Loss of Shp2 enhances the inhibitory function of IL-10 in macrophages. (A) IL-10 concentration was measured in culture supernatant from colon tissues. (B) Shp2^M WT and Shp2^M KO PMs were stimulated with LPS (100 ng/ml) in the absence or presence of IL-10 (10 ng/ml) or anti–IL-10 (αIL-10) for indicated time, the levels of TNF-α and IL-6 in culture supernatant were determined by ELISA. (C) PMs were stimulated with LPS (100 ng/ml) in the absence or presence of indicated concentrations of IL-10 for 12 h; the level of TNF-α in culture supernatant was determined by ELISA. Results were presented as relative cytokine values (value_{LPS + IL-10}/value_{LPS alone}). (D) PMs were stimulated with PGN or E. coli in the absence or presence of IL-10 for 12 h, relative TNF-α, and IL-6 levels were determined by ELISA. (E) PMs were stimulated with LPS in the absence or presence of IL-10 for 3 h; relative TNF-α mRNA level was determined by qPCR. (F) PMs were stimulated with IL-10 (10 ng/ml) for indicated time, the phosphorylation of STAT3, ERK, and p38 was determined by Western blot. Data are mean ± SEM and are representative of at least three independent experiments. (G) CLPMs isolated from IL-10^−/−Shp2^M WT and IL-10^−/−Shp2^M KO mice were stimulated with LPS in the absence or presence of IL-10 (5 ng/ml) for 12 h; the levels of TNF-α and IL-6 in culture supernatant were determined by ELISA. (H) CLPMs were stimulated with IL-10 (5 ng/ml) for 30 min; the phosphorylation of STAT3 was determined by Western blot. (I) CLPMs were stimulated with IL-10 (5 ng/ml) for 2 h; the level of SOCS3 was determined by qPCR. Data are mean ± SEM and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.