Allergen crosslinking of IgE on cells bearing high affinity IgE receptors (FcepsilonRI, alphabetagamma2), such as mast cells and basophils, triggers the release of mediators that initiate allergic inflammation. Recent studies have defined a role for IgE in the regulation of FcepsilonRI expression and function on basophils. Approaches that remove IgE, or prevent its attachment to the alpha subunit of FcepsilonRI, dramatically reduce levels of basophil FcepsilonRIalpha and result in reductions of antigen-specific histamine release. Other studies show that re-exposure to IgE in vitro or in vivo reverses these effects. In contrast, preliminary studies fail to show similar IgE-dependent FcepsilonRIalpha modulation on monocytes, which express a different form of FcepsilonRI lacking the beta subunit (alphagamma2). Further, preliminary data suggest that expression of monocyte FcepsilonRI only occurs in allergic donors, but unlike basophils, appears to be independent of serum IgE levels. The goal of this proposal is to enhance our understanding of the mechanisms of IgE- dependent and IgE-independent regulation of FcepsilonRI on human basophils and monocytes, with an emphasis on the role played by the beta chain of FcepsilonRI We hypothesize that IgE-dependent enhancement of the basophil beta subunit occurs with the alpha subunit. We further hypothesize that basophils, after IgE-induced receptor enhancement, will secrete increased amounts of IL-4. We also hypothesize that cytokines present in allergic disease are needed for monocyte FcepsilonRIalpha expression and that the lack of the beta subunit in monocytes reduces IgE-dependent receptor modulation. To test these hypotheses, specific aims will 1) determine whether IgE-dependent alteration of FcepsilonRIalpha expression on basophils alters transcription and translation of the beta subunit of FcepsilonRI; 2) test whether observed FcepsilonRIalpha receptor modulation alters IL-4 release from basophils; and 3) explore mechanisms regulating FcepsilonRI expression on monocytes. Experiments will use RT-PCR and Western blotting to monitor changes in beta subunit mRNA and protein expression in lysates from basophils cultured with or without IgE. Similar basophil cultures will be examined for changes in the magnitude or releasability of IL-4 protein secretion under various stimulation conditions. Finally, regulation of monocyte FcepsilonRI expression will be examined in cultures supplemented with or without IgE and selected cytokines. Using transfection, the introduction of the beta subunit into human monocytes or monocytic cell lines bearing alphagamma2 will be tested for its' effects on IgE or cytokine mediated expression of FcepsilonRI. The results of the planned studies will further define mechanisms of FcepsilonRI regulation on basophils and monocytes, and may lead to novel FcepsilonRI-based strategies for the treatment of allergic diseases.
