Modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-alpha production by omega-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10.
BACKGROUND
The role of omega-3 fatty acids (FA) as anti-inflammatory agents involves the inhibition of macrophage (Mphi) cytokine production, but the mechanisms involved are not well defined. The effects of omega-3 FA on the transcription and translation of cyclooxygenase-2 (COX-2), the production of prostaglandin E(2) (PGE(2)), and the production of interleukin-10 (IL-10) were investigated as potential mechanisms for the down-regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production.


METHODS
RAW 264.7 Mphi were incubated with Omegaven (10 mg% omega-3 FA), Lipovenos (10 mg% omega-6 FA), or DMEM for 4 h of pretreatment. The cells were then exposed to LPS (1 microg/ml) or medium alone for 3 h. COX-2 mRNA levels were determined by semi-quantitative reverse transcriptase polymerase chain reaction, and COX-2 protein levels were determined by Western blotting. The levels of PGE(2) and IL-10 proteins secreted into the medium were quantified using enzyme-linked immunosorbent assays.


RESULTS
Pretreatment with omega-3 FA increased Mphi COX-2 protein expression levels without altering the levels of COX-2 mRNA in response to LPS stimulation. In addition, pretreatment with omega-3 FA dramatically decreased the PGE(2) and IL-10 production induced by LPS, whereas pretreatment with an equivalent dose of omega-6 FA only resulted in a modest increase in PGE(2) and a slight decrease in IL-10 production compared to controls.


CONCLUSIONS
As COX-2 protein levels were increased without a change in COX-2 mRNA levels with omega-3 FA pretreatment, this suggested that omega-3 FA did not upregulate COX-2 at the transcriptional level. The omega-3 FA may instead posttranscriptionally stabilize existing COX-2 mRNA. The increased COX-2 expression may thus be explained by increased translation of COX-2 and/or decreased COX-2 degradation. The decreased PGE(2) production could be attributed to the replacement of Mphi membrane omega-6 FA substrates by omega-3 FA and the competitive inhibition of COX-2 enzyme by omega-3 FA. The reduction of active COX-2 product associated with an increase in COX-2 enzyme implies the existence of a negative feedback mechanism. Surprisingly, IL-10 production was decreased by omega-3 FA pretreatment, indicating that the reduced IL-10 inhibition of Mphi cytokine production was superceded by the other actions of omega-3 FA.
Babcock
Tricia A
TA
Laboratories of Surgical Metabolism, University of Illinois at Chicago, 60612, USA.
Novak
Todd
T
Ong
Evan
E
Jho
David H
DH
Helton
W Scott
WS
Espat
N Joseph
NJ
D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis; D015525:Fatty Acids, Omega-3 / Q000494:pharmacology*; D016753:Interleukin-10 / Q000037:antagonists & inhibitors / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D015525:Fatty Acids, Omega-3; D007527:Isoenzymes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone