REGULATION OF CC CHEMOKINES IN HUMAN AIRWAY EPITHELIUM

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DESCRIPTION (Adapted from the applicant's abstract): Previous work has demonstrated that airway epithelial cells are an important source of three major eosinophil-active chemokines, eotaxin, MCP-4, and RANTES, that glucocorticoids (GCs) inhibit production of these chemokines, and that both production and sensitivity to GCs differ among these chemokines. To explore these observations, the PI has proposed 3 specific aims: 1) To identify the molecular basis of the differential regulation by IL-4 of eotaxin and RANTES expression in human airway epithelial cells, specifically the transcriptional and post-transcriptional regulation of the expression of these chemokines. The work planned is based on the observation that in reporter gene constructs, IL-4 upregulates eotaxin promoter-driven constructs, an effect which appears to be dependent on STAT6, while it inhibits RANTES promoter activity. Transfection of BEAS-2B cells with overexpressed and repressed STAT6 is planned. Post-transcriptional pathways will be examined by determining the effect of IL-4 on eotaxin mRNA stability. 2) To study the molecular mechanisms by which glucocorticoids inhibit epithelial expression of eotaxin and RANTES. Based on the observation that GCs accelerate the decay of mRNA for eotaxin and MCP-4, but not RANTES, post-transcriptional mechanisms will be explored including the stimulus-specificity, time course and duration of mRNA destabilization, and the need for protein synthesis for these effects to occur. The PI hypothesizes that AU rich-elements in the 3' untranslated region of eotaxin but not RANTES are involved in this GC-induced mRNA destabilization. BEAS-2B cells will be transfected with a transiently infected with reporter constructs containing a transiently inducible c-fos/B-globulin gene fusion plasmid containing the 3'-UTR of eotaxin or RANTES. Finally, the mechanism(s) by which GCs regulate transcription will be determined based on the observation that GCs suppress both eotaxin and RANTES promoter-driven constructs. The effect of GCs on STAT6 expression and function will be determined using deletional and mutational analyses of reporter construct genes.3) To characterize the expression and function of CCR-3 on epithelial cells, including a determination of the factors modulating CCR3 expression in epithelium; the binding, signaling properties, and function of epithelial CCR3; and the expression of CCR3 in vivo in airway epithelial cells in normal and allergic subjects.