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1549078 
Journal Article 
Ceramide and Ceramide 1-Phosphate Are Negative Regulators of TNF-alpha Production Induced by Lipopolysaccharide 
Jozefowski, S; Czerkies, M; Lukasik, A; Bielawska, A; Bielawski, J; Kwiatkowska, K; Sobota, A 
2010 
Yes 
Journal of Immunology
ISSN: 0022-1767
EISSN: 1550-6606 
185 
11 
6960-6973 
21041721 
WOS:000284311500063 
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-alpha 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-alpha and MIP-2 production in J774 cells and macrophages.
Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-alpha 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-alpha production. The latter result indicates that ceramide rather than sphingosine
suppresses TNF-alpha and MIP-2 production. Of these two cytokines, only TNF-alpha was negatively
regulated by ceramide 1-phosphate as was indicated by upregulated TNF-alpha 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-alpha 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. The Journal of Immunology,
2010, 185: 6960-6973.