Flow Cytometric Analysis of Oxidase Activity of Neutrophils from Chronic Granulomatous Disease Patients

  • Nassef F. Hassan
  • Donald E. Campbell
  • Steven D. Douglas
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 239)


Phagocyte cells, neutrophils and macrophages constitute the main defense mechanisms of the human body against invading microorganisms. The bactericidal activity of polymorphonuclear leukocytes (PMNs) involves the activation of a cell membrane associated NADPH oxidase enzyme“. Following activation of the NADPH oxidase enzyme, a cascade of oxidation/reduction reactions occurs, with the generation of several oxygen radicals. Superoxide (0 2 ) hydrogen peroxide (H202), hydroxyl radical (HO) singlet oxygen (0) are the main oxygen by-products of the respiratory burst of PMNs2 and have an important role in killing of bacteria. The enzyme NADPH oxidase has a major function in the generation of O 2 radical while other enzymes including superoxide dismutase3, myeloperoxidase4, catalase and glutathione peroxidase5 serve a regulatory activity in the respiratory burst. NADPH oxidase differs from other cellular oxidase enzymes (i.e., mitochondrial), since it is a cyanide insensitive enzyme6. The activation of the NADPH oxidase enzyme with different soluble stimulants as phorbol esters7, formyl-methionyl-leucyl-phenylalanine8 and γ-hexachlorocyclohexane9 results in the generation of active oxygen radicals and mimics the biochemical events which occur during phagocytosis in vivo.


NADPH Oxidase Phorbol Myristate Acetate Respiratory Burst Chronic Granulomatous Disease Phorbol Myristate Acetate 
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  1. 1.
    B.M. Babior, J.T. Curnutte, and R.S. Kipnes, Pyridine nucleotide-dependent superoxide production by a cell-free system from human granulocytes, J. Clin. Invest., 56: 1035, 1975.PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    D.P. Clifford and J.E. Repine, Measurement of oxidizing radicals by polymorphonuclear leukocytes, Meth. in Enzymol., 105:393, 1984.Google Scholar
  3. 3.
    I. Fridovich, Superoxide radical and superoxide dismutase, Accts. Chem. Res., 5: 321, 1972.CrossRefGoogle Scholar
  4. 4.
    S.J. Klebanoff and C.B. Ramon, Role of myeloperoxidase-mediated antimicrobial systems in intact leukocytes, J. Retículoendothel. Soc., 12: 170, 1972.PubMedGoogle Scholar
  5. 5.
    P.C. Hinkle, R.A. Butow, E. Racker, and B. Chance, Partial resolution of the enzymes catalyzing oxidative phosphorylation, J. Biol. Chem., 242: 5169, 1967.PubMedGoogle Scholar
  6. 6.
    A.J. Sbarra and M.L. Karnovsky, The biochemical basis of phagocytosis. I: Metabolic changes during the ingestion of particles by polymorphonuclear leukocytes, J. Biol. Chem., 234: 1355, 1959.PubMedGoogle Scholar
  7. 7.
    J.E. Repine, J.G. White, and C.C. Clanson, and B.M. Holmes, The influence of phorbol myristate acetate on oxygen consumption by polymorphonuclear leukocyte, J. Lab. Clin. Med., 83: 911, 1974.Google Scholar
  8. 8.
    C.E. McCall, D.A. Bass, L.R. DeChatelet, A.R. Link, Jr., and M. Mann, In vitro responses of human neutrophils to N-formyl-methionylleucyl-phenylalanine: correlation with effects of acute bacterial infection, J. Infect. Dis., 140:277, 1979.Google Scholar
  9. 9.
    D.B. Kuhns, S.S. Kaplan, and R.E. Basford, Hexachlorocyclohexanes, potent stimuli of 02 production and calcium release in human polymorphonuclear leukocytes, Blood, 68: 535, 1986.PubMedGoogle Scholar
  10. 10.
    A.I Tauber, N. Borregaard, E. Simons, and J. Wright, Chronic granulomatous disease: a syndrome of phagocyte oxidase deficiencies, Medicine, 62: 286, 1983.PubMedCrossRefGoogle Scholar
  11. 11.
    J.I. Gallin, E.S. Buescher, B.E. Seligmann, J. Nath, T. Gaither, and P. Katz, Recent advances in chronic granulomatous disease, Ann. Int. Med., 99: 657, 1983.PubMedCrossRefGoogle Scholar
  12. 12.
    D.A. Bass, J.W. Parce, L.R. DeChatelet, P. Szejda, M.C. Seeds, and M. Thomas, Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation, J. Immunol., 130: 1910, 1983.PubMedGoogle Scholar
  13. 13.
    D.A. Bass, P. Olbrantz, P. Szejda, M.C. Seeds, and C.E. McCall, Sub-populations of neutrophils with increased oxidative product formation in blood of patients with infection, J. Immunol., 136: 860, 1986.PubMedGoogle Scholar
  14. 14.
    S.D. Douglas, Metabolic and immunologic studies of phagocytes with abnormal bactericidal function, 1973, pp. 392–398, in: Erythrocytes Thrombocytes Leukocytes, E. Gerlach, K. Moser, E. Deutsch, W. Wilmanns, ed., Georg Thieme, Stuttgart.Google Scholar
  15. 15.
    H.D. Ochs and R.P. Igo, The NBT slide test: a simple screening method for detecting chronic granulomatous disease and female carriers, J. Pediatrics, 83: 77, 1973.CrossRefGoogle Scholar
  16. 16.
    R.B. Johnston III, R.J. Harbeck, and R.B. Johnston, Jr., Recurrent severe infections in a girl with apparently variable expression of mosa-icism for chronic granulomatous disease, J. Pediatrics, 106: 50 1985.CrossRefGoogle Scholar
  17. 17.
    R.L. Baehner and D.G. Nathan, Quantitative nitroblue tetrazolium test in chronic granulomatous disease, N. Engl. J. Med., 278: 971, 1968.PubMedCrossRefGoogle Scholar
  18. 18.
    M.L. Salin and J.M. McCord, Superoxide dismutase in polymorphonuclear leukocytes, J. Clin. Invest., 54: 1005, 1974.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    N.F. Hassan, D.E. Campbell, and S.D. Douglas, 0-phenylenediamine oxidation by phorbol myristate acetate stimulated human polymorphonuclear leukocytes: Characterization of two distinct oxidative mechanisms, Clin. Imm. Immunopathol, In press.Google Scholar
  20. 20.
    R.K. Root, J. Metcalf, N. Oshino, and B. Chance, H202 release from human granulocytes during phagocytosis, J. Clin. Invest., 55: 945, 1975.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    W. Ruch, P.H. Cooper, and M. Gaggiolini, Assay of H202 production by macrophages and neutrophils with homovanilic acid and horse radish peroxidase, J. Immunol. Meth., 63: 347, 1983.CrossRefGoogle Scholar
  22. 22.
    N.F. Hassan, D.E. Campbell, and S.D. Douglas, Phorbol myristate acetate induced oxidation of 2’7-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease (submitted).Google Scholar

Copyright information

© Springer Science+Business Media New York 1988

Authors and Affiliations

  • Nassef F. Hassan
    • 1
  • Donald E. Campbell
    • 2
  • Steven D. Douglas
    • 1
  1. 1.Division of Allergy-Immunology, Department of PediatricsUniversity of Pennsylvania School of Medicine, The Joseph Stokes, Jr. Research InstitutePhiladelphiaUSA
  2. 2.Clinical Immunology LaboratoryChildren’s Hospital of PhiladelphiaPhiladelphiaUSA

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