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Killing activity of neutrophils is mediated through activation of proteases by K+ flux

Nature volume 416, pages 291–297 (2002)Cite this article

Abstract

According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.

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Figure 1: Susceptibility of protease-deficient mice to bacterial and fungal infection.
Figure 4: Vacuolar swelling and its control.
Figure 2: Appearance, concentration and buffering capacity of granule contents in the phagocytic vacuole.
Figure 3: K+ and 86Rb transport associated with the generation of O-2 in neutrophils.
Figure 5: Activation of the microbicidal proteases requires K+ and neutral pH.

References

  1. Metchnikoff, B. Immunity in Infective Diseases (Cambridge Univ. Press, 1905).

    Google Scholar 

  2. Sbarra, A. J. & Karnovsky, M. L. The biochemical basis of phagocytosis. 1. Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes. J. Biol. Chem. 234, 1355–1362 (1959).

    CAS  PubMed  Google Scholar 

  3. Mandell, G. L. Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect. Immun. 9, 337–341 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Babior, B. M., Curnutte, J. T. & Kipnes, R. S. Biological defense mechanisms. Evidence for the participation of superoxide in bacterial killing by xanthine oxidase. J. Lab. Clin. Med. 85, 235–244 (1975).

    CAS  PubMed  Google Scholar 

  5. Babior, B. M., Kipnes, R. S. & Curnutte, J. T. Biological defence mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52, 741–744 (1973).

    Article  CAS  Google Scholar 

  6. Thrasher, A. J., Keep, N. H., Wientjes, F. & Segal, A. W. Chronic granulomatous disease. Biochim. Biophys. Acta 1227, 1–24 (1994).

    Article  CAS  Google Scholar 

  7. Klebanoff, S. J. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12, 117–142 (1975).

    CAS  PubMed  Google Scholar 

  8. Belaaouaj, A. et al. Mice lacking neutrophil elastase reveal impaired host defense against Gram-negative bacterial sepsis. Nature Med. 4, 615–618 (1998).

    Article  CAS  Google Scholar 

  9. Tkalcevic, J. et al. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12, 201–210 (2000).

    Article  CAS  Google Scholar 

  10. Hampton, M. B., Kettle, A. J. & Winterbourn, C. C. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils. Infect. Immun. 64, 3512–3517 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Aratani, Y., Koyama, H., Nyui, S., Suzuki, K., Kura, F. & Maeda, N. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. Infect. Immun. 67, 1828–1836 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Cross, A. R. & Jones, O. T. The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase. Biochem. J. 237, 111–116 (1986).

    Article  CAS  Google Scholar 

  13. Segal, A. W., Geisow, M., Garcia, R., Harper, A. & Miller, R. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH. Nature 290, 406–409 (1981).

    Article  ADS  CAS  Google Scholar 

  14. Segal, A. W. & Coade, S. B. Kinetics of oxygen consumption by phagocytosing human neutrophils. Biochem. Biophys. Res. Commun. 84, 611–617 (1978).

    Article  CAS  Google Scholar 

  15. Hampton, M. B., Kettle, A. J. & Winterbourn, C. C. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, 3007–3017 (1998).

    CAS  PubMed  Google Scholar 

  16. Bu-Ghanim, H. N., Segal, A. W., Keep, N. H. & Casimir, C. M. Molecular analysis in three cases of X91-variant chronic granulomatous disease. Blood 86, 3575–3582 (1995).

    CAS  PubMed  Google Scholar 

  17. Jiang, Q., Griffin, D. A., Barofsky, D. F. & Hurst, J. K. Intraphagosomal chlorination dynamics and yields determined using unique fluorescent bacterial mimics. Chem. Res. Toxicol. 10, 1080–1089 (1997).

    Article  CAS  Google Scholar 

  18. Styrt, B. & Klempner, M. S. Internal pH of human neutrophil lysosomes. FEBS Lett. 149, 113–116 (1982).

    Article  CAS  Google Scholar 

  19. Henderson, L. M., Chappell, J. B. & Jones, O. T. The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel. Biochem. J. 246, 325–329 (1987).

    Article  CAS  Google Scholar 

  20. Nanda, A. & Grinstein, S. Protein kinase C activates an H+ (equivalent) conductance in the plasma membrane of human neutrophils. Proc. Natl Acad. Sci. USA 88, 10816–10820 (1991).

    Article  ADS  CAS  Google Scholar 

  21. DeCoursey, T. E., Cherny, V. V., Zhou, W. & Thomas, L. L. Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils. Proc. Natl Acad. Sci. USA 97, 6885–6889 (2000).

    Article  ADS  CAS  Google Scholar 

  22. Menegazzi, R., Busetto, S., Dri, P., Cramer, R. & Patriarca, P. Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-α (TNF) on biologic surfaces. J. Cell Biol. 135, 511–522 (1996).

    Article  CAS  Google Scholar 

  23. Clapp, L. H. & Tinker, A. Potassium channels in the vasculature. Curr. Opin. Nephrol. Hypertens. 7, 91–98 (1998).

    CAS  PubMed  Google Scholar 

  24. Love, W. D. & Burch, G. E. A comparison of potassium42, rubidium86, and cesium134 as traces of potassium in the study of cation metabolism of human erythrocytes in vitro. J. Lab. Clin. Med. 41, 351–362 (1953).

    CAS  PubMed  Google Scholar 

  25. Segal, A. W. & Meshulam, T. Production of superoxide by neutrophils: a reappraisal. FEBS Lett. 100, 27–32 (1979).

    Article  CAS  Google Scholar 

  26. Ince, C. et al. Intracellular K+, Na+ and Cl- concentrations and membrane potential in human monocytes. Biochim. Biophys. Acta 905, 195–204 (1987).

    Article  CAS  Google Scholar 

  27. Potma, E., de Boeij, W. P., van Haastert, P. J. & Wiersma, D. A. Real-time visualization hydrodynamics in single living cells. Proc. Natl Acad. Sci. USA 98, 1577–1582 (2001).

    Article  ADS  CAS  Google Scholar 

  28. Aderem, A. & Underhill, D. M. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17, 593–623 (1999).

    Article  CAS  Google Scholar 

  29. Rizoli, S. B., Rotstein, O. D., Parodo, J., Phillips, M. J. & Kapus, A. Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling. Am. J. Physiol. Cell Physiol. 279, C619–C633 (2000).

    Article  CAS  Google Scholar 

  30. Kolset, S. O. & Gallagher, J. T. Proteoglycans in haemopoietic cells. Biochim. Biophys. Acta 1032, 191–211 (1990).

    CAS  PubMed  Google Scholar 

  31. Odeberg, H. & Olsson, I. Antibacterial activity of cationic proteins from human granulocytes. J. Clin. Invest. 56, 1118–1124 (1975).

    Article  CAS  Google Scholar 

  32. Winterbourn, C. C., Garcia, R. C. & Segal, A. W. Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride. Biochem. J. 228, 583–592 (1985).

    Article  CAS  Google Scholar 

  33. Kettle, A. J. & Winterbourn, C. C. A kinetic analysis of the catalase activity of myeloperoxidase. Biochemistry 40, 10204–10212 (2001).

    Article  CAS  Google Scholar 

  34. Guerin, I. & de Chastellier, C. Pathogenic mycobacteria disrupt the macrophage actin filament network. Infect. Immun. 68, 2655–2662 (2000).

    Article  CAS  Google Scholar 

  35. Henderson, L. M. & Meech, R. W. Evidence that the product of the human X-linked CGD gene, gp91-phox, is a voltage-gated H+ pathway. J. Gen. Physiol. 114, 771–786 (1999).

    Article  CAS  Google Scholar 

  36. Maturana, A. et al. Heme histidine ligands within gp91phox modulate proton conduction by the phagocyte NADPH oxidase. J. Biol. Chem. 276, 30277–30284 (2001).

    Article  CAS  Google Scholar 

  37. Nanda, A., Romanek, R., Curnutte, J. T. & Grinstein, S. Assessment of the contribution of the cytochrome b moiety of the NADPH oxidase to the transmembrane H+ conductance of leukocytes. J. Biol. Chem. 269, 27280–27285 (1994).

    CAS  PubMed  Google Scholar 

  38. De Coursey, T. E., Cherny, V. V., Morgan, D., Katz, B. Z. & Dinauer, M. C. The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps. J. Biol. Chem. 276, 36063–36066 (2001).

    Article  CAS  Google Scholar 

  39. McCord, J. M. & Wong, K. in Oxygen Free Radicals and Tissue Damage (ed. Fitzsimons, D. W.) 343–360 (Excerpta Medica, Amsterdam, 1979).

    Google Scholar 

  40. Klebanoff, S. J. & Pincus, S. H. Hydrogen peroxide utilization in myeloperoxidase-deficient leukocytes: a possible microbicidal control mechanism. J. Clin. Invest. 50, 2226–2229 (1971).

    Article  CAS  Google Scholar 

  41. Brennan, M. L. et al. Increased atherosclerosis in myeloperoxidase-deficient mice. J. Clin. Invest. 107, 419–430 (2001).

    Article  CAS  Google Scholar 

  42. Klebanoff, S. J. & Clark, R. A. Iodination by human polymorphonuclear leukocytes: a re-evaluation. J. Lab. Clin. Med. 89, 675–686 (1977).

    CAS  PubMed  Google Scholar 

  43. Grogan, A. et al. Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J. Cell Sci. 110, 3071–3081 (1997).

    CAS  PubMed  Google Scholar 

  44. Hall, T. A. & Gupta, B. L. in Principles of Analytical Electron Microscopy (eds Joy, D. C., Romig, A. D. & Goldstein, J. I.) 219–248 (Plenum, London, 1986).

    Book  Google Scholar 

  45. Olsen, R. L. & Little, C. Purification and some properties of myeloperoxidase and eosinophil peroxidase from human blood. Biochem. J. 209, 781–787 (1983).

    Article  CAS  Google Scholar 

  46. Sadir, R., Baleux, F., Grosdidier, A., Imberty, A. & Lortat-Jacob, H. Characterization of the stromal cell-derived factor-1–α-heparin complex. J. Biol. Chem. 276, 8288–8296 (2001).

    Article  CAS  Google Scholar 

  47. Vita, F. et al. Preparation of membrane fractions from human neutrophil granules: A simple method. Methods Cell Sci. 19, 197–205 (1997).

    Article  Google Scholar 

  48. Barrett, A. J. Cathepsin G. Methods Enzymol. 80C, 561–565 (1981).

    Article  Google Scholar 

  49. Amos, B. J., Pocock, G. & Richards, C. D. On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones. Exp. Physiol. 81, 623–632 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank W. Gratzer, P. Rich, F. Ashcroft, M. Brand, R. Vaughan-Jones and M. Duchen for discussions; E. Hawe for statistical analysis; A. Hankin and E. C. Davis for technical assistance; and A. Scott for illustration and art work. The Wellcome Trust and Chronic Granulomatous Disease Research Trust provided financial support.

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Author notes

  1. Emer P. Reeves, Hui Lu and Jürgen Roes: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Molecular Medicine, University College London, 5 University Street, London, WC1E 6JJ, UK

    Emer P. Reeves, Hui Lu, Carlo G. M. Messina & Anthony W. Segal

  2. Centre for Molecular Medicine, Department of Physiology, University College London, 5 University Street, London, WC1E 6JJ, UK

    Steve Bolsover

  3. Centre for Molecular Medicine, Department of Anatomy, University College London, 5 University Street, London, WC1E 6JJ, UK

    Giorgio Gabella

  4. Windeyer Institute of Medical Sciences, University College London, 5 University Street, London, WC1E 6JJ, UK

    Jürgen Roes

  5. Institut de Biologie Structurale, 41 rue Horowitz, Grenoble, 38027, France

    Hugues Lortat Jacobs

  6. Ultrafast Laser and Spectroscopy Laboratory, Materials Science Centre, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, The Netherlands

    Eric O. Potma

  7. Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London, SE1 7EH, UK

    Alice Warley

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  1. Emer P. Reeves
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Correspondence to Anthony W. Segal.

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Reeves, E., Lu, H., Jacobs, H. et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416, 291–297 (2002). https://doi.org/10.1038/416291a

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