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. 1998 Jan 15;101(2):455–463. doi: 10.1172/JCI949

Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.

F R DeLeo 1, J Renee 1, S McCormick 1, M Nakamura 1, M Apicella 1, J P Weiss 1, W M Nauseef 1
PMCID: PMC508585  PMID: 9435318

Abstract

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

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Selected References

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  1. Abo A., Pick E. Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages. J Biol Chem. 1991 Dec 15;266(35):23577–23585. [PubMed] [Google Scholar]
  2. 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]
  3. BERENDES H., BRIDGES R. A., GOOD R. A. A fatal granulomatosus of childhood: the clinical study of a new syndrome. Minn Med. 1957 May;40(5):309–312. [PubMed] [Google Scholar]
  4. Borregaard N., Heiple J. M., Simons E. R., Clark R. A. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol. 1983 Jul;97(1):52–61. doi: 10.1083/jcb.97.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borregaard N., Lollike K., Kjeldsen L., Sengeløv H., Bastholm L., Nielsen M. H., Bainton D. F. Human neutrophil granules and secretory vesicles. Eur J Haematol. 1993 Oct;51(4):187–198. doi: 10.1111/j.1600-0609.1993.tb00629.x. [DOI] [PubMed] [Google Scholar]
  6. Boxer G. J., Curnutte J. T., Boxer L. A. Polymorphonuclear leukocyte function. Hosp Pract (Off Ed) 1985 Mar 15;20(3):69-73, 77, 80 passim. doi: 10.1080/21548331.1985.11703014. [DOI] [PubMed] [Google Scholar]
  7. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  8. Clark R. A., Leidal K. G., Pearson D. W., Nauseef W. M. NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. J Biol Chem. 1987 Mar 25;262(9):4065–4074. [PubMed] [Google Scholar]
  9. Clark R. A., Volpp B. D., Leidal K. G., Nauseef W. M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990 Mar;85(3):714–721. doi: 10.1172/JCI114496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Condliffe A. M., Chilvers E. R., Haslett C., Dransfield I. Priming differentially regulates neutrophil adhesion molecule expression/function. Immunology. 1996 Sep;89(1):105–111. doi: 10.1046/j.1365-2567.1996.d01-711.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Leo F. R., Ulman K. V., Davis A. R., Jutila K. L., Quinn M. T. Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox. J Biol Chem. 1996 Jul 19;271(29):17013–17020. doi: 10.1074/jbc.271.29.17013. [DOI] [PubMed] [Google Scholar]
  12. DeLeo F. R., Quinn M. T. Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. J Leukoc Biol. 1996 Dec;60(6):677–691. doi: 10.1002/jlb.60.6.677. [DOI] [PubMed] [Google Scholar]
  13. Doerfler M. E., Weiss J., Clark J. D., Elsbach P. Bacterial lipopolysaccharide primes human neutrophils for enhanced release of arachidonic acid and causes phosphorylation of an 85-kD cytosolic phospholipase A2. J Clin Invest. 1994 Apr;93(4):1583–1591. doi: 10.1172/JCI117138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dusi S., Della Bianca V., Grzeskowiak M., Rossi F. Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils. Biochem J. 1993 Feb 15;290(Pt 1):173–178. doi: 10.1042/bj2900173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. El Benna J., Han J., Park J. W., Schmid E., Ulevitch R. J., Babior B. M. Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK. Arch Biochem Biophys. 1996 Oct 15;334(2):395–400. doi: 10.1006/abbi.1996.0470. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Haslett C., Guthrie L. A., Kopaniak M. M., Johnston R. B., Jr, Henson P. M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. 1985 Apr;119(1):101–110. [PMC free article] [PubMed] [Google Scholar]
  19. Hayakawa T., Suzuki K., Suzuki S., Andrews P. C., Babior B. M. A possible role for protein phosphorylation in the activation of the respiratory burst in human neutrophils. Evidence from studies with cells from patients with chronic granulomatous disease. J Biol Chem. 1986 Jul 15;261(20):9109–9115. [PubMed] [Google Scholar]
  20. Heyworth P. G., Badwey J. A. Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils. Biochim Biophys Acta. 1990 May 2;1052(2):299–305. doi: 10.1016/0167-4889(90)90225-3. [DOI] [PubMed] [Google Scholar]
  21. Jesaitis A. J., Quinn M. T., Mukherjee G., Ward P. A., Dratz E. A. Death by oxygen: radical views. The molecular basis of oxidative damage by leukocytes: a Montana State University/Keystone Symposium, Big Sky, MT, USA, January 28-February 3, 1991. New Biol. 1991 Jul;3(7):651–655. [PubMed] [Google Scholar]
  22. Jesaitis A. J. Structure of human phagocyte cytochrome b and its relationship to microbicidal superoxide production. J Immunol. 1995 Oct 1;155(7):3286–3288. [PubMed] [Google Scholar]
  23. Johnson K. G., Perry M. B. Improved techniques for the preparation of bacterial lipopolysaccharides. Can J Microbiol. 1976 Jan;22(1):29–34. doi: 10.1139/m76-004. [DOI] [PubMed] [Google Scholar]
  24. Jones W. M., Walcheck B., Jutila M. A. Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family. J Immunol. 1996 May 15;156(10):3772–3779. [PubMed] [Google Scholar]
  25. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  26. Koshkin V., Pick E. Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators. FEBS Lett. 1993 Jul 19;327(1):57–62. doi: 10.1016/0014-5793(93)81039-3. [DOI] [PubMed] [Google Scholar]
  27. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M., Nauseef W. M., Clark R. A., Gallin J. I., Malech H. L. Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. Science. 1990 May 11;248(4956):727–730. doi: 10.1126/science.1692159. [DOI] [PubMed] [Google Scholar]
  28. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I., Malech H. L. Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease. Science. 1989 Jul 28;245(4916):409–412. doi: 10.1126/science.2547247. [DOI] [PubMed] [Google Scholar]
  29. Lundqvist H., Karlsson A., Follin P., Sjölin C., Dahlgren C. Phagocytosis following translocation of the neutrophil b-cytochrome from the specific granule to the plasma membrane is associated with an increased leakage of reactive oxygen species. Scand J Immunol. 1992 Dec;36(6):885–891. doi: 10.1111/j.1365-3083.1992.tb03151.x. [DOI] [PubMed] [Google Scholar]
  30. McPhail L. C., Qualliotine-Mann D., Waite K. A. Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7931–7935. doi: 10.1073/pnas.92.17.7931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mizuno Y., Hara T., Nakamura M., Ueda K., Minakami S., Take H. Classification of chronic granulomatous disease on the basis of monoclonal antibody-defined surface cytochrome b deficiency. J Pediatr. 1988 Sep;113(3):458–462. doi: 10.1016/s0022-3476(88)80628-0. [DOI] [PubMed] [Google Scholar]
  32. Nakamura M., Murakami M., Koga T., Tanaka Y., Minakami S. Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers. Blood. 1987 May;69(5):1404–1408. [PubMed] [Google Scholar]
  33. Nauseef W. M., McCormick S. J., Clark R. A. Calreticulin functions as a molecular chaperone in the biosynthesis of myeloperoxidase. J Biol Chem. 1995 Mar 3;270(9):4741–4747. doi: 10.1074/jbc.270.9.4741. [DOI] [PubMed] [Google Scholar]
  34. Nauseef W. M., Volpp B. D., Clark R. A. Immunochemical and electrophoretic analyses of phosphorylated native and recombinant neutrophil oxidase component p47-phox. Blood. 1990 Dec 15;76(12):2622–2629. [PubMed] [Google Scholar]
  35. Nick J. A., Avdi N. J., Gerwins P., Johnson G. L., Worthen G. S. Activation of a p38 mitogen-activated protein kinase in human neutrophils by lipopolysaccharide. J Immunol. 1996 Jun 15;156(12):4867–4875. [PubMed] [Google Scholar]
  36. Nunoi H., Rotrosen D., Gallin J. I., Malech H. L. Two forms of autosomal chronic granulomatous disease lack distinct neutrophil cytosol factors. Science. 1988 Dec 2;242(4883):1298–1301. doi: 10.1126/science.2848319. [DOI] [PubMed] [Google Scholar]
  37. Ohno Y., Seligmann B. E., Gallin J. I. Cytochrome b translocation to human neutrophil plasma membranes and superoxide release. Differential effects of N-formylmethionylleucylphenylalanine, phorbol myristate acetate, and A23187. J Biol Chem. 1985 Feb 25;260(4):2409–2414. [PubMed] [Google Scholar]
  38. Parkos C. A., Allen R. A., Cochrane C. G., Jesaitis A. J. Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest. 1987 Sep;80(3):732–742. doi: 10.1172/JCI113128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Qualliotine-Mann D., Agwu D. E., Ellenburg M. D., McCall C. E., McPhail L. C. Phosphatidic acid and diacylglycerol synergize in a cell-free system for activation of NADPH oxidase from human neutrophils. J Biol Chem. 1993 Nov 15;268(32):23843–23849. [PubMed] [Google Scholar]
  41. Quinn M. T., Evans T., Loetterle L. R., Jesaitis A. J., Bokoch G. M. Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components. J Biol Chem. 1993 Oct 5;268(28):20983–20987. [PubMed] [Google Scholar]
  42. Quinn M. T., Parkos C. A., Walker L., Orkin S. H., Dinauer M. C., Jesaitis A. J. Association of a Ras-related protein with cytochrome b of human neutrophils. Nature. 1989 Nov 9;342(6246):198–200. doi: 10.1038/342198a0. [DOI] [PubMed] [Google Scholar]
  43. Robinson J. M., Badwey J. A. The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view. Histochem Cell Biol. 1995 Mar;103(3):163–180. doi: 10.1007/BF01454021. [DOI] [PubMed] [Google Scholar]
  44. Robinson J. M., Heyworth P. G., Badwey J. A. Utility of staurosporine in uncovering differences in the signal transduction pathways for superoxide production in neutrophils. Biochim Biophys Acta. 1990 Oct 15;1055(1):55–62. doi: 10.1016/0167-4889(90)90090-z. [DOI] [PubMed] [Google Scholar]
  45. Rotrosen D., Leto T. L. Phosphorylation of neutrophil 47-kDa cytosolic oxidase factor. Translocation to membrane is associated with distinct phosphorylation events. J Biol Chem. 1990 Nov 15;265(32):19910–19915. [PubMed] [Google Scholar]
  46. Royer-Pokora B., Kunkel L. M., Monaco A. P., Goff S. C., Newburger P. E., Baehner R. L., Cole F. S., Curnutte J. T., Orkin S. H. Cloning the gene for an inherited human disorder--chronic granulomatous disease--on the basis of its chromosomal location. Nature. 1986 Jul 3;322(6074):32–38. doi: 10.1038/322032a0. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Sengeløv H., Follin P., Kjeldsen L., Lollike K., Dahlgren C., Borregaard N. Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol. 1995 Apr 15;154(8):4157–4165. [PubMed] [Google Scholar]
  49. Shapira L., Champagne C., Gordon B., Amar S., Van Dyke T. E. Lipopolysaccharide priming of superoxide release by human neutrophils: role of membrane CD14 and serum LPS binding protein. Inflammation. 1995 Jun;19(3):289–295. doi: 10.1007/BF01534388. [DOI] [PubMed] [Google Scholar]
  50. Thrasher A. J., Keep N. H., Wientjes F., Segal A. W. Chronic granulomatous disease. Biochim Biophys Acta. 1994 Oct 21;1227(1-2):1–24. doi: 10.1016/0925-4439(94)90100-7. [DOI] [PubMed] [Google Scholar]
  51. Volpp B. D., Nauseef W. M., Clark R. A. Two cytosolic neutrophil oxidase components absent in autosomal chronic granulomatous disease. Science. 1988 Dec 2;242(4883):1295–1297. doi: 10.1126/science.2848318. [DOI] [PubMed] [Google Scholar]
  52. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Wientjes F. B., Hsuan J. J., Totty N. F., Segal A. W. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993 Dec 15;296(Pt 3):557–561. doi: 10.1042/bj2960557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]

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