The tracheobronchial epithelium is a dynamic participant in lung physiology and responses, including clearance of inhaled materials, regulation of inflammatory responses, and orchestration of repair. It has been demonstrated by a number of laboratories that respiratory epithelial cells possess an inducible nitric oxide synthase (iNOS). In addition, it has been shown that severe damage to the epithelium results from exposure to an inflammatory cytokine, interleukin-1 (IL-1), and respiratory epithelial cells, in a fashion similar to macrophages, can produce IL-1 in response to inflammatory stimuli. It is now becoming apparent that the epithelial cytopathology caused by IL-1 is due to triggering of overproduction of nitric oxide (NO) by the respiratory epithelium itself. In asthma, inflammation is a critical determinant of disturbed airway function and is associated with widespread epithelial injury. This research plan is designed to evaluate the production of IL-1 and NO in asthmatic airways, the pathway and specificity of NO-mediated damage, the effects of IL-1 and NO on epithelial repair, and possibilities for therapeutic intervention. In testing the central hypothesis that IL-1 and NO are important mediators of airway epithelial damage in asthmatic inflammation, effort will be focused on the following four specific aims: I. Cellular sources of IL-1 and NO in asthmatic and normal airways. The experiments in this section will evaluate the hypothesis that IL-1 and NO production are upregulated in asthmatic inflammation. ELISA, biochemical assays, immunohistochemistry and in situ hybridization will be used to detect these mediators in epithelial and non-epithelial cells from mice, as well as in cell and tissue samples from asthmatic patients. II. Pathway of epithelial damage in asthmatic airways. The hypothesis to be tested is that damage to the respiratory epithelium in asthma is dependent on locally produced IL-1. The role of IL-1 will be studied in an animal model of asthma, taking particular advantage of IL-1-deficient mice. Tissues will be evaluated for the presence of necrosis or apoptosis to permit an assessment of the way in which ongoing cell injury may influence the nature and persistence of the IL-1 signal. III. Respiratory epithelial responses to IL-1. The hypothesis proposed in this section is that acute and/or chronic characteristics of asthmatic airway inflammation result from IL-1- triggered damage, which is significantly mediated by epithelial NO production. In vitro experiments with cultured cells and with explanted animal and human tracheal tissue will be used to evaluate cellular specificity of damage, the pathway of cell death, and epithelial repair responses. IV. Therapeutic intervention. Experiments are designed to test the hypothesis that inhibiting the production or effects of IL-1 and/or the generation of NO will significantly reduce airway epithelial damage in animal models of airway hypersensitivity, anticipating application to asthmatic patients.
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