Ifenprodil modulates IL-10 production of B cells. A) Ifenprodil reduces IL-10 mRNA transcripts in PMA/IO-activated B cells. Splenic B cells were left untreated or were activated with PMA and IO in the absence or presence of ifenprodil (10 and 30 μM, lanes 3 and 4) for 16 h. Cells were restimulated with PMA/IO and monensin for 6 h and RT-PCR for IL-10 and β-actin expression was performed. Data are the representative of two experiments. B-D) IL-10 production in BCR + CD40-stimulated B cells is enhanced upon ifenprodil treatment. B) B cells were activated with α-IgM + CD40, CD40 Abs alone or LPS for 2–3 days. Ifenprodil (10 μM) was added at day 1. The production of IFN-γ and IL-10 was analyzed by intracellular staining and flow cytometry. The percentage of cells positive for IL-10 (gates were set according to staining with isotype controls, right panel) is indicated in each dot plot. The graphs represent the relative + SD percentage of IL-10 expressing B cells in α-IgM + CD40, CD40 and LPS cultures treated with ifenprodil. For each stimulation condition, data were related to the percentage of IL-10+ B cells generated in the absence of ifenprodil, which was set as 1.0. Data for 48 h were calculated from three, for 72 h from two to four experiments. C, D) B cells from IL-10-GFP tiger mice were activated with α-IgM+CD40 or LPS. Ifenprodil (10 and 20 μM) was added after 21-25 h and expression of IL-10-GFP at C) day 2 or D) day 4 was determined with flow cytometry. Data are from two independent experiments.