Fig 2. E.coli K12 LPS induces Mφ subset-specific secreted IL-10 and endogenous IL-10 activity.
THP-1-derived M1 and M2 Mφs were generated by differentiating THP-1 monocytic cells with either 25 ng/ml phorbol 12-myristate 13-acetate (PMA) for 3 days or 10 nM 1,25-(OH)2 vitamin D3 for 7 days, respectively. M1 (bold) and M2 (shaded) Mφ subsets were stimulated with or without 100 ng/ml K12-LPS. IL-10 gene expression of mRNA (a) is presented as fold change (RQ) using GAPDH as reference gene and resting cells as a calibrator sample, as described in [16] using 2-ΔΔct method. Secretion of IL-10 (b) was measured by sandwich ELISA and presented as the mean ± SD in pg/ml. Endogenous cell-associated IL-10 activity was measured based upon the anti-inflammatory activity of IL-10 to suppress LPS-induced TNFα. Mφs were pre-treated with 10 μg/ml 9D7 neutralising anti-IL-10 antibody and compared to an irrelevant isotype-matched control antibody (IC) and is represented for K12-LPS-stimulated and unstimulated M1 (c) and M2 (d) macrophage TNFα secretion. TNFα is expressed as the mean± SD in pg/ml. Data displayed is representative of triplicate samples for n = 3 replicate experiments. Significant differences in cytokine expression and secretion between LPS-activated M1 and M2 Mφs and unstimulated controls and between isotype control and neutralising IL-10 antibody treatment are indicated as **p < 0.01, ***P < 0.001 and ns = not significant.