Mucosal Innate Immune Responses
In the mucosa, innate defense includes the physical barrier provided by epithelial cells and cilia movement, mucus production, secreted molecules with antibacterial activity, and the cytolytic activity of NK cells. Recent studies have demonstrated that a number of innate molecules produced at mucosal surfaces (including cytokines, chemokines, and defensins) can provide the necessary signals to enhance systemic or both systemic and mucosal immunity to antigens.
Mucosal surfaces are covered by a layer of epithelial cells that prevent the entry of exogenous antigens into the host. The physical protection of the largest mucosal surface, i.e., the GI tract, involves a monolayer of tightly joined absorptive epithelial cells termed enterocytes, which constitute a highly specialized selective barrier that allows the absorption of nutrients while preventing the entry of pathogens (2). The barrier effect of intestinal epithelial cells is facilitated by the mucus blanket that covers these cells and prevents the penetration of microorganisms and the diffusion of molecules toward the intestinal surface. Mucus resembles glycoprotein and glycolipid receptors that occur on enterocyte membranes, tending to interfere with the attachment of microorganisms. The barrier effect of the epithelial surface is ensured by the continuous renewal of the epithelial cell layer. By this process, which results in complete renewal of the absorptive enterocyte layer every 2-3 days, damaged or infected ente-rocytes are replaced by crypt epithelial cells, which differentiate into enterocytes as they migrate toward the desquamation zone at the villus tip. The epithelia of other mucosal surfaces (including the oral cavity, pharynx, tonsils, urethra, and vagina) are made of stratified epithelial cells that lack tight junctions. However, the renewal of exposed epithelial cell layers by cells from subjacent layers and mucus secretion contribute to the permeability barrier effect on these surfaces as well.
Epithelial cells also secrete antimicrobial peptides such as defensins, inflammatory cytokines, and chemokines, which contribute to mucosal innate immune responses. In this regard, the human intestinal a-defensins (HDs) HD-5 and HD-6 were identified in intestinal Paneth cells and in the human reproductive tract (58). The a-Defensin are also secreted by tracheal epithelial cells, and they are homologous to peptides that function as mediators of nonoxidative microbial cell killing in human neutrophils (termed human neutrophil petide [HNPs]) (59,60). The p-defensins, and in particular human p-defensin-1 (hBD-1), are expressed in the epithelial cells of the oral mucosa, trachea, and bronchi, as well as mammary and salivary glands in humans (61-63). Human intestinal epithelial cells were reported to express hBD-1 constitutively, whereas hBD-2 was only seen in inflamed colon or after bacterial infection of a colonic epithelial cell line (64). Secretory phospholipase A2 (S-PLA2) is an antimicrobial peptide present in granules of small intestinal Paneth cells and human polymorphonuclear neu-trophils (PMNs). The S-PLA2 molecule is released by Paneth cells upon exposure to cholinergic agonists, bacteria, or lipopolysaccharide (LPS). High concentrations of
S-PLA2 are also found in human tears. In contrast to other PLA2 molecules produced by mammalian cells, the S-PLA2 preferentially removes bacterial phosphatidyl glyc-erol and phosphatidyl ethanolamine, a property that can explain the potent antimicrobial activity of S-PLA2 (65,66). Other antimicrobials produced of mucosal surfaces include lysozyme, peroxidases, cathelin-associated peptides, and lactoferrin. In this regard, lactoferrin was recently reported to inhibit HIV-1 replication at the level of viral fusion/entry (67).
It is now well established that epithelial cells produce proinflammatory cytokines, including interleukin (IL)-1, IL-6, tumor necrosis factor-a (TNF-a), and granulo-cyte/macrophage colony-stimulating factor (GM-CSF) in response to pathogen invasion (68,69). Interestingly, epithelial cells also express CxC and CC chemokines. For example, bacterial or parasitic (i.e., Cryptosporidium parvum) infections of intestinal epithelial cells were shown to upregulate expression and secretion of the CxC chemokines IL-8 and GRO-a (70). Bacterial infection of intestinal epithelial cell lines was also reported to stimulate the expression of the CC chemokines monocyte chemo-tactic protein-1 (MCP-1), RANTES, and macrophage inflammatory protein-3a (MIP-3a) (71,72), and freshly isolated colon epithelial cells produced an array of chemokines similar to the cell lines, as well as MIP-1a and MIP-1p (71). More recently, inflammatory protein-10 (IP-10) and monokine inducible by interferon-7 (IFN-7) (MIG), which are CxC chemokines that are known to attract CD4+ T-cells, were detected in normal intestinal epithelial cells, and their expression was upregulated by infection with invasive bacteria or stimulation with proinflammatory cytokines (73). Furthermore, 78 T-cell receptor-positive (TCR+) (IELs) produce the C-type chemokine lym-photactin, which is chemotactic for T-cells and NK cells but not for monocytes, neutrophils, or dendritic cells (74,75). Taken together, these studies clearly indicate that the mucosal epithelium has the potential to produce a large spectrum of C, CC, and CxC chemokines and that both epithelial cells and intestinal lymphocytes can contribute to these innate responses.
NK cells are major players in the innate immune system, especially in the GI tract. NK cells occur in both the lamina propria and the intraepithelial compartment as large granular lymphocytes (76,77). Studies performed on human IELs have shown that the aEp7 integrin is the main surface molecule involved in the lysis process (77). Significant increases in intestinal IEL NK cell activity were seen during the early phase of secondary infection of chickens with the Eimeria parasite (78). Furthermore, nonspecific recruitment of cytotoxic effector cells into the intestinal mucosa of enteric virus-infected mice has been reported (79). Humans with inherited deficiency of NK cells experience more severe herpesvirus infections (80); however, these individuals clear the virus infection in a fashion comparable to that seen in immunocompetent subjects, suggesting that the role of NK cells may be to limit the extent of certain mucosal viral infections. Finally, NK cells are known to secrete interferon-7 (IFN-7) and IL-4 after infection. Thus, mucosal NK cells could be major players in the cytokine environment that influences the development of effector T-cells.
Mucosal Adaptive Immune Responses
It is now well accepted that the functional diversity of the immune response is exemplified by an inverse relationship between antibody and cell-mediated immune responses. This dichotomy is due to Th cell subsets, which are classified as either Th1 or Th2 according to the pattern of cytokines produced (81). Thus, Th1 cells produce IL-2, IFN-7 and lymphotoxin-a (LT-a, also known as TNF-p), LT-p and TNF-a, and Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL 10, and IL-13. The cytokine environment plays a key role in the differentiation of both Th cell subsets from precursor Th0 cells. IL-2 is produced by Th0 cells upon antigen exposure and serves as an important growth factor. IL-12 induces NK cells to produce IFN-7 (82,83), which, together with IL-12, triggers Th0 cells to differentiate along the Th1 pathway. Murine Thl-type responses are associated with development of cell-mediated immunity as manifested by delayed-type hypersensi-tivity (DTH) as well as by B-cell responses with characteristic IgG Ab subclass patterns.
For example, IFN-7 induces murine ^ ^ 72a switches (84) and production of complement-fixing IgG2a antibodies. On the other hand, IL-4 production induces Th0 ^ Th2-type development. The production of IL-4 by Th2 cells is supportive of B-cell switches from sIgM expression to SIgG1+ and to sIgE+ B-cells (85-87). Furthermore, the Th2 cell subset is an effective helper phenotype for supporting the IgA isotype in addition to IgG1, IgG2b, and IgE responses in the mouse system. Both Th1 and Th2 cells are also quite sensitive to cross-regulation. IFN-7 produced by Th1 cells inhibits both Th2 cell proliferation and B-cell isotype switching stimulated by IL-4 (88,89). Likewise, Th2 cells regulate Th1 cell effects by secreting IL-10, which inhibits IFN-7 secretion by Th1 cells. This decreased IFN-7 production allows development of Th2-type cells. It is also clear that Th1- and Th2-type cells express distinct patterns of chemokine receptors (90,91). Thus, CCR5 and the CxC chemokine receptors CxCR3 and CxCR5 are preferentially expressed by human Th1 cell clones, whereas Th2 cells express CCR4 and to a lesser extent CCR3 (91,92).
Studies in the last decade have shown that two Th2 cytokines, IL-5 and IL-6, are of particular importance for inducing SIgA+ B-cells to differentiate into IgA-producing plasma cells (93-95). In this regard, IL-6 induced strikingly high IgA responses in vitro in both mouse (93-95) and human (96) systems. However, the role of IL-6 in IgA responses in vivo remains to be demonstrated since both reduced (97) and normal IgA responses were reported in IL-6-/- mice (98). IL-10 has also been shown to play an important role in the induction of IgA synthesis, especially in humans (99-101). Finally, high frequencies of Th2 cells producing IL-5, IL-6, and IL-10 were shown in mucosal effector sites (e.g., the intestinal lamina propria and the salivary glands) where IgA responses predominate (102,103).
The S-IgA Abs constitute the predominant isotype present at mucosal surfaces, and they are the first Abs to come into contact with the microorganisms that have entered the host through the mucosae. Inhibition of microbial adherence is a critical initial step for the protection of the host and is mediated by both specific and nonspecific mechanisms. For instance, the agglutinating ability of S-IgA specific to capsular polysaccharide of Hemophilus influenzae seems to be crucial for avoiding colonization by H. influenzae (104). Finally, another nonspecific mechanism that inhibits microbial adherence is owing to the presence of carbohydrate chains on the S-IgA molecule that bind to bacteria or other antigens (105-107). The S-IgA Abs have been shown to be effective at neutralizing viruses at different steps in the infectious process. In particular, S-IgA specific for influenza hemagglutinin can interfere with the initial binding of influenza virus to target cells or with the internalization and the intracellular replication of the virus (108). The S-IgA can neutralize the catalytic activity of many enzymes of microbial origin (such as neuraminidase, hyaluronidase, glycosyltransferase and IgA-specific protease), as well as the toxic activity of bacterial enterotoxins (cholera toxin and the related heat-labile enterotoxin of E. coli). In vitro experiments employing murine polarized epithelial cells have demonstrated that antibodies specific to rotavirus and hepatitis virus can neutralize the respective viruses inside the epithelial cells (109,110), and evidence has been provided that similar mechanisms occur in vivo (111). Similarly, it has been shown that transcytosis of primary HIV isolates is blocked by polymeric IgA specific to HIV envelope proteins (112). These authors have shown that neutralization of HIV transcytosis occurs within the apical recycling endosome and that immune complexes are specifically recycled to the mucosal surface (112).
It should be mentioned that S-IgA appears to be important in limiting inflammation at mucosal surfaces. In fact, IgA Abs are unable to activate complement and interfere with IgM- and IgG-mediated complement activation (113,114). Furthermore, S-IgA inhibits phagocytosis, bactericidal activity, and chemotaxis by neutrophils, monocytes, and macrophages. In addition, IgA can downregulate the synthesis of TNF- a and IL-6 as well as enhance the production of IL-1R antagonists by LPS-activated human monocytes (115,116).
There is a clear demarcation between inductive sites, which harbor precursor (p)CTLs, and effector sites, which include the lamina propria and the epithelial cells where activated CD8+ CTLs function. it is now established that administration of virus into the GI tract results in a higher frequency of pCTL in Peyer's patches (117,118). For example, reovirus localizes to T-cell regions and is clearly associated with increased CD8+ pCTLs and memory B-cell responses (119). Oral administration of Vaccinia to rats resulted in the induction of virus-specific CTLs in Peyer's patches and mesenteric lymph nodes (120). These findings suggest that after enteric infection or immunization, antigen-stimulated CTLs are disseminated from Peyer's patches into mesenteric lymph nodes via the lymphatic drainage (120). Furthermore, virus-specific CTLs are also generated in mucosa-associated tissues by oral immunization with reovirus and rotavirus (117,118) and a high frequency of virus-specific CTLs is present in the Peyer's patches as early as 6 days after oral immunization. These studies suggest that oral immunization with live virus can induce antigen-specific CTLs in both mucosal inductive and effector tissues for mucosal responses and in systemic lymphoid tissues as well.
The vaginal infection model of rhesus macaques with SIV has been useful in studies of immunity to SIV in the female reproductive tract (121,122). Recent studies in this model have provided direct evidence that pCTLs occur in female macaque repro ductive tissues and that infection with SIV induces CTL responses (123). This important finding has now been extended to vaginal infection with an SIV/HIV-1 chimeric virus (SHIV) containing HIV-1 89.6 env gene (124). Interestingly, all macaques resisted two challenges with virulent SIV, and functional, gag-specific CTLs were present in the peripheral blood (124). Again, it should be emphasized that vaginal Abs were also induced; however, these results clearly indicate that mucosal CTL responses may be of importance in immunity to SIV infection. Recent work has shown that intranasal immunization with SIV/HIV components induces antibody responses in vaginal secretions (reviewed in ref. 125). It should be noted that intranasal immunization of mice with HIV-1 T-cell epitopes and the mucosal adjuvant CT induced functional CTLs (126). This evidence suggests that mucosal delivery of SIV/HIV components can induce mucosal CTLs that will contribute to immunity.
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