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Endocrinology Vol. 140, No. 9 3890-3903
Copyright © 1999 by The Endocrine Society

ARTICLES

Interleukin-6 Is a Needed Proinflammatory Cytokine in the Prolonged Neural Activity and Transcriptional Activation of Corticotropin-Releasing Factor during Endotoxemia1

Luc Vallières2 and Serge Rivest3

Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Sainte-Foy, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Serge Rivest, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier boulevard, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: serge.rivest{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukin-6 (IL-6) is a proinflammatory cytokine that plays multiple roles in the central nervous system during infections and injuries. Although this molecule is capable of stimulating the release of ACTH and glucocorticoids, it has been demonstrated that a single injection of IL-6 fails to activate the paraventricular nucleus (PVN) neurons that control the hypothalamic-pituitary-adrenal axis. The observation that IL-6 receptor (IL-6R) is up-regulated in the brain during endotoxemia led us to hypothesize that prior induction of IL-6R synthesis could amplify the effect of circulating IL-6 on the neuroendocrine response. Rats received a first iv injection of either bacterial lipopolysaccharide (LPS; 5 µg) or vehicle solution. After a 6-h waiting period, they received a second iv injection of either recombinant rat IL-6 or vehicle solution and were killed 1 h thereafter. Using in situ hybridization, we observed that IL-6R was barely expressed in the PVN under basal conditions, but was rapidly produced in response to LPS. IL-6 itself was also able to induce the synthesis of its own receptor along cerebral blood vessels, and this effect extended to several parenchymal structures, including the PVN, when the cytokine was administrated after LPS. In agreement with our hypothesis, we found that IL-6 injected in LPS-pretreated rats stimulated PVN neurons, as revealed by the expression of CRF primary transcript and c-fos messenger RNA, an immediate early gene used as a marker of cellular activation. A significant increase in plasma corticosterone levels was also found in animals that received iv IL-6 injection after being pretreated 6 h before with the very low dose of LPS. The fact that IL-6 alone or injected after LPS treatment was unable to induce cyclooxygenase-2 synthesis is an argument in favor of a PG-independent mechanism. The relative contribution of IL-6 in stimulating CRF expression in the PVN and neural activity throughout the brain during endotoxemia was also investigated in IL-6-deficient mice after an ip injection of LPS. The endotoxin induced similar c-fos and CRF expression patterns in knockout and wild-type mice, but the expression levels were generally higher and/or lasted longer in wild-type animals. Taken together, physiological changes that may include the induction of IL-6R synthesis seem to be necessary for IL-6 to activate PVN neurons. Moreover, although IL-6 does not appear essential during the early phases of endotoxemia, this cytokine is required during the later phases to prolong the activation of neural cells throughout the brain and to maintain CRF expression in the PVN neurons that control the hypothalamic-pituitary-adrenal axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BACTERIAL septic shock is a serious pathological state often caused by certain Gram-negative bacteria and characterized by numerous physiological changes, such as fever, hypotension, and secretion of a variety of hormones. This toxicity is due to a component of the bacterial cell wall, the endotoxin lipopolysaccharide (LPS), that stimulates monocytes and macrophages to produce proinflammatory cytokines. In addition to their classic roles in modulating the immune and inflammatory responses, these pleiotropic factors can also act within the nervous system to solicit local and systemic changes necessary to restore homeostasis. Recent evidence indicates that different cell populations in the brain can synthesize their own set of cytokines during endotoxemia, in particular interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-{alpha} (TNF{alpha}) (1, 2, 3). Among their central effects, the induction of fever and the activation of the hypothalamic-pituitary-adrenal (HPA) axis have been widely investigated because of their major impacts on the inflammatory processes (4, 5, 6).

IL-6 has been defined as one of the principal endogenous pyrogens from the observations that IL-6-deficient mice are unable to develop normal fever in response to both LPS and IL-1ß (7). Based on physiological and neuroanatomical studies, it has been proposed that IL-6 may induce fever by signaling thermoregulatory neurons of the anterior hypothalamic area through PG-dependent pathways (8, 9, 10). On the other hand, the relative contribution of IL-6 to modulating the HPA axis during endotoxemia and its exact mechanisms of action are not yet fully understood. It has been shown that IL-6 can increase glucocorticoid production by stimulating the secretion of ACTH from the anterior pituitary (11, 12) and by acting directly on the adrenal gland (13, 14, 15). Although IL-6 can also trigger the release of CRF into the hypophyseal portal system (16, 17, 18), this cytokine seems unable, in contrast to IL-1ß, to induce neuronal activation and CRF gene transcription in the hypothalamic paraventricular nucleus (PVN) (10, 19), a region that contains neuroendocrine CRF neurons. This lack of effect might, however, be explained by the fact that IL-6 receptor (IL-6R) is not expressed in this nucleus under basal conditions (1). It is plausible that the induction of IL-6R synthesis may be an essential step taking place early during inflammation to allow IL-6, when it becomes available in the blood circulation, to trigger neural responses. We, therefore, hypothesized that this event may potentiate and amplify the action of systemic IL-6 on the CRF-containing neurons that control the HPA axis.

Besides fever and CRF secretion, IL-6 may also influence a variety of other neural functions during endotoxemia. This attractive possibility is suggested by the observations that IL-6 is synthesized by the circumventricular organs and secreted into cerebrospinal fluid in response to systemic LPS administration (1, 20) and that an intracerebroventricular injection of IL-6 induces widespread cellular activation in the brain, particularly over the ependymal cell layer, the ventricular walls, and the meninges (10). One possible role of IL-6 is the modulation of certain properties of the endothelial and ependymal cells, which express the IL-6R subunits (1) and constitute natural barriers against immunological substances present in the circulation and cerebrospinal fluid, respectively. It has been demonstrated that IL-6 participates in the development of reactive gliosis affecting both microglia and astrocytes (21, 22, 23). This response may be essential to eliminate and restrain the penetration of circulating LPS into the nervous tissue. Furthermore, new findings suggest that IL-6 may play neuroprotective functions, as, for example, the repression of TNF{alpha}-induced neurotoxic inflammation and the enhancement of nerve growth factor signaling (24, 25).

In this study, we have evaluated the contribution of IL-6 in stimulating neural activity and CRF gene transcription in rodent brains during endotoxemia. Given that IL-6R has been shown to be up-regulated in response to endotoxin (1), we first characterized its expression pattern in the brain and verified whether CRF neurons of the PVN produced this receptor after different treatments with LPS and IL-6. To verify the possibility that preinduction of IL-6R synthesis may increase the influence of IL-6 on neuroendocrine functions, we further compared the effects of a systemic injection of IL-6 on the PVN when given under basal conditions and 6 h after pretreatment with low dose LPS. To confirm and complement the results obtained with this approach, we finally used IL-6-deficient mice to analyze the role of IL-6 in the activation of neural cells, a phenomenon commonly observed in the PVN and elsewhere in the brain during endotoxemia. We provide here evidence that IL-6 modulates, under LPS challenge, the expression of its own receptor, the neuroendocrine CRF, and the protooncogene c-fos, an immediate early gene used as a marker of cellular activation. In addition, we demonstrate that IL-6 is unable to stimulate the gene encoding the enzyme cyclooxygenase-2 (COX-2) normally required for PG production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The experiments were performed on adult male Sprague Dawley rats (275–300 g) and IL-6 knockout and wild-type mice of the C57BL/6 line (20–30 g). The mutant mice were provided by Dr. M. Kopf (Basel Institute for Immunology, Basel, Switzerland) and generated as previously described (26). All animals were housed individually under standard laboratory conditions (14-h light, 10-h dark cycle; lights on at 0600 h) with food and water available ad libitum. A total of 16 rats was assigned to 4 different treatments (injection of vehicle plus vehicle, LPS plus vehicle, vehicle plus IL-6, and LPS plus IL-6), and 24 mice were divided into 8 groups (knockout and wild-type mice killed before or 1, 4, and 8 h after LPS administration). The experiments were repeated at least twice to confirm the data, although animals of two different sets of studies were not pooled together. All protocols were approved by the Laval University’s animal welfare committee.

iv injection of LPS and IL-6 in rats
A catheter containing sterile pyrogen-free heparin-saline (5.0 U/ml) was implanted into the right jugular vein of rats anesthetized with an ip injection (300 µl) of a mixture of ketamine hydrochloride (91 mg/ml) and xylazine (9 mg/ml). The catheter was made from a piece of SILASTIC brand tubing (SILASTIC medical grade tubing, 0.50 mm i.d., 0.94 mm o.d.; Dow Corning Corp., Midland, MI) connected to Intramedic polyethylene tubing (id, 0.58 mm; od, 0.965 mm; PE-50, Clay Adams, Parsippany, NJ). The internal SILASTIC tip of the catheter was positioned at the atrium, and the outlet was placed at an interscapular position and sealed. After the surgery, the animals were kept individually for a 5-day recuperation period.

On the day of the experiment (0900 h), the outlet of the catheter was fastened to a truncated 27-gauge needle attached to PE-50 tubing. This connector was then fixed to a 1-ml syringe, and the rats were placed individually in a quiet room for at least 2 h before experimentation. This procedure was used to avoid disturbing the animals during the injections. Rats received first an iv injection of either LPS (5 µg dissolved in 300 µl 0.9% sterile pyrogen-free saline; from Escherichia coli, serotype 055:B5, catalog no. L2880, Sigma Chemical Co., Oakville, Canada) or vehicle solution. The animals were conscious and freely moving during the procedure. After a 6-h waiting period, they received a second iv injection of either recombinant rat IL-6 (1.5 µg dissolved in 300 µl sterile pyrogen-free distilled water; bioactivity, >=1 x 107 U/mg; endotoxin, <0.1 ng/µl; catalog no. PRC0065, lot H032506, BioSource Technologies, Inc., Camarillo, CA) or vehicle solution. The animals were killed 1 h after the second injection with an iv injection of ketamine-xylazine. Tissue processing was performed as described below.

ip injection of LPS in mice
In the morning (at ~0830 h), IL-6 knockout and wild-type mice received an ip injection of LPS (25 µg dissolved in 100 µl 0.9% sterile pyrogen-free saline). All animals were conscious and freely moving during the procedure and were kept individually in their cages. The mutant mice seemed less affected by LPS, showing higher locomotor activity than wild-type controls. The animals were killed 1, 4, and 8 h after LPS administration with an ip injection of ketamine-xylazine. Additional mice were not injected with LPS and were used as controls (time zero). Tissue processing was performed as described below.

Tissue processing
All animals were rapidly perfused transcardially with 0.9% saline followed by ice-cold 4% paraformaldehyde in 100 mM borax buffer (pH 9.5). Their brains were removed, postfixed with fresh 4% paraformaldehyde for 2–5 days at 4 C, and placed in the same fixative containing 10% sucrose overnight at 4 C. They were mounted onto a microtome (Reichert-Jung, Cambridge Instrument Co., Deerfield, IL), frozen with dry ice, and cut into 30-µm (rats) or 20-µm (mice) coronal sections from the olfactory bulb to the caudal medulla. The slices were collected in a cold cryoprotectant solution [50 mM sodium phosphate buffer (pH 7.3), 30% ethylene glycol, and 20% glycerol] and stored at -20 C until histochemical analysis.

Riboprobe synthesis and preparation
Details of the plasmids and enzymes used for complementary RNA (cRNA) probe synthesis are presented in Table 1Go. Rat c-fos complementary DNA (cDNA) was obtained from Dr. I. Verma (The Salk Institute, La Jolla, CA), rat CRF cDNA from Dr. K. Mayo (Northwestern University, Evanston, IL), rat intronic CRF cDNA from Dr. S. Watson (University of Michigan, Ann Arbor, MI), rat COX-2 cDNA from Dr. K. Peri (Sainte-Justine Hospital Research Center, Montréal, Canada), and rat IL-6 cDNA from American Type Culture Collection (Manassas, VA). All protocols were adapted from Simmons et al. (27). Sense and antisense radioactive cRNA copies were synthesized by incubating 250 ng linearized plasmid in 6 mM MgCl2, 40 mM Tris (pH 7.9), 2 mM spermidine, 10 mM NaCl, 10 mM dithiothreitol, 0.2 mM ATP/GTP/CTP, [{alpha}-35S]UTP, 40 U RNAsin (Promega Corp., Madison, WI), and 20 U of the appropriate RNA polymerase (Table 1Go) for 60 min at 37 C. The unincorporated nucleotides were removed by adding 100 µl deoxyribonuclease solution (1 µl deoxyribonuclease, 5 µl 5 mg/ml transfer RNA, and 94 µl 10 mM Tris-10 mM MgCl2) for 10 min, followed by phenol-chloroform extraction. The probes were precipitated with 80 µl 5 M ammonium acetate and 500 µl 95% ethanol for 20 min on dry ice. After centrifugation, the pellets were washed with 500 µl 70% ethanol, dried, and resuspended in 100 µl 10 mM Tris/1 mM EDTA. The probes (107 cpm/ml) were mixed into an appropriate volume of hybridization solution [822 µl solution 1 (500 µl formamide; 60 µl 5 M NaCl; 10 µl 1 M Tris, pH 8.0; 2 µl 0.5 M EDTA, pH 8.0; 20 µl 50 x Denhart’s solution; 200 µl 50% dextran sulfate; and 30 µl diethylpyrocarbonate (depc) water), 50 µl 10 mg/ml transfer RNA, and 10 µl 1 M dithiothreitol (118 µl depc water - volume of probe used)] and heated for 5 min at 65 C before being spotted on the slides (100 µl). Radioactive sense cRNA copies (controls) were also prepared to verify the specificity of each probe.


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  		Table 1. Plasmids and enzymes used for probe synthesis

 
In situ hybridization histochemistry
In situ hybridization was performed to localize each transcript [c-fos, CRF, heteronuclear (hn) CRF, IL-6R, and COX-2 messenger RNAs (mRNAs)] on every sixth section from the olfactory bulb to the end of the medulla. All solutions were treated with depc and sterilized to prevent RNA degradation. The slices were washed in 50 mM potassium PBS (KPBS), mounted onto gelatin- and poly-L-lysine-coated slides, and vacuum-dried. They were fixed in 4% paraformaldehyde for 20 min and digested by proteinase K (10 µg/ml in 100 mM Tris-HCl, pH 8.0, and 50 mM EDTA, pH 8.0) at 37 C for 25 min. The sections were then rinsed in sterile depc water followed by a solution of 100 mM triethanolamine (pH 8.0), acetylated in 0.25% acetic anhydride in 100 mM triethanolamine, and dehydrated through graded concentrations of alcohol (50%, 70%, 95%, and 100%). After vacuum drying, 100 µl hybridization mixture (106 cpm) were spotted on each slide, sealed under a coverslip, and incubated at 60 C for 15–20 h on a slide warmer. The coverslips were then removed, and the slides were rinsed four times in 4 x SSC (150 mM NaCl and 15 mM Tris-NaCl citrate buffer, pH 7.0) at room temperature. The sections were digested by ribonuclease A (20 µg/ml in a solution of 500 mM NaCl; 10 mM Tris-HCl, pH 8.0; and 1 mM EDTA, pH 8.0) at 37 C for 30 min, rinsed in descending concentrations of SSC (2, 1, and 0.5 x), washed in 0.1 x SSC for 30 min at 60 C, and dehydrated through graded concentrations of alcohol. After being vacuum-dried, the sections were exposed at 4 C to x-ray films (Eastman Kodak Co.) for 17 h (c-fos and CRF), 65 h (IL-6R and COX-2), or 96 h (hnCRF), defatted in xylene, and dipped into NTB2 nuclear emulsion (Eastman Kodak Co.; diluted 1:1 with distilled water). Slides were exposed for 7 days (c-fos and CRF), 14 days (IL-6R and COX-2), or 21 days (hnCRF); developed in D19 developer (Eastman Kodak Co.) for 3.5 min at 14–16 C; washed for 15 sec in water; and fixed in rapid fixer (Eastman Kodak Co.) for 5 min. Thereafter, the tissues were rinsed under running distilled water for 1 h, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX (BDH, Poole, UK).

Dual labeling
Immunohistochemistry and in situ hybridization were combined to determine whether CRF neurons of the PVN expressed IL-6R mRNA. Hypothalamic sections were stained for CRF using the avidin-biotin amplification bridge method with peroxidase as a substrate. The slices were washed in sterile depc-treated KPBS at room temperature and incubated for 2 h with rabbit antihuman/rat CRF serum (diluted 1:20,000; code PBL rC70; provided by Dr. W. Vale, Peptide Biology Laboratory, The Salk Institute, La Jolla, CA) mixed in KPBS containing 0.4% Triton X-100 and 0.25% heparin sodium salt USP. The sections were then washed in KPBS and incubated for 1 h with biotinylated goat antirabbit IgG (diluted 1:1500; Vector Laboratories, Inc. Burlingame, CA) mixed in KPBS containing 0.4% Triton X-100 and 0.25% heparin sodium salt USP. The slices were then rinsed with KPBS and incubated for 1 h with an avidin-biotin-peroxidase complex (Vectastain ABC elite kit, Vector Laboratories, Inc., Burlingame, CA). After several washes, the brain slices were reacted in KPBS containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.003% hydrogen peroxide (H2O2). Thereafter, the slices were rinsed with KPBS, mounted onto gelatin- and poly-L-lysine-coated slides, and vacuum-dried. In situ hybridization was performed as described above to localize IL-6R mRNA, except that the steps with alcohol were shortened (10 dips) to avoid decoloration of immunostained cells.

Blood sampling and corticosterone measurement in rats
Blood was collected quickly by cardiac puncture before perfusion, transferred in EDTA tubes (Becton Dickinson and Co., Franklin Lakes, NJ), and centrifuged for 10 min at 3000 rpm (4 C). Plasma was collected and stored at -20 C until the analysis. Corticosterone levels were measured by RIA (Immuchem corticosterone RIA kit for rats, ICN Biomedicals, Inc., Costa Mesa, CA) according to the manufacturer’s protocol. The intra- and interassay coefficients of variation were both 7%.

OD analysis
Semiquantitative analysis of hybridization signals for c-fos, CRF, hnCRF, and IL-6R mRNAs was carried out on x-ray films over at least two hypothalamic paraventricular nuclei (bilateral) for each animal. Transmittance values (referred here as OD) of the hybridization signals were measured on a Northern Light Desktop Illuminator (Imaging Research, Inc.) using a Sony Camera Video System fastened to a MicroNikkor 55-mm Vivitar extension tube set for Nikon lens and coupled to a Macintosh computer (Power Macintosh 7100/66) and the NIH Image software version 1.59/ppc (written by W. Rasband at the NIH and available from the internet by anonymous ftp from zippy.nih.gov). The OD values for each pixel were calculated using a known standard of intensity and distance measurements from a logarithmic specter adapted from Bioimage Visage 110s (Millipore Corp., Ann Arbor, MI). The wedge was calibrated before correcting for film saturation, which was determined by sampling darkest PVNs and adjusting the light source and exposure time. All samples displaying clear positive signals were evaluated within the linear OD curve to avoid pixel saturation and underestimation. Sections from experimental and control animals were digitized and subjected to densitometric analysis, yielding measurements of mean density per area. The OD of each side of the PVN (bilateral) was then corrected for the average background signal by subtracting the OD of areas without positive signal located immediately outside the digitized nucleus. Because of the lack of basal expression of c-fos mRNA and CRF hnRNA in the PVN, the stained slides were used to delineate the nucleus on the film. Data are expressed as the mean ± SEM and were analyzed by two-way ANOVA followed by the Bonferroni/Dunn test used for post-hoc comparisons when appropriate.

Qualitative analysis
Anatomical identification of brain structures was based on the Swanson’s atlas (28). The relative intensity of c-fos mRNA signals throughout mouse brains was assessed on x-ray film images and graded according to the scale of undetectable (-), low (+), moderate (2+), strong (2++), or very strong (2+2+).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-6R mRNA is up-regulated in response to LPS and IL-6
We have previously described the distribution of IL-6R mRNA in rat brain and its regulation during endotoxemia (1). To confirm these results and verify whether IL-6 can modulate the synthesis of its own receptor, we examined the expression of IL-6R mRNA by in situ hybridization after different treatments with LPS and IL-6. Consistent with our previous results, IL-6R mRNA was found in several brain regions under basal conditions, including the ependymal cell layer, vascular organ of the lamina terminalis (OVLT), cerebral cortex (CC), bed nucleus of the stria terminalis (BNST), subfornical organ (SFO), hippocampus, central nucleus of the amygdala (CeA), median eminence (ME), and area postrema (AP; Fig. 1Go, Veh+Veh). This transcript, however, was undetectable or very slightly expressed in the PVN of vehicle-treated animals. The IL-6R mRNA levels increased 7 h after LPS administration in the OVLT, CC, BNST, PVN, and CeA (Fig. 1Go, LPS+Veh). Interestingly, IL-6 was also capable of activating the gene encoding its receptor. This effect was restricted to blood vessels after a single injection of IL-6 (Fig. 1Go, Veh+IL-6, and Fig. 2Go), but extended to the above-mentioned structures when the cytokine was injected 6 h after LPS administration (Fig. 1Go, LPS+IL-6). Indeed, IL-6 injection induced a more pronounced expression of its receptor in several regions of the parenchyma of rats pretreated with the endotoxin. In particular, the OD analysis of hybridization signals revealed that the IL-6R mRNA levels in the PVN were significantly higher (P < 0.05) after treatment with LPS and IL-6 together than after LPS or IL-6 alone (Fig. 3Go). Adjacent sections hybridized with a sense probe did not exhibit positive signal in any of the regions expressing the transcript with the antisense probe.



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  		Figure 1. Distribution of IL-6R mRNA in rat brains after different treatments with LPS and IL-6. Rats first received an iv injection of either 5 µg LPS or vehicle solution. After a 6-h waiting period, they received a second iv injection of either 1.5 µg IL-6 or vehicle solution. The animals were killed 1 h afterward, and in situ hybridization was performed to detect IL-6R mRNA. bv, Blood vessel; CER, cerebellar cortex; epc, ependymal cell layer; HP, hippocampus; PIR2, layer 2 of the piriform area; Veh, vehicle.

 


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  		Figure 2. Localization of IL-6R mRNA in cerebral blood vessels after different treatments with LPS and IL-6. Rats first received an iv injection of either 5 µg LPS or vehicle solution. After a 6-h waiting period, they received a second iv injection of either 1.5 µg IL-6 or vehicle solution. The animals were killed 1 h afterward, and in situ hybridization was performed to detect IL-6R mRNA. These darkfield photomicrographs show representative examples of hybridization signals along vascular cells in rats treated with either IL-6 alone (C) or both LPS and IL-6 (D), whereas no positive cells were detected in vehicle-injected rats (A). Additional control animals were treated with LPS during 3 h, and strong hybridization signals for IL-6R mRNA were observed along blood vessels (B). The sections were taken from thalamic areas. Magnification, x25.

 


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  		Figure 3. Expression of IL-6R mRNA in the PVN after different treatments with LPS and IL-6. Rats first received an iv injection of either 5 µg LPS or vehicle solution. After a 6-h waiting period, they received a second iv injection of either 1.5 µg IL-6 or vehicle solution. The animals were killed 1 h afterward, and in situ hybridization was performed to detect IL-6R mRNA. OD analysis of hybridization signals was performed on x-ray films, and data represent the mean ± SEM of four animals per group. *, P < 0.05 compared with vehicle-treated animals. **, P < 0.05 compared with all other groups.

 
The expression pattern of IL-6R over the parvocellular division of the PVN (Fig. 4BGo) led us to believe that CRF-containing cells might express this receptor. The dual labeling experiment revealed that few CRF-labeled perikarya were positive for the IL-6R transcript in response to LPS and IL-6 (Fig. 4CGo). However, the IL-6R transcript was very sensitive to the immunohistochemical procedures, making evaluation of the exact proportion of double labeled cells difficult. The possibility that other cell types, such as microglia, express this receptor cannot be ruled out.



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  		Figure 4. Expression of IL-6R mRNA in CRF-containing neurons of the PVN during endotoxemia. These photomicrographs show hybridization signals for IL-6R mRNA in the PVN (B) and within CRF neurons (C) after treatment with both LPS and IL-6. This transcript was undetectable in the PVN of control animals (A). Magnification, x25 in A and B; x250 in C.

 
IL-6 stimulates the PVN and plasma corticosterone release during endotoxemia
Based on the above observations, we hypothesized that IL-6 signaling could be enhanced during endotoxemia after the induction of IL-6R synthesis. We therefore compared the effects of IL-6 on the expression of different genes in the PVN when injected alone and 6 h after induction of the immune response with LPS. To this end, c-fos mRNA was used as a marker of cellular activation, and CRF hnRNA was used as an index of de novo CRF gene transcription. The time was chosen on the basis of preliminary data showing that the levels of both transcripts increased 1 h after LPS injection and declined at 3 h, and CRF hnRNA returned to basal levels at 6 h (data not shown). As expected, c-fos mRNA and CRF hnRNA were expressed at very low levels in the PVN of vehicle- and IL-6-treated rats (Fig. 5Go). Moderate levels of c-fos mRNA were still observed in the PVN 7 h after LPS injection, whereas CRF hnRNA was barely detectable. Confirming our hypothesis, both transcripts were significantly (P < 0.05) higher in the PVN of rats treated with both LPS and IL-6 than in the animals treated with either LPS or IL-6 alone (Fig. 5Go, A and B). Plasma corticosterone levels were also significantly higher in animals presensitized and receiving a single iv bolus of recombinant IL-6 than vehicle- and LPS-injected rats (Fig. 5CGo). However, the Bonferroni post-hoc analysis did not reveal a significant difference between the circulating corticosterone concentrations of animals that were injected only with IL-6 (Veh+IL-6) and those of vehicle-treated rats (Veh+Veh).



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  		Figure 5. c-fos mRNA (A) and CRF heteronuclear (hn) RNA (B) expression in the PVN of the rat hypothalamus and plasma corticosterone levels (C) after different treatments with LPS and IL-6. Rats first received an iv injection of either 5 µg LPS or vehicle solution. After a 6-h waiting period, they received a second iv injection of either 1.5 µg IL-6 or vehicle solution. The animals were killed 1 h afterward, and in situ hybridization was performed to detect each transcript. OD analysis of hybridization signals was performed on x-ray films, and data represent the mean ± SEM of four animals per group. *, P < 0.05 compared with vehicle-treated animals. **, P < 0.05 compared with all other groups.

 
It has been demonstrated that IL-6 is insufficient in itself to induce the expression of the mRNA encoding the enzyme COX-2 necessary for PG synthesis during inflammation (29). Here we verified whether IL-6 can stimulate the synthesis of COX-2 mRNA after pretreatment with LPS. COX-2 mRNA was constitutively expressed in the hippocampus, increased along blood vessels 3 h after LPS injection, and returned to basal levels 6 h thereafter. In agreement with previous results, COX-2 mRNA was not induced by IL-6 when injected alone or in combination with LPS (data not shown).

IL-6 is necessary for the prolonged stimulation of neural activity during endotoxemia
It has been commonly observed that LPS induces widespread neural activation in both rat and mouse brains (30). To verify the contribution of IL-6 to this response and to confirm its involvement in stimulating the PVN during endotoxemia, we compared the effects of LPS on c-fos and CRF expression in the brains of IL-6-deficient and wild-type mice. Table 2Go describes the qualitative analysis of hybridization signals for c-fos mRNA in mice killed before or 1, 4, and 8 h after LPS administration. As shown in Fig. 6AGo, the distribution and the intensity of c-fos mRNA hybridization signals were similar in wild-type and knockout mice under basal conditions (0 h). One hour after LPS administration, increased levels of c-fos mRNA were observed in several nuclei without a difference between knockout mice and their controls, namely the CC, BNST, medial preoptic area (MPO)/OVLT, SFO, PVN, supraoptic nucleus (SON), ME, CeA, dorsomedial hypothalamic nucleus, locus coeruleus (LC), AP, nucleus of the solitary tract, blood vessels, choroid plexus, leptomeninges, and parabrachial nucleus (Fig. 6AGo). Interestingly, we found that c-fos mRNA expression was markedly reduced in knockout mice 4 h after LPS injection, but remained elevated in several brain regions of wild-type animals (Fig. 6BGo). These regions were the ependymal cell layer, which did not express c-fos mRNA at any time in IL-6-deficient mice, the PVN, CC, LC, MPO/OVLT, ME, leptomeninges, SFO, submeningual zone (sm), and SON (Table 2Go). In the PVN, the OD analysis indicated that the mRNA encoding c-fos was indeed significantly higher (P < 0.05) in wild-type mice than in knockout animals at 4 h post-LPS injection (Fig. 7AGo). In wild-type mice, c-fos mRNA levels decreased and reached levels observed in knockout animals at 8 h postinjection, except in the locus coeruleus, where the message remained elevated (Fig. 6BGo).


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  		Table 2. Expression of c-fos mRNA in brains of IL-6-knockout (KO) and wild-type (WT) mice before and after an ip injection of LPS

 



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  		Figure 6. Distribution of c-fos mRNA in brains of IL-6 knockout (KO) and wild-type (WT) mice during endotoxemia (A and B). The transcript was detected by in situ hybridization in brains collected before or 1, 4, and 8 h after an ip injection of 25 µg LPS. CER, cerebellar cortex; DMH, dorsomedial hypothalamic nucleus; epc, ependymal cell layer; HP, hippocampus; ICe, external nucleus of the inferior colliculus; NTS, nucleus of the solitary tract; PG, pontine gray; PIR2, layer 2 of the piriform area; PVT, thalamic paraventricular nucleus; sm, submeningual zone.

 


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  		Figure 7. Expression of c-fos (A) and CRF (B) mRNAs in the PVN of IL-6 knockout (KO) and wild-type (WT) mice during endotoxemia. Each transcript was detected by in situ hybridization in brains collected before or 1, 4, and 8 h after an ip injection of 25 µg LPS. OD analysis of hybridization signals was performed on x-ray films, and data represent the mean ± SEM of three or four animals per group.

 
On the other hand, CRF mRNA progressively increased in the PVN after LPS injection in both wild-type and knockout animals (Fig. 7BGo). Statistical analysis of OD data showed a significant difference (P = 0.0365) between the IL-6-deficient and control mice. Indeed, the CRF mRNA levels in the PVN were generally higher in wild-type than in knockout mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was undertaken to determine to what degree IL-6 contributes to the stimulation of CRF expression in the PVN and neural activity throughout the brain during endotoxemia. We first report that IL-6R is barely expressed in the PVN under basal conditions, but is constitutively produced in several other brain areas. Although IL-6R expression can be controlled by IL-6 itself, the synthesis of IL-6R mRNA is increased in parenchymal structures, such as the PVN, only in response to bacterial LPS. These observations suggest that IL-6 signaling is enhanced during endotoxemia and led us to hypothesize that IL-6 could modulate PVN functions after induction of its receptor. Indeed, our subsequent results confirm that IL-6 can activate PVN neurons after pretreatment with LPS by stimulating c-fos and CRF gene transcription as well as plasma corticosterone release. Using a dual labeling technique, we found that some CRF neurons express IL-6R, suggesting that IL-6 may directly target these cells to trigger neuronal activation and CRF secretion. The fact that IL-6 is unable to induce COX-2 synthesis during this process is an argument in favor of a PG-independent mechanism. In addition, evidence obtained from the experiment with mutant animals indicates that LPS induces similar CRF and c-fos expression patterns in wild-type and IL-6-deficient mice. However, the expression levels are generally higher and remain elevated longer in wild-type animals, particularly in the PVN and over structures close to the ventricles. We, therefore, propose that IL-6, although not involved during the initial phases of endotoxemia, seems necessary during the later phases for maintaining the stimulation of CRF neurons controlling the HPA axis and for prolonging the activation of neural cells throughout the brain. This phenomenon might be of great importance to protect the brain and to restore homeostasis during bacterial septic shock.

IL-6 signaling in rat brain
Our previous work (1) together with the present results suggest that neural cells become more sensitive to IL-6 during systemic immune challenges by increasing the number of receptor molecules on their surface. Although this modification seems to be essential, additional physiological changes might be required to allow such a permissive effect. The shedding of soluble IL-6R, the alteration of the blood-brain barrier (BBB), and the activation of transduction pathways cooperating with those solicited by IL-6 are examples of mechanisms that may amplify, widen, and prolong the IL-6 activities. On the one hand, these changes may be initiated by the direct action of LPS on endothelial and parenchymal cells after binding to its specific receptor CD14. Indeed, we have recently demonstrated that CD14 is distributed in brain regions accessible from the blood circulation under basal conditions and that scattered microglial cells produce this receptor within a few hours after LPS administration (31). Although the endothelial cells do not express CD14, they can respond to circulating endotoxin in a soluble CD14-dependent manner (32, 33, 34). On the other hand, the enhancement of IL-6 signaling may be induced by proinflammatory cytokines released by macrophages and glial cells in response to LPS. It has been shown that systemically injected IL-1ß is capable of stimulating IL-6R synthesis in cerebral blood vessels (1) and that TNF{alpha} can modulate the expression of both IL-6R and gp130 mRNAs in cultured neurons (35). Moreover, as observed in the present study, IL-6 can increase the levels of its receptor within perivascular and parenchymal elements of the brain, an effect consistent with the observation that IL-6 can up-regulate its receptor in the liver (36).

IL-6 and the HPA axis
The presence of IL-6 is essential for coordinating a variety of physiological responses during infections, but overexposition of the nervous tissue to IL-6 induces a pronounced reactive gliosis and leads to neurodegeneration associated with impaired learning capacities (22, 37, 38). Appropriate modulation of IL-6 signaling is thus not only important for enhancing the effects of IL-6, but also to avoid its deleterious actions. It is well known that IL-6 stimulates the HPA axis, which provides negative feedback signals acting to reduce the immune response in general and the secretion of IL-6 and other proinflammatory cytokines in particular. IL-6 is thought to be a long term regulator of plasma ACTH and glucocorticoid levels by acting directly on the anterior pituitary (11, 12) and the adrenal gland (13, 14, 15), respectively. Moreover, IL-6 can exert an acute stimulatory effect on the HPA axis by acting on the hypothalamus to induce infundibular CRF secretion (16, 17, 18). However, the importance of IL-6 in this process was recently challenged by the demonstration that iv injected IL-6 is unable by itself to activate PVN neurons and to stimulate CRF gene transcription (10, 19), contrasting with the capacity of IL-1ß (1) and TNF{alpha} (Nadeau, S., and S. Rivest, unpublished results) to induce these changes. We prove here that IL-6 can, in fact, activate PVN neurons, but the cytokine seems to require prior activation of the immune response and the synthesis of its receptor. The present study also demonstrated that the increase in plasma glucocorticoid levels at 1 h after iv IL-6 injection is significant only in animals that were pretreated 6 h before with a very low dose of LPS. It is interesting to note that coadministration of IL-6 and IL-1ß results in synergistic activation of ACTH and corticosterone release (39, 40, 41, 42). Although these effects may be caused by the interaction of both cytokines in the peripheral glands and despite IL-1ß is unable to induce IL-6R expression in the PVN (1), it cannot be excluded that IL-1ß and IL-6 may cooperate in the brain to modulate the HPA axis or other nervous functions. It will be exciting in future work to characterize clearly the nature of these interactions, but the fact that circulating IL-1ß is capable of inducing IL-6R in blood vessels of the brain may be a crucial event leading to such permissive action of the proinflammatory cytokine (1).

Possible mechanisms of action
The exact mechanisms by which IL-6 stimulates PVN neurons remain to be elucidate. The presence of IL-6R in some CRF neurons offers the possibility that IL-6 may directly target these cells. Alternatively, IL-6 may act on neurons devoid of IL-6R, but expressing the signal transducer gp130, after binding its soluble receptor. At first sight, these possibilities seem to be conflicting with the current view that circulating cytokines cannot cross the BBB, but a number of mechanisms may explain this discrepancy. First, PVN neurons project to the median eminence and to the neurohypophysis, thereby exposing their axon terminals to molecules circulating into the bloodstream or secreted locally from cells of myeloid lineage. It has been proposed that IL-6 may act within the median eminence to trigger the release of CRF into the hypophyseal portal system (43). This is supported by our recent observations that median eminence expresses the IL-6R subunits and that iv injected IL-6 induces c-fos expression in this region (1, 10). Second, excessive nitric oxide and IL-6 production caused by endotoxin can alter cerebral endothelium functions and disrupt the BBB (44). The resulting increase of permeability may then allow high weight molecules, including cytokines, to reach sites behind the BBB, but such a mechanism has been challenged by numerous studies. Third, it has been demonstrated that a small quantity of IL-6 can penetrate across the intact BBB via a transport system distinct from those for IL-1ß and TNF{alpha} (45). Some brain areas may, therefore, be equipped with an as yet uncharacterized active transport for IL-6, which might be activated under stressful circumstances. Forth, the choroid plexus is a cerebrospinal fluid-synthesizing structure that has been pointed out as one of the principal sources of central IL-6 during endotoxemia (1). After being released into the ventricular system, centrally produced IL-6 may circulate throughout the brain and reach PVN neurons through passive diffusion.

In addition to the above mechanisms and given that many CRF neurons seem not to express IL-6R, it is conceivable that IL-6 may stimulate intermediate cells, such as endothelial or microglial cells, to release paracrine factors that, in turn, could influence neuronal activity. As potential candidates, PGs and nitric oxide are key mediators in neuroimmune communication, and their production can be controlled by IL-1ß and TNF{alpha} (46, 47, 48, 49). Unfortunately, depending on the experimental procedure and the tissue studied, there are as many studies that prove their involvement in mediating the central effects of IL-6 as there are studies that are in disagreement with it. For examples, it has been demonstrated that PGs mediate IL-6-induced fever (8, 9, 50) and HPA axis stimulation (16, 17), but IL-6 is unable to activate PG formation in cerebral microvessels (51) and to induce COX-2 mRNA synthesis in rat brains (29) or in cultured microglial cells (52). It is clear, however, that IL-6 does not stimulate the production of PGs in peripheral organs, and that, conversely, its own synthesis is induced by them (53, 54). In agreement with the latter, it has recently been shown that IL-6 expression in astrocytes can be induced in vitro by PGE2 (55). Taking together, these observations suggest that IL-6 does not stimulate COX-2 gene transcription, but the possibility that IL-6 may influence PG synthesis at posttranscriptional levels or may cooperate with them to activate CRF neurons cannot be ruled out. On the other hand, much less information is available regarding the effect of IL-6 on nitric oxide production. It has been shown that IL-6 stimulates nitrite formation in cultured hippocampal slices (56). However, IL-6-induced glial cell growth (57) and neuroblastoma cell differentiation (49) are not mediated by nitric oxide, and transgenic mice overexpressing IL-6 in the brain do not manifest any increase in inducible nitric oxide synthase gene expression (22). In light of the above observations, the roles of PGs and nitric oxide in mediating the effects of IL-1ß and TNF-{alpha} cannot be generalized to IL-6, and more detailed studies will be needed to clarify the mechanisms by which this cytokine may affect the neuroendocrine response.

Relative importance of IL-6 during endotoxemia
It is generally believed that proinflammatory cytokines released by macrophages during endotoxemia are mainly responsible for the stimulation of the HPA axis. The relative contribution of IL-6 in this process is currently the subject of intense debates and investigations. Accumulating data are, however, in disagreement with this view and rather suggest that LPS may directly act within the hypothalamus to activate PVN neurons without any help of systemic cytokines during the early phases. Indeed, the levels of plasma ACTH and corticosterone are increased in response to LPS before the elevation of circulating IL-1ß, TNF{alpha}, and IL-6 (58). It is therefore not surprising that pretreatment with IL-1ß antagonist or anti-TNF{alpha} antibody does not abolish the LPS-induced HPA axis and that IL-6-deficient mice produce normal corticosterone levels during the first hour after LPS injection (41, 59, 60, 61). Thereafter, the increase in circulating corticosterone levels has recently been found to be lower in IL-6-/- than IL-6+/+ mice in response to ip LPS injection, but not during restraint stress (62). This suggests that the involvement of IL-6 in the control of the HPA axis is quite specific to the immune stimuli and not to neurogenic stresses. The participation of IL-6 is further suggested by the fact that pretreatment with anti-IL-6 antibody abrogates ACTH secretion both 2 and 4 h after LPS administration, but not at 1 h (41). This cytokine may be considered as a crucial modulator of the HPA axis, as anti-IL-6 antibody also abolishes the IL-1-induced increase in plasma ACTH (40). Interestingly, this determining role has been confirmed in models using inflammatory agents lacking the intrinsic capacity to stimulate the HPA axis, but able to induce cytokine production. It was concluded that IL-6 is an obligate factor to increase glucocorticoid production during cytomegalovirus infection or after the injection of a synthetic analog of viral nucleic acid (62).

The present study brings some light on the mechanisms by which IL-6 contributes to the regulation of the neuroendocrine response by demonstrating that IL-6 is required during the later phases of endotoxemia to sustain the activated state of CRF neurons controlling the HPA axis. It is interesting to mention that IL-6 can also induce the secretion of arginine vasopressin (AVP) (63, 64), an important ACTH and corticosteroid secretagogue, and that chronic cerebral expression of IL-6 modulates the stress-induced increase in plasma corticosterone via a mechanism involving AVP (65). Given that the activation of neural cells located in both parvocellular and magnocellular PVN was observed in the present study after cotreatment with LPS and IL-6, it is plausible that IL-6 stimulates both CRF and AVP neuroendocrine systems during endotoxemia. An important point to address in future investigations will be determination of the exact proportions of CRF-, AVP- and CRF/AVP-activated neurons during the acute phase response in both wild-type and IL-6-deficient mice.

Besides controlling the HPA axis, IL-6 induces a series of other brain-mediated responses and participates in the modulation of local inflammatory events. Over the past years, we used the protooncogene c-fos to identify within the brain the sites of action of the cytokine. We found that CVOs, the meninges, the ependymal cell layer covering the ventricular spaces, and the portion of parenchyma surrounding the ventricles are profoundly activated after an intracerebroventricular injection of IL-6 (10). More recently, we reported that IL-6 is not synthesized in the brain under basal conditions, but is rapidly expressed by the choroid plexus and other CVOs in response to systemically injected endotoxin. Furthermore, the present study shows that LPS-induced c-fos expression is markedly reduced in the brain of knockout animals, especially in structures that were responding to the intracerebroventricular IL-6 treatment. Taken together, these observations suggest that IL-6 may be secreted by the choroid plexus into cerebrospinal fluid, which diffuses across the brain through the ventricles. This may allow the cytokine to stimulate various neural cells, principally those associated with or close to the ventricular system. The presence of IL-6R in parvocellular neurons of the PVN is of great interest, as it suggests that IL-6 of central origin may bind to its cognate receptor directly within the endocrine hypothalamus. Such a mechanism obviously does not take place in the brain of IL-6-deficient mice, which may contribute to the reduced activity of PVN neurons during endotoxemia.

Conclusion
In summary, the following sequence of events presumably takes place in the central nervous system during endotoxemia. Bacterial endotoxin reaches the brain through the circulation and acts directly on nonparenchymal and parenchymal structures accessible from the blood, which leads to the activation of vascular-associated cells and development of the inflammatory response. PVN neurons are also activated in presence of LPS and respond by increasing the transcription of the gene encoding CRF, the neurosecretagogue that controls the HPA axis. A variety of neural cells become gradually more sensitive to IL-6, mainly by expressing more receptor molecules on their surface. When it becomes available, IL-6 maintains the activation state of the hypothalamic CRF neurons and participates in specific cellular responses necessary for eliminating the endotoxin. Centrally produced IL-6 may also play local roles in regulating inflammatory processes and can serve as a neuroprotective signal after being released into cerebrospinal fluid. As a consequence, the inflammation can be properly controlled by the inhibitory effects of glucocorticoids on cytokine expression.


   Acknowledgments
 
We thank Dr. M. Kopf (Basel Institute for Immunology, Basel, Switzerland) for the generous gift of IL-6-deficient mice, Dr. I. Verma (The Salk Institute, La Jolla, CA) for the rat c-fos cDNA, Dr. K. Mayo (Northwestern University, Evanston, IL) for the rat CRF cDNA, Dr. S. Watson (University of Michigan, Ann Arbor, MI) for the rat intronic CRF cDNA, and Dr. K. Peri (Sainte-Justine Hospital Research Center, Montreal, Canada) for the rat COX-2 cDNA. We also thank Nathalie Laflamme, Nicole Martel, and René Labrecque for invaluable technical assistance.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada. Back

2 Supported by Ph.D. studentships from the Natural Sciences and Engineering Research Council of Canada and the Fonds pour la Formation des Chercheurs et l’Aide à la Recherche du Québec. Presently a postdoctoral fellow at The Salk Institute (La Jolla, CA). Back

3 Medical Research Council Scientist. Back

Received December 1, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vallières L, Rivest S 1997 Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-1ß. J Neurochem 69:1668–1683[Medline]
  2. Quan N, Whiteside M, Herkenham M 1998 Time course and localization patterns of interleukin-1ß messenger RNA expression in brain and pituitary after peripheral administration of lipopolysaccharide. Neuroscience 83:281–293[CrossRef][Medline]
  3. Breder CD, Hazuka C, Ghayur T, Klug C, Huginin M, Yasuda K, Teng M, Saper CB 1994 Regional induction of tumor necrosis factor alpha expression in the mouse brain after systemic lipopolysaccharide administration. Proc Natl Acad Sci USA 91:11393–11397[Abstract/Free Full Text]
  4. Besedovsky HO, Del Rey A 1996 Immune-neuro-endocrine interactions: facts and hypotheses. Endocr Rev 17:64–102[CrossRef][Medline]
  5. Rothwell NJ, Hopkins SJ 1995 Cytokines and the nervous system. II. Actions and mechanisms of action. Trends Neurosci 18:130–136[CrossRef][Medline]
  6. Schöbitz B, De Kloet ER, Holsboer F 1994 Gene expression and function of interleukin 1, interleukin 6 and tumor necrosis factor in the brain. Prog Neurobiol 44:397–432[CrossRef][Medline]
  7. Chai Z, Gatti S, Toniatti C, Poli V, Bartfai T 1996 Interleukin (IL)-6 gene expression in the central nervous system is necessary for fever response to lipopolysaccharide or IL-1ß: a study on IL-6-deficient mice. J Exp Med 183:311–316[Abstract/Free Full Text]
  8. Dinarello CA, Cannon JG, Mancilla J, Bishai I, Lees J, Coceani F 1991 Interleukin-6 as an endogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral blood mononuclear cells. Brain Res 562:199–206[CrossRef][Medline]
  9. Fernandez-Alonso A, Benamar K, Sancibrian M, Lopez-Valpuesta FJ, Minano FJ 1996 Role of interleukin-1ß, interleukin-6 and macrophage inflammatory protein-1ß in prostaglandin-E2-induced hyperthermia in rats. Life Sci 59:L185–190
  10. Vallières L, Lacroix S, Rivest S 1997 Influence of interleukin-6 on neural activity and transcription of the gene encoding corticotropin-releasing factor in the rat brain: an effect depending upon the route of administration. Eur J Neurosci 9:1461–1472[CrossRef][Medline]
  11. Fukata J, Usui T, Naitoh Y, Nakai Y, Imura H 1989 Effects of recombinant human interleukin-1{alpha}, -1ß, 2 and 6 on ACTH synthesis and release in the mouse pituitary tumour cell line AtT-20. J Endocrinol 122:33–39[Abstract]
  12. Lyson K, McCann SM 1991 The effect of interleukin-6 on pituitary hormone release in vivo and in vitro. Neuroendocrinology 54:262–266[Medline]
  13. Salas MA, Evans SW, Levell MJ, Whicher JT 1990 Interleukin-6 and ACTH act synergistically to stimulate the release of corticosterone from adrenal gland cells. Clin Exp Immunol 79:470–473[Medline]
  14. Path G, Bornstein SR, Ehrhart B-M, Scherbaum WA 1997 Interleukin-6 and the interleukin-6 receptor in the human adrenal gland: expression and effects on steroidogenesis. J Clin Endocrinol Metab 82:2343–2349[Abstract/Free Full Text]
  15. Weber MM, Michl P, Auernhammer CJ, Engelhardt D 1997 Interleukin-3 and interleukin-6 stimulate cortisol secretion from adult human adrenocortical cells. Endocrinology 138:2207–2210[Abstract/Free Full Text]
  16. Navarra P, Tsagarakis S, Faria MS, Rees LH, Besser GM, Grossman AB 1991 Interleukins-1 and -6 stimulate the release of corticotropin-releasing hormone-41 from rat hypothalamus in vitro via the eicosanoid cyclooxygenase pathway. Endocrinology 128:37–44[Abstract]
  17. Lyson K, McCann SM 1992 Involvement of arachidonic acid cascade pathways in interleukin-6-stimulated corticotropin-releasing factor release in vitro. Neuroendocrinology 55:708–713[Medline]
  18. Naitoh Y, Fukata J, Tominaga T, Nakai Y, Tamai S, Mori K, Imura H 1988 Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 155:1459–1463[CrossRef][Medline]
  19. Harbuz MS, Stephanou A, Sarlis N, Lightman SL 1992 The effects of recombinant human interleukin (IL)-1{alpha}, IL-1ß or IL-6 on hypothalamo-pituitary-adrenal axis activation. J Endocrinol 133:349–355[Abstract]
  20. LeMay LG, Vander AJ, Kluger MJ 1990 Role of interleukin 6 in fever in rats. Am J Physiol 258:R798–R803
  21. Klein MA, Moller JC, Jones LL, Bluethmann H, Kreutzberg GW, Raivich G 1997 Impaired neuroglial activation in interleukin-6 deficient mice. Glia 19:227–233[CrossRef][Medline]
  22. Chiang CS, Stalder A, Samimi A, Campbell IL 1994 Reactive gliosis as a consequence of interleukin-6 expression in the brain: studies in transgenic mice. Dev Neurosci 16:212–221[Medline]
  23. Balasingam V, Tejada B-T, Wright E, Bouckova R, Yong VW 1994 Reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines. J Neurosci 14:846–856[Abstract]
  24. Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, Achong MK 1998 IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 101:311–320[Medline]
  25. Sterneck E, Kaplan DR, Johnson PF 1996 Interleukin-6 induces expression of peripherin and cooperates with Trk receptor signaling to promote neuronal differentiation in PC12 cells. J Neurochem 67:1365–1374[Medline]
  26. Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, Kohler G 1994 Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368:339–342[CrossRef][Medline]
  27. Simmons DM, Arriza JL, Swanson LW 1989 A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radio-labeled single-stranded RNA probes. J Histotechnol 12:169–181
  28. Swanson LW 1992 Brain Maps: Structure of the Rat Brain. Elsevier, New York, p 240
  29. Lacroix S, Rivest S 1998 Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain. J Neurochem 70:452–466[Medline]
  30. Rivest S, Laflamme N 1995 Neuronal activity and neuropeptide gene transcription in the brains of immune-challenged rats. J Neuroendocrinol 7:501–525[CrossRef][Medline]
  31. Lacroix S, Feinstein D, Rivest S 1998 The bacterial endotoxin lipopolysaccharide has the ability to target the brain in upregulating its membrane CD14 receptor within specific cellular populations. Brain Pathol 8:625–640[Medline]
  32. Pugin J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, Tobias PS 1993 Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90:2744–2748[Abstract/Free Full Text]
  33. Arditi M, Zhou J, Dorio R, Rong GW, Goyert SM, Kim KS 1993 Endotoxin-mediated endothelial cell injury and activation: role of soluble CD14. Infect Immun 61:3149–3156[Abstract/Free Full Text]
  34. Haziot A, Rong GW, Silver J, Goyert SM 1993 Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J Immunol 151:1500–1507[Abstract]
  35. Marz P, Gadient RA, Otten U 1996 Expression of interleukin-6 receptor (IL-6R) and gp130 mRNA in PC12 cells and sympathetic neurons: modulation by tumor necrosis factor {alpha} (TNF-{alpha}). Brain Res 706:71–79[CrossRef][Medline]
  36. Saito M, Yoshida K, Hibi M, Taga T, Kishimoto T 1992 Molecular cloning of a murine IL-6 receptor-associated signal transducer, gp130, and its regulated expression in vivo. J Immunol 148:4066–4071[Abstract]
  37. Campbell IL, Abraham CR, Masliah E, Kemper P, Inglis JD, Oldstone MB, Mucke L 1993 Neurologic disease induced in transgenic mice by cerebral overexpression of interleukin 6. Proc Natl Acad Sci USA 90:10061–10065[Abstract/Free Full Text]
  38. Heyser CJ, Masliah E, Samimi A, Campbell IL, Gold LH 1997 Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain. Proc Natl Acad Sci USA 94:1500–1505[Abstract/Free Full Text]
  39. Perlstein RS, Mougey EH, Jackson WE, Neta R 1991 Interleukin-1 and interleukin-6 act synergistically to stimulate the release of adrenocorticotropic hormone in vivo. Lymphokine Cytokine Res 10:141–146[Medline]
  40. Neta R, Perlstein R, Vogel SN, Ledney GD, Abrams J 1992 Role of interleukin 6 (IL-6) in protection from lethal irradiation and in endocrine responses to IL-1 and tumor necrosis factor. J Exp Med 175:689–694[Abstract/Free Full Text]
  41. Perlstein RS, Whitnall MH, Abrams JS, Mougey EH, Neta R 1993 Synergistic roles of interleukin-6, interleukin-1, and tumor necrosis factor in the adrenocorticotropin response to bacterial lipopolysaccharide in vivo. Endocrinology 132:946–952[Abstract]
  42. Zhou D, Shanks N, Riechman SE, Liang R, Kusnecov AW, Rabin BS 1996 Interleukin 6 modulates interleukin-1-and stress-induced activation of the hypothalamic-pituitary-adrenal axis in male rats. Neuroendocrinology 63:227–236[Medline]
  43. Spinedi E, Hadid R, Daneva T, Gaillard RC 1992 Cytokines stimulate the CRH but not the vasopressin neuronal system: evidence for a median eminence site of interleukin-6 action. Neuroendocrinology 56:46–53[Medline]
  44. Brett FM, Mizisin AP, Powell HC, Campbell IL 1995 Evolution of neuropathologic abnormalities associated with blood-brain barrier breakdown in transgenic mice expressing interleukin-6 in astrocytes. J Neuropathol Exp Neurol 54:766–775[Medline]
  45. Banks WA, Kastin AJ, Gutierrez EG 1994 Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 179:53–56[CrossRef][Medline]
  46. Ericsson A, Arias C, Sawchenko PE 1997 Evidence for an intramedullary prostaglandin-dependent mechanism in the activation of stress-related neuroendocrine circuitry by intravenous interleukin-1. J Neurosci 17:7166–7179[Abstract/Free Full Text]
  47. Dayer JM, Beutler B, Cerami A 1985 Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 162:2163–2168[Abstract/Free Full Text]
  48. Rivier C, Shen GH 1994 In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1ß, vasopressin, and oxytocin. J Neurosci 14:1985–1993[Abstract]
  49. Munoz-Fernandez MA, Cano E, O’Donnell CA, Doyle J, Liew FY, Fresno M 1994 Tumor necrosis factor-{alpha} (TNF-{alpha}), interferon-{gamma}, and interleukin-6 but not TNF-ß induce differentiation of neuroblastoma cells: the role of nitric oxide. J Neurochem 62:1330–1336[Medline]
  50. Xin L, Blatteis CM 1992 Hypothalamic neuronal responses to interleukin-6 in tissue slices: effects of indomethacin and naloxone. Brain Res Bull 29:27–35[CrossRef][Medline]
  51. Bishai I, Coceani F 1996 Differential effects of endotoxin and cytokines on prostaglandin E2 formation in cerebral microvessels and brain parenchyma: implications for the pathogenesis of fever. Cytokine 8:371–376[CrossRef][Medline]
  52. Bauer MK, Lieb K, Schulze-Osthoff K, Berger M, Gebicke-Haerter PJ, Bauer J, Fiebich BL 1997 Expression and regulation of cyclooxygenase-2 in rat microglia. Eur J Biochem 243:726–731[Medline]
  53. Hinson RM, Williams JA, Shacter E 1996 Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2. Proc Natl Acad Sci USA 93:4885–4890[Abstract/Free Full Text]
  54. Williams JA, Shacter E 1997 Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. J Biol Chem 272:25693–25699[Abstract/Free Full Text]
  55. Fiebich BL, Hull M, Lieb K, Gyufko K, Berger M, Bauer J 1997 Prostaglandin E2 induces interleukin-6 synthesis in human astrocytoma cells. J Neurochem 68:704–709[Medline]
  56. Ma T, Zhu X 1997 Interleukin-6 increases the levels of cyclic GMP and nitrite in rat hippocampal slices. Eur J Pharmacol 321:343–347[CrossRef][Medline]
  57. Munoz-Fernandez MA, Fresno M 1993 Involvement of nitric oxide on the cytokine induced growth of glial cell. Biochem Biophys Res Commun 194:319–325[CrossRef][Medline]
  58. Givalois L, Dornand J, Mekaouche M, Solier MD, Bristow AF, Ixart G, Siaud P, Assenmacher I, Barbanel G 1994 Temporal cascade of plasma level surges in ACTH, corticosterone, and cytokines in endotoxin-challenged rats. Am J Physiol 267:R164–R170
  59. Dunn AJ 1992 The role of interleukin-1 and tumor necrosis factor {alpha} in the neurochemical and neuroendocrine responses to endotoxin. Brain Res Bull 29:807–812[CrossRef][Medline]
  60. Paez Pereda M, Sauer J, Perez Castro C, Finkielman S, Stalla GK, Holsboer F, Arzt E 1995 Corticotropin-releasing hormone differentially modulates the interleukin-1 system according to the level of monocyte activation by endotoxin. Endocrinology 136:5504–5510[Abstract]
  61. Fattori E, Cappelletti M, Costa P, Sellitto C, Cantoni L, Carelli M, Faggioni R, Fantuzzi G, Ghezzi P, Poli V 1994 Defective inflammatory response in interleukin 6-deficient mice. J Exp Med 180:1243–1250[Abstract/Free Full Text]
  62. Ruzek MC, Miller AH, Opal SM, Pearce BD, Biron CA 1997 Characterization of early cytokine responses and an interleukin (IL)-6-dependent pathway of endogenous glucocorticoid induction during murine cytomegalovirus infection. J Exp Med 185:1185–1192[Abstract/Free Full Text]
  63. Mastorakos G, Weber JS, Magiakou MA, Gunn H, Chrousos GP 1994 Hypothalamic-pituitary-adrenal axis activation and stimulation of systemic vasopressin secretion by recombinant interleukin-6 in humans: potential implications for the syndrome of inappropriate vasopressin secretion. J Clin Endocrinol Metab 79:934–939[Abstract]
  64. Kageyama K, Watanobe H, Takebe K 1995 In vivo evidence that arginine vasopressin is involved in the adrenocorticotropin response induced by interleukin-6 but not by tumor necrosis factor-{alpha} in the rat. Neuroimmunomodulation 2:137–140[CrossRef][Medline]
  65. Raber J, O’Shea RD, Bloom FE, Campbell IL 1997 Modulation of hypothalamic-pituitary-adrenal function by transgenic expression of interleukin-6 in the CNS of mice. J Neurosci 17:9473–9480[Abstract/Free Full Text]



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K. Kageyama, K. Hanada, Y. Iwasaki, S. Sakihara, T. Nigawara, J. Kasckow, and T. Suda
Pituitary adenylate cyclase-activating polypeptide stimulates corticotropin-releasing factor, vasopressin and interleukin-6 gene transcription in hypothalami