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Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain
Neuroscience Letters, 136 (1992) 189-192 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
189
NSL 08427
Localization of interleukin 6 m R N A and interleukin 6 receptor m R N A in rat brain Bernd Sch6bitz, D o r o t h y A.M. Voorhuis and E. Ronald De Kloet Max-Planck-lnstitute for Psychiatry, Clinical Institute, Munich (FRG) and Center for Bio-Pharmaceutical Sciences, Division of Medical Pharmacology, University of Leiden, Leiden (Netherlands) (Received 6 October 1991; Revised version received 15 November 1991; Accepted 3 December 199 l)
Key words: Interleukin 6; Interleukin 6 receptor; In situ hybridization; Rat; Hippocampus; Hypothalamus; Glia The cytokine interleukin 6 (IL6) has several effects on the central nervous system in addition to the well established regulation of the acute phase inflammatory response. Therefore, the distribution of IL6- and IL6 receptor mRNA in the rat brain has been investigated by in situ hybridization using [35S]-labeled oligonucleotides. The messages of both genes were found in the CA1-CA4 regions as well as in the dentate gyms of the hippocampus, in the habenulae, the dorsomedial and the ventromedial hypothalamus, in the internal capsule, the optic tract and in the piriform cortex. These data indicate both neuronal and glial localization of IL6 and IL6 receptor and their involvement in an autocrine or paracrine action of the cytokine in centrally regulated functions including neuroendocrine control.
During inflammatory conditions, the cytokine interleukin 6 (IL6) is induced transiently and mediates hepatic acute phase protein synthesis, B cell growth and hematopoiesis [1]. Moreover, IL6 has prominent effects on the central nervous system (CNS). These central effects include stimulation of neuronal growth, reduction in food intake, induction of fever and activation of the hypothalamic-pituitary-adrenal (HPA) axis [1]. An interaction of IL 1, IL6 and other cytokines, with the limbic system and/ or the hypothalamus elevates serum adrenocorticotropic hormone (ACTH) and glucocorticoid levels during infections [1]. The resultant increase in glucocorticoid level is thought to protect the host against an overshoot of immune reactions. A direct transport of IL6 into the CNS seems unlikely in view of the hydrophilic nature and molecular weight of the protein. Two findings suggest that IL6 might be produced within the brain. Firstly, high brain IL6 levels have been demonstrated in the absence of elevated blood IL6 concentrations [6]. Secondly, astrocytes and microglia cells can be stimulated in vitro to produce IL6 [3]. The central effects of IL6 are supposed to depend on the presence of a membrane associated receptor. So far, binding of radiolabeled IL6 has only been shown for
Correspondence: B. Schrbitz, Center for Bio-Pharmaceutical Sciences, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands. Fax: (31) (71) 276292.
astrocytoma and glioblastoma cell lines [10] and not for untransformed cells and 'normal' brain tissues. Thus, in spite of this increasing evidence for the effects of IL6 on centrally regulated functions, little is known about the actual source and site of action of the cytokine in the brain. To address these issues, it is necessary to identify brain areas expressing IL6 and its receptor. In the present work, in situ hybridization has been employed to demonstrate that the mRNAs of IL6 and its receptor are codistributed in highly discrete rat brain regions. In situ hybridization was performed as described by Young [11]. In brief, male adult Wistar rats (n--5) were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde/PBS. Brains were frozen after incubation in 15% sucrose/PBS and 10 gm coronal sections were mounted on poly-L-lysine-coated glass slides. Four oligonucleotide probes of the IL6- and IL6 receptor cDNAs were synthesized. Probe A: base 144-183 of the published rat IL6 cDNA sequence [8]. Probe B and C: base 761-800 and 662-700 of the published rat IL6 receptor cDNA sequence [2], respectively. Probe D which is complementary to probe A served as a sense probe. The oligonucleotides were labeled with terminal transferase (Boehringer Mannheim, The Netherlands) using [35S]dATP (1000 Ci/mmol; Amersham, The Netherlands). A specific activity of 5x 10s cpm/gg DNA was obtained. Sections were hybridized with 1.5x 107 cpm/ml of the labeled probe at 40°C for 16 h. Thereafter, slides
190
IL6 mRNA
IL6 receptor mRNA
.A,
E
(2,
G
D
H
Fig. 1. Autoradiograms of coronal sections of brain areas 3 mm posterior to bregma following in situ hybridization with [3sS]-Iabeledoligonucleotides. A: IL6 mRNA detected with probe A as described in text. B: RNase pretreated section incubated with probe A. C: hybridization with probe A in the presence of excess unlabeled oligonucleotide. D: incubation of a section with the IL6 sense probe D, E: IL6 receptor mRNA detected with probe B. F: RNase pretreated section incubated with probe B. G: hybridization with probe B in the presence of excess unlabeled oligonucleotide. H: IL6 receptor mRNA detected with probe C. DG, dentate gyrus; Hb, habenular nucleus; opt, optic tract: ic, internal capsule; DH, dorsomedial hypothalamus; VH, ventromedial hypothalamus; Pir, piriform cortex.
191 were washed with 0.2×SSC/20 m M mercaptoethanol at 40°C and exposed to X-ray films for about 5 days. In control experiments sections were treated before prehybridization with 20 Bg/ml RNase A (Sigma Chemical Co.) in 2×SSC for 1 h at 37°C. Further controls were performed in the presence of 2 Bg/ml unlabeled IL6 sense oligonucleotides. The specificity of in situ hybridization was concluded from the following observations: (i) pretreatment of sections with RNase (Fig. 1B,F) or hybridization in the presence of an excess of unlabeled oligonucleotides (Fig. 1C,G) abolished the hybridization signal. (ii) Experiments performed with a labeled IL6 sense probe resulted only in non-specific binding of radioactive material (Fig. 1D). (iii) Identical patterns of IL6 receptor m R N A distribution were obtained using two different probes (Fig. 1E,H). Fig. 1A,E,H shows that the mRNAs of IL6 and the IL6 receptor are colocalized in brain sections of about 3 mm posterior to bregma. The autoradiographic signal was markedly above background in the hippocampal formation (CA1, CA2, CA3 and CA4 field) with highest m R N A levels in the dentate gyrus (DG). Moreover, both mRNAs were found in the habenular nucleus (Hb), the piriform cortex (Pir) and the dorsomedial and the ventromedial part of the hypothalamus (DH and VH, resp.) including the periventricular nucleus. Labeling was also observed in the internal capsule (ic) and the optic tract (opt). Other white matter areas, such as the corpus callosum, which contain only fiber tracts and glia cells, were not labeled at this brain level. The present results clearly show regional codistribution of IL6- and IL6 receptor mRNAs in the rat brain. Localization of the messages was found in discrete areas under highly specific experimental conditions. In view of this distinct regional distribution of IL6- and IL6 receptor mRNAs, hybridization to perivascular leucocytes or endothelial cells seems unlikely. The highly discrete density of signal in the granule cell layer of the dentate gyrus and the pyramidal cells of the CA1-CA4 fields of the hippocampus suggests neuronal rather than glial localization of the mRNAs in this brain region. However, since both IL6- and I6-receptor mRNAs were detected in the optic tract and the internal capsule, areas devoid of neurons, the message in these regions is present in glial cells. Several in vitro studies have already demonstrated gene expression of IL6 in microglia cells and astrocytes [3]. With respect to the distribution of IL6- and IL6 receptor mRNAs in the brain and the recently discovered central actions of IL6, 3 different functions can be considered: (i) the cytokine may act as B cell growth factor in the brain or stimulate T lymphocytes during inflamma-
tion or infections [1] of the brain. Analogous to the reaction in peripheral organs, microglia cells may function under those conditions as tissue macrophages activating lymphocytes, which invade the brain. (ii) IL6 like ILl may act as a neurotrophic factor by stimulating nerve growth factor release [3]. Recently, it has been reported that IL6 facilitates the growth of PC12 cells [1] as well as of catecholaminergic and cholinergic neurons [5]. Moreover, it has been suggested that IL6 mediates the synthesis of acute phase protease inhibitors in the brain and thus may be implicated in the etiology of Alzheimer's disease [4]. (iii) IL6 can be released in vitro from rat medial basal hypothalamus and stimulates the release of CRH [7, 9]. The pattern oflL6- and IL6 receptor m R N A distribution, as observed in this study, especially in limbic and hypothalamic brain areas, is also consistent with an important neuroendocrine role of IL6. Hippocampus and hypothalamus regulate the HPA axis, body temperature and food intake --- effects which can be caused by IL6 in a similar mode as by ILl [1]. The data of this study support the concept that under pathophysiological conditions of inflammation host defense supported by the central component of the acute phase reaction (fever, anorexia and release of corticotropin releasing hormone (CRH)) may be partly mediated by the interaction of brain-synthesized IL6 with its specific receptor. The coordinated action of CRH and cytokines in the brain might then cause the symptoms known as 'unspecific sickness behavior'. We express our appreciation to Mr. Mark Fluttert for his excellent technical assistance as well as to Dr. Matthias Baumann, Dr. Jurrien de Koning and Dr. Win Sutanto for expert advice. We gratefully acknowledge the synthesis of oligonucleotide probes by Dr. Paul Murphy and their purification by Dr. Piero Giordano. We wish to thank Prof. Dr. Florian Holsboer from the Max-Planck-Institute for Psychiatry, Munich, for critically reading the manuscript. 1 Akira, S., Hirano, T., Taga, T. and Kishimoto,T., Biologyof multifunctionalcytokines:IL 6 and related molecules(IL 1 and TNF), FASEBJ., 4 (1990) 2860-2867. 2 Baumann, M., Baumann, H. and Fey, G.H., Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor, J. Biol. Chem.,265 (1990) 19853-19862. 3 Frei, K., Malipiero, U.V., Leist, T.P., Zinkernagel,R.M., Schwab, M.E. and Fontana, A., On the cellularsource and functionof interleukin 6 produced in the central nervous system in viral diseases, Eur. J. Immunol., 19 (1989) 689-694. 4 Ganter. U., Strauss, S., Jonas, U., Weidemann,A., Beyreuther,K., Volk, B., Berger, M. and Bauer, J., Alpha 2-macroglobulinsynthesis in interleukin-6-stimulatedhuman neuronal (SH-SY5Y neuroblastoma) cells, FEBS Lett., 282 (1991) 127-131. 5 Hama, T., Kushima,Y., Miyamoto,M., Kubota, M., Takei,N. and
192 Hatanaka, H., lnterleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week old rats in cultures, Neuroscience, 40 (1991) 445 452. 6 Houssiau, F.A., Bukasa, K., Sindic, C.J.M., Van Damme, J. and Van Snick, J., Elevated levels of the 26K human hybridoma growth factor (interleukin 6) in cerebrospinal fluid of patients with acute infection of the central nervous system, Clin. Exp. Immunol.. 71 (1988) 320-323. 7 Navarra, L., Tsagarakis, S., Faria, M.S., Rees, L.H., Besser, G.M. and Grossman, A.B., lnterleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway, Endocrinology, 128 (1991) 3744.
8 Northemann, W., Braciak, T.A., Hattori, M., Lee, F. and Fey, G.H., Structure of the rat interleukin 6 gene and its expression in macrophage-derived cells, J. Biol. Chem., 264 (1989) 16072-16082. 9 Spangelo, B.L., Judd, A.M., MacLeod, R.M., Goodman, D.W. and Isakson, RC., Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami, Endocrinology, 127 (1990) 1779-1785. 10 Taga, T., Kawanashi, Y., Hardy, R.R., Hirano, T. and Kishimoto, T., Receptors for B cell stimulatory factor 2, J. Exp. Med., 166 (1987) 967-981. I 1 Young, W.S., Hormone Action. Methods in Enzymology, Vol. 168, Part K: Neuroendocrine Peptides, Academic Press, New York, 1989.
189
NSL 08427
Localization of interleukin 6 m R N A and interleukin 6 receptor m R N A in rat brain Bernd Sch6bitz, D o r o t h y A.M. Voorhuis and E. Ronald De Kloet Max-Planck-lnstitute for Psychiatry, Clinical Institute, Munich (FRG) and Center for Bio-Pharmaceutical Sciences, Division of Medical Pharmacology, University of Leiden, Leiden (Netherlands) (Received 6 October 1991; Revised version received 15 November 1991; Accepted 3 December 199 l)
Key words: Interleukin 6; Interleukin 6 receptor; In situ hybridization; Rat; Hippocampus; Hypothalamus; Glia The cytokine interleukin 6 (IL6) has several effects on the central nervous system in addition to the well established regulation of the acute phase inflammatory response. Therefore, the distribution of IL6- and IL6 receptor mRNA in the rat brain has been investigated by in situ hybridization using [35S]-labeled oligonucleotides. The messages of both genes were found in the CA1-CA4 regions as well as in the dentate gyms of the hippocampus, in the habenulae, the dorsomedial and the ventromedial hypothalamus, in the internal capsule, the optic tract and in the piriform cortex. These data indicate both neuronal and glial localization of IL6 and IL6 receptor and their involvement in an autocrine or paracrine action of the cytokine in centrally regulated functions including neuroendocrine control.
During inflammatory conditions, the cytokine interleukin 6 (IL6) is induced transiently and mediates hepatic acute phase protein synthesis, B cell growth and hematopoiesis [1]. Moreover, IL6 has prominent effects on the central nervous system (CNS). These central effects include stimulation of neuronal growth, reduction in food intake, induction of fever and activation of the hypothalamic-pituitary-adrenal (HPA) axis [1]. An interaction of IL 1, IL6 and other cytokines, with the limbic system and/ or the hypothalamus elevates serum adrenocorticotropic hormone (ACTH) and glucocorticoid levels during infections [1]. The resultant increase in glucocorticoid level is thought to protect the host against an overshoot of immune reactions. A direct transport of IL6 into the CNS seems unlikely in view of the hydrophilic nature and molecular weight of the protein. Two findings suggest that IL6 might be produced within the brain. Firstly, high brain IL6 levels have been demonstrated in the absence of elevated blood IL6 concentrations [6]. Secondly, astrocytes and microglia cells can be stimulated in vitro to produce IL6 [3]. The central effects of IL6 are supposed to depend on the presence of a membrane associated receptor. So far, binding of radiolabeled IL6 has only been shown for
Correspondence: B. Schrbitz, Center for Bio-Pharmaceutical Sciences, Leiden University, P.O. Box 9503, 2300 RA Leiden, The Netherlands. Fax: (31) (71) 276292.
astrocytoma and glioblastoma cell lines [10] and not for untransformed cells and 'normal' brain tissues. Thus, in spite of this increasing evidence for the effects of IL6 on centrally regulated functions, little is known about the actual source and site of action of the cytokine in the brain. To address these issues, it is necessary to identify brain areas expressing IL6 and its receptor. In the present work, in situ hybridization has been employed to demonstrate that the mRNAs of IL6 and its receptor are codistributed in highly discrete rat brain regions. In situ hybridization was performed as described by Young [11]. In brief, male adult Wistar rats (n--5) were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde/PBS. Brains were frozen after incubation in 15% sucrose/PBS and 10 gm coronal sections were mounted on poly-L-lysine-coated glass slides. Four oligonucleotide probes of the IL6- and IL6 receptor cDNAs were synthesized. Probe A: base 144-183 of the published rat IL6 cDNA sequence [8]. Probe B and C: base 761-800 and 662-700 of the published rat IL6 receptor cDNA sequence [2], respectively. Probe D which is complementary to probe A served as a sense probe. The oligonucleotides were labeled with terminal transferase (Boehringer Mannheim, The Netherlands) using [35S]dATP (1000 Ci/mmol; Amersham, The Netherlands). A specific activity of 5x 10s cpm/gg DNA was obtained. Sections were hybridized with 1.5x 107 cpm/ml of the labeled probe at 40°C for 16 h. Thereafter, slides
190
IL6 mRNA
IL6 receptor mRNA
.A,
E
(2,
G
D
H
Fig. 1. Autoradiograms of coronal sections of brain areas 3 mm posterior to bregma following in situ hybridization with [3sS]-Iabeledoligonucleotides. A: IL6 mRNA detected with probe A as described in text. B: RNase pretreated section incubated with probe A. C: hybridization with probe A in the presence of excess unlabeled oligonucleotide. D: incubation of a section with the IL6 sense probe D, E: IL6 receptor mRNA detected with probe B. F: RNase pretreated section incubated with probe B. G: hybridization with probe B in the presence of excess unlabeled oligonucleotide. H: IL6 receptor mRNA detected with probe C. DG, dentate gyrus; Hb, habenular nucleus; opt, optic tract: ic, internal capsule; DH, dorsomedial hypothalamus; VH, ventromedial hypothalamus; Pir, piriform cortex.
191 were washed with 0.2×SSC/20 m M mercaptoethanol at 40°C and exposed to X-ray films for about 5 days. In control experiments sections were treated before prehybridization with 20 Bg/ml RNase A (Sigma Chemical Co.) in 2×SSC for 1 h at 37°C. Further controls were performed in the presence of 2 Bg/ml unlabeled IL6 sense oligonucleotides. The specificity of in situ hybridization was concluded from the following observations: (i) pretreatment of sections with RNase (Fig. 1B,F) or hybridization in the presence of an excess of unlabeled oligonucleotides (Fig. 1C,G) abolished the hybridization signal. (ii) Experiments performed with a labeled IL6 sense probe resulted only in non-specific binding of radioactive material (Fig. 1D). (iii) Identical patterns of IL6 receptor m R N A distribution were obtained using two different probes (Fig. 1E,H). Fig. 1A,E,H shows that the mRNAs of IL6 and the IL6 receptor are colocalized in brain sections of about 3 mm posterior to bregma. The autoradiographic signal was markedly above background in the hippocampal formation (CA1, CA2, CA3 and CA4 field) with highest m R N A levels in the dentate gyrus (DG). Moreover, both mRNAs were found in the habenular nucleus (Hb), the piriform cortex (Pir) and the dorsomedial and the ventromedial part of the hypothalamus (DH and VH, resp.) including the periventricular nucleus. Labeling was also observed in the internal capsule (ic) and the optic tract (opt). Other white matter areas, such as the corpus callosum, which contain only fiber tracts and glia cells, were not labeled at this brain level. The present results clearly show regional codistribution of IL6- and IL6 receptor mRNAs in the rat brain. Localization of the messages was found in discrete areas under highly specific experimental conditions. In view of this distinct regional distribution of IL6- and IL6 receptor mRNAs, hybridization to perivascular leucocytes or endothelial cells seems unlikely. The highly discrete density of signal in the granule cell layer of the dentate gyrus and the pyramidal cells of the CA1-CA4 fields of the hippocampus suggests neuronal rather than glial localization of the mRNAs in this brain region. However, since both IL6- and I6-receptor mRNAs were detected in the optic tract and the internal capsule, areas devoid of neurons, the message in these regions is present in glial cells. Several in vitro studies have already demonstrated gene expression of IL6 in microglia cells and astrocytes [3]. With respect to the distribution of IL6- and IL6 receptor mRNAs in the brain and the recently discovered central actions of IL6, 3 different functions can be considered: (i) the cytokine may act as B cell growth factor in the brain or stimulate T lymphocytes during inflamma-
tion or infections [1] of the brain. Analogous to the reaction in peripheral organs, microglia cells may function under those conditions as tissue macrophages activating lymphocytes, which invade the brain. (ii) IL6 like ILl may act as a neurotrophic factor by stimulating nerve growth factor release [3]. Recently, it has been reported that IL6 facilitates the growth of PC12 cells [1] as well as of catecholaminergic and cholinergic neurons [5]. Moreover, it has been suggested that IL6 mediates the synthesis of acute phase protease inhibitors in the brain and thus may be implicated in the etiology of Alzheimer's disease [4]. (iii) IL6 can be released in vitro from rat medial basal hypothalamus and stimulates the release of CRH [7, 9]. The pattern oflL6- and IL6 receptor m R N A distribution, as observed in this study, especially in limbic and hypothalamic brain areas, is also consistent with an important neuroendocrine role of IL6. Hippocampus and hypothalamus regulate the HPA axis, body temperature and food intake --- effects which can be caused by IL6 in a similar mode as by ILl [1]. The data of this study support the concept that under pathophysiological conditions of inflammation host defense supported by the central component of the acute phase reaction (fever, anorexia and release of corticotropin releasing hormone (CRH)) may be partly mediated by the interaction of brain-synthesized IL6 with its specific receptor. The coordinated action of CRH and cytokines in the brain might then cause the symptoms known as 'unspecific sickness behavior'. We express our appreciation to Mr. Mark Fluttert for his excellent technical assistance as well as to Dr. Matthias Baumann, Dr. Jurrien de Koning and Dr. Win Sutanto for expert advice. We gratefully acknowledge the synthesis of oligonucleotide probes by Dr. Paul Murphy and their purification by Dr. Piero Giordano. We wish to thank Prof. Dr. Florian Holsboer from the Max-Planck-Institute for Psychiatry, Munich, for critically reading the manuscript. 1 Akira, S., Hirano, T., Taga, T. and Kishimoto,T., Biologyof multifunctionalcytokines:IL 6 and related molecules(IL 1 and TNF), FASEBJ., 4 (1990) 2860-2867. 2 Baumann, M., Baumann, H. and Fey, G.H., Molecular cloning, characterization and functional expression of the rat liver interleukin 6 receptor, J. Biol. Chem.,265 (1990) 19853-19862. 3 Frei, K., Malipiero, U.V., Leist, T.P., Zinkernagel,R.M., Schwab, M.E. and Fontana, A., On the cellularsource and functionof interleukin 6 produced in the central nervous system in viral diseases, Eur. J. Immunol., 19 (1989) 689-694. 4 Ganter. U., Strauss, S., Jonas, U., Weidemann,A., Beyreuther,K., Volk, B., Berger, M. and Bauer, J., Alpha 2-macroglobulinsynthesis in interleukin-6-stimulatedhuman neuronal (SH-SY5Y neuroblastoma) cells, FEBS Lett., 282 (1991) 127-131. 5 Hama, T., Kushima,Y., Miyamoto,M., Kubota, M., Takei,N. and
192 Hatanaka, H., lnterleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week old rats in cultures, Neuroscience, 40 (1991) 445 452. 6 Houssiau, F.A., Bukasa, K., Sindic, C.J.M., Van Damme, J. and Van Snick, J., Elevated levels of the 26K human hybridoma growth factor (interleukin 6) in cerebrospinal fluid of patients with acute infection of the central nervous system, Clin. Exp. Immunol.. 71 (1988) 320-323. 7 Navarra, L., Tsagarakis, S., Faria, M.S., Rees, L.H., Besser, G.M. and Grossman, A.B., lnterleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway, Endocrinology, 128 (1991) 3744.
8 Northemann, W., Braciak, T.A., Hattori, M., Lee, F. and Fey, G.H., Structure of the rat interleukin 6 gene and its expression in macrophage-derived cells, J. Biol. Chem., 264 (1989) 16072-16082. 9 Spangelo, B.L., Judd, A.M., MacLeod, R.M., Goodman, D.W. and Isakson, RC., Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami, Endocrinology, 127 (1990) 1779-1785. 10 Taga, T., Kawanashi, Y., Hardy, R.R., Hirano, T. and Kishimoto, T., Receptors for B cell stimulatory factor 2, J. Exp. Med., 166 (1987) 967-981. I 1 Young, W.S., Hormone Action. Methods in Enzymology, Vol. 168, Part K: Neuroendocrine Peptides, Academic Press, New York, 1989.