Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1993 Oct 1;178(4):1347–1355. doi: 10.1084/jem.178.4.1347

Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen

PMCID: PMC2191210  PMID: 7690833

Abstract

Stimulation of heparinized blood with 1 microM formyl-methionyl-leucyl- phenylalanine (FMLP) resulted in the formation of < 30 pmol/ml plasma of 5-lipoxygenase (5-LO) products. The preincubation of blood with 1 microgram/ml of lipopolysaccharide (LPS) (Escherichia coli 0111-B4) for 30 min before stimulation with FMLP resulted in the accumulation of 250- 300 pmol of 5-LO products per ml plasma. The major products detected were leukotriene B4 and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid which were produced in equivalent amounts. The priming activity was detectable with as little as 1-10 ng LPS per ml blood and was optimal using 1-10 micrograms LPS/ml blood. The priming for 5-LO product synthesis was optimal after 20-30 min of preincubation with LPS and declined at preincubation times > 30 min. The priming effect of LPS was also observed using the complement fragment C5a or interleukin 8 as agonists. Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells accounted for 80 and 20% of the synthesis of 5-LO products, respectively. The ability of LPS to prime isolated PMN was dependent on the presence of plasma and was inhibited by the anti-CD14 antibody IOM2, indicating a CD14-dependent priming mechanism. The priming of whole blood with tumor necrosis factor alpha (TNF-alpha) and LPS was additive and the presence of mononuclear cells did not enhance the ability of LPS to prime PMN, indicating that the priming activity of LPS is independent of LPS-induced TNF-alpha synthesis. The mechanism by which LPS enhance 5-LO product synthesis in PMN was investigated. Treatment of PMN with LPS strongly enhanced the release of arachidonic acid after stimulation with FMLP. The release of arachidonic acid was optimal 2-3 min after stimulation with FMLP, attaining levels 5-15-fold greater than those observed in unprimed cells stimulated with FMLP. These results demonstrate that LPS dramatically increases the ability of blood to generate 5-LO products, and support the putative role of leukotrienes in pathological states involving LPS.

Full Text

The Full Text of this article is available as a PDF (927.7 KB).

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. Aida Y., Pabst M. J. Neutrophil responses to lipopolysaccharide. Effect of adherence on triggering and priming of the respiratory burst. J Immunol. 1991 Feb 15;146(4):1271–1276. [PubMed] [Google Scholar]
  3. Aida Y., Pabst M. J. Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha. J Immunol. 1990 Nov 1;145(9):3017–3025. [PubMed] [Google Scholar]
  4. Allen J. N., Herzyk D. J., Allen E. D., Wewers M. D. Human whole blood interleukin-1-beta production: kinetics, cell source, and comparison with TNF-alpha. J Lab Clin Med. 1992 May;119(5):538–546. [PubMed] [Google Scholar]
  5. 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]
  6. Bayston K., Tomlinson M., Cohen J. In-vitro stimulation of TNF-alpha from human whole blood by cell-free supernatants of gram-positive bacteria. Cytokine. 1992 Sep;4(5):397–402. doi: 10.1016/1043-4666(92)90084-5. [DOI] [PubMed] [Google Scholar]
  7. Beutler B., Mahoney J., Le Trang N., Pekala P., Cerami A. Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J Exp Med. 1985 May 1;161(5):984–995. doi: 10.1084/jem.161.5.984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Billiau A., Vandekerckhove F. Cytokines and their interactions with other inflammatory mediators in the pathogenesis of sepsis and septic shock. Eur J Clin Invest. 1991 Dec;21(6):559–573. doi: 10.1111/j.1365-2362.1991.tb01410.x. [DOI] [PubMed] [Google Scholar]
  9. Bone R. C. The pathogenesis of sepsis. Ann Intern Med. 1991 Sep 15;115(6):457–469. doi: 10.7326/0003-4819-115-6-457. [DOI] [PubMed] [Google Scholar]
  10. Borgeat P., Naccache P. H. Biosynthesis and biological activity of leukotriene B4. Clin Biochem. 1990 Oct;23(5):459–468. doi: 10.1016/0009-9120(90)90272-v. [DOI] [PubMed] [Google Scholar]
  11. Borgeat P., Picard S., Vallerand P., Bourgoin S., Odeimat A., Sirois P., Poubelle P. E. Automated on-line extraction and profiling of lipoxygenase products of arachidonic acid by high-performance liquid chromatography. Methods Enzymol. 1990;187:98–116. doi: 10.1016/0076-6879(90)87014-t. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Coggeshall J. W., Christman B. W., Lefferts P. L., Serafin W. E., Blair I. A., Butterfield M. J., Snapper J. R. Effect of inhibition of 5-lipoxygenase metabolism of arachidonic acid on response to endotoxemia in sheep. J Appl Physiol (1985) 1988 Sep;65(3):1351–1359. doi: 10.1152/jappl.1988.65.3.1351. [DOI] [PubMed] [Google Scholar]
  14. Couturier C., Haeffner-Cavaillon N., Caroff M., Kazatchkine M. D. Binding sites for endotoxins (lipopolysaccharides) on human monocytes. J Immunol. 1991 Sep 15;147(6):1899–1904. [PubMed] [Google Scholar]
  15. Cybulsky M. I., Chan M. K., Movat H. Z. Acute inflammation and microthrombosis induced by endotoxin, interleukin-1, and tumor necrosis factor and their implication in gram-negative infection. Lab Invest. 1988 Apr;58(4):365–378. [PubMed] [Google Scholar]
  16. Darbonne W. C., Rice G. C., Mohler M. A., Apple T., Hébert C. A., Valente A. J., Baker J. B. Red blood cells are a sink for interleukin 8, a leukocyte chemotaxin. J Clin Invest. 1991 Oct;88(4):1362–1369. doi: 10.1172/JCI115442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Deitch E. A. Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg. 1992 Aug;216(2):117–134. doi: 10.1097/00000658-199208000-00002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. DiPersio J. F., Naccache P. H., Borgeat P., Gasson J. C., Nguyen M. H., McColl S. R. Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis. Prostaglandins. 1988 Nov;36(5):673–691. doi: 10.1016/0090-6980(88)90013-5. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Erzurum S. C., Downey G. P., Doherty D. E., Schwab B., 3rd, Elson E. L., Worthen G. S. Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties. J Immunol. 1992 Jul 1;149(1):154–162. [PubMed] [Google Scholar]
  21. Fiore S., Serhan C. N. Formation of lipoxins and leukotrienes during receptor-mediated interactions of human platelets and recombinant human granulocyte/macrophage colony-stimulating factor-primed neutrophils. J Exp Med. 1990 Nov 1;172(5):1451–1457. doi: 10.1084/jem.172.5.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fitzpatrick F. A., Morton D. R., Wynalda M. A. Albumin stabilizes leukotriene A4. J Biol Chem. 1982 May 10;257(9):4680–4683. [PubMed] [Google Scholar]
  23. 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]
  24. Fradin A., Zirrolli J. A., Maclouf J., Vausbinder L., Henson P. M., Murphy R. C. Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism. J Immunol. 1989 Dec 1;143(11):3680–3685. [PubMed] [Google Scholar]
  25. Fujimoto K., Kobayashi T. The role of leukotriene B4 in endotoxin-induced lung injury in unanesthetized sheep. Respir Physiol. 1988 Mar;71(3):259–268. doi: 10.1016/0034-5687(88)90020-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Hack C. E., Hart M., van Schijndel R. J., Eerenberg A. J., Nuijens J. H., Thijs L. G., Aarden L. A. Interleukin-8 in sepsis: relation to shock and inflammatory mediators. Infect Immun. 1992 Jul;60(7):2835–2842. doi: 10.1128/iai.60.7.2835-2842.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kreger B. E., Craven D. E., McCabe W. R. Gram-negative bacteremia. IV. Re-evaluation of clinical features and treatment in 612 patients. Am J Med. 1980 Mar;68(3):344–355. doi: 10.1016/0002-9343(80)90102-3. [DOI] [PubMed] [Google Scholar]
  30. Lin A. H., Morton D. R., Gorman R. R. Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis in human polymorphonuclear leukocytes. J Clin Invest. 1982 Nov;70(5):1058–1065. doi: 10.1172/JCI110693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Martin M. A., Silverman H. J. Gram-negative sepsis and the adult respiratory distress syndrome. Clin Infect Dis. 1992 Jun;14(6):1213–1228. doi: 10.1093/clinids/14.6.1213. [DOI] [PubMed] [Google Scholar]
  33. Matera G., Cook J. A., Hennigar R. A., Tempel G. E., Wise W. C., Oglesby T. D., Halushka P. V. Beneficial effects of a 5-lipoxygenase inhibitor in endotoxic shock in the rat. J Pharmacol Exp Ther. 1988 Oct;247(1):363–371. [PubMed] [Google Scholar]
  34. McColl S. R., Krump E., Naccache P. H., Poubelle P. E., Braquet P., Braquet M., Borgeat P. Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation. J Immunol. 1991 Feb 15;146(4):1204–1211. [PubMed] [Google Scholar]
  35. McDonald P. P., McColl S. R., Naccache P. H., Borgeat P. Studies on the activation of human neutrophil 5-lipoxygenase induced by natural agonists and Ca2+ ionophore A23187. Biochem J. 1991 Dec 1;280(Pt 2):379–385. doi: 10.1042/bj2800379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McGee J. E., Fitzpatrick F. A. Erythrocyte-neutrophil interactions: formation of leukotriene B4 by transcellular biosynthesis. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1349–1353. doi: 10.1073/pnas.83.5.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Meyrick B., Brigham K. L. Acute effects of Escherichia coli endotoxin on the pulmonary microcirculation of anesthetized sheep structure:function relationships. Lab Invest. 1983 Apr;48(4):458–470. [PubMed] [Google Scholar]
  38. Newton R. C. Human monocyte production of interleukin-1: parameters of the induction of interleukin-1 secretion by lipopolysaccharides. J Leukoc Biol. 1986 Mar;39(3):299–311. doi: 10.1002/jlb.39.3.299. [DOI] [PubMed] [Google Scholar]
  39. Nichols F. C., Garrison S. W., Davis H. W. Prostaglandin E2 and thromboxane B2 release from human monocytes treated with bacterial lipopolysaccharide. J Leukoc Biol. 1988 Nov;44(5):376–384. doi: 10.1002/jlb.44.5.376. [DOI] [PubMed] [Google Scholar]
  40. O'Sullivan M. G., Chilton F. H., Huggins E. M., Jr, McCall C. E. Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase. J Biol Chem. 1992 Jul 25;267(21):14547–14550. [PubMed] [Google Scholar]
  41. Palmantier R., Borgeat P. Thrombin-activated platelets promote leukotriene B4 synthesis in polymorphonuclear leucocytes stimulated by physiological agonists. Br J Pharmacol. 1991 Aug;103(4):1909–1916. doi: 10.1111/j.1476-5381.1991.tb12351.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Parsons P. E., Worthen G. S., Moore E. E., Tate R. M., Henson P. M. The association of circulating endotoxin with the development of the adult respiratory distress syndrome. Am Rev Respir Dis. 1989 Aug;140(2):294–301. doi: 10.1164/ajrccm/140.2.294. [DOI] [PubMed] [Google Scholar]
  43. Purdon A. D., Rao A. K. Interaction of albumin, arachidonic acid and prostanoids in platelets. Prostaglandins Leukot Essent Fatty Acids. 1989 Mar;35(4):213–218. doi: 10.1016/0952-3278(89)90004-5. [DOI] [PubMed] [Google Scholar]
  44. Schumann R. R., Leong S. R., Flaggs G. W., Gray P. W., Wright S. D., Mathison J. C., Tobias P. S., Ulevitch R. J. Structure and function of lipopolysaccharide binding protein. Science. 1990 Sep 21;249(4975):1429–1431. doi: 10.1126/science.2402637. [DOI] [PubMed] [Google Scholar]
  45. Sylvester I., Yoshimura T., Sticherling M., Schröder J. M., Ceska M., Peichl P., Leonard E. J. Neutrophil attractant protein-1-immunoglobulin G immune complexes and free anti-NAP-1 antibody in normal human serum. J Clin Invest. 1992 Aug;90(2):471–481. doi: 10.1172/JCI115883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tiegs G., Wendel A. Leukotriene-mediated liver injury. Biochem Pharmacol. 1988 Jul 1;37(13):2569–2573. doi: 10.1016/0006-2952(88)90248-1. [DOI] [PubMed] [Google Scholar]
  47. Watson F., Robinson J. J., Edwards S. W. Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines. Biosci Rep. 1992 Apr;12(2):123–133. doi: 10.1007/BF02351217. [DOI] [PubMed] [Google Scholar]
  48. Welbourn C. R., Young Y. Endotoxin, septic shock and acute lung injury: neutrophils, macrophages and inflammatory mediators. Br J Surg. 1992 Oct;79(10):998–1003. doi: 10.1002/bjs.1800791006. [DOI] [PubMed] [Google Scholar]
  49. Worthen G. S., Seccombe J. F., Clay K. L., Guthrie L. A., Johnston R. B., Jr The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor. Potential role in mediation of enhanced superoxide secretion. J Immunol. 1988 May 15;140(10):3553–3559. [PubMed] [Google Scholar]
  50. Wright S. D. Multiple receptors for endotoxin. Curr Opin Immunol. 1991 Feb;3(1):83–90. doi: 10.1016/0952-7915(91)90082-c. [DOI] [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., Patel M., Miller D. S. Septin: a factor in plasma that opsonizes lipopolysaccharide-bearing particles for recognition by CD14 on phagocytes. J Exp Med. 1992 Sep 1;176(3):719–727. doi: 10.1084/jem.176.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Yasui K., Becker E. L., Sha'afi R. I. Lipopolysaccharide and serum cause the translocation of G-protein to the membrane and prime neutrophils via CD14. Biochem Biophys Res Commun. 1992 Mar 31;183(3):1280–1286. doi: 10.1016/s0006-291x(05)80329-8. [DOI] [PubMed] [Google Scholar]
  55. Yoshikawa D., Goto F. Effect of platelet-activating factor antagonist and leukotriene antagonist on endotoxin shock in the rat: role of the leukocyte. Circ Shock. 1992 Sep;38(1):29–33. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES