0013-7227/90/1266-3053$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 126, No. 6 Printed in U.S.A.
Intrinsic Pituitary Interleukin-1/? Is Induced by Bacterial Lipopolysaccharide* JAMES I. KOENIGf, KENNETH SNOW, BURTON D. CLARK*, ROBERTO TONI, JOSEPH G. CANNON, ALAN R. SHAW, CHARLES A. DINARELLO, SEYMOUR REICHLIN, STEPHANIE L. LEE§, AND RONALD M. LECHAN Department of Medicine, Divisions of Endocrinology and Infectious Disease, New England Medical Center Hospitals, Boston, Massachusetts 02111; and Glaxo Institute for Molecular Biology (A.R.S.), Geneva, Switzerland
ABSTRACT. Using a specific antiserum recognizing recombinant rat interleukin-1/3 (IL-1/3), immunoreactive material was localized to cytoplasmic granules in anterior pituitary endocrine cells and colocalized with TSH in thyrotropes. Authenticity was established by Northern blot hybridization using a specific rat IL-1/3 cRNA probe, revealing a 1.8-kilobase mRNA identical to
that in the spleen. The marked increase in anterior pituitary IL1/3 message after the administration of bacterial lipopolysaccharide, raises the possibility that IL-1/3 may be involved in paracrine or autocrine regulation of pituitary function during infectious challenge. (Endocrinology 126: 3053-3058, 1990)
I
NTERLEUKIN-1/3 (IL-1/3), a cytokine released by activated macrophages, monocytes, and several other cell types (1), stimulates the secretion of ACTH (2, 3) and inhibits the secretion of GH, LH, and TSH in the intact rat (4, 5). Physiological experiments suggest that these effects may be exerted directly on the pituitary (610) or indirectly through the hypothalamus (11-17). Recent neuroanatomical studies by Breder et al. (18) showing the presence of immunoreactive (IR) IL-1/3 neuronal processes in the human hypothalamus and the finding of an extensive IR IL-l/3-positive neuronal system in the rat brain (19) suggest that IL-1/3 may be a hypophysiotropic factor. As a number of neuropeptides known to influence anterior pituitary secretion, such as vasoactive intestinal peptide, substance-P, neuropeptide-Y, and galanin (20-23), are synthesized by pituitary cells, and IL6, a cytokine which influences the secretion of several pituitary hormones, is localized to pituitary folliculostellate cells (24, 25), the possibility was considered that IL-1/3 might also be secreted by pituitary cells and form
part of an intrinsic paracrine control system. To determine whether IL-1/3 is an intrinsic pituitary secretory product, immunocytochemical staining and molecular hybridization of the rat pituitary gland were carried out using specific antisera and molecular probes for rat IL1/3. The response of pituitary IL-1/3 to treatment with bacterial lipopolysaccharide (LPS), a pyrogen known to activate monocyte IL-1/3 synthesis (26, 27), was also determined. Materials and Methods Animals and tissue preparation
Received January 3,1990. Address requests for reprints to: Ronald M. Lechan, M.D., Ph.D, Department of Medicine, Endocrinology Division, New England Medical Center Hospitals, Box 268, 750 Washington Street, Boston, Massachusetts 02111. * This work was supported by NIH Grants DK-37021, DK-16684, and AI-15614. f Current address: Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, D.C. t Recipient of a grant from the National Arthritis Foundation. § Recipient of a Charles A. King Trust Fellowship.
The pituitaries of normal male Sprague-Dawley rats were fixed by intracardiac perfusion with Bouin's solution for light microscopy or 4% paraformaldehyde-0.2% glutaraldehyde in 0.1 M PBS, pH 7.6, for electron microscopy and prepared for immunocytochemistry by the avidin-biotin-peroxidase complex technique (28). A second group of animals injected 4 h before study with normal saline or LPS (E. coli 055:B5 LPS; 25 mg/ kg, ip) were anesthetized with pentobarbital and, after clamping the descending aorta, perfused through the ascending aorta with 60 ml ice-cold saline to free the anterior pituitary of circulating blood products in preparation for Northern blot hybridizations. The pituitaries were rapidly removed, separated from the posterior pituitary and intermediate lobe, and frozen on dry ice. Sections of spleen, an organ that can be induced to synthesize an abundance of IL-1/3 message, and cerebral cortex were similarly prepared as positive and negative controls, respectively.
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IL-1/3 IN ANTERIOR PITUITARY
3054 Antiserum preparation and characterization
Antiserum to recombinant rat IL-1/3 was raised in New Zealand White rabbits, immunized with 100 ng recombinant rat IL-1/3 (Glaxo, Geneva, Switzerland), emulsified in complete Freund's adjuvant, and boosted at monthly intervals using incomplete adjuvant. After 12 weeks, one antiserum was demonstrated to recognize recombinant rat IL-1/3 by Western blot analysis at a titer of 1:2000 after resolving 50 ng recombinant rat IL-1/3 on a 15% sodium dodecyl sulfate-polyacrylamide gel (19). This antiserum was used at a titer of 1:750, diluted in 0.05 M Tris-buffered saline, pH 7.6, containing 0.5% Triton X-100 for light microscopic immunocytochemical studies and 0.02% Triton X-100 for ultrastructural studies. Immunocytochemical procedure To determine whether IR IL-1/3 was contained in one or more of the classical anterior pituitary cell types, serial 3-^m sections were reacted with antiserum to IL-1/3 (1:750) or with antiserum to PRL, GH, /3-lipotropin, LH, or TSH (1:750), respectively, using antisera from the National Pituitary Hormone Distribution Program of the NIH, diluted in 0.05 M Trisbuffered saline containing 0.3% Triton X-100. Identical regions in the serial tissue sections were photographed, and areas of IL-1/3 and anterior pituitary hormone immunoreactivity were compared by visual inspection. Ultrastructural analysis Tissues prepared for ultrastructural analysis were developed with 0.02% diaminobenzidine (DAB)-0.05% nickel ammonium sulfate, activated by 0.02% hydrogen peroxide to create an electron-dense immunoreaction product. Tissues were stained en bloc with an aqueous solution of 1% osmium tetraoxide-1.5% potassium ferrocyanide, followed by 1% uranyl acetate in 0.05 N maleate buffer, pH 6.0, and 1% tannic acid in 0.05 M cacodylate buffer, pH 7.4. After dehydration in ethanols and propylene oxide, the tissues were flat embedded in Epon 812Araldite 6005-DDSA (1:1:2.4) and polymerized with 2.4% DMP-30 (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin sections were cut on an MT-2000 ultramicrotome and examined under a Philips CM-10 transmission microscope (Philips Electronic Instruments, Mahwah, NJ).
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hybridization solution recommended by the manufacturer containing 10 millon cpm/ml [32P]IL-l/3 antisense RNA probe. After washing the blot in 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) containing 0.1% sodium dodecyl sulfate at 68 C, the membrane was exposed to Kodak XAR5 film at —70 C and developed after 1-7 days. The size of hybridized bands was estimated by comparison to a RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD). To demonstrate the specificity of the IL-1/3 mRNA response to LPS, the blots were reprobed with 10 million cpm/ml 32Plabeled /3-actin antisense probe after eluting the first radiolabeled probe by washing three times in 50% formamide-6 X SSPE (1 x SSPE = 0.18 M NaCl, 0.01 M sodium phosphate pH 7.7, 1 mM EDTA) at 65 C for 1-3 h. The adequacy of the elution of the previous probe was assessed by reexposure of the autoradiogram. After all counts were removed, the blots were prehybridized and hybridized following methods described above. Relative densities of hybridizing bands in the autoradiographs were determined by semiquantitative computer image analysis using Image (version 1.17) obtained from the NIH. Autoradiographs were uniformily illuminated using a fluorescent light source (Northern Light Precision Illiminator model B90, Imaging Research, Inc., St. Catherines, Ontario, Canada) and digitized with a MOS-5300 CCD video camera (General Imaging Corp., Gainesville, FL) and Quick Capture frame grabber board and software (Data Translation, Marlboro, MA) to a 512 X 512 pixel array. The integrated density of each hybridized band was analyzed using a Macintosh II computer. Antisense RNA probes A 216-basepair Pstl fragment of rat IL-1/3 cDNA was used as a template for the synthesis of the single stranded antisense hybridization probe. This sequence encodes the amino acid residues 125-196 of rat pro-IL-1/3, as described by Nishida et al. (29), and is directed toward the amino-terminal end of the mature IL-1/3 peptide. The construct was linearized by restriction with EcoRI to form the template for synthesis of 32Plabeled antisense RNA by SP6 polymerase. A chicken /3-actin (30) cDNA template was provided as a gift by Dr. Donna Chikaraishi. The /3-actin construction was linearized by BamHI before synthesis of 32P-labeled antisense RNA probes with T7 polymerase.
RNA analysis Total RNA was isolated by guanidine isothiocyanate lysis and centrifugation through a CsCl gradient. The RNA content was estimated by visual inspection of a nondenaturing ethidium bromide-1% agarose gel under UV illumination. The RNA was poly(A)+ selected on oligo-dT cellulose (Collaborative Research, Waltham, MA) columns or with Hybond paper (Amersham, Arlington Heights, IL) following the manufacturer's protocol. The purified mRNA was quantified by spectrophotometry, and equal amounts from control and LPS-treated tissues were size-fractionated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde. RNA was electrophoretically transferred to nylon membrane (Nytran, Schleicher and Schuell, Inc., Keene, NH) overnight and hybridized at 68 C in
Results IL-1/3 immunoreactivity was localized to polygonal and stellate cells with abundant cytoplasm, distributed singly and in clusters throughout the anterior pituitary, but was particularly prominent in anterior-ventral areas and adjacent to the intermediate lobe (Fig. 1, A and C). No immunoreaction product was present after immunoadsorption with recombinant rat IL-1/3, establishing the specificity of the immunostaining (Fig. IB). In serial tissue sections, a subpopulation of cells containing IL1/3 was also noted to be immunoreactive for TSH, located primarily in anterior-ventral areas (Fig. 1, C and D), but
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IL-1/3 IN ANTERIOR PITUITARY
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FlG. 1. A, Coronal sections through the rat anterior pituitary showing the presence of rat IL-1/3 (ABC technique) in numerous cells throughout the gland. Two prominent collections of cells (arrowheads) are seen in the antero-ventral portion and adjacent to the intermediate lobe (NIL). B, Reaction product is completely abolished when the antiserum is preadsorbed with 10~6 M recombinant rat IL-1/3 (Glaxo). C and D, Immunostaining of serial 3-^m sections of rat anterior pituitary with antiserum to recombinant rat IL-10 (1:750; C) and rat TSH (1:750; D). Arrows denote doubly immunolabeled cells. Original magnification, X79 in A and B and X500 in C and D.
many IR IL-1/3 and TSH cells were only singly labeled, particularly in central areas of the anterior pituitary. No other anterior pituitary hormone tested colocalized with IR IL-1/3. Ultrastructurally, immunoreactive IL-1/3 was associated with cytoplasmic granules measuring approximately 140 nm, located near the plasma membrane and extending into cytoplasmic processes (Fig. 2). To further establish the authenticity of anterior pituitary IR IL-1/3 and to determine whether rat IL-1/3 arises directly as a translation of anterior pituitary cells, Northern hybridizations were performed in three separate experiments. A single hybridizing band of approximately 1.8 kb in length, identical to that present in the spleen, was detectable in the normal pituitary, but only after loading large amounts of mRNA on the gel (Fig. 3A). In LPS-treated animals, however, IL-1/3 message was readily detected and was 15-fold more abundant than in the normal controls, as determined from integrated density measurements. In contrast, after reprobing the same blot with a radiolabeled /3-actin cRNA, fairly uniform hybridization densities were seen in the paired samples (Fig. 3B). No hybridization was present in RNA prepared from the posterior pituitary/intermediate lobe or the cerebral cortex obtained from control or LPS-treated rats.
Discussion These studies demonstrate that IL-1/3 is synthesized as an intrinsic product of the rat anterior pituitary gland. Its authenticity is established by the presence of a 1.8kb mRNA by Northern hybridization, identical to IL-1/3 mRNA extracted from rat spleen. A similar size RNA has been found in rat macrophages (31) and contrasts to the 2.2-kb mRNA of IL-la, a cytokine that shares bio-
logical activities with IL-1/3 (32). The morphological appearance of immunoreactive cells as polygonal and stellate shaped characterizes them as belonging to endocrine cells of the anterior pituitary, rather than endothelial cells or tissue macrophages, which are known peripheral sources of IL-1/3 (1). In addition, the presence of typical secretory granules in these cells, containing the majority of the immunoreactive material, distinguishes them from folliculo-stellate cells of the anterior pituitary, previously shown to produce and secrete IL-6 (24, 25). This distinct subcellular compartmentalization suggests that the process of intracellular transport and secretion of anterior pituitary IL-1/3 may differ from the mechanisms proposed for monocytes and fibroblasts (3335). The presence of IL-1/3 in the pituitary raises the possibility that it may have a physiological role in the control of anterior pituitary hormone secretion, either directly or by inducing the secretion of IL-6 from anterior pituitary folliculo-stellate cells (24, 25). Since the original observation by Wexler et al. (36) that bacterial endotoxin stimulates the pituitary-adrenal axis, numerous reports have now established potent effects of recombinant IL-1 on ACTH secretion (2, 6-17). Although most indicate that this may be primarily due to central actions of IL1/3 to stimulate CRH (11-17), direct effects on anterior pituitary corticotrophs have also been demonstrated (610). Furthermore, as IR IL-1/3 cells tend to cluster in the anterior-ventral area of the pituitary adjacent to LHand FSH-containing cells, a paracrine mechanism for control of gonadotropin secretion could also be proposed. LH secretion is stimulated from pituitary cells in culture after treatment with IL-1/3, but is reported to be inhibited after intraventricular administration (5). The latter re-
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IL-1/3 IN ANTERIOR PITUITARY
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A B
CD
4.4
B FIG. 3. A, Northern blot of IL-1/8 poly(A)+ mRNA extracted from the anterior pituitary (lanes A, B, E, and F) and spleen (lanes C and D) obtained from untreated and LPS-treated rats. A hybridizing band of identical size (~1.8 kb) is present in both spleen and pituitary. IL-1/3 mRNA in control pituitaries is seen after loading high concentrations of total message (lane E), but is markedly increased by LPS treatment (lanes B and F) and readily visable after loading smaller amounts of message (lane B). B, Same Northern blot as in A, but completely eluted of radioactivity and reprobed for /3-actin mRNA. Note the similar hybridization densities of the 2.2-kb band for each paired samples. Concentrations of poly(A)+ message per lane are 2 ng in A and B, 0.5 fig in C and D, and 10 jtg in E and F (bubble artifact in lane F).
FlG. 2. Electron micrographs showing the distribution of IR IL-1/3 in the rat anterior pituitary gland. A, Low power magnification of an immunopositive cell. Note the presence of immunoreactive material in the cytoplasm (arrows) extending into cellular processes (arrowheads). B, High power magnification showing immunoreactive material primarily confined to secretory granules (arrows). Original magnification, X8,9OO in A and X15.500 in B. Bar = 0.5 ^m. N, Nucleus.
sponse, however, is thought to be mediated by the central nervous system and not to occur at the level of the pituitary. An important role of anterior pituitary IL-1/3 may be the modulation of TSH secretion. Double immunolabeling studies demonstrate that IR IL-1/3 is at least partly a product of the anterior pituitary thyrotrope, in keeping with the ultrastructural features of the immunoreactive cells, including its stellate appearance and small secretory granules (37). This observation together with the marked increase in IL-1/3 mRNA after LPS administration raises the possibility that anterior pituitary IL-1/3 may modulate TSH secretion, particularly during infection. This suggestion is supported by the finding that bacterial endotoxin suppresses plasma concentrations of TSH (38) and that the systemic administration of IL-1/3 causes a rapid decline in thyroid function and fall in
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IL-10 IN ANTERIOR PITUITARY serum TSH (4). It is possible, therefore, that the socalled euthyroid sick syndrome, characterized by low thyroid hormone levels and decreased or inappropriately normal TSH levels in patients with infection, starvation, and chronic illness (39), may be partly mediated through an autocrine mechanism after the induction of endogenous IL-1/3 in anterior pituitary thyrotropes to depress biosynthesis of TSH or its secretion. TSH secretion may also be inhibited by increased secretion of hypothalamic somatostatin, shown to be brought about by direct effects of IL-ljS, and tumor necrosis factor-a (40, 41). The demonstration of intrinsic hypothalamic and pituitary IL-ljS-secreting cells suggests that anterior pituitary responses to inflammation and infection may be regulated by a family of cytokines having coordinated functions.
Acknowledgments We thank Jorge Tejada for technical assistance.
References 1. Dinarello CA 1985 An update on human interleukin-1: from molecular biology to clinical relevance. J Clin Immunol 5:287 2. Besedovsky H, del Rey A, Darkin E, Dinarello CA 1986 Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 233:652 3. Uehara A, Gottschall PE, Dahl RR, Arimura A 1987 Stimulation of ACTH release by human interleukin-1 beta, but not by interleukin-1 alpha, in conscious, freely-moving rats. Biochem Biophys Res Commun 146:1286 4. Dubois J-M, Dayer J-M, Siegrist-Kaiser CA, Burger AG 1988 Human recombinant interleukin-1/9 decreases plasma thyroid hormone and thyroid stimulating hormone levels in rats. Endocrinology 123:2175 5. Rivier C, Vale W 1989 In the rat, interleukin-1 alpha acts at the level of the brain and the gonads to interfere with gonadotropin and sex steroid secretion. Endocrinology 124:2105 6. Woloski BMRNJ, Smith EM, Meyer III WJ, Fuller GM, Blalock JE 1985 Corticotropin-releasing activity of monokines. Science 230:1035 7. Beach JE, Smallridge RC, Kinzer CA, Bernton EW, Holoday JW, Fein HG 1989 Rapid release of multiple hormones from rat pituitaries perifused with recombinant interleukin-1. Life Sci 44:1 8. Berton EW, Beach JE, Holaday JW, Smallridge RC, Fein HG 1987 Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 238:519 9. Brown SC, Smith LR, Blalock JE 1987 Interleukin 1 and interleukin 2 enhance proopiocortin gene expression in pituitary cells. J Immunol 139:3181 10. Kehrer P, Turnill D, Dayer JM, Muller AF, Gaillard RC 1988 Human recombinant interleukin-1 beta and -alpha, but not recombinant tumor necrosis factor alpha stimulates ACTH release from rat anterior pituitary cells in vitro in a prostaglandin E2 and cAMP independent manner. Neuroendocrinology 48:160 11. Berkenbosch F, Van Oers J, del Rey A, Tilders F, Besedovsky H 1987 Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-1. Science 238:524 12. Sapolsky R, Rivier C, Yamamoto G, Plotsky P, Vale W 1987 Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing facter. Science 238:522 13. Uehara A, Gillis S, Arimura A 1987 Effects of interleukin-1 on hormone release from normal rat pituitary cells in primary culture. Neuroendocrinology 45:343 14. Katsuura G, Gottschall PE, Dahl RR, Arimura A 1988 Adrenocorticotropin release induced by intracerebroventricular injection of
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recombinant human interleukin-1 in rats: possible involvement of prostaglandin. Endocrinology 122:1773 15. Katsuura G, Gottschall PE, Dahl PR, Arimura A 1989 Interleukin1/3 increases prostaglandin E2 in rat astrocyte cultures: modulatory effect of neuropeptides. Endocrinology 124:3125 16. Suda T, Tozawa F, Ushiyama T, Tomori N, Sumitomo T, Nakagami Y, Yamada M, Demura H, Shizume K 1989 Effects of protein kinase-C-related adrenocorticotropin secretagogues and interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells. Endocrinology 124:1444 17. Tsagarakis S, Gilles G, Rees LH, Besser M, Grossman A 1989 Interleukin-1 directly stimulates the release of corticotrophin releasing factor from rat hypothalamus. Neuroendocrinology 49:98 18. Breder CD, Dinarello CA, Saper CB 1988 Interleukin-1 immunoreactive innervation of the human hypothalamus. Science 240:321 19. Lechan RM, Toni R, Clark BA, Cannon JG, Shaw AR, Dinarello CA, Reichlin S, Immunoreactive interleukin-1 beta localization in the rat forebrain. Brain Res, in press 20. Segerson TP, Lam KSL, Cacicedo L, Minamitani N, Fink JS, Lechan RM, Reichlin S 1989 Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland. Endocrinology 125:2221 21. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, Martin JB, Chin WW 1988 Galanin is an estrogen-inducible secretory product of the rat anterior pituitary. Proc Natl Acad Sci USA 85:7408 22. Aronin N, Morency K, Leeman SE, Braverman LE, Coslovsky R 1984 Regulation by thyroid hormone of the concentration of substance P in the rat anterior pituitary. Endocrinology 114:2138 23. Jones PM, Ghatei MA, Steel J, O'Halloran D, Legon G, Burrin JM, Leonhardt U, Polak JM, Bloom SR 1989 Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125:334 24. Vankelecom H, Carmeliet P, Van Damme J, Billiau A, Denef C 1989 Production of interleukin-6 by folliculo-stellate cells of the anterior pituitary gland in a histiotypic cell aggregate culture system. Neuroendocrinology 49:102 25. Spangelo BL, Judd AM, Isakson PC, MacLeod RM 1989 Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 125:575 26. Fenton MJ, Clark BD, Collins KL, Webb AC, Rich A, Auron PE 1987 Transcriptional regulation of the human prointerleukin 1 beta gene. J Immunol 38:372 27. Fenton MJ, Vermoulen MW, Clark BD, Webb AC, Auron PE 1988 Human pro-IL-I beta gene expression in monocytic cells is regulated by two distinct pathways. J Immunol 140:2267 28. Hsu SM, Raine L 1981 Protein A, avidin, and biotin in immunohistochemistry. J Histochem Cytochem 29:460 29. Naishida T, Hirato T, Nishino N, Mizuno K, Sekiguchi Y, Takano M, Kawai K, Nakai S, Hirai Y 1988 Cloning of the cDNAs for rat interleukin-la and /9. In: Powanda MC, Oppenheim JJ, Kluger MJ, Dinarello CA (eds) Monokines and Other Non-Lymphocytic Cytokines. Liss, New York, pp 73-78 30. Cleveland DW, Lopata MA, MacDonald RJ, Cowan NJ, Rutter WJ, Kirschner WW 1980 Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma actin genes using specific cloned cDNA probes. Cell 20:95 31. Takaca L, Kovacs EJ, Smith MR, Young HA, Durum SK 1988 Detection of IL-1/3 and IL-1/3 gene expression by in situ hybridization. J Immunol 141:3081 32. Nishida T, Nishino N, Takano M, Sekiguchi Y, Kawai K, Mizuno K, Nakai S, Masui Y, Hirai Y 1989 Molecular cloning and expression of rat interleukin 1/3 convertase activity. Proc Natl Acad Sci USA 86:5227 33. Kostura MJ, Tocci MJ, Limjuco G, Chin J, Cameron P, Hillman AG, Chartrain NA, Schmidt JA 1989 Identification of a monocyte specific pre-interleukin 1/3 convertase activity. Proc Natl Acad Aci USA 86:5227 34. Hazuda DJ, Lees JC, Young PR 1988 The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta. J Biol Chem 264:843
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35. Young PR, Hazuda DJ, Simon P 1988 Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA. J Cell Biol 107:477 36. Wexler BC, Dolgin AE, Tryczunski EW 1957 Effects of a bacterial polysaccharide (Piromen) on the pituitary-adrenal axis: adrenal ascorbic acid, cholesterol and histologic alterations. Endocrinology 61:300 37. Nakane PK 1970 Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J Histochem Cytochem 18:9 38. Kasting NW, Martin JB 1982 Altered release of growth hormone
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and thyrtropin induced by endotoxin in the rat. Am J Physiol 243:E332 39. Tibaldi JM, Surks MI 1985 Animal models of nonthyroid disease. Endocr Rev 6:87 40. Scarborough DE, Lee SL, Dinarello CA, Reichlin S 1989 Interleukin-1/3 stimulates somatostatin biosynthesis in primary cultures of fetal rat brain. Endocrinology 124:549 41. Pang X-P, Hershman JM, Mirell CJ, Pekary AE 1989 Impairment of hypothalamic-pituitary-thyroid function in rats treated with human recombinant tumor necrosis factor-a (cachectin). Endocrinology 125:76
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Vol. 126, No. 6 Printed in U.S.A.
Intrinsic Pituitary Interleukin-1/? Is Induced by Bacterial Lipopolysaccharide* JAMES I. KOENIGf, KENNETH SNOW, BURTON D. CLARK*, ROBERTO TONI, JOSEPH G. CANNON, ALAN R. SHAW, CHARLES A. DINARELLO, SEYMOUR REICHLIN, STEPHANIE L. LEE§, AND RONALD M. LECHAN Department of Medicine, Divisions of Endocrinology and Infectious Disease, New England Medical Center Hospitals, Boston, Massachusetts 02111; and Glaxo Institute for Molecular Biology (A.R.S.), Geneva, Switzerland
ABSTRACT. Using a specific antiserum recognizing recombinant rat interleukin-1/3 (IL-1/3), immunoreactive material was localized to cytoplasmic granules in anterior pituitary endocrine cells and colocalized with TSH in thyrotropes. Authenticity was established by Northern blot hybridization using a specific rat IL-1/3 cRNA probe, revealing a 1.8-kilobase mRNA identical to
that in the spleen. The marked increase in anterior pituitary IL1/3 message after the administration of bacterial lipopolysaccharide, raises the possibility that IL-1/3 may be involved in paracrine or autocrine regulation of pituitary function during infectious challenge. (Endocrinology 126: 3053-3058, 1990)
I
NTERLEUKIN-1/3 (IL-1/3), a cytokine released by activated macrophages, monocytes, and several other cell types (1), stimulates the secretion of ACTH (2, 3) and inhibits the secretion of GH, LH, and TSH in the intact rat (4, 5). Physiological experiments suggest that these effects may be exerted directly on the pituitary (610) or indirectly through the hypothalamus (11-17). Recent neuroanatomical studies by Breder et al. (18) showing the presence of immunoreactive (IR) IL-1/3 neuronal processes in the human hypothalamus and the finding of an extensive IR IL-l/3-positive neuronal system in the rat brain (19) suggest that IL-1/3 may be a hypophysiotropic factor. As a number of neuropeptides known to influence anterior pituitary secretion, such as vasoactive intestinal peptide, substance-P, neuropeptide-Y, and galanin (20-23), are synthesized by pituitary cells, and IL6, a cytokine which influences the secretion of several pituitary hormones, is localized to pituitary folliculostellate cells (24, 25), the possibility was considered that IL-1/3 might also be secreted by pituitary cells and form
part of an intrinsic paracrine control system. To determine whether IL-1/3 is an intrinsic pituitary secretory product, immunocytochemical staining and molecular hybridization of the rat pituitary gland were carried out using specific antisera and molecular probes for rat IL1/3. The response of pituitary IL-1/3 to treatment with bacterial lipopolysaccharide (LPS), a pyrogen known to activate monocyte IL-1/3 synthesis (26, 27), was also determined. Materials and Methods Animals and tissue preparation
Received January 3,1990. Address requests for reprints to: Ronald M. Lechan, M.D., Ph.D, Department of Medicine, Endocrinology Division, New England Medical Center Hospitals, Box 268, 750 Washington Street, Boston, Massachusetts 02111. * This work was supported by NIH Grants DK-37021, DK-16684, and AI-15614. f Current address: Department of Physiology and Biophysics, Georgetown University School of Medicine, Washington, D.C. t Recipient of a grant from the National Arthritis Foundation. § Recipient of a Charles A. King Trust Fellowship.
The pituitaries of normal male Sprague-Dawley rats were fixed by intracardiac perfusion with Bouin's solution for light microscopy or 4% paraformaldehyde-0.2% glutaraldehyde in 0.1 M PBS, pH 7.6, for electron microscopy and prepared for immunocytochemistry by the avidin-biotin-peroxidase complex technique (28). A second group of animals injected 4 h before study with normal saline or LPS (E. coli 055:B5 LPS; 25 mg/ kg, ip) were anesthetized with pentobarbital and, after clamping the descending aorta, perfused through the ascending aorta with 60 ml ice-cold saline to free the anterior pituitary of circulating blood products in preparation for Northern blot hybridizations. The pituitaries were rapidly removed, separated from the posterior pituitary and intermediate lobe, and frozen on dry ice. Sections of spleen, an organ that can be induced to synthesize an abundance of IL-1/3 message, and cerebral cortex were similarly prepared as positive and negative controls, respectively.
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IL-1/3 IN ANTERIOR PITUITARY
3054 Antiserum preparation and characterization
Antiserum to recombinant rat IL-1/3 was raised in New Zealand White rabbits, immunized with 100 ng recombinant rat IL-1/3 (Glaxo, Geneva, Switzerland), emulsified in complete Freund's adjuvant, and boosted at monthly intervals using incomplete adjuvant. After 12 weeks, one antiserum was demonstrated to recognize recombinant rat IL-1/3 by Western blot analysis at a titer of 1:2000 after resolving 50 ng recombinant rat IL-1/3 on a 15% sodium dodecyl sulfate-polyacrylamide gel (19). This antiserum was used at a titer of 1:750, diluted in 0.05 M Tris-buffered saline, pH 7.6, containing 0.5% Triton X-100 for light microscopic immunocytochemical studies and 0.02% Triton X-100 for ultrastructural studies. Immunocytochemical procedure To determine whether IR IL-1/3 was contained in one or more of the classical anterior pituitary cell types, serial 3-^m sections were reacted with antiserum to IL-1/3 (1:750) or with antiserum to PRL, GH, /3-lipotropin, LH, or TSH (1:750), respectively, using antisera from the National Pituitary Hormone Distribution Program of the NIH, diluted in 0.05 M Trisbuffered saline containing 0.3% Triton X-100. Identical regions in the serial tissue sections were photographed, and areas of IL-1/3 and anterior pituitary hormone immunoreactivity were compared by visual inspection. Ultrastructural analysis Tissues prepared for ultrastructural analysis were developed with 0.02% diaminobenzidine (DAB)-0.05% nickel ammonium sulfate, activated by 0.02% hydrogen peroxide to create an electron-dense immunoreaction product. Tissues were stained en bloc with an aqueous solution of 1% osmium tetraoxide-1.5% potassium ferrocyanide, followed by 1% uranyl acetate in 0.05 N maleate buffer, pH 6.0, and 1% tannic acid in 0.05 M cacodylate buffer, pH 7.4. After dehydration in ethanols and propylene oxide, the tissues were flat embedded in Epon 812Araldite 6005-DDSA (1:1:2.4) and polymerized with 2.4% DMP-30 (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin sections were cut on an MT-2000 ultramicrotome and examined under a Philips CM-10 transmission microscope (Philips Electronic Instruments, Mahwah, NJ).
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hybridization solution recommended by the manufacturer containing 10 millon cpm/ml [32P]IL-l/3 antisense RNA probe. After washing the blot in 0.1 x SSC (1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7) containing 0.1% sodium dodecyl sulfate at 68 C, the membrane was exposed to Kodak XAR5 film at —70 C and developed after 1-7 days. The size of hybridized bands was estimated by comparison to a RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD). To demonstrate the specificity of the IL-1/3 mRNA response to LPS, the blots were reprobed with 10 million cpm/ml 32Plabeled /3-actin antisense probe after eluting the first radiolabeled probe by washing three times in 50% formamide-6 X SSPE (1 x SSPE = 0.18 M NaCl, 0.01 M sodium phosphate pH 7.7, 1 mM EDTA) at 65 C for 1-3 h. The adequacy of the elution of the previous probe was assessed by reexposure of the autoradiogram. After all counts were removed, the blots were prehybridized and hybridized following methods described above. Relative densities of hybridizing bands in the autoradiographs were determined by semiquantitative computer image analysis using Image (version 1.17) obtained from the NIH. Autoradiographs were uniformily illuminated using a fluorescent light source (Northern Light Precision Illiminator model B90, Imaging Research, Inc., St. Catherines, Ontario, Canada) and digitized with a MOS-5300 CCD video camera (General Imaging Corp., Gainesville, FL) and Quick Capture frame grabber board and software (Data Translation, Marlboro, MA) to a 512 X 512 pixel array. The integrated density of each hybridized band was analyzed using a Macintosh II computer. Antisense RNA probes A 216-basepair Pstl fragment of rat IL-1/3 cDNA was used as a template for the synthesis of the single stranded antisense hybridization probe. This sequence encodes the amino acid residues 125-196 of rat pro-IL-1/3, as described by Nishida et al. (29), and is directed toward the amino-terminal end of the mature IL-1/3 peptide. The construct was linearized by restriction with EcoRI to form the template for synthesis of 32Plabeled antisense RNA by SP6 polymerase. A chicken /3-actin (30) cDNA template was provided as a gift by Dr. Donna Chikaraishi. The /3-actin construction was linearized by BamHI before synthesis of 32P-labeled antisense RNA probes with T7 polymerase.
RNA analysis Total RNA was isolated by guanidine isothiocyanate lysis and centrifugation through a CsCl gradient. The RNA content was estimated by visual inspection of a nondenaturing ethidium bromide-1% agarose gel under UV illumination. The RNA was poly(A)+ selected on oligo-dT cellulose (Collaborative Research, Waltham, MA) columns or with Hybond paper (Amersham, Arlington Heights, IL) following the manufacturer's protocol. The purified mRNA was quantified by spectrophotometry, and equal amounts from control and LPS-treated tissues were size-fractionated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde. RNA was electrophoretically transferred to nylon membrane (Nytran, Schleicher and Schuell, Inc., Keene, NH) overnight and hybridized at 68 C in
Results IL-1/3 immunoreactivity was localized to polygonal and stellate cells with abundant cytoplasm, distributed singly and in clusters throughout the anterior pituitary, but was particularly prominent in anterior-ventral areas and adjacent to the intermediate lobe (Fig. 1, A and C). No immunoreaction product was present after immunoadsorption with recombinant rat IL-1/3, establishing the specificity of the immunostaining (Fig. IB). In serial tissue sections, a subpopulation of cells containing IL1/3 was also noted to be immunoreactive for TSH, located primarily in anterior-ventral areas (Fig. 1, C and D), but
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IL-1/3 IN ANTERIOR PITUITARY
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FlG. 1. A, Coronal sections through the rat anterior pituitary showing the presence of rat IL-1/3 (ABC technique) in numerous cells throughout the gland. Two prominent collections of cells (arrowheads) are seen in the antero-ventral portion and adjacent to the intermediate lobe (NIL). B, Reaction product is completely abolished when the antiserum is preadsorbed with 10~6 M recombinant rat IL-1/3 (Glaxo). C and D, Immunostaining of serial 3-^m sections of rat anterior pituitary with antiserum to recombinant rat IL-10 (1:750; C) and rat TSH (1:750; D). Arrows denote doubly immunolabeled cells. Original magnification, X79 in A and B and X500 in C and D.
many IR IL-1/3 and TSH cells were only singly labeled, particularly in central areas of the anterior pituitary. No other anterior pituitary hormone tested colocalized with IR IL-1/3. Ultrastructurally, immunoreactive IL-1/3 was associated with cytoplasmic granules measuring approximately 140 nm, located near the plasma membrane and extending into cytoplasmic processes (Fig. 2). To further establish the authenticity of anterior pituitary IR IL-1/3 and to determine whether rat IL-1/3 arises directly as a translation of anterior pituitary cells, Northern hybridizations were performed in three separate experiments. A single hybridizing band of approximately 1.8 kb in length, identical to that present in the spleen, was detectable in the normal pituitary, but only after loading large amounts of mRNA on the gel (Fig. 3A). In LPS-treated animals, however, IL-1/3 message was readily detected and was 15-fold more abundant than in the normal controls, as determined from integrated density measurements. In contrast, after reprobing the same blot with a radiolabeled /3-actin cRNA, fairly uniform hybridization densities were seen in the paired samples (Fig. 3B). No hybridization was present in RNA prepared from the posterior pituitary/intermediate lobe or the cerebral cortex obtained from control or LPS-treated rats.
Discussion These studies demonstrate that IL-1/3 is synthesized as an intrinsic product of the rat anterior pituitary gland. Its authenticity is established by the presence of a 1.8kb mRNA by Northern hybridization, identical to IL-1/3 mRNA extracted from rat spleen. A similar size RNA has been found in rat macrophages (31) and contrasts to the 2.2-kb mRNA of IL-la, a cytokine that shares bio-
logical activities with IL-1/3 (32). The morphological appearance of immunoreactive cells as polygonal and stellate shaped characterizes them as belonging to endocrine cells of the anterior pituitary, rather than endothelial cells or tissue macrophages, which are known peripheral sources of IL-1/3 (1). In addition, the presence of typical secretory granules in these cells, containing the majority of the immunoreactive material, distinguishes them from folliculo-stellate cells of the anterior pituitary, previously shown to produce and secrete IL-6 (24, 25). This distinct subcellular compartmentalization suggests that the process of intracellular transport and secretion of anterior pituitary IL-1/3 may differ from the mechanisms proposed for monocytes and fibroblasts (3335). The presence of IL-1/3 in the pituitary raises the possibility that it may have a physiological role in the control of anterior pituitary hormone secretion, either directly or by inducing the secretion of IL-6 from anterior pituitary folliculo-stellate cells (24, 25). Since the original observation by Wexler et al. (36) that bacterial endotoxin stimulates the pituitary-adrenal axis, numerous reports have now established potent effects of recombinant IL-1 on ACTH secretion (2, 6-17). Although most indicate that this may be primarily due to central actions of IL1/3 to stimulate CRH (11-17), direct effects on anterior pituitary corticotrophs have also been demonstrated (610). Furthermore, as IR IL-1/3 cells tend to cluster in the anterior-ventral area of the pituitary adjacent to LHand FSH-containing cells, a paracrine mechanism for control of gonadotropin secretion could also be proposed. LH secretion is stimulated from pituitary cells in culture after treatment with IL-1/3, but is reported to be inhibited after intraventricular administration (5). The latter re-
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IL-1/3 IN ANTERIOR PITUITARY
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A B
CD
4.4
B FIG. 3. A, Northern blot of IL-1/8 poly(A)+ mRNA extracted from the anterior pituitary (lanes A, B, E, and F) and spleen (lanes C and D) obtained from untreated and LPS-treated rats. A hybridizing band of identical size (~1.8 kb) is present in both spleen and pituitary. IL-1/3 mRNA in control pituitaries is seen after loading high concentrations of total message (lane E), but is markedly increased by LPS treatment (lanes B and F) and readily visable after loading smaller amounts of message (lane B). B, Same Northern blot as in A, but completely eluted of radioactivity and reprobed for /3-actin mRNA. Note the similar hybridization densities of the 2.2-kb band for each paired samples. Concentrations of poly(A)+ message per lane are 2 ng in A and B, 0.5 fig in C and D, and 10 jtg in E and F (bubble artifact in lane F).
FlG. 2. Electron micrographs showing the distribution of IR IL-1/3 in the rat anterior pituitary gland. A, Low power magnification of an immunopositive cell. Note the presence of immunoreactive material in the cytoplasm (arrows) extending into cellular processes (arrowheads). B, High power magnification showing immunoreactive material primarily confined to secretory granules (arrows). Original magnification, X8,9OO in A and X15.500 in B. Bar = 0.5 ^m. N, Nucleus.
sponse, however, is thought to be mediated by the central nervous system and not to occur at the level of the pituitary. An important role of anterior pituitary IL-1/3 may be the modulation of TSH secretion. Double immunolabeling studies demonstrate that IR IL-1/3 is at least partly a product of the anterior pituitary thyrotrope, in keeping with the ultrastructural features of the immunoreactive cells, including its stellate appearance and small secretory granules (37). This observation together with the marked increase in IL-1/3 mRNA after LPS administration raises the possibility that anterior pituitary IL-1/3 may modulate TSH secretion, particularly during infection. This suggestion is supported by the finding that bacterial endotoxin suppresses plasma concentrations of TSH (38) and that the systemic administration of IL-1/3 causes a rapid decline in thyroid function and fall in
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IL-10 IN ANTERIOR PITUITARY serum TSH (4). It is possible, therefore, that the socalled euthyroid sick syndrome, characterized by low thyroid hormone levels and decreased or inappropriately normal TSH levels in patients with infection, starvation, and chronic illness (39), may be partly mediated through an autocrine mechanism after the induction of endogenous IL-1/3 in anterior pituitary thyrotropes to depress biosynthesis of TSH or its secretion. TSH secretion may also be inhibited by increased secretion of hypothalamic somatostatin, shown to be brought about by direct effects of IL-ljS, and tumor necrosis factor-a (40, 41). The demonstration of intrinsic hypothalamic and pituitary IL-ljS-secreting cells suggests that anterior pituitary responses to inflammation and infection may be regulated by a family of cytokines having coordinated functions.
Acknowledgments We thank Jorge Tejada for technical assistance.
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35. Young PR, Hazuda DJ, Simon P 1988 Human interleukin 1 beta is not secreted from hamster fibroblasts when expressed constitutively from a transfected cDNA. J Cell Biol 107:477 36. Wexler BC, Dolgin AE, Tryczunski EW 1957 Effects of a bacterial polysaccharide (Piromen) on the pituitary-adrenal axis: adrenal ascorbic acid, cholesterol and histologic alterations. Endocrinology 61:300 37. Nakane PK 1970 Classifications of anterior pituitary cell types with immunoenzyme histochemistry. J Histochem Cytochem 18:9 38. Kasting NW, Martin JB 1982 Altered release of growth hormone
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and thyrtropin induced by endotoxin in the rat. Am J Physiol 243:E332 39. Tibaldi JM, Surks MI 1985 Animal models of nonthyroid disease. Endocr Rev 6:87 40. Scarborough DE, Lee SL, Dinarello CA, Reichlin S 1989 Interleukin-1/3 stimulates somatostatin biosynthesis in primary cultures of fetal rat brain. Endocrinology 124:549 41. Pang X-P, Hershman JM, Mirell CJ, Pekary AE 1989 Impairment of hypothalamic-pituitary-thyroid function in rats treated with human recombinant tumor necrosis factor-a (cachectin). Endocrinology 125:76
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