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Activation of Thymocyte Deoxyribonucleic Acid Degradation by Endogenous Glucocorticoids1
IMMUNOLOGY Activation of Thymocyte Deoxyribonucleic Acid Degradation by Endogenous Glucocorticoids 1 MARK M COMPTON,2 USA R. JOHNSON, and PENELOPE S. GIBBS Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication March 19, 1990)
1991 Poultry Science 70:521-529 INTRODUCTION
Plasma glucocorticoid levels are rapidly elevated when avian species are exposed to stressful stimuli (Beuving and Vonder, 1978). This adrenal response is activated by a cascade of hormonal signals that emanate from the hypothalamus and are mediated through the pituitary-adrenal axis (Frankel et al, 1967; Salem et al, 1970; Frankel, 1970). Other evidence indicates that adrenocorticotropic hormone (ACTH)-like peptides secreted from immune cells may also modulate adrenal function (Blalock and Smith, 1985; Westley et al, 1986). In rum, glucocorticoids activate gluconeogenic pathways, which ultimately result in an elevation of plasma glucose.
Supported by USDA Grant GEO-RC300-145 and state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. Portions of these data were presented at the 78th Annual Meeting of the Poultry Science Association in Madison, Wisconsin. Send correspondence to: Mark M. Compton, Department of Poultry Science, 144 Livestock-Poultry Building, University of Georgia, Athens, GA 30602. Phone: 404/ 542-1359, 404/542-0414.
In addition to their role as orchestrators of gluconeogenesis, glucocorticoids modulate the immune response (Munck and Young, 1975; Munck et al, 1984). They attenuate the inflammatory reaction and act directly on lymphoid cells to inhibit immune function. Indeed, the most profound immunosuppressive effect of glucocorticoids is the activation of lymphocytolysis. This process has been widely studied (for review see Munck and Crabtree, 1981) although the precise sequence of cellular events that culminate in this form of steroidinduced lymphocyte cell death has not been clearly delineated. Nevertheless, it is known that adrenal steroids a) inhibit numerous anabolic pathways involved in macromolecular biosynthesis, and b) stimulate cellular catabolism. In addition, other evidence indicates mat prior to cell death glucocorticoids activate a DNA degrading process in which the lymphocyte genome is cleaved at internucleosomal sites (Wyllie, 1980; Umansky et al., 1981; Vanderbilt et al, 1982; Vedeckis and Bradshaw, 1983; Cohen and Duke, 1984; Wyllie et al, 1984; Compton and Cidlowski, 1986, 1987; Compton et al, 1987). It is presumed that this nucleolytic response pro-
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ABSTRACT Immature lymphocytes in the thymus gland are killed by treatment with exogenous glucocorticoids. This steroid-mediated lymphocytolysis is preceded by numerous alterations in lymphocyte metabolism, including a DNA-degrading process in which the genome is cleaved at internucleosomal intervals. To date, this process has only been characterized by treating lymphocytes in vitro with glucocorticoids or by exogenous treatment of whole animals with adrenal steroids. To determine whether thymocyte DNA degradation could be activated by endogenous glucocorticoids, 4-wk-old chicks were treated with porcine adrenocorticotropic hormone (ACTH). This procedure elevated serum corticosterone levels approximately 80-fold within 2 h of hormone treatment Following ACTH administration, thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The ACTH activated a DNA-degrading process that generated internucleosomal fragments of DNA identical in size to those observed following exogenous treatment with synthetic or naturally occurring glucocorticoids. Furthermore, this response could be inhibited by the glucocorticoid antagonist RU486 (17fJ-hydroxy-lip, 4-dimethylaminophenyl-17oc-propynl-estra4,9,diene-3-one), indicating that adrenal steroids activate this process via the glucocorticoid receptor. These results demonstrate that lymphocyte DNA degradation does not result solely from exogenous glucocorticoid treatment; moreover, endogenous glucocorticoids can mediate this process and may thereby play an important role in thymic gland function. (Key words: thymus, glucocorticoids, adrenocorticotropic hormone, deoxyribonucleic acid degradation, programmed cell death)
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motes cell death by disrupting protein biosynthesis. This process has been studied by treating lymphocytes in vitro with glucocorticoids or by exogenous administration of these steroids to whole animals. However, activation of this process by endogenous adrenal steroids, to date, has not been demonstrated, hi the current study the authors have investigated whether lymphocyte DNA degradation can be triggered by elevating endogenous glucocorticoids via ACTH administration.
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ficity and assay variation, were similar to that which has been previously reported (Satterlee et al, 1980). Lymphocyte Preparation
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Avian lymphocytes were prepared from the thymic tissue of 4-wk-old male broiler chicks.7 Following treatment with glucocorticoids, ACTH, or a control vehicle, birds were sacrificed by rapid desanguination. Then the thymic tissue was removed and placed in ice MATERIALS AND METHODS coldPBS-EDTA (.01 Mphosphate, .15 MNaCl, 2 mM EDTA, pH 7.4). The tissue was minced with scissors and then homogenized using a Radioimmunoassay of loose-fitting Kontes Number 24 tapered glass Corticosterone and glass homogenizer.8 The cell suspension Quantitation of serum corticosterone by was filtered through 100-um Nitex mesh9 and radioimmunoassay (Satterlee et al., 1980) was centrifuged at 800 x g for 5 min. The supernatant performed according to the recommendations of was discarded, and the cell pellet was resus3 the supplier of the anticorticosterone antibody. pended in PBS-EDTA, refiltered, and centriSerum samples (.1 mL) were extracted first with fuged again as described. Cell concentration was 1.0 mL isooctane to remove progestins followed determined using a Coulter counter10 cell by 1.0 mL of methylene chloride to isolate glucocorticoids. The methylene chloride extract viability was ascertained using a fluorescein was evaporated to dryness and reconstituted in diacetate uptake assay (Rotman and Paper1.0 mL phosphate-buffered saline (.1 M phos- master, 1966). This procedure resulted in a phate, .15 M NaCl, pH 7.4) containing . 1 % suspension of thymocytes that were effectively gelatin (PBSG). Aliquots of the sample (.05 mL) separated from the thymic epithelium and or corticosterone standards were incubated connective tissue while maintaining a viability overnight at room temperature with .2 mL of a 1: of 85 to 90%. 64,000 dilution of anticorticosterone antibody The DNA was extracted from about 1 x 108 (B21-42) and approximately 45,000 dpm of thymocytes using a phenol-chloroform-enzy[1,3,6,7-3H] corticosterone/ Antibody-bound matic method (Compton and Cidlowski, 1986). and free hormone were separated by precipita- A .1-mL volume of cells in PBS-EDTA was tion with .25 mL of saturated ammonium added to .6 mL of lysis buffer (50 mM Tris, pH sulfate. A .4-mL aliquot of the supernatant was 8.0; 100mMEDTA,pH8.0; 100 mM NaCl; and added to 10 mL of liquid scintillation fluid5 and 1% SDS) in a 1.5-mL microfuge tube. Prothe radioactivity was quantitated using a Pack- teinase K (500 fig/mL) was added and the ard Tricarb Liquid Scintillation Counter.6 Se- solution incubated at 55 C for 1 h, followed by rum corticosterone levels were calculated using an overnight incubation at 37 C. The sample was a log-logit regression analysis. The characterisdialyzed against 10 mM Tris, pH 7.4; 1 mM tics of this corticosterone radioimmunoassay, in terms of extraction efficiency, sensitivity, speci- EDTA, pH 8.0 (TE) for 18 h at 4 C and then treated with ribonuclease A (500 |ig/mL) for 4 h (37 C) followed by proteinase K (500 ug/mL) at 55 C for 1 h. The sample was then extracted 3 twice with an equal volume of phenolxhloroEndocrine Sciences, Tarzana, CA 91356. "•New England Nuclear, Boston, MA 02118. form (1:1, vol/vol; plus 2% isoamyl alcohol) 5 Scintiverse n, Fisher Scientific, Springfield, NJ 07081. followed by an extraction with water-saturated "Packard Instrument Co., Downers Grove, IL 60515. 7 ether and dialysis overnight against TE. Final Seaboard Farms, Athens, GA 30601. 'Kontes Co., Vineland, NJ 08360. DNA concentrations were determined by 9 Tetko Inc., Elmsford, NY 10523. ultraviolet (UV) absorbance at 260 run using a 10 Coulter Electronics, Hialeah, FL 33012. 1 Lomb Spectronic 2000 spectropho'Bausch and Lomb Analytical Systems, Rochester, NY Bausch and tometer.11 14625.
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACID
Agarose Gel Electrophoresis and Quantification of Deoxyribonucleic Acid Degradation
Digestion of Chicken Red Blood Cell Nuclei with Micrococcal Nuclease Chicken red blood cell nuclei were prepared from heparinized blood collected from 4-wk-old chicks. Approximately 3 mL of packed red blood cells were washed twice with 50 mL of PBS-EDTA and then lysed with 50 mL of 10 mM MgCl2, . 1 % NP-40. The nuclei were pelleted by centrifugation (3,175 x g, 10 min), and washed twice with this lysis solution, followed by two washes with PBS-EDTA. The nuclear pellet was resuspended in .9% NaCl, 50% glycerol and stored at -20 C. Digestion of red blood cell nuclei was
"Polaroid Corp., Cambridge, MA 02139. 13 Helena Laboratories, Beaumont, TX 77704. 14 Lake Pharmaceuticals, Milwaukee, WI 53209. 15 The RU486 was kindly provided by R. Deraedt at the Centre de Recherches Roussel-UCLAF, Romainville, France.
performed in a .1-mL volume containing 5 x 107 nuclei, 100 ng of micrococcal nuclease, 10 mM Tris, pH 7.4; 5 mM MgCl 2 , and 5 mM CaCl2. Nuclei were incubated with micrococcal nuclease for 30 min at 37 C; the reaction was terminated by adding .01 mL of .5 M EDTA, pH 8.0. The DNA was extracted and analyzed by agarose gel electrophoresis as previously described. Experiment 1. To evaluate the in vivo effects of exogenously administered glucocorticoids on avian thymocyte DNA, birds were randomly assigned to treatment groups and subcutaneously injected with a vehicle control or 1 mg/kg body weight of dexamethasone solubilized in 1.0 mL of PBS-EDTA. (The dexamethasone was added to PBS-EDTA and sonicated briefly to generate a suspension.) The birds were killed 0, .5, 1, 2, 4, and 6 h later; 0-h data represent birds treated for 6 h with a control vehicle. Thymic tissue was removed, and the DNA was extracted from isolated thymocytes and then analyzed by agarose gel electrophoresis. Three birds per time point were employed in each experiment; this experiment was replicated three times. Experiment 2. To study the effects of endogenous glucocorticoids on thymocyte DNA, chicks were randomly assigned to treatment groups. The porcine ACTH 14 used in these experiments was dissolved in an aqueous solution of 16% gelatin at a concentration of 40 IU/mL. Birds were administered a vehicle control or 40 IU/kg body weight of the porcine ACTH 14 intramuscularly and 20 IU/kg body weight intravenously. In a single experiment, serum samples were collected from 10 birds per time point at 0, 2, 4, 6, and 8 h after ACTH administration (0-h data represent birds treated for 8 h with a control vehicle) and corticosterone levels were determined by radioimmunoassay (Satterlee et al., 1980). At the same time, thymus tissue was collected and thymocyte DNA analyzed by agarose gel electrophoresis. Experiment 3. To study the effects of a glucocorticoid antagonist on thymocyte DNA, me antiglucocorticoid RU486 (17pMiydroxy1 lp,4 - dimethylaminophenyl - 17a - propyrdestra - 4,9 - diene - 3 - one 15 was employed (Moguilewski and Philibert, 1984; Duval et al., 1984). Chicks were randomly assigned to treatment groups and treated for 6 h with a control vehicle, ACTH (as described previously), RU486 (two doses of 20 mgflcg body weight), or ACTH plus RU486. Birds treated
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The DNA content among treatment groups was adjusted to the same concentration (200 ug/ mL) and the DNA was then electrophoretically separated (approximately 16 ug DNA per lane) on 1.8% agarose gels containing ethidium bromide (.5 |ig/mL). The DNA was visualized by a UV (302 nm) transilluminator, and gels were photographed with a Polaroid MP-4 camera system.12 Relative amounts of DNA degradation from a representative replicate of each experiment were quantitated by densitometric scanning of the photographic negatives using a Helena Laboratories Quick Scan densitometer13 and integrating the area under the curve. The relative amount of DNA degradation was estimated by measuring the integrated area of densitometric scans representing discrete DNA fragments and expressing this quantity as a percentage of the total integrated scan area. Only the amount of fragmented DNA above random background DNA degradation was considered to represent internucleosomal DNA degradation. Thus, the DNA degradation values reported in the present experiment are used as a relative comparison between treatments rather than as absolute amounts of DNA degradation. A Hind m digest of X, DNA, or a Haem digest of * X 174 served as molecular weight standards.
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with ACTH plus RU486 were administered a predose of the antiglucocorticoid 16 h prior to ACTH treatment; a similar dose of RU486 was coinjected with ACTH. Other birds received the appropriate vehicle control injections. For this series of experiments RU486 was solubilized in dimethyl sulfoxide at a concentration of 20 mg/ mL due to its highly insoluble nature in aqueous buffers. Three birds per treatment were employed in each experiment; this experiment was replicated three times.
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Activation of Thymocyte Deoxyribonucleic Acid Degradation by Exogenous Glucocorticoids
FIGURE 1. Time course of glucocorticoid-mediated internucleosomal cleavage of avian thymocyte DNA. Chicks were treated with dexamethasone (1 mg/kg body weight) and killed 0, .5, 1, 2,4, and 6 h later. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis (upper panel). Molecular weight markers expressed in kiiobase pairs are indicated on the left; time in hours is indicated along the top. The lower panel is a densitometric scan of the photographic negative of the agarose gel above. These data are representative of three replicated experiments.
Experiment 1. Initial experiments were aimed at evaluating the in vivo effects of exogenously administered glucocorticoids on avian thymocyte DNA. Figure 1 shows that DNA isolated from thymocytes at 0, .5, or 1 h after steroid treatment appeared as a single high molecular weight species, indicative of intact DNA. However, beginning at 2 h and continuing through 6 h, the genomic DNA exhibited a pattern of degradation in which discrete DNA fragments were generated that consisted of multiples of approximately 200 base pairs (bp). Quantitation of these data revealed that prior to 1 h of steroid treatment, less than 1% of the DNA was fragmented. However, generation of internucleosomal DNA products increased as a function of time from 2 to 6 h. At 2 h, 7% of the DNA was fragmented, and at 4 and 6 h 15 and 17% of the DNA was degraded, respectively. Over this time course, thymocyte viability of control animals was not significantly different from that of birds treated with dexamethasone (85 to 90%).
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Experiment 4. To compare the effects of exogenous glucocorticoids on thymocyte DNA, chicks were randomly assigned to treatment groups and treated for 6 h with a control vehicle, dexamethasone (1 mg/kg body weight), corticosterone (20 mg/kg body weight), or ACTH (as described previously). Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. In addition, nuclei obtained from chicken red blood cells were treated in vitro with micrococcal nuclease to compare the patterns of DNA degradation. Three birds per treatment were employed in each experiment; this experiment was replicated three times.
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACTD
Activation of Thymocyte Deoxyribonucleic Acid Degradation by Endogenous Glucocorticoids
Inhibition of Adrenocorticotropic Hormone-Mediated Deoxyribonucleic Acid Degradation by the Glucocorticoid Antagonist RU486 Experiment 3. As shown in Figure 4, thymocyte DNA from control- and RU486treated birds was intact, whereas the DNA from ACTH-treated birds was degraded into internucleosomal fragments. However, when birds were treated with the glucocorticoid antagonist plus ACTH, there was a diminution in the amount of DNA fragmentation. Densitometric quantitation of these results showed that approximately 6% of thymocyte DNA from ACTHtreated birds contained internucleosomal fragments, whereas half this amount was detected in thymocytes from birds treated with ACTH plus the antiglucocorticoid. Thymocyte viability of control birds was similar to that of RU486- and ACTH-treated birds (85 to 90%). A Comparison of the Patterns of Deoxyribonucleic Acid Fragmentation Following Various Treatments Experiment 4. Figure 5 shows that thymocyte DNA from control birds migrated in the agarose gel as a single, high-molecular weight species, but DNA from chicks treated with dexamethasone, corticosterone, or ACTH was degraded at
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FIGURE 2. Effect of porcine adrenocorticotropic (ACTH) treatment on serum corticosterone levels. Chicks were treated with a control vehicle (0 h) or ACTH hormone. Serum samples were collected 0, 2, 4, 6, and 8 h later. Corticosterone levels were determined by radioimmunoassay (Satterlee et al., 1980). The data represent the x ± SE of 10 chicks per time point. Means having different letters are significantly different (P<05).
FIGURE 3. Time course of adrenocorticotropic hormone (ACTH)-mediated internucleosomal cleavage of avian thymocyte DNA. Chicks were treated with a control vehicle (0 h) or ACTH and killed 0,2,4,6, and 8 h later. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The figures above are densitometric scans of the photographic negative of the gel. These data are representative of three replicated experiments.
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Experiment 2. The data presented in Figure 2 show that initial levels of corticosterone were 1 + .4 ng/mL (x ± SE); however, 2 h after ACTH administration, serum corticosterone levels were elevated to 79 ± 8 ng/mL. Heightened levels of corticosterone were maintained up to 6 h after treatment (48 ± 7 ng/mL at 4 h and 18 ± 4 ng/mL at 6 h) before declining to 5 ± 1 ng/mL 8 h after treatment. These data, as well as data from several preliminary experiments (data not shown), indicate that administration of porcine ACTH to chicks effectively raised serum corticosterone levels, thus allowing analysis of their effect on thymocyte DNA. For up to 2 h following ACTH treatment, thymocyte DNA was undegraded and appeared as a high molecular weight species (Figure 3). However, beginning at 4 h and continuing through 8 h DNA degradation products were detectable. During this time period approximately 6 to 8% of the DNA appeared as internucleosomal DNA fragments, a value that is about half that observed following exogenous dexamethasone treatment over a similar time period. Throughout this time course, viability of thymocytes from control and ACTH-treated birds was the same (85 to 90%).
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red blood cell nuclei (or thymocyte nuclei, data not shown) with micrococcal nuclease also resulted in a "ladder" pattern of DNA fragmentation that was similar to that generated by the endogenous thymocyte nuclease. DISCUSSION
The data presented in the present study clearly demonstrate that exogenous administration of glucocorticoids in vivo activates a DNA-degrading process in avian thymic lym-
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FIGURE 4. Inhibition of adrenocorticotropic hormone (ACTH)-mediated degradation of avian thymocyte DNA by the glucocorticoid antagonist RU486. Chicks were treated for 6 h with a vehicle control (CON), RU486, ACTH, or RU486 plus ACTH. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. Molecular weight markers expressed as kilobase pairs are indicated on the left. These data are representative of three replicated experiments.
FIGURE 5. Comparison of the patterns of internucleosomal DNA degradation following in vivo glucocorticoid treatment or in vitro micrococcal nuclease treatment. Chicks were treated for 6 h with a control vehicle (CON), dexamethasone (DEX, 1 mg/kg body weight), corticosterone (20 mg/kg body weight), or adrenocorticotropic hormone (ACTH). Chicken red blood cell nuclei were treated in vitro with a control vehicle (Nuc) or 100 ng of micrococcal nuclease (Nuc + MN). The DNA was isolated and analyzed by agarose gel electrophoresis. Molecular weight markers expressed as kilobase pairs are indicated on the left These data are representative of three replicated experiments.
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internucleosomal sites. Exogenous treatment with dexamethasone (1 mg/kg body weight) resulted in approximately 18% of the DNA being degraded into internucleosomal fragments; treatment with a 20-fold greater dose of corticosterone (20 mg/kg body weight) degraded a similar amount of genomic DNA. These results are predictable based on the relative potencies of diese glucocorticoids (Munck and Young, 1975). The ACTH treatment, as previously shown (Figure 4), was less effective: approximately 10% of the DNA was degraded into internucleosomal fragments. Thymocyte viability of control birds was similar to that of dexamethasone-, corticosterone-, or ACTH-treated birds (85 to 90%). Treatment of
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACID
described in rodent models (Compton and Cidlowski, 1986). Likewise, the dose-response, steroid-specificity, and tissue-specificity of this in vivo response is comparable in avian (Compton et al., 1990a) and rodent species (Compton and Cidlowski, 1986). However, activation of this nucleolytic process via endogenous adrenal steroids has not been previously characterized in avian or rodent models. The current study demonstrates that administration of porcine ACTH elevates endogenous glucocorticoids (Figure 2). Other investigators (Beuving and Vonder, 1978; Siegel, 1980) have previously demonstrated a similar responsiveness of the chicken adrenal to porcine ACTH. This elevation of glucocorticoids apparently activates a thymocyte DNA degrading response mat is characterized by the generation of intemucleosomal fragments of DNA (Figure 3). This pattern of DNA fragmentation is identical to that generated by exogenous administration of dexamethasone or corticosterone (Figure 5). Moreover, this ACTH-induced response can be inhibited by the antiglucocorticoid RU486, indicating a glucocorticoid receptor-activated process that is mediated by adrenal steroids (Figure 4). Similar results were obtained when RU486 was used to block this effect following exogenous glucocorticoid treatment (Compton et al, 1990a). Interestingly, a comparison of the patterns of DNA degradation generated by the endogenous glucocorticoid-activated thymocyte nuclease and micrococcal nuclease indicate mat the two nucleases degrade DNA differently (Figure 5). On a temporal basis (data not shown), micrococcal nuclease initially degraded the genome into large intemucleosomal lengths of DNA (greater than 1 to 2 kbp) followed by cleavages that generated smaller DNA fragments (less than 1 to 2 kbp). This cleavage pattern is characterized by the conversion of high molecular weight DNA into progressively smaller and smaller intemucleosomal fragments of DNA. Similar observations have been previously reported (Noll, 1974). However, the glucocorticoid-activated thymocyte nuclease degraded only a limited portion of the genome into fragments of DNA that represented the entire intemucleosomal "ladder." Unlike micrococcal nuclease activity, a complete ladder of DNA fragments is clearly visible even though large amounts of genomic DNA appear to be intact. These results suggest
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phocytes. This form of DNA degradation is characterized by the generation of discrete DNA fragments that are multiples of approximately 200 bp. Such a pattern of DNA degradation is indicative of intemucleosomal cleavage where the genome has been degraded in the linker region between successive nucleosomal cores (Noll, 1974). This nucleolytic process is a hallmark of a form of cell death referred to as programmed cell death (Wyllie et al., 1980) by which cells die in response to a specific physiological cue. In the thymocyte model employed in the current studies, glucocorticoids are the hormonal signal that apparently activates a nuclease, which degrades the genome, a process that ultimately results in lymphocytolysis. This pattern of DNA degradation has been described previously by other investigators (Wyllie, 1980; Umansky et al., 1981; Vanderbilt et al., 1982; Vedeckis and Bradshaw, 1983; Cohen and Duke, 1984; Wyllie et al., 1984; Compton and Cidlowski, 1986, 1987; Compton et al., 1987) using rodent lymphocytes. More recently, the authors Compton et al. (1990a,b) have characterized this process using avian lymphocytes. Activation of programmed cell death in avian thymocytes occurs in viable cells prior to lymphocytolysis. In the present studies the viability of lymphocytes [as measured by fluorescene diacetate uptake and propidium iodide exclusion (Rotman and Papermaster, 1966)] treated with glucocorticoids or ACTH was unchanged. Similar observations have been reported by others (Gould and Siegel, 1981). Thus, DNA degradation and presumably other early cellular events associated with programmed cell death occur in a viable population of lymphocytes. This statement is further substantiated by other studies that have shown that inhibitors of protein biosynthesis will block glucocorticoid-mediated DNA degradation in lymphocytes (Wyllie et al., 1984; Compton et al., 1988). Apparently, this process can only be activated in viable cells where the cellular machinery of protein biosynthesis is intact. Therefore, this nucleolytic process is not a function of postmortem cytolysis. Rather, it is an initial cellular event characteristic of lymphocytes undergoing programmed cell death. The time course of dexamethasone-activated DNA degradation in avian thymocytes presented here (Figure 1) and elsewhere (Compton et al., 1990a) is similar to that
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lymphocytes from the immune cell pool This cellular suicide response may play a central role in the rapid cell turnover rate (Glick, 1976) of thymic lymphocytes, or perhaps it is the mode by which self-reactive thymocyte clones (Kappler et al., 1987; Fowlkes et al., 1988) are deleted. Thus, adrenal glucocorticoids can have acute effects on the immune system by modulating the inflammatory response and chronic long-term effects by activating clonal selection of immunocompetent lymphocytes. ACKNOWLEDGMENTS
The authors wish to thank R. Deraedt at the Centre de Recherches, Roussel-UCLAF, for the RU486 used in this study and Jane Blount for her excellent clerical assistance in preparing this manuscript. Special appreciation is expressed to Claiborne Glover and Joe Crimm for their critical review of this manuscript. This work was supported by USDA grants GEO-RC300-145 and GEO-00-519. REFERENCES Beuving, G., and G.M.A. Vonder, 1978. Effects of stressing factors on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 35: 153-159. Blalock, J. E., and E. M. Smith, 1985. Lymphocyte production of endorphin and ACTH-like peptides: A complete regulatory loop between the immune and neuroendocrine systems. Fed. Proc. 44:108-111. Cohen, J. J., and R. C. Duke, 1984. Glucocorticoid activation of a calcium dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132:38-42. Compton, M. M., L. M. Caron, and J. A. Cidlowski, 1987. Glucocorticoid action on the immune system. J. Steroid Biochem. 27:201-208. Compton, M. M., and J. A. Cidlowski, 1986. Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology 118:38-45. Compton, M. M., and J. A. Cidlowski, 1987. Identification of a glucocorticoid-induced nuclease in thymocytes: A potential "lysis gene product" J. Biol. Chem. 262: 8288-8292. Compton, M. M., P. S. Gibbs, and L. R. Swicegood, 1990a. Glucocorticoid mediated activation of DNA degradation in avian lymphocytes. Gen. Comp. Endocrinol. 80:68-79. Compton, M. M., P. S. Gibbs, and L. R. Johnson, 1990b. Glucocorticoid activation of DNA degradation in bursal lymphocytes. Poultry Sci. 69:1292-1298. Compton, M. M., J. S. Haskill, and J. A. Cidlowski, 1988. Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescene-activated flow cytometry. Endocrinology 122:2158-2164. Davison, T. F., B. M. Freeman, and J. Rea, 1985. Effects
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that, over the time period investigated, the thymocyte genome is differentially sensitive to endogenous nuclease attack. The vast majority of the genome appears resistant, whereas a smaller portion is more readily degraded. Similar observations have been reported for a Ca-Mg-dependent liver nuclease (Hewish and Burgoyne, 1973). Other investigators suggest that this nuclease-sensitive portion of the genome may represent regions of active gene transcription (Vanderbilt et al., 1982). The degree to which this glucocorticoidmediated nucleolytic response occurs under normal physiological conditions remains unknown. Obviously, in the current studies detection of genomic DNA degradation has been greatly amplified by employing thymocytes, i.e., immature lymphocytes, which are particularly sensitive to the lytic actions of glucocorticoids. Furthermore, relatively large doses of corticosterone (20 mg/kg body weight) or the potent glucocorticoid, dexamethasone, were administered to induce thymocyte DNA degradation. To detect this response when mediated by endogenous glucocorticoids, an approximate 80-fold elevation in plasma corticosterone level was required. Thus, it remains contentionable whether physiological levels of corticosterone are sufficient to activate detectable amounts of lymphocyte DNA degradation. However, it is well documented that chronic administration of adrenal steroids or ACl'H induces both thymic and bursal involution (Glick, 1960; Sato and Glick, 1970; Dieter and Breitenbach, 1970; Gross et al, 1980; Davison et al, 1985). This process is accompanied by lymphocyte cell death and presumably genomic DNA degradation. Experimentally induced glucocorticoid-mediated thymocyte DNA degradation may be an amplification of an ongoing physiological process that is virtually undetectable in lymphoid tissues, using the methodologies employed. Perhaps glucocorticoid-mediated lymphocytolysis plays an active role in the generation of immunocompetent, glucocorticoid-resistant lymphocytes. In support of this contention, Compton et al. (1990a) observed that exogenous glucocorticoid treatment readily induces DNA degradation in immature bursal and thymic lymphocytes, whereas mature splenocytes are virtually resistant to this nucleolytic effect of adrenal steroids. Furthermore, lymphocytolysis via DNA fragmentation may be a potential mechanism of eliminating
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A. Lockshin, ed. Chapman and Hall, New York, NY. Munck, A., P. M Guyre, and N. Holbrook, 1984. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr. Rev. 5:25-44. Munck, A., and D. A. Young, 1975. Glucocorticoids and lymphoid tissue. Pages 231-243 in: Handbook of Physiology. S. R. Geiger, ed. Vol. 6. Williams and Wilkins, Baltimore, MD. Noll, M., 1974. Subunit structure of chromatin. Nature 251:249-251. Rotman, B., and B. W. Papermaster, 1966. Membrane properties of living mammalian cells as studied by enzymatic hydrolysis of fluorogcnic esters. Proc. Natl. Acad. Sci. USA 55:135-141. Salem, M.H.M., H. W. Norton, and A. V. Nalbandov, 1970. A study of ACTH and CRF in chickens. Gea Comp. Endocrinol. 14:270-280. Sato, K., and B. Glick, 1970. Antibody and cell mediated immunity in corticosteroid-treated chicks. Poultry Sci. 49:982-986. Satterlee, D. G., R. B. Abdullah, and R. P. Gildersleeve, 1980. Plasma corticosterone radioimmunoassay and levels in the neonate chick. Poultry Sci. 59:900-905. Siegel, H. S., 1980. Physiological stress in birds. BioScience 30:529-534. Umansky, S. R., B. A. KoroL and P. A. Nelipovich, 1981. In vivo degradation in thymocytes of gammairradiated or hydrocortisone-treated rats. Biochim. Biophys. Acta 655:9-17. Vanderbilt, J. N., K. S. Bloom, and J. N. Anderson, 1982. Endogenous nuclease: Properties and effects on transcribed genes in chromatin. J. Biol. Chem. 257: 13009-13027. Vedeclds, W. V., and H. D. Biadshaw, 1983. DNA fragmentation in S49 lymphoma cells killed with glucocorticoids and other agents. Mol. Cell. Endocrinol. 30:215-227. Westley, J. H., A. J. Kleiss, K. W. Kelly, P.K.Y. Wang, and P. H. Yuen, 1986. Newcastle disease virusinfected splenocytes express the proopiomelanocortin gene. J. Exp. Med. 168:1589-1594. Wyllie, A. H., 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555-556. Wyllie, A. H., JJJR. Kerr, and A. R. Currie, 1980. Cell death: The significance of apoptosis. Int. Rev. Cytol. 68:251-306. Wyllie, A. H., R. G. Morris, A. L. Smith, and D. Dunlop, 1984. Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142: 67-77.
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of continuous treatment with synthetic ACTH1 or corticosterone on immature Gallus domesticus. Gen. Comp. Endocrinol. 59:416-423. Dieter, M. P., and R. P. Breitenbach, 1970. A comparison of the lympholytic effects of corticosterone and testosterone propionate in immature cockerels. Proc. Soc. Exp. Biol. Med. 133:357-364. Duval, D., S. Durant, and F. Homo-Delarche, 1984. Effect of antiglucocorticoids on dexamethasone-induced inhibition of uridine incorporation and cell lysis in isolated mouse thymocytes. J. Steroid Biochem. 20: 283-287. Frankel, A. I., 1970. Neurohumoral control of the avian adrenal: A review. Poultry Sci. 49:869-921. Frankel, A. I., J. W. Graber, and A. V. Nalbandov, 1967. The effect of hypothalamic lesions on adrenal function in intact and adenohypophysectomized cockerels. Gen. Comp. Endocrinol. 8:387-396. Fowlkes, B. J., R. H. Schwartz, and D. M. Pardoll, 1988. Deletion of self-reactive thymocytes occurs at a CD4 + 8 + precursor stage. Nature 334:620-623. Glick, B., 1960. The effect of bovine growth hormone, deoxycorticosterone, and cortisone on the weight of the bursa of Fabricius, adrenal glands, heart, and body weight of young chickens. Poultry Sci. 39: 1527-1533. Glick, B., 1976. Lymphocyte lifespan in chickens. Pages 237-245, in: Phylogeny of Thymus and Bone Marrow-bursa Cells. R. K. Wright and E. L. Cooper, ed.). North Holland Publishers, Amsterdam, The Netherlands. Gould, N. R., and H. S. Siegel 1981. Viability of and corticosteroid binding in lymphoid cells of various tissues after adrenocorticotropin injection. Poultry Sci. 60:891-393. Gross, W. B., P. B. Siegel, and R. T. DuBose, 1980. Some effects of feeding corticosterone to chickens. Poultry Sci. 59:516-522. Hewish, D. R., and L. A. Burgoyne, 1973. Chromatin substructure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem. Biophys. Res. Commun. 52: 504-510. Kappler, J. W., N. Roehm, and P. Marrack, 1987. T cell tolerance by clonal elimination in the thymus. Cell 49:273-280. Moguilewski, M., and D. Philibert, 1984. RU 38486: Potent antiglucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor followed by impaired activation. J. Steroid Biochem. 20:271-275. Munck, A., and G. R. Crabtree, 1981. Glucocorticoidinduced lymphocyte death. Pages 329—259 in: Cell Death in Biology and Pathology. I. D. Bowen and R.
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1991 Poultry Science 70:521-529 INTRODUCTION
Plasma glucocorticoid levels are rapidly elevated when avian species are exposed to stressful stimuli (Beuving and Vonder, 1978). This adrenal response is activated by a cascade of hormonal signals that emanate from the hypothalamus and are mediated through the pituitary-adrenal axis (Frankel et al, 1967; Salem et al, 1970; Frankel, 1970). Other evidence indicates that adrenocorticotropic hormone (ACTH)-like peptides secreted from immune cells may also modulate adrenal function (Blalock and Smith, 1985; Westley et al, 1986). In rum, glucocorticoids activate gluconeogenic pathways, which ultimately result in an elevation of plasma glucose.
Supported by USDA Grant GEO-RC300-145 and state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. Portions of these data were presented at the 78th Annual Meeting of the Poultry Science Association in Madison, Wisconsin. Send correspondence to: Mark M. Compton, Department of Poultry Science, 144 Livestock-Poultry Building, University of Georgia, Athens, GA 30602. Phone: 404/ 542-1359, 404/542-0414.
In addition to their role as orchestrators of gluconeogenesis, glucocorticoids modulate the immune response (Munck and Young, 1975; Munck et al, 1984). They attenuate the inflammatory reaction and act directly on lymphoid cells to inhibit immune function. Indeed, the most profound immunosuppressive effect of glucocorticoids is the activation of lymphocytolysis. This process has been widely studied (for review see Munck and Crabtree, 1981) although the precise sequence of cellular events that culminate in this form of steroidinduced lymphocyte cell death has not been clearly delineated. Nevertheless, it is known that adrenal steroids a) inhibit numerous anabolic pathways involved in macromolecular biosynthesis, and b) stimulate cellular catabolism. In addition, other evidence indicates mat prior to cell death glucocorticoids activate a DNA degrading process in which the lymphocyte genome is cleaved at internucleosomal sites (Wyllie, 1980; Umansky et al., 1981; Vanderbilt et al, 1982; Vedeckis and Bradshaw, 1983; Cohen and Duke, 1984; Wyllie et al, 1984; Compton and Cidlowski, 1986, 1987; Compton et al, 1987). It is presumed that this nucleolytic response pro-
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ABSTRACT Immature lymphocytes in the thymus gland are killed by treatment with exogenous glucocorticoids. This steroid-mediated lymphocytolysis is preceded by numerous alterations in lymphocyte metabolism, including a DNA-degrading process in which the genome is cleaved at internucleosomal intervals. To date, this process has only been characterized by treating lymphocytes in vitro with glucocorticoids or by exogenous treatment of whole animals with adrenal steroids. To determine whether thymocyte DNA degradation could be activated by endogenous glucocorticoids, 4-wk-old chicks were treated with porcine adrenocorticotropic hormone (ACTH). This procedure elevated serum corticosterone levels approximately 80-fold within 2 h of hormone treatment Following ACTH administration, thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The ACTH activated a DNA-degrading process that generated internucleosomal fragments of DNA identical in size to those observed following exogenous treatment with synthetic or naturally occurring glucocorticoids. Furthermore, this response could be inhibited by the glucocorticoid antagonist RU486 (17fJ-hydroxy-lip, 4-dimethylaminophenyl-17oc-propynl-estra4,9,diene-3-one), indicating that adrenal steroids activate this process via the glucocorticoid receptor. These results demonstrate that lymphocyte DNA degradation does not result solely from exogenous glucocorticoid treatment; moreover, endogenous glucocorticoids can mediate this process and may thereby play an important role in thymic gland function. (Key words: thymus, glucocorticoids, adrenocorticotropic hormone, deoxyribonucleic acid degradation, programmed cell death)
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motes cell death by disrupting protein biosynthesis. This process has been studied by treating lymphocytes in vitro with glucocorticoids or by exogenous administration of these steroids to whole animals. However, activation of this process by endogenous adrenal steroids, to date, has not been demonstrated, hi the current study the authors have investigated whether lymphocyte DNA degradation can be triggered by elevating endogenous glucocorticoids via ACTH administration.
ETAL.
ficity and assay variation, were similar to that which has been previously reported (Satterlee et al, 1980). Lymphocyte Preparation
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Avian lymphocytes were prepared from the thymic tissue of 4-wk-old male broiler chicks.7 Following treatment with glucocorticoids, ACTH, or a control vehicle, birds were sacrificed by rapid desanguination. Then the thymic tissue was removed and placed in ice MATERIALS AND METHODS coldPBS-EDTA (.01 Mphosphate, .15 MNaCl, 2 mM EDTA, pH 7.4). The tissue was minced with scissors and then homogenized using a Radioimmunoassay of loose-fitting Kontes Number 24 tapered glass Corticosterone and glass homogenizer.8 The cell suspension Quantitation of serum corticosterone by was filtered through 100-um Nitex mesh9 and radioimmunoassay (Satterlee et al., 1980) was centrifuged at 800 x g for 5 min. The supernatant performed according to the recommendations of was discarded, and the cell pellet was resus3 the supplier of the anticorticosterone antibody. pended in PBS-EDTA, refiltered, and centriSerum samples (.1 mL) were extracted first with fuged again as described. Cell concentration was 1.0 mL isooctane to remove progestins followed determined using a Coulter counter10 cell by 1.0 mL of methylene chloride to isolate glucocorticoids. The methylene chloride extract viability was ascertained using a fluorescein was evaporated to dryness and reconstituted in diacetate uptake assay (Rotman and Paper1.0 mL phosphate-buffered saline (.1 M phos- master, 1966). This procedure resulted in a phate, .15 M NaCl, pH 7.4) containing . 1 % suspension of thymocytes that were effectively gelatin (PBSG). Aliquots of the sample (.05 mL) separated from the thymic epithelium and or corticosterone standards were incubated connective tissue while maintaining a viability overnight at room temperature with .2 mL of a 1: of 85 to 90%. 64,000 dilution of anticorticosterone antibody The DNA was extracted from about 1 x 108 (B21-42) and approximately 45,000 dpm of thymocytes using a phenol-chloroform-enzy[1,3,6,7-3H] corticosterone/ Antibody-bound matic method (Compton and Cidlowski, 1986). and free hormone were separated by precipita- A .1-mL volume of cells in PBS-EDTA was tion with .25 mL of saturated ammonium added to .6 mL of lysis buffer (50 mM Tris, pH sulfate. A .4-mL aliquot of the supernatant was 8.0; 100mMEDTA,pH8.0; 100 mM NaCl; and added to 10 mL of liquid scintillation fluid5 and 1% SDS) in a 1.5-mL microfuge tube. Prothe radioactivity was quantitated using a Pack- teinase K (500 fig/mL) was added and the ard Tricarb Liquid Scintillation Counter.6 Se- solution incubated at 55 C for 1 h, followed by rum corticosterone levels were calculated using an overnight incubation at 37 C. The sample was a log-logit regression analysis. The characterisdialyzed against 10 mM Tris, pH 7.4; 1 mM tics of this corticosterone radioimmunoassay, in terms of extraction efficiency, sensitivity, speci- EDTA, pH 8.0 (TE) for 18 h at 4 C and then treated with ribonuclease A (500 |ig/mL) for 4 h (37 C) followed by proteinase K (500 ug/mL) at 55 C for 1 h. The sample was then extracted 3 twice with an equal volume of phenolxhloroEndocrine Sciences, Tarzana, CA 91356. "•New England Nuclear, Boston, MA 02118. form (1:1, vol/vol; plus 2% isoamyl alcohol) 5 Scintiverse n, Fisher Scientific, Springfield, NJ 07081. followed by an extraction with water-saturated "Packard Instrument Co., Downers Grove, IL 60515. 7 ether and dialysis overnight against TE. Final Seaboard Farms, Athens, GA 30601. 'Kontes Co., Vineland, NJ 08360. DNA concentrations were determined by 9 Tetko Inc., Elmsford, NY 10523. ultraviolet (UV) absorbance at 260 run using a 10 Coulter Electronics, Hialeah, FL 33012. 1 Lomb Spectronic 2000 spectropho'Bausch and Lomb Analytical Systems, Rochester, NY Bausch and tometer.11 14625.
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACID
Agarose Gel Electrophoresis and Quantification of Deoxyribonucleic Acid Degradation
Digestion of Chicken Red Blood Cell Nuclei with Micrococcal Nuclease Chicken red blood cell nuclei were prepared from heparinized blood collected from 4-wk-old chicks. Approximately 3 mL of packed red blood cells were washed twice with 50 mL of PBS-EDTA and then lysed with 50 mL of 10 mM MgCl2, . 1 % NP-40. The nuclei were pelleted by centrifugation (3,175 x g, 10 min), and washed twice with this lysis solution, followed by two washes with PBS-EDTA. The nuclear pellet was resuspended in .9% NaCl, 50% glycerol and stored at -20 C. Digestion of red blood cell nuclei was
"Polaroid Corp., Cambridge, MA 02139. 13 Helena Laboratories, Beaumont, TX 77704. 14 Lake Pharmaceuticals, Milwaukee, WI 53209. 15 The RU486 was kindly provided by R. Deraedt at the Centre de Recherches Roussel-UCLAF, Romainville, France.
performed in a .1-mL volume containing 5 x 107 nuclei, 100 ng of micrococcal nuclease, 10 mM Tris, pH 7.4; 5 mM MgCl 2 , and 5 mM CaCl2. Nuclei were incubated with micrococcal nuclease for 30 min at 37 C; the reaction was terminated by adding .01 mL of .5 M EDTA, pH 8.0. The DNA was extracted and analyzed by agarose gel electrophoresis as previously described. Experiment 1. To evaluate the in vivo effects of exogenously administered glucocorticoids on avian thymocyte DNA, birds were randomly assigned to treatment groups and subcutaneously injected with a vehicle control or 1 mg/kg body weight of dexamethasone solubilized in 1.0 mL of PBS-EDTA. (The dexamethasone was added to PBS-EDTA and sonicated briefly to generate a suspension.) The birds were killed 0, .5, 1, 2, 4, and 6 h later; 0-h data represent birds treated for 6 h with a control vehicle. Thymic tissue was removed, and the DNA was extracted from isolated thymocytes and then analyzed by agarose gel electrophoresis. Three birds per time point were employed in each experiment; this experiment was replicated three times. Experiment 2. To study the effects of endogenous glucocorticoids on thymocyte DNA, chicks were randomly assigned to treatment groups. The porcine ACTH 14 used in these experiments was dissolved in an aqueous solution of 16% gelatin at a concentration of 40 IU/mL. Birds were administered a vehicle control or 40 IU/kg body weight of the porcine ACTH 14 intramuscularly and 20 IU/kg body weight intravenously. In a single experiment, serum samples were collected from 10 birds per time point at 0, 2, 4, 6, and 8 h after ACTH administration (0-h data represent birds treated for 8 h with a control vehicle) and corticosterone levels were determined by radioimmunoassay (Satterlee et al., 1980). At the same time, thymus tissue was collected and thymocyte DNA analyzed by agarose gel electrophoresis. Experiment 3. To study the effects of a glucocorticoid antagonist on thymocyte DNA, me antiglucocorticoid RU486 (17pMiydroxy1 lp,4 - dimethylaminophenyl - 17a - propyrdestra - 4,9 - diene - 3 - one 15 was employed (Moguilewski and Philibert, 1984; Duval et al., 1984). Chicks were randomly assigned to treatment groups and treated for 6 h with a control vehicle, ACTH (as described previously), RU486 (two doses of 20 mgflcg body weight), or ACTH plus RU486. Birds treated
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The DNA content among treatment groups was adjusted to the same concentration (200 ug/ mL) and the DNA was then electrophoretically separated (approximately 16 ug DNA per lane) on 1.8% agarose gels containing ethidium bromide (.5 |ig/mL). The DNA was visualized by a UV (302 nm) transilluminator, and gels were photographed with a Polaroid MP-4 camera system.12 Relative amounts of DNA degradation from a representative replicate of each experiment were quantitated by densitometric scanning of the photographic negatives using a Helena Laboratories Quick Scan densitometer13 and integrating the area under the curve. The relative amount of DNA degradation was estimated by measuring the integrated area of densitometric scans representing discrete DNA fragments and expressing this quantity as a percentage of the total integrated scan area. Only the amount of fragmented DNA above random background DNA degradation was considered to represent internucleosomal DNA degradation. Thus, the DNA degradation values reported in the present experiment are used as a relative comparison between treatments rather than as absolute amounts of DNA degradation. A Hind m digest of X, DNA, or a Haem digest of * X 174 served as molecular weight standards.
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with ACTH plus RU486 were administered a predose of the antiglucocorticoid 16 h prior to ACTH treatment; a similar dose of RU486 was coinjected with ACTH. Other birds received the appropriate vehicle control injections. For this series of experiments RU486 was solubilized in dimethyl sulfoxide at a concentration of 20 mg/ mL due to its highly insoluble nature in aqueous buffers. Three birds per treatment were employed in each experiment; this experiment was replicated three times.
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Activation of Thymocyte Deoxyribonucleic Acid Degradation by Exogenous Glucocorticoids
FIGURE 1. Time course of glucocorticoid-mediated internucleosomal cleavage of avian thymocyte DNA. Chicks were treated with dexamethasone (1 mg/kg body weight) and killed 0, .5, 1, 2,4, and 6 h later. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis (upper panel). Molecular weight markers expressed in kiiobase pairs are indicated on the left; time in hours is indicated along the top. The lower panel is a densitometric scan of the photographic negative of the agarose gel above. These data are representative of three replicated experiments.
Experiment 1. Initial experiments were aimed at evaluating the in vivo effects of exogenously administered glucocorticoids on avian thymocyte DNA. Figure 1 shows that DNA isolated from thymocytes at 0, .5, or 1 h after steroid treatment appeared as a single high molecular weight species, indicative of intact DNA. However, beginning at 2 h and continuing through 6 h, the genomic DNA exhibited a pattern of degradation in which discrete DNA fragments were generated that consisted of multiples of approximately 200 base pairs (bp). Quantitation of these data revealed that prior to 1 h of steroid treatment, less than 1% of the DNA was fragmented. However, generation of internucleosomal DNA products increased as a function of time from 2 to 6 h. At 2 h, 7% of the DNA was fragmented, and at 4 and 6 h 15 and 17% of the DNA was degraded, respectively. Over this time course, thymocyte viability of control animals was not significantly different from that of birds treated with dexamethasone (85 to 90%).
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Experiment 4. To compare the effects of exogenous glucocorticoids on thymocyte DNA, chicks were randomly assigned to treatment groups and treated for 6 h with a control vehicle, dexamethasone (1 mg/kg body weight), corticosterone (20 mg/kg body weight), or ACTH (as described previously). Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. In addition, nuclei obtained from chicken red blood cells were treated in vitro with micrococcal nuclease to compare the patterns of DNA degradation. Three birds per treatment were employed in each experiment; this experiment was replicated three times.
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACTD
Activation of Thymocyte Deoxyribonucleic Acid Degradation by Endogenous Glucocorticoids
Inhibition of Adrenocorticotropic Hormone-Mediated Deoxyribonucleic Acid Degradation by the Glucocorticoid Antagonist RU486 Experiment 3. As shown in Figure 4, thymocyte DNA from control- and RU486treated birds was intact, whereas the DNA from ACTH-treated birds was degraded into internucleosomal fragments. However, when birds were treated with the glucocorticoid antagonist plus ACTH, there was a diminution in the amount of DNA fragmentation. Densitometric quantitation of these results showed that approximately 6% of thymocyte DNA from ACTHtreated birds contained internucleosomal fragments, whereas half this amount was detected in thymocytes from birds treated with ACTH plus the antiglucocorticoid. Thymocyte viability of control birds was similar to that of RU486- and ACTH-treated birds (85 to 90%). A Comparison of the Patterns of Deoxyribonucleic Acid Fragmentation Following Various Treatments Experiment 4. Figure 5 shows that thymocyte DNA from control birds migrated in the agarose gel as a single, high-molecular weight species, but DNA from chicks treated with dexamethasone, corticosterone, or ACTH was degraded at
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FIGURE 2. Effect of porcine adrenocorticotropic (ACTH) treatment on serum corticosterone levels. Chicks were treated with a control vehicle (0 h) or ACTH hormone. Serum samples were collected 0, 2, 4, 6, and 8 h later. Corticosterone levels were determined by radioimmunoassay (Satterlee et al., 1980). The data represent the x ± SE of 10 chicks per time point. Means having different letters are significantly different (P<05).
FIGURE 3. Time course of adrenocorticotropic hormone (ACTH)-mediated internucleosomal cleavage of avian thymocyte DNA. Chicks were treated with a control vehicle (0 h) or ACTH and killed 0,2,4,6, and 8 h later. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. The figures above are densitometric scans of the photographic negative of the gel. These data are representative of three replicated experiments.
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Experiment 2. The data presented in Figure 2 show that initial levels of corticosterone were 1 + .4 ng/mL (x ± SE); however, 2 h after ACTH administration, serum corticosterone levels were elevated to 79 ± 8 ng/mL. Heightened levels of corticosterone were maintained up to 6 h after treatment (48 ± 7 ng/mL at 4 h and 18 ± 4 ng/mL at 6 h) before declining to 5 ± 1 ng/mL 8 h after treatment. These data, as well as data from several preliminary experiments (data not shown), indicate that administration of porcine ACTH to chicks effectively raised serum corticosterone levels, thus allowing analysis of their effect on thymocyte DNA. For up to 2 h following ACTH treatment, thymocyte DNA was undegraded and appeared as a high molecular weight species (Figure 3). However, beginning at 4 h and continuing through 8 h DNA degradation products were detectable. During this time period approximately 6 to 8% of the DNA appeared as internucleosomal DNA fragments, a value that is about half that observed following exogenous dexamethasone treatment over a similar time period. Throughout this time course, viability of thymocytes from control and ACTH-treated birds was the same (85 to 90%).
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red blood cell nuclei (or thymocyte nuclei, data not shown) with micrococcal nuclease also resulted in a "ladder" pattern of DNA fragmentation that was similar to that generated by the endogenous thymocyte nuclease. DISCUSSION
The data presented in the present study clearly demonstrate that exogenous administration of glucocorticoids in vivo activates a DNA-degrading process in avian thymic lym-
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FIGURE 4. Inhibition of adrenocorticotropic hormone (ACTH)-mediated degradation of avian thymocyte DNA by the glucocorticoid antagonist RU486. Chicks were treated for 6 h with a vehicle control (CON), RU486, ACTH, or RU486 plus ACTH. Thymocyte DNA was isolated and analyzed by agarose gel electrophoresis. Molecular weight markers expressed as kilobase pairs are indicated on the left. These data are representative of three replicated experiments.
FIGURE 5. Comparison of the patterns of internucleosomal DNA degradation following in vivo glucocorticoid treatment or in vitro micrococcal nuclease treatment. Chicks were treated for 6 h with a control vehicle (CON), dexamethasone (DEX, 1 mg/kg body weight), corticosterone (20 mg/kg body weight), or adrenocorticotropic hormone (ACTH). Chicken red blood cell nuclei were treated in vitro with a control vehicle (Nuc) or 100 ng of micrococcal nuclease (Nuc + MN). The DNA was isolated and analyzed by agarose gel electrophoresis. Molecular weight markers expressed as kilobase pairs are indicated on the left These data are representative of three replicated experiments.
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internucleosomal sites. Exogenous treatment with dexamethasone (1 mg/kg body weight) resulted in approximately 18% of the DNA being degraded into internucleosomal fragments; treatment with a 20-fold greater dose of corticosterone (20 mg/kg body weight) degraded a similar amount of genomic DNA. These results are predictable based on the relative potencies of diese glucocorticoids (Munck and Young, 1975). The ACTH treatment, as previously shown (Figure 4), was less effective: approximately 10% of the DNA was degraded into internucleosomal fragments. Thymocyte viability of control birds was similar to that of dexamethasone-, corticosterone-, or ACTH-treated birds (85 to 90%). Treatment of
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACID
described in rodent models (Compton and Cidlowski, 1986). Likewise, the dose-response, steroid-specificity, and tissue-specificity of this in vivo response is comparable in avian (Compton et al., 1990a) and rodent species (Compton and Cidlowski, 1986). However, activation of this nucleolytic process via endogenous adrenal steroids has not been previously characterized in avian or rodent models. The current study demonstrates that administration of porcine ACTH elevates endogenous glucocorticoids (Figure 2). Other investigators (Beuving and Vonder, 1978; Siegel, 1980) have previously demonstrated a similar responsiveness of the chicken adrenal to porcine ACTH. This elevation of glucocorticoids apparently activates a thymocyte DNA degrading response mat is characterized by the generation of intemucleosomal fragments of DNA (Figure 3). This pattern of DNA fragmentation is identical to that generated by exogenous administration of dexamethasone or corticosterone (Figure 5). Moreover, this ACTH-induced response can be inhibited by the antiglucocorticoid RU486, indicating a glucocorticoid receptor-activated process that is mediated by adrenal steroids (Figure 4). Similar results were obtained when RU486 was used to block this effect following exogenous glucocorticoid treatment (Compton et al, 1990a). Interestingly, a comparison of the patterns of DNA degradation generated by the endogenous glucocorticoid-activated thymocyte nuclease and micrococcal nuclease indicate mat the two nucleases degrade DNA differently (Figure 5). On a temporal basis (data not shown), micrococcal nuclease initially degraded the genome into large intemucleosomal lengths of DNA (greater than 1 to 2 kbp) followed by cleavages that generated smaller DNA fragments (less than 1 to 2 kbp). This cleavage pattern is characterized by the conversion of high molecular weight DNA into progressively smaller and smaller intemucleosomal fragments of DNA. Similar observations have been previously reported (Noll, 1974). However, the glucocorticoid-activated thymocyte nuclease degraded only a limited portion of the genome into fragments of DNA that represented the entire intemucleosomal "ladder." Unlike micrococcal nuclease activity, a complete ladder of DNA fragments is clearly visible even though large amounts of genomic DNA appear to be intact. These results suggest
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phocytes. This form of DNA degradation is characterized by the generation of discrete DNA fragments that are multiples of approximately 200 bp. Such a pattern of DNA degradation is indicative of intemucleosomal cleavage where the genome has been degraded in the linker region between successive nucleosomal cores (Noll, 1974). This nucleolytic process is a hallmark of a form of cell death referred to as programmed cell death (Wyllie et al., 1980) by which cells die in response to a specific physiological cue. In the thymocyte model employed in the current studies, glucocorticoids are the hormonal signal that apparently activates a nuclease, which degrades the genome, a process that ultimately results in lymphocytolysis. This pattern of DNA degradation has been described previously by other investigators (Wyllie, 1980; Umansky et al., 1981; Vanderbilt et al., 1982; Vedeckis and Bradshaw, 1983; Cohen and Duke, 1984; Wyllie et al., 1984; Compton and Cidlowski, 1986, 1987; Compton et al., 1987) using rodent lymphocytes. More recently, the authors Compton et al. (1990a,b) have characterized this process using avian lymphocytes. Activation of programmed cell death in avian thymocytes occurs in viable cells prior to lymphocytolysis. In the present studies the viability of lymphocytes [as measured by fluorescene diacetate uptake and propidium iodide exclusion (Rotman and Papermaster, 1966)] treated with glucocorticoids or ACTH was unchanged. Similar observations have been reported by others (Gould and Siegel, 1981). Thus, DNA degradation and presumably other early cellular events associated with programmed cell death occur in a viable population of lymphocytes. This statement is further substantiated by other studies that have shown that inhibitors of protein biosynthesis will block glucocorticoid-mediated DNA degradation in lymphocytes (Wyllie et al., 1984; Compton et al., 1988). Apparently, this process can only be activated in viable cells where the cellular machinery of protein biosynthesis is intact. Therefore, this nucleolytic process is not a function of postmortem cytolysis. Rather, it is an initial cellular event characteristic of lymphocytes undergoing programmed cell death. The time course of dexamethasone-activated DNA degradation in avian thymocytes presented here (Figure 1) and elsewhere (Compton et al., 1990a) is similar to that
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lymphocytes from the immune cell pool This cellular suicide response may play a central role in the rapid cell turnover rate (Glick, 1976) of thymic lymphocytes, or perhaps it is the mode by which self-reactive thymocyte clones (Kappler et al., 1987; Fowlkes et al., 1988) are deleted. Thus, adrenal glucocorticoids can have acute effects on the immune system by modulating the inflammatory response and chronic long-term effects by activating clonal selection of immunocompetent lymphocytes. ACKNOWLEDGMENTS
The authors wish to thank R. Deraedt at the Centre de Recherches, Roussel-UCLAF, for the RU486 used in this study and Jane Blount for her excellent clerical assistance in preparing this manuscript. Special appreciation is expressed to Claiborne Glover and Joe Crimm for their critical review of this manuscript. This work was supported by USDA grants GEO-RC300-145 and GEO-00-519. REFERENCES Beuving, G., and G.M.A. Vonder, 1978. Effects of stressing factors on corticosterone levels in the plasma of laying hens. Gen. Comp. Endocrinol. 35: 153-159. Blalock, J. E., and E. M. Smith, 1985. Lymphocyte production of endorphin and ACTH-like peptides: A complete regulatory loop between the immune and neuroendocrine systems. Fed. Proc. 44:108-111. Cohen, J. J., and R. C. Duke, 1984. Glucocorticoid activation of a calcium dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132:38-42. Compton, M. M., L. M. Caron, and J. A. Cidlowski, 1987. Glucocorticoid action on the immune system. J. Steroid Biochem. 27:201-208. Compton, M. M., and J. A. Cidlowski, 1986. Rapid in vivo effects of glucocorticoids on the integrity of rat lymphocyte genomic deoxyribonucleic acid. Endocrinology 118:38-45. Compton, M. M., and J. A. Cidlowski, 1987. Identification of a glucocorticoid-induced nuclease in thymocytes: A potential "lysis gene product" J. Biol. Chem. 262: 8288-8292. Compton, M. M., P. S. Gibbs, and L. R. Swicegood, 1990a. Glucocorticoid mediated activation of DNA degradation in avian lymphocytes. Gen. Comp. Endocrinol. 80:68-79. Compton, M. M., P. S. Gibbs, and L. R. Johnson, 1990b. Glucocorticoid activation of DNA degradation in bursal lymphocytes. Poultry Sci. 69:1292-1298. Compton, M. M., J. S. Haskill, and J. A. Cidlowski, 1988. Analysis of glucocorticoid actions on rat thymocyte deoxyribonucleic acid by fluorescene-activated flow cytometry. Endocrinology 122:2158-2164. Davison, T. F., B. M. Freeman, and J. Rea, 1985. Effects
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that, over the time period investigated, the thymocyte genome is differentially sensitive to endogenous nuclease attack. The vast majority of the genome appears resistant, whereas a smaller portion is more readily degraded. Similar observations have been reported for a Ca-Mg-dependent liver nuclease (Hewish and Burgoyne, 1973). Other investigators suggest that this nuclease-sensitive portion of the genome may represent regions of active gene transcription (Vanderbilt et al., 1982). The degree to which this glucocorticoidmediated nucleolytic response occurs under normal physiological conditions remains unknown. Obviously, in the current studies detection of genomic DNA degradation has been greatly amplified by employing thymocytes, i.e., immature lymphocytes, which are particularly sensitive to the lytic actions of glucocorticoids. Furthermore, relatively large doses of corticosterone (20 mg/kg body weight) or the potent glucocorticoid, dexamethasone, were administered to induce thymocyte DNA degradation. To detect this response when mediated by endogenous glucocorticoids, an approximate 80-fold elevation in plasma corticosterone level was required. Thus, it remains contentionable whether physiological levels of corticosterone are sufficient to activate detectable amounts of lymphocyte DNA degradation. However, it is well documented that chronic administration of adrenal steroids or ACl'H induces both thymic and bursal involution (Glick, 1960; Sato and Glick, 1970; Dieter and Breitenbach, 1970; Gross et al, 1980; Davison et al, 1985). This process is accompanied by lymphocyte cell death and presumably genomic DNA degradation. Experimentally induced glucocorticoid-mediated thymocyte DNA degradation may be an amplification of an ongoing physiological process that is virtually undetectable in lymphoid tissues, using the methodologies employed. Perhaps glucocorticoid-mediated lymphocytolysis plays an active role in the generation of immunocompetent, glucocorticoid-resistant lymphocytes. In support of this contention, Compton et al. (1990a) observed that exogenous glucocorticoid treatment readily induces DNA degradation in immature bursal and thymic lymphocytes, whereas mature splenocytes are virtually resistant to this nucleolytic effect of adrenal steroids. Furthermore, lymphocytolysis via DNA fragmentation may be a potential mechanism of eliminating
DEGRADATION OF THYMOCYTE DEOXYRIBONUCLEIC ACID
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