Novel mechanism of U18666A‐induced tumour necrosis factor‐α 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)‐α production in mouse macrophage cell line, RAW 264·7 cells and peritoneal macrophages was examined. U18666A induced TNF‐α mRNA expression 48 h after the treatment, and TNF‐α 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‐α 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‐α production. Taken together, U18666A was suggested to induce TNF‐α production in RAW 264·7 cells via free cholesterol accumulation‐mediated ROS generation.