Clin. exp. Immunol. (1977) 28, 467-473.
The effect of acute and prolonged administration of prednisolone and ACTH on lymphocyte subpopulations D. A. COOPER, VALERIE PETTS, ELIZABETH LUCKHURST & R. PENNY Department of Immunology, St Vincent's Hospital, and the Department of Medicine, University of New South Wales, Sydney, Australia
(Received 18 January 1977) SUMMARY
The effect of acute and prolonged three week administration of prednisolone and ACTH on the numbers and function of T- and B-lymphocyte subpopulations in patients requiring corticosteroid therapy was studied. Prednisolone caused severe reduction in E-rosette-forming lymphocytes, phytohaemagglutinin response, EAC-rosette-forming lymphocytes and surface-membrane yupositive B-lymphocytes maximal at 4-6 hr after administration with reversal sometimes to supernormal levels by 24 hr. Prolonged administration resulted in a similar pattern of response. Acute but not prolonged prednisolone administration caused a reduction in the percentage of E-rosette-forming lymphocytes maximal at 4 hr. ACTH caused moderate reduction in these parameters at 4 and 6 hr which remained low at 24 hr after prolonged administration.
INTRODUCTION Corticosteroids have been used for almost 30 years in the treatment of a variety of inflammatory, allergic, hypersensitivity, autoimmune and neoplastic diseases. These agents cause immunosuppression by affecting numbers and reducing function of lymphoid-cell populations (Claman, 1972). Marked differences in corticosteroid sensitivity of such lymphoid cells in different mammalian species point to the error of extrapolation of results obtained from animal experiments to clinical practice (Claman, 1972). Subpopulations of lymphoid cells in man have been defined by the use of surface markers and lectin responses, and divided into thymus-derived or T lymphocytes and bone-marrow-derived or B lymphocytes. Within these major subpopulations further subclasses have been recognized. The effect of acute corticosteroid administration on these subpopulations in normal man has recently been studied (Fauci & Dale, 1974; Yu et al., 1974). Although ACTH therapy has been used as an alternative to corticosteroid therapy, there is little available data on the effect of ACTH on lymphocyte subpopulations or whether such effects differ from corticosteroids. This study compares the effects of acute and continued administration of prednisolone and ACTH on lymphocyte subpopulations. MATERIALS AND METHODS Patients. After obtaining informed consent for the study, sixteen patients were randomly allocated either prednisolone or ACTH therapy. The indications for therapy were iritis (six patients), retinal vasculitis (four), Sjogren's syndrome (two), immune haemolytic anaemia (one) and malignant lymphoma without circulating tumour cells (one). One patient with staphylococcal skin infections, and one patient with recurrent herpes simplex infection, who were otherwise immunologically normal, were used as controls together with two of the patients with iritis who were studied prior to the commencement of corticosteroid therapy. No patient had received prior corticosteroid therapy except for one with retinal vasculitis who had a short course of high-dose corticosteroid therapy for Guillain-Barre syndrome 4 years previously. Correspondence: Dr D. A. Cooper, Department of Immunology, St Vincent's Hospital, Victoria Street, Darlinghurst N.S.W. 2010, Australia.
467
468
D. A. Cooper et al.
Design of study. The patients were studied over a period of 24 hr on two separate occasions, 3 weeks apart, referred to as periods I and II. ACTH or prednisolone therapy was commenced in period I and continued for 3 weeks until period II. Efforts were made to select comparable high doses of ACTH and prednisolone. ACTH was administered as tetracosactrin (Synacthen Depot, Ciba-Geigy) in a dose of 0.5 mg i.m. on each of the first 2 days and then 0 5 mg on the second day. Prior to period II ACTH had not been administered in the previous 48 hr. Prednisolone obtained as 25 mg tablets was administered in a dose of 50 mg orally at the commencement of period I, then at a dose of 25 mg twice a day until the commencement of period II when another 50 mg dose was given. The first blood samples were collected between 8 a.m. and 9 a.m., the patients were put to bed rest and the allocated drug was administered. Apart from toilet privileges, the patients remained in bed for the duration of the period. Normal meals but no medications were permitted. During each period, approximately 300 ml of blood was collected from the patient without a tourniquet at 2, 4, 6, 8, 11 and 24 hr after administration of the drug. The four control patients were studied under similar conditions for one 24 hr period but no corticosteroid was administered. All five patients given ACTH and four of the patients given prednisolone therapy were studied over both periods but the other three patients taking prednisolone were studied during period I only. Controls. In order to control for the laboratory tests of T and B lymphocyte numbers and function, peripheral blood was obtained from normal adult hospital and laboratory staff. The same normal control was used for the one patient throughout the study. Lymphocyte counts. Total white cell counts were obtained using a Coulter S automated counter. Differentials were performed by the one observer (E.L.) on 200 white cells in Leishman-stained films. Lymphocyte Isolation. Peripheral blood was collected into sterile glass tubes (Vacutainer, Becton-Dickinson, U.S.A.) containing 10 u of preservative-free heparin (Commonwealth Serum Laboratories (C.S.L.) Melbourne, Australia) per ml of blood. The blood was diluted one part to two with calcium and magnesium-free Dulbecco phosphate-buffered saline (PBS) (C.S.L., Melbourne) and placed on a Ficoll-Hypaque (Pharmacia Fine Chemicals AB Uppsala, Sweden) (Winthrop Laboratories, Sydney Australia) separation mixture according to the method of Boyum (1968). The mononuclear cells were collected and washed three times with PBS. Mononuclear-cell preparations were at least 95%4 viable by trypan-blue exclusion. Serum. Serum from each patient was collected prior to period I and stored frozen at - 200C for use during period II. Serum collected prior to period I is referred to as pre-treatment serum. E-rosette-forming lymphocytes (E-RFL). T cells were determined by non-immune rosette formation (Special Technical Report, 1974), as modified by Cooper et al. (1975). Lymphocytes which bound three or more sheep red blood cells (SRBC) were counted as positive. Monocytes were excluded on morphological grounds. EAC-rosette-forming lymphocytes (EAC-RFL). Fresh SRBC in Alsever's solution (C.S.L., Melbourne) sensitized with rabbit antibody and mouse complement were used to identify the C3 receptor on B cells (Special Technical Report, 1974). A 0.500 suspension of 3-times-washed SRBC was incubated in 4 ml of a 1 in 3000 dilution of rabbit anti-SRBC antiserum (prepared in this laboratory) for 30 min at 370C. The cells were then washed twice in PBS and incubated in 0 1 ml of fresh mouse serum as the source of complement for 20 min at 370C. The sensitized cells were washed 3 times in PBS and resuspended at 0.5% in calcium and magnesium-free Hanks' balanced salt solution (BSS) (C.S.L. Melbourne) containing 10% foetal calf serum (FCS) (C.S.L. Melbourne). To 50 ul of the sensitized SRBC was added 5 x 105 3-times-washed lymphocytes in 100 j1 Hanks's BSS. The mixture was incubated for 10 min at 370C, centrifuged at 100 g for 5 min and reincubated at 370C for 30 min. The mixture was then stored overnight at 40C and vigourously resuspended on a vortex mixer to break up E-RFL prior to counting in a haemocytometer chamber. Lymphocytes which bound three or more SRBC were counted as positive. Monocytes were excluded on morphological grounds. Surface-membrane immunoglobulin (SmIg) staining. Monospecific solid-phase adsorbed sheep antisera to human IgA, IgG and IgM were obtained commercially from Wellcome Diagnostics, Beckenham, Kent (IP15 Batch No. 6768, IP16 Batch No. 8583, IP17 Batch No. 8678). Immunofluorescent (IF) staining for surface membrane immunoglobulin was carried out as previously described by Cooper et al. (1975). Lymphocyte blastogenesis to phytohaemagglutinin. The washed mononuclear-cell suspensions obtained from the FicollHypaque separation mixtures for each time interval were diluted in medium 199 (C.S.L., Melbourne) containing 20% autologous serum at a concentration of 5 x 105 lymphocytes/ml. To compare the effect of corticosteroid present in the serum on blastogenesis, the cells were cultured in serum collected at the corresponding time as well as in pre-treatment serum. The micro-method for measuring blastogenesis of lymphocytes to phytohaemagglutinin (PHA) has been previously described by Cooper et al. (1975). Radioactivity (expressed as disintegrations per minute, d/min) was measured in a liquid scintillation spectrometer (Phillips) with external standard and automatic quench correction. In vitro effect of ACTH and prednisolone. The effects of ACTH and prednisolone on rosette formation, immunofluorescent staining for SmIg and PHA blastogenesis were studied in vitro. Mononuclear-cell suspensions were incubated at 370C with ACTH (Synacthen Ciba-Geigy, Basel, Switzerland) or prednisolone (Solu-Dacortin, E. Merck Darmstadt, Germany) at concentrations ranging from 10- 4M to 10- 8M for periods of 1-2 hr. The cells were then either washed twice or left unwashed before adding SRBC, sensitized SRBC, fluoresceinated anti-immunoglobulin or PHA. The only effect observed which was different from the drug-free cultures was with prednisolone which caused significant inhibition of PHA blastogenesis at concentrations down to 10- 7M. Analysis of results. Initial studies indicated that the most significant effects of both drugs in both periods occurred at 4, 6 and 24 hr after administration. Hence these time intervals were selected for analysis. Results at 4, 6 and 24 hr for each
Effect of prednisolone and ACTH on lymphocytes
469
patient were directly related to the baseline recording at 0 hr because of the intrinsic variation in lymphocyte counts and mitogen responsiveness from patient to patient. In all cases the 0 hr result was assumed to be 1000 and the 4, 6 and 24 hr values were expressed as a percentage of the original 0 hour value. Hence values less than 100% for any parameter signify a decrease in that parameter whereas values greater than 1000% signify an increase. The patterns of changes in IF staining of Smy- and Sma-bearing lymphocytes did not differ significantly from those Smu. Hence only the results for Smp-IF staining are presented. The results of each parameter at these time intervals for the patients in the control group and the groups given ACTH or prednisolone during periods I and II were analysed separately. For the numbers of lymphocytes, E-RFL, EAC-RFL and Smp-positive B lymphocytes, the arithmetic mean + s.e.m. were taken. For the percentage of E-RFL, the 0 time result was not normalized to 1000% but was analysed as counted; the arithmetic mean + s.e.m. was taken. For PHA blastogenesis the geometric means + s.e. at the optimal dose of PHA (100 pg/ml) were taken as validated by Ziegler et al. (1974). Statistical analysis. Non-parametric tests of statistical significance were used to compare data obtained. These included the Wilcoxon signed-ranks test for matched pairs and the Wilcoxon rank-sum test for two samples of unequal size.
RESULTS The results of T-lymphocyte numbers and function in all groups are shown in Fig. 1 and the results for total lymphocyte and B-lymphocyte numbers are shown in Fig. 2. 100 _ (d) 150
(b)
(a)
10)50
50 {
250
002 200 -I
00oo
-I CII 1
oU
0,1500 0100
'-t---
500
0
4 6
/A
24
0
4 6
24
0
4 6
24
0
0
4 6
24
Time (hr) FIG. 1 Changes in PHA blastogenesis (autologous and pre-treatment serum), E-RFL and per cent E-RFL in controls and following administration of ACTH and prednisolone to patients. Mean + s.e.m. of per cent of0 hr value at 4, 6 and 24 hr (PHA, E-RFL) and mean + s.e.m. of per cent E-RFL at 0, 4, 6 and 24 hr are shown for controls (A . A), patients in period I (a - ) and in period II (* *). (a) PHA; (b) PHA in pretreatment serum; (c) E-RFL; (d) per cent E-RFL.
Control There was a wide scatter of the values of the results obtained in the control patients over the 24 hr period of study. A trend was noted for elevated T- and B-lymphocyte numbers up to 150% at 4 and 6 hr which returned to baseline at 24 hr. However, statistical significance was only obtained for numbers of lymphocytes (P
The effect of acute and prolonged administration of prednisolone and ACTH on lymphocyte subpopulations D. A. COOPER, VALERIE PETTS, ELIZABETH LUCKHURST & R. PENNY Department of Immunology, St Vincent's Hospital, and the Department of Medicine, University of New South Wales, Sydney, Australia
(Received 18 January 1977) SUMMARY
The effect of acute and prolonged three week administration of prednisolone and ACTH on the numbers and function of T- and B-lymphocyte subpopulations in patients requiring corticosteroid therapy was studied. Prednisolone caused severe reduction in E-rosette-forming lymphocytes, phytohaemagglutinin response, EAC-rosette-forming lymphocytes and surface-membrane yupositive B-lymphocytes maximal at 4-6 hr after administration with reversal sometimes to supernormal levels by 24 hr. Prolonged administration resulted in a similar pattern of response. Acute but not prolonged prednisolone administration caused a reduction in the percentage of E-rosette-forming lymphocytes maximal at 4 hr. ACTH caused moderate reduction in these parameters at 4 and 6 hr which remained low at 24 hr after prolonged administration.
INTRODUCTION Corticosteroids have been used for almost 30 years in the treatment of a variety of inflammatory, allergic, hypersensitivity, autoimmune and neoplastic diseases. These agents cause immunosuppression by affecting numbers and reducing function of lymphoid-cell populations (Claman, 1972). Marked differences in corticosteroid sensitivity of such lymphoid cells in different mammalian species point to the error of extrapolation of results obtained from animal experiments to clinical practice (Claman, 1972). Subpopulations of lymphoid cells in man have been defined by the use of surface markers and lectin responses, and divided into thymus-derived or T lymphocytes and bone-marrow-derived or B lymphocytes. Within these major subpopulations further subclasses have been recognized. The effect of acute corticosteroid administration on these subpopulations in normal man has recently been studied (Fauci & Dale, 1974; Yu et al., 1974). Although ACTH therapy has been used as an alternative to corticosteroid therapy, there is little available data on the effect of ACTH on lymphocyte subpopulations or whether such effects differ from corticosteroids. This study compares the effects of acute and continued administration of prednisolone and ACTH on lymphocyte subpopulations. MATERIALS AND METHODS Patients. After obtaining informed consent for the study, sixteen patients were randomly allocated either prednisolone or ACTH therapy. The indications for therapy were iritis (six patients), retinal vasculitis (four), Sjogren's syndrome (two), immune haemolytic anaemia (one) and malignant lymphoma without circulating tumour cells (one). One patient with staphylococcal skin infections, and one patient with recurrent herpes simplex infection, who were otherwise immunologically normal, were used as controls together with two of the patients with iritis who were studied prior to the commencement of corticosteroid therapy. No patient had received prior corticosteroid therapy except for one with retinal vasculitis who had a short course of high-dose corticosteroid therapy for Guillain-Barre syndrome 4 years previously. Correspondence: Dr D. A. Cooper, Department of Immunology, St Vincent's Hospital, Victoria Street, Darlinghurst N.S.W. 2010, Australia.
467
468
D. A. Cooper et al.
Design of study. The patients were studied over a period of 24 hr on two separate occasions, 3 weeks apart, referred to as periods I and II. ACTH or prednisolone therapy was commenced in period I and continued for 3 weeks until period II. Efforts were made to select comparable high doses of ACTH and prednisolone. ACTH was administered as tetracosactrin (Synacthen Depot, Ciba-Geigy) in a dose of 0.5 mg i.m. on each of the first 2 days and then 0 5 mg on the second day. Prior to period II ACTH had not been administered in the previous 48 hr. Prednisolone obtained as 25 mg tablets was administered in a dose of 50 mg orally at the commencement of period I, then at a dose of 25 mg twice a day until the commencement of period II when another 50 mg dose was given. The first blood samples were collected between 8 a.m. and 9 a.m., the patients were put to bed rest and the allocated drug was administered. Apart from toilet privileges, the patients remained in bed for the duration of the period. Normal meals but no medications were permitted. During each period, approximately 300 ml of blood was collected from the patient without a tourniquet at 2, 4, 6, 8, 11 and 24 hr after administration of the drug. The four control patients were studied under similar conditions for one 24 hr period but no corticosteroid was administered. All five patients given ACTH and four of the patients given prednisolone therapy were studied over both periods but the other three patients taking prednisolone were studied during period I only. Controls. In order to control for the laboratory tests of T and B lymphocyte numbers and function, peripheral blood was obtained from normal adult hospital and laboratory staff. The same normal control was used for the one patient throughout the study. Lymphocyte counts. Total white cell counts were obtained using a Coulter S automated counter. Differentials were performed by the one observer (E.L.) on 200 white cells in Leishman-stained films. Lymphocyte Isolation. Peripheral blood was collected into sterile glass tubes (Vacutainer, Becton-Dickinson, U.S.A.) containing 10 u of preservative-free heparin (Commonwealth Serum Laboratories (C.S.L.) Melbourne, Australia) per ml of blood. The blood was diluted one part to two with calcium and magnesium-free Dulbecco phosphate-buffered saline (PBS) (C.S.L., Melbourne) and placed on a Ficoll-Hypaque (Pharmacia Fine Chemicals AB Uppsala, Sweden) (Winthrop Laboratories, Sydney Australia) separation mixture according to the method of Boyum (1968). The mononuclear cells were collected and washed three times with PBS. Mononuclear-cell preparations were at least 95%4 viable by trypan-blue exclusion. Serum. Serum from each patient was collected prior to period I and stored frozen at - 200C for use during period II. Serum collected prior to period I is referred to as pre-treatment serum. E-rosette-forming lymphocytes (E-RFL). T cells were determined by non-immune rosette formation (Special Technical Report, 1974), as modified by Cooper et al. (1975). Lymphocytes which bound three or more sheep red blood cells (SRBC) were counted as positive. Monocytes were excluded on morphological grounds. EAC-rosette-forming lymphocytes (EAC-RFL). Fresh SRBC in Alsever's solution (C.S.L., Melbourne) sensitized with rabbit antibody and mouse complement were used to identify the C3 receptor on B cells (Special Technical Report, 1974). A 0.500 suspension of 3-times-washed SRBC was incubated in 4 ml of a 1 in 3000 dilution of rabbit anti-SRBC antiserum (prepared in this laboratory) for 30 min at 370C. The cells were then washed twice in PBS and incubated in 0 1 ml of fresh mouse serum as the source of complement for 20 min at 370C. The sensitized cells were washed 3 times in PBS and resuspended at 0.5% in calcium and magnesium-free Hanks' balanced salt solution (BSS) (C.S.L. Melbourne) containing 10% foetal calf serum (FCS) (C.S.L. Melbourne). To 50 ul of the sensitized SRBC was added 5 x 105 3-times-washed lymphocytes in 100 j1 Hanks's BSS. The mixture was incubated for 10 min at 370C, centrifuged at 100 g for 5 min and reincubated at 370C for 30 min. The mixture was then stored overnight at 40C and vigourously resuspended on a vortex mixer to break up E-RFL prior to counting in a haemocytometer chamber. Lymphocytes which bound three or more SRBC were counted as positive. Monocytes were excluded on morphological grounds. Surface-membrane immunoglobulin (SmIg) staining. Monospecific solid-phase adsorbed sheep antisera to human IgA, IgG and IgM were obtained commercially from Wellcome Diagnostics, Beckenham, Kent (IP15 Batch No. 6768, IP16 Batch No. 8583, IP17 Batch No. 8678). Immunofluorescent (IF) staining for surface membrane immunoglobulin was carried out as previously described by Cooper et al. (1975). Lymphocyte blastogenesis to phytohaemagglutinin. The washed mononuclear-cell suspensions obtained from the FicollHypaque separation mixtures for each time interval were diluted in medium 199 (C.S.L., Melbourne) containing 20% autologous serum at a concentration of 5 x 105 lymphocytes/ml. To compare the effect of corticosteroid present in the serum on blastogenesis, the cells were cultured in serum collected at the corresponding time as well as in pre-treatment serum. The micro-method for measuring blastogenesis of lymphocytes to phytohaemagglutinin (PHA) has been previously described by Cooper et al. (1975). Radioactivity (expressed as disintegrations per minute, d/min) was measured in a liquid scintillation spectrometer (Phillips) with external standard and automatic quench correction. In vitro effect of ACTH and prednisolone. The effects of ACTH and prednisolone on rosette formation, immunofluorescent staining for SmIg and PHA blastogenesis were studied in vitro. Mononuclear-cell suspensions were incubated at 370C with ACTH (Synacthen Ciba-Geigy, Basel, Switzerland) or prednisolone (Solu-Dacortin, E. Merck Darmstadt, Germany) at concentrations ranging from 10- 4M to 10- 8M for periods of 1-2 hr. The cells were then either washed twice or left unwashed before adding SRBC, sensitized SRBC, fluoresceinated anti-immunoglobulin or PHA. The only effect observed which was different from the drug-free cultures was with prednisolone which caused significant inhibition of PHA blastogenesis at concentrations down to 10- 7M. Analysis of results. Initial studies indicated that the most significant effects of both drugs in both periods occurred at 4, 6 and 24 hr after administration. Hence these time intervals were selected for analysis. Results at 4, 6 and 24 hr for each
Effect of prednisolone and ACTH on lymphocytes
469
patient were directly related to the baseline recording at 0 hr because of the intrinsic variation in lymphocyte counts and mitogen responsiveness from patient to patient. In all cases the 0 hr result was assumed to be 1000 and the 4, 6 and 24 hr values were expressed as a percentage of the original 0 hour value. Hence values less than 100% for any parameter signify a decrease in that parameter whereas values greater than 1000% signify an increase. The patterns of changes in IF staining of Smy- and Sma-bearing lymphocytes did not differ significantly from those Smu. Hence only the results for Smp-IF staining are presented. The results of each parameter at these time intervals for the patients in the control group and the groups given ACTH or prednisolone during periods I and II were analysed separately. For the numbers of lymphocytes, E-RFL, EAC-RFL and Smp-positive B lymphocytes, the arithmetic mean + s.e.m. were taken. For the percentage of E-RFL, the 0 time result was not normalized to 1000% but was analysed as counted; the arithmetic mean + s.e.m. was taken. For PHA blastogenesis the geometric means + s.e. at the optimal dose of PHA (100 pg/ml) were taken as validated by Ziegler et al. (1974). Statistical analysis. Non-parametric tests of statistical significance were used to compare data obtained. These included the Wilcoxon signed-ranks test for matched pairs and the Wilcoxon rank-sum test for two samples of unequal size.
RESULTS The results of T-lymphocyte numbers and function in all groups are shown in Fig. 1 and the results for total lymphocyte and B-lymphocyte numbers are shown in Fig. 2. 100 _ (d) 150
(b)
(a)
10)50
50 {
250
002 200 -I
00oo
-I CII 1
oU
0,1500 0100
'-t---
500
0
4 6
/A
24
0
4 6
24
0
4 6
24
0
0
4 6
24
Time (hr) FIG. 1 Changes in PHA blastogenesis (autologous and pre-treatment serum), E-RFL and per cent E-RFL in controls and following administration of ACTH and prednisolone to patients. Mean + s.e.m. of per cent of0 hr value at 4, 6 and 24 hr (PHA, E-RFL) and mean + s.e.m. of per cent E-RFL at 0, 4, 6 and 24 hr are shown for controls (A . A), patients in period I (a - ) and in period II (* *). (a) PHA; (b) PHA in pretreatment serum; (c) E-RFL; (d) per cent E-RFL.
Control There was a wide scatter of the values of the results obtained in the control patients over the 24 hr period of study. A trend was noted for elevated T- and B-lymphocyte numbers up to 150% at 4 and 6 hr which returned to baseline at 24 hr. However, statistical significance was only obtained for numbers of lymphocytes (P
