4A). We conclude that a strong passive saturating binding of IgE to basophils occurs in IgE knock-in mice in vivo. The central experiment to demonstrate a function of increased IgE in allergy is the analysis of anaphylaxis. Nutlin-3a datasheet The three genotypes (Fig. 3A) allowed a dissection of IgE versus IgG1 sensitizing capacity in an active anaphylaxis experiment. We used the same protocol for immunizing IgE knock-in mice as explained above (Fig. 3B), followed by an i.v. challenge with 30 μg TNP-OVA to induce systemic anaphylaxis (Fig. 4B). PBS-injected control mice did react with minimal body temperature drop of 0.5°C (Fig. 4B, panel2).

In sensitized IgEwt/ki and IgEki/ki mice, a comparable strong drop in body temperature of 6°C was observed, whereas WT mice reacted with moderate temperature drop of 4°C. It is important to note that the drop in body temperature in the IgE knock-in mice is more sustained compared with that in WT mice (Fig. 4B, panel 1). Surprisingly, only in the group of the

IgEki/ki mice, about 40%, died due to anaphylaxis (Fig. 4C panel 1). IgEki/ki p38 MAPK activity lack IgG1 and express high levels of antigen-specific IgE, yet are more susceptible to anaphylactic shock compared with WT mice, which express high levels of antigen-specific IgG1, but little IgE. Therefore, we suggest that antigen-specific IgE is a more potent inducer of anaphylaxis compared with antigen-specific IgG1. Mannose-binding protein-associated serine protease Importantly, while the IgEki/ki and the IgEwt/ki mice had similar temperature curves, death occurred only in the IgEki/ki mice, arguing for the strongest anaphylactic reaction in the IgEki/ki mice. These results argue against a major role for the alternative pathway of systemic anaphylaxis, which is mediated largely through IgG1 and FcγRIII and basophil activation in our model [8, 9]. In the following experiment, we addressed two questions, namely, whether CD23, the low affinity receptor for IgE, on B cells in conjunction with the IgE knock-in affects the

outcome of systemic anaphylaxis, and if basophil depletion influences IgE-mediated active anaphylaxis. First, we backcrossed the IgE knock-in mice to CD23-deficient mice [23]. No significant effect of a loss of CD23 on anaphylaxis in the IgEwt/wt animals was observed (Fig. 4B panel 2, open squares) when compared with the CD23 competent IgEwt/wt mice (Fig. 4B panel 1, open triangles). Also, no CD23-deficient mice died due to anaphylaxis (Fig. 4C panel 2), similar to wild-type animals (Fig. 4C panel 1). The double-mutant CD23−/− IgE knock-in heterozygous and homozygous mice respond to the anaphylactic challenge with faster and more sustained temperature drop and death (Fig. 4B and C, panels 3 and 4). Again, homozygous CD23−/− IgEki/ki mice display the strongest increase in lethality.