Direct effects of IL-4 on mast cells drive their intestinal expansion and increase susceptibility to anaphylaxis in a murine model of food allergy

Abstract

Interleukin (IL)-4 has critical roles in allergic disorders, including food hypersensitivity. The direct effects of the cytokine on the survival and function of mast cells, the key effectors of food anaphylaxis, have not been established. In this study, we demonstrate that IL-4 induces a marked intestinal mastocytosis in mice. This phenotype is reproduced in animals expressing Il4rαF709, an activating variant of the IL-4 receptor α-chain (IL-4Rα). Il4rαF709 mice exhibit enhanced anaphylactic reactions but unaltered physiological responses to vasoactive mediators. IL-4 induces Bcl-2 and Bcl-X(L) and enhances survival and stimulates proliferation in cultured bone marrow-derived mast cells (BMMC). These effects are STAT6 (signal transducer and activator of transcription factor 6)-dependent and are amplified in Il4rαF709 BMMC. In competitive bone marrow chimeras, Il4rαF709 mast cells display a substantial competitive advantage over wild-type mast cells, which, in turn, prevail over IL-4Rα⁻/⁻ mast cells in populating the intestine, establishing a cell-intrinsic effect of IL-4 in intestinal mast cell homeostasis. Our results demonstrate that IL-4-signaling is a key determinant of mast cell expansion in food allergy.

Figure 1. IL-4 drives intestinal mastocytosis

A) Chloroacetate esterase staining for mast cells (red, examples highlighted by red arrows) in jejunal sections in wild-type, IL-4-treated or Il4rαF709 mice. B) Representative flow cytometry diagrams and summary data of small intestinal mast cells (c-Kit⁺IgE⁺lin⁻CD45⁺) from wild-type and Il4rαF709 mice, gating on live lin⁻CD45⁺ cells (n=5). C) IL-4 treatment dramatically expands intestinal mast cells in wild-type mice. IL-4 was injected as a cytokine-antibody complex with 2μg IL-4 and 10μg anti-IL-4 i.p. on days −1, −4, −7. Statistical analysis by unpaired t test, two-tailed. Data are representative of at least three experiments. Mast cell frequencies are expressed as the fraction out of lin⁻CD45⁺ cells.

Figure 2. IL-4Rα-driven effects exacerbate anaphylaxis

A) Il4rαF709 mice show an increased loss of temperature and limited recovery from anaphylaxis. Wild-type and Il4rαF709 mice lacking endogenous IgE (IgE^−/−) were subjected to IgE-mediated passive anaphylaxis and core body temperatures were recorded to assess the severity of the response. Statistical analysis by two-way ANOVA. B) Il4rαF709 mice uniquely exhibit rapid-onset diarrhea during IgE-mediated passive anaphylaxis. P=0.0084 by log-rank test of Kaplan-Meier survival curves. C) Serum release of MMCP-1 is greatly enhanced in Il4rαF709 mice following passive anaphylaxis. Statistical analysis by one-way ANOVA. D) IL-4 release in Il4rαF709 mice is also enhanced following passive anaphylaxis. Statistical analysis by unpaired t test. Mice were injected r.o. with 20μg IgE anti-DNP (clone SPE-7) and challenged 16hrs later with 0.5mg DNP-BSA r.o. Data shown are from 4–7 mice per group and are representative of three experiments. *P<0.05, **P<0.01, ***P<0.0001 by Bonferroni post-test where indicated. Enhanced anaphylaxis in Il4rαF709 mice is not the result of altered vascular responsiveness. Wild-type and Il4rαF709 mice were injected r.o. with E) histamine (1.25mg), F) serotonin (1mg) or G) Leukotriene C₄ (LTC₄, 1μg) and core body temperatures were recorded to assess the severity of the response. Data shown are from 3–5 mice per group.

Figure 3. IL-4 drives mast cell activation, growth and survival and the IL-4Rα ITIM limits these effects

A) Il4rαF709 BMMC exhibit enhanced phosphorylation of STAT6 in response to IL-4 stimulation. B) IgE receptor (FcεRIα) upregulation by IL-4 is enhanced in Il4rαF709 BMMC at 24hrs. C) IL-4 (2ng/ml) but not IL-13 (10ng/ml) enhances mast cell growth in combination with IL-3 and SCF, particularly for Il4rαF709 BMMC. D) Mast cell death in the absence of IL-3 and SCF is prevented by IL-4 but not IL-13 and the effect is more evident in Il4rαF709 BMMC. E) IL-4 induces greater Bcl-2 and Bcl-X_L expression in Il4rαF709 than wild-type BMMC. Statistical analysis by two-way ANOVA.*P<0.05, **P<0.01, ***P<0.0001 by Bonferroni post-test where indicated. Data are representative of at least three experiments.

Figure 4. IL-4 drives mast cell activation, growth and survival largely via STAT6

A) IgE receptor (FcεRIα) upregulation by IL-4 in mast cells is STAT6-dependent. Wild-type and STAT6^−/− BMMC were fluorescently tagged and co-cultured in the presence of increasing amounts of IL-4. After 24hrs, cells were stained for FcεRIα and analyzed by flow cytometry. B) IL-4 enhances mast cell growth in combination with IL-3 and SCF only when STAT6 is expressed. BMMC were co-cultured as in A for 5 days. C) Mast cell death in the absence of IL-3 and SCF is prevented by IL-4 via STAT6. Data for day 5 of culture are shown. IL-4 induces Bcl-2 (D) and Bcl-X_L (E) expression in wild-type but not STAT6^−/− BMMC. Statistical analysis by two-way ANOVA.*P<0.05, **P<0.01, ***P<0.0001 by Bonferroni post-test where indicated. Data are representative of at least three experiments.

Figure 5. IL-4Rα signaling directly promotes mast cell expansion during food allergy in vivo

A) Experimental design: MLN cells from OVA-sensitized Il4rαF709 mice were adoptively transferred into naïve recipient mice. The recipients were radiation bone marrow chimeras, previously reconstituted with a mixture (50:50) of bone marrow from WT and either Il4rαF709 or IL-4Rα^−/− animals. They were then fed OVA over 10 days to drive mast cell expansion. Cells from saline-treated Il4rαF709 mice were separately transferred into control recipients. Intestinal mast cells were analyzed using flow cytometry. B) IL-4Rα is required for maximal mast cell expansion. Adoptive transfer of OVA-allergic cells into wild-type/IL-4Rα^−/− mixed bone marrow chimeras (n=4–5 per group) favors the outgrowth of IL-4Rα-sufficient mast cells. C) Expression of an activated IL-4Rα variant promotes mast cell expansion in a cell-intrinsic manner in wild-type/Il4rαF709 mixed bone marrow chimera recipients (n=6 per group). Statistical analysis by repeated measures two-way ANOVA with Bonferroni post-tests for B and C, ***P<0.0001.