Effect of cytokines, synergistically induced by combined TLR engagement, on CCL2 induction and regulation. (A) MD-DCs were treated with LPS (100 ng/mL) or R848 (2 μg/mL) alone or in combination, in the presence or in the absence of a neutralizing mAb to type I IFN receptor (20 μg/mL) or control antibody. Eighteen hours later, supernatants were collected and tested for CCL2 content. Results are presented as means ± SD of duplicate wells from 1 representative of 6 independent experiments. P values were calculated by Student t test, and statistical significance is indicated comparing results from cultures treated with LPS or R848 or their combination in the presence of type I IFN receptor (IFN-R)–blocking mAb with those achieved in the absence of the mAb. (B) MD-DCs were stimulated as described in (A). Eighteen hours later, supernatants were collected and the biologically active type I IFN content in the culture medium was titered as described in “Cytokine and chemokine determination.” The content of IFN-β was determined by the addition of a specific anti-IFN-β antibody in the titration assay. A representative experiment of 3 performed is shown. (C-D) MD-DCs were activated with LPS (100 ng/mL) or R848 (2 μg/mL) alone or in combination, in the presence or in the absence of a mAb to TNF-α (100 ng/mL) and its control antibody (C) or a mAb to IL-10 (500 ng/mL) and its control antibody (D). Eighteen hours later, supernatants were collected and tested for CCL2 content. Results are presented as means ± SD of duplicate wells from 1 representative of 3 independent experiments. P values were calculated by paired Student t test and indicate no significant changes in CCL2 content in cultures treated with LPS or R848 or their combination in the presence or absence of mAb to TNF-α or IL-10, respectively.