Novel mechanism of U18666A-induced tumour necrosis factor-alpha production in RAW 264.7 macrophage cells.

U18666A is a cholesterol transport-inhibiting agent that is used widely to mimic Niemann-Pick type C disease. The effect of U18666A on tumour necrosis factor (TNF)-alpha production in mouse macrophage cell line, RAW 264.7 cells and peritoneal macrophages was examined. U18666A induced TNF-alpha mRNA expression 48 h after the treatment, and TNF-alpha production 48 and 72 h after stimulation in RAW 264.7 cells. U18666A accumulated intracellular free cholesterol in the culture of normal medium but not cholesterol-free medium. U18666A also induced reactive oxygen species (ROS) generation in normal medium but much less in cholesterol-free medium. Anti-oxidant N-acetyl-L-cysteine (NAC) abolished U18666A-induced TNF-alpha production. U18666A led to the phosphorylation of p38 mitogen-activated protein kinase 24 and 48 h after the stimulation and the p38 activation was inhibited in presence of cholesterol-free medium or NAC. A p38 inhibitor reduced U18666A-induced TNF-alpha production. Taken together, U18666A was suggested to induce TNF-alpha production in RAW 264.7 cells via free cholesterol accumulation-mediated ROS generation.