Brain-specific endothelial induction of prostaglandin E2 synthesis enzymes and its temporal relation to fever

Neuroscience Research 44 (2002) 51 /61 www.elsevier.com/locate/neures

Brain-specific endothelial induction of prostaglandin E2 synthesis enzymes and its temporal relation to fever Wataru Inoue a, Kiyoshi Matsumura a,*, Kanato Yamagata b, Takako Takemiya b, Takuma Shiraki a, Shigeo Kobayashi a a

Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan b Department of Neuropharmacology, Tokyo Metropolitan Institute for Neuroscience, Fuchu 183-8526, Japan Received 11 April 2002; accepted 14 May 2002

Abstract Brain endothelial cells are hypothesized to be the major source of prostaglandin E2 (PGE2) responsible for fever because they express 2 PGE2-synthesizing enzymes (cyclooxygenase-2 and microsomal-type PGE synthase) in response to pyrogens. To further validate this hypothesis, we examined in rats whether endothelial expression of these enzymes occurs only in the brain, and whether the time course of enzyme expression in brain endothelial cells can explain the time courses of brain PGE2 level and fever. Intraperitoneal injection of lipopolysaccharide induced these enzymes only in brain endothelial cells, but not in those of peripheral organs including the neck, heart, lung, liver and kidney. Induction of these enzymes in brain endothelial cells was first noticed at 1.5 h after lipopolysaccharide injection, at which time elevation of PGE2 was also first detected. Fever started just after this time point. These results demonstrate the significance of brain endothelial cells in the PGE2 production during fever. Unexpectedly, PGE2 level markedly dropped at 5 h in spite of high levels of these enzymes, implicating the existence of an unknown mechanism that suppresses PGE2 level during the recovery phase of fever. # 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved. Keywords: Cyclooxygenase-2; PGE synthase; Cerebrospinal fluid; Lipopolysaccharide; Rat

1. Introduction Fever, a common symptom of various infectious diseases, is brought about as a result of immune-brain signaling. This signaling is now well documented as the following scenario: first, exogenous pyrogens activate peripheral monocytic cells and cause them to release proinflammatory cytokines as endogenous pyrogens. Second, these proinflammatory cytokines act somewhere in the brain and induce prostaglandin E2 (PGE2) biosynthesis. Third, the elevation of the brain PGE2 level brings about fever (Dinarello et al., 1988; Kluger, 1991; Rothwell, 1997; Matsumura et al., 1998b; Dinarello, 1999; Matsumura and Kobayashi, 2001). Among the components of these signaling cascades,

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PGE2, which directly acts on hypothalamic neurons, is thought to be the final mediator of fever. Although little had been known about the cellular source of brain PGE2, studies during the last decade have provided several important findings on this issue. We and other groups demonstrated the induction of the inducible-type of cyclooxygenase (COX), i.e. COX2, in brain endothelial cells or in brain perivascular cells in response to various pyrogenic stimuli (Cao et al., 1995, 1996, 1998, 2001; Breder and Saper, 1996; Elmquist et al., 1997; Lacroix and Rivest, 1998; Matsumura et al., 1998a; Quan et al., 1998; Laflamme et al., 1999). This fact led us to hypothesize that brain endothelial cells are the major source of the PGE2 that is responsible for fever. This hypothesis was further supported by our recent study on the cellular localization of microsomal-type prostaglandin E synthase (mPGES), a terminal enzyme for PGE2 biosynthesis (Yamagata et al., 2001). That study demonstrated that

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mPGES was coexpressed with COX-2 in brain endothelial cells after the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Furthermore, mPGES was colocalized with COX-2 in the perinuclear region of endothelial cells, implicating their tight functional coupling. Ek et al. (2001) also showed the coexpression of mPGES and COX-2 in brain blood vessels after the intravenous injection of an endogenous pyrogen, interleukin-1b (IL-1b). Although these studies strongly suggest that brain endothelial cells are the major source of PGE2 during fever, there still remain several points to be clarified. First, it is unclear whether brain endothelial cells are the sole source of PGE2 responsible for fever. In other words, we cannot exclude the possibility that PGE2 is also synthesized in endothelial cells of peripheral organs and transported to the brain by the circulation. In fact, several studies demonstrated that plasma PGE2 level also elevates during fever (Skarnes et al., 1981; Davidson et al., 1992, 2001; Morimoto et al., 1992). Second, it is still unclear whether the time course of COX-2 and mPGES induction in the brain endothelial cells can explain that of changes in the brain PGE2 level and that of fever. To answer these questions, first, we examined the expression of COX-2 and mPGES in both mRNA and protein levels in endothelial cells of the peripheral organs and compared it with that in the brain endothelial cells after i.p. injection of LPS. Second, we conducted a precise time course study on fever, the brain PGE2 level and the protein levels of both COX-2 and mPGES in brain endothelial cells after the i.p. injection of LPS.

2. Materials and methods 2.1. Materials Male Wistar rats (8-weeks-old) were purchased from Shizuoka Laboratory Animal Cooperative (Shizuoka, Japan). They were housed four or five to a cage in a room at 269/2 8C with a standard 12:12 h light:dark cycle, with free access to food and water. Other materials and their sources were as follows: LPS of Escherichia coli 026:B6 (Sigma, St. Louis, MO); rabbit polyclonal antibody against human mPGES (Cayman Chemical, Ann Arbor, MI); goat polyclonal antibody against rat COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA); sheep polyclonal antibody against rat von Willebrand factor (Affinity Biologicals, Ont., Canada); secondary antibodies of multiple labeling grade (Jackson Immuno Research, PN); PGE2 monoclonal enzyme immunoassay (EIA) kit (Cayman Chemical); digoxigenin RNA labeling mix (Roche Diagnostics); and T3

and T7 RNA synthesis set (Nippon Gene, Tokyo, Japan). 2.2. Temperature monitoring In conscious rats, abdominal temperature (Tab) was monitored with a telemetry system as described previously (Cao et al., 1997). At least 1 week before the day of an experiment, a temperature transmitter (MiniMitter, Sunriver, OR) was implanted into the abdominal cavity of each rat under pentobarbital anesthesia. The signal from the transmitter was detected by a receiver placed under the cage and was fed to an IBM personal computer through an appropriate interface. Temperature data were taken every 10 min and stored in the computer. 2.3. Sample preparation To study time courses of PGE2 concentration in the cerebrospinal fluid (CSF) and expressions of COX-2 and mPGES, we injected rats with LPS (100 mg/kg in 0.5 ml saline, i.p.) between 09:30 and 10:30 a.m. At six time points, i.e. 0.75, 1.5, 3, 5, 12 and 24 h after the LPS injection, the animals were anesthetized with pentobarbital (50 mg/kg, i.p.) and their heads were fixed in a stereotaxic apparatus. CSF was sampled from the cisterna magna with a 27-gauge needle connected to a microsyringe (0.25 ml) via PE20 tubing. The samples were immediately frozen in liquid nitrogen and stored at /80 8C for EIA. After the CSF sampling, the animals were perfused through the left ventricle with ice-cold 20 mM phosphate buffered saline (PBS; pH /7.4, 50 ml) to remove the blood. The brains and peripheral organs, including neck, heart, lung, liver and kidney, were quickly removed, freshly frozen in dry-ice powder and stored at /30 8C for immunohistochemistry. For each time point, three rats were prepared. In some cases, rats were injected with a higher dose of LPS (400 mg/kg in 500 ml saline, i.p.) and samples were taken 4 h after the injection for in situ hybridization study and 5 h after that for immunohistochemistry. Samples from untreated rats were used as the control. In addition, samples were taken from three rats that had been injected with saline and killed at 5 h after the injection. All experiments were carried out under the Guideline for Animal Experiments of Kyoto University. 2.4. Enzyme immunoassay for PGE2 in CSF The CSF samples were thawed on ice and PGE2 was extracted from 80 ml of CSF with organic solvent (ethylacetate). The extracted samples were assayed for PGE2 with an EIA kit (Cayman Chemical) according to the manufacturer’s instructions.

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2.5. In situ hybridization The detection of COX-2 and mPGES mRNA signals was carried out with digoxigenin (DIG)-labeled cRNA probes. Anti-sense riboprobes for COX-2 and mPGES were prepared from a 1-kb 3? cDNA sequence of rat COX-2 (Yamagata et al., 1993) and a full-length cDNA sequence of rat mPGES (Yamagata et al., 2001), respectively, that were subcloned into appropriately restricted pBS plasmids. Each sense riboprobe of identical length was also made in a similar way and used for the negative control experiment. Brain sections (14 mm thick) were hybridized in a humidified chamber for 12 h at 55 8C with 0.2 mg/ml DIG-labeled riboprobe diluted in hybridization buffer, which contained 50% formamide, 5/SSC (1 /SSC /0.15 M NaCl, 0.015 M sodium citrate), 5 /Denhart’s, 0.25 mg/ml yeast tRNA and 0.5 mg/ml herring sperm DNA. After removal of the probe solution, the sections were washed with 0.1 / SSC at 65 8C. Signals were developed by using alkaline phosphatase-conjugated anti-DIG antibody (1:1000 dilution) (Roche Diagnostics) and standard chromogenic substrates (Boehringer Mannheim). 2.6. Immunohistochemistry For detection of COX-2 and mPGES proteins, the frozen brains and other organs were cut at a thickness of 16 mm in a cryostat and thaw-mounted on glass slides. Subsequent procedures were carried out at room temperature unless stated otherwise. After air-drying, the sections were fixed for 10 min with 2% paraformaldehyde (Pfa) for COX-2 detection or with 0.1% sodium metaperiodate for mPGES detection. The sections were then incubated with 10% normal donkey serum (NDS) for 1 h and thereafter incubated with anti-COX-2 (1:2000 dilution) or anti-mPGES (1:500 dilution) antibody for 12 h at 24 8C. After removal of the primary antibody and a subsequent wash with 0.1 M PBS, the sections were incubated for 2 h with biotin-labeled antigoat IgG (1:500 dilution) for COX-2 detection or with anti-rabbit IgG (1:500 dilution) for mPGES detection. Finally, they were incubated with Cy3-labeled streptavidin D (1:2000 dilution). In the case of triple immunostaining for COX-2, mPGES and von Willebrand factor (v. W. factor), the sections were treated with 2% Pfa followed by 0.02% sodium metaperiodate for 10 min each. After the double-immunostaining of COX-2 and mPGES as described in a previous paper (Yamagata et al., 2001), the sections were further incubated with sheep anti-v. W. factor IgG (1:3000 dilution) for 1 h and then with Cy5-labeled anti-sheep IgG (1:500 dilution) for 1 h. The control experiment for the triple-immunostaining was performed in two ways. First, the staining was carried out in the same way except that the primary antibody solution was preabsorbed with either COX-2

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antigen peptide (1 mg/ml) or mPGES antigen peptide (1 mg/ml). Second, one of the three primary antibodies was substituted with nonimmunized IgG of the same animal species. In all cases, inappropriate cross-reactions among the antibodies were negligible. All antibodies were diluted with 10% NDS in 0.1 M PBS and all other chemicals were dissolved in 0.1 M PBS. Fluorescent images were captured by a confocal microscope (Radiance 2000; Bio-Rad, Hercules, CA). 2.7. Semiquantitative analysis of immunohistochemical images For time course analysis of the immunoreactivity, special care was taken to stain brain sections under the same conditions. This was accomplished by mounting six or seven brain sections from different rats on one glass slide. In this way, brain sections from 24 rats for the time course study were mounted on four glass slides. Two sets of these four glass slides were prepared for immunostaining of COX-2 and mPGES, respectively. For each enzyme immunostaining, these four glass slides were treated in the same way. Fluorescent images of blood vessels were captured by the confocal laserscanning microscope with /20 objective lens. Since these enzymes were induced in blood vessels of the entire brain with no regional specificity, the images for quantification were captured from the thalamic region. This region was suitable for the quantification because every coronal section of this region consistently contained multiple numbers of positively-stained blood vessels and there was no neuronal expression of COX2. In each side of the thalamus of coronal sections, a microscopic view containing the highest amount of immunoreactivity was captured. Sensitivity setting of the laser confocal microscope was kept the same during image capturing for each enzyme. The captured images were converted to 8-bit gray scale images and saved as bit map format files. These images were then analyzed with an image analysis program, Scion Image for Windows (Scion Corp., Frederick, MD). First, we set a threshold at a level slightly higher than the background levels of immunostaining, so that all pixels below this threshold were considered as the background, and excluded from the calculations of the area and intensity of immunostaining. The threshold value was set for COX-2 and mPGES separately and kept constant throughout the measurement of each enzyme. Then, regions of interest (ROIs) were set so that they enclosed all immuno-positive blood vessels in each image. Areas, i.e. the number of pixels above the threshold and their mean gray scale values, were measured within the ROIs. The products of area and mean gray scale of each image were calculated and used to represent the amount of immunoreactive protein in each image. Although it is not determined to what extent the product values are

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linearly proportional to the amounts of enzyme expressed, the values correctly tell us at least the order of enzyme levels expressed along the LPS treatment and should be more objective than evaluating them by visual inspection. The unit for the products is an arbitrary one. Two measurements were made in each rat. The mean value of this amount in each rat was further averaged among three rats for each time point. Data were expressed as the mean9/S.E.M. Student’s t-test was used to examine the statistical significance.

3. Results 3.1. COX-2 and mPGES induction: the central versus peripheral endothelial cells Fig. 1(a /e) shows the results of DIG-based in situ hybridization study for detection of COX-2 and mPGES mRNAs near the central sulcus, where blood vessels were consistently found. In agreement with previous studies by the 35S-based method (Cao et al., 1995; Yamagata et al., 2001), both COX-2 mRNA (Fig. 1b) and mPGES mRNA (Fig. 1d) were induced in blood vessels of the rat brain and subarachinoidal space 4 h after i.p. injection of LPS (400 mg/kg) but not of saline (Fig. 1a,c). The signals for mPGES were so intense that their localization in the blood vessels was unclear (Fig. 1d), but a less magnified view clearly indicated the localization of mPGES mRNA in blood vessels (Fig. 1e). These mRNAs were observed in venous- or venulelike blood vessels in the entire brain of the LPS-treated rats. Indeed, the large artery in the central sulcus (Fig. 1a /d) was consistently negative for both enzymes. On the other hand, in the same LPS-injected rats, none of the signals were observed in blood vessels of peripheral organs, including neck, heart, lung, liver and kidney (data not shown). None of the signals were observed when the sense RNA probes were hybridized with the brain sections (data not shown). The imunohistochemical study yielded essentially the same results as the in situ hybridization study with one

exception, i.e. a low level of mPGES protein was detected in a small number of brain blood vessels in untreated (data not shown) and saline-injected rats (Fig. 1f). On the other hand, no COX-2 expression was detected in brain blood vessels of untreated or salinetreated rats. LPS injection (400 mg/kg, i.p.) induced COX-2 and mPGES (Fig. 1g) proteins in the blood vessels throughout the brain 5 h after the injection. Triple immunostaining for COX-2 (Fig. 1h), mPGES (Fig. 1i) and v. W. factor (Fig. 1j), an endothelial marker, clearly demonstrated that COX-2 and mPGES proteins were coexpressed in the same intracellular compartment of endothelial cells (Fig. 1k). As shown in our previous study, COX-2 and mPGES were colocalized in the nuclear envelope. In contrast to the strong coexpression of COX-2 and mPGES in brain endothelial cells, neither COX-2 nor mPGES immunoreactivity was detected in endothelial cells of any of the peripheral organs we examined, including neck, heart, lung, liver and kidney. Fig. 1(l/q) shows double immunostaining for COX-2 and v. W. factor (Fig. 1l, n, p) and for mPGES and v. W. factor (Fig. 1m, o, q), in the peripheral organs of LPS-treated rats. In the neck (Fig. 1l, m), endothelial cells of carotid artery, jugular vein and small blood vessels were clearly visualized with antibodies against v. W. factor, but neither COX-2 nor mPGES was observed. This was also the case for pulmonary artery and small blood vessels in the lungs (Fig. 1n, o). In the kidney, COX-2 and mPGES were separately expressed (Fig. 1p, q). COX-2 was intensely stained in the macula densa (Fig. 1p); whereas mPGES was so in a portion of urinary tubules (Fig. 1q), which was recently reported to be the proximal urinary tubules (Guan et al., 2001). These expressions were seen even in untreated and salinetreated rats. Again, endothelial cells visualized with v. W. factor immunostaining expressed neither of the enzymes. These results were consistent in rats receiving a lower dose of LPS (100 mg/kg) and killed between 0.75 and 5 h after the LPS injection. This time period covers the peak time of brain PGE2 level and fever as shown later. Thus, these results exclude the possibility that

Fig. 1. Cellular expression of COX-2 and mPGES at the mRNA (a /e) and protein (f /q) levels. Rats were injected intraperitoneally with saline or LPS (400 mg/kg) and killed 4 h after the injection for the mRNA study and 5 h after the injection for the protein study. Neither COX-2 mRNA (a) nor mPGES mRNA (c) was observed in brain blood vessels in saline-injected rats. LPS injection induced mRNAs for both COX-2 (b) and mPGES (d, e) in brain blood vessels (indicated by arrows in b and d). Although vessel structures in (d) were difficult to see due to the intense signals of the mPGES mRNA, a less magnified view (e) clearly demonstrated that mPGES mRNA was expressed along blood vessels. mPGES protein was faintly expressed in a small number of brain blood vessels in untreated rats as well as in saline-injected rats (f) and was markedly upregulated by the LPS injection (g). Inset in (f) is a magnified view of a blood vessel indicated by the arrow. h /k: Triple immunostaining of a brain blood vessel for COX-2 (h), mPGES (i) and v. W. factor (j) 5 h after the LPS injection. The overlaid image (k) indicates that COX-2 (h) and mPGES (i) were colocalized in brain endothelial cells identified by the v. W. factor immunostaining (j). l /q: Double immunostaining of v. W. factor (green) with either COX-2 (red in l, n, p) or mPGES (red in m, o, q) in the neck (l, m), lung (n, o) and kidney (p, q) 5 h after the LPS injection. In the neck, neither COX-2 (l) nor mPGES (m) was detected in the endothelial cells of the carotid artery (arrow), jugular vein (arrowhead) or small blood vessels. Endothelial cells in the lung were also negative for COX-2 (n) and mPGES (o). In the kidney, COX-2 was negative in the endothelial cells of arteries (inset in p) and glomerulus (* in p), but was constitutively expressed in the macula densa cells. mPGES was also negative in the glomerular endothelial cells, but was constitutively expressed in a subset of urinary tubules. Scale bars: a /d: 100 mm; e: 200 mm; f, g: 100 mm; inset of f: 20 mm; h /k: 20 mm; l, m: 100 mm; n, o: 25 mm; p, q: 25 mm.

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Fig. 1

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peripheral endothelial cells produce a large amount of PGE2 as brain endothelial cells do in this time period. This consideration led us to ask whether the time course of enzyme expression in brain endothelial cells can explain the time courses of brain PGE2 level and fever. 3.2. Temporal relationships among fever, PGE2 in CSF and the enzyme expression To examine the precise temporal relationships among fever, PGE2 level in the CSF and expressions of COX-2 and mPGES, we collected CSF and brain samples at six time points after the LPS injection. These time points approximately correspond to the latent phase (0.75 h), onset (1.5 h), rising phase (3 h), maximum febrile phase (5 h), recovery phase (12 h) and complete recovery phase (24 h) of fever. Samples from untreated rats were used as the control (n /3). In addition, samples were taken from another three rats 5 h after an i.p. injection of saline as a second control. Fig. 2(A) shows the time course of fever. In both the LPS- and saline-injected rats, the Tab increased slightly (B/0.5 8C) immediately after the injection. This increase in Tab seemed to be caused by the stress associated with the injection. The Tab of the LPSinjected rats again started to increase between 1.5 and 3 h after the injection, reached its maximum at 5 h and then gradually declined. The Tab of saline-injected rats showed no increase until 8 h after the injection and then started to elevate. This elevation was caused by the circadian change in Tab because a similar elevation was also observed in untreated rats (data not shown). Fig. 2(B) shows the time course of PGE2 concentration in the CSF. The value at time point zero indicates that of untreated rats. At 0.75 h after the injection, the PGE2 level stayed at the level comparable to that in untreated rats. The PGE2 level started to rise between 0.75 and 1.5 h and further elevated by 3 h. Then, it dropped to half of the maximum level by 5 h and returned to the baseline level by 12 h. The PGE2 level in the saline-treated rats was the same as that of untreated rats. This figure points out an important relationship between fever and PGE2 level in the CSF. Fever followed the rise in the PGE2 level with a slight time lag. In particular, at 1.5 h, PGE2 was elevated while Tab was still at the baseline level. Just after this time point, fever started. On the contrary, by 5 h, PGE2 had markedly dropped, whereas Tab was still at the maximum level. This time lag is likely due to the heat capacity of the body. Thus, the relationship agrees well with the established theory for PGE2 as the central force of fever. Fig. 3 shows the appearances of COX-2 (Fig. 3A) and mPGES (Fig. 3B) protein expression in brain blood vessels at each time point. The photos represent the most intensely-stained blood vessels found in sections of

the thalamic region. Sensitivity setting of laser confocal microscope was kept the same during image acquisition for each enzyme. Fig. 4 shows the results of semiquantification of the enzyme levels in the confocal microscope images. At 0.75 h after LPS, there was no detectable change in the expression of either enzyme. At this time point, mPGES was faintly detectable in a small number of blood vessels, but its abundance and intensity did not differ from those in untreated rats. Elevation of the enzyme levels was first noticed at 1.5 h. After this time point, these two enzymes followed distinct time courses: COX-2 reached its maximum level by 3 h, stayed at a comparable level until 5 h, markedly dropped by 12 h and returned to the control level by 24 h. In contrast, mPGES changed more slowly. Its level continued to elevate until 12 h and dropped by 24 h, at which time, however, it was still expressed at an amount comparable to that at 5 h. Thus, there was a clear dissociation of COX-2 and mPGES levels during the later phase of fever. Fig. 5 summarizes temporal relationships among PGE2 level in CSF and the enzyme levels. The values in Fig. 5 are expressed as the ratio to their maximum values and time scale was expanded for the early time points to present their relationships more clearly. Fig. 5 points out two important relationships among them: (i) the initial elevation of PGE2 in the CSF coincided with the expression of enzymes in the brain endothelial cells, thus supporting the causal relationship between the enzymes in endothelial cells and PGE2 in the CSF; and (ii) however, the enzyme levels and PGE2 level obviously dissociated at 5 h. Both COX-2 and mPGES were at high levels, whereas the PGE2 level dropped markedly. Thus, the PGE2 level cannot be explained solely by the enzyme levels. This result suggests the presence of other factor(s) involved in the regulation of the PGE2 level during fever.

4. Discussion It is widely accepted that elevation of PGE2 in the brain triggers the febrile response and other neurological responses that accompany infection/inflammation. Recently, several groups including ours, provided evidence that brain endothelial cells are the major source of PGE2 that is responsible for the fever response (Cao et al., 1995; Matsumura et al., 1998a; Rivest, 1999; Ek et al., 2001; Yamagata et al., 2001). The present study further supports this idea by demonstrating the following two facts: (1) in contrast to brain endothelial cells, those in the peripheral organs neither expressed COX-2 nor mPGES in response to the i.p. injection of LPS, suggesting the dominant and specific role of brain endothelial cells in PGE2 production; and (2) the expression of COX-2 and mPGES in endothelial cells

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Fig. 2. (A) Time courses of fever (Tab change) after i.p. injection of LPS (100 mg/kg, thick gray line with open circles, n/5) or saline (thin black line with closed circles, n/5) at the time point zero. Although the Tab was measured every 10 min, error bars were shown only at seven time points, i.e. 0, 0.75, 1.5, 3, 5, 12 and 24 h after the injection. Asterisks indicate that the values were different from those at time point zero with statistical significance (P B/0.05). (B) Time course of PGE2 concentration in the CSF after i.p. injection of LPS (open circles) or saline (closed circle). Asterisks indicate that the values were different from those of untreated rats plotted at time point zero with statistical significance (P B/0.05). Dotted lines indicate 1.5 h, at which time elevation of PGE2 was first detected.

coincided with the elevation of PGE2 in the CSF and preceded the onset of fever, satisfying the temporal correlation between the molecular and physiological events.

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Fig. 4. Semiquantitative analysis of COX-2 (A) and mPGES (B) level induced in brain blood vessels after i.p. injection of LPS (100 mg/kg). The values are expressed as the percentage of the maximum values. Asterisks indicate that the values were different from those of untreated rats plotted at time point zero with statistical significance (P B/0.05).

4.1. Rationale to study COX-2 and mPGES PGE2 is produced from arachidonic acid through the enzymatic cascade of COX and PGES. At present, two isoforms of COX and PGES have been molecularly identified, i.e. for the former, COX-1 and COX-2 and for the latter, mPGES and cytosolic PGES (cPGES). Thus, PGE2 can be theoretically produced through various combinations of COX and PGES isoforms. However, during fever, COX-2 seems to play the essential role in PGE2 production. This is evidenced by the following two facts: a COX-2 inhibitor completely suppressed both elevation of PGE2 in the CSF (Yamagata et al., 2001) and fever (Cao et al., 1997). COX-2

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Fig. 3

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Fig. 5. Temporal relationships among PGE2 level in the CSF (closed circles) and expression of COX-2 (open squares) and mPGES (closed squares). Note that the time scale for the earlier time points was expanded for a better representation of their relationship.

gene-disrupted mice do not develop fever, whereas COX-1 gene-disrupted mice do develop fever (Li et al., 1999). As for PGES, there is neither a specific inhibitor nor gene-disrupted mice available at present and therefore, the exact roles of each PGES isoform in fever are not conclusively known. However, studies by Murakami et al. (2000) and Tanioka et al. (2000) demonstrated that COX-2 functionally couples with mPGES more tightly than with cPGES, whereas COX-1 does so with cPGES. Preferential coupling of COX-2 and mPGES seems to be supported at least by their intracellular colocalization at the nuclear envelope, as shown in our previous study (Yamagata et al., 2001) and in the present study. In addition, we could not find colocalization of COX-2 with the other PGES, i.e. cPGES, in rat brain (our unpublished observation). These facts provided us with the rationale to focus this study on COX-2 and mPGES. 4.2. Significance of brain endothelial cells as the source of PGE2 We previously showed that the expression of COX-2 and mPGES took place in blood vessels of the entire central nervous system in response to i.p. administered LPS (Cao et al., 1995; Yamagata et al., 2001). However, little was known about the peripheral endothelial cells as a possible source of PGE2. Several studies demonstrated that the blood level of PGE2 was elevated in response to LPS or endogenous pyrogens (Skarnes et al., 1981; Davidson et al., 1992, 2001; Morimoto et al., 1992) and

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suggested that PGE2 in the blood may enter the brain and evoke fever. These facts led us to ask whether peripheral endothelial cells also might produce PGE2 during fever. At 3/5 h after LPS injection, when the robust induction of COX-2 and mPGES was observed in brain endothelial cells, COX-2 and mPGES were hardly observed in endothelial cells of peripheral blood vessels including carotid artery, jugular vein and those in the lung, heart, liver and kidney. This was also the case for the earlier time points and for the mRNA levels as well. Although we cannot completely exclude the possibility that small amounts of these enzymes were expressed in the peripheral endothelial cells, we can conclusively state that brain endothelial cells are much more sensitive to LPS than peripheral cells in terms of the activation of PGE2 biosynthesis. Thus, peripheral endothelial cells unlikely contribute to the elevation of blood PGE2. Instead, elevation of plasma PGE2 during fever seems to be attributable to tissue macrophages, circulating blood cells or brain endothelial cells. The intravenous injection of PGE2 evokes fever under certain conditions (Eguchi et al., 1988; Romanovsky et al., 1999) and permeability of brain blood vessels to PGE2 is elevated by pretreatment with LPS or other pyrogenic cytokines (Davidson et al., 2001). Therefore, if plasma PGE2 level far exceeds brain PGE2 level, it may enter the brain from the circulation and evoke fever. However, available literature suggests PGE2 in the CSF is generally higher than that in the plasma. For example, Morimoto et al. (1992) showed that, in rabbits that had received an i.v. administration of IL-1b, the PGE2 level in push /pull perfusate of the third ventricle of the brain was comparable to or higher than the PGE2 level in plasma of the jugular vein and much higher than that of the carotid artery. Similarly, Davidson et al. (2001) found that the PGE2 level in the push /pull perfusate of the third ventricle was comparable to that in the plasma prepared from the marginal ear vein in rabbits that had been intravenously injected with LPS, double-stranded RNA or IL-1b. In both studies, this observation was consistent during the time period between 1 (or 1.5 h) and 5 h after the injection of pyrogens, the time mostly covering the fever period. Here, we must consider two issues: first, push /pull perfusion results in a dilution of CSF and therefore, actual concentration of PGE2 in the CSF should be much higher than that in the push /pull perfusate. In fact, we found over ten times higher concentration of PGE2 in undiluted CSF compared with that of venous plasma in rats 3 h after i.v. injection of LPS (25 mg/kg, i.v., our unpublished observation). Secondly, a large portion of blood PGE2 becomes inactive when it passes

Fig. 3. Expressions of COX-2 (A) and mPGES (B) in brain blood vessels after i.p. injection of LPS (100 mg/kg). The rats were killed at the indicated time after the LPS injection. The blood vessels shown here represent those most intensely stained in the diencephalic region of each section. Scale bar indicates 100 mm.

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through the lungs (Piper et al., 1970; Hamberg and Samuelsson, 1971). In addition, the post-lung circulatory system, which is composed of the left atrium, left ventricle and carotid artery, was negative for COX-2 and mPGES, as shown in the present study. This means that the PGE2 concentration in arterial blood, which perfuses the brain, should be much lower than that in the vein. Thus, it is very unlikely that PGE2 detected in the CSF was derived from the peripheral circulation in the time period later than 1 h after the injection. This, in turn, strongly implies the significance of brain endothelial cells as the source of brain (CSF) PGE2. One possible situation in which blood PGE2 enters the brain, if any, should be during the very early time period, i.e. around 30 min after the i.v. injection of pyrogen (Romanovsky et al., 1998). At this time point, PGE2 level is higher in the blood of marginal vein than in the CSF (Davidson et al., 2001). Thus, entry of PGE2 from the circulation to the brain may occur and support the very early phase of fever, at which time the expression of effective amounts of COX-2 and mPGES is hard to assume. 4.3. Temporal relationships among fever, PGE2 in the CSF and the enzyme expression This study is the first to provide detailed information on the temporal relationships among fever, PGE2 level in the CSF and the expression of COX-2 and mPGES. The results given here provide strong support for our current hypothesis and also represent some unexpected findings that gave us a deeper insight into the regulation of fever. In particular, we would like to emphasize that the first elevation of the PGE2 level in the CSF was observed at 1.5 h after the LPS injection and this was coincided with the first elevation of COX-2 and mPGES expression levels. Fever started just after this time point. These results constitute strong evidence for the causal relationship between the enzyme expression, the PGE2 level in the CSF and fever. At 3 h, COX-2 and PGE2 levels reached their peaks, while mPGES continued to elevate until 12 h. Thus, the COX-2 level coincided with the PGE2 level very well; and, at first glance, COX-2 likely played the rate-limiting role in PGE2 biosynthesis. However, these enzyme levels alone cannot explain the whole time course of the changes in the PGE2 level. In particular, at 5 h after the LPS injection, the COX-2 level was still high, almost comparable to that at 3 h and mPGES level was even higher than that at 3 h. In spite of the high levels of the enzymes, the PGE2 level dropped markedly at this time point. Thus, the PGE2 level is not a simple reflection of the enzyme levels, suggesting the presence of additional factors that influence the PGE2 level in the CSF. There are three possible mechanisms that can explain the dissociation

between PGE2 and enzyme levels: (i) reduced supply of the COX-2 substrate (arachidonic acid); (ii) inhibition of COX-2 and mPGES activity; and (iii) stimulated degradation of PGE2 or excretion of it from the brain to the circulation. At present, we have no solid evidence for any of these mechanisms. Elucidation of this mechanism will provide a new insight into the endogenous antipyretic mechanism.

Acknowledgements This study was performed through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government and supported partly by a grant from the program Grants-in Aid for Scientific Research (B) of the Japan Society for the Promotion of Science (to K.M.).

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