清野 宏

Background: FTY720 is a novel reagent that possesses potent immunosuppressive activity. The immunosuppression induced by FTY720 is mediated by completely different mechanisms from those of conventional immunosuppressants, that is, by altering the tissue distribution of lymphocytes rather than inhibiting activation. In this study, we examined the efficacy of FTY720 in the treatment of chronic colitis in an interleukin-10 gene-deficient (IL-10-/-) mouse model. Methods: FTY720 was administered orally for 4 weeks to IL-10-/- mice with clinical signs of colitis. The gross and histologic appearance of the colon and the numbers, phenotype, cytokine production, and apoptosis of lymphocytes were compared with those characteristics in a control group. Results: Single-dose administration of FTY720 resulted in the sequestration of circulating lymphocytes within the secondary lymphoid tissues. Four-week administration resulted in a significant reduction of the CD4+ T lymphocytes subpopulation in the colonic lamina propria and IFN-γ production of the colonic lymphocytes, accompanied by a significant decrease in the severity of colitis. Conclusions: Treatment of established colitis in IL-10-/- mice with FTY720 ameliorated the colitis, probably as a result of decreasing the number of lymphocytes in the colonic mucosa and an associated reduction in IFN-γ production. Copyright © 2004 by Lippincott Williams &amp Wilkins.

CD3−CD4+CD45+ cells are thought to be an inducer cell population for Peyer's patch (PP) and lymph node development. Nasopharyngeal-associated lymphoid tissue (NALT) is an organized lymphoid structure found on both sides of the nasopharyngeal duct dorsal to the cartilaginous soft palate. Although NALT is considered to be functionally analogous to PP in the intestinal tract, the association of CD3−CD4+CD45+ cells with the initiation of NALT development has not yet been clarified. To examine this issue further, we isolated CD3−CD4+CD45+ cells from the fetal intestines of wild-type mice. Id2−/− mice genetically deficient in NALT were adoptively transferred with purified CD3−CD4+CD45+ cells or fetal liver cells. After the adoptive transfer, we employed immunohistochemical analysis to examine the expression of PNAd, an important addressin for the recruitment of lymphocytes, in the nasal tissue of Id2−/− mice transferred with fetal liver cells. Though NALT-like structures were indeed induced in Id2−/− mice adoptively transferred with CD3−CD4+CD45+ cells or fetal liver cells, those structures were not observed to express PNAd. These findings indicate that CD3−CD4+CD45+ cells and Id2 genes are key to the initiation of NALT. However, they also suggest that an additional element is required for the induction of PNAd. © 2003, Elsevier B.V.

As mucosa-associated lymphoid tissues, nasopharyngeal-associated lymphoreticular tissues (NALT) and Peyer's patches (PP) are key inductive tissues for the generation of antigen-specific mucosal immune responses via the respiratory and intestinal tracts, respectively. In this study, we have established that both the IL-7R-and LTβ receptor (LTβR)-mediated cytokine-signaling pathways are essential for the tissue organogenesis of PP by demonstrating that the blockage of the LTβR signaling cascade by in utero treatment with the LTβR-Ig fusion protein resulted in deficient PP formation. Further, in utero co-administration of LTβR-Ig with TNFR-Ig resulted in the lack of both PP and mesenteric lymph node (MLN) formation. Our findings also constitute new evidence that NALT requires its own set of cytokines and its own cytokine receptor cascade. In contrast to organogenesis in PP and in other secondary lymphoid tissues, NALT formation does not appear to be dependent on the IL-7R and LTβR signaling pathways, known as the essential cytokine cascade. Instead, a population of inducer cells with a phenotype of CD3−CD4+CD45+ was found to be responsible for the initiation of NALT organogenesis. These findings demonstrate that the lymphoid tissue organogenesis of the respiratory tract relies on a distinct set of cytokine/cytokine receptor-signaling cascades from organogenesis in the digestive tract. © 2003 Elsevier B.V.

Expression of a distant homologue MHC class I molecule, MHC class I-related chain A (MICA), has been found to be stress inducible and limited to the intestinal epithelium. This nonclassical MHC molecule is associated with various carcinomas in humans. To understand the biological consequences of MICA expression in the gut, we generated transgenic (Tg) mice (T3b-MICA Tg) under the control of the T3b promoter. The T3b-MICA Tg mice expressed MICA selectively in the intestine and had an increased number of TCRαβ CD4CD8αα, double-positive (DP) intraepithelial lymphocytes (IELs) in the small bowel. These MICA-expanded DP IELs exhibited a bias to Vβ8.2 and overlapped motifs of the complementarity-determining region 3 region among various Tg mice. Hence, the overexpression of MICA resulted in a clonal expansion of DP IELs. Studies in model of inflammatory bowel disease showed that transgenic MICA was able to attenuate the acute colitis induced by dextran sodium sulfate administration. Therefore, this unique in vivo model will enable investigation of possible influences of stress-inducible MICA on the gut immune surveillance.

Gut-associated lymphoreticular tissues, such as Peyer's patches and cecal patches, are important inductive sites for mucosal immune responses. As such, gut-associated lymphoreticular tissues may have an epithelial barrier different from that of villous epithelium. In this study, we investigated the immunohistochemical distribution of the claudin family and occludin in the follicle-associated epithelium (FAE) of Peyer's patches and cecal patches of murine intestine. Unique profiles of claudin-2, -3, and -4 and occludin expression were noted in the tight junctions of the FAE: claudin-4 was preferentially expressed in the apex region claudin-2 was only weakly expressed on the crypt side of the FAE compared with stronger expression on the crypt side of villous epithelial cells and claudin-3 and occludin were found throughout the dome. These unique expression patterns were present also in cecal patch FAE. We also found that claudin-4 expression in the FAE of Peyer's patches and cecal patches correlated with the presence of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)-positive apoptotic cells, and Peyer's patch-deficient mice exhibited expression patterns of claudin and occludin in villous epithelia similar to those in wild-type mice. We conclude that claudin-4 expression was preferentially associated with the dome region of FAE, the mucosal inductive site of the murine intestine. In that location it might correlate with the cell life cycle, help maintain the apex configuration of the dome, or be a factor favoring the uptake of antigens by the FAE.

Cholera toxin (CT), a powerful mucosal adjuvant, is a potent inducer of Th2-type responses via activation of co-stimulatory molecules for the induction of IgA antibody responses. Less appreciated is the ability of CT to induce and regulate cytotoxic T lymphocyte (CTL) responses. In order to help for clarifying mechanisms underlying the CTL-inducing ability of CT, we have examined the effects of CT on dendritic cells (DCs) that could lead to the induction of cytotoxic CD8+ T cells. When bone marrow-derived DCs (BM-DCs) were cultured with CT in vitro, B7-1 but not B7-2 molecules were significantly enhanced and allogenic CTL responses were induced. Also, increased numbers of IFN-γ-secreting CD8+ T cells were elicited when CT-treated BM-DCs were co-cultured with allogenic CD8+ CTLs. Antibody blockade of B7-1 on CT-treated BM-DCs suppressed allogenic CTL responses, further indicating the importance of CT-induced B7-1 molecules on DCs for the acquisition of cytolytic function by CTL precursors. CD40 signaling was proven not necessary for the CT-induced CTL response since CT-treated CD40-/- BM-DCs developed CTL responses equivalent to those detected in CT-treated BM-DCs derived from normal mice. Our results suggest that CT-treated DCs are effective inducers of CD8+ CTL, and this induction is mediated through CT's ability to enhance B7-1 expression on DCs. © 2002 Elsevier Science Ltd. All rights reserved.

Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3 resulted in inflammatory cytokine production and development of chronic enterocolitis with enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 deficiency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of Statl, which is essential for IFN-γ signaling pathways, Stat3 mutant mice developed chronic enterocolitis. TNF-α/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1 cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocolitis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1 responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Furthermore, enterocolitis was significantly improved and IFN-γ production by T cells was reduced in TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for the development of Th1-dependent enterocolitis.

Forbidden CD4+ββ T cells, which produce interleukin (IL)-4 predominantly, are a pathological subset in the development of colitis in T-cell receptor α chain (TCRα)-deficient mice. Stimulation of naive CD4+ T cells with IL-4 induces Th2 development via the activation of signal transducers and activators of transcription (STAT) 6. In the present study, we had found that IL-4 enhanced the expression of STAT6 in CD4+ββ T cells isolated from TCRα-/- mice with colitis, suggesting that the IL-4 signal in the CD4+ κκ T cells is mediated by STAT6. To further investigate the role of STAT6 in the development of colitis induced by TCRα deficiency, we generated double-deficient mice by crossing TCRα-/- mice and STAT6-/- mice. Surprisingly, STAT6 deficiency did not result in decreased severity of colitis in TCRα-/- mice. STAT6-deficient CD4+ ββ T cells produced IL-4 and intraperitoneal injection of anti-IL-4 monoclonal antibody in the nondiseased TCRα-/- and STAT6 double-deficient mice prevented the colitis formation, thus indicating that the cells differentiated into the Th2 phenotype have the ability to mediate the development of the colitis in the absence of STAT6.

In the vaccine strategy against HIV, the bacillus Calmette-Guerin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of the potential vectors for mucosal delivery of antigens. We analyzed the induction of antigen-specific antibodies by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3JI) that secretes the V3 principal neutralizing epitope of HIV [Proc. Natl. Acad. Sci. U. S. A. 92 (1063) 1995]. C57BL/6 mice were nasally immunized with the rBCG-V3J1 (10 mug) four times by weekly intervals. After 1 week of the final immunization, V3J1-specific IgG was seen in the serum. However, antigen-specific secretary and serum IgA antibodies were not induced. High levels of antigen-specific serum IgG responses were maintained for 6 months following the nasal immunization. Antigen-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinized mice. Furthermore, antigen-specific IgG antibodies induced by nasal immunization with rBCG-V3JI possessed the ability to neutralize HIV in vitro. Collectively, nasal immunization together with rBCG-V3J1 is considered to be a powerful vaccination regimen for the induction of effective V3J1-specific immune responses. We are currently investigating antigen-specific CTL activity and cytokine production following nasal vaccination with rBCG-V3J1. (C) 2003 Published by Elsevier B.V.

Nasal immunization of normal mice with HIVgp160-encapsulated hemagglutinating virus of Japan (HVJ)-liposome induced high titers of gp160-specific neutralizing IgG in serum and IgA in nasal wash, saliva, fecal extract, and vaginal wash, along with both Th1- and Th2-type responses. HIVgp160-specific IgG- and IgA-producing cells were also detected in mononuclear cells isolated from spleen, nasal cavity, salivary gland, intestinal lamina propria, and vaginal tissue of nasally immunized mice. In addition, CD8(+) CTLs were induced in mice nasally immunized with gp160-HVJ-Iiposome. These findings suggest that two layers of effective HIV-specific Immoral and cellular immunity, in mucosal and systemic sites, were induced by this nasal vaccine. In immunodeficient mice, nasal immunization with gp160-HVJ-Iiposome induced Ag-specific immune responses for the systemic and mucosal compartments of both Th1 (IFN-gamma(-/-)) and Th2 (IL-4(-/-)). In vitro Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and IgG Abs that had been induced by nasal immunization with gp160-HVJ-Iiposome were able to neutralize a clinically isolated strain of HIV-MN strain isolated from Japanese hemophiliac patients. Taken together, these results suggest that, for the prevention and control of AIDS, nasally administered gp160-HVJ-Iiposome is a powerful immunization tool that induces necessary Ag-specific immune responses at different stages of HIV infection.

The development of a successful recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vector-based vaccine for human immunodeficiency virus type 1 (HIV-1) requires the induction of high levels of HIV-1-specific immunity while at the same time maintaining immunity to tuberculosis. To examine a combined vaccination strategy for enhancement of immune responses specific for HIV-1, guinea pigs were inoculated with either a single or combination intradermal (i.d.), intrarectal (i.r.) and intranasal (i.n.) administration of rBCG-pSOV3J1 which secretes a chimeric protein of HIV-1 V3J1 peptide and α-antigen. Significant level of delayed-type hypersensitivity to both V3J1 peptide and tuberculin was induced in guinea pigs inoculated with human doses of rBCG-pSOV3J1 by a combination of intrarectal and intradermal routes. Guinea pigs inoculated by combined routes also had significantly higher titers of HIV-1-specific serum IgG and IgA compared with those animals immunized only intrarectally, which led to the enhanced neutralization activity against HIV-1MN. In addition, the induction of high levels of IFNγ and interleukin-2 (IL-2) mRNA in PBMC, splenocytes, and intraepithelial lymphocytes from the immunized animals was detected until at least 110 weeks post-inoculation. These results suggest that enhanced immune responses specific for HIV-1 are efficiently induced by combined intrarectal and intradermal immunization with rBCG-HIV, and antigen-specific Th1-type memory cells are maintained for more than 2 years in the immunized animals. Thus, inoculation with rBCG-HIV by combined routes represents an effective vaccination strategy to elicit high levels of HIV-1-specific immune responses. © 2002 Elsevier Science Ltd. All rights reserved.

Immune responses induced by a nasal influenza vaccine with a mutant cholera toxin (CT112K), known to be a safe adjuvant, were characterized in BALB/c mice to confirm the most suitable regimen of this vaccine for humans. Mice received a primary intranasal administration of the adjuvant (0.1 mug)-combined PR8 vaccine (0.1 mug) and a secondary administration of the PR8 vaccine alone (0.1 mug) 4 weeks later. Two weeks after the secondary immunization, the mice were infected with a nonlethal or a lethal dose of PR8 viruses. Nasal and lung wash virus titers 1 or 3 days after infection indicated that complete protection could be provided by secondary immune responses, which had an immediate effect of preventing infection 2 weeks after the secondary immunization. In this two-dose regimen, high levels of secondary IgA, IgG and IgM antibody-forming cell (AFC) responses were induced in the nasal-associated lymphoid tissue and the spleen. In parallel with the AFC responses, high levels of nasal wash anti-PR8 HA IgA, and lung and serum IgG antibody (Ab) responses were induced 2 weeks after the secondary immunization. The two-dose regimen also induced accelerated delayed-type hypersensitivity responses, which exhibited almost the same peak height as that in the case of the primary response. In addition, the two-dose regimen induced a low memory cell activity of cytotoxic T lymphocytes, detected by in vitro culture of spleen cells. Thus, the immediate effect of preventing infection was mainly provided by the secondary Ab responses. Moreover, the levels of nasal wash IgA Abs correlated well with cross-protection against infection with variant viruses in the upper respiratory tract (RT). These results suggest that the major protective factors among Ab and T cell-mediated immune responses, which are induced by the two-dose regimen using CT112K-combined vaccines, are the cross-reactive IgA Abs in the upper RT and the less cross-reactive IgG Abs in the lower RT, and that the two-dose regimen is a suitable vaccination condition for humans. (C) 2002 Elsevier Science Ltd. All rights reserved.

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO2-/NO3-, a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium. (C) 2002 Elsevier Science (USA). All rights reserved.

In some cases of atopic dermatitis (AD), a possible pathological contribution to disease development by Candida albicans (C. albicans) has been suggested. AD patients with severe symptoms showing positive capsulated hydrolic carrier polymer radioallergosorbent test (CAP-RAST) against C. albicans demonstrated significantly higher levels of serum IgE Abs than did AD patients with mild symptoms. Based on the clinical facts, we have postulated that elimination of C. albicans by mucosal vaccination may lead to the restoration of severe symptoms in AD patients. For this purpose, we have developed an allergic murine model. Mice which were systemically challenged with C. albicans-associated antigen, manganese superoxide dismutase (MnSOD) or secreted aspartic proteases 2 (SAP2), together with alum, exhibited hyper IgE Abs. Systemically primed mice were then immunized with MnSOD or SAP2 plus cholera toxin (CT) as mucosal adjuvant through the nasal route. Interestingly, nasally immunized mice showed increased levels of Candida Ag-specific IgA Ab in fecal and nasal washes as well as in saliva samples but unchanged levels in Ag-specific IgE responses. Consistent with the Ab levels, high numbers of Candida Ag-specific IgA Ab-forming cells were induced in mononuclear cells isolated from intestinal lamina propria, nasal passages and salivary glands of nasally vaccinated mice with Ag plus CT. Furthermore, nasal immunization using MnSOD or SAP2 together with CT resulted in the elimination of colonized C. albicans from the intestinal tract. These results also suggest a potential role of mucosal vaccination in the control of C. albicans in patients with allergic diseases, including AD, although more research is needed to establish this therapeutic approach for mucosal vaccination. © 2002 Elsevier Science Ltd. All rights reserved.

To clarify the role of IL-15 at local sites, we engineered a transgenic (Tg) mouse (T3b-IL-15 Tg) to overexpress human IL-15 preferentially in intestinal epithelial cells by the use of T3b-promoter. Although IL-15 was expressed in the entire small intestine (SI) and large intestines of the Tg mice, localized inflammation developed in the proximal SI only. Histopathologic study revealed reduced villus length, marked infiltration of lymphocytes, and vacuolar degeneration of the villus epithelium, beginning at ∼3-4 mo of age. The numbers of CD8+ T cells, especially CD8αβ+ T cells expressing NK1.1, were dramatically increased in the lamina propria of the involved SI. The severity of inflammation corresponded to increased numbers of CD8αβ+NK1.1+ T cells and levels of production of the Thl-type cytokines IFN-γ and TNF-α. Locally overexpressed IL-15 was accompanied by increased resistance of CD8αβ+ NK1.1+ T cells to activation-induced cell death. Our results suggest that chronic inflammation in the SI in this murine model is mediated by dysregulation of epithelial cell-derived IL-15. The model may contribute to understanding the role of CD8+ T cells in human Crohn's disease involving the SI.

最近，IL-15は粘膜免疫関連サイトカインとして注目されている．そこで本総説では，最初に，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証拠を示す．腸管のB-1細胞にはIL-15レセプターが優先的に発現している．加えてIL-15は2つの異なった状態（sIgM⁻ sIgA⁺ and sIgM⁺ sIgA⁻）にあるIgA免疫応答誘導循環帰巣経路独立型のB-1細胞に働くのに対して，IL-5はsIgM⁻ sIgA⁺の状態のB-1細胞にのみ働く．一方，循環帰巣経路依存型のB-2細胞はIL-5とIL-6のレセプターを発現しておりIL-5とIL-6の作用でIgA産生細胞に分化する．次に，我々はマウスの腸管上皮細胞特異的にIL-15を強制発現させることによって小腸炎症モデルを確立した例を示す．このIL-15トランスジェニックマウスの中でIL-15によって誘導されたCD8αβ⁺ NK1.１⁺細胞は小腸炎症の発症に重要である．さらにこのT細胞はTh1タイプのサイトカインを産生しており，このT細胞の選択的な増加はIL-15の抗アポトーシス活性に帰するのであろう．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，その腸管免疫で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．<br>

Background and aims: Enteric bacterial and/or food antigens may be crucial in the development of colitis but little is known of the exact mechanism of antigen specific reactions in this condition. The aim of this study was to determine whether systemically primed antigen specific CD4(+) T cells containing both CD45RB(high) and CD45RB(low) populations participate as a pathogenic subset that in turn leads to inflammatory reactions selectively in the large intestine. Methods: SCID mice were reconstituted with splenic CD4(+) CD45RB(+) T cells or CD4(+) CD45RB(low) T cells isolated from donor mice systemically primed with ovalbumin (OVA) plus CFA. The reconstituted mice were then fed OVA for several weeks. Results: Reconstitution of SCID mice with OVA primed splenic CD4(+) T cells, containing populations of CD45RB(high) and CD45RB(low), resulted in the development of colitis by 4-5 weeks following repeated administration of oral OVA. Histopathological study revealed thickened wall, inflammatory cell infiltration, crypt elongation, and loss of goblet cells in the large intestine. The CD4(+) CD45RB(low) population of cells extracted from the affected large intestine secreted high levels of interferon gamma (IFN-gamma) and tumour necrosis factor a (TNF-alpha) at the protein and mRNA levels. Administration of neutralising antibodies to TNF-alpha, but not to IFN-gamma prevented the development of colitis. Furthermore, adoptive transfer with OVA, primed splenic CD4(+) CD45RB(low) T cells evoked severe colitis. Conclusions: These results demonstrate that systemically primed activated/memory CD4(+) CD45RB(low) T cells can mediate the development of specific antigen induced colitis in SCID mice, and also that TNF-alpha is critical in the induction of this type of colitis. Our results contrast with those from studies in some colitis models in which CD45RB(low) T cells appeared to prevent colitis through secretion of immunosuppressive cytokines.

An effective vaccine against sexual transmission of human immunodeficiency virus (HIV) should elicit both systemic and mucosal immune responses. In this study, to examine the possibility of using an attenuated virus for mucosal immunization, four female macaques were intranasally or intravenously administered with a chimeric simian-human immunodeficiency virus with a deleted nef gene (SHIV-dn). Although all the monkeys had anti-HIV-1 antibodies with neutralizing activity in the plasma, the intranasally immunized monkeys had much higher levels of HIV-1 Env-specific IgG and IgA antibodies in mucosal secretions compared with the intravenously immunized monkeys. Moreover, three of four intranasally immunized monkeys were completely protected from intravaginal challenge with a pathogenic virus, SHIV-89.6P, whereas only one intravenously immunized monkey was protected. Thus, intranasal immunization of an attenuated virus can induce the protective efficacy against intravaginal infection. © 2002 Elsevier Science (USA).

Initiation of nasopharyngeal-associated lymphoid tissue (NALT) development is independent of the programmed cytokine cascade necessary for the formation of Peyer's patches (PP) and peripheral lymph nodes (PLN), a cytokine cascade which consists of IL-7R, LTα1β2/LTβR, and NIK. However, the subsequent organization of NALT seems to be controlled by these cytokine signaling cascades since the maturation of NALT structure is generally incomplete in those cytokine cascade-deficient mice. NALT as well as PP and PLN are completely absent in Id2-/- mice. NALT organogenesis is initiated following the adoptive transfer of CD3-CD4+CD45+ cells into Id2-/- mice, constituting direct evidence that CD3-CD4+CD45+ inducer cells can provide an IL-7R-, LTα1β2/LTβR-, and NIK-independent tissue organogenesis pathway for secondary lymphoid tissue development.

In the vaccine strategy against HIV, bacillus Calmette-Guerin (BCG), a live attenuated strain of Mycobacterium bovis, is considered to be one of potential vectors for mucosal delivery of vaccine Ag. We analyzed the induction of the Ag-specific Ab response by nasal immunization with recombinant BCG vector-based vaccine (rBCG-V3J1) that can secrete the V3 principal neutralizing epitope of HIV. Alice were nasally immunized with rBCG-V3J1 (10 mug) three times at weekly intervals. Four weeks after the initial immunization, high titers of V3J1-specific IgG Abs were seen in serum. These high levels of HIV-specific serum IgG responses were maintained for &gt; 12 mo following nasal immunization without any booster immunization. V3J1-specific IgG-producing cells were detected in mononuclear cells isolated from spleen, nasal cavity, and salivary gland of the nasally vaccinated mice. Nasal rBCG-V3J1 also induced high levels of prolonged HIV-specific serum IgG responses in Th1 (IFN-gamma (-/-))- or Th2 (IL-4(-/-))-immunodeficient mice. Further, IgG3 was highest among V3 peptide-specific IgG subclass Ab responses in these immunodeficient mice as well as in wild-type mice. In addition, this Ag-specific serum IgG Abs induced by nasal immunization with rBCG-V3J1 possessed the ability to neutralize clinical isolate of HIV in vitro. These results suggested that the nasal rBCG-V3J1 system might be used as a therapeutic vaccine in addition to a prophylaxis vaccine for the control of AIDS.

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, elicits mucosal adjuvant activities by inducing antigen-specific CD4+ T cells secreting T helper type 2 (Th2) cytokines. Experimental autoimmune encephalomyelitis (EAE) is induced by Th1 cells specific for myelin-derived antigens. We induced EAE in C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55 and CT was nasally administered as an immunomodulator on day 7 following MOG challenge. Clinical severity in the CT-treated mice was milder when compared to PBS-treated mice, while the levels of expression of interleukin (IL)-12 and interferon (IFN)-γ in the central nervous system (CNS) of CT-treated mice were lower than PBS-treated mice. Thus, nasal administration of the mucosal immunomodulator CT ameliorated the severity of EAE, which was associated with the suppression of Th1 cell responses. © 2001 Elsevier Science Ltd. All rights reserved.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis in humans. EAE can be passively transferred into naive syngeneic animals by administration of MOG-specific T cell clones. Lymphocytes isolated from green fluorescent protein (GFP) - transgenic (Tg) mice can light up by emitting green fluorescence, thus making it feasible to use such animals in a passive transfer model for EAE. When MOG-sensitized splenic lymphocytes from GFP-Tg mice were adoptively transferred to irradiated, syngeneic C57BL/6 and RAG-1-/- mice, typical symptoms of EAE developed. Analysis of the reconstituted mice with EAE revealed prominent infiltration of fluorescing (GFP+), CD4+ T cells into the central nervous system (CNS). Real-time confocal imaging revealed these cells in the spinal cords and brains of recipient mice. This infiltration was also confirmed by anti-GFP monodonal antibodies. Furthermore, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation indicated that the infiltrating GFP+, CD4+ T cells exclusively produced T helper type 1 (Th1) cytokines, especially interferon-γ (IFN-γ). These results clearly show that MOG-specific CD4+ T cells preferentially invade into the CNS and mediate the development of EAE by producing Th1-biased cytokines. © 2001 Academic Press.

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4+ Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.

Pro-inflammatory cytokines have been shown to be involved in the genesis, persistence, and severity of neuropathic pain following nerve injury. The transcription factor, nuclear factor-kappa B (NF-kappaB), plays a pivotal role in regulating pro-inflammatory cytokine gene expression. To elucidate the role of NF-kappaB in the pathogenesis of neuropathic pain, using a gene-based approach of NF-kappaB decoy, we tested whether the activated NF-kappaB affected pain behavior via the expression of inflammatory mediators. Single endoneurial injections of NF-kappaB decoy, at the site of nerve lesion, significantly alleviated thermal hyperalgesia for up to 2 weeks and suppressed the expression of mRNA of the inflammatory cytokines, iNOS, and adhesion molecules at the site of nerve injury. This finding suggests that a perineural inflammatory cascade, that involves NF-kappaB, is involved in the pathogenesis of neuropathic pain.

The changes in T cell receptor (TCR) Vβ expression, use, and clonality in mice orally challenged with Escherichia coli heat-labile enterotoxin (LT) were assessed. Use of the TCR Vβ family and clonality were significantly changed at the single-cell level. In Peyer's patches of treated mice, use of TCR Vβ6, Vβ8, and Vβ14 increased in CD4+CD44+ T cells, compared with use in nontreated mice. On the other hand, use of TCR Vβ1 and Vβ8 was enhanced in splenic CD4+CD44+ T cells. Intraepithelial lymphocytes isolated from LT-challenged mice showed expanded clonality (e.g., Vβ1, Vβ2, Vβ9, and Vβ18) and altered TCR Vβ use (e.g., Vβ15, Vβ16, and Vβ17). These findings reveal that oral administration of LT has distinct effects on mucosal versus systemic αβ T cells for induction of CD4+ T cells with selected Vβ use. This most likely reflects the function of LT as a mucosal modulator.

The mucosal immune system is a first line of defense against foreign antigens, including microbial and dietary antigens. Under normal circumstances, the mucosal immune system employs tightly regulated dynamic mucosal intra- and internets consisting of inductive and effector sites for the induction of an appropriate immunological homeostasis between the host and mucosal environments. The common mucosal immune system (CMIS), which interconnects between inductive (e.g. Peyer patch) and effector (e.g. intestinal lamina propria) tissues for the induction of the IgA response, is well characterised. Recent results provide strong evidence for the presence of a CMIS-independent IgA induction pathway. Two distinct subsets of mucosal IgA-committed B cells, termed B-1 and B-2, are associated with CMIS-independent and CMIS-dependent cascades, respectively. In some cases, the breakdown of this tightly regulated mucosal immune system leads to pathological responses to different gut environmental antigens. As a result, disorders such as inflammatory bowel disease (e.g. IBD) and allergic gastroenteropathy can be evoked in the gastrointestinal tissues. Recently, many studies have described possible molecular and cellular mechanisms for this dysfunction in the gastrointestinal tissues by using murine models with specific gene manipulation. In this review, we summarise recent findings from our group concerning the CMIS-dependent and CMIS-independent IgA induction pathways and gastrointestinal diseases (IBD and intestinal allergic diseases). These observations may provide useful information for the development of new mucosal immune therapy.

OBJECTIVE: To investigate, in a rat model, the role of the Mac-1/ICAM-1 pathway and the anti-inflammatory activity of steroid in ventilator-induced lung injury. DESIGN: Prospective, randomized controlled study. SETTING: Animal investigation using Wistar rats. INTERVENTION: Rats in three randomly assigned groups of 18, a total of 54 animals, were subject to the following: Two groups received high peak inspiratory pressure (35 cm H2O) ventilation after pretreatment with methylprednisolone (high-methylprednisolone group) or pretreatment with methylprednisolone vehicle (high-vehicle group). The third group of animals received low peak inspiratory pressure (7 cm H2O) ventilation after pretreatment with methylprednisolone vehicle (low-vehicle group). Except for animals previously killed to establish baseline values, after 40 mins of mechanical ventilation, the animals in each group were killed. Some animals provided histological samples, and the rest received total lung lavage. MEASUREMENT: We measured flow cytometry of lavage fluid, cell counts of tissue samples, and pressure-volume curves before and after mechanical ventilation. RESULTS: In the groups that received high peak inspiratory pressure ventilation, both the number of neutrophils that infiltrated the lungs and the expression of Mac-1 and ICAM-1 on neutrophils and macrophages increased significantly more than in the low-vehicle group. Static lung compliance was reduced in the high peak inspiratory pressure groups. In the high peak inspiratory pressure groups, there were significantly fewer neutrophils in samples from the high-methylprednisolone group (0.412 +/- 0.1 x 10(5)) than from the high-vehicle group (1.10 +/- 0.1 x 10(5); p < .05). The high-vehicle group showed greater expression of CD11b on neutrophils, but this was significantly decreased by methylprednisolone (mean fluorescence intensity: high-vehicle, 118.4 +/- 34.3; high-methylprednisolone, 25.8 +/- 4.2; p < .05). The lung mechanics measured by pressure-volume curve analysis were deteriorated less in the high-methylprednisolone group. CONCLUSION: Our study suggests that a neutrophil-endothelium interaction via the Mac-1/ICAM-1 pathway is involved in the activation and recruitment of neutrophils in ventilator-induced lung injury. Activation and recruitment of neutrophils were lessened by pretreatment with methylprednisolone, which might have contributed to the improvement of lung dysfunction after mechanical ventilation.

The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.

Activated neutrophils contribute to the development of ventilator-induced lung injury (VILI) caused by high-pressure mechanical ventilation. However, exact cellular and molecular mechanisms have not been conclusively studied. Our investigation aimed to examine expression of adhesion molecules by both neutrophils and macrophages in lung lavage fluids of rats with VILI. Further, involvement of proinflammatory (tumor necrosis factor-alpha) and profibrogenetic (transforming growth factor-beta 1) mediators was analyzed at mRNA level in lung tissue. Wistar rats were ventilated by high pressure (45 cm H(2)O of peak inspiratory pressure, n = 23) or low pressure (7 cm H(2)O, n = 13) with 0 positive end-expiratory pressure. After 40 min of comparative ventilation, lung lavage was performed in 20 rats from the experimental group and 10 from the control for immunofluorescence analysis with anti-Mac-1 and anti-ICAM-1 monoclonal antibodies. The lung tissues from remaining rats were subjected to pathological and reverse transcription-polymerase chain reaction examinations. Although there was no significant change of PaO(2) in the low-pressure group, PaO(2) was decreased in the high-pressure group. The high-pressure group also had greater neutrophil infiltration into alveolar spaces, upregulation of CD54 and CD11b on alveolar macrophages, and more transforming growth factor-beta 1 mRNA in lung tissues. Tumor necrosis factor-alpha was not involved in the pathogenesis of the severe VILI observed. Histologic findings also demonstrated more infiltrating neutrophils, destructive change of the alveolar wall, and deposition of matrix in the high-pressure group. These results suggest that a series of proinflammatory reactions and profibrogenetic process may be involved in the course of VILI.

The gastrointestinal immune system is a major component of the mucosal barrier to foreign antigens including microbial and dietary antigens. Under normal circumstances, the mucosal immune system employs tightly regulated dynamic mucosal intra- and internets for the maintenance of an appropriate immunological balance between the host and gut environments. For example, mucosally induced tolerance is usually induced against enteric commensal bacterial and/or dietery antigens. However, in some cases, the break down of these tightly regulated mucosal immune responses led to hyper-responsiveness against these gut environmental antigens. To this end, numerous disorders could evoke in the gastrointestinal tissues, such as inflammatory bowel disease (IBD) and allergic gastroenteropathy. Recently, many studies have described possible molecular and cellular mechanisms of those dysfunctions in the gastrointestinal tissues. In this review, we have briefly summarized some of the current interesting and exciting findings for gastrointestinal diseases, especially IBD and intestinal allergic diseases from our group, which can be applied for the development of new mucosal immune therapy. Copyright © 2001 S. Karger AG, Basel.

We tested the possibility that heat-labile enterotoxin of Escherichia coli (LT) affects the development of extrathymic T cells in the intraepithelial lymphocyte (IEL) compartment. After oral administration of LT, the number of extrathymic CD8αα+ IEL was selectively and significantly diminished when compared with the corresponding cells in phosphate-buffered saline-fed control mice. To clarify the mechanism behind this selective reduction of CD8αα+ IEL, we analyzed the expression of essential cytokines and their corresponding receptors for the mucosal intranet formed by intestinal epithelial cells (IEC) and IEL. The expression levels of stem cell factor, interleukin (IL)-7, and IL-15 in IEC, and their corresponding receptors, i.e. c-kit, IL-7 receptor, and IL-15 receptor, in CD8αα+ IEL were reduced following oral feeding with LT. These findings suggest that LT negatively regulates development of CD8αα+ IEL via the disruption of mucosal intranet-associated cytokine and cytokine receptors, which are required for the development and/or expansion of extrathymically developed T cells. Further, LT-induced destruction of the mucosal intranet resulted in the impairment of IEC generation via an increase of apoptosis.

Determination of cytotoxic T lymphocyte (CTL) responses in humans by traditional assays have been restricted by the volumes of blood needed and the nature of the target cells used for the assays. A number of technological advances have recently been described which improve quantitation and sensitivity of the assays. We are developing the concept of using fibroblasts as permissive targets for respiratory viruses in CTL assays. Methods: Fibroblasts can be grown from dispersed nasal epithelium obtained from adenoids. These cells infected with influenza are used as a stimulator layer for lymphocytes. They also can form the target for CTLs in a chromium release assay. Results: Comparison showed fibroblasts to be comparable to influenza infected lymphocytes as stimulators and to EBV transformed lymphocytes as targets. Cells from 25 adenoids have been explored in the fibroblast CTL system with about half of the cryoperserved. peripheral blood lymphocytes showing CTL activity and mucosally associated CTLs being found in one patient. Conclusions: Exploration of new CTL targets that may be adapted not only to chromium release assays but to newer CTL assays offers the promise of developing a practical way of defining the CTL response in young children to natural infection and to varying immunization strategies. This may allow us to put in perspective CTLs as a correlate of immunity to influenza in humans. (C) 2001 Elsevier Science B.V. All rights reserved.