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Research Report

CCR1 and CCR5 chemokine receptors are involved in feverinduced by LPS (E. coli ) and RANTES in rats

Renes R. Machadoa, Denis M. Soaresa, Amanda E. Proudfootb, Glória E.P. Souzaa,⁎aLaboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Universidade de São Paulo, Ribeirão Preto, SP, BrazilbSerono Pharmaceutical Research Institute, Geneva, Switzerland

A R T I C L E I N F O

⁎ Corresponding author. Laboratório de FarmaAv. do Café, s/n-Campus USP, 14.040-903, Rib

E-mail address: gepsouza@fcfrp.usp.br (G.Abbreviations: AH/POA, preoptic area of th

nervous system; CCR, C–C chemokine recintravenous; LPS, lipopolysaccharide; MCP-1nuclear factor-κB; OVLT, Organum vasculosuregulated on activation, normal T cells expre

0006-8993/$ – see front matter © 2007 Publisdoi:10.1016/j.brainres.2007.05.054

A B S T R A C T

Article history:Accepted 8 May 2007Available online 9 June 2007

This study, besides examining the involvement of CCR1 and CCR5 receptors in the LPS-induced fever (lipopolysaccharide, Escherichia coli) in male Wistar rats, evaluated if RANTES(regulated on activation, normal T cells expressed and secreted) injected into the preopticarea of the anterior hypothalamus (AH/POA) would promote an integrated febrile responsevia these receptors. Moreover, the effects of selective and non-selective cyclooxygenaseblockers on both fever and the level of prostaglandin (PG)E2 in the cerebrospinal fluid (CSF)after injection of RANTES into the AH/POA were also investigated. Met-RANTES, CCR1 andCCR5 receptor antagonist, reduced LPS-evoked fever dose dependently. RANTESmicroinjected into the AH/POA increased the rectal temperature of rats dose dependentlyand caused a significant decrease in the tail skin temperature and an increase (at 2.5 and 5 h)of the levels of PGE2 in the CSF. Met-RANTES prevented the fever induced by RANTES.Ibuprofen abolished the fever caused by RANTES between 60 min and 2.5 h, and it reducedthe temperature until the end of observation period. Celecoxib blocked the RANTES-inducedfever, while indomethacin reduced it in the last 60 min of the experimental period. At 2.5and 5 h all antipyretics brought the CSF PGE2 level near to the control. These results indicatethat CCR1 and CCR5 receptors are involved in the fever induced by systemic LPS andintrahypothalamic RANTES. RANTES promotes an integrated febrile response accompaniedby an increase of CSF PGE2. The inhibitory effects of celecoxib and ibuprofen suggest thatPGE2 was generated via COX-2. As indomethacin dissociates fever and the decrease of PGE2level during the RANTES-induced fever, an alternative COX-2-independent pathway orother mechanisms of action of celecoxib and ibuprofen might be considered.

© 2007 Published by Elsevier B.V.

Keywords:RANTES/CCL5FeverLipopolysaccharideHypothalamusProstaglandin E2Cyclooxygenase inhibitor

cologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-Universidade de São Paulo,eirão Preto, SP, Brazil. Fax: +55 16 3602 4880.E.P. Souza).e anterior hypothalamus; BMAC, B cell- and monocyte-activating chemokine; CNS, centraleptor; ET, endothelin; i.p., intraperitoneal; IP-10, interferon inducible protein-10; i.v.,, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; NF-κB,m laminae terminalis; PFPF, pre-formed pyrogenic factor; PGE2, prostaglandin E2; RANTES,ssed and secreted

hed by Elsevier B.V.

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1. Introduction

A large body of evidence shows that fever induced by bacteriallipopolysaccharide (LPS) is mediated by a number of endo-genous pyrogens (PEs) produced either centrally or periph-erically. Among these pyrogens, there are interleukins IL-1αand β (Dinarello, 1984, 1994), IL-6 (Helle et al., 1988; Souza et al.,2002; Harre et al., 2002), tumor necrosis factor (TNF) (Dinarelloet al., 1986), pre-formed pyrogenic factor (PFPF) (Zampronio etal., 2000; Fabricio et al., 2006), corticotrophin-releasing factor(CRF) (Zampronio et al., 2000; Souza et al., 2002), endothelins(ETs) (Fabricio et al., 2005a; 2006), and prostaglandins (PGs)(Morimoto et al., 1992; Souza et al., 2002; Rummel et al., 2005).PEs can act either direct or indirectly on thermoregulatoryneurons in the preoptic area of the hypothalamus (POA) byaltering the activity of these neurons (Roth et al., 2006).

Alongside these established fever-inducer mediators, thechemokines have come into view as a pyrogenic mediator foryears (Davatelis et al., 1989; Miñano et al., 1990; Rothwell et al.,1990; Zampronio et al., 1994, 1995; Tavares and Miñano, 2000;Melo Soares et al., 2006). Chemokines are chemotacticcytokines that participate in immune and inflammatoryresponses by promoting leukocyte activation and migration(Luster, 1998; Baggiolini, 1998). On the basis of the relativeposition of their amino terminal cysteine residues (Rollins,1997; Zlotnik and Yoshie, 2000), chemokines are divided intofour subfamilies (CXC, CC, C and CX3C).

RANTES/CCL5, a member of the CC family of chemokines(8–10 kDa) promotes the recruitment and activation ofinflammatory cells such as lymphocytes (Schall et al., 1990),monocytes (Meurer et al., 1993), eosinophils (Dinarello, 1994),natural killer cells and basophils (Nelson and Krensky, 1998),by acting on CCR1, CCR3 and CCR5 receptors (Wells et al.,1998). These receptors belong to a family of seven-transmem-brane G-protein-coupled receptors leading to the activation ofintracellular signaling pathways (Thelen, 2001). Chemokinereceptors have been detected in the CNS on several cellsubsets, including, microglia, astrocytes and neurons in thehypothalamus during disease and after endotoxin adminis-tration (Boddeke et al., 1999; Dorf et al., 2000; Simpson et al.,2000).

Previous in vitro studies (Jang et al., 2002; Kremlev et al.,2004) demonstrated that endotoxin (LPS from Gram-negativebacteria) induces, via activation of the nuclear factor-kB (NF-κB) transcription, the expression of RANTES and macrophageinflammatory protein (MIP)-1α in microglial cells. Further-more, treatment of astrocytes with TNF-α or LPS inducesmRNA expression of RANTES, monocyte chemoattractantprotein (MCP)-1 and MIP-1β (Guo et al., 1998). In vivo, LPSinduces MIP-1α and MIP-1β mRNAs in rat brain (Gourmala etal., 1999). In addition, immunoreactivity to MIP-1β wasidentified in the OVLT and AH/POA of rats (Miñano et al.,1996). Other kinds of stimuli, such as West Nile virus, wereshown to increase mRNAs for RANTES, MIP-1α, MIP-1β, IP-10(interferon inducible protein 10 kDa) and BMAC (B cell- andmonocyte-activating chemokine) in mice brain homogenates(Shirato et al., 2004).

RANTES also promotes fever sensitive to steroidal and non-steroidal antipyretic drugs (Tavares and Miñano, 2000, 2002),

suggesting the involvement of prostaglandins in its pyreticeffect. Furthermore, the CCR5 receptor seems to be respon-sible for fever in response to RANTES, since a specific antibodyagainst this receptor blocked fever induced by the chemokine(Tavares and Miñano, 2004).

The addition of a single methionine on the amino terminalof RANTES resulted in the production of Met-RANTES, a potentantagonist of CCR1 and CCR5 receptors (Proudfoot et al.,1999a), which is effective in reducing inflammation in rodentmodels of renal inflammation (Grone et al., 1999), reducingcollagen-induced arthritis (Plater-Zyberk et al., 1997) andreducing significantly the colonic damage and bacterialtranslocation in experimental colitis (Kucuk et al., 2006).

In view of these considerations, the present study inves-tigated the involvement of CCR1 and CCR5 chemokinereceptors in the febrile response induced by LPS by treatingrats with Met-RANTES. It was also investigated whetherRANTES injected into the anterior hypothalamus preopticarea (AH/POA) would promote an integrated febrile responseand the contribution of CCR1 and CCR5 receptors to thisresponse. The effects of selective and non-selective cycloox-ygenase blockers both on fever and on the level of prosta-glandin (PG)E2 in the cerebrospinal fluid (CSF) after injection ofRANTES into the AH/POA were also investigated.

2. Results

2.1. Effect of intravenous injection of Met-RANTES onfever induced by LPS in rats

Fig. 1 shows that intravenous injectionof LPS (5μgkg−1) promoteda rapid raise on fever, peaking 2 h after administration.Intravenous administration of the dose of 5 μg kg−1 of Met-RANTES promoted no changes (Fig. 1A); the dose of 25 μg kg−1 ofthis antagonist reduced the fever response for a short period(from 75 to 150 min, F=35.43, P<0.01; Fig. 1B) while the dose of100μgkg−1markedly reduced the fever inducedby5μgkg−1 of LPS(F=1527,P<0.01; Fig. 1C). Therefore, this dose ofMet-RANTESwasselected for further experiments. The i.v. injection of 100 μg kg−1

Met-RANTES plus sterile saline, 0.2 ml, did not affect the bodytemperature of the animals (Fig. 1C).

2.2. Effect of RANTES microinjection into the AH/POA,on core and tail skin temperature of rats. Influence ofMet-RANTES on fever induced by RANTES

Themicroinjection directly into the AH/POA of 1, 5, 25 or 50 pg/500 nl of RANTES increased the body temperature of animalsin a dose-dependent way. One or five pg of RANTES increasedbody temperature, peaking at 1 or 2 h, respectively (F=116.3,P<0.05 and F=195.0, P<0.01, respectively; Fig. 2A); 25 and 50 pgof RANTES promoted a substantial and similar increase inbody temperature, peaking 2.5 h after its administration(F=859.9, P<0.001 and F=340.0, P<0.05, respectively; Fig. 2A).A gradual reduction of rectal temperature after the peak wasobserved during the 6-h experimental period in all dosesstudied. Since 25 or 50 pg doses of RANTES were equallyeffective in increasing body temperature, the former wasselected to be used in further experiments. The fever induced

Fig. 1 – Effect of Met-RANTES on fever induced by LPS. RatsreceivedMet-RANTESendovenously, 5μg kg−1 (A), 25μg kg−1

(B) and 100 μg kg−1 (C) or sterile saline, 15 min prior to LPS(5 μg kg−1, i.v.) or sterile saline (1 ml kg−1, i.v.). Bodytemperature was measured by radio-telemetry. Controlanimals received Met-RANTES 100 μg kg−1 or sterile saline.Values represent means±SEM of the variation in bodytemperature (ΔT, °C) observed in 6 animals of each group.Basal temperatures (mean±SEM; °C)were as follows: panel A:♦, 37.1±0.12; panel B: E, 37.0±0.10; panel C:●, 37.0±0.07; ▪,37.1±0.06;○, 37.2±0.06. **P<0.01, +P<0.001 when comparedwith the saline plus LPS group.

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by this dose of RANTES was significantly reduced by aprevious intravenous injection of 100 μg kg−1 of Met-RANTES(F=304.0, P<0.01; Fig. 2B). Representative photomicrograph ofcoronal sections illustrating the site of RANTESmicroinjection

into the POA and the region where the POA is located in thebrain is showed in the Fig. 2C (left and right, respectively). Theincrease in rectal temperature produced by the injection of25 pg of RANTES into the AH/POA (F=564.8, P<0.001; Fig. 3A)was accompanied by a significant reduction in the tail skintemperature (F=94.69, P<0.05; Fig. 3B). This result indicates alocal (tail) vasoconstriction, and it can be clearly showed bythe reduction in heat loss index (HLI) values (saline: 0.50±0.02;RANTES: 0.33±0.05; 1 h; F=65.85, P<0.05; Fig. 3C).

Sterile saline (500 nl) injected into AH/POA or Met-RANTESplus sterile saline, given i.v. did not alter the rectal and tailskin temperature or HLI above baseline values for up to 6 h.

2.3. Effect of non-selective and selective cyclooxygenaseinhibitors on fever induced by RANTES

Ibuprofen (10 mg kg−1 i.p.), indomethacin (2.0 mg kg−1, i.p.),and celecoxib (5 mg kg−1, p.o.) given 30 min before thebeginning of the experiment altered fever induced by RANTES(25 pg, AH/POA). Ibuprofen abolished the fever between 60minand 2.5 h after administration, in addition, it reduced thetemperature until the end of observation period (F=372.6,P<0.05; Fig. 4A). Indomethacin only reduced the feverresponse to RANTES significantly during the last 60 min(F=21.05, P<0.05; Fig. 4B); celecoxib abolished the feverresponse to the chemokine during the entire 6-h period ofobservation (F=1854, P<0.001; Fig. 4C). The vehicles (Tris–HCli.p. or sterile water p.o.) did not alter the pyrogenic effect ofRANTES and the antipyretic agents plus saline did not changethe basal temperatures of control animals.

2.4. Experiments on CSF PGs levels

Cisternal CSF of the animals was collected 2.5 and 5 h afterintrahypothalamic injection of 25 pg of RANTES or saline intoAH/POA. These time points were selected aiming to correlatethe antipyretic effect of the assayed drugs with the PGE2 levelin the CSF, since, as shown above, each drug showed differentprofile of fever reduction.

After intrahypothalamic injection of saline in the animalgroup receiving Tris i.p. the CSF levels of PGE2 at 2.5 and 5 hwere 432.5±165.9 pg ml-1 and 513.7±194.4 pg ml-1, respec-tively (Figs. 5A, B). RANTES induced 11- and 4.5-fold increase inPGE2 level in the CSF at 2.5 and 5 h, respectively, compared tocontrol animals (Figs. 5A, B). Indomethacin (2 mg kg−1, i.p.),ibuprofen (10mg kg−1, i.p.) and celecoxib (5mg kg−1, p.o.) givenbefore RANTES administration into the AH/POA brought theCSF PGE2 level near to control values at both time pointsstudied (Figs. 5A and B).

3. Discussion

The present study shows, for the first time, that Met-RANTES,a CCR1 and CCR5 receptor antagonist, reduced the febrileresponse induced by LPS in rats, suggesting a contribution ofthese receptors to this response. Furthermore, it shows thatthe injection of RANTES into the AH/POA of rats increases theCSF PGE2 level and promotes an integrated febrile responsesensitive to Met-RANTES, ibuprofen, celecoxib and in a feeble

Fig. 2 – Increase in rectal temperature induced by intrahypothalamic injection of RANTES and the effect of intravenoustreatment with Met-RANTES. RANTES (1, 5, 25 or 50 pg) or sterile saline was microinjected into AH/POA (A). Rats receivedMet-RANTES (100μg kg−1, i.v.) 15min before RANTES (25 pg, AH/POA) or sterile saline (B). The rectal temperaturewas evaluatedby telethermometry. Values represent themeans±SEM of variation in rectal temperature (ΔT, °C) observed in 7 animals in eachgroup. Basal temperatures (mean±SEM; °C) were the following: panel A:○, 36.9±0.04;E, 36.9±0.01; Δ, 36.9±0.02; ♦, 36.8±0.04;▪, 37.0±0.05; panel B:○, 37.0±0.03;●, 36.9±0.03; ▪, 37.0±0.02. *P<0.05, **P<0.01, +P<0.001when compared to control saline inpanel A; **P<0.01, +P<0.001 when compared to saline-treated group plus RANTES in panel B. Representative photomicrograph(left panel) and schematic representation (right panel) of coronal sections illustrating the site of microinjection into the POA (C).Abbreviations: 3V, third ventricle; LV, lateral ventricle; PaMC, paraventricular magnocellular nucleus; AH/POA, preoptic area ofthe anterior hypothalamus; so, supraoptic nucleus.

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Fig. 3 – Effect of intrahypothalamic injection of RANTES oncore and tail skin temperature of the rat. RANTES (25 pg) orsterile saline was microinjected into AH/POA in a volume of500 nl. Values shown represent means ± SEM of the variationin degree Celsius of rectal temperature (ΔTr) (A), tail skintemperature (ΔTtail) (B) and the heat loss index (HLI) (C)observed in 6 animals of each group. Rectal and tail skintemperatures were evaluated by telethermometry. Basaltemperatures (mean ± SEM, °C) were as follows: panel A: ○,37.0±0.07;●, 36.9±0.03; panel B:○, 32.8±0.23;●, 32.8±0.21.*P<0.05, **P<0.01, +P<0.001 when compared to the saline-treated control group.

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degree to indomethacin. The effect of all antipyretic drugs wasaccompanied by the inhibition in RANTES-stimulated PGE2levels in the CSF.

Tavares and Miñano (2000) showed for the first time thatRANTES, injected directly into the AH/POA, dose dependentlyincreased the rectal temperature of rats. Nonetheless, itremained to be determined whether the pyretic action ofRANTES would constitute an integrated fever response. Thecurrent study clears this issue by demonstrating that theincrease in core body temperature induced by RANTESinjected into the AH/POA was accompanied by significantreduction of heat loss index (HLI; Romanovsky et al., 2002).The reduction of heat loss index was reflected in the decreasein tail skin temperature, indicating vasoconstriction of thelocal (tail) vascular bed aimed to retain heat, which is acharacteristic feature of the febrile response (O'Leary et al.,1985). The rat's tail serves as a variable heat exchanger(O'Leary et al., 1985; Gordon et al., 2002), since its blood flowis regulated by the activity of sympathetic vasoconstrictornerves, specifically controlled by neurons of the raphé mag-nus/pallidum nucleus (Blessing and Nalivaiko, 2001). Thus, itseems that by stimulating vasoconstriction of cutaneousvessels, centrally administered RANTES evokes a concertedthermoregulatory response in which the reduced efficacy ofthe tail skin to dissipate body heat collaborates with thefever's progress.

In the present study it is also shown that Met-RANTES, aCCR1 and CCR5 receptor antagonist, dose dependentlyreduced fever induced by LPS and, at a dose that producesthe maximal inhibitory effect (100 μg kg−1), it also reduced thefever induced by RANTES. These findings clearly indicate thatthese receptors are involved in the fever induced by bothstimuli. Chemokines such as MIP-1α, RANTES and MCP-3 acton CCR1 receptor and MIP-1β, MIP-1α and RANTES act onCCR5 receptor (Proudfoot et al., 1999b). However, thesechemokines also bind to other receptors to exert theirbiological effects. For instance, MIP-1β binds to CCR9/10;MIP-1α to CCR4; RANTES to CCR3 and MCP-3 to CCR2/3.Except MCP-3, these chemokines are known as endogenouspyrogens (Tavares and Miñano, 2000, 2002; Melo Soares et al.,2006) and, as mentioned in the Introduction section, LPSinduces MIP-1α and MIP-1β mRNAs in rat brain (Gourmala etal., 1999). Moreover, immunoreactivity to MIP-1β was identi-fied in two important areas related to fever, the OVLT and theAH/POA of rats (Miñano et al., 1996). So, it is possible that theinhibitory effect of Met-RANTES on fever induced by LPS mayresult from the blockage of CCR1 and CCR5 receptors,impeding the chemokines to exert their fully pyrogeniceffects. However, in the fever induced by RANTES, whichbinds to CCR1, CCR3 and CCR5 receptors, the inhibitory effectof Met-RANTES observed here may result from the inhibitionof CCR1 and CCR5 receptors. This suggestion is corroboratedby previous results from Tavares and Miñano (2004) that haveshown the involvement of the CCR5 receptor in the feverinduced by intrahypothalamically injected RANTES (but notby MIP-1β), using a specific antibody against CCR5 injectedinto the same site.

Moreover, not only does the LPS seem to induce mRNA tochemokines in the brain. It has been shown that viral infectionby West Nile virus increases mRNA for RANTES, MIP-1α, MIP-1β, IP-10 and BMAC in mice brain homogenates (Shirato et al.,2004). Taken together with our present results, these findingsillustrate the ability of brain structures to synthesize chemo-

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kines, including RANTES, which could importantly contributeto febrile responses to exogenous pyrogens.

COX-2, the induced isoform of cyclooxygenase, seems to bethe enzyme responsible for the biosynthesis of prostanoidsduring inflammatory and febrile responses (Bakhle andBotting, 1996; Cao et al., 1995, 1996; Lugarini et al., 2002).Furthermore, induction of COX-2 mRNA expression occurs inthe inner surface of brain blood vessels, including the preopticarea and its vicinity during the time course of LPS-inducedfever in rats (Cao et al., 1995) and it mainly occurs in thesubfornical organ of guinea pig after intra-arterial or intraper-itoneal injection of LPS (Rummel et al., 2005). However, itseems that constitutive COX-2 gene expressionmay also occur

in various brain areas including the hypothalamus (Breder etal., 1995). These pieces of evidence led us to investigate theeffect of selective (celecoxib) and non-selective COX inhibitors(indomethacin, ibuprofen) on fever and on the increase of CSFPGE2 level induced by intrahypothalamic administration ofRANTES.

Indomethacin (2 mg kg−1) reduced the increase in the CSFPGE2 levels as early as 2.5 h after RANTES administration but,in contrast, indomethacin only significantly reduced the feverin the last 60min of the experimental period when the level ofPGE2 was the same as the level found at 2.5 h. A delayed effectof indomethacin was also observed in the fever response toLPS (Fabricio et al., 2005b). This response is difficult to explain;but it is believed that it is not due to a low amount ofindomethacin reaching the brain, because indomethacinreduced the early phase of fever induced by several cytokines,such as TNF-α, IL-1β and IL-6 (Souza et al., 2002). In our hands,indomethacin reduced the PGE2 concentration in the CSF afteri.c.v. injection of MIP-1α (Melo Soares et al., 2006), PFPF (pre-formed pyrogenic factor from LPS stimulated macrophages)(Veiga-Souza et al., unpublished results) or ET-1 (Fabricio et al.,2005b) without changing the fever, which therefore suggeststhat prostaglandins are not essential for the fever develop-ment for these agents.

The effectiveness of ibuprofen in reducing fever to RANTESwas firstly observed by Tavares and Miñano (2000). In thepresent study, ibuprofen (10 mg kg−1) abolished the feverresponse induced by RANTES from 1 to 2.5 h and significantlyreduced the remaining fever. At 2.5 and 5 h after RANTESinjection, ibuprofen reduced PGE2 concentration in the CSFalmost 10- and 5-fold, respectively. Our data corroborate withprevious find from Taniguchi et al. (1997) who showed thatibuprofen reduced the fever and hypothalamic PGE2 concen-trations in yeast-challenged rats, suggesting the effectivenessof ibuprofen in reducing fever dependent on PGE2. However,other mechanisms of action could be implicated in theantipyretic effect of ibuprofen (see below).

Celecoxib (5 mg kg−1) abolished the fever induced byRANTES and it also reduced, to the same levels of ibuprofen,the PGE2 concentration in the CSF. Since celecoxib was givenbefore RANTES injection into the AH/POA, we cannot distin-guish whether celecoxib (or even ibuprofen or indomethacin)

Fig. 4 – Effect of ibuprofen (IBU), indomethacin (INDO) orcelecoxib (CELEC) on fever induced by intrahypothalamicinjection of RANTES in rats. Ibuprofen (10 mg kg−1, i.p.)(A); indomethacin (2 mg kg−1, i.p.) (B); and celecoxib(5 mg kg−1, p.o.) (C); Tris–HCl (pH 8.2) or sterile water wereadministered 30 min prior to RANTES (25 pg) or sterile salineinjected into AH/POA. Rectal temperature was evaluated bytelethermometry. Control animals received Tris (A, B) orsterile water plus RANTES (C). Values represent means±SEMof the variation in rectal temperature (ΔT, °C) observed in 6animals of each group. Basal temperatures (mean±SEM, °C)were as follows: panels A and B: ○, 37.0±0.01; ●, 37.0±0.07;

▪, 37.0±0.03; E, 36.9±0.04; □, 36.9±0.04; panel C: ○,37.1±0.04; ●, 36.9±0.07; ♦, 37.0±0.08. *P<0.05, **P<0.01,+P<0.001 when compared to the respective vehicle treatedgroup plus RANTES.

Fig. 5 – Effect of IBU, INDO or CELEC on changes in CSF PGE2levels induced by RANTES in rats. Ibuprofen (10mg kg−1, i.p.),indomethacin (2 mg kg−1, i.p.), and celecoxib (5 mg kg−1, p.o.)or Tris–HCl (pH 8.2) was administered 30min prior to RANTES(25 pg) or sterile saline injected into AH/POA, as previouslydescribed. Control animals received Tris–HCl and sterilesaline. The CSF was harvested from cisterna magna of therats, 2.5 h (A) and 5 h (B) after AH/POA injection of RANTES.PGE2 levels were determined by ELISA. Values representmeans ± SEM of the concentration (pgml−1) of PGE2 in the CSFof 3–5 animals. **P<0.01, +P<0.001 vs. Tris/RANTES group.

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is acting on the constitutive or inducible isoform of COX-2. Atthe same dose used in the present study, celecoxib inhibitedthe fever and the increase in PGE2 concentration in the CSFafter i.c.v. injection of MIP-1α and ET-1 in rats; however, at adose of 1 mg kg−1, celecoxib does not change the fever but itstrongly reduced the PGE2 concentration in the CSF inresponse to i.c.v. ET-1 (Melo Soares et al., 2006; Fabricio et al.,2005b). Taken together, these findings and also the poorinhibitory effect of indomethacin on fever induced by RANTES,in spite of the reduction of PGE2 in the CSF (the present andother study from our laboratory), suggest that COX-2-inde-pendent pathwaysmay also be involved in the fever caused bythis chemokine.

The different antipyretic profiles of COX inhibitors on feverinduced by RANTES could also reflect distinct effects of thesedrugs on PG-synthesizing enzymes, on PG transporters and on

PGcatabolismat fever-related sites (Ivanovet al., 2002, 2003); so,additionalmechanisms resulting in the decrease of the synthe-sis and/or release of pyrogenicmediatorsmay also be involved.

In this context, it is now suggested that several non-steroidal anti-inflammatory drugs (NSAIDs), among them,ibuprofen and celecoxib (Tegeder et al., 2001; Takada et al.,2004), are able to inhibit NF-κB activation, one of the earliestevents during cell defense activation in response to threaten-ing stimuli (Müller et al., 1993; Bharti and Aggarwal, 2002), sostarting the synthesis and/or release of pyrogenic mediators.More recently, Miyamoto et al. (2006) showed that celecoxibprevents experimental autoimmune encephalomyelitis (EAE)in mice by blocking the synthesis of monocytes chemoattrac-tant peptide-1 (MCP-1), P-selectin and ICAM-1 (intercellularadhesion molecule) and consequently the infiltration ofinflammatory cells into the central nervous system. Worthyof note in Miyamoto's study is that the inhibitory effect ofcelecoxib on EAE was also observed in COX-2-deficient micereinforcing its COX-2-independent mechanism. Concerningindomethacin, its inhibitory effect on NF-κB activation iscontroversial (Tegeder et al., 2001; Takada et al., 2004). It istherefore plausible that the inhibition of the synthesis of othermediators involved in the fever promoted by RANTES is also atarget for the antipyretic effect of ibuprofen and celecoxib.

These results indicate that CCR1 and CCR5 receptors areinvolved in the fever induced by systemic LPS and intrahy-pothalamic RANTES. RANTES promotes an integrated febrileresponse accompanied by an increase of CSF PGE2 level. Theinhibitory effects of celecoxib and ibuprofen suggest that PGE2was generated via COX-2. As indomethacin dissociates feverand the decrease of CSF PGE2 level during the RANTES-inducedfever, an alternative COX-2-independent pathway, as sug-gested to occur in the fever to LPS (Fabricio et al., 2006), orothermechanisms of action of celecoxib and ibuprofen shouldbe considered. It now appears pertinent to assess the relation-ship between RANTES and known endogenous pyrogensrecruited by LPS in order to establish the relative position ofthis chemokine in the neural chain mechanism of fever.

4. Experimental procedure

4.1. Animals

Experiments were conducted using male 180–200 g, Wistarrats, individually housed at 24±1 °C under a 12:12-h light–darkcycle (lights on at 06:00 AM), with free access to chow and tapwater until the day of the experiment proper, when only waterwasmade available to them. Each animal was used only once.All experiments were previously approved by the institution'sethical committee for research on laboratory animals of theUniversity of São Paulo and were performed in accordancewith Brazilian legislation and the Guide for the Care and Use ofLaboratory Animals of the Institute for Laboratory Animal Research(1996).

4.2. Implantation of the intrahypothalamic cannula

Sodium pentobarbital (40 mg kg−1, i.p.)-anesthetized rats(Souza et al., 2002; Fabricio et al., 2005a) were stereotaxically

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unilaterally implanted with a permanent 24-gauge stainlesssteel guide cannula (0.55-mm outer diameter, 15 mm long),into the anterior hypothalamic preoptic area (AH/POA). Itsstereotaxic coordinates were: 0.6 mm lateral to the midline,7.7 mm anterior to the interaural line and 6.5 mm under thebrain surfaceand the incisor barwas lowered3.0mmbelowthehorizontal zero, according to Paxinos and Watson (1997).Cannulae were fixed to the skull with jeweller's screwsembedded in dental acrylic cement. All procedures wereconducted under aseptic conditions. Animals were treatedwith oxytetracycline hydrochloride (400 mg kg−1, i.m.) andallowed to recover for 1 week prior to experimental use (Souzaet al., 2002; Fabricio et al., 2005a). After each experiment, eachrat was microinjected into the POA (500 nl) with Evan's blue(2.5%). Immediately after dye microinjection, each rat wasgiven an overdose of pentobarbital, perfused transcardiallywith 0.9% saline, followed by 4%paraformaldehyde. Each brainwas removed, stored in the same fixative for 6 h, kept in 30%sucrose overnight, and cut at 40 μm on freezing microtome.From an analysis of the histological material under lightmicroscopy, the position of the cannulae and respective sitesof perfusion were subsequently verified and “mapped” anato-mically. Animals showing cannula misplacement or blockageupon injection, or abnormal gain patterns during the post-implantation period were excluded from the study.

4.3. Temperature measurements

Rectal temperature was measured in conscious and unrest-rained rats for 1 min every 30 min for up to 6 h, by gentlyinserting, 4 cm into the rectum, a vaseline-coated thermistorprobe (model 402 coupled to a model 46 telethermometer,Yellow Springs Instruments, Ohio, USA), without removingthe animals from their home cages.

Tail skin temperature was measured by a thermistor probe(model 402, coupled to a model 46 telethermometer, YellowSprings Instruments, Ohio, USA) attached with adhesive tapeto the lateral surface of the proximal segment of the tail (2 cmfrom its base).

Experimental measurements were conducted at the ther-moneutral zone for rats (Gordon, 1990), in a temperature-controlled room kept at 28±1 °C, following adaptation of theanimals to this environment for at least 1 h. After this period,baseline temperatures were determined 4 times at 30-minintervals prior to any injection; only animals displaying meanbasal rectal temperatures between 36.8 and 37.2 °C wereselected. To minimize core temperature changes due tohandling, animals were conditioned twice to this environmentand procedure on the preceding day.

Body temperature was also measured by using battery-operated biotelemetry transmitters (Data Science, St. Paul,MN, USA), implanted in the peritoneal cavity of each animal.Signals transmitters were delivered through a computer-linked receiver to the Dataquest data acquisition system(Dataquest A.R.T System). Preliminary experiments revealedthat LPS-induced fever recorded using the radio-telemetrysystemwas indistinguishable from that assessed by the rectalprobe method (Fabricio et al., 2005a).

The heat loss index (HLI), used to evaluate thermoeffectorresponses of tail skin vasculature, was calculated according to

the formula: HLI= (Tsk−Ta). (Tc−Ta)−1 where Tsk: tail skintemperature; Ta: ambient temperature; Tc: core body tempera-ture. The values of HLI will vary from 0 to 1.0, representingstates of maximum vasoconstriction to maximum vasodilata-tion, respectively (Romanovsky et al., 2002).

4.4. CSF sampling and determination of PG levels in theCSF

A single sample of CSF was collected from each animalaccording to the method described by Consiglio and Lucion(2000). Briefly, just prior to the CSF collection each rat wasanesthetized with pentobarbital sodium (40 mg kg−1, i.p.) andfixed to the stereotaxic apparatus, with its body flexed down-ward. The top of the head was trichotomized (to facilitate thevisualization of the area) and moistened with a cotton swabsoaked in ethanol to reveal a small depression between theoccipital protuberance and the atlas. A scalp (25-gauge)connected to a 1-ml syringe was then inserted vertically andcentrally through this depression into the cisterna magna and agentle aspirationmade the CSF flow through it resulting on 60-to 100-μl samples. Frequently, it is not necessary to aspiratewith the syringe, since the way the head is positioned exertsenough pressure to let the fluid flow spontaneously. Gentlemovements of the needle are necessary during collection inorder topreventbleeding. Then theCSF sampleswereplaced inEppendorf tubings containing 2 μl of indomethacin (2.5 μg μl-1),to stop PGs production. Samples were maintained in the darkunder ice until centrifugation at 1300×g for 15 min, and thenthey were immediately frozen to −20 °C until analysis. Whencontaminated with blood, samples were discarded.

PGE2wasmeasuredusingProstaglandinE2 ParameterAssayKit (R&D Systems, Minneapolis, MN, USA), which has a limit ofdetection of 10.1 pgml-1. Cross-reactivity datawere as follows:17.5%withPGE3, 11.9%withPGE1, 7%withPGF1α, 6%withPGF2α,2.5% with 6-Keto-PGF1α, less than 0.1% with all other prosta-noids tested. Intra- and inter-assay coefficients of variationwere <11%. All samples were assayed at optimal concentra-tions and according to the manufacture's instructions.

4.5. Drugs

The following drugs were used: human RANTES (CCL5) andMet-RANTES (CCR1 and CCR5 receptor antagonist), werepurified from E. coli inclusion bodies as previously described(Proudfoot et al., 1996; Proudfoot and Borlat, 2000); LPS (from E.coli 0111:B4), pentobarbital sodium and ibuprofen from Sigma(St. Louis, USA). Oxytetracycline hydrochloride (Terramicina®)was from Pfizer (São Paulo, Brazil); celecoxib (Celebra®) fromPharmacia (São Paulo, Brazil), and indomethacin was a giftfrom Merck, Sharp and Dohme (São Paulo, Brazil).

4.6. Experimental protocols

In a first set of experiments, the animals were treated witheither Met-RANTES, a CCR1 and CCR5 receptor antagonist, atdoses of 5, 25 and 100 μg kg−1, 0.2 ml, i.v., or sterile saline (1 mlkg−1), 15 min prior to intravenous injection of LPS (5 μg kg−1) orsterile saline (1 ml kg−1). In this set of experiments, bodytemperature was measured by radio-telemetry. LPS, Met-

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RANTES and saline were injected into lateral tail vein. For thisprocedure, the animalswere carefully immobilized and the tailintroduced in a recipient containing warm water (≅40 °C) topromote vasodilatation and facilitate the injection. After that,the tail was dried, sterilized with cotton soaked with alcoholand 0.2 ml of the solutions was injected by using 1-ml syringeand a stainless steel needle (26 gauge1/2).

The next series of experiments was aimed at the determi-nation of the dose of RANTES able to induce a febrile responseand the effect of Met-RANTES on it. To this effect, RANTES atdoses of 1, 5, 25 and 50 pg or sterile saline was microinjectedinto the AH/POA, and the animal's rectal temperature wasmonitored by telethermometry for up to 6 h. To verify ifRANTES caused an integrated febrile response, 25 pg of thechemokine or sterile salinewas given into the AH/POA and theanimal's rectal and tail skin temperatures monitored bytelethermometry at 30-min intervals, for the same period.Met-RANTES (100 μg kg−1, i.v.), or sterile saline were injected15 min prior to the microinjection of RANTES (25 pg, AH/POA)or sterile saline.

In another series of experiments we investigated theinvolvement of prostaglandins in the fever induced byRANTES. Animals were treated 30 min prior to the injectionof either RANTES (25 pg) or sterile saline with non-selectiveand selective cyclooxygenase inhibitors: ibuprofen (10mgkg−1;i.p.), indomethacin (2mg kg−1; i.p.) and celecoxib (5mg kg−1; bygavage).

In the final set of the experiments, we analyzed the effect ofthese drugs on the increase of CSF PGE2 levels induced byRANTES. In accordance with the fever alteration caused bythese drugs, we chose the 2.5- and 5-h time points to measurethe CSF PGE2 levels.

As Tris (tris [hydroxymethyl] aminomethane–HCl, pH 8.2),the vehicle for indomethacin, does not change the basaltemperature of rats (Fabricio et al., 2005b), the positive controlgroup Tris/RANTES was used to investigate the effect ofibuprofen and indomethacin on the fever induced byRANTES. The positive control for celecoxib received sterilewater only. Control animals received ibuprofen, indometha-cin or celecoxib 30 min before injection of sterile saline(500 nl, AH/POA). Oral administration of celecoxib (2 mg ml−1

solution) was made by gavage, through a polyethylenecannula (PE50, 4-cm length) connected to a needle (22gauge1/4) placed in a 1-ml plastic syringe. To ensure thatappropriate dose was administered the volume and thereactions of animals after drug administration were cau-tiously observed. Animals that present cough or respiratoryanguish (when the drug solution reaches the respiratory tract)were sacrificed and animals that regurgitate were excludedfrom the study.

The doses and routes of administration of all the anti-pyretic employed here had already been chosen for previousstudies in our laboratory (Zampronio et al., 1995; Souza et al.,2002; Fabricio et al., 2005b; Melo Soares et al., 2006; Pessiniet al., 2006).

All pyrogenic stimuli were injected between 10:00 and 11:00AM. Microinjections into the AH/POA were made aseptically.For that, a 30-gauge needle, connected by polyethylene (PE10)tube was used. The needle was protruded 2 mm beyond thecannula tip and a 500 nl volume was injected slowly (over

1 min) with a 25 μl Hamilton syringe coupled to a microinfu-sion pump (KD Scientific, model KDS101, EUA). After injection,the needle remained in place for 30 s before it was withdrawnto prevent backflow of the injection fluid through the cannula.

4.7. Data analysis

All variations in core body or tail skin temperature wereexpressed as changes from themean basal value (i.e., as ΔT, indegree Celsius). All results are presented as mean±standarderror of themean (SEM) andmean baseline temperatures werenot statistically different among the groups included in anyparticular set of experiments. The levels of PGs were analyzedby one-way ANOVA followed by Tukey's test. ΔT and HLIresponses were compared across treatments and time pointsanalyzed by two-way ANOVA for repeated measures followedby Bonferroni test. All data were analyzed using Prismcomputer software (Graph-Pad, San Diego, CA, USA). Differ-ences were considered significant when P<0.05.

Acknowledgments

We are most grateful to Miriam C.C. Melo, Juliana Vercesi,Giuliana Bertozi and Frédéric Borlat for their expert technicalassistance. This study was supported by Fundação de Amparoà Pesquisa do Estado de São Paulo (FAPESP, Proc. Nr. 97/09837-6; 01/11014-5; 03/04838-7 and 05/55717-0) and ConselhoNacional de Desenvolvimento Científico e Tecnológico (Proc.Nr. 305802/2004-6), Brazil.

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