메밀알레르기의 생쥐모델 개발과 비강내 면역요법 후 특이 IgE 생성의 변화
BACKGROUND: Buckwheat is one of the common food allergens in Korea. And it often causes severe and near fatal allergic reactions. At present allergen avoidance is the only therapeutic option for food allergies. PURPOSE: This study is designed to develop a murine model of IgE-mediated buckwheat hypersensitivity induced by intragastric sensitization with native buckwheat flour. During the procedure it was intended to determine not only the qualitative and quantitative differences of IgE production dependent upon amount of the sensitizing antigen, but also differences of cytokines produced by sensitizing antigen. At the same time, I evaluated a possibility of immunomodulatory effect of intranasal immunotherapy with buckwheat extract using established buckwheat allergy model in this study. MATERIALS & METHODS: Female C3H/HeJ mice(4～5 weeks old, 6～8 per group) were sensitized and challenged via intragastric route with various doses of fresh buckwheat flour(Group 1: 1 ㎎/dose, Group 2: 5 ㎎/dose, Group 3: 25 ㎎/dose of proteins) mixed with cholera toxin as adjuvant. Sham-sensitized(Group 4) mice were sensitized with cholera toxin alone. Naive(Group 5) mice were not sensitized with buckwheat flour or cholera toxin. Three weeks later, mice of Group 1, Group 2, Group 3, and Group 4 were then challenged via intragastric route three weeks later with fresh buckwheat flour extract containing cholera toxin. Anaphylactic reactions were evaluated 30 minutes after challenge, and then B- and T-cell responses to a crude extract of buckwheat flour were characterized by evaluating buckwheat-specific IgE, IgG₁, and IgG_(2a) antibody concentrations, by performing splenocyte proliferation assays and by assaying of various cytokines with antigen stimulation. In the second stage of experiment, all the mice were sensitized by introducing buckwheat antigen(1 ㎎/dose of proteins) via intragastric route on Day 1, 2, 3, and 7. From the sensitization day 14 on, different mice were administered intranasally buckwheat antigens(Group 6: 2 ㎍/dose/mouse, Group 7: 20 ㎍/dose/mouse), or PBS(Group 8) by the same route, three times per week for 2 weeks. After that I evaluated the concentrations of antigen specific IgE, IgG, the production of various cytokines and the level of splenocyte proliferation by stimulation in vitro with buckwheat allergen. RESULTS: Intragastric buckwheat challenges of sensitized mice provoked anaphylactic reactions such as severe scratch, perioral/periorbital swelling, or decreased activity. Reactions were associated with elevated levels of buckwheat-specific IgE antibodies. Splenocytes from buckwheat allergic mice exhibited significantly greater proliferative responses to buckwheat than non-allergic mice. Increased production of IL-4, IL-5, and IFN-γ by splenocyte induced by buckwheat were positively correlated with elevated levels of buckwheat-specific IgE and symptom scores in sensitized mice. In the second part, in the 4th week, which is two weeks after the intranasal immunotherapy, IgE production was blunted. The blunting of IgE production was correlated with diminished splenocytic IL-4 production and proliferative capacity. Also this finding was associated with increased IL-10 and IgG_(2a) production but any relationship was not revealed with the productive capability of IFN-γ, IL-5, or TGF-β. CONCLUSIONS: I developed IgE-mediated buckwheat allergic murine model by intragastric sensitization and challenge. And then in this model intranasal immunotherapy utilizing buckwheat antigen was performed and it was observed that a possibility of intranasal immunotherapy for modulation of food allergic reaction and IgE production to buckwheat.
메밀알레르기, 위장관을 통한 감작, 생쥐모델, 비강내 면역요법, Buckwheat allergy, Intragastric sensitization, Murine model, Intranasal immunotherapy
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