Priming of systemic and local delayed-type hypersensitivity responses by feeding low doses of ovalbumin to mice.

We have examined the effects on both systemic and intestinal immunity of feeding different doses of ovalbumin (OVA) to mice. A single feed of doses of more than 1 mg OVA produced significant suppression of subsequent delayed-type hypersensitivity (DTH) and IgG antibody responses. Feeding 100 micrograms-1 mg OVA had no net effect on systemic immunity, but mice fed 10-50 micrograms OVA had consistently enhanced systemic DTH responses when immunized subsequently with OVA in adjuvant. Oral challenge of these mice with OVA produced alterations in mucosal architecture and in intra-epithelial lymphocyte counts, consistent with the presence of an intestinal DTH response. Similar changes were not found in mice fed tolerogenic doses of OVA. Although feeding low doses of OVA primed both systemic and intestinal DTH responses, this had no effect on serum IgG responses and very little systemic DTH could be revealed in OVA-fed mice without systemic challenge with OVA in adjuvant. We conclude that feeding certain low doses of protein antigens can induce priming of local and systemic DTH responses rather than the immune tolerance which is normally found. The development of clinical food hypersensitivities may be highly dependent on the dose of dietary antigen at the time of first encounter.     

A genetically determined lack of oral tolerance to ovalbumin is due to failure of the immune system to respond to intestinally derived tolerogen

In this study we have examined whether differences between mouse strains in the induction of tolerance after feeding ovalbumin (OVA) are due to differences in intestinal processing of OVA or are determined by the systemic immune system. Compared with major histocompatibility complex (MHC)-congenic BALB/c mice, BALB/B mice develop much less tolerance of systemic delayed-type hypersensitivity (DTH) and humoral immunity after feeding OVA and this defect is also expressed partially in (BALB/B x BALB/c)F1 animals. Serum taken from either BALB/c or BALB/B mice fed OVA 1 h before produced significant suppression of systemic DTH responses in BALB/c, but not in BALB/B mice. Although OVA-fed BALB/B serum was slightly less tolerogenic than BALB/c serum, we conclude that the defective induction of oral tolerance in BALB/B mice is due primarily to a MHC-influenced defect with the immune system. These findings support the idea that clinical food-sensitive enteropathy reflects an immune response gene-controlled defect in tolerance to dietary proteins.     

Depletion of suppressor T cells by 2'-deoxyguanosine abrogates tolerance in mice fed ovalbumin and permits the induction of intestinal delayed-type hypersensitivity.

We have re-examined the role of suppressor T cells (Ts) in regulating immune responses to fed proteins by investigating the effect of 2'-deoxyguanosine (dGuo) on systemic and intestinal immunity in mice fed ovalbumin (OVA). Administration of dGuo for 10 days abrogated the suppression of systemic delayed-type hypersensitivity (DTH) and antibody responses normally found after feeding OVA, and also prevented the generation of OVA-specific Ts. In parallel, mice given dGuo and fed OVA developed sensitization to OVA in the gut-associated lymphoid tissues (GALT) after oral challenge with OVA and had increased intraepithelial lymphocyte (IEL) counts and crypt cell production rates (CCPR) in the jejunal mucosa, indicating the presence of a local DTH response. These findings confirm the importance of Ts in preventing hypersensitivity to dietary protein antigens and suggest that enteropathies associated with food hypersensitivity are due to a defect in Ts activity.     

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.     

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.     

Adjuvant actions of linear mannan-possessing lipopolysaccharide (LPS) in LPS-resistant C3H/HeJ mice.

Immunopotentiation has been demonstrated when Klebsiella O3 lipopolysaccharide (KO3 LPS), which possesses a linear mannan as the O-specific side chain, was injected subcutaneously into endotoxin resistant C3H/HeJ mice together with soluble protein antigens. The LPS exhibited significantly positive adjuvant effects on antibody responses in vivo after secondary antigen challenge and on delayed-type hypersensitivity (DTH) reactions against protein antigens. However, KO3 LPS was not a polyclonal B cell activator (PBA) in C3H/HeJ mice nor mitogenic in cultures of spleen cells of C3H/HeJ. Thus, the activity of the LPS in C3H/HeJ mice is confined to the potentiation of T-dependent immune responses. The contribution of the mannan O side chain to the adjuvant action of KO3 LPS was suggested.     

The kinetics of oral hyposensitization to a protein antigen are determined by immune status and the timing, dose and frequency of antigen administration.

We have investigated the immunological consequences of feeding a protein antigen to previously immunized animals. BALB/c mice were systemically primed with ovalbumin (OVA) in complete Freund's adjuvant (CFA) and fed with high (10 mg/g body weight), medium (1 mg/g body weight) or low (1 microgram/g body weight) doses of OVA once (Day 1, 7 or 14) or sequentially for 5 days (Days 1-5, 7-11, 14-18). The specific IgG antibody response was suppressed only by early feeds of high-dose OVA (Days 1-5). Medium-dose OVA fed on Day 14 or low-dose OVA fed at any stage after immunization enhanced the IgG antibody response. In contradistinction, systemic delayed-type hypersensitivity responses (DTH) were usually suppressed by early feeds of high or medium doses of OVA but never after feeding low-dose OVA. The results suggest that systemic DTH and IgG antibody responses to oral antigen are subject to different control mechanisms in previously primed animals. Such responses depend on the immune status of the animal and are controlled by antigen dose, time and frequency of feeding. The immunological effects observed are also demonstrable following adoptive transfer of spleen cells collected 14 days after multiple feeds of high-dose OVA to immunized mice. Our findings suggest that oral hyposensitization after systemic immunization is regulated by (suppressor) spleen cells which are activated by gut-processed antigen.     

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.     

Suppression of an established DTH response to ovalbumin in mice by feeding antigen after immunization

Experiments were designed to examine whether systemic delayed-type hypersensitivity responses (DTH) to ovalbumin (OVA) can be suppressed when antigen is fed after immunization, and to investigate the immunological mechanisms involved. A single 25 mg feed of OVA given 7 or 14 days after immunization with OVA in complete Freund's adjuvant (CFA) suppressed the DTH response of BDF1 mice, but had no significant effect on the serum IgG antibody response. DTH suppression was greatest when antigen was fed soon after immunization, and became less pronounced as the time interval between feeding and immunization increased. The phenomenon was also demonstrated in mice of the BALB/c strain. Cell transfer experiments suggested that the post-immunization suppression was not due to a population of suppressor cells that have been described previously in association with classical oral tolerance for DTH. We conclude that there are separate and distinct mechanisms for the prevention of induction of DTH by antigen feeding in naive mice and the suppression of expression of DTH in sensitized animals.     

Delayed-Type Hypersensitivity Response in Mice to Treponema Pallidum

C3H/HeJ mice which had been primed with either virulent or killed T. pailidum were studied for in vivo delayed-type hypersensitivity (DTH) responses to T. pallidum following local footpad challenge. Mice sustaining a chronic infection of 5 months duration failed to develop a DTH to treponemal antigens, whereas priming by a single intravenous injection with killed organisms resulted in a significant DTH response in mice when challenged 5 days later. Treatment of mice prior to priming with a single sublethal dose of cyclophosphamide (100 mg/kg body weight) not only failed to potentiate T. pallidum-DTH, but abrogated the response observed in untreated primed animals.     

Immunological responses of mice to lipopolysaccharide: lack of secondary responsiveness by C3H/HeJ mice.

Mice of the C3H/HeJ strain, which were unresponsive to the biological effects of bacterial lipopolysaccharide (LPS), could not be induced to make specific secondary immunological responses to LPS; they responded to two doses of LPS with a primary response. This lack of secondary responsiveness by C3H/HeJ mice was due to a defect in a single, autosomal, dominant gene. Thus, further evidence was provided that an intact second immunological signal and responsiveness thereto were required to trigger secondary antibody responses in primed animals.     

Oral tolerance in protein-deprived mice. I. Profound antibody tolerance but impaired DTH tolerance after antigen feeding.

We have examined the effects of protein deprivation on the induction of oral tolerance for systemic antibody and DTH responses to the protein antigen ovalbumin (OVA). Mice were fed 4% or 24% protein diets from weaning and given a single feed of OVA 2 weeks later (short-term deprivation) or after 10 weeks (long-term deprivation). Tolerance for serum antibody responses was more profound in protein-deprived animals than in 24% protein-fed control groups. Conversely, tolerance for DTH responses was impaired in protein-deprived mice. This was demonstrated both for short-term deprivation, where nutritional rehabilitation after OVA feeding was necessary to demonstrate this effect on DTH, and for long-term deprivation. Furthermore, the effect of short-term deprivation on tolerance for DTH responses was similar to that observed after cyclophosphamide pretreatment of OVA-fed mice. Protein deprivation has disparate effects on the humoral and cell-mediated limbs of oral tolerance, and our results support the hypothesis that this regime selectively depletes a population of suppressor T cells responsible for the fine control of DTH tolerance.     

Suppression of delayed-type hypersensitivity mediated by macrophage-like cells in mice with experimental liver injury.

Systemic hyporesponsiveness after ingestion of a protein antigen (oral tolerance) depends on antigen processing by the gut and the actions of immunoregulatory T cells. We have examined the effects of a graft-versus-host reaction (GvHR) on oral tolerance, since both immune status and intestinal function are altered in GvHR. The GvHR was induced in unirradiated (CBA X BALB/c)F1 mice by intraperitoneal injection of CBA spleen cells. The tolerance of systemic humoral immunity and of delayed-type hypersensitivity normally found in mice fed 25 mg ovalbumin (OVA) was partially abrogated from 1 to 3 weeks after induction of the GvHR. In addition, mice with GvHR had a persistent enhancement of systemic immunity to OVA, and this was associated with an augmented ability of spleen cells to present OVA to primed T cells. The phagocytic activity of the reticuloendothelial system, as established by carbon clearance tests, was not altered by the GvHR. These findings suggest that enhanced antigen-presenting cell activity interferes with the induction of oral tolerance, and may be another pathogenetic mechanism of intestinal hypersensitivity disease.     

Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea-denatured OVA (UD-OVA) and carboxymethylated UD-OVA (CM-OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed-type hypersensitivity (DTH) responses to OVA were not suppressed by CM-OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM-OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM-OVA were orally tolerized by OVA, but not by UD-OVA or CM-OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM-OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM-OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma-globulin (HGG), a well-known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.     

Enhancement of in Vitro Immune Responses of Murine Peyer's Patch Cultures by Concanavalin A, Muramyl Dipeptide and Lipopolysaccharide

Immunofluorcscent and esterase staining of Peyer's patch (PP) cell preparations from six mouse strains showed that each strain possessed approximately eqtial numbers of T and B cells but less than 1% esterase-positive cells (macrophages; Mψ). The addition of concanavalin A (Con A) to either lipopolysaccharide (LPS)-responsive C3H/HeN or LPS-non-responsive C3H/Hej PP cultures immunized with sheep erythrocytes (SRBC) resulted in good anti-SRBC plaque-forming cell responses, whereas the addition of Con A to PP cultures from SRBC orally primed C3H/HeJ mice resulted in significantly higher in vitro responses to trinitrophenyl-conjugated SRBC than similarly treated cultures from C3H/HeN mice. On the other hand, muramyl dipeptide (MDP) promoted higher responses in PP cultures derived from either normal or carrier-primed LPS-rcsponsive (C3H/HeN) mice than were observed with C3H/HeJ PP cultures. A similar pattern of responsiveness was seen when LPS prepared by a phenol-water extraction procedure (LPS(Ph)) was added to PP cultures. When LPS prepared by a bntanol-water extraction method was used, PP cultures from both C3H/HeN and C3H/HeJ mice were enhanced; however, the response of C3H/HeN PP cultures was significantly greater than that seen with C3H/HeJ PP cultures. The enhanced responsiveness of C3H/HeN PP cultures to MDP was probably due to an effect on the Mψ, since addition of MDP to either C3H/HeN or C3H/HeJ PP cultures containing C3H/HeN splenic adherent cells resulted in significantly enhanced immune responses. C3H/HeJ splenic Mψ did not promote adjuvant responses. LPS(Ph) augmentation of immune responses required that Mψ (spleen) and PP cultures be derived from LPS-re-sponsive C3H/HeN mice. These results demonstrated that Con A, MDP, and LPS promoted immune responses of PP cultures to the T-dependent antigen, SRBC. Evidence is presented that Con A enhanced T-helper-cell activity, whereas MDP and LPS required the presence of LPS-responsive Mψ for augmentation of immune responses.     

Oral tolerance in T cells is accompanied by induction of effector function in lymphoid organs after systemic immunization

The physiological ramifications of oral tolerance remain poorly understood. We report here that mice fed ovalbumin (OVA) exhibit oral tolerance to subsequent systemic immunization with OVA in adjuvant, and yet they clear systemic infection with a recombinant OVA-expressing strain of Salmonella enterica serovar Typhimurium better than unfed mice do. Mice fed a sonicated extract of S. enterica serovar Typhimurium are also protected against systemic bacterial challenge, and the protection is Th1 mediated, as feeding enhances clearance in interleukin-4-null (IL-4(-/-)) and IL-10(-/-) mice but not in gamma interferon-null (IFN-gamma(-/-)) mice. When T-cell priming in vivo is tracked temporally in T-cell receptor-transgenic mice fed a single low dose of OVA, CD4 T-cell activation and expansion are restricted largely to mucosal lymphoid organs. However, T cells from spleens and peripheral lymph nodes of fed mice proliferate and secrete IFN-gamma when restimulated with OVA in vitro, indicating the presence of primed T cells in systemic tissues following oral exposure to antigen. Nonetheless, oral tolerance can be observed in the fed mice as reduced recall responses following subsequent systemic immunization with OVA in adjuvant. Soluble OVA administered systemically has similar effects in vivo, and the "tolerance" seen in both cases can be partially reversed if the initial priming is made more immunogenic. Together, the results indicate that antigen exposure under poor adjuvantic conditions, whether oral or systemic, may lead to T-cell commitment to effector rather than proliferative capabilities, necessitating a reassessment of therapeutic modalities for induction of oral tolerance in allergic or autoimmune states.     

Delayed-type hypersensitivity and immunity to Salmonella typhimurium

Studies were carried out to correlate immunity and expression of delayed-type hypersensitivity (DTH) in mice of the C3H lineage immunized with an avirulent strain of Salmonella typhimurium (strain SL3235). This strain belongs to a class of aroA- organisms which are being considered as vaccine strains for humans and veterinary use. In a systematic study, the relationship between the mouse strain and the immunizing dose of strain SL3235 on the development of protective immunity and DTH was examined. It was found that in hypersusceptible C3H/HeJ and C3HeB/FeJ mice, several doses of strain SL3235 afforded protection against intravenous challenge doses as high as 1,300 50% lethal doses. Despite these significant levels of immunity to challenge, mice of these two strains never mounted significant DTH responses following immunization with the doses of strain SL3235 tested, which spanned 3 orders of magnitude. Nonresponsiveness was not due to antigen overload, as all of the mouse strains were comparably colonized with strain SL3235 at the time of DTH elicitation. Further, it was found that the ability of responsive C3H/HeNCrlBR mice to display DTH was dependent on the immunizing dose of strain SL3235 and that a dosage could be found that resulted in increased resistance to challenge in these mice without a concomitant display of DTH. Thus, while both induction of protective immunity and DTH were vaccine dosage dependent in the responsive mouse strain (C3H/HeNCrlBR), DTH was a less sensitive measure of protective immunity than survival. Vaccine dosages ranging over three orders of magnitude failed to yield positive footpads to the Salmonella elicitin in the nonresponsive mice. The data suggest that caution should be observed in interpreting Salmonella DTH tests that are used as screens of immune status to typhoid fever in humans, as the extent of discordance between immunity and DTH in humans is unknown.     

Immunological studies of NK cell-deficient beige mice. II. Analysis of T-lymphocyte functions in beige mice

Lymphocytes from natural killer (NK) cell-deficient beige mice have a poor ability to induce many different forms of graft-versus-host reaction (GvHR). In this study, we have examined whether this defect could reflect an associated abnormality in beige T-lymphocyte function. Compared with normal, congenic C57Bl/6 (B6) mice, beige mice had similar numbers and proportions of T-cell subsets and generated normal allospecific delayed-type hypersensitivity (DTH) responses in vivo. In addition, beige mice developed normal or enhanced DTH responses to the protein antigen, ovalbumin (OVA), and were fully susceptible to the induction of tolerance by feeding OVA. Beige lymphocytes recirculated normally in vivo and showed enhanced proliferative responses to mitogens and alloantigens in vitro. In contrast, beige responder cells generated poor cytotoxic T-lymphocyte (CTL) responses in vitro and had quantitatively identical defects in CTL and NK cell activation after alloimmunization in vivo. These results suggest that although many effector and regulatory T-cell functions are normal in beige mice, the NK-cell defect in these animals is paralleled by impaired CTL activity. We suggest that abnormal T-cell function accounts for the inability of beige lymphocytes to induce GvHR.     

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.     

Genetic control of delayed-type hypersensitivity in mice to Salmonella antigen

Genetic restriction on the expression of delayed-type hypersensitivity (DTH) to Salmonella typhimurium in mice transferred passively with immune spleen cells was studied. After the intravenous transfer of immune C3H/HeJ (H-2Ik) cells into A.TL (H-2Ik) or A.TH (H-2Is) mice, footpad DTH responses could be evoked in the A.TL recipients, but not in the A.TH mice. When the immune cells of BALB/c or C3H/He mice were intravenously-transferred into F1 hybrids produced by mating BALB/c and C57BL/6 or C3H/He and C57BL/6, respectively, no DTH response could be evoked in these F1 hybrids that received immune parental cells. Local transfer as well as systemic intravenous transfer of immune parental cells to F1 haplotype recipients did not cause any DTH. Previous treatment of the F1 hybrid recipients with cyclophosphamide did not result in the expression of the DTH response. Transfer of immune F1 spleen cells into parental strains also did not induce DTH. When the immune cells of parental strains were transferred into F2 mice and into back-cross mice, examination of the DTH response in these mice showed that some of them did not have any obvious footpad swelling, while others revealed various magnitudes of swelling. The resistance of F1 hybrids to transfer of DTH is discussed.