Objective: Dysregulated macrophage cytokine production may predispose to organ failure during sepsis. Macrophages pretreated in vitro with low-dose endotoxin (LPSp) become "tolerant" to subsequent lipopolysaccharide (LPS) activation (LPSa), characterized by inhibition of tumor necrosis factor (TNF) and augmentation of interleukin-1 (IL-1). To understand cytokine dysregulation we examined the Ca2+ dependence of TNF and IL-1 signal transduction to LPSa and whether it was altered by LPSp.
Methods: Murine peritoneal exudate macrophages received +/- 100 ng/mL of LPSp for 24 hours. Cultures were pretreated for 2 hours with specific signal transduction inhibitors (verapamil, a Ca2+ channel inhibitor; TMB-8, an inhibitor of intracellular Ca2+ release; U73122, an inhibitor of phospholipase C; or W7, a calmodulin inhibitor) before 24 hours LPSa-stimulation. TNF and IL-1 mRNA were estimated 6 hours after LPSa by using reverse transcriptase polymerase chain reaction. Supernatant TNF and IL-1 were measured by bioassay.
Results: Treatment with verapamil, TMB-8, U73122, or W7 markedly inhibited TNF release by LPSa, but had little effect on IL-1 release. Reprogramming by LPSp did not alter the Ca2+ signal transduction pathways for either cytokine. U73122 and verapamil did prevent the augmentation of IL-1 release seen after LPSp. TNF message was present after LPSa despite reprogrammed inhibition of TNF protein by LPSp. Signal transduction inhibitors that blocked Ca2+ altered TNF and IL-1 message in reprogrammed 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 LPSp reprogrammed cells, however LPSa-stimulated IL-1 release in peritoneal macrophages does not require Ca2+ dependent signaling pathways.