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Introduction

  1. Top of page
  2. Introduction
  3. Mucosal innate immunity
  4. Mucosal adaptive immunity
  5. Perspectives and concluding remarks
  6. Acknowledgements
  7. References

The mucosal surfaces of the respiratory, digestive and urogenital systems represent vast areas, i.e. 400 m2 for a human adult. These surfaces are covered by thin epi-thelial layers that are protected from potentially harmful microorganisms by innate and adaptive defence mechanisms. Intestinal mucosal surfaces are in continuous contact with a heterogeneous population of microorganisms of the endogenous flora (up to 1011–12 per g of lumenal material in the colon) and are exposed to food and microbes. A major role for the mucosal epithelia is in barrier function, essential for preventing colonization or invasion of the host by foreign microorganisms. Epithelial tissues also provide the mucosal immune system with a continuous stream of information about the external environment. Depending on the nature and the dose of the antigens transported from the gut lumen into mucosal lymphoid tissues, strong immune responses or unresponsiveness can be induced. Immune responses participate in the elimination of pathogenic microorganisms, while immune tolerance prevents harmful reactions against the gut flora and food antigens. The type of immune response triggered by environmental antigens appears to depend, in part, on initial recognition by the innate immune system. Recent data underscore the importance of innate immunity in sensing the microbial environment and the role of the epithelium in releasing signals that allow recruitment of pro-inflammatory leucocytes, immune cells, or both. We shall review these various aspects of mucosal immunity, with special emphasis on the cross talk that takes place between the microflora, the epithelium and the immune cells in the gut.

Mucosal innate immunity

  1. Top of page
  2. Introduction
  3. Mucosal innate immunity
  4. Mucosal adaptive immunity
  5. Perspectives and concluding remarks
  6. Acknowledgements
  7. References

Innate immunity provides broad protection against microorganisms without previous exposure. Various innate protective mechanisms are aimed at preventing direct interaction of microorganisms with the mucosal interstitium. These include the barrier function of epithelia that depends on specializations such as tight junctions (Madara et al., 1990), apical surface coats (Frey et al., 1996), and secretion products such as mucins (Corfield et al., 2000). In addition, mucosal epithelia maintain an antimicrobial environment on mucosal surfaces characterized by the production of a broad spectrum of antimicrobial agents that include defensins, cecropins, lactoferrin and lysozyme (for review see Boman, 2000).

Epithelial barrier function

Epithelia provide a barrier against both endogenous commensal microorganisms of the gut flora, and enteropathogenic bacteria and viruses. In contrast to commensal microbes, pathogenic microbes have acquired mechanisms to breach host innate defences and in some cases to poison epithelial cells. Multiple virulence mechanisms are involved in these processes; they are partially characterized for bacteria and poorly understood for viruses. For instance, the Gram-negative enteropathogenic bacteria Salmonella typhimurium, produce factors required for resistance to stresses, such as pH, oxygen tension or detergents, encountered in the gastrointestinal tract. S. typhimurium are also equipped with adhesion and invasion programs that allow them to cross the epithelial barrier on the villi or Peyer’s patches (for review see Lucas and Lee, 2000; Darwin and Miller, 1999).

In response to microbial injury, intestinal epithelial cells increase their barrier activity by up-regulating the production of antimicrobial agents, such as beta (O´Neil et al., 1999) or alpha defensins (Ayabe et al., 2000). Epithelial cells are also able to respond to specific microbial signals by releasing chemokines that recruit phagocytic cells to contain the infection and professional antigen presenting cells that trigger immune responses, as described for S.◊typhimurium (Eckmann et al., 1993; McCormick et al., 1993; Gewirtz et al., 2001b; Sierro et al., 2001). The recruited monocytes, macrophages and dendritic cells in turn release cytokines and chemokines that recruit additional leucocytes to clear the infection. Thus, epithelial cells act as watchdogs for the mucosal immune system and as links between innate and adaptive immunity.

Sensing the outside world

The activation of innate host defence mechanisms is based on specific recognition of signature molecules of microorganisms referred to as pathogen-associated molecular patterns (PAMPs) (for review see Aderem and Ulevitch, 2000). However, since some innocuous and endogenous microbes share similar signature molecules with their pathogenic counterparts, it is more appropriate to name them microbe-associated molecular patterns or MAMPs. MAMPs are usually shared by a large group of microorganisms. For example, peptidoglycans and lipoproteins are MAMPs found in most bacteria, lipopolysaccharides (LPS) are associated with Gramnegative bacteria, while Gram-positive bacteria share teichoic acids. Since different pathogen classes, i.e. Gram-negative, Gram-positive bacteria, mycobacteria and fungi, express distinct MAMPs, recognition of the motifs informs the host about the nature of the potentially threatening microbe.

Two cell types are specialized in sensing the outside world in mucosal tissues: the epithelial cells and the monocytic cells, especially the intraepithelial and sub-epithelial dendritic cells (DC) (Fig. 1). Both cell types are able to recognize MAMPs by their pattern-recognition receptors (PRR). PRRs form a heterogeneous family of proteins that are either secreted or expressed at the cell surface. They can also be found associated with intracellular organelles (Hemmi et al., 2000). PRRs identified to date consist of distinct types of recognition domains including C-type lectins, cysteine-rich domains and leucine-rich repeats (LRR) (Medzhitov and Janeway, 2000). Secreted PRRs, including mannose-binding lectin and surfactant, function as opsonins bridging microbial cell wall components to lectin receptors of the complement pathway, while surface receptors, i.e. the scavenger and mannose receptors, mediate microbial internalization and delivery to lysosomes for degradation (Hopken et al., 1996).

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Figure 1. Signalling mechanisms in gut innate immunity. In intestinal tissues, membrane (green bars) and cytosolic (red bars) pattern recognition receptors (PRR) of epithelial cells and/or dendritic cells are sensing the environment by interacting with the microbe-associated molecular patterns (MAMPs) depicted as green circle for extracellular MAMP and red triangle for cytosolic MAMP. Upon stimulation, different genes, especially genes encoding chemokines and defence molecules, are up-regulated which in turn triggers both inflammatory (black arrows) and antigen-specific reactions (blue arrows). The following epithelial PRRs have been described in epithelial cells: (1 and 3) apical or/and basolateral TLR5 signalling triggered by bacterial flagellins, and (2) Nod1/CARD4 signalling by cytosolic bacterial LPS. Subepithelial dendritic cells (DCs) and/or recruited DCs can also be activated by MAMPs after their translocation through the epithelial layer (4). Intraepithelial DCs could sense directly extracellular and cytosolic MAMPs in the lumen via dendrites (5). In turn, DCs and other recruited cells could influence the innate response of epithelial cells (dashed black arrows).

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During the past four years, the role of PRRs containing LRR domains in innate immunity has been elucidated. Two receptor families are instrumental in the recognition of microbial components and the signalling cascade that follows: the Toll family, i.e. Toll-like receptors (TLR), and the ‘nucleotide binding site plus LRR’ (NBS-LRR) plant disease resistance-like protein family, including CARD4/Nod1 and Nod2 (Aderem and Ulevitch, 2000; Dangl and Jones, 2001). Whereas TLRs are membrane proteins that sense extracellular MAMPs, NBS-LRR are cytosolic and respond to intracytoplasmic MAMPs. Human monocytes and DCs together express all known TLR, i.e. TLR1 to TLR10 (Muzio et al., 2000; Kadowaki et al., 2001; Visintin et al., 2001). For instance, human DCs express TLR4 and the molecules associated with LPS recognition. TLR2 is involved in recognition of lipoproteins and TLR3 for sensing double-stranded RNA (Alexopoulou et al., 2001), TLR5 interacts with bacterial flagellins (Gewirtz et al., 2001a; Hayashi et al., 2001) and TLR9 recognizes the unmethylated CpG motif of bac-terial DNA (Bauer et al., 2001). In addition, TLRs are able to discriminate among various MAMPs, and distinct TLRs can combine to generate additional MAMP specificities, as shown for TLR2/TLR6 heterodimers that sense peptidoglycans (Aderem and Ulevitch, 2000; Ozinsky et al., 2000). TLR expression patterns in vitro, however, depend on the type of progenitors used for DC preparation and are altered upon differentiation of DCs. Finally, the NBS-LRR molecule Nod2 is produced by monocytes (Ogura et al., 2001a). TLR and NB-LRR expression in human DCs has not yet been analyzed in situ and little is known about expression of these receptors by gut DCs.

The response of epithelial cells to microbial MAMPs is also mediated by PRR. Human epithelial intestinal cell lines, including Caco-2 and T84 cells, express TLR2, TLR3, TLR4 and TLR5 (Cario et al., 2000; Gewirtz et al., 2001a; Sierro et al., 2001). In the human gut, TLR3 and TLR5 are more highly produced than TLR2 and TLR4 on colonocytes and villi enterocytes (Cario and Podolsky, 2000). This pattern reflects the ability of the epithelium to sense infection mediated by flagellated bacteria, as well as components of enteropathogenic bacteria, including S. typhimurium, Escherichia coli, and Vibrio cholerae, and of dsRNA enteroviruses such as reovirus or rotavirus. In addition Card4/Nod1, which recognizes intracytosolic LPS, is expressed in human epithelial cell lines (Girardin et al., 2001).

MAMP-mediated signalling in intestinal epithelial cells

PRRs that contain LRR provide a link between recognition of MAMPs and signal transduction. The consensus effect is activation of the NFκB pathway. The NFκB transcription factor is essential for innate defences, since it regulates the expression of antimicrobial agents (Lemaitre et al., 1996), cytokines, and chemokines (Hoffmann et al., 1999). The effect of S. typhimurium on intestinal epithelial cells provides a good model for dissection of the signalling pathway. Inflammatory responses triggered by this bacte-rium have been intensively studied and are dependent on NFκB (Elewaut et al., 1999). Recently, flagellin, the subunit of Salmonella flagella, was found to be the major factor that triggers the pro-inflammatory gut epithelial cell response via TLR5 (Gewirtz et al., 2001a; b; Hayashi et al., 2001). The flagellin-mediated innate response in the epithelium is characterized by release of various chemokines, including IL-8 which recruits inflammatory cells such as neutrophils and macrophages (Gewirtz et al., 2000), and by up-regulation of antimicrobial factors, in-cluding human beta 2 defensin and nitric oxide synthase (Eaves-Pyles et al., 2001; Ogushi et al., 2001). The cytoplasmic Toll-interleukin 1 receptor (TIR) domain of TLR5, the myeloid differentiation (MyD) marker MyD88, and IRAK (interleukin-1 receptor-associated kinase) are required for the signal transduction elicited by flagellin (Gewirtz et al., 2001a; Hayashi et al., 2001; Moors et al., 2001). It is remarkable that flagellin induces innate defences in plants using similar mechanisms (Felix et al., 1999; Gomez-Gomez et al., 2001). Indeed, mammals, insects and plants share the molecules involved in innate immune signalling. In human intestinal epithelial cell line flagellin also induces the NFκB-dependent up-regulation of CCL20, the chemokine that recruits immature dendritic cells (Izadpanah et al., 2001; Sierro et al., 2001) (Fig. 2). Thus through MAMPs and TLRs, epithelial cells are able to link innate to adaptive immunity by attracting dendritic cells, the antigen-presenting cells that activate naïve T lymphocytes.

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Figure 2. Modulation of chemokine expression in intestine by microbial products. CCL20 is a chemokine involved in the trafficking of dendritic cells (DCs). Under steady-state conditions, CCL20 is expressed only in the epithelium associated to Peyer’s patches as illustrated in (A). Upon flagellin stimulation in a ligated ileal loop, expression of CCL20 is induced throughout the epithelium of the small intestine including villi (B and C) Transcription of CCL20 in mouse small intestine as detected by in situ hybridization with antisense RNA probe 2 h after luminal injection of flagellin. Arrows indicate the FAE.

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Discriminating MAMP of commensal from pathogenic bacteria

Why commensals, which can express MAMPs, do not trigger pro-inflammatory host responses under normal conditions is not yet understood, although several explanations can be considered. Firstly, the absence of inflammation could be due to a lack of MAMPs produced by the normal flora or their dilution in the intestinal lumen. Most commensal anaerobic bacteria in the gut, including Bacteroides, Bifidobacterium or Eubacterium species, are poorly characterized genetically and phenotypically (Berg, 1996), and thus their MAMPs have not been identified.

Secondly, gut epithelial cells may be relatively unresponsive, and may not produce PRRs or functional transduction complexes. The lack of epithelial membrane-linked CD14 and other molecules required for LPS recognition, and the low levels of TLR4 on epithelial cells probably explain the non-responsiveness of the gut to LPS. This is a highly desirable situation considering the abundance of LPS provided by commensal microorganisms or food (Funda et al., 2001; Cario and Podolsky, 2000).

Thirdly, the exclusion of commensal microorganisms from the apical surface membranes of epithelial cells may prevent epithelial responses. The fact that commensals are concentrated in the lumen or trapped in the thick mucus layer covering the intestinal mucosa together with the strong antimicrobial environment at epithelial surfaces support this hypothesis. Commensal-specific sIgA release in the intestinal lumen is probably instrumental in mediating the exclusion of bacteria. In contrast, pathogens are equipped with virulence factors that facilitate their penetration of mucus layers, their specific binding to epithelial surfaces, and their resistance to microbicidal components. The relative lack of response by epithelial cells may also result from the polarized expression of PRR on epithelial cell surfaces. For instance TLR5 expression is restricted to basolateral membranes of the human T-84 colonic epithelial cells, implying that flagellin has to be transported across the epithelial cell to trigger a chemokine response (Gewirtz et al., 2001b). This is, however, not the case in normal intestine, where TLR5 is equally distributed on apical and basolateral cell surfaces. Furthermore, signalling may require internalization of MAMPs, as shown for the LPS from the invasive entero-bacteria Shigella flexneri that recognize the cytosolic PRR CARD4/Nod1 (Girardin et al., 2001).

Finally, commensals may program epithelial cells to down-regulate epithelial pro-inflammatory responses by interfering with NFκB signalling, as recently reported (Neish et al., 2000). Clearly, the in vitro studies discussed above will have to be validated in animal models. Such models will also be useful for understanding the complex interactions between commensal bacteria and their hosts (Hooper et al., 1999; Hooper et al., 2001).

Dysregulation of MAMP-signalling

MAMP-signalling can be disturbed in infectious diseases and inflammatory bowel diseases (IBD). Most enteropathogenic bacteria release exotoxins or inject toxins or other virulence factors, directly in the cytoplasm of epithelial cells through type III secretion systems (for review see Finlay and Falkow, 1997). For example, Salmonella and pathogenic E. coli up-regulate MAMP-mediated inflammation by interfering with signalling pathways (Hardt et al., 1998; Norris et al., 1998; Savkovic et al., 1997), while Yersinia down-regulates MAMP signalling by disrupting NFκB pathways (Orth et al., 2000)

IBDs are characterized by an uncontrolled mucosal immune response that appears to be triggered by the bacterial microflora. It is thought that IBD is initiated by a break of oral tolerance, i.e. the loss of the down-regulation of T cell responses against the indigenous flora. The possibility that the break of tolerance is linked to alteration in MAMP/PRR signalling is suggested by the recent finding that susceptibility to Crohn’s disease is linked to the Nod2 leucine-rich repeat variants, an NBS-LRR-like molecule (Hugot et al., 2001; Ogura et al., 2001b). Since dysregulation of MAMP signalling appears to be linked to chronic inflammation of the gut, it will be important to determine the role of MAMP and PRR polymorphism in the triggering of inflammatory reactions, especially early in life during colonization of the host by the intestinal flora. Under inflammatory conditions, TLR expression can be modulated as described for TLR4 (Cario and Podolsky, 2000). Up-regulation of TLR4 and/or accessory molecules could confer hyper-responsiveness to LPS. Interestingly, fibronectin splice variants induced during tissue injury can also activate NFκB signalling via TLR4 (Okamura et al., 2001), and this could further maintain an inflammatory environment.

Mucosal adaptive immunity

  1. Top of page
  2. Introduction
  3. Mucosal innate immunity
  4. Mucosal adaptive immunity
  5. Perspectives and concluding remarks
  6. Acknowledgements
  7. References

The adaptive immune response plays an essential role in maintaining an intact epithelial barrier function and providing additional lines of defence. Immune cells act synergistically with epithelial innate immunity. The importance of adaptive immunity is highlighted by the size of the lymphocyte compartment in the gut. It is estimated that 70% of the total lymphocytes of the body are concentrated in the intestinal intra and subepithelial layers. The way an antigen interacts with the cells of the innate immune system determines the type of adaptive immune response that is triggered, i.e. active suppression versus robust immune response.

Sampling of antigens at mucosal sites

The induction of an immune response requires that antigens penetrate, and eventually cross, the epithelial barrier to reach the lymphoid tissues where they can interact with immune cells. Three pathways can mediate transport of antigens across epithelial barriers: the absorptive epithelial cell (enterocyte), the M cell, and the dendritic cell pathways (Neutra et al., 2001) (Fig. 3). The relative importance of these pathways probably varies along the length of the digestive tract and also changes during the maturation of the gut that takes place after birth.

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Figure 3. Antigen sampling and presentation in the gut. Antigens follow different pathways to cross the gut epithelial barriers and reach local organized gut-associated lymphoid tissue or draining mesenteric lymph nodes.

The dendritic cell pathway. Circulating monocytes are recruited and differentiate within the epithelial microenvironment into dendritic cells (red). These cells open epithelial tight junctions and capture and internalize antigenic macromolecules and microorganisms through dendrites. They subsequently migrate into local gut-associated lymphoid tissues (GALT), or via lymphatics into the draining mesenteric lymph nodes, where they process the antigens and present them to naïve lymphocytes. The dendritic cells can also present the native antigens to naïve lamina propria B cells (yellow). These B cells undergo IgA isotype switch in the absence of T cells and produce antibodies mainly directed against commensal bacteria.

The M cell pathway. Specialized enterocytes found in the follicle-associated epithelium (FAE) internalize macromolecules and microorganisms through fluid or receptor-mediated endocytosis and transport them into an intraepithelial pocket filled with antigen-presenting cells. If induction of the immune response occurs in GALT and lymph nodes associated with mucosal tissues, the effector and memory immune cells acquire a mucosal homing program (α4β7 integrin) and return to mucosal effector sites. If induction occurs in distant organized lymphoid tissues, the resulting effector and memory cells express a peripheral homing program.

The enterocyte pathway. Enterocytes are able to take up and present antigens that have diffused across the mucus layer and the glycocalyx.

The are equipped with conventional and non-conventional antigen processing and presentation machineries. They present antigens to intraepithelial lymphocytes that participate in the maintenance of the epithelial barrier function by stimulating repair processes and elimination of damaged epithelial cells. They are also thought to down-regulate Th1 immune responses.

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The enterocyte pathway. The absorptive enterocytes of the small intestine and the colonocytes of the large intestine represent by far the major surface area on which antigen contact can occur. These cells are able to internalize antigens via bulk uptake and receptor-mediated endocytosis, and present processed antigens to intra-epithelial lymphocytes (IEL) (Kaiserlian, 1999). Soluble antigens that diffuse through the glycocalyx and are endocytosed, enter intracellular degradative pathways that are known to intersect with antigen-presenting compartments (Blumberg et al., 1999). Following processing in endosomes, some antigens may be presented by the epithelial cells to IELs via conventional and non-conventional MHC class I and class II molecules (for review see Christ and Blumberg, 1997; Kaiserlian, 1999; Shao et al., 2001). Co-stimulatory molecules, including B7 family members and CD40 are not expressed by normal intestinal epi-thelial cells (Hershberg et al., 1997), suggesting that these cells may induce suppression rather than activation of naïve T cells. Exosomes, small membrane vesicles that are produced and released by epithelial cells, have also been proposed to play a role in the induction of unresponsiveness as discussed below.
The M cell pathway. The follicle-associated epithelium (FAE) over organized gut-associated lymphoid tissue (GALT) is phenotypically and functionally distinct from the rest of the gut epithelium. There are little mucus producing cells and defensin- and lysozyme-producing Paneth cells in the follicle-associated crypts (Giannasca et al., 1994). FAE enterocytes do not express polymeric Ig receptors (Pappo and Owen, 1988) and express only low levels of membrane-associated digestive hydrolases (Owen and Bhalla, 1983). These features tend to promote local contact of intact antigens and pathogens with the FAE surface. The most striking feature of the FAE is the presence of M cells, epithelial cells with the ability to efficiently deliver samples of foreign material by transepithelial transport from the lumen to organized lymphoid tissues within the mucosa (for review see Neutra et al., 2001; Kraehenbuhl and Neutra, 2000). Antigens transported across the FAE by M cells are captured by immature DCs in the subepithelial dome region, as shown for live attenuated Salmonella typhimurium in mouse Peyer’s patches after oral feeding (Hopkins et al., 2000) and orally administered Listeria monocytogenes (Pron et al.◊2001).
The dendritic cell pathway. DCs, the only professional cells able to present antigens to naïve T lymphocytes, derive from circulating monocytes and migrate into mucosal epithelia following epithelial-derived chemokine gradients (Cook et al., 2000). In vitro, immature DCs have been shown to be capable of migrating between epithelial cells, opening tight junctions that seal epithelial intestinal cells, and capturing antigens via their dendrites (Rescigno et al., 2001). This phenotype was associated with the up-regulation of DC genes encoding adhesion and tight junction proteins, such as E-cadherin, occludin, and ZO-1. In vivo, S. typhimurium or non-pathogenic E. coli were found to stimulate the recruitment of DCs into the gut epithelium, and this was followed by their uptake by the recruited cells (Rescigno et al., 2001). S. typhimurium administered by the oral route appear very rapidly in CD18+ cells, probably DCs, circulating in peri-pheral blood (Vazquez-Torres et al., 1999). The fact that no systemic infection occurs in CD18-deficient mice confirms the importance of CD18+ phagocytic cells in transport of bacteria from the intestine to other organs such as spleen. Once DCs have taken up an antigen or a micro-organism they are thought to leave the villus epithelium and migrate into the lamina propria and/or to a draining lymph node (Huang et al., 2000) (Fig. 3).

DCs are also involved in the sampling of antigens and bacteria that are transported through M cells. The FAE constitutively produces the chemokine CCL20, respon-sible for chemotactic migration of DCs into the sub-epithelial dome (SED) region of Peyer’s patches (Tanaka et al., 1999; Iwasaki and Kelsall, 2000) (Fig. 2). CCL20 binds to the chemokine receptor CCR6 expressed by immature DCs, naïve B lymphocytes, and memory T lymphocytes. In CCR6-deficient mice, myeloid DCs are absent from the subepithelial region of Peyer’s patches and the mucosal immune response to enteropathogens is impaired, although the size of their Peyer’s patches and the distribution of B and T cells is normal (Varona et al., 2001; Cook et al., 2000). Interestingly, CCL20 expression was found to be up-regulated in the epithelium on villi of the small intestine in response to challenge with flagellin (Fig. 2). The CCL20 response in the gut epithelium triggered by bacterial products is likely to be instrumental in recruiting DCs into the epithelium, thus providing another link between innate and adaptive immune responses.

Antigen-specific immune responses

Thymus-independent mucosal immunity. In mucosal tissues, particularly in the gut, two thymus-independent primitive immune mechanisms help to maintain the barrier function of the epithelium by eliminating infected epithelial cells, stimulating wound healing or neutralizing and eliminating invading microorganisms.
B cell responses.

The vast majority of intestinal secretory IgA is produced by lamina propria B220+IgM+ lymphocytes in a T cell-independent manner that is not dependent on organized lymphoid tissues with germinal centres and follicular dendritic cells (Fagarasan et al., 2001) (Fig. 3). This specific T cell-independent IgA antibody response is mainly directed against commensal bacteria of the intestinal flora (Macpherson et al., 2000). Intraepithelial dendritic cells that have sampled intestinal microorganisms on the villus surfaces, are known to migrate into the lamina propria where they could present native antigens to local naïve B cells (Fagarasan et al., 2001). The nature of this lamina propria B cell population remains to be characterized, especially in view of the fact that commensal specific IgA responses can occur in mice lacking IgM-expressing B cells (Macpherson et al., 2001).

These T cell-independent immune responses are thought to be an evolutionarily primitive form of specific immune defence designed to prevent the gut flora from invading the host. Indeed, commensals that have crossed the mucosal barrier would be coated by commensal-specific IgA and either be transported back into the lumen by polymeric-Ig receptor-mediated transcytosis (Kaetzel et al., 1991) or taken up locally and degraded by macrophages expressing μ/α Fc receptors (Shibuya et al., 2000; Sakamoto et al., 2001).

T cell responses.

Intraepithelial lymphocytes (IEL) form a heterogeneous cell population that consists of both CD4+ and CD8+ cells (MacDonald, 1999). In the upper airways most IELs are CD4+ cells, while in the gut CD8+ T that express αβ and γδ T cell receptors (TCR) represent the major IEL population (Lefrançois and Puddington, 1998). IELs expressing CD8α homodimers on their surface are believed to be thymus-independent deriving from cryptopatches in mice (Saito et al., 1998), but in man they are rare and there is limited evidence for extrathymic maturation. Recently, it has been shown that mouse CD8αα IELs derive from double negative thymic precursors (Guy-Grand et al., 2001). Both αβ and γδ TCR IELs (Arstila et al., 2000) display a restricted repertoire of TCR interacting with non-conventional MHC class I mole-cules on epithelial cells. These MHC molecules include MHC-linked (MICA, MICB, HLA-E) and unlinked (CD1d) class-I like molecules loaded with ‘degenerated’ antigens that do not require intracellular processing (Blumberg et al., 1999; Shao et al., 2001). The γδ TCR-bearing CD8αα IELs that recognize unique motifs presented by stressed epithelial cells have been shown to participate in epithelial monitoring and repair processes (Witherden et al., 2000), and may represent a primitive mechanism essential for the maintenance of the integrity of the epithelial barrier.

Thymus-dependent immunity. A major function of the mucosal immune system is the down-regulation or suppression of local and systemic immune responses specific for food antigens and commensal bacteria. However, the mucosal system is also capable of stimulating immunity specific for pathogen-derived antigens.
Oral tolerance.

The primary sources of foreign antigens in the gut are food and the commensal microbial flora. These antigens generally do not trigger defensive immune responses in spite of the fact that they enter the mucosa in measurable amounts. The exact sites or mechanisms of this ‘oral tolerance’ are still controversial and have been reviewed elsewhere (Garside and Mowat, 2001; Simmons et al., 2001). Unresponsiveness in mucosal tissues may be achieved by the functional or physical elimination of T cells through anergy, clonal deletion, or interaction with regulatory CD4+T cells or mediators including TGFβ and IL-10. TGFβ and IL-10 are produced by T cells and by gut epithelial cells (Xian et al., 1999), lamina propria stromal cells (Fagarasan et al., 2001), and Peyer’s patch dendritic cells (Iwasaki and Kelsall, 2000). These cytokines are known to induce the differentiation of T helper type 2 (Th2) cells and to promote immunoglobulin isotype switch both in Peyer’s patches and in the lamina propria. They also down regulate Th1 responses that could be harmful to mucosal tissues. Abrogation of TGFβ signalling in T cells leads to tissue inflammation, especially in the gut lamina propria (Gorelik and Flavell, 2000). Using TCR transgenic mice, Lefrançois and collaborators have shown that antigens specifically expressed in gut epithelium stimulate the expansion of antigen specific CD8 cells that are non-cytolytic for the epithelial cells (Vezys et al., 2000). Whether cytotoxic T lymphocyte (CTL) activity is inhibited by the TGFβ- and IL-10-rich microenvironment of the gut has not been examined.

Whether IELs participate in systemic (oral) tolerance is not known, but if so, they should be able to leave the epithelium and reach systemic lymphoid tissue to exert their suppressive function. There is evidence that IELs migrate in and out of a human intestinal epithelial monolayer in vitro (Shaw et al., 1998), but whether they migrate back into the lamina propria and return to lymphatics or the blood circulation in vivo is not known. Mucosal dendritic cells that are known to migrate into mesenteric lymph nodes, can induce systemic tolerance (Huang et al., 2000). Exosomes (MHC class I and II-containing vesicles) released by cells, may also participate in oral tolerance (Denzer et al., 2000). There is evidence that human intestinal cells can release exosomes loaded with food antigens basolaterally (M. Heyman, personal communication). Exosomes could be taken up by dendritic cells and carried into draining lymph nodes where they could induce tolerance if there is an absence of co-stimulatory molecules at their surfaces.

B cell responses.

In response to enteropathogenic microbes, antigen-specific lymphocytes are activated in the mucosa to eliminate the infection and to prevent secondary infection. The mechanisms of B cell activation in GALT, the migration of effector B cells and their differentiation into polymeric IgA producing cells have been extensively reviewed (Farstad et al., 2000). In mucosa-associated lymphoid tissue (MALT), stimulated B cells acquire mucosal homing receptors. The effector and memory lymphocytes lose their adhesion to stromal cells, leave organized MALT structures and enter the bloodstream via the lymph (Brandtzaeg et al., 1999). Virtually all IgA- and even IgG-antibody secreting cells detected after oral and rectal immunization expressed α4β7 integrin, the mucosal homing receptor, while only a minor fraction of these cells expressed L-selectin, the peripheral homing receptor. In contrast, circulating B cells induced by intranasal immunization expressed both L-selectin and α4β7 (Quiding-Jarbrink et al., 1995). The α4β7 memory/effector cells primed in MALT recognize the vascular addressin MadCAM-1 expressed on the lumenal surfaces of postcapillary venule endothelial cells in the gut (Briskin et al., 1993).

T cell responses.

CD8 T cell responses characterized by cytotoxic activity or secretion of IFN-γ are required for elimination of virus-infected cells and intracellular bacteria (Harty et al., 2000). CD8 effector cells are induced in systemic lymphoid compartments after immunization with intracellular pathogens. They can also be detected in mucosal tissue after acute infections with viruses specific for the airways (Bangham et al., 1985; Yap et al., 1978), or for intestines (Offit et al., 1991; London et al., 1987). In the mucosa, CD8 lymphocytes function like their systemic counterparts, via αβ TCR recognition of conventional MHC class I molecules and via a perforin/granzyme- or Fas-dependent pathways (Kagi et al., 1996), although perforin-independent pathways were also reported (Kerksiek and Pamer, 1999; Franco and Greenberg, 1999). Protozoan parasites like Toxoplasma gondii also induce specific CTLs in the gut (Buzoni-Gatel et al., 1999).

The use of MHC-class I tetramers and adoptive transfer of TCR transgenic cells allow direct quantification and phenotyping of antigen-specific CD8 T cells by flow cytometry and immunohistology (Kim et al., 1999). This approach has been applied to mucosal tissues (Haanen et al., 2001), to show that CTL responses are more intense in the intestine than in the spleen after oral immunization, although the kinetics of induction were similar in the two compartments (Huleatt et al., 2001; Pope et al., 2001). The CD8 intestinal response is also associated with higher effector capacities, with an oligoclonal TCR repertoire, and with persistent effector/ memory phenotypes (Huleatt et al., 2001; Marshall et al., 2001; Masopust et al., 2001; Chen et al., 1997). Moreover, intestinal CTL activation is dependent on a distinct pattern of co-stimulatory molecules (Kim et al., 1998). Maintenance of intestinal memory cells is prob-ably not due to antigen persistence, but rather to the nature of the mucosal microenvironment (Cerwenka et al., 1999; Ku et al., 2000). Expansion of CD8 cells following mucosal challenge is more rapid at the site of entry than in spleen (Kim et al., 1999; Marshall et al., 2001). Whether these cells proliferate locally or migrate back to mucosal tissues after expansion in draining lymph nodes is not yet known.

In contrast to B lymphocytes, the requirement of mucosal immunization for homing of CD8 T cells to the mucosa is not well established. In a rotavirus infection model, splenocytes from donors immunized systemically transferred protection against an intestinal infection (Kuklin et al., 2000). Similarly, CD8+ T cells from β7–/– mice prevented infection against rotavirus intestinal infection (Kuklin et al., 2000). Thus, α4β7-independent mechanisms might be involved in T cell trafficking to or retention at intestinal effector sites. In contrast, a recent study demonstrated that local T cell-mediated immunity in macaques was enhanced by mucosal immunization (Belyakov et al., 2001). Interestingly, both B and T lymphocytes express the CCR9/CCR10 chemokine receptors that respond to the epithelial chemokines CCL25 and MEC (mucosa-associated epithelial chemokine), respectively (Kunkel et al., 2000; Pan et al., 2000), allowing the cells to return to mucosal sites. Whether intestinal DCs are able to ‘imprint’ gut tropism on mucosal lymphocytes has not yet been demonstrated. It is tempting to speculate that in the special intestinal microenvironment mucosal DCs program lymphocytes to express homing and chemokine receptors that allow the lymphocytes to preferentially return to mucosal sites.

Perspectives and concluding remarks

  1. Top of page
  2. Introduction
  3. Mucosal innate immunity
  4. Mucosal adaptive immunity
  5. Perspectives and concluding remarks
  6. Acknowledgements
  7. References

The gut maintains a delicate balance between the down-regulation of inflammatory reactions to foreign materials and the capacity to respond to pathogens with vigorous cellular and humoral immune responses. The complex mechanisms that maintain this balance have yet to be clearly understood. In addition, recent work has elucidated the importance of the innate immune system in inducing adaptive responses. Effective mucosal vaccines will require not only efficient delivery across epithelial barriers, but also strategies that trigger protective immune responses in the absence of undesired inflammatory reactions. To this end, various bacteria and viruses are being genetically attenuated, engineered and harnessed to deliver antigens into mucosal inductive sites, and to provide appropriate signals to the epithelium and its associated antigen-presenting cells and lymphocytes. Elucidation of the earliest events in antigen and pathogen entry, and the cellular interactions that occur in and under the epithelium at specific sites in mucosal tissues, could greatly facilitate the design of effective mucosal vaccines in the future.

Acknowledgements

  1. Top of page
  2. Introduction
  3. Mucosal innate immunity
  4. Mucosal adaptive immunity
  5. Perspectives and concluding remarks
  6. Acknowledgements
  7. References

The authors are supported by the Swiss National Science Foundation Grant 31-56936-99 (JPK), the Swiss League against Cancer Grant SKL 635-2- 1998 (JPK), and by NIH Research Grants HD17557, AI34757, AI 35365, and NIH Center Grant DK34854 to the Harvard Digestive Diseases Center (MRN).

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  5. Perspectives and concluding remarks
  6. Acknowledgements
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