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Am J Physiol Regul Integr Comp Physiol 289: R340-R347, 2005. First published April 14, 2005; doi:10.1152/ajpregu.00104.2005
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Physiology and Pharmacology of Temperature Regulation

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

Christoph Rummel,1 Stephan W. Barth,2 Thilo Voss,1 Stefan Korte,1 Rüdiger Gerstberger,1 Thomas Hübschle,1 and Joachim Roth1

1Institut für Veterinär-Physiologie, Justus-Liebig-Universität Giessen, Giessen; and 2Institut für Ernährungsphysiologie, Bundesforschungsanstalt für Ernährung und Lebensmittel, Karlsruhe, Germany

Submitted 14 February 2005 ; accepted in final form 23 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 µg/kg) or a low (10 µg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E2 (PGE2) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 µg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE2 formation (5 or 500 µg/kg diclofenac). Even the lowest dose of diclofenac (5 µg/kg) attenuated fever in response to 10 µg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE2 at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 µg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 µg/kg LPS, whereas subcutaneous injections of 100 µg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 µg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.

lipopolysaccharide; febrile response; prostaglandin E2; cyclooxygenase-2; immune system-to-brain communication

MOST EXPERIMENTAL STUDIES employing bacterial lipopolysaccharide (LPS) to induce fever use a systemic route of administration, such as intraperitoneal, intravenous, or intra-arterial injections of the pyrogen. These treatments result in a generalized systemic inflammatory response, which is accompanied by increased measurable amounts of proinflammatory cytokines and LPS in the circulation (10, 14, 17, 28, 32). To study a cytokine release within a local area of inflammation, experimental models have been introduced in which LPS is administered locally, rather than systemically, into a subcutaneous air pouch in rats (8, 22, 23) or a subcutaneously implanted artificial Teflon chamber in guinea pigs (29, 30, 34). LPS that is injected into the air pouch (8) or the subcutaneous chamber (29) is not measurable in the systemic circulation 1 h after its injection. This observation does, however, not exclude the possibility that a small fraction of the injected LPS might have leaked from the air pouch or the chamber immediately after the injection procedure. Among the pyrogenic cytokines (TNF, IL-1, or IL-6) that are produced within the site of localized subcutaneous inflammation, IL-6 is the only putative endogenous pyrogen that enters the circulation in significant quantities. It has therefore been proposed that the increase of IL-6 in the bloodstream, albeit moderate, is involved in the manifestation of fever and other brain-controlled signs of illness that occur during localized inflammation (9, 34, 38).

However, the following observations from our previous studies (29, 30, 34) suggested that, in addition to a humoral signal provided by circulating IL-6, local mechanisms generated directly at the site of subcutaneous inflammation may participate in the transmission of febrile signals to parts of the brain that are involved in fever generation. 1) We tested the effects of two doses of LPS (100 or 10 µg/kg) injected into the subcutaneous chamber on body temperature. Both treatments resulted in fever, and the febrile response to the low dose of LPS was markedly reduced by coinjection of a local anesthetic, an effect for which a systemic action of the local anesthetic could be ruled out (30). 2) A direct LPS-induced and IL-6-mediated activation of brain cells could only be demonstrated when the amount of circulating IL-6 reached a critical threshold. Again, for the lower dose of LPS injected into the subcutaneous chamber, we failed to detect any activation of brain cells, most likely because of the rather moderate circulating concentrations of IL-6 induced by this LPS dose (34).

On the basis of these observations, we speculated that a localized formation of inflammatory mediators might contribute to the manifestation of fever in response to injection of the low LPS dose into the subcutaneous chamber. With regard to putative mediators that could be induced at the local site of inflammation and then activate pyrogenic mechanisms, we concentrated on the roles of TNF and the cyclooxygenase (COX) products, i.e., prostaglandins (29). Indeed, injection of the nonselective COX inhibitor diclofenac into the subcutaneous chamber at the rather high dose of 5 mg/kg abolished LPS-induced fever in this specific experimental model (29). This high dose of diclofenac might, however, have depressed the formation of prostaglandins also at other sites, for example, in the brain.

The aim of the present study was therefore to obtain further evidence for the participation of locally produced prostaglandins in the manifestation of fever during localized subcutaneous inflammation. For this purpose, we progressively reduced the dose of diclofenac that was injected along with LPS into the subcutaneous chamber and measured the resulting fever responses. We further tested the capacity of diclofenac to block the LPS-induced local formation of prostaglandin E2 (PGE2), which is regarded as a critical endogenous mediator of fever. Finally, for the first time in the guinea pig, we studied the expression of COX-2 mRNA in the brain during localized, as opposed to systemic, LPS-induced inflammation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male guinea pigs (Cavia aperea porcellus, 390–430 g body wt on the day of surgery) were housed in individual cages at 22°C and exposed to a 12:12-h light-dark cycle (lights off at 7 PM). The animals had access to food and water ad libitum. Twice a week the reservoirs were filled with fresh pellet food and water, and at the same time the cages were changed. At ~10 days before the fever experiment, the animals were prepared surgically (see below) and habituated to the handling procedures at least twice before the experiment. The national guidelines for experiments with vertebrate animals were followed, and approval for the experimental protocols was obtained from the local ethics committee (GI 18/2-42/00).

Surgery

At ~10 days before the experiment, the animals were chronically implanted with artificial subcutaneous Teflon (polytetrafluoroethene) chambers equipped with catheters for injection of drugs and collection of lavage and radio transmitters for measurement of body temperature.

Briefly, the guinea pigs were anesthetized with 100 mg/kg ketamine hydrochloride (Pharamcia Upjohn, Erlangen, Germany) and 0.25 mg/kg medetomidine hydrochloride (Pfizer, Karlsruhe, Germany). The Teflon chambers were implanted into subcutaneous cavities, which were formed with a cylindrical Plexiglas stick after a cutaneous incision. The cavities that remained after removal of the Plexiglas stick had about the same diameter and size as the artificial subcutaneous chamber, which was open at both ends. The open sides of the cylindrical chambers (10 mm ID) had close contact with the skin tissue. The subcutaneous chamber was placed laterally to the dorsal midline and caudally to the scapulae of the anesthetized guinea pigs. The chambers were equipped with catheters for administration of drugs and sampling of lavage fluid that were tunneled subcutaneously to the interscapular region of the back and sealed by heating. Then the skin was closed with sutures (for details about size and shape of the subcutaneous chambers see Refs. 38 and 39). After surgical placement of the subcutaneous chamber, a biotelemetry transmitter was implanted intraperitoneally for measurement of core temperature.

For experiment 3, some guinea pigs were implanted with intra-arterial, instead of subcutaneous, chambers (for details about the intra-arterial catheter see Refs. 29 and 30). All implanted materials were sterilized, and all the surgical procedures were conducted under aseptic conditions.

Substances

Bacterial LPS (derived from Escherichia coli, O111:B4; Sigma Chemical, St. Louis, MO) was suspended in sterile pyrogen-free 0.9% saline at 100 µg/ml (high dose) or 10 µg/ml (low dose). Doses of 100 or 10 µg/kg were used for injections into the subcutaneous chamber. In experiment 3, some animals received systemic injections of LPS at 10 µg/kg (intra-arterial route of administration; prepared from a 10 µg/ml LPS solution) or 30 µg/kg (intraperitoneal route of administration; prepared from a 30 µg/ml LPS solution). Equivalent volumes of the vehicle were injected in control groups. All LPS doses were chosen according to previous studies from this laboratory (11, 29–32, 34).

Diclofenac sodium (Calbiochem, La Jolla, CA) was dissolved at 5,000, 500, 50, or 5 µg/ml in vehicle, which consisted of 95% sterile saline and 5% ethanol. In these solutions, diclofenac was injected into the subcutaneous chamber, together with LPS or an equivalent volume of solvent (0.9% saline) at 5,000, 500, 50, or 5 µg/kg, corresponding to a consistent injection volume of 1 ml/kg. Control groups were injected with an equivalent volume of vehicle (1 ml vehicle/kg) together with LPS or saline.

Experimental Protocols

On the day of the experiment, each previously implanted subcutaneous chamber was tested for accumulation of fluid in the chamber by aspiration with a syringe through the catheter. The experiment was performed only if there was <0.1 ml of fluid in the syringe.

Experiment 1: dose-dependent attenuation of LPS fever by coinjection of diclofenac. The influence of four doses of diclofenac (5,000, 500, 50, or 5 µg/kg) injected into the subcutaneous chamber along with LPS (100 or 10 µg/kg) or with an equivalent volume of solvent on the manifestation of a febrile response was studied. In an additional control experiment, the lowest dose of diclofenac or solvent was injected subcutaneously into the site contralateral to the chamber simultaneously with administration of 10 µg/kg LPS or saline into the subcutaneous chamber. Each of the treatment groups consisted of five or six animals.

Experiment 2: measurement of PGE2 in lavage fluid of the subcutaneous chamber. In separate experiments, we collected lavage fluid from the subcutaneous chamber 120 min after the respective injections to study a localized formation of PGE2 and the influence of diclofenac on LPS-induced PGE2 production. This time point was chosen according to data from the literature on peripheral LPS-induced formation of PGE2 (36) and according to our own pilot measurements showing consistently elevated PGE2 concentrations in lavage fluid 2 h after LPS administration. Localized PGE2 formation was measured in response to both administered doses of LPS or saline along with solvent or with a high (500 µg/kg) or a low (5 µg/kg) dose of diclofenac.

To collect lavage fluid from the artificial subcutaneous chamber, 1 ml of sterile saline was slowly injected into the chamber through the catheter, left in the chamber for ~30 s, withdrawn again into a sterile syringe, and immediately centrifuged, so that a clear supernatant without cells was obtained. Thereafter, the catheter was flushed with a small volume (0.1 ml) of sterile pyrogen-free saline and closed by heating. The supernatant was stored at –70°C for later determination of PGE2.

Experiment 3: detection of COX-2 mRNA in the guinea pig brain during systemic or localized inflammation. Using radioactive in situ hybridization (see below), we studied the expression of COX-2 in the guinea pig brain during systemic or localized inflammation induced by intra-arterial (10 µg/kg, n = 2) or intraperitoneal (30 µg/kg, n = 2) injections of LPS vs. injections of LPS (100 or 10 µg/kg, n = 3 for each LPS dose) into the artificial subcutaneous chambers. Five control animals were injected with 0.9% saline instead of LPS: two intra-arterially, one intraperitoneally, and two subcutaneously. COX-2 expression was determined 180 min after the respective injections. This time point was chosen according to our own pilot experiment in guinea pigs and according to published studies on LPS-induced COX-2 mRNA expression in the rat or mouse brain (5–7, 25).

Measurement of Body Temperature

Intraperitoneally implanted (see above) battery-operated biotelemetry transmitters (VM-FH disks or PDT-4000 E-mitter, Mini-Mitter, Sunriver, OR) were used to measure core temperature. Output (frequency in Hz) was monitored with an antenna placed under each cage (RA 1000 or ER-4000 radio receivers, Mini-Mitter). A data acquisition system (Vital View, Mini-Mitter) was used for automatic control of data collection and analysis. Body temperature was monitored and recorded at 5-min intervals. For analysis and graphical documentation, temperature data collected at 15-min intervals were used.

PGE2 Assay

PGE2 in the collected lavage fluid was determined by use of a commercially available enzyme immunoassay (R & D Systems, Wiesbaden, Germany). The assay is based on the competitive binding technique in which PGE2 in a sample competes with a fixed amount of alkaline phosphatase-labeled PGE2 for sites on a mouse monoclonal antibody. During incubation, the mouse monoclonal antibody was bound to a goat anti-mouse antibody that coated the wells of a microplate. After the sample was washed once to remove excess conjugate and unbound sample, a substrate solution was added to the wells for determination of the bound enzyme activity. Immediately after color development, the absorbance was read at 405 nm on a microplate reader (Digiscan, Asys Hightech, Eugendorf, Austria). The color intensity inversely correlated with the PGE2 concentration in the sample. According to the manufacturer's product information, the sensitivity of the PGE2 assay was ~35 pg/ml.

COX-2 In Situ Hybridization

Animals were decapitated 180 min after local or systemic injections of LPS or 0.9% NaCl (controls), and the brains were removed quickly, frozen on dry ice, and stored at –70°C until further use. For COX-2 mRNA hybridization, coronal cryostat sections (20 µm; model HM 500-O, Microm, Walldorf, Germany) were obtained from brain regions containing the subfornical organ (SFO) and the vascular organ of the lamina terminalis, both of which are implicated in fever (34, 37), as well as other hypothalamic and extrahypothalamic brain areas. Sections were mounted and processed as previously described (1) and then hybridized with oligodeoxynucleotide probes complementary to nt 932 to 888, nt 1049 to 1005, and nt 1321 to 1277 of the guinea pig COX-2 cDNA (GenBank accession no. Y07896.1). Specificity of probe sequences for complementarity exclusively with COX-2 cDNA was checked vs. GenBank by blastN (National Center for Biotechnology Information) and sequence alignments (ClustalW 1.8, National Center for Biotechnology Information) with published partial sequence sets of guinea pig COX-1 cDNA (GenBank accession nos. AY757362 and AB054840). The probe labeling and high-stringency hybridization procedure followed a recently published protocol (1) using 50 µl/section of an equimolar mixture with the three oligodeoxynucleotide probes and a final probe concentration of 7.5–10 x 105 cpm/µl hybridization buffer. For autoradiographical detection, X-ray films (Kodak Biomax MR) were exposed to hybridized sections for 10–17 days.

Evaluation and Statistics

Body temperature changes in response to injections of LPS or saline combined with diclofenac or solvent of the different experimental groups were plotted against time and expressed as means ± SE at each time point. Changes of abdominal temperatures between groups were compared by two-way repeated-measures ANOVA followed by an all-pairwise Bonferroni's multiple comparison post hoc test (Sigmaplot/Sigmastat analysis software, Jandel Scientific, SPSS Science Software, Erkrath, Germany). Concentrations of PGE2 were compared between the experimental groups by one-way ANOVA followed by Scheffé's post hoc test (StatView, Abacus Concepts, Berkeley, CA). Statistical analyses resulting in P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiment 1: Dose-Dependent Attenuation of LPS-Induced Fever by Coinjection of Diclofenac

The effects of injections of 100 µg/kg LPS into the subcutaneous chamber along with solvent or different doses of diclofenac (5, 50, 500, or 5,000 µg/kg) on changes in abdominal temperature of guinea pigs are shown in Fig. 1.



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  		Fig. 1. Changes in abdominal temperatures [body (core) temperature (Tb)] of 5 groups of guinea pigs injected subcutaneously with 100 µg/kg LPS along with solvent (L100 + solvent) or different doses of diclofenac [5 and 50 µg/kg diclofenac (L100 + D5 and L100 + D50, respectively; top) and 500 and 5,000 µg/kg diclofenac (L100 + D500 and L100 + D5000, respectively; bottom)]. Ti, initial (time 0, i.e., before injection) temperature; n, number of animals. Dashed horizontal lines in Figs. 1–3 indicate mean baseline temperature that was calculated from all temperatures measured within the 2 h before injection.

 
At 100 µg/kg, LPS elicited two clearly distinct phases of fever, lasting 90 and 270 min, respectively. Treatment with 5 or 50 µg/kg diclofenac slightly delayed the onset of fever and reduced the magnitude, but not the course, of both fever phases (Fig. 1, top). At 30–150 min after injection, both doses of diclofenac significantly reduced fever in response to injections of 100 µg/kg LPS into the subcutaneous chamber (P < 0.05, 2-way repeated-measures ANOVA and Bonferroni's multiple comparison post hoc test).

Treatment with 500 or 5,000 µg/kg diclofenac depressed both fever phases (Fig. 1, bottom; for 500 µg/kg, P < 0.001 from 15 to 270 min after injection; for 5,000 µg/kg, P < 0.001 from 15 to 360 min after injection; 2-way ANOVA and Bonferroni's post hoc test). Subcutaneous treatment with 5,000 µg/kg diclofenac along with 100 µg/kg LPS even induced a hypothermic response. Pronounced alterations of abdominal temperatures were not observed in any of the control groups injected with saline instead of LPS (not shown).

Identical experiments were also performed with the low LPS dose (10 µg/kg), and the results are summarized in Fig. 2.



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  		Fig. 2. Changes in abdominal temperatures of 5 groups of guinea pigs injected subcutaneously with 10 µg/kg LPS along with solvent or different doses of diclofenac. See Fig. 1 legend for details.

 
Again, we observed a dose-dependent attenuation of fever by coinjections of diclofenac. When injected with LPS into the subcutaneous chamber, even the lowest dose of diclofenac (5 µg/kg) significantly reduced the mean LPS-induced increase of abdominal temperature from 165 to 360 min after the injection (Fig. 2, top; P < 0.05, 2-way repeated-measures ANOVA and Bonferroni's multiple comparison post hoc test). Diclofenac at 50 µg/kg significantly reduced fever from 120 to 360 min after injection (P < 0.01). The initial phase of fever induced by the lower LPS dose was, however, evidently not affected by treatment with 5 or 50 µg/kg diclofenac. Again the higher doses of diclofenac (500 and 5,000 µg/kg; Fig. 2, bottom) caused a more pronounced depression of fever. The onset of fever was delayed by 60 min in guinea pigs injected with 500 µg/kg diclofenac along with 10 µg/kg LPS into the subcutaneous chamber. The highest dose of the nonselective COX inhibitor even induced a slight hypothermia. The antipyretic effects of 500 or 5,000 µg/kg diclofenac were significant from 30 to 360 min after injection (P < 0.001, 2-way ANOVA and Bonferroni's post hoc test).

Because a systemic action of diclofenac by entry of the drug from the subcutaneous chamber into the blood could not be ruled out, we tested whether a subcutaneous injection of the lowest dose of the COX inhibitor reduced fever in response to 10 µg/kg LPS also when injected subcutaneously into the site contralateral to the chamber. The result of this experiment is shown in Fig. 3.



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  		Fig. 3. Top: Changes in abdominal temperatures of guinea pigs injected with 10 µg/kg LPS into the subcutaneous chamber and 5 µg/kg diclofenac or solvent subcutaneously into the site contralateral (cl) to the chamber (L10 + solvent cl and L10 + D5 cl, respectively). Bottom: Changes in abdominal temperatures of guinea pigs injected with saline into the subcutaneous chamber and 5 µg/kg diclofenac or solvent subcutaneously into the site contralateral to the chamber position (NaCl + solvent cl and NaCl + D5 cl, respectively). See Fig. 1 legend for details.

 
Injections of 10 µg/kg LPS into the subcutaneous chamber resulted in identical changes of abdominal temperature in guinea pigs, irrespective of simultaneous administration of 5 µg/kg diclofenac or an equivalent volume of solvent into the contralateral site of the chamber position (Fig. 3, top). This result indicates that subcutaneous injections of 5 µg/kg diclofenac may have no substantial systemic effect with regard to the manifestation of fever under these specific conditions.

Experiment 2: Measurement of PGE2 in Lavage Fluid of the Subcutaneous Chamber

The results of the PGE2 measurements in lavage collected 120 min after the different treatments are summarized in Fig. 4.



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  		Fig. 4. Top left: PGE2 concentration in lavage fluid collected from the subcutaneous chamber 120 min after injection of LPS [100 and 10 µg/kg (L100 and L10, respectively)] or saline (NaCl) along with solvent (solv) into the chamber. Top right and bottom, left and right: effects of administration of a low [5 µg/kg (D5)] or a high [500 µg/kg (D500)] dose of diclofenac along with LPS, instead of solvent. Bars with the same letter (a–c) are not significantly different from each other; bars with different letters are significantly different (P < 0.05, 1-way ANOVA with Scheffé's test). Values are means ± SE of the number of samples shown in parentheses.

 
Both doses of LPS injected into the chamber caused a significant rise of PGE2 in lavage fluid, which tended to be more pronounced in response to the high LPS dose (Fig. 4, top left). An almost complete suppression of LPS-induced local formation of PGE2 was achieved by injections of a high or low dose of diclofenac in both LPS-treated groups (Fig. 4, top right and bottom left). The moderate levels of PGE2, which were present in the control group, tended to be attenuated by treatment with diclofenac (Fig. 4, bottom right).

Experiment 3: Detection of COX-2 mRNA in Guinea Pig Brain During Systemic or Localized Inflammation

Experiment 3 was performed to determine whether localized subcutaneous stimulation with LPS stimulates the expression of COX-2 mRNA in the guinea pig brain, as previously demonstrated in other animal species after systemic LPS treatment (5–7). Using radioactive in situ hybridization, we analyzed COX-2 expression in the guinea pig brain 180 min after intraperitoneal or intra-arterial injections of LPS or after subcutaneous injections of 100 or 10 µg/kg LPS into the subcutaneous chamber. Control animals were injected intraperitoneally, intra-arterially, or subcutaneously with sterile saline. Although the resolution of X-ray autoradiograms is methodically limited, the pattern of COX-2-specific hybridization signals throughout the entire brain is strongly suggests that, also in the guinea pig brain, COX-2 mRNA signals are mainly associated with blood vessels in animals treated intra-arterially or intraperitoneally with LPS. A similar observation was made in animals injected subcutaneously with 100 µg/kg LPS. However, in none of the guinea pigs injected subcutaneously with the low LPS dose (10 µg/kg) or intra-arterially, intraperitoneally, or subcutaneously with saline, were any COX-2 mRNA signals observed. In treatment groups showing induced COX-2 gene expression, the most intense COX-2 specific labeling was observed in the SFO. Less pronounced COX-2 signals were also observed in the vascular organ of the lamina terminalis and in brain vessels distributed throughout the entire guinea pig brain. Brain sections containing the SFO of the different LPS-treated animals and of a control are shown in Fig. 5.



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  		Fig. 5. Cyclooxygenase-2 (COX-2) expression in the subfornical organ (SFO) of a saline-treated guinea pig (B, intra-arterial NaCl) and guinea pigs injected with LPS intra-arterially (10 µg/kg, L10 ia, C), intraperitoneally (30 µg/kg, L30 ip, D), or subcutaneously into the subcutaneous chamber [100 µg/kg, L100 sc (E) and 10 µg/kg, L10 sc (F)]. A: schematic brain map showing coronal section at the level of the SFO, which represents the site of the sections used for in situ hybridization (B–F). B–F: bright micrographs of emulsion-coated sections. Black labeling represents expression of COX-2 mRNA within SFO of guinea pigs injected intra-arterially, intraperitoneally, or subcutaneously (high dose) with LPS. Scale bar, 100 µm. The black color in C–E represents autoradiographically visualized radioactive in situ hybridization signals.

 
It thus seems that, during a moderate localized inflammatory response induced by injection of 10 µg/kg LPS into the subcutaneous chamber, a pronounced COX-2 induction in the guinea pig brain is absent, despite the manifestation of a moderate fever (Fig. 2).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Using the subcutaneous chamber as a model to induce a localized inflammatory response, we previously (30) analyzed the febrile responses of guinea pigs to injections of a high (100 µg/kg) or a low (10 µg/kg) dose of LPS into the chamber. In this study (30), we tested the effects of administration of the local anesthetic ropivacaine, along with LPS, into the subcutaneous chamber. Fever in response to the low, as opposed to the high, LPS dose was attenuated by treatment with the local anesthetic. A systemic action of ropivacaine could be ruled out, because when injected subcutaneously on the contralateral side of the animal in relation to the chamber at the same dose, it failed to alter fever induced by the low dose of LPS (30). This result suggests that local signals that were blocked by the local anesthetic in the chamber might have participated in the transmission of febrile signals from a localized site of peripheral inflammation, in this case, the subcutaneous chamber, to parts of the brain that are involved in the generation of fever. One local effect of ropivacaine was reduced formation of LPS-induced TNF at the site of inflammation (30). In another study, we therefore successfully neutralized the locally produced TNF by a synthetic TNF-binding protein and observed a reduction of the late phase of fever in response to injection of the low LPS dose into the subcutaneous chamber (29). In response to the high LPS dose, neither the local anesthetic nor treatment with TNF-binding protein had an effect on fever. We speculated that, in the case of the high LPS dose, humoral signals (i.e., IL-6) entering the systemic circulation from the chamber in sufficient amounts could totally override the local components for the induction of fever.

An obvious effect of the local anesthetic is a silencing of local afferent nerve fibers. Inflammation leads to the release of prostanoids, namely, PGE2, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity (21). It was therefore tempting to speculate that a peripheral, localized formation of PGE2 might also play a role in the manifestation of fever that develops in response to injections of LPS into the subcutaneous chamber, perhaps via stimulation of afferent nerve fibers. The results of the present study provide modest evidence for such a hypothesis. We clearly demonstrated a localized formation of PGE2 at the subcutaneous site of inflammation that could be blocked effectively even by the lowest tested dose of the nonselective COX inhibitor diclofenac (5 µg/kg) when injected into the chamber together with LPS. We further observed a dose-dependent attenuation or complete suppression of fever in this experimental model by local treatment with diclofenac. We were especially interested in the effect of a low dose of the COX inhibitor on fever in response to subcutaneous injections of 10 µg/kg LPS because of the previously described effect of the local anesthetic (30) and because of the almost complete lack of brain activation by circulating cytokines under these experimental conditions (34) (Fig. 5F). Indeed, a systemic effect of the lowest applied dose of diclofenac could be ruled out, because an injection of this dose of the COX inhibitor into the subcutaneous tissue contralateral to the chamber position did not attenuate fever (Fig. 3). Nevertheless, the following question arises: How might peripherally (locally) produced PGE2 contribute to the manifestation of fever? On the one hand, the effects of PGE2 could be mediated locally at the site of inflammation. From the skin, a predominant input of signals from cold fibers into the thermoregulatory system shows characteristic static and dynamic discharge patterns (4). An activation of cutaneous cold receptors results in characteristic cold defense reactions (vasoconstrictions and increased heat production), responses that are also observed during a febrile rise of body temperature. Interestingly, a preliminary report has been published (20) showing a specific activation of peripheral cold-sensitive neurons by PGE2. However, the activation of cold defense reactions is usually observed in the rising phase of fever, which was obviously unimpaired by treatment with the lowest dose of diclofenac (Fig. 2). Consistent with the rapid onset of cold defense responses during the initial phase of fever, it has been reported that neural signaling mechanisms are mostly involved in the early phases of a given febrile response (26). According to the data presented, we must admit that the local formation of PGE2 in our model has just a moderate effect and exclusively contributes to the maintenance of fever. This becomes obvious by the fact that fever in response to the low LPS dose was not attenuated within the first 2 h, despite the absence of PGE2 in the chamber at 120 min under the influence of 5 µg/kg diclofenac.

It thus seems difficult to explain how the rapid onset and the manifestation of the first 2 h of fever are mediated in our experimental model of a moderate local inflammation (subcutaneous injection of the low LPS dose). A possible mechanism for induction of fever after intraperitoneal injection of a low dose of LPS in guinea pigs has been suggested recently (3). The monophasic fever that develops under this condition seems to be initiated by an {alpha}1-adrenoreceptor-mediated and PGE2-independent mechanism within the preoptic-anterior hypothalamic area in response to vagally transmitted signals of peripheral LPS. According to this study, the later phase of fever is mediated by an {alpha}2-adrenoreceptor-mediated and PGE2-dependent mechanism (3). Perhaps a similar mechanism operated in our experimental model.

With regard to our experiments in which we used the high LPS dose (100 µg/kg) for injections into the subcutaneous chamber, it seems that distinct mechanisms were activated to establish a febrile response. The fever, in this case, was biphasic. The shape of the fever curve resembled a curve after systemic treatment with LPS (32, 34). Although the local formation of PGE2 within the subcutaneous chamber was similar in response to both LPS doses (Fig. 4), there was a pronounced difference in the expression of COX-2 in the brain, which is regarded as critical for the manifestation of fever by several authors (7, 12, 15, 18, 24, 39). Injections of the high (as opposed to the low) dose of LPS into the subcutaneous chamber caused expression of COX-2 in the guinea pig brain, which may have contributed to the development of the very pronounced second fever phase. Consistent with this observation, we previously reported that humoral mechanisms cause direct brain activation under these specific experimental conditions (34).

In conclusion, the mechanisms for the manifestation of fever in response to a localized peripheral inflammation seem to depend on the strength of the inflammatory stimulus. A local formation of PGE2 at the peripheral site of inflammation seems to participate in the maintenance of fever in response to a moderate inflammatory stimulus. If the inflammatory stimulus is strong enough to induce sufficient amounts of humoral mediators, such as cytokines, prostaglandins, or LPS itself (2, 13, 16, 19, 27, 28, 33, 35), expression of COX-2 is induced in the brain and contributes to the maintenance of fever under these conditions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Deutsche Forschungsgemeinschaft Graduiertenkolleg 455 Molekulare Veterinärmedizin and Deutsche Forschungsgemeinschaft Project RO 896/5-2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Roth, Institut für Veterinär-Physiologie, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, 35392 Giessen, Germany (E-mail: joachim.roth{at}vetmed.uni-giessen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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