The Role of Tumor Necrosis Factor Alpha in PAF-AH Expression

Objectives: Tumor necrosis factor alpha (TNF-α) is an important pro-inflammatory cytokine produced by macrophages in response to immunological challenges. Likewise, inflammatory challenges also cause the production of platelet-activating factor (PAF), a robust phospholipid derivative. When stimulated, TNF-α activates signal transduction pathways resulting in a mass production of PAF. PAF plays an integral role in the inflammatory response causing vasodilation, platelet adhesion, and degranulation and is inactivated by the degrading enzyme, PAF acetylhydrolase (PAF-AH). We investigated whether an inflammatory challenge with TNF-α would promote production of PAF-AH and initiated experiments to decipher the signaling mechanism(s) responsible.

Methods: A monocyte/macrophage cell line (MM6) was grown in RPMI media supplemented with 10% FBS. Twenty-four hours after seeding the cells at a density of 2 X 10^5 cells/mL, the cells were transferred to serum-free media. E. coli LPS (0-200 ng/ml) and/or TNF-α (0-5 ng/ml) was administered and PAF-AH mRNA levels were examined by quantitative real-time RT-PCR along with the controls 18S and cyclophilin. Pharmacological inhibitors of MAPK pathways were used to examine the pathways activated in response to LPS and TNF-α.

Results: TNF-α increased PAF-AH expression in a dose-dependent manner with peak expression of PAF-AH at 24 hours following administration. TNF-α was significantly less potent than LPS (4-fold vs. 10-fold). When added concomitantly with LPS, TNF-α further increased the expression of PAF-AH. While the p38 MAPK inhibitor, SB203580, blocked half of the LPS-induced up-regulation of PAF-AH, it had no effect on TNF-α -induced up-regulation of PAF-AH.

Conclusion: TNF-α is a crucial inflammatory cytokine which up-regulates the production of PAF-AH in MM6 cells upon its administration. The production of PAF-AH is pivotal in deactivating potent mediators of inflammation. Because the p38 MAPK inhibitor had no significant effect on TNF-α induced production of PAF-AH, we hypothesized TNF-α up-regulates the production of PAF-AH via the JNK pathway.