JOURNAL OF HEMATOTHERAPY 1:261-271 Mary Ann Lieben, Inc., Publishers
(1992)
Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Superoxide Anión and Hydrogen Peroxide Production in Human Neutrophils DIETER
DENNIG,1
MICHAEL
LAM,2 GOTTFRIED FISCHER,3 SCHARF,4 and WALTER KNAPP4
CHARLES
ABSTRACT The effect of purified recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the oxidative metabolism of human peripheral blood granulocytes was investigated. The respiratory burst of granulocytes was assessed in individual cells by flow cytometry utilizing the oxidation of the nonfluorescent 2',7'-dichlorofluorescein (DCFH) to the highly fluorescent DCF by hydrogen peroxide (H202). Treatment with GM-CSF caused granulocytes to produce H202 without addition of a second stimulus. The amount of H202 produced correlated with the concentration of GM-CSF administered. Also, GM-CSF did not prime the granulocytes for enhanced H202 production in response to N-formylmethionylleucyl-phenylalanine (f-MLP). Consecutive stimulation of granulocytes with GM-CSF and f-MLP resulted in additive production of H202. GM-CSF also induced granulocytes to release Superoxide anión (02~) in a dose-dependent manner, when the respiratory burst was assessed by a conventional cytochrome c reduction assay. In contrast to hydrogen Superoxide production, GM-CSF significantly (p < 0.001) enhanced f-MLP-stimulated release of superoxide anion over that expected from the additive effects of the two agonists.
INTRODUCTION of
cytokines, notably granulocyte colony-stimulating Thecyte-macrophage colony-stimulating (GM-CSF), suggested
factor (G-CSF) and granulohas been as an adjunct to bone marrow include an & benefits accelerated Possible recovery of neutrophils transplantation (Lieschke Burgess, 1992). after bone marrow transplantation, with an associated reduction in the incidence and duration of infection. G-CSF and GM-CSF have also been used to stimulate peripheralization of progenitors that can then be used administration
factor
'Sloan-Kettering Institute, New York, NY 10021. 2Sandoz Forschungsinstitute, A-1235 Vienna, Austria. 3Institute of Blood Group Serology, University of Vienna, A-1090 Vienna, Austria. 4Institute of Immunology, University of Vienna, A-1090 Vienna, Austria. 261
DENNIG ET AL.
supplement or replace autologous bone marrow support. The administration of G-CSF and GM-CSF is accompanied by an initial decrease in circulating leukocytes, with evidence of pulmonary neutrophil sequestration for GM-CSF (Devereux et al, 1987; Morstyn et al, 1989). In addition, there is an increase in baseline respiratory burst activity of neutrophils upon administration of GM-CSF in vivo (Sullivan et al, 1989). It is not known whether this decrease in the number of neutrophils, or their activation constitutes a possible risk to the patient. Unraveling the various effects of cytokines on the maturing hématologie compartment is essential to the rational application of these powerful biologic reagents. Human GM-CSF is a 22-kD T-lymphocyte-derived glycoprotein that promotes the in vitro growth and differentiation of granulocyte and macrophage myeloid progenitor cells (for review, see Metcalf, 1985; Clark and Kamen, 1987). GM-CSF binds to specific surface receptors on mature granulocytes (Walker and Burgess, 1985 ; Park et al, 1986), induces cell-to-cell adhesion by enhancing surface expression of adhesion molecules (Nicola etal, 1986; Amaoutefa/, 1987; Fischer et al, 1987; Cairo et al, 1991), and augments the migration of granulocytes (Wang et al, 1987). Although these findings indicate that GM-CSF directly modulates various functions of mature phagocytic cells, it has been reported that GM-CSF primes granulocytes for the stimulation by exogenous stimuli, such as the bacterial chemoattractant A/-formylmethionyl-leucyl-phenylto
alanine (f-MLP) in vitro and in vivo (Weisbart et al, 1985, 1987; Metcalf et al, 1986; Sullivan et al, 1989; Balazovich et al, 1991; Khwaja 1992). However, the effect of GM-CSF alone on the respiratory burst of granulocytes has not yet been demonstrated. A combined effect of GM-CSF and f-MLP on the respiratory burst of granulocytes has been reported utilizing a dichlorofluorescein oxidation assay (Khwaja et al, 1992). Also, a priming effect of GM-CSF on the respiratory burst of granulocytes in response to f-MLP has been reported, utilizing Superoxide dismutaseinhibitable reduction of ferricytochrome c (Metcalf et al, 1986; Weisbart et al, 1987; Sullivan et al, 1989; Balazovich et al, 1991). However, cytochrome c reduction is a reversible reaction, and is potentially susceptible to reoxidation, either by oxygen present in the incubation medium or by oxidizing metabolites produced by stimulated phagocytes (Arthur et al, 1987). In our study, we investigated the respiratory burst of peripheral blood granulocytes by flow cytometry at a single cell level, the oxidation of a nonfluorescent 2',7'-dichlorofluorescein (DCFH) to a fluorescent 2',7'-dichlorofluorescein (DCF) by intracellularly produced hydrogen Superoxide. In particular, we investigated whether GM-CSF alone can activate the oxidative metabolism of granulocytes, or can only elicit a priming effect on granulocytes for their further stimulation. In addition, we compared the fluorocytometric assay to the conventional cytochrome c reduction assay.
MATERIALS AND METHODS
Cytokines, stimulants,
and reagents
Purified recombinant human GM-CSF (lot PBS 13) was derived from Escherichia coli transfected with GM-CSF cDNA inserted in a plasmid vector. The GM-CSF was 99.97% pure, with a specific activity of 1.06 x 108 CML units/mg of protein. It was dissolved in phosphate-buffered saline (PBS) and stored at 20°C as a stock solution at 1 mg/ml. Working dilutions were freshly prepared in Hank's balanced salt solution (HBSS). f-MLP (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide, and stored at —20°C as a 10~2 M stock solution. Immediately before use, the samples were diluted in HBSS. Dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, OR) was dissolved in absolute ethanol, and stored in the dark at -20°C at a concentration of 5 x 10~3 M. Working dilutions were freshly prepared in HBSS without Ca2+ and —
Mg2+.
Isolation
of granulocytes
Granulocytes were isolated from freshly drawn, heparinized peripheral blood of healthy volunteers by sedimentation in 2% dextran, and subsequent centrifugation over a Ficoll-Hypaque gradient (Pharmacia, Sweden). The cell pellet was collected and resuspended in a 0.83% ammonium chloride solution for the hypotonie lysis of residual red blood cells. After washing, the granulocytes were resuspended in Ca2+ and —
262
EFFECT OF GM-CSF ON NEUTROPHILS x 107 cells/ml. The resulting suspension contained assessed by flow cytometry, with a viability of over 95% (ethidium bromide
Mg2+-free HBSS and adjusted to a concentration of 2 more
than 95%
granulocytes
as
exclusion). DCFH oxidation assay Intracellular H202 generation was assessed by flow cytometry utilizing the dye DCFH-DA as described elsewhere (Bass et al, 1983; Szejda et al, 1984). Briefly, the nonpolar substance DCFH-DA is incubated with granulocytes. It permeates via diffusion into the cell, where it is hydrolyzed by intracellular esterases to DCFH. The resultant polar fluorescein DCFH is trapped within the cell. If the granulocyte generates hydrogen peroxide (H202), the DCFH is oxidized to DCF, a highly fluorescent dye that can then be monitored via flow cytometry at a single cell level. Briefly, 50 pi ( 106 cells) of cell suspension and 50 pi of a solution containing 3.2 x 10"6 DCFH-DA were incubated with 100 pi of medium alone or with GM-CSF or f-MLP. After various incubation periods at 37°C, the supernatants were collected for spectrophotometric evaluations and the cells were prepared for flow cytometry. Intracellular fluorescence intensity, as a result of DCF formation, was monitored with a FACS 440 cell sorter (Becton Dickinson, Sunnyvale, CA) equipped with a 2W argon laser. Before the measurements were performed, the cell sorter was calibrated with fluorescein isothiocyanate (FITC)-labeled standard beads (Fluorospheres, Coulter, FL), and a standard curve relating the fluorescence intensity to the number of FITC-molecules on the beads was established. This curve was used to calculate the FITC equivalents from the mean value of fluorescence intensity measured on the sample. In the present study, the mean values of intracellular fluorescence intensity are given as FITC equivalents (xl0~5). The fluorescence intensity of cell supernatants was monitored using a spectrofluorimeter (Aminco SPF-500, Baxter), and the data have been presented in arbitrary units. Statistical analysis was performed using the Student's i-test for paired variance. To determine whether GM-CSF does prime granulocytes for increased activity in response to a second stimulus, the cells were preincubated with GM-CSF for 2 hours at 37°C and briefly agitated after 1 hour. Thereafter, cells were washed and incubated for 30 minutes with f-MLP as a second stimulus. Intracellular oxidation of DCFH was then assessed as described above.
Cytochrome c
reduction assay
To assess the release of Superoxide anión, purified granulocytes (0.2 ml, 5 x 106 cells/ml) suspended in HBSS containing 1 % fetal calf serum, were incubated for 2 hours at 37CC together with 100 pi of a GM-CSF, f-MLP dilution, or medium alone. Thereafter, 100 pi of freshly prepared cytochrome c (Sigma, type VI, 12 mg/ml) was added to each tube followed by 100 pi of either medium or 10~8 M f-MLP and 500 pi of HBSS. Control mixtures containing Superoxide dismutase (50 pg/ml) were set up in parallel. The cells were further incubated for 10 minutes at 37°C. To halt the metabolism, the cells were rapidly cooled ,-centrifuged for 5 minutes at 400 x g, and the supernatants were transferred into cuvettes. Superoxide-inhibitable reduction of cytochrome c was measured spectrophotometrically at 500 nm. Superoxide anión production (nanomoles/106 cells/10 minutes) was calculated from the reduced cytochrome c using an extinction coefficient of 21,100 m~ ' cm"1 (Van Gelder & Slater, 1962).
RESULTS GM-CSF stimulates
H202 production
in human
granulocytes
Assessing the DCF formation as a consequence of oxidation of intracellular DCFH by H202 has been established by Bass et al (1983) as a quantitative evaluation of the oxidative burst in individual granulocytes by flow cytometry. Utilizing this method, incubation of granulocytes for 45 minutes with 10~9 M (20 ng/ml) GM-CSF resulted in the oxidation of the nonfluorescent compound DCFH to the fluorescent dye DCF (Fig. 1 A). A similar response was observed by stimulating granulocytes with 10"8 M of the bacterial chemotactic 263
DENNIG ET AL.
B Medium
Medium o
E
13
f-MLP
GM-CSF
Fluorescence
intensity
FIG. 1. GM-CSF induces DCF formation in granulocytes. Cells were loaded with DCFH, which was converted to fluorescent DCF by intracellularly generated H202 in response to 10"9 M GM-CSF (A), or 10~8 M f-MLP (B). Arrows indicate the mean value of fluorescence intensity.
factor f-MLP (Fig. IB), or with phorbol ester (data not shown). These findings suggest that the effect of GM-CSF on the oxidative metabolism of granulocytes is similar to the effect of f-MLP.
Kinetics
of GM-CSF-induced H202 production
in
granulocytes
To determine the kinetics of the H202 production, granulocytes were preloaded with DCFH, and then stimulated with 10"9 M GM-CSF, 10-8 M f-MLP, or medium alone at 37°C. The oxidative metabolism was stopped by placing some samples on ice every 15 minutes for up to 2 hours. The formation of DCF in response to the production of H202 was assessed by flow cytometry. The maximal production of H202 was observed after 45 minutes for GM-CSF, and after 30 minutes for f-MLP (Fig. 2). Thereafter, the H202 production decreased over 2 hours to the level obtained after 15 minutes. These findings suggest that the maximal formation of H202 in granulocytes occurs within 30-45 minutes after exposure to GM-CSF or f-MLP.
O-O FMLP
ID U C 0) u
•—• GM-CSF
o
A-A
medium -i—i—i—i—i—i—i—i—i—i—i—T—i—i—r-
15 30 45 60 75 90 105 120
Time
(
min
)
FIG. 2. Kinetics of intracellular H202 production. Granulocytes were loaded with DCFH, which was converted to fluorescent DCF by intracellularly generated H202 in response to 10~9 M GM-CSF, or 10"8 M f-MLP, or medium alone. A maximal response was obtained after 30 to 45 minutes. Data of one representative experiment are depicted.
264
EFFECT OF GM-CSF ON NEUTROPHILS
Dose-response of the intracellular H202 production
upon stimulation with GM-CSF and f-MLP
' ' M (200 pg/ml) to 10"7 M (2 pg/ml) GM-CSF, or were incubated for 45 minutes with 10" Granulocytes " M minimal dose of f-MLP. The GM-CSF leading to a measurable effect on the H202 10" M to 10-7 formation was as little as 10"" M (Fig. 3). The maximal effective dose of GM-CSF was 10~8 M and the plateau level of the dose response curve was reached with 10_9M. In contrast, f-MLP was only effective in concentrations exceeding 10"9 M, and the plateau of the dose-response curve was still not reached using 10"7 M f-MLP (10"6 M f-MLP elicited much higher responses than obtained with 10~7 M, Dennig et al, data not shown). These findings suggest that the plateau level of intracellular oxidation activity obtained with GM-CSF was not due to limited intracellular availability of DCFH, but rather due to a maximal GM-CSF activity, which was below that of f-MLP.
GM-CSF induces
granulocytes
to
release
H202
into the supernatant
To determine whether the formation of intracellular hydrogen Superoxide is associated with the release of into the supernatant, granulocytes were incubated for 45 minutes with GM-CSF at different concentrations; thereafter, the supernatants were collected and analyzed spectrophotometrically. As measured by the oxidation of extracellular DCFH to DCF, stimulation of granulocytes with GM-CSF induced a release of H202 into the supernatant in a dose-dependent manner (Fig. 4). To determine whether induction of H202 secretion can also be found upon stimulation with f-MLP, granulocytes were stimulated with different concentrations of f-MLP. As shown for stimulation with 10"8 M peptide, f-MLP also induced a release of H202 into the supernatant (Fig. 4).
H202
Combined
effect of GM-CSF and f-MLP on H202 production
Granulocytes were loaded with DCFH, and the effect of simultaneous stimulation over 45 minutes with a minimal or a maximal effective dose of GM-CSF together with a subminimal or a minimal effective dose of f-MLP was determined. Hydrogen peroxide production did not exceed an additive effect of GM-CSF and f-MLP (Table 1). We then determined the effect of sequential stimulation of granulocytes with GM-CSF and f-MLP. The granulocytes were prestimulated with 10"12 M to 10"8 M GM-CSF for 2 hours followed by a 30-minute stimulation with a minimal effective dose of f-MLP (10"8 M). The H202 production obtained by sequential stimulation with GM-CSF followed by f-MLP again did not exceed an additive effect of these
J_I_I_1_I_I_I_I_I_I_L
0
10-11 10-10 10-9 10-8 10-7 M
FIG. 3. Dose-response curve of H202 formation. Granulocytes were loaded with DCFH, stimulated with various concentrations of GM-CSF or f-MLP, and after 45 minutes the H202 production was measured as fluorescence intensity of intracellularly generated DCF. The data represent mean values ± SE of three to eight independent experiments.
265
DENNIG ET AL. 10
GM-CSF
03 C
FMLP
®
CD
ü c ü
6
CO
CD i— O
«= c
4
ca
(1992)
Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Superoxide Anión and Hydrogen Peroxide Production in Human Neutrophils DIETER
DENNIG,1
MICHAEL
LAM,2 GOTTFRIED FISCHER,3 SCHARF,4 and WALTER KNAPP4
CHARLES
ABSTRACT The effect of purified recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the oxidative metabolism of human peripheral blood granulocytes was investigated. The respiratory burst of granulocytes was assessed in individual cells by flow cytometry utilizing the oxidation of the nonfluorescent 2',7'-dichlorofluorescein (DCFH) to the highly fluorescent DCF by hydrogen peroxide (H202). Treatment with GM-CSF caused granulocytes to produce H202 without addition of a second stimulus. The amount of H202 produced correlated with the concentration of GM-CSF administered. Also, GM-CSF did not prime the granulocytes for enhanced H202 production in response to N-formylmethionylleucyl-phenylalanine (f-MLP). Consecutive stimulation of granulocytes with GM-CSF and f-MLP resulted in additive production of H202. GM-CSF also induced granulocytes to release Superoxide anión (02~) in a dose-dependent manner, when the respiratory burst was assessed by a conventional cytochrome c reduction assay. In contrast to hydrogen Superoxide production, GM-CSF significantly (p < 0.001) enhanced f-MLP-stimulated release of superoxide anion over that expected from the additive effects of the two agonists.
INTRODUCTION of
cytokines, notably granulocyte colony-stimulating Thecyte-macrophage colony-stimulating (GM-CSF), suggested
factor (G-CSF) and granulohas been as an adjunct to bone marrow include an & benefits accelerated Possible recovery of neutrophils transplantation (Lieschke Burgess, 1992). after bone marrow transplantation, with an associated reduction in the incidence and duration of infection. G-CSF and GM-CSF have also been used to stimulate peripheralization of progenitors that can then be used administration
factor
'Sloan-Kettering Institute, New York, NY 10021. 2Sandoz Forschungsinstitute, A-1235 Vienna, Austria. 3Institute of Blood Group Serology, University of Vienna, A-1090 Vienna, Austria. 4Institute of Immunology, University of Vienna, A-1090 Vienna, Austria. 261
DENNIG ET AL.
supplement or replace autologous bone marrow support. The administration of G-CSF and GM-CSF is accompanied by an initial decrease in circulating leukocytes, with evidence of pulmonary neutrophil sequestration for GM-CSF (Devereux et al, 1987; Morstyn et al, 1989). In addition, there is an increase in baseline respiratory burst activity of neutrophils upon administration of GM-CSF in vivo (Sullivan et al, 1989). It is not known whether this decrease in the number of neutrophils, or their activation constitutes a possible risk to the patient. Unraveling the various effects of cytokines on the maturing hématologie compartment is essential to the rational application of these powerful biologic reagents. Human GM-CSF is a 22-kD T-lymphocyte-derived glycoprotein that promotes the in vitro growth and differentiation of granulocyte and macrophage myeloid progenitor cells (for review, see Metcalf, 1985; Clark and Kamen, 1987). GM-CSF binds to specific surface receptors on mature granulocytes (Walker and Burgess, 1985 ; Park et al, 1986), induces cell-to-cell adhesion by enhancing surface expression of adhesion molecules (Nicola etal, 1986; Amaoutefa/, 1987; Fischer et al, 1987; Cairo et al, 1991), and augments the migration of granulocytes (Wang et al, 1987). Although these findings indicate that GM-CSF directly modulates various functions of mature phagocytic cells, it has been reported that GM-CSF primes granulocytes for the stimulation by exogenous stimuli, such as the bacterial chemoattractant A/-formylmethionyl-leucyl-phenylto
alanine (f-MLP) in vitro and in vivo (Weisbart et al, 1985, 1987; Metcalf et al, 1986; Sullivan et al, 1989; Balazovich et al, 1991; Khwaja 1992). However, the effect of GM-CSF alone on the respiratory burst of granulocytes has not yet been demonstrated. A combined effect of GM-CSF and f-MLP on the respiratory burst of granulocytes has been reported utilizing a dichlorofluorescein oxidation assay (Khwaja et al, 1992). Also, a priming effect of GM-CSF on the respiratory burst of granulocytes in response to f-MLP has been reported, utilizing Superoxide dismutaseinhibitable reduction of ferricytochrome c (Metcalf et al, 1986; Weisbart et al, 1987; Sullivan et al, 1989; Balazovich et al, 1991). However, cytochrome c reduction is a reversible reaction, and is potentially susceptible to reoxidation, either by oxygen present in the incubation medium or by oxidizing metabolites produced by stimulated phagocytes (Arthur et al, 1987). In our study, we investigated the respiratory burst of peripheral blood granulocytes by flow cytometry at a single cell level, the oxidation of a nonfluorescent 2',7'-dichlorofluorescein (DCFH) to a fluorescent 2',7'-dichlorofluorescein (DCF) by intracellularly produced hydrogen Superoxide. In particular, we investigated whether GM-CSF alone can activate the oxidative metabolism of granulocytes, or can only elicit a priming effect on granulocytes for their further stimulation. In addition, we compared the fluorocytometric assay to the conventional cytochrome c reduction assay.
MATERIALS AND METHODS
Cytokines, stimulants,
and reagents
Purified recombinant human GM-CSF (lot PBS 13) was derived from Escherichia coli transfected with GM-CSF cDNA inserted in a plasmid vector. The GM-CSF was 99.97% pure, with a specific activity of 1.06 x 108 CML units/mg of protein. It was dissolved in phosphate-buffered saline (PBS) and stored at 20°C as a stock solution at 1 mg/ml. Working dilutions were freshly prepared in Hank's balanced salt solution (HBSS). f-MLP (Sigma, St. Louis, MO) was dissolved in dimethyl sulfoxide, and stored at —20°C as a 10~2 M stock solution. Immediately before use, the samples were diluted in HBSS. Dichlorofluorescein diacetate (DCFH-DA, Molecular Probes, OR) was dissolved in absolute ethanol, and stored in the dark at -20°C at a concentration of 5 x 10~3 M. Working dilutions were freshly prepared in HBSS without Ca2+ and —
Mg2+.
Isolation
of granulocytes
Granulocytes were isolated from freshly drawn, heparinized peripheral blood of healthy volunteers by sedimentation in 2% dextran, and subsequent centrifugation over a Ficoll-Hypaque gradient (Pharmacia, Sweden). The cell pellet was collected and resuspended in a 0.83% ammonium chloride solution for the hypotonie lysis of residual red blood cells. After washing, the granulocytes were resuspended in Ca2+ and —
262
EFFECT OF GM-CSF ON NEUTROPHILS x 107 cells/ml. The resulting suspension contained assessed by flow cytometry, with a viability of over 95% (ethidium bromide
Mg2+-free HBSS and adjusted to a concentration of 2 more
than 95%
granulocytes
as
exclusion). DCFH oxidation assay Intracellular H202 generation was assessed by flow cytometry utilizing the dye DCFH-DA as described elsewhere (Bass et al, 1983; Szejda et al, 1984). Briefly, the nonpolar substance DCFH-DA is incubated with granulocytes. It permeates via diffusion into the cell, where it is hydrolyzed by intracellular esterases to DCFH. The resultant polar fluorescein DCFH is trapped within the cell. If the granulocyte generates hydrogen peroxide (H202), the DCFH is oxidized to DCF, a highly fluorescent dye that can then be monitored via flow cytometry at a single cell level. Briefly, 50 pi ( 106 cells) of cell suspension and 50 pi of a solution containing 3.2 x 10"6 DCFH-DA were incubated with 100 pi of medium alone or with GM-CSF or f-MLP. After various incubation periods at 37°C, the supernatants were collected for spectrophotometric evaluations and the cells were prepared for flow cytometry. Intracellular fluorescence intensity, as a result of DCF formation, was monitored with a FACS 440 cell sorter (Becton Dickinson, Sunnyvale, CA) equipped with a 2W argon laser. Before the measurements were performed, the cell sorter was calibrated with fluorescein isothiocyanate (FITC)-labeled standard beads (Fluorospheres, Coulter, FL), and a standard curve relating the fluorescence intensity to the number of FITC-molecules on the beads was established. This curve was used to calculate the FITC equivalents from the mean value of fluorescence intensity measured on the sample. In the present study, the mean values of intracellular fluorescence intensity are given as FITC equivalents (xl0~5). The fluorescence intensity of cell supernatants was monitored using a spectrofluorimeter (Aminco SPF-500, Baxter), and the data have been presented in arbitrary units. Statistical analysis was performed using the Student's i-test for paired variance. To determine whether GM-CSF does prime granulocytes for increased activity in response to a second stimulus, the cells were preincubated with GM-CSF for 2 hours at 37°C and briefly agitated after 1 hour. Thereafter, cells were washed and incubated for 30 minutes with f-MLP as a second stimulus. Intracellular oxidation of DCFH was then assessed as described above.
Cytochrome c
reduction assay
To assess the release of Superoxide anión, purified granulocytes (0.2 ml, 5 x 106 cells/ml) suspended in HBSS containing 1 % fetal calf serum, were incubated for 2 hours at 37CC together with 100 pi of a GM-CSF, f-MLP dilution, or medium alone. Thereafter, 100 pi of freshly prepared cytochrome c (Sigma, type VI, 12 mg/ml) was added to each tube followed by 100 pi of either medium or 10~8 M f-MLP and 500 pi of HBSS. Control mixtures containing Superoxide dismutase (50 pg/ml) were set up in parallel. The cells were further incubated for 10 minutes at 37°C. To halt the metabolism, the cells were rapidly cooled ,-centrifuged for 5 minutes at 400 x g, and the supernatants were transferred into cuvettes. Superoxide-inhibitable reduction of cytochrome c was measured spectrophotometrically at 500 nm. Superoxide anión production (nanomoles/106 cells/10 minutes) was calculated from the reduced cytochrome c using an extinction coefficient of 21,100 m~ ' cm"1 (Van Gelder & Slater, 1962).
RESULTS GM-CSF stimulates
H202 production
in human
granulocytes
Assessing the DCF formation as a consequence of oxidation of intracellular DCFH by H202 has been established by Bass et al (1983) as a quantitative evaluation of the oxidative burst in individual granulocytes by flow cytometry. Utilizing this method, incubation of granulocytes for 45 minutes with 10~9 M (20 ng/ml) GM-CSF resulted in the oxidation of the nonfluorescent compound DCFH to the fluorescent dye DCF (Fig. 1 A). A similar response was observed by stimulating granulocytes with 10"8 M of the bacterial chemotactic 263
DENNIG ET AL.
B Medium
Medium o
E
13
f-MLP
GM-CSF
Fluorescence
intensity
FIG. 1. GM-CSF induces DCF formation in granulocytes. Cells were loaded with DCFH, which was converted to fluorescent DCF by intracellularly generated H202 in response to 10"9 M GM-CSF (A), or 10~8 M f-MLP (B). Arrows indicate the mean value of fluorescence intensity.
factor f-MLP (Fig. IB), or with phorbol ester (data not shown). These findings suggest that the effect of GM-CSF on the oxidative metabolism of granulocytes is similar to the effect of f-MLP.
Kinetics
of GM-CSF-induced H202 production
in
granulocytes
To determine the kinetics of the H202 production, granulocytes were preloaded with DCFH, and then stimulated with 10"9 M GM-CSF, 10-8 M f-MLP, or medium alone at 37°C. The oxidative metabolism was stopped by placing some samples on ice every 15 minutes for up to 2 hours. The formation of DCF in response to the production of H202 was assessed by flow cytometry. The maximal production of H202 was observed after 45 minutes for GM-CSF, and after 30 minutes for f-MLP (Fig. 2). Thereafter, the H202 production decreased over 2 hours to the level obtained after 15 minutes. These findings suggest that the maximal formation of H202 in granulocytes occurs within 30-45 minutes after exposure to GM-CSF or f-MLP.
O-O FMLP
ID U C 0) u
•—• GM-CSF
o
A-A
medium -i—i—i—i—i—i—i—i—i—i—i—T—i—i—r-
15 30 45 60 75 90 105 120
Time
(
min
)
FIG. 2. Kinetics of intracellular H202 production. Granulocytes were loaded with DCFH, which was converted to fluorescent DCF by intracellularly generated H202 in response to 10~9 M GM-CSF, or 10"8 M f-MLP, or medium alone. A maximal response was obtained after 30 to 45 minutes. Data of one representative experiment are depicted.
264
EFFECT OF GM-CSF ON NEUTROPHILS
Dose-response of the intracellular H202 production
upon stimulation with GM-CSF and f-MLP
' ' M (200 pg/ml) to 10"7 M (2 pg/ml) GM-CSF, or were incubated for 45 minutes with 10" Granulocytes " M minimal dose of f-MLP. The GM-CSF leading to a measurable effect on the H202 10" M to 10-7 formation was as little as 10"" M (Fig. 3). The maximal effective dose of GM-CSF was 10~8 M and the plateau level of the dose response curve was reached with 10_9M. In contrast, f-MLP was only effective in concentrations exceeding 10"9 M, and the plateau of the dose-response curve was still not reached using 10"7 M f-MLP (10"6 M f-MLP elicited much higher responses than obtained with 10~7 M, Dennig et al, data not shown). These findings suggest that the plateau level of intracellular oxidation activity obtained with GM-CSF was not due to limited intracellular availability of DCFH, but rather due to a maximal GM-CSF activity, which was below that of f-MLP.
GM-CSF induces
granulocytes
to
release
H202
into the supernatant
To determine whether the formation of intracellular hydrogen Superoxide is associated with the release of into the supernatant, granulocytes were incubated for 45 minutes with GM-CSF at different concentrations; thereafter, the supernatants were collected and analyzed spectrophotometrically. As measured by the oxidation of extracellular DCFH to DCF, stimulation of granulocytes with GM-CSF induced a release of H202 into the supernatant in a dose-dependent manner (Fig. 4). To determine whether induction of H202 secretion can also be found upon stimulation with f-MLP, granulocytes were stimulated with different concentrations of f-MLP. As shown for stimulation with 10"8 M peptide, f-MLP also induced a release of H202 into the supernatant (Fig. 4).
H202
Combined
effect of GM-CSF and f-MLP on H202 production
Granulocytes were loaded with DCFH, and the effect of simultaneous stimulation over 45 minutes with a minimal or a maximal effective dose of GM-CSF together with a subminimal or a minimal effective dose of f-MLP was determined. Hydrogen peroxide production did not exceed an additive effect of GM-CSF and f-MLP (Table 1). We then determined the effect of sequential stimulation of granulocytes with GM-CSF and f-MLP. The granulocytes were prestimulated with 10"12 M to 10"8 M GM-CSF for 2 hours followed by a 30-minute stimulation with a minimal effective dose of f-MLP (10"8 M). The H202 production obtained by sequential stimulation with GM-CSF followed by f-MLP again did not exceed an additive effect of these
J_I_I_1_I_I_I_I_I_I_L
0
10-11 10-10 10-9 10-8 10-7 M
FIG. 3. Dose-response curve of H202 formation. Granulocytes were loaded with DCFH, stimulated with various concentrations of GM-CSF or f-MLP, and after 45 minutes the H202 production was measured as fluorescence intensity of intracellularly generated DCF. The data represent mean values ± SE of three to eight independent experiments.
265
DENNIG ET AL. 10
GM-CSF
03 C
FMLP
®
CD
ü c ü
6
CO
CD i— O
«= c
4
ca
