Skip to main content
PMC home page
Mediators of Inflammation logoLink to Mediators of Inflammation
. 1997 Dec;6(5-6):327–333. doi: 10.1080/09629359791460

Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies

A Mouithys-Mickalad 1, G Deby-Dupont 1,2, M Hoebeke 3, M Mathy-Hartert 1, M Lamy 1,2, C Deby 1
PMCID: PMC2365874  PMID: 18472867

Abstract

Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 × 10-7M) stimulated PMN (6 × 106 cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8 × 10-6M), C2-ceramide (N-acetyl SPN) and C6-ceramide (N-hexanoyl SPN) at the final concentration of 2 × 10-5 and 2 × 10-4M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 × 10-6M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 × 10-6M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H2O2, 0.01 mM Fe2+ and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 × 10-6 to 10-5M), but N-hexanoyl SPN was less active (from 2 × 10-5 to 2 × 10-4M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals.

Full Text

The Full Text of this article is available as a PDF (208.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen R. C. Phagocytic leukocyte oxygenation activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol. 1986;133:449–493. doi: 10.1016/0076-6879(86)33085-4. [DOI] [PubMed] [Google Scholar]
  2. Augé N., Andrieu N., Nègre-Salvayre A., Thiers J. C., Levade T., Salvayre R. The sphingomyelin-ceramide signaling pathway is involved in oxidized low density lipoprotein-induced cell proliferation. J Biol Chem. 1996 Aug 9;271(32):19251–19255. doi: 10.1074/jbc.271.32.19251. [DOI] [PubMed] [Google Scholar]
  3. Bazzi M. D., Nelsestuen G. L. Mechanism of protein kinase C inhibition by sphingosine. Biochem Biophys Res Commun. 1987 Jul 15;146(1):203–207. doi: 10.1016/0006-291x(87)90711-x. [DOI] [PubMed] [Google Scholar]
  4. Bender J. G., Van Epps D. E. Analysis of the bimodal chemiluminescence pattern stimulated in human neutrophils by chemotactic factors. Infect Immun. 1983 Sep;41(3):1062–1070. doi: 10.1128/iai.41.3.1062-1070.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borgeat P., Samuelsson B. Arachidonic acid metabolism in polymorphonuclear leukocytes: effects of ionophore A23187. Proc Natl Acad Sci U S A. 1979 May;76(5):2148–2152. doi: 10.1073/pnas.76.5.2148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Britigan B. E., Coffman T. J., Buettner G. R. Spin trapping evidence for the lack of significant hydroxyl radical production during the respiration burst of human phagocytes using a spin adduct resistant to superoxide-mediated destruction. J Biol Chem. 1990 Feb 15;265(5):2650–2656. [PubMed] [Google Scholar]
  7. Britigan B. E., Rosen G. M., Thompson B. Y., Chai Y., Cohen M. S. Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques. J Biol Chem. 1986 Dec 25;261(36):17026–17032. [PubMed] [Google Scholar]
  8. Chao C. P., Laulederkind S. J., Ballou L. R. Sphingosine-mediated phosphatidylinositol metabolism and calcium mobilization. J Biol Chem. 1994 Feb 25;269(8):5849–5856. [PubMed] [Google Scholar]
  9. Dahlgren C., Aniansson H., Magnusson K. E. Pattern of formylmethionyl-leucyl-phenylalanine-induced luminol- and lucigenin-dependent chemiluminescence in human neutrophils. Infect Immun. 1985 Jan;47(1):326–328. doi: 10.1128/iai.47.1.326-328.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Easmon C. S., Cole P. J., Williams A. J., Hastings M. The measurement of opsonic and phagocytic function by Luminol-dependent chemiluminescence. Immunology. 1980 Sep;41(1):67–74. [PMC free article] [PubMed] [Google Scholar]
  11. Fatatis A., Miller R. J. Sphingosine and sphingosine 1-phosphate differentially modulate platelet-derived growth factor-BB-induced Ca2+ signaling in transformed oligodendrocytes. J Biol Chem. 1996 Jan 5;271(1):295–301. doi: 10.1074/jbc.271.1.295. [DOI] [PubMed] [Google Scholar]
  12. Fuortes M., Jin W., Nathan C. Ceramide selectively inhibits early events in the response of human neutrophils to tumor necrosis factor. J Leukoc Biol. 1996 Mar;59(3):451–460. doi: 10.1002/jlb.59.3.451. [DOI] [PubMed] [Google Scholar]
  13. Hannun Y. A., Linardic C. M. Sphingolipid breakdown products: anti-proliferative and tumor-suppressor lipids. Biochim Biophys Acta. 1993 Dec 21;1154(3-4):223–236. doi: 10.1016/0304-4157(93)90001-5. [DOI] [PubMed] [Google Scholar]
  14. Hannun Y. A., Loomis C. R., Merrill A. H., Jr, Bell R. M. Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem. 1986 Sep 25;261(27):12604–12609. [PubMed] [Google Scholar]
  15. Hannun Y. A. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem. 1994 Feb 4;269(5):3125–3128. [PubMed] [Google Scholar]
  16. Kimura S., Kawa S., Ruan F., Nisar M., Sadahira Y., Hakomori S., Igarashi Y. Effect of sphingosine and its N-methyl derivatives on oxidative burst, phagokinetic activity, and trans-endothelial migration of human neutrophils. Biochem Pharmacol. 1992 Oct 20;44(8):1585–1595. doi: 10.1016/0006-2952(92)90476-y. [DOI] [PubMed] [Google Scholar]
  17. Lai C. S., Piette L. H. Hydroxyl radical production involved in lipid peroxidation of rat liver microsomes. Biochem Biophys Res Commun. 1977 Sep 9;78(1):51–59. doi: 10.1016/0006-291x(77)91220-7. [DOI] [PubMed] [Google Scholar]
  18. Lee T. C., Ou M. C., Shinozaki K., Malone B., Snyder F. Biosynthesis of N-acetylsphingosine by platelet-activating factor: sphingosine CoA-independent transacetylase in HL-60 cels. J Biol Chem. 1996 Jan 5;271(1):209–217. doi: 10.1074/jbc.271.1.209. [DOI] [PubMed] [Google Scholar]
  19. Lehmeyer J. E., Snyderman R., Johnston R. B., Jr Stimulation of neutrophil oxidative metabolism by chemotactic peptides: influence of calcium ion concentration and cytochalasin B and comparison with stimulation by phorbol myristate acetate. Blood. 1979 Jul;54(1):35–45. [PubMed] [Google Scholar]
  20. Lundqvist H., Follin P., Khalfan L., Dahlgren C. Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told. J Leukoc Biol. 1996 Feb;59(2):270–279. doi: 10.1002/jlb.59.2.270. [DOI] [PubMed] [Google Scholar]
  21. Mason R. P., Hanna P. M., Burkitt M. J., Kadiiska M. B. Detection of oxygen-derived radicals in biological systems using electron spin resonance. Environ Health Perspect. 1994 Dec;102 (Suppl 10):33–36. doi: 10.1289/ehp.94102s1033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rodriguez L. V., Lapeyre J. N., Robberson D. L., Maizel A. L., Becker F. F. Hydrodynamic shearing by VirTis blending conserves nucleosome structure of rat liver chromatin. Arch Biochem Biophys. 1980 Mar;200(1):116–129. doi: 10.1016/0003-9861(80)90338-0. [DOI] [PubMed] [Google Scholar]
  23. Roschger P., Graninger W., Klima H. Simultaneous detection of native and luminol-dependent luminescence of stimulated human polymorphonuclear leukocytes. Biochem Biophys Res Commun. 1984 Sep 28;123(3):1047–1053. doi: 10.1016/s0006-291x(84)80239-9. [DOI] [PubMed] [Google Scholar]
  24. Rosen G. M., Britigan B. E., Cohen M. S., Ellington S. P., Barber M. J. Detection of phagocyte-derived free radicals with spin trapping techniques: effect of temperature and cellular metabolism. Biochim Biophys Acta. 1988 May 13;969(3):236–241. doi: 10.1016/0167-4889(88)90057-2. [DOI] [PubMed] [Google Scholar]
  25. Rosen G. M., Pou S., Ramos C. L., Cohen M. S., Britigan B. E. Free radicals and phagocytic cells. FASEB J. 1995 Feb;9(2):200–209. doi: 10.1096/fasebj.9.2.7540156. [DOI] [PubMed] [Google Scholar]
  26. Rothstein J. L., Lint T. F., Schreiber H. Tumor necrosis factor/cachectin. Induction of hemorrhagic necrosis in normal tissue requires the fifth component of complement (C5). J Exp Med. 1988 Dec 1;168(6):2007–2021. doi: 10.1084/jem.168.6.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rudkowski R., Ziegler J. B., Graham G. G., Joulianos G. Gold complexes and activation of human polymorphonuclear leukocytes. Dissociation of changes in membrane potential and oxidative burst. Biochem Pharmacol. 1992 Sep 25;44(6):1091–1098. doi: 10.1016/0006-2952(92)90372-p. [DOI] [PubMed] [Google Scholar]
  28. Samuni A., Black C. D., Krishna C. M., Malech H. L., Bernstein E. F., Russo A. Hydroxyl radical production by stimulated neutrophils reappraised. J Biol Chem. 1988 Sep 25;263(27):13797–13801. [PubMed] [Google Scholar]
  29. Sasaki J. I., Yamaguchi M., Saeki S., Yamane H., Okamura N., Ishibashi S. Sphingosine inhibition of NADPH oxidase activation in a cell-free system. J Biochem. 1996 Oct;120(4):705–709. doi: 10.1093/oxfordjournals.jbchem.a021468. [DOI] [PubMed] [Google Scholar]
  30. Tritsch G. L., Niswander P. W. Modulation of macrophage superoxide release by purine metabolism. Life Sci. 1983 Mar 21;32(12):1359–1362. doi: 10.1016/0024-3205(83)90811-1. [DOI] [PubMed] [Google Scholar]
  31. Tubaro E., Lotti B., Santiangeli C., Cavallo G. Xanthine oxidase: an enzyme playing a role in the killing mechanism of polymorphonuclear leucocytes. Biochem Pharmacol. 1980 Nov 1;29(21):3018–3020. doi: 10.1016/0006-2952(80)90053-2. [DOI] [PubMed] [Google Scholar]
  32. Williams A. J., Cole P. J. The onset of polymorphonuclear leucocyte membrane-stimulated metabolic activity. Immunology. 1981 Aug;43(4):733–739. [PMC free article] [PubMed] [Google Scholar]
  33. Wilson E., Olcott M. C., Bell R. M., Merrill A. H., Jr, Lambeth J. D. Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. J Biol Chem. 1986 Sep 25;261(27):12616–12623. [PubMed] [Google Scholar]
  34. Wilson E., Rice W. G., Kinkade J. M., Jr, Merrill A. H., Jr, Arnold R. R., Lambeth J. D. Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils. Arch Biochem Biophys. 1987 Nov 15;259(1):204–214. doi: 10.1016/0003-9861(87)90487-5. [DOI] [PubMed] [Google Scholar]
  35. Wong K., Li X. B., Hunchuk N. N-acetylsphingosine (C2-ceramide) inhibited neutrophil superoxide formation and calcium influx. J Biol Chem. 1995 Feb 17;270(7):3056–3062. doi: 10.1074/jbc.270.7.3056. [DOI] [PubMed] [Google Scholar]
  36. Zhang P., Liu B., Jenkins G. M., Hannun Y. A., Obeid L. M. Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis. J Biol Chem. 1997 Apr 11;272(15):9609–9612. doi: 10.1074/jbc.272.15.9609. [DOI] [PubMed] [Google Scholar]

Articles from Mediators of Inflammation are provided here courtesy of Wiley

RESOURCES