Psychoneuroendocrinology.Vol. 17, No. 2/3, pp. 145-152, 1992
0306-4530192 $5.00+0.00 @1992 Pergamon Press Ltd.
Printed in Great Britain
EFFECTS OF INTRAVENOUS A N D ORAL DEXAMETHASONE O N SELECTED LYMPHOCYTE SUBPOPULATIONS IN NORMAL SUBJECTS FRANCESCO CHIAPPELLI,1,2 GLENN J. GORMLEYr3 HARRY E. GWIRSTMAN,4 MARTIN T. LOWY,5 LINH-DAN NGUYEN, 1 LY NGUYEN, 1 IMU ESMAIL,2 MICHAEL STROBER,1,3 and HERBERT WEINER 1 1Department of Psychiatry and Biobehavioral Sciences, 2Center for Interdisciplinary Research in Immunology and Disease, Department of Microbiology and Immunology, and 3Department of Pediatrics, UCLA, School of Medicine, Los Angeles, California, U.S.A,; 6National Institute of Mental Health, Bethesda, Maryland, U.S.A., and 7I)epartment of Psychiatry, Case Western Reserve University, Cleveland, Ohio, U.S.A. (Received 12 December 1990; in final form 14 March 1991)
SUMMARY Our studies describe the effects of 1 mg oral (PO) and intravenous (IV) administration of dexamethasone (DEX) on certain subpopulations of circulating lymphocytes in normal subjects. We compared the outcomes of PO and 1V DEX administration because of individual differences in gastro-intestinal absorption of DEX and the issue of noncompliance in patients undergoing the dexamethasone suppression test (DST). Both routes of DEX administration were equally effective in suppressing plasma cortisol levels below 5 I.tg/dl, the customary criterion level. Both routes of DEX administration also significantly decreased the percent and absolute number of CD4+ cells, the CIM+/CD8+ ratio, and the percent and absolute number of virgin, but not of memory, CD4+ cells.
INTRODUCTION FOR 24 HR following the oral (PO) administration of the synthetic glucocorticoid, dexamethasone (DEX, 1-2 mg), plasma cortisol levels are normally suppressed below the criterion level of 5 gg/dl ( W o r d Health Organization, 1987). The failure of DEX to suppress cortisol levels occurs under many different conditions, including a variety of diseases, after weight loss, and in older subjects (Shapiro et al., 1983; Arana et al., 1985; World Health Organization, 1987). The criterion of what constitutes an adequate plasma DEX level in order for suppression of plasma cortisol to occur in normal subjects varies substantially among studies (Poland et al., 1987; Walsh et al., 1987; Weiner, 1989), and the meaning of the DEX suppression test outcome is not always clear. An inverse relationship has been reported between plasma cortisol and DEX levels in some (Arana et al., 1984; Holsboer et al., 1984; Johnson et al., 1984; Berger et al., 1985; Morris et al., 1986; Poland et al., 1987; Walsh et al., 1987) but not in all (Carroll et al., 1980) patient samples examined. Address correspondence and reprint requests to: Dr. Francesco Chiappelli, Department of Anatomy and Cell Biology, UCLA School of Medicine, CHS-1763, Los Angeles CA 90024, USA. 145
146
E CHIAPPELLIet al.
Other complicating factors in the interpretation of the DEX test include individual differences in gastro-intestinal absorption o f DEX and potential noncompliance. Intravenous (IV) administration of DEX may avert the possibility that its bioavailability varies between subjects. However, PO and IV DEX administration do not always lead to identical results, perhaps because of differences in DEX p h a r m a c o k i n e t i c s , in its conjugation, or in its disposition (Holsboer et al., 1986; Wiedemann & Holsboer, 1987). Several reports have described the effects of DEX on measures of cell-mediated immunity. For example, a statistically significant reduction in the proliferative response of peripheral blood m o n o n u c l e a r cells (PBMC) to T cell mitogens and in the n u m b e r o f glucocorticoid cytosolic receptor (GCCR) sites in PBMC of normal subjects occurred 10 hr after PO DEX administration (Lowy et al., 1984; 1988; Gormley et al., 1985a). Glucocorticoids play a significant immunoregulatory role, including influencing lymphocyte migration and distribution (Fauci & Dale, 1974; Haynes & Fauci, 1978; Onsrud & Thorsby, 1981; Cupps & Fauci, 1982). In control subjects, the circadian variation in cortisol levels is in a negative phase relationship to the cyclic variation in of circulating lymphocyte populations (Abo et al., 1981; Ritchie et al., 1983; Miyawaki et aL, 1984; Richardson & Martin, 1988). CD4+ lymphocyte, especially less mature populations of CD4+ ceils reportedly are more migratory (Yednock & Rosen, 1989) and thus potentially more subject to the influence of glucocorticoids. However, the effect of DEX administration on the distribution of CD4+ cells is not known. Our studies therefore were designed to characterize the effects of the administration of DEX IV and PO on several immunofunctional and immunophenotypic parameters in normal subjects. SUBJECTS AND METHODS Subjects All subjects completed and signed an Informed Consent form approved by the Human Subjects Protection Committee of the UCLA School of Medicine. The General Health Questionnaire, Hamilton Depression Rating Scale, and Beck Depression Inventory were used to screen the subjects. All subjects had scores within the normal range. The subjects' state of good physical health was established based on interviews and routine clinical laboratory tests. Subjects were requested to fast for 8-10 hr prior to admission to the UCLA Clinical Research Center on the morning of the study (0730h). Subjects were also requested to remain supine 1015 min prior to and following blood sampling. Protocols Nine subjects (8 women, 1 man; age range: 18-36 yr) were administered DEX PO or IV, and then recalled at least 4 wk later to receive DEX by the alternate route. The order of administration was randomized across subjects. Forty-five to sixty milliliters venous blood was drawn from the subjects at 0800h on day 1. At 2200h on the same day, the subjects received 1 mg DEX either PO (tablet) or IV (in sterile saline). At 0800h on the second day (i.e., 10 hr after DEX administration), another 45-60 ml venous blood was drawn. At 1600h on day 2 (i.e., 18 hr after DEX administration), another sample of 5-10 ml venous blood was obtained. Experimental Procedures The blood was centrifuged at 4°C within 10-30 min of collection (1200 rpm, 10 min). The plasma was frozen at -70 ° C in 1 ml aliquots, and the buffy coat was diluted with an equal volume of physiological saline at room temperature. PBMC were obtained from the 0800h blood samples. The 1600h samples were used for neuroendocrine measurements only. PBMC were obtained by standard buoyant density gradient centrifugation over Lymphoprep (Nycomed, Oslo) (2000 rpm, room temperature, 20 rain). Cells were counted with a Coulter counter, and differentials were obtained by light-microscopy after staining of cytospin slides with Cameo Quick Wright stain (American Scientific Products, McGraw Park, IL).
DEXAMETHASONE-INDUCED DECREASE IN VIRGIN C D 4 CELLS IN N O R M A L SUBJECTS
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For flow cytometric analyses, the PBMC were suspended in RPMI-1640 (Gibco, Grand Island, NY) supplemerited with 10% fetal calf serum (FCS, Gibco), and analyzed by standard dual-color fluorescence. In brief, PBMC (104 per 0.1 ml) were incubated (room temperature, 30 min) with fluorochrome-conjugated monoclonal antibodies (Beeton Dickinson, Mountain View, CA). The first antibody type was conjugated to fluorescein isothiocyanate, the second to phycoerythrin. Stained PBMC were washed twice in excess saline-5% FCS, freed from contaminating erythrocytes by osmotic shock lysis, and assessed by flow cytometry with a FACScan instrument (Becton Dickinson). Antibodies were directed against the CD3, CD4, CD45RA, CDS, CD20, CD16, and CD56 human lymphocyte markers. Plasma levels of ACTH and cortisol were measured with 125I-labelled radioimmunoassay (RIA) kits (Diagnostic Products, Los Angeles, CA). The detection limit of the cortisol solid phase RIA was 0.2 I,tg/di, the range was 1-50 Ixg/dl, the intraassay variability was 2% (at 5 Ixg/di) to 7.5%, and the interassay variability was 4.7-6.3%. The range of normal, morning plasma cortisol levels obtained by this assay is 5-25 Ixg/di, and the afternoon values are one-half of those levels. The double antibody ACTH RIA has very low cross-reactivity (
0306-4530192 $5.00+0.00 @1992 Pergamon Press Ltd.
Printed in Great Britain
EFFECTS OF INTRAVENOUS A N D ORAL DEXAMETHASONE O N SELECTED LYMPHOCYTE SUBPOPULATIONS IN NORMAL SUBJECTS FRANCESCO CHIAPPELLI,1,2 GLENN J. GORMLEYr3 HARRY E. GWIRSTMAN,4 MARTIN T. LOWY,5 LINH-DAN NGUYEN, 1 LY NGUYEN, 1 IMU ESMAIL,2 MICHAEL STROBER,1,3 and HERBERT WEINER 1 1Department of Psychiatry and Biobehavioral Sciences, 2Center for Interdisciplinary Research in Immunology and Disease, Department of Microbiology and Immunology, and 3Department of Pediatrics, UCLA, School of Medicine, Los Angeles, California, U.S.A,; 6National Institute of Mental Health, Bethesda, Maryland, U.S.A., and 7I)epartment of Psychiatry, Case Western Reserve University, Cleveland, Ohio, U.S.A. (Received 12 December 1990; in final form 14 March 1991)
SUMMARY Our studies describe the effects of 1 mg oral (PO) and intravenous (IV) administration of dexamethasone (DEX) on certain subpopulations of circulating lymphocytes in normal subjects. We compared the outcomes of PO and 1V DEX administration because of individual differences in gastro-intestinal absorption of DEX and the issue of noncompliance in patients undergoing the dexamethasone suppression test (DST). Both routes of DEX administration were equally effective in suppressing plasma cortisol levels below 5 I.tg/dl, the customary criterion level. Both routes of DEX administration also significantly decreased the percent and absolute number of CD4+ cells, the CIM+/CD8+ ratio, and the percent and absolute number of virgin, but not of memory, CD4+ cells.
INTRODUCTION FOR 24 HR following the oral (PO) administration of the synthetic glucocorticoid, dexamethasone (DEX, 1-2 mg), plasma cortisol levels are normally suppressed below the criterion level of 5 gg/dl ( W o r d Health Organization, 1987). The failure of DEX to suppress cortisol levels occurs under many different conditions, including a variety of diseases, after weight loss, and in older subjects (Shapiro et al., 1983; Arana et al., 1985; World Health Organization, 1987). The criterion of what constitutes an adequate plasma DEX level in order for suppression of plasma cortisol to occur in normal subjects varies substantially among studies (Poland et al., 1987; Walsh et al., 1987; Weiner, 1989), and the meaning of the DEX suppression test outcome is not always clear. An inverse relationship has been reported between plasma cortisol and DEX levels in some (Arana et al., 1984; Holsboer et al., 1984; Johnson et al., 1984; Berger et al., 1985; Morris et al., 1986; Poland et al., 1987; Walsh et al., 1987) but not in all (Carroll et al., 1980) patient samples examined. Address correspondence and reprint requests to: Dr. Francesco Chiappelli, Department of Anatomy and Cell Biology, UCLA School of Medicine, CHS-1763, Los Angeles CA 90024, USA. 145
146
E CHIAPPELLIet al.
Other complicating factors in the interpretation of the DEX test include individual differences in gastro-intestinal absorption o f DEX and potential noncompliance. Intravenous (IV) administration of DEX may avert the possibility that its bioavailability varies between subjects. However, PO and IV DEX administration do not always lead to identical results, perhaps because of differences in DEX p h a r m a c o k i n e t i c s , in its conjugation, or in its disposition (Holsboer et al., 1986; Wiedemann & Holsboer, 1987). Several reports have described the effects of DEX on measures of cell-mediated immunity. For example, a statistically significant reduction in the proliferative response of peripheral blood m o n o n u c l e a r cells (PBMC) to T cell mitogens and in the n u m b e r o f glucocorticoid cytosolic receptor (GCCR) sites in PBMC of normal subjects occurred 10 hr after PO DEX administration (Lowy et al., 1984; 1988; Gormley et al., 1985a). Glucocorticoids play a significant immunoregulatory role, including influencing lymphocyte migration and distribution (Fauci & Dale, 1974; Haynes & Fauci, 1978; Onsrud & Thorsby, 1981; Cupps & Fauci, 1982). In control subjects, the circadian variation in cortisol levels is in a negative phase relationship to the cyclic variation in of circulating lymphocyte populations (Abo et al., 1981; Ritchie et al., 1983; Miyawaki et aL, 1984; Richardson & Martin, 1988). CD4+ lymphocyte, especially less mature populations of CD4+ ceils reportedly are more migratory (Yednock & Rosen, 1989) and thus potentially more subject to the influence of glucocorticoids. However, the effect of DEX administration on the distribution of CD4+ cells is not known. Our studies therefore were designed to characterize the effects of the administration of DEX IV and PO on several immunofunctional and immunophenotypic parameters in normal subjects. SUBJECTS AND METHODS Subjects All subjects completed and signed an Informed Consent form approved by the Human Subjects Protection Committee of the UCLA School of Medicine. The General Health Questionnaire, Hamilton Depression Rating Scale, and Beck Depression Inventory were used to screen the subjects. All subjects had scores within the normal range. The subjects' state of good physical health was established based on interviews and routine clinical laboratory tests. Subjects were requested to fast for 8-10 hr prior to admission to the UCLA Clinical Research Center on the morning of the study (0730h). Subjects were also requested to remain supine 1015 min prior to and following blood sampling. Protocols Nine subjects (8 women, 1 man; age range: 18-36 yr) were administered DEX PO or IV, and then recalled at least 4 wk later to receive DEX by the alternate route. The order of administration was randomized across subjects. Forty-five to sixty milliliters venous blood was drawn from the subjects at 0800h on day 1. At 2200h on the same day, the subjects received 1 mg DEX either PO (tablet) or IV (in sterile saline). At 0800h on the second day (i.e., 10 hr after DEX administration), another 45-60 ml venous blood was drawn. At 1600h on day 2 (i.e., 18 hr after DEX administration), another sample of 5-10 ml venous blood was obtained. Experimental Procedures The blood was centrifuged at 4°C within 10-30 min of collection (1200 rpm, 10 min). The plasma was frozen at -70 ° C in 1 ml aliquots, and the buffy coat was diluted with an equal volume of physiological saline at room temperature. PBMC were obtained from the 0800h blood samples. The 1600h samples were used for neuroendocrine measurements only. PBMC were obtained by standard buoyant density gradient centrifugation over Lymphoprep (Nycomed, Oslo) (2000 rpm, room temperature, 20 rain). Cells were counted with a Coulter counter, and differentials were obtained by light-microscopy after staining of cytospin slides with Cameo Quick Wright stain (American Scientific Products, McGraw Park, IL).
DEXAMETHASONE-INDUCED DECREASE IN VIRGIN C D 4 CELLS IN N O R M A L SUBJECTS
147
For flow cytometric analyses, the PBMC were suspended in RPMI-1640 (Gibco, Grand Island, NY) supplemerited with 10% fetal calf serum (FCS, Gibco), and analyzed by standard dual-color fluorescence. In brief, PBMC (104 per 0.1 ml) were incubated (room temperature, 30 min) with fluorochrome-conjugated monoclonal antibodies (Beeton Dickinson, Mountain View, CA). The first antibody type was conjugated to fluorescein isothiocyanate, the second to phycoerythrin. Stained PBMC were washed twice in excess saline-5% FCS, freed from contaminating erythrocytes by osmotic shock lysis, and assessed by flow cytometry with a FACScan instrument (Becton Dickinson). Antibodies were directed against the CD3, CD4, CD45RA, CDS, CD20, CD16, and CD56 human lymphocyte markers. Plasma levels of ACTH and cortisol were measured with 125I-labelled radioimmunoassay (RIA) kits (Diagnostic Products, Los Angeles, CA). The detection limit of the cortisol solid phase RIA was 0.2 I,tg/di, the range was 1-50 Ixg/dl, the intraassay variability was 2% (at 5 Ixg/di) to 7.5%, and the interassay variability was 4.7-6.3%. The range of normal, morning plasma cortisol levels obtained by this assay is 5-25 Ixg/di, and the afternoon values are one-half of those levels. The double antibody ACTH RIA has very low cross-reactivity (
