Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1994 Apr;93(4):1583–1591. doi: 10.1172/JCI117138

Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2.

M E Doerfler 1, J Weiss 1, J D Clark 1, P Elsbach 1
PMCID: PMC294185  PMID: 7512985

Abstract

Production of leukotriene B4 (LTB4) by human neutrophils (PMN) in response to different stimuli is increased after pretreatment with lipopolysaccharides (LPS). We have analyzed the steps in arachidonic acid (AA) metabolism affected by LPS by examining release of AA and its metabolites from [3H]AA prelabeled PMN. Pretreatment of PMN for 60 min with up to 1 microgram/ml of LPS alone had no effect, but release of [3H]AA was stimulated up to fivefold during subsequent stimulation with a second agent. In the absence of LPS-binding protein (LBP), priming was maximal after pretreatment of PMN with 10 ng of LPS/ml for 60 min; in the presence of LBP maximal priming occurred within 45 min at 0.1 ng of LPS/ml and within 15 min at 100 ng of LPS/ml. Treatment of PMN with 10 ng of LPS/ml also increased uptake of opsonized zymosan by up to 60%. Phospholipids are the source of released [3H]AA. No release was observed from [14C]oleic acid (OA)-labeled PMN suggesting that phospholipolysis may be specific for [3H]AA-labeled phospholipid pools. Cytosol from PMN primed with LPS contains two to three times the phospholipase A2 (PLA2) activity of control PMN, against 1-palmitoyl-[2-14C]arachidonoyl-phosphatidylcholine. This activity is Ca2+ dependent and dithiothreitol resistant. LPS priming is accompanied by reduced migration during SDS-PAGE of an 85-kD protein, identified as a cytosolic PLA2. The extent and kinetics of this effect of LPS on cPLA2 parallel the priming of [3H]AA release, both depending on LPS concentration either with or without LBP. These findings suggest that priming by LPS of AA metabolism by PMN includes phosphorylation of an AA-phospholipid-selective cytosolic PLA2 that is dissociated from activation until a second stimulus is applied.

Full text

PDF
1583

Images in this article

1588Image
on p.1588
1588Image
on p.1588
1589Image
on p.1589

Selected References

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

  1. Aderem A. A., Cohen D. S., Wright S. D., Cohn Z. A. Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites. J Exp Med. 1986 Jul 1;164(1):165–179. doi: 10.1084/jem.164.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aderem A. A., Cohn Z. A. Calcium ionophore synergizes with bacterial lipopolysaccharides in activating macrophage arachidonic acid metabolism. J Exp Med. 1988 Feb 1;167(2):623–631. doi: 10.1084/jem.167.2.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alonso F., Henson P. M., Leslie C. C. A cytosolic phospholipase in human neutrophils that hydrolyzes arachidonoyl-containing phosphatidylcholine. Biochim Biophys Acta. 1986 Sep 12;878(2):273–280. doi: 10.1016/0005-2760(86)90156-6. [DOI] [PubMed] [Google Scholar]
  4. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  5. Balsinde J., Diez E., Mollinedo F. Arachidonic acid release from diacylglycerol in human neutrophils. Translocation of diacylglycerol-deacylating enzyme activities from an intracellular pool to plasma membrane upon cell activation. J Biol Chem. 1991 Aug 25;266(24):15638–15643. [PubMed] [Google Scholar]
  6. Balsinde J., Diez E., Schüller A., Mollinedo F. Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization. J Biol Chem. 1988 Feb 5;263(4):1929–1936. [PubMed] [Google Scholar]
  7. Bauldry S. A., McCall C. E., Cousart S. L., Bass D. A. Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming. J Immunol. 1991 Feb 15;146(4):1277–1285. [PubMed] [Google Scholar]
  8. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  9. Chilton F. H., Connell T. R. 1-ether-linked phosphoglycerides. Major endogenous sources of arachidonate in the human neutrophil. J Biol Chem. 1988 Apr 15;263(11):5260–5265. [PubMed] [Google Scholar]
  10. Chilton F. H. Potential phospholipid source(s) of arachidonate used for the synthesis of leukotrienes by the human neutrophil. Biochem J. 1989 Mar 1;258(2):327–333. doi: 10.1042/bj2580327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Clancy R. M., Dahinden C. A., Hugli T. E. Arachidonate metabolism by human polymorphonuclear leukocytes stimulated by N-formyl-Met-Leu-Phe or complement component C5a is independent of phospholipase activation. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7200–7204. doi: 10.1073/pnas.80.23.7200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  13. Clark J. D., Milona N., Knopf J. L. Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7708–7712. doi: 10.1073/pnas.87.19.7708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Diez E., Mong S. Purification of a phospholipase A2 from human monocytic leukemic U937 cells. Calcium-dependent activation and membrane association. J Biol Chem. 1990 Aug 25;265(24):14654–14661. [PubMed] [Google Scholar]
  15. Doerfler M. E., Danner R. L., Shelhamer J. H., Parrillo J. E. Bacterial lipopolysaccharides prime human neutrophils for enhanced production of leukotriene B4. J Clin Invest. 1989 Mar;83(3):970–977. doi: 10.1172/JCI113983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elsbach P., Pettis P., Beckerdite S., Franson R. Effects of phagocytosis by rabbit granulocytes on macromolecular synthesis and degradation in different species of bacteria. J Bacteriol. 1973 Aug;115(2):490–497. doi: 10.1128/jb.115.2.490-497.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ford-Hutchinson A. W., Bray M. A., Doig M. V., Shipley M. E., Smith M. J. Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature. 1980 Jul 17;286(5770):264–265. doi: 10.1038/286264a0. [DOI] [PubMed] [Google Scholar]
  18. Forehand J. R., Pabst M. J., Phillips W. A., Johnston R. B., Jr Lipopolysaccharide priming of human neutrophils for an enhanced respiratory burst. Role of intracellular free calcium. J Clin Invest. 1989 Jan;83(1):74–83. doi: 10.1172/JCI113887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Forst S., Weiss J., Elsbach P., Maraganore J. M., Reardon I., Heinrikson R. L. Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate. Biochemistry. 1986 Dec 30;25(26):8381–8385. doi: 10.1021/bi00374a008. [DOI] [PubMed] [Google Scholar]
  20. Franson R., Patriarca P., Elsbach P. Phospholipid metabolism by phagocytic cells. Phospholipases A2 associated with rabbit polymorphonuclear leukocyte granules. J Lipid Res. 1974 Jul;15(4):380–388. [PubMed] [Google Scholar]
  21. Fujimori Y., Murakami M., Kim D. K., Hara S., Takayama K., Kudo I., Inoue K. Immunochemical detection of arachidonoyl-preferential phospholipase A2. J Biochem. 1992 Jan;111(1):54–60. doi: 10.1093/oxfordjournals.jbchem.a123718. [DOI] [PubMed] [Google Scholar]
  22. Glaser K. B., Asmis R., Dennis E. A. Bacterial lipopolysaccharide priming of P388D1 macrophage-like cells for enhanced arachidonic acid metabolism. Platelet-activating factor receptor activation and regulation of phospholipase A2. J Biol Chem. 1990 May 25;265(15):8658–8664. [PubMed] [Google Scholar]
  23. Guthrie L. A., McPhail L. C., Henson P. M., Johnston R. B., Jr Priming of neutrophils for enhanced release of oxygen metabolites by bacterial lipopolysaccharide. Evidence for increased activity of the superoxide-producing enzyme. J Exp Med. 1984 Dec 1;160(6):1656–1671. doi: 10.1084/jem.160.6.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Haziot A., Chen S., Ferrero E., Low M. G., Silber R., Goyert S. M. The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol. 1988 Jul 15;141(2):547–552. [PubMed] [Google Scholar]
  25. Kaluzny M. A., Duncan L. A., Merritt M. V., Epps D. E. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res. 1985 Jan;26(1):135–140. [PubMed] [Google Scholar]
  26. Klein J. B., Payne V., Schepers T. M., McLeish K. R. Bacterial lipopolysaccharide enhances polymorphonuclear leukocyte function independent of changes in intracellular calcium. Inflammation. 1990 Oct;14(5):599–611. doi: 10.1007/BF00914279. [DOI] [PubMed] [Google Scholar]
  27. Korchak H. M., Vosshall L. B., Zagon G., Ljubich P., Rich A. M., Weissmann G. Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling. J Biol Chem. 1988 Aug 15;263(23):11090–11097. [PubMed] [Google Scholar]
  28. Kramer R. M., Roberts E. F., Manetta J., Putnam J. E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells. J Biol Chem. 1991 Mar 15;266(8):5268–5272. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lin L. L., Lin A. Y., Knopf J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6147–6151. doi: 10.1073/pnas.89.13.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  32. Lund-Johansen F., Olweus J., Aarli A., Bjerknes R. Signal transduction in human monocytes and granulocytes through the PI-linked antigen CD14. FEBS Lett. 1990 Oct 29;273(1-2):55–58. doi: 10.1016/0014-5793(90)81049-t. [DOI] [PubMed] [Google Scholar]
  33. Lynn W. A., Raetz C. R., Qureshi N., Golenbock D. T. Lipopolysaccharide-induced stimulation of CD11b/CD18 expression on neutrophils. Evidence of specific receptor-based response and inhibition by lipid A-based antagonists. J Immunol. 1991 Nov 1;147(9):3072–3079. [PubMed] [Google Scholar]
  34. Naccache P. H., Molski T. F., Borgeat P., White J. R., Sha'afi R. I. Phorbol esters inhibit the fMet-Leu-Phe- and leukotriene B4-stimulated calcium mobilization and enzyme secretion in rabbit neutrophils. J Biol Chem. 1985 Feb 25;260(4):2125–2131. [PubMed] [Google Scholar]
  35. Nemenoff R. A., Winitz S., Qian N. X., Van Putten V., Johnson G. L., Heasley L. E. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem. 1993 Jan 25;268(3):1960–1964. [PubMed] [Google Scholar]
  36. Palma C., Cassone A., Serbousek D., Pearson C. A., Djeu J. Y. Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans. Infect Immun. 1992 Nov;60(11):4604–4611. doi: 10.1128/iai.60.11.4604-4611.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Philips M. R., Abramson S. B., Kolasinski S. L., Haines K. A., Weissmann G., Rosenfeld M. G. Low molecular weight GTP-binding proteins in human neutrophil granule membranes. J Biol Chem. 1991 Jan 15;266(2):1289–1298. [PubMed] [Google Scholar]
  38. RAAFLAUB J. Applications of metal buffers and metal indicators in biochemistry. Methods Biochem Anal. 1956;3:301–325. doi: 10.1002/9780470110195.ch10. [DOI] [PubMed] [Google Scholar]
  39. Rouser G., Fkeischer S., Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May;5(5):494–496. doi: 10.1007/BF02531316. [DOI] [PubMed] [Google Scholar]
  40. Sha'afi R. I., White J. R., Molski T. F., Shefcyk J., Volpi M., Naccache P. H., Feinstein M. B. Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium. Biochem Biophys Res Commun. 1983 Jul 29;114(2):638–645. doi: 10.1016/0006-291x(83)90828-8. [DOI] [PubMed] [Google Scholar]
  41. Shak S. Leukotriene B4 catabolism: quantitation of leukotriene B4 and its omega-oxidation products by reversed-phase high-performance liquid chromatography. Methods Enzymol. 1987;141:355–371. doi: 10.1016/0076-6879(87)41083-5. [DOI] [PubMed] [Google Scholar]
  42. Stefanová I., Horejsí V., Ansotegui I. J., Knapp W., Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. doi: 10.1126/science.1719635. [DOI] [PubMed] [Google Scholar]
  43. Tobias P. S., Soldau K., Ulevitch R. J. Isolation of a lipopolysaccharide-binding acute phase reactant from rabbit serum. J Exp Med. 1986 Sep 1;164(3):777–793. doi: 10.1084/jem.164.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ulevitch R. J., Watanabe Y., Sano M., Lister M. D., Deems R. A., Dennis E. A. Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line. J Biol Chem. 1988 Mar 5;263(7):3079–3085. [PubMed] [Google Scholar]
  45. Victor M., Weiss J., Klempner M. S., Elsbach P. Phospholipase A2 activity in the plasma membrane of human polymorphonuclear leukocytes. FEBS Lett. 1981 Dec 28;136(2):298–300. doi: 10.1016/0014-5793(81)80639-4. [DOI] [PubMed] [Google Scholar]
  46. Vosbeck K., Tobias P., Mueller H., Allen R. A., Arfors K. E., Ulevitch R. J., Sklar L. A. Priming of polymorphonuclear granulocytes by lipopolysaccharides and its complexes with lipopolysaccharide binding protein and high density lipoprotein. J Leukoc Biol. 1990 Feb;47(2):97–104. doi: 10.1002/jlb.47.2.97. [DOI] [PubMed] [Google Scholar]
  47. Wedmore C. V., Williams T. J. Control of vascular permeability by polymorphonuclear leukocytes in inflammation. Nature. 1981 Feb 19;289(5799):646–650. doi: 10.1038/289646a0. [DOI] [PubMed] [Google Scholar]
  48. Weinstein S. L., Gold M. R., DeFranco A. L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4148–4152. doi: 10.1073/pnas.88.10.4148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wright G. C., Weiss J., Kim K. S., Verheij H., Elsbach P. Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases. J Clin Invest. 1990 Jun;85(6):1925–1935. doi: 10.1172/JCI114655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wright G. W., Ooi C. E., Weiss J., Elsbach P. Purification of a cellular (granulocyte) and an extracellular (serum) phospholipase A2 that participate in the destruction of Escherichia coli in a rabbit inflammatory exudate. J Biol Chem. 1990 Apr 25;265(12):6675–6681. [PubMed] [Google Scholar]
  51. Wright S. D., Ramos R. A., Hermanowski-Vosatka A., Rockwell P., Detmers P. A. Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14. J Exp Med. 1991 May 1;173(5):1281–1286. doi: 10.1084/jem.173.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wright S. D., Ramos R. A., Tobias P. S., Ulevitch R. J., Mathison J. C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science. 1990 Sep 21;249(4975):1431–1433. doi: 10.1126/science.1698311. [DOI] [PubMed] [Google Scholar]
  53. Xing M., Mattera R. Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes. J Biol Chem. 1992 Dec 25;267(36):25966–25975. [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES