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Am J Physiol Regul Integr Comp Physiol 276: R652-R658, 1999;
0363-6119/99 $5.00
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Vol. 276, Issue 3, R652-R658, March 1999

Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats

M. J. P. Lenczowski1, R.-M. Bluthé2, J. Roth3, G. S. Rees4, D. A. Rushforth5, A.-M. van Dam1, F. J. H. Tilders1, R. Dantzer2, N. J. Rothwell5, and G. N. Luheshi5

1 Department of Pharmacology, Faculty of Medicine, Research Institute Neurosciences Vrije Universiteit, Graduate School Neurosciences Amsterdam, 1081 BT Amsterdam, The Netherlands; 2 Institut François Magendie, Institut National de la Recherche Agronomique-Institut National de la Santé et de la Recherche Médicale U394, 33077 Bordeaux Cedex, France; 3 Klinikum Physiologisches Institut, Justus-Liebig Universität Giessen, D-35392 Giessen, Germany; 4 Division of Endocrinology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG; and 5 School of Biological Sciences, Manchester M13 9PT, United Kingdom


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Interleukin (IL)-6 has been proposed to mediate several sickness responses, including brain-mediated neuroendocrine, temperature, and behavioral changes. However, the exact mechanisms and sites of action of IL-6 are still poorly understood. In the present study, we describe the effects of central administration of species-homologous recombinant rat IL-6 (rrIL-6) on the induction of hypothalamic-pituitary-adrenal (HPA) activity, fever, social investigatory behavior, and immobility. After intracerebroventricular administration of rrIL-6 (50 or 100 ng/rat), rats demonstrated HPA and febrile responses. In contrast, rrIL-6 alone did not induce changes in social investigatory and locomotor behavior at doses of up to 400 ng/rat. Coadministration of rrIL-6 (100 ng/rat) and rrIL-1beta (40 ng/rat), which alone did not affect the behavioral responses, reduced social investigatory behavior and increased the duration of immobility. Compared with rhIL-6, intracerebroventricular administration of rrIL-6 (100 ng/rat) induced higher HPA responses and early-phase febrile responses. This is consistent with a higher potency of rrIL-6, compared with rhIL-6, in the murine B9 bioassay. We conclude that species-homologous rrIL-6 alone can act in the brain to induce HPA and febrile responses, whereas it only reduces social investigatory behavior and locomotor activity in the presence of IL-1beta .

adrenocorticotropic hormone; corticosterone; interleukin-1beta ; interleukin-6; brain; social behavior; locomotor behavior; hypothalamic-pituitary-adrenal activation


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

INFLAMMATION INDUCES an acute phase response that includes an array of metabolic, endocrine, and behavioral alterations. Among these are brain-mediated responses, such as activation of the hypothalamic-pituitary-adrenal (HPA) axis (41), fever (13), and sickness behavior (8). The induction of these brain-mediated responses involves peripheral and/or central actions of cytokines, including tumor necrosis factor (TNF)-alpha (6) and interleukin (IL)-1beta (1, 11, 16, 21, 22, 24, 29, 32, 40).

Results of recent experiments with IL-6-deficient mice indicate that IL-6 plays a role in such brain-mediated responses as well. For instance, IL-6-deficient mice could not mount a febrile response, whereas wild-type mice responded with fever to peripherally administered lipopolysaccharide (LPS) (2). In addition, ACTH (Van Dam, unpublished results) and corticosterone (Cort) (31) responses to LPS are attenuated in IL-6-deficient mice compared with those in wild-type mice. It remains to be established whether this relates to deficient IL-6 production in the brain or in peripheral tissues. Because LPS-induced IL-6 concentrations in plasma correlate with the amplitude of thermogenic and HPA responses (17, 19, 30, 37), it has been suggested that IL-6 produced outside the brain is involved in such brain-mediated responses, possibly by acting at sites outside the blood-brain barrier (18). Alternatively, IL-6 can be produced in the brain. Although not consistently shown (42), some in situ hybridization studies have demonstrated constitutive expression of IL-6 mRNA in discrete rat brain regions, including hippocampal and hypothalamic areas (33, 36). In addition, hypothalamic IL-6 mRNA expression is upregulated by LPS both in vitro (38) and in vivo (25). In line with these observations, LPS increases the levels of IL-6 protein in perfusates of the anterior hypothalamus (30) and in cerebrospinal fluid (3, 17) in vivo and increases the release of IL-6 from hypothalami (38) in vitro.

In addition to IL-6 production in the brain, possible central sites of action of IL-6 are supported by the demonstration of IL-6 receptor mRNA in hippocampal and hypothalamic areas (33, 36, 42) and by results showing IL-6 binding in hypothalamic membranes (7). In accordance with the view that IL-6 may induce brain-mediated symptoms of illness by acting at central sites, intracerebroventricular administration of IL-6 has been reported to induce ACTH and Cort secretion (23, 43) and fever (2, 17) and to decrease food intake and locomotor activity (34). Moreover, intrahypothalamic administration of neutralizing antibodies to IL-6 attenuates hyperthermic responses (9).

Most studies on the effects of central IL-6 on brain-mediated symptoms of illness in rats have been performed using species-heterologous recombinant human IL-6 (rhIL-6). It is unclear whether species-homologous IL-6 will induce the same biological activities in rats. In this context, it should be noted that species-heterologous rhIL-1beta induces febrile and behavioral responses at least quantitatively different from those induced by species-homologous recombinant rat IL-1beta (rrIL-1beta ) (Luheshi and Bluthé, unpublished results). Moreover, opposing actions of human and murine TNF-alpha have been reported on the febrile response in rats (39). These observations and the proposed central involvement of IL-6 in brain-mediated sickness responses led us to investigate the effects of central administration of newly produced species-homologous rrIL-6 (28) in rats on HPA activity, fever, social exploration, and locomotion.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Animals

Adult male Wistar rats were used unless otherwise stated. Wistar (Harlan, Zeist, The Netherlands and Charles River, St-Aubin-les-Elbeuf, France or Sulzfeld, Germany) and adult male Sprague-Dawley rats (Charles River, Kent, UK) were housed under a 12:12-h light-dark regime at 20-22°C. Food and water were available ad libitum. At the time of the experiments, the animals had body weights of 200-300 g. All experimental protocols had been approved by the respective Institutional Committees for Animal Health and Care.

Materials

rrIL-6. The production, purification, and characterization of rrIL-6 have recently been described in more detail (28). Briefly, cDNA coding for the mature protein was cloned between the Nco I (5'-end) and EcoR I (3'-end) restriction sites of the pET-21d expression vector, downstream of the T7 RNA polymerase promoter. The open reading frame encoded an additional NH2-terminal Met-Gly sequence to facilitate cloning at the Nco I site of the expression vector. Sequencing of the vector-insert construct revealed a single base change from the published sequence, resulting in a conservative amino acid substitution of threonine to alanine at position 182 in the recombinant product. The expression plasmid pET-21d-rat IL-6 was transformed into Escherichia coli, strain BL21 (DE3), after which induction with 1 mM isopropyl-beta -D-thiogalactopyranoside caused expression of a 21-kDa protein. Immunoblotting with use of an antibody raised against murine IL-6 confirmed that this protein was IL-6. After production, rrIL-6 was purified and shown to be >90% monomer, with the main impurity being rrIL-6 dimer. Electrospray mass spectrometric analysis of rrIL-6 showed the protein to have a molecular mass of 21,756.63 ± 0.25 Da. The endotoxin content of the purified rrIL-6 preparation was <4 ng endotoxin/mg rrIL-6, as measured by a Limulus amebocyte lysate assay. All present experiments were carried out using the same batch of rrIL-6 (batch 290696).

rhIL-6 and rrIL-1beta . rhIL-6 (batch 88/514) and rrIL-1beta (batch 041096, biological activity 317 IU/µg) were obtained from Dr. A. Bristow and Dr. S. Poole (National Institute for Biological Standards and Control, Potters Bar, UK). All cytokines were dissolved in pyrogen-free saline and stored at -80°C. Immediately before intracerebroventricular injection, appropriate dilutions were prepared in a vehicle of pyrogen-free saline containing 0.1% BSA (wt/vol; BSA A-8806, fatty acid-free, low endotoxin; Sigma, St. Louis, MO). Control animals received vehicle.

In Vitro Biological Activity of rrIL-6 and rhIL-6

The biological activities of the rrIL-6 and rhIL-6 preparations used in the present study were measured using the IL-6-sensitive B9 cell line, as described elsewhere (10). Using rhIL-6 (lot 1449-10-01, CLB, Amsterdam, The Netherlands) as a standard, we defined half-maximal stimulation of B9 cell proliferation as 1 U of IL-6 bioactivity.

Surgical Procedures

While rats were under general anesthesia [halothane (Fluothane) or a combination of 100 mg/kg ip ketamine (Kombivet, Etten-Leur, The Netherlands) and 2 mg/kg sc xylazine (Rompun; Bayer, Leverkusen, Germany)], a permanent guide cannula was implanted into the right lateral ventricle of the brain. The cannula was secured on the skull with two screws and dental cement. During the same procedure, animals used in temperature studies were implanted intraperitoneally with a battery-operated, temperature-sensitive radio transmitter (Data Sciences, St. Paul, MN). After surgery, rats were allowed to recover for at least 7 days. In some experiments, rats were housed individually (Wistar rats), and in other experiments, they were housed in groups of six until 24 h before the start of the experiment, after which they were housed individually (Sprague-Dawley rats). Correct placement of the cannula was verified post mortem.

Experimental Procedures

IL-6 effects on plasma ACTH and Cort concentrations. To reduce nonspecific activation of the HPA axis, rats were habituated to experimental procedures by manipulation two times daily for three successive days before the experiment. Three groups of rats (n = 5-6) were given 100 ng/rat rhIL-6, rrIL-6, or vehicle (10 µl) intracerebroventricularly. The injection fluid was administered to freely moving rats at a rate of 5 µl/min using a Hamilton microsyringe and a syringe infusion pump (Harvard Apparatus, South Natick, MA). The injection cannula was left in place for another 10 s to allow the injection fluid to diffuse away from the site of injection. One hour after the injection, when ACTH and Cort responses are known to be (near) maximal (43), rats were killed by decapitation and trunk blood was collected in ice-cold heparin-coated tubes (Sarstedt, Etten-Leur, The Netherlands). Blood samples were centrifuged (2,000 g for 10 min at 4°C), and aliquots of plasma were stored at -20°C until assayed. Plasma ACTH and Cort concentrations were determined as previously described (19).

IL-6 effects on core body temperature. Four groups of Sprague-Dawley rats (n = 4-5) were given 50 or 100 ng/rat rrIL-6, 100 ng/rat rhIL-6, or vehicle (2 µl) intracerebroventricularly within a period of 10-20 s. In a separate experiment, Wistar rats were given 100, 200, or 500 ng/rat rrIL-6 or vehicle (2 µl) intracerebroventricularly. The core body temperature was continuously monitored by the intraperitoneally implanted radio transmitters. The output from each of these transmitters (frequency in Hz) was monitored by an antenna (Data Sciences, St. Paul, MN) placed under each animal's cage and channeled into a consolidation matrix (BCM 100) connected to a PC. Core body temperature signals were sampled at 5-min intervals over a period of 6 h after cytokine administration and converted to degrees Celsius by the processor.

IL-6 effects on social investigatory behavior and duration of immobility. Animals were kept under reversed light-dark conditions (lights on 6 PM, lights off 6 AM). Four-week-old juvenile male Wistar rats, which were housed in groups of 10 animals in a different room, served as social stimuli. The behavioral tests were performed as previously described in detail (1). Briefly, IL-6 effects on social investigatory behavior and locomotor activity were studied by recording the cumulative duration of social investigation by the test animal of a conspecific juvenile rat introduced into the home cage as well as by measuring the duration of immobility of the test animal. Social investigatory behavior consisted of anogenital sniffing and close following of the juvenile. Behavioral studies were carried out during the first 8 h of the dark phase of the light-dark cycle under red light illumination. Rats were tested for 4 min in their home cage, immediately before (baseline) and 90, 180, and 360 min after cytokine injection. After each 4-min test, the conspecific juvenile rat was removed and returned to its group home cage. Different conspecifics were used in each test. To determine the dose-response relationship of centrally administered rrIL-6 on social investigatory behavior and duration of immobility, four groups of rats (n = 3-5) were given 100, 200, or 400 ng/rat rrIL-6, or vehicle (2 µl) intracerebroventricularly by gravity, using a 30-gauge needle.

To assess whether rrIL-6 interacts with rrIL-1beta , four groups of rats (n = 5-6) were given rrIL-6 (100 ng/rat) and rrIL-1beta (40 ng/rat) either alone or in combination. rrIL-6 or vehicle (1 µl) was injected immediately before rrIL-1beta or vehicle (1 µl). The dose of rrIL-1beta used (40 ng/rat) has previously been shown to have no effect on social investigation (Bluthé, unpublished results).

Statistical Analysis

Data were analyzed by one-way ANOVA (ACTH, Cort, and febrile response) to determine significance of variance between groups or by two- or three-way ANOVA with repeated measurements on the time factor (social exploration and duration of immobility). If necessary, ACTH and Cort data were log transformed before analysis to meet the criteria of ANOVA. The overall febrile response was assessed by calculating the area under the curve (AUC) for each animal (interval between 30 and 400 min after IL-6 injection), taking the lowest temperature measured in each individual rat as the baseline for that particular animal. Social exploration is expressed as percentage of baseline. Between-group differences were determined by post hoc multiple comparison (Tukey: ACTH, Cort, and febrile response; least significant difference test: behavioral parameters). The in vitro biological activities of rrIL-6 and rhIL-6 were compared using an independent-samples t-test.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

In Vitro Biological Activity of rrIL-6 and rhIL-6

The biological activities of rrIL-6 and rhIL-6, as shown in the B9 bioassay, were 0.56 ± 0.02 and 0.31 ± 0.05 U/pg, respectively (mean ± SE, n = 3). Comparison of these activities revealed that rrIL-6 was more potent than rhIL-6 (P < 0.01).

IL-6 Effects on Plasma ACTH and Cort Concentrations

As illustrated in Fig. 1, intracerebroventricular administration of rhIL-6 and rrIL-6 (100 ng/rat) induced plasma ACTH concentrations of 166 ± 56 and 340 ± 90 pg/ml, respectively. Corresponding plasma Cort concentrations were 215 ± 50 and 369 ± 54 ng/ml. Plasma ACTH and Cort concentrations of rrIL-6-injected rats were significantly higher than those of vehicle-injected control animals, whereas rhIL-6-induced plasma ACTH and Cort concentrations did not differ from control values (1-way ANOVA, Tukey's post hoc test, P < 0.05). Thus, although rrIL-6- and rhIL-6-treated groups were not statistically different from each other, rrIL-6 appeared more potent than rhIL-6 in stimulating ACTH and Cort secretion.


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  		Fig. 1.   Effect of intracerebroventricular administration of interleukin (IL)-6 to rats on hypothalamic-pituitary-adrenal system. Plasma ACTH (A) and corticosterone (Cort; B) concentrations were measured 90 min after intracerebroventricular administration (100 ng/rat) of recombinant human IL-6 (rhIL-6) or recombinant rat IL-6 (rrIL-6). Data are presented as means and SE (vehicle and rhIL-6, n = 5; rrIL-6, n = 6). * P < 0.05 vs. vehicle-treated animals.

IL-6 Effects on Core Body Temperature

As shown in Fig. 2A, intracerebroventricular injection of rrIL-6 (50 and 100 ng/rat) induced a hyperthermic response in a dose-dependent manner, whereas vehicle alone did not affect the core body temperature. At a dose of 100 ng/rat, rrIL-6 and rhIL-6 raised the body temperature to similar levels. However, rrIL-6-induced maximum values were reached much earlier (~2 h) than those induced by rhIL-6 (~5 h). The rrIL-6-induced hyperthermic responses in Wistar rats were similar to those observed in Sprague-Dawley rats (data not shown).


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  		Fig. 2.   Effect of intracerebroventricular administration of IL-6 to rats on core body temperature. A: core body temperature was measured continuously after intracerebroventricular administration of rhIL-6 (100 ng/rat, n = 5), rrIL-6 (50 or 100 ng/rat; n = 5 or 4, respectively), or vehicle (n = 4). B: alterations in overall core body temperature after intracerebroventricular administration of rhIL-6 or rrIL-6 are presented as area under the curve (AUC) values. AUC values were calculated from IL-6-induced hyperthermic responses shown in A. Data are presented as means and SE. * P < 0.05 vs. vehicle-treated animals.

As illustrated in Fig. 2B, the overall febrile responses to rrIL-6 showed a dose-dependent increase. The AUC values of rats treated with 100 ng/rat rrIL-6 were significantly higher than those of vehicle-treated control animals, whereas AUC values after an equal dose of rhIL-6 or after 50 ng/rat rrIL-6 did not differ from control values (1-way ANOVA, Tukey's post hoc test, P < 0.05). Thus, although rrIL-6- and rhIL-6-treated (100 ng/rat) groups were not statistically different from each other, rrIL-6 appeared more potent than rhIL-6 in inducing overall febrile responses.

IL-6 Effects on Social Investigatory Behavior and Duration of Immobility

Social investigatory behavior before intracerebroventricular injection of any substance was not different in rats from different groups (1-way ANOVA, data not shown). Figure 3 shows that, at doses of up to 400 ng/rat, intracerebroventricularly administered rrIL-6 did not significantly affect the duration of social investigatory behavior at any time interval tested. This applied for comparisons with individual scores before cytokine injection (baseline), as well as for comparisons with scores obtained from vehicle-treated animals (2-way ANOVA, post hoc least significant difference test). Moreover, central administration of rrIL-6 did not induce immobility at any time interval studied (data not shown).


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  		Fig. 3.   Effect of intracerebroventricular administration of rrIL-6 to rats on social investigatory behavior. Duration of social investigatory behavior was measured before and 1.5, 3, and 6 h after intracerebroventricular injection of 100 ng/rat (n = 3), 200 ng/rat (n = 4), or 400 ng/rat (n = 5) rrIL-6 or vehicle (0 ng/rat, n = 3). Duration of social investigatory behavior is expressed as percentage of duration of social investigatory behavior observed before injection of rrIL-6 or vehicle (baseline). Data are presented as means and SE.

Because of the ineffectiveness of intracerebroventricular administration of rrIL-6 alone, we decided to study the possible interactive effects of rrIL-6 with rrIL-1beta on social investigatory behavior and the duration of immobility. Figure 4A shows that intracerebroventricular administration of vehicle, rrIL-1beta (40 ng/rat), or rrIL-6 (100 ng/rat) alone did not affect baseline social investigatory behavior. However, administration of 100 ng/rat rrIL-6 together with 40 ng/rat rrIL-1beta markedly decreased social investigatory behavior, which was most prominent after 3 h (P < 0.001 vs. vehicle-, IL-1beta -, or IL-6-treated groups). As illustrated in Fig. 4B, intracerebroventricular administration of rrIL-6 together with rrIL-1beta also significantly suppressed locomotor activity, as indicated by a marked increase in the duration of immobility at 3 h postinjection (P < 0.001 vs. rrIL-1beta -treated group). Thus, although rrIL-6 alone was ineffective, intracerebroventricular administration of rrIL-6 could induce changes in social investigatory behavior and locomotion in the presence of rrIL-1beta .


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  		Fig. 4.   Effect of intracerebroventricular administration of rrIL-6 and/or IL-1beta to rats on social investigatory behavior and locomotor activity. Duration of social investigatory behavior (A) or immobility (B) was measured before and 1.5, 3, and 6 h after intracerebroventricular injection of 100 ng/rat rrIL-6 and/or 40 ng/rat rrIL-1beta . Control animals received vehicle (Veh) alone. Duration of social investigatory behavior is expressed as percentage of duration of social investigatory behavior observed before injection of cytokines or vehicle (baseline). Duration of immobility is given in seconds. Data are presented as means and SE. * P < 0.05, *** P < 0.001 vs. rrIL-1beta -treated rats.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

In the present study, we show that intracerebroventricular administration of species-homologous rrIL-6 to rats induces HPA and hyperthermic responses at doses that do not induce sickness behavior. At doses of up to 400 ng/rat, rrIL-6 had no effects on social investigatory behavior or immobility. Our findings and those of others (23, 43) showing that intracerebroventricular administration of IL-6 induces HPA activation support the hypothesis that IL-6 receptors in the brain may play a role in the HPA response to inflammatory stimuli. Indeed, increased levels of IL-6 receptor mRNA have been found in parvocellular neurons of the hypothalamic paraventricular nucleus after peripheral administration of LPS (42), suggesting that IL-6 may have direct actions on hypothalamic corticotropin-releasing hormone neurons. Because the hippocampus is also involved in HPA control (35), constitutive (36, 42) and endotoxin-induced (42) expression of IL-6 receptor mRNA in this structure may represent an additional level of IL-6 control over the HPA axis. Our present results also support a role for brain IL-6 receptors in febrile responses and are in accordance with observations of others (2, 17). It has been reported that for febrile responses, IL-6 actions in the hypothalamus may be of primary importance (12, 20). The observed expression of IL-6 receptor mRNA in the medial preoptic nucleus (33), which is considered to be a thermoregulatory center, is consistent with this idea.

The dissociation between the physiological and behavioral effects of rrIL-6 observed in our study was not due to the use of different rrIL-6 preparations, because all experiments were carried out using the same rrIL-6 batch. At first sight, the finding of this dissociation was unexpected, because intracerebroventricular administration of rhIL-6 to rats (doses of 10 and 100 ng/rat) had previously been claimed to decrease food intake and locomotor activity (34). However, closer examination of these findings revealed that the decreased locomotor activity started only 9 h after cytokine administration. These late effects on locomotor activity may represent a delayed effect of intracerebroventricularly administered rhIL-6 or, rather, an indirect effect of rhIL-6-induced early physiological alterations. Other studies confirm the lack of early direct behavioral effects of rhIL-6. For instance, intracerebroventricular administration of rhIL-6 does not impair spatial navigation learning, despite the fact that it does increase body temperature (26). Furthermore, intracerebroventricular administration of rhIL-6 does not affect behavioral responses on the elevated plus maze, whereas rhIL-1beta induces an anxiogeniclike response (4). Similarly, peripheral administration of recombinant mouse (rm) IL-6 does not alter milk intake, in contrast to rmIL-1beta , which suppresses this feeding behavior (40). The lack of short-term effects of IL-6 on behavioral parameters appears to be at variance with results obtained in IL-6-deficient mice. In contrast to wild-type mice, IL-6-deficient mice do not show turpentine-induced anorexia or cachexia (14). Similarly, IL-6-deficient mice are less sensitive to the centrally IL-1- or LPS-induced depressing effects on social behavior (Bluthé, unpublished results). Taken together, these results suggest that central IL-6 may affect behavioral responses only in the context of other proinflammatory cytokines, which is supported by our observation showing that intracerebroventricular coadministration of rrIL-6 (100 ng/rat) and a subthreshold dose of rrIL-1beta (40 ng/rat) markedly decreased social investigatory behavior and markedly increased the duration of immobility. Synergistic effects of IL-6 and the proinflammatory cytokine IL-1beta have previously been reported both in vivo (27, 44) and in vitro (5). The mechanisms by which these cytokines potentiate each others' actions are poorly understood. We postulate that central IL-1beta may induce IL-6 receptors in brain areas that are involved in social investigatory behavior and locomotor activity, thereby facilitating the actions of IL-6. In support of this, peripheral injection of IL-1beta resulted in an upregulation of IL-6 receptor mRNA in brain microvasculature that could be reached from the general circulation (42). In addition, central administration of soluble IL-6 receptors to rats markedly potentiated the inhibitory actions of IL-6 on locomotor activity (34). Thus, considering the ubiquitous and constitutive mRNA expression of the IL-6 signal transducer gp130 in the rat brain (42), condition-dependent upregulation of central IL-6 receptors may determine the response of a particular neural circuit to IL-6.

In the present study, we demonstrate that intracerebroventricular administration of species-homologous rrIL-6 appears more potent than species-heterologous rhIL-6 in inducing HPA and febrile responses. This apparent difference in biological activity of rrIL-6 and rhIL-6 may relate to species-related differences in affinity for rat IL-6 receptors or kinetic differences per se or to batch-related differences. With respect to this, the higher in vivo biological activities of rrIL-6 are in accordance with the observed twofold-higher biological activity of rrIL-6, compared with that of rhIL-6, on murine B9 cells (Ref. 28 and present study). This is in accordance with previous in vitro data showing that a hybrid of human IL-6 with the COOH terminus of rat IL-6 was two- to threefold more potent on a mouse hybridoma cell line than wild-type human IL-6 (15). Taken together, these results support the view that the differences in biological activity between rrIL-6 and rhIL-6 reported in the present study reflect species-related rather than batch-related differences. Furthermore, our results demonstrate that the relative biological potencies of IL-6 as measured on murine B9 cells are predictive for the relative in vivo biological potencies in rats, at least after intracerebroventricular administration.

In summary, our results show that intracerebroventricular administration of species-homologous rrIL-6 to rats activates the HPA axis and induces hyperthermia, thereby inducing two key features of the host defense response. In contrast, even at four-times higher doses than those needed for induction of febrile and HPA responses, rrIL-6 did not affect social investigatory behavior or immobility. This indicates that different IL-6 responsive substrates may underlie these physiological and behavioral responses to rrIL-6. After intracerebroventricular administration of a subthreshold dose of rrIL-1beta , rrIL-6 markedly affected behavioral responses. We hypothesize that intracerebroventricularly administered rrIL-6 may induce HPA and febrile responses by acting on brain structures in which IL-6 receptors are already present, whereas behavioral changes may be induced in brain structures that require the induction of IL-6 receptors.


    ACKNOWLEDGEMENTS

We thank R. Binnekade, J. Brevé, V. Tridon, and B. Stoerr for excellent technical assistance.


    FOOTNOTES

This study was supported by the BIOMED I program "Cytokines in the Brain" (PL-391450) of the commission of the European Communities.

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. §1734 solely to indicate this fact.

Address for reprint requests: F. J. H. Tilders, Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Medicine, Dept. of Pharmacology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

Received 18 August 1998; accepted in final form 7 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Regul Integr Compar Physiol 276(3):R652-R658
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