Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1977 Dec;60(6):1266–1279. doi: 10.1172/JCI108886

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

Richard K Root 1, Julia A Metcalf 1
PMCID: PMC372483  PMID: 199619

Abstract

Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosis-induced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H2O2 released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H2O2 in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H2O2-dependent protein iodination and more than doubled H2O2 release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H2O2 catabolism.

When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H2O2 production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H2O2 catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H2O2-dependent reaction, protein iodination, in both normal and cytochalasin B cells. When oxygen consumption, O2.−, and H2O2 release were compared in the presence of azide under identical incubation conditions, the molar relationships for normal cells were 1.00:0.34:0.51 and for cytochalasin B-treated cells 1.00:0.99:0.40, respectively. Nonopsonized, or opsonized but disrupted, bacteria did not stimulate any of these metabolic functions.

The results indicate that with normal cells approximately 50% of H2O2 released during phagocytosis is derived directly from O2.− by dismutation, the remainder appearing from an (intra)cellular source shared by azide-inhibitable heme enzymes. With cytochalasin B treatment the evidence is consistent with the derivation of all H2O2 from an O2.− precursor which is released from the cell surface. Furthermore, when activated by phagocytic particle binding, the neutrophil O2.− generating system appears to make more of this compound than can be accounted for by dismutation to H2O2. This establishes conditions for the direct participation of both compounds in the microbicidal and cytocidal activity of these cells.

Full text

PDF
1266

Images in this article

1272Image
on p.1272

Selected References

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

  1. 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. 1975 Feb;85(2):235–244. [PubMed] [Google Scholar]
  2. Babior B. M., Curnutte J. T., McMurrich B. J. The particulate superoxide-forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest. 1976 Oct;58(4):989–996. doi: 10.1172/JCI108553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babior B. M., Kipnes R. S., Curnutte J. T. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973 Mar;52(3):741–744. doi: 10.1172/JCI107236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baehner R. L., Gilman N., Karnovsky M. L. Respiration and glucose oxidation in human and guinea pig leukocytes: comparative studies. J Clin Invest. 1970 Apr;49(4):692–700. doi: 10.1172/JCI106281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baehner R. L., Murrmann S. K., Davis J., Johnston R. B., Jr The role of superoxide anion and hydrogen peroxide in phagocytosis-associated oxidative metabolic reactions. J Clin Invest. 1975 Sep;56(3):571–576. doi: 10.1172/JCI108126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Briggs R. T., Drath D. B., Karnovsky M. L., Karnovsky M. J. Localization of NADH oxidase on the surface of human polymorphonuclear leukocytes by a new cytochemical method. J Cell Biol. 1975 Dec;67(3):566–586. doi: 10.1083/jcb.67.3.566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. CHANCE B. Oxidase and peroxidase reactions in the presence of dihydroxymaleic acid. J Biol Chem. 1952 May;197(2):577–589. [PubMed] [Google Scholar]
  8. Curnutte J. T., Babior B. M. Effects of anaerobiosis and inhibitors on O2-production by human granulocytes. Blood. 1975 Jun;45(6):851–861. [PubMed] [Google Scholar]
  9. DeChatelet L. R., McPhail L. C., Mullikin D., McCall C. E. Reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate diaphorase activity in human polymorphonuclear leukocytes. Infect Immun. 1974 Sep;10(3):528–534. doi: 10.1128/iai.10.3.528-534.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ehlenberger A. G., Nussenzweig V. The role of membrane receptors for C3b and C3d in phagocytosis. J Exp Med. 1977 Feb 1;145(2):357–371. doi: 10.1084/jem.145.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldstein I. M., Cerqueira M., Lind S., Kaplan H. B. Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J Clin Invest. 1977 Feb;59(2):249–254. doi: 10.1172/JCI108635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein I. M., Roos D., Kaplan H. B., Weissmann G. Complement and immunoglobulins stimulate superoxide production by human leukocytes independently of phagocytosis. J Clin Invest. 1975 Nov;56(5):1155–1163. doi: 10.1172/JCI108191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gregory E. M., Yost F. J., Jr, Fridovich I. Superoxide dismutases of Escherichia coli: intracellular localization and functions. J Bacteriol. 1973 Sep;115(3):987–991. doi: 10.1128/jb.115.3.987-991.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hodgson E. K., Fridovich I. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry. 1975 Dec 2;14(24):5294–5299. doi: 10.1021/bi00695a010. [DOI] [PubMed] [Google Scholar]
  15. Hohn D. C., Lehrer R. I. NADPH oxidase deficiency in X-linked chronic granulomatous disease. J Clin Invest. 1975 Apr;55(4):707–713. doi: 10.1172/JCI107980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnston R. B., Jr, Keele B. B., Jr, Misra H. P., Lehmeyer J. E., Webb L. S., Baehner R. L., RaJagopalan K. V. The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes. J Clin Invest. 1975 Jun;55(6):1357–1372. doi: 10.1172/JCI108055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. KEILIN D., HARTREE E. F. Purification of horse-radish peroxidase and comparison of its properties with those of catalase and methaemoglobin. Biochem J. 1951 Jun;49(1):88–104. doi: 10.1042/bj0490088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Klebanoff S. J. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin Hematol. 1975 Apr;12(2):117–142. [PubMed] [Google Scholar]
  19. Klebanoff S. J., Hamon C. B. Role of myeloperoxidase-mediated antimicrobial systems in intact leukocytes. J Reticuloendothel Soc. 1972 Aug;12(2):170–196. [PubMed] [Google Scholar]
  20. Klebanoff S. J., Pincus S. H. Hydrogen peroxide utilization in myeloperoxidase-deficient leukocytes: a possible microbicidal control mechanism. J Clin Invest. 1971 Oct;50(10):2226–2229. doi: 10.1172/JCI106718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Klebanoff S. J. Role of the superoxide anion in the myeloperoxidase-mediated antimicrobial system. J Biol Chem. 1974 Jun 25;249(12):3724–3728. [PubMed] [Google Scholar]
  22. Krinsky N. I. Singlet excited oxygen as a mediator of the antibacterial action of leukocytes. Science. 1974 Oct 25;186(4161):363–365. doi: 10.1126/science.186.4161.363. [DOI] [PubMed] [Google Scholar]
  23. Malawista S. E., Gee J. B., Bensch K. G. Cytochalasin B reversibly inhibits phagocytosis: functional, metabolic, and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages. Yale J Biol Med. 1971 Dec;44(3):286–300. [PMC free article] [PubMed] [Google Scholar]
  24. Mandell G. L. Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal--leukocyte interaction. J Clin Invest. 1975 Mar;55(3):561–566. doi: 10.1172/JCI107963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. McCord J. M., Keele B. B., Jr, Fridovich I. An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc Natl Acad Sci U S A. 1971 May;68(5):1024–1027. doi: 10.1073/pnas.68.5.1024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McPhail L. C., DeChatelet L. R., Shirley P. S. Further characterization of NADPH oxidase activity of human polymorphonuclear leukocytes. J Clin Invest. 1976 Oct;58(4):774–780. doi: 10.1172/JCI108528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Messner R. P., Jelinek J. Receptors for human gamma G globulin on human neutrophils. J Clin Invest. 1970 Dec;49(12):2165–2171. doi: 10.1172/JCI106435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Paul B., Sbarra A. J. The role of the phagocyte in host-parasite interactions. 13. The direct quantitative estimation of H2O2 in phagocytizing cells. Biochim Biophys Acta. 1968 Feb 1;156(1):168–178. doi: 10.1016/0304-4165(68)90116-5. [DOI] [PubMed] [Google Scholar]
  29. Peterson P. K., Verhoef J., Sabath L. D., Quie P. G. Effect of protein A on staphylococcal opsonization. Infect Immun. 1977 Mar;15(3):760–764. doi: 10.1128/iai.15.3.760-764.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pincus S. H., Klebanoff S. J. Quantitative leukocyte iodination. N Engl J Med. 1971 Apr 8;284(14):744–750. doi: 10.1056/NEJM197104082841402. [DOI] [PubMed] [Google Scholar]
  31. Reed P. W. Glutathione and the hexose monophosphate shunt in phagocytizing and hydrogen peroxide-treated rat leukocytes. J Biol Chem. 1969 May 10;244(9):2459–2464. [PubMed] [Google Scholar]
  32. Roos D., Homan-Müller J. W., Weening R. S. Effect of cytochalasin B on the oxidative metabolism of human peripheral blood granulocytes. Biochem Biophys Res Commun. 1976 Jan 12;68(1):43–50. doi: 10.1016/0006-291x(76)90007-3. [DOI] [PubMed] [Google Scholar]
  33. Root R. K., Metcalf J., Oshino N., Chance B. H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors. J Clin Invest. 1975 May;55(5):945–955. doi: 10.1172/JCI108024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Root R. K., Stossel T. P. Myeloperoxidase-mediated iodination by granulocytes. Intracellular site of operation and some regulating factors. J Clin Invest. 1974 May;53(5):1207–1215. doi: 10.1172/JCI107667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rossi F., Romeo D., Patriarca P. Mechanism of phagocytosis-associated oxidative metabolism in polymorphonuclear leucocytes and macrophages. J Reticuloendothel Soc. 1972 Aug;12(2):127–149. [PubMed] [Google Scholar]
  36. Salin M. L., McCord J. M. Free radicals and inflammation. Protection of phagocytosine leukocytes by superoxide dismutase. J Clin Invest. 1975 Nov;56(5):1319–1323. doi: 10.1172/JCI108208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Segal A. W., Peters T. J. Characterisation of the enzyme defect in chronic granulomatous disease. Lancet. 1976 Jun 26;1(7974):1363–1365. doi: 10.1016/s0140-6736(76)93021-x. [DOI] [PubMed] [Google Scholar]
  38. Weening R. S., Wever R., Roos D. Quantitative aspects of the production of superoxide radicals by phagocytizing human granulocytes. J Lab Clin Med. 1975 Feb;85(2):245–252. [PubMed] [Google Scholar]
  39. Zigmond S. H., Hirsch J. G. Effects of cytochalasin B on polymorphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp Cell Res. 1972 Aug;73(2):383–393. doi: 10.1016/0014-4827(72)90062-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES