CD14 is required for rHagB to activate DC. DC (2×105 ) from WT (open bars) and CD14−/− (grey dotted bars) mice were stimulated with 40 µg/ml rHagB (shaded histograms), 100 ng/ml of E. coli K12 LPS (grey lines) or left untreated (black lines). (A and B) Culture supernatants were harvested 24 h post-stimulation and assayed for levels of TNF-α, IL-6, IL-12p40 and IL-10 by ELISA. Results are expressed as the mean ± standard error of triplicate cultures from one of three representative experiments. *** Significant differences at P < 0.001 compared to WT cultures stimulated with rHagB. (C, D and E) Cells were harvested 16 h post-stimulation and stained with fluorescent-labeled antibodies against CD11c, CD86 and CD40. Histogram plots were gated on CD11c+ cells (C) and bar graphs represent the percentage expression of CD86 and CD40 on CD11c+ cells (D) or the mean florescence intensity on CD11c+ cells (E). Results are expressed as the mean ± standard error of three independent experiments. ** Significant difference at P < 0.01 compared to WT cultures stimulated with rHagB.