Induction of oral tolerance in the primed immune system: influence of antigen persistence and adjuvant form

Oral tolerance is being promoted as a therapy for autoimmune diseases and therefore will need to be functional in a primed immune system. In previous studies, we found that although primed mice could be tolerized by feeding ovalbumin (OVA), the degree of the tolerance and its effects on individual components of the systemic immune response were more limited than that found in naive animals. Here we increased the dose and frequency of antigen feeding in an attempt to extend the effects of oral tolerance in primed mice and to understand why its effects are limited under these conditions. Increasing the amounts of OVA fed, up to a single dose of 400 mg, or using multiple feeds of 5 x 5 or 5 x 25 mg OVA, did not radically alter the extent of tolerance, with DTH responses, antigen-specific proliferation, and IL5 and IFN-gamma production still being tolerized, but antibody responses remaining generally resistant. The deficient tolerance in primed mice could not be overcome by waiting for maximum clonal expansion to wane and was not influenced by persistent release of antigen from a depot adjuvant. We conclude that the resistance of primed mice to oral tolerance may be due to the fact that antigen-experienced T cells may be inherently resistant to induction of tolerance, or that the microenvironment of the primed immune system inhibits the delivery of tolerogenic signals to antigen-specific T cells.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.     

Immunosuppressive activity of Semen Persicae ethanol extract on specific antibody and cellular response to ovalbumin in mice

The immunosuppressive activity of the ethanol extract of Semen Persicae (EESP) was studied with respect to specific antibody and cellular response to ovalbumin (OVA) in mice. The effects of EESP on mice splenocyte proliferation in vitro were measured. EESP significantly suppressed concanavalin A (ConA)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. Furthermore, the effects of EESP at three dose levels on the humoral and cellular immune responses in the OVA-immunized mice were examined. ICR Mice were immunized subcutaneously with OVA on day 0 and 14. Starting on the day of immunization, the mice were administered intraperitoneally with EESP at a single dose of 0.25, 0.5, and 1.0 mg, and cyclosporin A (CsA, positive drug) at a single dose of 0.1 mg at intervals of 7 days. On day 28, mitogen- and OVA-induced splenocyte proliferation and OVA-specific antibody level in serum were measured. EESP significantly decreased ConA-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at the dose of 1.0 mg. Meanwhile, the OVA-specific serum IgG, IgG1, and IgG2b antibody levels in the OVA-immunized mice were markedly reduced by EESP in a dose-dependent manner. The results suggest that EESP could suppress the cellular and humoral immune response in mice, and deserve further research to be developed as immunosuppressant.     

Immunosuppressive Activity of the Ethanol Extract of Sedum sarmentosum and Its Fractions on Specific Antibody and Cellular Responses to Ovalbumin in Mice

The immunosuppressive activity of the ethanol extract of Sedum sarmentosum (EESS) and its fractions was studied with respect to specific antibody and cellular response to ovalbumin (OVA) in mice. ICR Mice were immunized subcutaneously with OVA on days 0 and 14. Beginning on the day of immunization, the mice were administered intraperitoneally (ip) with EESS and it fractions at a single dose of 0.25, 0.5, and 1.0 mg, and cyclosporin A at a single dose of 0.1 mg at intervals of 7 days. On day 28, splenocyte proliferation and specific antibody level in serum were measured. EESS significantly suppressed concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and OVA‐induced splenocyte proliferation in the immunized mice in a dose‐dependent manner. The OVA‐specific serum IgG, IgG1, and IgG2b levels in the immunized mice were also markedly reduced by EESS. Among four fractions of EESS, the BuOH fraction consisting mainly of flavonoid glycosides showed the highest suppressive activity. The results suggest that EESS could suppress the cellular and humoral immune response in mice, and deserve further research to be developed as immunosuppressant.     

Suppression of oral tolerance by Lactococcus lactis in mice

Although oral ovabumin (OVA) administration suppressed the antibody (Ab) response in OVA-immunized mice, Lactococcus lactis increased OVA-specific IgG2a in these mice. L. lactis increased the casein-specific IgG level in NC/Nga mice fed on a casein diet. The percentage of CD4(+)CD25(+) cells was increased in DO11.10 mice orally given OVA, but this increase of CD4(+)CD25(+) cells were suppressed in L. lactis-fed DO11.10 mice.     

Immunosuppressive activity of the ethanol extract of Siegesbeckia orientalis on the immune responses to ovalbumin in mice

The in vitro and in vivo immunosuppressive activity of the ethanol extract of Siegesbeckia orientalis (EESO) was studied on the immune responses in mice. EESO significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. ICR Mice were immunized subcutaneously with ovalbumin (OVA) on days 0 and 14. Beginning on the day of immunization, the mice were administered intraperitoneally with EESO at a single dose of 0.25, 0.5, and 1.0 mg at intervals of 7 days. On day 28, OVA-specific antibodies in serum, and mitogen- and OVA-induced splenocyte proliferation were measured. EESO significantly suppressed Con A-, LPS- and OVA-induced splenocyte proliferation in the OVA-immunized mice in a dose-dependent manner. The OVA-specific serum IgG, IgG1, and IgG2b levels in the OVA-immunized mice were also significantly reduced by EESO. Moreover, reducing effect on the IgG1 antibody of EESO at the dose of 1.0 mg was more significant than that of cyclosporin A (CsA; positive drug). The results suggest that EESO could suppress the cellular and humoral response to ovalbumin in mice, and deserve further investigations to be developed as immunosuppressant.     

Oral-tolerance induction in diet-induced obese mice

OBJECTIVE: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. RESEARCH METHODS AND PROCEDURES: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. RESULTS: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. DISCUSSION: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

Inhibition of specific immune responses by feeding protein antigens. V. Induction of the tolerant state in the absence of specific suppressor T cells

The effect of CY pretreatment on the ability of OVA feeding to induce both tolerance and active suppression was examined in mice. CY-pretreated, OVA fed mice were fully unresponsive in both OVA-specific DTH and antibody responses, but, in contrast to untreated OVA-fed mice, did not transfer suppression to normal recipients via splenic lymphocytes. Restoration of Ts activity in CY-pretreated mice was accomplished by reconstitution with normal T cells before antigen feeding, indicating that the CY effect was at the Ts precursor level. In addition, it was found that certain OVA-specific immune parameters (DTH and splenic PFC responses) in recipient mice were susceptible to suppression by transfer of spleen cells from OVA-fed donors, whereas other measures (antigen-induced T cell proliferation and serum antibody titers) were not. The data suggest that CY-sensitive Ts are not necessary for either induction or maintenance of specific tolerance after OVA feeding.     

Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen

Induction of oral tolerance has long been considered a promising approach to the treatment of chronic autoimmune diseases, including rheumatoid arthritis (RA). Oral administration of type II collagen (CII) has been proven to improve signs and symptoms in RA patients without troublesome toxicity. To investigate the mechanism of immune suppression mediated by orally administered antigen, we examined changes in serum IgG subtypes and T-cell proliferative responses to CII, and generation of IL-10-producing CD4+CD25+ T-cell subsets in an animal model of collagen-induced arthritis (CIA). We found that joint inflammation in CIA mice peaked at 5 weeks after primary immunization with CII, which was significantly less in mice tolerized by repeated oral feeding of CII before CIA induction. Mice that had been fed with CII also exhibited increased serum IgG1 and decreased serum IgG2a as compared with nontolerized CIA animals. The T-cell proliferative response to CII was suppressed in lymph nodes of tolerized mice also. Production of IL-10 and of transforming growth factor-beta from mononuclear lymphocytes was increased in the tolerized animals, and CD4+ T cells isolated from tolerized mice did not respond with induction of IFN-gamma when stimulated in vitro with CII. We also observed greater induction of IL-10-producing CD4+CD25+ subsets among CII-stimulated splenic T cells from tolerized mice. These data suggest that when these IL-10-producing CD4+CD25+ T cells encounter CII antigen in affected joints they become activated to exert an anti-inflammatory effect.     

Oral tolerance revisited: prior oral tolerization abrogates cholera toxin-induced mucosal IgA responses

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced IgG 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 mucosal IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS. Furthermore, when OVA-specific Ab-forming cells (AFCs) in both systemic and mucosa-associated tissues were 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.     

Tolerizing effect of DNP-ficoll on IgE antibody production.

A/J and DBA/1 mice were infected with 750 third-stage larvae of Nippostrongylus brasiliensis and immunized with 1 mug dinitrophenylated N. brasiliensis extract (DNP-Nb) with 1 mg Al(OH)3 to produce high titers of anti-hapten IgG1 and IgE antibody. Partial tolerance to the production of anti-hapten IgG1 and IgE antibody could be induced by DNP-Ficoll from 5 weeks before to 1 week after the DNP-Nb immunization. The tolerized state persisted through the duration of the experiments. However, no tolerizing effect could be demonstrated on secondary antihapten IgE antibody production induced by DNP-Nb. Moreover, DNP-Ficoll failed to evoke anti-hapten IgG1 or IgE antibody production.     

Kinetic analysis of oral tolerance: memory lymphocytes are refractory to oral tolerance.

Oral administration of soluble Ag before immunization induces peripheral tolerance and is effective in suppressing animal models of autoimmune diseases. Although tolerance induction in primed animals is more clinically relevant, it is not well studied. Therefore, this study was designed to examine the feeding effects on different phases of the immune response. We observed that feeding a single high dose (250 mg) of OVA to OVA-primed BALB/c mice could induce OVA-specific suppression in the Ab production and T cell proliferation only at the naive and the activation phases of the immune response, whereas multiple high doses (100 mg/feed for 10 days) were effective at the effector phase. OVA-specific IL-4 production in culture supernatant was also suppressed in the tolerized groups. However, when the mice had resting memory lymphocytes, even multiple feeding regimens were not effective in tolerance induction, although multiple low doses (1 mg/feed for 10 days) partially suppressed Ab production. This phenomenon was confirmed by adoptive transfer study. Nevertheless, the reactivated memory response was suppressed partially by multiple high doses. Our findings have an important implication for understanding the mechanism of oral tolerance and for the therapeutic applications of oral tolerance to autoimmune diseases.     

Haemolytic activity and adjuvant effect of notoginsenoside K from the roots of Panax notoginseng

Notoginsenoside K (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD50 value) being 318+/-13 microg/ml, on a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1, especially at a dose of 25 mug compared to an OVA control group (P<0.001). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (AlOH gel; P<0.01). These results suggest that 1 exhibits a slight haemolytic activity and a significant adjuvant effect on specific antibody and cellular response against OVA in mice.     

Immunological adjuvant effect of ginsenoside Rh4 from the roots of Panax notoginseng on specific antibody and cellular response to ovalbumin in mice

Ginsenoside Rh(4) (1), a saponin isolated from the roots of Panax notoginseng (Burk.) F. H. Chen, was evaluated for its haemolytic activity and adjuvant potential on specific antibody and cellular response to ovalbumin (OVA) in mice. Compound 1 showed a slight haemolytic effect, its concentration inducing 50% of the maximum haemolysis (HD(50) value) being 407+/-12 microg/ml using a 0.5% suspension of red blood cells. Compound 1 significantly increased the concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-induced splenocyte proliferation in OVA-immunized mice especially at a dose of 25 microg (P<0.05, P<0.01, or P<0.001). The OVA-specific serum IgG, IgG1, and IgG2b antibody levels were also significantly enhanced by 1 at a dose of 25 microg compared to the OVA control group (P<0.05 or P<0.01). Moreover, the enhancing effect of 1 on the OVA-specific IgG2b antibody responses to OVA in mice was more significant than that of Alum (Al(OH)(3) gel; P<0.01). These results suggest that 1 could be safely used as adjuvant with low or non-haemolytic effect.     

Dietary melibiose regulates th cell response and enhances the induction of oral tolerance

We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.     

Immunological Adjuvant Effect of a Water‐Soluble Polysaccharide,CPP, from the Roots of Codonopsis pilosula on the Immune Responses to Ovalbumin in Mice

In this study, one water‐soluble polysaccharide, CPP, was purified from the root of Codonopsis pilosula. The immunomodulatory effect and the adjuvant potential of CPP on the cellular and humoral immune response of ICR mice against ovalbumin (OVA) were investigated. CPP was shown not to be lethal in vivo for mice in doses ranging from 0.5 to 4 mg. ICR Mice were immunized subcutaneously with 0.1 mg of OVA alone or with 0.1 mg of OVA dissolved in saline‐containing aluminum hydroxide gel (Alum) (0.2 mg), QuilA (0.01 and 0.02 mg) or CPP (0.5, 1 or 2 mg) on days 1 and 15. Two weeks later (day 28), concanavalin A (ConA)‐, lipopolysaccharide (LPS)‐, and OVA‐stimulated splenocyte proliferation, and OVA‐specific serum antibodies were measured. CPP significantly enhanced the ConA‐, LPS‐, or OVA‐induced splenocyte proliferation in the OVA‐immunized mice especially at a dose of 1 mg (P<0.05 or P<0.01). The OVA‐specific IgG, IgG1, and IgG2b antibody levels in serum were also significantly enhanced by CPP compared with OVA control group (P<0.05 or P<0.01). The results suggest that CPP could be a safe efficacious adjuvant for use in vaccines against both pathogens and cancer.     

Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.     

Dietary Melibiose Regulates Th Cell Response and Enhances the Induction of Oral Tolerance

We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.