CONTROL OF EOSINOPHIL APOPTOSIS BY INTRACELLULAR SIGNALS

DESCRIPTION One of the most efficacious treatments for chronic asthma is inhaled glucocorticoid therapy which reduces inflammation in the airways by inhibiting cytokine production and inducing eosinophil apoptosis. Although glucocorticoid therapy has proven to be effective for most asthmatics, there is a range of patient responsiveness, suggesting that variability may be due to underlying differences in intracellular signaling. The reduction for eosinophilia in asthmatics undergoing glucocorticoid therapy is strongly associated with improved airway functions, and glucocorticoids have been shown to induce apoptosis in human eosinophils in vitro and in vivo. Based on these observations, the investigators hypothesize that the quality and quantity of cytokine production in the lung, and differences in the eosinophil gene expression profile between individuals, are two key factors that modulate the potency of glucocorticoid therapy. Three specific aims are proposed to test this general hypothesis. 1) They have found that glucocorticoids induce human eosinophil apoptosis in primary cell cultures containing low levels of cytokines, but actually enhance eosinophil survival in cultures with high concentrations of cytokines. They will investigate the molecular basis for differential glucocorticoid effects in purified human eosinophils using cell extracts to monitor mitochondrial dysfunction, and kinase-specific inhibitors to determine which pathways contribute to this enhancement effect. 2) They will identify key signaling pathways required for anti- and pro-apoptotic signaling in the human eosinophil cell line model AML14.3D10. This will be done using molecular genetic approaches that exploit co-transfection assays containing the green fluorescent protein (GFP) gene and dominant negative gene variants of selected signaling molecules. 3) They will use cDNA microarray phenotyping of 20,000 human genes to identify and characterize expression patterns that associate with apoptotic sensitivity of primary human eosinophils, and with intracellular signaling pathways in AML14.3D10 cells grown under various culture conditions. This microarray analysis takes advantage of low-cost services and resources provided by the university-wide U. of Arizona Microarray Core Facility.