Vol. 25, No. 1

INFECTION AND IMMUNITY, July 1979, p. 473-476

0019-9567/79/07-0473/04$02.00/0

Enhancement of Granulocyte Chemiluminescence with Hydroxyl Radical Scavengers LIANA HARVATH West Side Veterans Administration Medical Center and the Departments ofMicrobiology and Medicine, University of Illinois Medical Center, Chicago, Illinois 60612 Received for publication 7 May 1979

Addition of hydroxyl radical scavengers (benzoate, ethanol, and mannitol) to granulocyte chemiluninescent reactions significantly enhanced light generation without altering granulocyte oxygen consumption or superoxide production.

Granulocyte (GR) light production or chemiluminescence (CL) occurs as an event associated with increased cellular oxidative metabolism and can be initiated by exposing GRs to opsonized particles (2) or soluble, nonparticulate agents (7, 8). The light-producing reactions of GRs have not yet been identified. Superoxide anion, 02 (5), hydrogen peroxide, H202 (13), and hydroxyl radical, OH. (16, 20), are generated by metabolically activated GRs; however, it is not known whether these oxidative intermediates directly participate in CL reactions. CL is produced from the reaction of the GR enzyme, myeloperoxidase, with H202 and chloride (1, 4), and there is evidence that singlet molecular oxygen, 102, is generated by the reaction (14). It has been postulated that reactions of 102 with organic molecules are sources of GR light production (2); however, it is also possible that 02, OH., and H202 may be involved in GR CL reactions. The present study examined the effect of OHscavengers on light generation, 02 consumption, and 02 production of metabolically activated GRs. Two chemical CL reactions were also examined for the effects of OH. scavengers on light production. Fenton's reaction, which consisted of FeSO4 + H202 (6, 18), and the reaction of NaOCl + H202 (10, 15) were studied. GRs were obtained from heparinized canine blood (10 U of heparin per ml of blood) by sedimentation with 5% dextran solution. Residual erythrocytes were removed by ammonium chloride lysis (19), and GRs were washed and suspended in Hanks balanced salt solution to a final concentration of 5 x 10' GRs per reaction vial. GR oxidative metabolism was stimulated with 20 mM sodium fluoride, NaF, (7) and GR CL was assayed in a CL spectrometer (3) in which reactions were continuously mixed at 37°C throughout the assay period. GR-emitted light is reported as counts per minute. Benzoate (20 mM), ethanol (40 mM), and mannitol (50 mM) were used at concentrations previously

described for scavenging OH. produced by GRs (16, 20). CL from Fenton's reaction (FeSO4 + H202) and the NaOCl + H202 reaction was assayed at 37°C, and emitted light is reported as counts per initial 4-s interval of the reaction. GR 02 consumption was measured with a biological 02 monitor and a Clark polarographic electrode (YSI model 53, Yellow Springs Instrument Co., Yellow Springs, Ohio). GR 02 production was assayed by using the method of Babior et al. (5) with modifications (7). Cell viability was assessed by trypan blue dye exclusion. OH. scavengers enhanced NaF-induced GR CL without altering the kinetics of the CL responses (Fig. la). Benzoate (20 mM) and ethanol (40 mM) were consistently the most effective of the scavengers in augmenting GR CL. When five experiments were analyzed by calculating the integral areas of CL curves (2), it was found that CL was augmented 10- to 25-fold with benzoate, 3- to 12-fold with mannitol, and 5- to 15-fold with ethanol. OH. scavengers did not alter baseline CL of GRs when the metabolic stimulus was absent. Sodium azide, an inhibitor of myeloperoxidase (11) and scavenger of 1O2 (9), prevented augmentation of CL by OH. scavengers. Figure lb illustrates the inhibitory effect of 0.1 mM azide on the benzoate-enhanced CL of NaF-stimulated GRs. The inhibitory effect of azide could not be explained by cytotoxicity, since >90% ofthe GRs were viable after exposure to 0.1 mM azide in the presence of 20 mM benzoate and 20 mM NaF. Azide similarly inhibited ethanol- and mannitol-enhanced CL. The suppressive effect of azide may have been due to its quenching of GR oxidative intermediates or to its suppression of myeloperoxidase, resulting in depressed oxidative intermediate production. GRs were examined for the effect of OHscavengers on 02 consumption and 02 production (two early events of oxidative metabolism). OH. scavengers did not significantly alter 02 473

474

INFECT. IMMUN.

NOTES

b. b BU 0. -, Lai

0.

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L'J (I

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z

z

i

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-i

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TIME (Mmn)

8 10 12 14 16 I8 20 TIME (Min)

FIG. 1. (a) Enhancement of NaF-induced GR CL with OH. scavengers. CL responses of GRs exposed to NaF alone were significantly amplified in the presence of 20 mM benzoate, 40 mM ethanol, and 50 mM mannitol. Results represent the means of five experiments with different GR donors. (b) Azide inhibition of OH. scavenger-enhanced GR CL. Benzoate-enhanced CL was suppressed with the addition of 0.1 mM azide to reactions. Results represent the means of three experiments with different GR donors.

consumption or O2 production of NaF-stimulated cells (Table 1), indicating that OH. scavenger-enhanced GR CL was not due to increased oxidative metabolism of GRs. These results suggest that enhanced CL may be due to secondary light-producing reactions between OH- scavengers and GR oxidative intermediates. Two chemical light-producing reactions were studied to determine whether OH. scavengers and oxidative intermediates may participate in secondary CL reactions. In one reaction, NaOCl + H202, '02 is the primary light-producing intermediate (10, 15). A second reaction, FeSO4 + H202 (Fenton's reaction), generates OH. (18) and light (B. R. Andersen and L. Harvath, Biochim. Biophys. Acta, in press). Both reactions contain H20 and H202 and form highly reactive products, and it is likely that more than one oxidative species is produced by each reaction. Benzoate and ethanol were evaluated for their effects on CL of both reactions (Table 2). Benzoate did not alter CL from Fenton's reaction, but it increased CL of the NaOCl + H202 reaction (significant, P < 0.05). Ethanol significantly enhanced CL of Fenton's reaction (P < 0.05), but modestly suppressed CL of the NaOCl

TABLE 1. Effect of OH. scavengers on fluoridestimulated Granulocyte 02 consumption and 02productiona Scavenger

02 Ctnio5n1bP

02 production'

Control (no scavenger)

2.9 ± 0.2

131.4 ± 3.5

20 mM benzoate

2.7 ± 0.2

116.4 ± 4.1

40 mM ethanol

2.8 ± 0.3

126.0 ± 3.9

2.5 ± 0.3 120.0 ± 4.5 50 mM mannitol 'Values are the means of four observations ± 1 standard deviation. b of 02 con02 consumption is reported as sumed per 5 x 106 GRs per 20 min. Data are values above nonstimulated cell levels. e 02- production is reported as nanomoles of cytochrome c reduced by 3 x 106 GRs per 20 min.

Aliters

+ H202 reaction. These results indicate that OH. scavengers have the potential of reacting with some oxidative intermediates to increase light production. Since neither of the chemical reactions studied produces a single product free of

VOL. 25, 1979

TABLE

NOTES

2. Effect of OH. scavengers on CL of chemical reactions

Scavenger Control (no scav-

CL' (counts/initial 4 s of reaction) Fenton's reacNaOCl + H202 tionb reaction' 5,219 ± 183 108,279 + 2,713

enger) 20 mM benzoate

5,681 ± 769

139,399 + 4,045d

40 mM ethanol 42,008 ± 1,165d 91,587 ± 3,381d a Values are the means of six observations ± 1 standard error of the mean. b Fenton's reaction consisted of 0.18 mM FeSO4 in distilled water + 120 mM H202. Reactions were initiated upon injection of 0.5 ml of H202. c NaOCl + H202 reaction consisted of 200 mM NaOCl in distilled water and 100 mM H202. Reactions were initiated upon injection of 0.5 ml of H202. dSignificant, P < 0.05 compared to control (Student's t test).

other intermediates, it is not possible to determine which intermediates react with the OH. scavengers to increase CL. There appear to be at least two mechanisms by which oxidative intermediates and OH. scavengers may react to directly alter CL: (1) Light Oxidative intermediates + OH. scavenger

O'

Scavenger*

-.

Light

(2)

In one mechanism, OH. scavengers could quench light-generating intermediates and impair CL (reaction 1). In the second mechanism, oxidative intermediates and OH. scavengers may react to form an excited-state scavenger molecule (scavenger*) which could emit light energy and enhance CL (reaction 2). The OHscavengers used in the present study may have reacted with GR oxidative products to generate secondary CL reactions via the second mechanism. It is also possible that OH scavengers indirectly affect CL by reacting with intermediates or reactants to alter oxidative product generation. If reactions are altered to favor the production of light-emitting species, CL of the reaction could be enhanced. OH. scavengers reacting via this postulated mechanism could potentially alter GR oxidative intermediate production without affecting initial cellular metabolic events. CL assays provide useful information about the oxidative metabolic activity of GRs. To accurately correlate CL with GR metabolism, however, it is important to determine the contribution of extracellular chemical components to the

475

CL reactions. Other investigators have shown that tryptophan (12) and indole acetic acid (17) augment CL of phagocytizing GRs. The present study demonstrates that OH. scavengers (benzoate, ethanol, and mannitol) can augment CL of non-phagocytizing GRs without altering initial events of cellular oxidative metabolism. The helpful suggestions of Burton Andersen and the excellent technical assistance of Lilia Kizlaitis and Harold J. Amirault are gratefully acknowledged. This work was supported by Public Health Service research funds from the Heart, Lung, and Blood Institute of the National Institutes of Health (1 R23 HL 21212) and research funds from the West Side Veterans Administration Medical Center (MRIS 0394). L.H. is the recipient of Public Health Service Young Investigator Research Award from the Heart, Lung, and Blood Institute.

LITERATURE CITED 1. Allen, R. C. 1975. Halide dependence of the myeloperoxidase-mediated antimicrobial system of the polymorphonuclear leukocyte in the phenomenon of electronic excitation. Biochem. Biophys. Res. Commun. 63:675683. 2. Allen, R. C., R. Stiernholm, and R. Steele. 1972. Evidence for the generation of an electronic excitation state(s) in human polymorphonuclear leukocytes and its participation in bactericidal activity. Biochem. Biophys. Res. Commun. 47:679-684. 3. Andersen, B. R., and A. M. Brendzel. 1978. Use of a unique chemiluminescence spectrometer in a study of factors influencing granulocyte light emission. J. Immunol. Methods 19:279-287. 4. Andersen, B. R., A. M. Brendzel, and T. F. Lint. 1977. Chemiluminescence spectra of human myeloperoxidase and polymorphonuclear leukocytes. Infect. Immun. 17: 62-66. 5. Babior, B. M., R. S. Kipnes, and J. T. Curnutte. 1973. Biological defense mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent. J. Clin. Invest. 52:741-744. 6. Fenton, H. 1894. Oxidation of tartaric acid in the presence of iron. J. Chem. Soc. 65:899-910. 7. Harvath, L., H. J. Amirault, and B. R. Andersen. 1978. Chemiluminescence of human and canine polymorphonuclear leukocytes in the absence of phagocytosis. J. Clin. Invest. 61:1145-1154. 8. Hatch, G. E., D. G. Gardner, and D. B. Menzel. 1978. Chemiluminescence of phagocytic cells caused by Nformylmethionyl peptides. J. Exp. Med. 147:182-195. 9. Hodgson, E. K., and I. Fridovich. 1974. The production of superoxide radical during the decomposition of potassium peroxochromate. Biochemistry 13:3811-3815. 10. Khan, A. V., and M. Kasha. 1964. Rotational structure in the chemiluminescence spectrum of molecular oxygen in aqueous systems. Nature (London) 204:241-243. 11. Klebanoff, S. J., and S. H. Pincus. 1971. Hydrogen peroxide utilization in myeloperoxidase deficient leukocytes: a possible microbicidal control mechanism. J. Clin. Invest. 50:2226-2229. 12. Nelson, R. D., M. J. Herron, J. R. Schmidtke, and R. L Simmons. 1977. Chemiluminescence response of human leukocytes: influence of medium components on light production. Infect. Immun. 17:513-520. 13. Root, R. K., J. Metcalf, N. Oshino, and B. Chance. 1975. H202 release from human granulocytes during phagocytosis. J. Clin. Invest. 55:945-955. 14. Rosen, H., and S. J. Klebanoff. 1977. Formation of singlet oxygen by the myeloperoxidase-mediated antimicrobial system. J. Biol. Chem. 252:4803-4810.

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NOTES

15. Seliger, H. H. 1960. A photoelectric method for the measurement of spectra of light sources of rapidly varying intensities. Anal. Biochem. 1:60-65. 16. Tauber, A. L, and B. M. Babior. 1977. Evidence for hydroxyl radical production by human neutrophils. J. Clin. Invest. 60:374-379. 17. Ushijima, Y., M. Nakano, Y. Tsuji, and H. Inabia. 1978. Excitation of indole analogs by phagocytizing leukocytes. Biochem. Biophys. Res. Commun. 82:853858.

INFECT. IMMUN. 18. Walling, C. 1975. Fenton's reagent revisited. Acc. Chem. Res. 8:125-131. 19. Weening, R., D. Roos, and J. Loos. 1974. Oxygen consumption of phagocytizing cells in human leukocyte and granulocyte preparations: a comparative study. J. Lab. Clin. Med. 83:570-576. 20. Weiss, S. J., P. K. Rustagi, and A. F. LoBuglio. 1978. Human granulocyte generation of hydroxyl radical. J. Exp. Med. 147:316-323.