Journal of Immtmologu'al Methods, 155 (1992) 49-55
49
.D Iq~2 ElsevierSciencePublishers B.V. All rights re.~rved 0022-1759/92/~'d)5.{10
JIM 06448
A peroxidase-independent method for the quantitation of extraceilular hydrogen peroxide ,generated by activated phagocytes in vitro R. A n d e r s o n Medical Research Unit for the Study of Phagocyte Function, Department of Immunology. tnstimte fin, Pathology, Umrersity of Pretoria, Pretoria. South Africa
(Received 13 December 1991,revisedreceived7 April 1992,accepted 15 May 1992)
A peroxidase-independent method for the determination of phagoojte-derived hydrogen peroxide in vitro is described. The method is based on the catalase-inhibitable oxidation of added cysteine. Human blood neutrophils (1 × 10~/ml) were coincubate6 with the myeloperoxidase (MPO) inhibitor, sodium azide (10/zg/ml), for 30 mia at 37°C followed by addition of phorbol 12-myristate 13-acetate (PMA, l0 ng/ml), a potent activator of membrane-associated oxidative metabolism. After incubation at 37°C the cells were removed and the supernatants aliquoted and incubated with and without catalase (500 U / m i ) for 10 min at room temperature followed by addition of cysteine (50 ~M). Thereafter the catalase-inhibitable, H eOe-mediated oxidation of added eysteine was assayed spectrophotometrically following the addition of the thiol-reactive agent 5,5'-dithiobis-(2-nitrobenzoic acid), and the H 2 0 2 concentration determined using a standard curve constructed from difference data based on the oxidation of cysteine by H , O 2 ( 0 - 2 0 0 nmol). The average amount of H202 produced by l06 PMA-activated neutrophils was 47 :t: 7 nmol/30 min. This sensitive assay procedure is likely to be particularly useful for investigating the effects of pharmacological agents and anti-oxidant nutrients on H202 production by activated phagocytes, since these agents are generally unsuited for use in peroxidase-based assays. Key words: Neutrophil;Phagocyte;Hydrogen peroxide; Supero~idc; NADPH-oxidasc
Introduction Although they are sensitive and useful, existing methods for the detection of micromolar quantities of hydrogen peroxide ( H 2 0 2) released by
Correspondence to: R. Anderson, Institute for Pathology, P.O. Box 2034, Pretoria 0O01.South Africa. Fax: 12-324-41'~86. Abbreuiations: MPO. myeloperoxidase; PMA, phorbol 12myristate 13-acetate, DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); LECL. luminol-enhancedchemiluminescence.
stimulated phagocytes have limited application because they are indirect and dependent on the conversion of H202 by myeloperoxida~ (MPO), or added peroxidase, to secondary, more reactive oxidising agents such as hypohalous acids. These in turn convert added substrates to products which can be detected using spectrofluorometric (Homan-Muller et al., 1975; Root et al, 1975; Nahum et al, 1990), or spectrophotometric methods (Pick and Mizel, 1981). These indirect, peroxidase-dependent methods are clearly unsuitable for the laboratory investigation of the effects of tcst
agents such as drugs or nutrients, which interfere with peroxidase activity and/or interact with hypohalous acids, on the production of H202 by activated phagocytes. For this reason an alternative, peroxidase-independent method of assaying phagocyte.derived H zO 2 has been devised. This method is based on the catalase-inhibitable, H 202-mediated oxidation of cysteine and may be particularly suited to immunopharmacological studies.
Materials and methods
Chemicals and reage:~ts Unless indicated, these were purchased from the Sigma Chemical Co., St. Louis, MO, USA.
Neutrophils Venous blood from adult human volunteers was treated with preservative-free heparin (5 U / ml). Neutrophi!s were separated from mononuclear leucocytes by centrifugation at 400 × g for 15 rain of hcparin-treated blood on cushions of Ficoli (Pharmacia, Uppsala, Sweden) metrizoate. The resultant erythrocyte/neutrophil fraction was sedimented with 3% gelatin for 30 rain at 37°C in order to remove most of the erythrocytes. The neutrophil-containing supernatant was centrifuged at 250 x g for 10 min and the residual erythrocytes in the cell pellet were lysed by exposure to 0.85% ammonium chloride. The neutrophils were centrifuged, washed once and resuspended to 1 × 10~/ml in indicator-free, Hepes (4.2 mM)-buffered Hanks' balanced salt solution (HBSS) pH 7.4 (Anderson et al., ~986). The neutrophils which were routinely of high purity (> 90%) and viability (> 95%) were used within 15 rain of completing the isolation procedure.
Measurement of H20 _, production by neutrophils Neutrophils (10*/ml) were preincubated for 30 rain at 37°C with 10 ~g/ml sodium azide, an inhibitor of MPO (IQebanoff, 1970), which was included to prevent conversion of H 202 to HOCI. in preliminary experiments, ~.hisconcentration (10 /~g/ml) of sodium azidc was found to cause almost complete inhibition of the MPO-mediated luminol-enhanced chemiluminescence responses
of activated neutrophils, while at the final concentration of 5 ~g/ml in the H20 2 assay system this agent did not prevent catalase-mediated elimination of the oxidant. Following preincubation the neutrophils were activated with PMA (10 ng/ml) or opsonised zymosan (500 p,g/ml). The final volume in each tube was 5 ml containing 5 x 10' neutrophils. After 20 min at 37°C the tubes were placed on ice and 10 rain later the neutrophils were removed by centrifugation and the supernatants diluted 1/2 to bring the azide concentration to 5 ~g/ml. The supernatants were then assayed for H202. To 1.8 ml of paired supernatants were added 100 tLI of catalase (from bovine liver, 500 U/ml final concentration) or 100 ~l HBSS, followed after 15 min at room temperature by the addition of 100 p,I cysteine (final concentration 50 p,M). After a further 30 rain period of incubation at room temperature 100 p,I of the thiol-reactive agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 53 /~M final concentration) were added to each tube and the remaining unoxidised cysteine assayed spectrophotometrically at a wavelength of 412 nm (Ellman, 1959; Riddles et al., 1979). The difference (decrease) in OD at 412 nm between matched tubes with and without catalase is attributed to the H202-mediated oxidation of ,~steine. The H20 2 concentration was determined using a standard curve constructed from the difference data based on the oxidation of cysteine by H20 2 (0-200 nmol)+catalase. Standard curves were performed for each experiment. Spontaneous oxidation of cysteine was undetectable during the 30 rain incubation period. In additional experiments the following were also investigated (a) the comparative kinetics of superoxide and H 2 0 , production by PMA-activated neutrophils, (b) the effects of exclusion of sodium azide on the levels of H20 2 in the supernatants of stimulated neutrophils, (c) the effects of inclusion with neutrophils of methionine (1 mM final concentration), a potent scavenger of hypochlorous acid (HOCI) (Winterbourn, 1985) on the oxidation of cysteine, (d) the effects of inclusion of catalase (500 U/ml) during PMA activation of neutrophils, and (e) the effects of the immunosuppressive oral gold compound auranofin [(2,3,4,6-tetra-O-acteyl-I -t hio-/3-D-glucopyranosa-
to-S) (triethylphosphine) gold (I)] at therapeutic (0.5 p.g/ml and i /~g/ml) concentrations (Gottlieb, 1982; Snyder et al., 1987). Auranofin is a potent inhibitor of NADPH-oxidase activity, oxygen consumption and superoxide generation by activated neutrophils (Anderson et al., 1991).
HzO2 scavenging by neutrophils The H20 2 scavenging potential of neutrophils was measured by c~oincubatingunstimulated cells (1 X 10*/ml) with 100 nmol of adl'ed H20 2 for 30 rain at either 4°C or 37°C in HBSS with sodium azide (10 /~g/ml). After incubation the cells were removed by centrifugation and the H20 2 remaining in the supernatants as.~yed as described above and compared with that in the corresponding cell-free, control systems, in additional experiments l-!eOe (100 nmol final concentration) was added to the cell-free supernatant fluids from neutrophils which had been incubated for 30 rain at 4°C or 37°C. After an additional incubation period of 30 rain at 4°C or 37°C the amount of H eOe was assayed as described above.
Measurement of superoxide generation This was assayed by a standard spectrophotometric technique based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c (cyt c) (Curnutte and Babior, 1974). Neutrophils (10') were preincubated for 30 rain at 37°C in 900/~l HBSS containing 100 nmol of horse heart cyt c with and without 100 U / m l of SOD, followed by the addition of 100 p.I of HBSS or PMA (final concentration 10 ng/ml) to control and experimental systems respectively. The tubes were then incubated at 37°C for 1, 5, 10, 15, 20, 30 and 60 min after which the reactions were terminated by the addition of 1 ml of ice-cold HBSS. After removal of the cells by centrifugation the supernatants were assayed spectrophotometrically at a wavelength of 550 nm and the amount of reduced cyt c calculated using an extinction coefficient of 21.1 mM at 550 nm (Van Gelder and Slater, 1962).
ng/ml) in the Wcsence or absence of sodium azide was measured using a luminol (5-amino-2.3-dihydro-l,4-phthalazinedione)-enhanced chcmilumincsecnce (LECL) method (Briheim ct al., 1984). To I ml of supern~-tant fluid containing 0.1 mM luminol was added 20 /zl of glucose oxidase (final concentration 25 mU/ml) and LECL was then recorded in an LKB Wallac 1251 chemiluminometcr (Turku, Finland). LECL readings were integrated for 5 s intervals and recorded as mV-s-~. Background, MPO-free, control terns contained HBSS, luminol and glucose oxidase.
Measurement of 1-1202 preduction by a cell-free, enzymatic glucose ~glucose oxidase system In these experiments 20/~1 of glucose oxidase (12.5, 25, 50, 100 and 200 mU final concentrations) was added to 980/LI of HBSS containing approximately 5 mM glucose. After 60 rain incubation at 37°C the mixtures were diluted 1/2 and 1/10 and the H20 2 concentrations measured as described above, in preliminary kinetic experiments it was establi.~hed that H20 2 generation was almost complete within 60 rain. In .~me experiments catalase (500 U/ml) was included with the enzymatic HzO2-generating system.
Measurement of the cytotoxic potential of sodium azide In these experiments neutrol~hils (2 × 10*/ml) were coincubated with i0 p.g/ml sodium azide at 37°C and the cytotoxic potential of the azide was measured spectrophotometrically according to the extent of extracellular release of cytosolic lactate dehydrogenase (LDH) in neutrophil-free supernatants (Beutler, 1984). Intracellular ATP levels were also measured in control and azide-treated neutrophils as an additional index of cytotoxic potential, using a luciferin/luciferase chemiluminescence method (Holmsen et al., 1972).
Expression of results Measurement of MPO activity MPO activity in the diluted (1/2) supernatant fluids from neutrophils activated with PMA (10
The results of each series of experiments are expressed as the mean values + the standard errors of the means (SEMs).
Results
Assay of HzOa (0-200 nmol) using c~,~talase-inhibitable oxidation of cysteine The results of a typical experiment are shown in Fig. 1. These demonstrate that the method is suitable for the detection of H202 at concentrations as low as 5 nmol.
Measurement of neutrophil-derived HzOz These results are shown in Table I. Activation of neutrophils with opsonised zymosan or PMA increased measurable H 2 0 2 from < 1 nmol to 4 3 ± 12 and 4 7 ± 7 nmol H202/106 neutro phils/20 rain which is well within the detection limits of the assay. The levels of H 2 0 2 in the supernatants of PMA-stimulated neutrophils were markedly decreased when sodium azide was omitted, presumably as a consequence of MPO-mediated conversion of H 2 0 2 to HOCI (Table I). Inclusion of the H O C | scavenger methionine with azide-treated, PMA-activated neutrophils had no detectable effects on the cysteine-oxidation based assay system, confirming that the azide effectively btocked conversion of H202 to HOCI, while inclusion of catalase neutralised the phagocyte-derived H 2 0 2 (Table I). Inclusion of auranofin (0.5 and 1 p,g/ml) with neutrophils inhibited PMA-
TABLE 1 MEASUREMENT OF H202 GENERATION BY OPSONISED ZYMOSAN- AND PMA-ACTIVATED NEUTROPHILS AND THE EFFECTS OF INCLUSION OF AZIDE, CATALASE,METHIONINE AND AURANOFIN
(I) Neutrophilsonly (2) Neutrophils-~azide (10 pg/ml) (3) Neutrophils+ azide (4) Neutrophilsonly (5) Neutrophils+ catalas¢ (500 U/ml) (6) Neutrophi|s+ azide (7) Neutrophils+ azide + methionine (i mM) (8) NeutTophils+ azide + auranofin (0.5 p,g/ml) (9) Neutrophils+ azide + auranofin (I p,g/ml)
Stimulant
H202 (p,M)
None
< 1a
None Opsonised zymosan PMA
49
.D Iq~2 ElsevierSciencePublishers B.V. All rights re.~rved 0022-1759/92/~'d)5.{10
JIM 06448
A peroxidase-independent method for the quantitation of extraceilular hydrogen peroxide ,generated by activated phagocytes in vitro R. A n d e r s o n Medical Research Unit for the Study of Phagocyte Function, Department of Immunology. tnstimte fin, Pathology, Umrersity of Pretoria, Pretoria. South Africa
(Received 13 December 1991,revisedreceived7 April 1992,accepted 15 May 1992)
A peroxidase-independent method for the determination of phagoojte-derived hydrogen peroxide in vitro is described. The method is based on the catalase-inhibitable oxidation of added cysteine. Human blood neutrophils (1 × 10~/ml) were coincubate6 with the myeloperoxidase (MPO) inhibitor, sodium azide (10/zg/ml), for 30 mia at 37°C followed by addition of phorbol 12-myristate 13-acetate (PMA, l0 ng/ml), a potent activator of membrane-associated oxidative metabolism. After incubation at 37°C the cells were removed and the supernatants aliquoted and incubated with and without catalase (500 U / m i ) for 10 min at room temperature followed by addition of cysteine (50 ~M). Thereafter the catalase-inhibitable, H eOe-mediated oxidation of added eysteine was assayed spectrophotometrically following the addition of the thiol-reactive agent 5,5'-dithiobis-(2-nitrobenzoic acid), and the H 2 0 2 concentration determined using a standard curve constructed from difference data based on the oxidation of cysteine by H , O 2 ( 0 - 2 0 0 nmol). The average amount of H202 produced by l06 PMA-activated neutrophils was 47 :t: 7 nmol/30 min. This sensitive assay procedure is likely to be particularly useful for investigating the effects of pharmacological agents and anti-oxidant nutrients on H202 production by activated phagocytes, since these agents are generally unsuited for use in peroxidase-based assays. Key words: Neutrophil;Phagocyte;Hydrogen peroxide; Supero~idc; NADPH-oxidasc
Introduction Although they are sensitive and useful, existing methods for the detection of micromolar quantities of hydrogen peroxide ( H 2 0 2) released by
Correspondence to: R. Anderson, Institute for Pathology, P.O. Box 2034, Pretoria 0O01.South Africa. Fax: 12-324-41'~86. Abbreuiations: MPO. myeloperoxidase; PMA, phorbol 12myristate 13-acetate, DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); LECL. luminol-enhancedchemiluminescence.
stimulated phagocytes have limited application because they are indirect and dependent on the conversion of H202 by myeloperoxida~ (MPO), or added peroxidase, to secondary, more reactive oxidising agents such as hypohalous acids. These in turn convert added substrates to products which can be detected using spectrofluorometric (Homan-Muller et al., 1975; Root et al, 1975; Nahum et al, 1990), or spectrophotometric methods (Pick and Mizel, 1981). These indirect, peroxidase-dependent methods are clearly unsuitable for the laboratory investigation of the effects of tcst
agents such as drugs or nutrients, which interfere with peroxidase activity and/or interact with hypohalous acids, on the production of H202 by activated phagocytes. For this reason an alternative, peroxidase-independent method of assaying phagocyte.derived H zO 2 has been devised. This method is based on the catalase-inhibitable, H 202-mediated oxidation of cysteine and may be particularly suited to immunopharmacological studies.
Materials and methods
Chemicals and reage:~ts Unless indicated, these were purchased from the Sigma Chemical Co., St. Louis, MO, USA.
Neutrophils Venous blood from adult human volunteers was treated with preservative-free heparin (5 U / ml). Neutrophi!s were separated from mononuclear leucocytes by centrifugation at 400 × g for 15 rain of hcparin-treated blood on cushions of Ficoli (Pharmacia, Uppsala, Sweden) metrizoate. The resultant erythrocyte/neutrophil fraction was sedimented with 3% gelatin for 30 rain at 37°C in order to remove most of the erythrocytes. The neutrophil-containing supernatant was centrifuged at 250 x g for 10 min and the residual erythrocytes in the cell pellet were lysed by exposure to 0.85% ammonium chloride. The neutrophils were centrifuged, washed once and resuspended to 1 × 10~/ml in indicator-free, Hepes (4.2 mM)-buffered Hanks' balanced salt solution (HBSS) pH 7.4 (Anderson et al., ~986). The neutrophils which were routinely of high purity (> 90%) and viability (> 95%) were used within 15 rain of completing the isolation procedure.
Measurement of H20 _, production by neutrophils Neutrophils (10*/ml) were preincubated for 30 rain at 37°C with 10 ~g/ml sodium azide, an inhibitor of MPO (IQebanoff, 1970), which was included to prevent conversion of H 202 to HOCI. in preliminary experiments, ~.hisconcentration (10 /~g/ml) of sodium azidc was found to cause almost complete inhibition of the MPO-mediated luminol-enhanced chemiluminescence responses
of activated neutrophils, while at the final concentration of 5 ~g/ml in the H20 2 assay system this agent did not prevent catalase-mediated elimination of the oxidant. Following preincubation the neutrophils were activated with PMA (10 ng/ml) or opsonised zymosan (500 p,g/ml). The final volume in each tube was 5 ml containing 5 x 10' neutrophils. After 20 min at 37°C the tubes were placed on ice and 10 rain later the neutrophils were removed by centrifugation and the supernatants diluted 1/2 to bring the azide concentration to 5 ~g/ml. The supernatants were then assayed for H202. To 1.8 ml of paired supernatants were added 100 tLI of catalase (from bovine liver, 500 U/ml final concentration) or 100 ~l HBSS, followed after 15 min at room temperature by the addition of 100 p,I cysteine (final concentration 50 p,M). After a further 30 rain period of incubation at room temperature 100 p,I of the thiol-reactive agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, 53 /~M final concentration) were added to each tube and the remaining unoxidised cysteine assayed spectrophotometrically at a wavelength of 412 nm (Ellman, 1959; Riddles et al., 1979). The difference (decrease) in OD at 412 nm between matched tubes with and without catalase is attributed to the H202-mediated oxidation of ,~steine. The H20 2 concentration was determined using a standard curve constructed from the difference data based on the oxidation of cysteine by H20 2 (0-200 nmol)+catalase. Standard curves were performed for each experiment. Spontaneous oxidation of cysteine was undetectable during the 30 rain incubation period. In additional experiments the following were also investigated (a) the comparative kinetics of superoxide and H 2 0 , production by PMA-activated neutrophils, (b) the effects of exclusion of sodium azide on the levels of H20 2 in the supernatants of stimulated neutrophils, (c) the effects of inclusion with neutrophils of methionine (1 mM final concentration), a potent scavenger of hypochlorous acid (HOCI) (Winterbourn, 1985) on the oxidation of cysteine, (d) the effects of inclusion of catalase (500 U/ml) during PMA activation of neutrophils, and (e) the effects of the immunosuppressive oral gold compound auranofin [(2,3,4,6-tetra-O-acteyl-I -t hio-/3-D-glucopyranosa-
to-S) (triethylphosphine) gold (I)] at therapeutic (0.5 p.g/ml and i /~g/ml) concentrations (Gottlieb, 1982; Snyder et al., 1987). Auranofin is a potent inhibitor of NADPH-oxidase activity, oxygen consumption and superoxide generation by activated neutrophils (Anderson et al., 1991).
HzO2 scavenging by neutrophils The H20 2 scavenging potential of neutrophils was measured by c~oincubatingunstimulated cells (1 X 10*/ml) with 100 nmol of adl'ed H20 2 for 30 rain at either 4°C or 37°C in HBSS with sodium azide (10 /~g/ml). After incubation the cells were removed by centrifugation and the H20 2 remaining in the supernatants as.~yed as described above and compared with that in the corresponding cell-free, control systems, in additional experiments l-!eOe (100 nmol final concentration) was added to the cell-free supernatant fluids from neutrophils which had been incubated for 30 rain at 4°C or 37°C. After an additional incubation period of 30 rain at 4°C or 37°C the amount of H eOe was assayed as described above.
Measurement of superoxide generation This was assayed by a standard spectrophotometric technique based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c (cyt c) (Curnutte and Babior, 1974). Neutrophils (10') were preincubated for 30 rain at 37°C in 900/~l HBSS containing 100 nmol of horse heart cyt c with and without 100 U / m l of SOD, followed by the addition of 100 p.I of HBSS or PMA (final concentration 10 ng/ml) to control and experimental systems respectively. The tubes were then incubated at 37°C for 1, 5, 10, 15, 20, 30 and 60 min after which the reactions were terminated by the addition of 1 ml of ice-cold HBSS. After removal of the cells by centrifugation the supernatants were assayed spectrophotometrically at a wavelength of 550 nm and the amount of reduced cyt c calculated using an extinction coefficient of 21.1 mM at 550 nm (Van Gelder and Slater, 1962).
ng/ml) in the Wcsence or absence of sodium azide was measured using a luminol (5-amino-2.3-dihydro-l,4-phthalazinedione)-enhanced chcmilumincsecnce (LECL) method (Briheim ct al., 1984). To I ml of supern~-tant fluid containing 0.1 mM luminol was added 20 /zl of glucose oxidase (final concentration 25 mU/ml) and LECL was then recorded in an LKB Wallac 1251 chemiluminometcr (Turku, Finland). LECL readings were integrated for 5 s intervals and recorded as mV-s-~. Background, MPO-free, control terns contained HBSS, luminol and glucose oxidase.
Measurement of 1-1202 preduction by a cell-free, enzymatic glucose ~glucose oxidase system In these experiments 20/~1 of glucose oxidase (12.5, 25, 50, 100 and 200 mU final concentrations) was added to 980/LI of HBSS containing approximately 5 mM glucose. After 60 rain incubation at 37°C the mixtures were diluted 1/2 and 1/10 and the H20 2 concentrations measured as described above, in preliminary kinetic experiments it was establi.~hed that H20 2 generation was almost complete within 60 rain. In .~me experiments catalase (500 U/ml) was included with the enzymatic HzO2-generating system.
Measurement of the cytotoxic potential of sodium azide In these experiments neutrol~hils (2 × 10*/ml) were coincubated with i0 p.g/ml sodium azide at 37°C and the cytotoxic potential of the azide was measured spectrophotometrically according to the extent of extracellular release of cytosolic lactate dehydrogenase (LDH) in neutrophil-free supernatants (Beutler, 1984). Intracellular ATP levels were also measured in control and azide-treated neutrophils as an additional index of cytotoxic potential, using a luciferin/luciferase chemiluminescence method (Holmsen et al., 1972).
Expression of results Measurement of MPO activity MPO activity in the diluted (1/2) supernatant fluids from neutrophils activated with PMA (10
The results of each series of experiments are expressed as the mean values + the standard errors of the means (SEMs).
Results
Assay of HzOa (0-200 nmol) using c~,~talase-inhibitable oxidation of cysteine The results of a typical experiment are shown in Fig. 1. These demonstrate that the method is suitable for the detection of H202 at concentrations as low as 5 nmol.
Measurement of neutrophil-derived HzOz These results are shown in Table I. Activation of neutrophils with opsonised zymosan or PMA increased measurable H 2 0 2 from < 1 nmol to 4 3 ± 12 and 4 7 ± 7 nmol H202/106 neutro phils/20 rain which is well within the detection limits of the assay. The levels of H 2 0 2 in the supernatants of PMA-stimulated neutrophils were markedly decreased when sodium azide was omitted, presumably as a consequence of MPO-mediated conversion of H 2 0 2 to HOCI (Table I). Inclusion of the H O C | scavenger methionine with azide-treated, PMA-activated neutrophils had no detectable effects on the cysteine-oxidation based assay system, confirming that the azide effectively btocked conversion of H202 to HOCI, while inclusion of catalase neutralised the phagocyte-derived H 2 0 2 (Table I). Inclusion of auranofin (0.5 and 1 p,g/ml) with neutrophils inhibited PMA-
TABLE 1 MEASUREMENT OF H202 GENERATION BY OPSONISED ZYMOSAN- AND PMA-ACTIVATED NEUTROPHILS AND THE EFFECTS OF INCLUSION OF AZIDE, CATALASE,METHIONINE AND AURANOFIN
(I) Neutrophilsonly (2) Neutrophils-~azide (10 pg/ml) (3) Neutrophils+ azide (4) Neutrophilsonly (5) Neutrophils+ catalas¢ (500 U/ml) (6) Neutrophi|s+ azide (7) Neutrophils+ azide + methionine (i mM) (8) NeutTophils+ azide + auranofin (0.5 p,g/ml) (9) Neutrophils+ azide + auranofin (I p,g/ml)
Stimulant
H202 (p,M)
None
< 1a
None Opsonised zymosan PMA
