Mandrekar Pranoti, Jeliazkova Valentina, Catalano Donna, Szabo Gyongyi
Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
J Immunol. 2007 Jun 15;178(12):7686-93. doi: 10.4049/jimmunol.178.12.7686.
Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.
急性酒精摄入与免疫反应受损及促炎细胞因子产生减少有关。我们早期的研究表明，急性酒精摄入以不依赖IκBα的方式抑制NF-κB与DNA的结合。我们报告，使用人外周血单核细胞和转染了CD14细胞的中国仓鼠卵巢细胞，体外急性酒精处理通过破坏p65的磷酸化来发挥NF-κB抑制作用。p65和IκBα的免疫沉淀显示，急性酒精暴露1小时可减少细胞质中NF-κB-IκBα复合物。p65在Ser(536)处的磷酸化由IκB激酶（IKK）β介导，是NF-κB依赖性细胞反应所必需的。我们发现急性酒精处理可降低脂多糖诱导的IKKα和IKKβ活性，导致p65在Ser(536)处的磷酸化减少。此外，酒精处理后IKKα的核表达增加，这可能有助于抑制NF-κB。核p65在Ser(276)处磷酸化减少可能不是由于酒精诱导的蛋白激酶A和丝裂原及应激激活蛋白激酶-1活性受到抑制。虽然急性酒精处理后IκBα磷酸化减少归因于IKKβ活性降低，但酒精暴露期间IκBα的降解不依赖于IKKβ。在存在26S蛋白酶体抑制剂的情况下，酒精诱导的IκBα降解提示IκBα降解不依赖于蛋白酶体。总的来说，我们的研究表明，急性酒精暴露主要通过影响p65的磷酸化来调节不依赖IκBα的NF-κB活性。这些发现进一步表明IKKβ在酒精对免疫细胞的急性作用中起重要作用。
