清野 宏

Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer's patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.

Type I (α/β) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-α (SasaIFN-α1 &amp 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-α1 (rSasaIFN-α1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-α subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-α, the mature form of the SasaIFN-α2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-α2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-α2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 × 106 U mg-1 against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-α2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-α2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity. © 2007 Elsevier Ltd. All rights reserved.

We previously reported that sphingosine 1-phosphate (S1P) regulates peritoneal B-cell trafficking and subsequent intestinal IgA production, but the underlying mechanisms remain obscure. We demonstrate here that nuclear factor kappa B-inducing kinase (NIK) is involved in the regulation of S1P-mediated trafficking of peritoneal B cells. Although peritoneal B cells from NIK-mutated alymphoplasia (aly) mice expressed type 1 S1P receptor (S1P(1)) at comparable levels and demonstrated normal migration toward S1P, aly peritoneal B cells showed decreased sensitivity to FTY720, an S1P(1) modulator. NIK-mutated stromal cells showed decreased levels of adhesion molecules (VCAM-1 and ICAM-1) and increased CXCL13 expressions, leading to impaired ability to support S1P-mediated emigration, but not immigration, of peritoneal B cells. Therefore, aly peritoneal B cells exhibited normal S1P-mediated peritoneal B-cell trafficking from peritoneum to intestine for IgA production when they were transferred into severe combined immunodeficient or wild-type mice. However, S1P-mediated emigration of wild-type B cells from the aly peritoneal cavity was impaired without affecting their immigration from the blood. Further, transfer of wild-type stromal cells into the peritoneum restored S1P-mediated trafficking of aly peritoneal B cells. These findings suggest that INK in stromal cells has a specific role in the regulation of S1P-mediated trafficking of peritoneal B cells.

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer's patch-null mice but abolished in the Peyer's patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after M. Unexpectedly, langerin(+)CD8 alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in MLN.

Although the mucosal and the systemic immune compartments are structurally and functionally independent, they engage in cross-talk under specific conditions. To investigate this cross-talk, we. vaccinated mice with tetanus toxoid together with cholera toxin with s.c. priming followed by intrarectal (IR) boosting. Interestingly, higher numbers of Ag-specific IgA and IgG Ab-secreting cells (ASCs) were detected in the lamina propria of the large intestine of mice vaccinated s.c.-IR. Ag-specific ASCs from the colon migrated to SDF-1 alpha/CXCL12 and mucosae-associated epithelial chemokine/CCL28, suggesting that CXCR4(+) and/or CCR10(+) IgA ASCs found in the large intestine after s.c.-IR are of systemic origin. In the colonic patches-null mice, IgA ASCs in the large intestine were completely depleted. Furthermore, the accumulation of IgA ASCs in the colonic patches by inhibition of their migration with FTY720 revealed that colonic patches are the IgA class-switching site after s.c.-IR. Most interestingly, s.c.-IR induced numbers of Ag-specific IgA ASCs in the large intestine of TLR2(-/-), TLR4(-/-), MyD88(-/-), and TRIF(-/-) mice that were comparable with those of wild-type mice. Taken together, our results suggest the possibility that cross-talk could occur between the large intestine and the systemic immune compartments via the colonic patches without the assistance of innate immunity.

In mucosa-associated lymphoid tissues (MALT), such as Peyer's patches and nasopharynx-associated lymphoid tissues, all types of immunocyte including dendritic cells, T cells, B cells and others are present and form the mucosal immune network against invasion of luminal pathogenic bacteria or virus. M cells are known to be antigen-sampling cells located in the follicle-associated epithelium (FAE) of MALT and play an important role as gateway of mucosal epithelial barrier. In this review, we shed light on the recent progress of protein antigen delivery system to MALT with M cell-targeting technology for the creation of new generation of mucosal vaccine. © 2008, THE JAPAN SOCIETY OF DRUG DELIVERY SYSTEM. All rights reserved.

Stimulation of mucosal immunity has great potential in vaccinology and immunotherapy. However, the mucosal immune system is more complex than the systemic counterpart, both in terms of anatomy (inductive and effector tissues) and effectors (cells and molecules). Therefore, immunologists entering this field need a precise terminology as a crucial means of communication. Abbreviations for mucosal immune-function molecules related to the secretory immunoglobulin A system were defined by the Society for Mucosal Immunolgy Nomenclature Committee in 1997, and are briefly recapitulated in this article. In addition, we recommend and justify standard nomenclature and abbreviations for discrete mucosal immune-cell compartments, belonging to, and beyond, mucosa-associated lymphoid tissue.

We examined the in vivo immunostimulatory effects of a recombinant Atlantic salmon (Salmo salar) interferon-α2 (rSasaIFN-α2). The mature rSasaIFN-α2, expressed and purified from Escherichia coli, was administered to rainbow trout (Oncorhynchus mykiss) via the oral, immersion, or intraperitoneal (IP) injection route. Injection of rSasaIFN-α2 at 0.1 μg/g fish gave significantly greater protection than a phosphate buffered saline (PBS) injection against a lethal challenge of infectious hematopoietic necrosis virus (IHNV), with a relative percent survival of 39%. Relative percent survival (RPS) increased significantly to 92% when the fish were injected with rSasaIFN-α2 at 1 μg/g fish. Antiviral protection was evident for up to 7 days post-injection of rSasaIFN-α2. Administration of rSasaIFN-α2 by the oral or immersion route was not protective, and the fish succumbed to virus infection. The level of systemic IFN-induced expression of the Mx1 gene was significantly greater (p&lt 0.01) in the IFN-injected group than in the PBS-injected group, and this was correlated with the fish survival rates in the challenge study. We used relative quantitative real-time polymerase chain reactions to examine the systemic expression of several other IFN-induced genes (including genes for IFN1, IFN regulatory factors 1 and 2, MHC-I, STAT1, vig-1, and GBP) and found that their expression was significantly increased 1-day post-rSasaIFN-α2 injection. Expression of the IFN-γ and interleukin-1β genes was not significantly increased. Thus, a salmonid rIFN-α can modulate the innate immune response of rainbow trout and mediate early antiviral protection against IHNV. © 2008 Elsevier Ltd. All rights reserved.

The lymphoid chemokines CCL19 and CCL21 are known to be crucial both for lymphoid cell trafficking and for the structural organization of lymphoid tissues such as nasopharynx-associated lymphoid tissue (NALT). However, their role in allergic responses remains unclear, and so our current study aims to shed light on the role of CCL19/CCL21 in the development of allergic hinitis. After nasal challenge with OVA, OVA-sensitized plt (paucity of lymph node T cells) mice, which are deficient in CCL19/ CCL21, showed more severe allergic symptoms than did identically treated wild-type mice. OVA-specific IgE production, eosinophil infiltration, and Th2 responses were enhanced in the upper airway of plt mice. Moreover, in plt mice, the number of CD4+CD25+ regulatory T cells declined in the secondary lymphold tissues, whereas the number of Th2-inducer-type CD8 alpha(-)CD11b(+) myeloid dendritic cells (m-DCs) increased in cervical lymph nodes and NALT. Nasal administration of the plasmid-encoding DNA of CCL19 resulted in the reduction of m-DCs in the secondary lymphoid tissues and the suppression of allergic responses in plt mice. These results suggest that CCL19/CCL21 act as regulatory chemokines for the control of airway allergic disease and so may offer a new strategy for the control of allergic disease.

It is well established that intraepithelial T lymphocytes ( IELs) are derived from conventional single- positive ( SP) thymocytes, as well as unconventional double- negative ( DN) thymocytes and CD103(+) CD8 alpha beta recent thymic emigrants ( RTEs). We show that IELs can be divided into two groups according to their dependency on sphingosine 1- phosphate ( S1P) for trafficking into the intestines. CD4 or CD8 alpha beta naive lymphocytes originating from SP thymocytes express high levels of type 1 S1P receptor ( S1P 1), and their preferential migration into the large intestine is regulated by S1P. In contrast, RTEs migrate exclusively into the small intestine, whereas DN thymic IEL precursors expressing either TCR alpha beta or TCR gamma delta migrate into both the small and large intestines. S1P does not play a role in the migration pathways of these unconventional thymic IEL precursors. Thus, down- regulation of S1P(1) expression or disruption of the S1P gradient halted conventional CD4 or CD8 alpha beta IEL trafficking into the intestines, but did not affect the trafficking of unconventional thymic IEL precursors. These data are the first to demonstrate that a lipid- mediated system discriminates IELs originating from conventional and unconventional thymic precursors.

<title>ABSTRACT</title> Poliovirus (PV) is easily transferred to humans orally; however, no rodent model for oral infections has been developed because of the alimentary tract's low sensitivity to the virus. Here we showed that PV is inactivated by the low pH of the gastric contents in mice. The addition of 3% NaHCO₃ to the viral inoculum increased the titer of virus reaching the small intestine through the stomach after intragastric inoculation of PV. Transgenic mice (Tg) carrying the human PV receptor (hPVR/CD155) gene and lacking the alpha/beta interferon receptor (IFNAR) gene (hPVR-Tg/<italic>Ifnar</italic>KO) were sensitive to the oral administration of PV with 3% NaHCO₃, whereas hPVR-Tg expressing IFNAR were much less sensitive. The virus was detected in the epithelia of the small intestine and proliferated in the alimentary tract of hPVR-Tg/<italic>Ifnar</italic>KO. By the ninth day after the administration of a virulent PV, the mice had died. These results suggest that IFNAR plays an important role in determining permissivity in the alimentary tract as well as the generation of virus-specific immune responses to PV via the oral route. Thus, hPVR-Tg/<italic>Ifnar</italic>KO are considered to be the first oral infection model for PV, although levels of anti-PV antibodies were not elevated dramatically in serum and intestinal secretions of surviving mice when hPVR-Tg/<italic>Ifnar</italic>KO were administered an attenuated PV.

Sphingosine I-phosphate (SIP) has been proposed as a regulator of lymphocyte trafficking, but its role in mucosa-associated diseases, such as in food allergies, remains to be elucidated. To examine the role of SIP in allergic diseases in the intestine, we used a Th2 cell-mediated Ag-specific allergic diarrhea model and demonstrated that type 1 SIP receptor (S1P(1)) expression was preferentially associated with pathogenic CD4(+) T cells for the development of allergic reactions. Consistent with this demonstration, treatment with FTY720, a modulator of the SIP,, prevented allergic diarrhea by inhibiting the migration of systemically primed pathogenic CD4(+) T cells induced by oral challenge with allergen into the large intestine. In addition, FTY720 hampered mast cell infiltration into the large intestine, whereas eosinophil infiltration into the large intestine and total and allergen-specific serum IgE production were comparable between mock- and FTY720-treated groups. These results suggest that modulation of the SIP-mediated pathway to inhibit the migration of pathogenic CD4(+) T cells and mast cells into the large intestine could be a novel strategy for preventing allergic diarrhea.

Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for &gt 1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism. © 2007 by The National Academy of Sciences of the USA.

Intestinal allergic diseases are initiated by aberrant Th2-type immune responses, including elevation of IgE antibodies (Abs) and infiltration of eosinophils. However, little is known about the rote of Peyer's patches (PP) in the control of allergic diseases. Using a mouse model for food allergy, we here show that mice tacking PP are more susceptible to disease development and show higher levels of antigen-specific IgE Abs than do PP-intact mice. In our study, we noted that high numbers of eosinophils infiltrated into the small intestine of PP-null mice. In contrast, the PP of intact mice contained regulatory CD4(+)CD25(+) Foxp3(+) T cells (Treg) that are known to produce high levels of IL-10, and inhibited the development of allergic diarrhea. PP-intact mice thus developed allergic diarrhea when treated with anti-CD25 or anti-IL-10 monoclonal antibody (mAb) in vivo. These studies demonstrate that PP, as the site where IL-10-producing Treg cells are created, mediate the mucosal regulatory network for the control of undesired allergic responses in the intestine. (C) 2007 Elsevier Inc. All rights reserved.

Background: A limited number of therapeutic strategies are currently available to treat patients with inflammatory bowel disease (IBD). Interleukin-10 (IL- 10)-deficient mice, well characterized as an experimental model of IBD, develop severe chronic colitis because of aberrant Th1 responses. Roxithromycin (RXM), a macrolide antibiotic, has received attention because it offers not only antibacterial but also immunosuppressive effects. We examined the immunosuppressive effect of RXM on the development of IBD. Methods: To test the efficacy of short-term administration of RXM, elder IL-10-deficient mice (16-20 weeks old) with established colitis were orally treated for 10 days with RXM (20 mg/kg 4-per day). To test the long-term preventive effects of RXM, for 20 weeks young adult IL-10-deficient mice (4-5 weeks old) also were administered RXM orally (20 mg/kg per day). Results: The short-term treatment-oriented administration of RXM reduced the degree of inflammatory change and lowered serum amyloid A in IL-10-deficient mice with severe colitis. Mononuclear cells from the lamina propria of RXM-treated large intestines showed lower production of IFN-gamma than did those from diseased mice that were untreated. Long-term prevention-oriented administration of RXM suppressed the development of severe colitis and decreased production of IFN-gamma and IL-12. In addition to its expected immunosuppressive effect, RXM treatment also decreased the level of Bacteroides vulgatus, a Gram-negative anaerobe. Conclusions: The anti-inflammatory changes observed in IL-10-deficient mice resulted from the efficacy of RXM as an immununo-suppressant as well as from its efficacy as an antibiotic. According to our findings, RXM would seem to have significant potential as a preventive and/or therapeutic agent for IBD.

Sphingosine 1-phosphate (S1P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S1P. All peritoneal B cells including B1a, B1b, and B2 B cells express comparable levels of the type 1 S1P receptor. Thus, treatment with FTY720, an S1P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heat-killed Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S1P in connecting peritoneal B cells with intestinal B-cell immunity (C) 2007 by The American Society of Hematology.

Probiotics have been considered as preventive agents for the control of inflammatory bowel disease (IBD). In this study, we assessed the immunomodulatory effect of Bifidobacterium bifidum BGN4 on the control of IBD using the CD4(+) CD45RB(high) T cell transfer disease model. The mice were fed for 4 weeks with either a conventional diet containing only skim milk or a diet containing skim milk with 0.3% (w/w) BGN4. The BGN4-fed mice showed normal weight growth, fewer clinical symptoms such as thickened watt and inflammatory cell infiltration, and tower levels of CD4(+) T lymphocyte infiltration and inflammatory cytokine productions than the skim milk-fed mice with IBD in the large intestine. Suppression of these cytokine productions, particularly IFN-gamma and MCP-1, through BGN4 treatment was also observed in the in vitro co-culture between intestinal epithelial. cells and T cells. These findings suggested that a BGN4 supplemented diet could be helpful for the control of aberrant immune responses in the intestinal tissue. (c) 2006 Elsevier Inc. All rights reserved.

The gastrointestinal immune system consists of immune cells in organized gut-associated lymphoreticular tissues (GALT) and diffuse lamina propria, which give rise to mucosal secretory IgA antibody responses. A recent study showed that the retinoic acid produced by GALT dendritic cells (DCs) imprints B cells for gut homing. Surprisingly, GALT DCs, together with interleukin-5 (IL-5) and IL-6, also provided a milieu for both B cell switching to IgA and IgA synthesis.

Phosphorylcholine (PC) is a structural component of a wide variety of pathogens including Streptococcus pneunioniae and Haemophilus influenzae, and anti-PC immune responses are known to protect mice against invasive bacterial diseases. The present study tested the capability of PC as an intranasal plurispecific vaccine against upper airway infections. BALB/c mice immunized with intranasal PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT) as a mucosal adjuvant showed increased PC-specific IgM in serum, IgA in nasal wash and saliva, and numbers of PC-specific nasal and splenic antibody producing cells. Enhanced production of IL-4 and IFN-gamma by CD4(+) T cells indicated the participation of Th2- and Th1-type cells. Salivary IgA antibodies produced by intranasal immunization with PC-KLH plus CT reacted to most strains of S. pneunioniae and H. influenzae. Further we demonstrated that the clearance of S. pnemoniae and H. influenzae from the nasal tract was significantly enhanced by nasal immunization with PC-KLH and CT. Thus, intranasal vaccination to induce PC-specific immune responses might help to prevent upper airway infections caused by S. pneumoniae and H. influenzae. (c) 2007 Published by Elsevier Ltd.

At the front line of the body's immunological defense system, the gastrointestinal tract faces a large number of food-derived antigens, allergens, and nutrients, as well as commensal and pathogenic microorganisms. To maintain intestinal homeostasis, the gut immune system regulates two opposite immunological reactions: immune activation and quiescence. With their versatile immunological features, intraepithelial lymphocytes (IELs) play an important role in this regulation. IELs are mainly composed of T cells, but these T cells are immunologically distinct from peripheral T cells. Not only do IELs differ immunologically from peripheral T cells but they are also comprised of heterogeneous populations showing different phenotypes and immunological functions, as well as trafficking and developmental pathways. Though IELs in the small and large intestine share common features, they have also developed differences as they adjust to the two different environments. This review seeks to shed light on the immunological diversity of small and large intestinal IELs.

The respiratory and digestive tracts represent major entry routes for pathogenic microorganisms from the lumen into an almost sterile environment of the body. Several physical and biological barriers associated with the innate immune system protect these sites from invasion and help to maintain mucosal homeostasis. The first physical defense line is a barrier structure made up of epithelial cells (ECs) joined firmly by tight junction proteins with brush-border microvilli and a dense layer of mucin (Berkes et al., 2003). Antimicrobial peptides such as defensins and type II phospholipase A2 produced by ECs and Paneth cells are additional molecules preventing the attachment and penetration of pathogenic microorganisms into mucosal tissues (Selsted and Ouellette, 2005).

CD3(-) CD4(+) CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-) CD4(+) CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

Although dendritic cells (DCs) located in the small intestinal lamina propria (LP-DCs) migrate to mesenteric lymph nodes (MLNs) constitutively, it is unclear which chemokines regulate their trafficking to MLNs. In this study we report that LP-DCs in unperturbed mice require CCR7 to migrate to MLNs. In vitro, LP-DCs expressing CCR7 migrated toward CCL21, although the LP-DCs appeared morphologically and phenotypically immature. In MLNs, DCs bearing the unique LP-DC phenotype (CD11chighCD8αintCD11b lowαL lowβ7 high and CD11chighCD8α-CD11b highαL lowβ7 high) were abundant in wild-type mice, but were markedly fewer in CCL19-, CCL21-Ser-deficient plt/plt mice and were almost absent in CCR7-deficient mice, indicating the critical importance of CCR7 in LP-DC trafficking to MLNs. Interestingly, CCR7+ DCs in MLNs with the unique LP-DC phenotype had numerous vacuoles containing cellular debris in the cytoplasm, although MLN-DCs themselves were poorly phagocytic, suggesting that the debris was derived from the LP, where the LP-DCs ingested apoptotic intestinal epithelial cells (IECs). Consistent with this, LP-DCs ingested IECs vigorously in vitro. By presenting IEC-associated Ag, the LP-DCs also induce T cells to produce IL-4 and IL-10. Collectively, these results strongly suggest that LP-DCs with unique immunomodulatory activities migrate to MLNs in a CCR7-dependent manner to engage in the presentation of IEC-associated Ags acquired in the LP. Copyright © 2006 by The American Association of Immunologists, Inc.

Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9-23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 μg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9-23(C19S) in CFA. Nasal administration of as high as 50 μg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 μg of the insulin peptide alone or 5 μg of insulin peptide with 25 μg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B.choshinensis-derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases. © 2005 Wiley Periodicals, Inc.

Peptide immunotherapy using multiple predominant allergen-specific T cell epitopes is a safe and promising strategy for the control of type I allergy. In this study, we developed transgenic rice plants expressing mouse dominant T cell epitope peptides of Cry j I and Cry j II allergens of Japanese cedar pollen as a fusion protein with the soybean seed storage protein glycinin. Under the control of the rice seed storage protein glutelin GluB-1 promoter, the fusion protein was specifically expressed and accumulated in seeds at a level of 0.5% of the total seed protein. Oral feeding to mice of transgenic rice seeds expressing the T cell epitope peptides of Cry j I and Cry j II before systemic challenge with total protein of cedar pollen inhibited the development of allergen-specific serum IgE and IgG antibody and CD4+ T cell proliferative responses. The levels of allergen-specific CD4+ T cell-derived allergy-associated T helper 2 cytokine production of IL-A, IL-5, and IL-13 and histamine release in serum were significantly decreased. Moreover, the development of pollen-induced clinical symptoms was inhibited in our experimental sneezing mouse model. These results indicate the potential of transgenic rice seeds in production and mucosal delivery of allergen-specific T cell epitope peptides for the induction of oral tolerance to pollen allergens. © 2005 by The National Academy of Sciences of the USA.

As inductive tissues for the initiation of antigen-specific T and B cell responses, the various mucosa-associated lymphoid tissues (MALT) of the aerodigestive tract, which include gut-associated lymphoid tissue (GALT), nasopharynx-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT), share many histological and immunological characteristics. However, recent advances in our molecular and cellular understanding of immunological development have revealed that the various types of MALT also exhibit different molecular and cellular interactions for their organogenesis. In this review, we delineate the distinctive features of GALT, NALT and BALT and seek to show the role played by those features in the regulation of mucosal tissue organogenesis, the mucosal immune system, and mucosal homeostasis, all in an attempt to provide insights which might lead to a prospective mucosal vaccine.

Shiga toxin (Stx) derivatives, such as the Stx1 B subunit (StxB1), which mediates toxin binding to the membrane, and mutant Stx1 (mStx1), which is a nontoxic doubly mutated Stx1 harboring amino acid substitutions in the A subunit, possess adjuvant activity via the activation of dendritic cells (DCs). Our results showed that StxB1 and mStx1, but not native Stx1 (nStx1), resulted in enhanced expression of CD86, CD40, and major histocompatibility complex (MHC) class II molecules and, to some extent, also enhanced the expression of CD80 on bone marrow-derived DCs. StxB1-treated DCs exhibited an increase in tumor necrosis factor alpha and interleukin-12 (IL-12) production, a stimulation of DO11.10 T-cell proliferation, and the production of both Th1 and Th2 cytokines, including gamma interferon (IFN-γ), IL-4, IL-5, IL-6, and IL-10. When mice were given StxB1 subcutaneously, the levels of CD80, CD86, and CD40, as well as MHC class II expression by splenic DCs, were enhanced. The subcutaneous immunization of mice with ovalbumin (OVA) plus mStx1 or StxB1 induced high titers of OVA-specific immunoglobulin M (IgM), IgG1, and IgG2a in serum. OVA-specific CD4+ T cells isolated from mice immunized with OVA plus mStx1 or StxB1 produced IFN-γ, IL-4, IL-5, IL-6, and IL-10, indicating that mStx1 and StxB1 elicit both Th1- and Th2-type responses. Importantly, mice immunized subcutaneously with tetanus toxoid plus mStx1 or StxB1 were protected from a lethal challenge with tetanus toxin. These results suggest that nontoxic Stx derivatives, including both StxB1 and mStx1, could be effective adjuvants for the induction of mixed Th-type CD4+ T-cell-mediated antigen-specific antibody responses via the activation of DCs. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The mucosal immune system acts as a first line of defense against bacterial and viral infections while also playing a crucial role in the establishment and maintenance of mucosal homeostasis between the host and the outside environment. In addition to epithelial cells and antigen-presenting cells (dendritic cells and macrophages), B and T lymphocytes form a dynamic mucosal network for the induction and regulation of secretory IgA (S-IgA) and cytotoxic T lymphocyte (CTL) responses. This review seeks to shed light on the pathways of induction and regulation of these responses and to elucidate the role they simultaneously play in fending off pathogen invasion and maintaining mucosal homeostasis.

Epithelial cells of the ocular surface are key in the first-line defense as a part of the mucosal immune system against pathogens. We investigated whether polyI:C induces the production by human corneal epithelial cells (HCEC) of pro-inflammatory cytokines and IFN-β, and whether Toll-like receptor (TLR)-3 expression is amplified by polyI:C. TLR3 was expressed on the surface of HCEC. Stimulation with polyI:C elicited the elevated production and mRNA expression of IL-6 and IL-8 in HCEC. While polyI:C induced IFN-β, far stronger than human fibroblasts, and TLR3 gene expression in HCEC, LPS stimulation did not. Similarly, polyI:C, but not LPS, induced the gene expression of IκBα and MAIL, members of the IκB family, in HCEC. The innate immune response of HCEC is distinct from that of immune-competent cells, and we suggest that this is indicative of the symbiotic relationship between corneal epithelium and microbes inhabiting the ocular surface. © 2005 Elsevier Inc. All rights reserved.

Signaling by lymphotoxin (LT) and TNF is essential for the organogenesis of secondary lymphoid tissues in systemic and mucosal compartments. In this study, we demonstrated that the progeny of mice treated with fusion protein of LTβR and IgGFc (LTβR-Ig) or LTβR-Ig plus TNFR55-Ig (double Ig) showed significantly increased numbers of isolated lymphoid follicles (ILF) in the large intestine. Interestingly, double Ig treatment accelerated the maturation of large intestinal ILF. Three-week-old progeny of double Ig-treated mice showed increased numbers of ILF in the large intestine, but not in the small intestine. Furthermore, alteration of intestinal microflora by feeding of antibiotic water did not affect the increased numbers of ILF in the large intestine of double Ig-treated mice. Most interestingly, mice that developed numerous ILF also had increased levels of activation-induced cytidine deaminase expression and numbers of IgA-expressing cells in the lamina propria of the large intestine. Taken together, these results suggest that ILF formation in the large intestine is accelerated by blockage of LTβR and TNFR55 signals in utero, and ILF, like colonic patches, might play a role in the induction of IgA response in the large intestine. Copyright © 2005 by The American Association of Immunologists, Inc.

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botalinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infections Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 × 103 LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 μg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.

Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer's patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-β receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP- mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although γδ-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of γδ-IEL was found to take place in the intestinal epithelia of nu/nu LNP- and nu/nu aly/aly mice. Notably, the population size of a major CD8αα+ γδ-IEL subset was maintained, the use of TCR-γ-chain variable gene segments by these γδ-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP- and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of γδ-IEL in athymic nu/nu mice.

This chapter reviews the role of mucosal effector cells, including eosinophils, mast cells, and Th2-type cells, in the development of allergic reactions in the gastrointestinal tract. Mucosal eosinophils, mast cells, and T cells play a critical role in the development of gastrointestinal allergic reactions. They have also elucidated the complex cell-to-cell interactions involved in the induction and regulation of mucosal cell-mediated allergic diseases. Eosinophils are multifunctional leukocytes involved in the pathogenesis of allergic disorders. Aberrant eosinophil accumulation in the gastrointestinal tissues is often associated with serious clinical symptoms such as-gastric dysmotility, malabsorption, cachexia, or diarrhea. Eosinophils can potentially initiate antigen-specific immune responses by mimicking antigen-presenting cells. In this regard, eosinophils express major histocompatibility (MHC) class II and costimulatory molecules, such as CD40, CD80, and CD86. Mast cells are the main effector cells in the development of IgE-mediated allergic responses. The pathologic processes of mucosal allergic reactions are known to be mediated by T helper (Th)2-type cells, which preferentially produce IgE-enhancing cytokines such as interleukin (IL)-4 and IL-13. © 2005 Elsevier Inc. All rights reserved.

The gut is an essential organ for the intake of nutrients, and even primitive animals with primitive brains and nervous systems have a well-developed intestine. The intestinal mucosa-continuously invaded by many food-derived extrinsic antigens, allergens, and by toxins and pathogenic organisms-is the highest risk site in the body. Therefore, it is defended by the intestinal immune tissue composed of large numbers of immunocompetent cells that represent 60% of the entire peripheral lymphocyte population. The gut is not only a digestive and absorptive organ but also the most advanced immune organ in the body. The gut mucosal immune system has developed uniquely organized lymphoid tissues such as-the Peyer's patches (PP), the isolated lymphoid follicles (ILF), and the cryptopatches (CP)-to deliver both innate and acquired immunity. Among the numerous luminal antigens, commensal microbes constitute the largest antigenic load in the gut. Even in mice reared under specific pathogen-free conditions, large numbers of T and B cells in the intestinal mucosa remain constantly activated, and the germinal center (GC) formation occurs constantly in PP and ILF lumen. © 2005 Elsevier Inc. All rights reserved.

Background &amp Aims: Mice that have a truncated mutation of the common cytokine receptor γ chain (CRγ-/Y) are known to spontaneously develop colitis. To identify the pathologic elements responsible for triggering this localized inflammatory disease, we elucidated and characterized aberrant T cells and their enteropathogenic cytokines in CRγ-/Y mice with colitis. Methods: The histologic appearance, cell population, T-cell receptor Vβ usage, and cytokine production of lamina propria lymphocytes were assessed. CRγ-/Y mice were treated with anti-interleukin (IL)-6 receptor monoclonal antibody to evaluate its ability to control colitis, and splenic CD4+ T cells from the same mouse model were adoptively transferred into SCID mice to see if they spurred the appearance of colitis. Results: We found marked thickening of the large intestine, an increase in crypt depth, and infiltration of the colonic lamina propria and submucosa with mononuclear cells in the euthymic CRγ-/Y mice, but not in the athymic CRγ-/Y mice, starting at the age of 8 weeks. Colonic CD4+ T cells with high expressions of antiapoptotic Bcl-x and Bcl-2 were found to use selected subsets (Vβ14) of T-cell receptor and to exclusively produce IL-6. Treatment of CRγ-/Y mice with anti-IL-6 receptor monoclonal antibody prevented the formation of colitis via the induction of apoptosis in IL-6-producing CD4+ T cells. Adoptive transfer of pathologic CD4 + T cells induced colitis in the recipient SCID mice. Conclusions: Colonic IL-6-producing thymus-derived CD4+ T cells are responsible for the development of colitis in CRγ-/Y mice. © 2005 by the American Gastroenterological Association.

Both B subunit of Shiga toxin 1 (Stx1-B), which mediates the binding of toxin to the membrane, and mutant Stx1 (mStx1), which is a non-toxic double-mutated Stx1 harboring double amino acid substitutions in the A subunit, possess potent mucosal adjuvant activity. Nasal immunization of mice with ovalbumin (OVA) plus the Stx1-B or mStx1 induced OVA-specific serum IgG and mucosal IgA responses. IgG subclass analysis revealed that mStx1 and Stx1-B as mucosal adjuvants supported Ag-specific IgG1 followed by IgG2b Abs. The co-administration of either mStx1 or Stx1-B with OVA enhanced the production of IL-4, IL-5, IL-6 and IL-10 with low IFN-γ, by OVA-specific CD4+ T cells. To better elucidate the mechanisms underlying mStx1's and Stx1-B's adjuvant activity, we next sought to examine whether or not dendritic cells (DC) residing in the nasopharyngeal-associated lymphoreticular tissue (NALT) were activated by nasal administration of Stx1-B or mStx1. We found that mice nasally administered with Stx1-B or mStx1 showed an up-regulation in the expression of CD80, CD86 and especially CD40 on NALT DCs. Taken together, these results suggest that non-toxic Stx derivatives could be effective mucosal adjuvants for the induction of Th2-type, CD4+ T cell mediated, antigen-specific mucosal IgA and systemic IgG Ab responses, and that they likely owe their adjuvant activity to the up-regulation of co-stimulatory molecules including CD80, CD86 and CD40 on NALT DCs. © 2004 Elsevier Ltd. All rights reserved.

Secretory IgA plays a crucial role in the host immune response as a first line of defense. A recent demonstration of in situ IgA class switching in intestinal lamina propria provided an opportunity to reconsider the model for the homing of IgA-committed B cells characterized by distinctive trafficking patterns to effector sites. Those effector sites depend on the organized mucosa-associated lymphoid tissues as their site of induction. In this report we show the preferential presence of IgM+B220+ and IgA+B220+ cells belonging to pre- and post-IgA isotype class-switched cells in the organized mucosa-associated lymphoid tissues, such as nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer's patches, and the defect of those populations in the diffuse effector tissues, such as the nasal passage and intestinal lamina propria. Consistent with these findings, the expressions of a series of IgA isotype class switch recombination-related molecules, including activation-induced cytidine deaminase, Iα-Cμ circle transcripts, and Iα-Cμ circle transcripts, were selectively detected in these organized mucosa-associated lymphoid structures, but not in the diffuse mucosal effector sites. Taken together, these findings suggest that IgA isotype class switching occurs only in the organized mucosa-associated lymphoid organs (e.g., nasopharynx-associated lymphoid tissues, isolated lymphoid follicles, and Peyer's patches), but not in the diffuse effector tissues of the upper respiratory and gastrointestinal tracts.