Cyclooxygenase-2-dependent generation of 8-epiprostaglandin F 2a by lipopolysaccharide-activated J774 macrophages

Objective: The generation of 8-epiprostaglandin F(2α)(8-epi-PGF(2α)) by arachidonic acid (AA)- and lipopolysaccharide (LPS)- stimulated J774 macrophages has been investigated. Material: Murine monocyte/macrophage J774 cell line. Methods: Cells were incubated with AA or LPS and the amount of 8-epi-PGF(2α), 6-ketoprostaglandin F(1α) (6-keto-PGF(1α)) and prostaglandin E2 (PGE2) released in the incubation media measured by radioimmunoassay (RIA) or, in some experiments, by enzyme immunoassay (EIA). The effect of dexamethasone (DXM), cycloheximide (CXM) and 5,5 dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU), a cyclooxygenase-2 (COX-2) selective inhibitor, on LPS-induced generation of AA metabolites was assessed. Results: AA induced a significant production of 6-keto-PGF(1α) and PGE2, whereas LPS caused a concentration- and time-dependent increase of 8-epi-PGF(2α), 6-keto-PGF(1α) and PGE2. DXM (2 μM) as well as CXM (1 μM) significantly decreased (p < 0.001; n = 4) the LPS-stimulated production of 8-epi-PGF(2α) (by 86% and 82%, respectively), 6-keto-PGF(1α) (by 78% and 74%, respectively) and PGE2 (by 83% and 78%, respectively). Immunostimulated production of AA metabolites was also inhibited by DFU (IC50 0.3 ± 0.04 μM; 0.16 ± 0.02 μM and 0.11 ± 0.05 μM for 8-epi-PGF(2α), 6-keto-PGF(1α) and PGE2, respectively. Conclusions: These results demonstrate the role of COX-2 in the generation of 8-epi-PGF(2α) by LPS-stimulated J774 macrophages. The relevance of these findings requires further elucidation.