Eosinophils are myeloid cells that play pivotal roles in the pathology associated with certain allergic, parasitic, neoplastic and inflammatory diseases. The large specific granule of the eosinophil contains a number of prominent low molecular weight proteins that include the eosinophil-cationic protein (ECP) and the eosinophil-derived neurotoxin (EDN). Particularly significant have been studies implicating these proteins in the destruction of larval stages of helminth parasites, the cardiovascular pathology characteristic of the hypereosinophilic syndrome, and in the pathogenesis of acute and chronic asthma and other allergic diseases. The Charcot-Leyden crystal (CLC) protein, a constituent of the eosinophil's primary granule, forms the hexagonal bipyramidal crystals (CLC) classically observed in tissues and secretions from sites of eosinophil-associated inflammation; however, its role in eosinophil function remains obscure. Progress in these areas is seriously hampered by the problems inherent in obtaining large quantities of eosinophils and eosinophil proteins in high purity and yield, and by limitations imposed by relying exclusively on the techniques of protein chemistry. We have cloned and sequenced cDNA's encoding EDN, ECP and CLC protein. The overall goal of this continuation proposal is to investigate, at the molecular level, the mechanisms by which eosinophils, through the activities of these proteins, mediate important cytotoxic and inflammatory functions in allergic and parasitic states. We propose (1) to express, purify and characterize wild type and mutant ECP, EDN and CLC proteins by recombinant methods to analyze the cytotoxic, inflammatory and enzymatic activities exhibited by these proteins by studying their mechanisms of action in vitro and in vivo using a variety of specific assays; (2) to study structure-function relationships for these proteins to define the molecular basis for their unique cytotoxic, helminthotoxic, enzymatic, and inflammatory functions to provide a rationale for the eventual development of specific antagonists or agonists of these activities for therapeutic uses; and (3) to analyze, at the molecular level, the basis for eosinophil heterogeneity and mechanisms of eosinophil activation/post-mitotic differentiation by studying the expression of ECP, EDN, and CLC messenger RNA's in normal and activated subpopulations of eosinophils from patients with diseases associated with eosinophilia and during the in vitro activation of normal eosinophils during culture with various hemopoietic growth factors.
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