Ethanol inhibits lipid raft-mediated TCR signaling and IL-2 expression: potential mechanism of alcohol-induced immune suppression.

Alcohol abuse imposes a heavy burden on the U.S. healthcare system. Immunosuppression associated with chronic alcohol abuse is an important predisposing factor to opportunistic infections and cancer. Clinical complications of alcohol abuse are associated with induction of immune dysfunction. The mechanisms of alcohol-induced immunosuppression include defects in both humoral and cell-mediated immunity (Adams and Jordan, 1984; Eckardt et al., 1981; MacGregor, 1986; Szabo and Mandrekar, 2009). Although many types of immune cells are affected by alcohol, including neutrophils, natural killer cells, and monocytes/macrophages, the effect of alcohol on thymus-derived lymphocytes (T lymphocytes) is a major factor that contributes to overall immune suppression (Bagasra et al., 1987; Glassman et al., 1985). Additionally, clinical and experimental data show that ethanol exposure leads to alterations in the extent and pattern of T-lymphocyte cytokine production, and T-lymphocyte hyporesponsiveness (Chadha et al., 1991; Jerrells et al., 1990; Laso et al., 1999; Mutchnick and Lee, 1988; Peterson et al., 1998; Spinozzi et al., 1991). These observations suggest that alcohol intake can adversely affect T lymphocytes, leading to multiple immune defects. However, the molecular mechanisms behind these alterations are not clearly understood. Stimulation of CD4+ T cells initiates an intracellular signaling cascade via the T-cell receptor (TCR), leading to T-cell activation and interleukin-2 (IL-2) production. These initial events take place in plasma membrane microdomains that are termed as lipid rafts. Several studies demonstrate that the clustering or aggregation of lipid rafts plays a crucial role in TCR-mediated signaling by providing a platform for recruitment of proteins involved in signal transduction (Chan et al., 1994; Hunter, 2000; Janes et al., 1999, 2000; Simons and Toomre, 2000). In the lipid rafts, TCR ligation results in the activation of a Src family kinase Lck (lymphocyte-specific protein tyrosine kinase), leading to phosphorylation of immunoreceptor-based tyrosine-activated motifs (ITAMS) on TCR-associated CD3 zeta chain (Moarefi et al., 1997; Reynolds et al., 1992). These phosphorylated ITAMS create a docking site for ZAP70 (zeta chain associated protein) to bind, which subsequently is activated via Lck phosphorylation and plays a critical role in coordinating the downstream molecular signaling essential for T-cell activation and function (Chan et al., 1992, 1995). Further, activated ZAP70 phosphorylates LAT (linker for activated T cells), an adapter protein which in turn recruits PLCγ1 (phospholipase C) to the raft assembly (Finco et al., 1998; Zhang et al., 1998a). These proximal plasma membrane lipid raft-associated signaling events initiated by TCR ligand binding lead to the activation of distal signaling components, including transcription factors like NFAT (nuclear factor for activated T cell), ultimately resulting in IL-2 gene expression (Chow et al., 1999; Porter and Clipstone, 2002). IL-2 is a critical lymphokine and growth factor for T cells; it plays a major role in regulating both immune activation and homeostasis (Bachmann and Oxenius, 2007; Malek, 2008). It exerts a wide spectrum of effects on the immune system including the expansion of activated T cells and effector T-cell function. Furthermore, clinical conditions leading to immunosuppression such as chronic alcohol abuse are characterized by a decrease in IL-2 expression. Serum levels of IL-2 (also, alpha-interferon and gamma-interferon) are significantly depressed in alcoholic cirrhotics compared with nonalcoholic subjects (Vicente-Gutierrez et al., 1991). CD4+ T-cell function in human immunodeficiency virus (HIV) disease is markedly impaired, and these defects are accompanied by reduced production of the immunoregulatory cytokine IL-2 (Sieg et al., 2001). Moreover, ethanol-induced hyporesponsiveness of T lymphocytes involves a membrane-specific interaction which affects transmembrane signaling upon ex vivo exposure to ethanol (Spinozzi et al., 1992), suggesting that ethanol can alter membrane-associated events. The present study investigates the mechanisms underlying the immunosuppressive effects of ethanol on proximal TCR-mediated lipid raft-associated events, as well as distal intracellular signaling events leading to IL-2 expression in CD4+ T lymphocytes. Our data show that physiologically relevant concentrations of ethanol significantly inhibited the lipid raft colocalization of essential signaling components and downregulated the activation/DNA binding of NFAT, and decreased IL-2 production in cultured as well as freshly isolated primary human T lymphocytes.