Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1

BACKGROUND: Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFβ1 may become activated in the alveolar space of the alcoholic lung. METHODS: Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague–Dawley rats. Expression of TGFβ1 and the epithelial integrin αvβ6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran. RESULTS: TGFβ1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFβ1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFβ1 antibody treatment, indicating the presence of bioactive TGFβ1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFβ1 and anti-αvβ6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFβ1 in vitro, increased the expression of αvβ6 integrin, which is known to activate TGFβ1, in alveolar epithelial cells. CONCLUSIONS: Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFβ1 at points of cell-cell contact.