Modulation of Lipopolysaccharide-Stimulated Macrophage Tumor Necrosis Factor-α Production by ω-3 Fatty Acid Is Associated with Differential Cyclooxygenase-2 Protein Expression and Is Independent of Interleukin-10

• DOI: 10.1006/jsre.2002.6498
• Publication Date: 2003-01-09T12:12:55Z
• Authors: Tricia A. Babcock; Todd Novak; Evan Ong; David H. Jho; W.Scott Helton; N.Joseph Espat
• Keywords: Lipopolysaccharides; 0301 basic medicine; Tumor Necrosis Factor-alpha; Macrophages; Dinoprostone; Cell Line; Interleukin-10; Isoenzymes; Mice; 03 medical and health sciences; Cyclooxygenase 2; Prostaglandin-Endoperoxide Synthases; Fatty Acids, Omega-3; Animals; RNA, Messenger

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.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.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.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.