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Veterinary-Physiology, Justus-Liebig-University Giessen, D-35392 Giessen, Germany
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Interleukin-6 (IL-6) is regarded as an endogenous mediator of lipopolysaccharide (LPS)-induced fever. IL-6 is thought to act on the brain at sites that lack a blood-brain barrier, the circumventricular organs (CVOs). Cells that are activated by IL-6 respond with nuclear translocation of the signal transducer and activator of transcription 3 molecule (STAT3) and can be detected by immunohistochemistry. We investigated whether the LPS-induced release of IL-6 into the systemic circulation was accompanied by a nuclear STAT3 translocation within the sensory CVOs. Treatment with LPS (100 µg/kg) led to a slight (1 h) and then a strong increase (2-8 h) in plasma IL-6 levels, which started to decline at the end of the febrile response. Administration of both pyrogens LPS and IL-6 (45 µg/kg) induced a febrile response with IL-6, causing a rather moderate fever compared with the LPS-induced fever. Nuclear STAT3 translocation in response to LPS was observed within the vascular organ of the lamina terminalis (OVLT) and the subfornical organ (SFO) 2 h after LPS treatment. To investigate whether this effect was mediated by IL-6, the cytokine itself was systemically applied and indeed an identical pattern of nuclear STAT3 translocation was observed. However, nuclear STAT3 translocation already occurred 1 h after IL-6 application and proved to be less effective compared with LPS treatment when analyzing OVLT and SFO cell numbers that showed nuclear STAT3 immunoreactivity after the respective pyrogen treatment. Our observations represent the first molecular evidence for an IL-6-induced STAT3-mediated genomic activation of OVLT and SFO cells and support the proposed role of these brain areas as sensory structures for humoral signals created by the activated immune system and resulting in the generation of fever.
cytokines; signal transducers and activators of transcription; vascular organ of the lamina terminalis; subfornical organ; area postrema
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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INFECTIOUS OR INFLAMMATORY stimulation in the
periphery of the body results in the generation of a number of
centrally mediated characteristic responses. These brain-mediated signs
of illness include the generation of fever (17, 37), the
activation of the hypothalamic-pituitary-adrenal axis
(34), the loss of appetite, and a number of behavioral
changes collectively termed sickness behavior (9). Under
such infectious conditions, cytokines with a molecular mass of
15-20 kDa are endogenously produced by monocytes, macrophages, and numerous other cell types. Among them, the
proinflammatory cytokines interleukin-1
(IL-1), interleukin-6
(IL-6), and tumor necrosis factor-
(TNF-) are regarded as key
players in the modification of the brain-controlled functions mentioned
above. With regard to the febrile response, these cytokines are called
endogenous pyrogens (10, 17). Increased release of these
cytokines into the general circulation can be experimentally achieved
by systemic injections of lipopolysaccharide (LPS) derived from the
outer membrane of gram-negative bacteria. Initially, it was thought that the large hydrophilic cytokine proteins could not pass the relatively impermeable blood-brain barrier (BBB) to influence brain
structures. However, there are some small brain areas that lack a tight
BBB, including the so-called sensory circumventricular organs (CVOs),
such as the vascular organ of the lamina terminalis (OVLT), the
subfornical organ (SFO), and the area postrema (AP).
First evidence for a role of these sensory CVOs as "windows to the brain" for inflammatory signal molecules is based on classical lesion studies. For example, it has been demonstrated in guinea pigs and sheep that electrolytic lesions of the anteroventral third ventricle area, which includes the OVLT, resulted in a suppression of fever (3, 4). In addition, an interruption of neuronal connections between the OVLT and the anterior hypothalamus, where the febrile resetting of body temperature is regulated, led to an attenuation of fever (12). More recently, it has been reported that electrolytic lesions of the SFO (32) or microinjection of the IL-1 receptor antagonist into the SFO (6) reduced febrile responses. Finally, removal of the AP abolished stimulatory cytokine effects on the hypothalamic-pituitary-adrenal axis activity (19).
Accepting that the sensory CVOs play a crucial role in mediating
circulating pyrogenic messages necessary for fever induction in turn
leads to two further prerequisites that should be fulfilled. First, a
putative endogenous humoral mediator of LPS-induced fever should be
measurable in the systemic circulation in close relation to the febrile
response. Second, an activation of neurons or other cellular elements
within the sensory CVOs needs to be demonstrated. TNF-, IL-1, and
IL-6 are produced in response to LPS and can be detected in the general
circulation with characteristic kinetics. Depending on the route of LPS
administration and the injected LPS dose, TNF- is the first cytokine
that appears in the bloodstream (21, 23), followed by very
small traces of IL-1 (16), which is frequently not
detected at all (17). IL-6, however, is produced during
the complete time course of LPS-induced fever, and the circulating
levels of IL-6 show an excellent correlation with the febrile changes
of body temperature (20, 23). Therefore, IL-6 seems to be
an appropriate candidate to act as a humoral signal that may be sensed
by the aforementioned CVOs. Measurement of electrical activity of OVLT
neurons in brain slices provided first evidence that the firing rates
are changed under TNF- treatment (28). The effect of
IL-6 on the electrical activity of neurons within the CVOs has not yet
been investigated; however, IL-6 decreased the activity of
warm-sensitive neurons in brain slices from the preoptic area
(36), an area involved in the central control of
temperature regulation.
The effects of IL-6 are mediated via the gp130 cytokine receptor family. Stimulation of these receptors activates a cytokine-specific signal transduction pathway, the so-called Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade. IL-6 is known to act through the STAT3 isoform, which gets phosphorylated, dimerizes, and then translocates into the nucleus, where it regulates gene expression by binding to specific gene promoters (33). We recently reported (14, 15) that intracerebroventricular microinjections of IL-6 caused a very characteristic pattern of STAT3 translocation into cell nuclei of neurons located in brain areas involved in the generation of fever. Thus STAT3 immunohistochemistry seems to be an excellent tool to demonstrate genomic activation of cells induced by IL-6. If IL-6 is a critical circulating mediator for an inflammatory stimulation of sensory CVOs, it should be possible to demonstrate, during LPS-induced fever, not only the increase of circulating IL-6 but also the parallel IL-6-induced genomic activation of CVOs. Indeed, a recent study reported that treatment with LPS resulted in a nuclear STAT3 translocation in cells of the OVLT (18). In line with this study, our aim was to investigate 1) the LPS-induced nuclear translocation of STAT3 not only in the OVLT but in all sensory CVOs and 2) the time dependency of this process. To verify whether LPS-induced nuclear STAT3 translocation might truly reflect a biological activity of IL-6, we injected the cytokine itself and investigated the sensory CVOs with the use of STAT3 immunohistochemistry at distinct time intervals after systemic IL-6 treatment. The results of this study should enable us to state whether there is a role for circulating IL-6 in genomic activation of cells within the sensory CVOs during fever.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
The study was performed in 99 male Wistar rats (Rattus spec.) with body weights of 225 ± 5 g. Experiments were carried out in accordance with the local Ethics Committee (ethics approval numbers GI 20/1-1/96 and GI 18/2-42/00). Animals were housed individually with free access to drinking water and standard laboratory chow. The room temperature (RT) was adjusted to 23 ± 1°C, and lights were on from 7:00 AM to 7:00 PM. The animals were surgically prepared for telemetric body temperature measurement at least 1 wk before the fever experiments.Substances
Animals were anesthetized with intraperitoneal injections of ketamine-xylazine solution [ketamine-hydrochloride: 100 mg/kg body wt Ketamin Gräub (Albrecht, Aulendorf, Germany); xylazine: 25 mg/kg body wt Rompun (Bayer Vital, Leverkusen, Germany)]. Bacterial LPS derived from Escherichia coli (serotype O111:B4, Sigma Chemical, Deisenhofen, Germany) was suspended in sterile pyrogen-free 0.9% saline. A dose of 100 µg/kg body wt was injected intraperitoneally. Controls were treated with the same volume of sterile pyrogen-free 0.9% saline (vehicle). Rat recombinant IL-6 (rrIL-6) was obtained from Dr. S. Poole (National Institute for Biological Standards and Control, Potters Bar, UK), stored at
70°C
in stock aliquots of 10 µg in 50 µl of pyrogen-free 0.9% saline,
and then injected intraperitoneally in a final volume of 500 µl of
sterile 0.9% saline per rat, corresponding to a dose of ~45 µg/kg
body wt.
Measurement of Body Temperature
Abdominal temperature was measured by use of biotelemetry transmitters (PDT-4000 E-Mitter, Mini-Mitter) implanted intraperitoneally under general anesthesia. Output (frequency in Hz) was monitored by a receiver placed under each cage (ER-4000 receiver, Mini-Mitter). A data acquisition system (Vital View, Mini-Mitter) was used for automatic control of data collection and analysis. Body temperature was continuously monitored and recorded at 5-min intervals. For analysis and graphical documentation, temperature data at time intervals of 15 min were used.Measurement of Bioactive IL-6
Determination of plasma IL-6 was performed by a bioassay based on the dose-dependent growth stimulation of IL-6 on the B9 hybridoma cell line (1). The assay was performed in sterile, 96-well microtiter plates. In each well, 5,000 B9 cells were incubated for 72 h with serial dilutions of biological samples or different concentrations of a human IL-6 standard (code 89/548, National Institute for Biological Standards and Control, South Mimms, UK). The number of living cells after 72 h was measured by use of the dimethylthiazol-diphenyl tetrazolium bromide (MTT) colorimetric assay (13). Plasma samples were prediluted to adjust them to the standard dilution curves. Considering the sample dilution within the assay, the detection limit of the assay was three international units (IU) of IL-6 per milliliter.Experimental Protocols
LPS-induced circulating IL-6.
At selected time points after intraperitoneal injection of 100 µg/kg
body wt LPS or vehicle, rats (n = 3-5 per group)
were killed for collection of blood via cardiac puncture. Blood samples were immediately centrifuged and stored at 70°C for later
measurement of IL-6.
LPS-induced fever. Two groups of rats (n = 7 in each group) were injected intraperitoneally with either 100 µg/kg body wt LPS or an equivalent volume of vehicle. Body temperature was evaluated from 2 h before until 8 h after the time of injection.
IL-6-induced fever. Two groups of rats (n = 5 in each group) were injected intraperitoneally with either 10 µg of rrIL-6 dissolved in 500 µl of sterile 0.9% saline or the solvent alone. Body temperature was evaluated from 2 h before until 8 h after the time of injection.
LPS- or IL-6-induced nuclear STAT3 translocation. To investigate LPS- or IL-6-induced nuclear STAT3 translocation and its putative time dependency, rats were separated into five groups with different postinjection times. Animals of the first group (n = 4 with two IL-6-treated and two vehicle-treated rats) were perfused 30 min, animals of the second group (n = 14, with four LPS-, five IL-6-, and five vehicle-treated animals) were perfused 1 h, animals of the third group (n = 14, with five LPS-, four IL-6-, and five vehicle-treated animals) were perfused 2 h, animals of the fourth group (n = 6, with three LPS-treated and three vehicle-treated animals) were perfused 3 h, and animals of the fifth group (n = 4, with two LPS-treated and two vehicle-treated animals) were perfused 4 h after treatment with the respective substances. Transcardial perfusions with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) were performed in animals that were deeply anesthetized with ketamine-xylazine. The brains were removed and postfixed in the same fixative for 1 h at RT. The tissue was cryoprotected in 20% sucrose in phosphate buffer overnight at 4°C. Tissue was cut the following day.
STAT3 Immunohistochemistry
To detect STAT3 signals in brain sections, a rabbit anti-STAT3 antibody (sc-482, Santa Cruz Biotechnology, Heidelberg, Germany) was used. The specificity of this STAT3 antibody has clearly been proven (30) and was also demonstrated in our own preabsorption control experiments (14). Previous studies investigating STAT3 distribution in the rat central nervous system have shown that amplification procedures are a helpful tool to investigate STAT3 immunoreactivity (15, 30). Therefore, a commercial tyramide amplification kit (NEL700, NEN Life Science Products, Köln, Germany), based on the catalyzed reporter deposition amplification method, was used.Coronal 40-µm brain sections were cut on a freezing microtome (model 1205, Jung, Heidelberg, Germany). Tyramide amplification staining was performed according to the kit description in a phosphate buffer system (pH 7.2). In detail, sections were placed into 10% normal horse serum containing 0.3% Triton X-100 for 1 h at RT. Next, sections were transferred into 0.5% blocking powder to block the unspecific tyramine binding sites. Primary STAT3 antibody incubation (1:12,000) was performed for 24-48 h at 4°C. The STAT3 antibody was then detected with a secondary biotinylated anti-rabbit antibody (1:200, Vector BA-1000, Linaris Biologische Produkte, Wertheim-Bettingen, Germany) for 1 h at RT. After amplification, the immunohistochemical processing was finished with an avidin biotin horseradish peroxidase complex (Vector Elite Kit, Linaris Biologische Produkte), which was visualized by diaminobenzidine hydrochloride (Sigma Chemical) reaction in the presence of hydrogen peroxide. Finally, sections were counterstained with cresyl violet and coverslipped with Entellan (Merck, Darmstadt, Germany) for the light microscopic analysis.
Microscopic and Quantitative Histological Analysis
Sections were analyzed with the use of an Olympus BX50 light microscope (Olympus Optical, Hamburg, Germany). Digital images were taken with an Olympus Camedia C-3030 camera, for which we used the Olympus Camedia Master 2.0 software package. Image editing software (Adobe Photoshop) was used to adjust brightness and contrast, to change the graphic mode to CMYK, and to combine the individual images into the figure plates.The numbers of STAT3-immunoreactive cell nuclei were quantitatively evaluated for the two CVOs (the OVLT and the SFO) 60 and 120 min after systemic treatment with the pyrogens (IL-6 or LPS) and compared with the vehicle control. For each animal, three rostral OVLT sections at similar stereotaxic coordinates and three SFO sections throughout the rostral-to-caudal SFO extension were selected. A microscopic counting grid (200 × 200 µm) was used at 400-fold magnification to determine the number of nuclei stained for STAT3 per three sections within the OVLT or the SFO of each animal investigated. Numbers of stained cell nuclei are represented as means ± SE per OVLT or SFO in four or five animals per group.
Statistics
All statistical calculations were carried out with the Sigmaplot/Sigmastat analysis software (Jandel Scientific, Corte Madera, CA) or Stat View (Abacus Concepts, Berkeley, CA). Mean levels of circulating IL-6 and mean abdominal temperatures of different animal groups are presented as means ± SE. IL-6 in plasma was compared between LPS- and vehicle-treated rats by one-way ANOVA followed by Scheffé's post hoc test. Abdominal temperatures of LPS-treated vs. vehicle-treated rats as well as IL-6-treated vs. vehicle-treated animals were compared by two-way repeated-measures ANOVA followed by an all pairwise Bonferroni's multiple comparison post hoc test. Statistical analysis for the quantitative histological evaluation was performed by one-way ANOVA with subsequent post hoc analysis. Statistical significance was accepted for all post hoc procedures at P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Role of Circulating IL-6 in LPS-Induced Fever
Circulating levels of bioactive IL-6 at distinct time intervals after intraperitoneal injection of LPS or pyrogen-free saline (vehicle) are shown in Fig. 1A. Immediately after injection of LPS or vehicle, basal IL-6 levels reached ~20 IU/ml. However, in response to LPS injection, bioactive plasma IL-6 rose to 425 ± 130 IU/ml at 60 min, to 3,010 ± 1,250 IU/ml at 120 min, to 5,360 ± 3,070 IU/ml at 180 min, and to 6,275 ± 2,240 IU/ml at 240 min, respectively. By 480 min postinjection, plasma IL-6 had already decreased to 1,030 ± 565 IU/ml. Injection of saline did not induce changes in basal plasma levels of IL-6, which remained at 21 ± 15 IU/ml. At 120 and 240 min postinjection, IL-6 in plasma of LPS-treated rats was significantly higher than in animals injected with sterile saline (P < 0.05).
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Changes in body temperature in response to LPS or vehicle are shown in Fig. 1B. The injection procedure itself caused a transient stress-induced increase in abdominal temperature of ~0.8°C in both groups. Thereafter, LPS-treated rats developed a robust biphasic fever, with the first peak reaching levels of 38.0°C at 180 min posttreatment and the second peak reaching ~38.8°C at 300-345 min posttreatment. In contrast, body temperature of control animals returned to the preinjection values of ~37.0°C. From 135 to 480 min after the time of injection, body temperature of LPS-treated rats was significantly elevated compared with animals injected with sterile saline (P < 0.05).
Figure 1C shows the thermal responses of two groups of rats injected with rrIL-6 or vehicle. Again, in both groups, a transient stress-induced rise of abdominal temperature occurred. In saline-treated rats, body temperature returned to the baseline value, whereas treatment with IL-6 caused a rather moderate elevation in body temperature (~37.8°C, lasting 3-4 h), which was less pronounced than that of the febrile response induced by LPS. From 120 to 270 min and again at 300 min after the time of injection, body temperature of IL-6-injected rats was significantly higher than the corresponding value of control rats injected with sterile saline (P < 0.05).
Distribution of Cytoplasmic and Nuclear STAT3 Immunoreactivity in the Rat Brain Analyzed Under Control and Pyrogen (IL-6 and LPS)- Stimulated Conditions
In control rats as well as in pyrogen-stimulated animals, basal cytoplasmic STAT3 expression was detected in various brain areas with a similar staining pattern, irrespective of the time point investigated (Table 1). The postinjection time points used for Table 1 (60 and 120 min) are those proven to be necessary to demonstrate pyrogen-induced nuclear STAT3 translocation (see also Figs. 2 and 3).
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Brain areas with a moderate to strong cytoplasmic STAT3 expression were predominantly found within the hypothalamus and the medulla. As for the hypothalamus, those areas included the arcuate nucleus, the periarcuate area, the lateral hypothalamic area, the magnocellular and ventromedial preoptic nucleus, the median eminence, the periventricular nucleus, the retrochiasmatic area, and the supraoptic nucleus. Within the medulla oblongata, intense cytoplasmic STAT3 signals were observed in the ambiguus nucleus, the cuneate nucleus, the dorsal motor nucleus of the vagus, the gracile nucleus, the hypoglossal nucleus, and the lateral reticular nucleus. As for those brain structures of particular interest for this study, the CVOs, moderate cytoplasmic STAT3 expression was found within the AP and low cytoplasmic STAT3 signals was found in the SFO and OVLT at all time points investigated and irrespective of the type of treatment.
In contrast, nuclear STAT3 immunoreactivity was only observed in a very
limited number of brain structures after both vehicle and pyrogen
application. The most intense nuclear signals were detected within the
OVLT and the SFO 60 or 120 min after IL-6 or LPS treatment, whereas the
respective controls showed no nuclear STAT3 immunoreactivity. This
proved to be different for the AP and in some respects also for the
solitary tract nuclei, in which at both time points and irrespective of
the type of treatment a low nuclear STAT3 expression was observed. As
for other brain structures showing nuclear STAT3 signals during
pyrogen-stimulated conditions, a low STAT3 expression was found in the
arcuate nucleus, the median eminence, the ventromedial preoptic
nucleus, the choroid plexus, the ependymal lining of the ventricles,
and the meninges. However, the numbers of cells (1-5)
that stained positively for STAT3 in these structures were of
magnitudes lower than those detected in the two CVOs (the OVLT and SFO)
(see Fig. 4).
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Systemic IL-6 and LPS Treatment Induces a Time-Dependent Nuclear Translocation of the Transcription Factor STAT3 in the OVLT and SFO
Cytoplasmic STAT3 expression in the OVLT under control conditions (Fig. 2, A-C, and Fig. 2, A and B, insets) as well as during pyrogen-stimulated conditions (Fig. 2, D-I, and Fig. 2, E and H, insets) was very weak and could hardly be separated from background labeling. In contrast, 120 min after LPS treatment (Fig. 2E and inset in Fig. 2E) or 60 min after IL-6 treatment (Fig. 2H and inset in Fig. 2H), a nuclear STAT3 translocation and an increase in STAT3 immunoreactivity were observed in cells within rostral aspects of the OVLT. The size and the round shape of the STAT3-labeled cell nuclei (insets in Fig. 2, E and H) compared with the cresyl violet-counterstained neuronal nuclei (insets in Fig. 2, A and B) indicate a predominant nuclear STAT3 translocation in OVLT neurons. All other time intervals investigated showed no difference in STAT3 immunoreactivity between pyrogen- and vehicle-treated animals (for details, see Fig. 2).Similar to the situation noted for the OVLT, cytoplasmic STAT3 expression within the SFO under control conditions (Fig. 3, A and C, and insets in Fig. 3, A and C) as well as during pyrogen-stimulated conditions (Fig. 3, B and D, and insets in Fig. 3, B and D) was low and could hardly be separated from background labeling. In contrast, 60 min after IL-6 treatment (Fig. 3B and inset in Fig. 3B) or 120 min after LPS treatment (Fig. 3D and inset in Fig. 3D), a nuclear STAT3 translocation and an increase in STAT3 immunoreactivity were observed in SFO cells, particularly in cells lying in the core of the SFO (Fig. 3, B and D). In the case of the SFO, the size and the shape of the STAT3-labeled cell nuclei (insets in Fig. 3, B and D) compared with the cresyl violet-counterstained neuronal nuclei (insets in Fig. 3, A and C) do not suggest a predominant nuclear STAT3 translocation in neurons. All other time intervals investigated showed no difference in STAT3 immunoreactivity between pyrogen- and vehicle-treated animals (data not shown).
Finally, the AP seemed to be the one exception out of the sensory CVOs that did not show a specific pyrogen-induced nuclear STAT3 translocation. Neither 60-min IL-6 (Fig. 3F) nor 120-min LPS treatment (Fig. 3H) induced an obvious difference in nuclear STAT3 signals compared with the respective controls (Fig. 3, E and G). However, irrespective of the postinjection time interval investigated and the type of treatment, a few AP cells just adjacent to the fourth ventricle displayed nuclear STAT3 labeling. This nuclear STAT3 labeling was often hidden in a line of cells along the fourth ventricle with moderate cytoplasmic STAT3 expression (Fig. 3, E-H).
The time dependency of specific nuclear STAT3 translocation was quantitatively verified by counting the STAT3-labeled cell nuclei in the OVLT and SFO (Fig. 4). Although in the vehicle-treated rats at 60 and 120 min none to two cell nuclei were positively labeled within the OVLT and the SFO, treatment with both pyrogens (IL-6 and LPS) significantly increased the number of STAT3-stained nuclei. Sixty minutes after systemic IL-6 application, an increase in STAT3-labeled nuclei up to a number of 56 ± 17 (OVLT) and 34 ± 16 (SFO) was observed, which almost disappeared 120 min after IL-6 treatment, with only 8 ± 4 (OVLT) and 1 ± 1 (SFO) cell nuclei being STAT3 labeled. With 3 ± 1 (OVLT) and 1 ± 1 (SFO) STAT3-labeled nuclei, no significant change was observed 60 min after LPS treatment, but the number of STAT3-positive nuclei rose up to 102 ± 13 (OVLT) and 348 ± 100 (SFO) 120 min after LPS application. This LPS-induced peak in nuclear STAT3 translocation proved to be gone 180 min after LPS application, with only one or two cell nuclei being labeled (not shown, n = 3).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Role of IL-6 and Sensory CVOs in Fever
IL-6 is regarded as one of the endogenous mediators of LPS-induced fever. In line with this concept, an excellent correlation of bioactive IL-6 in plasma with the febrile changes of body temperature was found during LPS-induced fever in the present investigation and various previous studies (20, 23, 24). This correlation between febrile body temperature and plasma IL-6 alone of course does not prove that fever is really caused by circulating IL-6. However, within the last years, some additional experimental evidence for a critical role of IL-6 in fever has accumulated. Thus it has been shown that the febrile response to LPS or IL-1 is completely abolished in
IL-6-deficient ("knockout") mice (8). These mice, on
the other hand, develop fever in response to central administration of
exogenous IL-6, suggestive of IL-6 gene expression being essential for
the manifestation of the febrile response (8). In
addition, circulating IL-6 has also been identified as a critical
component of fever in an experimental model of localized inflammation
in rats (7). However, the results shown in Fig. 1 indicate
that IL-6 may participate in, but is clearly not responsible alone for,
the febrile response of rats treated with an intraperitoneal injection
of LPS, since fever after administration of the relatively high dose of
rrIL-6 (45 µg/kg) was rather moderate. In this context, it has been
suggested that circulating IL-6 needs a still unidentified "cofactor" to elicit a more pronounced febrile response
(7).
If circulating IL-6 is involved in LPS-induced fever, then the question arises of how the pyrogenic message is transported to the thermoregulatory centers in the brain where the febrile response is generated. One current hypothesis is that central fever induction pathways involve the CVOs lacking a BBB as potential sites where circulating pyrogens interact with the brain via prostaglandin-dependent mechanisms (2, 26). Such critical pyrogenic zones in the brain, which were initially revealed in lesion and knife-cut studies, include the medial preoptic area of the rostral hypothalamus and all sensory CVOs (3, 4, 12, 19, 32).
In situ hybridization experiments investigating the constitutive expression of IL-6 receptor mRNA in the rat brain showed strong signals in neurons of the medial preoptic area (27) and low signals within the sensory CVOs (the AP, the OVLT, and the SFO) (35). This is in accordance with our laboratory's recent neuroanatomic results (14, 15) revealing that an IL-6-induced activation of the IL-6 receptor signaling cascade was more pronounced in ventromedial parts of the preoptic area compared with the OVLT or the SFO after central IL-6 application. No nuclear STAT3 translocation was found in the AP after central IL-6 treatment (14). This is once again in line with a recent study (35) showing an upregulation of IL-6-receptor mRNA expression from low to strong (OVLT) and from low to moderate levels (SFO) 3 h after LPS treatment, whereas no changes were induced within the AP. Therefore, in summary, this suggests that, of the sensory CVOs, the OVLT and the SFO seem particularly to represent the target structure for LPS-induced circulating IL-6. In addition, the close proximity of the OVLT to rostral parts of the ventromedial parts of the preoptic area makes both hypothalamic areas an attractive pyrogenic zone that initiates and/or mediates LPS-induced febrile responses with IL-6 as its endogenous pyrogen.
Molecular Aspects of STAT3-Induced Genomic Activation in IL-6- and LPS-Induced Fever
STAT3 is activated in response to ligand binding at the IL-6 cytokine receptor family. Activation of STAT3 involves cytokine-induced dimerization of the gp130 receptor, association of JAKs to the gp130 receptor complex, and JAK-mediated phosphorylation of STAT3. Then phospho-STAT3 dimerizes and translocates into the cell nucleus where it binds to specific response elements of target gene promoters (33). Konsman et al. (18) and also the present study have employed STAT3 immunohistochemistry after systemic LPS treatment as a molecular and neuroanatomic marker of direct IL-6-receptor activation and consequent genomic activation. In the assessment of functionally active central IL-6 target structures, a nuclear STAT3 translocation was predominantly observed within the OVLT and the SFO for both groups: the 60-min rrIL-6-treated and the 120-min LPS-treated group (Table 1 and Figs. 2-4). Although we have no exclusive proof for the involvement of IL-6 as the endogenous mediator of LPS-induced fever that induces nuclear STAT3 translocation in these two CVOs, the similarity of the nuclear STAT3 translocation pattern induced by the pyrogens IL-6 and LPS is striking. The peak in STAT3-labeled cell nuclei occurred 60 min after IL-6 treatment, whereas LPS needed an additional 60 min to exert its peak in nuclear STAT3 translocation. This correlates well with the idea of LPS-induced IL-6 formation (20, 23) and the plasma IL-6 levels being elevated already 1 h after LPS injection (Fig. 1).On the basis of the initial appearance of nuclear STAT3 translocation, there might seem to exist no obvious correlation between the time point of nuclear STAT3 translocation with the time course of the pyrogen-induced febrile response. However, according to the current concept of how fever is likely to be initiated in the brain, a cytokine-induced genomic activation of prostaglandin synthesis needs to take place (5, 11, 22, 25, 26). This is exactly the point at which STAT3 could exert its role as a transcription factor. Indeed, a gene sequence analysis of the inducible cyclooxygenase 2 (COX2) gene (Rattus norvegicus) revealed a STAT3 consensus sequence (29) in the promoter region (66 CTGGRAA 74) of the COX2 gene. Therefore, the time gap in between the pyrogen-induced nuclear STAT3 translocation (60 min for IL-6 and 120 min for LPS) and the maximum febrile response (~200 min for IL-6 and ~300 min for LPS) might be exactly the time necessary for transcription of the COX2 gene and consequent synthesis of prostaglandin E2.
Perspectives
Our results represent evidence for a genomic activation of important thermoregulatory brain structures by circulating cytokines via the transcription factor STAT3. Although this genomic activation seems to be mediated by blood-borne IL-6, other endogenous mediators of LPS-induced fever, such as TNF- and IL-1, also contribute to
fever responses induced by bacterial infection. These cytokines use
similar intracellular signaling pathways; however, other members of the
STAT family and other signaling pathways are thought to be involved. To
functionally map cytokine action on the brain, STAT
immunohistochemistry offers the opportunity to reveal a
cytokine-specific pattern of genomic activation in the brain with
distinct STAT molecules as different neuroanatomic markers.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Hopkins (University of Manchester, Salford, UK) for providing us with the B9 cell line and for the protocol of the IL-6 assay. We thank Dr. Stephan Barth (Federal Research Centre for Nutrition, Karlsruhe, Germany) for the STAT3 consensus sequence analysis of the COX2 gene. Rat recombinant IL-6 was provided by the BIOMED I program PL-391450 (Dr. Stephen Poole, Herts, UK).
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft and Research Grant I-572-22.2/1998 to R. Gerstberger by the German-Israeli Foundation.
Address for reprint requests and other correspondence: T. Hübschle, Veterinary-Physiology, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany (E-mail: thomas.huebschle{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.
First published February 8, 2002;10.1152/japplphysiol.00822.2001
Received 6 August 2001; accepted in final form 6 February 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Aarden, LA,
DeGroot ER,
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