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 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.
West
M A
MA
Department of Surgery, Hennepin Count Medical Center, University of Minnesota, Minneapolis 55415, USA. westx015@tc.umn.edu
Clair
L
L
Bellingham
J
J
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D019446:Endotoxemia / Q000276:immunology* / Q000503:physiopathology; D007375:Interleukin-1 / Q000096:biosynthesis / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D010537:Peritoneum / Q000166:cytology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D018805:Sepsis / Q000276:immunology / Q000503:physiopathology; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000276:immunology*
D007375:Interleukin-1; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium