Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation

Abstract

1. The isoprostane 8-epi-prostaglandin (PG)F(2α) is produced by free radical-catalyzed peroxidation of arachidonic acid. It may also be formed as a minor product of the cyclo-oxygenase activity of platelet PGH synthase (PGHS)-1. We investigated 8-epi-PGF(2α) production associated with induction of the human monocyte PGHS-2 and its pharmacological modulation. 2. Heparinized whole blood samples were drawn from healthy volunteers, 48 h following oral dosing with aspirin 300 mg to suppress platelet cyclo-oxygenase activity. One ml aliquots were incubated with lipopolysaccharide (LPS: 0.1-50 μg ml-1) for 0-24 h at 37°C. PGE2 and 8-epi-PGF(2α) were measured in separated plasma by radioimmunoassay and enzyme immunoassay techniques. 3. Levels of both eicosanoids were undetectable (i.e. < 60 pg ml-1) at time 0. LPS induced the formation of PGE2 and 8-epi-PGF(2α) in a time- and concentration-dependent fashion, coincident with the induction of PGHS-2 detected by Western blot analysis of monocyte lysates. After 24 h at 10 μg ml-1 LPS, immunoreactive PGE2 and 8-epi-PGF(2α) averaged 10,480 ± 4,643 and 295 ± 140 pg ml-1 (mean ± s.d., n = 6), respectively. 4. Dexamethasone and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indanone (L-745,337), a selective inhibitor of the cyclo-oxygenase activity of PGHS-2, reduced PGE2 and 8-epi-PGF(2α) production in response to LPS. 5. Isolated monocytes produced PGE2 and 8-epi-PGF(2α) in response to LPS (10 μg ml-1) in a time-dependent fashion. Monocyte PGE2 and 8-epi-PGF(2α) production was largely prevented by dexamethasone (2 μM) and cycloheximide (10 μg ml-1) in association with suppression of PGHS-2 but not of PGHS-1 expression. 6. We conclude that the induction of PGHS-2 in human monocytes is associated with cyclo-oxygenase-dependent generation of the vasoconstrictor and platelet-agonist 8-epi-PGF(2α).