Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages.
Ceramide is a well-known apoptotic agent that has been implicated in LPS signaling. Therefore, we examined whether LPS-induced macrophage cytotoxicity is mediated by mimicking ceramide. Both LPS and the cell-permeable ceramide analogue, C2 ceramide, induced significant cell death in IFN-gamma-activated, thioglycollate-elicited peritoneal macrophages after 48 and 24 h, respectively. Ceramide-induced cell death was neither accompanied by DNA fragmentation nor phosphatidyl serine externalization, characteristics of apoptosis. In contrast, LPS induced a significant fraction of cells to undergo apoptosis, as demonstrated by DNA fragmentation and quantified by DNA analysis on FACS, yet the majority of the cells died in a necrotic fashion. C3H/HeJ Lps(d) macrophages were resistant to LPS-induced cell death and less sensitive to C2 ceramide-evoked cytotoxicity, when compared with Lps(n) macrophages. C2 ceramide plus IFN-gamma failed to activate release of nitric oxide (NO.), whereas LPS-induced cell death, but not C2-induced cytotoxicity, was blocked by an inhibitor of inducible NO. synthase (iNOS), NG-monomethyl-L-arginine. Macrophages from IFN regulatory factor-1 (-/-) mice shown previously to respond marginally to LPS plus IFN-gamma to express iNOS mRNA and NO., were refractory to LPS plus IFN-gamma-induced cytotoxicity and apoptosis. These data suggest that although LPS may mimic certain ceramide effects, signal transduction events that lead to cytotoxicity, as well as the downstream mediators, diverge.
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Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D016923:Cell Death / Q000187:drug effects; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000502:physiology; D004357:Drug Synergism; D005260:Female; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000201:enzymology / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008809:Mice, Inbred C3H; D018345:Mice, Knockout; D009336:Necrosis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D008070:Lipopolysaccharides; C064769:N-acetylsphingosine; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D013110:Sphingosine