Effects of PGE2 on DC phenotype and CCL19-induced chemotaxis. CD11c+ DCs were treated with IFN-α (1000 IU/mL) plus TNF-α (20 ng/mL) with or without PGE2 (10⁻⁶ M) (A-C) or with different PGE2 concentrations (D) for 48 hours. Controls consisted of DCs cultured in medium, treated with PGE2 alone, or with LPS (1 μg/mL). (A) FACS analysis for MHCII, CD40, CD80, and CD86 expression. Dotted lines in the left panels represent isotype controls. (B) CCR7 expression was analyzed by FACS (24 hours for LPS and 48 hours for all other treatments) and real-time RT-PCR (12 hours for LPS and 24 hours for all other treatments). (C) DCs (10⁵ cells in 0.1 mL) were placed in the upper chambers of a 8.0-μm pore size Transwell plate. The bottom chambers were filled with serum-free medium with or without CCL19 (100 ng/mL and 300 ng/mL). After 3 hours of incubation at 37°C, the migrating cells were collected from the lower chambers and counted by FACS (60-second counts). (D) DCs were treated with TNF-α +IFN-α in the absence and presence of various PGE2 concentrations. Chemotaxis in response to CCL19 (300 ng/mL) was determined as described in panel C. Data are representative of 3 independent experiments.