清野 宏

The tyrosine kinase receptor RET regulates the intestinal nervous system. A recent paper by Veiga-Fernandes et al. (2007) demonstrates that RET is also involved in the intestinal immune system through the initiation of Peyer's-patch tissue genesis.

The mucosal immune system acts as the first line of defense against microbial infection through a dynamic immune network based on innate and acquired mucosal immunity. To prevent infectious diseases, it is pivotal to develop effective mucosal vaccines that can induce both mucosal and systemic immune responses, especially secretory IgA (S-IgA) and plasma IgG, against pathogens. Recent advances in medical and biomolecular engineering technology and progress in cellular and molecular immunology and infectious diseases have made it possible to develop versatile mucosal vaccine systems. In particular, mucosal vaccines have become more attractive due to recent development and adaptation of new types of drug delivery systems not only for the protection of antigens from the harsh conditions of the mucosal environment but also for effective antigen delivery to mucosa-associated lymphoid tissues such as Peyer's patches and nasopharynx-associated lymphoid tissue, the initiation site for the induction of the antigen-specific immune response. In this review, we shed light on the dynamics of the mucosal immune system and recent advances toward the development of prospective mucosal antigen delivery systems for vaccines.

The mucosal immune system is equipped with unique innate and acquired defense mechanisms which provide a first line of protection against ingested and inhaled infectious agents. Peyer's patches (PPs) and nasopharynx-associated lymphoid tissue (NALT) have been shown to be important inductive sites for the initiation of the acquired phase of antigen-specific immune responses. In addition, the mucosal innate immune system acts as both a physical and an immunological boundary, playing a key role in the sensing and eliminating of pathogens and in the creating of symbiosis. The mucus layer covering the mucosal epithelium acts as a first physical and biochemical barrier. An additional layer of physical protection against microorganisms is provided by a tightly interlaced cell-to-cell network of epithelial cells and intraepithelial lymphocytes. Various antimicrobial peptides produced by the epithelium and secreted into the mucosal lumen can directly kill the invading pathogenic bacteria. Finally, Toll-like receptors (TLRs) associated with the mucosal compartment have been shown to recognize the pathogen-associated molecular patterns (PAMPs) of a variety of pathogenic and commensal microorganisms. Therefore, a greater understanding of the immunological progression from mucosal innate to acquired immune systems should facilitate the development of new generation of mucosal vaccines to prevent and control infectious diseaseses. © 2006 Bentham Science Publishers Ltd.

The epithelium covering mucosal tissues consists of epithelial cells (ECs) and intraepithelial lymphocytes (IELs), which constitutes the first line of defense against infection by microorganisms, through mucosal innate and acquired immunity. One of the principal roles of the mucosal epithelium is to form a strong physical barrier such as tight junction to prevent microbial penetration. While adhesion mechanisms between ECs have been extensively studied, there are few investigations on the mechanisms between ECs and IELs. Our findings indicate that a homophylic adhesion molecule termed epithelial cell adhesion molecule (Ep-CAM) is also expressed in IELs in addition to ECs, thus forming a physical interaction between ECs and IELs. In addition, we also characterized the expression and function of Toll-like receptors (TLRs) in mucosal epithelium using human corneal epithelial cells (HCEs). TLRs are mainly expressed by antigen presenting cells (APCs) and recognize pathogen-associated molecular patterns (PAMPs). However, responsiveness via TLR2 and TLR4 in HCEs was impaired, presumably due to restricted expression of the TLRs in the cytoplasm. These findings suggest that mucosal epithelium may create a state of tolerance in order to avoid unnecessary response to environmental and commensal antigens via TLRs. However, the mucosal surfaces that cover gut and respiratory lymphoid tissues such as Peyer's patch and nasopharynx-associated lymphoid tissue (NALT) are equipped with the gateway system which effectively uptake the outside antigens via M cells to initiate both the positive and negative immune responses. We recently identified M cell-like cells that are located within the villous epithelium and involved in antigen-transport into the lamina propria. Therefore, we designated them as villous M cells. These findings suggest that mucosal immune system is equipped with multiple layers of induction/suppression mechanisms for the regulation of mucosal innate and acquired immunity. © 2005 Elsevier B.V. All rights reserved.

The mucosal epithelium including intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) provide a first line of defense in the gastrointestinal tract. However, limited information is currently available concerning the nature of the physical interaction molecule that interconnects IECs and IELs. Among the several monoclonal antibodies (mAbs) generated by immunizing porcine IECs, mAb (5-15-1) was shown to strongly react with IELs in addition to IECs. MALDI-TOF-MS and tandem MS analysis suggested that the antigen belongs to a family of human homophilic epithelial cell adhesion molecule (Ep-CAM). The amino acid sequence of porcine Ep-CAM showed 82.8%, 78.1%, and 76.8% homology compared to human, mouse, and rat Ep-CAM. Moreover, 5-15-1 specifically reacted with transfectant of porcine Ep-CAM. These data suggest that the Ep-CAM may act as a physical homophilic interaction molecule between IELs and IECs at the mucosal epithelium for providing immunological barrier as a first line of defense against mucosal infection. © 2004 Elsevier Inc. All rights reserved.

Recent studies indicate that the mechanism of nasopharynx-associated lymphoid tissue (NALT) organogenesis is different from that of other lymphoid tissues. NALT has an important role in the induction of mucosal immune responses, including the generation of T helper 1 and T helper 2 cells, and IgA-committed B cells. Moreover, intranasal immunization can lead to the induction of antigen-specific protective immunity in both the mucosal and systemic immune compartments. Therefore, a greater understanding of the differences between NALT and other organized lymphoid tissues, such as Peyer's patches, should facilitate the development of nasal vaccines.

Epithelial cells are key players in the first line of defense offered by the mucosal immune system against invading pathogens. In the present study we sought to determine whether human corneal epithelial cells expressing Toll-like receptors (TLRs) function as pattern-recognition receptors in the innate immune system and, if so, whether these TLRs act as a first line of defense in ocular mucosal immunity. Incubation of human primary corneal epithelial cells and the human corneal epithelial cell line (HCE-T) with peptidoglycan or LPS did not lead to activation, at the level of DNA transcription, of NF-κB or the secretion of inflammation-associated molecules such as EL-6, IL-8, and human β-defensin-2. However, when incubated with IL-1α to activate NF-κB, the production by these cells of such inflammatory mediators was enhanced. Human corneal epithelial cells were observed to express both TLR2- and TLR4-specific mRNA as well as their corresponding proteins intracellularly, but not at the cell surface. However, even when LPS was artificially introduced into the cytoplasm, it did not lead to the activation of epithelial cells. Taken together, our results demonstrate that the intracellular expression of TLR2 and TLR4 in human corneal epithelial cells fails to elicit innate immune responses and therefore, perhaps purposely, contributes to an immunosilent environment at the ocular mucosal epithelium.

M cells located in the follicle-associated epithelium of Peyer's patches (PP) are shown to be the principal sites for the sampling of gut luminal antigens. Thus, PP have long been considered the gatekeepers of the mucosal immune system. Here, we report a distinct gateway for the uptake of gut bacteria: clusters of non-follicle-associated epithelium-associated Ulex europaeus agglutinin (UEA)-1+cells, which we have designated intestinal villous M cells. Interestingly, villous M cells are developed in various PP [or gut-associated lymphoid tissue (GALT)]-null mice, such as in utero lymphotoxin β receptor (LTβR)-Ig-treated, lymphotoxin α (LTα)-/-, tumor necrosis factor/LTα-/-, and inhibition of differentiation 2 (Id2)-/-mice. Intestinal villous M cells have been observed to take up GFP-expressing Salmonella, Yersinia, and Escherichia coli-expressing invasin, as well as gut bacterial antigen for subsequent induction of antigen-specific immune responses. Thus, the identified villous M cells could be an alternative and PP-independent gateway for the induction of antigen-specific immune responses by means of the mucosal compartment.

Invasion of infectious agents through mucosal surfaces can be prevented by use of the common mucosal immune system (CMIS), which interconnects inductive tissues, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT), and effector tissues of the intestinal and respiratory tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant has been shown to be essential. When vaccine antigen is administered together with mucosal adjuvant, antigen-specific T-helper (Th) 1 and Th2 cells, cytotoxic T lymphocytes (CTLs) and IgA B cell responses are effectively induced by oral or nasal routes via the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium (FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying dendritic cells (DCs). Elucidation of the molecular/cellular characteristics of M cells and mucosal DCs will greatly facilitate the design of a new generation of effective mucosal adjuvants and of a vaccine delivery vehicle that maximises the use of the CMIS. Our recent efforts at mucosal vaccine development have focused on nasal administration of vaccine antigen together with nontoxic mutant-based or cytokine-/chemokine-based adjuvant for the induction of the protective immunity. To this end, a chimeric form of a nontoxic adjuvant combining the merits of mutant cholera toxin A subunit (mCT-A) and heat labile toxin B subunit (LT-B) was created as the second generation of detoxified toxin-based mucosal adjuvant. When a vaccine antigen was coexpressed together with an immune stimulatory/delivery molecule in crop seed, this edible vaccine is not only effective but also extremely practical in that it can be produced in huge quantities and preserved and shipped over long distances at room temperature without altering the quality of the vaccine. Because such qualities would greatly facilitate global vaccination, this new generation edible vaccines with a built-in adjuvant and/or M cell-targeted edible vaccine promises to be a powerful weapon for combating infectious diseases and bioterrorism. Copyright © 2003 John Wiley &amp Sons, Ltd.

腸管免疫機構は全身免疫機構とは似て非なるユニークなシステムと考えられているが,その特殊性は,消化管に寄生する免疫システムが消化管の栄養分の消化吸収という本来の働きとうまく折り合いをつけるために,生体にとって有用な食餌等の抗原に対しては免疫反応を極力差し控え,一方,有害な抗原に対してはこれを積極的に排除するという2通りの免疫応答を共進的に発達させた結果として形成されたものと考えられる.この粘膜免疫システムは極めて複雑・精巧である反面,砂上の楼閣のような側面を内包しており,致命的なシステムエラーを引き起こすことがある.その代表的な疾患として腸管粘膜組織に好発する炎症性腸疾患があげられる

Peyer's patches (PP) represent a well-characterized inductive site in gut-associated lymphoid tissue that actively acquires antigens from the intestinal lumen. It was reported that organized PP are not required for antigen-specific IgA responses induced by oral immunization with soluble antigen mixed with the mucosal adjuvant, cholera toxin. However, the role of PP in the induction of mucosal and systemic immune responses remains to be clarified in the case of particulate antigen. Here, we created PP-null mice by treating them with monoclonal anti-IL-7 receptor α chain (IL-7Rα) antibody during gestation and then immunized with antigen-encapsulated poly-lactic acid (PLA) microspheres. Brisk OVA-specific antibody responses were noted in serum and fecal extracts of normal mice following direct intestinal immunization with OVA in PBS (OVA-PBS) as well as in PLA-microspheres (OVA-MS). Antibody production was similarly elevated in PP-null mice immunized with OVA-PBS via direct injection into the intestinal tract. In contrast, OVA-specific antibody responses were dramatically decreased in both serum and fecal extracts collected from PP-null mice immunized intestinally with OVA-MS. These results were further supported by the number of OVA-specific antibody-forming cells detected in the spleen and intestinal lamina propria. PP deficiency also resulted in the reduction in OVA-specific Th1/Th2 cell responses in the spleen and mesenteric lymph nodes of mice intestinally immunized with OVA-MS. These results suggested that organized PP do, in fact, play a crucial role in the induction of antigen-specific immune responses against ingested particulate antigen.

IL-15は粘膜免疫のIgA免疫応答の制御において,重要なサイトカインであることが分かってきた.IL-15は,現在まで主流と考えられてきたIgA誘導のための循環帰巣経路から独立した形式で動いているB-1型細胞に作用している.このIL-15が腸管局所で異常発現するとCD8αβ+NK1.1+T細胞が増殖し,小腸炎症の発症に深くかかわっているらしい.つまり,このT細胞はTh1型のサイトカインを産生しており,このT細胞の優先的な増加をIL-15の抗アポトーシス活性に帰して異常を増殖しているらしい.これらの結果は,IL-15は粘膜免疫のIgA免疫応答に重要なサイトカインであるが,その制御異常が生体に不利な効果を与えることを示唆している

Cholera toxin (CT) and heat-labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co-administered protein antigen (Ag). Both LT and CT induce B7-2 expression on antigen-presenting cells (APCs) for subsequent co-stimulatory signalling to CD4 + T cells. CT directly affects CD4 + T cells activated via the TCR-CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)-4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL-4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4 + Th2 cells, which provided effective help for the Ag-specific mucosal immunoglobulin (Ig)A, as well as serum IgG1. IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7-2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4 + T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.

The common mucosal immune system (CMIS) consists of an integrated cross-communication pathway of lymphoid tissues made up of inductive and effector sites for host protection against pathogenic microorganisms. Major effector molecules of the CMIS include IgA antibodies and cytokines, chemokines and their corresponding receptors. Secretory IgA (S-IgA), the major immunoglobulin, is induced by gut-associated lymphoreticular tissue (GALT)-derived B cells with the help of Th1- and Th2-type CD4+ T lymphocytes. Cytotoxic T lymphocytes (CTLs) in the mucosal epithelium, a subpopulation of intraepithelial lymphocytes (IELs), also help maintain the mucosal barrier. The CMIS is unique in that it can provide both positive and negative signals for the induction and regulation of immune responses in both the mucosal and systemic compartments after oral or nasal antigen exposure. Prevention of infection through mucosal surfaces can be achieved by the CMIS through connections between inductive (e.g. GALT) and effector tissues. When vaccine antigens are enterically administered together with mucosal adjuvants [e.g. cholera toxin (CT), heat-labile toxin produced by Escherichia coli (LT) and IL-12], antigen-specific Th1/Th2 and IgA B cell responses are induced simultaneously in the mucosal effector compartment. Since these antigen-specific immune responses are not generated by oral vaccine without mucosal adjuvant, safe and effective adjuvants for the induction of antigen-specific S-IgA and CTL responses are essential for the development of mucosal vaccines for protection against infectious diseases. Finally, recent findings suggest the presence of a CMIS-independent IgA induction pathway, which also must be considered in the development of mucosal vaccines. Copyright © 2001 John Wiley &amp Sons, Ltd.