IL-4–induced TIMP-3 expression during DC differentiation was mediated by p38 MAPK pathway. (A) Monocytes were differentiated by standard dose of GM-CSF (1000 U/mL) in combination with gradient doses of IL-4 (0, 125, 250, 375, and 500 U/mL) for 5 days. (B) Alternatively, monocytes were stimulated by standard dose of IL-4 (500 U/mL) in combination with gradient doses of GM-CSF (0, 250, 500, 750, and 1000 U/mL) for 5 days. (C) Monocytes were preincubated with p38MAPK inhibitor SB203580 (10μM), JAK3 inhibitor ZM 39923 hydrochloride (5μM), or PI3K inhibitor wortmannin (100nM) for 1 hour. Then the cells were washed extensively and incubated with GM-CSF (1000 U/mL) and the indicated concentrations of IL-4 for 3 days. RNA extracts from the aforementioned cells differentiated under different conditions were analyzed for TIMP-3 mRNA by quantitative RT-PCR. (D) Monocytes were cultured with or without IL-4 or GM-CSF for the indicated times. Bottom insets: The quantitative analysis of p-p38/total p38 protein ratio, as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units. (E) Monocytes were incubated with or without SB203580 (10μM) for 60 minutes and then stimulated with IL-4 (500 U/mL) for 60 minutes; kinase activity of p38 MAPK was measured with ATF-2 as a substrate. Data are the mean ± SD of 3 independent experiments. *P < .05.