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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

2002; Elsevier BV; Volume: 107; Issue: 1 Linguagem: Inglês

10.1006/jsre.2002.6498

1095-8673

Tricia A. Babcock, Todd E. Novak, Evan Ong, David H. Jho, W. Scott Helton, N. Joseph Espat,

Eicosanoids and Hypertension Pharmacology

Background. The role of ω-3 fatty acids (FA) as anti-inflammatory agents involves the inhibition of macrophage (Mφ) cytokine production, but the mechanisms involved are not well defined. The effects of ω-3 FA on the transcription and translation of cyclooxygenase-2 (COX-2), the production of prostaglandin E2 (PGE2), and the production of interleukin-10 (IL-10) were investigated as potential mechanisms for the down-regulation of lipopolysaccharide (LPS)-induced tumor necrosis factor-α production. Methods. RAW 264.7 Mφ were incubated with OmegavenR (10 mg% ω-3 FA), LipovenosR (10 mg% ω-6 FA), or DMEM for 4 h of pretreatment. The cells were then exposed to LPS (1 μg/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 PGE2 and IL-10 proteins secreted into the medium were quantified using enzyme-linked immunosorbent assays. Results. Pretreatment with ω-3 FA increased Mφ COX-2 protein expression levels without altering the levels of COX-2 mRNA in response to LPS stimulation. In addition, pretreatment with ω-3 FA dramatically decreased the PGE2 and IL-10 production induced by LPS, whereas pretreatment with an equivalent dose of ω-6 FA only resulted in a modest increase in PGE2 and a slight decrease in IL-10 production compared to controls. Conclusion. As COX-2 protein levels were increased without a change in COX-2 mRNA levels with ω-3 FA pretreatment, this suggested that ω-3 FA did not upregulate COX-2 at the transcriptional level. The ω-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 PGE2 production could be attributed to the replacement of Mφ membrane ω-6 FA substrates by ω-3 FA and the competitive inhibition of COX-2 enzyme by ω-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 ω-3 FA pretreatment, indicating that the reduced IL-10 inhibition of Mφ cytokine production was superceded by the other actions of ω-3 FA.