Cystic fibrosis pulmonary disease is characterized by neutrophil-dominated airway inflammation, which eventually leads to fibrosis, bronchiectasis, and pulmonary failure. Pseudomonas aeruginosa infection is an important cause of this inflammatory process. In the experiments proposed, exactly how this organism stimulates inflammation in both normal and epithelial cells with mutant CFTR alleles will be delineated. Adherent P. aeruginosa stimulate epithelial cells to express large amounts of the neutrophil chemokine IL-8. This is accomplished through a complex signaling cascade. One of the earliest responses of the epithelial cell to P. aeruginosa is a brisk rise in intracellular calcium, which is followed by activation of the transcription factor of IL-9 and other pro-inflammatory cytokines. We will establish which bacterial gene products trigger the epithelial flux in calcium which is required to evoke this inflammatory response. The nature of the epithelial receptor, the kinases and phosphatases activated by the bacteria, and the regulation of this signaling pathway will be established. Endogenous stimuli such as the accumulation of mutant CFTR within the endoplasmic reticulum (ER overload) may also trigger a rise in intracellular calcium which similarly initiates activation of NF-kappaB By comparing the response of cells with specific types of CFTR mutations, it should be possible to determine how CFTR dysfunction affects the immune function of epithelial cells, as well as the electrophysiological properties. Analysis of the activation of NF-kapapB in cells with defined CFTR mutations from both transgenic mice, as well as from patients with CF may suggest therapeutic strategies to modulate the inflammatory response. Immunosuppressant drugs such as FK506 and cyclosporine A block the calcium dependent phosphatase calcineurin. It may be possible to use such drugs to diminish the inflammation provoke by P. aeruginosa as well as the endogenous stimulation of NF-kappaB produced in response to the cell stress initiated by the accumulation of mutant, mistrafficked CFTR in the endoplasmic reticulum.