The link between CFTR mutations and the chronic lung infections associated with CF has been elusive. Recent studies indicate that one result of CF mutations in pulmonary epithelia is an impaired ability to kill bacteria added to the mucosal surface. Elevations in airway surface fluid (ASF) Na/+ and Cl/- appear to impair the function of anti-microbial factors important in normal mucosal defenses. We propose that beta-defensins, cationic salt-sensitive anti-microbial peptides with broad spectrum activity, contribute to the bactericidal activity of the ASF. In this application we propose studies of human beta-defensins 1 and 2 (hBD-1 and 2) which were recently found to be expressed in epithelia of the lung. The following four specific aims are proposed for this studies: 1) What is the cell-specific localization of the hBD-1 and hbd-2 mRNAs and proteins in the lung? This question will be addressed using the combined approaches of in situ hybridization and immunocytochemistry. Identification of the cell types responsible for beta-defensin production will help frame further hypothesis regarding their rule in the pulmonary mucosal immune system. What are the anti-microbial properties of the human beta defensins? Using both synthetic and recombinant peptides in established and novel anti- microbial assays, we will determine the spectrum of anti-microbial activity and salt-sensitivity of the beta-defensins in in vitro and epithelial cell culture models. 3) What factors regulate the expression and secretion of beta-defensins in airway epithelia? To understand how beta-defensin gene expression may be regulated we will treat cultured epithelia with candidate regulatory factors including bacterial, pro- and anti-inflammatory cytokines, and glucocorticoids. Strategies including biochemical analysis of airway epithelia cell surface fluid and cell extracts, metabolic labeling, ELISA and immunoblot assays will allow localization of the peptides in intra-or extracellular compartments. 4) Are other human beta-defensins important in pulmonary epithelial defenses? Two approaches will be employed. First, we will analyze cell fractions from cultured airway epithelia for new cationic peptides using conventional biochemical means. The second approach takes advantage of the observation that the defensins are a gene cluster on chromosome 8p23. A bacterial artificial chromosome (BAC) contig from chromosome 8 containing the loci for the know defensins will be constructed and screened for defensin sequences using the technique for 3' exon trapping. An induced, size-selected cDNA library will be constructed from airway epithelia and screened for beta-defensin expression. From these studies we hope to understand the role beta-defensins play in the mucosal defenses of the lung and gain new insights into CF disease pathogenesis and new therapies.
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