Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1975 May;55(5):945–955. doi: 10.1172/JCI108024

H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors.

R K Root, J Metcalf, N Oshino, B Chance
PMCID: PMC301840  PMID: 1123431

Abstract

The extinction of fluorescence of scopoletin during its oxidation by horseradish peroxidase (HPO) provides a highly sensitive and specific assay for small quantities of peroxide in solution. With this assay, the release of free H2O2 into the extracellular medium by phagocytizing human granulocytes has been documented and quantitated, and some of the regulating factors have been determined. Under basal conditions granulocytes released less than 0.01 nmol/ml of H2O2 (2.5 X 10-6 polymorphonuclear leukocytes/ml). Upon the addition of phagocyte particles (latex, opsonized yeast, or staphylococci), an abrupt increase in extracellular peroxide concentration was observed (greater than 50-fold above basal levels) after latencies as short as 10 s. Release reflected increased intracellular H2O2 production during phagocytosis in that it paralleled the respiratory burst and was absent when phagocytosis was prevented or when cells from patients with chronic granulomatous disease were utilized. Evidence that scpoletin oxidation occurred predominantly in the extracellular medium was obtained by demonstrating a marked inhibition when HPO was omitted from the reaction mixture or when exogenous catalase was added. Similarly, it was found that exogenous serum also inhibited scopoletin oxidation, apparently because of the presence of competing hydrogen donors. H2O2 formation and release were observed at rates which closely paralleled those of phagocytosis. With O2 consumption as an approximate index of H2O2 formation, the fractions released during maximal rates of particle uptake were calculated as follows: for latex, 15.7%; for staphylococci, 10.3%; and for yeast, 4.9%. It is postulated that release is due to diffusion of free H2O2 from an expanded intracellular pool of this substance that develops during phagocytosis. This poos represents tha net of increased synthesis versus catabolism by various enxymatic pathways for H2O2 disposal within the cells. The close relationship between rates of H2O2 formation and rates of phagocytosis by human granulocytes suggests a role for specialized areas of the cell membrane, involved in particle ingestion, in the trigger mechanism for H2O2 synthesis. The consequences of H2O2 release to other cells or organisms in the immediate environment of phagocytizing granulocytes remain to be determined.

Full text

PDF
945

Selected References

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

  1. ANDREAE W. A. A sensitive method for the estimation of hydrogen peroxide in biological materials. Nature. 1955 May 14;175(4463):859–860. doi: 10.1038/175859a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Baehner R. L., Nathan D. G., Castle W. B. Oxidant injury of caucasian glucose-6-phosphate dehydrogenase-deficient red blood cells by phagocytosing leukocytes during infection. J Clin Invest. 1971 Dec;50(12):2466–2473. doi: 10.1172/JCI106747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang Y. H. Studies on phagocytosis. I. Uptake of radio-iodinated (131-I) human serum albumin as a measure of the degree of phagocytosis in vitro. Exp Cell Res. 1969 Jan;54(1):42–48. doi: 10.1016/0014-4827(69)90290-0. [DOI] [PubMed] [Google Scholar]
  6. Goetzl E. J., Austen K. F. Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors. J Clin Invest. 1974 Feb;53(2):591–599. doi: 10.1172/JCI107594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Graham R. C., Jr, Karnovsky M. J., Shafer A. W., Glass E. A., Karnovsky M. L. Metabolic and morphological observations on the effect of surface-active agents of leukocytes. J Cell Biol. 1967 Mar;32(3):629–647. doi: 10.1083/jcb.32.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harris M. B., Djerassi I., Schwartz E., Root R. K. Polymorphonuclear leukocytes prepared by continuous-flow filtration leukapheresis: viability and function. Blood. 1974 Nov;44(5):707–713. [PubMed] [Google Scholar]
  9. Karnovsky M. L. Chronic granulomatous disease--pieces of a cellular and molecular puzzle. Fed Proc. 1973 Apr;32(4):1527–1533. [PubMed] [Google Scholar]
  10. 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]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Lay W. H., Nussenzweig V. Receptors for complement of leukocytes. J Exp Med. 1968 Nov 1;128(5):991–1009. doi: 10.1084/jem.128.5.991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Loschen G., Flohé L., Chance B. Respiratory chain linked H(2)O(2) production in pigeon heart mitochondria. FEBS Lett. 1971 Nov 1;18(2):261–264. doi: 10.1016/0014-5793(71)80459-3. [DOI] [PubMed] [Google Scholar]
  14. MAEHLY A. C., CHANCE B. The assay of catalases and peroxidases. Methods Biochem Anal. 1954;1:357–424. doi: 10.1002/9780470110171.ch14. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Oshino N., Oshino R., Chance B. The characteristics of the "peroxidatic" reaction of catalase in ethanol oxidation. Biochem J. 1973 Mar;131(3):555–563. doi: 10.1042/bj1310555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Root R. K., Rosenthal A. S., Balestra D. J. Abnormal bactericidal, metabolic, and lysosomal functions of Chediak-Higashi Syndrome leukocytes. J Clin Invest. 1972 Mar;51(3):649–665. doi: 10.1172/JCI106854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Sbarra A. J., Paul B. B., Jacobs A. A., Strauss R. R., Mitchell G. W., Jr Biochemical aspects of phagocytic cells as related to bactericidal function. J Reticuloendothel Soc. 1972 May;11(5):492–502. [PubMed] [Google Scholar]
  22. Stossel T. P. Phagocytosis (third of three parts). N Engl J Med. 1974 Apr 11;290(15):833–839. doi: 10.1056/NEJM197404112901506. [DOI] [PubMed] [Google Scholar]
  23. Stossel T. P., Pollard T. D., Mason R. J., Vaughan M. Isolation and properties of phagocytic vesicles from polymorphonuclear leukocytes. J Clin Invest. 1971 Aug;50(8):1745–1747. doi: 10.1172/JCI106664. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES