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The Journal of Neuroscience, January 15, 1999, 19(2):716-725
Lipopolysaccharide Injected into the Cerebral Ventricle Evokes
Fever through Induction of Cyclooxygenase-2 in Brain Endothelial
Cells
Chunyu
Cao,
Kiyoshi
Matsumura,
Masashi
Ozaki, and
Yasuyoshi
Watanabe
Department of Neuroscience, Osaka Bioscience Institute, Furuedai
6-2-4, Suita, Osaka 565-0874, Japan
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ABSTRACT |
Activation of the arachidonic acid cascade is an essential step for
the development of fever during brain inflammation. We investigated the
brain sites where this activation takes place by use of a rat model of
brain inflammation. Intracerebroventricular administration of
lipopolysaccharide but not of its vehicle evoked fever. The fever was
markedly suppressed when the rats had been pretreated with a
cyclooxygenase-2-specific inhibitor. In situ hybridization and immunohistochemical studies revealed that
cyclooxygenase-2 mRNA and its protein were induced by
lipopolysaccharide in blood vessels near the cerebral ventricles and in
those in the subarachnoidal space. Double immunohistochemical staining
revealed that these cyclooxygenase-2-positive cells were mostly
endothelial cells. The time course of fever and that of
cyclooxygenase-2 induction in the endothelial cells were in parallel.
Cyclooxygenase-2 mRNA in a certain type of telencephalic neurons was
also upregulated by the intracerebroventricular administration, but
this neuronal response occurred both in vehicle-injected rats and in
lipopolysaccharide-injected ones to the same extent. Therefore, the
neuronal response was not essential to the development of fever. These
results suggest that brain endothelial cells play a crucial role in the
development of fever during brain inflammation by activating their
arachidonic acid cascade.
Key words:
brain inflammation; fever; meningitis; cyclooxygenase; prostaglandin; endothelial cells
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INTRODUCTION |
Fever is a common symptom associated
with various types of cerebral inflammation, including meningitis and
encephalitis. Under such inflammatory conditions, one essential process
leading the patients to fever is the activation of the arachidonic acid
cascade in the brain (for review, see Milton, 1982 ), in which membrane phospholipid is enzymatically converted to several kinds of bioactive prostaglandins (PGs). Among the PGs, PGE2 is most likely to
be the mediator of fever because it is highly pyrogenic when injected into the cerebral ventricle or into the preoptic area, the
thermoregulatory center and a presumed locus of PGE2 action
(Milton, 1982; Stitt, 1986). The PGE2 level in the
CSF indeed increases during fever (Bernheim et al., 1980;
Coceani et al., 1983). Little is known, however, where in the brain
such activation of the arachidonic acid cascade takes place during the
cerebral inflammation. Although there are several cell groups
potentially to be the source of PGs, including neurons (Fujimoto et
al., 1992; Van Dam et al., 1993), astrocytes (Katsuura et al., 1989;
O'Banion et al., 1996), microglia (Matsuo et al., 1995; Minghetti and
Levi, 1995; Fiebich et al., 1996; Bauer et al., 1997; Slepko et al.,
1997), and endothelial cells (Moore et al., 1988; Van Dam et al., 1993;
de Vries et al., 1995), it has not been clarified yet which of these
cell groups plays the major role in the production of PGs during
cerebral inflammation in vivo.
To answer the above question, we focused our attention on
cyclooxygenase-2 in the brain. Cyclooxygenases (COXs), i.e.,
cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), are
rate-limiting enzymes in the biosynthesis of PGs and are the targets of
the nonsteroidal anti-inflammatory drugs such as aspirin and
indomethacin (Vane, 1971; Goppelt-Struebe, 1995; Herschman, 1996).
COX-2 is distinct from COX-1 in that COX-2 is immediately induced by
inflammatory stimuli and, therefore, is considered to be involved in
the PG biosynthesis during inflammation (Goppelt-Struebe, 1995;
Herschman, 1996). In the present study, we used an experimental model
of fever associated with brain inflammation, in which rats received an
intracerebroventricular injection of lipopolysaccharide (LPS). The pathophysiological events occurring in this experimental model are
particularly relevant to those of meningitis (McAllister et al., 1975;
Sanna et al., 1995). In this experimental model, we examined whether
COX-2 is involved in this type of fever, and if so, where COX-2 is
induced in the brain.
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MATERIALS AND METHODS |
Materials. Male Wistar rats of 7 weeks of age were
purchased from Shizuoka Laboratory Animal Cooperative (Shizuoka,
Japan). They were housed four or five to a cage in the room at 26 ± 2°C with a standard 12 hr light/dark cycle until they had surgery. Other materials used and their sources are as follows: autoradiographic 14C microscales (RPA511 and RPA504);
[35S]UTP and x-ray films ( max) (Amersham,
Arlington Heights, IL); emulsion for microautoradiography (NTB2)
(Eastman Kodak, Rochester, NY); lipopolysaccharide of Escherichia
coli O26:B6 (Sigma, St. Louis, MO); RNA transcription kit
(Stratagene, La Jolla, CA); NS-398, a COX-2-specific inhibitor (a
generous gift from Dr. S. Higuchi of Taisho Pharmaceutical Co. Ltd,
Tokyo, Japan); rabbit polyclonal antibody against murine COX-2 (Cayman
Chemical); rabbit polyclonal antibody against human von Willebrand
(vW) factor (Dako, Carpinteria, CA); and mouse monoclonal
antibody OX-42 against rat microglia/macrophages (Serotec, Oxford, UK).
Surgery. Under pentobarbital anesthesia, temperature
transmitters (Mini-Mitter, Sunriver, OR) were implanted in the
abdominal cavity of the rats. Their heads were then fixed in a
stereotaxic apparatus (Narishige, Tokyo, Japan) according to a brain
atlas (Paxinos and Watson, 1986). The skin of the skull was incised in
the midline, and two holes (0.8 mm in diameter) for
intracerebroventricular injection were drilled into the skull so that
they were located above the right and left lateral ventricles (0.8 mm
posterior and 1.2 mm lateral to the bregma). The incision of the skin
was closed, and the rats were housed individually in cages placed on
the receivers of the temperature telemetry system (Data Sciences, St.
Paul, MN).
Experimental protocol. Intracerebroventricular injections
were made after a recovery period of at least 1 week. Between 9:00 and
9:30 A.M. on the day of the experiment, the rats were lightly anesthetized with halothane (3% in the air) and received an
intracerebroventricular injection of either LPS (50 ng/rat in 15 µl
of saline) or saline alone. The injections were made through one of the
holes in the skull by inserting a stainless steel needle (27 gauge)
that was connected to a 1 ml disposable syringe via a polyethylene
(PE) tubing (PE20, Cray Adams) of 50 cm length. The syringe, PE
tubing, and the needle were filled with either saline or LPS solution. Once the needle had been inserted into the brain 4 mm from the skull,
the syringe was removed from the PE tubing, and the free end of the PE
tubing was lifted up so that it was located 50 cm above the rat's
head. If the tip of the needle was properly located in the lateral
ventricle, the solution in the PE tubing was delivered into the
ventricle by hydrostatic pressure. This was confirmed by a drop in the
level of meniscus of the solution in the PE tubing. The volume of the
solution injected was checked with the precalibrated scale put on the
PE tubing. If the meniscus of the solution did not drop, the needle was
slightly moved until the meniscus started to drop. After the injection,
the rats were returned to their home cages. The whole process of this
injection took ~5 min for each rat. The other hole in the skull was
left unused. Abdominal temperature (Tab) of each rat
was monitored every 10 min during a 24 hr period before and after the injection.
To investigate the effect of COX-2 inhibitor on the febrile response,
we injected rats intraperitoneally with either NS-398 (4 mg/kg, i.p.)
or its vehicle [500 µl of 50% dimethyl sulfoxide (DMSO) in saline]
1 hr before the injection of LPS. NS-398 possesses a high selectivity
for COX-2 as shown by several in vitro and in
vivo studies (Futaki et al., 1993a,b, 1994; Masferrer et al., 1994; Gierse et al., 1995).
In situ hybridization and immunohistochemistry.
Based on the time course of fever, two time points, i.e., 1.5 and 3.5 hr after the intracerebroventricular injections, were chosen for
sacrificing the animals for in situ hybridization of COX-2
mRNA. For each time point, four or five rats were anesthetized with
diethylether and perfused via the left ventricle with 50 ml of ice-cold
saline followed by 200 ml of 4% paraformaldehyde solution, pH 6.5. The brains were put in a solution containing 20% sucrose and 4%
paraformaldehyde overnight, frozen in dry-ice powder, and stored at
 80°C until used. In situ hybridization of COX-2 was done
with 35S-labeled rat COX-2 antisense riboprobe as described
in our previous study (Cao et al., 1995, 1997). A sense strand
35S-labeled COX-2 probe of identical length was used for
negative-control experiments.
For immunohistochemical detection of COX-2 protein, four time
points, i.e., 2, 4, 6, and 24 hr after the intracerebroventricular injection, were chosen for killing the animals. At each time point, three rats were anesthetized with diethylether and perfused via the
left ventricle with 50 ml of ice-cold 20 mM PBS, pH
7.4. Immunostainings of COX-2, vW factor (an endothelial marker), and a
microglia/macrophage marker were performed as reported previously (Cao
et al., 1998; Matsumura et al., 1998a).
Analysis and statistics. Relative changes in the COX-2 mRNA
level in the telencephalic regions were analyzed on autoradiographic films by use of an image analysis program (NIH Image, version 1.55), as
described in a previous study (Cao et al., 1997). The number of COX-2
immunoreactive (COX-2-IR) cells in the blood vessels was counted
in three consecutive brain sections prepared from each rat. Coronal
sections used for this evaluation corresponded to the plane of bregma
around 0 mm according to a stereotaxic atlas (Paxinos and Watson,
1986). Data were expressed as mean ± SE. The Mann-Whitney
U test was used to evaluate the significance of differences.
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RESULTS |
Fever evoked by intracerebroventricular injection of LPS
Figure 1 shows a 14 hr recording of
the change in Tab after intracerebroventricular injection
of LPS or saline, as well as recordings under the untreated condition
of both groups 1 d before the injection. The average
Tabs at the time of injection, which was made between 9:00
and 9:30 A.M., were 37.0 ± 0.17°C for the saline-injected group
(n = 4) and 36.9 ± 0.18°C for the LPS-injected one (n = 5). These values were not significantly
different from their average Tab (36.9 ± 0.12°C)
measured at 9:00 A.M. 1 d before the injection. The
Tab of the untreated group showed a typical circadian
fluctuation, being low during the day and high during the night. When
the rats received the intracerebroventricular injections,
Tabs of both LPS-injected and saline-injected rats initially decreased because of the light halothane anesthesia and then
recovered to the preanesthesia level until 1.5 hr after the injection.
After this time point, the Tab of LPS-injected rats
continued to rise steeply until 4 hr after the injection and stayed at
this higher level (~1.5°C above the preinjection level) during the
next 6 hr, whereas the Tab of the saline-injected rats
increased slowly and reached a level only slightly higher than that
under the untreated condition.
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Figure 1.
Changes in abdominal temperature
(Tab) after intracerebroventricular injection of LPS
(closed circle) or saline (triangle). LPS
(50 ng/rat) or saline (15 µl) was injected at time point 0, which was
between 9:00 and 9:30 A.M. Each value represents the mean ± SE of
four (saline) or five (LPS) rats. The
change in Tab was significantly different between LPS- and
saline-injected rats in the time period denoted by the
asterisk (p < 0.05). Their
Tab 1 d before the injection was also shown as the
control (open circle).
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Involvement of COX-2 in the fever generation
To examine whether COX-2 is involved in fever evoked by
intracerebroventricular injection of LPS, we pretreated rats with either NS-398 (4 mg/kg), a COX-2-specific inhibitor, or its vehicle (500 µl of 50% DMSO in saline) intraperitoneally 1 hr before
intracerebroventricular injection of LPS. The average Tab
before the injection was 36.6 ± 0.19°C for the
NS-398-pretreated group (n = 4) and 36.9 ± 0.26°C for the vehicle-pretreated group (n = 5). In
the NS398-pretreated rats, the fever was significantly suppressed as
compared with that in the vehicle-pretreated rats (Fig.
2). This result implies that COX-2 is
involved in fever evoked by intracerebroventricular injection of
LPS.
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Figure 2.
Changes in Tab of NS-398- or
vehicle-pretreated rats after the intracerebroventricular injection of
LPS. NS-398 (4 mg/kg; n = 4) or its vehicle (500 µl of 50% DMSO; n = 5) was injected
intraperitoneally at time point 0. LPS (50 ng/rat) was injected via the
intracerebroventricular route at the time point of 60 min. Each value
represents the mean ± SE of four or five rats. The change in
Tab was significantly different between NS-398- and
vehicle-pretreated rats in the time period denoted by the
asterisk (p < 0.05).
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Induction of COX-2 mRNA
Figure 3 shows the distribution of
COX-2 mRNA in coronal brain sections of the rats under untreated (Fig.
3A1,A2), saline-injected (Fig.
3B1-C2), and LPS-injected (Fig.
3D1-E2) conditions. The coronal planes of Figure
3, A1-E1, contained the cerebral cortex, striatum, septum, the organum vasculosum laminae terminalis, the rostral part of the preoptic area, and optic chiasma, corresponding to
the stereotaxic plane of bregma 0 mm (Paxinos and Watson, 1986). Coronal planes of Figure 3, A2-E2, contained the
cerebral cortex, dorsal hippocampus, amygdala, thalamus, and mediobasal
hypothalamus, corresponding to the stereotaxic plane of bregma 3.6 mm.
Under the untreated condition (Fig.
3A1,A2), COX-2 mRNA was constitutively expressed in the neocortex (cingulate-frontal cortices, layers II-III
and V), piriform cortex, hippocampus, and lateral amygdala. Consistent
with previous reports (Yamagata et al., 1993; Cao et al., 1995), these
COX-2 mRNA signals were from neurons as revealed by light microscopic
examination of COX-2 mRNA signals (Fig.
4A). When saline was
injected intracerebroventricularly, the level of COX-2 mRNA increased
in the neocortex and piriform cortex on the injection side (right side
in each brain section) until 1.5 hr after the injection (Fig.
3B1,B2). Semiquantitative image analysis of the COX-2 mRNA level in these regions revealed a threefold to
fivefold increase in COX-2 mRNA from the untreated condition (Fig.
5). This increase was mainly caused by an
enhanced COX-2 mRNA induction in the neurons as revealed by light
microscopic examination (Fig. 4B). By 3.5 hr after
the saline injection, the COX-2 mRNA level had become reduced, although
it was still high compared with that on the opposite side of the cortex
or with that in the untreated cortex (Figs.
3C1,C2, 4C, 5). On the other hand,
COX-2 mRNA levels in the other side of the cerebral cortices did not
differ very much from those of untreated rats.
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Figure 3.
Macroautoradiographic images of COX-2 mRNA signals
in coronal sections of the rat brain. In
B1-E2, the sections were arranged so
that the side in which intracerebroventricular injection was made was
shown on the right. In the brain sections of untreated
rats (A1, A2), the COX-2 mRNA signal was
observed in the cingulate/frontal cortices, layer II-III and V of the
neocortex, piriform cortex, lateral amygdala, and hippocampus. The mRNA
signals were markedly enhanced in one side of the cerebral cortex where
the injection of either saline (B1, C2)
or LPS (D1, E2) had been made. Until 1.5 hr after LPS injection (D1, D2), but not
saline injection, the spot-like COX-2 mRNA signals appeared in the
brain parenchyma near the cerebral ventricles (arrows)
and in the subarachnoidal space (arrowheads). Until 3.5 hr after LPS injection (E1, E2), the
spot-like COX-2 mRNA signals markedly increased in the number and
intensity. OX, Optic chiasma; Cg,
cingulate cortex; Fr, frontal cortex;
Pir, piriform cortex;
II-III, the second and third layers of
the neocortex; V, the fifth layer of the neocortex;
LV, lateral ventricle; V3, third
ventricle; DG, dentate gyrus; CA3, field
CA3-in Ammon's horn; LA, lateral amygdala.
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Figure 4.
Light microscopic views of localization of
neuronal COX-2 mRNA signals in the untreated rats and saline- or
LPS-injected ones. A-E were obtained
from the layer II/III of the neocortex of injection side (except for
A) in the plane of Figure
2A1-E1. A,
Constitutive COX-2 mRNA signals were observed in neuron-like cells
(arrowheads). COX-2 mRNA signal was markedly
enhanced in the neurons 1.5 hr after saline
(B) or LPS (D) injection.
The mRNA signals decreased until 3.5 hr after the injections
(C, E). Scale bar
(A-E), 20 µm.
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Figure 5.
Change in COX-2 mRNA level in the cortical areas.
The optical densities of the autoradiographic films in each region were
converted to the relative amount of radioactivity using a standard
scale for 14C, the particles of which isotope possess
almost the same energy as those of 35S, allowing us to
quantitate the relative amount of 35S using the
14C scale as the reference. The value in each region and
time point was expressed as the ratio to that of untreated rats. The
values for the neocortex (NC), including the
cingulate/frontal cortex and the layers II-III of the neocortex, and
piriform cortex (PC) were obtained in the plane of
Figure 2A1-E1
(n = 4). Significant differences from the untreated
group (N) were denoted by
*p < 0.05; significant differences between the
injection side and the opposite side were denoted by
#p < 0.05.
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When LPS was injected via the intracerebroventricular route, the
cortical COX-2 mRNA level was elevated in a similar manner to that
observed after intracerebroventricular injection of saline in terms of
its distribution, magnitude, and time course (Figs. 3D1-E2,
4D,E, 5). Such elevation of
cortical COX-2 mRNA is unlikely to be essential to fever because
intracerebroventricular injection of saline did not result in fever. On
the other hand, another type of COX-2 mRNA signal appeared only after
intracerebroventricular injection of LPS. By 1.5 hr after the LPS
injection, spot-like COX-2 mRNA signals appeared in the brain
parenchyma near the cerebral ventricles (Fig.
3D1,D2, arrows) and in the
subarachnoidal space (Fig. 3D1,D2,
arrowheads). This type of signal had further increased in
number and intensity by 3.5 hr after the injection (Fig.
3E1,E2).
Figure 6 shows light microscopic views of
such spot-like COX-2 mRNA signals that were specifically induced by
intracerebroventricular injection of LPS. These signals were, in most
cases, associated with blood vessels near the lateral ventricle (Fig.
6A-D) and the third ventricle (Fig.
6E-G), and in the subarachinoidal space (Fig. 6H). The mRNA signals associated with blood
vessels were more intense at 3.5 hr after the LPS injection (Fig.
6D,F) than at 1.5 hr after
it (Fig. 6B,E) and were very faint,
if present at all, after the intracerebroventricular injection of
saline (Fig. 6A,C). Sometimes,
clusters of COX-2 mRNA signals were also found in sites where a
vascular structure was not apparent, for example in the bottom right of
Figure 6B, leaving it uncertain whether they were
associated with blood vessels. The following immunohistochemical study
provided more anatomical details of the COX-2-positive cells.
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Figure 6.
Light microscopic views of COX-2 mRNA signals in
the blood vessels. A small amount of COX-2 mRNA (arrows)
was induced in the cells of blood vessels near the lateral ventricles
1.5 (A) and 3.5 (C) hr
after saline injection. Marked COX-2 mRNA signals were induced in blood
vessels of the same area 1.5 (B) and 3.5 (D) hr after LSP injection. COX-2 mRNA
(arrows) was also induced in blood vessels near the
third ventricle 1.5 hr after LPS injection (E) and was
further enhanced 3.5 hr after LPS injection (F,
G). COX-2 mRNA was also induced in blood vessels of the
subarachnoidal space (H).
BV, Blood vessel; V3, third ventricle.
Scale bars: A-D, G, 50 µm; E, F, 100 µm; H,
25 µm.
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Induction of COX-2 protein
After intracerebroventricular injection of LPS, COX-2-IR
cells were found in the subarachinoidal space (Fig.
7A-C) and in the
brain parenchyma near the cerebral ventricles (Fig.
7G-I). The distribution of COX-2-positive
blood vessels, but not that of individual cells, at 4 hr after LPS
injection is summarized in Figure 8. In
the cisterna chiasmatis, where the subarachinoidal space is enlarged
lateral to the optic chiasma, COX-2-IR cells appeared by 2 hr after
intracerebroventricular injection of LPS (Fig. 7A),
increased in number and signal intensity until 4 hr (Fig.
7B), slightly decreased until 6 hr (Fig. 7C), and
almost disappeared by 24 hr after the injection (Fig. 7D).
In contrast, intracerebroventricular injection of saline did not induce
COX-2-IR cells in the same area until 4 hr (Fig. 7E) and 6 hr (Fig. 7F) after the injection. Essentially the
same response occurred in other parts of the subarachnoidal space,
although the number of blood vessels varied among them. COX-2-IR cells
also appeared in the brain parenchyma near the third ventricle (Fig.
7G) and the lateral ventricles (Fig.
7H,I) with a time course
similar to that in the subarachnoidal space. This time course was shown as the total number of COX-2-IR cells near the third ventricle and in
the cisterna of the chiasma (Fig.
9A) and as that near the
lateral ventricles (Fig. 9B). COX-2-IR cells were most
abundant at 4 hr after the LPS injection, at which time the fever was
just before the plateau. Although 24 hr after LPS injection there was still a small number of COX-2-IR cells only near the lateral ventricle, the staining was faint, and there was no apparent fever at this time
point. Injection of saline also induced weak COX-2-like
immunoreactivity in a small number of cells only in the brain
parenchyma near the lateral ventricle (Fig. 9B). The
intensity of COX-2 staining in the blood vessels was generally higher
in the subarachnoidal space than near the third ventricle.
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Figure 7.
COX-2-IR cells appeared in the cisterna chismatis
at 2 (A), 4 (B), 6 (C), and 24 (D) hr after
LPS injection. Little COX-2-IR cell was observed at 4 (E) and 6 (F) hr after
saline injection. COX-2-IR cells also appeared in the blood vessels
near the third ventricle (G) 4 hr after LPS
injection and in those near the lateral ventricle (H,
I) 4 and 6 hr after LPS injection, respectively.
OX, Optic chiasma; V3, third ventricle.
Scale bar, 50 µm.
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Figure 8.
Distribution of COX-2-IR blood vessels in
LPS-injected rats. Each symbol represents one blood
vessel associated with one to several COX-2-IR cells. Results from
three rats, which were killed 4 hr after LPS injection, were
plotted on the same plane. The three different symbols
represent the result from each rat. COX-2-positive blood vessel was
mainly observed the near lateral ventricles, third ventricle, and the
cisterna chismatis. The outline of the section represents the pia
mater.
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Figure 9.
Time course of the number of COX-2-IR cells after
LPS injection. The COX-2-IR cells were counted near the third
ventricle, including the preoptic area and cisterna chismatis
(A), and near lateral ventricles
(B). The numbers represent the
average number of cells in three consecutive brain sections.
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Identification of the COX-2-IR cells
To determine the type of cells that express COX-2 after
intracerebroventricular injection of LPS, we conducted
double-immunostaining of COX-2 and cell-specific markers. Figure
10, A-D, shows
double-immunostaining of COX-2 (red) and vW factor
(green) in blood vessels 4 hr after intracerebroventricular injection of LPS. In large subarachnoidal blood
vessels, as shown in Figure 10A, a COX-2-IR structure
(red) was surrounded by a vW factor-positive structure
(green). This was also the case for small and large
parenchymal blood vessels, as shown in Figure 10, B and
C1, respectively. An additional staining with a
nuclear-specific dye, TOTO-3, showed that the COX-2-IR structure
overlapped the cellular nucleus (Fig.
10C2,D2). Confocal microscopic imaging of
thinner optical sections further demonstrated that the COX-2-IR
structure was mainly located in the nuclear membrane but not inside the
nucleus (Fig. 10E). On the other hand, vW factor is a
protein specifically expressed in the endothelial cytosol (Wagner and
Marder, 1983). Taken together, these results indicate that COX-2 was
expressed in the nuclear membrane of the endothelial cells. Although
TOTO-3 staining showed a number of cells located near the blood vessel,
those positive for COX-2 were, in most cases, stained with anti-vW
factor (Fig. 10C2). However, little evidence was obtained
for expression of COX-2 in microglia/macrophage, as revealed by
double-immunostaining with OX-42, a monoclonal antibody that recognizes
rat complement receptor 3 (Fig. 10E).
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Figure 10.
Double immunostaining of COX-2
(red) and von Willebrand factor
(green), an endothelial cell marker in a large
subarachnoidal blood vessel (A), in a small
parenchymal blood vessel (B), and in a large
parenchymal blood vessel (C1, C2). An
additional staining for nucleus was done with TOTO-3
(blue) in C2, and the magnified views were shown in
D1 and D2. Note that COX-2-IR structure
overlapped in a large part with nuclear staining resulting in
pinkish color. Double immunostaining of COX-2
(red) and OX-42 (green) provided
little evidence for the expression of COX-2 in microglial/macrophages
(E). Scale bars: A, B, 10 µm; C1, C2, 25 µm; D1,
D2, 7.5 µm; E, 15 µm.
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DISCUSSION |
To unveil the molecular mechanism of fever accompanying cerebral
inflammation, the present study was aimed at elucidating the role of
COX-2 and sites of its induction in rats that had received
intracerebroventricular administration of LPS, resulting in
pathophysiological events relevant to those seen in central inflammation, particularly to those of meningitis (McAllister et al.,
1975; Sanna et al., 1995).
Involvement of COX-2 in fever evoked by intracerebroventricular
injection of LPS
It is highly likely that the small amount of LPS (50 ng) injected
via the intracerebroventricular route evoked fever through its action
within the brain, because the same amount of LPS, when injected
peripherally, did not evoke fever of similar magnitude (our unpublished
observations). The fever induced by the intracerebroventricular injection of LPS was markedly suppressed by pretreatment with a
COX-2-specific inhibitor, NS-398. These results suggest that NS-398
exerted its antipyretic action through an inhibition of COX-2 activity
within the brain. This idea is also in line with previous reports that
fever evoked by intracerebroventricular injection of LPS was suppressed
by intracerebroventricular injection of indomethacin (Morimoto et al.,
1987; Hashimoto et al., 1988), although indomethacin is a nonspecific
inhibitor for both COX-1 and COX-2. We therefore further searched for
the sites of COX-2 expression in the brain as the sites of activation
of the arachidonic acid cascade.
Upregulation of COX-2 mRNA in neuronal cells
In situ hybridization study for COX-2 mRNA showed that,
in both LPS- and saline-injected rats, COX-2 mRNA was elevated in the
neurons of the cerebral cortex on the injection side. Staining of
COX-2-like immunoreactivity was also enhanced on the same side of the
cerebral cortex (data not shown). The extent of this response was
similar in LPS- and saline-injected rats, whereas the fever was evident
only in the LPS-injected ones. These results clearly indicate that
COX-2 induction in the cortical neurons is not essential to the fever.
This conclusion is in line with the following two previous findings of
ours. In rats that had received intraperitoneal injection of LPS, the
level of COX-2 mRNA in the telencephalic neurons was not correlated
with the magnitude of fever (Cao et al., 1997). Furthermore, lowering
the level of neuronal COX-2 mRNA by a general anesthesia did not
prevent the development of fever (Cao et al., 1997).
At present, little is known about the physiological significance of
COX-2 induction in the cortical neurons after the
intracerebroventricular injection of either saline or LPS. As for its
mechanism, we currently speculate that mechanical stimulus in the
cortex evoked by insertion of the needle into the lateral ventricle
triggered a response known as cortical spreading depression, in which a
depolarizing wave spreads over one side of the cortex. Cortical
spreading depression is reported to enhance the level of COX-2-like
immunoreactivity in cortical neurons (Caggiano et al., 1996).
Induction of COX-2 in the endothelial cells
The sites where the intracerebroventricular injection of LPS, but
not that of saline, specifically induced COX-2 were blood vessels in
the periventricular parenchyma and those in the subarachnoidal space.
As revealed by the double immunostaining of COX-2 and vW factor, the
latter being a cytosolic marker protein of endothelial cells (Wagner
and Marder, 1983), most of these COX-2-positive cells were endothelial
cells. On the other hand, double immunostaining of COX-2 and
microglial/macrophage marker provided little evidence for the
expression of COX-2 in these phagocytic cells. Thus, under the present
experimental conditions, the endothelial cells seem to be the major
sites where activation of the arachidonic acid cascade took place in
response to intracerebroventricular administration of LPS in a
COX-2-dependent manner.
The above results were somewhat unexpected because a number of previous
in vitro studies had demonstrated that glial cells expressed
COX-2 in response to inflammatory stimuli. For example, astrocytes
(Katsuura et al., 1989; O'Banion et al., 1996) and microglia
(Minghetti and Levi, 1995; Bauer et al., 1997; Slepko et al., 1997) in
culture upregulated COX-2 in response to LPS or to IL-1 and enhanced
the release of PGE2 into the culture medium. Elmquist et
al. (1997) also demonstrated that COX-2 was induced in perivascular
microglia, but not in parenchymal ones, in response to intravenous
injection of LPS. To our knowledge, however, COX-2 expression in the
parenchymal glia has not been reported in in vivo
preparations yet. These results imply that glial cells may express
COX-2 under certain conditions but that, at least under the present
experimental conditions, the endothelial cells are more sensitive to
LPS in terms of COX-2 induction.
Significance of endothelial COX-2 in fever
The predominant expression of COX-2 in brain endothelial cells was
also the case in another experimental model of fever, in which LPS was
injected intraperitoneally (Cao et al., 1997; Matsumura et al., 1998a);
this model was considered to represent pathophysiological events
accompanying peritonitis. In both experimental models, i.e.,
intracerebroventricular and intraperitoneal administrations of LPS, the
time courses of COX-2 expression in brain endothelial cells were in
parallel with that of fever, and inhibition of COX-2 activity by its
specific inhibitor suppressed the fever (Cao et al., 1997). These
results strongly suggest that COX-2 in the brain endothelial cells
plays an essential role in the development of fever both in peritonitis
and in central inflammation.
How does the endothelial expression of COX-2 lead to the development of
fever? It was reported that brain endothelial cells produce and secrete
PGE2, the most pyrogenic PG, in response to LPS or
inflammatory cytokines (de Vries et al., 1995). We also have an
unpublished result that shows that the PGE2 level in the CSF dramatically increased when the brain endothelial cells expressed COX-2 in response to intraperitoneally administration of LPS and that
this increase in PGE2 as well as fever was suppressed by the COX-2-specific inhibitor. These lines of evidence, although still
fragmental, brought us to hypothesize that PGE2 is produced in the endothelial cells in a COX-2-dependent manner and is released into the brain parenchyma and/or into the CSF. The secreted
PGE2 presumably then acts on the neurons in the
thermoregulatory center to evoke fever.
Several lines of evidence have suggested that one of the brain loci at
which PGE2 acts to evoke fever is the anteroventral part of
the preoptic area. The evidence includes the following: (1) the density
of PGE2-binding sites, presumably its receptors, were found
to be particularly high in that region (Watanabe et al., 1988;
Matsumura et al., 1990, 1998b); (2) that region was most sensitive to
PGE2 in terms of fever response (Scammell et al., 1996);
and (3) microinjection of COX inhibitors into that region suppressed
fever that was evoked by systemic injection of LPS (Scammel et al.,
1998). In line with this idea, we found a large number of COX-2-IR
endothelial cells there. In particular, it should be emphasized that
the endothelial cells in the subarachnoidal space just ventral to the
anteroventral part of the preoptic area were higher in number and in
intensity of COX-2 staining than those in the parenchymal region.
Scammel et al. (1998) also demonstrated that microinjection of a COX
inhibitor into the subarachnoidal space ventral to the anteroventral
part of the preoptic area effectively suppressed fever. These results
suggest that COX-2-positive endothelial cells in the subarachnoidal
space play a significant role in the development of the fever during
central inflammation.
It is well agreed that fever is a consequence of immune-brain
communication. There are, however, several hypotheses as to how the
immune signals are transmitted to the brain (Tilders et al., 1994;
Watkins et al., 1995; Blatteis and Sehic, 1997). The present study as
well as our previous ones (Cao et al., 1995, 1997; Matsumura et al.,
1998a) strongly suggest that LPS signal is converted in the brain
endothelial cells to a PG signal, which could directly affect the
neuronal activity of the brain. Recently, another hypothesis for the
mechanism of immune-brain communication was proposed by Watkins et al.
(1995). Their hypothesis emphasizes the importance of vagal sensory
nerves in the transmission of peripheral immune signals to the brain
and, consequently, in the development of fever. Under the present
experimental condition, however, the involvement of vagal sensory
system is very unlikely because LPS was administrated within the brain,
where no vagal sensory system exists. Further studies will be required
to understand how LPS administered via the intracerebroventricular
route induced COX-2 in the endothelial cells. In other words, does LPS
act on the endothelial cells directly, or does it act via induction of cytokines, such as interleukin-1 and/or tumor necrosis factor? In fact,
brain endothelial cells, but not microglial cells, possess interleukin-1 type 1 receptor and also express COX-2 mRNA in response to these cytokines (Cunningham et al., 1992; Cao et al., 1996, 1998).
Fever is a common symptom associated with various types of inflammatory
diseases, as has been known since the age of Hippocrates. The
understanding of its mechanism at the molecular level was facilitated
by two important findings made in the early 1970s. Milton and Wendlandt
(1970) reported that PGE1 was pyrogenic when injected into
the cat brain. In 1971, Vane (1971) reported that nonsteroidal
anti-inflammatory drugs exert their antipyretic action through the
inhibition of PG biosynthesis, i.e., inhibition of cyclooxygenase.
After these findings, the mechanism and site of PGE2
biosynthesis have been two of the central issues of fever research. The
present study as well as our previous ones (Cao et al., 1995, 1996,
1997, 1998; Matsumura et al., 1998a) have provided answers to these
questions by demonstrating the seminal role of brain endothelial COX-2
in the development of fever. We believe this information to be crucial
for an understanding of fever at the molecular level and to be helpful
when considering the clinical management of fever.
| |
FOOTNOTES |
Received Sept. 8, 1998; accepted Oct. 23, 1998.
This work was supported in part by Research for the Future Program
JSPS-RFTF 98L00201 from the Japan Society for the Promotion of Science.
The initial phase of this work was supported in part by Subfemtomole
Biorecognition Project, ICORP, and Japan Science Technology
Corporation. We thank Dr. Larry D. Frye for editorial help with this
manuscript, and Dr. Kanato Yamagata for providing us with the rat COX-2 cDNA.
Correspondence should be addressed to Dr. Kiyoshi Matsumura, Department
of Neuroscience, Osaka Bioscience Institute, 6-2-4 Furue-dai, Suita,
Osaka 565, Japan.
| |
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Top
Abstract
Introduction
