There is convincing evidence that eosinophils (EOS) contribute to airway inflammation and, possibly, the pathogenesis of asthma. This position is supported by known effects of EOS on airway tissue, responses to cytokines, interactions with adhesion proteins, and histologic features of bronchial tissue. Yet to be established are mechanisms that ultimately determine the inflammatory function of EOS in airways and their contribution to bronchial hyperresponsiveness and asthma. The long-term goal of this project is to define mechanisms by which EOS cause asthma. To achieve this, evidence has been obtained that airway EOS are phenotypically distinct from their blood counterparts and are functionally upregulated. Therefore, it is hypothesized that the eventual phenotype and function of airway EOS, and hence their contribution to asthma, are determined by multiple events occurring during recruitment of cells from blood to the airways. These events include endothelial adhesion, migration through lung matrix, transepithelial movement, and exposure to pro- inflammatory molecules (i.e. cytokines).
Specific aims of this proposal are designed to determine the effects of adhesion to endothelium, matrix (fibronectin as a model), and epithelium on EOS function, signal transduction, and cytokine and adhesion protein expression. In addition, effects of cytokines on these EOS/adhesion substrate interactions will be determined. To accomplish these goals, EOS will be isolated from blood and cultured with endothelium (Human Umbilical Vein Endothelial Cells), fibronectin, or epithelium (primary human bronchial epithelial cells or airway epithelial cell lines). The effect of adherence on EOS function will be assessed by changes in gen.eration of superoxide anion and leukotriene C4 and degranulation of major basic protein and EOS derived neurotoxin. Moreover, the actions of selected cytokines (lL-5 [for EOS] and IL-1 (endothelial and epithelial monolayers]) on EOS adhesion and function will be determined. To establish mechanisms of EOS upregulation, the capacity of EOS to produce cytokine mRNA and protein (e.g. lL-5) will be determined by in situ hybridization, in vitro EOS survival essays, and ELISAs. Finally, EOS signal transduction will be assessed by intracellular calcium levels and protein phosphorylation, and correlated with functional responses and cytokine production. Changes in blood EOS phenotype (i.e. function, cytokine production and signal transduction) which result from these in vitro interactions will be compared to similar assessments on airway EOS from patients with allergic disease or asthma to correlate biological and clinical relevance. These observations will provide new information about EOS biology in the lung and insights into inflammatory mechanisms in the pathogenesis of asthma.
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