SEROUS CELL MALFUNCTION AND CYSTIC FIBROSIS LUNG DISEASE

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Most people with cystic fibrosis (CF) die from chronic lung infections, but it is unknown how CFTR mutations compromise mucosal defenses. The hypothesis to be tested is that CF lung disease begins because CFTR mutations disrupt serous cell secretion, thus depriving CF airways of the antibiotic-rich fluid secreted by serous cells, which express the highest levels of CFTR among airway cells. Preliminary data indicate that serous cells require CFTR for fluid secretion. The serous cell malfunction hypothesis will be directly tested by measuring antibiotic levels in secretions. This project is made possible by a model serous cell line (Calu-3 cells) and especially by improve human airway 1 degree cultures that retain a "pure" serous cell phenotype. Aim 1 test the hypothesis that serous cell 1 degree cultures and Calu-3 cells express abundant antimicrobials, including defensins and collectins. Semi-quantitative RT- PCR will be used to measure expression of mRNA for lysozyme, lactoferrin, secretory component, serum leukocyte protease inhibitor (SLPI), the human defensin molecules hBD-1 and hBD-2, and the collectin SP-A. Aim 2 will test the hypothesis that human airway serous cells do not secrete antibiotics in CF. ELISA will be used to quantify the release of antimicrobials from Calu-3 cells, from serous cell 1 degree cultures from control and CF subjects, and in nasal lavages from normal and CF subjects. Aim 3 tests the hypothesis that CF human airway serous cells do not secrete fluid. A double-sided capacitance probe method will be used to measure fluid secretion across the 1 degree serous cell monolayers from controls and CF individuals. Gland secretions will also be measured with constant bore capillaries in freshly excised trachea and bronchi from controls and CF individuals. Aim 4 will test the hypothesis that serous cells require CFTR to secrete in response to increase [Ca/2+]/i, and that they secrete a variable mixture of HCO/3 and Cl-. This will be tested by shot circuit current measurements, isotope fluxes and patch-clamping. The results expected for this project are that Ca/2+ dependent secretion of Cl and fluid by serous cells will be greatly diminished in CF, resulting in reduced volumes of antibiotics reaching the airway surface from the glands.