The findings reported here indicate that augmented IL-4 receptor signaling is singularly effective in promoting both the development of allergic sensitization to ingested protein antigens and the induction of anaphylactic responses upon enteral challenge. Studies in humans and mice have previously established that IL-4 and IL-13 play central roles in the pathogenesis of gastrointestinal allergic responses.32–38
Genetic polymorphisms affecting the IL-4/IL-13 axis are reported to be correlated with asthma susceptibility4, 9–11
and risk associations between polymorphisms in the IL-4, IL-4Rα and IL-13 genes and food allergy have been reported.39, 40
We therefore hypothesized that augmented IL-4 signaling could predispose to food allergy and tested this in F709 mice in which the sensitivity of IL-4Rα signaling is enhanced resulting in increased IL-4 induced STAT6 phosphorylation, Th2 induction and IgE production13
Our results demonstrate that the F709 genotype confers a very dramatic sensitivity to food allergen and that this phenotype is mediated primarily by IL-4R signaling effects in hematopoietic cells. Mice with this mutation exhibited not only increased susceptibility to immune sensitization, producing both specific IgE antibodies and Th2 responses, but also a marked intestinal mast cell expansion, enhance intestinal permeability and a capacity to mount intense anaphylactic responses upon enteral challenge. Perhaps the single most notable phenotype of these mice with respect to food allergy was their susceptibility both to immune sensitization and to enteral allergen-induced anaphylaxis even when exposed only to a protein antigen, OVA, in the absence of any immune stimulating adjuvant. Taken together, these observations indicate that IL-4 signaling plays critical roles in food allergy by enhancing allergic sensitization, promoting mast cell expansion and anaphylactic sensitivity and by interfering with the physiologic induction of tolerance to new protein antigens introduced via the gastrointestinal tract.
A number of groups have developed murine models to study food-allergen mediated hypersensitivity reactions in mice.19, 20, 27, 41–47
. Studies by Rothenberg and colleagues using an “allergic diarrhea” have established a central role for the IL-4/IL-13 axis in the development of food allergy. Our findings confirm and advance this observation by demonstrating that enhanced IL-4R signaling is permissive for purely enteral immune sensitization in the absence of parenteral priming with adjuvant as is used in the allergic diarrhea protocol. Furthermore, we find that the IL-4R effect also results in the manifestation of vascular responses (hypothermia) upon purely enteral challenge (as opposed to i.v
. challenge as in the allergic diarrhea system)..
Recently it was shown that IL-4 can divert developing Treg to a cytokine profile dominated by IL-9.48
IL-9, produced by epithelial and other cells, is an important mast cell growth and differentiation factor and has been implicated in intestinal anaphylaxis in an IgE-dependent manner.21, 49–51
Forbes and colleagues have elegantly demonstrated that IL-9-driven mast cell expansion in the intestine leads to enhanced intestinal permeability21
. This finding suggests a mechanism whereby the enhanced IL-4Rα signaling of F709 mice, which is accompanied by high levels of intestinal IL-9 transcripts and markedly enhanced mast cell numbers, might increase the intestinal permeability in these mice. We and others have observed that recruitment of mast cell progenitors to allergen challenged lung in sensitized mice is IL-9 dependent.52–54
Dramatically increased jejunal IL-9 levels were observed in the F709 mutants in our study (), suggesting pro-amplifying effects of the intestinal epithelium on mast cell accumulation. This is further borne out by the finding, that although the sensitivity to anaphylaxis is largely mediated by hematopoietic cells such as mast cells, non-hematopoietic cells also contribute.
IgE antibodies, which in combination with mast cells serve as exquisitely sensitive biological sensors for allergen, have long been implicated as the key trigger of anaphylactic reactions in allergic individuals. Osterfeld and colleagues have shown that reactions to food allergens in parenterally sensitized mice are IgE dependent23
. Thus, at first glance, our finding that the robust anaphylactic responses expressed by F709 mice in response to food allergen challenge were abrogated in IgE−/−
F709 animals might not seem surprising. It is important to note though that anaphylaxis in rodents can be driven by completely IgE-independent mechanisms and that the IgG isotype can substitute for IgE.16, 17, 28
Our data show for the first time that IgE is critical in the development of active anaphylaxis to food antigens using exclusively oral routes for both sensitization as and challenge. This is in contrast to our previous findings which showed that parenterally mediated systemic active anaphylaxis could occur in the absence of IgE 16
Strait and colleagues have shown that while the IgE pathway is more sensitive, requiring lower threshold levels of antigen for full activation, IgG-mediated responses, occurring at appropriate antibody to antigen ratios can nevertheless be lethal. Our observations regarding anaphylactic responses in response to enteral OVA in F709 mice provide important information regarding the relative contributions of IgE and IgG isotypes in the gut. Although OVA specific IgE responses were markedly higher in F709 mice than in Y709 controls, IgG1 antibodies to OVA were similar in the two groups. This finding indicates a preferential effect of IL-4Rα signaling on IgE vs. IgG production in intestinal immune responses to food allergens. More interesting though, was our finding that, in the absence of IgE, the presence of IgG1 responses in both Y709 and F709 animals was insufficient to drive anaphylactic reactions. Neither IgE−/− mice nor F709/IgE−/− double mutants displayed physiologic signs of anaphylaxis upon enteral allergen challenge despite having significant anti-OVA IgG1 titers. This observation suggests that either the amount of absorbed antigen in the gut is insufficient to trigger non-IgE pathways or that the local effector cells of anaphylaxis, likely the markedly expanded mast cells, are preferentially IgE responsive. This observation certainly lends support to the concept currently being evaluated in clinical trials that anti-IgE therapy might block food allergy responses.
Our bone marrow chimera data suggest a partial role of heightened target organ responsiveness to mast cell mediators in the anaphylaxis sensitivity of F709 mice. Finkelman and colleagues have demonstrated that administration of IL-4 immediately prior to anaphylactic challenge results in markedly stronger physiologic reactions because of enhanced tissue sensitivity to platelet activating factor, histamine, serotonin, and cysteinyl leukotrienes.14, 55
Nonhematopoietic cells express predominantly the Type II IL-4/13 receptor (IL-4Rα and IL-13Rα1).
In addition to providing important information regarding potential effects of amplified IL-4 signaling on immune sensitization, mast cell responses and anaphylaxis, we believe that the F709 mice will provide a powerful tool for investigators studying food allergy. Furthermore, the failure of enteral tolerance in these mice will permit not only investigations of physiologic tolerogenic mechanisms but may additionally provide a practical model for evaluating allergenicity of food proteins in the absence of any immune stimulating adjuvants. We anticipate that the observations presented in this report, along with the increasing availability of mouse strains with alterations in IL-4 receptor function will lead to fruitful future investigations of the pathogenesis of food allergy.