Interleukin 10 inhibits the expression of interleukin 17 in macrophages in vitro . (A) Thioglycollate-elicited peritoneal (TEP) macrophages from interleukin 10–knockout (IL-10−/−) mice were activated with or without 1 μg/ml lipopolysaccharide (LPS) in culture for 1 hour, followed by addition of 100 ng/ml IL-10, and then cultured for 2, 6 and 12 hours. Total RNA was extracted and analyzed for IL-17 mRNA by quantitative RT-PCR (qRT-PCR). (B) TEP macrophages from IL-10−/− mice were activated with or without LPS (1 μg/ml) for 1 hour, and then 1, 10 or 100 ng/ml IL-10 was added separately to cultures. The cells were cultured for another 6 hours, and total RNA was extracted and analyzed for IL-17 mRNA by qRT-PCR. (C) and (E) TEP F4/80+ macrophages from IL-10−/− mice and WT mice were separated by flow cytometry and then cultured and stimulated with LPS for 1 hour in the presence or absence of 100 ng/ml IL-10 for 6 hours. Total RNA was extracted and analyzed in qRT-PCR experiments for IL-17 mRNA (C) and retinoid-related orphan receptor γt (RORγt) mRNA (E). (D) and (F) TEP macrophages from IL-10−/− mice and WT mice were activated with 1 μg/ml LPS for 1 hour, F4/80+ macrophages were stained with fluorescein isothiocyanate-conjugated F4/80 (green) and AleaxFluor647-conjugated IL-17 (red) for IL-17 (D) and RORγt (F) expression and analyzed by immunofluorescence microscopy. Original magnification, ×200. Values are mean ± SEM (n = 3). *P < 0.05; **P < 0.01.