Interleukin-1, Interleukin-6: Messengers in the Neuroendocrine Immune System?

Path. Res. Pract. 187, 622-625 (1991)

Interleukin-l, Interleukin-6: Messengers in the Neuroendocrine Immune System? E. Hooghe-Peters, B. Velkeniers, L. Vanhaelst and R. Hooghe1 Department of Pharmacology, Faculty of Medicine, Vrije Universiteit Brussel, Brussels, Belgium and 1Department of Biology, Studiecentrum voor Kernenergie-Centre d'Etude de I'Energie nucleaire, Mol, Belgium

SUMMARY

Recent findings indicate that interleukin-1 (1L-1) and interleukin-6 (IL-6), cytokines secreted by immunologically activated monocytes and macrophages modulate neuroendocrine function. The site of production (centrally, peripherally), the site of action and the physiological significance of 1L-1 and IL-6 as "classical hormones" are questioned.

Introduction Until recently, the neuroendocrine system was thought to be relatively insensitive to the input of other, non-target, organ systems. Indeed, its activity is mainly regulated by neurohormonal feed-back. The same kind of concepts prevailed for the immune system, which is specifically trigerred by antigen and tuned by helper-suppressor cell circuits, idiotype-antiidiotype networks and "its own" regulatory cytokines, the interleukins. Whereas the immunosuppressive action of glucocorticoids and ACTH has been recognized for several decades, other neuroendocrine input into the immune system has been reported but usually not considered to be of great importance25 . Lymphocytes have receptors for glucocorticoids, for adrenergic ligands, but also for prolactin and for POMCderived peptides (ACTH, a-MSH, B-endorphin)25. It has now been shown that these agents do have immunomodulatory properties25 . Moreover, in response to nanomolar concentrations of CRF, some leucocytes synthesize ACTH and B-endorphin. This response can be regulated by dexamethasone, suggesting that the regulation of the POMC gene is similar in the pituitary and in the leukocyte. Now, the link between the immune system and the neuroendocrine system has become obvious to everyone. The relationship between the prolactin receptor and the receptors for several hemopoietic growth factors may mean more than common ancestry for these molecules 2 • Of 0344-0338/9110187-0622$3.50/0

even more direct significance for our subject, interleukin 1 (IL-l), a polypeptide monokine product of activated phagocytes that mediates many biological functions (including host organism's response to infection or inflammation, cell growth and differentiation) has been demonstrated to alter the adrenocorticotropic hormone (ACTH) release from ACTH secreting pituitary tumor cells (AtT20)34,38. Also, in 1985, Woloski et al. discovered a hepatocyte stimulating factor (later renamed IL-6) that modulated ACTH secretion from the same tumor cells (AtT -20). These observations were reinforcing the idea for a "cross-talk" between these two signalling pathways. A number of selected papers, addressing the issue of the site of synthesis and of action for both cytokines using similar experimental approaches, but with contradictory results will be discussed in an attempt to reconcile seemingly incompatible results reported in the literature and to formulate some hypotheses for future investigation. 1 Can IL-l Modulate Neuroendocrine Function? IN 1985, Woloski et al.3 8 were among the first to show that murine interleukin-l (IL-l) stimulated the ACTH release from mouse ACTH secreting pituitary tumor cells (AtT-20) suggesting that IL-l may be a CRF-like molecule. Since AtT-20 tumor cells exhibit characteristics other than those of normal pituitary corticotrophs32 , the effect of monokines was further investigated using primary mono© 1991 by Gustav Fischer Verlag, Stuttgart

Interleukins in the Neuroendocrine Immune System? . 623

layer cultures of rat anterior pituitary cells. Two distinct forms of IL-l have been characterized I2 ,23. They have different isoelectric points and share only 26% homology in their aminoacid sequence, IL-la and IL-l~. Nevertheless, both cytokines exert their effects through the same receptors. However, an equal binding affinity of the cytokines does not reflect an equal ability to induce a biological response 31 . Neither human IL-l a nor human IL-l ~ stimulated the ACTH release from normal rat pituitary cells in concentrations up to 10 mM. In contrast, a slight increase in ACTH release was observed at a (pharmacological) concentration of 100 mM for IL-l~. At the latter dosis IL1~ exhibited a slight synergistic action with CRF upon ACTH secretion. Both forms of IL-l failed to alter the secretion of other pituitary hormones. No sex difference in the pituitary response to IL-la or IL-l~ was observed, nor any influence of the duration of preincubation in serum-free medium 35 . Uehara 36 also reported that intravenously injected IL-l (2 !lgJrat) stimulated the secretion of ACTH in conscious, freely moving male rats. Since immunoneutralization of endogenous corticotropin-releasing hormone blocked the stimulatory action of IL-l, the authors suggested that IL-l was acting centrally in the brain and stimulated hypothalamic CRF secretion, that in turn increased the release of ACTH from the pituitary gland. In a very elegant study, Sapolsky and collaborators 26 demonstrated an increased concentration of CRF in the portal blood after rats were given an intravenous injection of 3 !lg huIL-l, but they also failed to demonstrate a direct effect of huIL-l on ACTH secretion by rat anterior pituitary cells in culture. Those results provide evidence that IL-l is acting at the hypothalamic level but has no direct acute stimulatory effect on ACTH secretion. In contrast, in the work presented by Bernton et al.5, evidence was provided that IL-l (from 10- 9 M to 10- 12 M) stimulated the secretion of ACTH, luteinizing hormone, growth hormone, and thyroid-stimulating hormone from dispersed anterior pituitary cells of female rats taken at random phases of the estrous cycle. To rule out any possibility of a paracrine effect resulting from the elevated levels of "release factors" that accumulate in a static monolayer system, and to better examine the dynamic response of hormone release elicited by IL-l, two groups conducted their studies on rat anterior pituitary tissue 3 or on dispersed rat anterior pituitary cells33 in a computer-controlled automated perifusion system. Various doses ofIL-l~3,33 and/or CRF-41 33 were administered as 10 min infusion in a randomized way. While Tsagarakis provided evidence that various doses of CRF-41 but not of IL-l had an effect on ACTH release from dispersed pituitary cells, the findings of Beach3, in contrast, pointed to an acute action of IL-l on ACTH release. The rapid response elicited suggested an effect on preformed hormone stores. Studies were therefore undertaken by Brown and co-workers9 and Suda et al.3° to analyse whether the IL-l-induced release of hypothalamic CRF and subsequently of ACTH from the pituitary gland were accompanied by changes in cytoplasmic CRF- and rOMC mRNA levels respectively. Rat hypothalamic tissue demonstrated and increased CRF mRNA in response to

intravenous IL-1 9,3o. In addition, rat pituitary cells30 and AtT-20 cells 9 also exhibited an increase in rOMC content in IL-l treated cells compared to untreated cells. The latter findings indicate that IL-l acted in a manner similar to CRP. Although contradictory results regarding the site of action for IL-l in the control of ACTH are demonstrated, all authors seemed to agree that IL-l can somehow stimulate ACTH release. The differences observed between the different groups may be explained by differences in the experimental design, in the use of pituitary tissue versus pituitary dispersed cells, in the time lapse between cell preparation and cell use (directly after dissociation or after 3 days in culture), in rat strains (Wistar rats, Sprague Dawley) and rat sex (female at random phases of estrous cycle or male rats) as source of cells, in the source of interleukin utilized (species: rat, mouse, human), in the form of interleukin used in the study (a form or ~ form), or in the medium used for performing the assay (synthetic medium or not, use of serum or not, use of BSA). All authors pointed to an action of IL-l on the hypothalamus; some of them suggested that IL-l might (in addition?) modulate directly anterior pituitary hormone(s) release. Whether IL-l exerts a direct effect on CRF release or whether this effect is mediated via another neurotransmitter remains also to be determined. Carmeliet et al. 10 have indeed shown that IL-l~ inhibits acetylcholine synthesis in transformed mouse corti co tropic cells (AtT 20), whereas Besedovsky et al. 6 have presented evidence that soluble products (which include IL-l) derived from stimulated rat spleen cells supernatant induced a decrease of noradrenaline synthesis in the hypothalamus. The activity of central nor adrenergic neurons in the rat and in the mouse were also shown to be lightly and transiently affected by IL-1 4, 13,21. IL-l could possibly affect presynaptic nerves, alternatively, the sympathic response may be mediated by central CRF projections known to be involved in the regulation of sympathic outflow 8. IL-l ~ immunoreactivity in different brain regions has been recently associated with hypophysiotropic functions in both anterior and posterior pituitary gland 22 . Of great pertinence is the fact that astroglial cells and microglial cells of the brain are present in large number in the median eminence region of the hypothalamus. Astroglial cells have been shown to produce IL-l in in vitro systems. Giulian et al. 18 have in addition reported stimulation of astrocyte proliferation in mixed rat glial cells culture and have ascribed this activity to IL_1 17- 19 . Those cells project their processes onto portal capillaries and it is tempting to hypothesize that they may release IL-l into the portal blood. IL-l would thus function as a new "releasing or inhibiting" factor 7,22. However, recent studies 7 have also demonstrated the presence of IL-l~ immunoreactivity (IR) in the brain, but the authors demonstrated IR in hypothalamic or thalamic axons rather than in astroglial cells. Lechan et al. 22 also demonstrated immunoreactive product in the periventricular medial hypothalamus, hippocampus and olfactory bulb. The label was preferentially associated with nerve processes and nerve terminals. The presence of immunoreactive IL-l~ in the normal rat hypothalamus (arcuate,

624 . E. Hooghe-Peters et al. periventricular areas) and in nuclei in which numerous neurons that contribute to the tubero infundibular system and median eminence do originate, place neurons IL-l~ IR in a strategic position to influence the secretion of hypophysiotropic hormones. This statement was reinforced by the observation that IL-l~, given intraventricularly, induced a prompt increase of CRF, gonadotropin RF4,7 and of SRIF mRNA. Another quite important observation is the stimulation of astrogliosis and of neovascularization at the site of injection in the rat brain 17. The detection of IL-l immunoreactivity in the central nervous system, and the different effects IL-l exerts make this cytokine a likely candidate for different functions. It seems reasonable to hypothesize that IL-l may be a trophic or growth factor but one could also postulate for IL-l a role as a neuromodulator molecule or consider it as new hypophysiotropic RF or IF. More work is still needed to better define the site of synthesis, the site of action, and the exact function of this cytokine in the neuroendocrine immune system.

2 Can IL-6 Modulate Neuroendocrine Function? IL-6 (BSF-2) is, as IL-l, a pleiotropic cytokine that mediates different host responses and regulates multiple cell types. The clear parallel between the activities of IL-l and IL-6 in the immune system has prompted several investigators to test possible IL-6 involvement in the neuroendocrine immune response. In 1988 Naitoh et al,24 demonstrated that intravenous injection of recombinant huIL-6 to conscious freely moving rats (1-5 flglrat) resulted in a rise of plasma ACTH level. They further showed that prior administration of antibody to CRF neutralized IL-6 activity. This observation suggested that the site of action of IL-6 was not at the pituitary level, but was mediated by CRF containing neurons located in the hypothalamus. Vankelecom et alY suggested that folliculostellate cells, a pituitary cell type sharing some characteristics with mononuclear phagocytic cells, and with a yet unknown function could be a pituitary source of IL-6. To substantiate their hypothesis, they presented some evidence suggesting that aggregate cultures of mouse or rat anterior pituitary cells produce IL-6, as measured by 7 TD 1 hybridoma cells proliferation. They demonstrated, in addition, that aggregated cultures enriched in folliculostellate cells produced more IL-6 than regular pituitary cell cultures. Although the hypothesis of a folliculostellate cell as a source of IL-6 production is tempting, other cells such as endothelial cells, histiocytes remain "candidates"; moreover a definitive proof for local synthesis (combined in situ hybridization and immunocytochemistry) is still mIssmg. In recent publications, Spangelo et a1. 29 reported that cultured anterior pituitary cells spontaneously secrete large quantities of IL-6 in vitro. The production of IL-6 could be further stimulated by LPS or PMA suggesting that in pathophysiological conditions, local pituitary as well as peripheral production of IL-6 may be elicited resulting in

an enhanced hormone release. Indeed, the same group has shown that IL-6 stimulates the release of GH, PRL and LH from the pituitary in vitro 29 . Yamaguchi et al. 39 demonstrated that IL-6, possibly induced by IL-l~ in the pituitary gland, stimulated the release of gonadotropin and of prolactin. Their findings suggested that IL-l exerted its effect on pituitary cells both directly and through IL-6 production. IL-6 has also shown to be active on cells of the nervous system. It was found to induce the differentiation of a rat pheochromocytoma cell line into neural cells, mimicking the effects observed in this system with nerve growth factor 27 . It also induces nerve growth factor secretion by astrocytes l5 . The local production by microglial cells and by astrocytes during viral infection of the central nervous system 15 suggest the IL-6 may thus not only serve to stimulate immune defences but also to activate tissue repaIr. Thus IL-6 seems not only to be a functional cytokine that regulates the growth and differentiation of various tissues, it is also known for its role in the acute phase response. Increased level of IL-6 in the central nervous system of experimental allergic encephalitis diseased rats 16 and in acute bacterial viral meningitis in the human were reported. IL-6 is rapidly cleared from the blood stream and is taken up by the liverll. This observation lightened up the reported absence of "elevated IL-6" in the cerebro-spinal fluid of multiple sclerosis patients l4,20 and favors the idea of IL-6 production during initiation or exacerbation of the disease. During the last few years, we have learned that a single cytokine can interact with more than one type of cell, that a single cytokine has multiple biological activities, that a single cell can interact with more than one cytokine, and that several cytokines have overlapping activities. Today little is yet known about the type of cells synthesizing them in the nervous and the endocrine systems. The identification of those cells synthesizing the monokine as well as the cells bearing receptors for IL-l and IL-6 will enable us to learn more about their effects and their interplay in the neuroendocrine immune system. References 1 Affolter HV, Reisin ET (1985) Corticotroph releasing factor increases proopiomelanocortin messenger RNA in mouse anterior pituitary tumor cells.] Bioi Chem 260: 15477 2 Bazan]F (1989) A novel family of growth-factor receptors A common binding domain in the growth-hormone, prolactin, erythropoietin and IL-6 receptors, and the P 75 IL-2 receptor ~-chain.] Biochem Biophys Res Commun 164: 788-795 3 Beach ]E, Smallridge RC, Kinzer CA, Bernton EW, Holiday ]W, Fein HG (1989) Rapid release of multiple hormones from rat pituitaries perifused with recombinant Interleukin-l. Life Sci 44: 1-7 4 Berkenbosch F, van Oers], del Rey A, Tilders F, Besedovsky H (1987) Corticotropin-releasing factor-producing neurons in the rat activated by interleukin-l. Science 238: 524-526 5 Bernton EW, Beach JE, Holaday JW, Smallridge Re, Fein HG (1987) Release of multiple hormones by a direct action of interleukin-1 on pituitary cells. Science 238: 519-521

Interleukins in the Neuroendocrine Immune System? . 625 6 Besedovsky HO, deiRey A, Sorkin E, Da Prada M, Burri R, Honegger C (1983) The immune response evokes changes in brain noradrenergic neurons. Science 221: 564-565 7 Breder CD, Dinarello CA, Safer CB (1988). Interleukin-l immunoreactive innervations of the human rat hypothalamus. Science 240: 321-324 8 Brown MR, Fisher LA, Spiess ]S, Rivier C, Rivier], Vale W (1982) Corticotropin releasing factor: actions in the sympathic nervous system and metabolism. Endocrinology 111: 928-932 9 Brown SL, Smith LR, Blalock]E (1987) Interleukin-l and interleukin-2 enhance proopiomelanocortin gene expression in pituitary cells.] Immunol139: 3181-3183 10 Carmeliet P, Van Damme], Denef C (1989) Interleukin-l~ inhibits acetylcholine synthesis in the pituitary corticotropic cell line AtT 20. Brain Res 491: 199-203 11 Castell]V, Geiger T, Gross V, Andus T, Walter E, Hirano T, Kishimoto T, Heinrich PC (1988) Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat. Eur] Biochem 177: 357-361 12 Dower SK, Kronheim SR, Hopp TP, Cantrell M, Deeley M, Gillis S, Henney CS, Urdal DL (1986) The cell surface receptors for interleukin-la and interleukin-l~ are identical. Nature 324: 266-268 13 Dunn A] (1988) Systemic interleukin-l administration stimulates hypothalamic norepinephrine metabolism parallelling the increased plasma cortisone. Life Sci 43: 429-435 14 Frei K, Leist TP, Meager A, Gallo P, Leppert D, Zinkernagel RM, Fontana A (1988) Production of B cell stimulatory factor-2 and interferon y in the central nervous system during viral meningitis and encephalitis.] Exp Med 168: 449-453 15 Frei K, Malipiero UV, Leist TP, Zinkernagel RM, Schwab ME, Fontana A (1989) On the cellular source and function of interleukin 6 produced in the central nervous system in viral diseases. Eur] Immunol19: 689-694 16 Gijbels K, Van Damme], Proost P, Put W, Carton H, Billiau A (1990) Interleukin-6 production in the central nervous system during experimental autoimmune encephalomyelitis. Eur ] Immunol 20: 223-235 17 Giulian D, Young DG, Woodward], Brown DC, Lachman LB (1988) Interleukin-l is an astroglial growth factor in developing brain. ] Neurosci 8: 709-714 18 Giulian D, Lachman LB (1985) Interleukin-l stimulation of astroglial proliferation after brain injury. Science 228: 497-499 19 Giulan D, Woodward], Young DG, Krebs ]F, Lachman LB (1988) Interleukin-l injected into mammalian brain stimulates astrogliosis and neovascularization. ] Neurosci 8: 2485-2490 20 Houssiau FA, Bukasa K, Sindic ClM, Van Damme], Van Snick] (1988) Elevated levels of 26 K human hybridoma growth factor (interleukin 6) in cerebrospinal fluid of patients with acute infection of the central nervous system. Clin exp Immunol 71: 320-323 21 Kabiersch A, Del Rey A, Honegger CG, Besedovsky HO (1988) Interleukin-1 induces changes in norepinephrine metabolism in the rat brain. Brain Behav Immunol 2: 267-274 22 Lechan RM, Toni R, Clark BD, Cannon ]G, Shawar, Dinarello CA, Reichlin S (1990) Immunoreactive interleukin-l~ localization in the rat forebrain. Brain Res 514: 135-140 23 March q, Mosley B, Larsen A, Ceretti DP, Braedt G, Price V, Gillis S, Henney CS, Kronheim SR, Grabstein K, Conlon P],

Hopp TP, Cosman D (1985) Cloning, sequence and expression of two distinct human interleukin-l complementary DNAs. Nature 315: 641-647 24 Naitoh Y, Fukata], Tominaga T, Nakai Y, Tarnai S, Mori K, Imura H (1988) Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 155: 1459-1463 25 Plaut M (1987) Lymphocyte hormone receptors. Ann Rev Immunol5: 621-629 26 Sapolsky R, Rivier C, Yamamoto G, Plotsky P, Vale W (1987) Interleukin-l stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 238: 522-524 27 Satoh T, Nakamura S, Taga T, Matsuda T, Hirano T, Kishimoto T, Kaziro Y (1988) Induction of neuronal differentiation in PC 12 cells by B-cell stimulatory factor l/interleukin-6. Molec Cell BioI 8: 3546-3549 28 Spangelo BL, Judd AM, Isakson PC, MacLeod RM (1989) Interleukin-6 stimulates anterior pituitary hormone release in vitro. Endocrinology 125: 575-577 29 Spangelo BL, MacLeod RM, Isakson PC (1990) Production of interleukin-6 by anterior pituitary cells in vitro. Endocrinology 126:582-586 30 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 adrenocorticotropic secretagogues and interleukin-1 on proopiomelanocortin gene expression in rat anterior pituitary cells. Endocrinology 124: 1444-1449 31 Thieme TR, Hefeneider SH, Wagner CR, Bach DR (1987) Recombinant murine and human IL-la bind to human endothelial cells with an equal affinity, but have an unequal ability to induce endothelial cell adherence of lymphocytes.] Immunol13 9: 1173-1178 32 Tilders F]H, Berkenbosch F, Vermes I, Lindon EA, Smelik PG (1985) Role of epinephrine and vasopressin in the control of the pituitary-adrenal response to stress. Fed ProclFed Am Soc exp BioI 44: 155-160 33 Tsagarakis S, Gillies G, Rees LH, Besser M, Grossman A (1989) Interleukin-1 directly stimulates the release of corticotropin releasing factor from rat hypothalamus. Neuroendocrinology 49: 98-101 34 Uehara A, Minamino N, Townsend MH, Arimura A (1986) Corticotropin-releasing activity in the rat gastrum antrum. Proc Soc exp BioI Med 183: 106-113 35 Uehara A, Gillis S, Arimura A (1987) Effects of interleukin-1 on hormone release from normal rat pituitary cells in primary culture. Neuroendocrinology 45: 343-347 36 Uehara A, Gottschall PE, Dahl RR, Arimura A (1987) Interleukin-1 stimulates ACTH release by an indirect action which requires endogenous corticotropin releasing factor. Endocrinology 121: 1580-1582 37 Vankelecom H, Carmeliet P, Van Damme], Billiau A, Denef C (1989) Production of interleukin-6 by folliculo-stellate cells and hormone-secreting cells in peri fused anterior pituitary cell aggregates. Neuroendocrinology 49: 102-106 38 Woloski BMRN], Smith EM, Meyer W] III, Fuller GM, Blalock]E (1985) Corticotropin-releasing activity of monokines. Science 230: 1035-1037 39 Yamaguchi M, Matsuzaki N, Hirota K, Miyake A, Tanizawa 0 (1990) Interleukin-6 possibly induced by interleukin-l~ in the pituitary gland stimulates the release of gonadotropins and prolactin. Acta Endocrinoll22: 201-205

Received December 15, 1990 . Accepted January 16, 1991

Key words: Interleukin-l - Interleukin-6 Prof. E. L. Hooghe-Peters, Farmacologie-VUB, Laarbeklaan 103, B-1090 Brussels, Belgium