1162 Identification of an IgG1-mediated anaphylaxis marker and its application in evaluating the antigenicity of infant formulas

Wednesday, 14 October 2015
Hall D1 Foyer (Floor 3) (Coex Convention Center)

Takeshi Matsubara, PhD , Morinaga Milk Industry Co., Ltd., Zama, Japan

Hiroshi Iwamoto , Morinaga Milk Industry Co., Ltd., Zama, Japan

Yuki Nakazato , Morinaga Milk Industry Co., Ltd., Zama, Japan

Kazuyoshi Namba , Morinaga Milk Industry Co., Ltd., Zama, Japan

Yasuhiro Takeda , Morinaga Milk Industry Co., Ltd., Zama, Japan

Background: In clinical practice, the basophil activation test (BAT) is thought to be a safe and sensitive test for diagnosing IgE-mediated allergic disease. We tested whether the BAT can be used in mice. In the course of developing a murine BAT using CD200R1 as an IgE-mediated activation marker, we found that the expression of a murine basophil identification marker, CD200R3, on antigen-sensitized basophils decreased following specific antigen challenge. Interestingly, this decrease did not always harmonize with increased CD200R1 expression. Since IgG, as well as IgE, contributes to mouse anaphylaxis, we hypothesized that the decrease in CD200R3 on basophils was induced by IgG-mediated cell activation.

Objective: We aimed to establish whether CD200R3 is a marker of IgG-mediated basophil activation and whether its expression is correlated with IgG-mediated anaphylaxis in a mouse model. Furthermore, we attempted to evaluate whether the BAT, using CD200R1 and CD200R3 as basophil activation markers, indicates the antigenicity of cow’s milk-based infant formulas, compared with a systemic anaphylaxis test in mice.

Methods: Mouse basophils were stimulated via FcεRI and FcγRs, and levels of CD200R1 and CD200R3 were analyzed by flow cytometry. Basophils derived from naïve mice were passively sensitized with antiserum for β-lactoglobulin (β-LG) or an IgG/IgG subclass-depleted antiserum, and challenged with β-LG. Systemic anaphylaxis was induced by i.v. injection of anti-FcγRIII/II monoclonal antibody, and CD200R3 expression on peripheral basophils was analyzed. To evaluate the antigenicity of infant formulas, basophils from mice actively sensitized to β-LG or casein were stimulated with serially diluted conventional, partially hydrolyzed, or extensively hydrolyzed infant formulas. In addition, systemic anaphylaxis was assessed following i.v. injection of formula into the immunized mice.

Results: Stimulation via FcεRI induced a significant increase in CD200R1 expression but had only a small effect on that of CD200R3. However, anti-FcγRIII/II stimulation strongly reduced CD200R3 expression. In passive sensitization experiments, the decrease in CD200R3 expression induced by antigen challenge was canceled by the depletion of IgG or IgG1. Intravenous injection of anti-FcγRIII/II led to CD200R3 down-regulation on basophils, accompanied by a drop in rectal temperature. Basophil activation was induced by the conventional but not the extensively-hydrolyzed formula. The partially hydrolyzed formula induced basophil activation somewhat weakly. Induction of systemic anaphylaxis by formula was correlated with the BAT results.

Conclusions: Use of CD200R1 and CD200R3 as activation markers enables the evaluation of IgE- and IgG-mediated murine basophil activation, respectively, and the BAT system would be useful for evaluating food antigenicity.