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Purpose of Review: The default response to protein antigens in the intestine is the induction of systemic and local hyporesponsiveness, ensuring the prevention of coeliac disease and food allergies. Interest is increasing in the role of dietary manipulation and probiotics in treating allergic and other diseases, but less is known about how these regimens might influence systemic and local immune responses. This paper addresses the mechanisms at the interface of innate and adaptive immunity that determine how the body responds to orally administered proteins and how local bacteria modify these., Recent Findings: This paper discusses evidence that dendritic cells in the intestinal mucosa are the critical cells that take up dietary proteins and migrate to the draining mesenteric lymph node, where they induce regulatory CD4 T-cell differentiation. The properties of tolerized T cells are discussed and it is proposed that the gut microenvironment maintains homeostasis by conditioning dendritic cells to remain in a quiescent state. Inhibitory signalling by commensal bacteria possibly contributes to this process., Summary: A regulatory network controls how dietary antigens are taken up and presented to T lymphocytes by specialized antigen-presenting cells. Elucidating their nature and how they are influenced by external factors such as probiotics may help develop novel therapies for allergy and help understand diseases such as coeliac disease.

The prevalence of food allergic diseases is rising and poses an increasing clinical problem. Peanut allergy affects around 1% of the population and is a common food allergy associated with severe clinical manifestations. The exact route of primary sensitization is unknown although the gastrointestinal immune system is likely to play an important role. Exposure of the gastrointestinal tract to soluble antigens normally leads to a state of antigen-specific systemic hyporesponsiveness (oral tolerance). A deviation from this process is thought to be responsible for food-allergic diseases. In this study, we have developed a murine model to investigate immunoregulatory processes after ingestion of peanut protein and compared this to a model of oral tolerance to chicken egg ovalbumin (OVA). We demonstrate that oral tolerance induction is highly dose dependent and differs for the allergenic proteins peanut and OVA. Tolerance to peanut requires a significantly higher oral dose than tolerance to OVA. Low doses of peanut are more likely to induce oral sensitization and increased production of interleukin-4 and specific immunoglobulin E upon challenge. When tolerance is induced both T helper 1 and 2 responses are suppressed. These results show that oral tolerance to peanut can be induced experimentally but that peanut proteins have a potent sensitizing effect. This model can now be used to define regulatory mechanisms following oral exposure to allergenic proteins on local, mucosal and systemic immunity and to investigate the immunomodulating effects of non-oral routes of allergen exposure on the development of allergic sensitization to peanut and other food allergens.

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.

Convincing clinical and experimental evidence suggests that the disturbance of important immunoregulatory and suppressive immunological events induced after oral (mucosal) antigen exposure (oral tolerance) may lead to allergic and autoimmune diseases. Within a variety of factors, age of the host and timing of antigen (food) administration are important characteristics in the development of food allergic disease. Induction of tolerance is seen as a Th2 skewed response, which on one side may prevent harmful mucosal immune reactions but on the other side may contribute to adverse responses in the susceptible individual. The primary mechanisms by which tolerance may be mediated include deletion, anergy, suppression, "ignorance," and apoptosis. Cell-mediated delayed hypersensitivity reactions (Th1), which are implicated in the development of autoimmune and gastrointestinal diseases, are particularly well suppressed. Regulatory events after mucosal exposure of antigen are not well characterized and remain controversial. The balance between tolerance (suppression) and sensitization (priming) is dependent on several factors, such as: (a) genetic background, (b) nature and dose of antigen, (c) frequency of administration, (d) age at first antigen exposure, (e) immunological status of the host, (f) antigen transmission via breast milk, and others. Overall there is evidence in rodents that multiple low-dose feeds are more likely to induce regulatory cytokines (e.g., TGF-beta, IL-10, IL-4) in part secreted by CD4+CD25+ T regulatory cells. Despite the powerful suppressive effects of oral autoantigen exposure observed in experimental models of autoimmune diseases (including bystander suppression), their translation into clinical trials of autoimmune diseases has not yet yielded the expected beneficial results.

There is good clinical and experimental evidence that oral tolerance exists in man and that the timing of antigen (food) administration is an important factor in the development of food allergic sensitisation and disease. Induction of tolerance is often seen as a T-helper 2-skewed response, which on one side may prevent harmful mucosal immune reactions, but on the other side may contribute to adverse responses in the susceptible individual. The primary mechanisms by which tolerance may be mediated include T-cell deletion, anergy, suppression 'ignorance' and apoptosis. Cell-mediated delayed hypersensitivity reactions (T-helper 1), which are implicated as a pathogenetic principle in the development of autoimmune and gastrointestinal inflammation are particularly well suppressed. Regulatory events during the induction of tolerance (or sensitisation) are not well characterised and remain at times controversial. The balance between tolerance (suppression) and sensitisation (priming) is dependent on several factors, such as: (a) genetic background; (b) nature of antigen and dose of antigen; (c) frequency of administration; (d) age (maturity v. immaturity) at first antigen exposure; (e) immunological status of the host (e.g. virus infection); dietary exposure of the mother; (g) antigen transmission via breast milk, and others. Overall, there is evidence in rodents that multiple low-dose feeds are more likely to induce regulatory cytokines (e.g. transforming growth factor-beta, interleukins 10 and 4) in part secreted by CD4+CD25+ T-regulatory cells. Despite the powerful suppressive effect of oral antigen exposure observed in experimental models, its application in clinical trials of autoimmune diseases has not yet yielded the expected beneficial results.

The concept of immunologically mediated tolerance to food antigens through exposure to mucosal antigen has been the subject of continuous scientific debate. After a decline in interest in the mid-1980s, oral tolerance has again attracted the attention of immunologists. Here, Stephan Strobel and Allan Mowat discuss how this central immunological principle has potential new therapeutic applications for the treatment of autoimmune, inflammatory and possibly food-allergic diseases.

Serum collected 60 min after feeding ovalbumin (OVA) to BALB/c mice transfers specific tolerance to naive recipients via an unknown mechanism. We have now identified a large fragment of the OVA molecule as the putative active moiety. Specific absorption of immunoreactive OVA from tolerogenic serum by immunoaffinity chromatography removed the tolerogenic activity; following elution of the bound OVA and subsequent injection into naive recipients it was possible to demonstrate tolerogenic activity equivalent to that seen with unmanipulated serum. Passage of OVA tolerogenic serum through a bovine serum albumin (BSA)-specific affinity column had no effect on in vivo OVA tolerogenic activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using anti-OVA antibodies demonstrated the presence of two bands with apparent molecular weights (MW) of 21,000 and 24,000 in the OVA-related material absorbed from the tolerogenic serum. The 24,000 MW moiety was also visualized by silver staining. These moieties are putative candidate tolerogens as they were absent from normal BALB/c mouse serum analysed 5 min after feeding OVA and from severe combined immunodeficiency (SCID) mouse serum--without tolerising activity--analysed 60 min after a similar feed.

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.

In an attempt to investigate the molecular basis of the mechanisms underlying oral tolerance, we have evaluated the molecular and biological features of ovalbumin subjected to intestinal processing. Immunoreactive ovalbumin absorbed by the gut was measured by a sandwich ELISA at different times after feeding 25 mg ovalbumin to adult mice. Ovalbumin was detected as early as 5 min after the feed (36.7 +/- 16 ng/ml; mean +/- 1 s.d.) and reached maximal levels at 1 h (73.3 +/- 20 ng/ml). Pooled mouse serum, collected 5 min or 1 h after the feed, was transferred intraperitoneally into the naive recipients. Suppression of systemic delayed-type hypersensitivity (DTH) was found in mice receiving 0.8 ml of serum obtained 1 h after ovalbumin feeding but not when using serum obtained 5 min after feeding. In order to transfer samples containing similar levels of ovalbumin, an increased amount (1.3 ml) of serum collected 5 min post-feed was used in further experiments but again failed to induce DTH tolerance. Serum samples obtained 5 and 60 min after ovalbumin feeding were analysed by fast-protein liquid chromatography (FPLC) fractionation followed by ELISA. Both the charge characteristics and molecular weight of intestinally absorbed ovalbumin were indistinguishable from native ovalbumin. Although intact native ovalbumin is the only molecular species detected by ELISA, the results suggest that this has no role in the suppression of DTH responses.

The normal effect of feeding an antigen, such as ovalbumin (hens' egg albumin), to adult animals is the induction of a state of specific nonreactivity of the lymphoid tissues when the same antigen is presented again (oral tolerance). We have carried out feeding experiments in neonatal mice to investigate subsequent immune responses after physiologic antigen exposure and to examine the role of the neonatal intestine. We demonstrate for the first time that feeding a weight related dose of ovalbumin within the first week of life results in priming for both humoral and cell-mediated immune responses, despite the profound tolerance found in adult animals when treated in the same way. When the time scale of antigen exposure was extended into the prenatal period, the enhancement of the immune response was even more pronounced. These effects are long lasting and effects on cell-mediated immune responses are still demonstrable 14 wk after the initial priming feed. We postulate that after an antigen feed in the neonatal period, immunologic and digestive immaturity lead to a net gain in T help which prevents the induction of systemic hyporesponsiveness (oral tolerance).

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.

Feeding of a protein antigen to normal adult mice results in systemic immunologic hyporesponsiveness (oral tolerance). Local mucosal cell mediated immunity is not usually elicited. The objectives of these experiments were to abrogate the induction of oral tolerance and concomitantly to induce a local mucosal cell mediated immune response in mice; and thereby to establish which facets of intestinal physiology or immunology are relevant to the induction of normal, mainly suppressor immune responses to a fed antigen. Protein antigen (ovalbumin) was fed to animals in whom immune status had been modulated by intraperitoneal injection of N-acetyl-muramyl dipeptide (MDP), by induction of a graft versus host reaction, or naturally, by virtue of immaturity. The induction of oral tolerance was prevented in all treatment groups. In a second series of experiments mice were orally immunised as before, rested for four weeks, and then challenged with ovalbumin in their drinking water for 10 days. Jejunal architecture was not altered by the antigen challenge, but MDP treated and immature animals which had been sensitised to ovalbumin and later re-exposed to the same antigen had significantly higher intraepithelial lymphocyte counts than appropriate controls. Factors which may lead to abrogation of oral tolerance and induction of intestinal hypersensitivity are discussed in relation to food allergic diseases in man.

'Intestinal antigen processing' is a function of the gastro-intestinal tract whereby shortly after an animal has been fed an immunogenic protein antigen, such as ovalbumin (OVA), a tolerogenic form of the protein is generated and can be detected in the circulation. The effect of damage to the intestinal epithelium on the processing of OVA has been examined in lethally irradiated mice. Irradiated animals were fed 25 mg OVA and their serum collected 1 h later. When this serum was transferred intraperitoneally into naive recipient mice, this did not induce the typical suppression of systemic delayed-type hypersensitivity. Results were similar when the serum donors were at 2 days after irradiation, with crypt hypoplasia, and at 5 days after irradiation when there was reactive crypt hyperplasia. However reconstitution of donors with normal spleen cells immediately after irradiation restored their capacity to generate a tolerogenic form of the antigen. Immunoreactive OVA was detected by ELISA in both tolerizing and non-tolerizing sera, and the immunological properties of these sera were not related to serum levels of OVA after feeding. Thus subtle immunochemical alterations in the nature of a protein antigen are likely to be more important than the quantity of absorbed antigen, in influencing systemic cell-mediated immune responses after feeding. The lack of generation of a tolerogenic form of the protein in irradiated mice, unrelated to the pattern of epithelial cell kinetics, and the restoration of this function by normal spleen cells, suggests that lymphoid cells may be involved in the phenomenon of antigen processing.

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.

We have examined the mechanisms that prevent the induction of oral tolerance to protein antigens in neonatal mice. Serum collected from adult mice 1 h after feeding ovalbumin (1 mg/g body wt) was adoptively transferred to mice aged 1, 3, and 42 d (40 microL/g body wt). Whereas delayed-type hypersensitivity was significantly suppressed in adult recipients relative to control groups, no suppression of systemic delayed-type hypersensitivity was found in neonatal recipients. In attempts to identify the immunologic deficiency that prevents mature reactivity to protein antigens in neonates, adult splenocytes were transferred intraperitoneally (10(8) cells/recipient) 24 h before a feed of OVA (1 mg/g body wt) to neonates. Significant suppression of their systemic DTH response, but not of their anti-ovalbumin IgG antibody response was observed, indicating that spleen cell transfer only partially confers adult-type reactivity. Similar results were obtained using a second protein antigen, BSA. Our observations suggest that the failure to induce oral tolerance to protein antigens in neonatal mice is not simply due to immature antigen processing by the gut, but probably reflects cellular and/or antigen handling immaturity of the neonatal immune system.

When the first encounter with an antigen is by the oral route several different immune responses can occur. The spectrum of effects on systemic immunity ranges from immunological hyporesponsiveness (oral tolerance) to active sensitisation (priming). In this paper, the immunological principles underlying these effects are discussed and circumstances which influence them are demonstrated, particular emphasis being placed on experimental work in adult and neonatal mice.

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.

Mice fed ovalbumin develop specific systemic hyporesponsiveness. This oral tolerance is abrogated by cyclophosphamide pretreatment, and the mechanism of abrogation could be either via T suppressor cells or via damage to the gut epithelium. A serum transfer protocol was used to examine the site of action of cyclophosphamide in this system. Serum was collected from ovalbumin-fed mice and transferred into recipients which were then parenterally immunized with ovalbumin in Freund's complete adjuvant. Serum transfer suppressed the delayed-type hypersensitivity (DTH) responses but not the antibody responses of the recipients. Cyclophosphamide pretreatment (100 mg/kg) of recipients (but not of donors) abrogated this suppressor effect. Parenteral administration of ovalbumin in a range of doses did not induce immunological hyporesponsiveness. It is suggested that absorption across the gut mucosa leads to generation of fragments of ovalbumin that induce suppressor cells selective for DTH.

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.