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Oral administration of a protein without adjuvant brings about oral tolerance (systemic hyporesponsiveness) to that protein by mechanisms such as antigen-induced apoptosis. We monitored the number and apoptosis induction of CD4+ T cells in antigen-specific T cell receptor transgenic mice fed the antigen ovalbumin to identify where events leading to oral tolerance occurred. The antigen was distributed throughout the body, causing apoptosis and a decrease in cell number of CD4+ T cells in most of the lymphoid system: the spleen, peripheral lymph nodes, and the thymus which was not previously reported to be affected. Although apoptosis was induced in the Peyer's patches, the cell number did not change. Unexpectedly, T cells in the mesenteric lymph nodes did not undergo apoptosis; instead, they were more numerous as compared to that in the case of control animals not administered the antigen. The results suggested that the orally administered antigen activated the intestinal immune system, while it induced immune tolerance in other sites.

We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) gammadelta+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCR alphabeta+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCR alphabeta+/TCR gammadelta+ ratio was mainly observed in a CD8 alphaalpha+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCR gammadelta+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCR gammadelta+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-gamma. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.

Background: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific Th1 and Th2 responses and IgE responses., Methods: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA., Results: The level of production of antigen-specific interferon-gamma by spleen cells was significantly higher in the OVA-TCR Tg mice fed the NT(+) diet than in the control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet., Conclusion: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response., (Copyright 2000 S. Karger AG, Basel)

Background: The mechanism by which orally ingested allergens elicit an IgE response remains unclear because there are few animal models available for investigation of this response., Objective: We tried to develop a murine model suitable for investigation of the IgE response to orally ingested allergens, which would allow us to identify T cells that could promote IgE production., Methods: Ovalbumin (OVA)-specific T-cell receptor transgenic mice were fed a diet containing OVA, and both the serum antibody response and cytokine production by splenocytes were examined., Results: Oral administration of OVA to transgenic mice led to an increase in the levels of both antigen-specific IgE and total IgE in the sera. Subsequent intravenous challenge of OVA-fed transgenic mice with OVA resulted in anaphylactic shock. Analysis of cytokine production by splenocytes revealed that high IL-4-producing T cells appeared in the spleen 1 week after the start of feeding the OVA diet. T cells from these mice were found to promote IgE secretion by BALB/c B cells in vitro. This helper activity and the levels of IL-4 secretion were diminished after long-term feeding. These findings suggest the possibility that the orally ingested antigen elicited a response by a subpopulation of T cells that produce high levels of T(H2)-type cytokines and that promote IgE secretion, and these same T cells were tolerized by the orally ingested antigen., Conclusion: This experimental model with transgenic mice may be a useful tool for further studies of the cellular and molecular mechanisms of the T-cell and IgE responses to orally ingested antigens.

Modulation of CD8(+) T-cell responses specific for an exogenous antigen by epitope variants would be advantageous to develop a novel means of antigen-specific immune regulation. We have analyzed CD8(+) T-cell responses to single amino acid-substituted variants of a peptide corresponding to residues 142-149 (p142-149; LAYFYPEL) of alphas1-casein, a major milk allergen, which is a dominant determinant restricted by H-2Kb. An analog peptide L142I with a substitution of Ile for Leu at the nonanchor N-terminal residue induced more IFN-gamma secretion than p142-149 from specific CD8(+) T cells. Furthermore, L142I could prime CD8(+) T cells more efficiently in vivo, and these L142I-primed cells secreted more IFN-gamma than p142-149-primed CD8(+) T cells upon stimulation with p142-149 in vitro. These findings are mainly explained by the greater ability of L142I to form stable Kb-peptide complexes. These findings indicate that appropriate analog peptides may be useful as efficient inducers of CD8(+) T cells which recognize the parent peptide and secrete IFN-gamma, a potent inhibitor of Th2-dependent events, including IgE production., (Copyright 1998 Academic Press.)

Collagen-induced arthritis (CIA) is an autoimmune animal model for some types of human rheumatoid arthritis (RA). We have evaluated the effectiveness of intranasal administration of antigen in inhibiting CIA in DBA/1 mice. The intranasal administration of heat-denatured or trypsin-digested bovine type II collagen (CII) before immunization with CII strongly delayed the onset of CIA, whereas administration of native CII did not do so. The mice administered denatured or digested CII possessed much lower titers of anti-CII IgG2a than the control mice, whereas titers of anti-CII IgG1 and IgG2b were unchanged or slightly decreased. Responding to CII and peptides containing immunodominant T cell determinants, lymph node cells from mice administered denatured CII produced less IFN-gamma. These results suggest that intranasal administration of antigen downregulated preferentially Th1-type responses, whereas an enhanced Th2-type response was not observed. We demonstrate that the methods shown here are a possible treatment for rheumatoid arthritis., (Copyright 1998 Academic Press.)

The participation of each lymphocyte compartment in the induction of oral tolerance for antibody response was investigated by means of a new cell-transfer experimental system, using severe combined immunodeficiency (SCID) mice. Various lymphocyte compartments from BALB/c mice were transferred into SCID mice and these mice were evaluated for oral tolerance induction. First, whole splenocytes from BALB/c mice were transferred into SCID mice and these mice were orally administered bovine alpha s1-casein. The specific antibody response in these mice after subsequent immunization with antigen was greatly reduced compared to controls which were not fed the antigen, and it was demonstrated that oral tolerance was induced in SCID mice bearing donor splenocytes. Oral tolerance was induced in SCID mice that were reconstituted with only T cells, revealing that B cells were not required for the induction of oral tolerance. Further, oral tolerance was induced in SCID mice reconstituted with CD8-depleted splenocytes but not in mice reconstituted with only CD8+ T cells. These results demonstrate that oral tolerance could be induced in SCID mice bearing normal splenocytes and that interaction of CD4+ T cells with antigen-presenting cells other than B cells are responsible for the induction of oral tolerance. Our experimental system may be useful for investigations with human lymphocytes.

Background: Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host., Methods: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice., Results: L. casei induced IFN-gamma, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-gamma. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody., Conclusions: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.

The CD8+ T cell clone 5F1 produces interleukin 10 (IL-10) and interferon gamma(IFN-gamma) in response to stimulation with a peptide corresponding to region 142-149 of bovine alpha(s1)-casein (p142-149). Ninety analog peptides derived from p142-149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL-10 and IFN-gamma by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL-10 production whereas others induced production of IFN-gamma alone or both of these cytokines. Peptides inducing IFN-gamma production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL-10 but not IFN-gamma triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL-10 production in CD8+ T cells differs from that required for IFN-gamma production. The distinct cellular signals for IL-10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.

Feeding of a whole casein diet, which abolished the α(s1)-casein-specific proliferation and IFN-γ productivity of CD(4+) T cells, did not affect the proliferative response of CD8(+) T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8(+) T cells, we established α(s1)-casein-specific CD8(+) T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8(+) T cells in oral tolerance is discussed.

The systemic immune response and tolerance induced by oral administration of various doses of bovine alpha s1-casein were examined, focusing on cytokine responses in this study. Spleen cells from mice fed low doses of antigen secreted interferon-gamma and interleukin-2 in response to in vitro antigen stimulation, indicating that a Th1-type response was induced. In these mice, the Th1 responses after subsequent immunization and boosting with the antigen were diminished. In the case of mice fed high doses of the antigen, secretion of Th1 cytokines was minimal, but systemic responses after subsequent immunization and boosting with the antigen were strongly inhibited. Active suppression was not observed at any dose. The results indicate that, in this system, low-dose feeding induced activation of Th1 cells followed by tolerization, while high-dose feeding induced profound tolerance without prior activation. Our results have implications for clinical application of oral tolerance to allergy and autoimmune disease.

Intestinal intraepithelial T cells (IELs) expressing either gammadelta TCR or alphabeta TCR have been proposed to play an important role in the regulation of intestinal epithelia by producing cytokines that directly influence the adjoining intestinal epithelial cell (IEC) functions. To illuminate this issue, we utilized TCR mutant mice to obtain gammadelta IELs, alphabeta IELs and mixed gammadelta and alphabeta IELs from corresponding alphabeta T-cell-deficient (beta-/-), gammadelta T-cell-deficient (delta-/-) and wild-type (WT) littermate mice. The production of IFN-gamma by these IELs as well as the mRNA for IFN-gamma, TGF-alpha, TGF-beta1, TNF-alpha and TNF-beta in these IELs, in conjunction with the effect of produced cytokines on the expression of class II MHC molecules by the in vitro cell line IEC-6, were investigated. IFN-gamma and TNF-alpha [corrected] specific mRNA were detectable in all freshly isolated gammadelta, alphabeta and WT IELs. In addition to the IFN-gamma and TNF-alpha [corrected] mRNA, alphabeta and WT IELs that had been activated in culture plates coated with anti-CD3 mAb contained mRNA for TGF-beta1 and TNF-beta proteins. In the cultured gammadelta IELs, however, the signals for IFN-gamma and TNF-alpha [corrected] transcripts were weak, and mRNA for the latter two cytokines was almost undetectable. Supernatants from in vitro culturing of alphabeta and WT IELs but not gammadelta IELs induced class II MHC gene expression in IEC-6, whereas, in the presence of anti-IFN-gamma mAb, the same culture supernatants failed to do so. In fact, the concentration of IFN-gamma in supernatants from alphabeta and WT IEL cultures was ten- to twentyfold higher than that in the supernatant from the gammadelta IEL culture. Finally, TGF-alpha specific mRNA was not detectable in the gammadelta and alphabeta IELs even after in vitro activation. These results indicate that alphabeta IELs are superior to gammadelta IELs in the ability to produce IFN-gamma, TGF-beta1, TNF-alpha [corrected] and TNF-beta through TCR crosslinking primary in vitro stimulation.

To study cow's milk allergy at the cellular level, we assessed the reactivity of peripheral blood mononuclear cells from patients allergic to cow's milk to alpha s1-casein, which is one of the major allergens in cow's milk. Proliferation of the cells to alpha s1-casein activation showed a rather weak response. Therefore to understand T-cell reactivity to alpha s1-casein in more detail, we prepared alpha s1-casein-specific T-cell lines from patients allergic to cow's milk and established 26 T-cell lines. These T-cell lines could be classified into three groups by analyzing their surface marker expression: those containing predominantly CD4+ CD8- T cells, those containing both CD4+CD8- and CD4-CD8+ T cells, and those containing predominantly CD4- CD8+ T cells. The CD8+ T cells were obtained at an unexpectedly higher frequency from the patients. These T-cell lines produced interferon-gamma and IL-4. These results suggest that CD8+ T cells specific for alpha s1-casein and CD4+ T cells were primed by the stimulation with alpha s1-casein in patients allergic to milk and that both T cells may play a key role in the onset, progression of, or recovery from cow's milk allergy.

Immunological tolerance of systemic immunity can be induced by the oral administration of an exogenous antigen, which is termed oral tolerance. We examined whether there was a difference in the degree of tolerance between individual antigenic determinants in oral tolerance. Feeding bovine alpha s1-casein, a major protein in cow's milk, as a constituent of the diet induces oral tolerance in mice. However, a weak antibody response can be elicited in the alpha s1-casein-fed animals by subsequent immunization with the antigen. We examined the fine specificity of such anti-alpha s1-casein antibodies produced in alpha s1-casein-fed mice. The results demonstrated that there was a difference in the inhibition of antibody response between different B cell determinants. Differential inhibition could also be observed for T cell responses. T cells specific for dominant determinants were preferentially tolerized, while those for cryptic determinants escaped oral tolerance. Our results imply the importance of antigen presentation for this differential inhibition of antigenic determinants. We suggest that orally administered antigen does not induce tolerance to some of the B and T lymphocyte repertoire which could potentially induce harmful food hypersensitivity.

Milk whey protein fed to mice as a constituent of the diet elicited a systemic humoral response. The serum antibodies were of the IgG class, which recognized mainly beta-lactoglobulin. Feeding whey protein diet also induced tolerance of systemic immunity (oral tolerance). However, when heat-denatured whey protein was fed, the animals showed only a poor serum response, while oral tolerance was induced to a similar degree as that of native whey protein diet. The results demonstrate the difference of antigen recognition between oral tolerance and oral immunization and suggests that our system using mice may be a useful animal model for food allergy.

Feeding mice with bovine whole casein as a diet constituent elicited an anti-alpha s1-casein (alpha s1-CN) systemic humoral response. The mice fed with the casein (CN) diet responded poorly to subsequent parenteral immunization with the antigen, indicating that oral tolerance was induced in the animals. The serum antibodies elicited by feeding CN were tested for binding to a panel of synthetic peptides and are shown to recognize only a limited number of potential antigenic determinants on alpha s1-CN. Our results suggest that a similar mechanism may be involved in the onset of milk allergy.

A tryptic peptide of bovine beta-casein (amino acid residues 184-202) was used as a model antigen to investigate how the relative position of the (helper) T cell and B cell determinants on a protein antigen influences the antibody response. Immunization with the peptide elicited a considerably higher anti-peptide response in the C3H/He strain than in the C57BL/6 strain, despite the fact that the C57BL/6 T cells showed higher reactivity than the C3H/He T cells. The T cell and B cell determinants of the peptide were identified in these two strains. Each strain recognized a single B cell determinant and a single T cell determinant. In the C3H/He strain, the T and B cell determinants were located apart from one another, while the T and B cell determinants of the C57BL/6 strain were located in a region close each other. The results suggest that the level of an antibody response depends on the topological relationship of the T cell and B cell determinants on the antigen molecule.

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.