Brain, Vol. 122, No. 9, 1697-1707,
September 1999
© 1999 Oxford University Press
The interleukin-1 type I receptor is expressed in human hypothalamus
1 Parke-Davis Neuroscience Research Centre, Cambridge University and 2 School of Biological Sciences, Manchester, UK
Correspondence to:
E. A. Hammond, Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge CB2 2QB, UK E-mail: Elizabeth.Hammond{at}wl.com
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Several lines of evidence suggest that interleukin-1 (IL-1) acts directly on the central nervous system, probably within the hypothalamus, causing effects such as fever, activation of the immune response and sickness behaviour. IL-1 has also been shown to be involved in the aetiology of several neuronal diseases, including neurodegeneration, stroke and Alzheimer's disease. However, the question as to whether the full-length type I IL-1 receptor (IL-1RI) is expressed in the human hypothalamus has yet to be addressed. Using the polymerase chain reaction, we cloned a full-length cDNA encoding the human hypothalamic IL-1RI from human hypothalamic cDNA. The DNA sequence of the human hypothalamic receptor was identical to that of the human fibroblast IL-1RI. The IL-1RI receptor protein was detected in astrocytes of normal human hypothalamic brain sections using immunocytochemical techniques. To ascertain that the cloned receptor was functional, Chinese hamster ovary (CHO) cells were transfected with a plasmid vector containing the IL-1RI coding region. IL-1RI-mediated-signal transduction was assessed by microphysiometry and activation of p38 MAP (mitogen-activated protein) kinase. We report the first demonstration that both the type I IL-1 transcript and the protein are expressed in the human hypothalamus. The receptor was expressed in a stable CHO cell line, providing a tool with which to embark on a thorough analysis of the signalling mechanisms mediated by IL-1 via this receptor.
IL-1RI; human; hypothalamus; cloning; immunocytochemistry
BSA = bovine serum albumin; CHO = Chinese hamster ovary; GFAP = glial fibrillary acid protein; GSIB4 = Griffonia simplicifolia isolectin B4; IL-1 = interleukin-1; IL-1RI = IL-1 receptor type I; PCR = polymerase chain reaction; SDS = sodium dodecyl sulphate; TBS = Tris-buffered saline
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Interleukin-1 (IL-1) is a member of the cytokine family of proteins, which mediate regulatory functions between leukocytes in the immune system. There are three known molecules in the IL-1 group, two acting as agonists (IL-1
and IL-1ß), whilst the third, the IL-1 receptor antagonist (IL-1ra), inhibits the activities of IL-1 and IL-1ß. The receptor antagonist elicits no biological signalling of its own (Dinarello, 1996
).
At present two distinct IL-1 binding glycoprotein receptors, of type I (IL-1RI) and type II, and one receptor accessory protein (Greenfeder et al., 1995) have been described. The IL-1RI consists of an 80 kDa single polypeptide chain and is generally considered to be the functional IL-1 receptor. This molecule spans the membrane once and possesses an extracellular ligand-binding domain and a cytoplasmic region of 213 amino acids (Sims et al., 1988). Following ligand binding to the type I receptor, a ternary complex, consisting of the IL-1RI/IL-1/IL-1RI accessory protein, is formed, leading to IL-1 signal transduction. Formation of this complex appears to be an absolute requirement for IL-1 signalling (Hofmeister et al., 1997; Wesche et al., 1997). Several different signalling cascades have been implicated in mediating IL-1 signal transduction, including the NF
B pathway and activation of the MAP (mitogen-activated protein) kinase homologue family (O'Neill, 1995 and references therein).
The enzyme p38 MAP kinase, also called re-activating kinase, p40 and CSBP (cytokine synthesis anti-inflammatory drug-binding protein) (Gould et al., 1995), is the mammalian homologue of the yeast HOG kinase and participates in a cascade controlling cellular responses to cytokines and stress. The kinase is activated by a variety of cellular stress inducing factors including osmotic shock, inflammatory cytokines, lipopolysaccharides, UV light and growth factors. Activation of p38 MAP kinase is achieved by phosphorylation of tyrosine and threonine residues within the enzyme (Freshney et al., 1994; Han et al., 1994; Lee et al., 1994; Rouse et al., 1994). Activated p38 MAP kinase has been shown to phosphorylate and activate MAPKAP kinase-2 (Rouse et al., 1994) and to phosphorylate the transcription factors ATF-2 (Raingeaud et al., 1995) and Max (Zervos et al., 1995). Furthermore, Freshney and co-workers (Freshney et al., 1994) demonstrated that IL-1 activated the p38 MAP kinase cascade, leading to phosphorylation of the 27 kDa heat shock protein Hsp27.
The second IL-1 receptor subtype (type II) has a tertiary structure largely similar to that of the type I receptor, and is expressed as a membrane-bound 60 kDa protein or a soluble 45 kDa moiety. A notable feature of the membrane-associated type II receptor is its short cytoplasmic domain, which consists of only 29 amino acids (McMahan et al., 1991). This molecule is able to bind IL-1 but is incapable of mediating signal transduction across the membrane. The soluble form of this molecule, shed from cells in response to various extracellular stimuli (Colotta et al., 1995, 1996; Sambo et al., 1996), is thought to play a role in regulating levels of circulating IL-1ß and has been named the `decoy' receptor. Furthermore, it has been suggested recently that the membrane-bound type II receptor interacts with the accessory protein to reduce the number of signalling-competent type I receptor–accessory protein complexes (Malinowsky et al., 1998).
IL-1 stimulates a diverse range of actions in vivo, including thermogenesis (Dinarello, 1991; Kluger, 1991), but it also triggers other responses, including activation of the hypothalamic–pituitary–adrenal axis and the acute-phase response (Rothwell, 1991, 1998 and references therein). This cytokine is one of the most prominent cytokines in the brain, where it is presumed to act within the area of the brain responsible for thermoregulation, i.e. the hypothalamus (Blatteis, 1988, 1990). IL-1 is expressed in the brain by neurons (Breder et al., 1988; Lechan et al., 1990), microglia (Sheng et al., 1998) and astrocytes (Pearson et al., 1999); aberrant synthesis of IL-1 in the brain is thought to contribute to the development of acute and chronic CNS pathologies such as Alzheimer's disease, Down syndrome, stroke and head injury (Griffin et al., 1989; Hallenbeck et al., 1988; Rothwell, 1991; Rothwell and Hopkins, 1995).
We decided to attempt to clone the brain IL-1RI cDNA from the human hypothalamus using polymerase chain reaction (PCR) techniques. Here we report on the cloning and expression of a IL-1RI from the human hypothalamus. In addition, evidence is provided for the first time that the receptor protein is expressed in astrocytes of the normal human hypothalamus.
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Material and methods |
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Molecular cloning and expression
Cloning of the human IL-1RI cDNA was achieved using a commercially available human hypothalamic cDNA (CLONTECH, Palo Alto, Calif., USA) as a template. Thirty cycles of PCR amplification were carried out using oligonucleotide primers complementary to the 5' and 3' regions flanking the coding sequence of human fibroblast IL1-RI cDNA (Chua and Gubler, 1989
). The primers were designed to incorporate convenient restriction enzyme sites (underlined) (hI-5': 5'-CGAATTCCCTTGGTAAAAGAC-3' EcoRI; hI-3': 5'-CCTCGAGCACCTAAAGAACTC-3' XhoI). The resulting 1.7 kb product was cloned into pBluescript (Stratagene, La Jolla, Calif., USA), and the construct was termed pBShIL-RI. Both strands of the entire cDNA insert were sequenced using Sequenase Version 2 (Amersham International, Amersham, UK) to confirm the identity of the clone. Subcloning of the IL-1RI cDNA into expression vector pcDNA3 (Invitrogen, Carlsbad, Calif., USA) was accomplished using the 1.7 kb EcoRI–XhoI restriction fragment excised from pBShIL-RI. The resulting plasmid was named pchIL-1RI.
Expression of pchIL-1RI in Chinese hamster ovary K1 (CHO.K1) cells
CHO.K1 cells (American Type Culture Collection, Manassas, Va., USA) were transfected using a CaPO4-mediated mammalian transfection kit (Stratagene). Transfectants were selected with 400 µg/ml G418 (Life Technologies Inc., Rockville, Md., USA) and clones originating from single colonies were subsequently isolated.
Cell culture
CHO cells were maintained in Nutrient Mixture Ham's F12 with 1 g/l Glutamax II supplemented with 50 µg/ml streptomycin, 50 µg units/ml benzylpenicillin and 10% (v/v) foetal calf serum (Life Technologies).
Radioligand binding analysis
Several G418-resistant clones were subjected to [125I]IL-1 binding analysis. Cells were harvested by trypsinization and resuspended in Dulbecco's modified Eagle's medium supplemented with 1% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich Company Ltd, Poole, UK). Cells were incubated with recombinant human [125I]IL-1 (NEN Life Science Products, Boston, Mass., USA) for 2 h at 22°C in the presence or absence of 50 nM recombinant human IL-1 (R & D Systems, UK) to define non-specific binding. Samples were filtered through GF/C filter papers (Whatman International Ltd, Maidstone, UK) and bound radioactivity measured in a 
-counter. Clones identified as expressing specific radiolabelled IL-1 binding were named CHO.hIL1RI.
Analysis of p38 MAP kinase activation
Cells were seeded into 30 mm dishes at a density of 3x105 cells per well 24 h prior to serum starvation overnight. Following stimulation with or without IL-1 or IL-1ß (R & D Systems, Abingdon, UK) for the durations and concentrations indicated, cells were washed with ice-cold PBS (phosphate-buffered saline). Cells were extracted with sodium dodecyl sulphate (SDS) gel-loading buffer (Laemmli, 1970), boiled for 5 min and analysed by SDS–polyacrylamide gel electrophoresis using precast Tris–glycine gels (Novex Experimental Technology, San Diego, Calif., USA) at 125 mA for 1.5 h at 25°C. Proteins were transferred onto nitrocellulose membranes (Novex Experimental Technology) according to the manufacturer's instructions. Non-specific sites on membranes were blocked with 5% (w/v) non-fat dry milk solution in 1 x TSTB [10 mM Tris–HCl, pH 7.5, 0.2% (v/v) Tween, 150 mM NaCl, 5% (w/v) BSA] for 1 h at room temperature. Membranes were incubated overnight at 4°C with either rabbit polyclonal p38 MAP kinase antibody or rabbit polyclonal phospho-p38 MAP kinase (Thr180/Tyr182) (New England Biolabs Inc., Beverly, Mass., USA) diluted 1 : 1000. Filters were washed three times in 1 x TSTB prior to incubation for 1 h at room temperature with goat anti-rabbit IgG-HRP (IgG–horseradish peroxidase; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) diluted 1 : 1000. Membranes were again washed three times in 1 x TSTB. Labelled proteins were detected by ECL as described by the manufacturer (Amersham International).
Microphysiometry
Transfected CHO clones, identified by radioligand binding analysis as expressing specific [125I]IL-1 binding sites, were analysed using a Cytosensor microphysiometer (Molecular Devices Ltd, Crawley, UK). Cells were cultured in Nutrient Mixture Ham's F12 supplemented as before (omitting antibiotics) and seeded into Cytosensor capsule cups as described previously (Jordan et al., 1998) at a density of 0.6 x 106 cells per cup. The cups were perfused in the Cytosensor at 120 µl/min with bicarbonate-free Nutrient Mixture Ham's F12 (pH 7.4) containing 1 g/l glutamine (Life Technologies) and the acidification rate was recorded every 2 min by stopping the perfusion for 15 s. Cells were stimulated with IL-1 (concentrations and time intervals indicated) in the presence or absence of the IL-1RI antagonist (IL-1ra; 5–200 ng/ml). Responses were measured as the area under the curve, and are presented as mean ± standard error of the mean unless otherwise stated.
Immunocytochemistry
Hypothalamus fragments were obtained post-mortem from a female, non-demented patient (supplied by Professor David Mann, Department of Pathological Sciences, University of Manchester, UK). Formalin-fixed hypothalamic pieces were washed thoroughly in several changes of 0.01 M PBS (Sigma Aldrich) at 4°C for 24 h, prior to being dehydrated in alcohols, cleared in xylene and infiltrated in paraffin at 60°C. Sections (5 µm thick) were prepared on a Reichert microtome and mounted on uncoated slides. The sections were deparaffinized in xylene and partially rehydrated in 99 and 95% alcohols prior to use.
IL-1RI
Sections were processed for immunocytochemistry using the avidin–biotin method (Lewis, 1991) with modifications. Briefly, sections were placed in methanol/0.3% (v/v) H2O2/1 M HCl for 30 min to block endogenous peroxidase activity. Sections were then incubated in 20% (v/v) goat serum (Sigma-Aldrich) for 90 min followed by polyclonal rabbit anti-IL-1RI [IL-1RI (N-20); Santa Cruz Biotechnology] diluted 1 : 300 overnight. According to the supplier, this primary antibody was raised to an epitope corresponding to amino acids KIILVSSANEIDVRPCPLNP mapping at the amino terminus of the human IL-1RI and is specific for IL-1RI; it does not cross-react with other IL receptor subunits. Sections were then incubated sequentially with (i) biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, Calif., USA) diluted 1 : 3000, for 90 min; (ii) 5 µg/ml avidin peroxidase (Sigma-Aldrich) for 1 h and (iii) 3,3-diaminobenzidine (Sigma-Aldrich) for 10 min. Sections were mounted on glass coverslips, dried overnight at room temperature, dehydrated in ascending alcohols, cleared in xylene and mounted in Depex (Gurr, BDH, Poole, Dorset, UK). Sections were washed three times for 5 min in Tris-buffered saline (TBS) [50 mM Tris–HCl (pH 7.2), 150 mM NaCl] after each step, except after incubation with goat serum. All steps were performed at room temperature except for incubation with the primary antibody, which was carried out at 4°C. All reagents (except avidin peroxidase and diaminobenzidine) were diluted in TBS/0.1% (w/v) BSA. Avidin peroxidase and diaminobenzidine were diluted in TBS only.
Controls were obtained by omission of primary or secondary antibody or by preincubation of primary antibody with 1 µM antigenic peptide (corresponding to amino acids KIILVSSANEIDVRPCPLNP of the human IL-1RI) (Santa Cruz Biotechnology) for 24 h at 4°C for preadsorption experiments.
Glial fibrillary acid protein
For the specific detection of astrocytes, consecutive sections were processed as described above, but using polyclonal rabbit anti-human glial fibrillary acid protein GFAP IgG as the primary antibody (Sigma-Aldrich) diluted 1 : 1000.
Lectin staining
Consecutive sections were trypsinized [trypsin type II, Sigma-Aldrich; 0.1% (w/v) in TBS/0.1% (w/v) CaCl2, 30 min at 37°C], washed in TBS and incubated with 10 µg/ml biotinylated Griffonia simplicifolia isolectin B4 (GSIB4) (Sigma-Aldrich) for 30 min at room temperature. GSIB4 binds to microglia, monocytes, macrophages and endothelial cells. Sections were washed in TBS with 0.1% (w/v) CaCl2 and incubated with avidin–peroxidase and diaminobenzidine before dehydration in alcohols and mounting as described above.
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Molecular cloning and expression of the human IL-1RI cDNA
A product of 1.7 kb was obtained by PCR amplification of a human hypothalamic cDNA, and ligated into pBluescript, creating the construct pBShIL-1RI. The size of this product corresponded to that predicted from the fibroblastic type I receptor (Chua and Gubler, 1989
). Complete sequence analysis of the cDNA revealed that it encoded a full-length IL-1RI. The sequence was identical to that of the published human fibroblast IL-1RI receptor (Chua and Gubler, 1989). Following confirmation of its identity, the IL-1RI cDNA was subcloned into expression vector pcDNA3, resulting in the construction of plasmid pchIL-1RI.
The human hypothalamic IL-1RI cDNA was expressed in CHO.K1 cells to facilitate analysis of IL-1 receptor pharmacology and signal transduction. CHO cells exhibiting stable expression of the IL-1RI were isolated by their expression of G418 resistance. Several clones were analysed for specific [125I]IL-1 binding. The data for one clone (CHO.hIL1RI) are presented, and it is representative of all those analysed.
Scatchard analysis using [125I]IL-1 revealed that CHO.hIL1RI cells expressed 7951 ± 1837 receptors per cell with a Kd of 0.59 ± 0.16 nM (mean ± SEM, n = 3) and a Bmax of 13.2 ± 3.05 (mean ± SEM) fmol/106 cells (Fig. 1).
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Functional studies
IL-1-stimulated p38 activation
Western blotting assays were performed to measure activation (by phosphorylation) of p38 MAP kinase by IL-1 in CHO cells (Fig. 2
). Both unphosphorylated and phosphorylated CHO p38 MAP kinase were recognized in this assay: the unphosphorylated isoform was expressed constitutively in these cells and the total amount of either form of the kinase did not change over time (data not shown). A time-course study with 10 ng/ml ligand demonstrated that p38 MAP kinase was phosphorylated after 10 min of exposure to human IL-1 in CHO.hIL1RI cells and peaked after 15 min of stimulation (Fig. 2A). After this time, the level of phosphorylation decreased, but the kinase did in fact remain phosphorylated over the course of the study, which was terminated after 60 min of stimulation. Greatly diminished, barely detectable activation was observed in CHO.K1 by human IL-1, presumably via low levels of endogenously expressed type I receptor (Fig. 2B).
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Cytosensor microphysiometry
Microphysiometry measures the rate of proton excretion from living cells. In general, increases in metabolism result in an increase in intracellular pH, giving rise to the transport of protons across the plasma membrane. This extrusion of protons acidifies the extracellular environment. Recordings of the rate of such proton excretion can be used to represent the response of cells to a variety of chemicals, including ligands for specific membrane receptors. The cytosensor was used to examine the pharmacology of transfected CHO cells that stably expressed IL-1RI (Figs 3 and 4
). The data presented are from the same clone subjected to Scatchard analysis (CHO.hIL1RI), but are representative of three individual clones of transfected CHO cells.
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Exposure of the CHO.hIL1RI cells for 5 min to 10 ng/ml IL-1
(Fig. 3A
) caused a delayed, broad, monophasic increase, 4–6 min after exposure began, in the acidification rate, which peaked at 16–22 min and returned to the basal level within 36–44 min (n = 15).
The acidification rate of the parental CHO.K1 cells did not alter in response to stimulation with IL-1 (Fig. 3B). Varying the length of exposure between 3 and 20 min had no effect on either the magnitude or the time course of the response (data not shown, n = 4). Addition of IL-1 in the range 0.1–100 ng/ml for 5 min induced a response which was concentration-dependent, with a pEC50 of 9.93 ± 0.08 (n = 6, Fig. 3B). The acidification rate of the parental CHO.K1 cells did not alter in response to stimulation with IL-1 (Fig. 3B). The IL-1RI antagonist, IL-1ra (5–200 ng/ml), had no effect on the basal acidification rate, but dose-dependently inhibited the response induced by IL-1 (10 ng/ml for 5 min) with an IC50 of 19.7 ng/ml (n = 4–8) (Fig. 4A). Complete blockade (98.8 ± 1.2%) occurred at 200 ng/ml. Activation of the IL-1RI by 10 ng/ml IL-1 for 5 min caused profound desensitization of the receptor; a subsequent challenge 4 h after the first elicited a reduced (<20%, n = 4) response (Fig. 4B). Desensitization was evident even after a 12-h interval between challenges (data not shown). This desensitization was specific, in that it appeared to affect the IL-1 receptor response alone and not the acidification response to stimulation of the endogenously expressed P2U receptor with 3 µM UTP (n = 3, Fig. 4B). The response to the first stimulation with UTP was similar in magnitude to that of the second stimulation. As a consequence, when stimulating with UTP the second stimulation is ~100% when expressed as a percentage of the first. However, when analysing responses to IL-1, the second stimulation was reduced to ~20% of the first. Furthermore, the reduction in response to successive applications of IL-1 was not due to cell death occurring during the course of the experiment, as evidenced once again from the reproducible effect of repeated stimulation with UTP (i.e. second stimulation/first stimulation was ~100%).
Immunocytochemistry studies of the human hypothalamus using hIL-1RI
Staining with a polyclonal antibody raised to the N-terminal 20 amino acids of the human IL-1RI revealed expression of the protein in astrocyte-like cells in normal human hypothalamic sections (Fig. 5A). The identification of IL-1RI protein-positive cells as astrocytes was confirmed by anti-GFAP immunostaining in consecutive sections (Fig. 5B). Antibody specificity was demonstrated by complete loss of staining after preincubation of the antibody with the control antigen peptide (Fig. 6). Staining with anti-IL-1RI (Fig. 7A) and biotinylated GSIB4 (Fig. 7B) in consecutive sections failed to reveal IL-1RI protein-positive microglia, macrophages or endothelial cells in the human hypothalamus.
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Discussion |
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The involvement of IL-1 in disorders such as neurodegeneration, stroke and Alzheimer's disease (Hallenbeck et al., 1988
; Griffin et al., 1989) makes this particular cytokine an attractive target for pharmaceutical intervention. Despite the intense interest in the role of IL-1 in neuronal disorders, the literature lacks conclusive evidence to support the existence of expression of IL-1RI mRNA or protein in the human brain.
Analysis using PCR and immunocytochemistry techniques demonstrated unequivocally that the human hypothalamus expresses both messenger RNA and protein of the IL-1RI. To the best of our knowledge, this work represents the first demonstration of a full-length IL-1RI expressed in the human brain. The only other publication detailing analysis of the expression of the human brain receptor is that of Hillier and colleagues, who have reported the presence of a 453 bp expressed sequence tag cloned from the frontal lobe of a male schizophrenic (Hillier et al., 1997). This expressed sequence tag has homology with the IL-1RI precursor, which includes the receptor signal peptide.
As a consequence of this work, a stable CHO cell line expressing functional human hypothalamic IL-1RI is now available, facilitating a thorough analysis of the signalling mechanisms mediated by IL-1 via this receptor. In addition, this cell line will provide a tool to aid the discovery of novel IL-1 therapeutic agents.
Activation of p38 MAP kinase was used as a measure of the ability of the CHO.hIL1RI cells to transduce an IL-1 signal. IL-1 caused an increase in phosphorylation of p38 MAP kinase in CHO cells transfected with the hIL-1RI. The kinetics of activation of this kinase in our study was in agreement with that demonstrated by Scherle and colleagues (Scherle et al., 1997).
The fact that IL-1 can activate signalling in CHO cells transfected with the type I receptor indicates that the parental CHO.K1 cells endogenously express the IL-1RI accessory protein (Hofmeister et al., 1997; Wesche et al., 1997). This assumption has been confirmed by immunoblotting CHO.K1 cells with an antibody raised against the IL-1RI accessory protein (E.A.H., unpublished observations).
The pharmacology of the CHO.hIL1RI cell line was examined using a Cytosensor microphysiometer. The microphysiometer measures the cellular acidification rate as an index of the integrated functional response to receptor activation, and is well suited to the pharmacological study of stably expressed recombinant receptors (Owicki et al., 1990; Jordan et al., 1998).
Both the EC50 (2.0 ng/ml) for IL-1 and the complete blockade of the IL-1 response with a 20-fold excess of the antagonist IL-1ra are consistent with established IL-1RI pharmacology (Bankers-Fulbright et al., 1996).
The desensitization of the receptor following its activation with IL-1 in the present study is particularly interesting as this has not been identified in other in vitro studies (Bankers-Fulbright et al., 1996) but has been reported in vivo in the mouse brain (Haour et al., 1995).
The IL-1RI protein is expressed in the human hypothalamus, as demonstrated by immunocytochemistry. Displacement studies involving the preadsorption of the primary anti-IL-1RI antibody with control antigenic peptide confirmed the specificity to the type I receptor. Examination of consecutive sections stained with anti-IL-1RI or anti-GFAP antibodies revealed expression of the IL-1 type I receptor in astrocytes. This observation is in agreement with previous studies showing that astrocytes in culture express IL-1 binding sites (Ban et al., 1993; Rubio et al., 1994) and IL-1RI mRNA (Tomozawa et al., 1995). Astrocytes express functional IL-1RIs, as demonstrated by the range of IL-1-stimulated biological responses in these cells, including proliferation (Giulian and Tapscott, 1988) and increased levels of expression of nerve growth factor mRNA (Spranger et al., 1990; Pshenichkin et al., 1994). The expression of the receptor in hypothalamic astrocytes is of interest in view of the roles of IL-1 and the hypothalamus in thermogenesis. In the brain, neuroglia, of which astrocytes are the most abundant cell type, are approximately nine times more numerous than neurons (Szelényi, 1998). Astrocytes possess both the substrates (glucose and glycogen) and the machinery (enzymes of glycogenolysis) to enable them to play a role in adaptive cerebral heat production. Vasoactive intestinal peptide stimulates glycogenolysis and, in addition, production of this peptide is known to be upregulated, at least in lymphocytes, by IL-1 (Delgado et al., 1999). Whether the cytokine is capable of increasing expression of vasoactive intestinal peptide in astrocytes, thereby implicating astrocytic IL-1 receptors in thermogenesis, is yet to be determined. Furthermore, prostaglandin release is recognized as a prominent mediator of thermoregulation, and the induction of prostaglandin E2 release by IL-1 has been demonstrated recently in human cultured astrocytes (Mollace et al., 1998). We therefore believe that evidence from the literature, combined with our demonstration of astrocytic expression of the IL-1RI in the hypothalamus, can in part account for the role of IL-1 in thermoregulation.
Further analysis of consecutive sections stained with anti-IL-1RI or biotinylated GSIB4 failed to show expression of IL-1RI in endothelial cells of the human hypothalamus. This finding is in contrast to that of Yabuuchi and colleagues, who observed expression of IL-1RI in neurons and endothelial cells of the rat brain (Yabuuchi et al., 1994). The reasons for this discrepancy are unknown, but they may reflect differences in expression between species or the fact that the level of IL-1RI protein expression in neurons and endothelial cells may be below the detection limit of the antibody at the dilution used for the current study. Yabuuchi and colleagues (Yabuuchi et al., 1994) did in fact refer to their unpublished observations of expression of IL-1RI mRNA in astrocyte cultures. Alternatively, although mRNA is expressed, translation into membrane-bound receptor protein may not occur.
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Acknowledgments |
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The authors wish to thank Ruth Franks, Sandra Duffy, Richard Bystry and Joanne Hunt for technical support, Professor David Mann for providing human hypothalamus and Drs A. T. McKnight, G. Luheshi and Professor N. J. Rothwell for contributing to useful scientific discussions. S.T. is a Royal Society Dorothy Hodgkin Research Fellow.
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Received December 17, 1998. Revised March 5, 1999. Accepted April 1, 1999.
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