Phosphatidylcholine-specific phospholipase C regulates activation of RAW264.7 macrophage-like cells by

Abstract: Phospholipase activities are thought to be involved in the activation of macrophages by lipopolysaccharide (LPS). Because our previous studies showed that the synthetic lipopeptide JBT3002 might activate macrophages via signaling pathways similar to those used by LPS, we investi-gated whether phospholipase activities are re-quired for activation of macrophages by JBT3002. Treatment of RAW264.7 murine macrophage-like cells with JBT3002 stimulated expression of both inducible nitric oxide synthase (iNOS) and tumor necrosis factor-a (TNF-a) in a dose-dependent manner. The JBT3002-induced production of ni-tric oxide and TNF-a was significantly inhibited by tricyclodecan-9-yl xanthogenate (D609), a selec-tive inhibitor of phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). JBT3002-induced ex-pression of steady-state mRNA for both iNOS and TNF-a was inhibited by D609. Cells treated with JBT3002 had greater production of diacylglycerol (DAG) in 2 min, which lasted for at least 30 min and could be blocked by D609. Activation of RAW264.7 cells was not affected by butanol, a PC-specific phospholipase D inhibitor, and treat-ment with JBT3002 did not affect phosphatidic acid formation. RAW264.7 cells treated with DAG analogue 1-oleoyl-2-acetyl-sn-glycerol, in the pres-ence of interferon-g, produced TNF-a. These re-sults suggested that activation of RAW264.7 cells by JBT3002 requires PC-PLC activity. J. Leukoc