Ceramide (a sphingolipid) and reactive oxygen species are each partly responsible for intracellular signal transduction in response to a variety of agents. It has been reported that ceramide and reactive oxygen species are intimately linked and show reciprocal regulation [Liu, Andreieu-Abadie, Levade, Zhang, Obeid and Hannun (1998) J. Biol. Chem. 273, 11313–11320]. Utilizing synthetic, short-chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide formation or using stimulation of CD95 to induce ceramide formation, we found that the principal redox-altering property of ceramide is to lower the [peroxide]cyt (cytosolic peroxide concentration). Apoptosis of Jurkat T-cells, primary resting and phytohaemagglutinin-activated human peripheral blood T-lymphocytes was preceded by a loss in [peroxide]cyt, as measured by the peroxide-sensitive probe 2′,7′-dichlorofluorescein diacetate (also reflected in a lower rate of superoxide dismutase-inhibitable cytochrome c reduction), and this was not associated with a loss of membrane integrity. Where growth arrest of U937 monocytes was observed without a loss of membrane integrity, the decrease in [peroxide]cyt was of a lower magnitude when compared with that preceding the onset of apoptosis in T-cells. Furthermore, decreasing the cytosolic peroxide level in U937 monocytes before the application of synthetic ceramide by pretreatment with either of the antioxidants N-acetyl cysteine or glutathione conferred apoptosis. However, N-acetyl cysteine or glutathione did not affect the kinetics or magnitude of ceramide-induced apoptosis of Jurkat T-cells. Therefore the primary redox effect of cellular ceramide accumulation is to lower the [peroxide]cyt of both primary and immortalized cells, the magnitude of which dictates the cellular response.
Abbreviations used: a.u., arbitrary units; C2-ceramide, N-acetyl-sphingosine; C6-ceramide, N-hexanoyl-sphingosine; CD95L, CD95 ligand; DAGK, diacylglycerol kinase; DCF, 2′,7′-dichlorofluorescein; DCFH, non-fluorescent DCF; DCFH-DA, DCF diacetate; DPI, diphenylene iodinium; FCS, foetal calf serum; FS, forward scatter; IL, interleukin; MdX, median X; NAC, N-acetyl cysteine; PBL, peripheral blood lymphocyte; PE, phycoerythrin; PHA, phytohaemagglutinin; PI, propidium iodide; P/S, penicillin/streptomycin; ROS, reactive oxygen species; SMase, sphingomyelinase; SOD, superoxide dismutase; SS, side scatter.
Present address: Division of Molecular Therapeutics, Department of Hematology–Oncology, St Jude Children's Research Hospital DT 5062, 332 North Lauderdale, Memphis, TN 38105, U.S.A.