Chronic Alcohol Ingestion Impairs Rat Alveolar Macrophage Phagocytosis via Disruption of RAGE Signaling.

BACKGROUND: Alcohol significantly impairs antioxidant defenses and innate immune function in the lung and increases matrix metalloproteinase 9 (MMP-9) activity. The receptor for advanced glycation end products (RAGE) is a well-characterized marker of lung injury that is cleaved by MMP-9 into soluble RAGE and has not yet been examined in the alcoholic lung. We hypothesized that chronic alcohol ingestion would impair RAGE signaling via MMP-9 in the alveolar macrophage and thereby impair innate immune function.

MATERIALS AND METHODS: Primary alveolar macrophages were isolated from control-fed or alcohol-fed rats. Real-time polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assays were performed to evaluate RAGE expression. Silencing of MMP-9 ribonucleic acid (RNA) in a rat alveolar macrophage cell line was confirmed by qRT-PCR, and immunofluorescence (IF) was used to assess the association between alcohol, MMP-9, and RAGE. Phagocytosis was assessed using flow cytometry. Sulforaphane and glutathione were used to assess the relationship between oxidative stress and RAGE.

RESULTS: RAGE messenger RNA expression was significantly increased in the alveolar macrophages of alcohol-fed rats, but IF showed that membrane-bound RAGE protein expression was decreased. Lavage fluid demonstrated increased levels of soluble RAGE (sRAGE). Decreasing MMP-9 expression using si-MMP-9 abrogated the effects of alcohol on RAGE protein. Phagocytic function was suppressed by direct RAGE inhibition, and the impairment was reversed by antioxidant treatment.

CONCLUSIONS: Chronic alcohol ingestion reduces RAGE protein expression and increases the amount of sRAGE in alveolar lavage fluid, likely via cleavage by MMP-9. In addition, it impairs phagocytic function. Antioxidants restore membrane-bound RAGE and phagocytic function.