Interleukin-1 receptor antagonist inhibits the release of glutamate, hydroxyl radicals, and prostaglandin E2 in the hypothalamus during pyrogen-induced fever in rabbits

European Journal of Pharmacology 629 (2010) 125–131

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Immunopharmacology and Inflammation

Interleukin-1 receptor antagonist inhibits the release of glutamate, hydroxyl radicals, and prostaglandin E2 in the hypothalamus during pyrogen-induced fever in rabbits Kuo-Feng Huang a, Wu-Tein Huang b, Kao-Chang Lin c, Mao-Tsun Lin d,⁎, Ching-Ping Chang e,⁎ a

Department of Plastic Surgery, Chi Mei Medical Center, Tainan, 710, Taiwan Department of Sport and Health Sciences, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan Department of Neurology, Chi Mei Medical Center, Tainan 710, Taiwan d Department of Medical Research, Chi Mei Medical Center, Tainan 710, Taiwan e Department of Biotechnology, Southern Taiwan University, Tainan, 710, Taiwan b c

a r t i c l e

i n f o

Article history: Received 6 August 2009 Received in revised form 11 November 2009 Accepted 23 November 2009 Available online 1 December 2009 Keywords: Fever Lipopolysaccharide Glutamate Hydroxyl radical Interleukin-1 receptor antagonist Antipyresis Prostaglandin E2

a b s t r a c t The present study was attempted to determine whether interleukin-1 receptor antagonist (IL-1ra) pretreatment exerts its antipyresis by reducing organum vasculosum laminae terminalis (OVLT) release of glutamate, hydroxyl radicals and prostaglandin E2 in rabbits. It was found that systemic administration of lipopolysaccharide induced increased levels of both core temperature and OVLT levels of glutamate, hydroxyl radicals, and prostaglandin E2. The rise in both the core temperature and OVLT glutamate, hydroxyl radicals and prostaglandin E2 could also be induced by intracerebroventricular injection of interleukin-1β. Pretreatment with an intracerebroventricular dose of IL-1ra significantly prevented the lipopolysaccharide or IL-1β-induced overproduction of glutamate, hydroxyl radicals, and prostaglandin E2 in OVLT of rabbit's brain. The febrile response caused by systemic administration of lipopolysaccharide or central injection of interleukin-1β could also be IL-1ra pretreatment-ameliorated. These results indicate that IL-1ra pretreatment may exert its antipyresis by inhibiting the glutamate–hydroxyl radicals–prostaglandin E2 pathways in the OVLT of rabbit's brain during lipopolysaccharide fever. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The proinflammatory cytokine interleukin-1β (IL-1β), a major mediator of lipopolysaccharide (LPS)-induced fever, is known to elicit febrile responses via cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E2 (PGE2) in the brain (Kluger, 1991; Li et al., 2001). Studies have provided considerable evidence that IL-1 acts directly in the brain to cause fever. For example, peripheral injection of LPS causes synthesis and production of IL-1 in the brain (mainly in the hypothalamus) (Bandtlow et al., 1990; Ban et al., 1992; Nguyen et al., 1998). Intracerebral injection of recombinant IL-1 elicits marked fever in rodents (Anforth et al., 1998). The activity of hypothalamic thermosensitive neurons is altered by IL-1 administration (in vivo and in vitro) the characteristics of which are consistent with the fever development (Hori et al., 1988). The IL-1-induced fever can be attenuated by central administration of neutralizing anti-IL-1β antiserum (Klir et al., 1994; Gourine et al., 1998) or the naturally occurring interleukin-1 receptor antagonist (IL-1ra) (Luheshi et al., 1996; Miller et al., 1997). ⁎ Corresponding authors. M.-T. Lin is to be contacted at Department of Medical Research, Chi Mei Medical Center 710, Tainan, Taiwan. Tel./fax: +886 6 2517850. Chang, Department of Biotechnology, Southern Taiwan University, Tainan, 710, Taiwan. E-mail address: [email protected] (C.-P. Chang). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.11.060

Other line of evidence has accumulated to propose that a glutamate–hydroxyl radical–PGE2 pathway exists in the rabbit's brain (Huang et al., 2006; Kao et al., 2007a,b) and mediates pyrogen-induced fever. This raises the possibility that recombinant IL-1ra may exert its antipyretic action during pyrogen-induced fever in rabbits by inhibiting the glutamate–hydroxyl radical–PGE2 pathway in the brain. The aim of the present investigation, therefore, was to assess the time course change in both the core temperature and the brain levels of glutamate, hydroxyl radical, and PGE2 during the fever caused by systemic delivery of LPS or central administration of IL-1β in rabbits treated with or without IL-1ra. 2. Materials and methods 2.1. Experimental animals Clinically, pyrogen test is plied in rabbits which in the present, therefore, were used for pyrogen-induced fever test. One hundred and twenty adult male New Zealand white rabbits, aged between 5.2 and 6.1 years old and weighing between 2.3 and 3.1 kg at the study start were applied. The pyrogen assay with unanesthetized animals restrained in rabbit stocks. Between experiments the animals were housed individually at an ambient temperature of 22 ± 1 °C

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with a 12-h light–dark cycle, with the lights switched on at 06:00 h. Animal chow and water were allowed ad libitum. Experiments were between 09:00 and 19:00 h, with each animal manipulated at an interval of not less than 7 days. Throughout the experiment, core temperature was measured every 5 min with a copper constantan thermocouple inserted into the rectum and connected to a thermometer (HR 1300, Yokogawa, Tokyo, Japan). The core temperature of each animal was allowed to stabilize for at least 90 min before any injections. Only animals whose core temperature was stable and in the range of 38.1 to 39.2 °C were utilized to determine the effect of drug application. All the animals were acquired from the animal center of Chi Mei Medical Center (Tainan, Taiwan, ROC). The animal protocol accounted here was approved by the Animal Care Committee of Chi Mei Medical Center. 2.2. Surgical techniques

In experiment 2, an i.c.v. dose of IL-ra (10 ng, 50 ng, or 200 ng in 10 μl) or normal saline (10 μl) was randomly administered into rabbits (n = 16) 1 h before an i.v. dose of LPS (2 μg/kg) and their effects on OVLT levels of PGE2 were assessed. In experiment 3, an i.c.v. dose of IL-ra (10 ng, 50 ng, or 200 ng in 10 μl) or normal saline (10 μl) was randomly administered into rabbits (n = 16) 1 h before an i.c.v. dose of IL-1β (20 ng in 10 μl) and their effects on both core temperature and OVLT levels of both glutamate (Glu) and 2,3-DHBA were assessed. In experiment 4, an i.c.v. dose of IL-ra (10 ng, 50 ng, or 200 ng in 10 μl) or normal saline (10 μl) was randomly administered into rabbits (n = 16) 1 h before an i.c.v. dose of IL-1β (20 ng in 10 μl) and their effects on OVLT levels of PGE2 were assessed. 2.5. Microdialysis for detection of extracellular glutamate and hydroxyl radicals

An intracerebral or intracerebroventricular probe guide cannula was implanted into each animal under general anesthesia (sodium pentobarbital, 30 mg/kg, i.v.). Standard aseptic techniques were employed; so were the stereotoxic atlas and coordinates of Sawyer et al. (1954). The cannula was located in the left OVLT (A: 4.5 mm, L: 0 mm, and V: 14 mm) or lateral cerebral ventricle (P: 4 mm, R: 3 mm, and V: 5 mm). The animals were placed in the stereotoxic apparatus, and the frontal and parietal bones were exposed by a midline incision into the scalp. After appropriately located craniotomy has been trephined, two self-tapping screws were inserted into the parietal or frontal bones and the cannula was inserted to the depth through the craniotomy hole. The cannula was anchored with dental acrylic cement to the calvarium surface, which had been scraped clean of periosteum. The reflected muscles and skin were replaced around the acrylic mound containing the cannula and screws and were sutured with chromic gut (000). Postoperatively, the guide cannula was plugged with a stylet, and animals were returned to their cages for a minimal recovery period of 1 week.

For measuring extracellular levels of glutamate in OVLT of rabbit brain, the dialysates were collected every 20 min in a CMA 140 fraction collector. Aliquots of dialysates (2 μl) were injected into a CMA600 Microdialysis analyzer for measuring glutamate. Glutamate is enzymatically oxidized by glutamate oxidase. The hydrogen peroxide formed reacts with N-ethyl-N-(2-hydroxy-3sulfopropyl)m-toluidine and 4-amino-antipyrine. This reaction if catalyzed by peroxidase and yields the red–violet colored quinonediimine. The formation rate is measured photometrically at 546 nm and is proportional to the glutamate. For measuring extracellular levels of hydroxyl radicals in the OVLT, a probe guide cannula was planted in the OVLT. The morning before an experiment, after a microdialysis probe into the OVLT, it was perfused with artificial cerebrospinal fluid (149 mM NaCl2; 2.8 mM KCl, 1.3 mM CaCl2, 1.2 mM Cl2, 0.125 mM ascorbic acid, and 5.4 mM D-glucose, pH 7.2–7.4) containing 10 mM salicylic acid by a high pressure pump (CMA/Microdialysis; Ros Lagsvägen, Stockholm, Sweden) at a flow of 1.2 μl/min (Huang et al., 2006). The dialysis probe is a CMA-12 Elite microdialysis probe (Solna, Swlden).

2.3. Drug

2.6. Measuring PGE2 in the OVLT

All drug solutions were prepared in pyrogen-free glassware that was heated for 5 h at 180 °C before use. All drug solutions were prepared in pyrogen-free saline and passed through 0.22-μm Millipore bacterial filters. The LPS used in this study, which was derived from Escherichia coli serotype 026:B6 (Sigma-Aldrich, Chemical Co., St. Louis, MO, USA) was dissolved in sterile saline. LPS (2 μg/kg), recombinant human interleukin-1β (R&D, Minneapolis, Minn; IL-1, USA), and recombinant human interleukin-1 receptor antagonist (IL-1ra, Synergen, Boulder, CO, USA) were used for intracerebroventricular injection. Drugs for determining glutamate, 2,3-dihydroxylbenzoic acid (DHBA), salicylic acid, monochloracetic acid, and prostaglandin E2 (PGE2) were procured from SigmaAldrich, Chemical Co., St. Louis, MO, USA.

For measuring OVLT PGE2, the dialysis system was connected to a microdialysis pump and perfused with artificial cerebrospinal fluid at a flow rate of 1.2 μl/min. The unanesthetized animals were restrained in rabbit stocks for at least 90 min for a stable dialysis level of PGE2. Dialysis samples from the OVLT were collected into a microdialysis vial at 60-min intervals for 8 h, and they were stored at −80 °C until analyzed within 7 days. Immunoreactive PGE2 concentrations in dialysates were determined using commercially available enzyme immunoassay kits (Cayman Chemicals Co, Ann Arbor, MI, USA). Triplicate aliquots of 50 μl samples were added to each well of plate and each sample was assayed at a minimum of two dilutions. The quantitation limit for PGE2 was 20 pg/ml. 2.7. Data presentation

2.4. Experimental groups Preexperimentally, the indwelling stylet of the guide cannula was replaced by a CMA-12 microdialysis probe purchased from CMA/ Microdialysis ROS-Lagsvägen, Stockholm, Sweden, so that the membrane tip protruded exactly 1.5 mm beyond the guide tube was analyzed. The microdialysis probes and perfusion procedures (1.2 μl/min) used in this study have been described previously (Huang et al., 2006, 2008). In experiment 1, an intracerebroventricular (i.c.v.) dose of IL-ra (10 ng, 50 ng, or 200 ng in 10 μl) or normal saline (10 μl) was randomly administered into rabbits (n = 16) 1 h before an intravenous (i.v.) dose of LPS (2 μg/kg) and their effects on both core temperature and OVLT levels of both glutamate (Glu) and 2,3-DHBA were assessed.

At the end of an experiment, the animals were sacrificed with an overdose of anesthetics and decapitated with a guillotine. Their brains were removed and stored in 10% phosphate-buffered formalin for histologically verifying the placement of the dialysis probe tips. Only experiments in which the OVLT localization of the microdialysis probes was confirmed histologically were included in the results. The operation success-rate based on histology was about 90%. 2.8. Statistical analysis Temperature response was assessed as changes from pre-injection values (Δ°C) and the fever index (FI), which was the area under the

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curve produced in the 5-h period after the injection of LPS or IL-1β in terms of degrees centigrade per 5 h, were calculated (Huang et al., 2006). The glutamate and 2,3-DHBA levels of samples were expressed as a percentage of three consecutive mean baseline values. Results were expressed as the mean ± S.E.M. for n experiments. Two way analysis of variance (ANOVA) for repeated measurements (in the same animals) was used for the factorial experiment, whereas Dunnett's test was used for post hoc multiple comparisons among means. A P value less than 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1. LPS increased Tco and the OVLT levels of glutamate and hydroxyl radical Intravenously administrating 2 μg/kg of LPS produced a biphasic febrile response, with the Tco maximal at 80 and 200 min postinjection (Fig. 1 and Table 1). Each Tco elevation was accompanied by a distinct wave of OVLT levels of both glutamate and 2,3-DHBA (Fig. 1). If the temperature response was assessed as changes in terms of fever index, intravenous administration of LPS had significantly higher values of fever index as compared to those of vehicle controls (Table 2). 3.2. IL-1β increased Tco and the OVLT levels of glutamate and hydroxyl radical Fig. 2 depicts the effects of intracerebroventricular administration of IL-1β (20 ng per rabbit) on Tco and the OVLT levels of glutamate (B) and 2,3-DHBA (C) in rabbits. It can be seen from both the Fig. 2 and Table 3 that central injection of IL-1β caused higher levels of both Tco and the OVLT levels of glutamate and hydroxyl radicals as compared to those of vehicle controls. An i.v. dose of IL-1β (200 ng per rabbit) caused an insignificant effect (P N 0.05) on both the core temperature and the OVLT levels of both glutamate and hydroxyl radicals (the data are unshown here). 3.3. Both LPS and IL-1β increased the OVLT levels of PGE2 Figs. 3 and 4 depict the effects of intravenously administrating LPS and intracerebroventricularly administrating IL-1β, respectively, on the OVLT levels of PGE2. It can be seen from the figures that both LPS and IL-1β caused significantly higher levels of OVLT PGE2 as compared to those of vehicle controls. An i.v. dose of IL-1β (200 ng per rabbit) caused an insignificant effect (P N 0.05) on the OVLT levels of PGE2 (the data are unshown here). 3.4. IL-1ra reduced the increased levels of Tco, and OVLT levels of glutamate, hydroxyl radicals, and PGE2 following LPS or IL-1β injection Effects of intracerebroventricular administration of IL-1ra (10– 200 ng per rabbit) 1 h before the LPS or IL-1β injection on the Tco elevation, increased fever index, and OVLT glutamate, hydroxyl radical, and PGE elevation were assessed in rabbits. It was found that the core temperature elevations, the increased fever index, and the increased OVLT levels of glutamate, hydroxyl radical, and PGE2 induced by LPS or IL-1β were dose-dependently reduced with IL-1ra pretreatmentally (10–200 ng, i.c.v.) 1 h before the LPS or IL-1β injection (Figs. 1–4 and Tables 1–3). Again, the core temperatures, the increased fever index, and the increased OVLT levels of glutamate, hydroxyl radical, and PGE2 caused by LPS or IL-1β were insignificantly affected with an intravenous dose of 200 ng of IL-1ra pretreatmentally 1 h before the LPS or IL-1β injection the (data are unshown here).

Fig. 1. The effect of an intracerebroventricular (i.c.v.) dose of IL-1ra or normal saline 1 h before an intravenous (i.v.) dose of LPS on both core temperature and OVLT levels of both glutamate (Glu) and 2,3-DHBA. Mean ± S.E.M. changes in core temperature and glutamate and 2,3-DHBA in OVLT in rabbits injected with either saline (i.c.v.) plus saline (i.v.) (○) (n = 8), saline (i.c.v.) plus LPS (2 μg/kg, i.v.) (●) (n = 8), IL-1ra (10 ng, i.c.v.) plus LPS (2 μg/kg, i.v.) (▲) (n = 8), IL-1ra (50 ng, i.c.v.) plus LPS (2 μg/kg, i.v.) (■) (n = 8), or IL-1ra (200 ng, i.c.v.) plus LPS (2 μg/kg, i.v.) (△) (n = 8). *P b 0.05, significantly different from corresponding control values (saline + saline group); † P b 0.05, significantly different from corresponding control values (saline + LPS group) (Dunnett's test-followed ANOVA).

4. Discussion The organum vasculum laminal terminalis (OVLT), a circumventricular organ in the region anterolateral to the third cerebral ventricle, has been thought to possess a role for passage and/or production of interleukin-1 (IL-1). The febrile response induced by lipopolysaccharide (LPS) is significantly reduced after OVLT ablation (Blatteis et al., 1983). The rabbit OVLT has many perivascular spaces around well-developed fenestrated capillaries (Morimoto et al., 1990)

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Table 1 Effects of centrally administrating the IL-1 receptor antagonist 1 h before the LPS injection on peak Tco, glutamate, and hydroxyl radical elevation in POAH in response to LPS in rabbits. Treatments

Saline + saline Saline + LPS (2 μg/kg) IL-1 receptor antagonist (10 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (50 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (200 ng, i.c.v.) + LPS (2 μg/kg)

Response Early phase

Late phase

Peak Tco

(°C)

0.23 ± 0.08 1.57 ± 0.14a 0.81 ± 0.14

0.25 ± 0.08 2.10 ± 0.12a 0.92 ± 0.11b

0.76 ± 0.14b

0.74 ± 0.12b

0.57 ± 0.04b

0.44 ± 0.13b

% of mean basal levels of glutamate Saline + saline Saline + LPS (2 μg/kg) IL-1 receptor antagonist (10 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (50 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (200 ng, i.c.v.) + LPS (2 μg/kg)

101 ± 2 165 ± 12a 135 ± 6b

98 ± 4 215 ± 10a 162 ± 5b

112 ± 3b

125 ± 6b

101 ± 7b

97 ± 4b

% of mean basal levels of hydroxyl radical Saline + saline Saline + LPS (2 μg/kg) IL-1 receptor antagonist (10 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (50 ng, i.c.v.) + LPS (2 μg/kg) IL-1 receptor antagonist (200 ng, i.c.v.) + LPS (2 μg/kg)

98 ± 5 123 ± 5a 118 ± 4

99 ± 2 142 ± 1a 132 ± 4

110 ± 6b

120 ± 4b

109 ± 5b

110 ± 6b

The values are means ± S.E.M. of 8 rabbits per group. Rabbits injected LPS (2 μg/kg) produced a biphasic fever which peaked at 80 min (early phase) and 200 min (late phase) after LPS injection. a Significantly different from corresponding control values (vehicle plus vehicle group) (P b 0.05; two way analysis of variance followed by Dunnett's test). b Significantly different from corresponding control values (vehicle plus LPS group) (P b 0.05; Dunnett's test-followed two way analysis of variance).

containing reticuloendothelial cells (particularly, macrophages) (Yamaguchi et al., 1993). In situ hybridization and immunohistochemical studies reveal that systemically administrating LPS induces IL-1β production in the OVLT of rabbit brain (Nakamori et al., 1994). The cell type which produced IL-1β in the OVLT following LPS injection was confirmed to be a macrophage by electron microscopy. The cells producing IL-1β in the parenchyma were determined to be microglial cells. Interleukin-1 receptor antagonist (IL-1ra) identified inhibits the action of IL-1 by competing for its receptor (Eisenberg et al., 1990; Hannum et al., 1990); systemic IL-1ra inhibits endotoxin

Table 2 Effects of centrally administrating the IL-1 receptor antagonist 1 h before the LPS injection on the febrile response to intravenously injecting LPS in rabbits. Treatments

Fever index (FI, °C h)

Saline + saline Saline + LPS (2 μg/kg) IL-1 receptor antagonist (10 ng, i. c. v.) + LPS (2 μg/kg) IL-1 receptor antagonist (50 ng, i. c. v.) + LPS (2 μg/kg) IL-1 receptor antagonist (200 ng, i. c. v.) + LPS (2 μg/kg)

FI = 0.98 ± 0.05 FI = 7.63 ± 0.13a FI = 4.25 ± 0.17b FI = 3.27 ± 0.11b FI = 2.18 ± 0.05b

The values are means ± S.E.M. of 8 rabbits per group. FI represent fever index for 5 h experimental observation. a Significantly different from corresponding control values (vehicle plus vehicle group) (P b 0.05; two way analysis of variance followed by Dunnett's test). b Significantly different from corresponding control value (vehicle plus LPS) (P b 0.05; Dunnett's test-followed two way analysis of variance).

Fig. 2. The effect of an intracerebroventricular (i.c.v.) dose of IL-1ra or normal saline 1 h before an i.c.v. dose of IL-1β on both core temperature and OVLT levels of both glutamate (Glu) and 2,3-DHBA. Mean± S.E.M. changes in core temperature and glutamate (Glu) and 2,3-DHBA in OVLT in rabbits injected with either saline (i.c.v.) plus saline (i.c.v.) (○) (n = 8), saline (i.c.v.) plus IL-1β (20 ng, i.c.v.) (●) (n = 8), IL-1ra (10 ng, i.c.v.) + IL-1β (20 ng, i.c.v.) (▲) (n = 8), IL-1ra (50 ng, i.c.v.) + IL-1β (20 ng, i.c.v.) (■) (n = 8), or IL-1ra (200 ng, i.c.v.) + IL-1β (20 ng, i.c.v.) (△) (n = 8). *P b 0.05, significantly different from corresponding control values (saline + saline group); †P b 0.05, significantly different from corresponding control values (saline + IL-1β group) (Dunnett's test-followed ANOVA).

fever and systemic interleukin-6 (IL-6) induction in the rat (Luheshi et al., 1996). Intravenous delivery of IL-1ra leads to cerebrospinal fluid (CSF) concentrations in patients with subarachnoid hemorrhage comparable to those that are neuroprotective in rats, and might therefore be of therapeutic benefit (Clark et al., 2008). In addition to fever, the levels of IL-1β in the OVLT of rabbit brain have been elevated remarkably following systemic delivery of LPS (Nakamori et al., 1994). Our results also show that direct injection of IL-1β into the OVLT causes febrile response in rabbits, which can be

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Table 3 Effects of centrally administrating the IL-1 receptor antagonist 1 h before the IL-1β injection on the febrile response to intracerebroventricularly injecting IL-1 β in rabbits. Treatments

Fever index (FI, °C h)

Saline (i. c. v.) + saline (i. c. v.) Saline (i. c. v.) + IL-1 β (20 ng, i. c. v.) IL-1 receptor antagonist (10 ng, i. c. v.) + IL-1 β (20 ng, i. c. v.) IL-1 receptor antagonist (50 ng, i. c. v.) + IL-1 β (20 ng, i. c. v.) IL-1 receptor antagonist (200 ng, i. c. v.) + IL-1 β (20 ng, i. c. v.)

FI = 0.98 ± 0.03 FI = 7.38 ± 0.08a FI = 5.71 ± 0.15b FI = 4.71 ± 0.13b FI = 3.70 ± 0.08b

The values are means ± S.E.M. of 8 rabbits per group. FI represent fever index for 5 h experimental observation. a Significantly different from corresponding control value (vehicle plus vehicle group) (P b 0.05; two way analysis of variance followed by Dunnett's test). b Significantly different from corresponding control value (vehicle plus IL-1β) (P b 0.05; Dunnett's test-followed two way analysis of variance).

attenuated with an intracerebroventricular dose of IL-1ra pretreatmentally. It is likely that IL-1ra may exert its antipyresis by reducing LPS-induced overproduction of IL-1β in the OVLT of rabbit's brain. The hypothesis is partly supported by several previous findings. For example, IL-1β is induced rapidly in response to experimental brain injury (Rothwell et al., 1997a,b; Allan et al., 2005). IL-1ra is induced by tissue injury and is able to block all actions of IL-1 (Hannum et al., 1990; Loddick et al., 1997; Pinteaux et al., 2006). The anterior hypothalamus is the essential thermoregulatory center in the brain (Kluger, 1991), and is believed to be a primary target of IL-1 (Klir et al., 1994; Gourine et al., 1998). According to the findings of Cartmell et al. (1999), i.c.v. injection of IL-1ra significantly attenuated LPS fever. Unilateral microinjection of IL-1ra into the anterior hypothalamus, paraventricular hypothalamic nucleus, perisubfornical organ, subfornical organ or CA3-hippocampus also significantly reduced the LPS-induced fever. The same dose of IL-1ra had no effect on fever when administered into the ventromedial hypothalamus, OVLT, CA1 hippocampus, striatum or cortex. It should be stressed that the OVLT of rabbit's brain is very close to the anterior hypothalamus. Therefore, it cannot be ruled out that IL-1ra may also exert its antipyretic action by reducing IL-1β production in the anterior hypothalamus.

Fig. 3. The effect of an intracerebroventricular (i.c.v.) dose of IL-1ra 1 h before an intravenous (i.v.) dose of LPS on the OVLT levels of PGE2. Mean ± S.E.M. changes in PGE2 in OVLT in rabbits injected with either saline (i.c.v.) plus saline (i.c.v.) (○) (n = 8), saline (i.c.v.) plus LPS (2 μg/kg, i.v.) (●) (n = 8), IL-1ra (20 ng, i.c.v.) plus LPS (2 μg/kg, i.v.) (△) (n = 8), IL-1ra (50 ng, i.c.v.) plus LPS (2 ng/kg, i.v.) (■) (n = 8), or IL-1ra (200 ng, i.c.v.) plus LPS (2 μg/kg, i.v.) (▲) (n = 8). *P b 0.05, significantly different from corresponding control values (saline + saline group); †P b 0.05, significantly different from corresponding control values (saline + LPS group) (ANOVA followed by Dunnett's test).

Fig. 4. The effect of an intracerebroventricular (i.c.v.) dose of IL-1ra 1 h before an i.c.v. dose of IL-1β on the OVLT levels of PGE2. Mean ± S.E.M. changes in PGE2 in OVLT in rabbits injected with either saline (i.c.v.) plus saline (i.c.v.) (○) (n = 8), saline (i.c.v.) plus IL-1β (20 ng, i.c.v.) (●) (n = 8), IL-1ra (10 ng, i.c.v.) plus IL-1β (20 ng, i.c.v.) (△) (n = 8), IL-1ra (50 ng, i.c.v.) plus IL-1β (20 ng, i.c.v.) (■) (n = 8), or IL-1ra (200 ng, i.c.v.) plus IL-1β (20 ng, i.c.v.) (▲) (n = 8). *P b 0.05, significantly different from corresponding control values (saline + saline group); †P b 0.05, significantly different from corresponding control values (saline + IL-1β group) (ANOVA followed by Dunnett's test).

Our previous study has demonstrated that systemically delivering LPS (2 μg/kg, i.v.) causes a biphasic core temperature rise, with the core temperature maxima at 80–90 and 180–210 min post-injection (Huang et al., 2006, 2008; Niu et al., 2009). The early phase of the fever is associated with elevation of both tumor necrosis factor-alpha (TNF-α) and IL-1β in the serum, while the late phase of the fever is related to rise in serum level of IL-6. It seems that both the early and the late phases of the fever are associated with the increased serum levels of TNF-α plus IL-1β and IL-6, respectively. Both TNF-α and IL-1β may be responsible for the remarkable rise of IL-6 (Pedersen et al., 2001). Both the early and the late phases of the fever induced by systemic injection of LPS are significantly attenuated by priorly administrating IL-1ra (as shown in the present results). In addition, our results show that the fever caused by centrally injecting IL-1β is significantly reduced by prior administration of IL-1ra. These observations suggest that antagonism of ILreceptors may reduce LPS-induced fever by attenuating the pyrogenic actions exerted by these proinflammatory cytokines including TNF-α, IL-1β, and IL-6. The hypothesis is several studies-supported partially. For example, it has been shown that peripheral IL-1 somewhat induce both fever and the rise in plasma IL-6 that precedes it, and that IL-1 within the brain is also important in the induction of fever by LPS (Luheshi et al., 1996). Interleukin-1β and IL-6 act synergistically within the brain to induce fever in rats (Harden et al., 2008). Proteins of the IL-1 system include the secreted agonist IL-1β, and the IL-1ra, both competing for binding to the IL-1 receptor (Bartfai et al., 2007). Both IL-1β and IL-1ra are highly inducible in infection and inflammation. Consequently, IL-1β induces the elevation of other proinflammatory molecules, including IL6, cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS), as well as of IL-1ra. Elevating IL-1ra is of key importance for quenching the fever and inflammatory response. Our previous results (Huang et al., 2006; Tsai et al., 2006) have proposed that a glutamate–hydroxyl radical–PGE2 pathway in the OVLT of rabbit brain may mediate LPS fever. As shown in the present results, an intravenous dose of LPS or an intracerebroventricular dose of IL-1β causes an increase in both the core temperature and the OVLT levels of glutamate, hydroxyl radical, and PGE2 in rabbits. Both the core temperature rise and the increased levels of glutamate, hydroxyl radical, and PGE2 in the OVLT of rabbit brain following LPS or IL-1β administration are significantly ameliorated with an intracerebroventricular dose of IL-1ra pretreatmentally. Our data indicate that IL-1ra may cause attenuation of the LPS-induced fever by inhibiting the

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had little effect on these fever states. In the present study, since the rabbits were housed individually between the experiments, this might have produced stress-induced hyperthermia. But this is not expected because stress-induced hyperthermia cannot be blocked by PGE-synthesis blocking drug. In the present study, for determining the cellular levels of PGE2, glutamate, and hydroxyl radical in the OVLT of rabbit brain, a CMA12 Elite Microdialysis probe was implanted into the OVLT for collecting microdialysates. The stereotoxic atlas and coordinates of Sawyer et al. (1954) were used. The probe has a polyarylethersulfone membrane with a molecular cut-off of 20,000 Da (outer diameter 0.5 mm; length 4 mm). The probe was located in the OVLT (A: 4.5 mm, L: 0.0 mm, and V: 14 mm). The stereotoxic coordinates for preoptic anterior hypothalamus (POAH) were A: 2.5 mm, L: 2 mm, and V: 15 mm. It is likely that the microdialysates obtained from the OVLT region and likely from other close tissues such as POAH may be not limited only to OVLT.

Acknowledgment This work was supported partially by the National Science Council of the Republic of China (Taipei, Taiwan) (grant no. NSC96-2320-B218-001-MY2).

References

Fig. 5. Proposed scheme of the interacting sequence of events occurring from the beginning of exposure to LPS or IL-1β injection to fever occurrence in rabbits. (−) and (+) indicate “inhibiting” and “stimulating,” respectively. LPS, lipopolysaccharide; NFκB, nuclear factor-kappa B; IL-1β, interleukin-1 beta; TNF-α, tumor necrosis factor-alpha; IL-6, interleukin-6; IL-1ra, interleukin-1 receptor antagonist; COX-2, cyclooxygenase-2; PGE2, prostaglandin E2; BBB, blood-brain-barrier; and OVLT, organovasculosum laminae terminalis.

glutamate–hydroxyl radical–PGE2 pathway in the OVLT of rabbit brain. The results are consistent with the findings of many other investigators. For example, the fever induced by intracerebroventricular endothelin-1-induced fever is intracerebroventricular IL-1ra blocked completely (Fabricio et al., 2006a). Endothelin-1 increases IL1 production in the anterior hypothalamus, and this effect appears to be correlated to endothelin-1-induced fever in vivo, as well as PGE2 production in vitro (Fabricio et al., 2006b). Interleukin-1 receptor antagonist inhibits endotoxin fever and systemic IL-6 induction in the rat (Luheshi et al., 1996). Fig. 5 depicts the proposed scheme of the interacting sequence of events occurring from the beginning of exposure to a LPS injection to fever occurrence in rabbits. The link between circulating cytokines and fever is well established with IL-1β, IL-6, and TNF-α, implicated in triggering the fever response (Conti et al., 2004; Konsman et al., 2002; Luheshi, 1998). These cytokines activate nuclear factor-kappa B (NFκB). The above-mentioned pathways lead to the transcription and induction of COX-2 (Dawn et al., 2004; Nadjar et al., 2005; Rummel et al., 2006), the rate-limiting enzyme for PGE2 synthesis, and fever genesis (Ivanov and Romanovsky, 2004; Li et al., 1999). According to our present results, it seems that systemic or central administration of IL-1ra may exert its antipyretic action by inhibiting overproduction of IL-1β, TNF-α, and IL-6 and OVLT glutamate– hydroxyl radical–PGE2 pathways in rabbit's brain. A more recent report (Vinkers et al., 2009) has proposed that stress-induced hyperthermia and infection-induced fever are two distinct processes mediated largely by different neurobiological mechanisms. The benzodiazepine diazepam but not PGE-synthesis blocking drug aspirin dose-dependently attenuated the stressinduced hyperthermia response in both rats and mice. In contrast, aspirin reduced both LPS and IL-1β induced fever, whereas diazepam

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