Modulation of Macrophage Nitric Oxide Production by Prostaglandin D₂

Background: Nitric oxide and prostaglandins readily become activated in response to inflammatory events. The overproduction of nitric oxide is detrimental to the host. The present study was conducted to examine whether prostaglandin D₂ (PGD₂) modulates nitric oxide production in macrophages in response to an inflammatory stimulus. Methods: Cultures of RAW 264.7 murine macrophages were exposed to Escherichia coli lipopolysaccharide (LPS, 0.01 and 1.0 μg/ml) before and after exposure to PGD₂ (0.01 to 10 nmol). After 24-h incubation, supernatants were collected and nitrite was quantitated by Greiss reaction as a measure of nitric oxide synthesis. Inducible nitric oxide synthase (iNOS) protein was measured by Western blot analysis. Results: Macrophages exposed to 0.01 and 1.0 μg/ml LPS produced 8.3 ± 0.2 and 15.0 ± 1.4 nmol/1.1 × 10⁶ cells/24 h of nitrite, respectively. The simultaneous addition of PGD₂ with LPS inhibited nitrite production in a dose-dependent fashion and suppressed iNOS protein expression. A strong time effect was also exhibited when macrophages were incubated with PGD₂ 1 hour before as compared to 7 hours after the addition of LPS (0.01 or 1.0 μg/ml), indicating that the earlier the time PGD₂ was added to the culture media, the greater the inhibition. Prostaglandin D₂ had the capacity to block nitrite synthesis even when added as much as 7 hours after an LPS challenge. Blocking endogenous prostaglandins, using indomethacin (10 μΜ), suppressed nitrite production. Conclusion: Exogenous PGD₂ caused dose- and time-dependent decreases in LPS-stimulated nitrite production by RAW 264.7 macrophages by hindering iNOS protein expression. Conversely, the endogenous prostaglandins released by these same cells in response to an LPS challenge stimulated nitrite production, which may consequently dampen the inhibitory actions of exogenous PGD₂.