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Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes

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Neutrophil Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1124))

Abstract

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 −) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst is of great importance, and a large number of methods have been used for this purpose. A selection of methods, including chemiluminescence amplified by luminol or isoluminol, the absorbance change following reduction of cytochrome c, and the fluorescence increase upon oxidation of PHPA, are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to more clinically oriented research on innate immune mechanisms and inflammation.

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References

  1. Quie PG, White JG, Holmes B et al (1967) In vitro bactericidal capacity of human polymorphonuclear leukocytes: diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46:668–679

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Segal AW (2005) How neutrophils kill microbes. Ann Rev Immunol 23:197–223

    Article  CAS  Google Scholar 

  3. Quinn MT, Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases. J Leukoc Biol 76:760–781

    Article  CAS  PubMed  Google Scholar 

  4. Bylund J, Brown KL, Movitz C et al (2010) Intracellular generation of superoxide by the phagocyte NADPH oxidase: how, where, and what for? Free Radic Biol Med 49:1834–1845

    Article  CAS  PubMed  Google Scholar 

  5. Borregaard N, Heiple JM, Simons ER et al (1983) Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J Cell Biol 97:52–61

    Article  CAS  PubMed  Google Scholar 

  6. Hager M, Cowland JB, Borregaard N (2010) Neutrophil granules in health and disease. J Internal Med 268:25–34

    CAS  PubMed  Google Scholar 

  7. Dahlgren C, Johansson A, Lundqvist H et al (1992) Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin). Biochim Biophys Acta 1137:182–188

    Article  CAS  PubMed  Google Scholar 

  8. Karlsson A, Dahlgren C (2002) Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxid Redox Signal 4:49–60

    Article  CAS  PubMed  Google Scholar 

  9. Matute JD, Arias AA, Wright NA et al (2009) A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 114:3309–3315

    Article  CAS  PubMed  Google Scholar 

  10. Björkman L, Dahlgren C, Karlsson A et al (2008) Phagocyte-derived reactive oxygen species as suppressors of inflammatory disease. Arthritis Rheum 58:2931–2935

    Article  PubMed  Google Scholar 

  11. Ferguson PJ, Lokuta MA, El-Shanti HI et al (2008) Neutrophil dysfunction in a family with a SAPHO syndrome-like phenotype. Arthritis Rheum 58:3264–3269

    Article  CAS  PubMed  Google Scholar 

  12. Dahlgren C, Karlsson A (2002) Ionomycin-induced neutrophil NADPH oxidase activity is selectively inhibited by the serine protease inhibitor diisopropyl fluorophosphate. Antioxid Redox Signal 4:17–25

    Article  CAS  PubMed  Google Scholar 

  13. Karlsson A, Follin P, Leffler H et al (1998) Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils. Blood 91:3430–3438

    CAS  PubMed  Google Scholar 

  14. Karlsson A, Nixon JB, McPhail LC (2000) Phorbol myristate acetate induces neutrophil NADPH-oxidase activity by two separate signal transduction pathways: dependent or independent of phosphatidylinositol 3-kinase. J Leukoc Biol 67:396–404

    CAS  PubMed  Google Scholar 

  15. Lock R, Dahlgren C, Linden M et al (1990) Neutrophil killing of two type 1 fimbria-bearing Escherichia coli strains: dependence on respiratory burst activation. Infect Immun 58:37–42

    CAS  PubMed Central  PubMed  Google Scholar 

  16. Serrander L, Larsson J, Lundqvist H et al (1999) Particles binding beta(2)-integrins mediate intracellular production of oxidative metabolites in human neutrophils independently of phagocytosis. Biochim Biophys Acta 1452:133–144

    Article  CAS  PubMed  Google Scholar 

  17. Brown GE, Stewart MQ, Liu H et al (2003) A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC delta in intracellular production of reactive oxygen species by the NADPH oxidase. Mol Cell 11:35–47

    Article  CAS  PubMed  Google Scholar 

  18. Kent JD, Sergeant S, Burns DJ et al (1996) Identification and regulation of protein kinase C-delta in human neutrophils. J Immunol 157:4641–4647

    CAS  PubMed  Google Scholar 

  19. Sergeant S, McPhail LC (1997) Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils. J Immunol 159:2877–2885

    CAS  PubMed  Google Scholar 

  20. Murphy MP, Holmgren A, Larsson NG et al (2011) Unraveling the biological roles of reactive oxygen species. Cell Metab 13:361–366

    Article  CAS  PubMed  Google Scholar 

  21. Hancock JT, Jones OT (1987) The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages. Biochem J 242:103–107

    CAS  PubMed  Google Scholar 

  22. Hampton MB, Kettle AJ, Winterbourn CC (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92:3007–3017

    CAS  PubMed  Google Scholar 

  23. Kobayashi T, Robinson JM, Seguchi H (1998) Identification of intracellular sites of superoxide production in stimulated neutrophils. J Cell Sci 111:81–91

    CAS  PubMed  Google Scholar 

  24. Dahlgren C, Karlsson A (1999) Respiratory burst in human neutrophils. J Immunol Methods 232:3–14

    Article  CAS  PubMed  Google Scholar 

  25. Lundqvist H, Dahlgren C (1996) Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils. Free Radic Biol Med 20:785–792

    Article  CAS  PubMed  Google Scholar 

  26. Halliwell B, Gutteridge JM (1985) The importance of free radicals and catalytic metal ions in human diseases. Mol Aspects Med 8:89–193

    Article  CAS  PubMed  Google Scholar 

  27. Root RK, Metcalf J, Oshino N et al (1975) H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J Clin Invest 55:945–955

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Root RK, Metcalf JA (1977) H2O2 release from human granulocytes during phagocytosis. Relationship to superoxide anion formation and cellular catabolism of H2O2: studies with normal and cytochalasin B-treated cells. J Clin Invest 60:1266–1279

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. van Pelt LJ, van Zwieten R, Weening RS et al (1996) Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst. J Immunol Methods 191:187–196

    Article  PubMed  Google Scholar 

  30. Wardman P (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 43:995–1022

    Article  CAS  PubMed  Google Scholar 

  31. Beckman JS, Koppenol WH (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 271:C1424–C1437

    CAS  PubMed  Google Scholar 

  32. Klebanoff SJ (2005) Myeloperoxidase: friend and foe. J Leukoc Biol 77:598–625

    Article  CAS  PubMed  Google Scholar 

  33. Holland PC, Sherratt HS (1972) Biochemical effects of the hypoglycaemic compound diphenyleneiodonnium. Catalysis of anion-hydroxyl ion exchange across the inner membrane of rat liver mitochondria and effects on oxygen uptake. Biochem J 129:39–54

    CAS  PubMed  Google Scholar 

  34. Lundqvist H, Follin P, Khalfan L et al (1996) Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told. J Leukoc Biol 59:270–279

    CAS  PubMed  Google Scholar 

  35. Dahlgren C, Stendahl O (1983) Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes. Infect Immun 39:736–741

    CAS  PubMed Central  PubMed  Google Scholar 

  36. Björnsdottir H, Granfeldt D, Welin A et al (2013) Inhibition of phospholipase A(2) abrogates intracellular processing of NADPH-oxidase derived reactive oxygen species in human neutrophils. Exp Cell Res 319:761–774

    Article  PubMed  Google Scholar 

  37. Imada I, Sato EF, Miyamoto M et al (1999) Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012. Anal Biochem 271:53–58

    Article  CAS  PubMed  Google Scholar 

  38. Nishinaka Y, Aramaki Y, Yoshida H et al (1993) A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells. Biochem Biophys Res Commun 193:554–559

    Article  CAS  PubMed  Google Scholar 

  39. Kielland A, Blom T, Nandakumar KS et al (2009) In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012. Free Radic Biol Med 47:760–766

    Article  CAS  PubMed  Google Scholar 

  40. Almkvist J, Dahlgren C, Leffler H et al (2002) Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1. J Immunol 168:4034–4041

    CAS  PubMed  Google Scholar 

  41. Fu H, Belaaouaj AA, Dahlgren C et al (2003) Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides. Microbes Infect 5:781–788

    Article  CAS  PubMed  Google Scholar 

  42. Fu H, Karlsson J, Bylund J et al (2006) Ligand recognition and activation of formyl peptide receptors in neutrophils. J Leukoc Biol 79:247–256

    Article  CAS  PubMed  Google Scholar 

  43. Karlsson J, Fu H, Boulay F et al (2005) Neutrophil NADPH-oxidase activation by an annexin AI peptide is transduced by the formyl peptide receptor (FPR), whereas an inhibitory signal is generated independently of the FPR family receptors. J Leukoc Biol 78:762–771

    Article  CAS  PubMed  Google Scholar 

  44. Thoren F, Romero A, Lindh M et al (2004) A hepatitis C virus-encoded, nonstructural protein (NS3) triggers dysfunction and apoptosis in lymphocytes: role of NADPH oxidase-derived oxygen radicals. J Leukoc Biol 76:1180–1186

    Article  CAS  PubMed  Google Scholar 

  45. Foyouzi-Youssefi R, Petersson F, Lew DP et al (1997) Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal. Biochem J 322:709–718

    CAS  PubMed  Google Scholar 

  46. Boveris A, Martino E, Stoppani AO (1977) Evaluation of the horseradish peroxidase-scopoletin method for the measurement of hydrogen peroxide formation in biological systems. Anal Biochem 80:145–158

    Article  CAS  PubMed  Google Scholar 

  47. Faurschou M, Borregaard N (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect 5:1317–1327

    Article  CAS  PubMed  Google Scholar 

  48. Zarember KA, Kuhns DB (2011) Editorial: will the real neutrophil please stand up? J Leukoc Biol 90:1039–1041

    Article  CAS  PubMed  Google Scholar 

  49. Fäldt J, Ridell M, Karlsson A et al (1999) The phagocyte chemiluminescence paradox: luminol can act as an inhibitor of neutrophil NADPH-oxidase activity. Luminescence 14:153–160

    Article  PubMed  Google Scholar 

  50. Leutner S, Schindowski K, Frolich L et al (2005) Enhanced ROS-generation in lymphocytes from Alzheimer’s patients. Pharmacopsychiatry 38:312–315

    Article  CAS  PubMed  Google Scholar 

  51. Mauch L, Lun A, O’Gorman MR et al (2007) Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD. Clin Chem 53:890–896

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Swedish Medical Research Council, the Swedish Society for Medical Research, the IngaBritt and Arne Lundberg Research Foundation, the Swedish state under the LUA-ALF agreement, and the King Gustaf V Memorial Foundation. We thank Maria Hjulström and Hülya Çevik-Aras for performing chemiluminescence determinations with the National diagnostic kit and L-012, respectively.

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Authors and Affiliations

  1. The Phagocyte Research Group, Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden

    Johan Bylund, Halla Björnsdottir, Martina Sundqvist, Anna Karlsson & Claes Dahlgren

Authors
  1. Johan Bylund
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  2. Halla Björnsdottir
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  3. Martina Sundqvist
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  4. Anna Karlsson
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  5. Claes Dahlgren
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Editor information

Editors and Affiliations

  1. Montana State University Dept. Veterinary Molecular Biology, Bozeman, Montana, USA

    Mark T. Quinn

  2. Lab. Human Bacterial Pathogenesis, National Institutes of Health Nat. Inst. Allergy & Infectious Diseases, Hamilton, Montana, USA

    Frank R. DeLeo

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Bylund, J., Björnsdottir, H., Sundqvist, M., Karlsson, A., Dahlgren, C. (2014). Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes. In: Quinn, M., DeLeo, F. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology, vol 1124. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-845-4_21

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  • DOI: https://doi.org/10.1007/978-1-62703-845-4_21

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-844-7

  • Online ISBN: 978-1-62703-845-4

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