Role of PKA and Epac in in vitro macrophage maturation. (A) PKA inhibits, whereas Epac promotes, macrophage maturation. Freshly flushed bone marrow cells isolated from EP2^−/− and WT mice were cultured in 30% L929 cell supernatant in the presence of Epac or PKA agonist (500μM each). After 3 days, cell culture was supplemented with new medium containing Epac or PKA agonist totaling 50% of original volume; and after 6 days, cells were collected and stained with CD11b-FITC and F4/80-APC. The dot-plot shown is from a single experiment representative of 3 independent experiments, each using a different mouse. Percentages of CD11b^pos/CD115^pos cells in different conditions were compared. Data are expressed as the mean ± SEM from 3 experiments, each using a separate mouse (*P < .05, **P < .01, ***P < .001). (B) PKA agonist reduces numbers of macrophages during in vitro macrophage maturation. Cells were cultured as described in panel A. After 6 days of culture floating cells were washed away, and adherent cells were counted by light microscopy. Data are expressed as the mean ± SEM from 3 experiments, each using a different mouse (*P < .05, **P < .01, ***P < .001). (C) PKA agonist diminishes M-CSFR (CD115) expression on the cell surface. Cells were cultured as described in panels A-B. After 6 days of culture, adherent CD11b^pos/CD115^pos cells were stained with CD115-PE. MFI is expressed as the mean ± SEM from 3 experiments, each using a different mouse (**P < .01, ***P < .001). PKA agonist N6-benzoyladenosine-3′,5′-cyclic monophosphate (6-Bnz-cAMP) and Epac agonist 8-4-chlorophenylthio-2′-O-methyladenosine-3′,5′-cyclicmonophosphate (8-pCPT-2′-O-Me-cAMP).