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

Abstract

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.

Fig. 1

Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells. RAW 264·7 cells and peritoneal macrophages were incubated with U18666A (1 µg/ml) for various hours. (a) TNF-α production was determined with enzyme-linked immunosorbent assay. *P < 0·01 versus none (vehicle control). (b) TNF-α mRNA expression was analysed with real-time polymerase chain reaction. *P < 0·01 versus 0 h.

Fig. 2

Intracellular free cholesterol accumulation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. *P < 0·01 versus none (vehicle control). (b) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for 24 h and stained with filipin. Original magnification × 40.

Fig. 3

Reactive oxygen species generation in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the culture with medium X or medium Y. *P < 0·01 versus medium X.

Fig. 4

Tumour necrosis factor (TNF)-α production in U18666A-treated RAW 264·7 cells in medium X and Y. RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence or absence of N-acetyl-L-cysteine (5 mM) in medium X and Y. *P < 0·01 versus none (vehicle control).

Fig. 5

Oxidative stress-responsive signal transduction in U18666A-treated RAW 264·7 cells. (a) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours. (b) RAW 264·7 cells were incubated with U18666A (1 µg/ml) for various hours in the presence of N-acetyl-L-cysteine (5 mM) or medium Y. The phosphorylation was detected by immunoblotting.

Fig. 6

Participation of p38 in U18666A-induced tumour necrosis factor (TNF)-α production. RAW 264·7 cells were incubated with U18666A (1 µg/ml) in the presence of SB203580 (10 µM) or PD98058 (10 µM). In one of the experimental groups SB203580 was added into the culture 12 h after U18666A treatment. TNF-α production was determined with enzyme-linked immunosorbent assay. *P < 0·01 versus none (vehicle control).