Ceramide regulates oxidant release in adherent human neutrophils.
We investigated the role of sphingolipids in regulating oxidant release in adherent human neutrophils. Stimulation of adherent neutrophils with formyl-Met-Leu-Phe (fMLP) resulted in the accumulation of ceramide at a time when H2O2 release is terminated. H2O2 release in fMLP-stimulated neutrophils was suppressed in a concentration-dependent manner by the exogenous addition of several free sphingoid amines and short chain ceramides. Sphingosine, dihydrosphingosine, phytosphingosine, N-acetylsphingosine, and N-acetylphytosphingosine, but not N-acetyldihydrosphingosine, inhibited formyl peptide-stimulated oxidant release. The half-maximal inhibitory concentrations of N-acetylsphingosine and N-acetylphytosphingosine were 0.51 and 0.38 microM, respectively. Sphingosine, dihydrosphingosine, and phytosphingosine were less potent inhibitors with half-maximal inhibitory concentrations of 1.78, 15.4, and 1.48 microM, respectively. The 4 beta-phorbol 12 beta-myristate 13 alpha-acetate-induced respiratory burst was inhibited by 5 microM of sphingosine but not by 5 microM of N-acetylsphingosine. The effects of N-acetyl-conjugated sphingols (C2 ceramides) on phosphatidylcholine-specific phospholipase D and phosphatidic acid phosphohydrolase were markedly different from the effects of the related sphingoid bases. Both C2 ceramides and sphingoid bases partially inhibited the diradylglycerol formation by the phosphatidylcholine-specific phospholipase D pathway. Under the same conditions, however, N-acetyldihydrosphingosine and dihydrosphingosine failed to suppress H2O2 release in fMLP-stimulated neutrophils. These findings demonstrate that C2 ceramides inhibit H2O2 generation in fMLP-stimulated neutrophils via protein kinase C- or sphingoid base-independent mechanisms. The effect of ceramide in inhibiting the respiratory burst is structurally specific, because either a 4,5-trans double bond or 4-hydroxyl group is required for the inhibition. Therefore, ceramides may regulate oxidant release in adherent neutrophils.
Department of Pediatrics, University of Michigan, Ann Arbor 48109.
T
T
Nakamura
A
A
Abe
K J
KJ
Balazovich
D
D
Wu
S J
SJ
Suchard
L A
LA
Boxer
J A
JA
Shayman
D002448:Cell Adhesion; D002518:Ceramides / Q000097:blood* / Q000494:pharmacology*; D004075:Diglycerides / Q000097:blood; D005340:Fibrinogen; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood*; D066298:In Vitro Techniques; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D016877:Oxidants / Q000378:metabolism; D013107:Sphingolipids / Q000494:pharmacology
D002518:Ceramides; D004075:Diglycerides; D016877:Oxidants; D013107:Sphingolipids; D009240:N-Formylmethionine Leucyl-Phenylalanine; C035561:diarachidonyl diglyceride; D005340:Fibrinogen; D006861:Hydrogen Peroxide