Inhibition of expression of DC markers by PGE₂. (a) Characterization of EP2 receptor expression in bone marrow progenitors of wild-type and EP2^–/– mice. Total RNA was extracted from bone marrow progenitors, and cDNA was amplified by RT-PCR. Primers specific for the EP2 receptor were used. Lane 1, DNA marker; lane 2, wild type; lane 3, EP2^–/–; lane 4 and 5, no reverse transcriptase control for wild type and EP2^–/–, respectively. (b) DC differentiation to CD11c⁺, I-Ad⁺, CD86⁺, and CD40⁺ cells was significantly inhibited after PGE₂ treatment. Bone marrow progenitors from EP2^–/– (white bars) and wild-type (black bars) mice were cultured in GM-CSF/IL-4 medium with increasing concentrations of PGE₂. At day 9, FACScan flow cytometer was used to analyze the phenotype of DCs. The percentage of expression is normalized relative to untreated cells from wild-type mice. Each surface marker was plotted on a bar graph as shown in b. Graphs depict: (c) CD11c; (d) CD86; (e) MHC class II; and (f) CD40. Untreated wild-type cells (black bars), wild-type treated with 100 nM PGE₂ (gray bars), untreated EP2^–/– cells (white bars), EP2^–/– cells treated with 100 nM PGE₂ (dark gray bars). The PGE₂ dose response for CD11c expression is a representative experiment from two independent experiments with similar results. All others are from three to four independent experiments. For each graph there is a statistically significant difference between wild-type/no PGE₂ and wild-type/PGE₂ (P < 0.05); there are no statistically significant differences between EP2^–/–/no PGE₂ and EP2^–/–/PGE₂ (P > 0.05).