67 Horm. Metab. Res. 8 (1976) 67-70
© Georg Thieme Verlag Stuttgart
The Effect of ACTH Administration on the Phagocytic and Bactericidal Activities of Human Polymorphonuclear leukocytes P.G. Shilotri and J. Siva Prasad
Summary
Materials and Methods
Metabolie and bacterieial activities of leukoeytes obtained from 5 normal volunteers receiving 1 mg of synthetie BI-24 ACTH intra-museularly daily for 7 days, were studied. Baeterieidal activity and phagocytosis indueed hexosemonophosphate shunt activity of leukoeytes were found to be depressed following ACTH administration. However, glyeoIytie aetivity, whieh provides the necessary energy for particle uptake by leukocytes, was not altered. These findings indieate that a moderately prolonged exposure to elevated levels of plasma eortisol does not affeet the phagoeytie aetivity of leukoeytes (as indieated by glyeolytie aetivity) but significantly impairs their ability to destroy the ingested baeteria. It is suggested that the impaired baetericidal activity of leukocytes reported in ehildren suffering from proteincalorie malnutrition may partly be due to elevated plasma cortisol levels seen in them.
Experimental subjects: Five apparently normal adult male volunteers were the subjeets of the study. It was ensured that they were not suffering from any infection either at the time of the study or immediately prior to it.
Key·Words: ACTH - Plasma Cortisol - Phagocytosis &ctericidal Activity - Glycolytic Activity - Hexosemono· phosphate Shunt Activity - Protein·calorie Malnutrition
Introduction An increased susceptibility to bacterial infections has been amp1y demonstrated du ring therapy with ACTH and glucocorticoids (Thamas 1952). Plasma cortisol levels have been reported to be high in advanced states of protein-calorie malnutrition (AI/eyne and Young 1966, Jaya Raa, Srikantia and Gopa/an 1968). Various immune mechanisms have been shown to be impaired in this condition (Scrimshaw, Taylar and Gordan 1968), inc\uding phagocytic and bactericidal activities (Selvaraj and Bhat 1972, Seth and Chandra 1972). Hydrocortisone has been shown to inhibit leukocyte migration (Baggs, A thens, Cartwright and Wintrobe 1964), release of lysosomal enzymes (Weismann 1964) and to increase immunoglobulin catabolism (Levy and Waldmann 1970). Information on the effect of glucocorticoids on phagocytosis and bactericidal activity is, however, scanty and contlicting (Crepea, Magnin and Seastane 1951, Hirsch and Church 1961, Chretien and Garagusi 1972). Phagocytosis and intracellular destruction of bacteria have been shown to be accompanied by an increase in glycolysis and oxidative metabolism of glucose (Selvaraj and Sbarra 1966, Sbarra, Jacabs, Strauss, Paul and Mitchell Jr. 1971). A study was therefore undertaken to investigate the effect of ACTH administration on leukocyte metabolism in normal subjects. Received: 27 May 1975
Aeeepted: 6 Oet. 1975
Experimental design: A sampie of venous blood was obtained from eaeh subjeet under basal conditions at the beginning of the study. Each subjeet then reeeived daily intramuscular injeetions of 1 mg of a long-acting preparation of synthetie ACTH (BI-24 Synacthen Depot, Ciba), for 7 days. Twenty four hours following the last injection, a repeat sampie of blood was obtained. Blood was eollected under heparin and leukocytes were immediately isolated by dextran flotation technique (Christ· lieb, Sbarra and Bardawil 1962). The cells were treated with 3 ml iee-eold distilled water for 20 seconds to Iyse erythrocytes followed by 1 ml 3.6% sodium chloride to restore isotonicity. The cells, eolleeted after light centrifugation, were suspended in a known volume of Krebs-Ringer phosphate buffeT. Bactericidal activity of leukocytes, using Escherichio coli as the test organisms was measured as described by Selvaraj and Bhat (1972). Hexosemonophosphate shunt (HMS) aetivity was assayed by the eonversion of glucose-l-14 C to 14C0 2 by leukoeytes under resting and phagocytizing conditions (Selvara; and Bhat 1972). Glycolytic activity was measured by estimating lactate in the protein-free filtrate following the procedure of Barker and Summerson (1941). Plasma eortisol was estimated by the fluorometric method of Mattingly (1962). Glucose-J.l4C (Sp. Act. 50.0 mCi/mmole) was obtained from BARC, Bombay, India. Synacthen Depot. was the kind gift of Ciba, Switzerland.
Results The mean plasma cortisol level at the beginning of the study was 13.9 ± l.l0 J.Lg/100 ml. After the 7-day treatment with ACTH, the level increased significantly to 19.6 ± 1.25 J.Lg/100 ml (P < 0.001). Data on the bactericidal activity of leukocytes are presented in Figure 1. Initially, the mean number of viable bacteria at the end of 30 and 60 minutes of incubation were 5.6 x 10 3 ± 3.4 and 2.8 x 10 3 ± 1.3 respectively. At the end of 7 days of ACTH administration, the numbers were significantly increased to 122.4 ± 103 ± 2l.l (P< 0.01) and 118.0 ± 103 ± 25.9 (P < 0.02) respectively, indicating a reduced bactericidal activi ty. The results of lactate production and conversion of glucose-l-1 4C to 14C02 du ring resting and phagocytizing conditions, are presented in Table 1. ACTH administration did not bring about any significant al-
Downloaded by: University of British Columbia. Copyrighted material.
National Institute 01 Nutrition, Indian Council of Medical Research, Hyderabad, India
68
P.G. Shilotri and J. Siva Prasad
Crepea et al. used the more direct method of particle ingestion by leucycytes, the dose of ACTH used was much higher.
101
10'
PLASMA CONTROL
The observed effects may be assumed to have been brought about by the elevated plasma cortisol levels, though the direct effect of ACTH cannot be ruled out (Rebuck, Smith and Margulis 1951). The synthetic preparation of ACTH used in this study has been found to raise plasma cortisol levels 2-3 fold and even at the end of 24 hours after administration, levels were found to be much higher than the initial concentration (Besser, Butler and Plumpton 1967, Nelson, Neill, Montigomery, Mackay, Sheridan and Weaver 1968). In the present study too, significantly elevated levels of plasma cortisol were observed 24 hours after the injection of the hormone preparation.
C
...
ii
t
10 5
AfTER AcrH
~
...... •c;;
10'
~
..."' !
..
::I
101
Data reported here indicate that ACTH administration does not significantly affect phagocytosis by leucocytes. This is in contrast to the observation of Crepea, Magnin and Seastone (I951). However, it may be noted here that in the present study phagocytosis was investigated by using glycolysis as a parameter. Though
In children suffering from severe forms of proteincalorie mal nutrition elevated levels of plasma cortisol have been reported (Alleyne and Young 1966, Jaya Rao, Srikantia and Gopa/an 1968), also bactericidal activity of leukocytes has been demonstrated to be low (Selvaraj and Bhat 1972, Seth and Chandra 1972).
L-----------r-----------.---~ 30 TIME IN M'HUTES
Fig. 1. Effect of AcrH on the bactericidal activity of leukocytes. The assay system consisted of leukocytes and bacteria (E. coli) in the ratio of 1:2 and 20% heat-inactivated (56 0 C, 30 min.) autologous plasma in Krebs-Ringer phosphate medium. The plasma control did not contain leukocytes.
teration in the production of C0 1 during resting condition; however, a significant depression was observed during phagocytizing conditions (P < 0.05). Lactate production on the other hand, showed no significance alteration during either situation.
Table 1. Effect of synthetic AcrH on metabolie activities of leukocytes in five human subjects (Mean ± SEM). Values are for 10 6 leukocytes for a 30-minutes incubation period with 5 mM glucose-1 4C (0.2 llCi/ml) and 10% heat-inactivated autologous plasma in Krebs-Ringer phosphate medium, pH 7.4. Phagocytosis was induced with heat-ki11ed E. coJi at a phagocyte: particle ratio of 1: 100.
Downloaded by: University of British Columbia. Copyrighted material.
Discussion
Steroid therapy has been found not to alter phagocytosis (Hirsch and C/zurch 1961, Clzretien and Garagusi 1972). On the other hand, glucocorticoids added in vitro have been shown to inhibit bactericidal activity and phagocytosis induced HMS activity (Mandeli, Rubin and Hook 1970, Cooper, Dechatelet and McCall 1972). However Olds, Reed, Elberle and Kisch (1974) have suggested that since these effects are brought about only in the presence of very high concentrations of the steroid, short-term steroid therapy is unlikely to produce significant impairment of intraleukocytic bacterial killing. Observations presen ted here, show that the phagocytosis stimulated HMS activity and bactericidal activity are significantly depressed in leukocytes exposed to moderately high levels of cortisol over as short aperiod as 7 days.
Ir
R = Resting, P = Phagocytizing, P/R = Phagocytic effect Description
Initial Range: After AcrH (1 mg/day for 7 days) administration Range:
Glucose-l-14 C ........... 14C01 (JnIlIßoles) R P 0.55 ± 0.05 (0.43 - 0.64)
0.66 ± 0.09 (0.42 - 1.00)
3.05 ± 0.35 (2.15 - 4.10)
1.71 ± 0.29* (1.05 - 2.76)
P/R
5.60 ± 0.36 (4.71 - 6.78)
2.70 ±0.31'" (1.52 - 3.24)
R
Lactate production (JnIlIßoles) P
P/R
36.0 ± 4.76 (28.7 53.3)
41.5 ± 5.16 (28.7 - 58.0)
1.20 ± 0.05 (1.00 - 1.32)
30.0 ± 4.85
37.7 ± 5.58
1.10 ± 0.04
(17.9-47.4
(20.3 - 52.5)
(0.94 - 1.18) -------
*
P
© Georg Thieme Verlag Stuttgart
The Effect of ACTH Administration on the Phagocytic and Bactericidal Activities of Human Polymorphonuclear leukocytes P.G. Shilotri and J. Siva Prasad
Summary
Materials and Methods
Metabolie and bacterieial activities of leukoeytes obtained from 5 normal volunteers receiving 1 mg of synthetie BI-24 ACTH intra-museularly daily for 7 days, were studied. Baeterieidal activity and phagocytosis indueed hexosemonophosphate shunt activity of leukoeytes were found to be depressed following ACTH administration. However, glyeoIytie aetivity, whieh provides the necessary energy for particle uptake by leukocytes, was not altered. These findings indieate that a moderately prolonged exposure to elevated levels of plasma eortisol does not affeet the phagoeytie aetivity of leukoeytes (as indieated by glyeolytie aetivity) but significantly impairs their ability to destroy the ingested baeteria. It is suggested that the impaired baetericidal activity of leukocytes reported in ehildren suffering from proteincalorie malnutrition may partly be due to elevated plasma cortisol levels seen in them.
Experimental subjects: Five apparently normal adult male volunteers were the subjeets of the study. It was ensured that they were not suffering from any infection either at the time of the study or immediately prior to it.
Key·Words: ACTH - Plasma Cortisol - Phagocytosis &ctericidal Activity - Glycolytic Activity - Hexosemono· phosphate Shunt Activity - Protein·calorie Malnutrition
Introduction An increased susceptibility to bacterial infections has been amp1y demonstrated du ring therapy with ACTH and glucocorticoids (Thamas 1952). Plasma cortisol levels have been reported to be high in advanced states of protein-calorie malnutrition (AI/eyne and Young 1966, Jaya Raa, Srikantia and Gopa/an 1968). Various immune mechanisms have been shown to be impaired in this condition (Scrimshaw, Taylar and Gordan 1968), inc\uding phagocytic and bactericidal activities (Selvaraj and Bhat 1972, Seth and Chandra 1972). Hydrocortisone has been shown to inhibit leukocyte migration (Baggs, A thens, Cartwright and Wintrobe 1964), release of lysosomal enzymes (Weismann 1964) and to increase immunoglobulin catabolism (Levy and Waldmann 1970). Information on the effect of glucocorticoids on phagocytosis and bactericidal activity is, however, scanty and contlicting (Crepea, Magnin and Seastane 1951, Hirsch and Church 1961, Chretien and Garagusi 1972). Phagocytosis and intracellular destruction of bacteria have been shown to be accompanied by an increase in glycolysis and oxidative metabolism of glucose (Selvaraj and Sbarra 1966, Sbarra, Jacabs, Strauss, Paul and Mitchell Jr. 1971). A study was therefore undertaken to investigate the effect of ACTH administration on leukocyte metabolism in normal subjects. Received: 27 May 1975
Aeeepted: 6 Oet. 1975
Experimental design: A sampie of venous blood was obtained from eaeh subjeet under basal conditions at the beginning of the study. Each subjeet then reeeived daily intramuscular injeetions of 1 mg of a long-acting preparation of synthetie ACTH (BI-24 Synacthen Depot, Ciba), for 7 days. Twenty four hours following the last injection, a repeat sampie of blood was obtained. Blood was eollected under heparin and leukocytes were immediately isolated by dextran flotation technique (Christ· lieb, Sbarra and Bardawil 1962). The cells were treated with 3 ml iee-eold distilled water for 20 seconds to Iyse erythrocytes followed by 1 ml 3.6% sodium chloride to restore isotonicity. The cells, eolleeted after light centrifugation, were suspended in a known volume of Krebs-Ringer phosphate buffeT. Bactericidal activity of leukocytes, using Escherichio coli as the test organisms was measured as described by Selvaraj and Bhat (1972). Hexosemonophosphate shunt (HMS) aetivity was assayed by the eonversion of glucose-l-14 C to 14C0 2 by leukoeytes under resting and phagocytizing conditions (Selvara; and Bhat 1972). Glycolytic activity was measured by estimating lactate in the protein-free filtrate following the procedure of Barker and Summerson (1941). Plasma eortisol was estimated by the fluorometric method of Mattingly (1962). Glucose-J.l4C (Sp. Act. 50.0 mCi/mmole) was obtained from BARC, Bombay, India. Synacthen Depot. was the kind gift of Ciba, Switzerland.
Results The mean plasma cortisol level at the beginning of the study was 13.9 ± l.l0 J.Lg/100 ml. After the 7-day treatment with ACTH, the level increased significantly to 19.6 ± 1.25 J.Lg/100 ml (P < 0.001). Data on the bactericidal activity of leukocytes are presented in Figure 1. Initially, the mean number of viable bacteria at the end of 30 and 60 minutes of incubation were 5.6 x 10 3 ± 3.4 and 2.8 x 10 3 ± 1.3 respectively. At the end of 7 days of ACTH administration, the numbers were significantly increased to 122.4 ± 103 ± 2l.l (P< 0.01) and 118.0 ± 103 ± 25.9 (P < 0.02) respectively, indicating a reduced bactericidal activi ty. The results of lactate production and conversion of glucose-l-1 4C to 14C02 du ring resting and phagocytizing conditions, are presented in Table 1. ACTH administration did not bring about any significant al-
Downloaded by: University of British Columbia. Copyrighted material.
National Institute 01 Nutrition, Indian Council of Medical Research, Hyderabad, India
68
P.G. Shilotri and J. Siva Prasad
Crepea et al. used the more direct method of particle ingestion by leucycytes, the dose of ACTH used was much higher.
101
10'
PLASMA CONTROL
The observed effects may be assumed to have been brought about by the elevated plasma cortisol levels, though the direct effect of ACTH cannot be ruled out (Rebuck, Smith and Margulis 1951). The synthetic preparation of ACTH used in this study has been found to raise plasma cortisol levels 2-3 fold and even at the end of 24 hours after administration, levels were found to be much higher than the initial concentration (Besser, Butler and Plumpton 1967, Nelson, Neill, Montigomery, Mackay, Sheridan and Weaver 1968). In the present study too, significantly elevated levels of plasma cortisol were observed 24 hours after the injection of the hormone preparation.
C
...
ii
t
10 5
AfTER AcrH
~
...... •c;;
10'
~
..."' !
..
::I
101
Data reported here indicate that ACTH administration does not significantly affect phagocytosis by leucocytes. This is in contrast to the observation of Crepea, Magnin and Seastone (I951). However, it may be noted here that in the present study phagocytosis was investigated by using glycolysis as a parameter. Though
In children suffering from severe forms of proteincalorie mal nutrition elevated levels of plasma cortisol have been reported (Alleyne and Young 1966, Jaya Rao, Srikantia and Gopa/an 1968), also bactericidal activity of leukocytes has been demonstrated to be low (Selvaraj and Bhat 1972, Seth and Chandra 1972).
L-----------r-----------.---~ 30 TIME IN M'HUTES
Fig. 1. Effect of AcrH on the bactericidal activity of leukocytes. The assay system consisted of leukocytes and bacteria (E. coli) in the ratio of 1:2 and 20% heat-inactivated (56 0 C, 30 min.) autologous plasma in Krebs-Ringer phosphate medium. The plasma control did not contain leukocytes.
teration in the production of C0 1 during resting condition; however, a significant depression was observed during phagocytizing conditions (P < 0.05). Lactate production on the other hand, showed no significance alteration during either situation.
Table 1. Effect of synthetic AcrH on metabolie activities of leukocytes in five human subjects (Mean ± SEM). Values are for 10 6 leukocytes for a 30-minutes incubation period with 5 mM glucose-1 4C (0.2 llCi/ml) and 10% heat-inactivated autologous plasma in Krebs-Ringer phosphate medium, pH 7.4. Phagocytosis was induced with heat-ki11ed E. coJi at a phagocyte: particle ratio of 1: 100.
Downloaded by: University of British Columbia. Copyrighted material.
Discussion
Steroid therapy has been found not to alter phagocytosis (Hirsch and C/zurch 1961, Clzretien and Garagusi 1972). On the other hand, glucocorticoids added in vitro have been shown to inhibit bactericidal activity and phagocytosis induced HMS activity (Mandeli, Rubin and Hook 1970, Cooper, Dechatelet and McCall 1972). However Olds, Reed, Elberle and Kisch (1974) have suggested that since these effects are brought about only in the presence of very high concentrations of the steroid, short-term steroid therapy is unlikely to produce significant impairment of intraleukocytic bacterial killing. Observations presen ted here, show that the phagocytosis stimulated HMS activity and bactericidal activity are significantly depressed in leukocytes exposed to moderately high levels of cortisol over as short aperiod as 7 days.
Ir
R = Resting, P = Phagocytizing, P/R = Phagocytic effect Description
Initial Range: After AcrH (1 mg/day for 7 days) administration Range:
Glucose-l-14 C ........... 14C01 (JnIlIßoles) R P 0.55 ± 0.05 (0.43 - 0.64)
0.66 ± 0.09 (0.42 - 1.00)
3.05 ± 0.35 (2.15 - 4.10)
1.71 ± 0.29* (1.05 - 2.76)
P/R
5.60 ± 0.36 (4.71 - 6.78)
2.70 ±0.31'" (1.52 - 3.24)
R
Lactate production (JnIlIßoles) P
P/R
36.0 ± 4.76 (28.7 53.3)
41.5 ± 5.16 (28.7 - 58.0)
1.20 ± 0.05 (1.00 - 1.32)
30.0 ± 4.85
37.7 ± 5.58
1.10 ± 0.04
(17.9-47.4
(20.3 - 52.5)
(0.94 - 1.18) -------
*
P
