Scientific Review

Inflammatory bowel disease: dysfunction of GALT and gut bacterial flora (I)

P. Chandran* S. Satthaporn* A. Robins** O. Eremin*
*Department of Surgery and **Department of Immunology, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Correspondence to: P. Chandran, Department of Surgery, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH

Introduction GALT Homing of T cells Dendritic cells and gut immunity

Cytokines and gut immunity Regulatory T cells Conclusion References

Gut-associated lymphoid tissue (GALT) is the largest lymphoid organ in the body. This is not surprising considering the huge load of antigens (Ags) from food and commensal bacteria with which it interacts on a daily basis. Gut-associated lymphoid tissue has to recognise and allow the transfer of beneficial Ags whilst concurrently dealing with and successfully removing putative and overtly harmful Ags. This distinctive biological feature of GALT is believed to be crucial to good health. Deregulation or dysfunction of GALT is thought to predispose to inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn’s disease. The exact mechanism(s) underlying the pathogenesis of IBD is (are) poorly understood and the immunological defects in GALT are poorly documented. Advances in immunology have highlighted the importance of dendritic cells (DCs), which are the key Ag presenting cells in tissues and lymphoid compartments. Their crucial role in GALT, in health and disease is discussed in this review. Interaction of DCs with T cells in the gut produces a subset of T lymphocytes, which have immunosuppressive function. Inappropriate Ag uptake and presentation to naïve T cells in mesenteric lymph nodes may lead to T cell tolerance in GALT. These various complex factors in the gut are discussed and their possible relevance to IBD evaluated

Keywords: Gut-associated lymphoid tissue, immune tolerance, commensal bacteria, dendritic cell, inflammatory bowel disease
Surg J R Coll Surg Edinb Irel., 1 April 2003, 63-75

INTRODUCTION
Numerous and varied bacterial organisms inhabit the gastro-intestinal (GI) tract. These organisms exist in a symbiotic relationship with the host. As these bacterial organisms colonise the gut after birth they are foreign to the tissues in the host and could induce an immunological reaction by the host. Fortunately, this does not happen and the human body accepts them as immunologically inert. Also, a vast range and amount of food Ags are ingested regularly against which the human body does not normally produce a harmful immune reaction. This immunological acceptance and failure of induction of a deleterious response is called tolerance.

The exact mechanisms by which this immunological tolerance is maintained in the gut are not clear. There is evidence to suggest that it is an active and complex process mediated by more than one mechanism.^1,2  Various mechanisms have been postulated to be operable:

• T cells may never encounter the Ag in an immunologically relevant form and, thus, are ignorant of its presence

• T cells may encounter the Ag in circumstances that result in subsequent functional anergy (tolerance) or cell elimination (apoptosis)

• Regulatory cells or mediators may be induced that can modify and suppress the necessary specific responses

Before discussing these various possible mechanisms of immunological tolerance and/or suppression it is important to understand the microanatomy of the lymphoid tissue in the gut.

GUT-ASSOCIATED LYMPHOID TISSUE (GALT)
Gut-associated lymphoid tissue is the largest lymphoid organ in the body and is in close proximity with a vast array of Ags and mitogens in the gut lumen. It is significantly different from the systemic immune system. Gut-associated lymphoid tissue contains specialised immune cells, such as intraepithelial lymphocytes (IELs) and Ag-presenting epithelial cells, which are not found elsewhere in the body.³ In contrast to the systemic immune system, the inflammatory response is down-regulated and suppressed within the GI tract. The gut immune system has evolved to protect epithelial surfaces and the underlying tissues from potentially harmful environmental agents and microbial organisms. The organisation of GALT is characteristic and is composed of Peyer’s patches, lamina propria lymphocytes (LPLs) and IELs. Peyer’s patches contain five or more lymphoid follicles and are found predominantly in the terminal ileum. The centre of the follicle consists of B lymphocytes surrounded by mantles of mixed cellularity. The interfollicular region contains T lymphocytes. The mucosa overlying the Peyer’s patches is composed of specialised epithelial cells called M cells (Figure 1). They differ from the rest of the epithelial cells by the absence of mucus, due to lack of adjacent goblet cells, which secrete mucus. The mucus that covers normal epithelial cells acts as a physical barrier preventing attachment of luminal Ags. Unlike the normal gut epithelial cells these M cells lack the IgA transporting capacity.⁴ Absence of goblet cells and their secretory products overlying the M cells allows the ready attachment of luminal particulate Ags to M cell surfaces. The attachment of Ags and their subsequent endocytosis by M cells appears to operate selectively; this is not surprising considering the large amount of dietary Ags that pass through the gut lumen every day. The normal commensal bacteria fail to adhere to the M cells. Pathogenic bacteria like Vibrio cholerae are readily taken up by the M cells but the heat-inactivated organisms are not.⁵ The IELs are situated between the epithelial cells and above the lamina propria. Unlike the peripheral T cells, 75% to 85% of IELs express the CD8 class I major histocompatibilty (MHC) - restricted phenotype and only 5% to 15% express the CD4 class II MHC restricted phenotype.^6-8 The IELs can be further subdivided according to the type of T cell receptor (TCR) expression. Seventy-five per cent of the cells found in the compartment express aß TCR and 10% to 15% express yð TCR.^9-11 The majority of T cells in the compartment also express the CD45RO memory phenotype, suggesting that these cells have probably already encountered Ags.

Figure 1: Schematic diagram of intestinal epithelium showing M cells, Peyer’s patches, intestinal epithelial cells and pathway of Ag transport. DC: dendritic cells, IEC: intestinal epithelial cell (Nu-nucleus,Mv:microvilli), MC: M cell, IEL: intra epithelial lymphocytes, PP: Peyer’s patches, M¯: macrophages, Pv: particulate Ag in pinocytic vesicle of M cell

The lamina propria lymphoid tissue exists as a diffuse collection of cells including T lymphocytes, B lymphocytes, plasma cells, macrophages, mast cells and small numbers of eosinophils and neutrophils. The vast majority of the B cells are IgA producing cells and the remainder are IgM producing cells. Lamina propria T cells have a similar CD4/CD8 ratio as found in peripheral blood.8 As with the IELs the majority of CD4⁺ cells in the lamina propria express CD45RO, a marker of memory cells.¹²

HOMING OF T CELLS IN THE GALT
Mucosal adressin cell adhesion molecules (MAdCAM-1) are selectively expressed by high endothelial cells in venules in GALT, Peyer’s patches and mesenteric lymph nodes.^13-15 The ligand for the MAdCAM-1 is integrina4ß7, which is expressed on memory T lymphocytes. The engagement of this receptor-ligand is important for homing of specialised memory T cells into the GALT. The T cells which express integrina₄ß₇, when they traffic through the endothelial venules in GALT, bind to MAdCAM-1 on the venule cells. The T cells which lack expression of integrina₄ß₇ are excluded from the GALT.¹⁶ How integrina₄ß₇ is expressed on naïve lymphocytes which allow them to localise in the GALT is poorly understood. But in vitro studies have shown that intestinal DCs can induce the expression of integrina4ß7 on naïve T cells. Dendritic cells isolated from non intestinal sites were unable to induce integrina₄ß₇ expression on T cells.¹⁷ Animal studies have shown that monoclonal antibodies (mAbs) which block MAdCAM-1-integrina4ß7 interaction prevent the homing of T cells to the GALT. These antibodies also modulate the inflammation in animal models of chronic IBD.^18-21 Briskin et al (1997) studied the MAdCAM-1 expression in the venules in the intestinal mucosa in patients with ulcerative colitis and Crohn’s disease and found increased expression in comparison with the controls.¹⁵ Aihiro et al (2002) postulate that increased expression of MAdCAM-1 in the mucosal lymphoid tissue is the result of breakdown of gut tolerance and predisposes to chronic IBD.²² The T lymphocytes once they leave the intestinal vasculature, adhere to the intestinal epithelial cells by another integrin expressed on the T cell surface called a_Eß₇. The corresponding receptor on the epithelial cells is E-cadherin.²³ In experimental animal models with negative expression of a_Eß₇, reduced numbers of intra-epithelial lymphocytes were noted.²⁴

CD8+ T CELLS AND IBD
Intraepithelial lymphocytes in the gut mucosa are predominantly (75%-85%) CD8⁺ T cells. The function and the role of these cells in gut immunity and diseases like ulcerative colitis and Crohn’s disease are not well-established. These cells closely interact with intestinal epithelial cells (IECs) and have been shown to proliferate on receiving appropriate signals from IECs. The latter screte glycoprotein 180 (gp180), a member of the carcinoma embryonic antigen (CEA) family of proteins expressed on IECs, which acts as a ligand for CD8⁺ T cells.²⁵ Intestinal epithelial cells in patients with IBD have defective expression of gp180 and fail to induce the proliferation of CD8⁺T cells.²⁵ T CD4⁺ helper cells have two subsets of regulatory cells which modulate mucosal inflammation; one subset of T cells secrete immunoinhibitory cytokines such as interleukin-10 (IL-10) and transforming gastric factor-ß (TGF-ß), and another subset induces suppression by contact with T cells. Allez et al (2002) have shown in in vitro studies that epithelial cells, when cocultured with T cells, induce a subset of CD8+ T cells which have immunosuppressive function.²⁵ In a comparable study Mayer et al (1990) showed that in patients with IBD, IECs fail to induce the production of suppressor CD8⁺ T cells.²⁶ These experiments suggest that CD8⁺ T cells, on interacting with IECs, generate immune T cells necessary for gut homeostasis; failure to do may predispose to IBD.

CD4+ T CELLS AND IBD
From animal models of colitis it is evident that CD4⁺ T cells are essential for tissue damaging inflammation in the bowel. Animal models of colitis, as seen in severe combined immunodefecient (SCID) mice, when adoptively transferred with CD4⁺ T cells from genetically-related mice, undergo repopulation of the gut and develop colitis.^27,28 In the 2,4-dinitrobenzene sulfonic acid-induced colitis model, isolation and transfer of CD4⁺ T cells to naïve genetically identical mice led to the migration and localisation in the gut, followed by inflammation in the recipient’s colon. This migration is probably caused by T cell recognition of the colonic bacterial flora.²⁹ Leith per thousand user et al (2001) developed a transgenic animal model where the CD4⁺ T cells express a stable marker called enhanced green fluorescent protein (eGFP). By transferring these T cells in immunodeficient mice they were able to follow the migration of the cells from their site and time of administration to the small and large bowel before the development of colitis. The eGFP⁺ T cells cluster in the mucosal and submucosal junction of the bowel in association with submucosal aggregates of DCs. These T cells were also noted to be actively proliferating. These T cell aggregations occurred at the sites where subsequently colitis developed and the degree of aggregation was directly linked with the severity of the inflammatiory process. This prominent influx and proliferation of T cells, close to junctional DC aggregates, suggests that in situ T cell priming and or restimulation may be a key event in the pathogenesis of colitis.³⁰

HUMORAL IMMUNITY
Various autoantibodies have been detected in patients with IBD. These include antipancreatic antibodies, perinuclear antineutrophil cytoplasmic autoantibodies (pANCAs), anti-Saccharomyces cerevisiae mannan antibodies (ASCAs), antierythrocyte antibodies, antiendothelial cell antibodies, antibactericidal/permeability-increasing protein antibodies and antip40 antibodies.³¹ Of these, pANCAs and ASCAs are the more extensively studied autoantibodies in ulcerative colitis and Crohn’s disease, respectively.

Perinuclear antineutrophil cytoplasmic autoantibodies are detected in the serum of about 50-80% of patients with ulcerative colitis and a similar proportion of patients with Crohn’s disease have ASCAs in their serum. Cohavy et al (2000), in screening for Ags that cross-react with pANCAs, documented cross-reacting gut bacterial Ags-Bacteroides (B) caccae and Escherichia (E) coli. Isolation and partial sequencing of the B. caccae Ag identified a 100-kDa protein and the E. coli protein was biochemically and genetically identified as the outer membrane porin OmpC.³² The sensitivity of pANCAs or ASCAs are too low to be used as the only diagnostic tools in IBD. However, a combined measurement of pANCAs and ASCAs was found advantageous for subclassfying patients with indeterminate colitis. The clinical course of ulcerative colitis or Crohn’s disease has been predicted using pANCAs or ASCAs in indeterminate colitis, respectively.³³ Lombardi et al (2000) showed that pANCAs were significantly more frequent in patients with ulcerative colitis with high relapse rates and were also significantly less frequent in patients with ulcerative colitis in remission.³⁴ The pANCAs were found in a significantly higher proportion of patients with left-sided colitis and who were resistant to medical treatment.³⁵ These antibodies were also found in a higher proportion of first degree relatives of patients with ulcerative colitis.²⁵ Anti-Saccharomyces cerevisiae mannan was found in a higher proportion of relatives of patients with Crohn’s disease than unrelated controls.^36,37 This suggests that the presence of ASCAs in the serum of healthy relatives is a marker for possible future development of Crohn’s disease.

DENDRITIC CELLS AND GUT IMMUNITY
B cells, which are precursors for antibody-producing plasma cells, can directly recognise native Ag through specific B cell immunoglobulin receptors. T lymphocytes, however, need the Ag to be processed and presented to them by an antigen presenting cell (APC). The TCRs recognise fragments of Ags bound to MHC molecules on the surface of APCs. The peptides, bound to MHC class I or II molecules, stimulate the activation and proliferation of CD8⁺ cytotoxic T cells (CTLs) and CD4⁺ helper T (Th) cells, respectively. Cellular Ags are phagocytosed and cleaved into peptides within the cytoplasm of the APC. They are re-expressed on the cell surface bound to class I molecules and are recognised by CTL precursors (naïve CD8⁺T cells), which, once activated, can directly kill a target cell expressing the identical cell membrane bound peptide-MHC product. Extra-cellular Ags that have entered the cytoplasm by the endocytic pathway of the APC are processed there and are re-presented bound to MHC class II molecules to CD4⁺Th cells, which on activation secrete cytokines which have important immune-regulatory function.

Dendritic cells are bone-marrow-derived APCs that are found both in lymphoid and non-lymphoid tissues, where they exert a sentinel-like function. They are the most effective APCs and are crucial to the induction of the immune response.^38-40 In mucosal-associated lymphoid tissues, DCs lie beneath the epithelial M cells and, thus, are ideally placed to capture Ags transported across the mucosal barrier.⁴¹ Dendritic cells express tight junction proteins and penetrate the mucosal barrier to interact with luminal bacterial Ags.⁴²

Based on functional and phenotypic characteristics, DCs can be classified as immature/inactive and mature/activated. Immature DCs exist in peripheral tissues including the intestinal mucosa and are equipped for phagocytosis of Ags and bacteria. They do not express the necessary co-stimulatory and adhesion molecules CD80/CD86 and CD40, respectively, which are required for stimulating T cells. After taking up Ags and receiving appropriate activation signals they migrate to the paracortical area of the regional mesenteric lymph nodes. During migration they lose their phagocytic capacity and the processed Ag is re-expressed in a stable form in association with MHC proteins, at the cell surface for naïve T cell recognition and intimate interaction. Concurrently, they express the requisite accessory molecules including CD40, CD80, CD86 and CD54. The DCs are now activated and ready for T cell receptor interaction and effective T cell stimulation. Absence of accessory signals or blockage of receptors with antibodies or immune complexes inhibits T cell activation and can lead to tolerance (Figure 2). The costimulatory molecules can interact with receptors on T cells called cytotoxic lymphocytic antigen-4 (CTLA-4), which inhibits their proliferation.

Figure 2: Schematic diagram illustrating the two-signal postulate. Signal 1 is Ag presentation in association with MHCII to TCR. Signal 2 is co-stimulatory molecule CD80/86 expression and interaction with CD28 and adhesion molecule (CD40) expression and interaction with naïve T cells leading to activation and proliferation

This receptor helps to terminate T cell proliferation at the end of effective Ag elimination. The importance of DCs in oral tolerance is evident from experiments carried out by Viney et al (1998). Expanding the DC population by administering haemopoietic growth factor Ftl3L to mice makes it easier to induce oral tolerance than in untreated mice.⁴³ Failure of appropriate expression of costimulatory molecules on DCs induces T cell tolerance and failure of activation of CD4+ and CD8+ T cells. The suppressed release of key immunostimulatory molecules (e.g. interferon-. (INF-.), IL-12) further potentiates the immuno-inhibitory state.

Prevention of DC activation and maturation may be an important mechanism of inducing peripheral tolerance. There is evidence that persistent infection, such as occurs with herpes simplex, cytomegalovirus and plasmodium falciparum, decreases the efficacy of the host immune response. These persistent pathogens inhibit the maturation of DCs.^44-46 Steinman et al (2002) propose that the persistent exposure of food Ags and the commensal bacteria to mucosal DCs prevent them undergoing activation and maturation and hence leads to the creation of a tolerant environment in the gut.⁴⁷ The ability of pathogenic bacteria to induce immunity, as opposed to tolerance in the gut, depends on the expression of pathogen-associated molecular patterns (PAMPs). These are recognised by highly conserved Toll like receptors (TLRs) expressed by DCs. Activation of these receptors induce the maturation of DCs and expression of co-stimulatory molecules which are essential for activation of T cells.^48,49 Non-pathogenic commensal bacteria by not expressing a PAMP, are unable to activate the DCs through interaction with TLRs, an essential maturation process for T cell activation.

Figure 3: Schematic diagram showing the ratio of Bcl-2 (antiapoptotic protein) and Bax (proapoptotic protein) in T cell cytoplasm of normal and IBD mucosa

Notch is a highly conserved trans-membrane protein that was first described in Drosophilae. Delta and Serrate are two separate ligands in vertebrates which can bind with the Notch receptor.^50,51 The receptor and its ligands are co-expressed within the same cells. When Notch binds its ligand, Delta or Serrate, this induces cleavage of the Notch intracellular domain which then migrates to the nucleus where it associates with transcription factors that bind to the promoter region that regulate their expression. Splenic and lymph node DCs, B cells, as well as CD4⁺ and CD8⁺ T cells, all express transcripts for Notch1and 2, and the ligands Delta1 and Serrate1.52 Murine DCs that overexpress Serrate 1 ligand, when bound to naïve T cells induce tolerance.⁵³ Various signals can modulate the expression of Notch receptors and ligands on immune cells. For example, feeding high doses of Ag to mice leads to over expression of Serrate1 ligand in the mesenteric lymph node DCs. Hence, it is possible that various Ags and/or differing amounts of these Ags can modulate the expression of the Notch receptors and their ligands and, thereby, determine whether immunity or tolerance is induced.⁵⁴

Studies involving DCs in IBD are very limited. Sally et al (2001) isolated DCs from colonic biopsies and studied them using flow cytometry. The number of DCs isolated from patients with IBD and healthy controls were not different and there was also no difference in the number of DCs isolated between the inflamed and non-inflamed mucosa. When the phenotype was analysed these freshly isolated DCs were shown to be immature.⁵⁵ Onji et al (2001) and Vukovic et al (2001) studied the DCs isolated from the peripheral blood of patients with IBD. They found that patients with IBD had significantly increased numbers of mature DCs expressing co-stimulatory molecules and they induced enhanced proliferation of T cells in allogeneic mixed lymphocyte responses (MLRs) in vitro, compared with DCs isolated from healthy controls.^56,57

APOPTOSIS AND IMMUNE TOLERANCE
Apoptosis is another important mechanism by which immune tolerance may be induced in the gut. T cells in the mucosa of the gut are constantly undergoing apoptosis. Lamina propria T cells exhibit increased spontaneous apoptosis, compared with peripheral blood cells. Similarly, after stimulation of LP T cells with antiCD2 and antiCD28 antibodies, apoptosis is further augmented, compared with stimulated peripheral blood T cells.⁵⁸ To investigate whether clonal anergy/deletion was occurring in orally immunised transgenic mice, the frequency of T cells undergoing apoptosis in Peyer’s patches and the spleen was determined. Following ovalbumin feeding, specific T cells were induced in Peyer’s patches and the spleen, and were more susceptible to Fas- mediated apoptosis following restimulation in vitro. Lymphocytes from mice tolerised with a single feed of 25mg of ovalbumin, when cultured in vitro without ovalbumin, displayed an enhanced mortality in comparison with cells from nonimmunised control animals. This increased cell death affected both CD4+ and CD8+ T lymphocyte subsets, and it occurred via enhanced apoptosis. All of the changes associated with the propensity of tolerant cells to die by apoptosis in vitro was reduced by the inclusion of the ovalbumin in the cultures.⁵⁹

Experiments carried out in humans have shown that at least 15% of the LPLs from normal bowel are undergoing apoptosis. In the case of IBD, there is a lower proportion of cells undergoing apoptosis.⁶⁰ These various studies suggest that apoptosis may be an important component of induction of immunological tolerance and that disturbances in the normal apoptotic mechanisms could help initiate IBD. Apoptosis in T cells is controlled by various intracellular proteins which have antiapoptotic and proapoptotic thrusts, of which Bcl-2 and Bax are two key examples. The ratio of these two and related homologous intracellular proteins determines whether the cell will survive or undergo apoptosis.^61-63 T cells isolated from areas of inflammation in patients with IBD were resistant to apoptosis and have elevated Bcl-2 and increase in the Bcl-2/Bax ratio (Figure 3).^64,65 Cytokines such as IL-2 and IL-7 are known to increase the level of Bcl-2 in T cells, thereby, inducing resistance to apoptosis.⁶⁶

Interleukin-6 is a proinflammatory cytokine which enhances T cell resistance against apoptosis. Atreya et al (2000) have shown in their in vivo studies that administration of antiIL-6 mAbs will suppress T cell resistance against apoptosis in IBD.⁶⁷ One of the mechanisms of action of Infliximab in treating patients with Crohn’s disease is by inducing apoptosis of the T cells which they do by decreasing the Bcl-2/Bax ratio.⁶⁴

CYTOKINES AND GUT IMMUNITY
CD4⁺ T cells may be divided functionally into Th1 and Th2 subsets, based on their functional capabilities and the cytokines they produce. T helper 1 cells produce IFN-.y and tumour necrosis factor-a (TNF-a) and are responsible for cellmediated immunity and Th2 cells produce cytokines such as IL-4 and IL-5 and mediate humoral immunity. In the gut, the Th2 response plays an important role in immunoregulation.⁶⁸ It has been proposed that the induction of oral tolerance may reflect a preferential activation of Th2 cells with down regulation of the Th1 subtype of cells.⁶⁹ The Th1 and Th2 subsets are derived from common T cell precursors. The type of Ag, dose and the route of administration determine the development of these distinct Th subsets and their pathways of activation.^70-72 The cytokine microenvironment under which the Ag binds to the TCR also determines whether the immune response will be type 1 or 2.71,70 In the presence of IL-12 and IFN-.y ligation of TCR will drive the immune response along a Th1 pathway. By contrast, the presence of IL-4 will drive the immune response towards the Th2 pathway.⁷³ The cells of innate immunity, such as macrophages and natural killer (NK) cells, produce IL-12; mast cells and eosinophils secrete IL-4. Dendritic cells produce IL-12 when CD40 and class II peptides on the cell surface are bound by corresponding ligands and, thereby, drive the immune response towards a type 1 pathway.⁷⁴ Interleukin-4 and IL-5 have the ability to inhibit the DCs from producing IL-12, thereby, suppressing the development of Th1 cells.⁷⁵ A key immunosuppressive and regulatory cytokine produced by Th2 cells is IL-10.

Interleukin-10 plays an important role in regulating DC function. Immature DCs release small amounts of IL-10. On incubation with bacteria or bacterial products, however, there is increased release of IL-10. The latter blocks the up regulation of costimulatory molecules and IL-12 production and, thus, impairs the ability of DCs to generate Th1 responses.⁷⁶ Gasche et al (2000) studied the amount of IL-10 produced by lamina propria mononuclear cells from the inflamed and normal mucosa of patients with IBD and found low levels of IL-10 in the inflamed mucosa, compared with normal mucosa.⁷⁷ Hence, low levels of IL-10 induce maturation of DCs, which in turn stimulate the Th1 cells to produce proinflammtory cytokines. Induction of tolerance is due to the generation of T cells in the Peyer’s patches which secrete IL-10 and TGF-ß and this tolerance induction can be abolished by administering antiIL-10 and antiTGF-ß mAbs.⁷⁸ Interferon-. is up-regulated in murine models of IBD and in patients with IBD. The importance of this cytokine in IBD is further supported by the efficacy of antiIFN-a mAbs in reducing the pathophysiological disturbances in animal models of IBD.⁷⁹ Animal models with targeted deletion of IL-10 secreting genes develop intestinal inflammation. The inflammation is due to unregulated proliferation of T cells in the gut and related Th1 cytokines (such as IFN-y).⁸⁰ Chemically-induced colitis in animal models can be prevented by concurrent administration of IL-10.81 Dendritic cells isolated from Peyer’s patches of mice secrete IL-10 but the DCs isolated from the spleen did not. In the allogeneic MLRs, DCs from Peyer’s patches induced T cells to secrete Th2 cytokines and the splenic DCs to induce Th1 proliferation and associated cytokine secretion.⁸²

Another important regulatory cytokine is TGF-ß, which has immunomodulatory functions. In ovalbumin TCR transgenic mice, a subset of T cells isolated following feeding with ovalbumin secreted three times more TGF-ß than the same cells from unfed mice. These cells were labelled Th3 cells and may be a type of regulatory T cell (see below).⁸³ When SCID mice were adoptively transferred with CD4+ T cells the animals did not develop colitis. However, they did develop colitis if antiTGF-ß mAb was administered at the same time.⁸⁴ Crosslinkingof CTLA-4 induced TGF-ß production by murine CD4+ T cells. When CTLA-4 was cross-linked with T cells from TGF-ß gene-deleted (TGF-ß [-/-]) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Thus, secretion of TGF-ß beta by Th3 cells is another possible mechanism of maintaining tolerance.⁸⁵

Loss of genes for TGF-ß in mice results in the spontaneous development of colitis.⁸⁶ The cytokine binds to its receptor on the cell membrane; signals are then relayed to the nucleus using a cohort of proteins, termed Smads. To date, nine Smad proteins have been discovered. Of these, Smad 6 and Smad 7 are inhibitory proteins and up-regulation of Smad 7 has been associated with an inhibition of TGF-ß1-induced signaling.^87,88 The protein Smad 7 plays a central role as an inhibitory autoregulatory molecule in TGF-ß/ Smad signalling. Studies in animal models have shown that inflammation of the bowel is due to an imbalance between proinflammatory cytokines and TGF-ß. Monteleone et al (2001) showed that in ulcerative colitis and Crohn’s disease there is overexpression of Smad 7 protein and, hence, inhibition of the signalling of TGF-ß.⁸⁹ They postulated that Smad 7 blockade of TGF-ß signalling enhanced the proinflammtory cytokine cascade, thereby, inducing the inflammatory process in IBD. The factors responsible for this overexpression of Smad 7 protein is/are unknown. They also postulated that proinflammatory cytokine such as INF-y and TNF-a up-regulate the expression of Smad 7. Wiercinska-Drapalo et al (2001) and others measured the concentration of TGF-ß in the serum of patients with ulcerative colitis and documented higher concentrations in controls and showed a correlation with severity of the mucosal inflammation.^90,91 This raised level is due to the autoregulatory feedback loop where defective signalling due to inhibitory Smad 7 increases the production of TGF-ß.

The increased mRNA expression for IL-1ß, IL-6, and TNF-a were noted by Funakoshi et al (2001) when they analysed the mucosal samples from patients with IBD.⁹² These are proinflammatory cytokines postulated to be involved in the pathogenesis of IBD. The expression of TNF-a was analysed in a number of studies using mucosal biopsies from patients with IBD. The latter used RT-PCR techniques and found increased TNF-a mRNA in the inflamed mucosa, compared with mucosa obtained from healthy individuals.^92-94 Lamina propria mononuclear cells isolated from the involved IBD mucosa spontaneously produced TNF-a and IL-1ß, when compared with non IBD mucosa.⁹⁵ Mucosal biopsies from patients with IBD, when cultured in vitro, spontaneously produce TNF-a and IL-1ß compared with normal mucosa.⁹⁶ Variable results were found when INF-y was analysed from the mucosa of patients with IBD.^93,97

Interleukin-16 is a proinflammatory cytokine secreted by T cells, eosinophils, mast cells, and non-immune epithelial cells upon activation with Ags or mitogens. This cytokine acts as a chemoattractant for CD4⁺ T cells. This cytokine is increased in the mucosa of patients with IBD and helps to attract T cells to the inflammatory site. The source of this cytokine in IBD is from eosinophils and CD4⁺ T cells.⁹⁸

Interleukin -18 is a primary cytokine that shifts the immune reaction to a Th-1 type. It stimulates T cells to produce IFN-y, further reinforcing the shift towards a Th-1 response.^99,100

Interleukin-18 is synthesised as a precursor protein called pro IL-18. Interleukin-1 converting enzyme (ICE caspase) cleaves immature IL-18 to a functional mature protein, which is stored in the cytoplasm until the cell receives an activation signal for it to be released. Once released, its activity is further regulated by a naturally occurring serum protein called IL-18 blocking protein (IL-18bp). Interleukin-18 is mainly produced and released by APCs such as activated macrophages, B cells, Kupffer cells and DCs.¹⁰¹ This suggests that IL-18 acts at the early phase of the immune response and accounts for the polarisation of the responses elicited.

REGULATORY T CELLS
An important mechanism of tolerance induction is by the generation and activation of specific subsets of T cells, which have suppressive effects on Ag-specific T cells. CD45RB is a phenotypic marker of activation used for the discrimination of different CD4⁺T cell subsets. This surface molecule is upregulated during thymic development, and its expression on naïve CD4⁺T cells decreases upon activation. T lymphocytes can be classified into CD45RB^high and CDRB45^low representing naïve and activated/memory T cells, respectively.^102,103 Severe combined immune deficiency mice develop severe colitis when populated with CD4⁺/CD45RB^high T cells. Transfer of CD4⁺T/CD45RBlow cells into mice from normal donors did not produce IBD. When both CD45RB^low/CD4⁺T cells and CD45RB^high/CD4⁺T cells were transferred into SCID mice the latter did not develop IBD. These animal experiments suggest that CD45RB^high/CD4⁺T cells play a key role in inducing colitis, whilst CD45RB^low/CD4⁺T cells have a protective role.^104-108 When the mechanism of inhibition of colitis was analysed it was found to be due to a subset of T cells within the CD45RB^low /CD4+T cell subgroup expressing the CD25 phenotypic marker.

Administration of oral Ag leads to the generation of natural regulatory CD25^- T cells. Ovalbumin transgenic mice, when fed with ovalbumin, resulted in the expansion of CD4⁺/CD25^- T cells in the mesenteric and inguinal lymph nodes. Concurrently, there was a decrease in the number of CD4⁺/ CD25^- T cells. When lymph node cells were isolated and stimulated separately in vitro with ovalbumin and APCs, the CD4⁺/CD25⁺ T cells were anergic but the CD4⁺/CD25^- T cells proliferated. When ovalbumin was added to the co-cultures, the CD4⁺/CD25⁺ T cells inhibited the proliferation of the CD4⁺/CD25^-T cell subset. CD4⁺/CD25⁺ T cells isolated from the ovalbumin fed mice also expressed more of the CTLA-4⁺ phenotype.¹⁰⁹ Before the importance of CD4+/CD25⁺ T cells in the regulation of immunity was realised, Zeitz et al (1988) described that the lamina propria in non-human primate intestines contained more of these cells than in any other anatomical site.¹¹⁰

Kojima and Prehn (1981) discovered that mice thymectomised shortly after birth led to the development of multi-organ autoimmune disease.¹¹¹ Subsequently, it was shown that the thymectomised mice lacked T cells which constitutively expressed the CD25 phenotypic marker and the autoimmune disease was due to absence of CD4⁺ /CD25⁺ T cells. Adoptive transfer of CD4⁺/CD25⁺ T cells into the mice on day three or co-transfer with CD4⁺/CD25^-T cells into nude mice prevented autoimmune disease.^111-113  These CD4⁺/CD25⁺ T cells have been the subject of intense study because of their possible critical role in maintaining self-tolerance. T cell-mediated autoimmune diseases (such as insulin-dependent diabetes, thyroiditis and gastritis) can be produced in normal mice by eliminating the circulating CD4⁺/CD25⁺ T cell subpopulation.^112,114-117 Also, the various models of autoimmune disease can be prevented by transfer of CD4+/ CD25+ T cells.^118-122 Because of their ability to prevent autoimmune disease they are called regulatory T cells.

One important morphological feature of CD4⁺/CD25⁺ T cells is that they express CTLA-4 (cytotoxic lymphocyte-associated antigen) constitutively.¹²³ Animals with targeted deletion of the CTLA-4 coding gene will die of autoimmune disease.¹²⁴ Greenwald et al (2001) studied the tolerance induction in CTLA-4 (CTLA-4^-/-) deficient mice. These mice were resistant to tolerance induction, as shown by their proliferative responses. T cells from wild type animals, after receiving tolerogenic stimuli in vivo, when isolated and stimulated in vitro, showed arrest in progression of the cell cycle. But the T cells isolated from (CTLA-4^-/-) animals, which had the same tolerogenic stimuli showed no arrest in the T cell cycle, when stimulated in vitro.¹²⁵ Administration of antiCTLA-4 mAb abrogated the tolerance induction.¹²⁶

It is unclear how CTLA-4 regulates peripheral tolerance. It is possible that CTLA-4 may act through its effect on IL-2 production. Blockage of CTLA-4 enhances IL-2 production, an important growth factor for T cells.¹²⁷ In co-culture experiments involving CD4⁺/CD25- T cells and CD4⁺/CD25- T cells, suppressive responses could be reversed by exogenous addition of IL-2.128-130 Since CD4⁺/ CD25⁺ T cells express constitutively CTLA-4 and the elimination of CD4⁺/CD25⁺ T cells and CTLA-4 produce identical autoimmune disease it is postulated that the cell functions are mediated through modulation of CTLA-4 expression.¹²³ That the natural regulatory T cells require CTLA-4 for their function is evident from animal experiments. When SCID mice were adoptively transferred CD4⁺/CD45RB^high with CD4+/CD45RB^low T cells they did not develop colitis. The protective effect of the CD4⁺/CD45RB^low T cells was abrogated when antiCTLA-4 mAb was administered.⁸⁴

CONCLUSION
Various complex mechanisms operate in the GALT modulating immune function and inducing tolerance. There is an intricate balance between the immune system not reacting to commensal bacteria and food Ags and mounting an immune and inflammatory response to protect the host from pathogenic microorganisms and other tissue damaging agents. Tolerance is probably induced by the manner in which Ags are processed and presented by DCs to naïve T cells. It is also probable that a subset of T regulatory cells are generated that have immunosuppressive effects, either through inhibitory effects on DC interactions or secretion of immunosuppressive factors. The production of specific cytokines (e.g. IL-10, TGF-ß), as well as failure of production of other key immunoregulatory cytokines (e.g. IL-2, IL-12, INF-y) in GALT, further helps to create anergy and induce tolerance. Animal experiments and preliminary data from Ermann suggest that deregulation of this delicate balance and the resulting dysfunction induces the pathobiological process of IBD.

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Copyright: 7 March 2003