清野 宏

免疫系は通常、生体にとって有害な異物を排除するための生体防御システムとして機能しているが、稀に生体にとって有益な異物に対する過剰な反応を示すことがある。その代表的な免疫反応が免疫毒性とも呼ばれるアレルギー反応である。我々はこうしたアレルギー疾患のうち食物アレルギーに焦点を絞り、その発症メカニズムの解析を通じた食物アレルギーの予防・治療法の確立を目指している。<br> 食物アレルギーの病態形成部位である腸管には粘膜免疫システムと呼ばれる精密な免疫システムが構築されており、食餌性成分や腸内細菌など生体にとって有益な異物に対しては抑制型の免疫システムを示すことで、恒常性を維持したまま有益な異物の摂取を可能にしている。腸管免疫システムの恒常性維持機構の破綻により引き起こされる食物アレルギーは近年、乳幼児を中心に患者数が増加しているが、現時点での対処法は原因アレルゲンを含む食物を摂取しないという方法であり、Quality of Lifeの観点からも予防・治療法の開発が待望されている。我々は卵白アルブミンをアレルゲンとして用いた食物アレルギーモデルマウスを樹立し、免疫学的解析を進めている。本アレルギーモデルにおいては、卵白アルブミンで感作したマウスに卵白アルブミンを頻回経口投与することでアレルギー性の下痢が観察される。この下痢症状は一過性の症状であり、感作に用いたアレルゲンと同一のアレルゲンの経口投与が必要なアレルゲン特異的反応であり、その反応には2型ヘルパーT細胞により誘導されるアレルゲン特異的IgEが必須である。さらにはアレルギー性下痢を呈したマウスの腸管組織、特に粘膜固有層には活性化したT細胞やマスト細胞の増加が観察され、その増加を抑制することによりアレルギー性下痢の発症が抑制出来ることも示している。これらの結果から、本アレルギーモデルはヒトの食物アレルギーと非常に近い１型アレルギー疾患モデルとして広く使用されている。<br> 本シンポジウムでは我々がこれまで蓄積してきた食物アレルギーを用いた免疫学的解析について、特にマスト細胞の役割について紹介すると共に、マスト細胞特異的な疾患治療の可能性について報告したい。

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp alpha 1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH alpha 1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH beta 1 alpha 1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages.We revealed the presence of respiratoryMcells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2+ mice. The titers of Ag-specific Abs were as high in Id2-/- mice as in Id2+/- mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract. Copyright © 2011 by The American Association of Immunologists, Inc.

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer's patch M cells and F-ECs was distinctly regulated by alpha(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells. (C) 2010 Elsevier Inc. All rights reserved.

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyer's patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c+ dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-β, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c+ DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (Muco-Rice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates. Copyright © 2009 by The American Association of Immunologists, Inc.

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyers patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyers patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines. © 2009 Macmillan Publishers Limited. All rights reserved.

The eye is protected by the ocular immunosurveillance system. We show that tear duct-associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid-related orphan receptor γt, lymphotoxin (LT) α1β2-LTβR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity. © 2009 Nagatake et al.

Rice-expressed cholera toxin B (CTB) subunit is a cold-chain-free oral vaccine that effectively induces enterotoxin-neutralising immunity. We created another rice-based vaccine, MucoRice, expressing nontoxic double-mutant cholera toxin (dmCT) with CTA and CTB subunits. Western-blot analysis suggested that MucoRice-dmCT had the shape of a multicomponent vaccine. Oral administration of MucoRice-dmCT induced CTB- but not CTA-specific serum IgG and mucosal IgA antibodies, generating protective immunity against cholera toxin without inducing rice-protein-specific antibody responses. The potency of MucoRice-dmCT was equal to that of MucoRice-CTB vaccine. MucoRice has the potential to be used as a safe multicomponent vaccine expression system. © 2009 Elsevier Ltd. All rights reserved.

Mucosal vaccines are considered the most suitable type of vaccines to combat emerging and re-emerging infectious diseases because of their ability to induce both mucosal and systemic immunity. Considerable advances have been made toward the development of mucosal vaccines against influenza virus and rotavirus. Many additional mucosal vaccines are in development, including vaccines against cholera, typhoid, traveler's diarrhea and respiratory infections. In addition to oral and nasal vaccines, transcutaneous (or skin patch) and sublingual immunizations are now part of a new generation of mucosal vaccines. Furthermore, a rice-based oral vaccine (MucoRice (TM)) has been receiving global attention as a new form of cold chain-free vaccine, because it is stable at room temperature for a prolonged period. This review describes recent developments in mucosal vaccines with promising preclinical and clinical results.

Progress in the past quarter-century on understanding the molecular, cellular, and in vivo components of the mucosal immune system have allowed us to develop a practical strategy for a novel mucosal vaccine. The mucosal immune system can induce secretory IgA (SIgA) and serum IgG responses to provide two layers of defense against mucosal pathogens. For SIgA-mediated immunity in the gastrointestinal tract, the gut-associated lymphoid tissue contains both the tissue-dependent and tissue-independent IgA components. Harnessing the mucosal immune system for vaccine development may help prevent the global health problems caused by enteric infectious diseases. We have therefore combined mucosal immunology and plant biology to create a rice-based mucosal vaccine that requires neither needles and syringes nor refrigeration. © 2009 Elsevier Ltd. All rights reserved.

Mucosal surface performs various vital functions, such as respiration, absorption, excretion, and reproduction. At the same time, the mucosal surface is inevitably and continuously exposed to various kinds of external foreign antigens derived from food materials, and both commensal and pathogenic bacteria. Generally, tolerance is induced to beneficial antigens including food proteins and commensal bacteria, while protective immunity is induced against pathogenic bacteria and viruses to exclude these harmful pathogens from the entering into our body. Allergic disease is caused by the unexpected hyper-responsiveness against the unharmful antigens which induce generally tolerance. To establish new generation of the preventive and therapeutic strategies for the control of the allergic diseases, the molecular and cellular understanding of the induction of allergic responses at the mucosal compartment of aero-digestive tract needs to be accomplished. In this review, we focus on the recent progress in our understanding of how allergic diseases are developed in the respiratory and intestinal mucosae and applicable strategy for the mucosal allergy vaccine development and immunotherapy.

Airway and digestive tissues are the frontlines of the body's defense, being continuously exposed to the outside environment and encountering large numbers of antigens and microorganisms. To achieve immunosurveillance and immunological homeostasis in the harsh environments of the mucosal surfaces, the mucosal immune system tightly regulates a state of opposing but harmonized immune activation and quiescence. Recently, accumulating evidence has revealed that although the respiratory and intestinal immune systems share common mucosa-associated immunological features that are different from those of the systemic immune system, they also show distinctive immunological phenotypes, functions, and developmental pathways. We describe here the common and distinct immunological features of respiratory and intestinal immune systems and its application to the development of mucosal vaccines. © 2008 Elsevier Ltd. All rights reserved.

Previous studies have shown that Peyer's patches (PP) are not required for intestinal immunoglobulin A (IgA) responses to orally administered soluble protein. However, the roles of PP in regulation of mucosal immune responses against bacterial antigen remain to be clarified. In the present study, we generated several gut-associated lymphoreticular tissue-null mice by treatment with anti-interleukin-7 receptor antibody, the fusion protein of lymphotoxin β receptor and IgG Fc, and/or tumor necrosis factor receptor p55 and IgG Fc. These mice were then immunized with recombinant Salmonella expressing the C fragment of the tetanus toxin (rSalmonella-Tox C). Orally immunized PP-null mice as well as isolated lymphoid follicle (ILF)-null, PP/ILF-null, and PP/ILF/mesenteric lymph node-null mice induced identical levels of tetanus toxoid (TT)-specific systemic IgG responses to those of control mice. However, PP-null mice, but not ILF-null mice, failed to induce TT-specific intestinal IgA antibodies. Analysis of TT-specific CD4+ T-cell responses showed a reduction of gamma interferon (IFN-γ) synthesis in the intestinal lamina propriae of PP-null mice given oral rSalmonella-Tox C. In contrast, TT-specific IFN-γ responses in the spleen and delayed-type hypersensitivity responses were intact in those immunized mice. Interestingly, Salmonella lipopolysaccharide (LPS)-specific fecal IgA responses were not elicited in PP-null mice, while serum IgG anti-LPS antibodies were identical to those of control mice. These results suggest that while none of the gut-associated lymphoreticular tissues are required for the induction of systemic immune responses, PP are an essential lymphoid tissue for induction and regulation of intestinal IgA immunity against orally administered rSalmonella. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Parenteral vaccines are used commonly against most of infectious diseases. It is noted that these injection type vaccines are meant to induce protective immunity in the systemic compartment but not aimed at use of the benefits of mucosal immunity as a first line of defense against mucosal infectious diseases such as AIDS, SARS and Influenza. In addition, one of major practical obstacles to current vaccination is storage of the vaccine under refrigeration (or cold-chain) in the developing countries. To overcome these concerns, a plant-based vaccine is considered to be an attractive strategy. Currently, we have developed a rice-based oral vaccine that offers significant advantages over available vaccines. In the rice-based vaccine MucoRice, cholera toxin B subunit (CTB) as the vaccine antigen was accumulated in protein bodies as rice seed storage organella. When orally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches (PPs), and inducing toxin-specific serum IgG and mucosal IgA antibodies with neutralizing activity. Further, MuocRice CTB remained stable state and maintained immunogenicity at room temperature for 1.5 years and was protected from pepsin digestion in vitro. Taken together, these findings suggest that MucoRice does not require needle/syringe and cold-chain but induces two layers of immunity in both mucosal and systemic compartments, which is the most effective and highly practical global vaccine to combat emerging and re-emerging infectious diseases.

Mucosally ingested and inhaled antigens are taken up by membranous or microfold cells (M cells) in the follicle-associated epithelium of Peyer's patches or nasopharynx-associated lymphoid tissue. We established a novel M cell-specific monoclonal antibody (mAb NKM 16-2-4) as a carrier for M cell-targeted mucosal vaccine. mAb NKM 16-2-4 also reacted with the recently discovered villous M cells, but not with epithelial cells or goblet cells. Oral administration of tetanus toxoid (TT)-or botulinum toxoid (BT)-conjugated NKM 16-2-4, together with the mucosal adjuvant cholera toxin, induced high-level, antigen-specific serum immunoglobulin (Ig) G and mucosal IgA responses. In addition, an oral vaccine formulation of BT-conjugated NKM 16-2-4 induced protective immunity against lethal challenge with botulinum toxin. An epitope analysis of NKM 16-2-4 revealed specificity to an α(1,2)-fucose-containing carbohydrate moiety, and reactivity was enhanced under sialic acid-lacking conditions. This suggests that NKM 16-2-4 distinguishes α(1,2)-fucosylated M cells from goblet cells containing abundant sialic acids neighboring the α(1,2) fucose moiety and from non-α(1,2)-fucosylated epithelial cells. The use of NKM 16-2-4 to target vaccine antigens to the M cell-specific carbohydrate moiety is a new strategy for developing highly effective mucosal vaccines. JEM © The Rockefeller University Press.

The mucosal immune system acts as a first line of defense against infection caused by luminal pathogens. Because HIV is transmitted primarily via mucosal-associated tissues, particularly with sexual transmission, understanding antiviral immunity present at these sites is important. HIV infection results in depletion of gut-associated lymphoid tissue (GALT) and in this sense can be considered to be a disease of the mucosal immune system. A stumbling block for efforts to develop a vaccine against this disease has been the escape of vaccine-induced neutralizing antibodies and cytotoxic T lymphocytes (CTLs) at mucosal compartments and the resulting viral spread. To avoid these problems, the ideal mucosal vaccine would induce HIV-specific secretory IgA (S-IgA) and mucosal CD8(+) CTL as a first line of defense at a very early stage of HIV infection, before the virus can seed into the secondary lymphoid organs in mucosal and systemic tissues. In this review, we provide an overview of mucosal vaccine concepts and vaccination strategies that have been proposed for the development of an HIV mucosal vaccine, including live recombinant vaccines, peptide-based vaccines, virus-like particles (VLP), subunit vaccines and DNA vaccines. (c) 2007 Elsevier Ltd. All rights reserved.

Helicobacter pylori is a Gram-negative spiral bacterium that causes gastritis and peptic ulcer and has been implicated in the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Although Th1 immunity is involved in gastritis and the accumulation of H. pylori-specific CD4+ T cells in the H. pylori-infected gastric mucosa in human patients, how T cells are primed with H. pylori antigens is unknown because no apparent lymphoid tissues are present in the stomach. We demonstrate here that Peyer's patches (PPs) in the small intestine play critical roles in H. pylori-induced gastritis; no gastritis is induced in H. pylori-infected mice lacking PPs. We also observed that the coccoid form of H. pylori is phagocytosed by dendritic cells in PPs. We propose that H. pylori converts to the coccoid form in the anaerobic small intestine and stimulates the host immune system through PPs. © 2007 by The National Academy of Sciences of the USA.