Immunology, 1975, 28, 669.
Mechanisms of Corticosteroid Action on Lymphocyte Subpopulations I. REDISTRIBUTION OF CIRCULATING T AND B LYMPHOCYTES TO THE BONE MARROW A. S. FAUCI Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.
(Received 1st August 1974; acceptedfor publication 7th October 1974) Summary. The effect of corticosteroid administration on the redistribution of circulating lymphocytes was studied in the guinea-pig, since this species closely resembles man in its relative resistance to the lymphopenic effect of corticosteroids. A single intravenous injection of hydrocortisone (either 10 mg or 100 mg/kg) caused a profound but transient lymphocytopenia which was maximal at 4 hours following injection, with a return to normal counts by 24 hours. There was a proportionately greater decrease in circulating T lymphocytes compared to B lymphocytes, although both populations were diminished. Chronic cortisone acetate treatment (100 mg/kg subcutaneously for 7 days) caused a similar pattern of lymphocytopenia except that it was sustained during the period of chronically elevated plasma cortisol levels. The lymphocytes remaining in the circulation during the period of lymphocytopenia responded normally in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. There was very little effect of corticosteroid administration on the numbers, proportions, or mitogenic response of splenic lymphocytes. There was a dramatic increase in the bone marrow of proportions and absolute numbers of lymphocytes bearing surface T- and B-cell markers, as well as a marked increase in response of bone marrow lymphocytes to mitogenic stimulation during the period of maximal circulating lymphocytopenia caused by the administration of corticosteroids, especially chronic cortisone acetate. There was a preferential homing of reinfused 51Cr-labelled syngeneic peripheral blood lymphocytes to the bone marrow of corticosteroid-treated recipients. These studies demonstrate a redistribution of circulating lymphocytes to the bone marrow during corticosteroid treatment, resulting in an increase in immunocompetence ofthis compartment, while the peripheral blood lymphocyte compartment is quantitatively immunosuppressed due to a lymphocytopenia. INTRODUCTION The administration of corticosteroids to normal humans results in a profound but transient lymphocytopenia of both thymus-derived (T) and bursa equivalent (B) lymphoCorrespondence: Dr A. S. Fauci, Clinical Physiology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
669
670 A. S. Fauci cytes with a proportionately greater depletion of T lymphocytes (Fauci and Dale, 1974; Yu, Clements, Paulus, Peter, Levy and Barnett, 1974). The fate of these lymphocytes is unknown. It has been suggested that they are not destroyed in vivo, but are redistributed to other body compartments, particularly the bone marrow (Fauci and Dale, 1974; Yu et al., 1974). There is convincing evidence in the mouse model that the corticosteroidinduced lymphocytopenia is explained by a redistribution of T lymphocytes into the bone marrow, as measured by enhanced bone marrow responsiveness to mitogenic stimuli (Levine and Claman, 1970), enhanced capacity for helper cell function (Cohen, 1972), and for inducing graft-versus-host reactions (Cohen, Fischbach and Claman, 1970). It has been pointed out, however, that the mouse is a 'steroid-sensitive' species in that it is extremely susceptible to the lymphopenic effects of systemically administered corticosteroids (Claman, 1972). On the other hand, the guinea-pig more closely resembles man in this regard since both species are relatively resistant to corticosteroid-induced lymphopenia. Hence, the present study was designed to investigate the mechanisms and kinetics of the effects of both acutely and chronically administered corticosteroids on the redistribution between body compartments of guinea-pig lymphocyte subpopulations defined functionally and by surface markers. In addition, the effect of corticosteroid administration on the distribution of reinfused radioactive 51Cr-labelled syngeneic lymphocytes was studied. MATERIALS AND METHODS
Animals Inbred strain 13 female guinea-pigs (300-400 g) obtained from the Division of Research Services, National Institutes of Health, Bethesda, Maryland, were used throughout the study. Corticosteroid treatment For the acutely administered corticosteroid experiments, groups of non-immune animals were given a single injection intravenously (i.v.) in the hind foot vein of either 1 ml of normal saline (NaCl), 10 mg/kg, or 100 mg/kg of hydrocortisone sodium succinate (OHC) (The Upjohn Company, Kalamazoo, Michigan). For the chronically administered corticosteroid experiments, groups of non-immune animals were given subcutaneously (s.c.) either 1 ml of NaCl or 100 mg/kg of cortisone acetate (The Upjohn Company) daily for 7 days. The animals receiving chronic corticosteroid administration gained weight and thrived no differently from controls. Leucocyte counts and differentials In the group of guinea-pigs receiving a single i.v. dose of either NaCl or OHC, separate animals were bled at 0, 1, 4, 6 and 24 hours following injection and white blood cell (WBC) and differential counts were done in addition to plasma cortisol levels (Loriaux, Guy and Lipsett, 1973). In the group of guinea-pigs receiving 7 days of cortisone acetate, WBC and differential counts were done during treatment and at the end of the 7-day treatment period, 24 hours following the last dose of cortisone acetate. In addition, plasma cortisol levels were done daily throughout the 7 days of cortisone acetate treatment.
Preparation and purification of lymphocytes Four hours (point of maximal lymphocytopenia) after acutely administered NaCl or OHC and 24 hours following 7 days of chronically administered NaCl or cortisone acetate,
Corticosteroid Action on Lymphocytes. I. 671 the following studies were done: animals were exsanguinated by cardiac puncture and separation of peripheral blood lymphocytes was performed on acid-citrate-anticoagulated blood by Ficoll-Hypaque gradient centrifugation (Boyum, 1968). The spleens were aseptically removed and placed in a sterile Petri dish containing Eagle's minimum essential media modified for suspension culture (MEM-S) (Grand Island Biological Company, Grand Island, New York). Purified suspensions of splenic mononuclear cells were obtained by teasing the spleens apart, passing the suspension through a fine (number 100) wire mesh screen (W. S. Tyler Company, Mentor, Ohio) and layering the suspension over Ficoll-Hypaque as described above for the peripheral blood lymphocytes. Two femura were then removed and the bone marrow cells obtained by cutting off both ends of the bone and flushing the shaft contents into a Petri dish with a syringe containing 10 cc of MEM-S through a 20-gauge needle. Clumps were dispersed by repeated aspirations through the needle and syringe. Cells- from both femura were pooled, washed with MEM-S, and subjected to hypotonic lysis to remove erythrocytes. Differential counts were done and the suspensions were layered on linear sucrose density gradients to obtain lymphocyte-rich suspensions by a slight modification of a method previously described (Osmond, 1967). In brief, five sucrose gradients of 15-35 per cent (increments of 5 per cent) in Hanks's balanced salt solution (HBSS) and 20 per cent foetal calf serum (FCS) (Industrial Biological Laboratories, Incorporated, Rockville, Maryland) were prepared. A gradient of 35 per cent through 15 per cent (3 ml of each 5 per cent increment) was layered in a 1-5 x 15 cm glass test tube to give a total of 15 ml. Four millilitres of bone marrow cells (40 x 106/ml) were layered over each sucrose gradient tube and the tubes were centrifuged at 100 g for 12 minutes at 200. The upper 7 cm of the gradient containing the lymphocyte-rich suspension was collected and washed in MEM-S. The proportion of lymphocytes obtained in this manner was 61 (±2-1) per cent of the nucleated cells as compared to 15-4 (± 1.6) per cent in unfractionated marrow. The lymphocyte yield was 72 1 (± 4.7) per cent of the total lymphocytes put on the gradient. If the erythrocytes are not hypotonically lysed prior to layering cells on the gradient, the lymphocyte yield is considerably less. The lymphocyte-enriched cell suspensions were greater than 95 per cent viable as tested by the trypan blue dye exclusion test.
Proportion of thymus-derived (T) and bone marrow-derived (B) lymphocytes The ability of rabbit red blood cells (RRBC) to bind to guinea-pig lymphocytes to form rosettes has been shown to be a characteristic of T lymphocytes (Wilson and Coombs, 1973; Stadecker, Bishop and Wortis, 1973). In the present study, New Zealand white rabbits were bled from the heart into a 35-ml syringe containing 3 ml of acid-citrateanticoagulant. The RRBC were washed three times in HBSS and brought to a concentration of 0 5 per cent in HBSS. Guinea-pig peripheral blood, spleen, and bone marrow lymphocytes were brought to a concentration of 4x 106/ml. To 0-25 ml of the RRBC suspension was added 0-25 ml of the different lymphocyte suspensions. The mixture was incubated for 5 minutes at 370, centrifuged at 200 g for 5 minutes at 40 and kept at 40 overnight. The pellet was then resuspended by gentle shaking. One drop of cell suspension was put on a glass slide and a coverslip applied. Four hundred lymphocytes were counted by the same observer throughout the study using phase contrast optics at a magnification of x 400 on a Zeiss microscope (Carl Zeiss, Oberkochen, West Germany). All lymphocytes binding more than three RRBC were considered positive. Monocytes were distinguished by their phagocytosis of latex particles.
A. S. Faucl B lymphocytes were identified by their ability to bind to antibody-complement-coated sheep red blood cells (SRBC) (Bianco, Patrick and Nussenzweig, 1970). SRBC coated with rabbit IgM antibodies (EA) were a gift from Dr Michael M. Frank and were prepared as previously described (Frank and Gaither, 1970). Fresh mouse serum served as the source of complement (C). One millilitre of mouse serum diluted 1:10 in veronal-buffered saline containing optimal concentrations of calcium and magnesium in 0f1 per cent gelatin was incubated with 1 ml of IgM EA (1 x 108/ml in phosphate-buffered saline) for 40 minutes at 300. The resulting EAC were washed three times in 0*01 M EDTAbuffered saline and resuspended in 5 ml of 0-01 M EDTA-buffered saline. To 0-25 ml of the EAC was added 0-25 ml of the different lymphocyte suspensions. The mixture was incubated on a rotator at 370 for 40 minutes. A drop of the suspension was placed on a glass slide and rosettes were counted in the same manner as described above for RRBC rosettes. During several experiments various concentrations of OHC (0.1 pg/ml to 100 pug/ml) were incubated with the cell suspensions for 60 minutes at 370 prior to preparing the rosettes in order to determine if OHC itself interferes with or enhances rosette formation.
672
672A.SFac
In vitro lymphocyte blastogenic responses Purified peripheral blood, splenic, and bone marrow lymphocyte suspensions prepared as described above were obtained for in vitro culture from the groups receiving single dose NaCl or 100 mg/kg OHC at 4 hours after injection (point of maximal lymphocytopenia). For the chronic cortisone acetate experiments, cells were obtained for culture 24 hours after the last of seven daily injections of NaCl or cortisone acetate 100 mg/kg s.c. For culture, MEM-S was supplemented with 0-02 M L-glutamine, penicillin 100 u/ml, streptomycin sulphate (100 ug/ml), and 10 per cent FCS. Cultures were done in microtitre plates (Cooke Engineering Company, Alexandria, Virginia). Each well contained 0-2 ml of the cell suspensions in a concentration of 0 5 x 106 lymphocytes per millilitre. Quadruplicate cultures were incubated at 370 in 5 per cent CO2 in air, at 100 per cent humidity. Ten microlitres of mitogen were added to each well and cultures were incubated for three days. The mitogens used were: phytohaemagglutinin (PHA) MR 68 (Wellcome Reagents, Ltd, Beckenham) which had an optimal stimulatory dose for peripheral blood and splenic lymphocytes of 1 yg/ml of culture, while optimal dose for bone marrow lymphocytes was 2-5 jug/ml of culture; concanavalin A (con A) (Nutritional Biochemicals Corporation, Cleveland, Ohio), optimal dose of 10 pg/ml of culture for all cell suspensions; and pokeweed mitogen (PWM) (Grand Island Biological Company, Grand Island, New York), optimal dose of 10 ,l of 1:10 dilution in distilled water per 0-2 ml of culture for all cell suspensions. Sixteen hours before harvesting, 0 4 1Ci of tritiated thymidine (6-7 Ci/mM, New England Nuclear, Boston, Massachusetts) were added to each well. The cells were then collected from the wells on to fibreglass filters using a semi-automated microharvesting device (Harzman, Bach, Bach, Thurman and Sell, 1972). The filters were washed with 10 per cent trichloroacetic acid (TCA) and 95 per cent ethanol, and placed in 10 ml of Aquasol® (New England Nuclear). The TCA-precipitable radioactivity was counted in a liquid scintillation counter (model LS-350, Beckman Instruments, Incorporated, Fullerton, California). The arithmetic mean of counts per minute (ct/min) of quadruplicate cultures was determined and the degree of stimulation of PBL and splenic lymphocytes was expressed both as the difference in ct/min per 106 lymphocytes between stimulated and unstimulated (control) cultures (Act/min), as well as the ratio of experimental (E)
Corticosteroid Action on Lymphocytes. I. 673 or stimulated cultures to control (C) or unstimulated cultures (E/C). Because the background counts of unstimulated bone marrow were rather high relative to the counts of stimulated cultures, the gross counts after stimulation (E), the background control counts (C), as well as the stimulated to control ratios (E/C) of bone marrow counts are given in order to more accurately assess any changes in stimulation. In each experiment, for every corticosteroid-treated animal tested, a saline-treated control was simultaneously tested.
51Cr-labelled lymphocytes Peripheral blood was obtained from strain 13 guinea-pig donors by cardiac puncture using a 20-gauge needle and a syringe containing acid-citrate-anticoagulant. Pooled peripheral blood from two donors was used in each separate experiment. Lymphocytes were obtained by Hypaque-Ficoll gradient centrifugation. Lymphocytes were washed three times in HBSS. Labelling of lymphocytes with radioactive chromium (5"Cr) was carried out as previously described (Hershey, 1971). In brief, cells were suspended in HBSS at a concentration of 20 x 1 06/ml, and 100 1Ci of 51Cr (sodium chromate, Amersham/Searle Corporation, Arlington Heights, Illinois) per millilitre of cell suspension was added. Cells were incubated at 370 for 60 minutes and subsequently washed four times with HBSS. Each syngeneic recipient received 5 x 106 51Cr-labelled lymphocytes, contained in 1 ml HBSS, i.v. via the hind foot vein. The viability of the injected cells was greater than 90 per cent in every case (trypan blue exclusion). Recipients received cortisone acetate (100 mg/kg) s.c. every 12 hours for the 24 hours prior to infusion of labelled cells and subsequently every 12 hours for the duration of the study. All experiments were done in pairs; for every animal receiving cortisone acetate treatment, a salinetreated control was simultaneously assayed. Groups of animals were killed at 4, 24 and 48 hours following infusion of labelled cells, organs were removed, and radioactivity was counted in an automatic gamma counter (1185 Series, Nuclear-Chicago, Des Plaines, Illinois). Bone marrow radioactivity was determined by counting all eight long bones of the limbs in each animal after removing skin and muscle. For each organ the data is given as percentage of total injected radioactivity. In order to rule out the possibility that corticosteroid-induced redistribution of lymphocytes was due to a damaging or killing of cells, the distribution of heat-killed cells was determined. Labelled cells were incubated at 560 for 1 hour prior to infusion. More than 98 per cent of the cells were killed by this procedure. These cells were then infused into untreated syngeneic recipients and organ activity was assayed at 4 and 24 hours.
Statistical analysis of data Mean (±s.e.m.) values for saline and corticosteroid-treated groups were compared using a Student's t-test (Dixon and Massey, 1957). RESULTS PLASMA CORTISOL LEVELS
The normal 8 a.m. mean plasma cortisol level of four guinea-pigs tested was 20 1 (±+55) ug/100 ml. Following a single injection of 100 mg/kg OHC, the mean plasma cortisol level measured in four animals at each time point was 291 (±90) ug/l00 ml at 1 hour and 110 (± 19) pg/100 ml at 4 hours. By 24 hours following injection, the levels had returned to normal-26 (± 6) ,ug/ 100 ml. With chronically administered cortisone acetate,
674 A. S. Fauci the levels gradually reached a plateau after 3 days such that they were sustained at a mean level of 384 (± 74) yg/ 100 ml 24 hours after each daily injection of this depot preparation. TOTAL LYMPHOCYTE COUNTS
There was a significant decrease in circulating lymphocytes maximal at 4 hours following both 10 mg/kg OHC (P
Mechanisms of Corticosteroid Action on Lymphocyte Subpopulations I. REDISTRIBUTION OF CIRCULATING T AND B LYMPHOCYTES TO THE BONE MARROW A. S. FAUCI Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A.
(Received 1st August 1974; acceptedfor publication 7th October 1974) Summary. The effect of corticosteroid administration on the redistribution of circulating lymphocytes was studied in the guinea-pig, since this species closely resembles man in its relative resistance to the lymphopenic effect of corticosteroids. A single intravenous injection of hydrocortisone (either 10 mg or 100 mg/kg) caused a profound but transient lymphocytopenia which was maximal at 4 hours following injection, with a return to normal counts by 24 hours. There was a proportionately greater decrease in circulating T lymphocytes compared to B lymphocytes, although both populations were diminished. Chronic cortisone acetate treatment (100 mg/kg subcutaneously for 7 days) caused a similar pattern of lymphocytopenia except that it was sustained during the period of chronically elevated plasma cortisol levels. The lymphocytes remaining in the circulation during the period of lymphocytopenia responded normally in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. There was very little effect of corticosteroid administration on the numbers, proportions, or mitogenic response of splenic lymphocytes. There was a dramatic increase in the bone marrow of proportions and absolute numbers of lymphocytes bearing surface T- and B-cell markers, as well as a marked increase in response of bone marrow lymphocytes to mitogenic stimulation during the period of maximal circulating lymphocytopenia caused by the administration of corticosteroids, especially chronic cortisone acetate. There was a preferential homing of reinfused 51Cr-labelled syngeneic peripheral blood lymphocytes to the bone marrow of corticosteroid-treated recipients. These studies demonstrate a redistribution of circulating lymphocytes to the bone marrow during corticosteroid treatment, resulting in an increase in immunocompetence ofthis compartment, while the peripheral blood lymphocyte compartment is quantitatively immunosuppressed due to a lymphocytopenia. INTRODUCTION The administration of corticosteroids to normal humans results in a profound but transient lymphocytopenia of both thymus-derived (T) and bursa equivalent (B) lymphoCorrespondence: Dr A. S. Fauci, Clinical Physiology Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A.
669
670 A. S. Fauci cytes with a proportionately greater depletion of T lymphocytes (Fauci and Dale, 1974; Yu, Clements, Paulus, Peter, Levy and Barnett, 1974). The fate of these lymphocytes is unknown. It has been suggested that they are not destroyed in vivo, but are redistributed to other body compartments, particularly the bone marrow (Fauci and Dale, 1974; Yu et al., 1974). There is convincing evidence in the mouse model that the corticosteroidinduced lymphocytopenia is explained by a redistribution of T lymphocytes into the bone marrow, as measured by enhanced bone marrow responsiveness to mitogenic stimuli (Levine and Claman, 1970), enhanced capacity for helper cell function (Cohen, 1972), and for inducing graft-versus-host reactions (Cohen, Fischbach and Claman, 1970). It has been pointed out, however, that the mouse is a 'steroid-sensitive' species in that it is extremely susceptible to the lymphopenic effects of systemically administered corticosteroids (Claman, 1972). On the other hand, the guinea-pig more closely resembles man in this regard since both species are relatively resistant to corticosteroid-induced lymphopenia. Hence, the present study was designed to investigate the mechanisms and kinetics of the effects of both acutely and chronically administered corticosteroids on the redistribution between body compartments of guinea-pig lymphocyte subpopulations defined functionally and by surface markers. In addition, the effect of corticosteroid administration on the distribution of reinfused radioactive 51Cr-labelled syngeneic lymphocytes was studied. MATERIALS AND METHODS
Animals Inbred strain 13 female guinea-pigs (300-400 g) obtained from the Division of Research Services, National Institutes of Health, Bethesda, Maryland, were used throughout the study. Corticosteroid treatment For the acutely administered corticosteroid experiments, groups of non-immune animals were given a single injection intravenously (i.v.) in the hind foot vein of either 1 ml of normal saline (NaCl), 10 mg/kg, or 100 mg/kg of hydrocortisone sodium succinate (OHC) (The Upjohn Company, Kalamazoo, Michigan). For the chronically administered corticosteroid experiments, groups of non-immune animals were given subcutaneously (s.c.) either 1 ml of NaCl or 100 mg/kg of cortisone acetate (The Upjohn Company) daily for 7 days. The animals receiving chronic corticosteroid administration gained weight and thrived no differently from controls. Leucocyte counts and differentials In the group of guinea-pigs receiving a single i.v. dose of either NaCl or OHC, separate animals were bled at 0, 1, 4, 6 and 24 hours following injection and white blood cell (WBC) and differential counts were done in addition to plasma cortisol levels (Loriaux, Guy and Lipsett, 1973). In the group of guinea-pigs receiving 7 days of cortisone acetate, WBC and differential counts were done during treatment and at the end of the 7-day treatment period, 24 hours following the last dose of cortisone acetate. In addition, plasma cortisol levels were done daily throughout the 7 days of cortisone acetate treatment.
Preparation and purification of lymphocytes Four hours (point of maximal lymphocytopenia) after acutely administered NaCl or OHC and 24 hours following 7 days of chronically administered NaCl or cortisone acetate,
Corticosteroid Action on Lymphocytes. I. 671 the following studies were done: animals were exsanguinated by cardiac puncture and separation of peripheral blood lymphocytes was performed on acid-citrate-anticoagulated blood by Ficoll-Hypaque gradient centrifugation (Boyum, 1968). The spleens were aseptically removed and placed in a sterile Petri dish containing Eagle's minimum essential media modified for suspension culture (MEM-S) (Grand Island Biological Company, Grand Island, New York). Purified suspensions of splenic mononuclear cells were obtained by teasing the spleens apart, passing the suspension through a fine (number 100) wire mesh screen (W. S. Tyler Company, Mentor, Ohio) and layering the suspension over Ficoll-Hypaque as described above for the peripheral blood lymphocytes. Two femura were then removed and the bone marrow cells obtained by cutting off both ends of the bone and flushing the shaft contents into a Petri dish with a syringe containing 10 cc of MEM-S through a 20-gauge needle. Clumps were dispersed by repeated aspirations through the needle and syringe. Cells- from both femura were pooled, washed with MEM-S, and subjected to hypotonic lysis to remove erythrocytes. Differential counts were done and the suspensions were layered on linear sucrose density gradients to obtain lymphocyte-rich suspensions by a slight modification of a method previously described (Osmond, 1967). In brief, five sucrose gradients of 15-35 per cent (increments of 5 per cent) in Hanks's balanced salt solution (HBSS) and 20 per cent foetal calf serum (FCS) (Industrial Biological Laboratories, Incorporated, Rockville, Maryland) were prepared. A gradient of 35 per cent through 15 per cent (3 ml of each 5 per cent increment) was layered in a 1-5 x 15 cm glass test tube to give a total of 15 ml. Four millilitres of bone marrow cells (40 x 106/ml) were layered over each sucrose gradient tube and the tubes were centrifuged at 100 g for 12 minutes at 200. The upper 7 cm of the gradient containing the lymphocyte-rich suspension was collected and washed in MEM-S. The proportion of lymphocytes obtained in this manner was 61 (±2-1) per cent of the nucleated cells as compared to 15-4 (± 1.6) per cent in unfractionated marrow. The lymphocyte yield was 72 1 (± 4.7) per cent of the total lymphocytes put on the gradient. If the erythrocytes are not hypotonically lysed prior to layering cells on the gradient, the lymphocyte yield is considerably less. The lymphocyte-enriched cell suspensions were greater than 95 per cent viable as tested by the trypan blue dye exclusion test.
Proportion of thymus-derived (T) and bone marrow-derived (B) lymphocytes The ability of rabbit red blood cells (RRBC) to bind to guinea-pig lymphocytes to form rosettes has been shown to be a characteristic of T lymphocytes (Wilson and Coombs, 1973; Stadecker, Bishop and Wortis, 1973). In the present study, New Zealand white rabbits were bled from the heart into a 35-ml syringe containing 3 ml of acid-citrateanticoagulant. The RRBC were washed three times in HBSS and brought to a concentration of 0 5 per cent in HBSS. Guinea-pig peripheral blood, spleen, and bone marrow lymphocytes were brought to a concentration of 4x 106/ml. To 0-25 ml of the RRBC suspension was added 0-25 ml of the different lymphocyte suspensions. The mixture was incubated for 5 minutes at 370, centrifuged at 200 g for 5 minutes at 40 and kept at 40 overnight. The pellet was then resuspended by gentle shaking. One drop of cell suspension was put on a glass slide and a coverslip applied. Four hundred lymphocytes were counted by the same observer throughout the study using phase contrast optics at a magnification of x 400 on a Zeiss microscope (Carl Zeiss, Oberkochen, West Germany). All lymphocytes binding more than three RRBC were considered positive. Monocytes were distinguished by their phagocytosis of latex particles.
A. S. Faucl B lymphocytes were identified by their ability to bind to antibody-complement-coated sheep red blood cells (SRBC) (Bianco, Patrick and Nussenzweig, 1970). SRBC coated with rabbit IgM antibodies (EA) were a gift from Dr Michael M. Frank and were prepared as previously described (Frank and Gaither, 1970). Fresh mouse serum served as the source of complement (C). One millilitre of mouse serum diluted 1:10 in veronal-buffered saline containing optimal concentrations of calcium and magnesium in 0f1 per cent gelatin was incubated with 1 ml of IgM EA (1 x 108/ml in phosphate-buffered saline) for 40 minutes at 300. The resulting EAC were washed three times in 0*01 M EDTAbuffered saline and resuspended in 5 ml of 0-01 M EDTA-buffered saline. To 0-25 ml of the EAC was added 0-25 ml of the different lymphocyte suspensions. The mixture was incubated on a rotator at 370 for 40 minutes. A drop of the suspension was placed on a glass slide and rosettes were counted in the same manner as described above for RRBC rosettes. During several experiments various concentrations of OHC (0.1 pg/ml to 100 pug/ml) were incubated with the cell suspensions for 60 minutes at 370 prior to preparing the rosettes in order to determine if OHC itself interferes with or enhances rosette formation.
672
672A.SFac
In vitro lymphocyte blastogenic responses Purified peripheral blood, splenic, and bone marrow lymphocyte suspensions prepared as described above were obtained for in vitro culture from the groups receiving single dose NaCl or 100 mg/kg OHC at 4 hours after injection (point of maximal lymphocytopenia). For the chronic cortisone acetate experiments, cells were obtained for culture 24 hours after the last of seven daily injections of NaCl or cortisone acetate 100 mg/kg s.c. For culture, MEM-S was supplemented with 0-02 M L-glutamine, penicillin 100 u/ml, streptomycin sulphate (100 ug/ml), and 10 per cent FCS. Cultures were done in microtitre plates (Cooke Engineering Company, Alexandria, Virginia). Each well contained 0-2 ml of the cell suspensions in a concentration of 0 5 x 106 lymphocytes per millilitre. Quadruplicate cultures were incubated at 370 in 5 per cent CO2 in air, at 100 per cent humidity. Ten microlitres of mitogen were added to each well and cultures were incubated for three days. The mitogens used were: phytohaemagglutinin (PHA) MR 68 (Wellcome Reagents, Ltd, Beckenham) which had an optimal stimulatory dose for peripheral blood and splenic lymphocytes of 1 yg/ml of culture, while optimal dose for bone marrow lymphocytes was 2-5 jug/ml of culture; concanavalin A (con A) (Nutritional Biochemicals Corporation, Cleveland, Ohio), optimal dose of 10 pg/ml of culture for all cell suspensions; and pokeweed mitogen (PWM) (Grand Island Biological Company, Grand Island, New York), optimal dose of 10 ,l of 1:10 dilution in distilled water per 0-2 ml of culture for all cell suspensions. Sixteen hours before harvesting, 0 4 1Ci of tritiated thymidine (6-7 Ci/mM, New England Nuclear, Boston, Massachusetts) were added to each well. The cells were then collected from the wells on to fibreglass filters using a semi-automated microharvesting device (Harzman, Bach, Bach, Thurman and Sell, 1972). The filters were washed with 10 per cent trichloroacetic acid (TCA) and 95 per cent ethanol, and placed in 10 ml of Aquasol® (New England Nuclear). The TCA-precipitable radioactivity was counted in a liquid scintillation counter (model LS-350, Beckman Instruments, Incorporated, Fullerton, California). The arithmetic mean of counts per minute (ct/min) of quadruplicate cultures was determined and the degree of stimulation of PBL and splenic lymphocytes was expressed both as the difference in ct/min per 106 lymphocytes between stimulated and unstimulated (control) cultures (Act/min), as well as the ratio of experimental (E)
Corticosteroid Action on Lymphocytes. I. 673 or stimulated cultures to control (C) or unstimulated cultures (E/C). Because the background counts of unstimulated bone marrow were rather high relative to the counts of stimulated cultures, the gross counts after stimulation (E), the background control counts (C), as well as the stimulated to control ratios (E/C) of bone marrow counts are given in order to more accurately assess any changes in stimulation. In each experiment, for every corticosteroid-treated animal tested, a saline-treated control was simultaneously tested.
51Cr-labelled lymphocytes Peripheral blood was obtained from strain 13 guinea-pig donors by cardiac puncture using a 20-gauge needle and a syringe containing acid-citrate-anticoagulant. Pooled peripheral blood from two donors was used in each separate experiment. Lymphocytes were obtained by Hypaque-Ficoll gradient centrifugation. Lymphocytes were washed three times in HBSS. Labelling of lymphocytes with radioactive chromium (5"Cr) was carried out as previously described (Hershey, 1971). In brief, cells were suspended in HBSS at a concentration of 20 x 1 06/ml, and 100 1Ci of 51Cr (sodium chromate, Amersham/Searle Corporation, Arlington Heights, Illinois) per millilitre of cell suspension was added. Cells were incubated at 370 for 60 minutes and subsequently washed four times with HBSS. Each syngeneic recipient received 5 x 106 51Cr-labelled lymphocytes, contained in 1 ml HBSS, i.v. via the hind foot vein. The viability of the injected cells was greater than 90 per cent in every case (trypan blue exclusion). Recipients received cortisone acetate (100 mg/kg) s.c. every 12 hours for the 24 hours prior to infusion of labelled cells and subsequently every 12 hours for the duration of the study. All experiments were done in pairs; for every animal receiving cortisone acetate treatment, a salinetreated control was simultaneously assayed. Groups of animals were killed at 4, 24 and 48 hours following infusion of labelled cells, organs were removed, and radioactivity was counted in an automatic gamma counter (1185 Series, Nuclear-Chicago, Des Plaines, Illinois). Bone marrow radioactivity was determined by counting all eight long bones of the limbs in each animal after removing skin and muscle. For each organ the data is given as percentage of total injected radioactivity. In order to rule out the possibility that corticosteroid-induced redistribution of lymphocytes was due to a damaging or killing of cells, the distribution of heat-killed cells was determined. Labelled cells were incubated at 560 for 1 hour prior to infusion. More than 98 per cent of the cells were killed by this procedure. These cells were then infused into untreated syngeneic recipients and organ activity was assayed at 4 and 24 hours.
Statistical analysis of data Mean (±s.e.m.) values for saline and corticosteroid-treated groups were compared using a Student's t-test (Dixon and Massey, 1957). RESULTS PLASMA CORTISOL LEVELS
The normal 8 a.m. mean plasma cortisol level of four guinea-pigs tested was 20 1 (±+55) ug/100 ml. Following a single injection of 100 mg/kg OHC, the mean plasma cortisol level measured in four animals at each time point was 291 (±90) ug/l00 ml at 1 hour and 110 (± 19) pg/100 ml at 4 hours. By 24 hours following injection, the levels had returned to normal-26 (± 6) ,ug/ 100 ml. With chronically administered cortisone acetate,
674 A. S. Fauci the levels gradually reached a plateau after 3 days such that they were sustained at a mean level of 384 (± 74) yg/ 100 ml 24 hours after each daily injection of this depot preparation. TOTAL LYMPHOCYTE COUNTS
There was a significant decrease in circulating lymphocytes maximal at 4 hours following both 10 mg/kg OHC (P
