Abstract
BackgroundChronic 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.MethodsPrimary 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.ResultsTGFβ1 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.ConclusionsTaken 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.
Highlights
Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury
We have previously shown that chronic alcohol ingestion in rats increases oxidative stress [3] and transforming growth factor 1 (TGFβ1) expression in the lung [4], and inflammatory insults such as sepsis release activated transforming growth factor β1 (TGFβ1) into the alveolar space, which can intensify lung injury by further disrupting alveolar barrier function
The data from panels A-D suggest that the TGFβ1 gene expression was induced only in the alveolar macrophages by alcohol and significantly more TGFβ1 protein was present on macrophages from alcohol-fed rats as compared to controls
Summary
Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; we do not know the precise contribution of various alveolar cells in this process. We have previously shown that chronic alcohol ingestion in rats increases oxidative stress [3] and transforming growth factor 1 (TGFβ1) expression in the lung [4], and inflammatory insults such as sepsis release activated TGFβ1 into the alveolar space, which can intensify lung injury by further disrupting alveolar barrier function. We have previously shown that ethanol ingestion decreases another antioxidant, micronutrient zinc, in the lower airway [8] and adversely affects zinc importers through down-regulation of Kruppel-like transcription factor 4 by active TGFβ1 [9,10]. TGFβ1 is immunosuppressant [11,12], and decreases immune function of alveolar macrophages by dampening GM-CSF receptors on these cells [13]
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