Functional interleukin-6 receptor-α is located in tanycytes at the base of the third ventricle

Abstract

Interleukin (IL)-6^- /^- mice develop mature onset obesity, whereas i.c.v. injection of IL-6 decreases obesity in rodents. Moreover, levels of IL-6 in cerebrospinal fluid (CSF) were reported to be inversely correlated with obesity in humans. Tanycytes lining the base of the third ventricle (3V) in the hypothalamus have recently been reported to be of importance for metabolism. In the present study, we investigated whether tanycytes could respond to IL-6 in the CSF. With immunohistochemistry using a well characterised antibody directed against the ligand binding receptor for IL-6, IL-6 receptor α (IL-6Rα), it was found that tanycytes, identified by the two markers, vimentin and dopamine- and cAMP-regulated phosphoprotein of 32 kDa, contained IL-6Rα. There were fewer IL-6Rα on another type of ventricle-lining cells, ependymal cells, as identified by the marker glucose transporter-1. To demonstrate that the immunoreactive IL-6Rα were responsive to IL-6, we injected IL-6 i.c.v. This treatment increased immunoreactive phosphorylated signal transducer and activator of transcription-3 (pSTAT3) in tanycytes after 5 minutes and in cells in the medial part of the arcuate nucleus after 5 and 15 minutes. Intracerebroventricular injection of leptin exerted similar effects. As expected, i.p. injection of leptin also induced pSTAT3 staining in the hypothalamus, whereas i.p. IL-6 injection had little effect on this parameter. Intracerebroventricular or i.p. injection of vehicle only had no effect on pSTAT3-immunoreactivity. In summary, there are functional IL-6Rα on tanycytes at the bottom of the 3V, in agreement with the possibility that ventricular administration of IL-6 decreases obesity in mice via an effect on this cell type.

Keywords: IL-6; IL-6Rα; hypothalamus; tanycytes; third ventricle.

Figure 1

Interleukin (IL)‐6Rα immunoreactivity is located in tanycytes bordering the median eminence. Immunohistochemistry showing IL‐6 receptor α‐ (IL‐6Rα) immunoreactivity in green, vimentin‐ (A‐D) or dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa (DARPP‐32) (E‐G) immunoreactivity in red and 4’,6‐diamidino‐2‐phenylindole (DAPI) (nuclear staining) in blue, shown in coronal cross‐sections of parts of the third ventricle (3V) of the mouse brain. An overview of the area (A, E) and magnifications of parts of the 3V facing the median eminence (ME) (C, F) and arcuate nucleus (ARC) (B, D, G) showed IL‐6Rα‐immunoreactivity on vimentin‐ or DARPP‐32‐immunoreactive cells. White arrowheads (B‐D, F‐G) indicate examples of such cells displaying both vimentin‐ or DARPP‐32‐ and IL‐6Rα‐immunoreactivity. Approximately 90% of DARPP‐32‐immunoreactive cells also showed IL‐6Rα‐immunoreactivity, whereas only approximately 35% of GLUT1‐immunoreactive cells showed IL‐6Rα‐immunoreactivity (H). Images were obtained using the confocal microscope system described in the Materials and methods. Cell counting was performed from two animals using two slices per animal as described in the Materials and methods. Scale bars (overview)=100 μm, close‐up=25 μm (B, C, F, G) or 10 μm (D)

Figure 2

Interleukin (IL)‐6Rα immunoreactivity is located in some glucose transporter‐1 (GLUT‐1)‐positive ependymal cells. Immunohistochemistry showing IL‐6 receptor α‐ (IL‐6rα) immunoreactivity in green, GLUT‐1 (a marker for ependymal cells) immunoreactivity in red and 4’,6‐diamidino‐2‐phenylindole (DAPI) (nuclear staining) in blue, shown in coronal cross‐sections of parts of the third ventricle (3V) of the mouse brain. Overview of the area (A) and magnifications of parts of the 3V facing the arcuate nucleus (ARC) (B) and lateral 3V wall (C) showed IL‐6Rα immunoreactivity on some of the GLUT‐1 immunoreactive cells. Red arrowheads (C) indicate examples of such cells with both GLUT‐1‐ and IL‐6Rα‐immunoreactivity, whereas white arrowheads indicate cells with only IL‐6Rα‐immunoreactivity (B, C). Images were obtained using the confocal microscope system described in the Materials and methods. Scale bars (overview)=100 μm, close‐up=10 μm

Figure 3

Intracerebroventricular administration of leptin or interleukin (IL)‐6 induces pSTAT3 immunoreactivity in tanycytes after 5 minutes. Immunohistochemistry showing phosphorylated signal transducer and activator of transcription‐3 (pSTAT3)‐immunoreactivity in red, vimentin‐ (a tanycyte marker) immunoreactivity in green and 4’,6‐diamidino‐2‐phenylindole (DAPI) (nuclear staining) in blue, shown in coronal cross‐sections of parts of the third ventricle (3V) of the mouse brain. Images were obtained from mice that were killed 5 minute after i.c.v. injection of IL‐6 (A‐C) or leptin (D‐F). White arrowheads indicate examples of pSTAT3‐immunoreactivity. Images were obtained using the confocal microscope system described in the Materials and methods. Scale bars for overview=100 μm, close‐up=10 μm

Figure 4

Quantification of phosphorylated signal transducer and activator of transcription‐3 (pSTAT3)‐immunoreactivity after i.c.v. and i.p. administration of interleukin (IL)‐6, leptin or vehicle. Mice killed 5 minutes after i.c.v. administration of IL‐6 or leptin (A) show pSTAT3‐immunoreactivity in approximately 52% or 49% of ventricle‐lining vimentin‐positive cells, respectively. This immunoreactivity is absent in mice killed 15 minutes after i.c.v. administration of these substances (B). In the arcuate nucleus (ARC), i.c.v. IL‐6 administration induces pSTAT3‐immunoreactivity in approximately 43% of cells after 5 minutes (A) and 72% (B) after 15 minutes, whereas leptin similarly induces pSTAT3‐immunoreactivity in approximately 35% of cells after 5 minutes (A) and 71% of cells after 15 minutes (B). Intraperitoneal administration of IL‐6 does not induce pSTAT3‐immunoreactivity in the ARC after 15 minutes, whereas leptin induces pSTAT3‐immunoreactivity in approximately 68% of ARC cells (C). Articifial cerebrospinal fluid (aCSF) (A‐B) or saline (C) does not induce pSTAT3‐immunoreactivity in either ventricle‐lining vimentin‐immunoreactive cells or ARC cells. Cell counting was performed from two animals using two slices per animal as described in the Materials and methods

Figure 5

Intracerebroventricular administration of leptin or interleukin (IL)‐6 induces phosphorylated signal transducer and activator of transcription‐3 (pSTAT3)‐immunoreactivity in the arcuate nucleus (ARC) after 15 minutes. Immunohistochemistry showing pSTAT3‐immunoreactivity in red, vimentin‐ (a tanycyte marker) immunoreactivity in green and 4’,6‐diamidino‐2‐phenylindole (DAPI) (nuclear staining) in blue, shown in coronal cross‐sections of parts of the third ventricle (3V) of the mouse brain. Images were obtained from mice that were killed 15 minutes after i.c.v. injection of IL‐6 (A‐C) or leptin (D‐F). White arrowheads indicate examples of pSTAT3‐immunoreactivity in the ARC. Images were obtained using the confocal microscope system described in the Materials and methods. Scale bars (overview)=100 μm, close‐up=10 μm

Figure 6

Systemic leptin, but not interleukin (IL)‐6, induces phosphorylated signal transducer and activator of transcription‐3 (pSTAT3) activation in the arcuate nucleus (ARC) after 15 minutes. Immunohistochemistry showing pSTAT3‐immunoreactivity in red, vimentin‐ (a tanycyte marker) immunoreactivity in green and 4’,6‐diamidino‐2‐phenylindole (DAPI) (nuclear staining) in blue, shown in coronal cross‐sections of parts of the third ventricle (3V) of the mouse brain. Images were obtained from mice that were killed 15 minutes after i.c.v. injection of IL‐6 (A‐C) or leptin (D‐F). White arrowheads indicate examples of pSTAT3‐immunoreactive cells. Images were obtained using the confocal microscope system described in the Materials and methods. Scale bars (overview)=100 μm, close‐up=10 μm2