The goal of this project is to examine the interactions between eosinophils and adhesive extracellular proteins occurring during transit of eosinophils from blood stream to airway and learn how the interactions relate to the alteration of eosinophil phenotype. We hypothesize that a vicious cycle may develop that contributes to asthma whereby eosinophilic infiltration causes deposition of abnormal extracellular matrix that in turn causes more infiltration and functional upregulation of eosinophils.
The specific aims of this proposal are to: (A) develop models of adhesion and migration of eosinophils with an emphasis on cell function; (B) determine that distribution of relevant matrix proteins in the models vis-a-vis normal, non-asthmatic allergic and asthmatic bronchial mucosa; (C) determine the role of integrins in models established in specific aim A; (D) determine the role of cytokines and chemokines in eosinophil-matrix interactions; and (E) test whether products of activated eosinophils alter deposition of extracellular matrix in cultures of lung fibroblasts. To accomplish these goals, peripheral blood eosinophils will be studied in tow-and three-dimensional systems of cell adhesion, spreading, and migration. The production of superoxide anion, leukotriene C4, and cytokines, e.g., GM-DSF, extent of degranulation, and cellular survival will be assessed to determine alteration of cell function after various incubations with adhesive endothelial cell (VCAM-1) and matrix proteins (fibronectin, laminin isoforms). The effects of cytokines, chemokines, and antiintegrin antibodies will be examined in these assays to identify key interactions that could occur within the tissue. Expression of adhesive proteins in asthmatic airways will be determined by immunocytochemistry and in situ hybridization or reverse transcription-PCR. Projects of activated eosinophils will be tested for their ability to cause increased deposition of matrix proteins as occurs in airways of asthmatics. Changes in phenotype of blood eosinophils that result from these in vitro conditions will be compared to similar assessments on eosinophils from airways of patients with asthma to correlate biological and clinical relevance. These observations should provide new insights into eosinophil biology and mechanisms of persistent airway damage in asthma.
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