Clin. exp. Immunol. (1976) 25, 472-479.
Human lymphocyte subpopulations effect of epinephrine D. T. Y. YU & P. J. CLEMENTS Department of Medicine, UCLA School of Medicine, Los Angeles, California, U.S.A.
(Received 1 March 1976) SUMMARY
The effect of administration of 0-2 mg of epinephrine (Parke-Davis) on circulatory lymphocytes was investigated in fifteen normal subjects. Blood samples were taken prior to, 10 and 20 min after, subcutaneous injections. Thymus-derived (T) cells were assayed by sheep red blood cell rosettes, bone marrow-derived (B) cells by their complement receptors and immunofluorescent detection of Fc receptors plus surface immunoglobulins. Their percentages prior to injections were 72-2+1-4, 13-8+1P1, and 20-3+1 3. Ten minutes after injections, the absolute concentrations of these cells increased to 164+ 14%, 326+ 57%, and 272+45% respectively of the values prior to injection (averages+ standard errors). Further, when cells with receptors for both sheep red blood cells and complement were assayed by simultaneous rosette technique, they increased from 2-5+0 4% to 10-5+ 1.3% of the lymphocytes. Such changes were also observed in three subjects who had undergone splenectomy more than 2 years previously, but not in four subjects receiving injections of saline instead of epinephrine.
INTRODUCTION Peripheral blood lymphocytes are often the focus of study in evaluating patients who have immunologic diseases or who are receiving immunologic therapies. The parameters being measured include the lymphocyte concentration, the T and B cell ratio, and responses to mitogenic and antigenic stimulation. One difficulty often encountered when interpreting these assays is the enormous differences which exist among normal subjects and the variation in the same subject at different times (Allen, 1974; Gary & Bryan, 1935; Steel, Evans & Smith, 1974). The factors governing such variations are not certain. Variation in corticosteroid levels may be a determining factor. Several investigators have shown that administration of corticosteroids produced changes in all the three parameters (Fauci & Dale, 1974; Webel et al., 1974; Yu et al., 1974). In a recent study corticosteroid was not administered. Blood samples were obtained from normal subjects every 4 hr for 36 hr. Serial determinations of their peripheral blood concentrations and responses of the lymphocytes to phytohaemagglutinin revealed that a circadian rhythm existed which closely paralleled the corticosteroid concentration in the blood (Tavadia et al., 1975). This showed that the endogenous level of corticosteroid hormone was an important physiological factor governing the concentration and functional capacity of peripheral blood lymphocytes. The present study concerns another group of normally occurring hormones, the blood levels of which also fluctuate widely during the day. This is the adrenergic group of hormones. In this paper the authors demonstrate that 0-2 mg of epinephrine (Adrenaline®), administered subcutaneously could induce a significant lymphocytosis. The characterization of these as T or B cells and the assessment of their ability to respond to phytohaemagglutinin in vitro are the subjects of this study. MATERIALS AND METHODS Subjects. Fifteen subjects, 22-35 years of age, were studied. Ten subjects were male while five were female. These subjects Correspondence: Dr D. T. Y. Yu, Rehabilitation Center, 1000 Veteran Avenue, Los Angeles, California 90024, U.S.A.
472
Lymphocytes and epinephrine
473
had no history of cardiac or pulmonary diseases, and were not on any medications. The purpose and procedures were explained to them, and consent obtained prior to each experiment. After a rest period of 15 min, 0-2 mg of epinephrine (Parke-Davis) in 0-1 ml volume was injected subcutaneously into the arm. Most subjects experienced palpitations 5 min after the injections, lasting about 15 sec. Blood samples were taken into heparinized tubes just prior to injections and also 10 and 20 min afterward. For convenience of description, this group of subjects will be designated as Group A subjects. Three other male subjects, 22-29 years of age, who had undergone splenectomy previously were also studied in a similar manner. One subject underwent splenectomy during infancy for unknown reasons. The other two underwent splenectomy 2 years previously for trauma and hereditary spherocytosis respectively. The subject with hereditary spherocytosis was asymptomatic and did not have any anaemia prior to or after splenectomy. This group of subjects will be designated as Group B subjects. Group C consisted of one female and four male subjects from Group A. These were given injections of 0-2 ml of saline subcutaneously. The saline injections were given at least 1 week after the epinephrine injections. Lymphocyte counts. White cell concentrations were measured using white cell counting pipettes and haemocytometers. Differential counts were made from blood smears stained with Wright's stain. Two hundred white cells were counted on each smear. Assays of lymphocyte subpopulations. Mononuclear cells were isolated by Ficoll-Hypaque gradient (Boyum, 1968). Assays for lymphocytes with receptors for sheep red blood cells (SRBC) rosette-forming cells and aggregated gammaglobulin (AggG) have been published previously (Yu et al., 1974). Briefly, SRBC rosettes were prepared by light centrifugation of mixtures of SRBC and lymphocytes and keeping the pellets overnight at 4VC prior to resuspension. To detect AggC, lymphocytes were first incubated with aggregated human globulin fraction II and then stained with fluorescein isothiocyanateconjugated anti-human gammaglobulin. This method actually identified cells that have receptors for AggG as well as cells with surface immuncglobulins. Complement receptor lymphocytes (CRL) were recognized by their ability to bind to complement-sensitized zymosan granules (Huber & Wigzell, 1975; Mendes, Miki & Peixinho, 1974). Zymosan granules (Sigma, Missouri) were washed in saline and resuspended in 1 mg per ml concentration in Hanks's Balanced Salt Solution (HBSS). Each ml of suspension was then incubated with 0 5 ml of freshly frozen human sera at 370C for 30 min, washed three times with HBSS, and resuspended in HBSS at 1 x 108 granules per ml. To form rosettes, 0-1 ml of sensitized zymosan was mixed with 0-1 of 5 x 106 per ml of lymphocytes in HBSS in plastic tubes (Falcon, No. 2038). These were centrifuged at 200 g for 10 min and kept at 4°C overnight. The pellets were then gently resuspended by Pasteur pipettes and read in haemocytometers. A CRL was defined as a lymphocyte surrounded by two or more sensitized zymosan granules. Most zymosan rosettes had more than two zymosan granules per rosette. In several experiments CRL and AggC were assayed simultaneously. These showed that 80% of CRL were also AggC-positive, confirming the validity of this method of assaying B cells (Table 6). Lymphocytes with receptors for both SRBC and complement (mixed rosettes) were assayed by mixing 0-1 ml of the lymphocyte suspension with 0-1 ml of 0.5% SRBC suspension and 0-1 ml of sensitized zymosan granules. These were centrifuged at 200 g for 10 min and read after overnight incubation at 4°C. Mixed rosettes were arbitrarily defined as one with . 2 SRBC+ . 2 zymosan granules per rosette. Three hundred lymphocytes were counted in each rosette assay. Monocytes were recognized either by their morphological appearance or by their ability to ingest sensitized zymosan particles or latex beads. The latter has been described in previous publications (Yu et al., 1974). Phytohaemagglutinin stimulation. Phytohaemagglutinin (PHA-M, lot no. 05-28-56) was purchased from Difco, Michigan. Lymphocyte cultures were carried out for 72 h at 37°C in RPMI-5% heat-inactivated foetal calf serum as described previously (Yu et al., 1973). Four hours prior to harvesting, 0 1 ml of tritiated thymidine (2 pCi, TRA 61 specificity 5 Ci/mmol) (Amersham, Bucks.), was added to each culture tube. The amount ofradioactivity incorporated was assessed by a scintillation counter as described previously (Yu et al., 1973). Statistics. Results were expressed in averages+ standard errors of means. The proportionate increase in lymphocyte subpopulation shown in Table 4 was derived from the formula: absolute concentrations of the subpopulation after injection 100 absolute concentrations of the subpopulation prior to injection Statistical comparisons were made by the Student's t-test and the paired observation t-test.
RESULTS Pulse rates Pulse rates were recorded in eleven group A subjects. The average rates at 0, 10, and 20 min after epinephrine injections were 72-2+2-6, 79-0+2-9, and 76-3+3-0 per min respectively. The increase was statistically significant by paired observation t-test at 10 min but not at 20 min (P< 0005 and > 005 respectively). Leucocyte and lymphocyte concentrations Peripheral blood concentrations of leucocytes and lymphocytes were quantitated in the three groups of
474 D. T. Y. Yu & P. Clements subjects and compared by paired observation t-test. Significant leucocytosis developed in group A subjects at 10 and 20 mins (P
Human lymphocyte subpopulations effect of epinephrine D. T. Y. YU & P. J. CLEMENTS Department of Medicine, UCLA School of Medicine, Los Angeles, California, U.S.A.
(Received 1 March 1976) SUMMARY
The effect of administration of 0-2 mg of epinephrine (Parke-Davis) on circulatory lymphocytes was investigated in fifteen normal subjects. Blood samples were taken prior to, 10 and 20 min after, subcutaneous injections. Thymus-derived (T) cells were assayed by sheep red blood cell rosettes, bone marrow-derived (B) cells by their complement receptors and immunofluorescent detection of Fc receptors plus surface immunoglobulins. Their percentages prior to injections were 72-2+1-4, 13-8+1P1, and 20-3+1 3. Ten minutes after injections, the absolute concentrations of these cells increased to 164+ 14%, 326+ 57%, and 272+45% respectively of the values prior to injection (averages+ standard errors). Further, when cells with receptors for both sheep red blood cells and complement were assayed by simultaneous rosette technique, they increased from 2-5+0 4% to 10-5+ 1.3% of the lymphocytes. Such changes were also observed in three subjects who had undergone splenectomy more than 2 years previously, but not in four subjects receiving injections of saline instead of epinephrine.
INTRODUCTION Peripheral blood lymphocytes are often the focus of study in evaluating patients who have immunologic diseases or who are receiving immunologic therapies. The parameters being measured include the lymphocyte concentration, the T and B cell ratio, and responses to mitogenic and antigenic stimulation. One difficulty often encountered when interpreting these assays is the enormous differences which exist among normal subjects and the variation in the same subject at different times (Allen, 1974; Gary & Bryan, 1935; Steel, Evans & Smith, 1974). The factors governing such variations are not certain. Variation in corticosteroid levels may be a determining factor. Several investigators have shown that administration of corticosteroids produced changes in all the three parameters (Fauci & Dale, 1974; Webel et al., 1974; Yu et al., 1974). In a recent study corticosteroid was not administered. Blood samples were obtained from normal subjects every 4 hr for 36 hr. Serial determinations of their peripheral blood concentrations and responses of the lymphocytes to phytohaemagglutinin revealed that a circadian rhythm existed which closely paralleled the corticosteroid concentration in the blood (Tavadia et al., 1975). This showed that the endogenous level of corticosteroid hormone was an important physiological factor governing the concentration and functional capacity of peripheral blood lymphocytes. The present study concerns another group of normally occurring hormones, the blood levels of which also fluctuate widely during the day. This is the adrenergic group of hormones. In this paper the authors demonstrate that 0-2 mg of epinephrine (Adrenaline®), administered subcutaneously could induce a significant lymphocytosis. The characterization of these as T or B cells and the assessment of their ability to respond to phytohaemagglutinin in vitro are the subjects of this study. MATERIALS AND METHODS Subjects. Fifteen subjects, 22-35 years of age, were studied. Ten subjects were male while five were female. These subjects Correspondence: Dr D. T. Y. Yu, Rehabilitation Center, 1000 Veteran Avenue, Los Angeles, California 90024, U.S.A.
472
Lymphocytes and epinephrine
473
had no history of cardiac or pulmonary diseases, and were not on any medications. The purpose and procedures were explained to them, and consent obtained prior to each experiment. After a rest period of 15 min, 0-2 mg of epinephrine (Parke-Davis) in 0-1 ml volume was injected subcutaneously into the arm. Most subjects experienced palpitations 5 min after the injections, lasting about 15 sec. Blood samples were taken into heparinized tubes just prior to injections and also 10 and 20 min afterward. For convenience of description, this group of subjects will be designated as Group A subjects. Three other male subjects, 22-29 years of age, who had undergone splenectomy previously were also studied in a similar manner. One subject underwent splenectomy during infancy for unknown reasons. The other two underwent splenectomy 2 years previously for trauma and hereditary spherocytosis respectively. The subject with hereditary spherocytosis was asymptomatic and did not have any anaemia prior to or after splenectomy. This group of subjects will be designated as Group B subjects. Group C consisted of one female and four male subjects from Group A. These were given injections of 0-2 ml of saline subcutaneously. The saline injections were given at least 1 week after the epinephrine injections. Lymphocyte counts. White cell concentrations were measured using white cell counting pipettes and haemocytometers. Differential counts were made from blood smears stained with Wright's stain. Two hundred white cells were counted on each smear. Assays of lymphocyte subpopulations. Mononuclear cells were isolated by Ficoll-Hypaque gradient (Boyum, 1968). Assays for lymphocytes with receptors for sheep red blood cells (SRBC) rosette-forming cells and aggregated gammaglobulin (AggG) have been published previously (Yu et al., 1974). Briefly, SRBC rosettes were prepared by light centrifugation of mixtures of SRBC and lymphocytes and keeping the pellets overnight at 4VC prior to resuspension. To detect AggC, lymphocytes were first incubated with aggregated human globulin fraction II and then stained with fluorescein isothiocyanateconjugated anti-human gammaglobulin. This method actually identified cells that have receptors for AggG as well as cells with surface immuncglobulins. Complement receptor lymphocytes (CRL) were recognized by their ability to bind to complement-sensitized zymosan granules (Huber & Wigzell, 1975; Mendes, Miki & Peixinho, 1974). Zymosan granules (Sigma, Missouri) were washed in saline and resuspended in 1 mg per ml concentration in Hanks's Balanced Salt Solution (HBSS). Each ml of suspension was then incubated with 0 5 ml of freshly frozen human sera at 370C for 30 min, washed three times with HBSS, and resuspended in HBSS at 1 x 108 granules per ml. To form rosettes, 0-1 ml of sensitized zymosan was mixed with 0-1 of 5 x 106 per ml of lymphocytes in HBSS in plastic tubes (Falcon, No. 2038). These were centrifuged at 200 g for 10 min and kept at 4°C overnight. The pellets were then gently resuspended by Pasteur pipettes and read in haemocytometers. A CRL was defined as a lymphocyte surrounded by two or more sensitized zymosan granules. Most zymosan rosettes had more than two zymosan granules per rosette. In several experiments CRL and AggC were assayed simultaneously. These showed that 80% of CRL were also AggC-positive, confirming the validity of this method of assaying B cells (Table 6). Lymphocytes with receptors for both SRBC and complement (mixed rosettes) were assayed by mixing 0-1 ml of the lymphocyte suspension with 0-1 ml of 0.5% SRBC suspension and 0-1 ml of sensitized zymosan granules. These were centrifuged at 200 g for 10 min and read after overnight incubation at 4°C. Mixed rosettes were arbitrarily defined as one with . 2 SRBC+ . 2 zymosan granules per rosette. Three hundred lymphocytes were counted in each rosette assay. Monocytes were recognized either by their morphological appearance or by their ability to ingest sensitized zymosan particles or latex beads. The latter has been described in previous publications (Yu et al., 1974). Phytohaemagglutinin stimulation. Phytohaemagglutinin (PHA-M, lot no. 05-28-56) was purchased from Difco, Michigan. Lymphocyte cultures were carried out for 72 h at 37°C in RPMI-5% heat-inactivated foetal calf serum as described previously (Yu et al., 1973). Four hours prior to harvesting, 0 1 ml of tritiated thymidine (2 pCi, TRA 61 specificity 5 Ci/mmol) (Amersham, Bucks.), was added to each culture tube. The amount ofradioactivity incorporated was assessed by a scintillation counter as described previously (Yu et al., 1973). Statistics. Results were expressed in averages+ standard errors of means. The proportionate increase in lymphocyte subpopulation shown in Table 4 was derived from the formula: absolute concentrations of the subpopulation after injection 100 absolute concentrations of the subpopulation prior to injection Statistical comparisons were made by the Student's t-test and the paired observation t-test.
RESULTS Pulse rates Pulse rates were recorded in eleven group A subjects. The average rates at 0, 10, and 20 min after epinephrine injections were 72-2+2-6, 79-0+2-9, and 76-3+3-0 per min respectively. The increase was statistically significant by paired observation t-test at 10 min but not at 20 min (P< 0005 and > 005 respectively). Leucocyte and lymphocyte concentrations Peripheral blood concentrations of leucocytes and lymphocytes were quantitated in the three groups of
474 D. T. Y. Yu & P. Clements subjects and compared by paired observation t-test. Significant leucocytosis developed in group A subjects at 10 and 20 mins (P
