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Activation of neutrophils NADPH oxidase by PMA: Cytosol activity is translocated in phorbol-primed neutrophils
Int. J. Btochem. Vol. 25, No. 5, pp. 631-633, 1993 Printed in Great Britain.All rights reserved
0020-71IX/93 $6.00+ 0.00 Copyright© 1993PergamonPress Ltd
ACTIVATION OF NEUTROPHILS NADPH OXIDASE BY PMA: CYTOSOL ACTIVITY IS TRANSLOCATED IN PHORBOL-PRIMED NEUTROPHILS . .
TOSHIHIKOUMEI,t* NORIHIKO OHHARA,t SEIICHIOKAMURA,2 MINE HARADA,3 MASAYUKINAKAO, l TArd~m SHIRAI 1 and YosmrtJrd NIHO3 tDepartment of Medicine, Fukuoka Dental College, Fukuoka 814-01, Japan, 2Cancer Center and 3First Department of Internal Medicine, Kyushu University School of Medicine, Fukuoka 812, Japan (Received 19 November 1992) Al~tat't~l. Translocation of cytosol activity in phorbol-primed neutrophils was studied. 2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA. 3. In FMLP-primed neutrophils, the cytosoi had almost the same activity as resting one and cytosol activity was not eluted from the membrane. 4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was cor~spondingly e|uted from the membrane. 5. These observations suggested that cytosol activity was translocated in PMA-primed cells.
INTRODUCTION
and hypotonic lysis of erythrocytes (Umei et al., 1991). Isolated neutrophils were suspended in PBS.
Neutrophils play an important role in microbicidal activity (Badwey and Karnovsky, 1980; Babior, 1984). The central armory they utilize is N A D P H oxidase, which catalyzes the formation of superoxide anions (Curnutte and Babior, 1987; Babior and Woodman, 1990). The enzyme system is dormant until the cells are stimulated by an appropriate agent. Prior exposure to one stimulus can potentiate the oxidative response by subsequent heterogeneous stimulus. This phenomenon is called "priming" (McPhail et al., 1984, Weisbert et al., 1985; Karnad et al., 1989). The enzyme is a multiple component electron transfer system, which is composed of membrane and cytosol components (Parkos et al., 1987; Lomax et al., 1989; Leto et al., 1990). Cytosol components are present in active membrane and the translocation of cytosol components to membrane is essential for the activation (Clark et al., 1990). In this study, translocation of cytosol activity was studied in the primed cells. In the PMA-primed cells, cytosol activity was already translocated while it was, however, not translocated in the FMLPprimed cells. MATERIALS AND METHODS Preparation of ~eutrophils Human nentrophils were isolated from venous blood by dextran sedimentation, centrifugation over Ficoll-Paque, *To whom correspondence should be addressed. Abbreviotion~: FMLP, f-Met-Leu-Ala; PBS, phosphatebuffered saline; PMA, phorbol myristate acetate. 631
Preparation of particulate fractiom and cytosol eluates Neutrophils were stimulated with PMA or FMLP under various conditions and sonicated. Particulate fractious were isolated as previously described (Umei et al., 1991). For stimulation, neutrophils were suspended at I × l0~ in the presence of I mM NAN3. Cytosol components were eluted from membrane by the method as previously described (Umei et al., 1991). Assay of NADPH oxkta~e and cytosol activity Superoxide production was assayed spectrophotometrically as previously described (Umei et al., 1991). Assay mixture contained a particulate fraction in 50ram phosphate buffer pH 6.8, containing 75 ~tM ferricytochrome c, 0.1 mM SDS, 5/~M FAD, I mM MgCl and 2raM NaN 3 in a total volume of I ml. The reaction was started by the addition of NADPH (final concentration, 0.2 raM), and the ferricytuchrome c reduction was followed at 550 nm. Cell-free activation system consisted of unstimulated membrane and cytosol in the assay mixture (Fujita et al., 1987). For the assay of cytosoi activity, eluted membrane (1/~M PMA-stimulated) was used instead of unstimulated membrane. Cytosol activity was expressed as the enhancement of superoxide production by eluates. Proteins were assayed by the method of Bradford (Bradford, 1976). RF-,~ULT~AND DISCUSSION Membranes and cytosols were prepared from the cells stimulated by a variety of concentrations of P M A or FMLP. Membranes prepared from unstimulated cells showed no superoxide formation. Membranes from stimulated cells produced superoxide
632
Tosmm~o U E et al.
formation in proportion to the concentration of PMA [Fig. I(A)] or FMLP [Fig. I(B)]. Cytosols from PMA- or FMLP.stimulated cells had less activity in cell-free activation system using
A) 200
100 o
0 0.001
I 0.01
0.1
1
10
P M A (ttM)
(B)
unstimulated membrane (Figs 1 and 2). Cytosois from 1/~M PMA or 1/~M FMLP-stimulated cells had less activity than resting cytosol. While the cytosol from 0.1 ~tM PMA-stimulated cells had about the half of resting cytosol activity, the cytosoi from 0.1/~M FMLP-stimulated cells had almost the same activity of resting cytosol. The decrease was generally in inverse proportion to the activity of membrane prepared from stimulated cells. These observations support that the translocation of cytosol components to membrane is necessary for the activation. The priming effect of PMA and FMLP were then studied. Although membranes stimulated by 0.1/~ M PMA had little superoxide-generating activity itself [Fig. I(A)], the membrane was primed because the membrane stimulated by 0.1/~M PMA and then by 1 # M FMLP showed enhanced activity (Fig. 2). The maximal priming effect by PMA was achieved at the concentration of 0.1 #M and the priming effect by FMLP was plateau more than 0.1 #M. The ED~0 for priming by PMA or FMLP was at least 10-fold lower than the EDge for activation. Cytosol activity ehited from activated membrane with salt treatment was studied for the clarification of the translocation. Eluates were prepared from active membranes stimulated by a variety of concentrations
200 200
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FMLP (ttM) Fig. 1. (A) Dose dependency of cytosol activity and particulate superoxide-generating activity by PMA. Cells were exposed to the indicated concentration of PMA and then sonicated. C~toaols and particulate fractious were prepared and assayed as described under "Materials and Methods". [2, Cytosol activity in cell-free activation system. , , Superoxide-generating activity of particulate fractions. (B) Dose dependency of cytosol activity and particulate superoxidegenerating activity by FMLP. Cells were exposed to the indicated concentration of FMLP in the presence of cytochalasin B and then sonicated. Cytosuls and particulate fractions were prepared and assayed as dmcribed under "Materials and Methods". t-I, Cytosol activity in cell-free activation system. ~, Superoxide-gcnerating activity of particulate fractions.
O_ 0.001
..I 0.01
0.1
1
S t i m u l a n t (ttM) Fig. 2. Comparison o f dose-response curves for priming. Cells were incubated with the various concentrations o f
FMLP or PMA in the presence of cytochalasin B, and particulate fractions were prepared and assayed as described under "Materials and Methods". The values u~d for the priming activity were the .activities obtained with the second stimulus in primed cells after subtraction of the activity obtained with that stimulus in unprimed (control) cells and subtraction of any activity obtained with the priming stimulus alone. I~, Priming was with various doses of FMLP, followed by 1 #M P M A . . , Priming was with various doses of PMA, followed by !/~M FMLP.
Activation of NADPH oxidase
'~.,~. 200
I
to FMLP-priming (e.g. intracellular Ca, release of diacylglycerol). But no studies have been reported about N A D P H oxidase components. In this study, we sho~ed that cytosol activity was translocated in PMA-primed membranes and that it was not translocared in FMLP-primed membranes. These observations may suggest that the activation system by P M A could be separated into two successive steps. Clarification of each activation step is now under investigation.
e~
.~ ~ [
633
100
Acknowledgement--This work was supported in part by .m l~
Grant.in-Aid for Scientific Research from the Ministry of Education, Science and Culture.
m
I~FERENCES
o~
~ 0.001
I 0.01
0.1
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I 10
Stimulant (ttM) Fig. 3. Elution of cytosol activity from PMA- or FMLPprimed membrane. Cells were incubated with the various concentrations of FMLP or PMA, and particulate fractious were p t ~ x e d . Eluates were prepared from the membranes and cytosol activity was assayed as described under "Materials and Methods". I~, Eluates from PMA-primed memb r a n e s . . , Eluates from FMLP-primed membranes. of P M A or FMLP. Eluates alone were not sufficient in cell-free activation system using unstimulated membrane (data not shown). The cytosol activity eluted by salt treatment was detected also in the PMA-primed membranes which itself had little superoxide-generating activity (Fig. 3). Moreover, cytosol activity eluted from the membrane primed by 0.1/~ M P M A was almost the same with that from the membrane stimulated by 1/~M P M A although each superoxide-generating activity was much different. The cytosol activity in eluates was parallel with the priming effect by PMA. On the contrary, cytosol activity eluted from FMLP-primed membranes was much less than that from PMA-primed membranes and the cytosol activity in eluates was not parallel with the priming effect by FMLP. Priming effects by P M A and F M L P have been studied so far (Finkel et al., 1987; Bass et ai., 1989). These studies have demonstrated that intracellular responses to PMA-priming were different from those
Babior B. M. (1984) Blood 64, 959-966. Babior B. M. and Woodman R. C. (1990) Semin. Hematol. 27, 247-259. Badwey J. M. and Karnovsky M. L. (1980) A. Rev. Biochem. 49, 695-726. Bass D. A., McPhail L. C., Schmitt J. D., Morris-Natschke S., McCall C. E. and Wykle R. L. (1989) J. biol. Chem. 264, 19610-19617. Bradford M. M. (1976) Analyt. Biochem. 72, 248-254. Clark R. A., Volpp B. D., Leidal K. G. and Nauseef W. M. (1990) J. clin. Invest. 85, 714-721. Curnette J. T. and Babior B. M. (1987) Adv. Hum. Genet. 16, 229-297. Finkel T. H., Pabst M. J., Suzuki H., Guthrie L. A., Forehand J. R., Phillips W. A. and Johnston R. B. (1987) Y. biol. Chem. 262, 12589-12596. Fujita I., Takeshige K. and Minakami S. (1987) Biochem. biophys. Acta 931, 41-48. Karnad A. B., Hartshorn K. L., Wright J., Myers J. B., Schwartz J. H. and Tauber A. I. (1989) Blood 2519-2526. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M. G., Nauseef W. M., Clark R. A., Gallin J. I. and Malech H. L. (1990) Science 248, 727-730. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I. and Malech H. L. 0989) Science 245, 409-412. McPbail L. C., Clayton C. C. and Snyderman R. (1984) J. biol. Chem. 259, 5768-5775. Parkos C. A., Allen R. A., Cochrane C. G. and Jesaitis A. J. (1987) J. clin. Invest. 80, 732-742. Umei T., Babior B. M., Curnutte J. T. and Smith R. M. (1991) J. biol. Chem. 266, 6019-6022. Weisbert R. H., Goide D. W., Clark S. C., Wong G. G. and Gasson J. C. (1985) Nature 314, 361-362.
74,
0020-71IX/93 $6.00+ 0.00 Copyright© 1993PergamonPress Ltd
ACTIVATION OF NEUTROPHILS NADPH OXIDASE BY PMA: CYTOSOL ACTIVITY IS TRANSLOCATED IN PHORBOL-PRIMED NEUTROPHILS . .
TOSHIHIKOUMEI,t* NORIHIKO OHHARA,t SEIICHIOKAMURA,2 MINE HARADA,3 MASAYUKINAKAO, l TArd~m SHIRAI 1 and YosmrtJrd NIHO3 tDepartment of Medicine, Fukuoka Dental College, Fukuoka 814-01, Japan, 2Cancer Center and 3First Department of Internal Medicine, Kyushu University School of Medicine, Fukuoka 812, Japan (Received 19 November 1992) Al~tat't~l. Translocation of cytosol activity in phorbol-primed neutrophils was studied. 2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA. 3. In FMLP-primed neutrophils, the cytosoi had almost the same activity as resting one and cytosol activity was not eluted from the membrane. 4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was cor~spondingly e|uted from the membrane. 5. These observations suggested that cytosol activity was translocated in PMA-primed cells.
INTRODUCTION
and hypotonic lysis of erythrocytes (Umei et al., 1991). Isolated neutrophils were suspended in PBS.
Neutrophils play an important role in microbicidal activity (Badwey and Karnovsky, 1980; Babior, 1984). The central armory they utilize is N A D P H oxidase, which catalyzes the formation of superoxide anions (Curnutte and Babior, 1987; Babior and Woodman, 1990). The enzyme system is dormant until the cells are stimulated by an appropriate agent. Prior exposure to one stimulus can potentiate the oxidative response by subsequent heterogeneous stimulus. This phenomenon is called "priming" (McPhail et al., 1984, Weisbert et al., 1985; Karnad et al., 1989). The enzyme is a multiple component electron transfer system, which is composed of membrane and cytosol components (Parkos et al., 1987; Lomax et al., 1989; Leto et al., 1990). Cytosol components are present in active membrane and the translocation of cytosol components to membrane is essential for the activation (Clark et al., 1990). In this study, translocation of cytosol activity was studied in the primed cells. In the PMA-primed cells, cytosol activity was already translocated while it was, however, not translocated in the FMLPprimed cells. MATERIALS AND METHODS Preparation of ~eutrophils Human nentrophils were isolated from venous blood by dextran sedimentation, centrifugation over Ficoll-Paque, *To whom correspondence should be addressed. Abbreviotion~: FMLP, f-Met-Leu-Ala; PBS, phosphatebuffered saline; PMA, phorbol myristate acetate. 631
Preparation of particulate fractiom and cytosol eluates Neutrophils were stimulated with PMA or FMLP under various conditions and sonicated. Particulate fractious were isolated as previously described (Umei et al., 1991). For stimulation, neutrophils were suspended at I × l0~ in the presence of I mM NAN3. Cytosol components were eluted from membrane by the method as previously described (Umei et al., 1991). Assay of NADPH oxkta~e and cytosol activity Superoxide production was assayed spectrophotometrically as previously described (Umei et al., 1991). Assay mixture contained a particulate fraction in 50ram phosphate buffer pH 6.8, containing 75 ~tM ferricytochrome c, 0.1 mM SDS, 5/~M FAD, I mM MgCl and 2raM NaN 3 in a total volume of I ml. The reaction was started by the addition of NADPH (final concentration, 0.2 raM), and the ferricytuchrome c reduction was followed at 550 nm. Cell-free activation system consisted of unstimulated membrane and cytosol in the assay mixture (Fujita et al., 1987). For the assay of cytosoi activity, eluted membrane (1/~M PMA-stimulated) was used instead of unstimulated membrane. Cytosol activity was expressed as the enhancement of superoxide production by eluates. Proteins were assayed by the method of Bradford (Bradford, 1976). RF-,~ULT~AND DISCUSSION Membranes and cytosols were prepared from the cells stimulated by a variety of concentrations of P M A or FMLP. Membranes prepared from unstimulated cells showed no superoxide formation. Membranes from stimulated cells produced superoxide
632
Tosmm~o U E et al.
formation in proportion to the concentration of PMA [Fig. I(A)] or FMLP [Fig. I(B)]. Cytosols from PMA- or FMLP.stimulated cells had less activity in cell-free activation system using
A) 200
100 o
0 0.001
I 0.01
0.1
1
10
P M A (ttM)
(B)
unstimulated membrane (Figs 1 and 2). Cytosois from 1/~M PMA or 1/~M FMLP-stimulated cells had less activity than resting cytosol. While the cytosol from 0.1 ~tM PMA-stimulated cells had about the half of resting cytosol activity, the cytosoi from 0.1/~M FMLP-stimulated cells had almost the same activity of resting cytosol. The decrease was generally in inverse proportion to the activity of membrane prepared from stimulated cells. These observations support that the translocation of cytosol components to membrane is necessary for the activation. The priming effect of PMA and FMLP were then studied. Although membranes stimulated by 0.1/~ M PMA had little superoxide-generating activity itself [Fig. I(A)], the membrane was primed because the membrane stimulated by 0.1/~M PMA and then by 1 # M FMLP showed enhanced activity (Fig. 2). The maximal priming effect by PMA was achieved at the concentration of 0.1 #M and the priming effect by FMLP was plateau more than 0.1 #M. The ED~0 for priming by PMA or FMLP was at least 10-fold lower than the EDge for activation. Cytosol activity ehited from activated membrane with salt treatment was studied for the clarification of the translocation. Eluates were prepared from active membranes stimulated by a variety of concentrations
200 200
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~.,,
o
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FMLP (ttM) Fig. 1. (A) Dose dependency of cytosol activity and particulate superoxide-generating activity by PMA. Cells were exposed to the indicated concentration of PMA and then sonicated. C~toaols and particulate fractious were prepared and assayed as described under "Materials and Methods". [2, Cytosol activity in cell-free activation system. , , Superoxide-generating activity of particulate fractions. (B) Dose dependency of cytosol activity and particulate superoxidegenerating activity by FMLP. Cells were exposed to the indicated concentration of FMLP in the presence of cytochalasin B and then sonicated. Cytosuls and particulate fractions were prepared and assayed as dmcribed under "Materials and Methods". t-I, Cytosol activity in cell-free activation system. ~, Superoxide-gcnerating activity of particulate fractions.
O_ 0.001
..I 0.01
0.1
1
S t i m u l a n t (ttM) Fig. 2. Comparison o f dose-response curves for priming. Cells were incubated with the various concentrations o f
FMLP or PMA in the presence of cytochalasin B, and particulate fractions were prepared and assayed as described under "Materials and Methods". The values u~d for the priming activity were the .activities obtained with the second stimulus in primed cells after subtraction of the activity obtained with that stimulus in unprimed (control) cells and subtraction of any activity obtained with the priming stimulus alone. I~, Priming was with various doses of FMLP, followed by 1 #M P M A . . , Priming was with various doses of PMA, followed by !/~M FMLP.
Activation of NADPH oxidase
'~.,~. 200
I
to FMLP-priming (e.g. intracellular Ca, release of diacylglycerol). But no studies have been reported about N A D P H oxidase components. In this study, we sho~ed that cytosol activity was translocated in PMA-primed membranes and that it was not translocared in FMLP-primed membranes. These observations may suggest that the activation system by P M A could be separated into two successive steps. Clarification of each activation step is now under investigation.
e~
.~ ~ [
633
100
Acknowledgement--This work was supported in part by .m l~
Grant.in-Aid for Scientific Research from the Ministry of Education, Science and Culture.
m
I~FERENCES
o~
~ 0.001
I 0.01
0.1
I 1
I 10
Stimulant (ttM) Fig. 3. Elution of cytosol activity from PMA- or FMLPprimed membrane. Cells were incubated with the various concentrations of FMLP or PMA, and particulate fractious were p t ~ x e d . Eluates were prepared from the membranes and cytosol activity was assayed as described under "Materials and Methods". I~, Eluates from PMA-primed memb r a n e s . . , Eluates from FMLP-primed membranes. of P M A or FMLP. Eluates alone were not sufficient in cell-free activation system using unstimulated membrane (data not shown). The cytosol activity eluted by salt treatment was detected also in the PMA-primed membranes which itself had little superoxide-generating activity (Fig. 3). Moreover, cytosol activity eluted from the membrane primed by 0.1/~ M P M A was almost the same with that from the membrane stimulated by 1/~M P M A although each superoxide-generating activity was much different. The cytosol activity in eluates was parallel with the priming effect by PMA. On the contrary, cytosol activity eluted from FMLP-primed membranes was much less than that from PMA-primed membranes and the cytosol activity in eluates was not parallel with the priming effect by FMLP. Priming effects by P M A and F M L P have been studied so far (Finkel et al., 1987; Bass et ai., 1989). These studies have demonstrated that intracellular responses to PMA-priming were different from those
Babior B. M. (1984) Blood 64, 959-966. Babior B. M. and Woodman R. C. (1990) Semin. Hematol. 27, 247-259. Badwey J. M. and Karnovsky M. L. (1980) A. Rev. Biochem. 49, 695-726. Bass D. A., McPhail L. C., Schmitt J. D., Morris-Natschke S., McCall C. E. and Wykle R. L. (1989) J. biol. Chem. 264, 19610-19617. Bradford M. M. (1976) Analyt. Biochem. 72, 248-254. Clark R. A., Volpp B. D., Leidal K. G. and Nauseef W. M. (1990) J. clin. Invest. 85, 714-721. Curnette J. T. and Babior B. M. (1987) Adv. Hum. Genet. 16, 229-297. Finkel T. H., Pabst M. J., Suzuki H., Guthrie L. A., Forehand J. R., Phillips W. A. and Johnston R. B. (1987) Y. biol. Chem. 262, 12589-12596. Fujita I., Takeshige K. and Minakami S. (1987) Biochem. biophys. Acta 931, 41-48. Karnad A. B., Hartshorn K. L., Wright J., Myers J. B., Schwartz J. H. and Tauber A. I. (1989) Blood 2519-2526. Leto T. L., Lomax K. J., Volpp B. D., Nunoi H., Sechler J. M. G., Nauseef W. M., Clark R. A., Gallin J. I. and Malech H. L. (1990) Science 248, 727-730. Lomax K. J., Leto T. L., Nunoi H., Gallin J. I. and Malech H. L. 0989) Science 245, 409-412. McPbail L. C., Clayton C. C. and Snyderman R. (1984) J. biol. Chem. 259, 5768-5775. Parkos C. A., Allen R. A., Cochrane C. G. and Jesaitis A. J. (1987) J. clin. Invest. 80, 732-742. Umei T., Babior B. M., Curnutte J. T. and Smith R. M. (1991) J. biol. Chem. 266, 6019-6022. Weisbert R. H., Goide D. W., Clark S. C., Wong G. G. and Gasson J. C. (1985) Nature 314, 361-362.
74,