Arachidonic Acid Mediates Interferon-γ–Induced Sphingomyelin Hydrolysis and Monocytic Marker Expression in HL-60 Cell Line

Abstract

The biochemical signaling mechanisms involved in transducing the effects of interferon-γ (IFN-γ) on human leukemia-derived HL-60 cell differentiation are not completely understood. Recent studies established the existence of a sphyngomyelin (SM) cycle that operates in response to the action of IFN-γ on HL-60 cells, but the mechanisms by which IFN-γ induces the SM hydrolysis remain unexplored. In this study, biochemical events mediating IFN-γ effects on SM turnover and their specificity and role in HL-60 differentiation were investigated. The activation of the SM cycle by IFN-γ occurred rapidly, with a decrease of approximately 20% in the SM level observed after 60 minutes with a concomitant increase in ceramide level. Treatment of HL-60 cells with IFN-γ did not influence the 1,2-diacylglycerol concentration, intracellular Ca2+ concentration, or phospholipase D activity. IFN-γ 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-γ–mediated activation of phospholipase A2 (PLA2 ). At 4 to 120 hours after the stimulation of the cells with IFN-γ, a significant increase in the particulate and soluble PLA2 activity was observed, corresponding to an increase in the level of immunoreactive cPLA2 in both cytosol and membrane fractions. The treatment of cells with tyrosine kinase inhibitor herbimycin A completely abolished the effect of IFN-γ on PLA2 activity in membrane and cytosolic fractions, but had no effect on IFN-γ–mediated early AA release suggesting dual mechanism of PLA2 activation. Melittin, potent activator of PLA2 , and AA mimicked the effect of IFN-γ on SM hydrolysis. Pretreatment of HL-60 cells with the PLA2 inhibitor, bromophenacyl bromide (BPB), or pertussis toxin abolished the effect of IFN-γ on SM hydrolysis; exogenous addition of AA overcame the effects of BPB and pertussis toxin. Long-term exposure (5 days) of HL-60 cells to IFN-γ caused an increase in nitroblue tetrazolium (NBT)-reducing and nonspecific esterase (NSE) activity and induced expression of FcγRI (CD64) without significant effects on cell number, adherence, or fagocytic activity. The treatment of cells with AA or melittin induced NBT, NSE, and CD64 expression to the level similar to that observed with IFN-γ, and no further increase was observed with the combination of IFN-γ and AA or IFN-γ and melittin. Treatment of HL-60 cells with indomethacin, an inhibitor of cyclo-oxygenase, and nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, had no effects on IFN-γ–mediated induction of CD64 expression. These studies indicate a key role for the phospholipase A2/AA pathway, as an early biochemical signal elicited by the occupation of IFN-γ–receptor, in mediating IFN-γ induction of the SM cycle and phenotypic changes associated with differentiation of HL-60 along monocytic lineage.