Biochimica et Biophysica Acta, 1052(1990) 379-385
379
Elsevier BBAMCR12694
Superoxide enhances hypochlorous acid production by stimulated human neutrophils Anthony J. Kettle and Christine C. Winterboum Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, Christchurch (New Zealand)
(Received8 November1989)
Key words: Hypoeb_lorousacid; Myeloperoxidase;Superoxide;(Humanneutrophils)
Stimulated neutrophils undergo a respiratory burst discharging large quantifies of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and C!- to hypochlorous acid. Human neutrophfls stimulated with opsonized zymosan promoted the loss of monochiorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in O--free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalasc, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorboi myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.
Introduction When stimulated, neutrophils discharge large quantities of highly reactive species of oxygen which undoubtably play a pivotal role in inflammatory tissue damage [1,2]. Superoxide (02) is the primary radical formed by the respiratory burst oxidase of neutrophils [3] but it is unlikely to damage tissue directly because of its limited reactivity. Much attention has been focused on the hydroxyl radical ('OH) as the damaging oxidant in inflammation. It is extremely reactive, and is formed through the interaction of O~- and H20 2 in the metalcatalysed Haber-Weiss reaction [4]. Recent work, how-
Abbreviations: 02, superoxide; HOCI; hypoehlorous acid; H202, hydrogen peroxide; "OH, hydroxyl radical; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaaceticacid; Hepes, N-2-hydroxyethylpiperazine-n'-2-ethanesulfonic acid; Me2SO, dimethylsulphoxide; OZ, opsonizedzymosan;PBS,phosphate-bufferedsaline; PMA, phorbol myristate acetate; PMN, polymorphonuclearnew trophils. Correspondence: A.J. Kettle, Departmentof Pathology,Christchurch School of Medicine,ChristchurchHospital, Christchurch,New Zealand.
ever, has shed considerable doubt on the capability of neutrophils to produce "OH [5-7] and its contribution to the inflammatory response has been re-evaluated [8]. A second likely candidate is the potent oxidant hypochlorous acid (HOC1). It reacts readily with biological molecules, inactivating al-proteinase inhibitor, other neutrophil enzymes and inflammatory molecules, and is cytotoxic to a wide variety of mammalian cells (reviewed in Ref. 1). Since HOC1 can account for at least 30% of the 0 2 generated during the respiratory burst [9,10], it may be responsible for the majority of oxidant damage mediated by neutrophils. Myeloperoxidase, the most abundant neutrophil protein, catalyses the production of HOCI from H20 2 and C1- [11]. H20 2 is supplied as a secondary product by the respiratory burst through the dismutation of 0 2 [3]. However, O~- also reacts directly with myeloperoxidase [12] and modulates the chlorinating activity of the purified enzyme [13,14]. Under conditions where inactive compound II of myeloperoxidase accumulates, O~- can treble the production of HOC1 by reducing compound II back to active ferric myeloperoxidase [13]. This result leads to the interesting possibility that 0 2 may exert a deleterious effect in inflammation by boosting myeloperoxidase-dependent tissue damage.
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)
380 Although this mechanism has been demonstrated with the purified enzyme [13,14], there is no evidence from previous studies [9,15,16] that it operates in the neutrophil. We have therefore examined in detail the effect of 0 2 on HOC1 production by neutrophils stimulated with opsonized zymosan and phorbol myristate acetate (PMA). Materials and Methods
Materials Neutrophils were isolated from the blood of healthy donors by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of contaminating red cells [17]. Myeloperoxidase was purified from human neutrophils to a purity index (A43o/A2so) of more than 0.7, and its concentration was determined using c430 = 91000 M -1. cm -1 [18]. Zymosan was boiled for 20 min, washed twice in 10 mM phosphate buffer (pH 7.5) containing 0.138 mM NaC1 and 10 mM KC1 (PBS), and incubated at 5 mg. ml -a in 30% human plasma with end over end rotation for 30 rain at 37 o C. The opsonized zymosan was then washed twice with PBS and suspended at 50 nag. ml-1. Superoxide dismutase (SOD), catalase, monochlorodimedon, zymosan, phorbol myrisate acetate (PMA) and were purchased from Sigma. SOD was free of catalase activity. This was demonstrated by showing that 20 /~g. m1-1 of SOD was unable to degrade 100 #M H202, as measured with a H202 electrode [14]. All other chemicals were of the highest grade available.
Production of hypochlorous acid by neutrophils stimulated with opsonized zymosan Reactions were carried out in PBS containing 1 mM CaC12, 0.5 mM MgC12 and 1 mg.m1-1 of glucose. When incubations were carried out in the absence of CI-, the buffer was 10 mM phosphate (pH 7.5), containing 1.0 mM MgSO4, 47 mM Na2SO4 and 1 mg. nil-~ of glucose. Neutrophils were incubated in a total volume of 1, ml in 1.5 ml sealed microfuge tubes with end over end rotation for 15 minutes at 37°C. At the end of this period tubes were placed in melting ice for 10 minutes and centrifuged to pellet neutrophils and zymosan. The production of HOC1 was based on the loss of monochlorodimedon. This was determined by measuring the difference between A290 (E29019000 M-1 • cm -1) [19] of the supernatant of reaction mixtures with MCD only, and those with MCD plus stimulated cells. This value was adjusted for the contribution of proteins released by stimulated cells, and for the addition of exogenous proteins, by measuring AA290 in the absence of monochlorodimedon (see Table I). Myeloperoxidase was assayed in supernatants using otolidine [20].
Production of hypochlorous acid by neutrophils stimulated with PMA Neutrophils were incubated in PBS, or PBS containing 10 mM Hepes (pH 7.5), with 200/~M monochlorodimedon, and 100 nM myeloperoxidase in a total volume of 1 ml in 1.5 ml microfuge tubes at 37°C. Cells were stimulated with 100 ng of PMA in dimethylsulfoxide (Me2SO). At a prescribed time reactions were stopped by adding 125 #g. m1-1 of catalase and the tubes were placed in melting ice for 10 min, then centrifuged to pellet the neutrophils. The supernatants were diluted 1:1 in PBS and the loss of monochlorodimedon was calculated by measuring AA290between supernatants from unstimulated and stimulated cells. AA29o was corrected for the contribution made by proteins released from stimulated cells (typically 0.010). With 10 mM Hepes and 200 /xM monochlorodimedon 53.6% of reagent HOC1 was found to react with monochlorodimedon. Therefore in all experiments with neutrophils stimulated in Hepes buffer AA29o was corrected accordingly to account for the scavenging of HOC1 by Hepes.
The chlorination assay for purified myeloperoxidase The chlorinating activity of purified myeloperoxidase (100 nM) was determined at 25°C by measuring the rate of loss of monochlorodimedon at 290 nm with 100 #M monochlorodimedon and 200 #M H202.
Superoxide production O f production was measured as SOD-inhibitable cytochrome c reduction (%50 ~r~a,c,.a-o,ad~.a)21 100 M -1. cm -1) [21]. Neutrophils stimulated with opsonized zymosan were incubated with 250 ~tM cytochrome c and 100 #g. m1-1 of catalase plus or minus 20/~g. ml -~ of SOD. After 15 min at 37 o C, neutrophils and zymosan were pelleted and AA550 of the supematants were measured. With PMA stimulated cells, 1 • 106 cells, ml- 1 were incubated with 100 #M cytochrome c and 100 #g. ml -a of catalase. Cells were stimulated with 100 ng-m1-1 of PMA in Me2SO and A550 was recorded continuously. Results
Production of hypochlorous acid by neutrophils stimulated with opsonized zymosan The rapid reaction of monochlorodimedon with HOC1 to form dichlorodimedon [19] was used to determine the activity of neutrophil myeloperoxidase. Neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon, whereas unstimulated cells did not (Table I). Since catalase in the medium inhibited totally the loss of monochlorodimedon (see below) and little catalase is taken up into phagocytic vacuoles [22], most of the monochlorodimedon loss was due to extracellular production of HOC1.
381 TABLE I
Loss of monochlorodimedon mediated by neutrophils stimulated with opsonized zymosan 4 × 106 Neutrophils (PMN) were incubated with 5 nag of opsonized z y m o s a n (OZ) and monochlorodimedon ( M C D ) in 1 ml at 37 ° C for 15 min. AA29o represent the combination of (a) a decrease due to monoehlorodimedon loss a n d (b) an increase due to proteins released from the cells (0.055 for stimulated cells, zero for resting cells). To calculate the loss of monochlorodimedon, 0.055 was added to the AA29o values shown in the table for stimulated cells. With 200/~M monoehlorodimedon supernatants were diluted 1 : 1 with PBS before reading Az90. O~- production was measured in the presence of monoclalorodimedon by the discontinuous cytochrome c assay. Values represent m e a n s and ranges of duplicate experiments. Conditions
A 290
A A290 (No P M N - P M N )
Loss of M C D (#M)
O~- Production (btM/15 rain)
70/~M M C D 70/~M M C D + P M N 70 v M M C D + P M N + OZ 200/~M M C D 200/~M M C D + P M N + OZ PMN P M N + OZ
1.330+0.002 1.313 + 0.010 0.896 + 0.018 3.838 + 0.017 3.482 + 0.021 0.000 0.055
0.017 + 0.012 0.434 + 0.020 0.356 + 0.038 -
0.9 + 0.6 25.7 + 1.3 21.6 + 2.3 -
180 + 1 179 + 2 175 + 2
O~- production was measured with cytochrome c which detects only extracellular O~-. On this basis, assuming that two molecules of O~- give rise to one molecule of H202, and consequently one molecule of HOC1, the loss of monochlorodimedon corresponds to approx. 30% conversion of extracellular generated O~- to HOC1. Both the stoichiometry of this reaction, and the amount of O~- produced were independent of the concentration of monochlorodimedon (Table I).
Effects of superoxide dismutase and catalase When neutrophils were stimulated with opsonized zymosan, catalase and SOD inhibited the loss of monochlorodimedon by 93 and 73% respectively (Fig. 1). The effects of these proteins were due to their enzymatic activity since the heat-inactivated enzymes and bovine serum albumin were ineffective.
30 PMN
H.I. CAT
25
a
BSA H.I.
SOD
5 0
•
"
f
o --, r-.. u i •
0
.-I
300
z
20
b_
0
Increasing the concentration of opsonized zymosan from 0 to 5 mg. ml- ] resulted in increased rates of O~production and increased release of myeloperoxidase, until the response was saturated at 3 mg. ml-1. Results of typical experiment are given in Fig. 2. O~- production showed a similar dependence on the concentration of opsonized zymosan with other neutrophil preparations, but the maximum amount produced in 15 min ranged between 120 and 180 gM. The loss of monochlorodimedon in the absence of SOD was directly proportional to the production of O~- (Fig. 3), which was used as a measure of total oxidant (O~- + H202) production. In the presence of SOD the loss of MCD plateaued, so that SOD gave greatest inhibition at the highest rates of O~- production but did not inhibit at lower rates. SOD did not affect the release of myeloperoxidase (Fig. 2). Thus, it can be concluded that in the presence of SOD the increased production of H202 eventually saturated the activity of myeloperoxidase. However, in the absence of SOD, O~- was responsible for maintaining the
®
Fig. 1. The effects of catalase and superoxide dismutase on the loss of monochlorodimedon mediated by neutrophils stimulated with opsonized zymosan. Neutrophils (4.106 ml - l ) were incubated with 5 m g . m 1 - 1 of opsonized zymosan a n d 7 0 / x M monochlorodimedon in a total volume of 1 ml at 3 7 ° C for 15 rain. Concentrations of catalase (CAT), SOD and bovine serum albumin (BSA) were 100/~g.m1-1, 20 /~g.m1-1 and 100 # g - m l -~, respectively. Enzymes were heat inactivated (H.I.) by boiling for 15 min. Superoxide production was 170 # M in 15 rain. Values represent m e a n s and ranges of duplicate experiments.
250
--
"--~--
0.15
24 /
~ "
379
Elsevier BBAMCR12694
Superoxide enhances hypochlorous acid production by stimulated human neutrophils Anthony J. Kettle and Christine C. Winterboum Department of Pathology, Christchurch School of Medicine, Christchurch Hospital, Christchurch (New Zealand)
(Received8 November1989)
Key words: Hypoeb_lorousacid; Myeloperoxidase;Superoxide;(Humanneutrophils)
Stimulated neutrophils undergo a respiratory burst discharging large quantifies of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and C!- to hypochlorous acid. Human neutrophfls stimulated with opsonized zymosan promoted the loss of monochiorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in O--free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalasc, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorboi myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.
Introduction When stimulated, neutrophils discharge large quantities of highly reactive species of oxygen which undoubtably play a pivotal role in inflammatory tissue damage [1,2]. Superoxide (02) is the primary radical formed by the respiratory burst oxidase of neutrophils [3] but it is unlikely to damage tissue directly because of its limited reactivity. Much attention has been focused on the hydroxyl radical ('OH) as the damaging oxidant in inflammation. It is extremely reactive, and is formed through the interaction of O~- and H20 2 in the metalcatalysed Haber-Weiss reaction [4]. Recent work, how-
Abbreviations: 02, superoxide; HOCI; hypoehlorous acid; H202, hydrogen peroxide; "OH, hydroxyl radical; SOD, superoxide dismutase; DTPA, diethylenetriaminepentaaceticacid; Hepes, N-2-hydroxyethylpiperazine-n'-2-ethanesulfonic acid; Me2SO, dimethylsulphoxide; OZ, opsonizedzymosan;PBS,phosphate-bufferedsaline; PMA, phorbol myristate acetate; PMN, polymorphonuclearnew trophils. Correspondence: A.J. Kettle, Departmentof Pathology,Christchurch School of Medicine,ChristchurchHospital, Christchurch,New Zealand.
ever, has shed considerable doubt on the capability of neutrophils to produce "OH [5-7] and its contribution to the inflammatory response has been re-evaluated [8]. A second likely candidate is the potent oxidant hypochlorous acid (HOC1). It reacts readily with biological molecules, inactivating al-proteinase inhibitor, other neutrophil enzymes and inflammatory molecules, and is cytotoxic to a wide variety of mammalian cells (reviewed in Ref. 1). Since HOC1 can account for at least 30% of the 0 2 generated during the respiratory burst [9,10], it may be responsible for the majority of oxidant damage mediated by neutrophils. Myeloperoxidase, the most abundant neutrophil protein, catalyses the production of HOCI from H20 2 and C1- [11]. H20 2 is supplied as a secondary product by the respiratory burst through the dismutation of 0 2 [3]. However, O~- also reacts directly with myeloperoxidase [12] and modulates the chlorinating activity of the purified enzyme [13,14]. Under conditions where inactive compound II of myeloperoxidase accumulates, O~- can treble the production of HOC1 by reducing compound II back to active ferric myeloperoxidase [13]. This result leads to the interesting possibility that 0 2 may exert a deleterious effect in inflammation by boosting myeloperoxidase-dependent tissue damage.
0167-4889/90/$03.50 © 1990 ElsevierSciencePublishersB.V.(BiomedicalDivision)
380 Although this mechanism has been demonstrated with the purified enzyme [13,14], there is no evidence from previous studies [9,15,16] that it operates in the neutrophil. We have therefore examined in detail the effect of 0 2 on HOC1 production by neutrophils stimulated with opsonized zymosan and phorbol myristate acetate (PMA). Materials and Methods
Materials Neutrophils were isolated from the blood of healthy donors by Ficoll-Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of contaminating red cells [17]. Myeloperoxidase was purified from human neutrophils to a purity index (A43o/A2so) of more than 0.7, and its concentration was determined using c430 = 91000 M -1. cm -1 [18]. Zymosan was boiled for 20 min, washed twice in 10 mM phosphate buffer (pH 7.5) containing 0.138 mM NaC1 and 10 mM KC1 (PBS), and incubated at 5 mg. ml -a in 30% human plasma with end over end rotation for 30 rain at 37 o C. The opsonized zymosan was then washed twice with PBS and suspended at 50 nag. ml-1. Superoxide dismutase (SOD), catalase, monochlorodimedon, zymosan, phorbol myrisate acetate (PMA) and were purchased from Sigma. SOD was free of catalase activity. This was demonstrated by showing that 20 /~g. m1-1 of SOD was unable to degrade 100 #M H202, as measured with a H202 electrode [14]. All other chemicals were of the highest grade available.
Production of hypochlorous acid by neutrophils stimulated with opsonized zymosan Reactions were carried out in PBS containing 1 mM CaC12, 0.5 mM MgC12 and 1 mg.m1-1 of glucose. When incubations were carried out in the absence of CI-, the buffer was 10 mM phosphate (pH 7.5), containing 1.0 mM MgSO4, 47 mM Na2SO4 and 1 mg. nil-~ of glucose. Neutrophils were incubated in a total volume of 1, ml in 1.5 ml sealed microfuge tubes with end over end rotation for 15 minutes at 37°C. At the end of this period tubes were placed in melting ice for 10 minutes and centrifuged to pellet neutrophils and zymosan. The production of HOC1 was based on the loss of monochlorodimedon. This was determined by measuring the difference between A290 (E29019000 M-1 • cm -1) [19] of the supernatant of reaction mixtures with MCD only, and those with MCD plus stimulated cells. This value was adjusted for the contribution of proteins released by stimulated cells, and for the addition of exogenous proteins, by measuring AA290 in the absence of monochlorodimedon (see Table I). Myeloperoxidase was assayed in supernatants using otolidine [20].
Production of hypochlorous acid by neutrophils stimulated with PMA Neutrophils were incubated in PBS, or PBS containing 10 mM Hepes (pH 7.5), with 200/~M monochlorodimedon, and 100 nM myeloperoxidase in a total volume of 1 ml in 1.5 ml microfuge tubes at 37°C. Cells were stimulated with 100 ng of PMA in dimethylsulfoxide (Me2SO). At a prescribed time reactions were stopped by adding 125 #g. m1-1 of catalase and the tubes were placed in melting ice for 10 min, then centrifuged to pellet the neutrophils. The supernatants were diluted 1:1 in PBS and the loss of monochlorodimedon was calculated by measuring AA290between supernatants from unstimulated and stimulated cells. AA29o was corrected for the contribution made by proteins released from stimulated cells (typically 0.010). With 10 mM Hepes and 200 /xM monochlorodimedon 53.6% of reagent HOC1 was found to react with monochlorodimedon. Therefore in all experiments with neutrophils stimulated in Hepes buffer AA29o was corrected accordingly to account for the scavenging of HOC1 by Hepes.
The chlorination assay for purified myeloperoxidase The chlorinating activity of purified myeloperoxidase (100 nM) was determined at 25°C by measuring the rate of loss of monochlorodimedon at 290 nm with 100 #M monochlorodimedon and 200 #M H202.
Superoxide production O f production was measured as SOD-inhibitable cytochrome c reduction (%50 ~r~a,c,.a-o,ad~.a)21 100 M -1. cm -1) [21]. Neutrophils stimulated with opsonized zymosan were incubated with 250 ~tM cytochrome c and 100 #g. m1-1 of catalase plus or minus 20/~g. ml -~ of SOD. After 15 min at 37 o C, neutrophils and zymosan were pelleted and AA550 of the supematants were measured. With PMA stimulated cells, 1 • 106 cells, ml- 1 were incubated with 100 #M cytochrome c and 100 #g. ml -a of catalase. Cells were stimulated with 100 ng-m1-1 of PMA in Me2SO and A550 was recorded continuously. Results
Production of hypochlorous acid by neutrophils stimulated with opsonized zymosan The rapid reaction of monochlorodimedon with HOC1 to form dichlorodimedon [19] was used to determine the activity of neutrophil myeloperoxidase. Neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon, whereas unstimulated cells did not (Table I). Since catalase in the medium inhibited totally the loss of monochlorodimedon (see below) and little catalase is taken up into phagocytic vacuoles [22], most of the monochlorodimedon loss was due to extracellular production of HOC1.
381 TABLE I
Loss of monochlorodimedon mediated by neutrophils stimulated with opsonized zymosan 4 × 106 Neutrophils (PMN) were incubated with 5 nag of opsonized z y m o s a n (OZ) and monochlorodimedon ( M C D ) in 1 ml at 37 ° C for 15 min. AA29o represent the combination of (a) a decrease due to monoehlorodimedon loss a n d (b) an increase due to proteins released from the cells (0.055 for stimulated cells, zero for resting cells). To calculate the loss of monochlorodimedon, 0.055 was added to the AA29o values shown in the table for stimulated cells. With 200/~M monoehlorodimedon supernatants were diluted 1 : 1 with PBS before reading Az90. O~- production was measured in the presence of monoclalorodimedon by the discontinuous cytochrome c assay. Values represent m e a n s and ranges of duplicate experiments. Conditions
A 290
A A290 (No P M N - P M N )
Loss of M C D (#M)
O~- Production (btM/15 rain)
70/~M M C D 70/~M M C D + P M N 70 v M M C D + P M N + OZ 200/~M M C D 200/~M M C D + P M N + OZ PMN P M N + OZ
1.330+0.002 1.313 + 0.010 0.896 + 0.018 3.838 + 0.017 3.482 + 0.021 0.000 0.055
0.017 + 0.012 0.434 + 0.020 0.356 + 0.038 -
0.9 + 0.6 25.7 + 1.3 21.6 + 2.3 -
180 + 1 179 + 2 175 + 2
O~- production was measured with cytochrome c which detects only extracellular O~-. On this basis, assuming that two molecules of O~- give rise to one molecule of H202, and consequently one molecule of HOC1, the loss of monochlorodimedon corresponds to approx. 30% conversion of extracellular generated O~- to HOC1. Both the stoichiometry of this reaction, and the amount of O~- produced were independent of the concentration of monochlorodimedon (Table I).
Effects of superoxide dismutase and catalase When neutrophils were stimulated with opsonized zymosan, catalase and SOD inhibited the loss of monochlorodimedon by 93 and 73% respectively (Fig. 1). The effects of these proteins were due to their enzymatic activity since the heat-inactivated enzymes and bovine serum albumin were ineffective.
30 PMN
H.I. CAT
25
a
BSA H.I.
SOD
5 0
•
"
f
o --, r-.. u i •
0
.-I
300
z
20
b_
0
Increasing the concentration of opsonized zymosan from 0 to 5 mg. ml- ] resulted in increased rates of O~production and increased release of myeloperoxidase, until the response was saturated at 3 mg. ml-1. Results of typical experiment are given in Fig. 2. O~- production showed a similar dependence on the concentration of opsonized zymosan with other neutrophil preparations, but the maximum amount produced in 15 min ranged between 120 and 180 gM. The loss of monochlorodimedon in the absence of SOD was directly proportional to the production of O~- (Fig. 3), which was used as a measure of total oxidant (O~- + H202) production. In the presence of SOD the loss of MCD plateaued, so that SOD gave greatest inhibition at the highest rates of O~- production but did not inhibit at lower rates. SOD did not affect the release of myeloperoxidase (Fig. 2). Thus, it can be concluded that in the presence of SOD the increased production of H202 eventually saturated the activity of myeloperoxidase. However, in the absence of SOD, O~- was responsible for maintaining the
®
Fig. 1. The effects of catalase and superoxide dismutase on the loss of monochlorodimedon mediated by neutrophils stimulated with opsonized zymosan. Neutrophils (4.106 ml - l ) were incubated with 5 m g . m 1 - 1 of opsonized zymosan a n d 7 0 / x M monochlorodimedon in a total volume of 1 ml at 3 7 ° C for 15 rain. Concentrations of catalase (CAT), SOD and bovine serum albumin (BSA) were 100/~g.m1-1, 20 /~g.m1-1 and 100 # g - m l -~, respectively. Enzymes were heat inactivated (H.I.) by boiling for 15 min. Superoxide production was 170 # M in 15 rain. Values represent m e a n s and ranges of duplicate experiments.
250
--
"--~--
0.15
24 /
~ "
