediated tissueinjury was also associated with markedly increased levels ofactivated interstitial collagenase and stromelysin-1 after PWMstimulation and that the tissue injury was inhibited by a syntheticMMP inhibitor, CT1399. Most importantly, nanomolar concen-trations of recombinant stromelysin-1 (but not interstitial col-lagenase or gelatinases) caused severe tissue injury in 24 h [62].Stromal cells are the major cellular sources of stromelysin-1 andstromal cell lines rapidly upregulate MMP1 and MMP3 whencultured with TNF-a or IL-1b [62, 63]. Consistent with this,PWM-induced tissue injury can be inhibited by a soluble p55TNF-a receptor human immunoglobulin G1 fusion protein. Thisp55 TNFR-IgG fusion protein did not only inhibit tissue injurybut also 95% of stromelysin-1 production (but not collagenase orgelatinases) [64]. Figure 2 illustrates the cascade of events fromelevated local cytokines to excess MMPs and tissue degradation.

Epithelial growth factors

One of the most frequent manifestations of mild gut inflamma-tion in IBD is lengthening of the crypts and increased epithelialproliferation. There is now some evidence that this may bemediated at least in part by increased production of Keratinocytegrowth factor (KGF) from gut stromal cells activated by proin-flammatory cytokines. KGF is a good candidate for a moleculewhich can link immune reactions in the lamina propria withepithelial proliferation because its receptor, a splice variant ofFGFRII, is expressed only on epithelial cells. KGF is over-expressed in active IBD stromal cells [65–67] and functionalstudies in the foetal gut model described above indicate that it isalso upregulated in explants following T-cell activation withbacterial superantigens, coincident with crypt hyperplasia [68].Inhibition of KGF in this model decreases T cell mediated crypt

hyperplasia. Figure 3 illustrates how KGF produced by cytokineactivates stromal cells may mediate crypt hyperplasia. Crypthyperplasia may have evolved as a mechanism to eliminatepathogens which live inside epithelial cells or colonize theepithelial surface. In idiopathic diseases however, it serves toreduce the gut absorptive surface area and increased migration isassociated with more leaky tight junction so there will beincreased fluid, electrolyte and protein loss into the gut lumen.It is likely that KGF is only one of a number of molecules such asTGF-a, HGF and the other FGF’s which may play a role inregulating epithelial proliferation during inflammation. How-ever, it serves to further emphasize the point that the tissuedamaging effects of inflammatory cells and cytokines are asmuch to do with their ability to activate endogenous mechanismsof tissue injury than direct effects on gut cells themselves.



Fig. 2. Diagrammatic illustration of how TNF-a produced in excessby activated T cells and macrophages can drive the production of thematrix degrading enzyme stromelysin-1 (MMP3) by gut stromal cells.

Fig. 3. Diagrammatic illustration of how TNF-a produced in excessby activated T cells and macrophages can drive the excess productionof the epithelial mitogen KGF by gut stromal cells.

CONCLUSIONS

In this article we have concentrated mostly on the inflammatoryresponses in the gut in IBD, the antigens of the flora which drivesthese responses, and the downstream tissue damaging events.There has been a lot of progress in these areas but they are not yetfully linked. In animal models we know that the gut flora drivesIBD and this is also likely to be the case in man. However even inthe models in inbred mice, penetrance can be incomplete, and thedevelopment of disease in an individual mouse can be unpre-dictable. The overall ‘cleanliness’ of individual mouse facilitiesdetermines the penetration as well as the severity of the disease.We do not know whether the T-cell responses which drive theselesions in mice are generated in the Peyer’s patches, wherephysiological T cell responses in the gut occur, or whether thehyper-response is generated in the lamina propria. There is someevidence that removal of the lymphoid follicle at the tip of thecaecum slows down the development of IBD in T cell receptor-a

knockout mice [69], which has been touted as being of relevanceto the fact that prior appendectomy appears to protect againstulcerative colitis [70], but the immunological basis for these twophenomena are still very unclear. There clearly is a very complexrelationship between the gut immune system and the bacterialflora which deserves extensive study in the future.

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Recent Developments in the Immunology of Inflammatory Bowel Disease