Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-α Production Induced by Lipopolysaccharide

Abstract

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1–2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

Dietary Milk Sphingomyelin Reduces Systemic Inflammation in Diet-Induced Obese Mice and Inhibits LPS Activity in Macrophages

“…Sphingosine could display anti-inflammatory effects through the activation of the nuclear receptor, peroxisome proliferator-activate receptor-gamma (PPARγ) [22]. In support of our findings, exogenous C8-ceramide and sphingosine have been previously reported to reduce TNF-α secretion from LPS-stimulated macrophages [63]. Furthermore, both C16-ceramide and membrane-permeable C2-ceramide have been shown to reduce TNF-α secretion from LPS-stimulated macrophages by post-translational mechanisms [64].…”

Ceramide 1-Phosphate Mediates Endothelial Cell Invasion via the Annexin a2-p11 Heterotetrameric Protein Complex

“…Additionally, recent evidence documents the direct inhibition of TNF-␣-converting enzyme by C-1-P binding (11). These data supported previous research demonstrating that C-1-P, produced via ceramide generated from acid sphingomyelinase activity, could negatively modulate TNF-␣ release (12,13).…”

Bioactive Sphingolipids in Response to Chemotherapy: A Scope on Leukemias

“…On the other hand, C1P analogs were documented to exert anti-inflammatory functions through suppression of TNF-and induction of IL-10 production in macrophages [68]. This role was confirmed by various other groups as well, attributing the importance to C1P in the regulation of inflammatory response [69][70][71]. Besides this, C1P is also thought to have roles in the regulation of intracellular Ca 2+ levels [72][73][74].…”