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In vivo evidence that activation of tyrosine kinase is a trigger for lipopolysaccharide-induced fever in rats
Brain Research 852 Ž2000. 367–373 www.elsevier.comrlocaterbres
Research report
In vivo evidence that activation of tyrosine kinase is a trigger for lipopolysaccharide-induced fever in rats Hiromi Tsushima ) , Mayumi Mori Department of Pharmacology, Nagoya City UniÕersity Medical School, Kawasumi, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan Accepted 28 September 1999
Abstract We measured the rectal temperature of free-moving, conscious rats after intracerebroventricular Ži.c.v.. injections of lipopolysaccharide ŽLPS. and interleukin-1b ŽIL-1b . with or without various antagonists to investigate the mechanisms involved in LPS-induced fever. LPS Ž3 mg. elicited significant increases in rectal temperature, which lasted from 0.5 h to more than 8 h after administration. This febrile response was inhibited by pretreatment with L-nitro-arginine ŽLNA., indomethacin ŽIND., genistein ŽGEN., tyrphostin 46 and anti-rat IL-1b antibody Žanti-IL-1b Ab., but was not inhibited by pretreatment with daidzein or chelerythrine ŽCHE. into the ventricle. LPS Ž0.3 mg. following orthovanadate Ži.c.v.. produced fever, although the small amount of LPS Ž0.3 mg. or orthovanadate alone showed no effect on rectal temperature. I.c.v. injections of IL-1b also induced fever of approximately 4-h duration. This effect was inhibited by pretreatment with IND and anti-IL-1b Ab, but was not inhibited by pretreatment with LNA, GEN or CHE into the ventricle. These findings demonstrate that in the central nervous system, LPS increases IL-1b production after activation of tyrosine kinase and NO synthase, and IL-1b promotes prostaglandin production resulting in increased rectal temperature. Activation of tyrosine kinase in the central nervous system is probably a trigger for the febrile response induced by LPS. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Fever; Tyrosine kinase; Lipopolysaccharide; Interleukin-1b; Genistein; Intracerebroventricular injection
1. Introduction It is well known that lipopolysaccharide ŽLPS., one of the components of the cell wall of gram-negative bacteria, is a powerful pyrogenic agent w8,15x. Recent studies have shown that the mechanisms of LPS-induced fever involve increased production of certain cytokines. The LPS-induced fever is blocked by a IL-1b receptor antagonist and antibodies to interleukin ŽIL.-1b, IL-1b receptors w13,21,22x and macrophage inflammatory protein-1b w23x. Moreover, LPS increases the concentration of IL-1b, IL-6 and tumor necrosis factor-alpha ŽTNF-alpha. in the plasma, the cerebrospinal fluid or the brain w5,8,10,12–14,32,36x. LPS and these cytokines elicit induction of phospholipase A 2 or cyclo-oxygenase ŽCOX. w2,3,6,8x, which increases production of an endogenous thermogenetic substance, prostaglandin ŽPG. E 2 , in the hypothalamus w27,40x. It is generally agreed that the generation of PGE 2 is the final step and that of the cytokines, IL-1b plays an important role in fever induced by LPS. However, intracellular signal
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transductions involved in the LPS-induced fever remain to be clarified. Investigations of intracellular signal transductions activated by LPS are carried out using in vitro experimental systems with cultured cells, mostly cultured blood cells. Tyrosine phosphorylation and activation of mitogenactivated protein kinase in neutrophils, macrophages and vascular endothelial cells are demonstrated to be stimulated by LPS w7,26,34x. In human monocytes, increased production of IL-1b and TNF-alpha by LPS are mediated through two pathways, tyrosine kinase ŽTK. after LPS binds to LPS receptors ŽCD14. on the cell surface w44x and protein kinase ŽPK. C without activation of TK w35x. Also, CD14 has been found to associate with TK w37x. However, there is no evidence that these signal transductions are linked to the LPS-induced fever. The purpose of this study is to investigate signal transductions involved in the LPS-induced fever. For this purpose, we set up an in vivo experimental system using free-moving animals. This is the first in vivo evidence demonstrating that activation of TK in the central nervous system by LPS leads to fever generation and probably is the trigger step for IL-1b biosynthesis.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 1 7 7 - 0
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2. Materials and methods
Rats were anesthetized with pentobarbiturate Ž40 mgrkg, i.p.. and a guide cannula ŽAG-8, Eicom, Kyoto, Japan. was implanted in the right lateral ventricle with dental cement and small screws, as described previously w24x. A dummy cannula was placed in the guide cannula in order to prevent clogging of the injection sites. The site of the guide cannula was determined according to the atlas of Konig and Klippel Ž3.8 mm anterior to lambda, 1.8 mm ¨ lateral to the midline and 2.8 mm ventral to the skull surface. w17x. After surgery, the animals were kept in individual cages for at least 1 week to recover.
microinjection cannula was connected to a microsyringe by a polyethylene tube, which contained 5 ml of a drug solution or vehicle for pretreatment and 5 ml of a solution of LPS, cytokines or vehicle. A small air bubble divided the two kinds of solution in the tube. The tip of the microinjection cannula was located 1 mm beyond the tip of the guide cannula. After 1 h, tap water Ž5% of b.wt.. was orally administered, and immediately after that, the pretreatment drug was injected into the ventricle. The rat was moved to a metabolic cage Ža 25 = 30 = 40 cm3 box made of acrylic panels.. Another injection of LPS, cytokines or vehicle into the ventricle was performed 45 min later. Rectal temperature and volume of urine outflow were monitored at intervals of 15 and 30 min, respectively. The results of urinary volume are published elsewhere. In one animal, three or four experiments, including two experiments of LPS or cytokine administration and one or two experiments of administration of vehicle or antagonist alone, were carried out at intervals of 4 days. The basal rectal temperature of each group ranged from 36.44 " 0.278C to 37.05 " 0.148C and did not differ significantly from one group to another. When all of the experiments were finished, the site of the guide cannula was histologically verified under a microscope w38,39x.
2.3. Experimental procedure
2.4. Statistical analysis
The rats were acclimated to handling and experimental conditions during the recovery period. The experiments were started between 1000 and 1030 h. Under light anesthesia with ether, a cannula for drug microinjection was inserted into the guide cannula and a thermister probe with a thermometer ŽVX-310A, Matsushita Communication Industrial, Tokyo, Japan. was inserted in the rectum. The
Changes in rectal temperature were expressed as means " S.E.M. of differences from the basal rectal temperature immediately before intracerebroventricular Ži.c.v.. injections of LPS or cytokine. Statistical comparisons were made by One-way analysis of variance ŽANOVA. followed by Fisher’s PLSD test. Differences were considered statistically significant when the P value was under 0.05.
2.1. Animals Male Wistar rats weighing 280–350 g ŽJapan SLC, Hamamatsu, Japan. were used in this study. The animals were housed in a temperature-controlled room at 23–248C with a 12-h lightrdark cycle and allowed free access to commercial food and water. The experiments were carried out according to the Guidelines for Animal Care and Use of our university. 2.2. Surgical procedure
Fig. 1. Change in rectal temperature after i.c.v. injection of LPS Ž3 mg.. The symbols and bars indicate the means" S.E.M. from nine Žclosed circles: vehicle. or seven Žopen circles: LPS. experiments. U P - 0.05 vs. the vehicle-injected group at the corresponding time points ŽOne-way ANOVA followed by Fisher’s PLSD test..
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taining 0.1% bovine serum albumin was used as vehicle for the cytokines and the antibody. The other drugs were dissolved in sterile physiological saline.
3. Results 3.1. LPS-induced effects on rectal temperature I.c.v. injections of 3 mg of LPS produced significant increases in rectal temperature from 0.5 h to more than 8 h after administration, when compared to rectal temperature after i.c.v. injections of vehicle. The febrile response reached a plateau at 1.5–2.0 h after LPS injection. The first and second injections of LPS in one rat showed fever of a similar degree Ž0.97 " 0.248C and 1.08 " 0.218C at 2 h, 1.00 " 0.278C and 0.98 " 0.218C at 6 h after administration for the first and second injections, respectively; n s 5.. Fig. 1 shows the time course of the rise of rectal temperature after i.c.v. injections of 3 mg of LPS. The fever induced by LPS Ž3 mg, i.c.v.. over a period of 2.0–8.0 h after administration was completely blocked by pretreatment with anti-IL-1b antibody Žanti-IL-1b Ab; 20 mg, i.c.v.. ŽFig. 2 shows the result 3 h after administration.. However, the inhibition by anti-IL-1b Ab was not very intense at the stage when the rectal temperature was increasing Ž0.5–2.0 h after administration. wchanges in rectal temperature induced by LPS with anti-IL-1b Ab, 8C: 0.33 " 0.10 Ž1.0 h., 0.52 " 0.16U Ž1.25 h., 0.69 " 0.18 Ž1.5 h., 0.78 " 0.21 Ž1.75 h. and 0.48 " 0.22U Ž2.0 h after administration.; n s 5; U P - 0.05 compared with the LPS-injected group at the corresponding time pointsx. The pretreatment with a NO synthase inhibitor, L-nitro-arginine ŽLNA; 6 mg, i.c.v.., attenuated the LPS-induced fever over the entire period ŽFig. 2.. On the other hand, the inhibitory effect of a COX inhibitor, indomethacin ŽIND; 15 mg, i.c.v.., on the febrile response was similar to the inhibition produced by anti-IL-1b Ab ŽFig. 2.. IND did not inhibit a portion of the LPS-induced temperature increase during
Fig. 2. Effects of various antagonists on the LPS Ž3 mg.-induced fever. Each pretreatment Žanti-IL-1b Ab: 20 mg; IND: 15 mg; LNA: 6 mg; CHE: 3 mg; GEN: 3.4 mg. was performed by i.c.v. injection 45 min before LPS administration. The columns and bars indicate the means" S.E.M. of changes in rectal temperature 3 h after LPS administration. N in the figure shows the number of experiments. Superscripts a and b refer to P - 0.05 vs. the vehicle- and the LPS-injected groups, respectively ŽOne-way ANOVA followed by Fisher’s PLSD test..
2.5. Drugs The following drugs were used: LPS Žfrom Escherichia coli serotype 0111: B4., tyrphostin 46, indomethacin ŽSigma, St. Louis, MO., recombinant rat IL-1b, monoclonal anti-rat IL-1b antibody ŽR & D Systems, Minneapolis, MN., recombinant human TNF-alpha ŽPepro Tech EC, London, UK., C 2-ceramide ŽCayman Chemical, Place Ann Arbor, MI., L-nitro-arginine ŽPeptide Research Institute, Osaka, Japan., daidzein ŽWako, Osaka, Japan., genistein ŽGEN., sodium orthovanadate ŽSeikagaku, Tokyo, Japan. and chelerythrine chloride ŽResearch Biochemicals International, Natick, MA.. Indomethacin was dissolved in a minimum volume of 0.1 N NaOH and diluted with sterile phosphate buffered saline ŽpH 7.4. to the concentration injected. GEN, tyrphostin ŽTYR. 46, daidzein and C 2ceramide dissolved in ethanol were diluted with sterile physiological saline. Sterile phosphate buffered saline con-
Table 1 Changes in rectal temperature induced by i.c.v. injections of various antagonists Each of the antagonists Žindomethacin: 15 mg; LNA: 6 mg; anti-IL-1b Ab: 20 mg; chelerythrine: 3 mg; daidzen: 2 mg; genistein: 3.4 mg; orthovanadate: 100 mg. was injected into the lateral ventricle 45 min before administration of vehicle. The values indicate the means" S.E.M. of changes in rectal temperature Ž8C.. The numbers of experiments are shown in the parentheses. Drugs
Vehicleq vehicle Ž9. Indomethacinq vehicle Ž8. LNA q vehicle Ž6. Anti-IL-1b Ab q vehicle Ž5. Chelerythrineq vehicle Ž3. Daidzeinq vehicle Ž5. Genisteinq vehicle Ž3. Orthovanadateq vehicle Ž5.
Time after administration Žh. 1
2
3
4
5
6
0.15 " 0.08 0.27 " 0.12 0.15 " 0.12 0.15 " 0.11 0.06 " 0.09 0.46 " 0.12 0.03 " 0.13 y0.02 " 0.06
0.36 " 0.12 0.39 " 0.17 0.32 " 0.22 0.27 " 0.21 0.23 " 0.18 0.55 " 0.12 y0.03 " 0.23 y0.32 " 0.12
0.24 " 0.21 0.46 " 0.17 0.08 " 0.27 0.17 " 0.20 0.37 " 0.25 0.24 " 0.30 0.14 " 0.13 y0.28 " 0.09
0.35 " 0.14 0.47 " 0.19 0.08 " 0.25 0.22 " 0.21 0.17 " 0.06 0.31 " 0.26 0.14 " 0.19 y0.21 " 0.24
0.31 " 0.14 0.46 " 0.14 0.12 " 0.22 0.12 " 0.16 0.06 " 0.03 0.19 " 0.31 0.23 " 0.24 y0.31 " 0.19
0.14 " 0.12 0.32 " 0.13 0.04 " 0.22 y0.05 " 0.25 0.09 " 0.06 0.08 " 0.22 0.34 " 0.16 y0.19 " 0.18
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Fig. 3. Effect of LPS Ž0.3 mg. on rectal temperature in the presence of orthovanadate. VAN Ž100 mg. was injected into the lateral ventricle 45 min before LPS administration. The columns and bars indicate the means"S.E.M. of changes in rectal temperature 3 h after LPS administration. N in the figure shows the number of experiments. U P - 0.05 vs. the VAN-injected groups ŽOne-way ANOVA followed by Fisher’s PLSD test..
the stage when the temperature was increasing Ž0.5–2.0 h after administration. wchanges in rectal temperature induced by LPS with IND inhibition, 8C: 0.38 " 0.14 Ž1.0 h., 0.52 " 0.17 a Ž1.25 h., 0.75 " 0.19 b Ž1.5 h., 0.71 " 0.21 Ž1.75 h. and 0.60 " 0.23 Ž2.0 h after administration.; n s 5; superscripts a and b indicate P - 0.05 when compared with the LPS- and vehicle-injected groups at the corresponding time pointsx. Anti-IL-1b Ab, IND or LNA alone did not have any effect on rectal temperature ŽTable 1.. To investigate intracellular signal transductions involved in the LPS-induced fever, a PKC inhibitor, chelerythrine ŽCHE, 3 mg., the TK inhibitors, GEN Ž3.4 mg. and TYR 46 Ž3 mg., and a tyrosine phosphatase inhibitor, orthovanadate ŽVAN, 100 mg., were pre-injected into the ventricle. As shown in Fig. 2, GEN completely blocked the LPS-induced fever. The inhibition of the fever by GEN
was dose-dependent Ž8C, no GEN: 0.82 " 0.18, n s 4; 0.1 mg of GEN: 0.95, n s 2; 0.2 mg of GEN: 0.84 " 0.22, n s 3; 0.3 mg of GEN: 0.29 " 0.35, n s 3 and 0.5 mg of GEN: y0.04 " 0.21, n s 5 at 3 h after LPS administration.. A negative control agent of GEN, daidzein ŽDAI, 2 mg., did not influence the LPS-induced fever Žthe change in rectal temperature induced by LPS with DAI 3 h after administration was 0.858C; n s 2.. Another TK inhibitor, TYR 46 Ž3 mg., also completely blocked the fever Ž0.22 " 0.128C 3 h after administration; n s 6.. CHE did not affect the LPS-induced fever ŽFig. 2.. Because these results suggested the involvement of TK in the LPS-induced fever, the effect of LPS on rectal temperature in the presence of VAN was examined. A small amount of LPS Ž0.3 mg, i.c.v.. or VAN individually showed no effect on basal rectal temperature ŽFig. 3 and Table 1.. However, i.c.v. injections of 0.3 mg of LPS after pretreatment with VAN produced a small increase in the rectal temperature ŽFig. 3.. 3.2. IL-1b-induced effects on rectal temperature I.c.v. injections of 50 ng of IL-1b elevated the rectal temperature from 0.75 to 4.75 h after administration ŽFig. 4.. The fever induced by the first and second injections was not statistically different Ž1.25 " 0.138C and 1.09 " 0.118C at 3 h after administration, n s 3.. The increasing curve and the maximum response of the IL-1b-induced fever resembled those of the LPS-induced fever, but the duration was shorter than for the LPS-induced effect. Pre-injections of 2 mg of anti-IL-1b Ab into the ventricle did not influence the IL-1b-induced fever Ž n s 3; data not shown.. A higher dose of 20 mg of anti-IL-1b Ab attenuated the IL-1b-induced effects at all time points, although the changes in rectal temperature at 1.25 Ž0.56 " 0.198CU ., 1.5 Ž0.66 " 0.168CU . and 1.75 h Ž0.52 "
Fig. 4. Change in rectal temperature after i.c.v. injection of IL-1b Ž50 ng. and TNF-alpha Ž50 ng.. The symbols and bars indicate the means" S.E.M. of nine Žclosed circles: vehicle., six Žopen circles: IL-1b . or eight Žopen triangles: TNF-alpha. experiments. U P - 0.05 vs. the vehicle-injected group at the corresponding time points ŽOne-way ANOVA followed by Fisher’s PLSD test..
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0.168CU ; n s 5; U P - 0.05 when compared with both the vehicle- and IL-1b-injected groups at the corresponding time points. after administration showed slight increases even after pretreatment. Also, pretreatment with IND partially attenuated the IL-1b-induced effect and the changes in rectal temperature at 1.0 Ž0.61 " 0.238C. and 1.25 h Ž0.63 " 0.228C; n s 5. after administration were significantly different, compared with the vehicle- and IL-1b-injected groups Ž P - 0.05.. On the other hand, LNA did not attenuate the IL-1b-induced effect, and even tended to potentiate it. Neither CHE Ž3.0 mg. nor GEN Ž3.4 mg. influenced the IL-1b-induced fever. The effects of the various antagonists on the IL-1b-induced fever 3 h after the IL-1b administration are summarized in Fig. 5.
systems after LPS administration w5,32x. I.c.v. injections of 50 ng of TNF-alpha ŽFig. 4. or of 500 ng Ž n s 3; data not shown. did not show any significant effect on rectal temperature. To examine the interaction between IL-1b and TNF-alpha on the regulation of rectal temperature, these cytokines were simultaneously injected into the ventricle. However, the increase in rectal temperature after simultaneous injections of IL-1b and TNF-alpha of 50 ng each was not significantly different from that after injection of IL-1b alone Ž n s 3; data not shown..
3.3. C2-ceramide-induced effects on rectal temperature
The pharmacological characteristics of the LPS-induced fever in this study are consistent with those that have already been reported, such as the time course of the febrile response, as well as the inhibition of the fever by NO synthase inhibitors, COX inhibitors and anti-IL-1b Ab w8,13,20,30,41x. Also, the involvements of NO, PGs and IL-1b in the LPS-induced effect are supported by the following findings: Ž1. increases in the mRNAs, proteins or activities of NO synthase and COX w3,4,18,28x, Ž2. histochemical studies showing increased numbers of cells positive to anti-NO synthase Ab or anti-COX Ab in the central nervous system w8,16x, Ž3. increased production of NO, PGs or IL-1b after LPS administration w4,5,8,12, 16,29,42,43x and Ž4. disappearance of the LPS-induced fever and IL-1b-induced fever in PG EP3 receptor knockout mice w40x. Moreover, there is a report demonstrating a resistant component in the LPS-induced fever to a COX inhibitor, and the authors conclude that at least two mechanisms are involved in it w9x. Also, the LPS-induced fever in this study had the component resistant to IND. LNA blocked only the LPS-induced fever, suggesting that NO synthesis is necessary in the process before IL-1b production in the LPS-induced fever and that the IL-1b-induced fever does not involve NO production. In vitro experiments demonstrate IL-1b-induced increases in NO synthesis w6,29x or blockade of NO synthase inhibitors on IL-1b-induced PGE 2 production w25x. On the other hand, there is a report that IL-1b-induced fever is blocked by a NO synthase inhibitor in rabbits w19x. The reasons for this discrepancy are not clear, but may reflect the different animal species involved. Klir et al. w14x demonstrate that the relation between dose and effect induced by central injections of TNF-alpha is a polynomial and that i.p., but not i.c.v., injections of TNF-alpha are antipyrogenic. In general, both pyrogenic and cryogenic effects of TNF-alpha are known. The effects of TNF-alpha on body temperature are variable, probably due to interactions with other substances. In this study, i.c.v. injections of TNF-alpha elicited no significant effect on the basal rectal temperature nor on the IL-1b-induced fever. Although LPS raises the concentration of TNF-alpha
The results with GEN and CHE suggested that the IL-1b-induced fever did not involve either TK or PKC. Recently, IL-1b was reported to activate sphingomyelinase and to increase ceramide production in various cultured cells, which enhances PGE 2 synthesis mediated through activation of COX w1,2,6x. Therefore, the effect of C 2ceramide on rectal temperature was examined. I.c.v. injections of 7.7 mg of C 2-ceramide did not increase rectal temperature at all w8C; 0.28 " 0.07 Ž1 h., 0.22 " 0.08 Ž2 h., y0.06 " 0.04 Ž3 h., y0.44 " 0.22 Ž4 h., y0.32 " 0.32 Ž5 h. and y0.32 " 0.31 Ž6 h after administration.; n s 3x. 3.4. TNF-alpha-induced effects on rectal temperature TNF-alpha has been reported to be a cytokine whose production increases in both peripheral and central nervous
Fig. 5. Effects of various antagonists on the IL-1b Ž50 ng.-induced fever. Each of the antagonists Žanti-IL-1b Ab: 20 mg; IND: 15 mg; LNA: 6 mg; CHE: 3 mg; GEN: 3.4 mg. was injected into the lateral ventricle 45 min before IL-1b administration. The columns and bars indicate the means" S.E.M. of changes in rectal temperature 3 h after IL-1b administration. N in the figure shows the number of experiments. Superscripts a and b refer to P - 0.05 vs. the vehicle- and the IL-1b-injected groups, respectively ŽOne-way ANOVA followed by Fisher’s PLSD test..
4. Discussion
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along with other cytokines — IL-1b, IL-6 and so on — in the plasma or the central nervous system w5,12,32x, TNFalpha in the central nervous system did not play any role in the LPS-induced fever or IL-1b-induced fever under our experimental conditions. The findings that the TK inhibitors, GEN and TYR 46, attenuated the LPS-induced fever, but did not attenuate the fever induced by IL-1b suggest that TK is involved in the mechanisms of IL-1b production by LPS. This is the first evidence that the activation of TK in the central nervous system is a mechanism directly related to the LPS-induced fever, although activation of TK is observed in cultured cells stimulated by LPS w4,8,16,34,35x. It is unclear which kind of TK is involved and where TK is activated by LPS in the central nervous system. CD14 is recognized as a binding site for LPS with LPS-binding protein on blood cells w44x. Recently, it was demonstrated that cultured brain glial cells express CD14 protein w11x. Moreover, glial cells produce various cytokines in the central nervous system w33x and express IL-1b mRNA after LPS administration w8,43x. Taken together, these findings suggest that TK in glial cells is responsible for LPS-induced fever. Interestingly, human monocytes have two mechanisms for production of IL-1b induced by LPS: one is mediated through activation of TK after LPS binds to CD14, while the other is mediated through activation of PKC, but not TK or CD14 w35x. LPS stimulates CD14-associated TK after binding to CD14 w37x. Participation of TK in the LPS-induced fever is supported by the synergetic effect of VAN and LPS shown in this study. PKC is not involved in the febrile response induced by LPS or IL-1b, because CHE did not influence the LPSand IL-1b-induced effects. The dose of CHE used in this study is appropriate for inhibition of PKC w31x. In cultured blood cells, PKC is activated after LPS stimulation w4,35x. This mechanism was not found in the febrile response observed in this study. Many intracellular signal transductions are activated after IL-1b binds to IL-1 receptors w2,6x. It is unclear which of these function in the febrile response. It is obvious that the IL-1b-induced fever is not mediated through TK or PKC, because neither GEN nor CHE changed the IL-1b-induced fever in this study. Ceramide, which was recently shown to be a possible second messenger for IL-1 receptors w2,6x, increases PG production resulting from the activation of COX w1x. However, this did not raise the rectal temperature. Therefore, the signal transduction for the IL-1b-induced fever still remains unclear. In conclusion, Ž1. LPS increases IL-1b production after stimulation of TK and NO synthesis, and then promotes PG synthesis resulting in fever; Ž2. IL-1b raises the rectal temperature due to increases in synthesis of PGs mediated by unknown mechanisms, excepting TK, PKC, NO production or sphingomyelinase; Ž3. C 2-ceramide does not increase the rectal temperature, and Ž4. TNF-alpha does not play any role in regulating the rectal temperature. The
activation of TK in the central nervous system is probably a trigger for the febrile response induced by LPS.
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Research report
In vivo evidence that activation of tyrosine kinase is a trigger for lipopolysaccharide-induced fever in rats Hiromi Tsushima ) , Mayumi Mori Department of Pharmacology, Nagoya City UniÕersity Medical School, Kawasumi, Mizuho-ku, Nagoya, Aichi, 467-8601, Japan Accepted 28 September 1999
Abstract We measured the rectal temperature of free-moving, conscious rats after intracerebroventricular Ži.c.v.. injections of lipopolysaccharide ŽLPS. and interleukin-1b ŽIL-1b . with or without various antagonists to investigate the mechanisms involved in LPS-induced fever. LPS Ž3 mg. elicited significant increases in rectal temperature, which lasted from 0.5 h to more than 8 h after administration. This febrile response was inhibited by pretreatment with L-nitro-arginine ŽLNA., indomethacin ŽIND., genistein ŽGEN., tyrphostin 46 and anti-rat IL-1b antibody Žanti-IL-1b Ab., but was not inhibited by pretreatment with daidzein or chelerythrine ŽCHE. into the ventricle. LPS Ž0.3 mg. following orthovanadate Ži.c.v.. produced fever, although the small amount of LPS Ž0.3 mg. or orthovanadate alone showed no effect on rectal temperature. I.c.v. injections of IL-1b also induced fever of approximately 4-h duration. This effect was inhibited by pretreatment with IND and anti-IL-1b Ab, but was not inhibited by pretreatment with LNA, GEN or CHE into the ventricle. These findings demonstrate that in the central nervous system, LPS increases IL-1b production after activation of tyrosine kinase and NO synthase, and IL-1b promotes prostaglandin production resulting in increased rectal temperature. Activation of tyrosine kinase in the central nervous system is probably a trigger for the febrile response induced by LPS. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Fever; Tyrosine kinase; Lipopolysaccharide; Interleukin-1b; Genistein; Intracerebroventricular injection
1. Introduction It is well known that lipopolysaccharide ŽLPS., one of the components of the cell wall of gram-negative bacteria, is a powerful pyrogenic agent w8,15x. Recent studies have shown that the mechanisms of LPS-induced fever involve increased production of certain cytokines. The LPS-induced fever is blocked by a IL-1b receptor antagonist and antibodies to interleukin ŽIL.-1b, IL-1b receptors w13,21,22x and macrophage inflammatory protein-1b w23x. Moreover, LPS increases the concentration of IL-1b, IL-6 and tumor necrosis factor-alpha ŽTNF-alpha. in the plasma, the cerebrospinal fluid or the brain w5,8,10,12–14,32,36x. LPS and these cytokines elicit induction of phospholipase A 2 or cyclo-oxygenase ŽCOX. w2,3,6,8x, which increases production of an endogenous thermogenetic substance, prostaglandin ŽPG. E 2 , in the hypothalamus w27,40x. It is generally agreed that the generation of PGE 2 is the final step and that of the cytokines, IL-1b plays an important role in fever induced by LPS. However, intracellular signal
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Corresponding author. Fax: q81-52-842-0863
transductions involved in the LPS-induced fever remain to be clarified. Investigations of intracellular signal transductions activated by LPS are carried out using in vitro experimental systems with cultured cells, mostly cultured blood cells. Tyrosine phosphorylation and activation of mitogenactivated protein kinase in neutrophils, macrophages and vascular endothelial cells are demonstrated to be stimulated by LPS w7,26,34x. In human monocytes, increased production of IL-1b and TNF-alpha by LPS are mediated through two pathways, tyrosine kinase ŽTK. after LPS binds to LPS receptors ŽCD14. on the cell surface w44x and protein kinase ŽPK. C without activation of TK w35x. Also, CD14 has been found to associate with TK w37x. However, there is no evidence that these signal transductions are linked to the LPS-induced fever. The purpose of this study is to investigate signal transductions involved in the LPS-induced fever. For this purpose, we set up an in vivo experimental system using free-moving animals. This is the first in vivo evidence demonstrating that activation of TK in the central nervous system by LPS leads to fever generation and probably is the trigger step for IL-1b biosynthesis.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 1 7 7 - 0
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2. Materials and methods
Rats were anesthetized with pentobarbiturate Ž40 mgrkg, i.p.. and a guide cannula ŽAG-8, Eicom, Kyoto, Japan. was implanted in the right lateral ventricle with dental cement and small screws, as described previously w24x. A dummy cannula was placed in the guide cannula in order to prevent clogging of the injection sites. The site of the guide cannula was determined according to the atlas of Konig and Klippel Ž3.8 mm anterior to lambda, 1.8 mm ¨ lateral to the midline and 2.8 mm ventral to the skull surface. w17x. After surgery, the animals were kept in individual cages for at least 1 week to recover.
microinjection cannula was connected to a microsyringe by a polyethylene tube, which contained 5 ml of a drug solution or vehicle for pretreatment and 5 ml of a solution of LPS, cytokines or vehicle. A small air bubble divided the two kinds of solution in the tube. The tip of the microinjection cannula was located 1 mm beyond the tip of the guide cannula. After 1 h, tap water Ž5% of b.wt.. was orally administered, and immediately after that, the pretreatment drug was injected into the ventricle. The rat was moved to a metabolic cage Ža 25 = 30 = 40 cm3 box made of acrylic panels.. Another injection of LPS, cytokines or vehicle into the ventricle was performed 45 min later. Rectal temperature and volume of urine outflow were monitored at intervals of 15 and 30 min, respectively. The results of urinary volume are published elsewhere. In one animal, three or four experiments, including two experiments of LPS or cytokine administration and one or two experiments of administration of vehicle or antagonist alone, were carried out at intervals of 4 days. The basal rectal temperature of each group ranged from 36.44 " 0.278C to 37.05 " 0.148C and did not differ significantly from one group to another. When all of the experiments were finished, the site of the guide cannula was histologically verified under a microscope w38,39x.
2.3. Experimental procedure
2.4. Statistical analysis
The rats were acclimated to handling and experimental conditions during the recovery period. The experiments were started between 1000 and 1030 h. Under light anesthesia with ether, a cannula for drug microinjection was inserted into the guide cannula and a thermister probe with a thermometer ŽVX-310A, Matsushita Communication Industrial, Tokyo, Japan. was inserted in the rectum. The
Changes in rectal temperature were expressed as means " S.E.M. of differences from the basal rectal temperature immediately before intracerebroventricular Ži.c.v.. injections of LPS or cytokine. Statistical comparisons were made by One-way analysis of variance ŽANOVA. followed by Fisher’s PLSD test. Differences were considered statistically significant when the P value was under 0.05.
2.1. Animals Male Wistar rats weighing 280–350 g ŽJapan SLC, Hamamatsu, Japan. were used in this study. The animals were housed in a temperature-controlled room at 23–248C with a 12-h lightrdark cycle and allowed free access to commercial food and water. The experiments were carried out according to the Guidelines for Animal Care and Use of our university. 2.2. Surgical procedure
Fig. 1. Change in rectal temperature after i.c.v. injection of LPS Ž3 mg.. The symbols and bars indicate the means" S.E.M. from nine Žclosed circles: vehicle. or seven Žopen circles: LPS. experiments. U P - 0.05 vs. the vehicle-injected group at the corresponding time points ŽOne-way ANOVA followed by Fisher’s PLSD test..
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taining 0.1% bovine serum albumin was used as vehicle for the cytokines and the antibody. The other drugs were dissolved in sterile physiological saline.
3. Results 3.1. LPS-induced effects on rectal temperature I.c.v. injections of 3 mg of LPS produced significant increases in rectal temperature from 0.5 h to more than 8 h after administration, when compared to rectal temperature after i.c.v. injections of vehicle. The febrile response reached a plateau at 1.5–2.0 h after LPS injection. The first and second injections of LPS in one rat showed fever of a similar degree Ž0.97 " 0.248C and 1.08 " 0.218C at 2 h, 1.00 " 0.278C and 0.98 " 0.218C at 6 h after administration for the first and second injections, respectively; n s 5.. Fig. 1 shows the time course of the rise of rectal temperature after i.c.v. injections of 3 mg of LPS. The fever induced by LPS Ž3 mg, i.c.v.. over a period of 2.0–8.0 h after administration was completely blocked by pretreatment with anti-IL-1b antibody Žanti-IL-1b Ab; 20 mg, i.c.v.. ŽFig. 2 shows the result 3 h after administration.. However, the inhibition by anti-IL-1b Ab was not very intense at the stage when the rectal temperature was increasing Ž0.5–2.0 h after administration. wchanges in rectal temperature induced by LPS with anti-IL-1b Ab, 8C: 0.33 " 0.10 Ž1.0 h., 0.52 " 0.16U Ž1.25 h., 0.69 " 0.18 Ž1.5 h., 0.78 " 0.21 Ž1.75 h. and 0.48 " 0.22U Ž2.0 h after administration.; n s 5; U P - 0.05 compared with the LPS-injected group at the corresponding time pointsx. The pretreatment with a NO synthase inhibitor, L-nitro-arginine ŽLNA; 6 mg, i.c.v.., attenuated the LPS-induced fever over the entire period ŽFig. 2.. On the other hand, the inhibitory effect of a COX inhibitor, indomethacin ŽIND; 15 mg, i.c.v.., on the febrile response was similar to the inhibition produced by anti-IL-1b Ab ŽFig. 2.. IND did not inhibit a portion of the LPS-induced temperature increase during
Fig. 2. Effects of various antagonists on the LPS Ž3 mg.-induced fever. Each pretreatment Žanti-IL-1b Ab: 20 mg; IND: 15 mg; LNA: 6 mg; CHE: 3 mg; GEN: 3.4 mg. was performed by i.c.v. injection 45 min before LPS administration. The columns and bars indicate the means" S.E.M. of changes in rectal temperature 3 h after LPS administration. N in the figure shows the number of experiments. Superscripts a and b refer to P - 0.05 vs. the vehicle- and the LPS-injected groups, respectively ŽOne-way ANOVA followed by Fisher’s PLSD test..
2.5. Drugs The following drugs were used: LPS Žfrom Escherichia coli serotype 0111: B4., tyrphostin 46, indomethacin ŽSigma, St. Louis, MO., recombinant rat IL-1b, monoclonal anti-rat IL-1b antibody ŽR & D Systems, Minneapolis, MN., recombinant human TNF-alpha ŽPepro Tech EC, London, UK., C 2-ceramide ŽCayman Chemical, Place Ann Arbor, MI., L-nitro-arginine ŽPeptide Research Institute, Osaka, Japan., daidzein ŽWako, Osaka, Japan., genistein ŽGEN., sodium orthovanadate ŽSeikagaku, Tokyo, Japan. and chelerythrine chloride ŽResearch Biochemicals International, Natick, MA.. Indomethacin was dissolved in a minimum volume of 0.1 N NaOH and diluted with sterile phosphate buffered saline ŽpH 7.4. to the concentration injected. GEN, tyrphostin ŽTYR. 46, daidzein and C 2ceramide dissolved in ethanol were diluted with sterile physiological saline. Sterile phosphate buffered saline con-
Table 1 Changes in rectal temperature induced by i.c.v. injections of various antagonists Each of the antagonists Žindomethacin: 15 mg; LNA: 6 mg; anti-IL-1b Ab: 20 mg; chelerythrine: 3 mg; daidzen: 2 mg; genistein: 3.4 mg; orthovanadate: 100 mg. was injected into the lateral ventricle 45 min before administration of vehicle. The values indicate the means" S.E.M. of changes in rectal temperature Ž8C.. The numbers of experiments are shown in the parentheses. Drugs
Vehicleq vehicle Ž9. Indomethacinq vehicle Ž8. LNA q vehicle Ž6. Anti-IL-1b Ab q vehicle Ž5. Chelerythrineq vehicle Ž3. Daidzeinq vehicle Ž5. Genisteinq vehicle Ž3. Orthovanadateq vehicle Ž5.
Time after administration Žh. 1
2
3
4
5
6
0.15 " 0.08 0.27 " 0.12 0.15 " 0.12 0.15 " 0.11 0.06 " 0.09 0.46 " 0.12 0.03 " 0.13 y0.02 " 0.06
0.36 " 0.12 0.39 " 0.17 0.32 " 0.22 0.27 " 0.21 0.23 " 0.18 0.55 " 0.12 y0.03 " 0.23 y0.32 " 0.12
0.24 " 0.21 0.46 " 0.17 0.08 " 0.27 0.17 " 0.20 0.37 " 0.25 0.24 " 0.30 0.14 " 0.13 y0.28 " 0.09
0.35 " 0.14 0.47 " 0.19 0.08 " 0.25 0.22 " 0.21 0.17 " 0.06 0.31 " 0.26 0.14 " 0.19 y0.21 " 0.24
0.31 " 0.14 0.46 " 0.14 0.12 " 0.22 0.12 " 0.16 0.06 " 0.03 0.19 " 0.31 0.23 " 0.24 y0.31 " 0.19
0.14 " 0.12 0.32 " 0.13 0.04 " 0.22 y0.05 " 0.25 0.09 " 0.06 0.08 " 0.22 0.34 " 0.16 y0.19 " 0.18
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Fig. 3. Effect of LPS Ž0.3 mg. on rectal temperature in the presence of orthovanadate. VAN Ž100 mg. was injected into the lateral ventricle 45 min before LPS administration. The columns and bars indicate the means"S.E.M. of changes in rectal temperature 3 h after LPS administration. N in the figure shows the number of experiments. U P - 0.05 vs. the VAN-injected groups ŽOne-way ANOVA followed by Fisher’s PLSD test..
the stage when the temperature was increasing Ž0.5–2.0 h after administration. wchanges in rectal temperature induced by LPS with IND inhibition, 8C: 0.38 " 0.14 Ž1.0 h., 0.52 " 0.17 a Ž1.25 h., 0.75 " 0.19 b Ž1.5 h., 0.71 " 0.21 Ž1.75 h. and 0.60 " 0.23 Ž2.0 h after administration.; n s 5; superscripts a and b indicate P - 0.05 when compared with the LPS- and vehicle-injected groups at the corresponding time pointsx. Anti-IL-1b Ab, IND or LNA alone did not have any effect on rectal temperature ŽTable 1.. To investigate intracellular signal transductions involved in the LPS-induced fever, a PKC inhibitor, chelerythrine ŽCHE, 3 mg., the TK inhibitors, GEN Ž3.4 mg. and TYR 46 Ž3 mg., and a tyrosine phosphatase inhibitor, orthovanadate ŽVAN, 100 mg., were pre-injected into the ventricle. As shown in Fig. 2, GEN completely blocked the LPS-induced fever. The inhibition of the fever by GEN
was dose-dependent Ž8C, no GEN: 0.82 " 0.18, n s 4; 0.1 mg of GEN: 0.95, n s 2; 0.2 mg of GEN: 0.84 " 0.22, n s 3; 0.3 mg of GEN: 0.29 " 0.35, n s 3 and 0.5 mg of GEN: y0.04 " 0.21, n s 5 at 3 h after LPS administration.. A negative control agent of GEN, daidzein ŽDAI, 2 mg., did not influence the LPS-induced fever Žthe change in rectal temperature induced by LPS with DAI 3 h after administration was 0.858C; n s 2.. Another TK inhibitor, TYR 46 Ž3 mg., also completely blocked the fever Ž0.22 " 0.128C 3 h after administration; n s 6.. CHE did not affect the LPS-induced fever ŽFig. 2.. Because these results suggested the involvement of TK in the LPS-induced fever, the effect of LPS on rectal temperature in the presence of VAN was examined. A small amount of LPS Ž0.3 mg, i.c.v.. or VAN individually showed no effect on basal rectal temperature ŽFig. 3 and Table 1.. However, i.c.v. injections of 0.3 mg of LPS after pretreatment with VAN produced a small increase in the rectal temperature ŽFig. 3.. 3.2. IL-1b-induced effects on rectal temperature I.c.v. injections of 50 ng of IL-1b elevated the rectal temperature from 0.75 to 4.75 h after administration ŽFig. 4.. The fever induced by the first and second injections was not statistically different Ž1.25 " 0.138C and 1.09 " 0.118C at 3 h after administration, n s 3.. The increasing curve and the maximum response of the IL-1b-induced fever resembled those of the LPS-induced fever, but the duration was shorter than for the LPS-induced effect. Pre-injections of 2 mg of anti-IL-1b Ab into the ventricle did not influence the IL-1b-induced fever Ž n s 3; data not shown.. A higher dose of 20 mg of anti-IL-1b Ab attenuated the IL-1b-induced effects at all time points, although the changes in rectal temperature at 1.25 Ž0.56 " 0.198CU ., 1.5 Ž0.66 " 0.168CU . and 1.75 h Ž0.52 "
Fig. 4. Change in rectal temperature after i.c.v. injection of IL-1b Ž50 ng. and TNF-alpha Ž50 ng.. The symbols and bars indicate the means" S.E.M. of nine Žclosed circles: vehicle., six Žopen circles: IL-1b . or eight Žopen triangles: TNF-alpha. experiments. U P - 0.05 vs. the vehicle-injected group at the corresponding time points ŽOne-way ANOVA followed by Fisher’s PLSD test..
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0.168CU ; n s 5; U P - 0.05 when compared with both the vehicle- and IL-1b-injected groups at the corresponding time points. after administration showed slight increases even after pretreatment. Also, pretreatment with IND partially attenuated the IL-1b-induced effect and the changes in rectal temperature at 1.0 Ž0.61 " 0.238C. and 1.25 h Ž0.63 " 0.228C; n s 5. after administration were significantly different, compared with the vehicle- and IL-1b-injected groups Ž P - 0.05.. On the other hand, LNA did not attenuate the IL-1b-induced effect, and even tended to potentiate it. Neither CHE Ž3.0 mg. nor GEN Ž3.4 mg. influenced the IL-1b-induced fever. The effects of the various antagonists on the IL-1b-induced fever 3 h after the IL-1b administration are summarized in Fig. 5.
systems after LPS administration w5,32x. I.c.v. injections of 50 ng of TNF-alpha ŽFig. 4. or of 500 ng Ž n s 3; data not shown. did not show any significant effect on rectal temperature. To examine the interaction between IL-1b and TNF-alpha on the regulation of rectal temperature, these cytokines were simultaneously injected into the ventricle. However, the increase in rectal temperature after simultaneous injections of IL-1b and TNF-alpha of 50 ng each was not significantly different from that after injection of IL-1b alone Ž n s 3; data not shown..
3.3. C2-ceramide-induced effects on rectal temperature
The pharmacological characteristics of the LPS-induced fever in this study are consistent with those that have already been reported, such as the time course of the febrile response, as well as the inhibition of the fever by NO synthase inhibitors, COX inhibitors and anti-IL-1b Ab w8,13,20,30,41x. Also, the involvements of NO, PGs and IL-1b in the LPS-induced effect are supported by the following findings: Ž1. increases in the mRNAs, proteins or activities of NO synthase and COX w3,4,18,28x, Ž2. histochemical studies showing increased numbers of cells positive to anti-NO synthase Ab or anti-COX Ab in the central nervous system w8,16x, Ž3. increased production of NO, PGs or IL-1b after LPS administration w4,5,8,12, 16,29,42,43x and Ž4. disappearance of the LPS-induced fever and IL-1b-induced fever in PG EP3 receptor knockout mice w40x. Moreover, there is a report demonstrating a resistant component in the LPS-induced fever to a COX inhibitor, and the authors conclude that at least two mechanisms are involved in it w9x. Also, the LPS-induced fever in this study had the component resistant to IND. LNA blocked only the LPS-induced fever, suggesting that NO synthesis is necessary in the process before IL-1b production in the LPS-induced fever and that the IL-1b-induced fever does not involve NO production. In vitro experiments demonstrate IL-1b-induced increases in NO synthesis w6,29x or blockade of NO synthase inhibitors on IL-1b-induced PGE 2 production w25x. On the other hand, there is a report that IL-1b-induced fever is blocked by a NO synthase inhibitor in rabbits w19x. The reasons for this discrepancy are not clear, but may reflect the different animal species involved. Klir et al. w14x demonstrate that the relation between dose and effect induced by central injections of TNF-alpha is a polynomial and that i.p., but not i.c.v., injections of TNF-alpha are antipyrogenic. In general, both pyrogenic and cryogenic effects of TNF-alpha are known. The effects of TNF-alpha on body temperature are variable, probably due to interactions with other substances. In this study, i.c.v. injections of TNF-alpha elicited no significant effect on the basal rectal temperature nor on the IL-1b-induced fever. Although LPS raises the concentration of TNF-alpha
The results with GEN and CHE suggested that the IL-1b-induced fever did not involve either TK or PKC. Recently, IL-1b was reported to activate sphingomyelinase and to increase ceramide production in various cultured cells, which enhances PGE 2 synthesis mediated through activation of COX w1,2,6x. Therefore, the effect of C 2ceramide on rectal temperature was examined. I.c.v. injections of 7.7 mg of C 2-ceramide did not increase rectal temperature at all w8C; 0.28 " 0.07 Ž1 h., 0.22 " 0.08 Ž2 h., y0.06 " 0.04 Ž3 h., y0.44 " 0.22 Ž4 h., y0.32 " 0.32 Ž5 h. and y0.32 " 0.31 Ž6 h after administration.; n s 3x. 3.4. TNF-alpha-induced effects on rectal temperature TNF-alpha has been reported to be a cytokine whose production increases in both peripheral and central nervous
Fig. 5. Effects of various antagonists on the IL-1b Ž50 ng.-induced fever. Each of the antagonists Žanti-IL-1b Ab: 20 mg; IND: 15 mg; LNA: 6 mg; CHE: 3 mg; GEN: 3.4 mg. was injected into the lateral ventricle 45 min before IL-1b administration. The columns and bars indicate the means" S.E.M. of changes in rectal temperature 3 h after IL-1b administration. N in the figure shows the number of experiments. Superscripts a and b refer to P - 0.05 vs. the vehicle- and the IL-1b-injected groups, respectively ŽOne-way ANOVA followed by Fisher’s PLSD test..
4. Discussion
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along with other cytokines — IL-1b, IL-6 and so on — in the plasma or the central nervous system w5,12,32x, TNFalpha in the central nervous system did not play any role in the LPS-induced fever or IL-1b-induced fever under our experimental conditions. The findings that the TK inhibitors, GEN and TYR 46, attenuated the LPS-induced fever, but did not attenuate the fever induced by IL-1b suggest that TK is involved in the mechanisms of IL-1b production by LPS. This is the first evidence that the activation of TK in the central nervous system is a mechanism directly related to the LPS-induced fever, although activation of TK is observed in cultured cells stimulated by LPS w4,8,16,34,35x. It is unclear which kind of TK is involved and where TK is activated by LPS in the central nervous system. CD14 is recognized as a binding site for LPS with LPS-binding protein on blood cells w44x. Recently, it was demonstrated that cultured brain glial cells express CD14 protein w11x. Moreover, glial cells produce various cytokines in the central nervous system w33x and express IL-1b mRNA after LPS administration w8,43x. Taken together, these findings suggest that TK in glial cells is responsible for LPS-induced fever. Interestingly, human monocytes have two mechanisms for production of IL-1b induced by LPS: one is mediated through activation of TK after LPS binds to CD14, while the other is mediated through activation of PKC, but not TK or CD14 w35x. LPS stimulates CD14-associated TK after binding to CD14 w37x. Participation of TK in the LPS-induced fever is supported by the synergetic effect of VAN and LPS shown in this study. PKC is not involved in the febrile response induced by LPS or IL-1b, because CHE did not influence the LPSand IL-1b-induced effects. The dose of CHE used in this study is appropriate for inhibition of PKC w31x. In cultured blood cells, PKC is activated after LPS stimulation w4,35x. This mechanism was not found in the febrile response observed in this study. Many intracellular signal transductions are activated after IL-1b binds to IL-1 receptors w2,6x. It is unclear which of these function in the febrile response. It is obvious that the IL-1b-induced fever is not mediated through TK or PKC, because neither GEN nor CHE changed the IL-1b-induced fever in this study. Ceramide, which was recently shown to be a possible second messenger for IL-1 receptors w2,6x, increases PG production resulting from the activation of COX w1x. However, this did not raise the rectal temperature. Therefore, the signal transduction for the IL-1b-induced fever still remains unclear. In conclusion, Ž1. LPS increases IL-1b production after stimulation of TK and NO synthesis, and then promotes PG synthesis resulting in fever; Ž2. IL-1b raises the rectal temperature due to increases in synthesis of PGs mediated by unknown mechanisms, excepting TK, PKC, NO production or sphingomyelinase; Ž3. C 2-ceramide does not increase the rectal temperature, and Ž4. TNF-alpha does not play any role in regulating the rectal temperature. The
activation of TK in the central nervous system is probably a trigger for the febrile response induced by LPS.
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