Interleukin-l-induced corticosterone release occurs by an adrenergic mechanism from rat adrenal gland A. R. GWOSDOW, N. A. O’CONNELL, J. A. SPENCER, M. S. A. KUMAR, R. K. AGARWAL, H. H. BODE, AND A. B. ABOU-SAMRA Endocrine Unit of Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Institute, Boston, Massachusetts 02114 Gwosdow, A. R., N. A. O’Connell, J. A. Spencer, M. S. A. Kumar, R. K. Agarwal, H. H. Bode, and A. B. AbouSamra. Interleukinl-induced corticosterone release occurs by an adrenergic mechanism from rat adrenal gland. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E461-E466, 1992.Interleukin-1 (IL-l) has been shown to stimulate corticosterone release from the adrenal gland directly, and indirectly through activation of the hypothalamic-pituitary-adrenal axis. The aim of this paper was to determine whether IL-l-stimulated corticosterone release occurs indirectly through the local release of catecholamines from the rat adrenal gland. To accomplish this, experiments were conducted on both quartered rat adrenal glands and primary cultures of dispersed adrenal cells. Incubation of quartered adrenals with adrenocorticotropic hormone (ACTH, lo-‘:! to 10mH M) or IL-10 (10-l” to 10eH M) resulted in dose-dependent increases (P < 0.05) in corticosterone release. Corticosterone release stimulated by 10eH M doses of ACTH and IL-lp began to rise 30 min after incubation and peaked at 2 h. In primary cultures of adrenal cells, IL-la and IL-16 elevated corticosterone release after a 24-h incubation period. ACTH elevated corticosterone levels at 4 and 24 h. The stimulatory effect of IL-l on corticosterone release was mimicked by epinephrine (lo-(’ M), and was selectively blocked by the cyadrenergic antagonist phentolamine (lo-” M). The P-adrenergic antagonist propranolol (lo-” M) did not change IL-l-induced corticosterone release. Neither phentolamine nor propranolol had an effect on ACTH-stimulated corticosterone release. Both IL-la and IL-l@ significantly increased (P < 0.05) epinephrine levels after a 24-h incubation period compared with media-treated controls. These observations suggest that locally released catecholamines mediate the stimulatory effect of IL-l on corticosterone release through an cu-adrenergic receptor in the rat adrenal gland. epinephrine; phentolamine; propranolol
(IL-l) has been shown to stimulate the secretion of corticotropin-releasing factor (CRF) from the hypothalamus (2, 2 1), adrenocorticotropic hormone (ACTH) from the pituitary gland (3) and AtT-20 cells (31), and glucocorticoids from the adrenal gland (1, 19, 24, 29, 30). In the adrenal gland, IL-l stimulates glucocorticoid release from human (29), bovine (30), and rat (1, 24) adrenocortical cells. Current data suggest several different mechanisms of IL-l action on glucocorticoid release from the adrenal gland. Involvement of prostaglandin-dependent mechanisms in IL- I -stimulated glucocorticoid secretion have been demonstrated in bovine (30) and rat (24) primary adrenocortical cells. Additionally, the secretory effect of IL-l on the adrenal gland may involve the activation of an intra-adrenal CRF-ACTH system (1). An interaction between adrenal cells is supported by the fact that the adrenal medulla is required for IL-l stimulation of corticosterone release (1). In addition, IL-1 has been found colocalized with catecholamines in INTERLEUKIN-1
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chromaffin cells of the adrenal medulla (22). Because catecholamines are stimulators of glucocorticoid secretion (27), we hypothesized that IL-l stimulates corticosterone release through these adrenal medullary hormones. The data are consistent with a major role for catecholamines in mediating the effect of IL-1 on corticosterone release through a-adrenergic receptors. MATERIALS
AND METHODS
Animals. Adult male Sprague-Dawley rats (Taconic Farms) weighing 200-250 g were used in this study. Rats were housed in standard rodent shoe box cages and fed a pelleted Chow diet (Laboratory Rodent Chow 5001, Ralston Purina). Bottles containing water were attached to each cage. Food and water were available ad libitum. The cages were housed in a controlled environmental room at the dry-bulb temperature of 24°C. A relative humidity of 50% and a 12:12-h light-dark photoperiod (light from 0700 to 1900 h) were maintained. Organ cultures. Two fresh adrenal glands were collected and prepared as previously described (8). Briefly, after removal from the animal, each adrenal gland was weighed on an analytic balance (model B6, Mettler), quartered, and preincubated individually in a 35 x 10 mm Petri dish with 1 ml of Krebs-Ringer buffer without bicarbonate containing glucose (1 mg/ml, pH 7.4; catalog no. K 4002, Sigma) and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; H 0887, Sigma) at 37°C for 2.5 h. At the end of this preincubation period, the supernatant was discarded and replaced with sterile medium 199 (M-199) with Earle’s salts (catalog no. 320-1151 AJ, GIBCO) containing 25 mM HEPES, 0.1% bovine serum albumin (BSA; B 8894, Sigma), 20 pg/ml ascorbic acid, and 100 KIU/ml aprotinin (A 6279, Sigma). To this serum-free medium, the appropriate substance, such as ACTH, IL-l& or catecholamine antagonist, was added. Cells were incubated with catecholamine antagonist for 30 min before the addition of other stimulants. At the desired time after incubation, one 300~~1 aliquot was removed from each culture. For time course studies, a 50-~1 aliquot was removed from each culture and replaced with 50 ~1 of incubation medium at 0.25, 0.5, 1, 2, 4, and 24 h after incubation. The collected samples were frozen at -80°C until assayed for corticosterone concentration. The adrenal glands from each animal were paired so that one adrenal gland served as the control and the other received the experimental treatment. Corticosterone concentration from the control gland was subtracted from that of the paired treated gland before statistical analyses were performed. Corticosterone concentration was expressed per milligram of tissue. Adrenal cell cultures. Primary adrenal cells were prepared by a modification of the method previously described (18). Briefly, 30 rats were decapitated and the adrenal glands were removed from the surrounding fat. The entire adrenal gland was minced in a IO-cm Petri dish containing 20 ml of sterile M-199 with Earle’s salts. Collagenase (2 mg/ml; CLSIV, Cooper Biomedical), DNase (0.1 mg/ml; D 0876, Sigma), and trypsin inhibitor (20 mg/ml; T 9003, Sigma) were added, and the solution was
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transferred to a sterile 50-ml conical tube. The tube was capped and placed in a shaking water bath at 37°C for 20 min. After this incubation period, the tissue fragments were allowed to settle, and the medium containing dispersed cells was collected and spun, and the cell pellet was resuspended and saved on ice. The tissue fragments were dispersed mechanically by repeated aspiration with a sterile transfer pipette. The fragments were allowed to settle, and the rinse medium was replaced with sterile M-199. Each time the rinse medium was removed, it was saved on ice. This procedure was repeated until the tissue was completely dissociated. When all of the tissue was dissociated, the supernatant and saved rinse medium were combined and filtered through nylon mesh. The cells were collected by centrifugation at 1,000 revolutions/min for 12 min; resuspended in M-199 containing 10% fetal bovine serum, gentamycin (40 pg/ml), and mycostatin (20 II-J/ml); plated in 48-well plates at a density of 100,000 cells/O.5 ml in each well; and incubated at 37°C in 5% C02-95% air. The medium was replaced every 2-3 days. Five to seven days after preparation, the primary adrenal cells were used in experiments. All experiments were conducted in triplicate wells; each well received only one treatment. Each experiment was repeated at least three times. Before each experiment, the cells were rinsed with sterile phosphate-buffered saline (PBS) containing 0.1% BSA. The cells were incubated in serum-free M-199 containing 25 mM HEPES, 0.1% BSA, 20 pg/ml ascorbic acid, and 106 KIU/ml aprotinin with the desired stimulants. At the appropriate time (4, 24 h) after incubation, the 48-well plates were centrifuged at ~,~~o revolutiondmin for 5 min and a 3ohl aliquot of supernatant was collected and frozen at -80°C until assayed for hormone concentration. Test substances. Recombinant human IL-la with an activity measured by the DlO assay of 2 x 10s units/ml was used (7). Recombinant human IL-l@ with a specific activity X0* thymocyte mitogenesis U/mg protein was used (13). Doses of IL-la, IL-lp, ACTH (PACT 100, Bachem), propranolol hydrochloride (P 0884, Sigma), phentolamine (P 7547, Sigma), and (-)-epinephrine (E 4250, Sigma) were prepared in sterile PBS containing 0.1% BSA. Adrenal organ cultures and primary adrenal cells were incubated with the appropriate dose of each substance in a total volume of 0.5 ml. Corticosterone assay. Corticosterone levels were determined by a competitive protein-binding assay (16). Monkey plasma was the source of the binding protein. [3H]corticosterone (catalog no. NET-399, New England Nuclear) was used as the radioactive tracer, and corticosterone (C 2505, Sigma) was used as the corticosterone standard. The sensitivity of the corticosterone assay is 0.2 pg/tube. Analyses of biogenic amines. The high-performance liquid chromatography-electrochemical detection (HPLC-EC) system consisted of a pump (model 110, Beckman), sample injector (model 210, Beckman), microsorb reverse-phase short column (Cl8 3 pm, Rainin) connected in series with glassy carbon electrode (Bioanalytical Systems), amperometric detector (BAS LC-4B, Bioanalytical Systems), and integrator (model 3390A, Hewlett-Packard). Biogenic amines were fractionated over the microsorb reverse-phase column at a flow rate of 1.5 ml/min. The mobile phase formulation was as follows: 0.01 M NaOH, 0.07 M citric acid, 1.6% (wt/vol) disodium EDTA, 0.033% (wt/ vol) sodium dodecyl sulfate (SDS), 0.425% (vol/vol) diethanolamine, and 10% (vol/vol) acetonitrile, with the pH adjusted to 3.15. The working electrode potential was set at +0.7 V vs. an Ag-AgCl reference electrode. The internal standard used was 3,4-dihydroxybenzylamine (DHBA). Further details of this HPLC-EC procedure have been published (14). Statistica analysis. Data are expressed as percent increase (1 equals basal) to correct for variability between assay plates,
CATECHOLAMINES
and to allow for direct comparisons of dataobtained from quartered adrenals and primary adrenal cells. Analysis of variance was performed to determine differences between treatments. Duncan’s multiple range test was used to compare the means. Significance was assumed when P < 0.05. RESULTS
Quartered adrenal glands were used to determine the effect of IL-l on corticosterone release in freshly prepared adrenal tissues. The time course of corticosterone release from quartered adrenals incubated with IL-l@ (10B8 M) or ACTH (10B8 M) for 15 min to 24 h was rapid (Fig. 1). Corticosterone levels were significantly (P < 0.05) elevated after 30 min and plateaued at 2 h. Dose-dependent increases (P < 0.05) in corticosterone concentration were observed when quartered adrenals were incubated with different doses (lo-l2 to 10B8 M) of ACTH or IL-l/? (Fig. 2). The least effective concentration of ACTH and IL-l@ was 10-l’ M. The ability of IL-l to stimulate corticosterone release from primary adrenal cells was determined. Five to seven days after preparation, the primary adrenal cells were challenged with a 10 -8 M dose of either IL-k, IL-l& or ACTH. After a 4-h incubation period, ACTH significantly (P < 0.05) elevated corticosterone levels, while IL-1 had no effect at this time point (Fig. 3). After a 24-h incubation period, IL-la, IL-l& and ACTH significantly (P < 0.05) increased corticosterone
release. Because con-
sistently elevated corticosterone concentrations occurred at 24 h, this time point was chosen for experiments with
primary adrenalcells
To examine whether catecholamines were involved in IL- 1 -induced corticosterone release, both cell preparations were treated with IL-1 in the presence and absence of the a- and ,&catecholamine receptor antagonists phentolamine and propranolol, respectively. Significantly 5
4
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* *
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1
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~. 4 -, mg. 1. xlme course of corticosterone release from adrenal organ cultures incubated with equivalent doses ( lOBa M) of ACTH or IL-l& Each point represents mean value of 4-8 cultures; vertical bar represents SD. *Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline values for controls: time 0, 6.36 k 1.27 (SD) ngaml-l .mg-l, n = 6; 0.25 h, 8.89 I~I1.94; 0.5 h, 10.21 zk 0.99; 1 h, 13.16 t 1.12; 2 h, 14.88 zk 1.28; 4 h, 16.89 k 1.58; and 24 h, 19.53 t 1.82.
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ble for IL-1 action, similar experiments were repeated with either phentolamine or propranolol. Phentolamine prevented (P c 0.05)the IL-la-induced rise in corticosterone concentration (Fig. 4). In contrast, propranolol did not change the significantly (P < 0.05) elevated corticosterone levels observed with IL-la. Because catecholamine involvement was suggested in IL- 1-induced corticosterone secretion, epinephrine concentrations were measured in the supernatant of primary adrenal cells. After a 24-h incubation period, both IL-la! and IL-l/? significantly elevated (P < 0.05) epinephrine levels compared with media-treated controls (Fig. 6). IL-1 did not stimulate epinephrine release after a 4-h incubation period.
ACTH IL-16
DISCUSSION .OOl
.Ol Dose
1 .o
10
(nW)
Fig. 2. Corticosterone release from adrenal organ cultures incubated with different doses of ACTH or IL-l@ for 4 h. Each point represents mean of 4-7 organ cultures; vertical bar represents SD. Data are reported as percent increase, where one equals basal. *Significant (P < 0.05) differences from basal. Baseline for 4 h control, 19.15 t 3.19 (SD) ngeml-l+rng-l, n = 6.
24 lncu bati
me
(hr)
Fig. 3. Corticosterone release from primary adrenal cells incubated with 10B8 M of ACTH, IL-k, or IL-l@. Each point represents mean of 9-12 wells; vertical bar represents SD. *Significant (P < 0.05) differences from baseline. Data are reported as percent increase, where one equals basal. Baseline values for controls: 4 h, 13.77 t 1.38 (SD) rig/well, n = 10; 24 h, 13.91 2 0.11, n = 6.
(P c 0.05) elevated corticosterone levels were observed when primary adrenal cells were incubated with epinephrine ( 10H6 M), ACTH, or IL-la (Fig. 4). Simultaneous incubation with both phentolamine and propranolol together (10 -5 M each) blocked (P < 0.05) the effects of IL-l and epinephrine on corticosterone release, and had no effect on ACTH-stimulated corticosterone release. Cells treated with propranolol and phentolamine alone produced no change in corticosterone concentration above control levels. A similar response was observed in quartered adrenal glands (Fig. 5). To determine the type of adrenergic receptor responsi-
The present experiments examined the effect of catecholamines on IL- 1 -stimulated corticosterone release from the rat adrenal gland in two in vitro systems: quartered rat adrenal glands and primary cultures of dispersed adrenal cells. Using both of these cell preparations, we demonstrated that IL- 1 stimulates corticosterone release through catecholamines by activating an a-adrenergic receptor. These data confirm previous in vivo (1,19) and in vitro (1,24) observations that IL-1 acts on the adrenal gland to stimulate corticosterone secretion. Another investigator (25) observed that lymphokine-containing supernatants stimulated corticosterone release from perifused rat adrenocortical cells. Others have demonstrated that IL- 1 stimulates cortisol release from adult human (29) and bovine (30) adrenocortical cells. In contrast, it has been reported that IL-1 did not stimulate corticosterone release from human fetal adrenal cells (9) and the mouse adrenal tumor cell line Y -1 (31). We have previously shown that hypophysectomized 1 rats administered IL-l exhibited no change in plasma corticosterone levels (8). Histological examination of individual adrenal glands from these animals revealed marked atrophy. Because adrenal atrophy may have influenced the effect of IL-1 on the adrenal gland, quartered adrenal organ cultures were used. In these quartered adrenals, IL-l (lo- l1 M) did not stimulate corticosterone secretion. Because the present data shows that the least effective concentration of IL-l is 10-l* M (Fig. 2), the discrepancy between these data may be due to the difference in concentrations of IL-l used. In the experiments reported herein, the stimulatory effect of IL-l on quartered adrenals was rapid, beginning 30 min after incubation with IL-l and peaking at 2 h. In comparison, the response of primary adrenal cells to IL-l was longer, occurring 24 h later. This difference in response time may indicate that the structure of the adrenal gland may be important in the early response to IL-l. The close anatomical relationship between the adrenal medulla and the adrenal cortex suggests that these tissues may influence the function of each other. Blood is known to flow from the adrenal cortex to the adrenal medulla (26), and adrenocortical steroids reach the adrenal medulla by this route. For example, glucocorticoids from the adrenal cortex are known to influence the synthesis (32)
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Control
m byyy
ACTH IL-ICC
m
Epinephrine
Fig. 4. Effect of catecholamine antagonists on corticosterone release from primary adrenal cells incubated with ACTH (10D8 M), IL-la (10e8 M), or epinephrine (low6 M) in the presence and absence of propranolol (10V5 M) and phentolamine (10 -5 M) for 24 h. Each point represents mean of 9-12 wells; vertical bar represents SD. * Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for control, 35.00 k 3.50 (SD) rig/well, n = 12.
+ +
Propranolol Phentolamine
+
Phentolamine
and secretion (4) of catecholamines from the adrenal medulla. Conversely, acetylcholine, the major stimulator of catecholamine secretion from the adrenal medulla, has a stimulatory effect on adrenocortical function (20). It has also been shown that dopamine, originating from the adrenal medulla, regulates aldosterone secretion (11). These observations are consistent with our finding that epinephrine regulates corticosterone secretion. Our data demonstrate that catecholamines can influence the function of the adrenal cortex. Alternatively, shared innervation between the adrenal cortex and the adrenal medulla has been shown (10, 12, 15). Adrenergic (10, 23) and dopaminergic (5) receptors have been identified within the adrenal cortex. Addi-
+
Propranolol
tional evidence suggests that catecholamine stores may be localized in this part of the adrenal gland (6, 12). Substantial amounts of epinephrine and norepinephrine were reported in rat adrenal capsule and medulla (17). Andreis et al. (1) observedthat the adrenal medulla is required for IL- 1 to stimulate corticosterone secretion. The current study extends this finding by demonstrating that catecholamines are directly involved in IL-l -induced corticosterone release. Additionally, IL-l stimulation of epinephrine and corticosterone release from primary cultures of rat adrenal cells indicates that these cultures contained both adrenocortical- and epinephrine-producing cells, which may originate in the adrenal medulla. This is supported by the fact that 1) IL-l had no effect on a murine adrenocortical cell line, Y-l (31) and 2) IL-l dramatically increased catecholamine release from the murine medullary cell line
1
+ +
Propranolol Phentolamine
Fig. 5. Corticosterone release from quartered adrenals incubated with low8 M of ACTH or IL-lp in the presence and absence of propranolol (10D5 M) and phentolamine (10 -5 M) for 24 h. Each point represents mean value of 6 cultures; vertical bar represents SD. *Significant (P c 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for control, 16.60 k 3.20 (SD) ngsml-l*rng-l, n = 6.
0
4 Incubation
time
24 (hr)
Fig. 6. Epinephrine concentrations from primary adrenal cells incubated with low8 M of IL-la! or IL-10 for 4 or 24 h. Each point represents mean value of 3 wells; vertical bar represents SD. *Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for controls: 4 h, 2.47 zk 0.53 (SD) rig/well, n= 3; 24 h, 2.71 t 0.82, n = 3.
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PC-12 (unpublished data). These observations suggest that the primary target cell for IL-l is most likely an epinephrine-producing cell in the adrenal medulla. However, interactions between IL-1 and other adrenal cell types cannot be excluded. IL-l immunoreactivity has been found in chromaffin cells of the adrenal medulla, nerve fibers of the adrenal capsule, blood vessels of the adrenal gland, and the adrenal cortex of the rat (22). Activation of these IL-l stores may constitute autocrine and/or paracrine regulatory mechanisms of glucocorticoid secretion. We propose that once released, IL- 1 stimulates epinephrine secretion, which, in turn, activates glucocorticoid synthesis and secretion. The experiments reported herein demonstrate that IL- 1 stimulates epinephrine release directly from primary adrenal cells. Our data suggest that epinephrine, in turn, stimulates corticosterone release from adrenocortical cells. Catecholamines, such as epinephrine, have been shown to stimulate glucocorticoid release in vitro from primary cultures of adrenocortical cells (10, 28). In vivo studies have reported an increase in plasma glucocorticoid levels after administration of epinephrine (27). Our data suggest that the catecholamine receptor responsible for IL- 1 stimulation of corticosterone secretion is an cu-adrenergic receptor. In contrast, catecholamine stimulation of glucocorticoid release from bovine adrenocortical cells was found to be through a ,&adrenergic receptor (28). This difference in adrenergic receptor specificity may be due to experimental variation or species specificity in the mechanism of catecholamine action on rat adrenal cells. Other mechanisms for IL-l action in the adrenal gland have been proposed. IL- 1 -stimulated corticosterone release was inhibited by CRF and ACTH antagonists, suggesting an intra-adrenal CRF-ACTH system (1). IL-l has also been reported to stimulate prostaglandin production, a response that is inhibited by antagonists of prostaglandin synthesis (24, 30). The present report examined the adrenergic mechanism of IL-l action in the adrenal gland. Other factors such as prostaglandins, CRF, and ACTH may also be intracellular mediators of IL-l action in the adrenal gland. The observations presented herein indicate that IL-l stimulates epinephrine secretion, which, in turn, causes glucocorticoid release. These locally released catecholamines mediate the stimulatory effect of IL-l on corticosterone release through an a-adrenergic receptor in the rat adrenal gland. We gratefully acknowledge the generous donation of recombinant IL-la by Dr. Peter Lomedico, Dept. of Molecular Genetics, HoffmannLaRoche, Nutley, NJ; the generous donation of recombinant IL-lfl by Dr. S. Gillis, Immunex Corp., Seattle, WA; and the generous donation of rhesus monkey plasma by Dr. P. K. Sehgal, New England Regional Primate Center, Harvard Medical School, Southborough, MA. The technical assistance of Baqiyyat Ramza was greatly appreciated. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases First Award No. 1 R29-DK-41419-OlAl and Shriners Hospitals for Crippled Children Project Nos. 15854 and 15867. Present address of M. S. A. Kumar and R. K. Agarwal: Dept. of Anatomy and Cellular Biology, Tufts Univ. School of Veterinary Medicine. 200 Westboro Rd.. North Grafton. MA 01536: and of H. H. Bode:
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Prince of Wales Children’s Hospital, High St., Randwick, New South Wales 203 1, Australia. Address for reprint requests: A. R. Gwosdow, Endocrine Unit, Bulfinch 3, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. Received
12 December
1991; accepted
in final
form
9 April
1992.
REFERENCES 1. Andreis, P. G., G. Neri, A. S. Belloni, G. Mazzocchi, A. Kasprzak, and G. G. Nussdorfer. Interleukin-l@ enhances corticosterone secretion by acting directly on the rat adrenal gland. Endocrinology 129: 53-57, 1991. 2. Berkenbosch, F., J. van Oers, A. de1 Rey, F. Tilders, and H. Besedovsky. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science Wash. DC 238: 524-526, 1987. 3. Bernton, E. W., J. E. Beach, J. W. Holaday, R. C. Smallridge, and H. G. Fein. Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science Wash. DC 238: 519-521, 1987. 4. Critchley, J. A. J. H., P. Ellis, C. G. Henderson, and A. Ungar. The role of the pituitary-adrenocortical axis in reflex responses of the adrenal medulla of the dog. J. Physiol. Lond. 323: 533-541, 1982. 5. Dunn, M. G., and H. B. Bosmann. Peripheral dopamine receptor identification: properties of a specific dopamine receptor in the rat adrenal zona glomerulosa. Biochem. Biophys. Res. Commun. 99: 1081-1087, 1981. 6. Gallo-Payet, N., P. Potheir, and H. Isler. On the presence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function. Biochem. Cell Biol. 65: 588-592, 1987. U., A. 0. Chua, A. S. Stern, C. P. Hellmann, M. P. 7. Gubler, Vitek, T. M. Dechiara, W. R. Benjamin, K. J. Collier, M. Dukovich, P. C. Familletti, C. Fiedler-Nagy, J. Jenson, K. Kaffka, P. L. Kilian, D. Stremlo, B. H. Wittreich, D. Woehle, S. B. Mizel, and P. T. Lomedico. Recombinant human interleukin-lcu: purification and biological characterization. J. Immunol. 136: 2492-2497, 1986. 8. Gwosdow, A. R., M. S. A. Kumar, and H. H. Bode. Interleukin-1 stimulation of the hypothalamic-pituitary-adrenal axis. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E65-E70, 1990. 9. Harlan, C. A., and C. R. Parker. Investigation of the effect of interleukin-l@ on steroidogenesis in the human fetal adrenal gland. Steroids 56: 72-76, 1991. 10. Holzwarth, M. A., L. A. Cunningham, and N. Kleitman. The role of adrenal nerves in the regulation of adrenocortical functions. Ann. NY Acad. Sci. 512: 449-464, 1987. 11. Jungmann, E., G. German, I. Austin, B. Mack, D. StorpWenke, W. Fassbinder, U. Schwedes, K. H. Usadel, A. Encke, and K. Schoffling. The role of adrenal medulla in endogenous dopaminergic inhibition of aldosterone secretion. Res. Exp. Med. 186: 427-434, 1986. 12. Kleitman, N., and M. A. Holzwarth. Catecholaminergic innervation of the rat adrenal cortex. Cell Tissue Res. 241: 139-147, 1985. 13. Kronheim, S. R., C. J. March, S. K. Erb, P. J. Conlon, D. Y. Mochizuki, and T. P. Hopp. Human interleukin 1: purification to homogeneity. J. Exp. Med. 161: 490-502, 1985. 14. Marchand, J. E., K. Hershman, M. S. A. Kumar, M. L. Thompson, and R. M. Kream. Disulfiram administration affects substance P-like immunoreactive and monoaminergic neural systems in rodent brain. J. Biol. Chem. 265: 264-273, 1990. 15. Migally, N. The innervation of the mouse adrenal cortex. Anat. Rec. 194: 105-112, 1979. 16. Murphy, B. E. P. Some studies of protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive proteinbinding radioassay. J. Clin. Endocrinol. 27: 973-990, 1967. 17. Pratt, J. H., D. A. Turner, J. A. McAteer, andD. P. Henry. P-Adrenergic stimulation of aldosterone production by rat adrenal capsular explants. Endocrinology 117: 1189-l 194, 1985. J., and A. T. Suyama. Inhibition of repli18. Ramanchandran, cation of normal adrenocortical cells in culture by adrenocorticotropin. Proc. Natl. Acad. Sci. USA 72: 113-117, 1975.
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19. Roh, M. S., K. A. Drazenovich, J. J. Barbose, C. A. Dinarello, and C. F. Cobb. Direct stimulation of the adrenal cortex by interleukin-1. Surgery 102: 140-146, 1987. 20. Rosenfeld, G. Stimulative effect of acetylcholine on the adrenocortical function of isolated perfused calf adrenals. Am. J. Physiol. 183: 272-278, 1955. 21. Sapolsky, R., C. Rivier, G. Yamamoto, P. Plotsky, and W. Vale. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science Wash. DC 238: 522-524, 1987. 22. Schultzberg, M., C. Andersson, A. Unden, M. TroyeBlomberg, S. B. Svenson, and T. Bartfai. Interleukin-1 in adrenal chromaffin cells. Neuroscience 30: 805-810, 1989. 23. Shima, S., K. Komoriyama, M. Hirai, and H. Kouyama. Studies on cyclic nucleotides in the adrenal gland. XI. Adrenergic regulation of adenylate cyclase activity in the adrenal cortex. Endocrinology 114: 325-329, 1984. 24. Tominaga, T., J. Fukata, Y. Naito, T. Usui, N. Murakami, M. Fukushima, Y. Nakai, Y. Hirai, and H. Imura. Prostaglandin-dependent in vitro stimulation of adrenocortical steroidogenesis by interleukins. Endocrinology 128: 526-531, 1991. 25. Torres-Aleman, I., I. Barasoain, J. Borrell, and C. Guaza. Immune activation and psychoneurogenic stress modulate corticosterone-releasing effects of lymphokines and ACTH. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R839R845, 1988.
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26. Vinson, G. P., J. A. Pudney, and B. J. Whitehouse. The mammalian adrenal circulation and the relationship between adrenal blood flow and steroidogenesis. J. Endocrinol. 105: 285-294, 1985. 27. Vogt, M. Observations on some conditions affecting the rate of hormone output by the suprarenal cortex. J. Physiol. Land. 103: 317-332, 1944. 28. Walker, S. W., E. R. T. Lightly, S. W. Milner, and B. C. Williams. Catecholamine stimulation of cortisol secretion by 3-day primary cultures of purified zona fasciculata/reticularis cells isolated from bovine adrenal cortex. Mol. Cell. Endocrinol. 57: 139-147, 1988. 29. Whitcomb, R. W., W. M. Linehan, L. M. Wahl, and R. A. Knazek. Monocytes stimulate cortisol production by cultured human adrenocortical cells. J. Clin. Endocrinol. Metab. 66: 33-38, 1988. 30. Winter, J. S. D., K. W. Gow, Y. S. Perry, and A. H. Greenberg. A stimulatory effect of interleukin-1 on adrenocortical cortisol secretion mediated by prostaglandins. Endocrinology 127: 1904-1909, 1990. 31. Woloski, B. M. R. N. J., E. M. Smith, W. J. Meyer, G. M. Fuller, and J. E. Blalock. Corticotropin-releasing activity of monokines. Science Wash. DC 230: 1035-1037, 1985. 32. Wurtman, R. J., and J. Axelrod. Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticoids. Science Wash. DC 150: 1464-1465, 1965.
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(IL-l) has been shown to stimulate the secretion of corticotropin-releasing factor (CRF) from the hypothalamus (2, 2 1), adrenocorticotropic hormone (ACTH) from the pituitary gland (3) and AtT-20 cells (31), and glucocorticoids from the adrenal gland (1, 19, 24, 29, 30). In the adrenal gland, IL-l stimulates glucocorticoid release from human (29), bovine (30), and rat (1, 24) adrenocortical cells. Current data suggest several different mechanisms of IL-l action on glucocorticoid release from the adrenal gland. Involvement of prostaglandin-dependent mechanisms in IL- I -stimulated glucocorticoid secretion have been demonstrated in bovine (30) and rat (24) primary adrenocortical cells. Additionally, the secretory effect of IL-l on the adrenal gland may involve the activation of an intra-adrenal CRF-ACTH system (1). An interaction between adrenal cells is supported by the fact that the adrenal medulla is required for IL-l stimulation of corticosterone release (1). In addition, IL-1 has been found colocalized with catecholamines in INTERLEUKIN-1
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chromaffin cells of the adrenal medulla (22). Because catecholamines are stimulators of glucocorticoid secretion (27), we hypothesized that IL-l stimulates corticosterone release through these adrenal medullary hormones. The data are consistent with a major role for catecholamines in mediating the effect of IL-1 on corticosterone release through a-adrenergic receptors. MATERIALS
AND METHODS
Animals. Adult male Sprague-Dawley rats (Taconic Farms) weighing 200-250 g were used in this study. Rats were housed in standard rodent shoe box cages and fed a pelleted Chow diet (Laboratory Rodent Chow 5001, Ralston Purina). Bottles containing water were attached to each cage. Food and water were available ad libitum. The cages were housed in a controlled environmental room at the dry-bulb temperature of 24°C. A relative humidity of 50% and a 12:12-h light-dark photoperiod (light from 0700 to 1900 h) were maintained. Organ cultures. Two fresh adrenal glands were collected and prepared as previously described (8). Briefly, after removal from the animal, each adrenal gland was weighed on an analytic balance (model B6, Mettler), quartered, and preincubated individually in a 35 x 10 mm Petri dish with 1 ml of Krebs-Ringer buffer without bicarbonate containing glucose (1 mg/ml, pH 7.4; catalog no. K 4002, Sigma) and 10 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; H 0887, Sigma) at 37°C for 2.5 h. At the end of this preincubation period, the supernatant was discarded and replaced with sterile medium 199 (M-199) with Earle’s salts (catalog no. 320-1151 AJ, GIBCO) containing 25 mM HEPES, 0.1% bovine serum albumin (BSA; B 8894, Sigma), 20 pg/ml ascorbic acid, and 100 KIU/ml aprotinin (A 6279, Sigma). To this serum-free medium, the appropriate substance, such as ACTH, IL-l& or catecholamine antagonist, was added. Cells were incubated with catecholamine antagonist for 30 min before the addition of other stimulants. At the desired time after incubation, one 300~~1 aliquot was removed from each culture. For time course studies, a 50-~1 aliquot was removed from each culture and replaced with 50 ~1 of incubation medium at 0.25, 0.5, 1, 2, 4, and 24 h after incubation. The collected samples were frozen at -80°C until assayed for corticosterone concentration. The adrenal glands from each animal were paired so that one adrenal gland served as the control and the other received the experimental treatment. Corticosterone concentration from the control gland was subtracted from that of the paired treated gland before statistical analyses were performed. Corticosterone concentration was expressed per milligram of tissue. Adrenal cell cultures. Primary adrenal cells were prepared by a modification of the method previously described (18). Briefly, 30 rats were decapitated and the adrenal glands were removed from the surrounding fat. The entire adrenal gland was minced in a IO-cm Petri dish containing 20 ml of sterile M-199 with Earle’s salts. Collagenase (2 mg/ml; CLSIV, Cooper Biomedical), DNase (0.1 mg/ml; D 0876, Sigma), and trypsin inhibitor (20 mg/ml; T 9003, Sigma) were added, and the solution was
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transferred to a sterile 50-ml conical tube. The tube was capped and placed in a shaking water bath at 37°C for 20 min. After this incubation period, the tissue fragments were allowed to settle, and the medium containing dispersed cells was collected and spun, and the cell pellet was resuspended and saved on ice. The tissue fragments were dispersed mechanically by repeated aspiration with a sterile transfer pipette. The fragments were allowed to settle, and the rinse medium was replaced with sterile M-199. Each time the rinse medium was removed, it was saved on ice. This procedure was repeated until the tissue was completely dissociated. When all of the tissue was dissociated, the supernatant and saved rinse medium were combined and filtered through nylon mesh. The cells were collected by centrifugation at 1,000 revolutions/min for 12 min; resuspended in M-199 containing 10% fetal bovine serum, gentamycin (40 pg/ml), and mycostatin (20 II-J/ml); plated in 48-well plates at a density of 100,000 cells/O.5 ml in each well; and incubated at 37°C in 5% C02-95% air. The medium was replaced every 2-3 days. Five to seven days after preparation, the primary adrenal cells were used in experiments. All experiments were conducted in triplicate wells; each well received only one treatment. Each experiment was repeated at least three times. Before each experiment, the cells were rinsed with sterile phosphate-buffered saline (PBS) containing 0.1% BSA. The cells were incubated in serum-free M-199 containing 25 mM HEPES, 0.1% BSA, 20 pg/ml ascorbic acid, and 106 KIU/ml aprotinin with the desired stimulants. At the appropriate time (4, 24 h) after incubation, the 48-well plates were centrifuged at ~,~~o revolutiondmin for 5 min and a 3ohl aliquot of supernatant was collected and frozen at -80°C until assayed for hormone concentration. Test substances. Recombinant human IL-la with an activity measured by the DlO assay of 2 x 10s units/ml was used (7). Recombinant human IL-l@ with a specific activity X0* thymocyte mitogenesis U/mg protein was used (13). Doses of IL-la, IL-lp, ACTH (PACT 100, Bachem), propranolol hydrochloride (P 0884, Sigma), phentolamine (P 7547, Sigma), and (-)-epinephrine (E 4250, Sigma) were prepared in sterile PBS containing 0.1% BSA. Adrenal organ cultures and primary adrenal cells were incubated with the appropriate dose of each substance in a total volume of 0.5 ml. Corticosterone assay. Corticosterone levels were determined by a competitive protein-binding assay (16). Monkey plasma was the source of the binding protein. [3H]corticosterone (catalog no. NET-399, New England Nuclear) was used as the radioactive tracer, and corticosterone (C 2505, Sigma) was used as the corticosterone standard. The sensitivity of the corticosterone assay is 0.2 pg/tube. Analyses of biogenic amines. The high-performance liquid chromatography-electrochemical detection (HPLC-EC) system consisted of a pump (model 110, Beckman), sample injector (model 210, Beckman), microsorb reverse-phase short column (Cl8 3 pm, Rainin) connected in series with glassy carbon electrode (Bioanalytical Systems), amperometric detector (BAS LC-4B, Bioanalytical Systems), and integrator (model 3390A, Hewlett-Packard). Biogenic amines were fractionated over the microsorb reverse-phase column at a flow rate of 1.5 ml/min. The mobile phase formulation was as follows: 0.01 M NaOH, 0.07 M citric acid, 1.6% (wt/vol) disodium EDTA, 0.033% (wt/ vol) sodium dodecyl sulfate (SDS), 0.425% (vol/vol) diethanolamine, and 10% (vol/vol) acetonitrile, with the pH adjusted to 3.15. The working electrode potential was set at +0.7 V vs. an Ag-AgCl reference electrode. The internal standard used was 3,4-dihydroxybenzylamine (DHBA). Further details of this HPLC-EC procedure have been published (14). Statistica analysis. Data are expressed as percent increase (1 equals basal) to correct for variability between assay plates,
CATECHOLAMINES
and to allow for direct comparisons of dataobtained from quartered adrenals and primary adrenal cells. Analysis of variance was performed to determine differences between treatments. Duncan’s multiple range test was used to compare the means. Significance was assumed when P < 0.05. RESULTS
Quartered adrenal glands were used to determine the effect of IL-l on corticosterone release in freshly prepared adrenal tissues. The time course of corticosterone release from quartered adrenals incubated with IL-l@ (10B8 M) or ACTH (10B8 M) for 15 min to 24 h was rapid (Fig. 1). Corticosterone levels were significantly (P < 0.05) elevated after 30 min and plateaued at 2 h. Dose-dependent increases (P < 0.05) in corticosterone concentration were observed when quartered adrenals were incubated with different doses (lo-l2 to 10B8 M) of ACTH or IL-l/? (Fig. 2). The least effective concentration of ACTH and IL-l@ was 10-l’ M. The ability of IL-l to stimulate corticosterone release from primary adrenal cells was determined. Five to seven days after preparation, the primary adrenal cells were challenged with a 10 -8 M dose of either IL-k, IL-l& or ACTH. After a 4-h incubation period, ACTH significantly (P < 0.05) elevated corticosterone levels, while IL-1 had no effect at this time point (Fig. 3). After a 24-h incubation period, IL-la, IL-l& and ACTH significantly (P < 0.05) increased corticosterone
release. Because con-
sistently elevated corticosterone concentrations occurred at 24 h, this time point was chosen for experiments with
primary adrenalcells
To examine whether catecholamines were involved in IL- 1 -induced corticosterone release, both cell preparations were treated with IL-1 in the presence and absence of the a- and ,&catecholamine receptor antagonists phentolamine and propranolol, respectively. Significantly 5
4
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1
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~. 4 -, mg. 1. xlme course of corticosterone release from adrenal organ cultures incubated with equivalent doses ( lOBa M) of ACTH or IL-l& Each point represents mean value of 4-8 cultures; vertical bar represents SD. *Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline values for controls: time 0, 6.36 k 1.27 (SD) ngaml-l .mg-l, n = 6; 0.25 h, 8.89 I~I1.94; 0.5 h, 10.21 zk 0.99; 1 h, 13.16 t 1.12; 2 h, 14.88 zk 1.28; 4 h, 16.89 k 1.58; and 24 h, 19.53 t 1.82.
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ble for IL-1 action, similar experiments were repeated with either phentolamine or propranolol. Phentolamine prevented (P c 0.05)the IL-la-induced rise in corticosterone concentration (Fig. 4). In contrast, propranolol did not change the significantly (P < 0.05) elevated corticosterone levels observed with IL-la. Because catecholamine involvement was suggested in IL- 1-induced corticosterone secretion, epinephrine concentrations were measured in the supernatant of primary adrenal cells. After a 24-h incubation period, both IL-la! and IL-l/? significantly elevated (P < 0.05) epinephrine levels compared with media-treated controls (Fig. 6). IL-1 did not stimulate epinephrine release after a 4-h incubation period.
ACTH IL-16
DISCUSSION .OOl
.Ol Dose
1 .o
10
(nW)
Fig. 2. Corticosterone release from adrenal organ cultures incubated with different doses of ACTH or IL-l@ for 4 h. Each point represents mean of 4-7 organ cultures; vertical bar represents SD. Data are reported as percent increase, where one equals basal. *Significant (P < 0.05) differences from basal. Baseline for 4 h control, 19.15 t 3.19 (SD) ngeml-l+rng-l, n = 6.
24 lncu bati
me
(hr)
Fig. 3. Corticosterone release from primary adrenal cells incubated with 10B8 M of ACTH, IL-k, or IL-l@. Each point represents mean of 9-12 wells; vertical bar represents SD. *Significant (P < 0.05) differences from baseline. Data are reported as percent increase, where one equals basal. Baseline values for controls: 4 h, 13.77 t 1.38 (SD) rig/well, n = 10; 24 h, 13.91 2 0.11, n = 6.
(P c 0.05) elevated corticosterone levels were observed when primary adrenal cells were incubated with epinephrine ( 10H6 M), ACTH, or IL-la (Fig. 4). Simultaneous incubation with both phentolamine and propranolol together (10 -5 M each) blocked (P < 0.05) the effects of IL-l and epinephrine on corticosterone release, and had no effect on ACTH-stimulated corticosterone release. Cells treated with propranolol and phentolamine alone produced no change in corticosterone concentration above control levels. A similar response was observed in quartered adrenal glands (Fig. 5). To determine the type of adrenergic receptor responsi-
The present experiments examined the effect of catecholamines on IL- 1 -stimulated corticosterone release from the rat adrenal gland in two in vitro systems: quartered rat adrenal glands and primary cultures of dispersed adrenal cells. Using both of these cell preparations, we demonstrated that IL- 1 stimulates corticosterone release through catecholamines by activating an a-adrenergic receptor. These data confirm previous in vivo (1,19) and in vitro (1,24) observations that IL-1 acts on the adrenal gland to stimulate corticosterone secretion. Another investigator (25) observed that lymphokine-containing supernatants stimulated corticosterone release from perifused rat adrenocortical cells. Others have demonstrated that IL- 1 stimulates cortisol release from adult human (29) and bovine (30) adrenocortical cells. In contrast, it has been reported that IL-1 did not stimulate corticosterone release from human fetal adrenal cells (9) and the mouse adrenal tumor cell line Y -1 (31). We have previously shown that hypophysectomized 1 rats administered IL-l exhibited no change in plasma corticosterone levels (8). Histological examination of individual adrenal glands from these animals revealed marked atrophy. Because adrenal atrophy may have influenced the effect of IL-1 on the adrenal gland, quartered adrenal organ cultures were used. In these quartered adrenals, IL-l (lo- l1 M) did not stimulate corticosterone secretion. Because the present data shows that the least effective concentration of IL-l is 10-l* M (Fig. 2), the discrepancy between these data may be due to the difference in concentrations of IL-l used. In the experiments reported herein, the stimulatory effect of IL-l on quartered adrenals was rapid, beginning 30 min after incubation with IL-l and peaking at 2 h. In comparison, the response of primary adrenal cells to IL-l was longer, occurring 24 h later. This difference in response time may indicate that the structure of the adrenal gland may be important in the early response to IL-l. The close anatomical relationship between the adrenal medulla and the adrenal cortex suggests that these tissues may influence the function of each other. Blood is known to flow from the adrenal cortex to the adrenal medulla (26), and adrenocortical steroids reach the adrenal medulla by this route. For example, glucocorticoids from the adrenal cortex are known to influence the synthesis (32)
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m byyy
ACTH IL-ICC
m
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Fig. 4. Effect of catecholamine antagonists on corticosterone release from primary adrenal cells incubated with ACTH (10D8 M), IL-la (10e8 M), or epinephrine (low6 M) in the presence and absence of propranolol (10V5 M) and phentolamine (10 -5 M) for 24 h. Each point represents mean of 9-12 wells; vertical bar represents SD. * Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for control, 35.00 k 3.50 (SD) rig/well, n = 12.
+ +
Propranolol Phentolamine
+
Phentolamine
and secretion (4) of catecholamines from the adrenal medulla. Conversely, acetylcholine, the major stimulator of catecholamine secretion from the adrenal medulla, has a stimulatory effect on adrenocortical function (20). It has also been shown that dopamine, originating from the adrenal medulla, regulates aldosterone secretion (11). These observations are consistent with our finding that epinephrine regulates corticosterone secretion. Our data demonstrate that catecholamines can influence the function of the adrenal cortex. Alternatively, shared innervation between the adrenal cortex and the adrenal medulla has been shown (10, 12, 15). Adrenergic (10, 23) and dopaminergic (5) receptors have been identified within the adrenal cortex. Addi-
+
Propranolol
tional evidence suggests that catecholamine stores may be localized in this part of the adrenal gland (6, 12). Substantial amounts of epinephrine and norepinephrine were reported in rat adrenal capsule and medulla (17). Andreis et al. (1) observedthat the adrenal medulla is required for IL- 1 to stimulate corticosterone secretion. The current study extends this finding by demonstrating that catecholamines are directly involved in IL-l -induced corticosterone release. Additionally, IL-l stimulation of epinephrine and corticosterone release from primary cultures of rat adrenal cells indicates that these cultures contained both adrenocortical- and epinephrine-producing cells, which may originate in the adrenal medulla. This is supported by the fact that 1) IL-l had no effect on a murine adrenocortical cell line, Y-l (31) and 2) IL-l dramatically increased catecholamine release from the murine medullary cell line
1
+ +
Propranolol Phentolamine
Fig. 5. Corticosterone release from quartered adrenals incubated with low8 M of ACTH or IL-lp in the presence and absence of propranolol (10D5 M) and phentolamine (10 -5 M) for 24 h. Each point represents mean value of 6 cultures; vertical bar represents SD. *Significant (P c 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for control, 16.60 k 3.20 (SD) ngsml-l*rng-l, n = 6.
0
4 Incubation
time
24 (hr)
Fig. 6. Epinephrine concentrations from primary adrenal cells incubated with low8 M of IL-la! or IL-10 for 4 or 24 h. Each point represents mean value of 3 wells; vertical bar represents SD. *Significant (P < 0.05) differences from basal. Data are reported as percent increase, where one equals basal. Baseline for controls: 4 h, 2.47 zk 0.53 (SD) rig/well, n= 3; 24 h, 2.71 t 0.82, n = 3.
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PC-12 (unpublished data). These observations suggest that the primary target cell for IL-l is most likely an epinephrine-producing cell in the adrenal medulla. However, interactions between IL-1 and other adrenal cell types cannot be excluded. IL-l immunoreactivity has been found in chromaffin cells of the adrenal medulla, nerve fibers of the adrenal capsule, blood vessels of the adrenal gland, and the adrenal cortex of the rat (22). Activation of these IL-l stores may constitute autocrine and/or paracrine regulatory mechanisms of glucocorticoid secretion. We propose that once released, IL- 1 stimulates epinephrine secretion, which, in turn, activates glucocorticoid synthesis and secretion. The experiments reported herein demonstrate that IL- 1 stimulates epinephrine release directly from primary adrenal cells. Our data suggest that epinephrine, in turn, stimulates corticosterone release from adrenocortical cells. Catecholamines, such as epinephrine, have been shown to stimulate glucocorticoid release in vitro from primary cultures of adrenocortical cells (10, 28). In vivo studies have reported an increase in plasma glucocorticoid levels after administration of epinephrine (27). Our data suggest that the catecholamine receptor responsible for IL- 1 stimulation of corticosterone secretion is an cu-adrenergic receptor. In contrast, catecholamine stimulation of glucocorticoid release from bovine adrenocortical cells was found to be through a ,&adrenergic receptor (28). This difference in adrenergic receptor specificity may be due to experimental variation or species specificity in the mechanism of catecholamine action on rat adrenal cells. Other mechanisms for IL-l action in the adrenal gland have been proposed. IL- 1 -stimulated corticosterone release was inhibited by CRF and ACTH antagonists, suggesting an intra-adrenal CRF-ACTH system (1). IL-l has also been reported to stimulate prostaglandin production, a response that is inhibited by antagonists of prostaglandin synthesis (24, 30). The present report examined the adrenergic mechanism of IL-l action in the adrenal gland. Other factors such as prostaglandins, CRF, and ACTH may also be intracellular mediators of IL-l action in the adrenal gland. The observations presented herein indicate that IL-l stimulates epinephrine secretion, which, in turn, causes glucocorticoid release. These locally released catecholamines mediate the stimulatory effect of IL-l on corticosterone release through an a-adrenergic receptor in the rat adrenal gland. We gratefully acknowledge the generous donation of recombinant IL-la by Dr. Peter Lomedico, Dept. of Molecular Genetics, HoffmannLaRoche, Nutley, NJ; the generous donation of recombinant IL-lfl by Dr. S. Gillis, Immunex Corp., Seattle, WA; and the generous donation of rhesus monkey plasma by Dr. P. K. Sehgal, New England Regional Primate Center, Harvard Medical School, Southborough, MA. The technical assistance of Baqiyyat Ramza was greatly appreciated. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases First Award No. 1 R29-DK-41419-OlAl and Shriners Hospitals for Crippled Children Project Nos. 15854 and 15867. Present address of M. S. A. Kumar and R. K. Agarwal: Dept. of Anatomy and Cellular Biology, Tufts Univ. School of Veterinary Medicine. 200 Westboro Rd.. North Grafton. MA 01536: and of H. H. Bode:
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Prince of Wales Children’s Hospital, High St., Randwick, New South Wales 203 1, Australia. Address for reprint requests: A. R. Gwosdow, Endocrine Unit, Bulfinch 3, Massachusetts General Hospital, Fruit Street, Boston, MA 02114. Received
12 December
1991; accepted
in final
form
9 April
1992.
REFERENCES 1. Andreis, P. G., G. Neri, A. S. Belloni, G. Mazzocchi, A. Kasprzak, and G. G. Nussdorfer. Interleukin-l@ enhances corticosterone secretion by acting directly on the rat adrenal gland. Endocrinology 129: 53-57, 1991. 2. Berkenbosch, F., J. van Oers, A. de1 Rey, F. Tilders, and H. Besedovsky. Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science Wash. DC 238: 524-526, 1987. 3. Bernton, E. W., J. E. Beach, J. W. Holaday, R. C. Smallridge, and H. G. Fein. Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science Wash. DC 238: 519-521, 1987. 4. Critchley, J. A. J. H., P. Ellis, C. G. Henderson, and A. Ungar. The role of the pituitary-adrenocortical axis in reflex responses of the adrenal medulla of the dog. J. Physiol. Lond. 323: 533-541, 1982. 5. Dunn, M. G., and H. B. Bosmann. Peripheral dopamine receptor identification: properties of a specific dopamine receptor in the rat adrenal zona glomerulosa. Biochem. Biophys. Res. Commun. 99: 1081-1087, 1981. 6. Gallo-Payet, N., P. Potheir, and H. Isler. On the presence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function. Biochem. Cell Biol. 65: 588-592, 1987. U., A. 0. Chua, A. S. Stern, C. P. Hellmann, M. P. 7. Gubler, Vitek, T. M. Dechiara, W. R. Benjamin, K. J. Collier, M. Dukovich, P. C. Familletti, C. Fiedler-Nagy, J. Jenson, K. Kaffka, P. L. Kilian, D. Stremlo, B. H. Wittreich, D. Woehle, S. B. Mizel, and P. T. Lomedico. Recombinant human interleukin-lcu: purification and biological characterization. J. Immunol. 136: 2492-2497, 1986. 8. Gwosdow, A. R., M. S. A. Kumar, and H. H. Bode. Interleukin-1 stimulation of the hypothalamic-pituitary-adrenal axis. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E65-E70, 1990. 9. Harlan, C. A., and C. R. Parker. Investigation of the effect of interleukin-l@ on steroidogenesis in the human fetal adrenal gland. Steroids 56: 72-76, 1991. 10. Holzwarth, M. A., L. A. Cunningham, and N. Kleitman. The role of adrenal nerves in the regulation of adrenocortical functions. Ann. NY Acad. Sci. 512: 449-464, 1987. 11. Jungmann, E., G. German, I. Austin, B. Mack, D. StorpWenke, W. Fassbinder, U. Schwedes, K. H. Usadel, A. Encke, and K. Schoffling. The role of adrenal medulla in endogenous dopaminergic inhibition of aldosterone secretion. Res. Exp. Med. 186: 427-434, 1986. 12. Kleitman, N., and M. A. Holzwarth. Catecholaminergic innervation of the rat adrenal cortex. Cell Tissue Res. 241: 139-147, 1985. 13. Kronheim, S. R., C. J. March, S. K. Erb, P. J. Conlon, D. Y. Mochizuki, and T. P. Hopp. Human interleukin 1: purification to homogeneity. J. Exp. Med. 161: 490-502, 1985. 14. Marchand, J. E., K. Hershman, M. S. A. Kumar, M. L. Thompson, and R. M. Kream. Disulfiram administration affects substance P-like immunoreactive and monoaminergic neural systems in rodent brain. J. Biol. Chem. 265: 264-273, 1990. 15. Migally, N. The innervation of the mouse adrenal cortex. Anat. Rec. 194: 105-112, 1979. 16. Murphy, B. E. P. Some studies of protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive proteinbinding radioassay. J. Clin. Endocrinol. 27: 973-990, 1967. 17. Pratt, J. H., D. A. Turner, J. A. McAteer, andD. P. Henry. P-Adrenergic stimulation of aldosterone production by rat adrenal capsular explants. Endocrinology 117: 1189-l 194, 1985. J., and A. T. Suyama. Inhibition of repli18. Ramanchandran, cation of normal adrenocortical cells in culture by adrenocorticotropin. Proc. Natl. Acad. Sci. USA 72: 113-117, 1975.
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19. Roh, M. S., K. A. Drazenovich, J. J. Barbose, C. A. Dinarello, and C. F. Cobb. Direct stimulation of the adrenal cortex by interleukin-1. Surgery 102: 140-146, 1987. 20. Rosenfeld, G. Stimulative effect of acetylcholine on the adrenocortical function of isolated perfused calf adrenals. Am. J. Physiol. 183: 272-278, 1955. 21. Sapolsky, R., C. Rivier, G. Yamamoto, P. Plotsky, and W. Vale. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science Wash. DC 238: 522-524, 1987. 22. Schultzberg, M., C. Andersson, A. Unden, M. TroyeBlomberg, S. B. Svenson, and T. Bartfai. Interleukin-1 in adrenal chromaffin cells. Neuroscience 30: 805-810, 1989. 23. Shima, S., K. Komoriyama, M. Hirai, and H. Kouyama. Studies on cyclic nucleotides in the adrenal gland. XI. Adrenergic regulation of adenylate cyclase activity in the adrenal cortex. Endocrinology 114: 325-329, 1984. 24. Tominaga, T., J. Fukata, Y. Naito, T. Usui, N. Murakami, M. Fukushima, Y. Nakai, Y. Hirai, and H. Imura. Prostaglandin-dependent in vitro stimulation of adrenocortical steroidogenesis by interleukins. Endocrinology 128: 526-531, 1991. 25. Torres-Aleman, I., I. Barasoain, J. Borrell, and C. Guaza. Immune activation and psychoneurogenic stress modulate corticosterone-releasing effects of lymphokines and ACTH. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): R839R845, 1988.
CATECHOLAMINES
26. Vinson, G. P., J. A. Pudney, and B. J. Whitehouse. The mammalian adrenal circulation and the relationship between adrenal blood flow and steroidogenesis. J. Endocrinol. 105: 285-294, 1985. 27. Vogt, M. Observations on some conditions affecting the rate of hormone output by the suprarenal cortex. J. Physiol. Land. 103: 317-332, 1944. 28. Walker, S. W., E. R. T. Lightly, S. W. Milner, and B. C. Williams. Catecholamine stimulation of cortisol secretion by 3-day primary cultures of purified zona fasciculata/reticularis cells isolated from bovine adrenal cortex. Mol. Cell. Endocrinol. 57: 139-147, 1988. 29. Whitcomb, R. W., W. M. Linehan, L. M. Wahl, and R. A. Knazek. Monocytes stimulate cortisol production by cultured human adrenocortical cells. J. Clin. Endocrinol. Metab. 66: 33-38, 1988. 30. Winter, J. S. D., K. W. Gow, Y. S. Perry, and A. H. Greenberg. A stimulatory effect of interleukin-1 on adrenocortical cortisol secretion mediated by prostaglandins. Endocrinology 127: 1904-1909, 1990. 31. Woloski, B. M. R. N. J., E. M. Smith, W. J. Meyer, G. M. Fuller, and J. E. Blalock. Corticotropin-releasing activity of monokines. Science Wash. DC 230: 1035-1037, 1985. 32. Wurtman, R. J., and J. Axelrod. Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticoids. Science Wash. DC 150: 1464-1465, 1965.
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