Insights from mouse models of colitis

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1 Insights from mouse models of colitis Richard Boismenu and Yaping Chen Department of Immunology and Strohm Inflammatory Bowel Disease Center, The Scripps Research Institute, La Jolla, California Abstract: Emerging studies using mouse models of experimental colitis are defining the nature of the immunological disturbances that initiate inflammation and destruction of the intestine. A better understanding of disease-promoting and -suppressing CD4 T cells is providing insight into the mechanisms controlling immune responses within the intestinal compartment. Moreover, a role for distinct T cell populations, including intraepithelial T cells, in maintaining the physical integrity of the intestine was suggested by recent studies. Cytokine gene-knockout mice and anti-cytokine treatments remain important tools to define the pro- and anti-inflammatory functions of cytokines. These advances are fostering the design and evaluation of new therapeutic approaches that may eventually be applied to treat human inflammatory bowel disease. J. Leukoc. Biol. 67: ; Key Words: cytokines inflammation intestine keratinocyte growth factor T cells tumor necrosis factor INTRODUCTION Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of unknown origin. IBD comprises two main disease conditions, ulcerative colitis (UC) and Crohn s disease (CD). UC affects mostly the mucosal layer of the large intestine or colon. In contrast, CD is defined as a transmural granulomatous inflammation that can involve segments of the small and large intestine. An estimated one to two million Americans suffer from IBD, this number almost evenly split between cases of UC and CD. Although UC has been known since the late 19th century [1], CD was described only in the early 1930s [2]. This finding may be explained by historical evidence suggesting that CD is a more recent disease [3]. The distinction between UC and CD was clearly established in 1960 [4]. Traditionally, IBD was believed to be an infectious disease and for the last 50 years numerous studies have attempted to identify the responsible microorganism(s). Despite intense efforts, a causative organism for IBD remains to be identified [5]. The effectiveness of steroid therapy on IBD and the failure to find a causative microbe led to the currently prevailing view that IBD may be an immunological or autoimmune disorder. As summarized in Table 1, IBD is proposed to occur in genetically predisposed individuals that develop an abnormal immune response after an environmental insult affecting the intestinal mucosa. Progress in understanding the mechanism(s) leading to IBD has been slowed by the limited number of experimental model systems available up until the 1980s. The last decade, however, has witnessed a remarkable increase in new, mostly mouse, IBD model systems available to test the various hypotheses concerning the etiology and pathogenesis of IBD [6, 7]. As could be expected, these model systems have led to an unprecedented increase in our understanding of mechanisms involved in IBD. One of the first and most important lessons learned early-on from these model systems was that an emerging mucosal immune response against gut constituents is of critical importance for intestinal inflammation and the subsequent destruction of the mucosa [8 10]. For this reason, IBD research has focused intensely on the immune- and non-immune-cell subsets as well as the soluble mediators involved in normal and dysregulated immune responses. The primary goal of this review is to provide an overview of current concepts and hypotheses driving IBD research that are being addressed through the use of mouse model systems. We will also highlight the usefulness of these mouse IBD model systems for drug development and describe emerging therapeutic alternatives to the more conventional IBD treatment options resulting from that research. Additional reviews should be consulted for a more exhaustive description of experimental IBD model systems [5, 6, 11 14] and available IBD treatments [5, 15 18]. THE MODELS As summarized in Table 2, mouse models of intestinal inflammation can be classified into four main categories. First, intestinal inflammation and tissue damage can be induced in mice by administration of specific chemical agents such as acetic acid [19], dextran sulfate sodium (DSS) [20], trinitrobenzene sulfonic acid (TNBS) [21, 22], and oxazolone [23]. Second, colitis develops spontaneously in a few naturally occurring mutant mouse strains such as C3H/HeBir [24] and SAMP1/Yit [25]. Third, several gene-knockout and transgenic strains develop colitis [26 31]. Fourth, intestinal inflammation is observed following reconstitution of immunodeficient mice with CD4 T cells [32, 33]. The two most common chemically induced colitis models are Correspondence: Richard Boismenu, Department of Immunology, The Scripps Research Institute, IMM-8, North Torrey Pines Road, La Jolla, CA boismenu@scripps.edu Received January 12, 2000; accepted January 12, Journal of Leukocyte Biology Volume 67, March

2 Pathology Initiation TABLE 1. Probable Sequence of Events Leading to IBD Microbes Toxins Genes Potential effector Present and future therapies Antibiotics Vaccination Gene therapy < Perpetuation Microbial antigens Antibiotics, < Vaccination Hyperimmunity Activated immune cells Steroids Immunomodulators < Inflammation < Tissue injury T and B lymphocytes Macrophages Neutrophils Erosion Ulceration Fistula Fibrosis Steroids Immunomodulators Cytokine/chemokine inhibitors Growth factors those employing DSS and the haptenating compound TNBS. Administration of DSS leads to a UC-like colitis, whereas TNBS treatments initiate a CD-like intestinal inflammation. These models recapitulate many of the events proposed to initiate and sustain human IBD, as shown in Table 3 for DSS-induced colitis. Oral administration of DSS for several days leads to colonic epithelial cell lesions and acute inflammation characterized by the presence of neutrophils and macrophages within damaged segments. Termination of DSS treatment is followed by a chronic colitis characterized by large numbers of activated T cells near diseased segments. In addition, regeneration of the eroded epithelium over the course of several days to weeks is observed after termination of DSS treatment. The reason for the deleterious effects of DSS is not well understood, however, epithelial cell toxicity, increased intestinal permeability, and macrophage activation have been proposed as potential mechanisms [34, 35]. Similarly, rectal administration of TNBS dissolved in ethanol initiates a severe inflammatory response and usually transmural tissue necrosis that can be followed by regeneration [6]. In this model, ethanol is proposed to elicit a transient increase in intestinal permeability, allowing TNBS to reach the subepithelial space and haptenate tissue and microbial proteins. The intense and sustained inflammatory response that ensues apparently breaks T cell tolerance to mucosal antigens. The mucosal immune response initiated in both model systems involves T cells localized in the lamina propria, intraepithelial space, and gut-associated lymphoreticular tissue (GALT). Mouse models of inflammatory colitis such as DSSinduced and TNBS-induced colitis have been used to study various aspects of acute and chronic inflammation as well as mechanisms involved in colonic healing [6]. In addition, different inbred mouse strains showing differential susceptibility to chemically induced colitis have been used for research into the genetic basis of IBD [7]. Several new mouse IBD model systems derived from geneknockout and transgene technology were described in 1993 that more clearly defined a role for the immune system in IBD. A spontaneous chronic intestinal inflammation was the surprising outcome in mouse strains having targeted deletions of interleukin-2 (IL-2) [26], IL-10 [27], or T cell receptor (TCR) chains [28]. The same year, an additional mouse IBD model system was described in which intestinal inflammation developed spontaneously after reconstitution of SCID mice with CD4 T cells [32, 33]. This and other adoptive cell transfer models [36, 37] defined an important role for specific CD4 T cell populations in the development of IBD. It is important to note that the colitis-inducing potency of CD4 T cells in these adoptive cell transfer models is determined by various parameters, including their activation state, CD45RB phenotype, and IL-12 responsiveness [38]. Together, the mouse models discussed here have provided incontrovertible evidence that a dysregulated immune response and an abnormal cytokine production pattern is pivotal for the development of IBD. Since the introduction of these models, several new mouse models of intestinal inflammation have been described that are beginning to provide additional insights into events contributing to the pathogenesis of IBD [36, 39 43]. Other applications for chemically induced colitis model TABLE 2. Some Useful Mouse Colitis Model Systems Category Area(s) involved Inflammation Similarity References Chemically induced Acetic acid Descending colon/rectum Acute UC [19] DSS Colon Acute/chronic UC [20] Oxazolone Descending colon Acute/chronic UC [23] TNBS/ethanol Colon Acute/chronic CD [21, 22] Spontaneous C3H/HeBir Descending colon/rectum Acute/chronic [24] SAMP1/Yit Ileum/cecum Acute/chronic CD [25] Transgenic/gene deletion o/o G i Colon Chronic acute UC [30] IL-2 o/o Colon Acute/chronic UC [26] IL-10 o/o Small intestine/colon Chronic UC [27] Keratin 8 o/o Colon Chronic [29] N-cadherin Small intestine Chronic CD [31] TCR- o/o Colon Chronic UC [28] Adoptive cell transfer CD4 T cells/scid Colon ileum Chronic acute CD [32, 33, 38] CD3 (Tg 26) Colon Acute/chronic CD [36] 268 Journal of Leukocyte Biology Volume 67, March

3 TABLE 3. Features of Human CD and UC Compared to Mouse DSS-Induced Colitis Feature CD UC DSS-induced colitis Location Small intestine/colon Colon Colon Depth Transmural Mucosal Mucosal Extent Discontinuous Continuous Continuous Symptoms Non-bloody diarrhea, fistula Bloody diarrhea, no fistula Bloody diarrhea, no fistula Granuloma Yes No No Genetic Yes Yes Yes Microbial flora Yes Yes Yes Immunological Yes Yes Yes Inflammation Transmural Epithelium Epithelium TNF- Elevated Elevated Elevated KGF Elevated Elevated Elevated during regeneration systems have been reported more recently. For instance, administration of chemical agents such as DSS to geneknockout mice strains is often used as a means to reveal a potential role in IBD for gene products whose absence do not by themselves give rise to intestinal abnormalities. The rationale here being that the role of such genes in maintaining intestinal homeostasis may only manifest itself under stress or disease conditions. One example is mice having a targeted disruption of intestinal trefoil factor (ITF). The ITF is an abundant protein found at the intestinal mucosal surface [44]. ITF is overexpressed after injury in acetic acid-induced colitis [45] and is known to stimulate epithelial cell migration as determined by an in vitro wounding assay [46]. These observations suggested a role for ITF in the maintenance and repair of the intestinal mucosa. Unexpectedly, ITF o/o mice showed little intrinsic defect of the intestinal compartment [47]. This finding may be explained by functional compensation from other ITF family members or alternately, if ITF is only required following intestinal stress, damage or disease. A role for ITF under stress or injury conditions appears likely because ITF o/o mice were found to be markedly more sensitive to DSS-induced colitis than control mice [47]. Another example is provided by transforming growth factor o/o (TGF- o/o ) mice and mice carrying the waved 1 (wa1) mutation [48]. The wa1 mutation is allelic with the TGF- gene and results in a severe reduction in TGF- expression at the mrna and protein levels. Like ITF, TGF- has been proposed to have a role in preserving the integrity of the gastrointestinal tract [49, 50]. Egger et al. [48] found that TGF- o/o mice and wa1 mice do not have obvious intestinal disease. However, for reasons that remain unclear, TGF- -deficient mice were more susceptible to DSS-induced colitis, supporting a role for TGF- in maintaining intestinal homeostasis [48]. In this context it is interesting to note that TGF- may be involved in the regulation of ITF expression [48]. Thus, subjecting relevant geneknockout strains to chemically induced colitis represents one approach that may help to identify the complicated network of molecular and cellular interactions that maintain intestinal integrity. THE CELLS The intestine is an epithelial tissue consisting of several functionally specialized epithelial cell types, including enterocytes, goblet cells, and M cells. However, nonepithelial cell types reside within or can travel through the intestine. Thus, physical and functional interelationships between epithelial and nonepithelial cell types must occur normally as well as under disease states. Among the nonepithelial cell types are cells of the innate and acquired immune system such as T/B lymphocytes, natural killer (NK) cells, monocytes/macrophages, neutrophils, eosinophils, and mast cells. Additional nonepithelial, nonimmune cell types are found within the intestine, including endothelial cells, nerve cells, and fibroblasts. All cells found within the intestine can communicate with each other either locally or globally through the release of soluble mediators and cell-mediated contacts. Soluble mediators consist of cytokines, growth factors, antibodies, eicosanoids, neuropeptides, proteases, and nitric oxides to name a few. In evaluated cases, many of the cell types and factors mentioned previously have been implicated in the promotion or suppression of intestinal inflammation [for review see ref. 5]. Clearly, global approaches emerging from genomic, proteomic, and bioinformatic research are required to fully understand physiological responses within the intestinal tract. In this section we will review the contribution of T cells to IBD pathogenesis, primarily because mouse models have recently provided elegant approaches for deciphering the role of this cell type in the disease process. The crucial role of T cells, but not B cells, in IBD pathogenesis is supported by studies performed in several different mouse colitis models. For example, IL-2 o/o mice bred to B cell-deficient mice but not to T/B cell-deficient mice still develop colitis, further indicating a minor role for B lymphocytes in the initiation of intestinal inflammation [51]. Similar conclusions were reached after analysis of IL-10 o/o /B celldeficient mice [52]. In most mouse IBD model systems chronic intestinal inflammation appears to be critically dependent on a Th1 cell response. In these models, IL-12 is proposed to enhance the Th1 cell response by acting as a T cell growth factor. Administration of anti-il-12 antibodies in CD-like models such as TNBS-induced colitis prevents disease initiation and blocks ongoing disease [22]. Neutralization of IL-12 appears to be associated with deletion of pathogenic Th1 cells [13]. CD45RB hi CD4 T cells having a Th1 cytokine profile are the most potent phenotypically defined T cell subset capable of transferring colitis to imunodeficient recipient mice [38]. In most cases, transfer of other CD4 T cell subsets such as CD45RB lo cells induces little or late-onset disease [32, 38]. As Boismenu and Chen Mouse colitis models 269

4 described below, transfer of CD45RB lo along with CD45RB hi cells blocks the potential of the later CD4 cell population to induce severe intestinal inflammation [32, 53, 54]. Such findings imply the existence of regulatory mechanisms that limit the duration and/or amplitude of mucosal T cell responses. In contrast to findings in most mouse models, the UC-like oxazolone-induced colitis and the spontaneous colitis developing in TCR- o/o mice is mediated by a Th2 cell response characterized by IL-4 and TGF- overexpression [23, 55]. In these models, IL-4 has a pathogenic role, whereas TGF- limits the severity of the inflammation. As expected, anti-il-4 antibody treatment but not administration of anti-il-12 antibodies was found to diminish the severity of colitis in these Th2-driven model systems. In an interesting twist, Dohi et al. [56] reported that under certain experimental conditions TNBS-induced colitis is characterized by Th2 cells expressing IL-4 and thus resembles UC more than CD. Anti-IL-4 antibody treatment could reduce the severity of colitis in this model system. It was proposed that TNBS treatment may lead to a polarized Th1 or Th2 response depending on the mouse strain used and the dose of TNBS. Another possibility is that Th1 and Th2 responses co-exist during colitis with one type of response predominating at a certain stage of disease. It may thus be more appropriate to refer to intestinal Th responses as Th1- or Th2-dominated responses. By varying experimental conditions used to induce TNBS colitis, Dohi et al. [56] may have favored a dominant Th2 response. It would be interesting to pursue this idea in other mouse models traditionally associated with a Th1 or Th2 response because evidence for mixed Th responses was also obtained in DSS-induced colitis [57] and in IL-2 o/o mice [58]. These observations provide an important lesson, mainly that the underlying events responsible for the generation of CD- versus UC-like immune responses are not simply historical but dynamic. As a consequence, the nature of the ongoing intestinal immune response may undergo important qualitative changes during disease. A clinical correlate to this observation may be provided by IBD patients diagnosed with indeterminate colitis as well as by those patients whose disease phenotype changes over time. THE CYTOKINES A large number of cytokines, growth factors, and other soluble mediators are known to be up/down-regulated during inflammatory colitis in mouse models. In general, a similar pattern of expression has also been described for the human homologs when measured in intestinal tissue obtained from IBD patients. It is beyond the scope of this review to be comprehensive in this area. Instead, soluble factors currently considered of pivotal importance in IBD pathogenesis will be highlighted. Tumor necrosis factor (TNF- ) is a well-studied proinflammatory cytokine that is produced as both a soluble and a cell-associated molecule. The recent introduction of anti- TNF- antibody to the therapeutic armamentarium available to treat CD emphasizes the important role of TNF- and/or TNF- -producing cells in the disease process. TNF- is overexpressed during the intestinal inflammation developing in most mouse IBD models [43, 59 63]. Moreover, forced overexpression of TNF- in transgenic mice can lead to a more severe intestinal inflammation after TNBS treatment, whereas TNF- o/o mice are relatively protected from TNBS-induced colitis. In the adoptive CD4 T cell transfer IBD model, TNF- production is associated early on with lamina propria macrophages located near areas of intestinal lesions [63]. This study found that at later stages of disease epithelial cells expressed TNF- at low levels [63]. Macrophages were also found to represent the major source of TNF- in TNBS-induced colitis [59]. When examined in mouse IBD models, inhibition of TNF- with specific antibodies or pharmacological agents resulted in a much less severe colitis [59]. The relationship between cell types infiltrating the intestine and TNF- expression is being illuminated by elegant experiments exploiting mouse IBD model systems. For example, transfer of CDRB hi CD4 T cells overexpressing a TNF- transgene into RAG-2 o/o mice was found to lead to a more severe colitis [64]. However, transfer of TNF- / o/o CD45RB hi CD4 T cells was sufficient to upregulate TNF- expression in colonic non-t cells and to induce colitis. Taken together, these results suggest that TNF- production by both T cells and non-t cells is important for the development of intestinal inflammation. This hypothesis was supported by the demonstration that only a mild form of colitis developed after transfer of CD45RB hi T cells into TNF- / o/o / RAG-2 o/o mice [64]. IL-12 is mainly produced by activated macrophages and is involved in the induction of Th1 responses and interferon- (IFN- ) production. Up-regulation of IL-12 production in lamina propria mononuclear cells occurs in CD but not in UC patients [65]. An important role of IL-12, but not IFN-, in sustaining colonic inflammation has been demonstrated in TNBS-induced colitis [22], IL-10 o/o mice [66], and IL-2 o/o mice [67] by in vivo administration of neutralizing anti-cytokine antibody preparations. In contrast to these observations, anti- IL-12 antibody treatment only partially blocked the colitis developing after transfer of CD45RB hi CD4 T cells into immunodeficient mice [41]. This finding suggested that other cytokines may be required for the development of a pathogenic Th1 response. IL-18 has received considerable attention as one such potential cytokine because it is expressed at the highest levels by mouse and human intestinal epithelial cells and has been implicated in the development of polarized Th1 responses [68 71]. However, there is evidence that IL-18 may behave as a switch cytokine inducing a strong Th1 or Th2 response depending on the cytokine environment [72]. The availability of IL-12 o/o and IL-18 o/o mice [73] as well as IL-18-receptor o/o mice [74] should help unravel the role of these cytokines in the control of mucosal immune responses. The role of IL-4 in intestinal inflammation remains obscure. In mouse models there is no definitive evidence that IL-4 is involved in the counter-regulation of Th1 responses. However, under certain conditions IL-4 may increase the proliferation of TGF- -producing counter-regulatory cells. As a caveat, it is also clear that transfer of IL-4 o/o CD45RB lo cells into immunodeficient mice can still protect from colonic pathology mediated by CD45RB hi cells [75]. In contrast, a pathogenic role for IL-4-driven colonic inflammation in TCR- o/o mice and oxazolone-mediated colitis is more firmly established. This was 270 Journal of Leukocyte Biology Volume 67, March

5 demonstrated by the reduced incidence of disease in IL-4 o/o / TCR- o/o mice and the successful treatment of oxazoloneinduced colitis with anti-il-4 antibodies [23, 55]. As a final caveat, several studies have firmly established that IL-4 overexpression in intestinal epithelial, lamina propria, and submucosal cells is not associated with human UC and CD [76 79]. THE GENES Epidemiological data have suggested a genetic susceptibility to IBD. DSS-induced colitis in particular is being exploited for the identification of IBD susceptibility genes based on the varying sensitivity of genetically different mouse strains to chemically induced colitis [80]. Among the strains particularly sensitive to DSS-induced colitis is the C3H/HeJBir strain, which develops a spontaneous colitis early in life, and the parental C3H/HeJ strain that does not. The spontaneous colitis in the C3H/HeJBir is characterized by acute and chronic inflammation of the cecum and colon. Inflammation usually resolves within a few months, a process associated with regeneration of the epithelium. The mechanism responsible for the spontaneous colitis in C3H/HeJBir mouse remains unclear, but it appears that CD4 T cells from C3H/HeJBir can be more easily activated to produce a Th1-type response than similar T cells isolated from the parental strain [81]. Both strains have a mutation that provides resistance to bacterial endotoxin, however, it remains to be determined how this mutation affects susceptibility to DSS-induced colitis. Identification of polymorphic alleles between these strains may allow the identification of additional genes predisposing to intestinal inflammation [82]. It is interesting to note that C3H/HeJ mice are more susceptible than C57BL/6 mice to TNBS-induced colitis, suggesting that similar genes may predispose to or protect from colitis initiated by different means [83]. Other ongoing efforts into the genetic basis of susceptibility to experimental colitis include the generation of genetic crosses between IBD-susceptible mouse strains such as C3H/HeJ and partially IBD-resistant mouse strains such as C57BL/6, as well as the use of congenic strains [82]. Interestingly, this study found that both parental genomes contribute susceptibility loci to their progeny. Several loci located on chromosomes 5 and 2 appear to increase susceptibility to DSS-induced colitis, whereas potential resistance alleles may be present on chromosomes 2 and 9. Attractive candidate genes in those regions include the A chain of the chemotactic growth factor platelet-derived growth factor (PDGF), the integrin 4 subunit, as well as the proinflammatory cytokines IL-1 and IL-1. Such approaches should ultimately lead to the identification of genes involved in intestinal inflammation. They also convincingly demonstrate that experimental colitis is under complex genetic control reminiscent of the human disease. THE MICROBES When examined, germ-free conditions do not support the development of disease in the mouse colitis models discussed previously [25, 84 86]. Similarly, broad-spectrum antibiotic treatment appears to prevent colitis and resolve active inflammation in at least some rodent colitis models [39, 87]. One exception appears to be IL-2 o/o mice that can still develop delayed focal intestinal inflammation under germ-free conditions [88]. Microbial recolonization of the intestinal tract normally restores susceptibility to experimental colitis. Further studies have shown that recolonization with defined bacterial species such as Helicobacter hepaticus is sufficient to cause intestinal inflammation in several gene-deficient mouse colitis model systems [89, 90]. Clearly, however, the presence of specific organisms is not absolutely required for intestinal inflammation in wild-type unmanipulated mice or in mouse model systems. What these results suggest quite convincingly is that a microflora is necessary for the development of experimental IBD. However, the mechanism by which the microflora influences mucosal immune responses is unclear. One hypothesis is that microbial antigens induce a strong polyclonal T cell response that may lead to activation of pathogenic mucosal Th1 cells that cannot be brought under control by anti-inflammatory cytokines or regulatory T cells. Bacterial products such as intimins may favor such responses by reducing the threshold of activation of lamina propria and GALT-associated T cells [91]. Thus, some bacterial species may be highly potent inducers of Th1 responses. However, microbes may influence T cell responses in a variety of other ways. For example, a recent study has shown that the microbial flora is required for migration of adaptively transferred T cells into the intestinal microenvironment of SCID mice [92, 93]. This finding may indicate that interaction of microbes with colonic cells is required for production of T cell chemotactic factors. The response of colonic cells to microbial colonization may also induce the expression of adhesion proteins and other surface molecules required for T cell emigration and activity. It is becoming apparent that the gastrointestinal tract needs to be viewed as a highly integrated system where every cell has a role to play in ensuring a rapid and appropriate response to an emerging threat or danger signal [94, 95]. Thus, the normal response of a tissue such as the intestine to danger is to allow for an effective localized inflammatory response that minimizes collateral damage. Chronic disease-causing inflammation appears to result under conditions that favor the inappropriate expression of cellular or cytokine responses as demonstrated by nearly every mouse model of colitis. The high microbial load within the intestinal tract may explain why inflammation in spontaneous mouse IBD models is mostly restricted to the intestine and no other tissues. Differences in microbial flora found in several vivaria have been proposed to explain the observation of more/less severe intestinal inflammation or even the occurrence of inflammation in tissues other than the intestine that have been reported to occur in some of the mouse IBD model systems [96]. One implication of these studies is that, as shown by Duchmann et al. [97], mucosal T cells in normal unmanipulated mice are unresponsive to their microbial flora. Studies using haptenated colonic proteins provide support for the notion that a cross-reactive immune response can break this state of tolerance [67]. In contrast, oral feeding of haptenated colonic proteins can Boismenu and Chen Mouse colitis models 271

6 prevent colitis induced by a subsequent challenge, as demonstrated using the TNBS-induced colitis model [83, 98]. Oral administration of haptenated proteins is believed to suppress the development of colitis by eliciting mucosal T cells capable of expressing the Th3-type cytokines TGF- and the Th2-type cytokines IL-4 and IL-10. However, the precise mechanism of action and antigen specificity of T cells expressing antiinflammatory cytokines remains to be determined. CONTROL OF INFLAMMATION AND TISSUE REPAIR THROUGH CELLULAR IMMUNE MECHANISMS At least three immunologically relevant cell populations, CD45RB lo CD4 T cells, NK cells, and intraepithelial lymphocytes (IEL), can help control the symptoms of colitis by apparently different mechanisms. Further functional characterization of these cells and delineation of their role in the intestine under normal non-disease conditions remain important issues to be addressed in the future. CD45RB lo Several studies have shown that reconstitution of immunodeficient mice with CD45RB hi CD4 T cells initiate intestinal inflammation [96]. This colitis could be prevented by cotransfer of CD45RB lo CD4 T cells [99]. Disease inhibition required production of TGF- and IL-10 but was independent of IL-4, suggesting that this inhibitory T cell population is not identical to a classically defined Th2 cell subset. Further phenotypic characterization using cell culture systems identified the regulatory T cell subset as being CD38 [100]. Functionally, CD38 T cells have the capacity to inhibit proliferation and cytokine secretion by CD38 T cells. However, Powrie et al. found that these in vitro effects did not completely mimic in vivo observations because they apparently were not dependent on TGF- or IL-10 [ ]. It is interesting that CD38 T cell-mediated inhibition of CD38 T cells required cell-cell contact as revealed by using Transwell inserts [100]. Similar regulatory cells, termed T regulatory 1 (Tr1) cells, have been described following in vitro activation of CD4 T cells in the presence of IL-10 that could protect from colitis when administered to SCID mice reconstituted with CD45RB hi CD4 T cells [102, 103]. The antigen specificity and mode of action of these Tr1 cells remain to be defined to better understand this regulatory mechanism. NK cells Intestinal inflammation in IL-10 o/o mice is characterized by Th1 cells and IFN- production [104]. Transfer of purified CD4 Th1 cells from IL-10 o/o mice to immunodeficient RAG o/o mice leads to chronic colitis [52]. Fort et al. [105] postulated that NK cells in recipient mice could be an important source of IFN- and contribute to disease. Thus, NK cell-depleted RAG o/o mice reconstituted with CD4 T cells from IL-10 o/o mice would be expected to be less sensitive to colitis. Surprisingly, such mice developed colitis at a faster rate [105]. Moreover, usually nonpathogenic wild-type CD4 T cells, a population comprised of CD45RB hi and CD45RB lo cells, also caused colitis when transferred to NK cell-depleted RAG o/o mice. Further experiments revealed that depletion of NK cells did not affect the engraftment of CD45RB lo cells, ruling out this possible scenario. Reconstitution of perforin o/o /RAG o/o mice revealed that the regulatory role of NK cells was perforin dependent. Thus, it appears that NK cells can limit the response of pathogenic CD45RB hi CD4 T cells to mucosal antigen using a perforindependant mechanism (i.e., cytotoxic). It remains to be determined whether NK cells establish direct contact with CD45RB hi cells or whether they instead target other cells important for the activation or survival of CD45RB hi cells. IEL Human and mouse IEL can be differentiated from blood and lymphoid T cells based on phenotypic differences including expression of specific TCR and V-gene segments [106, 107]. IEL populations have been identified in all examined tissues thus far [108]. It is interesting to note that increased numbers of intestinal IEL have been observed near intestinal lesions in patients with IBD [109]. Elevated numbers of T cells having the phenotype usually associated with intestinal IEL can also be detected in peripheral blood of IBD patients, which are believed to represent activated cells migrating out of the intestine or an expansion of the few such cells normally present in peripheral blood [110, 111]. The significance of this increased number of intestinal IEL in IBD patients remains ill-defined. Intestinal IEL have cytotoxic activities as judged from in vitro redirected lysis assay. However, it is unclear whether intestinal IEL normally perform such a function in vivo. Intestinal epithelial cells in T cell-deficient (TCR- o/o ) mice have a reduced rate of proliferation and differentiation defects, suggesting instead a role for intestinal IEL in the maintenance of the intestinal tract [112]. We have described production of the epithelial cell growth factor FGF-7 (KGF) by activated but not resting intestinal IEL [113]. Resident IEL in epithelial tissues other than the intestine were also found to produce FGF-7 upon stimulation. Surprisingly, all T cells tested as well as lymphoid T cells failed to express FGF-7. We evaluated the hypothesis that intestinal IEL and FGF-7 have a role to play in maintaining intestinal homeostasis using the DSS-induced colitis model system [Y. Chen, E. Fuchs, W. L. Havran, and R. Boismenu, unpublished results]. A marked increase in the number of IEL localized within damaged colonic segments was detected during the regeneration period that follows termination of DSS treatment. Moreover, intestinal IEL isolated from DSStreated mice but not those from control animals, expressed FGF-7. Subsequent experiments determined that TCR- o/o mice are more susceptible to acute DSS-induced intestinal injury and have a sharply decreased rate of tissue repair after termination of DSS treatment. A similar phenotype was revealed in FGF-7 o/o mice [114] subjected to DSS treatment. It is interesting that the skin of FGF-7 o/o mice does not show such an obvious wound healing defect, suggesting differences between epithelial tissues in the growth factors that are required for efficient repair. Nonetheless, these results are consistent with the general 272 Journal of Leukocyte Biology Volume 67, March

7 notion that IEL have a specialized function involving expression of epithelial growth factors that is aimed at preserving the integrity of stressed or injured epithelial cells. It is unclear whether the activation of intestinal IEL resulting from DSS treatment is dependent on antigen recognition through the TCR, or if other signals, such as cytokines, are sufficient. The nature of the antigens recognized by mouse intestinal IEL remain unknown. Until mouse IEL antigens become better defined, we believe that the oligoclonality of intestinal IEL responses after infectious and noninfectious mucosal perturbations and the apparent specificity of cells in general for conserved stress-induced self-antigens is consistent with a requirement for TCR-dependent activation. Our findings on intestinal IEL and FGF-7 using the mouse DSS-induced colitis model may be relevent to understanding repair of intestinal lesions observed in IBD patients achieving clinical remission. Several reports have demonstrated increased FGF-7 expression in the intestine of IBD patients even though the cellular source for FGF-7 was not completely elucidated in these studies [ ]. One study identified fibroblasts and lymphocytes as sources of FGF-7 [115]. Our ongoing clinical studies confirm these results and provide some additional evidence that T cells found within inflamed intestinal tissue can express FGF-7 [Y. Chen, M. H. Poleski, W. L. Havran, and R. Boismenu, unpublished results]. RELEVANCE Conventional therapy consisting of drugs such as corticosteroid and 5-aminosalicylic acid derivatives are not successful in inducing clinical remission or maintaining long-term remission in about 30% of IBD patients [118]. This fact and the often severe complications associated with steroid therapy have propelled the search for additional treatment options [18]. One advantage of mouse IBD model systems is their ability to rapidly evaluate the potential protective or pathogenic roles of soluble mediators and cell types. In turn, this information allows for the design of more precise clinical investigations and the rational design of better more powerful therapeutic approaches. We will discuss a few of the approaches being pursued in an attempt to illustrate the synergy currently developing between basic and clinical research in the IBD field. Several studies have implicated TNF- as a dominant player in a cytokine network that promotes colonic inflammation and tissue damage in human IBD [ ]. The view that TNF- is important in the pathogenesis of IBD was reinforced by results from recent studies showing that anti-tnf- antibody therapy (i.e., infliximab, Remicade) is effective in patients with moderately to severely active CD that failed to respond to conventional therapies [ ]. It is important to note that the clinical response to anti-tnf- antibody treatment appears to be strongly associated with histological evidence of reduced inflammation [128] and mucosal healing [129]. However, the mode of action of anti-tnf- antibody therapy remains largely unknown. It has been proposed that the benefit provided by anti-tnf- antibody treatment results from the neutralization of soluble and membrane-associated TNF-, as well as from the lysis of TNF- -expressing cells by antibody-dependent cellular cytotoxicity (ADCC) and complement fixation [18]. Anti-TNF- antibody treatment has also been shown to be of significant benefit in several mouse IBD systems, including DSS-induced and TNBS-induced colitis [61]. Because of potential complications that may arise from systemic and repeated antibody administration, the search for alternative ways of controlling TNF- expression, inflammation, and intestinal damage seems warranted. Several additional anti-tnf- agents have been described for the treatment of IBD and other conditions associated with TNF- overproduction [18]. Many of these anti-tnf- drugs are being evaluated in mouse IBD models. For example, a soluble p75 TNF receptor-fc fusion molecule known to neutralize TNF- improved chronic but not acute DSS-induced colitis [130]. In addition, a novel TNF- inhibitor, RDP58 [131], was successfully used in our laboratory to treat acute and chronic DSS-induced colitis [Y. Chen, K. Chou, A. El-Sheikh, J. Roberts, S. Iyer, R. Buelow, and R. Boismenu, unpublished results]. This decapeptide composed of d-isomer amino acids effectively inhibits TNF- mrna translation [S. Iyer, D. Kontoyiannis, D. Chevrier, J. Woo, N. Mori, M. Cornejo, G. Kollias, and R. Buelow, unpublished results]. RDP58 given orally or intraperitonealy at pharmacologically relevent doses reduced the severity of acute DSS-mediated colitis as judged from clinical and histological improvements. RDP58 treatment reduced neutrophil infiltration within the colon and blunted the increase in TNF- mrna and protein levels normally observed after DSS administration. Thus, the effect of RDP58 on TNF- production may result from a direct inhibition of TNF- expression, from a reduction in TNF- -expressing cells infiltrating the colon, or both. It also remains possible that the compelling clinical benefit seen with RDP58 in DSS-colitis may derive from a spectrum of physiologically relevant effects [132]. In any event, BrdU-labeling and TUNEL studies indicated that RDP58 protects the intestinal mucosa from DSSinduced epithelial cell death. RDP58 was also effective at enhancing intestinal regeneration after termination of DSS treatment when administered using a therapeutic regimen. Future studies will aim to evaluate the effects of RDP58 in well-established mouse IBD models other than DSS colitis and to investigate the mechanism of action by which RDP58 protects the intestinal mucosa from colitis. Preliminary data from our laboratory suggest that orally administered RDP58 is of clinical benefit in non-human primates suffering from a chronic UC-like colitis. Our preliminary results in mouse and primate IBD model systems are very encouraging and suggest that additional studies with RDP58 may lead to an entirely new form of IBD therapy. IL-12 has been implicated as a central cytokine mediator in several mouse IBD models and, in particular, TNBS-induced colitis [13]. Anti-IL-12 antibody treatment was successfully shown to prevent TBNS-induced colitis and, most dramatically, could reverse disease in mice with established colonic inflammation [22]. Thus, humanized anti-il-12 antibody preparations may follow in the steps of infliximab as an antibody-based therapy for CD. The spontaneous colitis developing in IL-10 o/o mice as well Boismenu and Chen Mouse colitis models 273

8 as the role of IL-10 in the generation of disease-preventing T cells convincingly established the counter-regulatory role of this cytokine [133]. The anti-inflammatory effects of IL-10 are believed to be mediated through inhibition of cytokine expression in activated macrophages. Administration of recombinant IL-10 (ril-10) prevents colitis occurring in RAG o/o mice subjected to microbial colonization with H. hepaticus [134]. Moreover, treatment with ril-10 ameliorates colitis in other mouse IBD models [60, 135]. On the strength of these observations, ongoing clinical trials are evaluating ril-10 for the treatment of CD [17, 136]. In addition to protein/peptide-based therapeutic approaches, gene therapy approaches are being entertained for the treatment of IBD. An example is provided by recent studies on TGF-. This cytokine has important anti-inflammatory properties and was demonstrated to counter-regulate TNBS-induced colitis. The exact mode of action of TGF- in the intestine remains unclear, but it appears to involve increased production of IL-10 and down-regulation of IL-12 receptor expression [W. Strober, personal communication]. An interesting approach developed by Fuss et al. [137] consists of delivering a plasmid coding for active TGF- intranasally concurrent with intrarectal administration of TNBS. The results of these studies showed that expression of TGF- could be increased dramatically within stimulated lamina propria cells. It is important that administration of TGF- DNA prevented almost completely the development of TNBS-induced colitis. Several important questions remain to be answered concerning the safety of these therapeutic protocols. For instance, the mechanism of plasmid uptake and expression by probably various cell types needs to be defined more accurately. In addition, it is unclear whether plasmid-derived TGF- expression is transient or long-lasting. In this context, it remains possible that plasmid-derived TGF- may stimulate production of endogenous TGF-. However, these are mostly technical issues that should be easily resolved. It is important to note that the successful application of a gene therapy-based treatment to treat intestinal inflammation in a mouse model suggests that similar protocols could be effectively applied to human IBD patients. Another approach using a recombinant human type 5 adenovirus vector expressing IL-4 has shown some beneficial effect in TNBS-induced colitis in rats [138]. Antisense oligonucleotides have been successfully used to modulate the expression of genes involved in intestinal inflammation [139]. For example, intercellular adhesion molecule-1 (ICAM-1) and the transcription factor NF- B are strongly up-regulated in human IBD as well as in mouse models of intestinal inflammation [ ]. Administration of ICAM-1 or NF- B antisense oligonucleotides can prevent and reverse DSS-induced or TNBS-induced colitis, respectively [140, 141]. Thus, antisense oligonucleotides may represent an attractive treatment strategy as a complement or alternative to those discussed previously. Therapeutic approaches are also being considered that aim to promote intestinal regeneration. FGF-7 (KGF or KGF-1) and more recently FGF-10 (KGF-2) have been described as potent mitogen and differentiation factors for epithelial cells acting through an identical FGF receptor [145]. Administration of FGF-7 to rats led to striking epithelial cell proliferation throughout the intestinal tract, suggesting that FGF-7 may be therapeutically useful to promote the repair of intestinal lesions [146]. This hypothesis is supported by the finding that administration of FGF-7 to mice before and after whole-body irradiation significantly reduced radiation toxicity to the intestine [147]. When evaluated in models of mouse colitis under prophylactic and therapeutic regimens, both growth factors were shown to be beneficial in limiting tissue damage [148, 149]. Additional epithelial cell growth factors including TGF- [150], epidermal growth factor (EGF) [151], and FGF-18 [152] may also find application in the development of an optimal IBD treatment that should be capable of suppressing inflammation while promoting intestinal repair. CONCLUSIONS AND FUTURE Even though our understanding of IBD has advanced considerably in the last few years, the increasing number of published mouse IBD studies indicate that we are only beginning to fully exploit the potential presented by these model systems. Increased knowledge of intestinal immune responses in health and disease will continue to rely on in vivo models to validate in vitro experiments. It is clear from current studies that under defined conditions both Th1 and Th2 cells can mediate pathological inflammation of the intestine. Paradoxically, these same cells are presumably required to protect the intestine from pathogenic organisms and cancerous cells. An emerging answer to this problem is that additional immune and non-immune cell types perform regulatory or protective roles that limit the severity of intestinal inflammation and tissue damage. Cytokines, chemokines, and growth factors produced by these cells are pivotal mediators of the positive and negative mechanisms that control the inflammatory response. It is now appreciated that every soluble factor has multiple and diverse functions [153]. A better understanding of complex diseases such as IBD will thus require more detailed information on the expression pattern of soluble factors and their receptors during the many phases of the inflammatory response. The rapid development of sophisticated computerized techniques to analyze DNA, RNA, and protein data should only further enhance the rate of such discoveries in the years to come. A more complete delineation of the molecules involved in intestinal inflammation and their functional relationship is likely to help identify additional key regulatory steps and to suggest new therapeutic approaches for the treatment of IBD. ACKNOWLEDGMENTS We thank Dr. Wendy Havran for her comments and suggestions regarding the manuscript. We also thank the National Institutes of Health and the Crohn s and Colitis Foundation of America for financial support. This is manuscript IMM from The Scripps Research Institute. 274 Journal of Leukocyte Biology Volume 67, March

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