清野 宏

A major pathogenic factor for the development of inflammatory bowel disease (IBD) is the breakdown of the intestinal homeostasis between the host immune system and the luminal microenvironment. To assess the potential influence of luminal Ags on the development of IBD, we fed TCR α(-/-) mice an elemental diet (ED). ED-fed TCR α(-/-) mice showed no pathologic features of IBD, and their aberrant mucosal B cell responses were suppressed. Similar numbers of CD4+, TCR ββ homodimer T cells (ββ T cells) were developed in the colonic mucosa of ED-fed mice however, Th2-type cytokine productions were lower than those seen in diseased regular diet (RD)-fed mice. The higher cytokine production in diseased RD-fed mice could be attributed to the high incidence of Bacteroides vulgatus (recovered in 80% of these mice), which can induce Th2-type responses of colonic CD4+, ββ T cells. In contrast, ED-fed TCR α(-/-) mice exhibited a diversification of Vβ usage of ββT cell populations from the dominant Vβ8 one associated with B. vulgatus in cecal flora to Vβ6, Vβ11, and Vβ14. Rectal administration of disease-free ED-fed mice with B. vulgatus resulted in the development of Th2-type CD4+, ββ T cell-induced colitis. These findings suggest that the ED-induced alteration of intestinal microenvironments such as the enteric flora prevented the development of IBD in TCR α(-/-) mice via the immunologic quiescence of CD4+, ββ T cells.

We show in this report a new regulatory role for IL-15 and IL-15R in the development of B-1 cells and their differentiation into IgA-producing cells. Mucosal IgA levels were found to be inhibited by anti-IL-15 mAb treatment in vivo, but enhanced by administration of rIL-15, while serum IgA levels remained unaffected. Mucosal B-1 cells preferentially proliferated in response to IL-15 in vitro. When mucosal B-1 and B-2 cells were separated into surface (s)IgM+sIgA- and sIgM-sIgA+ fractions, IL-15R-specific mRNA was found to be predominant in both sIgM+sIgA- and sIgM-sIgA+ B-1 cells at a much higher level than B-2 cells. Further, incubation of these different subsets of B-1 and B-2 cells with IL-15 resulted in greater enhancement of the corresponding receptor expression by B-1 subset when compared with B-2 fraction. Interestingly, de novo isolated sIgM+sIgA- B-1, but not sIgM+sIgA- B-2, cells were already class-switched cells because the germline Cα transcript was detected and was then further enhanced by IL-15. IL-15 also supported differentiation of both sIgM+sIgA- and sIgM-sIgA+ B-1 cells into IgA-producing cells. Taken together, these findings suggest that IL-15 is a critically important cytokine for the differentiation of both sIgM+,IgA- and sIgM-sIgA+ B-1 cells expressing IL-15R into IgA-producing cells in mucosal tissues.

In the present study, mucoadhesive polymer-dispersed microspheres (MS) were examined as a potential mucosal vaccine carrier. A major focus of the study was aimed at directly assessing the influence of antigen release and persistence in the mouse small intestine for the induction of mucosal and systemic immune responses. BALB/c mice were immunized with various forms of MS containing chicken egg ovalbumin (OVA) by administration into the duodenum. No detectable anti-OVA immune responses were observed following the administration of OVA alone or that of MS without mucoadhesive polymer (MS- 0). MS-10 containing 10% mucoadhesive polymer rapidly released OVA and hardly induced anti-OVA antibody responses in either serum or fecal extracts. In contrast, MS-8 and MS-6 (with 8 and 6% mucoadhesive polymer) showed controlled release of OVA, which elicited strong OVA-specific IgG and IgA responses in serum and fecal extracts, respectively. Additionally, the strongest immune responses were induced in mice immunized with MS-8, which had both the optimal release-profile of OVA and the longest persistence in the small intestine. These findings indicate that antigen movement in the small intestine is an important factor and that appropriate microsphere forms with mucoadhesive polymers might be useful candidates as mucosal vaccine carriers. (C) 2000 Elsevier Science Ltd.

Systemically primed BALB/c mice developed severe diarrhea after repeated oral administration of ovalbumin (OVA). Histological analysis demonstrated that dramatic infiltration of eosinophils and mast cells occurred in the large intestine but not in the small intestine of mice with diarrhea. Interestingly, CD4(+) alpha beta T cells of the large intestine secreted IL-4 and IL-13 at high levels. Identically treated STAT6 gene-disrupted mice failed to develop OVA-induced diarrhea. Further, treatment of BALB/c mice with monoclonal anti-IL-4 antibody prevented the development of allergic diarrhea. An adoptive transfer study showed that systemically primed splenic CD4(+) T cells were preferentially recruited into the large intestine upon exposure to oral OVA. These results strongly suggest that systemically derived CD4(+) alpha beta T cells of the large intestine play a critical role in the onset of Th2-mediated intestinal allergic disorders via STAT6 signal transduction.

The purpose of the present study was to determine the immunologic responses, particularly immunopathologic reactions, associated with nasal immunization with the mucosal adjuvant, cholera toxin (CT). BALB/c mice were nasally immunized with tetanus toxoid (TT) combined with CT, and the responses of these mice were determined. After nasal immunization, mice produce a serum antibody response, primarily of the immunoglobulin G (IGG) isotype of predominantly IgG1 subclass, against both TT and CT. Along with the antibody responses, we also found that inflammatory reactions, which could be potentially fatal, developed within the lung. Furthermore, IgE responses were also induced after nasal immunization, and these responses were associated with the detection of interleukin 5 in the serum. Thus, nasal immunization with TT plus CT likely results in the activation of Th2 cells, which may contribute to serious immunopathologic reactions in the lung.

PURPOSE. Immunologic characterization of IgA-committed B-1 and B-2 cells, and unique subsets of T cells isolated from the murine lacrimal gland (LG), the primary exocrine tissue for the ocular surface, which is considered to be a part of the mucosal immune system. METHODS. Single cells were obtained from LGs of C57BL/6 mice by the enzyme dissociation method using collagenase type IV. Samples underwent flow cytometric analysis to characterize the unique subsets of T and B cells. To test the effectiveness of ocular vaccination, mice were immunized ocularly or nasally with cholera toxin (CT 10 μg/mouse) suspended in phosphate-buffered saline. Antigen- specific immune responses were determined by isotype and CT-specific enzyme- linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. RESULTS. When mononuclear cells (MC) isolated from LG samples were examined by flow cytometry, approximately 28% of cells were characterized as B220+ B cells. Because surface IgA+ (sIgA+) B cells develop from B-1 and B-2 lineages, it was important to examine which subset of B cells gives rise to LG sIgA+ B cells. Examination of the MC isolated from LG samples showed that approximately 4% of cells were sIgA+ B cells. Furthermore, nearly all these sIgA+ B cells (97.5%) belonged to the B-1 lineage, especially the B-1a cell line (B220(low), CD5+). Of the isolated CD3+ T cells, 75% were αβ and 25% were γδ T-cell receptor positive. The proportion of NK1.1+ αβ T cells was higher (3%) in LG samples than in sub-mandibular gland samples (0.5%). Ocular immunization with CT-induced antigen-specific mucosal (e.g., found in tear-wash and saliva samples) and systemic (e.g., serum) immune responses. The magnitude of antigen-specific antibody responses was comparable to those induced by nasal immunization. CONCLUSIONS. These results show that LG contains unique subsets of B (e.g., sIgA+ B-1 cells) and T (e.g., NK1.1+ αβ T cells) cells. Furthermore, as a part of the mucosal immune barrier, the LG is an important immunologic tissue for the ocular surface.

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The host is continuously exposed to the environment via the mucosal surface. To this end, a large number of infectious agents, allergens and foreign proteins enter the inside of our bodies via the oral region, nasal and upper respiratory tracts, intestine and reproductive tract. The total area of these mucosal surfaces, which cover these tube-like tissues are at least two hundred times larger than those of skin. To provide an optimal first line of defense for these large surface areas, the mucosal immune system including secretory immunoglobulin A (S-IgA), mucosal αβ and γδ T-cells and epithelium play an essential role. The goal of our research is aimed at understanding the molecular and cellular aspects of the mucosal immune system and their defense against infectious diseases, inflammation and immunological disorders. Further, it is important to quickly apply our fundamental findings of the mucosal immune system to the development of mucosal vaccines.

T cell receptor alpha chain-deficient (TCR-alpha(-/-)) mice are known to spontaneously develop inflammatory bowel disease (IBD). The colitis that develops in these mice is associated with increased numbers of T helper cell (Th)2-type CD4(+)TCR-beta beta (CD4(+)beta beta) T cells producing predominantly interleukin (IL)-4. To investigate the role of these Th2-type CD4(+)beta beta T cells, we treated TCR-alpha(-/-) mice with anti-IL-4 monoclonal antibody (mAb). Approximately 60% of TCR-alpha(-/-) mice, including those treated with mock Ab and those left untreated, spontaneously developed IBD. However, anti-IL-4 mAb-treated mice exhibited no clinical or histological signs of IBD, and their levels of mucosal and systemic Ab responses were lower than those of mock Ab-treated mice. Although TCR-alpha(-/-) mice treated with either specific or mock Ab developed CD4(+)beta beta T cells, only those treated with anti-IL-4 mAb showed a decrease in Th2-type cytokine production at the level of mRNA and protein and an increase in interferon gamma-specific expression. These findings suggest that IL-4-producing Th2-type CD4(+)beta beta T cells play a major immunopathological role in the induction of IBD in TCR-alpha(-/-) mice, a role that anti-IL-4 mAb inhibits by causing Th2-type CD4(+)beta beta T cells to shift to the Th1 type.

The present study has elucidated two distinct mechanisms that may explain how a mutant of cholera toxin (mCT), E112K, retains adjuvant effects though it lacks ADP-ribosyltransferase activity and associated toxicity. In the first mechanism, we show that taCT E112K, like native cholera toxin (nCT), enhances B7-2 expression, but, to some extent, also enhances B7-1 on Peyer's patch B cells and macrophages. Cocultivation of CD4+ T cells with E112K- or nCT-treated B cells and macrophages in the presence of anti-CD3 stimulation resulted in the induction of T cell-proliferative responses. Further, the responses were blocked by mAbs to B7-1 and/or B7-2 however, the effect of anti-B7-1 was minimal. In the second mechanism, addition of taCT E112K or nCT to anti-CD3 mAb-stimulated Peyer's patch CD4+ T cells inhibited proliferative responses, while recombinant CT-B subunit (rCT-B) did not. Analysis of cytokine responses showed that both taCT E112K and nCT preferentially inhibited IFN-γ, production. Interestingly, however, nCT, but not mCT E112K, induced apoptosis in CD4+ T cells activated via the TCR-CD3 complex. These results indicate that CT uses at least two pathways for inhibition of Th1 responses and that, while nCT induces cAMP accumulation that in turn leads to apoptosis in Th1-type cells, mCT E112K, which lacks ADP-ribosyltransferase activity, inhibits IFN-γ, synthesis by a separate mechanism. Thus, mCT E112K, like nCT, induces adjuvant responses via up- regulation of mainly B7-2 on APCs and through preferential inhibition of Th1- type CD4+ T cell responses in the absence of ADP-ribosyltransferase activity.

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To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 mu g) and CT (1 mu g) induced Ag-specific Th1/Th2-type response in CD4(+) T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P, gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.

A population of CD4(+) alpha(-)beta(+) T cells increases in the mucosal and peripheral lymphoid tissues of TCR alpha-chain-deficient mice with inflammatory bowel disease. The alpha-beta+ T cells, which produce predominantly IL-4, mediate the proliferation of colonic epithelial crypts and the infiltration of large numbers of IgA-producing plasma cells into the lamina propria of the colon. To examine whether enteric Ags were recognized by a population of monoclonal a-pf T cells leading to the intestinal inflammation, we examined the usage and clonotypes of TCR expressed by the alpha(-)beta(+) T cells in TCR alpha-chain-deficient mice with inflammatory bowel disease. Analyses of immunoprecipitates by two dimensional electrophoresis and single-cell RT-PCR revealed that TCR of the alpha(-)beta(+) T cells was a homodimer of beta-chains that was capable of recognizing luminal bacterial Ags. PCR single-strand conformation polymorphism analysis of TCR V beta transcripts revealed monoclonal accumulation of the alpha(-)beta(+) T cells in the colonic lamina propria of the diseased mice. DNA sequencing revealed the accumulation of the alpha(-)beta(+) T cells with the same CDR3 sequences in the colon. These findings suggest that the pathogenic CD4(+) alpha(-)beta(+) T cells expressing a homodimeric form of the TCR beta-chains can be clonally expanded upon the stimulation with gut-derived Ags.

We addressed the effects of two cytokines, IL-6 and IL-12, derived from APCs, for the development of mucosal IgA Ab responses following their nasal delivery with the protein vaccine tetanus toxoid (TT). Mice treated nasally with IL-6 and TT showed higher TT-specific serum IgG (mainly IgG1 and IgG2b) Ab responses than did control mice, but exhibited no IgE and negligible secretory IgA (S-IgA) Ab responses. In contrast, IL-12 administered nasally with TT not only induced sharp increases in TT-specific serum IgG (mainly IgG1 and IgG2b) and IgA, but also elevated mucosal S-IgA Ab responses. Coadministration of IL-6 and IL-12 with TT did not enhance the mucosal or serum Ab responses over those seen with IL-12 alone. TT-specific CD4+ T cells from mice given TT with IL-6 or IL-12 produced higher levels of IFN-γ IL-6, and IL-10 than did those from control mice, but only negligible levels of IL-4 and IL-5. In summary, both intranasal IL-6 and IL-12 induced serum Abs that protected mice from systemic challenge with TT, whereas only IL-12 induced mucosal S-IgA Ab responses. The significance of IL-12-induced Th1- type responses for regulation of both mucosal and systemic immunity is discussed.

Background and Objectives. Clinical evidence suggests that stellate ganglion block (SGB) might modulate the immune system. Little is known, however, about the immunologic effects of SGB. We examined how SGB affected immune functions by analyzing the activation response of lymphocytes during SGB. Methods. Twenty-four volunteers were randomly subdivided into three groups. The SGB group (SGB n = 9) received 6 mL 1% lidocaine at the sixth cervical vertebra (C6) transverse process and showed Homer's sign and elevation of ipsilateral facial and upper limb temperature. The lidocaine group (n = 7) had 6 mL 1% lidocaine injected into subcutaneous tissue at the neck and showed no remarkable clinical effects. The saline group (placebo n = 8) received 6 mL saline solution injected at approximately the C6 transverse process and showed no remarkable clinical effects. Peripheral blood samples were drawn before and 30 minutes after drug administration. Samples were incubated for 4 hours under the stimulation of mitogen. Using flow cytometry, we measured the de novo expression of CD69, which is one of the initial markers of lymphocyte activation and which reflects the cell activation process. The changes in pre- and post-values were calculated and compared among the three groups. Results. In only the SGB group, the helper T-cell activation was significantly reduced, and the cytotoxic T-cell activation also tended to decrease after SGB. Conclusions. SGB may depress immune system activity for a short time, as reflected in the T-cell activation response.

A population of CD4(+) T cells with TCR beta-chain without TCR alpha-chain (CD4(+), beta beta T cells) producing Th2-type cytokines increased in the mucosal and peripheral tissues of TCR alpha-chain deficient mice with inflammatory bowel disease (IBD). Analysis of TCR-beta immunoprecipitates by two-dimensional electrophoresis and RT-PCR revealed TCR of the CD4(+) T cells was a homodimer of TCR beta-chains. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses of TCR V beta-chain transcripts of the beta beta T cells revealed monoclonal to oligoclonal accumulation of the cells in the colon, suggesting clonal expansion of the mucosal beta beta T cells upon the stimulation with gut-derived antigens. The homodimer of TCR beta-chains on the beta beta T cells was a biologically functional receptor that transduced activation signals provided by MHC-class II-associated peptidic antigens and superantigens. Treatments of the mutant mice with mAb against TCR beta or IL-4 suppressed the onset of IBD. These findings suggest that the generation of oligoclonal Th2-type beta beta T cells plays a critical role for the development of IBD.

The effects of arginine on cell proliferation and subsequent T helper (Th) 1 and Th 2 cytokine synthesis by murine Peyer's patch (PP) Th cells in vitro and the influence of arginine on the induction of antigen-specific mucosal and systemic immune responses in vivo were examined. When the PP T cells were stimulated with the anti-αβ T cell receptor (TCR) antibody in the presence of different concentrations of arginine, a higher proliferative response was observed in the culture with an optimal concentration of arginine compared with that with a minimum amount of this amino acid. The concentration of cytokines in the supernatant, the number of cytokine-producing cells and the cytokine-specific mRNA expression of PP T cells were also increased in a dose-dependent fashion. Furthermore, when mice fed on an arginine-supplemented liquid diet were orally immunized with tetanus toxoid plus cholera toxin as a mucosal adjuvant, a higher level of antigen-specific fecal IgA was observed when compared with the response in mice fed on an arginine-free diet. Taken together, these results suggest that arginine enhanced antigen-specific mucosal immune response resulting from the supporting activation of cell proliferation and subsequent cytokine synthesis of PP Th cells.

UNLABELLED: For the control of postoperative infection, it may be important to understand the possible influences of surgical stress on the host immune system. To this end, we examined how the early phase of lymphocyte activation was affected in patients after major surgery (eight patients with esophageal carcinoma and six undergoing cardiac surgery) using a flow cytometric assay based on expression of the early activation antigen, CD69. Freshly isolated T cell in preoperative and postoperative samples did not express CD69. When peripheral blood mononuclear cells were stimulated in vitro, the expression of CD69 was greatly enhanced in both CD4 and CD8 T cells, compared with the preoperative samples. The proportion of de novo CD69-expressing cells in the CD4 subset was approximately 3 times (Postoperative Day 1) and 4 times (Postoperative Days 2, 3, 5, and 7) greater than those preoperatively, whereas the proportion of de novo CD69-expressing cells in the CD8 subset was approximately 1.5 times (Postoperative Days 2 and 5) and 2 times (Postoperative Day 3) greater than those preoperatively. The proportion of CD69+ cells was significantly greater in the CD4+ subset than in the CD8+ subset during the postoperative period. IMPLICATIONS: Our results show that major surgical stress enhances the early phase of lymphocyte activation. The augmentation of activation was greater in CD4 (helper) T cells than in CD8 (cytotoxic) T cells.

BACKGROUND & AIMS: Cholera toxin (CT) has been shown to be a strong mucosal adjuvant for the induction of antigen-specific secretory immunoglobulin A (IgA). The mechanism of adjuvant activity of CT is still unknown. The aim of this study was to examine the immunomodulatory function of CT on mucosal T cells using staphylococcal enterotoxin B (SEB) as coadministered oral antigen, because SEB has been shown to directly regulate alpha beta T-cell responses. METHODS: C3H/HeN mice were orally or systemically immunized with SEB and/or CT. The levels of SEB-specific antibodies and frequencies of CD4(+)Vbeta8(+) T cells were analyzed. SEB-specific T-cell proliferation and cytokine production were also determined. RESULTS: Neither SEB-specific IgA nor IgG antibodies were induced in feces when SEB was administered alone. This was a result of the clonal deletion and partial unresponsiveness of CD4(+)Vbeta8(+)T cells in Peyer's patches. On the other hand, SEB-specific antibodies were induced by oral immunization with SEB and CT. Although some degree of clonal deletion was induced by oral immunization with SEB and CT, coadministered CT prevented the induction of anergy for CD4(+)Vbeta8(+) T cells in Peyer's patches. CONCLUSIONS: CT is a powerful immunomodulatory molecule that prevents mucosal T cells from SEB-induced anergy.

Bone marrow stromal cell Ag-1 (BST-1 CD157)-deficient mice were generated to examine the immunologic roles of the molecule in vivo, In BST- 1(-/-) mice, the development of peritoneal B-1 cells was delayed, and CD38(low/-) B-lineage cells were increased in the bone marrow and spleen. Partial impairment of thymus-independent (TI-2) and thymus-dependent (TD) Ag- specific immune responses was noted in the systemic and mucosal compartments of BST-1(-/-) mice, respectively. Although serum Ig levels as well as TD and TI-1 Ag-specific systemic immune responses were normal, the TI-2 Ag-induced IgG3 response was selectively impaired. Oral immunization of BST-1(-/-) mice with cholera toxin, a potent TD Ag for the induction of IgA response, resulted in the poor production of Ag-specific Abs at the intestinal mucosa accompanied by the reduced number of Ag-specific IgA-producing cells in the lamina propria. These results indicate that BST-1 has roles in B cell development and Ab production in vivo.

Background & Aims: Intraepithelial lymphocytes (IELs) are located between epithelial cells that are thought to display unique features and functions at the small intestinal villus tip and crypt levels. We have addressed whether the spatial differences in the intestinal epithelium extend to IELs and subsequent cross-talk between IELs and epithelial cells. Methods: IELs were isolated from villus tip and crypt portions of mouse small intestine and then compared for spontaneous cytokine production and responsiveness to interleukin (IL)-2 and/or IL-7. Results: No difference was observed between number of alpha beta IELs in villus tips and crypts, whereas a trend toward increased frequencies of IELs bearing the gamma delta form of T-cell receptor was noted in villus tips. interestingly, the number of alpha beta IELs producing interferon gamma and IL-5 was significantly reduced in the cells from crypts compared with villus tips. Furthermore, villus tip alpha beta IELs exhibited higher responses to stimulation signals provided by IL-2 and/or IL-7 than their crypt counterpart, Such functional differences were not observed with gamma delta IELs from the two intestinal sites. Conclusions: Distinct molecular cross-talk between IELs and epithelial cells occurs in intestinal villus tips and crypts.

Based upon the prosthodontic literature, subjects who are at the transition stage between natural dentition and edentulism are called "terminal dentition" (TD) cases. The aim of the present cross-sectional investigation was to characterize the local and systemic inflammatory responses in 2 groups of patients with terminal dentition periodontitis. Eight severe adult periodontitis terminal dentition (AP-TD subjects and 8 early onset periodontitis terminal dentition (EOP-TD) subjects were entered into the study. Our purpose was to measure an extended battery of cytokines in the gingival crevicular fluid (GCF) and in lipopolysaccharide (LPS)-stimulated monocytic culture supernatants as well as gingival mononuclear cell messenger RNA (mRNA) transcripts determined from biopsy samples. Within the GCF there were 3 tiers (levels) of mediators based upon approximate 10-fold differences in concentration. The highest tier included prostaglandin E-2 (PGE(2)), interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2), the intermediate tier included tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN-gamma) and at the lowest concentration level were interleukin-4 (IL-4) and interleukin-6 (IL-6). Thus, the GCF analysis clearly indicated that in both AP-TD and EOP-TD groups the monocytic, i.e. IL-1 beta and PGE(2) and Th1, i.e. IL-2 and IFN-gamma, inflammatory mediator levels quantitatively dominated over the Th2 mediators, i.e. IL-4 and IL-6. LPS-stimulated monocytic release of IL-1 beta, PGE(2) and TNF alpha was significantly elevated in both AP-TD and EOP-TD groups compared to those of a control group of 21 subjects with moderate to advanced adult periodontitis. The cytokine mRNA expression of isolated gingival mononuclear cells showed that in both the AP-TD and the EOP-TD groups Th1 and Th2 cytokines were expressed, with low levels of IL-4 and IL-12. In conclusion, our data suggest that this cross-sectional TD periodontitis model may reflect progressive periodontal disease associated with tooth loss. Furthermore. although Th1 cytokine levels in the GCF dominate over the Th2 response, monocytic activation provides the main source of proinflammatory mediators. In addition, LPS-stimulated peripheral blood monocytes demonstrate an upregulated inflammatory mediator secretion in the terminal dentition.

To determine if there is an association between the isotype of simian immunodeficiency virus (SIV)-specific B cell responses and the profile of Th1 and Th2 cytokine expression, rhesus macaques were immunized with SIV antigens via the iliac lymph nodes, using a targeted lymph node (TLN) immunization procedure, When CD4(+) T cells purified from antigen-stimulated peripheral blood mononuclear cells were analyzed, the levels of Th2 cytokine production were gradually increased after the second and third immunizations, However, interferon-gamma production did not change, Analysis of SIV-specific B cell responses revealed that the main isotype was IgG after the second and third immunizations, In addition, a peak of SIV-specific IgA B cell responses was noted following the third immunization, These findings suggest that the induction of Th2 type responses in TLN-immunized rhesus macaques reflects the sequence of initial induction of SIV-specific IgG-producing cells followed by IgA-secreting cells.

To elucidate role of Th1 type responses in mucosally-induced tolerance, IFN-gamma deficient (IFN-gamma(-/-)) and background control mice were given an oral dose of 25 mg of OVA prior to systemic immunization. OVA-specific IgG antibody (Ab) responses in serum and splenic Ab-forming cells were reduced in IFN-gamma(+/+) but not in IFN-gamma(-/-) mice. Further, requirement of costimulatory signals for the induction of oral tolerance was provided by results obtained through a series of experiments using CD40L(-/-) mice. It was shown that any reductions in antigen (Ag)-specific T cell responses, including delayed type hypersensitivity (DTH) and proliferative responses were not detected. On the other hand, CD40L(+/+) mice underwent reduced T cell responses following oral delivery of OVA. These findings indicate that IFN-gamma synthesis and CD40L-CD40 interactions are essential for the induction of systemic unresponsiveness to orally administered Ag.

Our past studies have shown that the mucosal adjuvant cholera toxin (CT) induces T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (S-IgA) antibodies (Abs). In this study, recombinant murine IL-12 (rmIL-12) was given either parenterally or orally to mice orally immunized with tetanus toroid (TT) and CT to determine whether this cytokine could redirect the CT-induced Th2-type responses and what effect this shift would have on S-IgA Ab responses. Intraperitoneal administration of rmIL-12 shifted TT-specific responses toward Th1-type and resulted in CD4(+) T cells producing IFN-gamma and IL-2 with markedly reduced levels of Th2-type cytokines. This cytokine profile was accompanied by increased delayed-type hypersensitivity (DTH) and shifts in serum IgG1 to IgG2a and IgG3 anti-TT Ab responses. Further, serum IgE and S-IgA Ab responses were markedly reduced by parenteral IL-12. When IL-12 complexed to liposomes was given orally both shifts to IgG2a and IgG3 and low IgE Abs again occurred concomitant with enhanced serum IFN-gamma and DTH responses. Interestingly, oral rmIL-12 did not result in significant levels of serum IL-12 nor altered S-IgA Ab responses and resulted in higher levels of some Th2-type cytokines both in vitro and in vivo when compared with parenteral IL-12. Our results show that the shifts in systemic immune responses with intact S-IgA Abs which occur after oral delivery of IL-12-liposomes are due to cytokine effects in the Peyer's patches and suggest new strategies for the targeted manipulation of Th1- and Th2-type responses to mucosal vaccines.

We report here a murine model for experimental chronic colitis where administration of trinitrobenzene sulfonic acid (TNBS) in 50% ethanol induced inflammation of large intestine in susceptible (C3H/HeJ and BALB/c) but not resistant (C57BL/6 and DBA/2) mouse strains. We queried whether mucosal trinitrophenyl (TNP)-specific B cell responses were induced in mice with TNBS-induced colitis, and if induction of tolerance to TNBS by oral administration of this hapten protected mice from development of colitis. Isotypes and subclasses of polyclonal and TNP-specific Ab-forming cells (AFC) were assessed in mucosal and peripheral lymphoid tissues of C3H/HeJ mice with TNBS-induced colitis. Increased numbers of IgA- and IgG-secreting cells were found in the inflamed colon lamina propria. Inflamed colonic tissue also contained high frequencies of IgG anti-TNP AFC (predominantly of IgG1, IgG2a, and IgG2b subclasses) however, anti-TNP responses in noninflammed mucosal tissues of mice with colitis exhibited dominant IgA and IgM with low IgG anti-TNP responses. CD4+ T cells stimulated with TNP-splenocytes produced more IFN- γ and less IL-4, suggesting a Th1-type response. Oral administration of TNBS before induction of colitis markedly decreased mucosal anti-TNP responses and completely inhibited anti-TNP IgG2a and IgG2b responses. Control mice did not show inhibition of anti-TNP AFC responses or TNBS-induced colitis. Intracolonic sensitization of susceptible C3H/HeJ mice with TNBS induces a localized IgG anti-TNP B cell response in the inflamed tissue, whereas prior oral administration of TNBS results in unresponsiveness to this agent and protects mice from development of TNBS-induced colitis.

Mucosal immunoglobulin A (IgA) responses are often associated with Th2-type cells and derived cytokines, and interleukin-4 (IL-4) knockout (IL-4(-/-)) mice with impaired Th2 cells respond poorly to oral antigens. However, we have noted that IL-4(-/-) mice have normal mucosal IgA levels, which led us to query whether different oral delivery systems could elicit mucosal immunity. Two oral regimens were used: (i) a live recombinant Salmonella strain which expresses fragment C (ToxC) of tetanus toxin, and (ii) soluble tetanus toxoid (TT) with cholera toxin (CT) as an adjuvant. Oral immunization of IL-4(-/-) mice with recombinant Salmonella vaccine expressing ToxC induced brisk mucosal IgA and serum IgG (mainly IgG2a) anti-TT antibody responses. TT-specific CD4(+) T cells from spleen or Peyer's patches produced gamma interferon, indicative of Th1 responses; however, IL-6 and IL-10 were also seen. Oral immunization of IL-4(-/-) mice with TT and CT induced weak mucosal IgA to TT; however, brisk IgA anti-CT-B responses and CT-B-specific CD4(+) T cells producing IL-6 and IL-10 were also noted. These results show that although IL-4-dependent antibody responses are impaired, mucosal IgA responses are induced in IL-4(-/-) mice. These results suggest that certain cytokines, i.e., IL-6 and IL-10 from Th2-type cells, play an important compensatory role in induction and regulation of mucosal IgA responses.

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