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 D(2) (PGD(2)) 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 microg/ml) before and after exposure to PGD(2) (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 microg/ml LPS produced 8.3 +/- 0.2 and 15.0 +/- 1.4 nmol/1.1 x 10(6) cells/24 h of nitrite, respectively. The simultaneous addition of PGD(2) 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 PGD(2) 1 hour before as compared to 7 hours after the addition of LPS (0.01 or 1.0 microg/ml), indicating that the earlier the time PGD(2) was added to the culture media, the greater the inhibition. Prostaglandin D(2) 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 microM), suppressed nitrite production.


CONCLUSIONS
Exogenous PGD(2) 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 PGD(2).
Bellows
Charles F
CF
Department of Surgery, SL22, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA.
Alder
Adam
A
Wludyka
Peter
P
Jaffe
Bernard M
BM
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D015230:Prostaglandin D2 / Q000494:pharmacology*
D008070:Lipopolysaccharides; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D015230:Prostaglandin D2