Studies are proposed to examine the role of the epithelial cells of central airways in the pathogenesis of airway hyperreactivity. Recent observations suggest a potential role for epithelial cells in increasing airway tone by secretion of mediators that up-regulate airway smooth muscle responsiveness. Other observations suggest that inflammatory cells such as eosinophils may alter the growth and function of airway epithelial cells and thereby change epithelial cell modulation of airway tone. Experiments are proposed to determine the modulating influences of airway epithelium on airway smooth muscle responsiveness and to determine the mechanisms by which inflammatory cells alter epithelial cell modulation of airway smooth muscle. Guinea pig trachea] epithelial cells in primary culture will be co-cultured with human peripheral blood eosinophils or eosinophil protein mediators. Airway epithelial cell growth will be assessed by incorporation of 3H-thymidine and bromo- deoxyuridine into the cell DNA. Airway epithelial cell secretion of eicosanoids will be measured by enzyme-linked immunosorbent assay. Airway smooth muscle responsiveness to epithelial cell mediators will be assessed in situ in a newly developed guinea pig """"""""living explant"""""""" preparation of tracheal smooth muscle. Positive results and hypotheses in experiments using guinea pig epithelial cells will be confirmed using human bronchial airway epithelial cells. Human bronchial epithelial cells also will be used to study topographical differences in airway epithelial cell regulation of airway smooth muscle responsiveness. Activation and degranulation of eosinophils in these studies will be assessed in vitro by a newly developed kinetic assay for eosinophil peroxidase and by measurement of leukotriene and superoxide radical production. Two major goals are identified in this proposal: 1) Determine the modulatory role of eosinophils on airway epithelial cell growth, function, secretion of eicosanoid mediators. These studies will test the hypothesis that inflammatory cells such as eosinophils i) stimulate the secretion of bronchoactive eicosanoid mediators from the epithelial cells of central airways, and ii) may elicit changes in epithelial cell function and eicosanoid secretion without causing cell death. These studies will demonstrate potential mechanisms by which the epithelium can serve as a target of eosinophils and other inflammatory cells, and suggest mechanisms by which both cells potentiate the early stage (i.e., before death and cell sloughing) inflammation of the asthmatic state. 2) Determine the modulatory role of airway epithelial cells on the mechanical response of airway smooth muscle and the effect of inflammatory cells upon this modulatory role. These studies will test the hypothesis that airway epithelial cells secrete mediators that contract airway smooth muscle and that replacement of these factors will augment bronchomotor tone in airways in which the epithelium has been removed. These studies will test the further hypothesis that inflammatory cells such as eosinophils alter production of these factors by epithelial cells and thus alter bronchomotor tone. Data derived form these studies should provide insight into the mechanism by which airway epithelial cells modulate airway responsiveness in conditions such as human asthma.
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