藤橋 浩太郎

In the development of mucosal vaccines, cholera toxin (CT) has been shown to be an effective adjuvant and to induce both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its innate toxicity. Recent vaccine development efforts have emphasized nasal application of antigen and CT for the induction of mucosal IgA responses. When we examined potential toxicity of CT for the central nervous system (CNS), both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses the,e potential problems for effective mucosal vaccine development. (C) 2002 Elsevier Science Ltd. All rights reserved.

We demonstrated that the mutant of cholera toxin (mCT) E112K which, was LPS-free supported the induction of protective immunity in mucosal (e.g. lung lavage) and systemic (e.g. serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice. Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K. or native CT (nCT) was co-administered nasally with DT. The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands. Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT. The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4(+) T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT. The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective. Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity. These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g. lung lavages and the lungs). (C) 2001 Elsevier Science Ltd. All rights reserved.

Conditions consistent with tolerance or immunoregulation have been observed in experimental Candida albicans vaginal infections. The present study investigated the role of gamma/delta T cells in experimental vaginal candidiasis. Results showed that T-cell receptor delta -chain-knockout mice had significantly less vaginal fungal burden when compared to wild-type mice, suggesting an immunoregulatoty role for gamma/delta T cells in Candida vaginitis.

The purpose of this study was to determine the nature of the CD4(+) Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary, inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4+, and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-gamma, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-gamma) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4(+) Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1 beta expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.

Induction of mucosal immunity by oral immunization with protein antigen alone is difficult: potent mucosal adjuvants vectors, or other special delivery systems are required. Cholera toxin (CT) has been shown to be an effective adjuvant for the development of mucosal vaccines and, when given with vaccine, induces both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, and in addition to potential type-I hypersensitivity, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its inherent toxicity. When we examined the potential toxicity of CT for the central nervous system, both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development. Tolerance represents the most common and important response of the host to environmental antigens, including food and commensal bacterial components, for the maintenance of an appropriate immunological homeostasis. We have examined whether Peyer patches could play a more important role for the maintenance of oral tolerance. Using Peyer patch-null mice, we found that mice lacking this gut-associated lymphoid tissue retained their capability to produce secretory IgA antibodies but did not develop normal oral tolerance to protein antigens.

Past studies have shown that colonic patches, which are the gut-associated lymphoreticular tissues (GALT) in the colon, become much more pronounced in hapten-induced murine colitis, and this was associated with Th2-type T cell responses. To address the role of GALT in colonic inflammation, experimental colitis was induced in mice either lacking organized GALT or with altered GALT structures. Trinitrobenzene sulfonic acid was used to induce colitis in mice given lymphotoxin-beta receptor-Ig fusion protein (LT betaR-Ig) in utero, a treatment that blocked the formation of both Peyer's and colonic patches. Mice deficient in colonic patches developed focal acute ulcers with Th1-type responses, whereas lesions in normal mice were of a diffuse mucosal type with both Th1- and Th2-type cytokine production. We next determined whether LT betaR-Ig could be used to treat colitis in normal or Th2-dominant, IFN-gamma gene knockout (IFN-gamma (-/-)) mice. Four weekly treatments with LT betaR-Ig resulted in deletion of Peyer's and colonic patches with significant decreases in numbers of dendritic cells. This pretreatment protected IFN-gamma (-/-) mice from trinitrobenzene sulfonic acid-induced colitis; however, in normal mice this weekly treatment was less protective. In these mice hypertrophy of colonic patches was seen after induction of colitis. We conclude that Th2-type colitis is dependent upon the presence of colonic patches. The effect of LT betaR-Ig was mediated through prevention of colonic patch hypertrophy in the absence of IFN-gamma. Thus, LT betaR-Ig may offer a possible treatment for the Th2-dominant form of colitis.

Mucosal administration of experimental autoimmune encephalomyelitis (EAE)-specific autoantigens can reduce the onset of disease. To examine whether cholera toxin-B-subunit (CTB)-conjugated EAE-specific T-cell epitope can reduce development of the autoimmune disease in mice, we produced a recombinant hybrid molecule of CTB fusion protein linked with proteolipid-protein (PLP)-peptide139-151(C140S) at levels up to 0.1 gram per liter culture media in Bacillus brevis as a secretion-expression system. Amino acid sequencing and GM1-receptor binding assay showed that this expression system produced a uniformed recombinant hybrid protein. EAE was induced in SJL/J mice by systemic administration with the PLP-peptide. When nasally immunized 5 times with 70 mug rCTB PLP-peptide hybrid protein, mice showed a significantly suppressed development of ongoing EAE and an inhibition of both the PLP-peptide-specific delayed-type hypersensitivity (DTH) responses and leukocyte infiltration into the spinal cord. In contrast, all mice given the PLP-peptide alone or the PLP-peptide with the free form of CTB did not suppress the development of EAE and DTH responses. These results suggest that nasal treatment with the recombinant B. brevis-derived hybrid protein of CTB and autoantigen peptide could prove useful in the control of multiple sclerosis. (C) 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 62-69, 2001.

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced Ige and IgE Ab- and Ag specific CD4+ T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosa) IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS, Furthermore, when OVA-specific Ah-forming cells (AFCs) in both systemic and mucosa-associated tissues mere examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4+ T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance,

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin P-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Fever's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4(+) T-cell-derived IFN-gamma, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT1B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor. (C) 2001 Elsevier Science Ltd. All rights reserved.

Subepithelial and intraepithelial lymphocytes of human adenoids and tonsils were characterized and directly compared to determine the potential contribution of these tissues to mucosal and systemic immune responses. The distribution of T and B cell subsets, cytokine patterns, and antibody (Ab) isotype profiles were similar for adenoids and tonsils. Both tissues contained predominantly B cells (similar to 65%), approximately 5% macrophages, and 30% CD3(+) T cells. The T cells were primarily of the CD4(+) subset (similar to 80%). Tonsillar intraepithelial lymphocytes were also enriched in B cells. The analysis of dispersed cells revealed a higher frequency of cells secreting IgG than IgA and the predominant Ig subclass profiles were IgG1 > IgG3 and IgA1 > IgA2, respectively. In situ analysis also revealed higher numbers of IgG- than IgA-positive cells. These IgG-positive cells were present in the epithelium and in the subepithelial zones of both tonsils and adenoids. Mitogen-triggered T cells from tonsils and adenoids produced both Th1- and Th2-type cytokines, clearly exhibiting their pluripotentiality for support of cell-mediated and Ab responses. Interestingly, antigen-specific T cells produced interferon-gamma and lower levels of interleukin-5. These results suggest that adenoids and tonsils of the nasopharyngeal-associated lymphoreticular tissues represent a distinct component of the mucosal-associated lymphoreticular tissues with features of both systemic and mucosal compartments.

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 mug of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21, An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level, When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4(+) T cells from spleen and Peyer's patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s.c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice, These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.

Background & Aims: Most experimental models for inflammatory bowel disease in mice are associated with production of interferon (IFN)-gamma and other proinflammatory cytokines. We hypothesized that T-helper 2 (Th2)-type cells could also contribute to the colitis and cause inflammation different than that mediated by Th1-type cells. Methods: Trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6 background mice genetically deficient in interleukin (IL)-12 p40 (IL-12(-/-)), IFN-gamma (IFN-gamma(-/-)), or IL-4 (IL-4(-/-)) was examined in comparison with control mice (C57BL/6(+/+)). Results: C57BL/6(+/+), IFN-gamma(-/-), and IL-12(-/-) mice developed patterns of colitis characterized by distortion of crypts, loss of goblet cells, and mononuclear cell infiltration with fibrosis of the mucosal layer, IL-4(-/-) mice had greater mortality than other groups because of penetrating ulcers; however, survivors developed milder lesions that were limited to focal acute ulceration, Colonic CD4(+) T cells from normal, IFN-gamma(-/-), or IL-12(-/-) mice produced both IL-4 and IL-5. Conclusions: In TNBS colitis, Th1-like cytokine responses induce fatal, acute, transmural, and focal types of lesions, whereas Th2-like cytokine responses play a significant role in the diffuse atrophic changes in crypts and the mucosal layer that occur in the late stages of this disease.

In an in vitro study, Escherichia coli heat-labile toxin (LT) was shown to directly affect activated CD4(+) T cells and support interleukin (IL)-5 production in IL-4-deficient (IL-4(-/-)) mice, whereas cholera toxin (CT) did not. Both LT and CT enhanced B7-2 expression on B cells and macrophages. These effects were not influenced by CD40-CD40 ligand cosignaling. Addition of LT- or CT-treated antigen-presenting cells to anti-CD3-triggered CD4(+) T cells resulted in the induction of T cell proliferative responses. Further, these responses were inhibited by anti-B7-2 monoclonal antibody. Cocultivation of CD4(+) T cells with LT- or CT-treated antigen-presenting cells and anti-CD3 enhanced Th1- and IL-4-mediated Th2-type cytokine production, The results from in vitro studies were supported by in vivo studies in IL-4(-/-) mice, in which LT induced mucosal IgA responses but CT did not. Thus, although both LT and CT induce mucosal adjuvant responses via IL-4-dependent Th2-type responses, LT also elicits Th1- and IL-4-independent Th2-type responses.

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LT beta R) and Ig (LT beta R-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN), In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LT beta R-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA(+)) plasma cells in the intestinal lamina propria, Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4(+) T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha(-/-)) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LT beta R-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alpha beta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.

Objective. Administration of bovine type Il collagen (CII) or of its peptide either orally or nasally has been reported to suppress the development of collagen induced arthritis (CLA) in mice and rats. We examined the inhibitory effects of CII delivered by each route on CIA in DBA/IJ mice to determine which route was superior, Methods. Male mice were injected. twice with CII in Freund's complete adjuvant to induce CIA, Before induction of CIA, 1, 10, or 40 mu g of CII were administered nasally 15 times and 10, 100, 500, or 1000 mu g of CII were given 10 times orally. The development: of arthritis, arthritis score, CII-specific delayed-type hypersensitivity (DTH) response, and CII-specific antibody levels were examined. Results. Nasal administration of 10 mu g of CII 15 times had the most prominent suppressive effects, reducing disease incidence by 50% and inhibiting both CII-specific IgG antibody and DTH: responses. Of all the mice undergoing oral administration, those receiving 500 mu g of CII 10 times showed the greatest suppressive potential, However, the treatment only delayed disease onset for roughly 3 weeks, lowering CII-specific IgG antibody levels but failing to suppress DTH responses. Conclusion. Nasal administration of CII reduced CIA development and inhibited Cn-specific T cell and antibody responses to a greater degree than did oral administration.

To develop a mucosal vaccine strategy for the elderly, we have compared mucosal and systemic immune responses between aged (12-14 months) and young adult (8-12 weeks) mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of ovalbumin (OVA) and 10 mu g of cholera toxin as adjuvant. Although elevated levels of OVA-specific systemic IgG and mucosal IgA Ab responses were seen in young mice, aged mice showed impaired antigen-specific mucosal and systemic immune responses. These results suggest that mucosal immunity is down regulated in aged mice. (C) 2000 Elsevier Science Ltd. All rights reserved.

Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract. Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced, To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract. By convention, oral immunizations with Salmonella vectors induce CD4+ T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses, In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses. As expected, mice orally immunized with an E. coli-CFA/I construct elicited poor anti-CFA/I Ab responses. In fact, the addition of cholera toxin during oral E, coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses. Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CEA/I Abs, By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses. This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs.

We used gamma delta TCR-deficient (TCR delta(-/-)) mice to examine the role of gamma delta T cells for induction of mucosal responses and systemic tolerance to high versus low doses of oral antigen. When either TCR delta(-/-) or TCR delta(+/+) mice were immunized orally with a high dose of ovalbumin (OVA) prior to parenteral challenge, systemic IgG and IgE antibody responses were markedly reduced in both types of mice, while mucosal IgA responses were reduced only in the TCR delta(-/-) mice. Reduced T cell proliferative responses and delayed-type hypersensitivity were seen in TCR delta(-/-) and TCR delta(+/+) mice given the high dose of OVA, Antigen-induced T(h)1 and T(h)2,cytokine production by splenic CD4(+) T cells was severely inhibited in orally tolerized TCR delta(-/-) and TCR delta(+/+) mice. In contrast, while oral tolerance associated with increased levels of IL-10 synthesis was induced by a low dose of OVA in TCR delta(+/+) mice, the TCR delta(-/-) mice were not tolerized and failed to produce IL-10, Our findings indicate that gamma delta T cells play a significant immunoregulatory role in IL-10-mediated, low-dose oral tolerance induction, but are not essential participants in the induction of systemic tolerance to orally introduced antigens given in larger doses.

Interferon (IFN)-gamma synthesis of CD45RO(+) (memory) and CD45RA(+) (naive) CD8(+) cytotoxic T lymphocytes (CTLs) and the role of interleukin (IL)-12 in the regulation of human CTL functions in virus-specific immunity were investigated. After culture with influenza virus, CD45RO(+) CD8(+) T cells from human peripheral blood mononuclear cells increased in frequency and exhibited significant major histocompatibility complex class I-mediated CTL activity, whereas CD45RA(+) CD8(+) T cells did not. Influenza virus-stimulated CD45RO(+) CD8(+) T cells contained significantly higher levels of IFN-gamma-producing cells and IFN-gamma-specific mRNA than did CD45RA(+) CD8(+) T cells. Recombinant human IL-12 further enhanced CTL activity and IFN-gamma production by CD45RO(+) CD8(+) T cells. These data clearly show that human virus-specific CTL activity and coproduction of IFN-gamma are associated with the CD45RO(+) CD8(+) T cells that are modulated by the cell-mediated, immunity-inducible cytokine IL-12 in humans.

It has been reported that intranasal immunization can induce mucosal immune responses. However, the efficacy of intranasal immunization on otitis media caused by non-typeable Haemophilus influenzae (NTHi) is not yet elucidated. Mice were intranasally, orally, intratracheally or intraperitoneally immunized with outer membrane protein (OMP) isolated from NTHi, and antigen-specific immune responses were determined by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immune-spot assay (ELISPOT). Cytokine production from splenic CD4(+) T cells was examined by ELISA. Following the immunization, the clearance of NTHi from the nasal and nasopharyngeal cavity was examined. OMP-specific IgA antibody titers in nasal washes and the numbers of specific IgA-producing cells in nasal passages were significantly increased in intranasally immunized mice. Cytokine analysis showed that interferon-gamma (IFN-gamma) and interleukins IL-6 and IL-10 were predominantly produced from CD4(+) T cells. The clearance of NTHi was significantly enhanced in the intranasal immunization group. Intranasal immunization is an effective vaccination regimen for the induction of OMP-specific mucosal immune responses. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1(-/-)) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IBA AFC, A lack of IgA-committed B cells was seen in TGF-beta 1(-/-) mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1(-/-) mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1(-/-) mice acid likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.

To determine the efficacy of a mucosal vaccine against nontypeable Haemophilus influenzae (NTHi), mice were immunized nasally, orally, intratracheally, or intraperitoneally with NTHi antigen together with cholera toxin, Antigen-specific IgA antibody titers in nasal washes and the numbers of antigen-specific IgA-producing cells in nasal passages showed the greatest increases in mice immunized nasally. Cytokine analysis showed that interferon-gamma, interleukin (IL)-2, IL-5,IL-6, and IL-10 were induced by nasal immunization, suggesting that Th2- and Th1-type cells were generated. Furthermore, bacterial clearance of a homologous strain of NTHi from the nasal tract was significantly enhanced in the nasal immunization group. These findings suggest that nasal immunization is an effective vaccination regimen for the induction of antigen-specific mucosal immune responses, which reduce the colonization of NTHi in the nasal tract.

Simian immunodeficiency virus (SIV)-specific B cell responses and the Th1- or Th2-type profiles of cytokine expression were determined for rhesus macaques immunized with SIV antigens via the iliac lymph nodes (by use of a targeted lymph node [TLN] procedure) or orally with SIV p55(gag) plus cholera toxin (CT) as a mucosal adjuvant. Analysis of CD4(+) T cells purified from SIV-stimulated peripheral blood mononuclear cells of immunized macaques revealed that Th2 cytokine production gradually increased after the second and third TLN immunization, Analysis of m-specific B cell responses revealed that peak. SIV-specific IgA B cell responses followed the third TLN immunization and occurred during peak Th2-type T cell responses. OL al immunization of macaques with p55(gag) plus CT induced interferon-gamma-secreting Th1-type and select Th2-type cytokine-producing CDC T helper cells, which most likely accounted for the induction of p55-specific systemic IgG and mucosal IgA responses.

To investigate the potential involvement of T helper (Th)2-type responses in murine models of intestinal inflammation, we used trinitrobenzene sulfonic acid (TNBS)-hapten to induce inflammatory bowel disease in situations where Th1-type responses with interferon (IFN)-gamma synthesis are either diminished or do not occur. Intracolonic administration of TNBS to either normal (IFN-gamma(+/+)) or Th1-deficient IFN-gamma knockout (IFN-gamma(-/-)) BALB/c mice resulted in significant colitis. In IFN-gamma(-/-) mice, crypt inflammation was more severe than in IFN-gamma(+/+) mice and was accompanied by hypertrophy of colonic patches with a lymphoepithelium containing M cells and distinct B and T cell zones resembling Peyer's patches. Hapten-specific, colonic patch T cells from both mouse soups exhibited a Th2 phenotype with interleukin (IL)-4 and IL-5 production. TNBS colitis in normal mice treated with anti-IL-4 antibodies or in IL-4(-/-) mice was less severe than in either IFN-gamma(+/+) or IFN-gamma(-/-) mice. Our findings now show that the Th2-type responses in TNBS colitis are associated with colonic patch enlargement and inflammation of the mucosal layer and may represent a model for ulcerative colitis.

Vaccines able to induce both secretory IgA for protection of mucosal surfaces and systemic immunity to pathogens invading the host are of great interest in the war against infectious diseases. Mucosal vaccines trigger immune cells in mucosal inductive sites and thus can induce immunity in both the mucosal and systemic compartments. This review presents a critical survey of adjuvants and delivery systems currently being tested for mucosal immunization. A better understanding of cellular and molecular factors involved in the regulation of mucosal immunity will help in the design of safer mucosal vaccines to elicit the appropriate protective immune response to a given pathogen.

CD40 ligand (CD40L) gene-disrupted (CD40L(-/-)) mice were employed to examine the role of costimulatory signals via CD40L-CD40 interactions in mucosally induced tolerance. CD40L(-/-) and control (CD40L(+/+)) mice of the same C57BL/6 x 129/J background were immunized orally with 25 mg of OVA before systemic challenge with OVA in CFA, While CD40L(+/+) mice showed reductions in Ag-specific T cell responses including delayed-type hypersensitivity (DTII) and proliferative responses, CD40L(-/-) mice underwent normal T cell responses. Further, cytokine analysis of splenic CD4(+) T cells showed that both Th1-type (e.g., IFN-gamma and IL-2) and Th2-type (e.g., IL-4, IL-5, IL-6, and IL-10) responses were maintained in CD40L(-/-) mice orally immunized with OVA, whereas these cytokine responses in CD40L(+/+) mice were significantly reduced. In addition, splenic CD4(+) T cells from CD40L(-/-) mice orally immunized with OVA provided B cell help in Ag-specific Ab-forming cells when the cells were cultured with naive B cells in the presence of Ag and CD40L-transfected cell lines. In contrast, an identical culture condition containing splenic CD4(+) T cells from orally tolerized CD40L(+/+) mice did not exhibit helper activity. Taken together, these findings indicate that CD40L and CD40 interactions are essential for the induction of systemic T cell unresponsiveness to orally administered Ag.

Female rhesus macaques were nasally immunized with p55(gag) (p55) Of SN and cholera toxin as a mucosal adjuvant, Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum, Furthermore, high numbers of p55-specific IgA and IgG Ah-forming cells mere induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4(+) T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA, Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.

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.

Oral administration of large doses of protein antigen generally induces a state of systemic unresponsiveness currently termed mucosally induced tolerance. In this study, we used human milk protein (HMP) without casein as a multi-protein antigen for the study of mucosally induced tolerance. The HMP utilized in this study mainly contained secretory (S) IgA, lactoferrin (Lf) and alpha-lactalbumin (Lact), When mice were given 1 or 25 mg of HMP orally 3 times or 25 mg orally four consecutive weeks prior to systemic immunization, antigen-specific serum IgG responses to HMP were induced by subsequent parenteral immunization with 100 mu g of HMP. Analysis of IgG subclasses revealed that IgG1 followed by IgG2b accounted for the IgG responses noted. When both HMP and ovalbumin (OVA) were fed to mice, tolerance developed to OVA but not to HMP. To further investigate the nature of immune responses seen following oral gavage of HMP, we examined responses to individual protein of HMP. Brisk serum IgG1 and IgG2b responses to both S-IgA and if were induced by oral followed by systemic immunization with HMP. Analysis of splenic CD4(+) T cells from mice given oral HMP revealed production of T(h)2- but not T(h)1-type cytokines. These results show that oral administration of HMP preferentially induces exclusive T(h)2-type immune responses, which may prevent the development of HMP (S-IgA and Lf)-specific mucosally induced tolerance.

Epithelial cells and lymphocytes, including gamma delta and alpha beta T cells, in the gastrointestinal tract epithelium represent a major host defense intranet that is incompletely understood. Cell-to-cell interactions between intraepithelial lymphocytes (IELs) and intestinal epithelial cells (IECs) comprise this intranet, and we have assessed the role of IECs in the regulation of gamma delta and alpha beta T cell responses. When highly purified CD3(+) IEL T cells were stimulated via the TCR-CD3 complex, high proliferative responses and cytokine synthesis were induced. However, the addition of viable IECs or purified IEC membranes (mIEC) down-regulated T cell proliferative and cytokine responses. Further, the inhibitory effect of mIEC was not restored by antibodies to TGF-beta, CD1d, E-cadherin, or MHC class I or II. This inhibitory effect was noted for both gamma delta and alpha beta T cell subsets from IELs, and mRNA levels were reduced for both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-5) cytokines in gamma delta and alpha beta IELs. In contrast, a purified membrane fraction obtained from thymocytes did not inhibit IEL proliferative responses, Further, mIEC did not inhibit splenic alpha beta T cell proliferative responses. These findings show that cell-to-cell interactions between intraepithelial gamma delta and alpha beta T cells and IECs occur via cell surface molecules, suggesting an intranet to prevent potential inflammatory responses at the intestinal mucosal surface.

Splenic T cells isolated from BALB/c mice that had been mucosally tolerized by oral administration of 15 mg of OVA revealed selective increases in IFN-gamma production with impaired levels of IL-2, IL-4, IL-5, and IL-10, These mice possessed reduced splenic OVA-specific T cell proliferative and delayed-type hypersensitivity responses when compared with nontolerized controls. Further, OVA-specific IgG Ab responses in serum and the numbers of IgG Ab-forming cells in spleen mere significantly diminished following systemic challenge with OVA in CFA, When IFN-gamma-deficient (IFN-gamma(-/-)) mice of the same genetic background were given an oral dose of 25 mg of OVA before systemic immunization, no reduction in OVA-specific IgG Ab responses in serum and spleen was seen. Furthermore, the serum IgG Ab responses were restricted to IgG1 and IgG2b subclasses. Interestingly, although IFN-gamma(-/-) mice displayed a partial diminishment of T cell proliferative and delayed-type hypersensitivity responses to OVA, significant responses were still present when compared with the low responses noted in IFN-gamma(+/+) mice, In addition, OVA-specific T cells from IFN-gamma(-/-) mice produced Th2-type cytokines (e.g., IL-4), which provided help for systemic OVA-specific serum IgG1 and IgG2b Ab responses. These findings clearly indicate a central role for IFN-gamma in the induction and maintenance of mucosally induced tolerance.

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.

The isolation of intestinal intraepithelial lymphocytes (IEL) is a major prerequisite for the investigation of cellular and molecular cross-talk in the intestinal mucosa. Since intestinal epithelial cells exhibit distinct functional features at the villi tip and crypt levels, such differences could extend to IEL. We developed a mechanical procedure for isolation of IEL from these distinct epithelial sites to test our hypothesis. Cells isolated from the intestinal epithelium by sequential incubations under stirring were segregated based upon their alkaline phosphatase (AP) activity since villi tip and crypt fractions expressed high and low AP activity, respectively. IEL preparations obtained after a further purification step in Percoll gradient contained >90% Integrin alpha(IEL) chain(+), CD3(+) T cells, and no Igf cells. Villi tip IEL preparations possessed increased numbers of low density IEL when compared to crypt IEL, suggesting that distinct IEL-epithelial cell interactions occur at the intestinal villi tip and crypt levels. (C) 1997 Academic Press.

Both interleukin-7 (IL-7) and IL-7 receptor (R) gene knockout (IL-7(-/-) and IL-7R(-/-)) mice were employed in order to directly investigate the importance of the IL-7 and IL-7R signaling pathway for the development of intestinal intraepithelial lymphocytes (IEL). Loss of the IL-7R-specific gene resulted in complete deficiency of the gamma delta T cell lineage with lack of V gamma 4- and V gamma 7-specific messages in the epithelium of the gastrointestinal (GI) tract in comparison to control mice of the same genetic background (similar to 40 %). Disruption of the IL-7-specific gene resulted in marked, but not complete depletion of gamma delta T cells (2-3 %) in IEL. Furthermore, mRNA for both V gamma 4 and V gamma 7 genes were detected in the gamma delta IEL subset of IL-7(-/-) mice. The subtle differences between IL-7(-/-) and IL-7R(-/-) mice suggest that although IL-7 controls most of the expansion and/or development of gamma delta IEL, another ligand binding to the IL-7R also plays a discernable role. Furthermore, alpha beta IEL developed more slowly in IL-7R(-/-) mice when compared with ligand knockouts; however, the frequency of IEL T cells subsequently increased with age and normal levels of CD3(+) T cells expressing the alpha beta TCR were detected by 2 and 3 months of age in IL-7(-/-) and IL-7R(-/-) mice, respectively. The direct comparison of IL-7(-/-) and IL-7R(-/-) mice clearly supports the hypothesis that both IL-7 and another IL-7R binding molecule can influence the development of gamma delta T cells in the intestinal epithelium.

Rhesus macaques were orally immunized with a mucosal vaccine consisting of two different concentrations (1 mg vs 250 mu g) of recombinant SIV p55(gag) (p55) with or without cholera toxin (CT, 50 mu g) as a mucosal adjuvant. The plasma from macaques receiving the higher dose of p55 (1 mg) and CT had higher p55-specific IgG and IgA Ab titers compared with macaques that received the lower dose of p55 (250 mu g) and CT. Further, high levels of p55-specific IgC and IgA Abs were present in external secretions from both groups. The level of p55-induced T cell responses was elevated in PBMCs isolated from the high dose group compared with the low dose group, When culture supernatants from these p55-stimulated PBMCs were examined for Th1 (IFN-gamma) and Th2 (IL-4 and IL-10) cytokines, both IFN-gamma and IL-10 were present, but IL-4 was absent. CD4(+) T cells isolated from these p55-stimulated PBMCs contained IFN-gamma spot-forming cells (SFCs) but not IL-4 SFCs. These results were further confirmed by cytokine-specific reverse transcriptase PCR analysis, where p55-specific CD4(+) T cells expressed mRNA for IFN-gamma, IL-6, and IL-10, but not IL-4. These findings suggest that oral immunization of nonhuman primates induced both IFN-gamma-secreting Th1 and select Th2 cytokine (e.g., IL-6 and IL-10)-producing CD4(+) Th cells, which accounted for the generation of p55-specific systemic and mucosal Ab responses.

We have characterized a nontoxic mutant of cholera toxin (CT) as a mucosal adjuvant in mice. The mutant CT was made by substitution of serine with phenylalanine at position 61 of the A subunit (S61F), which resulted in loss of ADP ribosyltransferase activity and toxicity. Mice were intranasally immunized with ovalbumin, tetanus toxoid, or influenza virus either alone or together with mutant CT S61F, native CT, or recombinant CT-B. Mice immunized with these proteins plus S61F showed high serum titers of protein-specific IgG and IgA antibodies that were comparable to those induced by native CT. Further, high protein-specific IgA antibody responses were observed in nasal and vaginal washes, saliva, and fecal extracts as well as increased numbers of IgG and IgA antibody forming cells in cervical lymph nodes and lung tissues of mice intranasally immunized with these proteins and S61F or native CT, but not with recombinant CT-B or protein alone. Both S61F and native CT enhanced the induction of ovalbumin-specific CD4(+) T cells in lung and splenic tissues, and these T cells produced a Th2-type cytokine pattern of interleukin 4 (IL-4), IL-5, IL-6, and IL-10 as determined by analysis of secreted proteins and by quantitation of cytokine-specific mRNA. These results have shown that mutant CT S61F is an effective mucosal adjuvant when administrated intranasally and induces mucosal and systemic antibody responses which are mediated by CD4(+) Th2-type cells.

Cholera toxin (CT), the most commonly used mucosal adjuvant in experimental animals, is unsuitable for humans because of potent diarrhea-inducing properties. We have constructed two CT-A subunit mutants, e.g., serine-->phenylalanine at position 61 (S61F), and glutamic acid-->lysine at 112 (E112K) by site-directed mutagenesis. Neither mutant CT (mCT), in contrast to native CT (nCT), induced adenosine diphosphate-ribosylation, cyclic adenosine monophosphate formation, or fluid accumulation in ligated mouse ileal loops. Both mCTs retained adjuvant properties, since mice given ovalbumin (OVA) subcutaneously with mCTs or nCT, but not OVA alone developed high-titered serum anti-OVA immunoglobulin G (IgG) antibodies (Abs) which were largely of IgG1 and IgG2b subclasses. Although nCT induced brisk IgE Ab responses, both mCTs elicited lower anti-OVA IgE Abs. OVA-specific CD4(+) T cells were induced by nCT and by mCTs, and quantitative analysis of secreted cytokines and mRNA revealed a T helper cell 2 (Th2)-type response. These results now show that the toxic properties of CT can be separated from adjuvanticity, and the mCTs induce Ab responses via a Th2 cell pathway.

An accumulation of elevated numbers of macrophages (M Phi) and Ig producing cells is associated with localized and chronically inflamed gingiva of patients with adult periodontitis. When gingival lymphocytes were isolated from inflamed tissues and examined by flow cytometry, approximately 20-30% of lymphocytes were CD4(+) T cells. For the analysis of Th1 and Th2 cytokine expression by these CD4(+) T cells, RNA was extracted and reverse transcriptase polymerase chain reaction (RT-PCR) was performed by using specific 5' and 3' primers for IFN-gamma and IL-2 (Th1), IL-4, IL-5, IL-6, IL-10 and IL-13 (Th2) and p-actin (housekeeping gene). Two distinct cytokine profiles were noted based on the expression of selected Th1 and Th2 cytokines. Thus, one pattern was represented by the expression of mRNA for IFN-gamma, IL-6, IL-10 and IL-13, while the other case consisted of mRNA for IFN-gamma, IL-6 and IL-13. Except for a few cases, messages for IL-2, IL-4 and IL-5 were not detected by cytokine-specific RT-PCR. The predominant expression of Th2 cytokines (e.g. IL-6, IL-10 and IL-13) may contribute to the induction of high B cell responses in local disease sites. On the other hand, lack of IL-4 may be responsible for the accumulation of M Phi in diseased periodontium. We also investigated whether a relationship exists between IL-4 receptor (IL-4R) expression and M Phi persistence in the absence of exogenous IL-4. Gingival M Phi, when compared with monocytes (MN)/M Phi from peripheral blood mononuclear cells (PBMC), expressed high levels of IL-4R mRNA. When gingival M Phi were incubated with recombinant IL-4 (rIL-4), the cell viability was dramatically reduced by apoptosis. These findings clearly show that the lack of IL-4 may contribute to the persistant occurrence of M Phi at the disease site and addition of exogenous rIL-4 to gingival M Phi cultures leads to cell death by apoptosis.

Background: Antiviral cellular immune responses may influence immunological homeostasis in HIV-infected persons. Recent data indicate that V gamma 9/V delta 2 T lymphocytes display potent cytotoxic activities against human cells infected with certain viruses including HIV. Understanding the role of ya T cells in the course of HIV infection may be helpful for designing novel treatment strategies for HIV-associated disorders. Materials and Methods: The constitutive recognition of Daudi cells and monoethyl pyrophosphate (Etpp) by peripheral blood V gamma 9/V delta 2 T cells was assessed using a proliferation assay. The cytotoxicity of Daudi-stimulated lymphocyte populations was measured by chromium release assays. The HIV infectivity for gamma delta T cell clones was determined by measuring the levels of HIV p24 in cell supernatants. The effect of in vitro HIV-infection on cytokine mRNA production by gamma delta T cell clones was assessed by PCR. Results: The constitutive proliferative responses of peripheral blood V gamma 9/V delta 2 T cells and the lytic functions of Daudi-expanded lymphoid cells from HIV+ persons were substantially diminished in comparison with those of HIV-seronegative persons. These alterations were present in asymptomatic HIV+ persons prior to substantial alpha beta CD4(+) T cell loss. Productive HIV infection of gamma delta T cells in vitro had no measurable effect either on their proliferative response to Daudi stimuli or on the expression of cytokine mRNAs for IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-13. Conclusions: The constitutive responsiveness of V gamma 9/V delta 2 T lymphocytes to Daudi and Etpp is severely altered in HIV+ persons. HIV infection of gamma delta T cells in vitro does not substantially change their cytokine expression or antigenic response.

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 acid 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 IgC2b 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- gamma 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.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum Ige anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.

Mucosal tissues of mice are enriched in T cells that express the gamma/delta T cell receptor. Since the function of these cells remains unclear, we have compared mucosal immune responses in gamma/delta T cell receptor-deficient (TCR delta(-/-)) mice versus control mice of the same genetic background. The frequency of intestinal immunoglobulin (Ig) A plasma cells as well as IgA levels in serum, bile, saliva, and fecal samples were markedly reduced in TCR delta(-/-) mice. The TCR delta(-/-) mice produced much lower levels of IgA antibodies when immunized orally with a vaccine of tetanus toroid plus cholera toxin as adjuvant. Conversely, the antigen-specific IgM and IgG antibody responses were comparable to orally immunized control mice. Direct assessment of the cells forming antibodies against the tetanus toroid and cholera toxin antigens indicated that significantly lower numbers of IgA antibody-producing cells were present in the intestinal lamina propria and Peyer's patches of TCR delta(-/-) mice compared with dir orally immunized control mice. The selective reduction of IgA responses to ingested antigens in the absence of gamma/delta T cells suggests a specialized role for gamma/delta T cells in mucosal immunity.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells), gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta(Dim) and gamma delta(Bright) fractions according to the intensity of gamma delta T-cell receptor expression, The gamma delta(Dim) T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta(Bright) T cells did not express either receptor, Our study also revealed that recombinant murine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta(Dim) T cells but not on gamma delta(Bright) IELs. Thus, treatment of gamma delta(Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta(Bright) T cells did not respond to these two cytokines, The sources of these two cytokines for gamma delta(Dim) T cells were neighboring epithelial cells (IL-7) and alpha beta IELs (IL-2 and IL-7), Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta(Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.