Persistence of oral tolerance in mice fed ovalbumin is different for humoral and cell-mediated immune responses.

The duration of oral tolerance after a single feed of OVA at the age of 6 weeks was studied in BDF1 mice. Significant suppression of systemic antibody responses was present 3 months later, but not at 6.5 months; in contrast, at all times studied from 2 weeks to 17 months after an OVA feed there was suppression of systemic CMI to OVA as measured by an in vivo skin test. This indicates that the two limbs of the immune response differ in the factors responsible for the maintenance of oral tolerance.     

Genetic control of oral tolerance to ovalbumin in mice.

We have investigated the genetic basis of oral tolerance to OVA in a number of inbred mouse strains. Our results emphasise the efficiency of the oral route for inducing tolerance and provide evidence for both MHC and non-MHC linked control of oral tolerance.     

Failure of SCID mice to generate an oral tolerogen after a feed of ovalbumin: a role for a functioning gut-associated lymphoid system.

The role of the mucosal immune system in the generation of circulating tolerogenic ovalbumin (OVA) moieties has been investigated after a single feed of the protein. Serum collected from SCID mice 1 hr after a 25-mg feed of OVA was unable to transfer tolerance of delayed-type hypersensitivity (DTH) into naive BALB/c recipients. This is in contrast to serum collected from BALB/c mice which was able to transfer DTH tolerance to naive BALB/c recipients. The levels of circulating OVA detected in the serum of SCID mice 60 min after feeding OVA were approximately half those detected in the serum of BALB/c mice at the same time-point. However even dose adjustment of SCID mouse serum to a level of immunoreactive OVA equivalent to that found in BALB/c serum was unable to induce DTH tolerance in BALB/c recipients. This failure of SCID serum to transfer tolerance was shown to be unrelated to the germ-free conditions under which SCID mice are kept. Serum from OVA-fed germ-free BALB/c mice transferred DTH tolerance at equivalent levels to serum from conventionally reared BALB/c mice. When the intestinal morphology and intraepithelial lymphocyte (IEL) numbers in the duodenum of SCID mice were compared to conventionally reared and germ-free BALB/c controls, SCID mice were characterized by a lower number of IEL with a different morphology from the majority of IEL found in BALB/c mice.     

Covalent coupling of palmitate to ovalbumin inhibits and blocks the induction of oral tolerance

Oral tolerance is a phenomenon that may occur in animals exposed to soluble antigens for the first time by the oral route. In the present study, we show that oral tolerance against ovalbumin (Ova) can be obtained after intragastric administration of the antigen in the presence of free residues of palmitate. On the other hand, oral tolerance induction is blocked when the residues of palmitate are covalently bound to the antigen (Ova‐palmitate conjugates). We have also noticed that oral administration of Ova‐palmitate conjugates can boost and/or prime experimental animals for Ova‐specific cellular and humoral systemic immune responses. Oral treatment with the conjugates also induces the production of local secretory immunoglobulin A (IgA) as measured in intestinal washes. Furthermore, Ova‐palmitate given orally can inhibit oral tolerance induction by naïve Ova.     

The genetically determined production of the alarmin eosinophil‐derived neurotoxin is reduced in visceral leishmaniasis

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis. Recent findings indicate that dendritic cells have a key role in the defense against the Leishmania parasite and that the activity of this cell may be modified by the eosinophil secretory protein eosinophil‐derived neurotoxin (EDN). We hypothesized that the interactions between dendritic cells and EDN might be of importance in the disease development. Cellular content of EDN was analyzed by ELISA. The single‐nucleotide polymorphisms at positions 405, 416, and 1122 in the EDN gene were analyzed by real‐time PCR with TaqMan^® reagents. The study cohorts comprised 239 Sudanese subjects (65 healthy controls and 174 with VL) and 300 healthy Swedish controls. The eosinophil content of EDN was lower in VL as compared with controls (p < 0.0001). The EDN405 (G>C) genotype distribution was similar among Swedish and Sudanese controls, whereas VL subjects had a higher prevalence of the EDN405‐GG genotype (p < 0.0001). The content of EDN in the eosinophils was closely linked to the EDN405 polymorphism (p = 0.0002). Our findings suggest that the predisposition to acquire VL is related to the genetic polymorphism of the EDN gene and the reduced production by the eosinophil of this gene product.     

A method for estimation of circulating immune complexes after oral challenge with ovalbumin

Sera from forty-two children orally challenged with ovalbumin were analysed for the presence of IgG-ovalbumin immune complexes. The technique used is based on the adsorption of the IgG of the complex to immobilized protein-A and detection of the ovalbumin of the complex on the solid phase, using isotope-labelled, pepsin-digested antibodies to ovalbumin. Children with high serum levels of antibody to ovalbumin had significantly higher concentrations of IgG-ovalbumin complexes than children with low antibody levels. In both groups the appearance of the complexes in the serum was accompanied by a significant decrease of the complement factor C3 (P < 0.05). The duration of the decrease in C3 was correlated to the amount of IgG-ovalbumin complexes, suggesting a consumption of C3 by these immune complexes.     

Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit.

Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).     

Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions

Our past studies showed that Peyer's patches were required for the induction of oral tolerance to the protein antigen ovalbumin (OVA), but not to the hapten 2,4,6-trinitrobenzene sulfonic acid (TNBS). In the present study, the effects of immunosenescence on oral tolerance induction were assessed with these two toleragens. Significant reductions in OVA-specific serum IgG antibody and CD4(+) T cell responses to subsequent challenge were observed in OVA-fed, young adult mice. Importantly, these reduced anti-OVA antibody responses were associated with delayed-type hypersensitivity, and antigen-induced CD4(+) T(h)1- and T(h)2-type cytokine responses. On the other hand, aged mice fed OVA failed to develop oral tolerance. Thus, CD4(+) T cells from Peyer's patches produced selected T(h)2- but no T(h)1-type cytokines. The TNP-specific serum IgG antibody and T cell responses were significantly diminished by prior TNBS feeding in young adult, 6- to 8-month-old and 12- to 14-month-old, but not in senescent, 2-year-old mice. Finally, we have directly assessed dendritic cell subsets and T cell responses in Peyer's patches, and their function in tolerance induction was impaired at an earlier stage of life. These results suggest that lack of oral tolerance to the protein OVA during aging is the result of dysfunctional Peyer's patches.     

Differences in the target cells for tolerance induction in relation to the dose of tolerogen

Bone marrow cells and thymus cells were observed in cell transfer experiments to collaborate in the production of anti-BSA antibodies. The target cells for tolerance induction either with a `low dose' of antigen (100 μg of deaggregated BSA once a week × 5) or with a `high dose' (a single injection of 5000 μg) were identified by the same technique. In `low dose' tolerance, some indication was obtained that thymus-derived cells in peripheral lymphoid systems were the target cells; bone marrow-derived cells appeared not to be so susceptible, and the cells residing in thymus or bone marrow seemed to remain unimpaired. In contrast, the injection of a `high dose' of tolerogen rendered both types of cells in spleen, thymus cells and bone marrow cells, unresponsive or hyporesponsive in parallel with one another.     

The role of antigen recognition and suppressor cells in mice with oral tolerance to ovalbumin.

The induction of tolerance by feeding proteins may prevent potentially harmful delayed-type hypersensitivity (DTH) reactions to food antigens. Suppressor T cells (Ts) are present in mice with tolerance of systemic DTH after feeding ovalbumin (OVA) but, as other immunoregulatory mechanisms have also been described, the exact role of Ts in maintaining tolerance is not known. In this study, we have used the ability of native and denaturated OVA to cross-react at the level of helper/effector T cells, but not Ts, to re-examine the role of Ts in oral tolerance to OVA. Mice immunized with native OVA (nOVA) or denatured OVA (dOVA) in adjuvant had fully cross-reacting DTH to either nOVA or dOVA, but intravenous administration of antigen induced Ts which were specific for the appropriate form. Mice fed nOVA or dOVA had identical tolerance of systemic DTH to both forms of OVA, and feeding nOVA induced splenic Ts which suppressed the DTH response to both nOVA and dOVA. Splenic Ts could not be detected in mice fed dOVA. The results support the hypothesis that tolerance of systemic DTH in mice fed native proteins is due to Ts. Although, for the moment, there is no complementary evidence for a role for Ts in oral tolerance to denatured proteins, this study is consistent with the idea that Ts are the mechanism which normally prevent enteropathy due to DTH against dietary proteins. In addition, our study underlines the differences between orally and parenterally induced Ts and reinforces the view that fed proteins induce Ts after processing by the gut or its lymphoid accessory cells.     

Suppressor T cells, antigen-presenting cells and the role of I-J restriction in oral tolerance to ovalbumin.

Suppressor T cells (Ts) and antigen-presenting cell (APC) activity are both important for the induction of systemic tolerance after feeding protein antigens to mice. In this report, we have examined further the nature of the inter-relationship between Ts and APC in oral tolerance to ovalbumin (OVA). We found previously that oral tolerance to OVA could prevented by treating mice with oestradiol, and we now report that oestradiol enhances the ability of spleen APC to present OVA to T cells. In parallel, mice treated with oestradiol do not generate the Ts activity normally found after feeding OVA. Treatment of mice with anti-I-J antiserum prevents the induction of both tolerance and Ts activity after feeding OVA, but the suppressor effector cells generated by feeding OVA can not be depleted in vitro by treatment with anti-I-J antibody plus complement. In vivo administration of monoclonal anti-I-A antibody had no effect on oral tolerance to OVA. Our results show that induction of oral tolerance to OVA is an I-J-restricted phenomenon and we propose that this reflects an interaction between specific Ts cells and a population of I-J+ cells which we suggest are APC.     

An immune response to ovalbumin covalently coupled to liposomes is prevented when the liposomes used contain doxorubicin

It is now well established that liposomes with surface associated proteins are immunogenic. Repeated administration of protein coated liposomes elicits the generation of antibodies and the elimination of proteoliposome increases markedly in animals ‘immunized' with such liposomes. This immune response compromises the therapeutic potential of liposomal formulations that rely on the use of protein- or peptide-based targeting ligands to enhance cell specificity. Strategies to suppress or inhibit such immune responses must be developed if this technology is going to prove therapeutically viable. This study evaluates whether an immune response to a protein, covalently attached to liposomes by a thioether bond between N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP)-modified-protein and N-(4-(P-maleimidophenyl)butyryl) (MPB)-activated lipids, can be suppressed when the liposomes used contain the anti-cancer drug doxorubicin. To assess this, the highly immunogenic protein ovalbumin was conjugated onto liposomes composed of distearoylphosphatidylcholine/cholesterol (DSPC/Chol) with sufficient poly(ethylene glycol)-modified distearoyl phosphatidylethanolamine (PEG-DSPE) (2 mol%) to prevent liposome aggregation during protein coupling and to engender increased circulation lifetimes. The immune response to these liposomes with and without encapsulated doxorubicin was measured by: (1) monitoring liposome elimination after 3 weekly i.v. injections in C3H/HeJ mice and (2) measuring the anti-ovalbumin antibody levels by an ELISA assay. One week after a single dose of ovalbumin-coated PEG liposomes (50 μg protein/mouse) the immune response resulted in rapid elimination of a second dose of ovalbumin-coated PEG liposomes. Rapid liposome elimination was correlated to generation of high levels (>9 μg/ml plasma) of circulating anti-ovalbumin IgG. In contrast, anti-ovalbumin antibodies were not detected when the liposomes used contained doxorubicin. Plasma elimination of these drug loaded protein coated liposomes decreased following repeated weekly i.v. doses, an effect that is consistent with liposomal doxorubicin mediated suppression of phagocytic cells in the liver.     

Immunological responses to fed protein antigens in mice. IV. Effects of stimulating the reticuloendothelial system on oral tolerance and intestinal immunity to ovalbumin.

We have studied the role of the reticuloendothelial system (RES) in intestinal and systemic immunity in mice immunized orally with ovalbumin (OVA). Stimulation of the RES by oestradiol completely prevented the induction of systemic tolerance normally found in mice fed 25 mg OVA and this applied both to humoral immunity and delayed-type hypersensitivity (DTH). In addition, an active DTH response could be detected in the mucosa and mesenteric lymph nodes (MLN) of oestradiol-treated, OVA-fed mice on oral challenge with OVA. Oestradiol had no direct effect on lymphocyte function and we propose that RES activation may be one mechanism which predisposes to small intestinal disease associated with food hypersensitivity.     

Defective immune response and failure to induce oral tolerance following enterai exposure to antigen in broilers afflicted with stunting syndrome.

Infectious stunting syndrome (SS) in broilers is a multi-symptomatic disease that includes lesions in the intestinal tract. We investigated whether these lesions impeded functions of the intestinal immune system. Two functions were studied: the capacity to generate 1) immune responses to a resident pathogen .(E. coli) of the gut and to a parenterally administered antigen (ss-casein), and 2) tolerance to an orally administered antigen (ss-casein). SS was induced in day-old broilers by an inoculum prepared from SS afflicted broilers. After onset of SS, immune responses (or absence of, in the case of tolerance) were studied by specific antibody production and T lymphocyte proliferation. Immune responses were induced by subcutaneous immunization of broilers against ss-casein or following natural exposure to enteric .E. coli. Oral tolerance was induced by a single feeding of ss-casein in gelatine capsules. Both enterai anti-E. .coli and parenteral anti-ss-casein responses were significantly reduced in SS birds. SS afflicted broilers did not develop ss-casein-specific oral tolerance. These results indicate dysfunction of both the intestinal immune system and that of systemic acquired immune responses in SS.     

Induction of oral tolerance to cellular immune responses in the absence of Peyer's patches

Systemic hyporesponsiveness occurs following oral administration of antigen (oral tolerance) and involves the uptake and processing of antigen by the gut-associated lymphoid tissue (GALT), which includes Peyer's patches (PP) lamina propria lymphocytes and mesenteric lymph nodes (MLN). Animals with targeted mutations of genes in the tumor necrosis factor (TNF) family have differential defects in the development of peripheral lymphoid organs including PP and MLN, and provide a unique opportunity to investigate the role of GALT structures in the induction of oral tolerance. Oral tolerance could not be induced in TNF/lymphotoxin (LT) alpha-/- mice, which are devoid of both PP and MLN, although these animals could be tolerized by intraperitoneal administration of antigen, demonstrating the requirement for GALT for oral tolerance induction. LTbeta-/- mice and LTalpha/LTbeta+/- animals do not have PP but could be orally tolerized, as measured by IFN-gamma production and delayed-type hypersensitivity responses by administration of both low or high doses of ovalbumin. To further investigate the requirement for PP, we tested the progeny of LTbeta-receptor-IgG-fusion-protein (LTbetaRigG)-treated mice, which do not form PP but have an otherwise intact immune system. Although these animals had decreased fecal IgA production, they could be orally tolerized. Our results demonstrate that PP are not an absolute requirement for the induction of either high- or low-dose oral tolerance, although oral tolerance could not be induced in animals devoid of both PP and MLN.     

A specific immune tolerance toward offspring cells is to exist after the mother lymphocyte infusion

PurposeTo examine immune tolerance between maternal lymphocytes and offspring tissue after a donor lymphocyte infusion.MethodsMouse models were established by mating female BALB/c mice with male C57BL mice. Splenic lymphocytes from donors of different genetic backgrounds were labeled with carboxyfluorescein succinimidyl ester (CFSE), and 1 × 10⁷ of the labeled cells were intravenously injected into a recipient. At 6 h, 24 h, 72 h and 120 h after the infusion, mononuclear cells in recipient spleen, liver, thymus, lymph nodes, and peripheral blood were collected. CFSE+, CFSE-, CD3+, CD8+, CD4+, CD19+, NK1.1+, CD25+, and CD127+ lymphocytes in those samples were analyzed by flow cytometry. The distribution of donor T cells, B cells, NK cells, helper T cells, cytotoxic T cells, and recipient regulatory T cells in the tissues were then analyzed.ResultsMaternal lymphocytes were more likely to survive in offspring. At 120 h after infusion, the percentages of maternal cells in the offspring were 0.52 ± 0.11% in lymph nodes, 0.97 ± 0.04% in peripheral blood, and 0.97 ± 0.11% in the spleen. Few donor cells, if any, were detected in these tissues at 120 h after aunt to child, father to child, and unrelated allogeneic infusions were performed. The subtype proportion of donor lymphocytes changed significantly in the recipient tissues. Recipient Treg cells increased in the mother to child group, but not in the aunt to child, father to child, and unrelated allogeneic groups, suggesting a decreased cellular immune response to allogeneic cells in the mother to child group. At 120 h after the infusion, no donor cells were detected in the recipient livers and thymuses of all groups, implying that donor cells were barely able to colonize in the liver and thymus.ConclusionSpecific immune tolerance to maternal lymphocytes exists in offspring. An infusion of maternal donor lymphocytes may produce a relatively persistent effect of adoptive immunotherapy with reduced side-effects.     

Immunological responses to fed protein antigens in mice. I. Reversal of oral tolerance to ovalbumin by cyclophosphamide

Feeding ovalbumin over a wide range of doses is known to reduce subsequent systemic immune responses to parenteral immunization. In the present study, we have fed mice 2 mg and 25 mg ovalbumin (OVA) 2 weeks before systemic immunization and followed the resulting humoral antibody and cell-mediated immune (CMI) responses. The results indicate that while 25 mg OVA will reduce subsequent IgM, IgG and CMI responses to OVA, feeding 2 mg OVA will only suppress CMI responses and to a lesser extent the IgM response. Furthermore, the tolerant state induced by feeding 25 mg OVA was only partially prevented by 100 mg/kg cyclophosphamide (CY) while the suppressed CMI after feeding 2 mg OVA was completely blocked by CY pretreatment. These findings suggest that the humoral and cell-mediated limbs of the immune response may be controlled by different regulatory systems after feeding antigen, and that activation of these systems is dependent on the dose of oral antigen use. In addition, the results are in agreement with our previous finding that CY pretreatment will allow the development of CMI in the gut and gut-associated lymphoid tissue (GALT) after oral OVA and suggest that this phenomenon is related to breakdown of oral tolerance induction.     

Oral tolerance is inefficient in neonatal mice due to a physiological vitamin A deficiency

Increased risk of allergy during early life indicates deficient immune regulation in this period of life. To date, the cause for inefficient neonatal immune regulation has never been elucidated. We aimed to define the ontogeny of oral tolerance and to identify necessary conditions specific for this stage of life. Ovalbumin (OVA) was administered orally to mice through breast milk and efficiency of systemic tolerance to OVA was assessed in adulthood using a model of allergic airway inflammation. Oral tolerance induction was fully efficient starting third week of life. Inefficiency in neonates was a consequence of abnormal antigen transfer across the gut barrier and retinaldehyde dehydrogenase expression by mesenteric lymph node CD103⁺ neonatal dendritic cells, resulting in inefficient T-cell activation. Neonates' serum retinol levels were three times lower than in adult mice, and vitamin A supplementation was sufficient to rescue neonatal defects and allow tolerance induction from birth. The establishment of oral tolerance required the differentiation of Th1 lymphocytes in both vitamin A-supplemented neonates and 3-week-old unsupplemented mice. This knowledge should guide the design of interventions for allergy prevention that are adapted to the neonatal stage of life such as vitamin A supplementation.     

Ultrastructural studies of rat arteriosclerosis induced by stimulation of the immune system with ovalbumin

Intimal thickening in the aorta and carotid artery of rats was induced by repeated intraperitoneal injections of ovalbumin, 2.5 mg/kg BW, given weekly 5 times after initial subcutaneous sensitization, and/or feeding with a cholesterol-rich diet. The intimal thickening was apparent in immune-challenged rats fed with either a cholesterol-rich or a basal diet (p less than 0.01), whereas it was mild in non-immunized rats fed a cholesterol-rich diet. The ultrastructural changes in the thickened intima were characterized by leukocytic (mainly monocytic) adhesion and migration, and minor endothelial cell damage. Morphometric evaluation of leukocyte adhesion to the intima of the thoracic aorta revealed that the immunized rats fed either a cholesterol-rich or a basal diet showed greater leukocytic adhesion (p less than 0.01 and p less than 0.001, respectively) than that in non-immunized rats fed a cholesterol-rich diet, which in turn also showed an increased degree of leukocyte adhesion (p less than 0.05) than control rats. This immunological approach to the arteriosclerotic process could explain the earlier and more severe arteriosclerosis found in patients with immunological disorders, and the development of arteriosclerosis in the absence of hypercholesterolemia, hypertension and other risk factors.