Spin trapping of superoxide radicals following stimulation of neutrophils with fMLP is temperature dependent

Free Radical Biology & Medicine, Vol. 15, pp. 425-433, 1993 Printed in the USA. All rights reserved.

0891-5849/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd.

Original Contribution SPIN T R A P P I N G

OF SUPEROXIDE RADICALS FOLLOWING STIMULATION OF NEUTROPHILS WITH fMLP IS T E M P E R A T U R E DEPENDENT

T O R U TANIGAWA,* YASHIGE KOTAKE,* and LESTER A. REINKE t *National Biomedical Center for Spin Trapping and Free Radicals, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA; and +Department of Pharmacology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA (Received 4 December 1992; Revised 2 March 1993; Accepted 12 April 1993) Abstract--Oxygen radical formation by human neutrophils stimulated with a chemotactic peptide, formyl-methionylqeucylphenylalanine (fMLP), was studied through the use of spin trapping and superoxide dismutase-inhibitable reduction of oxidized cytochrome c. Both methods provided comparable data on temperature-dependent kinetics ofsuperoxide radical formation, but hydroxyl radicals were also detected in spin-trapping experiments. When superoxide generation was monitored at 37°C, the respiratory burst lasted only a few minutes. If the neutrophils were stimulated at 37°C, but superoxide measurements were done at room temperature, the respiratory burst was again transient. However, neutrophils persistently generated superoxide when both stimulation and subsequent measurements were performed at room temperature. In the presence of the actin polymerization inhibitor, cytochalasin B, superoxide generation was persistent, even when measurements were conducted at 37°C. A possible explanation for these observations is that the fMLP receptor complexes quickly aggregate and are internalized at physiological temperature, but not at room temperature. Very little superoxide was formed if cells were kept at a temperature of 4°C for l h prior to fMLP addition, which is consistent with decreased expression of the fMLP receptor at cold temperatures. Keywords--Neutrophils, Superoxide, fMLP, Spin trapping, Respiratory burst, Free radicals

The spin-trapping method has been used in a number of studies to detect oxygen radical formation by stimulated neutrophilsfl 4-22 In spin trapping, highly reactive free radicals are allowed to react with a suitable spin-trapping agent to form a more stable secondary radical which can be detected by electron paramagnetic resonance (EPR) s p e c t r o s c o p y . 23 Both superoxide radicals and hydroxyl radicals have been detected during the respiratory burst of neutrophils after stimulation with agents such as phorbol- 12-myristate-1 3-acetate (PMA) and opsonized z y m o s a n . 14-22 However, no radicals were detected when fMLP was used as a stimulus,24 in spite of the well-known ability of this agent to initiate a respiratory burst. The current study compares the methods of spin trapping and reduction of oxidized cytochrome c to measure superoxide generation following stimulation of human neutrophils with fMLP. The data indicate that fMLP-stimulated superoxide generation is strongly influenced by the temperature at which cells are exposed to fMLP, as well as the temperature at which superoxide formation is measured. Further-

INTRODUCTION

N-Formyl-methionyl-leucyl-phenylalanine(fMLP) is one of the most widely studied of the synthetic chemotactic N-formyl oligopeptides 1-5 which can stimulate neutrophils to produce superoxide radicals in a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent respiratory burst. Neutrophil cell membranes contain fMLP receptors which appear to be responsible for both the chemotactic response and the respiratory b u r s t . 6'7 The exact sequence of events between fMLP binding and superoxide formation is not completely understood, but extensive studies on this topic are ongoing,s-~° The respiratory burst, particularly when initiated with fMLP, is a relatively transient phenomenon, 11-13 but the factors which terminate this response are also uncertain. Address correspondence to: Yashige Kotake, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 N.E. 13th St. Oklahoma City, OK 73104, USA. 425

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more, these observations are consistent with known temperature-dependent expression of fMLP receptors, or aggregation and subsequent internalization of the ligand-receptor complexes. MATERIALS AND M E T H O D S

Preparation of neutrophils Heparized venous blood was obtained from healthy human volunteers and was layered on an equal volume of Ficoll-Hypaque solution with a density of 1.114 g/ml. 26 The solution was centrifuged at 400 × g for 30 min, and the granulocyte layer was removed and suspended in Hanks' balanced salt solution (HBSS). Erythrocytes in the suspension were lysed by suspending the cells in 0.2% saline for 30 s, followed by washing with HBSS. The viability of the cells immediately after isolation was found to be greater than 98% by Trypan Blue exclusion. All steps of neutrophil preparation were normally performed at room temperature (21 _+ 2°C).

Spin-trapping experiments The spin-trapping agent used in these experiments was 5,5-dimethylpyrroline N-oxide (DMPO), which forms secondary radicals (spin adducts) with both superoxide and hydroxyl radicalsfl7,28DMPO ( 100 mM) and diethylenetriaminepentaacetic acid (DETAPAC, 0.1 mM) were mixed with 0.2 ml of neutrophils suspended in HBSS before addition of fMLP (10 -5 M). The concentration of neutrophils in the suspension was adjusted to 2 × 10 6 cells/ml, because preliminary experiments indicated that higher concentrations of cells paradoxically decreased the intensity of the EPR signals. When fMLP was not added at room temperature, the neutrophil suspensions were briefly preincubated at the desired temperature and were kept at that temperature for 20 s after fMLP addition before transferring the mixtures to the EPR cell. In some experiments, PMA (100 ng/ml) was also used as a stimulus to compare with fMLP stimulation. Superoxide dismutase (SOD, 103 units/ml) was added in some experiments to determine effects of this enzyme on the EPR spectra. In experiments utilizing cytochalasin B ( 10-5 M), the neutrophils were preincubated with this agent for 3 min prior to fMLP addition. The cells were transferred to an flat quartz EPR cell (Wilmad WG814), which was placed into the spectrometer cavity in a horizontal orientation. Most EPR measurements were conducted at room temperature, but for measurements obtained at 37°C the temperature was kept constant by circulating temperaturecontrolled water through the jacket around the cavity.

The temperature of the cell was continuously monitored with a copper-constantan thermocouple attached to the flat part of the cell. EPR spectra were recorded using a Bruker ER300E EPR spectrometer (Bruker, Karlsruhe, Germany) equipped with 100kHz field modulation. Other typical EPR operating conditions were microwave power, 20 mW; modulation width, 1.0 gauss; field sweep, 100 gauss/84 s; time constant, 160 ms.

Cytochrome c reduction Superoxide generation by fMLP-stimulated neutrophils was also measured by the SOD-sensitive reduction of oxidized cytochrome c. In these experiments, neutrophils were mixed with cytochrome c (0.1 mM) and the absorbance increase at 550 nm was monitored with a Perkin Elmer Lambda 4B UV/VIS Spectrophotometer (Perkin Elmer, Norwalk, CT). The temperature in the cuvette was controlled at the temperature indicated in figure legends by circulating water from a constant temperature bath through the sample compartment. Other conditions were as described for the spin-trapping experiments, except that DMPO was omitted. In these experiments, cytochrome c reduction was completely inhibited in the presence of SOD.

Materials DMPO was purchased from the Sigma Chemical Co. (St. Louis, MO) and was purified by adding an equal volume of HBSS and washing twice with an equal volume of benzene. The aqueous layer was filtered through a column of activated charcoal (15 mm i.d., 20 mm long) which had previously been wet with HBSS. Cytochrome c (horse heart, type IV) and all other biochemicals were purchased from Sigma in the highest grade available. RESULTS

Spin-trapping experiments with fMLP-stimulated neutrophils When neutrophils were stimulated with fMLP at room temperature, transferred into a flat EPR cell, and the resulting EPR spectra were also recorded at room temperature, results of the type shown in Fig. 1 were obtained. The first EPR spectrum was obtained 2 min after stimulation and contained evidence of both the DMPO spin adducts of hydroxyl radical (DMPO-OH) and superoxide radical (DMPOOOH). The maximum signal intensity was observed

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OOH component decayed rapidly with ensuing scans of the same preparation, so that DMPO-OH provided the major spectral component 20 min after stimulation (Fig. 2). The apparent decay of DMPO-OOH signal was even more rapid when EPR measurements were also made at 37°C (data not shown). The data shown in Figs. 1 and 2 differ only in the temperature of the cells at the time of fMLP addition, and suggested that the generation of DMPO-oxy adducts are remarkably sensitive to the temperature of

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Fig. 1. EPR spectra of DMPO spin adducts obtained following stimulation of neutrophils with fMLP at room temperature. Neutrophils (2 × 106 cells/ml) suspended in HBSS containing DMPO (0.1 M) were stimulated with fMLP ( 10 5 M). The addition offMLP and all subsequent experimental producers were performed at room temperature. The time indicated in the spectra represents the elapsed time after stimulation. The stick spectrum at the bottom of the figure indicates the hypertine splitting patterns of DMPO-OOH ( I) and DMPO-OH (:).

after 6 min, and the characteristics of the spectrum changed very little over the next 14 min (Fig. 1). In contrast, if the same neutrophils were warmed to 37°C before stimulation with fMLP, and then were transferred to the flat cell for room temperature EPR measurements, striking differences in the results were obtained (Fig. 2). The initial spectrum obtained 2 min after stimulation still contained evidence of both DMPO-OOH and DMPO-OH, but the signals were more intense than when fMLP had been added at room temperature (compare to corresponding spectrum in Fig. 1). However, the intensity of the DMPO-

Fig. 2. EPR spectra of spin adducts of DMPO obtained following the stimulation of neutrophils with fMLP at 37°C. fMLP was added to neutrophils which had been preincubated at 37°C, and this temperature was maintained for an additional 20 s. The suspensions were subsequently transferred to a flat cell for room-temperature EPR measurements. All other conditions are as indicated in the legend to Fig. 1.

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Fig. 3. Time course of the relative EPR signal intensity of D M P O - O O H observed at room temperature after tMLP stimulation of neutrophils. The squares (11) indicate cells stimulated at room temperature, while the closed circles (O) indicate cells stimulated at 37°C.

the cells at the time of stimulation. Figure 3 summarizes the relative EPR signal intensity of D M P O O O H at various times after fMLP addition to cells at room temperature, or at 37°C. In these experiments, all EPR measurements were made at room temperature. The EPR signal intensity of D M P O - O O H reached a m a x i m u m at about 6 min after stimulation of neutrophils at room temperature and remained relatively stable over the next 20 min (Fig. 3). As already indicated in Fig. 2, the D M P O - O O H signal intensity was maximal in the first EPR spectrum obtained after stimulation of neutrophils at physiological temperature and declined rapidly thereafter (Fig. 3). Determination of cell viability at the end of these exposures showed that the neutrophils were not killed by the fMLP-induced respiratory burst. Additional experiments were conducted to test the effect of cold on the response ofneutrophils to stimulation with fMLP. Neutrophils were stored at 4°C for 1 h, fMLP was added, and room-temperature EPR measurements were begun after 20 s o f additional cold exposure. In these experiments, very weak EPR signals of D M P O - O O H and D M P O - O H were observed 10 min after fMLP addition (Fig. 4A). However, if the chilled cells were returned to room temperature for 2 h prior to stimulation with fMLP, typical EPR spectra were observed (Fig. 4B), indicating that the effects of cold were reversible. In comparison, when PMA was used as a stimulus for these cells, the temperature of storage did not affect the intensity and time course of D M P O - O O H generation (Figs. 4C, 4D). When SOD was added to neutrophils either before or immediately after fMLP addition, no EPR signals were observed (data not shown). These results are

consistent with previous reports which indicated loss of both D M P O - O O H and D M P O - O H in the presence of SOD. 17,22,25

Superoxide measurements utilizing cytochrome c reduction Results obtained from fMLP-stimulated neutrophils with the spin-trapping method were compared to the more widely utilized method of SOD-inhibitable cytochrome c reduction. In these experiments, neutrophils were either stimulated in a cuvette which contained cytochrome c, or fMLP was added to cells in a 37°C water bath and the suspensions were subsequently transferred to a cuvette containing cytochrome c. In both cases the increase in absorption of reduced cytochrome c was measured at room temperature. The results indicated that the cells which had been stimulated at 37 °C produced superoxide for less than 2 min, while cells stimulated at room temperature maintained superoxide production for a longer period of time (Fig. 5). The total a m o u n t of cytochrome c reduced, and therefore superoxide generated, was greater in cells that had been stimulated at room temperature. The use o f oxygen-saturated buffer did not prolong superoxide generation in cells that had been stimulated at 37°C (Fig. 5C).

Effects of cytochalasin B preincubation The observation that superoxide generation from fMLP-stimulated neutrophils is a transient phenomenon (e.g., Fig. 5B) has been reported by other investigators.11-13 However, preincubation of the cells with

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Fig. 4. EPR spectra of spin adducts of DMPO obtained from neutrophils isolated at room temperature but stored at 4°C for 1 h. In (a), neutrophils were stimulated with fMLP immediately after warming the cells to room temperature. In (b), neutrophils from the same preparation were preincubated at room temperature for 2 h prior to fMLP stimulation. In (c) and (d), the cells were stimulated in the same conditions as (a) and (b), respectively, but PMA (50 #g/ml) was used as a stimulant. All spectra were obtained 8 min after stimulation. EPR spectrometer sensitivity for (c) and (d) was a factor of 2 less than that for (a) and (b). Other EPR conditions are as indicated in the legend to Fig. 1.

Fig. 5. Temperature-dependent rates of cytochrome c reduction after stimulation of neutrophils with fMLP. (a) Stimulated at room temperature; (b) stimulated at 37°C; (c) stimulated at 37°C and transferred into a cuvette containing oxygen-saturated buffer, with continuous supply of oxygen above the suspension in the cuvette. In these observations, the absorbance increase at 550 n m was measured at room temperature.

decreased, but superoxide formation continued for a longer time, so that eventually more cytochrome c was reduced (Fig. 6). Comparable results were obtained with experiments utilizing spin trapping as a detection method (Fig. 7). As indicated previously, the EPR signal from D M P O - O O H decayed rapidly when the EPR measurements were made at 37°C, so that D M P O - O H was the major component of the EPR spectrum 2 min after fMLP addition. By 8 min, the EPR signal of D M P O - O O H was barely detectable (Fig. 7). In contrast, the EPR signal intensity of D M P O - O O H from the same cell preparation that was preincubated with

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the actin polymerization inhibitor cytochalasin B causes the superoxide generation to become more persistent. ~ For this reason, it was of interest to determine the effects of cytochalasin B on superoxide radicals that could be measured by either cytochrome c reduction or in spin-trapping experiments with DMPO. When neutrophils were stimulated with fMLP and superoxide generation was measured at 37°C, cytochrome c reduction was observed only for about 2 min (Fig. 6A). This result is similar to those obtained when cells were stimulated at 37°C and superoxide was measured at room temperature (Fig. 5B). In contrast, when the cells were preincubated with cytochalasin B, the initial rate of superoxide formation was

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time/mln Fig. 6. The effect o f cytochalasin B on cytochrome c reduction by neutrophils after stimulation with fMLP. The increase in reduced cytochrome c absorbance was measured at 37°C in the absence (a) or presence (b) of cytochalasin B (10 -5 M). In (b), the cells were preincubated with cytochalasin B for 3 min prior to fMLP addition.

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Fig. 7. EPR spectra obtained from neutrophils after stimulation with fMLP in the presenceand absenceofcytochalasinB. In (a),the spectra represent 2 min (top) and 8 min (bottom) after stimulation in the absence of cytochalasinB. In (b), the spectra wereobtained 2 min (top) and 8 rain (bottom) after stimulation in the presence of cytochalasin B (10 s M). Stimulation with fMLP and subsequent measurements were performed at 37°C, and identical EPR instrumental conditions were used for all spectra.

cytochalasin B was readily discernable in the first EPR scan and further increased in intensity during the first 8 min following fMLP addition (Fig. 7).

DISCUSSION The results of these experiments provide the first spin-trapping data for radicals generated following a well-defined receptor-ligand interaction at the neutrophil cell membrane. Although spin trapping has been used in a n u m b e r o f similar studies, '4-2z the stimulants used have typically been PMA, (which directly stimulates protein kinase C) or opsonized zymosan

al.

(which elicits a phagocytic response). When fMLP was tested in previous experiments, no spin adducts were detected, and these negative data were attributed to cytotoxic effects of DMPO. 24 We have also observed that the n u m b e r of cells utilized is a critical variable in spin-trapping experiments with fMLP stimulation (data not shown), which may also contribute to the negative data in the previous report. 24 Although possible toxic effects of D M P O 22'24are of concern, the results of the current experiments clearly demonstrate that both D M P O - O O H and D M P O O H can be detected from fMLP-stimulated neutrophils. A disadvantage of spin trapping for the measurement of superoxide is the relatively short half-life of D M P O - O O H , which is approximately 1 min. 29 This explains the results shown in Fig. 2, where the DMPO-superoxide EPR signal decayed rapidly after the initial observation. Because superoxide generation by the neutrophils is transient under these conditions (Fig. 5B), the adduct detected in the initial scan subsequently decayed, so that only the EPR signal of the more persistent D M P O - O H was apparent (Fig. 2). In contrast, the EPR signal of D M P O - O O H remained a dominant feature of the spectra when both stimulation with fMLP and observation was performed at room temperature (Fig. l). This result can only be explained by continuous superoxide generation, which was confirmed in experiments involving cytochrome c reduction (Fig. 5A). The results of these studies indicate that the fMLPinduced respiratory burst is highly dependent on the temperature at which stimulation occurred. For example, both cytochrome c reduction (Fig. 5B) and spin trapping (Fig. 2) indicated that the respiratory burst was transient when fMLP was added to cells at 37°C, and measurements were then made at room temperature. In contrast, the respiratory burst monitored by both methods was persistent when both fMLP addition and subsequent measurements were performed at room temperature (Figs. 1,5A). Furthermore, stimulation of cells which had been kept on ice resulted in a weak respiratory burst (Fig. 4A), even though room temperature was used for the spin-trapping experiments. A n u m b e r of explanations could be postulated for the transient respiratory burst when fMLP addition and/or subsequent measurements were done at 37°C 1~-~3 (Figs. 2, 3, 5B, 6A, 7A). First, it is conceivable that the cells could become deficient of oxygen or some essential nutrient under these conditions. However, additional oxygen failed to maintain the respiratory burst of cells which had been stimulated at 37°C (Fig. 5C). Furthermore, a persistent respiratory burst was observed at 37°C in the presence of cytochalasin

fMLP-stimulatedneutrophils B (Figs. 6B, 7B). If the explanation of the transient respiratory burst involved consumption of available oxygen, lack of an essential nutrient, or death of the cells, these problems could not be overcome by changing the stimulation temperature or adding cytochalasin B. In addition, the respiratory burst elicited by PMA under the identical experimental conditions was more pronounced (about three times) than observed with fMLP (Fig. 4), was uniformly persistent, and was relatively unaffected by the stimulation temperature. These effects of PMA also argue against limitation of oxygen or some other component of the medium in the fMLP-stimulated respiratory burst. The temperature-dependent effects on the neutrophil respiratory burst can most likely be explained by changes in fMLP receptors which have been documented in other types of experiments. When the cells were stored at 4°C prior to stimulation, relatively little superoxide was generated (Fig. 4A). This finding is consistent with the observation that the number of fMLP receptors expressed per neutrophil was decreased by two thirds when the cells were exposed to cold temperatures. 3°'3~ However, the expression of receptors increases when the cells are returned to room temperature, resulting in typical production of oxygen metabolites (Fig. 4B). As opposed to this observation, when PMA was used as a stimulus the intensity of DMPO-OOH was not dependent on the cell storage temperature (Figs. 4C, 4D). Because PMA is a stimulant which bypasses receptors in induction of superoxide formation, 8 these results support the hypothesis that the temperature dependence of the fMLP-stimulated respiratory burst is the result of altered expression of fMLP receptors. Other investigators have reported transient superoxide generation following fMLP stimulation of neutrophils when assays were conducted at physiological temperature, t~-~3 and data obtained with either spin trapping (Fig. 7) or cytochrome c reduction (Fig. 6) confirm these results. Optical fluorescence studies have indicated that fMLP ligand-receptor complexes aggregate within 2 min after stimulation at 37°C, and these aggregates subsequently enter the cell. 32 Studies using tritium-labeled fMLP also indicated that internalization of fMLP receptors did not occur at 15°C. 33 Thus, the transient burst of superoxide formation may be explained by an initial response to the binding of fMLP to its receptor, which is terminated by aggregation or internalization of the receptor-fMLP complexes. In contrast, when fMLP was added to neutrophils at room temperature, and measurements were also conducted at room temperature, superoxide generation became persistent (Figs. 1, 3, 5) rather than transient. This observation may be explained by pro-

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longed message transduction from the fMLP-receptor complex, if the aggregation or internalization of the complexes is slowed at room temperature, as it is at 1 5 ° C . 33 The hypothesis that the transient superoxide burst is related to aggregation or internalization of the fMLP receptor-ligand complexes is supported by experiments with cytochalasin B. When cells were preincubated with cytochalasin B, superoxide generation became persistent with both the cytochrome c (Fig. 6) and spin trapping (Fig. 7) assays, even when these measurements were conducted at 37°C. This effect of cytochalasin B has been studied previously 11'34 and is thought to result from inhibition of actin polymerization in the neutrophil outer membrane. Thus, cytochalasin B inhibits the formation of aggregates of fMLP receptor-ligand complexes and their subsequent internalization. 34 Temperature-dependent effects in spin-trapping experiments have previously been reported following stimulation of neutrophils with PMA 35'36 or opsonized zymosan.36DMPO-OOH was readily detected at room temperature, but DMPO-OH dominated the EPR spectrum at 37°C. The explanation which is usually given for these observations involves temperature-dependent decomposition of DMPO-OOH. The half-life of DMPO-OOH appears to decrease as the temperature increases. 35 DMPO-OOH decomposes to products which are thought to include DMPO-OH and undefined EPR-silent products. 35'36 Based on these observations, loss of the DMPO-OOH signal with concomitant formation of the DMPO-OH signal would be expected, especially as the temperature is increased. However, two observations from the current experiments indicate that this hypothesis may be overly simplistic. First, when neutrophils were stimulated at 37°C, the EPR signal of DMPO-OOH was lost rapidly even when EPR measurements were conducted at room temperature (Figs. 2, 3). Second, the EPR signal intensity of DMPO-OOH increased with time in the presence of cytochalasin B, even at 37°C (Fig. 7). Both of these observations are better explained by changes in superoxide formation and are opposite to results which would be predicted from rapid loss of the DMPO-OOH signal at 37°C, but not at room temperature. These findings suggest that the types of EPR signals observed may be strongly influenced by changes which occur in the neutrophils, in addition to decay of DMPO-OOH. Unlike the cytochrome c method, spin trapping also can provide information about hydroxyl radical generation during the respiratory burst (Figs. 1, 2, 4, 7). DMPO-OH was detected under all conditions tested, and its formation was enhanced when stimula-

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tion or observation were conducted at 37°C (Figs. 2, 7). Hydroxyl radical generation during the respiratory burst ofneutrophils has been highly controversial. Although DMPO-OH has been detected in previous spin trapping experiments, ~4-22 it has often been ascribed to decomposition products of DMPOO O H . 35-37 However, other experiments have indicated that only about 3% of the DMPO-OOH adduct decays to DMPO-OH, 37 so this explanation seems inconsistent with the large DMPO-OH signals observed in some experiments (Figs. 2, 7). Recently, Ramos et al. utilized another spin-trapping agent (4-pyridyl 1oxide N-tert-butylnitrone, 4-POBN) to test for hydroxyl radical formation by neutrophils and concluded that hydroxyl radicals are formed through a myeloperoxidase-dependent reactionY In their proposed mechanism, hypochlorous acid formed by myeloperoxidase reacts with superoxide to produce oxygen, chloride ion, and hydroxyl radical. If this hypothesis is correct, it would explain the observation that addition of superoxide dismutase to the neutrophil suspensions eliminated signals of DMPO-OH, as well as DMPO-OOH. 25 The current studies were not designed to permit conclusions to be drawn about hydroxyl radical formation following fMLP stimulation. This is an area of obvious interest, and additional experimentation is required.

7. 8.

9. 10. 11.

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Acknowledgements - - The National Biomedical Center for Spin Trapping and Free Radicals is supported by the Biomedical Research Technology Program of the National Center for Research Resources in the National Institute of Health; Grant #RR0 551701A 1. Partial support for this work was provided by the grant from the Oklahoma Center for Advancement of Science and Technology (HR2-093). The authors thank Danny R. Moore for his excellent technical assistance.

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