Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1977 Dec 1;146(6):1648–1662. doi: 10.1084/jem.146.6.1648

Hydrogen peroxide release from mouse peritoneal macrophages: dependence on sequential activation and triggering

PMCID: PMC2181906  PMID: 925614

Abstract

Using a specific and sensitive fluorometric assay, the H2O2 release from as few as 2 X 10(5) mouse peritoneal macrophages could be detected continuously and quantitated. It is emphasized that the assay measured H2O2 release, not production. Induction of H2O2 release required sequential application of two stimuli: the administration of an activating agent to the mice from 4 days to 10 wk before all harvest, and the exposure of the cells in vitro to a triggering agent. BCG was most effective as an activating agent, resulting in peritoneal macrophages which could be triggered to release H2O2 almost as copiously (8 nmol/10(6) macrophages per 5 min) as mouse peritoneal PMN (9 NMOL/10(6) PMN per 5 min). Casein and C. parvum could also serve as activators, but thioglycollate and FCS were ineffective after single injections. PMA was a potent triggering agent, resulting in a maximal rate of H2O2 release after a latency of about 40 s for cells in suspension. Other triggering agents included the ionophore A23187, concanavalin A in the presence of cytochalasin B, and phagocytosis. H2O2 release could be attributed to macrophages and PMN in peritoneal cell suspensions or in preparations of adherent peritoneal cells, but not to lymphocytes. Indirect evidence suggested that the H2O2 detected was formed from superoxide anion. These observations appear to justify renewed interest in the idea that H2O2 may be important in macrohpage antimicrobial and antitumor mechanisms.

Full Text

The Full Text of this article is available as a PDF (1,022.2 KB).

Selected References

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

  1. Allen J. M., Cook G. M., Poole A. R. Action of concanavalin A on the attachment stage of phagocytosis by macrophages. Exp Cell Res. 1971 Oct;68(2):466–467. doi: 10.1016/0014-4827(71)90178-9. [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., Johnston R. B., Jr Monocyte function in children with neutropenia and chronic infections. Blood. 1972 Jul;40(1):31–41. [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. Bianco C., Griffin F. M., Jr, Silverstein S. C. Studies of the macrophage complement receptor. Alteration of receptor function upon macrophage activation. J Exp Med. 1975 Jun 1;141(6):1278–1290. doi: 10.1084/jem.141.6.1278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark R. A., Klebanoff S. J. Neutrophil-mediated tumor cell cytotoxicity: role of the peroxidase system. J Exp Med. 1975 Jun 1;141(6):1442–1447. doi: 10.1084/jem.141.6.1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Drath D. B., Karnovsky M. L. Superoxide production by phagocytic leukocytes. J Exp Med. 1975 Jan 1;141(1):257–262. doi: 10.1084/jem.141.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Edelson P. J., Zwiebel R., Cohn Z. A. The pinocytic rate of activated macrophages. J Exp Med. 1975 Nov 1;142(5):1150–1164. doi: 10.1084/jem.142.5.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gee J. B., Vassallo C. L., Bell P., Kaskin J., Basford R. E., Field J. B. Catalase-dependent peroxidative metabolism in the alveolar macrophage during phagocytosis. J Clin Invest. 1970 Jun;49(6):1280–1287. doi: 10.1172/JCI106340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Germain R. N., Williams R. M., Benacerraf B. Specific and nonspecific antitumor immunity. II. Macrophage-mediated nonspecific effector activity induced by BCG and similar agents. J Natl Cancer Inst. 1975 Mar;54(3):709–720. [PubMed] [Google Scholar]
  12. Gordon S., Unkeless J. C., Cohn Z. A. Induction of macrophage plasminogen activator by endotoxin stimulation and phagocytosis: evidence for a two-stage process. J Exp Med. 1974 Oct 1;140(4):995–1010. doi: 10.1084/jem.140.4.995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Klebanoff S. J. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin Hematol. 1975 Apr;12(2):117–142. [PubMed] [Google Scholar]
  15. McRipley R. J., Sbarra A. J. Role of the phagocyte in host-parasite interactions. XI. Relationship between stimulated oxidative metabolism and hydrogen peroxide formation, and intracellular killing. J Bacteriol. 1967 Nov;94(5):1417–1424. doi: 10.1128/jb.94.5.1417-1424.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Meltzer M. S., Oppenheim J. J. Bidirectional amplification of macrophage-lymphocyte interactions: enhanced lymphocyte activation factor production by activated adherent mouse peritoneal cells. J Immunol. 1977 Jan;118(1):77–82. [PubMed] [Google Scholar]
  17. Michl J., Ohlbaum D. J., Silverstein S. C. 2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages II. Dissociation of the inhibitory effects of 2-deoxyglucose on phagocytosis and ATP generation. J Exp Med. 1976 Dec 1;144(6):1484–1493. doi: 10.1084/jem.144.6.1484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morahan P. S., Glasgow L. A., Crane J. L., Jr, Kern E. R. Comparison of antiviral and antitumor activity of activated macrophages. Cell Immunol. 1977 Feb;28(2):404–415. doi: 10.1016/0008-8749(77)90122-8. [DOI] [PubMed] [Google Scholar]
  19. Nathan C. F., Asofsky R., Terry W. D. Characterization of the nonphagocytic adherent cell from the peritoneal cavity of normal and BCG-treated mice. J Immunol. 1977 May;118(5):1612–1621. [PubMed] [Google Scholar]
  20. Nathan C. F., Karnovsky M. L., David J. R. Alterations of macrophage functions by mediators from lymphocytes. J Exp Med. 1971 Jun 1;133(6):1356–1376. doi: 10.1084/jem.133.6.1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nathan C. F., Terry W. D. Decreased phagocytosis by peritoneal macrophages from BCG-treated mice: induction of the phagocytic defect in normal macrophages with BCG in vitro. Cell Immunol. 1977 Mar 15;29(2):295–311. doi: 10.1016/0008-8749(77)90324-0. [DOI] [PubMed] [Google Scholar]
  22. Nathan C. F., Terry W. D. Differential stimulation of murine lymphoma growth in vitro by normal and BCG-activated macrophages. J Exp Med. 1975 Oct 1;142(4):887–902. doi: 10.1084/jem.142.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. OREN R., FARNHAM A. E., SAITO K., MILOFSKY E., KARNOVSKY M. L. Metabolic patterns in three types of phagocytizing cells. J Cell Biol. 1963 Jun;17:487–501. doi: 10.1083/jcb.17.3.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Olivotto M., Bomford R. In vitro inhibition of tumour cell growth and DNA synthesis by peritoneal and lung macrophages from mice injected with Corynebacterium parvum. Int J Cancer. 1974 Apr 15;13(4):478–488. doi: 10.1002/ijc.2910130406. [DOI] [PubMed] [Google Scholar]
  25. Paul B. B., Strauss R. R., Jacobs A. A., Sbarra A. J. Function of h(2)o(2), myeloperoxidase, and hexose monophosphate shunt enzymes in phagocytizing cells from different species. Infect Immun. 1970 Apr;1(4):338–344. doi: 10.1128/iai.1.4.338-344.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rabinovitch M., DeStefano M. J. Use of the local anesthetic lidocaine for cell harvesting and subcultivation. In Vitro. 1975 Nov-Dec;11(6):379–381. doi: 10.1007/BF02616374. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Romeo D., Zabucchi G., Rossi F. Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by concanavalin A. Nat New Biol. 1973 May 23;243(125):111–112. [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Stubbs M., Kühner A. V., Glass E. A., David J. R., Karnovsky M. L. Metabolic and functonal studies on activated mouse macrophages. J Exp Med. 1973 Feb 1;137(2):537–542. doi: 10.1084/jem.137.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Unkeless J. C., Gordon S., Reich E. Secretion of plasminogen activator by stimulated macrophages. J Exp Med. 1974 Apr 1;139(4):834–850. doi: 10.1084/jem.139.4.834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Werb Z., Gordon S. Elastase secretion by stimulated macrophages. Characterization and regulation. J Exp Med. 1975 Aug 1;142(2):361–377. doi: 10.1084/jem.142.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Werb Z., Gordon S. Secretion of a specific collagenase by stimulated macrophages. J Exp Med. 1975 Aug 1;142(2):346–360. doi: 10.1084/jem.142.2.346. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES