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Interleukin-6 receptor expression and localization after transient global ischemia in gerbil hippocampus
Neuroscience Letters 341 (2003) 49–52 www.elsevier.com/locate/neulet
Interleukin-6 receptor expression and localization after transient global ischemia in gerbil hippocampus Florence Vollenweidera,*, Martina Herrmanna, Uwe Ottenb, Cordula Nitscha a
Institute of Anatomy, Basel University, Pestalozzistrasse 20, CH-4056 Basel, Switzerland b Department of Physiology, Vesalianum, Vesalgasse 1, CH-4056 Basel, Switzerland
Received 13 December 2002; received in revised form 23 January 2003; accepted 27 January 2003
Abstract Ischemia results in increased interleukin-6 (IL-6) expression in the brain. To prove a connection between IL-6 upregulation and IL-6 receptor (IL-6R) expression following ischemia, we analyzed cell-type specific expression changes of IL-6R using transient global ischemia in the gerbil as a model. In sham operated animals, IL-6R mRNA and protein were mainly detected in hippocampal pyramidal cells and interneurons. After ischemia, IL-6R was expressed in neurons but there was no increase during the peak IL-6 expression. Neuronal IL-6R mRNA and protein decreased in parallel with pyramidal cell death, starting 2 days after ischemia. Double-labeling experiments revealed that in postischemic hippocampus IL-6R was not present in GFAP-reactive astrocytes but that the surviving parvalbumin containing interneurons expressed IL-6R mRNA. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Interleukin-6; Stroke; Hippocampus; Cornu amonis-1; Cytokine; Neuroprotection; Parvalbumin
Ischemic brain injury is characterized by apoptotic and necrotic events. In addition, recent evidence suggests that the inflammatory response plays an important role in the outcome of the insult, in particular with respect to neuronal survival. Much of this inflammatory response appears to be mediated by interleukins, a multifunctional subclass of cytokines. The cytokine Interleukin-6 (IL-6), a B-cell stimulating factor, is one of the major cytokines expressed in the brain [9]. Although the principal source of IL-6 has been considered to be microglia and/or astrocytes, it has been clearly shown that it is also expressed in neurons [5,6, 18]. Recent studies demonstrated the ability of IL-6 to exert neuroprotective effects [7,10,11,13]. In IL-6 deficient-mice, however, the infarct size was identical to normal mice after focal central nervous system (CNS) ischemia [4], and severe neurologic impairment was observed in transgenic mice overexpressing IL-6. Taken together, these findings suggest a complex role for IL-6 in the pathology of CNS disorders [3]. To exert its effects, IL-6 must be bound to its receptor. The IL-6 receptor (IL-6R) is composed of 2 glycoproteins: a * Corresponding author. Tel.: þ 41-61-2672729; fax: þ 41-61-2673959. E-mail address: [email protected] (F. Vollenweider).
transmembrane glycoprotein (Gp130), the signal transducing subunit and the IL-6 receptor, which exists in a membrane bound and a soluble form. Cellular activation of IL-6 signaling mediates phosphorylation of Gp130-associated cytoplasmic tyrosine kinases and phosphorylation and activation of transcriptional signals [8]. Whether the increase in IL-6 levels after ischemia is accompanied by an augmentation of its receptor expression remains unclear. An enhancement of receptor expression was observed between 4 and 24 h after a mechanical lesion in the rat striatum [20]. Elevated soluble receptor has been observed in a number of diseases [8]. However, it has also been shown by reverse transcription polymerase chain reaction (RT-PCR) that focal cerebral ischemia in rats upregulated the expression of IL-6 mRNA in cortex and striatum without affecting the transcription of its receptor [1]. The present study was undertaken to clarify the timecourse of expression and localization of the IL-6R after transient forebrain ischemia in gerbils. In particular, we determined which types of cells express the protein and therefore could interact with the cytokine IL-6. Finally, we compared the expression levels with the neuropathological changes provoked by the insult.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00136-8
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F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
Two to 6 month-old male Mongolian gerbils (50 – 70 g) were subjected to a 7 min bilateral forebrain ischemia or sham operation. Five and 12 h, 2 and 4 days after recirculation, animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). At least three animals per time-point were used. The brains were post-fixed in PFA, cryoprotected in 30% sucrose and frozen in isopentane. Serial sagittal 35 mm sections were cut with a cryostat. Neuronal cell loss was estimated by cresyl violet staining. For immunoreaction, free-floating sections were treated with H2O2 and PBS/0.1% saponin was then used as buffer. After blocking in normal goat serum (5%) for 90 min, sections were then incubated with a polyclonal rat antibody against IL-6R (1:50; Santa Cruz, USA) for 48 h at room temperature. Incubation with secondary antibody and detection with the avidin-biotin complex (Vector) and the diaminobenzidine reaction were performed. To provide a non-specific control, the primary antibody was omitted. IL6R riboprobe was transcribed from a pSPT19 vector containing the rat IL-6 receptor [5] in the presence of digoxigenin-UTP (Roche Molecular Biochemicals). In situ hybridization was performed as previously described [2]. Briefly, for hybridization, digoxygenin labeled probes (antisense or sense as control) were added (1:300), and sections were then incubated at 53 8C overnight. mRNA was detected with a digoxigenin antibody conjugated to alkaline phosphatase and staining was carried out using NBT/BCIPsubstrates (Roche Molecular Biochemicals). A doublelabeling technique was used to further characterize the localization of IL-6R [2] either with antibodies against parvalbumin (PV; 1:2500; Swant, Switzerland) or the astrocyte marker glial fibrillary acidic protein (GFAP; 1:1000; DAKO, Denmark). All animal experimentation was performed with permission of the local animal care committee and according to present Swiss law. Transient forebrain ischemia in the Mongolian gerbil is a reliable model for cerebral ischemia. Due to its incomplete circle of Willis, bilateral transient occlusion of the common carotids arteries produces a highly reproducible forebrain ischemia, resulting in delayed cell death in cornu amonis-1 (CA1) hippocampal neurons. The CA1 pyramidal cells start dying 2 –3 days after the restoration of circulation. After 4 days, most CA1 neurons are degenerated, with the exception of a PV containing subgroup of inhibitory interneurons [15]. In hippocampus, IL-6R was found both in the perikaryon and outlining the apical dendrites of virtually all neurons: it labeled densely CA1, CA2 and CA3 pyramidal cells as well as the granular cells of the dentate gyrus. IL-6R immunoreaction did not differ 5 or 12 h- after ischemia from sham operated animals (Fig. 1A – C). Localization of IL-6R mRNA detected by in situ hybridization correlated with results for the protein obtained by immunoreaction, but was restricted to the perikaryon. The cell body layers of CA, dentate gyrus and subiculum were densely stained. Similar to IL-6R protein, the IL-6R mRNA did not show any significant change early after the ischemic insult (Fig.
Fig. 1. Time-course of IL-6R expression after ischemia: by immunoreaction (IM) left panels (A –E) and by in situ hybridization (ISH) right panels (F– J); Sham and 5, 12 h; 2, 4 days after ischemia. Scale bar ¼ 0.5 mm.
1F– H). IL-6R mRNA data comprise both soluble and membrane-bound receptor forms. A sense probe used as control did not detect any stained cells (data not shown). While already the localization of the mRNA in the hippocampal cell body layers indicated the preferential neuronal expression of IL-6R, we studied this further by double staining with PV as marker of interneurons, and GFAP as marker for astrocytes. Double staining with PV showed preference of IL-6R for interneurons (Fig. 2C). No colocalization of the IL-6R with GFAP was detected in hippocampus, indicating that the major cell type expressing the IL-6R are neurons (Fig. 2A). Identical observations were obtained with double immunofluorescence (data not shown). Two days after transient ischemia, when neuronal cell loss has started in CA1, a decrease in protein expression in the CA1 pyramidal cells was observed in some cases (Fig. 1D). At the same time point, some upregulation of the protein in the neuropil layer was detected. In parallel to the immunohistochemical data, a decrease of mRNA was
F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
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Fig. 2. Colocalization of IL-6R mRNA and GFAP or PV in CA1. Cells are double-stained for GFAP (A,B) or PV (C,D) (brown staining product) and IL-6R mRNA (blue staining product). (A,C) sham operated animal and (B,D) 4 days after ischemia. Arrows show surviving interneurons double-labeled with PV and IL-6R mRNA. Scale bar ¼ 0.1 mm (D).
observed in CA1 two days after ischemia (Fig. 1I). Four days after the ischemic insult and recirculation, when neuronal death was clearly observed in CA1 and sometimes also in CA3 pyramidal cells, IL-6R expression was absent in the pyramidal cell layer (Fig. 1E). A comparison of serial sections in individual animals showed that loss of perikaryal IL-6R staining correlated with neuronal cell death. On the other hand, there was always an upregulation of the IL-6 receptor in the neuropil layer. The in situ hybridization data showed that expression was nearly absent in the CA1 field with the exception of a few single cells located in the pyramidal cell layer and in the stratum oriens (Fig. 1J). Double labeling experiments revealed that these cells were not GFAP-reactive astrocytes (Fig. 2B) but that the surviving PV containing interneurons possessed IL-6R mRNA (Fig. 2D: arrows). Previous studies have shown a peak of IL-6 expression as soon as 1 h after ischemia, which persisted for about 24 h [17,19]. A recent study demonstrated that production of IL6 is increased 24 h up to 7 days after the insult [16]. Our data show that these increases are not paralleled by increases in mRNA or protein levels of the IL-6R. Neither the insult nor the presence of increased IL-6 protein appeared to modulate IL-6R expression 5 or 12 h after ischemia, a time when a peak of IL-6 production was detected and when the delayed neuronal cell death was still reversible. This is in agreement with observations obtained from tissue extract (cortex and
striatum) by RT-PCR [1]. Further, our data suggest that both receptor forms behave similarly. With immunostaining only the membrane-bound receptor is detected. In situ hybridization identifies both receptor forms. However, no increase of IL-6R mRNA was observed after ischemia. The discrepancy between formation of IL-6R mRNA and declining levels of protein could be due to inhibition of overall cerebral protein synthesis which precedes neuronal cell death following ischemia. Thus, neuronal expression of IL-6R decreased together with pyramidal cell death, starting 2 days after ischemia. Some upregulation of IL-6R protein was observed 3 and 4 days post ischemia in the neuropil layer by immunohistoreaction in reactive glial cells. We were unable to detect IL6R in GFAP-reactive astrocytes. Absence of astrocytic expression of the IL-6R suggested that an astrocytic response to injury was unlikely to result from the early production of IL-6. Upregulation of IL-6 as observed by immunoreaction probably occurred mainly in microglia [19]. On the other hand, in vitro studies demonstrated production of IL-6 by astrocytes in response to diverse stimuli. For example, Maeda et al. showed that astrocytes produced IL-6 when exposed to hypoxia followed by a period of reoxygenation [11]. It is nevertheless important to note that in these cases expression of IL-6 and IL-6R was detected in primary cultures of astrocytes or in an astroglioma cell line.
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F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
Colocalization of IL-6R with PV was observed in a certain number of cells providing evidence that not only hippocampal pyramidal cells but also interneurons express the receptor (Fig. 2C,D). Our data demonstrate that after ischemia surviving interneurons expressed the IL-6R (Fig. 2D: arrows). These interneurons have been shown to participate in synaptic rearrangement taking place after ischemic nerve cell death [14]. Lesion-induced reorganization processes require trophic factors. Ma¨rz et al. [12] demonstrated that addition of the soluble IL-6R and the cytokine IL-6 enhanced survival of primary sympathetic neurons in the absence of the nerve growth factor. In this case, IL-6 acts as a trophic factor i.e. has the capacity to promote neuronal survival and differentiation. A prerequisite for these effects is the presence of the soluble IL-6R. The same phenomenon was observed for dorsal root ganglion neurons suggesting that IL-6 bound to the soluble IL-6R has neurotrophic potential [20]. It remains possible that the surviving interneurons produced additional soluble IL-6R, yet the sensitivity of our detection method did not allow to determine this unequivocally and would require additional investigations. IL-6 has been implicated in a number of neuropathological reactions, including acute ischemia. To elicit a direct effect on neurons, receptors for IL-6 must be expressed. In the present study we showed that the IL-6R is expressed in principal neurons and interneurons of the hippocampus; no IL-6R was observed in astrocytes. There was no change in expression during the early time points after transient cerebral ischemia in gerbils, when pyramidal cells were still viable. After delayed pyramidal cell death, IL-6R was still present in surviving interneurons. In summary, our results suggest that IL-6R is expressed constitutively in hippocampal neurons and that the increased production of IL-6 early after an ischemic insult does not induce a change in its receptor expression. In our model, there exists no indication for a neuroprotective effect of IL-6.
Acknowledgements The authors wish to thank Prof. C. Grothe and K. Kuhlemann for advice with the in situ hybridization, O. Bollag and G. Kalt for expert technical assistance and Dr K. Adcock and K. Bendfeldt for critical reading of the manuscript. This work was funded by the University of Basel.
References [1] C. Ali, O. Nicole, F. Docagne, S. Lesne, E.T. MacKenzie, A. Nouvelot, A. Buisson, D. Vivien, Ischemia-induced interleukin-6 as a potentiel endogenous neuroprotective cytokine against NMDA receptor-mediated excitotoxicity in the brain, J. Cereb. Blood Flow Metab. 20 (2000) 956–966.
[2] R. Bender, M.C. Hoffmann, M. Frotscher, C. Nitsch, Species-specific expression of parvalbumin in the entorhinal cortex of the Mongolian gerbil: dependence on local activity but not extrinsic afferents, Neuroscience 99 (2000) 423–431. [3] I.L. Campbell, C.R. Abraham, E. Masliah, P. Kemper, J.D. Inglis, M.B. Oldstone, L. Mucke, Neurologic disease induced in transgenic mice by cerebral overexpression interleukin 6, Proc. Natl. Acad. Sci. USA 90 (1993) 10061–10065. [4] W.M. Clark, L.G. Rinker, N.S. Lessov, K. Hazel, J.K. Hill, M. Stenzel-Poore, F. Eckenstein, Lack of interleukin-6 expression is not protective against focal central nervous system ischemia, Stroke 31 (1999) 1715–1720. [5] R.A. Gadient, U. Otten, Differential expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) mRNAs in rat hypothalamus, Neurosci. Lett. 153 (1993) 13–16. [6] R.A. Gadient, U. Otten, Identification of interleukin-6 (IL-6)expressing neurons in the cerebellum and hippocampus of normal adult rats, Neurosci. Lett. 182 (1994) 243 –246. [7] H. Hirota, H. Kiyama, T. Kishimoto, T. Taga, Accelerated nerve regeneration in mice by upregulated expression of interleukin IL-6 and IL-6 receptor after trauma, J. Exp. Med. 183 (1996) 2627– 2634. [8] S.A. Jones, S. Horiuchi, N. Topley, N. Yamamoto, G.M. Fuller, The soluble interleukin 6 receptor: mechanisms of production and implications in disease, FASEB J. 15 (2001) 43–58. [9] E. Juttler, V. Tarabin, M. Schwaninger, Interleukin-6 (IL-6): a possible neuromodulator induced by neuronal activity, Neuroscientist 8 (2002) 268 –275. [10] S.A. Loddick, A.V. Turnbull, N.J. Rothwell, Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat, J. Cereb. Blood Flow Metab. 18 (1998) 176 –179. [11] Y. Maeda, M. Matsumoto, O. Hori, K. Kuwabara, S. Ogawa, S. DuYan, T. Ohtsuki, T. Kinoshita, T. Kamada, D.M. Stern, Hypoxia/ reoxygenation-mediated induction of astrocyte interleukin-6: a paracrine mechanism potentially enhancing neuron survival, J. Exp. Med. 180 (1994) 2297–2308. [12] P. Ma¨rz, J-G. Cheng, R. Gadient, P.H. Patterson, T. Stoyan, U. Otten, S. Rose-John, Sympathetic neurons can produce and respond to interleukin 6, Proc. Natl. Acad. Sci. USA 95 (1998) 3251–3256. [13] S. Matsuda, T-C. Wen, F. Morita, H. Otsuka, K. Igase, H. Yoshimura, M. Sakanaka, Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils, Neurosci. Lett. 204 (1996) 109–112. [14] C. Nitsch, G. Goping, I. Klatzo, Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field, Brain Res. 495 (1989) 243–252. [15] C. Nitsch, A.L. Scotti, A. Sommacal, G. Kalt, GABAergic hippocampal neurons resistant to ischemia-induced neuronal death contain the Ca2þ-binding protein parvalbumin, Neurosci. Lett. 105 (1989) 263–268. [16] F. Perini, M. Morra, M. Alecci, E. Galloni, M. March, V. Toso, Temporal profile of serum anti-inflammatory and proinflammatory interleukins in acute ischemic stroke patients, Neurol. Sci. 22 (2001) 289 –296. [17] K. Saito, K. Suyama, K. Nishida, Y. Sei, A.S. Basile, Early increases in TNF-a IL-6 and IL-1a levels following transient cerebral ischemia in gerbil brain, Neurosci. Lett. 206 (1996) 149 –152. [18] B. Scho¨bitz, D.A.M. Voorhuis, E.R. de Kloet, Localization on interleukin-6 mRNA and interleukin-6 receptor mRNA in rat brain, Neurosci. Lett. 136 (1992) 189–192. [19] S. Suzuki, K. Tanaka, E. Nagata, D. Ito, T. Dembo, Y. Fukuuchi, Cerebral neurons express Interleukin-6 after transient forebrain ischemia in gerbils, Neurosci. Lett. 262 (1999) 117– 120. [20] M. Thier, P. Ma¨rz, U. Otten, J. Weis, S. Rose-John, Interleukin-6 IL-6 and its soluble receptor support survival of sensory neurons, J. Neurosci. Res. 55 (1999) 411 –422.
Interleukin-6 receptor expression and localization after transient global ischemia in gerbil hippocampus Florence Vollenweidera,*, Martina Herrmanna, Uwe Ottenb, Cordula Nitscha a
Institute of Anatomy, Basel University, Pestalozzistrasse 20, CH-4056 Basel, Switzerland b Department of Physiology, Vesalianum, Vesalgasse 1, CH-4056 Basel, Switzerland
Received 13 December 2002; received in revised form 23 January 2003; accepted 27 January 2003
Abstract Ischemia results in increased interleukin-6 (IL-6) expression in the brain. To prove a connection between IL-6 upregulation and IL-6 receptor (IL-6R) expression following ischemia, we analyzed cell-type specific expression changes of IL-6R using transient global ischemia in the gerbil as a model. In sham operated animals, IL-6R mRNA and protein were mainly detected in hippocampal pyramidal cells and interneurons. After ischemia, IL-6R was expressed in neurons but there was no increase during the peak IL-6 expression. Neuronal IL-6R mRNA and protein decreased in parallel with pyramidal cell death, starting 2 days after ischemia. Double-labeling experiments revealed that in postischemic hippocampus IL-6R was not present in GFAP-reactive astrocytes but that the surviving parvalbumin containing interneurons expressed IL-6R mRNA. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Interleukin-6; Stroke; Hippocampus; Cornu amonis-1; Cytokine; Neuroprotection; Parvalbumin
Ischemic brain injury is characterized by apoptotic and necrotic events. In addition, recent evidence suggests that the inflammatory response plays an important role in the outcome of the insult, in particular with respect to neuronal survival. Much of this inflammatory response appears to be mediated by interleukins, a multifunctional subclass of cytokines. The cytokine Interleukin-6 (IL-6), a B-cell stimulating factor, is one of the major cytokines expressed in the brain [9]. Although the principal source of IL-6 has been considered to be microglia and/or astrocytes, it has been clearly shown that it is also expressed in neurons [5,6, 18]. Recent studies demonstrated the ability of IL-6 to exert neuroprotective effects [7,10,11,13]. In IL-6 deficient-mice, however, the infarct size was identical to normal mice after focal central nervous system (CNS) ischemia [4], and severe neurologic impairment was observed in transgenic mice overexpressing IL-6. Taken together, these findings suggest a complex role for IL-6 in the pathology of CNS disorders [3]. To exert its effects, IL-6 must be bound to its receptor. The IL-6 receptor (IL-6R) is composed of 2 glycoproteins: a * Corresponding author. Tel.: þ 41-61-2672729; fax: þ 41-61-2673959. E-mail address: [email protected] (F. Vollenweider).
transmembrane glycoprotein (Gp130), the signal transducing subunit and the IL-6 receptor, which exists in a membrane bound and a soluble form. Cellular activation of IL-6 signaling mediates phosphorylation of Gp130-associated cytoplasmic tyrosine kinases and phosphorylation and activation of transcriptional signals [8]. Whether the increase in IL-6 levels after ischemia is accompanied by an augmentation of its receptor expression remains unclear. An enhancement of receptor expression was observed between 4 and 24 h after a mechanical lesion in the rat striatum [20]. Elevated soluble receptor has been observed in a number of diseases [8]. However, it has also been shown by reverse transcription polymerase chain reaction (RT-PCR) that focal cerebral ischemia in rats upregulated the expression of IL-6 mRNA in cortex and striatum without affecting the transcription of its receptor [1]. The present study was undertaken to clarify the timecourse of expression and localization of the IL-6R after transient forebrain ischemia in gerbils. In particular, we determined which types of cells express the protein and therefore could interact with the cytokine IL-6. Finally, we compared the expression levels with the neuropathological changes provoked by the insult.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00136-8
50
F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
Two to 6 month-old male Mongolian gerbils (50 – 70 g) were subjected to a 7 min bilateral forebrain ischemia or sham operation. Five and 12 h, 2 and 4 days after recirculation, animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA). At least three animals per time-point were used. The brains were post-fixed in PFA, cryoprotected in 30% sucrose and frozen in isopentane. Serial sagittal 35 mm sections were cut with a cryostat. Neuronal cell loss was estimated by cresyl violet staining. For immunoreaction, free-floating sections were treated with H2O2 and PBS/0.1% saponin was then used as buffer. After blocking in normal goat serum (5%) for 90 min, sections were then incubated with a polyclonal rat antibody against IL-6R (1:50; Santa Cruz, USA) for 48 h at room temperature. Incubation with secondary antibody and detection with the avidin-biotin complex (Vector) and the diaminobenzidine reaction were performed. To provide a non-specific control, the primary antibody was omitted. IL6R riboprobe was transcribed from a pSPT19 vector containing the rat IL-6 receptor [5] in the presence of digoxigenin-UTP (Roche Molecular Biochemicals). In situ hybridization was performed as previously described [2]. Briefly, for hybridization, digoxygenin labeled probes (antisense or sense as control) were added (1:300), and sections were then incubated at 53 8C overnight. mRNA was detected with a digoxigenin antibody conjugated to alkaline phosphatase and staining was carried out using NBT/BCIPsubstrates (Roche Molecular Biochemicals). A doublelabeling technique was used to further characterize the localization of IL-6R [2] either with antibodies against parvalbumin (PV; 1:2500; Swant, Switzerland) or the astrocyte marker glial fibrillary acidic protein (GFAP; 1:1000; DAKO, Denmark). All animal experimentation was performed with permission of the local animal care committee and according to present Swiss law. Transient forebrain ischemia in the Mongolian gerbil is a reliable model for cerebral ischemia. Due to its incomplete circle of Willis, bilateral transient occlusion of the common carotids arteries produces a highly reproducible forebrain ischemia, resulting in delayed cell death in cornu amonis-1 (CA1) hippocampal neurons. The CA1 pyramidal cells start dying 2 –3 days after the restoration of circulation. After 4 days, most CA1 neurons are degenerated, with the exception of a PV containing subgroup of inhibitory interneurons [15]. In hippocampus, IL-6R was found both in the perikaryon and outlining the apical dendrites of virtually all neurons: it labeled densely CA1, CA2 and CA3 pyramidal cells as well as the granular cells of the dentate gyrus. IL-6R immunoreaction did not differ 5 or 12 h- after ischemia from sham operated animals (Fig. 1A – C). Localization of IL-6R mRNA detected by in situ hybridization correlated with results for the protein obtained by immunoreaction, but was restricted to the perikaryon. The cell body layers of CA, dentate gyrus and subiculum were densely stained. Similar to IL-6R protein, the IL-6R mRNA did not show any significant change early after the ischemic insult (Fig.
Fig. 1. Time-course of IL-6R expression after ischemia: by immunoreaction (IM) left panels (A –E) and by in situ hybridization (ISH) right panels (F– J); Sham and 5, 12 h; 2, 4 days after ischemia. Scale bar ¼ 0.5 mm.
1F– H). IL-6R mRNA data comprise both soluble and membrane-bound receptor forms. A sense probe used as control did not detect any stained cells (data not shown). While already the localization of the mRNA in the hippocampal cell body layers indicated the preferential neuronal expression of IL-6R, we studied this further by double staining with PV as marker of interneurons, and GFAP as marker for astrocytes. Double staining with PV showed preference of IL-6R for interneurons (Fig. 2C). No colocalization of the IL-6R with GFAP was detected in hippocampus, indicating that the major cell type expressing the IL-6R are neurons (Fig. 2A). Identical observations were obtained with double immunofluorescence (data not shown). Two days after transient ischemia, when neuronal cell loss has started in CA1, a decrease in protein expression in the CA1 pyramidal cells was observed in some cases (Fig. 1D). At the same time point, some upregulation of the protein in the neuropil layer was detected. In parallel to the immunohistochemical data, a decrease of mRNA was
F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
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Fig. 2. Colocalization of IL-6R mRNA and GFAP or PV in CA1. Cells are double-stained for GFAP (A,B) or PV (C,D) (brown staining product) and IL-6R mRNA (blue staining product). (A,C) sham operated animal and (B,D) 4 days after ischemia. Arrows show surviving interneurons double-labeled with PV and IL-6R mRNA. Scale bar ¼ 0.1 mm (D).
observed in CA1 two days after ischemia (Fig. 1I). Four days after the ischemic insult and recirculation, when neuronal death was clearly observed in CA1 and sometimes also in CA3 pyramidal cells, IL-6R expression was absent in the pyramidal cell layer (Fig. 1E). A comparison of serial sections in individual animals showed that loss of perikaryal IL-6R staining correlated with neuronal cell death. On the other hand, there was always an upregulation of the IL-6 receptor in the neuropil layer. The in situ hybridization data showed that expression was nearly absent in the CA1 field with the exception of a few single cells located in the pyramidal cell layer and in the stratum oriens (Fig. 1J). Double labeling experiments revealed that these cells were not GFAP-reactive astrocytes (Fig. 2B) but that the surviving PV containing interneurons possessed IL-6R mRNA (Fig. 2D: arrows). Previous studies have shown a peak of IL-6 expression as soon as 1 h after ischemia, which persisted for about 24 h [17,19]. A recent study demonstrated that production of IL6 is increased 24 h up to 7 days after the insult [16]. Our data show that these increases are not paralleled by increases in mRNA or protein levels of the IL-6R. Neither the insult nor the presence of increased IL-6 protein appeared to modulate IL-6R expression 5 or 12 h after ischemia, a time when a peak of IL-6 production was detected and when the delayed neuronal cell death was still reversible. This is in agreement with observations obtained from tissue extract (cortex and
striatum) by RT-PCR [1]. Further, our data suggest that both receptor forms behave similarly. With immunostaining only the membrane-bound receptor is detected. In situ hybridization identifies both receptor forms. However, no increase of IL-6R mRNA was observed after ischemia. The discrepancy between formation of IL-6R mRNA and declining levels of protein could be due to inhibition of overall cerebral protein synthesis which precedes neuronal cell death following ischemia. Thus, neuronal expression of IL-6R decreased together with pyramidal cell death, starting 2 days after ischemia. Some upregulation of IL-6R protein was observed 3 and 4 days post ischemia in the neuropil layer by immunohistoreaction in reactive glial cells. We were unable to detect IL6R in GFAP-reactive astrocytes. Absence of astrocytic expression of the IL-6R suggested that an astrocytic response to injury was unlikely to result from the early production of IL-6. Upregulation of IL-6 as observed by immunoreaction probably occurred mainly in microglia [19]. On the other hand, in vitro studies demonstrated production of IL-6 by astrocytes in response to diverse stimuli. For example, Maeda et al. showed that astrocytes produced IL-6 when exposed to hypoxia followed by a period of reoxygenation [11]. It is nevertheless important to note that in these cases expression of IL-6 and IL-6R was detected in primary cultures of astrocytes or in an astroglioma cell line.
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F. Vollenweider et al. / Neuroscience Letters 341 (2003) 49–52
Colocalization of IL-6R with PV was observed in a certain number of cells providing evidence that not only hippocampal pyramidal cells but also interneurons express the receptor (Fig. 2C,D). Our data demonstrate that after ischemia surviving interneurons expressed the IL-6R (Fig. 2D: arrows). These interneurons have been shown to participate in synaptic rearrangement taking place after ischemic nerve cell death [14]. Lesion-induced reorganization processes require trophic factors. Ma¨rz et al. [12] demonstrated that addition of the soluble IL-6R and the cytokine IL-6 enhanced survival of primary sympathetic neurons in the absence of the nerve growth factor. In this case, IL-6 acts as a trophic factor i.e. has the capacity to promote neuronal survival and differentiation. A prerequisite for these effects is the presence of the soluble IL-6R. The same phenomenon was observed for dorsal root ganglion neurons suggesting that IL-6 bound to the soluble IL-6R has neurotrophic potential [20]. It remains possible that the surviving interneurons produced additional soluble IL-6R, yet the sensitivity of our detection method did not allow to determine this unequivocally and would require additional investigations. IL-6 has been implicated in a number of neuropathological reactions, including acute ischemia. To elicit a direct effect on neurons, receptors for IL-6 must be expressed. In the present study we showed that the IL-6R is expressed in principal neurons and interneurons of the hippocampus; no IL-6R was observed in astrocytes. There was no change in expression during the early time points after transient cerebral ischemia in gerbils, when pyramidal cells were still viable. After delayed pyramidal cell death, IL-6R was still present in surviving interneurons. In summary, our results suggest that IL-6R is expressed constitutively in hippocampal neurons and that the increased production of IL-6 early after an ischemic insult does not induce a change in its receptor expression. In our model, there exists no indication for a neuroprotective effect of IL-6.
Acknowledgements The authors wish to thank Prof. C. Grothe and K. Kuhlemann for advice with the in situ hybridization, O. Bollag and G. Kalt for expert technical assistance and Dr K. Adcock and K. Bendfeldt for critical reading of the manuscript. This work was funded by the University of Basel.
References [1] C. Ali, O. Nicole, F. Docagne, S. Lesne, E.T. MacKenzie, A. Nouvelot, A. Buisson, D. Vivien, Ischemia-induced interleukin-6 as a potentiel endogenous neuroprotective cytokine against NMDA receptor-mediated excitotoxicity in the brain, J. Cereb. Blood Flow Metab. 20 (2000) 956–966.
[2] R. Bender, M.C. Hoffmann, M. Frotscher, C. Nitsch, Species-specific expression of parvalbumin in the entorhinal cortex of the Mongolian gerbil: dependence on local activity but not extrinsic afferents, Neuroscience 99 (2000) 423–431. [3] I.L. Campbell, C.R. Abraham, E. Masliah, P. Kemper, J.D. Inglis, M.B. Oldstone, L. Mucke, Neurologic disease induced in transgenic mice by cerebral overexpression interleukin 6, Proc. Natl. Acad. Sci. USA 90 (1993) 10061–10065. [4] W.M. Clark, L.G. Rinker, N.S. Lessov, K. Hazel, J.K. Hill, M. Stenzel-Poore, F. Eckenstein, Lack of interleukin-6 expression is not protective against focal central nervous system ischemia, Stroke 31 (1999) 1715–1720. [5] R.A. Gadient, U. Otten, Differential expression of interleukin-6 (IL-6) and interleukin-6 receptor (IL-6R) mRNAs in rat hypothalamus, Neurosci. Lett. 153 (1993) 13–16. [6] R.A. Gadient, U. Otten, Identification of interleukin-6 (IL-6)expressing neurons in the cerebellum and hippocampus of normal adult rats, Neurosci. Lett. 182 (1994) 243 –246. [7] H. Hirota, H. Kiyama, T. Kishimoto, T. Taga, Accelerated nerve regeneration in mice by upregulated expression of interleukin IL-6 and IL-6 receptor after trauma, J. Exp. Med. 183 (1996) 2627– 2634. [8] S.A. Jones, S. Horiuchi, N. Topley, N. Yamamoto, G.M. Fuller, The soluble interleukin 6 receptor: mechanisms of production and implications in disease, FASEB J. 15 (2001) 43–58. [9] E. Juttler, V. Tarabin, M. Schwaninger, Interleukin-6 (IL-6): a possible neuromodulator induced by neuronal activity, Neuroscientist 8 (2002) 268 –275. [10] S.A. Loddick, A.V. Turnbull, N.J. Rothwell, Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat, J. Cereb. Blood Flow Metab. 18 (1998) 176 –179. [11] Y. Maeda, M. Matsumoto, O. Hori, K. Kuwabara, S. Ogawa, S. DuYan, T. Ohtsuki, T. Kinoshita, T. Kamada, D.M. Stern, Hypoxia/ reoxygenation-mediated induction of astrocyte interleukin-6: a paracrine mechanism potentially enhancing neuron survival, J. Exp. Med. 180 (1994) 2297–2308. [12] P. Ma¨rz, J-G. Cheng, R. Gadient, P.H. Patterson, T. Stoyan, U. Otten, S. Rose-John, Sympathetic neurons can produce and respond to interleukin 6, Proc. Natl. Acad. Sci. USA 95 (1998) 3251–3256. [13] S. Matsuda, T-C. Wen, F. Morita, H. Otsuka, K. Igase, H. Yoshimura, M. Sakanaka, Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils, Neurosci. Lett. 204 (1996) 109–112. [14] C. Nitsch, G. Goping, I. Klatzo, Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field, Brain Res. 495 (1989) 243–252. [15] C. Nitsch, A.L. Scotti, A. Sommacal, G. Kalt, GABAergic hippocampal neurons resistant to ischemia-induced neuronal death contain the Ca2þ-binding protein parvalbumin, Neurosci. Lett. 105 (1989) 263–268. [16] F. Perini, M. Morra, M. Alecci, E. Galloni, M. March, V. Toso, Temporal profile of serum anti-inflammatory and proinflammatory interleukins in acute ischemic stroke patients, Neurol. Sci. 22 (2001) 289 –296. [17] K. Saito, K. Suyama, K. Nishida, Y. Sei, A.S. Basile, Early increases in TNF-a IL-6 and IL-1a levels following transient cerebral ischemia in gerbil brain, Neurosci. Lett. 206 (1996) 149 –152. [18] B. Scho¨bitz, D.A.M. Voorhuis, E.R. de Kloet, Localization on interleukin-6 mRNA and interleukin-6 receptor mRNA in rat brain, Neurosci. Lett. 136 (1992) 189–192. [19] S. Suzuki, K. Tanaka, E. Nagata, D. Ito, T. Dembo, Y. Fukuuchi, Cerebral neurons express Interleukin-6 after transient forebrain ischemia in gerbils, Neurosci. Lett. 262 (1999) 117– 120. [20] M. Thier, P. Ma¨rz, U. Otten, J. Weis, S. Rose-John, Interleukin-6 IL-6 and its soluble receptor support survival of sensory neurons, J. Neurosci. Res. 55 (1999) 411 –422.