The in vitro effects of cigarette smoke on models of epithelial injury

Cigarette smoking has been shown to increase air space epithelial permeability, which is thought to be an early indication of air space epithelial injury. A large number of toxic compounds, in particular oxygen radicals, are produced by cigarette smoke, and it may be the imbalance between these and the intra- and extracellular antioxidants present in the lungs which is critical in inducing lung injury.Both superoxide dismutase and catalase have been shown to increase intracellularly in hamsters exposed to cigarette smoke. Reduced glutathione, another important antioxidant, is also present in increased concentrations in erythrocytes and in the lung epithelial lining fluid of cigarette smokers. This may be in response to the increased radical burden in smoking in an attempt to confer protection to the cells in the airway and alveoli of the lung. However the acute effects of smoking have received less attention.In this study the attachment, detachment and proliferation of the A549 human alveolar epithelial cell line was examined in an in vitro model of cell injury and the effects of exposure to cigarette smoke and its condensates on these assays of epithelial function were assessed. In addition a comparison of the effects of cigarette smoke on the bronchial cell line, BEA -2B or freshly isolated rat type II cells, and the protective effects of the antioxidants, were also studied.Whole and vapour phase cigarette smoke decreased 51Cr- labelled A549 epithelial cell attachment in a dose -dependent manner. The condensates prepared from both types of cigarette smoke increased cell detachment, and decreased cell proliferation, as assessed by the uptake of [3H] thymidine by A549 epithelial cells. Both freshly isolated rat type II alveolar epithelial cells and cells of the bronchial epithelial cell line, BEA- 2B, showed enhanced susceptibility to the injurious effects of cigarette smoke condensates when compared with the A549 epithelial cell line. The enzymatic antioxidants superoxide dismutase and catalase, when present extracellularly, gave partial protection against the effects of cigarette smoke or condensate exposure on cell attachment, detachment and proliferation. However extracellular reduced glutathione, in physiological concentrations (400μM), conferred total protection against cigarette smoke or condensate induced cell injury. Depletion of intracellular reduced glutathione also enhanced the epithelial cell detachment produced by cigarette smoke condensates. The oxidant hydrogen peroxide also decreased A549 epithelial cell attachment and increased cell detachment, which was prevented by the addition of reduced glutathione.To examine possible mechanisms of cigarette smoke induced changes in epithelial cell attachment and detachment the role of the cell surface adhesion molecules, and the cell cytoskeleton and changes in intracellular signalling were studied. Both the A549 and the BEA -2B cell lines stained positive for integrins for the extracellular matrix. Cigarette smoke exposure of these cells did not effect integrin expression, but this does not rule out changes in integrin function. Surface adhesion molecule function is cationic dependent and chelation of the cations was found to cause epithelial detachment.Activation of protein kinase C or increasing intracellular calcium also increased epithelial detachment, whereas inhibition of these effects reduced the level of epithelial cell injury produced by cigarette smoke condensate. This suggests that both protein kinase C and intracellular calcium play a role in cigarette smoke condensate induced epithelial cell iv injury. The cytoskeleton is also involved in epithelial cell detachment since disruption of the microfilaments of the epithelial cell cytoskeleton with cytochalasin also lead to increased detachment.These studies indicate that exposure of epithelial cells to cigarette smoke or its condensates in vitro causes cell injury. The mechanism of this effect appears to be oxidant mediated and may be initiated through a variety of intracellular signalling pathways