In vivo tolerization of Th1 lymphocytes following a single feeding with ovalbumin: Anergy in the absence of suppression

Oral tolerance is a biologically relevant pathway for inducing peripheral tolerance to foreign antigens. The mechanisms responsible for the tolerant state following feeding with antigen have been shown to involve both anergy and suppression. The demonstration of anergic T lymphocytes following oral tolerance has so far been limited in in vitro systems, and a primary objective of the present study was to provide evidence, in vivo, for the existence of a state of anergy in mice orally fed with ovalbumin (OVA). In addition, it has been shown that peripheral anergy following the intravenous administration of antigen is selectively induced in Th1 lymphocytes. Thus, a second objective of this study was to investigate whether tolerance induced by a feeding regimen known to cause anergy could be selectively limited to Th1 lymphocytes, and whether tolerance induction could be explained by antigen absorption from the gut into the circulation. Oral tolerance was induced by a single feeding with OVA, and was demonstrated by diminished antibody production in vivo, and by reduced cytokine secretion or proliferation in vitro. Anergy, as a mechanism for tolerance, was demonstrated by the ability to reverse the tolerant state after culturing tolerant cells in recombinant interleukin-2 (rIL-2). Reversal of the tolerant state in vivo was established by antibody production in irradiated mice adoptively transferred with cells cultured in the presence of rIL-2. The possibility that suppression was also an in vivo mechanism for tolerance was studied by adoptive transfer experiments. Our results show: 1) that a single dose of orally administered OVA leads to the selective tolerization of Th1 responses (diminished IgG2a, IL-2 and interferon-γ production) with intact Th2 responses (IgG1, and IL-4), 2) that tolerance in vivo is explained by anergy in the absence of active suppression, 3) that exposure of tolerant cells to rIL-2 in vitro abrogates the anergic state both in vitro (proliferation and cytokine secretion) and in vivo (IgG2a production), and 4) that the induction of oral tolerance is inhibited by the presence of antibodies specific for the tolerizing antigen. These findings indicate that the induction of anergy via the oral route might depend on the dissemination of antigen absorbed from the gut. It is suggested that tolerance is guaranteed by the fact that this absorbed antigen is presented to Th1 lymphocytes in the absence of inflammatory and co-stimulatory molecules; these foreign antigens are thus not different from self antigens.     

Systemic effects of oral tolerance on inflammation: mobilization of lymphocytes and bone marrow eosinopoiesis

Oral tolerance is a T-cell mediated phenomenon defined by inhibition of immune responsiveness to a protein previously contacted by the oral route. Oral tolerance may prevent autoimmune and allergic diseases that involve the recruitment and/or activation of different cell types including mast cells, neutrophils, eosinophils, monocytes and lymphocytes. The mechanisms by which oral tolerance avoids these immunological disorders are still controversial. Herein we used a murine model of ovalbumin (OVA)-induced peritonitis to investigate the effect of oral tolerance on allergic inflammation. Frequency of leucocyte subpopulations was evaluated by global and differential cell counts in peritoneal lavage fluid, peripheral blood, and bone marrow. Changes on lymphocyte subsets and adhesion molecules expression by these cells were analysed by flow cytometry. As compared with OVA-immune mice, intraperitoneal challenge of tolerant animals with OVA resulted in a significantly milder peritonitis, mostly affecting neutrophils and eosinophils; a concomitant reduction in total white blood cell counts was also observed, mainly because of lower neutrophil and eosinophil counts. Eosinophils, but not neutrophils, were also reduced in the bone-marrow of OVA-challenged tolerant mice. No changes occurred in total peritoneal lymphocyte counts in OVA-tolerant mice, however, there was a significant decrease in CD3+ CD8+ T cells and an increase in B cells (CD45R+) in these animals as compared to immune OVA-challenged animals. Altered expression of CD18 and CD54, respectively, in blood and peritoneal lymphocytes was also noted. These results suggest that, in addition to local specific effects, oral tolerance has systemic effects on the mobilization of leucocytes and bone-marrow eosinopoiesis.     

Ovalbumin-specific IgE modulates ovalbumin-specific T-cell response after repetitive oral antigen administration

BACKGROUND: Some patients outgrow their food allergies even though their serum antigen-specific IgE levels remain high. OBJECTIVE: To elucidate the role of T cells in outgrowing food allergies in the presence of antigen-specific IgE, we tracked antigen-specific T-cell responses after oral antigen administration. METHODS: Ovalbumin (OVA)-specific T-cell receptor (TCR) and OVA-specific IgE transgenic (Tg) mice (OVA-TCR/IgE-Tg) and OVA-specific TCR Tg (OVA-TCR-Tg) mice were fed with high doses of OVA or PBS every other day. After 7 administrations, OVA-specific proliferation and cytokine production of mononuclear cells of the spleen, mesenteric lymph nodes, and Peyer's patches and the number of splenic CD4 + CD25 + T cells were analyzed. RESULTS: Without OVA administration, the splenocytes from OVA-TCR/IgE-Tg mice exhibited a higher proliferative response and produced more IL-4 and IL-10 and less IFN-gamma than those from OVA-TCR-Tg mice. The proliferative responses of the splenocytes from either OVA-TCR/IgE-Tg mice or OVA-TCR-Tg mice fed with OVA were significantly reduced compared with those from PBS-fed mice. The number of OVA-specific TCR + T cells decreased in the spleen from OVA-fed mice, whereas the number of CD4 + CD25 + T cells increased. The suppressed proliferation of splenocytes of OVA-fed mice was partially resumed by neutralization of TGF-beta1, but not of IL-10. CONCLUSION: The presence of OVA-specific IgE modulated the OVA-specific responses of the splenocytes. Irrespective of the presence of OVA-specific IgE, repetitive oral administration of OVA induced tolerance, which seems to be composed of clonal deletion/anergy and TGF-beta1-mediated active suppression.     

Oral administration of bovine whey proteins to mice elicits opposing immunoregulatory responses and is adjuvant dependent

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.     

Indirect effects of immunological tolerance to a regular dietary protein reduce cutaneous scar formation

Oral tolerance refers to the specific inhibition of immune responsiveness to T‐cell‐dependent antigens contacted through the oral route before parenteral immunization. Oral tolerance to one protein does not inhibit immune responses to other unrelated proteins, but parenteral injection of tolerated antigens plus adjuvant into tolerant, but not normal, mice inhibits immune responses to antigens injected concomitantly or soon thereafter. The inhibitory effect triggered by parenteral injection of tolerated proteins is known as bystander suppression or indirect effects of oral tolerance. Intraperitoneal injection of ovalbumin (OVA) plus alum adjuvant in OVA‐tolerant mice soon before skin injury inhibits inflammation and improves cutaneous wound healing. However, as OVA is not a regular component of mouse chow, we tested whether indirect effects could be triggered by zein, the main protein of corn that is regularly present in mouse chow. We show that intraperitoneal injection of a single dose (10 μg) of zein plus alum adjuvant soon before skin injury in mice reduces leucocyte infiltration but increase the number of T cells and the expression of resistin‐like molecule‐α (a marker of alternatively activated macrophages) in the wound bed, increases the expression of transforming growth factor‐β₃ in the newly formed epidermis and reduces cutaneous scar formation. These results suggest that indirect effects of oral tolerance triggered by parenteral injection of regular dietary components may be further explored as one alternative way to promote scarless wound healing.     

Oral antigen induces antigen-specific activation of intraepithelial CD4+ lymphocytes but suppresses their activation in spleen

Intraepithelial lymphocytes (IELs) are considered to drive immune surveillance of the epithelial layer to the mucosa, which is initially exposed to exogenous antigens. However, how IELs are activated by orally administered antigens remains unclear. To clarify this mechanism, we fed ovalbumin (OVA) to T cell receptor transgenic (TCR-Tg) mice with OVA-specific MHC class II-restricted TCR and found that the cytotoxic activity of IELs was increased against both NK and LAK target cells, but notably reduced after depleting CD8 + IELs. Cytoplasmic staining showed that the production of IFN-gamma and IL-2 was increased in mice fed with OVA both in the supernatant of cultured IELs with immobilized anti-CD3 mAb and in fresh CD4+ IELs. In contrast, the cytotoxic activity against NK and LAK target cells and the production of IL-2 and IFN-gamma was decreased in splenic T cells from mice fed with OVA. However, when the splenic T cells from these mice were cultured with OVA and IL-2, IFN-gamma production recovered. The decreased response demonstrated the clonal anergy of T cells. Furthermore, tumor growth was enhanced in TCR-Tg mice carrying an OVA-transfected counterpart A20 B cell lymphoma (OVA-A20) and fed with OVA. These results indicate that the oral administration of soluble antigens can activate CD4+ IELs in an antigen-specific manner but induces hyporesponsiveness in the spleen. In addition, Th1-type cytokines produced by activated CD4+ IEL might provide a bystander effect on the cytotoxic activity of IELs.     

NKT cells are dispensable in the induction of oral tolerance but are indispensable in the abrogation of oral tolerance by prostaglandin E

NK1.1(+) alpha beta T cells (NKT cells) regulate the Th1/Th2 balance in response to dietary Ag, which may be involved in regulation of oral tolerance. OVA-specific IgE and IgG(1) Ab levels were significantly lower following an i.p. injection of OVA (in CFA) in C57BL/6 mice orally given a single, high dose (25 mg) of OVA than in those orally given PBS. The oral tolerance was normally induced in Jalpha281(-/-) mice which lack Valpha14(+) NKT cells, suggesting that NKT cells are dispensable for induction of oral tolerance. Treatment with PGE(1) or PGE(2 )abrogated the oral tolerance in Jalpha281(+/+) mice; this abrogation was accompanied by an OVA-specific Th2-dominant response. The abrogation of oral tolerance by PGE(1 )was not evident in Jalpha281(-/-) mice. Treatment with PGE(1) induced an early increase in IL-4 production by liver NKT cells in normal mice and neutralization of the early IL-4 by administration of anti-IL-4 mAb abolished PGE(1)-induced abrogation of oral tolerance. These results suggest that liver NKT cells producing IL-4 are responsible for the down-regulation of oral tolerance that is caused by the PGE molecules.     

特异性免疫治疗对哮喘小鼠的作用及机制的初步研究

目的　探讨特异性免疫治疗对哮喘小鼠的作用及其可能机制。方法　通过卵蛋白 (OVA)皮下注射的方法对致敏小鼠进行特异性免疫治疗 ,观察肺组织病理、支气管肺泡灌洗液 (BALF)细胞计数及分类、ELISA检测血清OVA特异性IgE(sIgE)及脾脏T淋巴细胞IL 2和IL 4的分泌 ,3H TdR掺入法检测T淋巴细胞的增殖反应 ,并与OVA致敏及激发的哮喘小鼠相比较。结果　哮喘特异性免疫治疗明显抑制小鼠肺组织炎症病理改变 ;BALF中细胞总数及嗜酸性粒细胞 (EOS)数显著减少 (P <0 .0 5 ) ;血清sIgE显著降低 (P <0 .0 5 ) ;T淋巴细胞IL 2和IL 4的分泌显著降低 (P <0 .0 5 ) ;T淋巴细胞对OVA的特异性刺激的反应显著降低 (P <0 .0 5 )。结论　特异性免疫治疗可显著抑制哮喘小鼠的炎症反应 ;诱导T淋巴细胞无能可能是特异性免疫治疗减轻哮喘相关炎症反应的机制之一     

Immune effector cells induced by complete Freund's adjuvant exert an inhibitory effect on antigen-specific type 2 T helper responses

Background II has hecn well documented that environmental factors such as antigenpresenting cells and related cytokines could affect the development of T helper cells. Objective The purpose of this study is to investigate the effect of different adjuvants on T cell development. Methods Ovalbumin (OVA) combined with aluminum hydroxide (Alum) plus pertussis toxin (PT) or complete Freund's adjuvant (CFA) were used to sensitize mice; the production of IgG and IgE anli-OVA antibodies was then followed. In addition, OVA-specific proliferative responses and cytokine production by spleen cells were also investigated. Results The data showed that the adjuvants themselves could modify the pattern of immune response: (1) IgG_2a > anti-OVA antibody was higher in mice sensitized with OVA + CFA compared to that of mice sensitized with OVA + Alum + PT; (2) the ratio of IFN-γ/IL-4 produced by OVA-stimutated spleen cells was higher in mice sensitized with OVA + CFA than that of mice sensitized with OVA + Alum + PT; (3) increased percentage of γδ T cells was noted in the peritoneal exudate cells of OVA + CFA immunized mice; and (4) the immune response of mice sensitized wilh OVA + Alum+ PT was inhibited by the adoptively transferred ascitic cells from OVA + CFA immunized mice. Conclusion In general, the data suggested higher IgG2a and the ratio of IFN-γ/IL-4 was noted In mice sensitized with OVA + CFA. Further elucidation of the regulatory mechanism of allergen-specific T helper cells development and exploration of possible agents for inmiunotherapy might shed light on the management of atopic diseases.     

Orally induced tolerance. Definition at the cellular level

Several elements of the phenomenon of oral tolerance were examined. It was shown that whereas intragastric (i.g.) exposure of mice to the T-dependent antigens ovalbumin (OVA), bovine serum albumin, and human gamma globulin severely compromised the ability to respond to a subsequent challenge with the homologous antigen, i.g. treatment with T-independent antigens such as dinitrophenylated Ficoll, polyvinylpyrrolidone and bacterial lipopolysaccharide (LPS) did not induce anergy. Furthermore, mice parenterally primed to OVA were not only refractory to the induction of oral tolerance with OVA, but displayed an anamnestic response following i.g. treatment with the antigen. In a final line of investigation, it was shown that whereas mice administered OVA orally lost specific T cell functions such as the ability to (a) provide helper activity; (b) proliferate in response to antigen, and (c) mediate delayed-type hypersensitivity, such animals possessed OVA-specific functional B cells as evidenced by the ability to respond to OVA when the antigen was administered either linked to a recognizable carrier or in conjunction with LPS.     

Chronic inhaled ovalbumin exposure induces antigen-dependent but not antigen-specific inhalational tolerance in a murine model of allergic airway disease

Sensitized mice acutely challenged with inhaled ovalbumin (OVA) develop allergic airway inflammation, characterized by OVA-specific IgE production, airway eosinophilia, increased pulmonary B and T lymphocytes, and airway hyperreactivity. In this study, a chronic exposure model was developed and two distinct patterns of response were observed. Discontinuous inhalational exposure to OVA (6 weeks) produced airway inflammation and hyperreactivity that were similar to acute (10 days) responses. Continuous inhalational exposure to OVA (6 or 11 weeks) resulted in attenuation of airway eosinophilia and hyperresponsiveness without reduction of OVA-specific IgE and IgG(1) levels. The inhibition of airway inflammation was dependent on continuous exposure to antigen, because continuously exposed mice with attenuated inflammatory responses redeveloped allergic airway disease if the OVA aerosols were interrupted and then restarted (11-week-discontinuous). Inhalational tolerance induced by continuous OVA exposure demonstrated bystander suppression of cockroach allergen-mediated airway eosinophilia. These findings may be attributed to changes in production of the anti-inflammatory cytokine interleukin-10 during continuous OVA aerosol exposure. The symptomatic and asymptomatic allergic responses in human asthmatics could be explained by similar variable or discontinuous exposures to aeroantigens.     

Induction of systemic tolerance in normal but not in transgenic mice through continuous feeding of ovalbumin

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2 (IL‐4 and IL‐10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T‐cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)‐β secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF‐β as a regulatory cytokine in oral tolerance in the murine model.     

Transforming growth factor-beta secreted from CD4(+) T cells ameliorates antigen-induced eosinophilic inflammation. A novel high-dose tolerance in the trachea.

The induction of peripheral tolerance is one of the feasible approaches for the control of autoimmunities and allergies. Tolerance induction in the intestine has been studied extensively and therapeutic applications to autoimmunities are in progress, whereas tolerance in the respiratory tract is poorly investigated. We examined the immunoregulatory mechanisms for evading exaggerated inflammatory responses in the murine airway mucosa. Administration of an optimal dose of ovalbumin (OVA) to the trachea elicited eosinophilic inflammation in the trachea of OVA/aluminum hydroxide-sensitized BALB/c mice, whereas higher doses were unable to do so. This failure paralleled the downregulation of interleukin-4 production by mediastinal lymph node (LN) T cells. This high-dose tolerance was attributable to the mechanisms of antigen (Ag)-specific suppression, because the adoptive transfer of CD4(+) LN T cells from the OVA-tolerant mice inhibited the OVA-specific, but not irrelevant Ag KLH-specific, eosinophilic responses. The inhibitory effects were neutralized by the intratracheal administration of anti-transforming growth factor (TGF)-beta, but not that of anti-interferon (IFN)-gamma, monoclonal antibodies, indicating that the high-dose tolerance was mediated by secreted TGF-beta, but not by the dominance of transferred T helper (Th)1 cells over Th2 cells. The pivotal role of TGF-beta was reinforced by the finding that the LN cells from the OVA-tolerant mice produced TGF-beta in response to the in vitro Ag stimulation. These results demonstrate a novel regulatory mechanism in the airway: that TGF-beta secreted by T cells plays an important role in the downmodulation of the immune responses to high doses of Ag which might otherwise induce deleterious inflammation in the airway mucosal tissues.     

Neonatal exposure to staphylococcal superantigen improves induction of oral tolerance in a mouse model of airway allergy

The hygiene hypothesis suggests that lack of microbial stimulation in early infancy may lead to allergy, but it has been difficult to identify particular protective microbial exposures. We have observed that infants colonised in the first week(s) of life with Staphylococcus aureus have lower risk of developing food allergy. As many S. aureus strains produce superantigens with T‐cell stimulating properties, we here investigate whether neonatal mucosal exposure to superantigen could influence the capacity to develop oral tolerance and reduce sensitisation and allergy. BALB/c mice were exposed to staphylococcal enterotoxin A (SEA) as neonates and fed with OVA as adults, prior to sensitisation and i.n. OVA challenge. Our results show that SEA pre‐treated mice are more efficiently tolerised by OVA feeding, as shown by lower lung‐cell infiltration and antigen‐specific IgE response in the SEA pre‐treated mice, compared with sham‐treated mice. This was not due to deletion or anergy of lymphocytes by SEA treatment, because the SEA pre‐treated mice that were fed with PBS showed similar inflammatory response as the sham‐treated PBS‐fed mice. Our results suggest that strong T‐cell activation in infancy conditions the mucosal immune system and promotes development of oral tolerance.     

Different kinetic patterns of cytokine gene expression in vivo in orally tolerant mice

A biologically relevant mechanism of generating peripheral tolerance in T cells that have escaped thymic deletion is by the oral administration of soluble antigens. Oral tolerance occurs by two distinct mechanisms. Feeding a single high dose of antigen induces anergy of antigen-specific TH1 cells, while multiple low doses of antigen induce regulatory Tcells that mediate suppression by producing immunosuppressive cytokines. Although cytokine production in orally tolerant animals has been well studied utilizing in vitro assay systems, semi-quantitative characterization of cytokine production in oral tolerance in vivo has not been carried out. In this paper we have developed a system using semi-quantitative RNA polymerase chain reaction to characterize cytokine gene expression in vivo in mice orally tolerized by feeding either a single high dose or multiple low dosages of antigen. We find that measurable differences in interleukin-4 (IL-4) and interferon-γ (IFN-γ) gene expression occurred between the tolerized and non-tolerized groups. Qualitatively, IL-4 mRNA was increased in both orally tolerized groups. However, significant differences in IL-4 gene expression between the two groups in both magnitude and kinetics were found. A large but short-lived increase in IL-4 was produced in mice fed a single high dose, while a relatively more moderate, longer-lived increase was produced in mice fed multiple low doses. The increase in IL-4 gene expression was specific only to the draining lymph node following antigen administration. Expression of IFN-γ was decreased in both orally tolerant groups. These results indicate that tolerance in TH1 cells is induced by both of these feeding regimens while TH2 responses are intact and amplified upon reexposure to antigen.     

CpG-DNA stimulates cellular and humoral immunity and promotes Th1 differentiation in aged BALB/c mice

We examined whether CpG-DNA could be used as adjuvant to induce a T helper cell type-1 (Th1) immunity in aged BALB/c mice that showed a Th2 polarization. Bordetella pertussis and complete Freund's adjuvant (CFA) were used as well. Immunization with ovalbumin (OVA)/CpG-DNA showed that the immunoglobulin G (IgG)2a/IgG1 ratio and OVA-specific T cell response were similar in young and aged mice. OVA/CpG-DNA induced the secretion of interferon-gamma (IFN-gamma) and absence of interleukin (IL)-5. Similar results were found in mice immunized with OVA/CFA. When mice were immunized with OVA/B. pertussis, we found that the IgG2a/IgG1 ratio and OVA-specific T cell response were lower in aged mice and elicited IFN-gamma and IL-5. In vitro CpG-DNA stimulated antigen-presenting cells to display IL-12 and up-regulate the expression of major histocompatibility complex class II and B7-2 on B cells as efficiently in aged as in young mice, but the up-regulation of B7-1 was stronger in aged mice. The findings demonstrate that CpG-DNA is able to induce a young-like Th1 specific immune response in aged mice.     

Prolonged inhibition of an antigen-specific IgE response in vivo by monoclonal antibody against lymphocyte function-associated antigen-1

Intraperitoneal injection of ovalbumin (OVA) into BALB/c mice caused the induction of OVA-specific IgE production in vivo. However, administration of monoclonal antibody against lymphocyte function-associated antigen-1 (anti-LFA-1 mAb) at days 0 and 1 after OVA immunization resulted in an inhibition of OVA-specific primary and secondary IgE production in a dose-dependent manner. The inhibition of the antigen-specific IgE response due to anti-LFA-1 mAb was seen up to 8 weeks after anti-LFA-1 mAb administration. The OVA-specific IgG1 response was also blocked by anti-LFA-1 mAb. The spleen cells obtained from OVA-immunized mice showed enhanced proliferation against secondary stimulation with OVA in vitro. However, the spleen cells obtained from the mice treated with both OVA and anti-LFA-1 mAb revealed a markedly decreased proliferative responses to OVA, while they showed no reduced responses against keyhole limpet hemocyanin stimulation, indicating that anti-LFA-1 mAb might induce antigen-specific anergy in vivo. It was also demonstrated that treatment of the mice with anti-LFA-1 mAb significantly inhibited the interleukin-4-producing ability of OVA-immunized mouse spleen cells. These results demonstrated that LFA-1-dependent cell-cell interaction is essential for the production of IgE in vivo and may be important in IgE-dependent allergic disease.     

Superantigens and conventional antigens induce different responses in alpha beta T-cell receptor transgenic mice.

While superantigens such as staphylococcal enterotoxin B (SEB) have been shown to induce both clonal deletion and clonal anergy, it is still not known why tolerance rather than memory is induced. To address this issue, we tested the proliferative capacity of T cells from ovalbumin (OVA)-specific alpha beta T-cell receptor transgenic mice primed with either SEB emulsified in complete Freund's adjuvant (CFA) or with OVA peptide, the specific antigen, in CFA. By contrast cells from mice primed with SEB in CFA appeared to be anergic in that they were hyporesponsive to OVA peptide as well as to SEB. The anergic cells could respond to phorbol myristate acetate (PMA) and ionomycin, suggesting that a proximal signal transduction step was affected. Cells from transgenic mice primed with OVA peptide and CFA were not anergic and in fact displayed an enhanced response when they were challenged with OVA in vitro. Thus, when the two antigens are emulsified in CFA and then injected subcutaneously, they behave very differently: the superantigen SEB induces anergy whereas the conventional antigen OVA induces a memory type of response.     

Complementary role of CD4+CD25+ regulatory T cells and TGF-beta in oral tolerance

CD4(+)CD25(+) regulatory T cells are thought to be generated in the periphery as well as in the thymus. We sought to determine the roles played by CD4(+)CD25(+) T cells and transforming growth factor-beta (TGF-beta) in the induction and maintenance of tolerance generated by oral antigens in BALB/c mice. We found that oral administration of a high dose of ovalbumin (OVA) suppressed OVA-specific proliferation and antibody production in BALB/c mice depleted of CD25(+) cells. In contrast, the unresponsiveness induced by lower doses of OVA was only partially blocked by CD25 depletion prior to feeding. Depletion of CD4(+)CD25(+) cells after mice were orally tolerized did not reverse the tolerant status, indicating that these cells were not required to maintain the established tolerance. Furthermore, the induction of oral tolerance was not hampered by the administration of TGF-beta-neutralizing antibodies. However, in mice depleted of CD25(+) cells, anti-TGF-beta-neutralizing antibodies blocked the induction of tolerance, regardless of whether the mice followed the high- or low-dose regimens of oral OVA. CD25 depletion together with TGF-beta neutralization led the expansion of OVA-specific CD4 T cells against the subsequent antigen challenge, and each treatment alone did not. Our findings indicate that CD4(+)CD25(+) T cells and TGF-beta play a complementary role in the induction of oral tolerance, at least in part, by regulating the expansion of antigen-specific CD4 T cells.     

Independent genetic control of B- and T-cell tolerance in strains of mouse selected for extreme phenotypes of oral tolerance

Two strains of mice were genetically selected for susceptibility (TS-Ab/HetS strain) or resistance (TR-Ab/HetS strain) to oral tolerance of the humoral response by using ovalbumin (OVA). The progressive interstrain divergence produced by bi-directional selective breeding during 15 generations demonstrated the polygenic nature of oral tolerance. This paper shows the humoral and delayed-type hypersensitivity (DTH) responses, after intragastric administration of OVA and subsequent immunization with that immunogen in complete Freund adjuvant (CFA). Only the TS-Ab/HetS mice were tolerant for immunoglobulin (Ig)G production with its tolerance degree being the same as that obtained when Al ((OH)(3)) was employed. The DTH reactivity was not correlated to the antibody responsiveness, because both the TS-Ab/HetS and TR-Ab/HetS strains had their DTH reactions suppressed. The cyclophosphamide (Cy) pretreatment prevented DTH suppression on TR-Ab/HetS but do not in TS-Ab/HetS mice. Interstrain difference was also observed for the splenic index in the Cy-treated groups, although the number of splenocytes was the same. Flux cytometry cell analysis showed the recovery of CD3(+) cell numbers in both strains, but only the TR-Ab/HetS mice had their CD4/CD8 pattern restored. These results suggest: firstly, the independent control of DTH and humoral tolerance responsiveness; secondly no support for the clonal anergy concept; and thirdly the matrix proteins neo-synthesis after Cy treatment may facilitate the tolerance abrogation.