ROLE OF CALCIUM IN LIPOPOLYSACCHARIDE-STIMULATED TUMOR-NECROSIS-FACTOR AND INTERLEUKIN-1 SIGNAL-TRANSDUCTION IN NAIVE AND ENDOTOXIN-TOLERANT MURINE MACROPHAGES

Objective: Dysregulated macrophage cytokine production may predispose to organ failure during sepsis, Macrophages pretreated in vitro with l ow-dose endotoxin (LPS(p)) become ''tolerant'' to subsequent lipopolys accharide (LPS) activation (LPS(a)), characterized by inhibition of tu mor necrosis factor (TNF) and augmentation of interleukin-l (IL-1), To understand cytokine dysregulation we examined the Ca2+ dependence of TNF and IL-1 signal transduction to LPS(a) and whether it was altered by LPS(p). Methods: Murine peritoneal exudate macrophages received +/- 100 ng/mL of LPS(p) for 24 hours, Cultures were pretreated for 2 hour s with specific signal transduction inhibitors (verapamil, a Ca2+ chan nel inhibitor; TMB-8, an inhibitor of intracellular Ca2+ release; U731 22, an inhibitor of phospholipase C; or W7, a calmodulin inhibitor) be fore 24 hours LPS(a)-stimulation, TNF and IL-1 mRNA were estimated 6 h ours after LPS(a) by using reverse transcriptase polymerase chain reac tion, Supernatant TNF and IL-I were measured by bioassay. Results: Tre atment with verapamil, TMB-8, U73122, or W7 markedly inhibited TNF rel ease by LPS(a), but had little effect on IL-1 release, Reprogramming b y LPS(p) did not alter the Ca2+ signal transduction pathways for eithe r cytokine, U73122 and verapamil did prevent the augmentation of IL-1 release seen after LPS(p). TNF message was present after LPS(a) despit e reprogrammed inhibition of TNF protein by LPS(p), Signal transductio n inhibitors that blocked Ca2+ altered TNF and IL-1 message in reprogr ammed macrophages in a pattern similar to their effects on naive cells , Conclusions: Intracellular Ca2+ is required for TNF protein release by naive macrophages and TNF mRNA transcription of both naive and LPS( p) reprogrammed cells, however LPS(a)-stimulated IL-1 release in perit oneal macrophages does not require Ca2+ dependent signaling pathways.