Pflfigers Arch (1991) 419:616-621

Journal of Physiology 9 Springer-Verlag1991

Characterization and mechanism of fever induction by interleukin-1 beta Masaaki Hashimoto Department of Physiology, Yamanashi Medical College, Tamaho, Nakakoma, Yamanashi, 409-38, Japan Received May 22, 1991/Received after revision August 12, 1991/Accepted September 17, 1991

Abstract. To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracerebroventricular (icv) or a subcutaneous (sc) route in conscious rabbits. Fever induced by icv administration of recombinant human interleukin-lfl (rhIL-lfl) was depressed by either icv or sc pretreatment with indomethacin. Fever induced by intravenous (iv) administration of rhIL-lfl was significantly inhibited, though initial small increase in colonic temperature still remained, and was completely depressed by combination of icy and sc pretreatment with indomethacin. Intracerebroventricularly administered recombinant rabbit IL-lfl (rrlL-lfl) induced dose-dependent increases in colonic temperature, which was depressed by sc pretreatment with indomethacin. There is little species specificity between human and rabbit IL-lfl, in terms of the pyrogenic potency and the inhibitory effect of sc indomethacin on fever induced by icy IL-lfl. Further, fever caused by icv administration of sodium arachidonate was significantly depressed by sc pretreatment with indomethacin. These results show that the inhibitory effect of indomethacin, administered either icy or sc, on IL-lfl-induced fever is similar to that of IL-la-induced fever reported previously [11]. This suggests that the site of arachidonate metabolism significantly involved in the mechanism of fever induction by IL-1 is easily accessible to the brain from the blood. Key words: Recombinant human interleukin-lfl (rhIL-lfl) - Recombinant rabbit interleukin-lfl (rrIL-lfl) - Endogenous pyrogen Blood brain barrier Arachidonate metabolism - Prostaglandin - Species specificity

Introduction Fever-induced by the administration of bacterial endotoxin appears to be mediated by several kinds of endogenous Offprint requests to: M. Hashimoto

pyrogenic substances, e.g., interleukin-I (IL-1), tumor necrosis factor, and interferon [7]. IL-1 has been thought to be a predominant component in endogenous pyrogenic cytokines produced by macrophage-monocyte cell lineage [3]. Since IL-1 is produced not only by peripheral leucocytes, but also by the glial cells in the central nervous system (CNS) [9], these data support the hypothesis that I b l in the CNS is involved in the mechanism of fever induced by endotoxin. Possibly an independent humoral mechanism involved in endotoxin fever may exist both inside and outside of the blood brain barrier (BBB) [22]. We reported previously that the fever induced by intracerebroventricular administration of recombinant human IL-la (rhIL-la) was significantly inhibited by sucutaneous (sc) pretreatment with indomethacin, and fever induced by intravenous (iv) administration of rhIL-la in rabbits was depressed by icv pretreatment with indomethacin [11]. The inhibitory effect of pretreatment with indomethacin on one side of the BBB on fever induced by administration of rhIL-la on the opposite side of the BBB seems to be curious; because indomethacin pretreatment on one side showed no effect on the fever induced by an administration of endogenous pyrogen, derived from cultured leukocytes, on the other side of the BBB [22, 25]. If IL-1 is the predominant ingredient of naturally derived endogenous pyrogen from cultured leucocytes, the question remains as to why the antipyretic effect of indomethacin on IL-I fever differed from that on fever induced by the endogenous pyrogen derived from cultured leucocytes. Recently two subtypes of IL-1 were cloned and purified, namely, IL-la and IL-lfl [13, 17]. Further, reports showed that more than 90% of IL-1, produced by human or rabbit leucocyte, was of the beta type [18]. It is hypothesized that the discrepancy mentioned above originated from the difference between two subtypes of IL-1, or the difference between human IL-1 and rabbit IL-I. To investigate these hypotheses, in this study the inhibitory effect of indomethacin in each administration route on I~lfl-induced fever was determined, and the possible mechanism to induce fever by IL-I was discussed.

617 To determine dose/response relationships, a total of 6 rabbits were injected with varying doses of rhIL-lfl and 5 for rrIL-tfl. Each rabbit was not used more than 3 times in order to prevent tolerance [11]. After determining the appropriate doses, 24 rabbits were divided in to 6 groups of 4, to evaluate the effect of indomethacin on IL-lfl-induced fever, as described previously [11], and an additional 4 rabbits were used to evaluate the effect on arachidonate fever. Pyrogens were either injected by an iv route into a marginal ear vein or directly into the lateral cerebral ventricle. Indomethacin, or vehicles for indomethacin as a control experiment, were administered via icy and/or sc routes 15 rain prior to the administration of pyrogens. Control experiment was performed in the same group of rabbits. Statistical significance of the temperature changes were evaluated usign 2-way ANOVA of repeated measures design.

Materials and methods

Drugs. rhIL-1/? (17,376 Da, Ohtsuka Pharmaceutical, Osaka, Japan) [13] was dissolved in phosphate buffered saline (PBS, pH 7.4) at a concentration of 1 mg/ml. The solution of rhIL-1/? was divided into several vials and stored at - 2 0 ~ The frozen stock solutions were thawed, diluted with 0.1%-gelatin containing Dulbecco's PBS (pH 7.4), and stored at 4 ~ throughout the experiment. IL-l solutions lost their pyrogenieity after moderate heating (80~ 30 min), which meant a negligible contamination of the solution by heat-stable endotoxin. The injection volume of the IL-1 solution was not more than 3 gl for icy administration, and not more than 0.5 ml for iv administration. The icv injection of IL-lfl was followed by a flush of sterile saline (5 ~xl). The pyrogenicity of the given volume of PBS and saline was negligible. Indomethacin (Sigma, St. Louis, USA) was dissolved in sterile castor oil in a concentration of 20 mg/ml for sc pretreatment, or dissolved in 99% ethanol (20 mg/ml) for icv pretreatment. Indomethacin was given at a dose of 400 gg/rabbit (icv), independent of the body weight, or 20 mg/kg (sc) [22, 25]. Injection of the vehicle for indomethacin in the given volume, or the given dose of indomethacin administered by each route did not affect the body temperature of afebrile rabbits [11]. Sodium arachidonate (Sigma) was dissolved in sterile saline in the concentration of 5mg/ml, and divided into 100 pg/vial and stored at - 2 0 ~ The stock solution was thawed and used for icv injection. Second use of the remaining portion in each vial was avoided. The dose for icv adminstration was 20 gg/kg, which increased colonic temperature by about 1 ~ [15]. Recombinant rabbit interleukin-1 beta (rrIL-l[}) was expressed from E. coli (HB101) and purified by successive chromatographies on Sephadex G-75, C-9 (Dainippon Pharmaceutical, Osaka, Japan; an Lipopolysaccharide removing agent) and IEX 530 KS (Toyo Soda, Tokyo, Japan; a cation exchanging high pressure liquid chromatography gel) in that order. The resulting purified material gave a single band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) and analytical isoelectrofocusing on PAG plate (LKB Tokyo, Japan). The rrIL-1]~ showed a molecular weight of 17,390 Da (152 amino acid residues) and pI value of 7.2. The specific activity for thymocyte comitogenesis [19] was 4.4x 107 units/mg protein.

Results

Dose~response relationship o f f e v e r o f icy adminsitration o f rhlL-1B T h e t e m p e r a t u r e o f t h e e a r s k i n a n d r e s p i r a t o r y rate b o t h d e c r e a s e d d u r i n g r h I L - l f l - i n d u c e d fever o v e r a s i m i l a r t i m e c o u r s e to t h e c h a n g e s in c o l o n i c t e m p e r a t u r e . C o l o n ic t e m p e r a t u r e i n c r e a s e d in r e s p o n s e to icv a d m i n i s t r a t i o n o f r h I L - l f l . T h e t i m e c o u r s e o f t h e fever i n d u c e d by r h I L - l ~ in d o s e s b e t w e e n 1 0 p g / k g a n d 1 0 0 0 p g / k g is s h o w n in Fig. 1 A . T h e fever b e g a n w i t h i n 20 m i n o f r h I L - l f l i n j e c t i o n , r e g a r d l e s s o f t h e dose. H o w e v e r , t h e p e a k a m p l i t u d e o f t h e fever c h a n g e d d o s e d e p e n d e n t l y , b e t w e e n 0.5 ~ (10 p g / k g ) a n d 2.7 ~ (1000 p g / k g ) , a n d t h e t i m e for t h e fever to p e a k r a n g e d 1 . 7 5 - 3 . 5 h (Fig. 1 A). T h e t o t a l t i m e c o u r s e o f t h e fever was l o n g e r t h a n 6 h, even w i t h t h e s m a l l e s t d o s e used, in this study. T h e d o s e / r e s p o n s e r e l a t i o n s h i p is s h o w n in Fig. 1 B.

Effects o f icv indomethacin on the f e v e r induced by icy rhIL-lfl

Procedures. Male Japanese white rabbits (2.0-3.0 kg) were used for the experiments. At least t 0 days prior to the day of experiment, a polyethylene guide cannula (0.82 mm o.d.) was implanted stereotaxically into the lateral cerebral ventricle for the icv administration of drugs in the 26 rabbits during deep anaesthesia (sodium pentobarbitone, 35 mg/kg, iv). Rabbits were allowed to recover from the surgery in feeding cages, according to the guide lines for animal experiments given by Yamanashi Medical College. The rabbits had been trained for several days prior to the day of experiment to adapt to the experimental conditions. Upon the day of the experiment, the rabbits were lightly restrained by the neck in an experimental rabbit chamber, at a temperature of 25_+ 1 ~ and relative humidity of 50%. A

T h e d o s e o f 250 p g / k g o f r h I L - l f l u s e d was e s t i m a t e d as the minimum effective dose for inducing a peak increase in c o l o n i c t e m p e r a t u r e o f a b o u t 2 ~ ( a c c o r d i n g to t h e d o s e / r e s p o n s e c u r v e s h o w n a b o v e ) at a b o u t 3 h. P r e t r e a t m e n t w i t h i n d o m e t h a c i n s t r o n g l y d e p r e s s e d t h e fever ind u c e d by icv r h I L - l f l (Fig. 2, crosses). T h e m a x i m u m c h a n g e in t h e c o l o n i c t e m p e r a t u r e o f t h e i n d o m e t h a c i n t r e a t e d g r o u p was 0.43 _+0.19 ~ ( m e a n + SE, n = 4) at 2 h

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Fig. 1A, B. Dose/response relationship of fewer to intracerebroventricular (icv) administration of recombinant human interleukin-lfl (rhIL-lfl). A Dose-dependent changes in colonic temperature (ATc) induced by icv administration of rhIL-lp at various doses (filled circles, 1000 pg/kg; open circles, 500 pg/kg; filled squares, 100 pg/kg; open squares, 50 pg/kg; filled triangles, 10 pg/kg), rhIL-lfl was injected at time 0 in this and the following figures. The lowest curve (open triangles) shows the response to heated rhIL-lfl (80 ~ 30 min) at a dose of 250 pg/kg. Each curve shows the mean of the peak temperature changes obtained from 3 rabbits. Tc before injection was 39.14_+0.06 ~ (mean+SE, n = 18). B Dose/response curve. Peak changes in Tc (A Tc max) were plotted against the dose of rhIL-lfl. The means are shwon as open circles connected by solid lines

618 Tc (*C) 41.5

The fever induced by iv rhlL-lfl was depressed by pretreatment with sc indomethacin, except for an initial increase in colonic temperature o f 0.72+0.13 ~ (n -- 14) (Fig. 3 A ) which peaked at 55.0_+3.5 rain after rhIL-lfl injection. This temperature depression was significant ( P < 0.01). The peak time o f the fever was not significantly different from control rabbits.

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Fig. 2. Effects of icv pretreatment with indomethacin on the fever induced by icv administration of rhIL-lp. Changes (mean_+SE) in Tc in 4 rabbits are shown against the time after icv administration of rhIL-lfl (250 pg/kg). Indomethacin (IM, 400 gg/rabbit, crosses) or its vehicle only (99% ethanol, 20 gl/rabbit, filled circles) were injected intracerebroventricularly 15 rain prior to rhIL-lfl, at the time indicated by an arrow in this and the following figures after rhlL-lfl injection. Depression o f the fever was statistically significant in c o m p a r i s o n to the results obtained in control rabbits pretreated with the vehicle only (Fig. 2, filled circles) ( P < 0.01).

Effect o f sc indomethacin on the fever induced by icv rM~IB

Effects o f sc indomethacin on the fever induced by iv rhlL-l# A n iv dose o f 1 g g / k g o f rhlL-lfl was used to induce fevers, similar to that induced by 250 p g / k g o f icv rhlL-lfl. Colonic temperature was increased by 1 . 8 4 + 0 . 1 0 ~ (n = 12) and peaked 165.5 +_12.2 min after rhlL-lfl administration.

A

Following icv pretreatment with indomethacin, the fever induced by iv rhIL-lfl was also markedly depressed, except for the initial phase (Fig. 3 B). The m a x i m u m colonic temperature increase was 0.86_+0.08 ~ at 49.0___4.6 rain after r h I L - l p , but only 0.22_+0.12~ at 3 h. The difference at the first peak was not significant, but the difference at 3 h was ( P < 0 . 0 1 ) . With indomethacin pretreatment administered t h r o u g h b o t h sc and icv routes, the r h I L - l f l fever was completely depressed (Fig. 3 C, crosses) c o m p a r e d to the fever course o f the control rabbits (Fig. 3 C, filled circles).

The fever induced by icv rhlL-lfl was also partially depressed by sc pretreatment with indomethacin. The peak o f the fever in the pretreated rabbits occurred at 90.0+1.8 min after rhIL-lfl (Fig. 4). The average peak temperature increase was 0 . 5 + 0 . 0 8 ~ ( n = 4 ) , and 0 . 1 + 0 . 1 4 ~ at 3 h after r h l L - l f l injection.

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Fig. 3A-C. Effects of subcutaneous (sc) and/or icv pretreatment with indomethacin on the fever induced by intravenously (iv) administered rhIL-lp. Changes (mean_+SE) in Tc in 4 rabbits are shown against the time after iv administration of rhIL-lfl (1 gg/kg). A Indomethacin (20mg/kg, crosses) or its vehicle (castor oil, 1 ml/kg, filled circles) were injected subcutaneously 15 min prior to the administration of

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rhIL-lfl. B Indomethacin (400 p.g/rabbit, crosses) or vehicle (99% ethanol, 20 ~tl/rabbit, filled circles) were injected intracerebroventricularly 15min prior to rhIL-lfl. C Indomethacin (400~tg/rabbit, icv and 20 mg/kg, sc, crosses) or vehicles (99% ethanol, 20 Ixl/rabbit, icv and castor oil, 1 ml/kg, sc, filled circles) were injected 15 min prior to rhIL-lfl

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Fig. 4. Effects of sc pretreatment with [M on the fever induced by icy administration of rhIL-lfl. This figure is similar to Fig. 2, but IM (20 mg/kg, crosses) or its vehicle (castor oil, 1 ml/kg, filled circles) were injected sc 15 rain prior to rhIL-lfl

Fig. 6. Effects of sc pretreatment with IM on fever induced by icv administration of rrlL-lfl. Changes (mean+SE) in Tc in 4 rabbits are shown against the time after the administration of rrlL-lfl (333 pg/kg). IM (20 mg/kg, crosses) or its vehicle only (castor oil, 1 ml/kg, filled circles) were injected subcutaneously 15 rain prior to rrlL-lfl

Dose~response relationship of the fever induced by icv administration of rrlL-lfl

duced by icv rrlL-lfl was strongly depressed by sc pretreatment with indomethacin. The peak of the fever, in the pretreated rabbits, was at 80.0+3.5min after rrlL-lfl (Fig. 6, crosses), and in the control rabbits at 167.8_+ 19.0 min (Fig. 6, filled circles). The average peak temperature increase was 0.04-+0.13~ (n =4) in the pretreated rabbits, and 1.64+_0.28 ~ (n = 4) in the control rabbits. The peak difference was significant (P

Characterization and mechanism of fever induction by interleukin-1 beta.

To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracer...
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