Mechanisms by which endotoxin-stimulated alveolar macrophages impair lung epithelial sodium transport

grantor: University of TorontoOur laboratory previously demonstrated that a vital component of the alveolar-capillary barrier, distal lung epithelial cell (DLEC) amiloride-sensitive Na+ transport, could be impaired by 'in vitro' coculture with endotoxin (LPS)-stimulated alveolar macrophages (AM) through an L-arginine dependent mechanism. A similar, L-arginine independent impairment was seen resulting from exposure to the cell-free supernatant of LPS-stimulated AM. It was hypothesized that AM coculture might result in modulation of DLEC mRNA expression for the specific epithelial Na+ channel, rENaC. The role of peroxynitrite in the L-arginine dependent impairment of DLEC Na + transport resulting from activated AM exposure was investigated. The ability of the thiol antioxidant N-acetylcysteine (NAC) to influence the changes in functional DLEC Na+ transport resulting from exposure to cell-free AM supernatant was also examined. LPS-stimulated AM resulted in a significant decrease in mRNA expression for all subunits of rENaC. Further experiments showed that this process was not L-arginine dependent. Immunofluorescence studies using anti-nitrotyrosine antibodies failed to support an important role for peroxynitrite in the effect of activated AM on DLEC Na+ transport. Furthermore, it was shown that the impairment of DLEC Na+ transport resulting from exposure to LPS-stimulated AM supernatant was significantly modulated by NAC, which increases intracellular glutathione levels, but not by dithiothreitol (DTT), which does not specifically influence cell thiol levels. These studies, then, elucidate mechanisms by which activated AM impair alveolar epithelial barrier function in an 'in vitro' model of acute lung injury.M.Sc