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

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

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.

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.

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.