from Patients with Allergy? Does IgE Bind to and Activate Eosinophils

Human eosinophils have been reported to express both the mRNA and protein for the high afﬁnity IgE receptor (Fc e RI); it is speculated that this receptor plays a role in eosinophil mediator release in allergic diseases. However, questions still remain. How much of the Fc e RI protein is actually expressed on the cell surface of the eosinophil? If they are present, are these IgE receptors associated with effector functions of eosinophils? To address these issues, we studied blood eosinophils from patients with ragweed hay fever. A high level of low afﬁnity IgG receptor (Fc g RII, CD32), but no expression of Fc e RI, was detectable on the eosinophil surface by standard FACS analysis. However, after in vitro sensitization with biotinylated chimeric IgE (cIgE), cell-bound cIgE was detected by PE-conjugated streptavidin. This cIgE binding was partially inhibited by anti-Fc e RI mAb, suggesting that eosinophils do express minimal amounts of Fc e RI detectable only by a sensitive method. Indeed, FACS analysis of whole blood showed that eosinophils express ; 0.5% of the Fc e RI that basophils express. When stimulated with human IgE or anti-human IgE, these eosinophils did not exert effector functions; there was neither production of leukotriene C 4 or superoxide anion nor any detectable degranulation response. In contrast, eosinophils possessed membrane-bound human IgG and showed functional responses when stimulated with human IgG or anti-human IgG. Thus, IgG and/or cytokines, such as IL-5, appear to be more important for eosinophil activation in allergic diseases than IgE. Journal Immunology, 1999, 162: 6901–6911. MFC between the test Ab (e.g., anti-human IgE) and control Ab by Becton Dickinson Lysis II software. To analyze binding of cIgE to individual cell populations of buffy coat leukocytes, cells were electroni- cally gated based on their unique green autoﬂuorescence and side scatter characteristics. Two-dimensional dot-plot analysis of the green ﬂuores- cence intensity and side scatter of buffy coat leukocytes showed four distinct populations, including lymphocytes, monocytes, neutrophils, and eo- sinophils (see Results for detail). Basophils fell within the population of lymphocytes. However, when basophils were stained with biotinylated cIgE and PE-conjugated streptavidin, they formed one unique population in green ﬂuorescence/side scatter dot-plot analysis due to their extremely strong PE ﬂuorescence. Thus, individual cell populations were easily identiﬁed by their unique green autoﬂuorescence and side scatter characteris- tics; appropriateness of the identiﬁcation of each population had been conﬁrmed by positive staining for appropriate Ab (CD9 for eosinophils and basophils, CD16 for neutrophils, and CD14 for monocytes) as well as morphological analysis of cytospin preparations of sorted cells. The cells were electronically gated, and the binding of cIgE was measured by examining the intensity of PE ﬂuorescence. Fluorescence intensity was de- termined on 50,000 total cells from each sample using logarithmic ampliﬁcation, which was converted to the linear equivalent by Becton Dickinson Lysis II software. Data are presented as the D MFI between test samples (e.g., biotinylated cIgE plus PE-conjugated streptavidin) and control (PE- conjugated streptavidin alone).