engleski

Arachidonic Acid Mediates Interferon-gamma-induced Sphingomyelin Hydrolysis and Monocytic Marker Expression in HL-60 Cell Line

The biochemical signaling mechanisms involved in transducing the effects of interferon-gamma (IFN-gamma) on human leukemia-derived HL-60 cell differentiation are incompletely understood. Recent studies established the existence of a sphingomyelin (SM) cycle that operates in response to the action of IFN-gamma on HL-60 cells, but the mechanisms by which IFN-gamma induced the SM-hydrolysis remain unexplored. In this study, biochemical events mediating IFN-gamma effects on SM turnover, and their specificity and role in HL-60 differentiation were investigated. Activation of the SM cycle by IFN-gamma occurred rapidly, with peak levels of approximately 30% SM hydrolysis observed after 60 min. Treatment of HL-60 cells with IFN- did not influence the 1,2-diacylglycerol concentration, the intracellular Ca^2^+ concentration or phospholipase D activity. IFN-gamma stimulated a rapid release of arachidonic acid (AA) from HL-60 cells; the effect was abolished by the pretreatment of cells with pertussis toxin, suggesting a role for a pertussis-toxin-sensitive G-protein in IFN-gamma-mediated activation of phospholipase A_2 (PLA_2). At 4-120 h after the stimulation of the cells with IFN-gamma, a significant increase in the particulate and soluble PLA_2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA_2 in both cytosol and membrane fractions. Melittin, a potent activator of PLA_2, and AA mimicked the effect of IFN-gamma on SM hydrolysis. Pre-treatment of HL-60 cells with the PLA_2 inhibitor, bromophenacyl bromide (BPB), abolished the effect of IFN-gamma on SM hydrolysis; exogenous addition of AA overcame the effect of BPB. Long-term exposure (5 days) of HL-60 cells to IFN-gamma cause an increase in NBT-reducing and non-specific esterase (NSE) activity and induced expression of FcgammaR1 (CD64) without significant effects on cell number, adherence or phagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE and CD64 expression to the level similar to that observed with IFN-gamma, and no further increase was observed with the combination of IFN-gamma and AA or IFN-gamma and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxigenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-gamma-mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A_2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-gamma-receptor, in mediating IFN-gamma induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.

arachidonic acid; interferon-gamma;sphingomyelin; monocytic marker; HL-60 cell line

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