Over the past two decades, information concerning the role of eosinophils in human disease has dramatically increased. The evidence now available suggests that elaboration of inflammatory mediators, in particular toxic cationic proteins, by eosinophils plays a critical role in the pathophysiology of diseases. We wish to investigate the mechanisms of eosinophil degranulation in human diseases by studying the mechanism of allergic inflammation, which is one of the most common and established manifestations of eosinophil degranulation in vivo. We will establish two in vitro models and one in vivo model, and determine factor(s) and cell(s) which play a major role in the induction of eosinophil degranulation. First, a model of lgE-dependent eosinophil degranulation in vitro will be established by co-incubation of eosinophils with anti-lgE antibody stimulated peripheral blood mononuclear cells (PBMC). Preliminary results indicate that lgE-mediated eosinophil degranulation requires accessory cells (namely PBMC), suggesting the necessity of soluble factor(s) and/or cellular adhesion for eosinophil activation. Therefore, the production of cytokines and lipid mediators and the requirement for specific cell types, cellular contact, and cytokines will be analyzed. Second, a model of allergen-dependent eosinophil degranulation in vitro will be established and analyzed by co-incubation of eosinophils with ragweed antigen, sera from patients with ragweed-sensitive asthma, and PBMC. Although this model is similar to IgE-dependent eosinophil degranulation as described above, it is different in that this model does not require cell-bound lgE and that not only lgE but also IgG and/or lgA may participate. Third, the modulators of eosinophil activation in vitro will be identified and their interaction will be investigated by studying the effects of cytokines, extracellular matrix proteins, adhesion molecules, and immobilized immunoglobulins. Here, we wish to utilized the insights of goal l and 2 and to reconstitute the system using purified molecules. Fourth, a model of murine allergic eosinophilic peritonitis will be established and investigated to determine and characterize critical factor(s) for eosinophil degranulation in vivo. This project will integrate the above 4 goals to investigate the mechanism of eosinophil degranulation in human diseases. Goal l and 2 are designed to understand and mimic the in vivo environment using anti-IgE-induced eosinophil degranulation (goal l) and allergen-induced eosinophil degranulation (goal 2). The physiologic factors for initiation and modulation of eosinophil degranulation in vitro will be identified and reconstituted to mimic anti-lgE- and allergen- induced eosinophil degranulation using purified molecules (goal 3). The murine in vivo model of eosinophilic peritonitis will be analyzed as an in vivo system based on and compared to the in vitro experiments (goal 4). The achievement of these goals may results in better understanding of the mechanism of eosinophil degranulation in diseases and provide new approaches in the treatment of patients with allergic and inflammatory diseases.
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