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Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, Québec, Canada G1V 4G2
1Correspondence: Laboratory of Molecular Endocrinology, CHUL Research Center and Department of Anatomy and Physiology, Laval University, 2705, boul. Laurier, Québec, Canada G1V 4G2. E-mail: Serge.Rivest{at}crchul.ulaval.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
activity and proinflammatory gene transcription during innate immune
response. Indeed, CD14 is not thought to participate directly in the
cell signaling, but rather one or more of the mammalian Toll-like
receptors (TLRs) acts in concert with the lipopolysaccharide (LPS)
receptor to discriminate between microbial pathogens or their products
and initiate transmembrane signaling. Mammalian cells may express as
many as 10 distinct TLRs, although the importance of TLR4 in response
to gram-negative bacteria and LPS is now supported by the fact that
TLR4-mutated mice are LPS resistant. We investigated the expression of
TLR4 across the rat brain under basal conditions and in response to
systemic LPS and IL-1ß injection. We first cloned the rat TLR4 cDNA
via RNA isolation and polymerase chain reaction (PCR) amplification
with a proofreading polymerase. Total RNA was isolated from the rat
liver tissue using Tri-Reagent and reverse transcribed into cDNA using
Superscript II reverse transcriptase and an oligonucleotide primer with
a degenerate 3' end of sequence 5'-T12(GAC)N-3'. Positive hybridization
signal was found in the leptomeninges, choroid plexus (chp),
subfornical organ, organum vasculosum of the lamina terminalis, median
eminence, and area postrema. Scattered small cells also displayed a
convincing hybridization signal within the brain parenchyma. Few
well-defined nuclei exhibited positive TLR4 transcript: the
supramamillary nucleus, cochlear nucleus, and the lateral reticular
nucleus. The circumventricular organs, the leptomeninges, and chp also
exhibited constitutive expression of the LPS receptor mCD14. In
contrast to the strong up-regulation of the gene encoding mCD14 during
endotoxemia, neither LPS nor IL-1ß caused a convincing increase in
the TLR4 mRNA levels across the CNS. A down-regulation of the gene
encoding TLR4 was found in the cerebral tissue of immune-challenged
animals. The constitutive expression of both mCD14 and TLR4 may explain
the innate immune response in the brain, which originates from the
structures devoid of blood–brain barrier in presence of circulating
LPS.—Laflamme, N., Rivest, S. Toll-like receptor 4: the missing link
of the cerebral innate immune response triggered by circulating
gram-negative bacterial cell wall components.
Key Words: circumventricular organs • innate immune response • in situ hybridization histochemistry • inflammation • lipopolysaccharide • proinflammatory cytokines • microglia • macrophages • NF-B • septic shock
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. These elements are called the pathogen-associated
molecular patterns (PAMPs), which are recognized by specific cells of
the immune system as innate mechanisms to mount a rapid response to
bacterial infection. The endotoxin lipopolysaccharide (LPS) is a major
component of the outer membranes of gram-negative bacteria, which is
the best-characterized example of innate recognition associated with a
robust inflammatory response by phagocytic cells (2)
.
Secretion of cytokines by circulating monocytes/neutrophils and tissue
macrophages by LPS requires a series of mechanisms in cascade, the
first step being binding of the endotoxin with the serum proteins LPS
binding protein (LBP) or septins. The newly formed complex may then
activate different populations of cells in binding to its receptor in
two forms: the membrane CD14 (mCD14) and soluble CD14 (sCD14)
(3)
.
mCD14 is present at the surface of myeloid cells and acts as a
glycosylphosphatidylinositol (GPI) -anchored membrane glycoprotein,
whereas sCD14 lacks the GPI properties but can bind LPS to activate
cells devoid of mCD14, such as endothelial cells (4)
.
Until recently, the exact mechanisms involved in the activation of the
proinflammatory signal transduction pathways after binding between the
LBP–LPS complex and the GPI-anchored mCD14 were unknown. Indeed,
studies in CD14-deficient mice suggested the existence of a coreceptor
to mediate LPS-induced nuclear factor kappa B (NF-B) activity and
cytokine gene transcription (5
, 6)
. The recent
characterization of human homologues of Toll may be the missing link
for the transduction events leading to NF-B activity and cytokine
production in response to the bacterial cell wall components. A large
family of Toll-like receptors (TLRs) has already been characterized
that share similar extracellular and cytoplasmic domains
(1)
. The extracellular domains include 18–31 leucine-rich
repeats, whereas the cytoplasmic domains are similar to the cytoplasmic
portion of the interleukin 1 (IL-1) receptor and is named the
Toll/IL-1-receptor homologous region (1
, 7)
. Distinct TLRs
have now been proposed as the key molecules to recognize quite
selectively one of the major PAMPs produced by either gram-negative or
gram-positive bacteria. The data that mutation of the mouse
Lps locus abolishes the LPS response and that Lps
encodes the TLR4 provided the first evidence that this particular
receptor may play a key role in the innate immune response to
gram-negative bacteria (for a review, see ref 8
). Further
supporting this concept are the TLR4-deficient mice, which are
unresponsive to LPS, whereas TLR2-deficient mice exhibit a normal
inflammatory response to the endotoxin (9)
. These results
demonstrate that whereas TLR2 makes no contribution to LPS signaling,
TLR4 is critical to recognize the PAMP produced by gram-negative
bacterial cell wall components.
It is not yet known how LBP, CD14, and TLR4 interact to function as the
LPS signal transducer leading to activation of NF-B and
mitogen-activated protein kinases. It is possible that CD14 acts as the
principal LPS binding protein on the surface of monocytic cells and the
newly formed complex reaches adjacent TLR4 receptors that transduce the
LPS signal via the general adaptor protein MyD88 (8
, 10)
.
These events may also take place in the brain because CD14 mRNA is
constitutively expressed in the circumventricular organs (CVOs), brain
regions that contain a rich vascular plexus with specialized
arrangements of the blood vessels (11)
. The tight
junctions normally present between the endothelial cells are shifted in
part to the ventricular surface and partly to the boundary between the
CVOs and the adjacent structures, explaining the diffusion of large
molecules into the perivascular region (12)
. Circulating
LPS causes a rapid increase in CD14 transcription in these leaky
structures, whereas a delayed response can be found in parenchymal
cells located in the anatomical boundaries of the CVOs and thereafter
in microglia across the brain parenchyma (11
, 13)
.
These results strongly suggest that the endotoxin first reaches organs
devoid of the blood–brain barrier to induce the transcription of its
own receptor and thereafter increases CD14 biosynthesis within
parenchymal structures surrounding the CVOs and then the entire brain
of severely challenged animals. TLR4 is likely to play a key role in
the LPS signaling and the innate immune response that is triggered in a
very well-organized manner from specific structures of the brain during
endotoxemia (13
, 14)
. The purpose of this study was
therefore to investigate whether TLR4 is present in the brain and
determine the fine cellular distribution and regulation of the gene
encoding TLR4 in the rat brain under basal and immune-challenged
conditions.
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MATERIALS AND METHODS |
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230–260 g b.w.) were
acclimated to standard laboratory conditions (14 h light, 10 h
dark cycle; lights on at 0600 and off at 2000) with free access to rat
chow and water. Each rat was used only once for experimentation and all
protocols were approved by the Laval University’s Animal Welfare
Committee. Sixty-four rats were assigned to different protocols each
corresponding to different treatments, which were further subdivided
into different postinjection times (15 min to 24 h, depending on
the challenge). Paired vehicle-treated rats were also killed at
corresponding times after the injection.
Surgeries
Rats were anesthetized with an intraperitoneal injection of a
mixture (1 ml/kg b.w.) of ketamine hydrochloride (91 mg/ml) and
xylazine (9 mg/ml) and implanted with a catheter into the jugular vein.
Catheters were made from a piece of silastic tubing (silastic medical
grade tubing: ID 0.020 in, OD 0.037 in; Dow Corning, Midland, Mich.)
connected to an intramedic polyethylene tubing (PE-50, ID 0.023 in, OD
0.038 in, Caly Adams, Becton Dickinson, Rutherford, N.J.). Outlets of
cannulas were placed at the level of the neck, and rats were housed
individually in plastic cages for a recuperation period of 3–5 days.
Experimental protocols
On the day of the experiment (0830 in the morning), the
outlet portion of the intravenous (i.v.) catheter was fixed to a
truncated 22 gauge needle, which was attached to a PE-50 tubing. These
connectors were then fixed to a 1 cc syringe and rats were placed
individually in a quiet room for at least 2 h before the
injections, which allows injections without disturbing the animals and
induction of genes that may be activated after the stress of handling.
LPS (40–100 µg/kg; from Escherichia coli, Serotype
055:B5, Sigma, L-2880, lot #127H4097), recombinant rat IL-1ß (1.8
µg/kg; kindly provided by Dr. R. Hart, The State University of
New-Jersey Rutgers, Newark, N.J.), or the vehicle solution (200 µl of
sterile pyrogen-free saline) was injected i.v. through the right
jugular vein.
At different times after the systemic injections (from 15 min to 24 h, depending on the challenge), animals were deeply anesthetized via an i.v. injection of a mixture of ketamine hydrochloride and xylazine, then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer (pH 9.5 at 4°C). Brains were removed swiftly from the skulls, postfixed for 2 to 8 days, and 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 Company, Deerfield, Ill.) and cut into 30 µm coronal sections from the olfactory bulb to the end of the medulla. The slices were collected in a cold cryoprotectant solution (0.05M sodium phosphate buffer, pH 7.3, 30% ethylene glycol, 20% glycerol) and stored at -20°C.
In situ hybridization histochemistry
Hybridization histochemical localization of TLR4, CD14, and
IB
mRNA was carried out on every sixth section of the whole
rostro-caudal 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 poly-L-lysine-coated slides
were desiccated overnight under vacuum, fixed in 4% paraformaldehyde
for 30 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). 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
2 h, 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 h) in a slide warmer.
Coverslips were then removed and the slides were rinsed in 4x standard
saline citrate (SSC) at room temperature. Sections were digested by
RNase A (20 µg/ml, 37°C, 30 min), rinsed in descending
concentrations of SSC (2x, 1x, 0.5x SSC), washed in 0.1x SSC for 30
min at 60°C (1x SSC: 0.15 M NaCl, 15 mM trisodium citrate buffer, pH
7.0), and dehydrated through graded concentrations of alcohol. After
being dried for 2 h under vacuum, the sections were exposed at
4°C to X-ray films (Biomax, Kodak, Rochester, N.Y.) for 1–3 days.
The slides were defatted in xylene, dipped in NTB-2 nuclear emulsion
(Kodak; diluted 1:1 with distilled water), exposed for 7 days (IB
transcript), 14 days (CD14 transcript), or 19 days (TLR4 transcript),
developed in D19 developer (Kodak) for 3.5 min at 14–15°C, washed
15 s in water, and fixed in rapid fixer (Kodak) for 5 min. Tissues
were then rinsed in running distilled water for 1–2 h, counterstained
with thionin (0.25%), dehydrated through graded concentrations of
alcohol, cleared in xylene, and coverslipped with distrene plasticizer
xylene mounting medium.
Cloning of the full-length rat TLR4 cDNA
Tri-Reagent used for RNA isolation was purchased from Molecular
Research Center (Cincinnati, Ohio). Superscript II reverse
transcriptase and oligo (dt) primer was purchased from Life
Technologies, Inc (Gaithersburg, Md.). Synthetic oligonucleotides were
synthesized in our laboratory using an applied Biosystems Model 394 DNA
synthesizer (Foster City, Calif.). Pfu DNA polymerase and Robocycler
Gradient 40 were purchased from Stratagene Cloning Systems (La Jolla,
Calif.). Zero blunt topo PCR cloning kit was purchased from Invitrogen
(Carlsbad, Calif.). The Qiagen plasmid kit and Qiaquick PCR
purification kit were purchased from Qiagen Inc. (Chatsworth, Calif.).
T7 sequencing kit was purchased from USB Corporation (Cleveland, Ohio).
Total RNA was isolated from the rat liver tissue using Tri-Reagent and
reverse transcribed into cDNA using Superscript II reverse
transcriptase and an oligonucleotide primer with a degenerate 3' end of
sequence 5'-T12 (GAC)N-3'. A DNA fragment of 2.5 kb corresponding to
the complete coding sequence of the reported rat Toll receptor cDNA
(nucleotide 260 to 2767, Genbank accession #AF057025) was amplified by
PCR from a cDNA library using a pair of 23 bp oligonucleotide primers
complementary to nucleotides 259 to 282
(5'-ATGATGCCTCTCTTGCATCTGGC-3') and 2745 to 2767
(5'-TCAGGTCAAAGTTGTTGCTTCTT-3'). The proofreading polymerase pfu,
generating blunt-end extremities, was used to permit direct insertion
of the PCR product into the plasmid vector PCR-Blunt II Topo (Zero
blunt topo PCR cloning kit, Invitrogen). To confirm the success of the
insertion, 130 bp of both PCR product extremities were sequenced
using a T7 sequencing kit.
cRNA probe synthesis and preparation
The PCR-Blunt II topo plasmid containing the rat TLR4 cDNA
fragment of 2.5 kb was linearized with PstI and
KpnI for the antisense and sense riboprobes, respectively.
The pBlueScript SK plasmids containing the full-length coding sequence
of the rat CD14 cDNA (kindly provided by Dr. Doug Feinstein, Cornell
University Medical College, New York; ref 11
) or the mouse
IB cDNA (kindly provided by Dr. Alain Israël, Institut
Pasteur, Paris, France; ref 15
) were linearized with
SacI and KpnI (CD14) or BamHI and
HindIII (IB) for the antisense and sense riboprobes,
respectively. Radioactive cRNA copies were synthesized by incubation of
250 ng 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, 100 µCi of -35S-UTP (DuPont
NEN, #NEG 039H), 20 U RNAsin (Promega, Madison, Wis.), and 10 U of
either T7 (CD14 and IB antisense probe, TLR4 sense probe), SP6
(TLR4 antisense probe), or T3 (CD14 and IB sense probe) RNA
polymerase for 60 min at 37°C. Unincorporated nucleotides were
removed using an ammonium-acetate 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; 10 min later, a
phenol-chloroform extraction was performed. The cRNA was precipitated
with 80 µl of 5M ammonium acetate and 500 µl of 100% ethanol for
20 min on dry ice. The pellet was dried and resuspended in 50 µl of
10 mM Tris/1 mM EDTA. A concentration of 107 cpm
probe was mixed into 1 ml of hybridization solution (500 µl
formamide, 60 µl 5 M NaCl, 10 µl 1 M Tris-pH 8.0, 2 µl 0.5 M
EDTA-pH 8.0, 50 µl 20x Denhart’s solution, 200 µl 50% dextran
sulfate, 50 µl 10 mg/ml tRNA, 10 µl 1M DTT; 118 µl Depc water
minus volume of probe used). This solution was mixed and heated for 5
min at 65°C before being spotted on slides.
Qualitative analysis
The relative intensity of TLR4 mRNA signal throughout the brain
of each animal was assessed on dipped emulsion slides under microscopic
evaluation and graded according to the scale of undetectable (-), low
(+), moderate (++), strong (+++), or very strong signal (++++).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows representative examples of hybridized brain sections with either
sense (control) and antisense probes. Although a positive hybridization
signal can be detected with the antisense probe (right section), the
same adjacent sections did not display any positive signal when
hybridized with the sense probes (left section). These results provide
the evidence of the selectivity of the probes used in this experiment.
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Constitutive expression of the gene encoding TLR4 in the rat brain
A low to moderate TLR4 mRNA hybridization signal was found in the
CVO organum vasculosum of the lamina terminalis, subfornical organ,
median eminence, and the area postrema of vehicle-administered rats
(Fig. 2
, left column). The message was quite diffuse across the organs, but
also specific inasmuch as adjacent tissues hybridized with the sense
probe were completely devoid of positive labeling. Two other groups of
nonparenchymal cells also exhibited a moderate to strong hybridization
signal for the gene encoding TLR4. Indeed, TLR4-expressing cells were
present along the leptomeninges and choroid plexus (chp) of
vehicle-injected rats (Fig. 3
). Of interest is the constitutive expression of the LPS receptor mCD14
in the CVOs, chp, and the leptomeninges, a pattern that seemed
comparable to that of TLR4 transcript under basal conditions (Figs. 2
and 3)
.
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Positive TLR4-expressing cells were found in a limited number of
parenchymal structures: the posterior hypothalamic nucleus,
supramamillary nucleus, cochlear nucleus, and the ventrolateral
medulla. A few isolated cells were also detected in the caudal medulla
at the level of the gigantocellular and parvicellular reticular nuclei.
As shown by Fig. 4
, the pattern of expression suggests that these cells are likely to be
neurons, although the possibility remains that a mixed population of
cells have the TLR4 biosynthetic machinery in these nuclei. A different
type of distribution was observed on emulsion-dipped slides in other
parenchymal structures, especially within the regions lining the
cerebroventricular system. This signal for TLR4 transcript was
characterized by a homogeneous and diffused distribution of silver
grains over one or several adjacent brain areas. These include the
medial preoptic area (MPOA), paraventricular nucleus of the
hypothalamus, dorsomedial hypothalamic nucleus, arcuate nucleus, the
ventromedial hypothalamus surrounding the aqueduct and central canal,
and the dorsovagal complex. Figure 5
(top right) shows a representative example of such phenomenon in the
MPOA.
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TLR4 and CD14 mRNA in the brain of immune-challenged animals
Systemic LPS treatment caused a robust increase in the CD14 mRNA
levels in all the CVOs (11
and Fig. 2
, right column). The
CD14 mRNA signal reached a peak at 3 h postinjection, declined at
6 h, and returned to basal levels 24 h after LPS treatment.
CD14-positive cells spread over the anatomical boundaries of these
organs in a migratory-like pattern 3 h after i.v. LPS
administration (Fig. 6
, right column). At that time, small positive cells were found
throughout the entire brain parenchyma, and dual labeling procedure
indicated that different cells of myeloid origin have the ability to
express CD14 in response to systemic LPS (11)
.
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The robust increase in CD14 expression in the brain of LPS-treated rats
was not associated with a transcriptional activation of the gene
encoding TLR4, but a decrease in the relative mRNA levels (Fig. 2)
. The
hybridization signal was actually barely detectable in the brain of
rats that were killed 3 h after being injected with the endotoxin
(Table 1
). Although variability occurred among animals, the hybridization signal
was generally lower in both parenchymal and nonparenchymal structures
of the brain. The message was lower in numerous regions already at 30
min postinjection, although the decrease was apparent in all animals
3 h after the single bolus of LPS. The endotoxin failed to
activate the gene encoding TLR4 in any regions of the brain, a
phenomenon that contrasts with CD14 expression and NF-B activity. It
is interesting to note the robust activation of IB transcript
(index of NF-B activity) in the blood vessels and across the brain
parenchyma, whereas the TLR4 message essentially vanished in adjacent
sections of the MPOA (Fig. 5)
.
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Circulating IL-1ß also caused a profound NF-B activity that was
associated with a decrease in TLR4 mRNA in the MPOA and other regions
surrounding the ventral third ventricle (Table 2
). Once again, the lower relative levels of TLR4 transcript correlated
with the strong expression of the gene encoding IB in the
cerebral endothelium and the parenchymal microglia (Table 2
, Fig. 5
).
As for the LPS treatment, the proinflammatory cytokine failed to
activate TLR4 gene in both parenchymal and nonparenchymal structures of
the central nervous system (CNS).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activity, IB, the
endotoxin and circulating IL-1ß caused a significant decrease of TLR4
transcript in most of the constitutively expressing parenchymal and
nonparenchymal regions of the brain. Basal expression of CD14 and TLR4
in the CVOs is likely to be a key mechanism in the proinflammatory
signal transduction events that originate from these structures during
innate immune response. Indeed, cell wall components of the
gram-negative bacteria may be selectively recognized by the
TLR4/CD14-bearing cells of the CVOs, which allows LPS signaling and
then the rapid transcription of proinflammatory cytokines, first within
these organs and thereafter across the brain parenchyma during severe
endotoxemia.
The rapid induction of IL-1ß, IL-6, and tumor necrosis factor
(TNF-) mRNA in the CVOs, chp, and the leptomeninges by systemic LPS
treatment clearly indicates that such events occur in these specific
populations of cells in the brain (13
, 14
, 16
, 17)
.
Microscopic analysis of emulsion-dipped slides revealed that
TNF-positive cells spread over the anatomical boundaries of the CVOs in
a migratory-like pattern during the course of the endotoxemia
(14)
. A similar pattern of de novo expression
was observed for the gene encoding CD14, but not TLR4, in response to
circulating LPS. LPS-induced CD14 transcription in parenchymal
microglia is dependent on the centrally produced TNF-, which
actually plays an essential autocrine/paracrine role in triggering
parenchymal microglial cells during severe endotoxemia
(13)
. The coexistence of both TLR4 and CD14 receptors in
the CVOs may be the recognizing molecules for the endotoxin to trigger
the proinflammatory signal transduction events in structures that can
be reached from the systemic circulation, whereas subsequent microglial
activation in the brain parenchyma is dependent on TNF-. Therefore,
TLR4 may be essential in this innate immune reaction that originates
from the CVOs in response to cell wall components of gram-negative
bacteria.
Although a strong increase in CD14 transcription is generally detected after systemic LPS injection, the endotoxin failed to stimulate the gene encoding TLR4. CD14-expressing cells were clearly devoid of TLR4 transcript in microglia across the brain parenchyma during moderate and severe endotoxemia. It is possible that TLR4 is the recognizing molecule for gram-negative bacterial components only in response to systemic infection, whereas CD14 has a more complex role in the proinflammatory signal transduction events in the brain parenchyma. These events may be determinant for orchestrating the neuroinflammatory responses that take place in a well-coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS likely include a rapid elimination of LPS particles via an increased opsonic activity of the transmembrane CD14 receptor to prevent potential detrimental consequences on neuronal elements during blood sepsis.
Nomura and colleagues have recently reported that TLR4 mRNA expression
in mouse peritoneal macrophages significantly decreased within a few
hours of LPS treatment and returned to the original level at 24 h
(18)
. A rapid decrease in surface TLR4 expression was seen
as early as 1 h and remained suppressed over 24 h in cells
pre-exposed with LPS. These authors suggested that down-regulation of
the surface TLR4 expression may be responsible for the decrease in
inflammatory cytokine production in tolerant macrophages, which may
explain one of the mechanisms for LPS tolerance (18)
.
These data obtained from systemic macrophages agree completely with the
present study, which shows convincing down-regulation of TLR4 gene in
response to a single LPS bolus. The phenotype of TLR4 cells in the CVOs
was not determined in the present study due to the rather low levels of
TLR4 transcript, making interpretation of the agglomeration of silver
grains within immunoreactive cells arbitrary. Because LPS has the
ability to increase CD14 mRNA in these organs, it was possible to
perform the dual labeling for the LPS receptor, and numerous resident
macrophages were positive for the transcript (11)
.
Although both transcripts may not be expressed in the same cells, we
speculate here that TLR4 is located at the surface of the phagocytic
population of cells of the CVOs, chp, and leptomeninges.
As depicted by different figures, TLR4 transcript levels were low in
the cerebral tissue under basal conditions. The signal was nevertheless
specific, as we did perform numerous controls to ensure that what was
being seen may not be related to an artifact of the in situ
hybridization procedure. We had to adjust and maximize the
hybridization conditions to detect this transcript in situ
by generating the riboprobe just after the prehybridization step on
freshly mounted brain sections. This very low level in the brain,
however, fits quite well with the fact that the copy number of TLR4 is
extremely low in systemic phagocytes compared to the more abundant
membrane protein CD14 (8)
. It is nevertheless remarkable
that so few TLR4 receptors (perhaps 1000 or fewer per cell) residing on
macrophages alone have such an important influence in the LPS signaling
and the coordination of the biological responses to gram-negative
infections (8)
. It is expected that CVO TLR4 acts as a
sensor for engaging the cerebral innate immune response in the case of
invasion during such systemic bacterial infection, which may have
detrimental consequences for the neuronal material.
Of interest is the constitutive expression of TLR4 in different nuclei and areas of the brain suggesting a potential role of this membrane-spanning component in the parenchymal elements of the brain. However, TLR4 seems quite specific to cell wall components of gram-negative bacteria; what, then, will a receptor do without ligand? This question obviously is difficult to answer: we are just at the embryonic stage of the mammalian Toll biology, and this is the first report showing evidence that TLR4 may be a key element of the well-organized innate immune response that takes place in the CNS.
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ACKNOWLEDGMENTS |
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B cDNA and CD14 cDNA, respectively.
Received for publication May 9, 2000.
Revision received July 5, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B activity in the brain during endotoxemia. J. Neurosci. 20,3456-3468B activity and COX-2 transcription in cells of the blood–brain barrier in response to systemic and localized inflammation, but not during endotoxemia. J. Neurosci. 19,10923-10930This article has been cited by other articles:
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J. M. Millward, M. Caruso, I. L. Campbell, J. Gauldie, and T. Owens IFN-{gamma}-Induced Chemokines Synergize with Pertussis Toxin to Promote T Cell Entry to the Central Nervous System J. Immunol., June 15, 2007; 178(12): 8175 - 8182. [Abstract] [Full Text] [PDF]
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M. Sanchez-Alavez and T. Bartfai It all happens between Toll receptors and Caspase 1 PNAS, May 8, 2007; 104(19): 7733 - 7734. [Full Text] [PDF]
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 J. R. Caso, J. M. Pradillo, O. Hurtado, P. Lorenzo, M. A. Moro, and I. Lizasoain Toll-Like Receptor 4 Is Involved in Brain Damage and Inflammation After Experimental Stroke Circulation, March 27, 2007; 115(12): 1599 - 1608. [Abstract] [Full Text] [PDF]
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 L. Elander, L. Engstrom, M. Hallbeck, and A. Blomqvist IL-1beta and LPS induce anorexia by distinct mechanisms differentially dependent on microsomal prostaglandin E synthase-1 Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R258 - R267. [Abstract] [Full Text] [PDF]
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 A. Zeold, M. Doleschall, M. C. Haffner, L. P. Capelo, J. Menyhert, Z. Liposits, W. S. da Silva, A. C. Bianco, I. Kacskovics, C. Fekete, et al. Characterization of the Nuclear Factor-{kappa}B Responsiveness of the Human dio2 Gene Endocrinology, September 1, 2006; 147(9): 4419 - 4429. [Abstract] [Full Text] [PDF]
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A. S. C. Fabricio, G. Tringali, G. Pozzoli, M. C. Melo, J. A. Vercesi, G. E. P. Souza, and P. Navarra Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1515 - R1523. [Abstract] [Full Text] [PDF]
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T. Hubschle, J. Mutze, P. F. Muhlradt, S. Korte, R. Gerstberger, and J. Roth Pyrexia, anorexia, adipsia, and depressed motor activity in rats during systemic inflammation induced by the Toll-like receptors-2 and -6 agonists MALP-2 and FSL-1 Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R180 - R187. [Abstract] [Full Text] [PDF]
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 R. D. Folkerth Neuropathologic Substrate of Cerebral Palsy J Child Neurol, December 1, 2005; 20(12): 940 - 949. [Abstract] [PDF]
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 A. Razmara, D. N. Krause, and S. P. Duckles Testosterone augments endotoxin-mediated cerebrovascular inflammation in male rats Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1843 - H1850. [Abstract] [Full Text] [PDF]
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 K. B. Goralski, D. Abdulla, C. J. Sinal, A. Arsenault, and K. W. Renton Toll-like receptor-4 regulation of hepatic Cyp3a11 metabolism in a mouse model of LPS-induced CNS inflammation Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G434 - G443. [Abstract] [Full Text] [PDF]
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G. Soucy, G. Boivin, F. Labrie, and S. Rivest Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain J. Immunol., May 15, 2005; 174(10): 6391 - 6398. [Abstract] [Full Text] [PDF]
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S. Saha, L. Engstrom, L. Mackerlova, P.-J. Jakobsson, and A. Blomqvist Impaired febrile responses to immune challenge in mice deficient in microsomal prostaglandin E synthase-1 Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2005; 288(5): R1100 - R1107. [Abstract] [Full Text] [PDF]
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F. Y. Tanga, N. Nutile-McMenemy, and J. A. DeLeo The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy PNAS, April 19, 2005; 102(16): 5856 - 5861. [Abstract] [Full Text] [PDF]
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 S. Chakravarty and M. Herkenham Toll-Like Receptor 4 on Nonhematopoietic Cells Sustains CNS Inflammation during Endotoxemia, Independent of Systemic Cytokines J. Neurosci., February 16, 2005; 25(7): 1788 - 1796. [Abstract] [Full Text] [PDF]
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 I. Glezer and S. Rivest Glucocorticoids: Protectors of the Brain during Innate Immune Responses Neuroscientist, December 1, 2004; 10(6): 538 - 552. [Abstract] [PDF]
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 A. CAMPBELL Inflammation, Neurodegenerative Diseases, and Environmental Exposures Ann. N.Y. Acad. Sci., December 1, 2004; 1035(1): 117 - 132. [Abstract] [Full Text] [PDF]
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 N. P. Turrin and S. Rivest Unraveling the Molecular Details Involved in the Intimate Link between the Immune and Neuroendocrine Systems |
