0013-7227/90/1266-2876$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
Interactions between Tumor Necrosis Factor-a, Hypothalamic Corticotropin-Releasing Hormone, and Adrenocorticotropin Secretion in the Rat* RENATO BERNARDINI, THEMIS C. KAMILARIS, ALDO E. CALOGERO, ELIZABETH 0. JOHNSON, M. TERESA GOMEZ, PHILIP W. GOLD, AND GEORGE P. CHROUSOS Developmental Endocrinology Branch, National Institute of Child Health and Human Development (R.B., T.C.K., A.E.C., M.T.G., G.P.C.J, and the Clinical Neuroendocrinology Branch, National Institute of Mental Health (R.B., T.C.K., E.O.J., P.W.G.), National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. We studied the effects of tumor necrosis factored (TNFa), a macrophage-derived pleiotropic cytokine produced during the inflammatory/immune response, on the function of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Intravenous injections of TNFa stimulated plasma ACTH and corticosterone secretion in a dose-dependent fashion. This effect was inhibited by a rat CRH antiserum that was administered to the rats 1 h before the TNFa injections. This suggested that CRH is a major mediator of the HPA axis response to TNFa. We subsequently evaluated the ability of TNFa to influence CRH and ACTH secretion in vitro by explanted rat hypothalami in organ culture and by dispersed rat anterior pituicytes in primary culture respectively. Hypothalami were incubated for 40 min with graded concentrations of TNFa (10 pM to 1 fiM). This cytokine stimulated CRH secretion in a dose-dependent fashion, with an ECB0 of 6.7 x 10 pM (P < 0.05). Preincubation
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ACHECTIN/tumor necrosis factor-a (TNFa) is a macrophage-derived pleiotropic cytokine involved in the acute phase response to inflammatory stimuli (1, 2). It is also one of the major immune/endocrine mediators of gram-negative endotoxic shock (3) and has been proposed as a cachexia-inducing agent in experimental animals and in the course of many chronic diseases, including cancer (4-7). In addition to producing cachexia, TNFa has also been associated with a series of diverse biological effects, including bone resorption, promotion of angiogenesis, and mobilization of body fat (4, 8, 9). In cachexia-associated states, such as malnutrition, anorexia nervosa, melancholic depression, and severe physical illness, anorexia and weight loss are associated with a hyperactive hypothalamic-pituitary-adrenal Received December 18,1989. Address all correspondence and requests for reprints to: Renato Bernardini, M.D., Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892. * Presented in part at the 18th Annual Meeting of the Society for Neurosciences, Toronto, Ontario, Canada, 1988.
of hypothalamic explants with dexamethasone, indomethacin (1 nM), eicosatetraynoic acid (10 pM), or nordihydroguaiaretic acid (30 fiM) resulted in inhibition of TNFa-stimulated CRH secretion (P < 0.05). Interestingly, 4-h incubation with TNFa had no effect on ACTH secretion from rat anterior pituicytes at a concentration of 10 nM. Higher concentrations of TNFa (100 nM and 1 fiM), however, elicited a dose-dependent increase in the ACTH concentration in the medium. Our results suggest that TNFa represents one of the immune response mediators that directly or via stimulation of other cytokines act as activators of the HPA axis during immune/inflammatory reactions. This effect appears to be glucocorticoid suppressible and eicosanoid mediated. The primary site of action of TNFa appears to be the hypothalamic CRH-secreting neuron. Some pituitary and adrenal effects of TNFa, however, cannot be excluded. {Endocrinology 126: 2876-2881, 1990)
(HPA) axis, presumably the result of increased secretion of CRH by the CRH neuron (10-12). Interestingly, the latter not only stimulates ACTH and glucocorticoid secretion, but also reproduces the suppression of appetite and loss of weight of animals that are given TNFa (47). The purpose of this study was to examine the effects of TNFa on the HPA axis. We examined the ability of TNFa to stimulate plasma ACTH and corticosterone secretion in vivo and whether this effect was mediated by CRH. Since cytokines have been reported to have central nervous system effects, we studied whether TNFa could directly influence CRH secretion by explanted rat hypothalami in vitro (1-7, 13). In addition, we investigated the possibility of a direct effect of TNFa on the pituitary corticotroph, using primary cultures of rat anterior pituicytes.
Materials and Methods Male Sprague-Dawley rats from Taconic Farms (150-200 g for the in vitro experiments; 350-400 g for the in vivo experi-
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ENDOCRINE EFFECTS OF TNFa ments) were housed under standard conditions (24 ± 1 C; 12-h light-dark cycle, commercial rat chow and tap water ad libitum). Animal studies were conducted in accord with the highest standards of humane care. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Human recombinant TNFa (hrTNFa) was a gift from Dr. M. Sheperd, Genentech (South San Francisco, CA). In vivo injection of TNF-a Cannulas were prepared from 35-cm lengths of polyethylene tubing (PE50; id, 0.58 mm; od, 0.965 mm; Clay Adams, Parsippany, NJ). A 3-cm piece of Silastic tubing (id, 0.98 mm; od, 0.185 mm; Dow-Corning, Medfield, MA) was inserted under methoxyflurane inhalation anesthesia into the internal jugular vein and then implanted into the right atrium. The cannula was run under the skin of the back, externalized at the nape of the neck, and sheathed in a metal stainless steel spring coil (Alice King Chatman, Los Angeles, CA). The latter was anchored at the base of the neck and then extended through the top of the cage. The free end of the catheter, extended from the coil by approximately 5 cm, was flushed with saline solution containing 20 U/ml heparin. After cannulation, animals were allowed to awaken from anesthesia and then were individually caged in Plexiglass cages where they could move freely. Animals were divided in groups of six and given an injection of hrTNFa in saline (0, 0.5, 5, and 25 Mg/kg) through the cannula. Blood samples of 0.5 ml were withdrawn through the catheter 30 min before and 0, 5, 15, 30, 60, 90, and 120 min after hrTNFa administration. Blood volume was maintained by injecting an equal volume of sterile 0.9% saline. Blood samples were collected in prechilled tubes containing EDTA and spun at 4 C for 15 min. Plasma was separated and frozen at —80 C until assayed for ACTH and corticosterone. Explanted rat hypothalamic in vitro system A detailed description of the methods employed, including validation and quality control, has been reported (14, 15). Briefly, male Sprague-Dawley rats, weighing 200-250 g, were killed by decapitation. Brains were removed rapidly. Hypothalamic tissues, delineated by the posterior margin of the optic chiasm, the anterior margin of the mammillary bodies, and lateral hypothalamic sulci, were excised. Dorsally, the cut was performed about 3 mm from the ventral surface. The culture medium used was medium 199 with modified Earle's salt (Gibco, Grand Island, NY), containing 0.1% BSA and 20 nM bacitracin (Aldrich Chemical Co., Milwaukee, WI). The explants were preincubated overnight in a water-jacketed incubator at 37 C under a 5% CO2 atmosphere. The experimental design consisted of serial passages of the hypothalamic explants in 6 different wells (48-multiwell plates, Costar Corp., Cambridge, MA) for 20 min each. The hypothalami were placed on a 3 x 3-mm square nylon mesh grid (Small Parts, Miami, FL), which was used to transfer the explants from well to well. Hypothalami were incubated in plain medium in the first 3 wells. The mean immunoreactive CRH (iCRH) secretion of these wells was used as basal secretion for a given hypothalamus. In the fourth and fifth wells the hypothalami were exposed to vehicle or graded concentrations of TNFa, and the mean iCRH secretion in these wells was used as stimulated
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iCRH secretion for a given hypothalamus. In the sixth and last well, each hypothalamus used was exposed to 60 mM potassium chloride (KC1) to test tissue responsiveness to membrane depolarization. In a second set of experiments, indomethacin (INDO; 1 MM), eicosatetraynoic acid (ETYA; Cayman Chemicals, Ann Arbor, MI; 10 nM), and nordihydroguaiaretic acid (NDGA; 30 ^M) were added 1 h before and throughout the experiment in which the hypothalami were stimulated in the fourth and fifth wells with 1 nM hrTNFa. After the experiment, media were frozen at —20 C until assayed for CRH. Primary cultures of rat pituitary Pituitaries were removed with sterile procedure from the sella turcica of decapitated rats and placed in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 0.3% collagenase, 0.1% hyaluronidase, 0.1% DNAase (Copper, Malvern, PA), and 0.5% BSA in a shaking bath at 37 C for 15 min. After mechanical dispersion, pituicytes were centrifuged for 10 min at 750 rpm at room temperature, and supernatants were collected. This procedure was repeated on the resultant pellet. Supernatants were then pooled, washed twice, and resuspended in 10% fetal calf serum (Gibco) DMEM. Combined cell suspension was counted, plated 1-1.5 x 105 cells/ml-well in a 48-well plastic plate (Costar), and placed in an incubator (37 C; 7.5% CO2 atmosphere). The trypan blue exclusion test was performed on a 50-^1 sample of cells. Typically, more than 98% of the pituicytes were viable. After 48 h, fresh medium was added to the cultures. Twenty-four hours later, graded concentrations of CRH (1 nM to 10 pM) or hrTNFa (10 pM to 1 juM) were prepared in fetal calf serum-free DMEM and added to the cultures. Four hours later, media were collected and frozen at —80 C until assayed for ACTH. All experiments were run at least in triplicate in two different cultures. Plasma and medium hormone measurements For measurement of plasma ACTH, 150-/xl aliquots of plasma were passed through C18 Sep-Pak cartridges (Waters Associates, Milford, MA) and washed with 0.1% trifluoroacetic acid buffer; ACTH was eluted in 5 ml of a 6:4 (vol/vol) acetonitriletrifluoroacetic acid mixture. Solvent was then evaporated in a Savant lyophilizer (Hicksville, NY), and samples were reconstituted in assay buffer. Samples were assayed for ACTH in duplicate by means of a specific RIA. The RIA procedures from this point have been described previously (16). Anti-ACTH antibody (IgG Corp., Nashville, TN) was used at a final dilution of 1:21,000, (assay volume, 300 ixi). Inter- and intraassay coefficients of variation (CVs) were 6.1% and 1.1 ± 0.2%, respectively. Plasma corticosterone was measured directly, without extraction, by RIA, using an [125I]corticosterone kit from Radioimmunoassay System Laboratories, Inc. (Carson, CA). Interand intraassay CVs were 8% and 11%, respectively. iCRH was measured in the media by means of a specific RIA, using a rabbit antirat CRH serum (TS-3) developed in our laboratory (14). TS-3 was used at a final dilution of 1:60,000 (assay volume, 300 n\). The detection limit of the assay was 20 pg/ml. Inter- and intraassay CVs were 6.5% and 4.5 ± 1.2%, respectively.
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Endo• 1990 Voll26«No6
ENDOCRINE EFFECTS OF TNFa
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Analysis of data
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In the hypothalamic explant procedure, basal iCRH secretion was the mean of the values calculated, considering the mean iCRH values of the first three wells as iCRH basal secretion. The mean iCRH values of wells 4 and 5 were considered stimulated iCRH secretion. The percent increase expressed in the figure represents the ratio between basal and stimulated iCRH secretion, according to the following formula:
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We investigated whether TNFa could also stimulate CRH secretion directly by explanted rat hypothalami. Mean (±SEM) basal secretion for all experiments was 40.5 ± 4.3 pg/ml (16.2 ± 1.7 pg/hypothalamus • 0.4 ml/20 min; n = 5 experiments; 132 measurements). The proportion of the hypothalami responding to 60 mM KC1 was 74% for the hypothalami incubated with plain medium and ranged between 74-81% for all of the other
4
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MINUTES
-300 5
Effect of TNFa on hypothalamic CRH secretion in vitro
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60
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90
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MINUTES
FIG. 2. Effect of the iv injection of a CRH antiserum (•) on TNFa (5 ^g/kg, iv)-stimulated (O) ACTH (A) and corticosterone (B) secretion in the rat. Vertical bars represent the mean ± SEM. *, P < 0.05 (by twoway analysis of variance).
groups treated with TNFa, INDO, ETYA, NDGA, or dexamethasone. TNFa caused a 10-fold increase in hypothalamic CRH secretion over baseline (P < 0.05). The stimulatory effect was observed at TNFa concentrations
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ENDOCRINE EFFECTS OF TNFa
ranging between 10 pM and 1 /xM. The EC50 was 6.7 X 10 PM (Fig. 3). To investigate whether eicosanoids could be involved in TNFa-stimulated CRH secretion, we used INDO (1 MM), ETYA (10 fiM), and NDGA (30 M M). INDO, ETYA, and NDGA all blocked TNFa-stimulated CRH secretion (P < 0.05; Fig. 4A). In addition, we explored the effect of dexamethasone on the same parameter. We found that dexamethasone inhibited TNFa-stimulated CRH secretion (P < 0.05; Fig. 4B). Effect of TNFa on ACTH release by primary cultures of rat pituicytes To investigate whether the stimulating effect of TNFa on the HPA axis could be at least in part directly mediated by the pituitary corticotroph, we explored the effect of TNFa on ACTH release by primary cultures of rat pituicytes. Stimulation of ACTH release occurred only at TNFa concentrations of 100 nM and 1 yuM. On the other hand, the addition of graded concentrations of CRH to the cultures resulted in a significant increase in ACTH release in the medium (P < 0.05; Fig. 5).
[TUMOR NECROSIS FACTOR-a] (Log M)
FIG. 3. Dose-dependent effect of TNFa (10 pM to 100 nM) on rat hypothalamic CRH secretion in vitro. The dashed line intercepts the curve at the level of the EC6o value. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's multiple range test).
Z
B
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*
CTL TNF TNF INDO TNF ETYA TNF NDGA + + + INDO ETYA NDGA
CTL TNF TNF + DEX
FIG. 4. Effect of eicosanoid synthesis inhibitors INDO (1 fiM), ETYA (10 jiM), and NDGA (30 J*M) (A) and dexamethasone (DEX; 100 pM; B) on TNFa (10 nM)-induced CRH secretion. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's test). CTL, Controls.
2879 *
2000*
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800 700 600 500 400 300 200 100
*
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10nM CRH
10nM TNF-a
100nM TNF-a
TNF-a
FlG. 5. Effects of TNFa (10 pM to 1 /iM) and CRH (10 nM) on ACTH secretion by rat anterior pituicytes in primary culture. The dashed line represents basal secretion. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's multiple range test). CTL, Controls.
Discussion Intravenously injected TNFa-stimulated secretion of ACTH and corticosterone in a dose-dependent fashion. The stimulatory effect on ACTH release was completely inhibited by previous injection of CRH antiserum, suggesting that endogenous CRH serves as a mediator of this response. The hypothesis that TNFa stimulates the HPA axis by causing direct hypothalamic CRH secretion was supported, but not proved, by our in vitro studies, showing that TNFa is capable of stimulating CRH secretion by explanted rat hypothalami. Alternatively, TNFa could activate the HPA axis by stimulating local secretion of interleukin-1 (IL-1), IL-2, or IL-6 (18, 19). Differently from that to ACTH, the corticosterone response to TNFa in rats pretreated with anti-CRH serum was only blunted, suggesting that TNFa may have a direct effect at the adrenal level. This hypothesis is strengthened by the prompt corticosterone response to TNFa. In light of the role that has been proposed for CRH in cachectic states, such as anorexia nervosa, malnutrition, and melancholic depression, it is of interest that a cytokine known to produce cachexia and loss of weight is at the same time able to stimulate the HPA axis (4, 11, 12). The involvement of TNFa in regulation of the HPA axis fits into the general concept that mediators of the inflammatory/immune response produce counterregulatory elevations in the secretion of glucocorticoids (13, 20, 21). The latter restrain the immune response and, consequently, prevent tissue damage (21). Dexamethasone, on the other hand, inhibited TNFa-stimulated hypothalamic CRH secretion, suggesting that the HPA axis exerts its own counterregulatory suppressive effect directly at the site of TNFa action. Interestingly, there is evidence that TNFa inhibits corticosterone secretion by
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ENDOCRINE EFFECTS OF TNFa
rat adrenocortical cells in culture (22), suggesting that there is a regulatory effect of TNFa at the level of the adrenal cortex also. A number of TNFa-induced responses are known to be associated with eicosanoid secretion (23). Not surprisingly, the TNFa-induced stimulation of hypothalamic CRH by the explanted hypothalami was inhibited by the arachidonic acid metabolism inhibitors INDO (for cyclooxygenase), NDGA (for lipoxygenases), or ETYA (for both enzymes). These results suggest that the effect of TNFa on hypothalamic CRH secretion may be mediated by cyclooxygenase (prostaglandins and thromboxanes) and, to a lesser extent, lipoxygenase (leukotrienes) metabolites. In this line, eicosanoids have been shown to influence HPA axis activity in vivo (24) as well as at the level of the hypothalamic CRH neuron in vitro (15). It is not known whether TNFa produced peripherally is able to cross the blood-brain-barrier (BBB) to exert a direct stimulatory effect on the CRH neuron. In the case of an impermeable BBB, TNFa stimulation of the HPA axis could be due to the release of CRH induced at the terminals of the CRH neurons that lie at the median eminence, a hypothalamic structure not protected by the BBB (25). It is also noteworthy that, similarly to IL-1, TNFa is produced locally in the brain by glial cells, which could act in a paracrine fashion, directly at the level of hypothalamic CRH neuron in the paraventricular nucleus (26). This is compatible with our findings, provided that peripherally administered TNFa has access to TNFa receptors in the central nervous system. TNFa had a relatively weak effect on ACTH secretion by cultured rat anterior pituitary cells. Milenkovic et al. (27), on the other hand, have shown that TNFa stimulates ACTH secretion by rat hemipituitaries cultured in vitro with considerably smaller (at least 10-fold) ED50 values compared to the effect on dispersed anterior pituicytes. This suggests that the integrity of the pituitary tissue may be crucial in attaining responsiveness to TNFa. If this hypothesis is correct, the physiological relevance of the results from the dispersed pituicyte culture might be questionable. Further studies are necessary to clarify this issue. In summary, we have shown that TNFa is a potent in vivo activator of the HPA axis. The hypothalamic CRH neuron is involved in such an activation, whereas pituitary corticotrophs and the adrenal gland may play a lesser role in the phenomenon. These findings provide further evidence that the hypothalamic CRH neuron represents a key site of interaction between the immune and central nervous systems as well as additional evidence for the pleiotropic role of TNFa during inflammatory/immune responses.
Endo • 1990 Vol 126 «No 6
Acknowledgments The authors wish to thank Dr. Andrea De Bartolomeis, Department of Psychiatry, Second Medical School (Naples, Italy), for his helpful suggestions and critiques, and Dr. M. Sheperd, Genentech Inc. (South San Francisco, CA), for the gift of the human recombinant TNFa.
References 1. Beutler B, Cerami A 1988 Tumor necrosis, cachexia, shock and inflammation: a common mediator. Annu Rev Biochem 57:505 2. Sherry B, Cerami A 1988 Cachectin/tumor necrosis factor exerts endocrine, paracrine and autocrine control of inflammatory responses. J Cell Biol 107:1269 3. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A 1987 Anti cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature (Lond) 330:662 4. Oliff A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, Socher SH 1987 Tumors secreting human TNF/ cachectin induce cachexia in mice. Cell 50:555 5. Cerami A, Ikeda Y, Trang Le N, Hotez PJ, Beutler B 1985 Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor). Immunol Lett 11:173 6. Scuderi P, Lam KS, Ryan KJ, Petersen E, Sterling KE, Finley PR, Ray GC, Sljmen DJ, Salmon SE 1986 Raised serum levels of tumor necrosis factor in parasitic infection. Lancet 2:1364 7. Vlassara H, Spiegel RJ, Doval DS, Cerami A 1986 Reduced plasma lipoprotein lipase activity in patients with malignancy associated with weight loss. Horm Metab Res 18:698 8. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR 1986 Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factor. Nature (Lond) 319:516 9. Frater-Schroeder M, Risau W, Hallmann R, Gautschi P, Bohlen B 1987 Tumor necrosis factor type a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci USA 84:5277 10. Smith SR, Bledsoe T, Chhetri MK 1975 Cortisol metabolism and the pituitary-adrenal axis in adults with protein-calorie malnutrition. J Clin Endocrinol Metab 40:43 11. Gold PW, Gwirtsman H, Avgerinos PC, Nieman LK, Gallucci WT, Kaye W, Jimerson D, Ebert M, Rittmaster R, Loriaux DL, Chrousos GP 1986 Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. Pathophysiologic mechanisms in underweight and weight-corrected patients. N Engl J Med 314:1335 12. Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellner CH, Nieman LK, Post RM, Pickar D, Gallucci WT, Avgerinos P, Paul S, Oldfield EH, Cutler GB Jr, Chrousos GP 1986 Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushing's disease. N Engl J Med 314:1329 13. Bernardini R, Calogero AE, Gold PW, Chrousos GP 1988 Hypothalamic-pituitary-adrenal axis activity during the inflammatory/ immune response. In: Maggi M, Johnston CA (eds) Horizons in Endocrinology. Serono Symp, Raven Press, New York, vol 50:197 14. Calogero AE, Bernardini R, Margioris AN, Bagdy G, Gallucci WT, Munson PJ, Tamarkin L, Tomai TP, Brady L, Gold PW, Chrousos GP 1989 Effects of serotonergic agonists and antagonists on hypothalamic corticotropin-releasing hormone secretion by explanted rat hypothalami. Peptides 10:189 15. Bernardini R, Chiarenza A, Calogero AE, Gold PW, Chrousos GP 1989 Arachidonic acid metabolites modulate rat hypothalamic corticotropin releasing hormone secretion in vitro. Neuroendocrinology 50:708 16. Chrousos GP, Schulte HM, Oldfield EH, Gold PW, Cutler Jr GB, Loriaux DL 1984 The corticotropin-releasing factor stimulation test: an aid in the evaluation of patients with Cushing's syndrome. N Engl J Med 310:622 17. DeLean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand
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ENDOCRINE EFFECTS OF TNFa
18. 19.
20. 21. 22.
assay and physiological dose-response curves. Am J Physiol 235:E97 Sapolsky R, Rivier C, Yamamoto G, Plotsky P, Vale W 1987 Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 238:522 Jablons DM, Mule JJ, Mclntosh JK, Sehgal PB, May LT, Huang CM, Rosenberg SA, Lotze MT 1989 IL-6/IFN-/3-2 as circulating hormone: induction by cytokine administration in humans. J Immunol 142:1542 Wolonsky BRMNJ, Smith EM, Meyer III WJ, Fuller GM, Blalock JE 1985 Corticotropin-releasing activity of monokines. Science 230:1035 Munck A, McGuyre PM 1986 Glucocorticoid physiology, pharmacology and stress. Adv Exp Med Biol 196:81 Brennan MJ, Betz JA, Poth M, Tumor necrosis factor inhibits ACTH-stimulated corticosterone secretion by rat adrenal cortical
23. 24. 25. 26. 27.
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cells. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 386 (Abstract 1453) Elias JA, Gustilo K, Baeder W, Freunlich B 1987 Synergistic stimulation of fibroblast prostaglandin release by recombinant interleukin-1 and tumor necrosis factor. J Immunol 138:3812 Hedge GA 1977 Roles for the prostaglandins in the regulation of anterior pituitary secretion. Life Sci 20:17 Moore RY 1978 Neuroendocrine regulation of reproduction. In: Yen SSC, Jaffe R (eds) Reproductive Endocrinology. Saunders, Philadelphia, p 3 Giulian D, Baker TJ, Shih LN, Lachman LB 1986 Interleukin 1 of the central nervous system is produced by ameboid microglia. J Exp Med 164:594 Milenkovic L, Rettori V, Snyder GD, Beutler B, McCann SM 1989 Cachectin alters anterior pituitary hormone release by a direct action in vitro. Proc Natl Acad Sci USA 86:2418
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Vol. 126, No. 6 Printed in U.S.A.
Interactions between Tumor Necrosis Factor-a, Hypothalamic Corticotropin-Releasing Hormone, and Adrenocorticotropin Secretion in the Rat* RENATO BERNARDINI, THEMIS C. KAMILARIS, ALDO E. CALOGERO, ELIZABETH 0. JOHNSON, M. TERESA GOMEZ, PHILIP W. GOLD, AND GEORGE P. CHROUSOS Developmental Endocrinology Branch, National Institute of Child Health and Human Development (R.B., T.C.K., A.E.C., M.T.G., G.P.C.J, and the Clinical Neuroendocrinology Branch, National Institute of Mental Health (R.B., T.C.K., E.O.J., P.W.G.), National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. We studied the effects of tumor necrosis factored (TNFa), a macrophage-derived pleiotropic cytokine produced during the inflammatory/immune response, on the function of the hypothalamic-pituitary-adrenal (HPA) axis of the rat. Intravenous injections of TNFa stimulated plasma ACTH and corticosterone secretion in a dose-dependent fashion. This effect was inhibited by a rat CRH antiserum that was administered to the rats 1 h before the TNFa injections. This suggested that CRH is a major mediator of the HPA axis response to TNFa. We subsequently evaluated the ability of TNFa to influence CRH and ACTH secretion in vitro by explanted rat hypothalami in organ culture and by dispersed rat anterior pituicytes in primary culture respectively. Hypothalami were incubated for 40 min with graded concentrations of TNFa (10 pM to 1 fiM). This cytokine stimulated CRH secretion in a dose-dependent fashion, with an ECB0 of 6.7 x 10 pM (P < 0.05). Preincubation
C
ACHECTIN/tumor necrosis factor-a (TNFa) is a macrophage-derived pleiotropic cytokine involved in the acute phase response to inflammatory stimuli (1, 2). It is also one of the major immune/endocrine mediators of gram-negative endotoxic shock (3) and has been proposed as a cachexia-inducing agent in experimental animals and in the course of many chronic diseases, including cancer (4-7). In addition to producing cachexia, TNFa has also been associated with a series of diverse biological effects, including bone resorption, promotion of angiogenesis, and mobilization of body fat (4, 8, 9). In cachexia-associated states, such as malnutrition, anorexia nervosa, melancholic depression, and severe physical illness, anorexia and weight loss are associated with a hyperactive hypothalamic-pituitary-adrenal Received December 18,1989. Address all correspondence and requests for reprints to: Renato Bernardini, M.D., Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, Bethesda, Maryland 20892. * Presented in part at the 18th Annual Meeting of the Society for Neurosciences, Toronto, Ontario, Canada, 1988.
of hypothalamic explants with dexamethasone, indomethacin (1 nM), eicosatetraynoic acid (10 pM), or nordihydroguaiaretic acid (30 fiM) resulted in inhibition of TNFa-stimulated CRH secretion (P < 0.05). Interestingly, 4-h incubation with TNFa had no effect on ACTH secretion from rat anterior pituicytes at a concentration of 10 nM. Higher concentrations of TNFa (100 nM and 1 fiM), however, elicited a dose-dependent increase in the ACTH concentration in the medium. Our results suggest that TNFa represents one of the immune response mediators that directly or via stimulation of other cytokines act as activators of the HPA axis during immune/inflammatory reactions. This effect appears to be glucocorticoid suppressible and eicosanoid mediated. The primary site of action of TNFa appears to be the hypothalamic CRH-secreting neuron. Some pituitary and adrenal effects of TNFa, however, cannot be excluded. {Endocrinology 126: 2876-2881, 1990)
(HPA) axis, presumably the result of increased secretion of CRH by the CRH neuron (10-12). Interestingly, the latter not only stimulates ACTH and glucocorticoid secretion, but also reproduces the suppression of appetite and loss of weight of animals that are given TNFa (47). The purpose of this study was to examine the effects of TNFa on the HPA axis. We examined the ability of TNFa to stimulate plasma ACTH and corticosterone secretion in vivo and whether this effect was mediated by CRH. Since cytokines have been reported to have central nervous system effects, we studied whether TNFa could directly influence CRH secretion by explanted rat hypothalami in vitro (1-7, 13). In addition, we investigated the possibility of a direct effect of TNFa on the pituitary corticotroph, using primary cultures of rat anterior pituicytes.
Materials and Methods Male Sprague-Dawley rats from Taconic Farms (150-200 g for the in vitro experiments; 350-400 g for the in vivo experi-
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ENDOCRINE EFFECTS OF TNFa ments) were housed under standard conditions (24 ± 1 C; 12-h light-dark cycle, commercial rat chow and tap water ad libitum). Animal studies were conducted in accord with the highest standards of humane care. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Human recombinant TNFa (hrTNFa) was a gift from Dr. M. Sheperd, Genentech (South San Francisco, CA). In vivo injection of TNF-a Cannulas were prepared from 35-cm lengths of polyethylene tubing (PE50; id, 0.58 mm; od, 0.965 mm; Clay Adams, Parsippany, NJ). A 3-cm piece of Silastic tubing (id, 0.98 mm; od, 0.185 mm; Dow-Corning, Medfield, MA) was inserted under methoxyflurane inhalation anesthesia into the internal jugular vein and then implanted into the right atrium. The cannula was run under the skin of the back, externalized at the nape of the neck, and sheathed in a metal stainless steel spring coil (Alice King Chatman, Los Angeles, CA). The latter was anchored at the base of the neck and then extended through the top of the cage. The free end of the catheter, extended from the coil by approximately 5 cm, was flushed with saline solution containing 20 U/ml heparin. After cannulation, animals were allowed to awaken from anesthesia and then were individually caged in Plexiglass cages where they could move freely. Animals were divided in groups of six and given an injection of hrTNFa in saline (0, 0.5, 5, and 25 Mg/kg) through the cannula. Blood samples of 0.5 ml were withdrawn through the catheter 30 min before and 0, 5, 15, 30, 60, 90, and 120 min after hrTNFa administration. Blood volume was maintained by injecting an equal volume of sterile 0.9% saline. Blood samples were collected in prechilled tubes containing EDTA and spun at 4 C for 15 min. Plasma was separated and frozen at —80 C until assayed for ACTH and corticosterone. Explanted rat hypothalamic in vitro system A detailed description of the methods employed, including validation and quality control, has been reported (14, 15). Briefly, male Sprague-Dawley rats, weighing 200-250 g, were killed by decapitation. Brains were removed rapidly. Hypothalamic tissues, delineated by the posterior margin of the optic chiasm, the anterior margin of the mammillary bodies, and lateral hypothalamic sulci, were excised. Dorsally, the cut was performed about 3 mm from the ventral surface. The culture medium used was medium 199 with modified Earle's salt (Gibco, Grand Island, NY), containing 0.1% BSA and 20 nM bacitracin (Aldrich Chemical Co., Milwaukee, WI). The explants were preincubated overnight in a water-jacketed incubator at 37 C under a 5% CO2 atmosphere. The experimental design consisted of serial passages of the hypothalamic explants in 6 different wells (48-multiwell plates, Costar Corp., Cambridge, MA) for 20 min each. The hypothalami were placed on a 3 x 3-mm square nylon mesh grid (Small Parts, Miami, FL), which was used to transfer the explants from well to well. Hypothalami were incubated in plain medium in the first 3 wells. The mean immunoreactive CRH (iCRH) secretion of these wells was used as basal secretion for a given hypothalamus. In the fourth and fifth wells the hypothalami were exposed to vehicle or graded concentrations of TNFa, and the mean iCRH secretion in these wells was used as stimulated
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iCRH secretion for a given hypothalamus. In the sixth and last well, each hypothalamus used was exposed to 60 mM potassium chloride (KC1) to test tissue responsiveness to membrane depolarization. In a second set of experiments, indomethacin (INDO; 1 MM), eicosatetraynoic acid (ETYA; Cayman Chemicals, Ann Arbor, MI; 10 nM), and nordihydroguaiaretic acid (NDGA; 30 ^M) were added 1 h before and throughout the experiment in which the hypothalami were stimulated in the fourth and fifth wells with 1 nM hrTNFa. After the experiment, media were frozen at —20 C until assayed for CRH. Primary cultures of rat pituitary Pituitaries were removed with sterile procedure from the sella turcica of decapitated rats and placed in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 0.3% collagenase, 0.1% hyaluronidase, 0.1% DNAase (Copper, Malvern, PA), and 0.5% BSA in a shaking bath at 37 C for 15 min. After mechanical dispersion, pituicytes were centrifuged for 10 min at 750 rpm at room temperature, and supernatants were collected. This procedure was repeated on the resultant pellet. Supernatants were then pooled, washed twice, and resuspended in 10% fetal calf serum (Gibco) DMEM. Combined cell suspension was counted, plated 1-1.5 x 105 cells/ml-well in a 48-well plastic plate (Costar), and placed in an incubator (37 C; 7.5% CO2 atmosphere). The trypan blue exclusion test was performed on a 50-^1 sample of cells. Typically, more than 98% of the pituicytes were viable. After 48 h, fresh medium was added to the cultures. Twenty-four hours later, graded concentrations of CRH (1 nM to 10 pM) or hrTNFa (10 pM to 1 juM) were prepared in fetal calf serum-free DMEM and added to the cultures. Four hours later, media were collected and frozen at —80 C until assayed for ACTH. All experiments were run at least in triplicate in two different cultures. Plasma and medium hormone measurements For measurement of plasma ACTH, 150-/xl aliquots of plasma were passed through C18 Sep-Pak cartridges (Waters Associates, Milford, MA) and washed with 0.1% trifluoroacetic acid buffer; ACTH was eluted in 5 ml of a 6:4 (vol/vol) acetonitriletrifluoroacetic acid mixture. Solvent was then evaporated in a Savant lyophilizer (Hicksville, NY), and samples were reconstituted in assay buffer. Samples were assayed for ACTH in duplicate by means of a specific RIA. The RIA procedures from this point have been described previously (16). Anti-ACTH antibody (IgG Corp., Nashville, TN) was used at a final dilution of 1:21,000, (assay volume, 300 ixi). Inter- and intraassay coefficients of variation (CVs) were 6.1% and 1.1 ± 0.2%, respectively. Plasma corticosterone was measured directly, without extraction, by RIA, using an [125I]corticosterone kit from Radioimmunoassay System Laboratories, Inc. (Carson, CA). Interand intraassay CVs were 8% and 11%, respectively. iCRH was measured in the media by means of a specific RIA, using a rabbit antirat CRH serum (TS-3) developed in our laboratory (14). TS-3 was used at a final dilution of 1:60,000 (assay volume, 300 n\). The detection limit of the assay was 20 pg/ml. Inter- and intraassay CVs were 6.5% and 4.5 ± 1.2%, respectively.
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Endo• 1990 Voll26«No6
ENDOCRINE EFFECTS OF TNFa
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Analysis of data
700 -
In the hypothalamic explant procedure, basal iCRH secretion was the mean of the values calculated, considering the mean iCRH values of the first three wells as iCRH basal secretion. The mean iCRH values of wells 4 and 5 were considered stimulated iCRH secretion. The percent increase expressed in the figure represents the ratio between basal and stimulated iCRH secretion, according to the following formula:
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We investigated whether TNFa could also stimulate CRH secretion directly by explanted rat hypothalami. Mean (±SEM) basal secretion for all experiments was 40.5 ± 4.3 pg/ml (16.2 ± 1.7 pg/hypothalamus • 0.4 ml/20 min; n = 5 experiments; 132 measurements). The proportion of the hypothalami responding to 60 mM KC1 was 74% for the hypothalami incubated with plain medium and ranged between 74-81% for all of the other
4
i
MINUTES
-300 5
Effect of TNFa on hypothalamic CRH secretion in vitro
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i
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MINUTES
FIG. 2. Effect of the iv injection of a CRH antiserum (•) on TNFa (5 ^g/kg, iv)-stimulated (O) ACTH (A) and corticosterone (B) secretion in the rat. Vertical bars represent the mean ± SEM. *, P < 0.05 (by twoway analysis of variance).
groups treated with TNFa, INDO, ETYA, NDGA, or dexamethasone. TNFa caused a 10-fold increase in hypothalamic CRH secretion over baseline (P < 0.05). The stimulatory effect was observed at TNFa concentrations
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ENDOCRINE EFFECTS OF TNFa
ranging between 10 pM and 1 /xM. The EC50 was 6.7 X 10 PM (Fig. 3). To investigate whether eicosanoids could be involved in TNFa-stimulated CRH secretion, we used INDO (1 MM), ETYA (10 fiM), and NDGA (30 M M). INDO, ETYA, and NDGA all blocked TNFa-stimulated CRH secretion (P < 0.05; Fig. 4A). In addition, we explored the effect of dexamethasone on the same parameter. We found that dexamethasone inhibited TNFa-stimulated CRH secretion (P < 0.05; Fig. 4B). Effect of TNFa on ACTH release by primary cultures of rat pituicytes To investigate whether the stimulating effect of TNFa on the HPA axis could be at least in part directly mediated by the pituitary corticotroph, we explored the effect of TNFa on ACTH release by primary cultures of rat pituicytes. Stimulation of ACTH release occurred only at TNFa concentrations of 100 nM and 1 yuM. On the other hand, the addition of graded concentrations of CRH to the cultures resulted in a significant increase in ACTH release in the medium (P < 0.05; Fig. 5).
[TUMOR NECROSIS FACTOR-a] (Log M)
FIG. 3. Dose-dependent effect of TNFa (10 pM to 100 nM) on rat hypothalamic CRH secretion in vitro. The dashed line intercepts the curve at the level of the EC6o value. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's multiple range test).
Z
B
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CTL TNF TNF INDO TNF ETYA TNF NDGA + + + INDO ETYA NDGA
CTL TNF TNF + DEX
FIG. 4. Effect of eicosanoid synthesis inhibitors INDO (1 fiM), ETYA (10 jiM), and NDGA (30 J*M) (A) and dexamethasone (DEX; 100 pM; B) on TNFa (10 nM)-induced CRH secretion. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's test). CTL, Controls.
2879 *
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10nM CRH
10nM TNF-a
100nM TNF-a
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FlG. 5. Effects of TNFa (10 pM to 1 /iM) and CRH (10 nM) on ACTH secretion by rat anterior pituicytes in primary culture. The dashed line represents basal secretion. Vertical bars represent the mean ± SEM. *, P < 0.05 (by Duncan's multiple range test). CTL, Controls.
Discussion Intravenously injected TNFa-stimulated secretion of ACTH and corticosterone in a dose-dependent fashion. The stimulatory effect on ACTH release was completely inhibited by previous injection of CRH antiserum, suggesting that endogenous CRH serves as a mediator of this response. The hypothesis that TNFa stimulates the HPA axis by causing direct hypothalamic CRH secretion was supported, but not proved, by our in vitro studies, showing that TNFa is capable of stimulating CRH secretion by explanted rat hypothalami. Alternatively, TNFa could activate the HPA axis by stimulating local secretion of interleukin-1 (IL-1), IL-2, or IL-6 (18, 19). Differently from that to ACTH, the corticosterone response to TNFa in rats pretreated with anti-CRH serum was only blunted, suggesting that TNFa may have a direct effect at the adrenal level. This hypothesis is strengthened by the prompt corticosterone response to TNFa. In light of the role that has been proposed for CRH in cachectic states, such as anorexia nervosa, malnutrition, and melancholic depression, it is of interest that a cytokine known to produce cachexia and loss of weight is at the same time able to stimulate the HPA axis (4, 11, 12). The involvement of TNFa in regulation of the HPA axis fits into the general concept that mediators of the inflammatory/immune response produce counterregulatory elevations in the secretion of glucocorticoids (13, 20, 21). The latter restrain the immune response and, consequently, prevent tissue damage (21). Dexamethasone, on the other hand, inhibited TNFa-stimulated hypothalamic CRH secretion, suggesting that the HPA axis exerts its own counterregulatory suppressive effect directly at the site of TNFa action. Interestingly, there is evidence that TNFa inhibits corticosterone secretion by
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ENDOCRINE EFFECTS OF TNFa
rat adrenocortical cells in culture (22), suggesting that there is a regulatory effect of TNFa at the level of the adrenal cortex also. A number of TNFa-induced responses are known to be associated with eicosanoid secretion (23). Not surprisingly, the TNFa-induced stimulation of hypothalamic CRH by the explanted hypothalami was inhibited by the arachidonic acid metabolism inhibitors INDO (for cyclooxygenase), NDGA (for lipoxygenases), or ETYA (for both enzymes). These results suggest that the effect of TNFa on hypothalamic CRH secretion may be mediated by cyclooxygenase (prostaglandins and thromboxanes) and, to a lesser extent, lipoxygenase (leukotrienes) metabolites. In this line, eicosanoids have been shown to influence HPA axis activity in vivo (24) as well as at the level of the hypothalamic CRH neuron in vitro (15). It is not known whether TNFa produced peripherally is able to cross the blood-brain-barrier (BBB) to exert a direct stimulatory effect on the CRH neuron. In the case of an impermeable BBB, TNFa stimulation of the HPA axis could be due to the release of CRH induced at the terminals of the CRH neurons that lie at the median eminence, a hypothalamic structure not protected by the BBB (25). It is also noteworthy that, similarly to IL-1, TNFa is produced locally in the brain by glial cells, which could act in a paracrine fashion, directly at the level of hypothalamic CRH neuron in the paraventricular nucleus (26). This is compatible with our findings, provided that peripherally administered TNFa has access to TNFa receptors in the central nervous system. TNFa had a relatively weak effect on ACTH secretion by cultured rat anterior pituitary cells. Milenkovic et al. (27), on the other hand, have shown that TNFa stimulates ACTH secretion by rat hemipituitaries cultured in vitro with considerably smaller (at least 10-fold) ED50 values compared to the effect on dispersed anterior pituicytes. This suggests that the integrity of the pituitary tissue may be crucial in attaining responsiveness to TNFa. If this hypothesis is correct, the physiological relevance of the results from the dispersed pituicyte culture might be questionable. Further studies are necessary to clarify this issue. In summary, we have shown that TNFa is a potent in vivo activator of the HPA axis. The hypothalamic CRH neuron is involved in such an activation, whereas pituitary corticotrophs and the adrenal gland may play a lesser role in the phenomenon. These findings provide further evidence that the hypothalamic CRH neuron represents a key site of interaction between the immune and central nervous systems as well as additional evidence for the pleiotropic role of TNFa during inflammatory/immune responses.
Endo • 1990 Vol 126 «No 6
Acknowledgments The authors wish to thank Dr. Andrea De Bartolomeis, Department of Psychiatry, Second Medical School (Naples, Italy), for his helpful suggestions and critiques, and Dr. M. Sheperd, Genentech Inc. (South San Francisco, CA), for the gift of the human recombinant TNFa.
References 1. Beutler B, Cerami A 1988 Tumor necrosis, cachexia, shock and inflammation: a common mediator. Annu Rev Biochem 57:505 2. Sherry B, Cerami A 1988 Cachectin/tumor necrosis factor exerts endocrine, paracrine and autocrine control of inflammatory responses. J Cell Biol 107:1269 3. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A 1987 Anti cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia. Nature (Lond) 330:662 4. Oliff A, Defeo-Jones D, Boyer M, Martinez D, Kiefer D, Vuocolo G, Wolfe A, Socher SH 1987 Tumors secreting human TNF/ cachectin induce cachexia in mice. Cell 50:555 5. Cerami A, Ikeda Y, Trang Le N, Hotez PJ, Beutler B 1985 Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: the role of cachectin (tumor necrosis factor). Immunol Lett 11:173 6. Scuderi P, Lam KS, Ryan KJ, Petersen E, Sterling KE, Finley PR, Ray GC, Sljmen DJ, Salmon SE 1986 Raised serum levels of tumor necrosis factor in parasitic infection. Lancet 2:1364 7. Vlassara H, Spiegel RJ, Doval DS, Cerami A 1986 Reduced plasma lipoprotein lipase activity in patients with malignancy associated with weight loss. Horm Metab Res 18:698 8. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR 1986 Stimulation of bone resorption and inhibition of bone formation in vitro by human tumor necrosis factor. Nature (Lond) 319:516 9. Frater-Schroeder M, Risau W, Hallmann R, Gautschi P, Bohlen B 1987 Tumor necrosis factor type a, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo. Proc Natl Acad Sci USA 84:5277 10. Smith SR, Bledsoe T, Chhetri MK 1975 Cortisol metabolism and the pituitary-adrenal axis in adults with protein-calorie malnutrition. J Clin Endocrinol Metab 40:43 11. Gold PW, Gwirtsman H, Avgerinos PC, Nieman LK, Gallucci WT, Kaye W, Jimerson D, Ebert M, Rittmaster R, Loriaux DL, Chrousos GP 1986 Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. Pathophysiologic mechanisms in underweight and weight-corrected patients. N Engl J Med 314:1335 12. Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellner CH, Nieman LK, Post RM, Pickar D, Gallucci WT, Avgerinos P, Paul S, Oldfield EH, Cutler GB Jr, Chrousos GP 1986 Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushing's disease. N Engl J Med 314:1329 13. Bernardini R, Calogero AE, Gold PW, Chrousos GP 1988 Hypothalamic-pituitary-adrenal axis activity during the inflammatory/ immune response. In: Maggi M, Johnston CA (eds) Horizons in Endocrinology. Serono Symp, Raven Press, New York, vol 50:197 14. Calogero AE, Bernardini R, Margioris AN, Bagdy G, Gallucci WT, Munson PJ, Tamarkin L, Tomai TP, Brady L, Gold PW, Chrousos GP 1989 Effects of serotonergic agonists and antagonists on hypothalamic corticotropin-releasing hormone secretion by explanted rat hypothalami. Peptides 10:189 15. Bernardini R, Chiarenza A, Calogero AE, Gold PW, Chrousos GP 1989 Arachidonic acid metabolites modulate rat hypothalamic corticotropin releasing hormone secretion in vitro. Neuroendocrinology 50:708 16. Chrousos GP, Schulte HM, Oldfield EH, Gold PW, Cutler Jr GB, Loriaux DL 1984 The corticotropin-releasing factor stimulation test: an aid in the evaluation of patients with Cushing's syndrome. N Engl J Med 310:622 17. DeLean A, Munson PJ, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand
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ENDOCRINE EFFECTS OF TNFa
18. 19.
20. 21. 22.
assay and physiological dose-response curves. Am J Physiol 235:E97 Sapolsky R, Rivier C, Yamamoto G, Plotsky P, Vale W 1987 Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 238:522 Jablons DM, Mule JJ, Mclntosh JK, Sehgal PB, May LT, Huang CM, Rosenberg SA, Lotze MT 1989 IL-6/IFN-/3-2 as circulating hormone: induction by cytokine administration in humans. J Immunol 142:1542 Wolonsky BRMNJ, Smith EM, Meyer III WJ, Fuller GM, Blalock JE 1985 Corticotropin-releasing activity of monokines. Science 230:1035 Munck A, McGuyre PM 1986 Glucocorticoid physiology, pharmacology and stress. Adv Exp Med Biol 196:81 Brennan MJ, Betz JA, Poth M, Tumor necrosis factor inhibits ACTH-stimulated corticosterone secretion by rat adrenal cortical
23. 24. 25. 26. 27.
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cells. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 386 (Abstract 1453) Elias JA, Gustilo K, Baeder W, Freunlich B 1987 Synergistic stimulation of fibroblast prostaglandin release by recombinant interleukin-1 and tumor necrosis factor. J Immunol 138:3812 Hedge GA 1977 Roles for the prostaglandins in the regulation of anterior pituitary secretion. Life Sci 20:17 Moore RY 1978 Neuroendocrine regulation of reproduction. In: Yen SSC, Jaffe R (eds) Reproductive Endocrinology. Saunders, Philadelphia, p 3 Giulian D, Baker TJ, Shih LN, Lachman LB 1986 Interleukin 1 of the central nervous system is produced by ameboid microglia. J Exp Med 164:594 Milenkovic L, Rettori V, Snyder GD, Beutler B, McCann SM 1989 Cachectin alters anterior pituitary hormone release by a direct action in vitro. Proc Natl Acad Sci USA 86:2418
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