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Chemiluminescence in activated human neutrophils

Role of buffers and scavengers


Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H2O2, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H2O2 was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn2+, Fe2+, Cu2+, and Co2+ very markedly enhanced CL induced by mixtures of H2O2 and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H2O2 probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.

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  1. 1.

    Babior, B. M. 1984. Oxidants from phagocytes: Agents of defence and destraction.Blood 64:959–966.

  2. 2.

    Klebanoff, S. J. 1988. Phatocytic cells: Products of oxygen metabolism.In Inflammation Basic Principles and Clinical Correlates. J. I. Gallin, I. M. Goldstein, and R. Snyderman, editors. Raven Press, New York. 391–444.

  3. 3.

    Bellavite, P. 1988. The superoxide-forming enzymatic system of phagocytes.Free Radical Biol. Med. 4:225–261.

  4. 4.

    Borregard, N. 1988. The respiratory burst; an overview.In The Respiratory Burst and Its Physiological Significance. A. J. Sbarra and R. Strauss, editors. Plenum Publishing, New York. 1–31.

  5. 5.

    Babior, B. M. 1988. Microbicidal oxidant production by phagocytes.In Oxyradicals in Molecular Biology and Pathology. P. A. Cerutti, editors. Allan R. Liss, New York. 39–51.

  6. 6.

    Halliwell, B. 1989. Current status review: Free radicals, reactive oxygen species and human disease: A critical evaluation with special references to artherosclerosis.Br. J. Exp. Pathol. 70:737–757.

  7. 7.

    Allen, R. C., andLoose, L. D. 1976. Phagocytic activation of luminol-dependent chemi luminescence in rabbit alveolar and peritoneal macrophages.Biochem. Biophys. Res. Commun. 69:245–252.

  8. 8.

    Trush, M. A., M. E. Wilson, andK. van Dyke. 1978. Generation of chemiluminescence (CL) by phagocytic cells.Methods Enzymol. 57:462.

  9. 9.

    Westrick, M. A., P. Shirley, andL. R. DeChatelet. 1980. Generation of chemiluminescence by neutrophils exposed to soluble stimuli of oxidative metabolism.Infect. Immun. 30:385–390.

  10. 10.

    DeChatelet, L. R., G. D. Long, P. S. Shirley, D. A. Bass, M. J. Thomas, F. W. Henderson, andM. S. Cohen. 1982. Mechanisms of the luminol dependent chemiluminescence of human neutrophils.J. Immunol. 129:1589–1593.

  11. 11.

    Dahlgren, C., andO. Stendahl. 1983. Role of myeloperoxidase in luminol dependent chemiluminescence of polymorphonuclear leukocytes.Infect. Immun. 39:736–741.

  12. 12.

    Muller-Peddinghaus, R. 1984. In vitro determination of phagocyte activity by luminol and lucigenin-amplified chemiluminescence.Int. J. Immunopharmacol. 6:455–466.

  13. 13.

    van Dyke, K., andV. Castranova. 1987. Cellular chemiluminescence, Vols. I and II. CRC Press, Boca Raton, Florida.

  14. 14.

    Dahlgren, C. 1989. Is lysosome fusion required for the granulocyte chemiluminescence reaction?Free Radical Biol. 6:399–403.

  15. 15.

    Vilimm, V., andJ. Wilhelm. 1989. What do we measure by a luminol-dependent chemiluminescence of phagocytes?Free Radical Biol. 6:623–629.

  16. 16.

    Ginsburg, I., R. Borinski, M. Lahav, K. E. Gillert, M. Winkler, andS. Muller. 1982. Bacteria and zymozan opsonized with histone and polyanethole sulfonate trigger intense chemiluminescence in human blood leukocytes, platelets and in mouse peritoneal macrophages: Modulation by metabolic inhibitors in relation to leukocyte-bacteria interactions in inflammatory sites.Inflammation 6:343–364.

  17. 17.

    Ginsburg, I., R. Borinski, M. Lahav, Y. Matzner, I. Eliasson, P. Christensen, andD. Mallamud. 1984. Poly-L-arginine and N-formylated chemotactic peptide act synergistically with lectins and calcium ionophore to induce intense chemiluminescence and Superoxide production in human blood leukocytes. Modulation by metabolic inhibitors, sugars and polyelectrolytes.Inflammation 8:1–26.

  18. 18.

    Ginsburg, I., R. Borinski, D. Malamud, F. Struckmayer, andV. Kilmetzek. 1985. Chemiluminescence and Superoxide generation by leukocytes stimulated by polyelectrolyte opsonized streptococci: Role of polyargine, polylysine, polyhistidine, cytochalasins and inflammatory exudates as modulators of the oxygen burst.Inflammation 9:245–271.

  19. 19.

    Ginsburg, I., andR. Borinski. 1987. “Cocktails” of soluble ligands and bacteria “opsonized” with cationic or anionic polyelectrolytes trigger intense chemiluminescence and Superoxide production by leukocytes.In Cellular Chemiluminescence, Vol. II. K. van Dyke and V. Castranova, editors. CRC Press, Boca Raton, Florida. 121–156.

  20. 20.

    Ginsburg, I., R. Borinski.M. Sadovnic, Y. Eilam, andK. Rainsord. 1987. Poly-l-histidine, a potent stimulator of Superoxide generation by human blood leukocytes.Inflammation 11:253–277.

  21. 21.

    Ginsburg, I. 1987. Cationic polyelectrolytes: A new look at their possible role as opsonins as stimulators of the respiratory bursts in leukocytes in bacteriolysis and as modulators of immune complex disease (a review hypothesis).Inflammation 11:489–515.

  22. 22.

    Ginsburg, I. 1989. Cationic polyelectrolytes: Potent opsonic agents which activate the respiratory burst in leukocytes.Free Radical Res. Commun. 8:11–26.

  23. 23.

    Thurman, R. G., H. G. Leyland, andR. Scholz. 1972. Hepatic microsomal ethanol oxidation hydrogen peroxidase formation and the role of catalase.Eur. J. Biochm. 25:420–430.

  24. 24.

    Klebanoff, S. J. 1968. Myeloperoxidase halide-hydrogen peroxide antibacterial system.J. Bacteriol. 95:2131–2138.

  25. 25.

    Margoliash, E., andA. Novogrodsky. 1958. A study of the inhabit of catalase by 3-amino1,2,4-triazole.Biochem. J. 68:468.

  26. 26.

    Lock, R., A. Johansson, K. Orselius, andC. Dahlgren. 1988. Analysis of horseradish peroxidase-amplified chemiluminescence production by human neutrophils reveals a role for the Superoxide anion in the light emitting reaction.Anal. Biochem. 173:450–455.

  27. 27.

    Toth, K. M., J. M. Harlan, C. J. Beehler, L. M. Berger, N. B. Parker, S. L. Linas, andJ. E. Repine. 1989. Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cellsin vitro and disappears in the process.J. Clin. Invest. 50:2226–2229.

  28. 28.

    McCord, J. M., andE. D. J. Day. 1989. Superoxide dependent production of hydroxyl radical catalyzed by iron-EDTA complex.FEBS Lett. 80:130–136.

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Ginsburg, I., Misgav, R., Gibbs, D.F. et al. Chemiluminescence in activated human neutrophils. Inflammation 17, 227–243 (1993).

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  • Azide
  • Cimetidine
  • NADPH Oxidase
  • Human Neutrophil
  • Deferoxamine