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The Journal of Neuroscience, December 15, 1999, 19(24):10923-10930
An Essential Role of Interleukin-1 in Mediating NF- B
Activity and COX-2 Transcription in Cells of the Blood-Brain Barrier
in Response to a Systemic and Localized Inflammation But Not During
Endotoxemia
Nathalie
Laflamme,
Steve
Lacroix, and
Serge
Rivest
Laboratory of Molecular Endocrinology, Centre de Recherche de
l'Université Laval Research Center and Department of Anatomy and
Physiology, Laval University, Québec, Canada G1V 4G2
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ABSTRACT |
When released into the bloodstream, proinflammatory cytokines have
the ability to trigger the transcription of different genes in cells of
the blood-brain barrier (BBB), including members of the nuclear factor
kappa B (NF- B) family and cyclooxygenase-2 (COX-2), the limiting
enzyme for the formation of prostaglandins (PGs). The present study
investigated the possibility that interleukin-1 (IL-1) plays an
essential role in these events during a systemic inflammatory response.
Both wild-type and IL-1-deficient mice were killed at different
times after two different immunogenic stimuli, i.e.,
intraperitoneal lipopolysaccharide (LPS) injection and
intramuscular turpentine injection, used here as a model of systemic
localized inflammatory insult. The inhibitory factor B (IB,
index of NF-B activity) and COX-2 transcripts were detected
throughout the brain by means of in situ hybridization. Systemic LPS injection caused a strong and rapid expression of IB
in endothelial cells lining the BBB of large and small blood vessels
and thereafter within parenchymal microglia across the brain. This
treatment also provoked a transient expression of COX-2 along cells of
the vascular system, and the expression pattern and intensity of the
signal for both transcripts were essentially the same in wild-type and
IL-1-deficient animals. In contrast, the induction of these genes
that was quite selective to the cells of the BBB in response to
intramuscularly turpentine insult was completely abolished in
IL-1-deficient mice. Indeed, a late and prolonged expression of
IB and COX-2 mRNAs was found along the cerebral blood vessels in
response to the sterile and localized inflammation in wild-type mice,
whereas such induction was absent in the brain of IL-1-deficient
animals. These results indicate that IL-1 has an obligatory role in
the activation of NF-B molecules and PGs within endothelial cells of
the BBB in an experimental model of intramuscularly turpentine-induced
inflammation but not during endotoxemia.
Key words:
blood vessels; circumventricular organs; endothelial
cells; in situ hybridization histochemistry; inflammation; interleukin-1-deficient mice; immunocytochemistry; lipopolysaccharide; proinflammatory cytokines; microglia; macrophages; septic shock; transcription factor
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INTRODUCTION |
Systemic administration of the
endotoxin lipopolysaccharide (LPS), a component of the outer membrane
of Gram-negative bacteria, is a powerful immune challenge associated
with an increase in the circulating levels of different proinflammatory
cytokines, such as tumor necrosis factor (TNF-),
interleukin-1 (IL-1), and IL-6 (Andersson et al., 1992 ). It is
not well known how cell activation is triggered after binding between
the LPS binding protein-LPS complex and the
glycosyl-phosphatidylinositol-anchored membrane CD14
(mCD14), although there is now evidence that activation of
tyrosine kinase leads to transduction signal and cytokine gene transcription through the nuclear factor kappa B (NF-B). NF-B is
normally present in the cytoplasm forming an inactive complex with an
inhibitor known as IB. After extracellular stimulation by growth
factors, mitogens and cytokines that activate mitogen-activated protein
(MAP) kinases, IB is phosphorylated by NF-B-inducible kinase
(NIK)/IB kinase (IKK), ubiquitinated and degraded by
cytoplasmic proteasome (Baeuerle, 1998; Baeuerle and Baltimore, 1996).
Free active NF-B (the most common complex is the p50/p65
heterodimer) is then translocated into the nucleus, where it is able to
regulate transcription of various genes in binding to its B
consensus sequence. After its degradation, IB is rapidly
resynthesized to act as an endogenous inhibitory signal for NF-B,
and monitoring IB expression is an effective tool to investigate
the activity of the transcription factor within the CNS.
Circulating LPS and proinflammatory cytokines have the ability to cause
robust transcriptional activity of IB in different structures of
the brain (Quan et al., 1997; Laflamme and Rivest, 1999). The endotoxin
injected either intravenously or intraperitoneally caused a rapid and
prolonged expression of IB mRNA in the endothelium of the brain
blood vessels and parenchymal microglia, whereas the effects of
circulating IL-1 and TNF- were rapid (within 30-60 min) and
vanished 3 hr after the injection (Laflamme and Rivest, 1999). On the
other hand, a selective expression of IB was detected along the
cerebral endothelium of intramuscular turpentine-injected rats
(Laflamme and Rivest, 1999), a model of sterile and localized inflammatory insult that provokes a robust swelling at the site of
injection (Fantuzzi and Dinarello, 1996). The rapid and transient induction of IB indicates a strong NF-B activity in cells of the blood-brain barrier (BBB) by circulating proinflammatory
cytokines, which may lead to the transcription of target genes.
One potential candidate is the gene encoding cyclooxygenase-2 (COX-2),
the limiting enzyme for the formation of prostaglandins (PGs). Two
putative NF-B motifs from the COX-2 promoter were found to bind
p50/p65 NF-B heterodimers in an IL-1-dependent manner, and the
two NF-B subunits synergistically activate a  917/+49 COX-2
promoter construct (Crofford et al., 1997; Sorli et al., 1998). The
NF-B site (nucleotide 223 to 214) is also involved in
LPS-induced expression of COX-2 in monocytic differentiated U937 cells
(Inoue and Tanabe, 1998), whereas hypoxia induces COX-2 transcription
via p65 binding to 3' NF-B consensus element in the enzyme upstream
promoter region in vascular endothelial cells (Schmedtje et al., 1997).
Of interest is the fact that systemic LPS, intravenous injection of
IL-1 and TNF-, and intramuscular turpentine insult cause a
profound transcriptional activation of the gene encoding COX-2 along
the brain vascular cells (Lacroix and Rivest, 1998). It is therefore
likely that circulating proinflammatory molecules stimulate PG
production via transcriptional activation of COX-2 through NF-B
signaling pathways within cells of the BBB.
Although the release of proinflammatory cytokines by systemic
phagocytes may contribute to activate the cerebral endothelial cells in
response to systemic LPS, the effects of the endotoxin may be direct
and independent of the production of other circulating molecules. The
endothelium does not express mCD14, but these cells were shown to play
a major role in the pathogenesis of Gram-negative bacterial sepsis via
free soluble CD14. In fact, LPS can trigger tyrosine phosphorylation of
MAP kinases within endothelial cells despite the lack of mCD14 (Arditi
et al., 1995). However, a direct action on cells of the BBB may not
take place during a systemic and localized inflammatory response, such
as intramuscular turpentine injection. This model of localized tissue
damage provokes a specific induction of IL-1 and IL-6 without any
detectable IL-1 and TNF- production, suggesting the existence of
a common cascade of cytokine release, characteristic of sterile
inflammation in which IL-1 and IL-6 might play a critical role
(Fantuzzi and Dinarello, 1996). Interestingly, IL-1-deficient mice
respond normally after systemic LPS injection, whereas the mutant mice
exhibit an impaired acute-phase inflammatory response and are
completely resistant to fever development when challenged with
turpentine (Zheng et al., 1995). Because intravenous IL-1 injection
is capable of triggering the transcription of IB and COX-2 in
cells of the BBB, whereas intravenous injection of high doses of IL-6
has no effect (Lacroix and Rivest, 1998; Laflamme and Rivest, 1999), we
hypothesized that IL-1 has a leading role in triggering NF-B
signaling events in cells lining the vascular wall of the CNS
irrigating system. We show here an elegant pattern of IB
expression in the mouse brain in response to different systemic
immunogenic challenges and that IL-1 is responsible for the effects
of intramuscular turpentine insult but not endotoxemia.
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MATERIALS AND METHODS |
Animals. Interleukin-1 knock-out mice were
generously provided by Dr. H. Zheng (Merck Research Laboratories,
Rahway, NJ) and generated with their control littermates as described
previously (Zheng et al., 1995). Adult male mice of both groups
[~20-30 gm body weight (b.w.)] were acclimated to standard
laboratory conditions (14/10 light/dark cycle; lights on at 6:00
A.M. and off at 8:00 P.M.; room temperature at 23 ± 1°C)
with access to mouse chow and water ad libitum. Each mouse
was only used once for experimentation, and all protocols were approved
by the Animal Welfare Committee of Laval University. A total of
53 mice were assigned to different protocols each, corresponding to
different treatments and postinjection times. Paired vehicle-treated
mice of both wild-type and mutant groups were also killed at
corresponding times after the injection.
Experimental protocols. The bacterial endotoxin LPS (100 µg/100 gm) diluted in 100 µl of sterile pyrogen-free saline or the vehicle solution was administered into the peritoneal cavity. Two other
groups of mice were injected into the left thigh muscle with 50 µl/100 gm b.w. of turpentine (TU 109, CAS 8006-64-2; Spectrum Chemical, Gandena, CA) or 0.9% of sterile pyrogen-free saline. This
experimental model of sterile inflammation (i.e., an inflammatory response developing in the absence of any microbial stimulus) induces a
local tissue damage that is responsible for the development of a
systemic acute-phase response.
At various times after the systemic treatments (from 30 min to 12 hr,
depending on the challenge), animals were deeply anesthetized via an
intraperitoneal injection of a mixture of ketamine hydrochloride (91 mg/ml) and xylazine (9 mg/ml) and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer, pH 9.5, at 4°C. For the
combination of immunocytochemistry [especially for
COX-2-immunoreactive (IR) cells] with in situ hybridization, another group of mice was perfused with saline, followed
by 4% paraformaldehyde in 0.1 M sodium
phosphate, pH 7.2. In this particular case, brains were removed from
the skull, post-fixed for 2 hr, and then placed in 20% sucrose diluted
in 4% paraformaldehyde-sodium phosphate buffer for 12-15 hr.
Single in situ hybridization histochemistry.
Rapidly after the transcardiac perfusions, brains were removed from the
skulls, post-fixed for 2-8 d, and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at
4°C. The frozen brains were mounted on a microtome (Reichert-Jung; Cambridge Instruments, Deerfield, IL), and cut into 20 µm coronal sections from the olfactory bulb to the end of the medulla. The slices
were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer, pH 7.3, 30% ethylene
glycol, and 20% glycerol) and stored at 20°C. Hybridization
histochemical localization of each transcript was performed on every
fifth section of the whole rostrocaudal extent of each brain using
35S-labeled cRNA probes. All solutions
were treated with diethylpyrocarbonate (Depc) and sterilized to prevent
RNA degradation. Tissue sections mounted onto gelatin and
poly-L-lysine-coated slides were desiccated overnight under vacuum, fixed in 4% paraformaldehyde for 20 min, and
digested with proteinase K (10 µg/ml in 0.1 M
tris HCl, pH 8.0, and 50 mM EDTA, pH 8.0, at
37°C for 25 min). Thereafter, the brain sections were rinsed in
sterile Depc water, followed by a solution of 0.1 M triethanolamine (TEA), pH 8.0, acetylated in
0.25% acetic anhydride in 0.1 M TEA, and
dehydrated through graded concentrations of alcohol (50, 70, 95, and
100%). After vacuum drying for a minimum of 30 min, 90 µl of
hybridization mixture (107 cpm/ml) was
spotted on each slide, sealed under a coverslip, and incubated at
60°C overnight (~15-20 hr) in a slide warmer. Coverslips were then
removed, and the slides were rinsed in 4× SSC at room
temperature. Sections were digested by RNase A (20 µg/ml, 37°C, 30 min), rinsed in descending concentrations of SSC (2, 1, and 0.5×),
washed in 0.1× SSC for 30 min at 60°C (1× SSC: 0.15 M NaCl and 15 mM sodium
citrate buffer, pH 7.0), and dehydrated through graded concentrations
of alcohol. After being dried for 2 hr under vacuum, the sections were
exposed at 4°C to x-ray films (Eastman Kodak, Rochester, NY)
for 17-65 hr (depending on the probe), defatted in xylene, and dipped
into NTB2 nuclear emulsion (diluted 1:1 with distilled water; Eastman
Kodak). Slides were exposed for 7 (IB mRNA) or 20 (COX-2 mRNA) d,
developed in D19 developer (Eastman Kodak) for 3.5 min at 14-15°C,
washed 15 sec in water, and fixed in rapid fixer (Eastman Kodak) for 5 min. Tissues were then rinsed in running distilled water for 1-2 hr, counterstained with thionin (0.25%), dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with
distrene plasticizen xylene (DPX) mounting medium.
cRNA probe synthesis and preparation. The Bluescript SK
plasmid containing the 1.114 kb full-length coding sequence of the mouse IB cDNA (kindly provided by Dr. Alain Israel, Institut Pasteur, Paris, France) was linearized with BamHI and
HindIII for the antisense and sense riboprobes,
respectively. The pGEM4 plasmid containing the COX-2 cDNA fragment
(kindly provided by Dr. K. Peri, Ste-Justine Hospital Research Center,
Montreal, Canada) was linearized with HindIII and
EcoRI for the antisense and sense riboprobes, respectively.
The length of COX-2 cDNA fragment was 176 bp consisting of nucleotides
124-300 of the complete published cDNA sequence (Feng et al., 1993).
Radioactive cRNA copies were synthesized by incubation of 250 ng of
linearized plasmid in 6 mM
MgCl2, 40 mM Tris, pH 7.9, 2 mM spermidine, 10 mM
NaCl, 10 mM dithiothreitol, 0.2 mM ATP-GTP-CTP, 200 µCi of
-35S-UTP (catalog #NEG 039H;
DuPont NEN, Boston, MA), 40 U of RNAsin (Promega, Madison, WI), and 20 U of T7 (IB antisense and COX-2 sense probes), SP6 (COX-2
antisense probe), or T3 (IB sense probe) RNA polymerase for 60 min at 37°C. Unincorporated nucleotides were removed using
ammonium-acetate precipitation method; 100 µl of DNase solution (1 µl DNase, 5 µl of 5 mg/ml tRNA, 94 µl of 10 mM Tris/10 mM
MgCl2) was added, and 10 min later, a
phenol-chloroform extraction was performed. The cRNA was precipitated
with 80 µl of 5 M ammonium acetate and 500 µl
of 100% ethanol for 20 min on dry ice. The pellet was washed with 500 µl of 70% ethanol, dried, and resuspended in 100 µl of 10 mM Tris/1 mM EDTA. A
concentration of 107 cpm probe was mixed
into 1 ml of hybridization solution [500 µl of formamide, 60 µl of
5 M NaCl, 10 µl of 1 M
Tris, pH 8.0, 2 µl of 0.5 M EDTA, pH 8.0, 50 µl of 20× Denhart's solution, 200 µl of 50% dextran sulfate, 50 µl of 10 mg/ml tRNA, 10 µl of 1 M DTT, (118 µl Depc water; volume of probe used)]. This solution was mixed and
heated for 5-10 min at 65°C before being spotted on slides.
Combination of immunocytochemistry with in situ
hybridization. Immunocytochemistry was combined with the
in situ hybridization histochemistry protocol to determine
the types of cells that express IB and COX-2 transcript and
protein. Among the antibodies selected for this study, anti-von
Willebrand factor (vWF) was used to stain the endothelial cells of the
vasculature, whereas anti-ionized calcium binding adapter molecule 1 (iba1) labeled cells of myeloid lineage (macrophages and
microglia). Dual labeling for COX-2 protein and IB or COX-2 mRNA
was accomplished using an affinity-purified polyclonal antibody raised
against the mouse COX-2 peptide. Every fifth brain section was
processed by using the avidin-biotin amplification bridge method with
peroxidase as a substrate. Briefly, slices were washed in sterile
Depc-treated 50 mM potassium PBS (KPBS) and incubated 2 hr at room temperature with either vWF, iba1, or COX-2
antibody diluted in sterile KPBS plus 0.4% Triton X-100 plus 1% bovine serum albumin (BSA) (fraction V; Sigma, St. Louis, MO)
plus 0.25% heparin sodium salt USP (ICN Biomedicals, Aurora, OH).
Sheep anti-vWF (catalog #CL20176A-R, lot AB22-74; Cederlane Laboratory, Hornby, Ontario, Canada), rabbit ant-iba1
(generously provided by Dr. Y. Imai, National Institute of
Neuroscience, Kodaira, Tokyo, Japan) (Imai et al., 1996) and COX-2
polyclonal antibody raised in goat (catalog #SC1747, lot L148; Santa
Cruz Biotechnology, Santa Cruz, CA) were diluted at 1:1000 or 1:8000
(iba1). After incubation with the primary antibodies, brain slices were
rinsed in sterile KPBS and incubated with a mixture of KPBS plus 0.4% Triton X-100 plus 1% BSA plus 0.25% heparin plus biotinylated secondary antibodies (rabbit anti-sheep IgG for vWF, goat anti-rabbit for iba1, and horse anti-goat IgG for COX-2; 1:1500 dilution) (Vector
Laboratories, Burlingame, CA) for 60 min. Sections were then rinsed
with KPBS and incubated at room temperature for 60 min with an
avidin-biotin-peroxidase complex (Vectastain ABC elite kit; Vector
Laboratories). After several rinses in sterile KPBS, the brain slices
were reacted in a mixture containing sterile KPBS, the chromogen
3,3'-diaminobenzidine tetrahydrochloride (0.05%), and 0.003%
H2O2.
Thereafter, tissues were rinsed in sterile KPBS, immediately mounted
onto gelatin and poly-L-lysine-coated slides, desiccated under vacuum for 30 min, fixed in 4% paraformaldehyde for 20 min, and
digested by proteinase K (10 µg/ml in 100 mM Tris HCl, pH 8.0, and 50 mM EDTA, pH 8.0) at 37°C for 25 min.
Prehybridization, hybridization, and posthybridization steps were
performed according to the above description with the difference of
dehydration (50, 70, 95, 100% alcohol), which was shortened to avoid
decoloration of immunoreactive cells (brown staining). After being
dried for 2 hr under the vacuum, sections were exposed at 4°C to
x-ray film (Eastman Kodak) for 17 hr, defatted in xylene, and dipped
into NTB2 nuclear emulsion (diluted 1:1 with distilled water; Eastman Kodak). Slides were exposed for 9 (IB mRNA) or 20 (COX-2 mRNA) d,
developed in D19 developer (Eastman Kodak) for 3.5 min at 15°C, and
fixed in rapid fixer (Eastman Kodak) for 5 min. Tissues were then
rinsed in running distilled water for 1-2 hr, rapidly dehydrated through graded concentrations of alcohol, cleared in xylene, and coverslipped with DPX. The presence of IB and COX-2 transcript was detected by the agglomeration of silver grains in perikarya, whereas vWF, iba1, and COX-2 immunoreactivity within the cell cytoplasm
and ramifications (microglia) was indicated by a brown homogeneous
coloration. Determination of the double-labeled cells was performed
visually for each cell exhibiting clear brown cytoplasm and a number of
silver grains within the cell body delineating convincing hybridized message.
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RESULTS |
Time-related induction of IB by intraperitoneal LPS
A robust hybridization signal was detected over the large blood
vessels of the leptomeninges, choroid plexus, circumventricular organs,
and those penetrating the brain parenchyma 30 min after the
intraperitoneal LPS injection (Fig. 1).
However, small capillaries also exhibited IB-positive cells at
that time, although the intensity of the signal varied among
capillaries and regions of the brain. Ninety minutes after LPS
injection, the message became very strong within the whole brain
vasculature and within scattered small cells across the brain
parenchyma. We have reported previously that these cells were microglia
because they were immunoreactive to the complement receptor type 3 on
phagocytes (CD11b/c) (Laflamme and Rivest, 1999). The intensity of the
signal declined along the endothelium of the blood vessels 3 hr after
the endotoxin administration, but it remained high within the small
scattered cells of the brain parenchyma. At 6 hr, the mRNA encoding
IB returned to basal levels in arteries, capillaries, and
venules, although small scattered positive cells were still detected
across the brain (Fig. 1, bottom panels). The message was
undetectable in the cerebral vascular elements and parenchymal
microglia 12 hr after the single injection with the bacterial endotoxin
(data not shown) and at all postinjection times with the vehicle
solution (Fig. 1, top panels).
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Figure 1.
Time-related influence of endotoxin LPS
injection on the expression of the mRNA encoding IB in the blood
vessels and parenchymal elements of the mouse brain. Animals were
killed 0.5, 1.5, 3, and 6 hr after intraperitoneal treatment with LPS
(100 µg/100 gm b.w.) or the vehicle (Veh) solution.
These dark-field photomicrographs of dipped coronal sections (20 µm)
into nuclear emulsion NTB2 exhibit positive signal first within blood
vessels and then within small cells scattered across the brain
parenchyma. Magnification: left panels, 25×;
right panels, 50×.
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The pattern of IB expression and the intensity of the signal was
essentially the same in wild-type and IL-1-deficient mice in all of
the animals evaluated in this study (Table
1). Figure 2 shows representative examples of
IB expression in cells of the vascular and parenchymal elements
of the brain in wild-type and IL-1-deficient mice 3 hr after
intraperitoneal LPS injection (middle panels). Both mouse
groups exhibited a moderate-to-strong signal for the inhibitory factor
transcript along the endothelium, as well as within small scattered
cells across the brain parenchyma, a message that returned to basal
levels at 12 hr after injection in wild-type as well as
IL-1-deficient mice.
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Table 1.
Qualitative analysis of the hybridization signal for
IB and COX-2 transcripts in the brain of IL-1-deficient (KO)
and wild-type (WT) mice in response to intraperitoneal LPS injection
and intramuscular turpentine
abscess
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Figure 2.
Effects of the bacterial endotoxin LPS or
turpentine injection on the expression of IB mRNA in the brain of
wild-type (WT) and IL-1-deficient
(KO) mice. These dark-field photomicrographs of dipped
NTB2 emulsion slides depict IB mRNA induction in vascular and
nonvascular elements of the brain in mice killed 3 hr after
intraperitoneal LPS injection or 7.5 hr after intramuscular turpentine
administration. Note that LPS caused induction of the inhibitory factor
within blood vessels as well as dispersed small cells in the brain of
both mouse groups (middle panels), whereas the systemic
and localized inflammatory insult provoked IB transcription only
in cells associated with the blood-brain barrier in wild-type
but not IL-1 knock-out mice (right panels).
Magnification, 25×.
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Effect of intramuscular turpentine injection on
IB expression
In contrast to systemic LPS administration, intramuscular
turpentine insult stimulated IB transcription only in cells
associated with the BBB and not within scattered small cells across the
brain parenchyma as seen in LPS-challenged mice (Fig. 2, top
right panel). We have observed similar selective IB
mRNA induction in vascular cells of the brain in the rats killed up to
2 d after intramuscular turpentine injection in which the signal
peaked at 6 and 12 hr (Laflamme and Rivest, 1999). Of interest is the
lack of positive signal in vascular elements of the brain in
IL-1-deficient mice after the sterile and localized systemic
inflammatory insult, whereas the signal was intense along the cerebral
endothelium of wild-type animals (Fig. 2, right panels).
Indeed, the mRNA encoding IB was comparable with background
levels in cells of the BBB of all the IL-1-deficient animals that
received the same dose of turpentine and were killed together with
their wild-type littermates. Injection of sterile saline in the left
thigh muscle (control treatment) did not activate transcription of
IB in the brain of either wild-type or IL-1 knock-out mice
(data not shown).
COX-2 expression in response to systemic inflammation
Although intraperitoneal LPS caused NF-B activation in both
vascular- and parenchymal-associated cells of the brain of both wild-type and IL-1-deficient mice, COX-2 mRNA was induced only within cells of the BBB during endotoxemia (Table 1, Fig.
3). Indeed, a positive signal was
detected along the leptomeninges, choroid plexus, as well as large and
small blood vessels penetrating the different regions of the brain 3 hr
after the systemic injection with the endotoxin. The pattern of
expression and the intensity of the COX-2 hybridization signal in these
structures were comparable in wild-type and IL-1-deficient mice in
response to intraperitoneal LPS challenge (Fig. 3, middle
panels). In contrast, intramuscular turpentine insult caused COX-2
transcriptional activation only in vascular-associated cells of
wild-type and not in the brain of IL-1-deficient animals (Fig. 3,
right panels). The systemic and sterile inflammatory
challenge induced COX-2 expression in the cerebral endothelium of
control littermates, whereas no positive cells were found along the BBB
of all IL-1 knock-out mice. Only constitutive expression was
observed in the brain of these latter and vehicle-injected mice that
exhibited positive signal in neurons of different regions, such as the
hippocampal formation, piriform cortex, and amygdala (data not
shown).
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Figure 3.
Representative examples of COX-2 expression in
blood vessels of wild-type (WT) and
IL-1-deficient (KO) mice in response to systemic LPS
or turpentine injection. Animals were killed 3 hr after intraperitoneal
LPS injection (middle panels) and 7.5 hr post-turpentine
injection into the left hind paw (right panels). These
dark-field photomicrographs show in situ hybridization
signals for COX-2 mRNA through the brain microvasculature in wild-type
mice receiving either LPS or turpentine challenge and in knock-out
animals during endotoxemia. The mRNA encoding COX-2 was undetectable in
the cerebral vascular cells of IL-1 knock-out mice in response to
the systemic and localized inflammatory insult (right bottom
panel). Magnification, 25×.
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IB and COX-2 transcripts are expressed in
endothelial cells
To determine the phenotype of cells expressing the induced genes
in vascular-associated elements, immunocytochemistry was combined with
in situ hybridization on the same coronal sections of
wild-type mice. Figure 4 shows different
examples of such dual-labeling procedures and presents evidence that
IB and COX-2 transcripts are colocalized within vWF-IR cells.
Indeed, the large majority of IB- and COX-2-positive cells were
found over those that stained for endothelial markers (Fig. 4,
top and middle panels), although isolated
IB cells were also detected across the brain parenchyma and these
were immunoreactive to a microglial marker (Laflamme and Rivest, 1999).
Of interest is the fact that COX-2-IR cells were positive for IB
mRNA in response to LPS administration (Fig. 4, middle right
panel), which suggests a potential interaction between
NF-B and COX-2 molecules in the endothelium of the brain blood
vessels.
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Figure 4.
Phenotype of IB- and COX-2-expressing cells
during endotoxemia. Endothelial cells were labeled by
immunohistochemistry using an antisera against the von Willebrand
factor (vWF-ir), whereas a COX-2 antibody
(COX-2-ir) was used to perform the dual-labeling with
IB-expressing cells (middle right panel).
Cells of myeloid origin were labeled by the immunoperoxidase technique
using an antisera directed against ionized calcium binding adapter
molecule (iba1-ir, bottom panels).
IB or COX-2 mRNA was thereafter hybridized on the same sections
by means of a radioactive in situ hybridization
technique (silver grains). Note the presence of the mRNA
encoding COX-2 within endothelial cells (top panels,
black arrowheads) but not those positive for iba1
molecule (bottom panels, white
arrowheads). Note also the expression of IB mRNA in the
endothelium of the brain microvasculature and within cells positive for
COX-2 protein (middle panels). Black
arrowheads, Dual-labeled cells (endothelial-COX-2 mRNA,
top panels; endothelial-IB mRNA, middle
left panel; COX-2-IR-IB mRNA, middle right
panel); white arrowheads, single iba1-IR
cells along the vascular walls (perivascular microglia); white
arrows, single iba1-IR cells within the brain parenchyma
(parenchymal microglia); black arrows, single
COX-2-expressing cells along the cerebral endothelium.
bv, Blood vessel. Magnification, 100×.
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A polyclonal antibody directed against iba1 molecule was used to label
cells of myeloid origin, such as small ramified microglia that were
found across the brain parenchyma (Fig. 4, bottom panels, white arrows). Positive iba1-IR cells were also present
along blood vessels, although there was no clear overlap with
COX-2-expressing cells (silver grains, black
arrows). Indeed, the expression pattern of the mRNA encoding COX-2
was different from the perivascular microglial cells that were
dispersed along the vascular walls (white arrowheads). Other
antibodies (Mac-1 and F4/80) were used to label these cells, which also
failed to show convincing colocalization with COX-2 transcript (data
not shown).
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DISCUSSION |
The present study shows a strong transcriptional activation of
both IB and COX-2 genes in the mouse brain in response to different models of systemic immunogenic stimuli. The bacterial endotoxin caused an interesting pattern of IB expression; a robust signal was first detected in blood vessels and then within microglial cells across the brain parenchyma, whereas COX-2 was only
induced along vascular cells of the brain. Dual-labeling procedure
provided the anatomical evidence that both IB and COX-2
transcripts were expressed over cells that stained for endothelial markers. These results contrast with the report showing that COX-2-IR proteins were present essentially within perivascular microglia of the
rat brain (Elmquist et al., 1997a). We have used different antibodies (Mac-1, F4/80, and iba1) to label cells of myeloid lineage
in the mouse brain and found very few perivascular microglia with
convincing hybridization signal for COX-2 mRNA. It is possible that
antibodies and the technique used in this study were not sensitive
enough to detect all perivascular microglial cells or that species
differences exist between mice and rats. In agreement with the present
study, however, are the recent reports showing that LPS induces COX-2
mRNA in cerebral endothelial cells and not those of myeloid origin in
the rats (Matsumura et al., 1998a,b; Quan et al., 1998; Cao et al.,
1999). The dosage or strain of LPS may be important factors in
determining the cell type(s) in which COX-2 is induced, and the
distribution of the protein may not entirely match the mRNA.
The patterns of expression and intensity of signal in response to
intraperitoneal LPS injection were similar in IL-1-deficient mice
and their control littermates. In contrast to the effects of the
endotoxin that induced of IB in vascular and parenchymal elements
of the brain, intramuscular turpentine injection stimulated the
inhibitory factor only within cells of the BBB in wild-type mice. This
model of sterile and localized inflammation also provoked a
transcriptional activation of the gene encoding COX-2 in the endothelium of the brain blood vessels in wild type, whereas
IL-1-deficient mice did not exhibit notable induction of both
IB and COX-2 genes. Together, these data provide the evidence
that IL-1 is responsible for triggering the endothelium of the brain
capillaries during a systemic and localized inflammatory response and
not during endotoxemia. The fact that both IB and COX-2 genes are induced within the same cells of the BBB suggests that IL-1 may stimulate COX-2 transcription through NF-B signaling pathways, an
event that may be of great importance in the production of PGs in the
brain during the acute-phase response.
We have reported previously a robust and transient transcriptional
activation of both COX-2 and IB genes in cells of the BBB by an
intravenous IL-1 injection (Lacroix and Rivest, 1998; Laflamme and
Rivest, 1999). The binding of IL-1 to its cognate type I receptor
leads to the formation of the IL-1 receptor-associated kinase
(IRAK)/TNF receptor-associated factor 6 (TRAF6) complex, which
activates NIK/IKK kinases involved in the phosphorylation and
degradation of IB (Baeuerle, 1998). NF-B is then translocated into the nucleus and may bind to its B consensus sequence on target
genes (Baeuerle and Baltimore, 1996). The nuclear factor binding to the
COX-2 promoter is able to influence the enzyme transcription in
response to different immunogenic ligands, including IL-1 (Crofford
et al., 1997; Sorli et al., 1998). Here, we show that both IB and
COX-2 are expressed within the same cells, indicating a potential
interaction between the transcription factor and COX-2 expression in
the cerebral endothelium of immune-challenged animals. Obviously, this
remains speculative from these anatomical data, and the signaling
pathways that lead to the NF-B nuclear translocation and COX-2
transcription have yet to be determined in the cells of the BBB.
In vivo approaches are quite limited in the investigation of
the intracellular events taking place within specific cellular
populations of the CNS, but they provide an essential integration of
the systems that interact during the acute-phase response. In this
regard, the results that IB and COX-2 transcripts were induced
only in wild-type and not in IL-1-deficient mice in response to
intramuscular turpentine injection strongly support the hypothesis that
the circulating proinflammatory cytokine, when released by the site of
inflammation (here the left hind paw), has a key role in leading to the
NF-B signaling pathway and COX-2 transcription in the brain
endothelial cells.
The physiological outcomes of such molecular mechanisms are numerous,
and the well characterized role of PGs in mediating fever during the
acute-phase response and circulating IL-1 has been studied in
various species (for review, see Elmquist et al., 1997b). Of
interest is the data that IL-1-deficient mice respond normally after
systemic LPS injection, whereas the mutant mice exhibit an impaired
acute-phase inflammatory response and are completely resistant to fever
development when challenged with turpentine (Zheng et al., 1995). The
data presented in this study are in agreement with this original
report, because COX-2 and NF-B activity is maintained in
IL-1-deficient mice that received the bacterial endotoxin but not
after intramuscular turpentine injection. It is possible that the
effects of the bacterial endotoxin in IL-1-deficient mice are
mediated by the production of other proinflammatory cytokines, such as
TNF-, which has the ability to stimulate IB and COX-2 in
cerebral endothelial cells (Lacroix and Rivest, 1998; Laflamme and
Rivest, 1999). However, cytokine production induced by LPS appears
essentially intact in TNF / mice, and
these animals show the same symptoms of toxicity as wild-type mice
after high doses of LPS (Marino et al., 1997). It would then be
surprising to see a suppressed NF-B/COX-2 induction within the
endothelium of the brain blood vessels in
TNF/ mice treated systemically with
LPS. The bacterial endotoxin may in fact activate directly endothelial
cells via its soluble receptor CD14 and trigger tyrosine
phosphorylation of MAP kinases despite the lack of mCD14 (Arditi et
al., 1995). Such a mechanism may obviously not take place in response
to a localized systemic insult, and intermediate molecules produced at
the site of inflammation and released into the bloodstream must
transmit the information to the brain.
The present results highlight a central role for IL-1 as a mediator
of the acute-phase response in an experimental model of localized
inflammation associated with a increase in the circulating levels of
IL-1 and IL-6 but not IL-1 and TNF-. Circulating IL-6 may not
contribute to the induction of COX-2 and IB during systemic
inflammation, because high doses of recombinant IL-6 failed to
stimulate these genes in cerebral vascular cells (Lacroix and Rivest,
1998; Laflamme and Rivest, 1999). Moreover, mice deficient in either
IL-1 or IL-1 receptor antagonist genes did not exhibit a
suppressed acute-phase response upon injection with turpentine (Horai
et al., 1998). This latter study also reported regional differences in
the induction of COX-2 in large regions of the brain by Northern blot
analysis (Horai et al., 1998). As shown here and in other reports (Cao
et al.,1996; Elmquist et al., 1997a; Lacroix and Rivest, 1998),
COX-2 mRNA and protein are induced only in cells associated with the
brain vasculature in response to systemic immunogenic challenges, and
regional changes may simply correspond to the structures that are
either highly or poorly vascularized.
Together, these data suggest that circulating IL-1 targets
directly the cerebral endothelium to induce NF-B nuclear
translocation in response systemic and localized inflammatory
processes. Induction of the IL-1R1-Toll/IRAK/TRAF6/NIK/IKK pathway may
then lead to COX-2 gene transcription and brain
PGE2 production by microvascular-associated cells. These intracellular mechanisms are likely to be the critical link between the circulating immunogenic molecules and parenchymal elements of the brain to activate the neuronal circuits needed to
restore the homeostatic balance. Induction of fever and autonomic and
neuroendocrine functions, such as the increase in plasma glucocorticoid levels, may be the determinant functional consequences of the molecular mechanisms that take place in cells of the BBB.
| |
FOOTNOTES |
Received Aug. 18, 1999; revised Sept. 30, 1999; accepted Oct. 20, 1999.
This research was supported by the Medical Research Council of Canada
(MRCC). S.R. is an MRCC Scientist. S.L. is currently an MRCC
Postdoctoral Fellow in the Department of Neurosciences at the
University of California, San Diego (La Jolla, CA). We thank Dr. Alain
Israel (Institut Pasteur, Paris) for the gift of the plasmid containing
the mouse IB cDNA, Dr. K. Peri (Ste-Justine Hospital Research
Center, Montreal, Canada) for COX-2 cDNA, and Dr. H. Zheng (Merck
Research Laboratories, Rahway, NJ) for the generous gift of
IL-1-deficient mice.
Correspondence should be addressed to Dr. Serge Rivest, Laboratory of
Molecular Endocrinology, Centre de Recherche de l'Université Laval Research Center and Department of Anatomy and Physiology, Laval
University, 2705, boulevard Laurier, Québec, Canada G1V 4G2. E-mail: serge.rivest{at}crchul.ulaval.ca.
| |
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ABSTRACT
INTRODUCTION
