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The Journal of Neuroscience, August 15, 1998, 18(16):6279-6289
Brain Endothelial Cells Express Cyclooxygenase-2 during
Lipopolysaccharide-Induced Fever: Light and Electron Microscopic
Immunocytochemical Studies
Kiyoshi
Matsumura1, 2,
Chunyu
Cao2,
Masashi
Ozaki2,
Hiroshi
Morii1, 2,
Kazuhiko
Nakadate2, and
Yasuyoshi
Watanabe1, 2
1 Subfemtomole Biorecognition Project, Japan
Science and Technology Corporation, Suita, Osaka 565, Japan, and
2 Department of Neuroscience, Osaka Bioscience Institute,
Suita, Osaka 565, Japan
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ABSTRACT |
Cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of
prostaglandins, is induced in brain blood vessels by pyrogens, and its
essential role in fever has been hypothesized. In this study, we
determined (1) the type of cells that express cyclooxygenase-2 in brain
blood vessels of lipopolysaccharide-treated rats, and (2) the precise
relationship between the time course of fever and that of
cyclooxygenase-2 protein expression in these cells. Five hours after
the lipopolysaccharide injection (100 µg/kg, i.p.),
cyclooxygenase-2-like immunoreactive cells were found in the
parenchymal and subarachnoidal blood vessels. In these blood vessels,
the cyclooxygenase-2-like immunoreactivity was restricted to the
perinuclear region of the endothelial cells as revealed by a laser
confocal microscopy, double-immunofluorescence staining with an
endothelial marker, and immunoelectron microscopy. On the other hand,
the cyclooxygenase-2-like immunoreactive cells were distinct from
microglia or perivascular/meningeal macrophages as revealed by double
immunostaining with macrophage/microglia-specific antibodies.
Cyclooxygenase-2-like immunoreactive cells were first found at 1.5 hr
after the lipopolysaccharide injection, at which time the fever had not
been developed. After that, the number of cyclooxygenase-2-like
immunoreactive cells and fever followed a similar time course, both
being highest at 5 hr after the lipopolysaccharide injection and both
returning to the baseline by 24 hr. These results demonstrate that
brain endothelial cells are the primary sites where the activation of
arachidonic acid cascade takes place during fever after intraperitoneal
injection of lipopolysaccharide.
Key words:
cyclooxygenase-2; brain endothelial cells; fever; prostaglandins; lipopolysaccharide; immune-CNS communication
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INTRODUCTION |
When animals become infected, a
series of pathological responses occur. These responses, known
collectively as the acute-phase response, include fever, activation of
the hypothalamo-pituitary-adrenal axis, anorexia, hyperalgesia, and
changes in the sleep-wake pattern, most of which are under the control
of the CNS (Kent et al., 1992 ). These facts imply that the immune
system somehow communicates with the CNS. The mechanism(s) of this
immune-CNS communication is not fully understood, although
accumulating data suggest that cytokines and prostaglandins (PGs) play
essential roles in it (Milton, 1982; Dinarello et al., 1988; Blalock,
1989; Tilders et al., 1994; Watkins et al., 1995; Matsumura et al.,
1998). Until recently, however, little is known about where in
the brain the activation of the arachidonic acid cascade (i.e., the
biosynthesis of PGs) takes place during infection.
To answer this question, we have been focusing our attention on brain
cyclooxygenase-2 (COX-2) in a rat model of fever, one of the
consequences of immune-CNS communication. COX-2 is an isoform of
cyclooxygenase (Goppelt-Struebe, 1995; Herschman, 1996) that is
immediately induced by inflammatory stimuli such as lipopolysaccharides (LPSs) and cytokines, and it enhances the biosynthesis of PGs (Goppelt-Struebe, 1995; Herschman, 1996). Using a COX-2-specific inhibitor, we showed that COX-2 played an essential role in fever that
was evoked by an intraperitoneal injection of LPS in rats (Cao et al.,
1997). As for the localization of COX-2, we (Cao et al., 1995) and
other groups (Breder and Saper, 1996) showed that COX-2 mRNA was
strongly induced in the brain blood vessels during fever. Its induction
was quantitatively correlated with the magnitude of fever but was not a
result of the fever (Cao et al., 1997). Furthermore, COX-2 mRNA in the
brain blood vessels was also induced by other pyrogenic substances,
such as interleukin-1  (IL-1) (Cao et al., 1996) and tumor
necrosis factor  (TNF-) (our unpublished observation). On the
basis of these findings, we proposed a hypothesis that during infection
the activation of the arachidonic acid cascade takes place in the brain
blood vessels, where the newly induced COX-2 accelerates the
biosynthesis of prostaglandin E2 (PGE2),
which after being released into the brain acts on the CNS neurons to
evoke fever.
This hypothesis, however, was based on the observation of COX-2
mRNA, not that of COX-2 protein. The latter obviously represents the
enzymatic activity of cyclooxygenase more directly than does its mRNA.
Thus, it is still not clear whether expression of COX-2 protein
precedes the onset of fever, a condition that is essential for the
hypothesis. Furthermore, the cellular identity of COX-2 mRNA-expressing
cells was hard to determine in the in situ hybridization study because of the difficulty in conducting a double staining with
appropriate cell markers. To determine definitely the cell type and the
precise relationship between the time course of fever and that of COX-2
protein expression in these cells, we here studied expression of COX-2
protein in LPS-treated rats by using double immunostaining and
immunoelectron microscopic techniques. This study has provided data of
importance to facilitate our understanding not only of the mechanism of
fever but also of that of immune-CNS communication.
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MATERIALS AND METHODS |
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 26 ± 2°C with a standard 12 hr light/dark cycle, with free access to food
and water. LPS of Escherichia coli O26:B6 and
3-aminopropyltriethoxysilane (3-APTES) for coating glass slides were
purchased from Sigma (St. Louis, MO). Rabbit anti-murine COX-2
polyclonal antibody was purchased from Cayman Chemical (Ann Arbor, MI).
This antibody was raised against a synthetic 17-mer peptide that is
unique to the C terminus of this protein. The synthetic peptide antigen
for preabsorption test was obtained as a generous gift from Dr. Jeff
Johnson (Cayman Chemical). Mouse anti-rat COX-2 monoclonal antibody was
purchased from Transduction Laboratories (Lexington, KY). This antibody was raised against a C-terminal protein fragment corresponding to amino
acids 368-604 of rat COX-2. For preabsorption of this monoclonal
antibody, COX-2 protein purified from sheep placenta (Cayman Chemical)
was used. Other antibodies used and their sources were as follows:
OX-42 (Serotec Ltd, Oxford, UK), ED2 (BMA Biomedicals Ltd, Augst,
Switzerland), rabbit anti-human von Willebrand (vW) factor polyclonal
antibody (DAKO, Carpinteria, CA), and biotinylated goat anti-rabbit IgG
and biotinylated goat anti-mouse IgG (Vector Laboratories, Burlingame,
CA).
Western blot analysis. For Western blot analysis, samples
rich in brain blood vessels were prepared from a normal rat as well as
from an LPS-treated rat that had been injected with LPS (100 µg/kg,
i.p.) and killed 5 hr later. The rats were decapitated under
deep anesthesia with diethyl-ether, and the brains were quickly
removed. Each brain was homogenized in 10 vol of 50 mM Tris-HCl, pH 7.5, 0.32 M sucrose, 5 mM EDTA, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 10 µg/ml
leupeptin, and 1 µg/ml E64 with a Teflon homogenizer, and the
homogenate was filtered two times through a nylon mesh (mesh size = 200 µm) with a sufficient amount of PBS (20 mM,
pH 7.4). The residue on the nylon mesh was highly enriched in blood vessels. These samples were further homogenized with a sonicator, mixed
with 2× concentrated SDS sample buffer (1× 125 mM
Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, and
1% -mercaptoethanol), and boiled for 5 min. The samples were then
applied to a polyacrylamide gel (10-20% gradient), electrophoresed,
and transferred to a polyvinylidene difluoride membrane (Pall Co., Port
Washington, NY). The membrane was blocked for 1 hr at room temperature
with a blocking buffer containing 0.5% casein and 0.3% Tween 20 in
PBS and incubated overnight at 4°C with the anti-COX-2 antibodies
(1:2000 dilution for the monoclonal antibody and 1:3000 for the
polyclonal one). After the membrane had been washed with the blocking
buffer, it was incubated with alkaline phosphatase-conjugated second
antibody for 1 hr at room temperature. Immunoreactive proteins were
detected with a Phototope-Star Western blot detection kit (New England Biolabs, Beverly, MA).
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
(Mini-Mitter, Sunriver, OR) was implanted into the abdominal cavity of
each rat under pentobarbital anesthesia. The signal from the
transmitter was detected by a receiver that had been placed under the
rat cage and was fed to an IBM personal computer through an appropriate interface. The temperature data were stored in the computer every 10 min.
Tissue preparation. To study the time course of COX-2
expression, we injected rats with LPS (100 µg/kg in 0.5 ml saline,
i.p.) between 9:30 and 10:30 A.M. and killed them at seven time points, i.e., 0.75, 1.5, 3, 5, 8, 12, and 24 hr after the LPS injection. For
each time point, at least four rats were prepared. As a negative control, rats were injected with saline (0.5 ml, i.p.) and killed 3 or
5 hr after the injection. Under diethyl-ether anesthesia, the rats were
perfused through the left ventricle with 20 mM PBS (50 ml)
to remove the blood, and their brains were immediately removed and
freshly frozen in dry-ice powder and stored at  80°C until use. The
brains were also used for double immunohistochemistry.
For immunoelectron microscopy, three rats were injected with LPS (100 µg/kg in 0.5 ml saline, i.p.), and two rats were injected with
saline. Four hours after the injection, they were perfused under deep
pentobarbital with 0.1 M phosphate buffer (PB), pH 7.4, followed by a fixative containing 4% paraformaldehyde in 0.1 M PB, pH 7.4, and 0.1% glutaraldehyde. The brains were
removed and stored in the same fixative at 4°C until use.
Immunohistochemistry. Both antibodies to COX-2, one
monoclonal and the other polyclonal, stained the same population of
COX-2-like immunoreactive (COX-2-ir) cells, although the two antibodies
had a distinct preference for a given type of fixation. That is, the polyclonal anti-COX-2 antibody yielded a better staining of sections made from the freshly frozen brains with a short post-fixation. On the
other hand, the monoclonal antibody yielded better results in vibratome
sections made from the brain fixed by perfusion. Thus, the two
antibodies were used properly according to the purpose of the
experiments.
For the quantitative analysis of the time course of COX-2 expression,
the freshly frozen brains were used to avoid possible variation of the
staining attributable to varied fixation conditions among the rats. The
freshly frozen brains were cut at a thickness of 14 µm in a cryostat
and thaw-mounted on 3-APTES-coated glass slides. After having been
air-dried at room temperature for 30 min, the sections were fixed with
2% paraformaldehyde in 0.1 M PBS, pH 7.4, for 10 min at
room temperature. After a rinse with PBS, they were treated with 0.3%
H2O2 in the PBS for 30 min followed by 3%
normal goat serum (NGS) (Vector Laboratories) and 0.25% Triton X-100
in the PBS. Endogenous biotin activity was blocked with a blocking kit
(Vector Laboratories) according to the manufacturer's instructions.
The sections were then incubated with rabbit anti-murine COX-2
polyclonal antibody diluted 2400 times in PBS containing 3% NGS and
0.25% Triton X-100 for 64 hr at 4°C followed by biotinylated goat
anti-rabbit IgG (200× dilution) for 1 hr at room temperature. COX-2-like immunoreactivity was visualized with a Vectastain Elite ABC
kit using diaminobenzidine (DAB) as a chromogen. Control staining was
conducted without the primary antibody or with preabsorbed antibody, in
which the diluted primary antibody and the antigen peptide (1 µg/ml)
had been mixed and incubated at 4°C overnight.
For immunoelectron microscopy, the sections with 50 µm thickness were
made with a microslicer (Dosaka EM, Kyoto, Japan). All of the following
protocol were conducted with the sections in a free-floating condition
with gentle shaking at room temperature unless stated otherwise. The
sections were first treated with 0.3% H2O2 in
PBS (0.1 M, pH 7.4) for 30 min followed by 10% NGS in PBS.
They were then incubated with mouse anti-rat COX-2 monoclonal antibody
diluted 1:600 (0.4 µg/ml) in the PBS containing 10% NGS at 4°C for
48 hr, and thereafter incubated for 1 hr with biotinylated goat
anti-rabbit IgG diluted 1:200. Control staining was conducted without
the primary antibody or with preabsorbed antibody in which the diluted
primary antibody and the purified sheep COX-2 (4 µg/ml) had been
mixed and incubated at 4°C overnight. The immunoreactivity was
visualized with a Vectastain Elite ABC kit. Some of the sections were
mounted on 3-APTES-coated glass slides and used for light microscopic
examination, and other sections were processed for electron microscopic
examination. The latter were post-fixed with 2% paraformaldehyde and
2% glutaraldehyde in PBS at 4°C overnight and then with 2%
osmiumtetroxide in 0.2 M PB at 4°C for 90 min. After they
were washed with distilled water, the sections were dehydrated in an
ascending series of ethanol and embedded in Epon. Ultrathin sections
were cut and picked up on mesh grids (no. 100). The sections were
stained with lead citrate and uranyl acetate.
Double immunostaining. For identification of the type of
COX-2-positive cells in the blood vessels, COX-2 staining was performed in combination with one of three cell-type markers, including anti-vW
factor (endothelial cell marker), OX-42 (parenchymal microglia and
perivascular/meningeal macrophage marker), and ED2
(perivascular/meningeal macrophage marker). Freshly frozen brain
sections post-fixed with 2% paraformaldehyde were suitable for the
double immunostaining. In the case of double staining for COX-2 and vW
factor (both antibodies were polyclonal from rabbits), the brain
sections were first incubated with anti-COX-2, followed by biotinylated
goat anti-rabbit IgG. The immunoreactivity was visualized with
Cy3-streptavidin (Amersham Life Science, Arlington Heights, IL). The
sections were again treated with 3% NGS and then incubated with
anti-human vW factor for 1 hr at room temperature followed by
FITC-labeled goat anti-rabbit IgG (Zymed, South San Francisco, CA). In
some sections that had been stained for COX-2, FITC-labeled goat
anti-rabbit IgG was applied without incubation with anti-human vW
factor to confirm that cross-binding of FITC-labeled goat anti-rabbit
IgG to the first primary antibody (rabbit anti-COX-2 antibody) was
negligible.
In the case of double immunostaining with anti-COX-2 and OX-42 or ED2
(the latter two are monoclonal antibodies), the brain sections were
incubated with anti-COX-2 polyclonal antibody premixed with either
OX-42 (final dilution ×1000) or ED2 (final dilution ×100) for 64 hr
at 4°C followed by biotinylated goat anti-rabbit IgG. COX-2
immunoreactivity was visualized with Cy3-streptavidin, and then the
sections were treated with an avidin-biotin blocking kit and incubated
with biotinylated goat anti-mouse IgG. The immunoreactivities were
visualized with Cy2-streptavidin. Cross-binding of Cy2-streptavidin to
the biotinylated goat anti-rabbit IgG was negligible. In some cases,
the double-stained sections were further stained with TOTO-3 (Molecular
Probes, Eugene, OR) to visualize the nuclei.
Microscopy. The sections colored with DAB were examined with
a microscope (Olympus), and the images were captured by a
high-resolution CCD video camera system, digitized to 32-bit color
images, fed into a Macintosh computer, and stored as PICT files for
further analysis and presentation. The sections stained with
fluorescent dyes were examined with a confocal laser-scanning
microscope (Bio-Rad MRC-1024; Bio-Rad, Hercules, CA). Electron
microscopic examination was made with a Hitachi H-7000.
Quantitative analysis and statistics. In the time-course
study, numbers of COX-2-ir cells in the subarachnoidal space lateral to
the optic chiasma were counted in at least four brain sections from
each rat. The mean value of this number in each rat was further averaged among four or five rats in each time point. Data were expressed as mean ± SE.
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RESULTS |
Western blot analysis
Western blot analysis was performed to examine the specificity of
antibodies to COX-2. In the blood vessel-enriched brain samples, both
monoclonal and polyclonal antibodies raised against COX-2 recognized a
protein band of ~70 kDa, which is consistent with the molecular
weight of rat COX-2 (Fig. 1, lanes
a and b) (Herschman, 1996). This signal was enhanced
when a rat had been injected with LPS (100 µg/kg, i.p.) and killed 5 hr later (Fig. 1, lane c), a result in good agreement with
the previous finding that COX-2 mRNA was strongly induced in the brain
blood vessels by the LPS treatment. The protein band corresponding to
COX-2 was eliminated when preabsorbed antibodies were used as the
primary antibodies (Fig. 1, lanes d and e).
Although the polyclonal antibody stained two additional bands of larger
molecular sizes, these bands were not eliminated with the preabsorbed
antibody (data not shown). Thus, possible nonspecific stainings with
the polyclonal antibody were distinguishable from specific ones by
using the preabsorbed antibody as a negative control.
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Figure 1.
Western blot analysis of COX-2 in the blood
vessel-enriched brain samples from rats that had been left untreated
(b, d) or injected with LPS (100 µg/kg,
i.p.) 5 hr before being killed (a, c, e). Both the
monoclonal antibody (a) and polyclonal antibody
(b, c) recognized a protein band, the molecular weight
of which corresponded well to that of COX-2. The COX-2 signal was
enhanced in the sample from an LPS-treated rat
(c) compared with that from the untreated rat
(b). Preabsorbed polyclonal antibody did not
stain the protein band corresponding to COX-2 (d, e).
The position of arrowheads indicates a molecular size of
68 kDa.
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COX-2 immunostaining
The two antibodies used in the present study revealed COX-2-like
immunoreactivity in a subset of telencephalic neurons in the brain
tissue from normal as well as LPS-treated rats. Figure 2a shows the COX-2-ir
neurons in the hippocampal pyramidal cell layer of a saline-injected
rat stained with the monoclonal antibody. This staining was completely
eliminated when the preabsorbed antibody was used as the primary
antibody (Fig. 2b). Figure 2c
shows the staining pattern with the polyclonal antibody of COX-2-ir
neurons in the cingulate cortex and COX-2-ir non-neuronal cells in the blood vessels in brain tissue from an LPS-treated rat. These neuronal and non-neuronal COX-2-like immunoreactivities were also eliminated when the preabsorbed polyclonal antibody was used as the primary antibody (Fig. 2d). These results together with
those of Western blot analysis thus indicated that the two antibodies
specifically recognized constitutively expressed COX-2-like
immunoreactivity in the neurons and LPS-induced COX-2-like
immunoreactivity in the non-neuronal cells in the blood vessels.
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Figure 2.
COX-2-ir cells in the brains of rats injected with
either saline (a, b, l) or LPS (100 µg/kg,
i.p.) (c-k, m-p) 5 hr before being killed.
a, COX-2-ir neurons in the pyramidal cell layer of the
hippocampus stained with the monoclonal antibody; b, the
same area as in a stained with the preabsorbed
monoclonal antibody; c, COX-2-ir cells in the cingulate
cortex and in the blood vessels penetrating into the brain
(arrows); d, the same area as in
b stained with the preabsorbed polyclonal antibody;
e-h, parenchymal blood vessels stained with the
polyclonal antibody; i, j, COX-2-ir cells in the blood
vessels stained with the monoclonal antibody; k, a
higher magnified view of the same blood vessel as in j;
l, no COX-2-ir cells are found in a blood vessel of a
saline-treated rat; m, COX-2-ir cells in the
subarachnoidal space lateral to the optic chiasma. The
arrowheads and asterisk indicate the
arachnoid membrane and the basilar artery, respectively.
OX, Optic chiasma; n, COX-2-ir cells in
the subarachnoidal space dorsal to the third ventricle. The
subarachnoidal space is separated from the third ventricle (*) by the
velum interpositum (arrowheads). chp,
Choroid plexus; o, COX-2-ir cells in the subarachnoidal
space between the cerebellum (*) and dorsal medulla; ap,
area postrema; p, COX-2-ir cells in the subarachnoidal
space formed by the cortex (ctx), the hippocampus
(hippo), and the dorsolateral brain stem, which is a
transitional area between the superior colliculus and thalamus;
bsc, brachium of the superior colliculus. Note that the
blood vessel with a thick wall (denoted by *) contained fewer COX-2-ir
cells than those with a thin wall (veins). Scale bars: a, b,
e-i, o, 50 µm; c, d, j, l, m, n, p, 100 µm;
k, 10 µm.
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The distribution of COX-2-ir neurons in the telencephalon was similar
to that reported previously by Breder et al. (1995) and was not
influenced very much by the LPS treatment. On the other hand, the
non-neuronal COX-2-ir cells were observed only in LPS-treated rats and
in almost all cases were associated with blood vessels in the brain
parenchyma and subarachnoidal space (Fig. 2e-p)
and in the spinal cord. The COX-2 staining in these cells was most
prominent at the time points of 3 and 5 hr after LPS injection (the
detailed time course of COX-2 expression is given in the last part of
Results). Figure 2e-h shows COX-2-ir cells in the
parenchymal blood vessels stained with the polyclonal antibody. The
COX-2-ir structures were oval or spindle-shaped and were situated
linearly along the vessel wall. Figure 2i-l shows
the results with the monoclonal antibody. In these preparations, probably because of the paraformaldehyde/glutaraldehyde fixation, the
COX-2-ir structures were more flattened than those in the freshly
frozen and lightly post-fixed samples (Fig. 2e-h).
Nevertheless, their oval shape and localization in the vessel wall were
essentially the same as those in the former samples. Those blood
vessels with COX-2-ir cells were apparently larger in diameter than the
capillaries.
COX-2-ir cells were also abundant in subarachnoidal spaces of the
LPS-treated rats (Fig. 2m-p). There the COX-2-ir
cells lined membranous structures that, in most cases, formed a closed
circle, indicating that the membranous structures were blood vessels
(further evidence for this point is presented in the following part). A number of COX-2-ir cells were found in blood vessels having a thin
wall, whereas few COX-2-ir cells were found in those with a thick wall
(Fig. 2m,p). These results indicate that COX-2-ir cells were located predominantly in veins or venules rather than in
arteries or arterioles. In the choroid plexus and other parts of the
cerebroventricular system, COX-2-ir cells were absent, although the
positive cells were present in the blood vessels located close to the
ventricular system (Fig. 2n). The examination of
COX-2-ir cells in the subarachnoidal space was made only in thaw-mounted brain sections in which the structures of subarachnoidal constituents were better conserved than in those processed by the
free-floating method, because the nonparenchymal blood vessels and
membranous structures were easily removed or distorted by the latter
staining procedure.
Identification of the COX-2-ir cells in the
blood vessels
To clarify which subcellular component is responsible
for the COX-2-like immunoreactivity, a nuclear-specific fluorescent dye, TOTO-3, was used. Figure
3a1-a2 shows laser confocal
microscopic views of COX-2-ir cells (red) in a
subarachnoidal blood vessel (Fig. 3a1) and those overlaid
with TOTO-3 nuclear staining (blue) (Fig. 3a2).
It is clear that the COX-2-like immunoreactivity was located mainly in
the structure surrounding the nucleus, most likely the nuclear
envelope.
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Figure 3.
Laser confocal immunofluorescent views of
COX-2-positive cells in the brains of LPS-treated rats.
a1, COX-2-ir structure (arrows) in the
cells of a blood vessel; a2, the COX-2 staining in
a1 was overlaid with TOTO-3 nuclear staining
(blue). Note that the COX-2-like immunoreactivity was
restricted to the surface of the nucleus. COX-2-ir cells in parenchymal
(b1) and subarachnoidal (c1, d1) blood
vessels. The same blood vessels in b1, c1, and
d1 were stained with anti-von Willebrand factor
(b2, c2, and d2, respectively). The COX-2
immunostaining (red) was overlaid with von Willebrand
factor immunostaining (green) (b3,
c3, and d3). d4, The double
immunostaining in d3 overlaid with TOTO-3 nuclear
staining (blue); e, triple staining of
COX-2 (red), von Willebrand factor
(green), and nucleus (blue). Note
that a neuron was positive for COX-2 but negative for von Willebrand
factor; a capillary was positive for von Willebrand factor. COX-2-like
immunoreactivity in the neuron was diffusely distributed in
the soma and a process. f, A brain section
was first stained for COX-2 (f1) and then
incubated with FITC-labeled goat anti-rabbit IgG without a previous
incubation with anti-von Willebrand factor (f2).
Note that fluorescent signal of FITC was very low compared with that in
the adjacent section, which had been first stained for COX-2 and then
stained for anti-von Willebrand factor as described in Materials and
Methods (f3), indicating that cross-binding of
FITC-labeled goat anti-rabbit IgG to anti-COX-2 was negligible. Double
immunostaining of COX-2-ir cells (red) and ED2-positive
macrophages (green) in parenchymal
(g) and subarachnoidal (h)
blood vessels. COX-2-ir cells (i1) and OX-42-positive
cells (i2) were located close to each other but were
distinct from one another (i3). Scale bars: 10 µm;
b, 20 µm; f, 50 µm.
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Figure 3b1-b3 shows double immunostaining for COX-2-like
immunoreactivity and vW factor, an endothelial cell marker. All of the
COX-2-ir structures (red) were embedded in vW
factor-positive structures (green). Also in a larger
blood vessel in the subarachnoidal space (Fig. 3c1-c3), all
of the COX-2-ir structures were surrounded by vW factor-positive
structures. Because vW factor is a cytosolic protein that is
specifically present in the endothelial cells (Jaffe et al., 1973;
Wagner and Marder, 1983; Theilen and Kuschinsky, 1992), this staining
pattern indicates that the COX-2-like immunoreactivity is located in
the nucleus of endothelial cells; in other words, COX-2-ir cells are
endothelial cells. By overlaying the pattern of TOTO-3 nuclear staining
on that of the double immunostaining for COX-2 and vW factor (Fig.
3d1-d4), we further confirmed that among the cells
associated with the blood vessels, the COX-2-ir cells were the ones
that were located closest to the lumen, supporting the idea that the
COX-2-ir cells are endothelial cells.
Finally, their endothelial nature was confirmed by the immunoelectron
microscopic study (Fig. 4). The
COX-2-like immunoreactivity was located mainly in the nuclear envelope
of the endothelial cells of LPS-treated rats (Fig. 4a,d).
The immunoreactivity was distributed in an intermittent manner on the
nuclear envelope from which less intense but distinct immunoreactivity
extended to the cytosol and reached the plasma membrane (Fig.
4b,c). Consistent with the light-microscopic study, little
COX-2-like immunoreactivity was detected in the capillaries (Fig.
4e). Incubation without the primary antibody did not yield
such staining (Fig. 4f).
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Figure 4.
Immunoelectron microscopic observation of
COX-2-like immunoreactivity in endothelial cells of LPS-treated rats.
COX-2-ir signals were located in the nuclear envelope and the cytosol
nearby (a, d). b, c, Magnified views of
the regions indicated in a with the white
arrows and black arrows, respectively. The
black and the white arrowheads in
b and c indicate plasma membrane and
nuclear membrane, respectively. The asterisk in
c indicates the luminal side. Minimal COX-2-ir signal
was found in capillaries (e). No COX-2-ir signal
was observed in the samples incubated without the primary antibody
(f). Scale bars: a, d-f, 1 µm; b, c, 0.1 µm.
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Because the primary antibodies to COX-2 and vW factor were both of
rabbit origin, there was a possibility that the FITC-labeled second
antibody, which was expected to bind to anti-vW factor rabbit IgG, also
might have bound to anti-COX-2 rabbit IgG, which had been added to the
section first. This possibility was clearly excluded by the following
two results (Fig. 3e,f1-f3). First, in Figure
3e, a triple staining for COX-2 (red), vW factor
(green), and nucleic acid (blue) was made
in the cerebral cortex. The neuron was positive for COX-2 but negative
for vW factor (whereas a small blood vessel was vW factor positive),
indicating that the FITC-labeled second antibody did not bind to
anti-COX-2 rabbit IgG. Second, when a section was first stained for
COX-2 (Fig. 3f1) and then further incubated with
FITC-labeled goat anti-rabbit IgG without a previous incubation with
anti-vW factor, fluorescent signal of FITC was negligible (Fig.
3f2). In contrast, when an adjacent section that had been first stained for COX-2 was further incubated with anti-vW factor followed by FITC-labeled goat anti-rabbit IgG, the
fluorescent signal was evident (Fig.
3f3).
In contrast to our findings, Elmquist et al. (1997) reported that
perivascular microglia and meningeal macrophages were the cells that
expressed COX-2 in rats that had been injected with LPS intravenously.
Therefore, we examined whether the microglia or macrophages also
expressed COX-2-like immunoreactivity in addition to the endothelial
cells. Double immunostaining with anti-COX-2 and ED2, which recognizes
perivascular and meningeal macrophages, showed that the COX-2-ir cells
were distinct from those stained with ED2 in both parenchymal blood
vessels (Fig. 3g) and subarachnoidal blood vessels (Fig.
3h). Double staining with anti-COX-2 and OX-42, which
recognizes the complement 3 receptor expressed in parenchymal and
perivascular microglia, also showed that COX-2-ir cells and OX-42-positive cells were distinct from each other although they were sometimes located close to one another (Fig. 3i1-i3).
These results indicate that at least in our experimental condition
COX-2-like immunoreactivity was induced mainly in the brain endothelial
cells and only minutely in the perivascular microglia/meningeal
macrophages.
Timing of COX-2 induction in the course of fever
Figure 5 shows the time course of
fever after injection of LPS (100 µg/kg, i.p.). Both in LPS-
and saline-injected rats, the Tab (<0.5°C)
increased slightly immediately after the injections. This increase in
Tab seemed to be caused by the stress associated with the injection. The Tab of the LPS-injected
rats, but not that of the saline-injected ones, started to rise again
1.5 hr after the injection, showed a maximum increase of 2°C
approximately 5 hr after the injection, and then gradually declined,
representing a typical LPS-induced fever. On the other hand, the
Tab of the saline-injected rats started to
increase 8 hr after the saline injection. This increase was caused by
the circadian change in Tab because a similar
increase in Tab was also observed in untreated rats.
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Figure 5.
Changes in abdominal temperature
(Tab) of LPS-injected rats
(squares), saline-injected rats
(circles), and untreated rats
(triangles). Tab of five rats
was measured with a telemetry system without any treatment on the first
day. On the second and fourth days, they were injected with saline (0.5 ml, i.p.) and LPS (100 µg/kg, 0.5 ml, i.p.), respectively. All of the
injections were made between 9:30 and 10:00 A.M., and the values are
expressed as the difference from the preinjection level. The time of
injection was set to 0 on the x-axis. For the untreated
group, the Tab at 10:00 A.M. was used as the
baseline. The downward arrows and the numbers
above indicate the time the rats were killed, which was
conducted in a separate group of rats (see Fig. 6).
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For the evaluation of the amount of COX-2 induction during the
time course of fever, we selected seven time points (Fig. 5, arrows) for killing additional rats, the time points
approximately corresponding to the latent phase of fever (45 min and
1.5 hr after LPS): rising phase of fever (3 hr), maximal febrile phase (5 hr), early recovery phase (8 hr), late recovery phase (12 hr), and
complete recovery phase (24 hr). As a negative control, four rats were
injected with saline and killed 3-5 hr later. At each time point, we
counted the number of COX-2-ir cells in a restricted region of the
subarachnoidal space, which was lateral to the optic chiasma and
ventral to the rostral part of the preoptic area (Fig. 6). We chose this region because it is
close to the anteroventral preoptic area, which was reported to be
highly sensitive to PGE2 in terms of the febrile response
(Stitt, 1986; Scammell et al., 1996) and to possess a high density of
PGE2 receptors (Matsumura et al., 1990, 1992). Figure 6
shows COX-2-ir cells in this subarachnoidal space at six of the seven
time points after intraperitoneal injection of LPS, and Figure
7 summarizes the time course of COX-2-ir
cell counts and that of fever. At 45 min after the LPS injection, there were no COX-2-ir cells (Fig. 6a). COX-2-ir cells were first
observed at 1.5 hr after the injection (Fig. 6b); their
number and intensity of staining further increased until 5 hr (Fig.
6c,d) and then gradually declined (Fig. 6e) and
disappeared by 24 hr after the injection (Fig. 6f).
The important observation here was that by 1.5 hr after the LPS
injection, a number of COX-2-ir cells had appeared, at which time the
fever was still in the latent phase, for the Tab
had not yet increased (Fig. 7). This is strong evidence that the
appearance of COX-2-ir cells preceded the onset of fever. Thus, the
induced COX-2 could be the cause of fever. Except for this earlier
appearance of COX-2-ir cells than of fever, the number of COX-2-ir
cells and fever followed a similar time course up to 24 hr, both being
highest at 5 hr after the LPS injection and both returning to the
baseline level by 24 hr after the injection. Although not
quantitatively analyzed, the time course of COX-2 induction in the
endothelial cells of other brain regions was similar to that observed
in this subarachnoidal space.
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Figure 6.
Time-dependent changes in the COX-2-ir cells in
the subarachnoidal space lateral to the optic chiasma and ventral to
the anteroventral preoptic area. After LPS injection, rats were killed
at 45 min (a), 1.5 hr (b),
3 hr (c), 5 hr (d), 12 hr
(e), and 24 hr
(f).
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Figure 7.
Time courses of the number of COX-2-ir cells ( )
and change in Tab ( ). The data on the
numbers of COX-2-ir cells were counted in the same subarachnoidal space
as shown in Figure 6.
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DISCUSSION |
Technical considerations
Several lines of evidence indicated that COX-2-like
immunoreactivity visualized in the present study represented the COX-2 protein. First, Western blot analysis showed that the two antibodies properly recognized the band corresponding to the size of COX-2 protein
and that the preabsorption test discriminated specific from nonspecific
staining. Second, the two different antibodies used here yielded
essentially the same staining pattern, which was also consistent with
that reported for normal rats (i.e., staining in the telencephalic
neurons) (Breder et al., 1995) and LPS-treated rats (i.e., staining in
blood vessels) (Elmquist et al., 1997). Third, the distribution of
COX-2-ir cells here was in good agreement with that of COX-2 mRNA
reported previously in both normal (Yamagata et al., 1993) and
LPS-treated rats (Cao et al., 1995, 1997).
Three combinations of double immunostaining, i.e., with anti-COX-2 and
anti-vW factor, OX-42, or ED2, were conducted only with freshly
frozen brain samples. This was mainly because the antigenicities of
these cell markers were well conserved in freshly frozen samples.
Possible cross-staining, especially in the combination of two rabbit
polyclonal antibodies, i.e., anti-COX-2 and anti-vW factor, was
negligible, as shown in Figure 3, e and f1-f3.
Another advantage of using the thaw-mounted thin sections was that this preparation retained the anatomical details of the subarachnoidal space
better than free-floating sections, thereby making it easy to
discriminate arachnoid membrane, pial membrane, and other cell components at the brain surface.
Identity of the COX-2-ir cells
The present result showing that systemic administration of LPS
induced COX-2-like immunoreactivity in the cells close to the parenchymal blood vessels is in line with the in situ
hybridization studies of COX-2 mRNA by our group (Cao et al., 1995,
1997) and also agrees well with the previous work by Elmquist et al.
(1997). As for the localization of COX-2-ir cells in the subarachnoidal space, however, this study demonstrated for the first time that the
COX-2-ir cells there lined a membranous structure that in most cases
formed a closed circle (ring shape) and was separated from the
arachnoidal membrane, indicating that the COX-2-ir cells were in the
blood vessel wall. If the membranous structures had been the meningeal
membrane, they would not have formed a closed circle. This is in
contrast to the interpretation by Elmquist et al. (1997) that the
COX-2-ir cells are meningeal macrophages. The possible reasons for
the discrepancy will be discussed in the following paragraph.
The present study provided definitive evidence for the presence of
COX-2 in the endothelial cells by double-immunohistochemical and
immunoelectron microscopic methods. In contrast, Elmquist et al. (1997)
proposed a different view: the COX-2-ir cells in the blood vessels were
perivascular microglia and those in the subarachnoidal space were
meningeal macrophages. This idea was based on their
double-immunohistochemical finding that the COX-2-ir cells were stained
with a monoclonal antibody that was reported to specifically recognize
activated microglia/macrophages. One possible reason for the
discrepancy would be the different experimental conditions between the
two studies. We injected LPS intraperitoneally, whereas Elmquist et al.
(1997) did so intravenously with a smaller amount. Although COX-2 is
potentially inducible in both endothelial cells and
microglia/macrophages, as reported in a number of in vitro
studies (Goppelt-Struebe, 1995; Herschman, 1996), the mode of its
regulation may not always be identical between the two cell groups
(Koll et al., 1997). Thus, different routes and amounts of LPS
administration may differentially influence the induction of COX-2 in
the two cell groups. Another possibility would be that COX-2 was
expressed in both endothelial cells and a subset of microglial
cells/macrophages, which were not recognized by the conventional
microglial/macrophage markers used in the present study (i.e., OX-42
and ED2) but were only stained with the antibody against the marker for
activated microglia used by them. In addition, the present study showed
little induction of COX-2 in the choroid plexus. Our previous studies
(Cao et al., 1995, 1997) also showed that COX-2 mRNA was induced only
minimally in the choroid plexus after intraperitoneal injection
of LPS. On the other hand, inductions of COX-2 mRNA and its protein in
the choroid plexus were reported by Breder et al. (Breder and Saper,
1996) and Elmquist et al. (1997), respectively. The reason for this
discrepancy is not clear at present.
Timing of COX-2 expression and its
physiological relevance
The fact that fever induced by intraperitoneal injection of LPS
was suppressed by a COX-2-specific inhibitor (Cao et al., 1997) and
brain endothelial cells were the major cell population expressing COX-2
in response to LPS suggests that COX-2 in the brain endothelial cells
is responsible for fever. However, some points still remained to be
clarified before the validity of this hypothesis was established. One
of the points was to show that expression of COX-2 protein, which
directly reflects potency of the cells to biosynthesize PGs, precedes
the occurrence of fever. The present study clearly showed that this is
the case. At 1.5 hr after LPS injection, when the
Tab had not yet increased, COX-2-positive cells
were already observed in some brain blood vessels. Unexpectedly, the
Western blot analysis showed that the brain blood vessels expressed a
low but significant amount of COX-2 protein in the absence of pyrogenic
stimuli. Probably, the histochemical techniques, including in
situ hybridization technique, were not sensitive enough to detect
such a low amount of COX-2 protein constitutively expressed in the
blood vessels under the normal condition. The COX-2 constitutively
expressed in the brain blood vessels may explain the early onset of
fever (10-30 min) that was reported after intravenous administration
of LPSs or cytokines (Kluger, 1991).
Another essential issue to be clarified for the understanding of the
role of brain endothelial COX-2 in fever is whether
PGH2, the product of COX-2, is further converted to
PGE2, the fever mediator, in the endothelial cells,
and whether PGE2 is secreted into the CSF-brain
compartment. Demonstration of PGE2 synthase colocalizing
with COX-2 in the endothelial cells would be of importance. However,
PGE2 synthase has not yet been identified at the molecular level; therefore, appropriate probes for histochemical study of PGE2 synthase are not available at present. On the other
hand, the following results suggest that the endothelial cells do
synthesize PGE2 and secrete it into the CSF-brain
compartment. First, PGE2-like immunoreactivity was detected
in the brain blood vessels after intravenous injection of a high dose
of LPS (Van Dam et al., 1993), and second, PGE2 was
secreted from cultured brain endothelial cells in a higher amount than
PGI2 in response to LPS or cytokine stimuli (de Vries et
al., 1995). Third, PGE2 level in the CSF was increased
after intraperitoneal injection of LPS, and this response was dependent
on COX-2 because pretreatment with a COX-2-specific inhibitor
significantly suppressed it (our unpublished observation), suggesting that PGE2 in the CSF is secreted from
COX-2-bearing cells close to the CSF compartment. Thus, endothelial
cells in the subarachnoidal space could well be the source of
PGE2 in the CSF.
COX-2-like immunoreactivity was predominantly expressed in veins
and venules rather than in arteries or capillaries. The mechanism of
this restricted induction and its physiological significance are not
clear at present. It was reported, however, that interleukin-1 type-1
receptor mRNA was also predominantly expressed in venules of the brain
(Cunningham et al., 1992; Yabuuchi et al., 1994). Thus, sensitivity to
the cytokines or LPS might be higher in the venules and veins than in
arteries or capillaries. As for its physiological significance, PGs
produced in the endothelial cells in veins may gain access to the CSF
more easily than those from the endothelial cells in arteries because
of the undeveloped smooth muscle layer in the former.
The induction of COX-2 in the brain endothelial cells was not
restricted to the specific brain regions related to fever but was
distributed throughout the brain, spinal cord, and subarachnoidal space. What would be the physiological significance of this widespread induction of COX-2? One possibility is that the products of COX-2 exert
their action locally, i.e., in the vicinity of their production. Recently, Ericsson et al. (1997) reported that there is an
intramedullary prostaglandin-dependent mechanism in the activation of
neuroendocrine circuitry by intravenous IL-1. They assumed that
circulating IL-1 activates perivascular cells possessing IL-1 receptor
to activate arachidonic acid cascade, the product of which acts on
medullary neurons. In relation to this notion, we have reported that
receptors for PGs are widely distributed throughout the brain and
spinal cord (Matsumura et al., 1990, 1992, 1995; Takechi et al., 1996; Matsumura et al., 1998). Another possibility is that the PGs
produced are first conveyed to the CSF and transported to the specific brain sites at a distance by a bulk flow of, or diffusion in, the CSF
and then they act there to evoke fever and other acute-phase responses.
This issue should be clarified in the future.
There has been a long discussion (Tilders et al., 1994; Watkins
et al., 1995; Blatteis and Sehic, 1997) as to how the immune system
communicates with the CNS. The present study, together with our
previous ones (Cao et al., 1996, 1997; Matsumura et al., 1998),
suggests that at least under the present experimental conditions, brain
endothelial cells constitute one of the routes for immune-CNS communication, in which the endothelial cells transform the blood-borne immune signal into a prostaglandin signal, which in turn acts on the
CNS neurons to evoke fever and other acute-phase responses. In this
sense, brain endothelial cells, although they construct a barrier
between blood and brain, function as an immune-CNS interface as
well.
| |
FOOTNOTES |
Received Jan. 5, 1998; revised May 26, 1998; accepted May 27, 1998.
We thank Dr. Jeff Johnson for providing the COX-2 antigen peptide, and
Dr. Kanato Yamagata for his helpful discussion.
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
Substance via MeSH
