Lipopolysaccharide and Ceramide Use Divergent Signaling Pathways to Induce Cell Death in Murine Macrophages

Abstract

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-γ-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 Lpsd macrophages were resistant to LPS-induced cell death and less sensitive to C2 ceramide-evoked cytotoxicity, when compared with Lpsn macrophages. C2 ceramide plus IFN-γ 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-γ to express iNOS mRNA and NO·, were refractory to LPS plus IFN-γ-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.