The airway epithelium exists at the interface with the external environment, so epithelial cells are strategically positioned to respond to environmental irritants (e.g., bacteria, viruses, pollutants, cigarette smoke). Neutrophil products are implicated in various inflammatory responses in airways, and work supported by Project D-3 has recently shown that neutrophil proteases are potent gland secretagogues. Airway cells (including epithelial cells) produce a cytokine, interleukin-8 (IL-8), which is a potent neutrophil chemoattractant. Studies supported by Project D-3 have provided evidence that IL-8 exists in high concentrations in sputum of patients with cystic fibrosis (CF), bronchiectasis, chronic bronchitis, and in subsets of patients with asthma, suggesting that IL-8 induction by airway cells is implicated in neutrophil recruitment. This project will utilize molecular, cellular, biochemical and physiologic tools to study the role of airway epithelial cell IL-8 in airway inflammatory responses. Because patients with CF often become infected with Pseudomonas aeruginosa (PA) bacteria, and because recent studies in Project D-3 showed that a product from PA induces IL-8 expression and secretion, studies are proposed to examine PA induction of IL-8 in epithelial and other cells in airways. Studies in humans will utilize tissue obtained from donors and recipients of lung transplants. Studies in humans must be limited, so the investigators in Project D-3 have recently cloned dog IL-8. The first goal in the project will be to express dog recombinant IL-8, to purify the recombinant protein, and to verify its activity. Antibodies to dog IL-8 will be raised, and neutralizing antibodies will be selected. Using these materials, an ELISA for dog IL-8 will be developed, so IL-8 activity can be measured. A second goal is to determine which cells in airways express IL-8 in response to PA supernatant delivered into the airways in dogs in vivo and in vitro. When neutralizing antibodies are available, its inhibitory effect on neutrophil recruitment and on airway secretion following PA supernatant will be examined. Evaluation of potential mechanisms for inhibiting IL-8 (e.g., corticosteroids) will be evaluated. The isolation, structural analysis and synthesis of the product of Pseudomonas aeruginosa bacteria that is responsible for IL-8 production in airway epithelial cells will be undertaken, in collaboration with expert bioorganic chemists in the area of carbohydrate and protein chemistry of microbes. In the future, studies will focus on other bacteria, viruses and pollutants on IL-8 production in airways. The studies have important pathophysiologic and therapeutic implications: they suggest mechanisms that underlie chronic inflammation and airway hypersecretion in diseases of airways. In CF and bronchiectasis, they suggest methods of intervention. In asthma, they suggest mechanisms that might explain the mucous plugging that is reported in pathologic studies of patients dying of asthma. In chronic bronchitis, they may provide mechanisms to explain some of the symptoms and inflammatory effects in the airways.
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