Biochimica et Biophysica Acta, 1052 (1990) 211-215
211
Elsevier BBAMCR 12657
Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems Shigenobu Umeki and Rinzo Soejima Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama (Japan)
(Received 21 July 1989)
Key words: Steroid;NADPH oxidase; Cell-freesystem; Respiratoryburst; (Human neutrophil) The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 p M. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the K m of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.
Introduction Human neutrophils have a bactericidal action attributable to their generation of oxygen free radicals including 0 2 , H202, and "OH [1]. Superoxide is produced primarily through the activation of plasma membrane-bound N A D P H oxidase (EC 1.6.99.6) by stimulation with phagocytizable particles [2] or soluble agents [3,4]. The major contribution of superoxide and its dismutation products to the bactericidal capacity of the body is readily demonstrated by the susceptibility to infection shown by patients with chronic granulomatous disease (CGD), in which there is a defect in the respiratory burst oxidase or its activating apparatus [1,5,6]. It is well known that steroids modify the biochemical events associated with phagocytosis [7-15]. A number of phagocytic mechanisms are inhibited by steroids, and there is good evidence that the clearance of particulate substances by the reticuloendothelial system in steroid-
treated animals is impaired [12]. In some instances the processing of antigens by macrophages is abnormal. Engulfed antigens accumulate in macrophages, but are not well digested [12,13]. Superoxide generation by phagocytes such as neutrophils [16] and macrophages [17] depends on the lipoxygenase activity, and steroids inhibit the phospholipase activity and the production of prostaglandins [18,19] and leukotriene B4 [20] via the synthesis and release of lipocortin. Buyon et al. [9] and Nelson et al. [15] have reported that steroids inhibit superoxide production of stimulated neutrophils in whole-cell systems. However, little information about effects of steroids on the activation of N A D P H oxidase in cell-free systems is available. In the present study, therefore, the effect of steroids on oxygen free radical formation, and especially on the NADPH-dependent superoxide-generating oxidase, of human neutrophils, was investigated in both whole-cell and cell-free activation systems. Materials and Methods
Abbreviations: Pipes, 1,4-piperazinediethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate; FAD, flavin adenine dinucleotide; SOD, superoxide dismutase; SDS, sodium dodecyl sulfate; DMSO, dimetliylsulfoxide;HBSS, Hanks' balanced salt solution. Correspondence: S. Umeki, Divisionof RespiratoryDiseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama701-01, Japan.
The following chemicals were obtained from commercial sources: bovine erythrocyte superoxide dismutase (SOD), cytochrome c (type III), fl-NADPH (type I), sodium deoxycholate, phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), flavin adenine dinucleotide (FAD), ATP, EGTA, Pipes,
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
212 glycerol, sucrose and hydrocortisone-21-sodium succinate (Sigma, St. Louis, MO); Ficoll-Paque (Pharmacia P-L Biochemicals, Piscataway, NJ); Hanks’ balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, NY). Sodium deoxycholate was recrystallized from ethanol before use. Other chemicals were of the highest purity available from commercial sources. Solubilized membranes and cytosol were prepared from resting human neutrophils as previously described [21,22]. Protein concentrations determined using the bicinchoninic acid protein assay reagent [23] (Pierce, Rockford, IL) were as follows: solubilized membranes, 33.8 _t 2.1 S.D. pg/lO’ cell eq (n = 5); and cytosol, 192 + 15 S.D. pg/107 cell eq (n = 5). Superoxide production by intact PMA-stimulated neutrophils (wholecell system) was measured by a discontinuous assay method in which the SOD-inhibitable reduction of cytochrome c was monitored by scanning between 530 and 570 nm, as previously described 1241. Cells (2. 10’ cell eq/cuvette) were incubated in an HBSS assay medium containing 0.12 mM cytochrome c and the desired concentrations of hydrocortisone/ methanol solution (l-50 mM stock solution) for 2 min at 37 o C before the reactions were initiated by adding PMA (0.3 pg/ml in DMSO). Assay mixtures were incubated for 4 min at 37 o C, in a total volume of 1.0 ml. The reference cuvette also received 20 pg of SOD. Superoxide production in the cell-free system was assayed by a modification [22] of the method of Curnutte et al. [21]. Assay mixtures contained 0.1 mM cytochrome c, 3.6 mM MgCl,, 89 mM KCl, 2.7 mM NaCl, 0.5 mM Pipes (pH 7.3), 0.9 mM ATP, 1.2 mM EGTA, 0.5 PM FAD, 6. lo6 cell eq of cytosol, 1.5 .lo6cell eq of membranes solubilized in deoxycholate (0.94 mM), the desired concentrations of hydrocortisone (l-50 mM stock hydrocortisone/ methanol solution), 0.04 mM sodium dodecyl sulfate (SDS) and 0.16 mM NADPH, with alterations as noted in the figure and table legends, in a total volume of 0.75 ml. The reference cuvette contained 40 pg of SOD. Basically, all of the constituents except NADPH were mixed in the cuvette and were then placed in the reference and sample cuvettes. Absorbance at 550 nm was followed for 3 min at room temperature (23-24” C). Then the reactions were started by adding 25 ~1 of NADPH to each cuvette, and the change in absorbance at 550 nm was followed for 3-5 min on a Cary Model118 double-beam spectrophotometer. Superoxide production was calculated using an extinction coefficient of = 19.6 mM_’ . cm-’ [25]. E $F",,, Results and Discussion It has been shown in previous studies that the mechanism by which glucocorticoids inhibit phagocytosis and associated biochemical events involves steroid-receptor interaction followed by RNA and protein
synthesis [7-lo]. The effects of hydrocortisone on the superoxide generation of intact neutrophils stimulated by PMA were investigated (Fig. 1). Hydrocortisone dose-dependently inhibited NADPH oxidase, and the concentration of the drug which produced 50% inhibition was 50 PM. However, the 50% inhibition of oxidase by methanol alone required a concentration of more than 500 mM. The exact mechanism of action of antiinflammatory steroids on oxygen free radical production by phagocytes is still unknown, and contradictory results have been reported. Nelson et al. [15] have reported an effect of corticosteroids to alter the phospholipid composition of leukocytes at the same time that superoxide production by these cells is decreased. Nakagawara et al. [lo] demonstrated that inhibition by dexamethasone of H,O, release from PMA-stimulated human mononuclear cells was time-dependent with maximal effect after 3 days of exposure. The need for such prolonged incubations suggests that the mechanism of action of the steroids involves a receptor-mediated process. However, the chemotactic peptide-dependent generation of superoxide by neutrophils has been partly inhibited by 10 PM dexamethasone after only 25-min preincubation [9], suggesting a direct effect of the steroid on the cell membrane. In our results, the inhibition by hydrocortisone of superoxide release from PMA-stimulated neutrophils was dose-dependent with
Methanol 0
50 I
100
50
100
concentration 150
200
CmM) 250
-
, 400 ,* I
500
400
500
I
16
20
’ 0
Hydrocortisone
150
200
concentration
250
( orM) W--C
Fig. 1. Dose-dependent changes by hydrocortisone in superoxide generation of intact neutrophils stimulated by PMA. The assay method is described in Materials and Methods. Concentrations of l-50 mM stock hydrocortisone/methanol solution were used in the assays. In assay mixtures containing concentrations of hydrocortisone of 250 PM and less than 250 PM, each final methanol concentration was 100 mM (4 pl/cuvette). In assay mixture containing 400 or 500 PM hydrocortisone, final methanol concentration was 200 (8 ~1 of 50 mM stock solution/cuvette) or 250 mM (10 pl/cuvette). The results shown are the means+ S.D. of three different experiments. ., only methanol treatment: o, hydrocortisone treatment.
213 Methanol 50
1O0
concentration (mM) 150
200
250
= 400
i
;
i
i
i
1
i
80
500
80
70
A
7O
E -~
E
~ g
-.. 60
~ g iu
.~_
60
=
~5o --r ~ z
40
D z< ~ 4 0
~ 30 -6 E c 20
" 3O
10
20 i
0
50
1O0
Hydrocortisone
150
200
concentration
250
400
0
i
1
0
1
2
0
1
3
0
500
Time (rain) Fig. 3. Time-dependent changes in the activation of the NADPH oxidase in the cell-free system after preincubation with hydrocortisone but before the addition of SDS and NADPH. The assay method is described in Materials and Methods. 50 #M hydrocortisone which required for 50% inhibition of oxidase in this cell-free system was added to each cuvette. The results shown are the mean ± S.D. of three different experiments, o, only methanol (100 raM) treatment; ©, hydrocortisone treatment.
(laM)
Fig. 2. Dose-dependent changes by hydrocortisone in the activation of the NADPH oxidase in the cell-free system. The assay method is described in Materials and Methods. Concentrations of 1-50 mM stock hydrocortisone/methanol solution were used in the assays. In assay mixtures containing hydrocortisone concentrations of 250 #M and less than 250 #M, each final methanol concentration was 100 mM (3 #l/cuvette). In assay mixture containing 400 or 500 #M hydrocortisone, final methanol concentration was 200 (6 #1 of 50 mM stock hydrocortisone/methanol solution/cuvette) or 250 mM (7.5 #l/cuvette). The results shown are the means ± S.D. of three different experiments, o, only methanol treatment; (3, hydrocortisone treatment.
2-rain preincubation before the start of the reaction, suggesting a direct effect of the steroid on the cell membrane or cytosol factors. An assay method using a cell-free system for the NADPH oxidase activation is most suitable for a better understanding of the precise mechanisms that regulate the enzyme activation system. Fig. 2 shows the effects of hydrocortisone on neutrophil NADPH oxidase activation in the cell-free system. In this system, concentration of hydrocortisone required for 50% inhibition of oxidase was 40 #M. In contrast, no significant reduction in oxidase activity was caused by methanol concentrations of less than 500 mM. Fig. 3 shows time-dependent changes in the activation of the oxidase in the cell-free system after preincubation of hydrocortisone (50 #M) or methanol (100 mM) alone but before the addition of SDS and NADPH. Hydrocortisone time-dependently inhibited the NADPH oxidase. There was, however, no significant change in the NADPH oxidase by methanol alone. Furthermore, in order to better understand the effects of hydrocortisone on activation mechanisms of NADPH oxidase in cell-free systems, first, the effect of hydrocortisone on K m and Vm,x for NADPH of the oxidase was investigated (Table I). Although the mean Vmax for NADPH of the oxidase after hydrocortisone treatment was more than 2-fold lower than that in the
control assay, hydrocortisone did not change the K m value for NADPH of the oxidase. The results suggest that the drug may not change the affinity of NADPH oxidase to NADPH. Fig. 4 shows the effect of adding hydrocortisone on the activation of superoxide-generating NADPH oxidase induced by SDS in cell-free systems. In Curve 1, the solubilized membranes were exposed to SDS. After the addition of NADPH to this mixture, oxidase activity was immediately apparent (control assay). When hydrocortisone was added after adding NADPH (Curve 2) or done together with NADPH (Curve 3), the maximal activation of oxidase by SDS was the same as that in Curve 1. In Curve 5,
TABLE I Effect of hydrocortisone on the Krn and Vmax for NADPH in a cell-free system Incubation method is described in Materials and Methods except that NADPH, at varying concentrations, was used in these experiments. Kinetic constants were calculated by linear regression analysis of Lineweaver-Burk plots. 40 #M of hydrocortisone which required for 50% inhibition of oxidase was used. n, the number of experiments performed. Additions
(n)
K m (#M)
None Hydrocortisone
5 3
37.6+2.1 a 36.8+1.9
Vn~x (nmo! O2/107 cell eq./min) 101+16 42+ 7 *
mean + S.D. * P < 0.001, compared with the value 101 ± 16.
a
214 the K, and V,, for NADPH of NADPH oxidase were extensively studied, we suggested that changes in concentrations of an active form of cytosolic activation factors may modify and regulate the affinity of oxidase to NADPH and oxidase activation in cell-free systems [22]. Based on these results, it is considered that steroids may inhibit the activation of a cytosolic stabilizing factor by SDS.
lmin
TimeCmin)
Fig. 4. Curves of neutrophil NADPH oxidase activated by SDS in the cell-free system and the effect of hydrocortisone on their oxidase activations. The assay method is described in Materials and Methods. In Curve 1, the solubilized membranes were exposed to SDS and thereafter NADPH (control assay). In Curve 2, 80 gM hydrocortisone which can sufficiently inhibit the superoxide production of NADPH oxidase in cell-free systems was added to the cuvette after adding SDS and subsequently NADPH. In Curve 3, 80 gM hydrocortisone and NADPH were simultaneously added to the cuvette after adding SDS. In Curve 4, 80 gM hydrocortisone and SDS were simultaneously added to the cuvette before adding NADPH. In Curve 5, 80 pM hydrocortisone was added before the addition of SDS and NADPH. One experiment. HC, hydrocortisone.
Steroids have been shown to inhibit the release of arachidonic acid and its products of conversion, thromboxane B, and leukotriene B4, from phagocytes in vitro (transduction) [18-201, as well as activation of the respiratory burst system (activation phase) [8-111. Oxygen free radicals derived from superoxide generated by neutrophils are essential to kill exogenous bacteria and maintain homeostasis of the body. In the inhibition of superoxide generation in neutrophil NADPH oxidase, hydrocortisone was active at concentrations lower than 25 PM, which corresponds to the plasma levels currently reached when this glucocorticoid is administered for therapeutic purposes [27]. The inhibitory effect of steroids on superoxide production may reduce the bactericidal action of neutrophils, i.e., the defense mechanism of the body against many kinds of pathogens. Acknowledgements We wish to thank Drs. B.M. Babior and J.T. Cumutte of the Research Institute of Scripps Clinic, La Jolla, for the encouragement given to undertake these studies and for their critical advice and review. We also would like to thank Dr. S. Fujimoto and Mrs. L.A. Mayo of the same laboratory for their excellent technical assistance. References
where hydrocortisone was added to the cuvette before the addition of NADPH and SDS, the maximal oxidase activity was greatly reduced by hydrocortisone in comparison with that in Curve 1, to which no hydrocortisone was added. In addition, a simultaneous addition of hydrocortisone and SDS also significantly inhibited the activation of oxidase in the cell-free system (Curve 4). The results obtained in Figs. 2, 3 and 4 suggest that hydrocortisone dose- and time-dependently inhibits the activation (reconstitution) of solubilized oxidase enzyme induced by SDS in the cell-free system. Babior et al. [26] have recently demonstrated that a cytosolic stabilizing component activated by SDS in cell-free systems activates the NADPH oxidase and constructs its highaffinity enzyme form (showing a low K, for NADPH) in the presence of other cytosolic activation (regulation) factors. In experiments where the effects of various concentrations of solubilized membranes and cytosol on
1 Babior, B.M. (1978) N. Engl. J. Med. 298, 659-668. 2 Rossi, F., Romeo, D. and Patriarca, P. (1972) J. Reticuloendothel. Sot. 12, 127-149. 3 Repine, J.E., White, J.G. Clawson, C.C. and Holmes, B.M. (1974) J. Lab. Clin. Med. 83, 911-920. 4 Kakinuma, K. (1974) B&him. Biophys. Acta 348, 76-85. 5 Tauber, A.I., Borregaard, N., Simons, E. and Wright, J. (1983) Medicine 62, 286-309. 6 Cumutte, J.T. and Babior, B.M. (1987) in Advances in Human Genetics (Harris, H. and Hirschhom, K., eds.), Vol. 16, pp. 229-297, Plenum, New York. 7 Jones, C.J.P., Morris, K.J. and Jayson, M.I.V. (1983) Ann. Rheum. Dis. 42, 56-62. 8 Baud, L., Perez, J. and Ardaillou, R. (1966) Am. J. Physiol. 250, F596-F604. 9 Buyon, J.P., Korchack, H.M., Rutherford, L.E., Ganguly, M. and Weissman, G. (1984) Arthritis Rheum. 27, 623-630. 10 Nakagawara, A., De Santis, N.M., Nogueira, N. and Nathan, C.F. (1982) J. Clin. Invest. 70, 1042-1048. 11 Goldstein, I.M., Roos, M.D., Weissman, G. and Kaplan, H.B. (1976) Inflammation 1, 305-315.
215 12 Gabrielson, A.E. and Good, R.A. (1967) Advances Immunol. 6, 91-229. 13 Craddock, C.G., Winkelstein, A., Masuyuki, Y. and Lawrence, J.S. (1967) J. Exp. Med. 125, 1149-1172. 14 Rossi, F. (1986) Biochirn. Biophys. Acta 853, 65-89. 15 Nelson, D.H., Wermhold, A.R. and Murray, D.K. (1981) J. Steroid Biochem. 14, 321-325. 16 Korchak, H.M., Vienne, K., Rutherford, L.E. and Weissman, G. (1984) Federation Proc. 43, 2749-2754. 17 Lim, L.K., Hunt, N.H. and Weissman, M.J. (1983) Bioehem. Biophys. Res. Commun. 114, 549-555. 18 Blackwell, G.J., Carnuccio, R., Di Rosa, M., Flower, R.J., Parente, L. and Persico, P. (1980) Nature (Lond.) 287, 147-149. 19 Hirata, F., Schiffmann, E., Venkatasubramanian, K., Salomon, D. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 2533-2536.
20 Fuller, R.W., Kelsey, C.R., Cole, P.J., Dollery, C.T. and MacDermet, J. (1984) Clin. Sci. 67, 653-656. 21 Curnutte, J.T., Kuver, R. and Babior, B.M. (1987) J. Biol. Chem. 262, 6450-6452. 22 Umeki, S. (1990) J. Biol. Chem., in press. 23 Smith, P.K., Krohn, R.I., Hermanson, G.T. MaUia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Anal. Biochem. 150, 76-85. 24 Markert, M., Andrews, P.C. and Babior, B.M. (1984) Methods Enzymol. 105, 358-365. 25 Yonetani, T. (1965) J. Biol. Chem. 240, 4509-4514. 26 Babior, B.M., Kuver, R. and Curnutte, J.T. (1988) J. Biol. Chem. 263, 1713-1718. 27 Stubbs, S.S. (1975) Transplant. Proc. 7, 11-19.
211
Elsevier BBAMCR 12657
Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems Shigenobu Umeki and Rinzo Soejima Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama (Japan)
(Received 21 July 1989)
Key words: Steroid;NADPH oxidase; Cell-freesystem; Respiratoryburst; (Human neutrophil) The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 p M. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the K m of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.
Introduction Human neutrophils have a bactericidal action attributable to their generation of oxygen free radicals including 0 2 , H202, and "OH [1]. Superoxide is produced primarily through the activation of plasma membrane-bound N A D P H oxidase (EC 1.6.99.6) by stimulation with phagocytizable particles [2] or soluble agents [3,4]. The major contribution of superoxide and its dismutation products to the bactericidal capacity of the body is readily demonstrated by the susceptibility to infection shown by patients with chronic granulomatous disease (CGD), in which there is a defect in the respiratory burst oxidase or its activating apparatus [1,5,6]. It is well known that steroids modify the biochemical events associated with phagocytosis [7-15]. A number of phagocytic mechanisms are inhibited by steroids, and there is good evidence that the clearance of particulate substances by the reticuloendothelial system in steroid-
treated animals is impaired [12]. In some instances the processing of antigens by macrophages is abnormal. Engulfed antigens accumulate in macrophages, but are not well digested [12,13]. Superoxide generation by phagocytes such as neutrophils [16] and macrophages [17] depends on the lipoxygenase activity, and steroids inhibit the phospholipase activity and the production of prostaglandins [18,19] and leukotriene B4 [20] via the synthesis and release of lipocortin. Buyon et al. [9] and Nelson et al. [15] have reported that steroids inhibit superoxide production of stimulated neutrophils in whole-cell systems. However, little information about effects of steroids on the activation of N A D P H oxidase in cell-free systems is available. In the present study, therefore, the effect of steroids on oxygen free radical formation, and especially on the NADPH-dependent superoxide-generating oxidase, of human neutrophils, was investigated in both whole-cell and cell-free activation systems. Materials and Methods
Abbreviations: Pipes, 1,4-piperazinediethanesulfonic acid; PMA, phorbol 12-myristate 13-acetate; FAD, flavin adenine dinucleotide; SOD, superoxide dismutase; SDS, sodium dodecyl sulfate; DMSO, dimetliylsulfoxide;HBSS, Hanks' balanced salt solution. Correspondence: S. Umeki, Divisionof RespiratoryDiseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama701-01, Japan.
The following chemicals were obtained from commercial sources: bovine erythrocyte superoxide dismutase (SOD), cytochrome c (type III), fl-NADPH (type I), sodium deoxycholate, phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), flavin adenine dinucleotide (FAD), ATP, EGTA, Pipes,
0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
212 glycerol, sucrose and hydrocortisone-21-sodium succinate (Sigma, St. Louis, MO); Ficoll-Paque (Pharmacia P-L Biochemicals, Piscataway, NJ); Hanks’ balanced salt solution (HBSS) (Gibco Laboratories, Grand Island, NY). Sodium deoxycholate was recrystallized from ethanol before use. Other chemicals were of the highest purity available from commercial sources. Solubilized membranes and cytosol were prepared from resting human neutrophils as previously described [21,22]. Protein concentrations determined using the bicinchoninic acid protein assay reagent [23] (Pierce, Rockford, IL) were as follows: solubilized membranes, 33.8 _t 2.1 S.D. pg/lO’ cell eq (n = 5); and cytosol, 192 + 15 S.D. pg/107 cell eq (n = 5). Superoxide production by intact PMA-stimulated neutrophils (wholecell system) was measured by a discontinuous assay method in which the SOD-inhibitable reduction of cytochrome c was monitored by scanning between 530 and 570 nm, as previously described 1241. Cells (2. 10’ cell eq/cuvette) were incubated in an HBSS assay medium containing 0.12 mM cytochrome c and the desired concentrations of hydrocortisone/ methanol solution (l-50 mM stock solution) for 2 min at 37 o C before the reactions were initiated by adding PMA (0.3 pg/ml in DMSO). Assay mixtures were incubated for 4 min at 37 o C, in a total volume of 1.0 ml. The reference cuvette also received 20 pg of SOD. Superoxide production in the cell-free system was assayed by a modification [22] of the method of Curnutte et al. [21]. Assay mixtures contained 0.1 mM cytochrome c, 3.6 mM MgCl,, 89 mM KCl, 2.7 mM NaCl, 0.5 mM Pipes (pH 7.3), 0.9 mM ATP, 1.2 mM EGTA, 0.5 PM FAD, 6. lo6 cell eq of cytosol, 1.5 .lo6cell eq of membranes solubilized in deoxycholate (0.94 mM), the desired concentrations of hydrocortisone (l-50 mM stock hydrocortisone/ methanol solution), 0.04 mM sodium dodecyl sulfate (SDS) and 0.16 mM NADPH, with alterations as noted in the figure and table legends, in a total volume of 0.75 ml. The reference cuvette contained 40 pg of SOD. Basically, all of the constituents except NADPH were mixed in the cuvette and were then placed in the reference and sample cuvettes. Absorbance at 550 nm was followed for 3 min at room temperature (23-24” C). Then the reactions were started by adding 25 ~1 of NADPH to each cuvette, and the change in absorbance at 550 nm was followed for 3-5 min on a Cary Model118 double-beam spectrophotometer. Superoxide production was calculated using an extinction coefficient of = 19.6 mM_’ . cm-’ [25]. E $F",,, Results and Discussion It has been shown in previous studies that the mechanism by which glucocorticoids inhibit phagocytosis and associated biochemical events involves steroid-receptor interaction followed by RNA and protein
synthesis [7-lo]. The effects of hydrocortisone on the superoxide generation of intact neutrophils stimulated by PMA were investigated (Fig. 1). Hydrocortisone dose-dependently inhibited NADPH oxidase, and the concentration of the drug which produced 50% inhibition was 50 PM. However, the 50% inhibition of oxidase by methanol alone required a concentration of more than 500 mM. The exact mechanism of action of antiinflammatory steroids on oxygen free radical production by phagocytes is still unknown, and contradictory results have been reported. Nelson et al. [15] have reported an effect of corticosteroids to alter the phospholipid composition of leukocytes at the same time that superoxide production by these cells is decreased. Nakagawara et al. [lo] demonstrated that inhibition by dexamethasone of H,O, release from PMA-stimulated human mononuclear cells was time-dependent with maximal effect after 3 days of exposure. The need for such prolonged incubations suggests that the mechanism of action of the steroids involves a receptor-mediated process. However, the chemotactic peptide-dependent generation of superoxide by neutrophils has been partly inhibited by 10 PM dexamethasone after only 25-min preincubation [9], suggesting a direct effect of the steroid on the cell membrane. In our results, the inhibition by hydrocortisone of superoxide release from PMA-stimulated neutrophils was dose-dependent with
Methanol 0
50 I
100
50
100
concentration 150
200
CmM) 250
-
, 400 ,* I
500
400
500
I
16
20
’ 0
Hydrocortisone
150
200
concentration
250
( orM) W--C
Fig. 1. Dose-dependent changes by hydrocortisone in superoxide generation of intact neutrophils stimulated by PMA. The assay method is described in Materials and Methods. Concentrations of l-50 mM stock hydrocortisone/methanol solution were used in the assays. In assay mixtures containing concentrations of hydrocortisone of 250 PM and less than 250 PM, each final methanol concentration was 100 mM (4 pl/cuvette). In assay mixture containing 400 or 500 PM hydrocortisone, final methanol concentration was 200 (8 ~1 of 50 mM stock solution/cuvette) or 250 mM (10 pl/cuvette). The results shown are the means+ S.D. of three different experiments. ., only methanol treatment: o, hydrocortisone treatment.
213 Methanol 50
1O0
concentration (mM) 150
200
250
= 400
i
;
i
i
i
1
i
80
500
80
70
A
7O
E -~
E
~ g
-.. 60
~ g iu
.~_
60
=
~5o --r ~ z
40
D z< ~ 4 0
~ 30 -6 E c 20
" 3O
10
20 i
0
50
1O0
Hydrocortisone
150
200
concentration
250
400
0
i
1
0
1
2
0
1
3
0
500
Time (rain) Fig. 3. Time-dependent changes in the activation of the NADPH oxidase in the cell-free system after preincubation with hydrocortisone but before the addition of SDS and NADPH. The assay method is described in Materials and Methods. 50 #M hydrocortisone which required for 50% inhibition of oxidase in this cell-free system was added to each cuvette. The results shown are the mean ± S.D. of three different experiments, o, only methanol (100 raM) treatment; ©, hydrocortisone treatment.
(laM)
Fig. 2. Dose-dependent changes by hydrocortisone in the activation of the NADPH oxidase in the cell-free system. The assay method is described in Materials and Methods. Concentrations of 1-50 mM stock hydrocortisone/methanol solution were used in the assays. In assay mixtures containing hydrocortisone concentrations of 250 #M and less than 250 #M, each final methanol concentration was 100 mM (3 #l/cuvette). In assay mixture containing 400 or 500 #M hydrocortisone, final methanol concentration was 200 (6 #1 of 50 mM stock hydrocortisone/methanol solution/cuvette) or 250 mM (7.5 #l/cuvette). The results shown are the means ± S.D. of three different experiments, o, only methanol treatment; (3, hydrocortisone treatment.
2-rain preincubation before the start of the reaction, suggesting a direct effect of the steroid on the cell membrane or cytosol factors. An assay method using a cell-free system for the NADPH oxidase activation is most suitable for a better understanding of the precise mechanisms that regulate the enzyme activation system. Fig. 2 shows the effects of hydrocortisone on neutrophil NADPH oxidase activation in the cell-free system. In this system, concentration of hydrocortisone required for 50% inhibition of oxidase was 40 #M. In contrast, no significant reduction in oxidase activity was caused by methanol concentrations of less than 500 mM. Fig. 3 shows time-dependent changes in the activation of the oxidase in the cell-free system after preincubation of hydrocortisone (50 #M) or methanol (100 mM) alone but before the addition of SDS and NADPH. Hydrocortisone time-dependently inhibited the NADPH oxidase. There was, however, no significant change in the NADPH oxidase by methanol alone. Furthermore, in order to better understand the effects of hydrocortisone on activation mechanisms of NADPH oxidase in cell-free systems, first, the effect of hydrocortisone on K m and Vm,x for NADPH of the oxidase was investigated (Table I). Although the mean Vmax for NADPH of the oxidase after hydrocortisone treatment was more than 2-fold lower than that in the
control assay, hydrocortisone did not change the K m value for NADPH of the oxidase. The results suggest that the drug may not change the affinity of NADPH oxidase to NADPH. Fig. 4 shows the effect of adding hydrocortisone on the activation of superoxide-generating NADPH oxidase induced by SDS in cell-free systems. In Curve 1, the solubilized membranes were exposed to SDS. After the addition of NADPH to this mixture, oxidase activity was immediately apparent (control assay). When hydrocortisone was added after adding NADPH (Curve 2) or done together with NADPH (Curve 3), the maximal activation of oxidase by SDS was the same as that in Curve 1. In Curve 5,
TABLE I Effect of hydrocortisone on the Krn and Vmax for NADPH in a cell-free system Incubation method is described in Materials and Methods except that NADPH, at varying concentrations, was used in these experiments. Kinetic constants were calculated by linear regression analysis of Lineweaver-Burk plots. 40 #M of hydrocortisone which required for 50% inhibition of oxidase was used. n, the number of experiments performed. Additions
(n)
K m (#M)
None Hydrocortisone
5 3
37.6+2.1 a 36.8+1.9
Vn~x (nmo! O2/107 cell eq./min) 101+16 42+ 7 *
mean + S.D. * P < 0.001, compared with the value 101 ± 16.
a
214 the K, and V,, for NADPH of NADPH oxidase were extensively studied, we suggested that changes in concentrations of an active form of cytosolic activation factors may modify and regulate the affinity of oxidase to NADPH and oxidase activation in cell-free systems [22]. Based on these results, it is considered that steroids may inhibit the activation of a cytosolic stabilizing factor by SDS.
lmin
TimeCmin)
Fig. 4. Curves of neutrophil NADPH oxidase activated by SDS in the cell-free system and the effect of hydrocortisone on their oxidase activations. The assay method is described in Materials and Methods. In Curve 1, the solubilized membranes were exposed to SDS and thereafter NADPH (control assay). In Curve 2, 80 gM hydrocortisone which can sufficiently inhibit the superoxide production of NADPH oxidase in cell-free systems was added to the cuvette after adding SDS and subsequently NADPH. In Curve 3, 80 gM hydrocortisone and NADPH were simultaneously added to the cuvette after adding SDS. In Curve 4, 80 gM hydrocortisone and SDS were simultaneously added to the cuvette before adding NADPH. In Curve 5, 80 pM hydrocortisone was added before the addition of SDS and NADPH. One experiment. HC, hydrocortisone.
Steroids have been shown to inhibit the release of arachidonic acid and its products of conversion, thromboxane B, and leukotriene B4, from phagocytes in vitro (transduction) [18-201, as well as activation of the respiratory burst system (activation phase) [8-111. Oxygen free radicals derived from superoxide generated by neutrophils are essential to kill exogenous bacteria and maintain homeostasis of the body. In the inhibition of superoxide generation in neutrophil NADPH oxidase, hydrocortisone was active at concentrations lower than 25 PM, which corresponds to the plasma levels currently reached when this glucocorticoid is administered for therapeutic purposes [27]. The inhibitory effect of steroids on superoxide production may reduce the bactericidal action of neutrophils, i.e., the defense mechanism of the body against many kinds of pathogens. Acknowledgements We wish to thank Drs. B.M. Babior and J.T. Cumutte of the Research Institute of Scripps Clinic, La Jolla, for the encouragement given to undertake these studies and for their critical advice and review. We also would like to thank Dr. S. Fujimoto and Mrs. L.A. Mayo of the same laboratory for their excellent technical assistance. References
where hydrocortisone was added to the cuvette before the addition of NADPH and SDS, the maximal oxidase activity was greatly reduced by hydrocortisone in comparison with that in Curve 1, to which no hydrocortisone was added. In addition, a simultaneous addition of hydrocortisone and SDS also significantly inhibited the activation of oxidase in the cell-free system (Curve 4). The results obtained in Figs. 2, 3 and 4 suggest that hydrocortisone dose- and time-dependently inhibits the activation (reconstitution) of solubilized oxidase enzyme induced by SDS in the cell-free system. Babior et al. [26] have recently demonstrated that a cytosolic stabilizing component activated by SDS in cell-free systems activates the NADPH oxidase and constructs its highaffinity enzyme form (showing a low K, for NADPH) in the presence of other cytosolic activation (regulation) factors. In experiments where the effects of various concentrations of solubilized membranes and cytosol on
1 Babior, B.M. (1978) N. Engl. J. Med. 298, 659-668. 2 Rossi, F., Romeo, D. and Patriarca, P. (1972) J. Reticuloendothel. Sot. 12, 127-149. 3 Repine, J.E., White, J.G. Clawson, C.C. and Holmes, B.M. (1974) J. Lab. Clin. Med. 83, 911-920. 4 Kakinuma, K. (1974) B&him. Biophys. Acta 348, 76-85. 5 Tauber, A.I., Borregaard, N., Simons, E. and Wright, J. (1983) Medicine 62, 286-309. 6 Cumutte, J.T. and Babior, B.M. (1987) in Advances in Human Genetics (Harris, H. and Hirschhom, K., eds.), Vol. 16, pp. 229-297, Plenum, New York. 7 Jones, C.J.P., Morris, K.J. and Jayson, M.I.V. (1983) Ann. Rheum. Dis. 42, 56-62. 8 Baud, L., Perez, J. and Ardaillou, R. (1966) Am. J. Physiol. 250, F596-F604. 9 Buyon, J.P., Korchack, H.M., Rutherford, L.E., Ganguly, M. and Weissman, G. (1984) Arthritis Rheum. 27, 623-630. 10 Nakagawara, A., De Santis, N.M., Nogueira, N. and Nathan, C.F. (1982) J. Clin. Invest. 70, 1042-1048. 11 Goldstein, I.M., Roos, M.D., Weissman, G. and Kaplan, H.B. (1976) Inflammation 1, 305-315.
215 12 Gabrielson, A.E. and Good, R.A. (1967) Advances Immunol. 6, 91-229. 13 Craddock, C.G., Winkelstein, A., Masuyuki, Y. and Lawrence, J.S. (1967) J. Exp. Med. 125, 1149-1172. 14 Rossi, F. (1986) Biochirn. Biophys. Acta 853, 65-89. 15 Nelson, D.H., Wermhold, A.R. and Murray, D.K. (1981) J. Steroid Biochem. 14, 321-325. 16 Korchak, H.M., Vienne, K., Rutherford, L.E. and Weissman, G. (1984) Federation Proc. 43, 2749-2754. 17 Lim, L.K., Hunt, N.H. and Weissman, M.J. (1983) Bioehem. Biophys. Res. Commun. 114, 549-555. 18 Blackwell, G.J., Carnuccio, R., Di Rosa, M., Flower, R.J., Parente, L. and Persico, P. (1980) Nature (Lond.) 287, 147-149. 19 Hirata, F., Schiffmann, E., Venkatasubramanian, K., Salomon, D. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 2533-2536.
20 Fuller, R.W., Kelsey, C.R., Cole, P.J., Dollery, C.T. and MacDermet, J. (1984) Clin. Sci. 67, 653-656. 21 Curnutte, J.T., Kuver, R. and Babior, B.M. (1987) J. Biol. Chem. 262, 6450-6452. 22 Umeki, S. (1990) J. Biol. Chem., in press. 23 Smith, P.K., Krohn, R.I., Hermanson, G.T. MaUia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Anal. Biochem. 150, 76-85. 24 Markert, M., Andrews, P.C. and Babior, B.M. (1984) Methods Enzymol. 105, 358-365. 25 Yonetani, T. (1965) J. Biol. Chem. 240, 4509-4514. 26 Babior, B.M., Kuver, R. and Curnutte, J.T. (1988) J. Biol. Chem. 263, 1713-1718. 27 Stubbs, S.S. (1975) Transplant. Proc. 7, 11-19.
