Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 4 Printed in U.S.A.
INHIBITION OF LEUKOCYTE SUPEROXIDE ANION PRODUCTION BY CORTISOL ADMINISTRATION TO NORMAL SUBJECTS Don H. Nelson and Ann Ruhmann-Wennhold Departments of Medicine and Anatomy, University of Utah College of Medicine, Salt Lake City, Utah 84112 ABSTRACT. Superoxide anion production was suppressed in leukocytes obtained at two or four hours following oral administration of 50 to 100 mg of cortisol to normal subjects. The suppression lasted at least 4 hours but was not present at 24 hours. The measurement of superoxide anion production by leukocytes appears to be a convenient method for the serial estimation of an important biologic effect of corticosteroids in human subjects. Corticosteroid suppression of inflammation and bactericidal activity is widely accepted, but the mechanism is incompletely understood and methods for estimation of the effect are not readily available. Studies have indicated that corticosteroids inhibit leukocyte functions such as nitroblue tetrazolium reduction or bactericidal killing in vitro, but they have employed as much as one mg corticosteroid per ml and the measurements have been difficult to perform or lacking in precision and reproducibility (1-3). The production of superoxide anion has been suggested to be a major factor in the bactericidal action of leukocytes (4). The present study was conducted to determine whether quantities of corticosteroids often administered to patients would have an influence upon this parameter of leukocyte function. MATERIALS AND METHODS Methods for preparation of human leukocytes and measurement of superoxide anion by reduction of cytochrome £ were similar to those of Goldstein et al. (5). Fifteen ml of whole blood was obtained by venipuncture from normal control subjects. The whole blood was immediately added to a mixture of 4 ml acid citrate dextrose and 10 ml of 6% dextran in a 50 ml graduated cylinder. The blood was gently mixed and allowed to settle at Submitted: November 23, 1977
room temperature for 30 minutes. Approximately 15 ml of the supernatant was removed by aspiration and divided into two 7.5 ml aliquots in 50 ml centrifuge tubes. Seventeen ml of 0.87% ammonium chloride was inverted with each and they were centrifuged for 10 min at 155g. The cells were then mixed with 10 ml of Dulbecco's buffer (8g NaCl, 200 mg KC1, 1.15 g Na2HPO4, 200 mg KH2PO4, 67 mg anhydrous CaCl2, 203 mg MgCl2'6 H 2 0 in a liter of distilled H2O) and centrifuged at 155g for ten minutes. This wash was repeated once. At the conclusion of the second centrifugation 1 ml of Dulbecco's buffer was added to resuspend the two buttons which were combined into one sample. A 1:100 dilution was made in dilute bovine serum albumin (20 mg albumin per ml Dulbecco's buffer) and the cells were counted in a hemocytometer. The original cell suspension was diluted to make a final concentration of 4 x 10° cells per ml incubation mixture. Incubation of the cells was carried out at 37° in 75 \M cytochrome £ (Sigma Chemical Co., superoxide dismutase free), concanavalin A (Sigma Chemical Co) at a concentration of 30 yg per ml, and superoxide dismutase (obtained from Truett Laboratories, Dallas, Texas) in concentrations of 25 or 250 yg per ml. After a 30 min incubation the tubes were placed in ice and centrifuged for 10 min at 4° and 755g. Two hundred yl of the supernatant was diluted
702
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RAPID COMMUNICATIONS
J C E & M • 1978 Vol 46 • No 4
to 2.8 ml with phosphate buffer in a cuvette and the amount of cytochrome £ reduced measured in a Cary Model 15 spectophotometer as the AOD 550-540. A few grains of sodium dithionite were added to the sample cuvettes to confirm the total amount of cytochrome c^ in the incubation. Nmoles of cytochrome c^ were calculated using an extinction coefficient of 21,000 M" 1 cm"1 (6). Cortisol determinations were made using the BectonDickinson Immunodiagnosties automated radioimmunoassay instrument, Aria 120. Cortisol was given by mouth to normal subjects from whom informed consent was obtained and sampling commenced at 9-9:30 am. Statistical analysis was by student's t test for paired samples. RESULTS Superoxide anion production by isolated human leukocytes and plasma cortisol levels following the oral administration of 100 mg cortisol to a normal subject are illustrated in Fig. 1. The sampling does not demonstrate that the peak level of cortisol probably occurred at one hour, but clearly shows the very high levels of the hormone at 2 hrs followed by a significant decrease at 4 hours and return to normal by 24 hrs following administration. Continued decrease in the production of the anion at 4 hours as the level of corticosteroid decreased probably represents the expected lag in biologic effect as compared with the changing hormone levels. Although no attempt was made in this study to estimate the total length of the observed effect, return to essentially normal levels of superoxide anion production at 24 hours suggests an action of a few hours consistent with its known length of action in the suppression of plasma ACTH (7). Four subjects, to whom no cortisol was administered, showed no significant change in superoxide anion production by leukocytes obtained at 9 am, 11 am, and 1 pm (mean nmol cytochrome c_ reduced = 1.26, 1.36, and 1.40).
703 -90 -SO -70 -60 ?
6 -50 ~ -40 -30 -20 -10
Figure 1. Plasma cortisol levels and superoxide anion production (nmol cytochrome £ reduced per 10" cells) by human leukocytes prior to, and 2, 4, and 24 hours following the oral administration of 100 mg cortisol to a normal subject. Table 1 presents superoxide anion production by leukocytes obtained from seven normal subjects receiving 50 to 100 mg of cortisol orally. Concanavalin A significantly stimulated production of superoxide anion at each time interval whether or not the cells were suppressed by corticosteroid (P < 0.05 - P < 0.005). There was a significant decrease in superoxide anion production by the control cells, which were not stimulated by concanavalin A, at both 2 and 4 hours following cortisol administration (P < 0.05 and < 0.025). Cells were stimulated by concanavalin A to determine whether corticosteroid would suppress activated cells and the difference was significant only at 4 hours (P < 0.05). The increased cytochrome c_ reduction following stimulation by concanavalin A was shown to be secondary to superoxide anion by a marked suppression of the reduction by superoxide dismutase (SOD) (1.92 nmoles cytochrome c_ were reduced without SOD, and 0.39 with SOD, n=6, P < 0.025) (4).
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704
RAPID COMMUNICATIONS
Table 1. Effect of cortisol given orally upon superoxide anion production by human leukocytes with and without concanavalin A (con A) stimulation.
xp.
1 2 3
4 5 6 7
Cortisol dose (mg orally)
100 50 50 50 50 100 100
Without con A
meaniSEM
1
2 3 4 5 6 7
100 50 50
With con A
50
50 100 100 meaniSEM
Hours after cortisol
0
4
2
24
(nmol cytochrome c reduced/10° leukocytes) 0.64 0.10 0.29 0.90 1.64 0.98 0.76 1.36 3.96 2.92 2.38 2.19 0.62 0.26 0.57 0.75 0.86 0.31 0.25 0.78 3.47 3.52 3.43 3.37 0.96 0.77 0.52 1.06 1.74±0.53 #1.13±0.48 *1.31±0 .51 1.4910.37
1.94 1.65 6.11 1.55 1.45 6.69 3.72 3.30±0.85
1.12 1.38
0.80
1.55
3.70
8.53 1.52
6.53
4.06 1.26 1.40 6.60 3.19 3.1810.70
0.81
0.73
5.67 2.94 3.13±1 .1
Plasma cortisol (yg/dl) 11.4+0.98 56.5+6.8 Significantly different from zero hour * P < 0.05, DISCUSSION It is generally accepted that a primary response of human polymorphonuclear leukocytes during phagocytosis is the release of superoxide anion produced by the one electron reduction of oxygen. This production is related to the increase in oxygen consumption during phagocytosis although it is only one of a number of oxygen species produced. Polymorphonuclear leukocytes, and also mononuclear phagocytes have been shown to produce superoxide anion (8). In vivo studies in man have demonstrated decreased granulocyte adherence and suppression of specific granule secretion in patients receiving 20 mg prednisone a day or 400 mg IV cortisol (9,10). In the latter study the intravenous cortisol produced suppression for up to 48 hours. Granulocyte adherence was significantly affected by 40 mg of prednisone orally after 2 and 4 hours. Neutrophils obtained two hours after large doses of dexamethasone have been re-
0.96 5.57 2.04 *2.61+0.91 34.5+5.1 # < 0.025.
2.04
9.911.4
ported to have normal bactericidal killing activity, but incubation of neutrophils with cortisol for 30 min was associated with decreased killing of Staphylococcus aureus (11,1). Decreased superoxide anion production by the leukocytes of patients with chronic granulomatous disease whose leukocytes have a defect in bacterial killing also suggests an important action of the free radical (12). Further studies will be necessary to establish the importance of the cortisol suppression of superoxide anion demonstrated here and the relative susceptibility to infection of patients receiving large amounts of corticosteroids. Suppression of this anion may be one factor in the action of large amounts of corticosteroids to increase susceptibility to infection and suppress inflammation. REFERENCES 1.
Mandell, G.L., W. Rubin, E. W. Hook, The effect of an NADH oxidase inhibitor (hydrocortisone) on
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 August 2015. at 22:40 For personal use only. No other uses without permission. . All rights reserved.
KAFID (JUMMUJN1UA11UJNS
2.
3.
4.
5.
6.
7.
polymorphonuclear leukocyte bactericidal activity. J Clin Invest 49:1381, 1970. Chretien, J.H., V.F. Garagusi, Corticosteroid effect on phagocytosis and NBT reduction by human polymorphonuclear neutrophils. J. Reticulendothelial Soc 11:358, 1972. Spirer, Z., V. Zakuth, Y. Weisman et al., The effect of corticosteroids on the bactericidal activity of leucocytes. Experientia 30:303, 1974. Fridovich, I., Superoxide radical and the bactericidal action of phagocytes. N Engl J Med 290: 624, 1974. Goldstein, I.M., M. Cerqueira, S. Lind, et al, Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J Clin Invest 59:249, 1977. Massey, V., The microestimation of succinate and the extinction coefficient of cytochrome c^. Biochem Biophys Acta 34:255, 1959. Bethune, J.E., D.H. Nelson, and G.W. Thorn, Plasma ACTH in Addison's disease and its modification by the administration of adrenal steroids. J Clin Invest 36:1701, 1957.
7U5
8.
Drath, D.B., M.L. Karnovsky, Superoxide production by phagocytic leukocytes. J Experimental Med 141:257, 1975. 9. MacGregor R.R., P.J. Spagnuolo, A.L. Lentnek, Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N Eng J Med 291:642, 1974. 10. Wright, D.G., D.A. Bralove, J.I. Gallin, Neutrophil specific granule secretion: Effects of corticosteroids and colchicine in vivo. Clin Res 25:351A, 1977. 11. Glasser, L., D.W. Huestis, J.F. Jones, Functional capabilities of steroid-recruited neutrophils harvested for clinical transfusion. N Engl J Med 297:1033, 1977. 12. DeChatelet, L.R., Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J of Inf Dis 131:295, 1975. ACKNOWLEDGMENTS
Supported by the Kroc Foundation and USPH Grant No. GM23095. The authors wish to thank Mr. Chad Milne for his technical assistance and Mrs. Judy Burnham for her secretarial assistance.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 August 2015. at 22:40 For personal use only. No other uses without permission. . All rights reserved.
Vol. 46, No. 4 Printed in U.S.A.
INHIBITION OF LEUKOCYTE SUPEROXIDE ANION PRODUCTION BY CORTISOL ADMINISTRATION TO NORMAL SUBJECTS Don H. Nelson and Ann Ruhmann-Wennhold Departments of Medicine and Anatomy, University of Utah College of Medicine, Salt Lake City, Utah 84112 ABSTRACT. Superoxide anion production was suppressed in leukocytes obtained at two or four hours following oral administration of 50 to 100 mg of cortisol to normal subjects. The suppression lasted at least 4 hours but was not present at 24 hours. The measurement of superoxide anion production by leukocytes appears to be a convenient method for the serial estimation of an important biologic effect of corticosteroids in human subjects. Corticosteroid suppression of inflammation and bactericidal activity is widely accepted, but the mechanism is incompletely understood and methods for estimation of the effect are not readily available. Studies have indicated that corticosteroids inhibit leukocyte functions such as nitroblue tetrazolium reduction or bactericidal killing in vitro, but they have employed as much as one mg corticosteroid per ml and the measurements have been difficult to perform or lacking in precision and reproducibility (1-3). The production of superoxide anion has been suggested to be a major factor in the bactericidal action of leukocytes (4). The present study was conducted to determine whether quantities of corticosteroids often administered to patients would have an influence upon this parameter of leukocyte function. MATERIALS AND METHODS Methods for preparation of human leukocytes and measurement of superoxide anion by reduction of cytochrome £ were similar to those of Goldstein et al. (5). Fifteen ml of whole blood was obtained by venipuncture from normal control subjects. The whole blood was immediately added to a mixture of 4 ml acid citrate dextrose and 10 ml of 6% dextran in a 50 ml graduated cylinder. The blood was gently mixed and allowed to settle at Submitted: November 23, 1977
room temperature for 30 minutes. Approximately 15 ml of the supernatant was removed by aspiration and divided into two 7.5 ml aliquots in 50 ml centrifuge tubes. Seventeen ml of 0.87% ammonium chloride was inverted with each and they were centrifuged for 10 min at 155g. The cells were then mixed with 10 ml of Dulbecco's buffer (8g NaCl, 200 mg KC1, 1.15 g Na2HPO4, 200 mg KH2PO4, 67 mg anhydrous CaCl2, 203 mg MgCl2'6 H 2 0 in a liter of distilled H2O) and centrifuged at 155g for ten minutes. This wash was repeated once. At the conclusion of the second centrifugation 1 ml of Dulbecco's buffer was added to resuspend the two buttons which were combined into one sample. A 1:100 dilution was made in dilute bovine serum albumin (20 mg albumin per ml Dulbecco's buffer) and the cells were counted in a hemocytometer. The original cell suspension was diluted to make a final concentration of 4 x 10° cells per ml incubation mixture. Incubation of the cells was carried out at 37° in 75 \M cytochrome £ (Sigma Chemical Co., superoxide dismutase free), concanavalin A (Sigma Chemical Co) at a concentration of 30 yg per ml, and superoxide dismutase (obtained from Truett Laboratories, Dallas, Texas) in concentrations of 25 or 250 yg per ml. After a 30 min incubation the tubes were placed in ice and centrifuged for 10 min at 4° and 755g. Two hundred yl of the supernatant was diluted
702
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 August 2015. at 22:40 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS
J C E & M • 1978 Vol 46 • No 4
to 2.8 ml with phosphate buffer in a cuvette and the amount of cytochrome £ reduced measured in a Cary Model 15 spectophotometer as the AOD 550-540. A few grains of sodium dithionite were added to the sample cuvettes to confirm the total amount of cytochrome c^ in the incubation. Nmoles of cytochrome c^ were calculated using an extinction coefficient of 21,000 M" 1 cm"1 (6). Cortisol determinations were made using the BectonDickinson Immunodiagnosties automated radioimmunoassay instrument, Aria 120. Cortisol was given by mouth to normal subjects from whom informed consent was obtained and sampling commenced at 9-9:30 am. Statistical analysis was by student's t test for paired samples. RESULTS Superoxide anion production by isolated human leukocytes and plasma cortisol levels following the oral administration of 100 mg cortisol to a normal subject are illustrated in Fig. 1. The sampling does not demonstrate that the peak level of cortisol probably occurred at one hour, but clearly shows the very high levels of the hormone at 2 hrs followed by a significant decrease at 4 hours and return to normal by 24 hrs following administration. Continued decrease in the production of the anion at 4 hours as the level of corticosteroid decreased probably represents the expected lag in biologic effect as compared with the changing hormone levels. Although no attempt was made in this study to estimate the total length of the observed effect, return to essentially normal levels of superoxide anion production at 24 hours suggests an action of a few hours consistent with its known length of action in the suppression of plasma ACTH (7). Four subjects, to whom no cortisol was administered, showed no significant change in superoxide anion production by leukocytes obtained at 9 am, 11 am, and 1 pm (mean nmol cytochrome c_ reduced = 1.26, 1.36, and 1.40).
703 -90 -SO -70 -60 ?
6 -50 ~ -40 -30 -20 -10
Figure 1. Plasma cortisol levels and superoxide anion production (nmol cytochrome £ reduced per 10" cells) by human leukocytes prior to, and 2, 4, and 24 hours following the oral administration of 100 mg cortisol to a normal subject. Table 1 presents superoxide anion production by leukocytes obtained from seven normal subjects receiving 50 to 100 mg of cortisol orally. Concanavalin A significantly stimulated production of superoxide anion at each time interval whether or not the cells were suppressed by corticosteroid (P < 0.05 - P < 0.005). There was a significant decrease in superoxide anion production by the control cells, which were not stimulated by concanavalin A, at both 2 and 4 hours following cortisol administration (P < 0.05 and < 0.025). Cells were stimulated by concanavalin A to determine whether corticosteroid would suppress activated cells and the difference was significant only at 4 hours (P < 0.05). The increased cytochrome c_ reduction following stimulation by concanavalin A was shown to be secondary to superoxide anion by a marked suppression of the reduction by superoxide dismutase (SOD) (1.92 nmoles cytochrome c_ were reduced without SOD, and 0.39 with SOD, n=6, P < 0.025) (4).
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704
RAPID COMMUNICATIONS
Table 1. Effect of cortisol given orally upon superoxide anion production by human leukocytes with and without concanavalin A (con A) stimulation.
xp.
1 2 3
4 5 6 7
Cortisol dose (mg orally)
100 50 50 50 50 100 100
Without con A
meaniSEM
1
2 3 4 5 6 7
100 50 50
With con A
50
50 100 100 meaniSEM
Hours after cortisol
0
4
2
24
(nmol cytochrome c reduced/10° leukocytes) 0.64 0.10 0.29 0.90 1.64 0.98 0.76 1.36 3.96 2.92 2.38 2.19 0.62 0.26 0.57 0.75 0.86 0.31 0.25 0.78 3.47 3.52 3.43 3.37 0.96 0.77 0.52 1.06 1.74±0.53 #1.13±0.48 *1.31±0 .51 1.4910.37
1.94 1.65 6.11 1.55 1.45 6.69 3.72 3.30±0.85
1.12 1.38
0.80
1.55
3.70
8.53 1.52
6.53
4.06 1.26 1.40 6.60 3.19 3.1810.70
0.81
0.73
5.67 2.94 3.13±1 .1
Plasma cortisol (yg/dl) 11.4+0.98 56.5+6.8 Significantly different from zero hour * P < 0.05, DISCUSSION It is generally accepted that a primary response of human polymorphonuclear leukocytes during phagocytosis is the release of superoxide anion produced by the one electron reduction of oxygen. This production is related to the increase in oxygen consumption during phagocytosis although it is only one of a number of oxygen species produced. Polymorphonuclear leukocytes, and also mononuclear phagocytes have been shown to produce superoxide anion (8). In vivo studies in man have demonstrated decreased granulocyte adherence and suppression of specific granule secretion in patients receiving 20 mg prednisone a day or 400 mg IV cortisol (9,10). In the latter study the intravenous cortisol produced suppression for up to 48 hours. Granulocyte adherence was significantly affected by 40 mg of prednisone orally after 2 and 4 hours. Neutrophils obtained two hours after large doses of dexamethasone have been re-
0.96 5.57 2.04 *2.61+0.91 34.5+5.1 # < 0.025.
2.04
9.911.4
ported to have normal bactericidal killing activity, but incubation of neutrophils with cortisol for 30 min was associated with decreased killing of Staphylococcus aureus (11,1). Decreased superoxide anion production by the leukocytes of patients with chronic granulomatous disease whose leukocytes have a defect in bacterial killing also suggests an important action of the free radical (12). Further studies will be necessary to establish the importance of the cortisol suppression of superoxide anion demonstrated here and the relative susceptibility to infection of patients receiving large amounts of corticosteroids. Suppression of this anion may be one factor in the action of large amounts of corticosteroids to increase susceptibility to infection and suppress inflammation. REFERENCES 1.
Mandell, G.L., W. Rubin, E. W. Hook, The effect of an NADH oxidase inhibitor (hydrocortisone) on
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 August 2015. at 22:40 For personal use only. No other uses without permission. . All rights reserved.
KAFID (JUMMUJN1UA11UJNS
2.
3.
4.
5.
6.
7.
polymorphonuclear leukocyte bactericidal activity. J Clin Invest 49:1381, 1970. Chretien, J.H., V.F. Garagusi, Corticosteroid effect on phagocytosis and NBT reduction by human polymorphonuclear neutrophils. J. Reticulendothelial Soc 11:358, 1972. Spirer, Z., V. Zakuth, Y. Weisman et al., The effect of corticosteroids on the bactericidal activity of leucocytes. Experientia 30:303, 1974. Fridovich, I., Superoxide radical and the bactericidal action of phagocytes. N Engl J Med 290: 624, 1974. Goldstein, I.M., M. Cerqueira, S. Lind, et al, Evidence that the superoxide-generating system of human leukocytes is associated with the cell surface. J Clin Invest 59:249, 1977. Massey, V., The microestimation of succinate and the extinction coefficient of cytochrome c^. Biochem Biophys Acta 34:255, 1959. Bethune, J.E., D.H. Nelson, and G.W. Thorn, Plasma ACTH in Addison's disease and its modification by the administration of adrenal steroids. J Clin Invest 36:1701, 1957.
7U5
8.
Drath, D.B., M.L. Karnovsky, Superoxide production by phagocytic leukocytes. J Experimental Med 141:257, 1975. 9. MacGregor R.R., P.J. Spagnuolo, A.L. Lentnek, Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin, measured with an assay system. N Eng J Med 291:642, 1974. 10. Wright, D.G., D.A. Bralove, J.I. Gallin, Neutrophil specific granule secretion: Effects of corticosteroids and colchicine in vivo. Clin Res 25:351A, 1977. 11. Glasser, L., D.W. Huestis, J.F. Jones, Functional capabilities of steroid-recruited neutrophils harvested for clinical transfusion. N Engl J Med 297:1033, 1977. 12. DeChatelet, L.R., Oxidative bactericidal mechanisms of polymorphonuclear leukocytes. J of Inf Dis 131:295, 1975. ACKNOWLEDGMENTS
Supported by the Kroc Foundation and USPH Grant No. GM23095. The authors wish to thank Mr. Chad Milne for his technical assistance and Mrs. Judy Burnham for her secretarial assistance.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 August 2015. at 22:40 For personal use only. No other uses without permission. . All rights reserved.
