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Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center, and Department of Anatomy and Physiology, Laval University, Québec City, Québec, Canada.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B
signaling events and transcriptional activation of genes that encode
TNF-
and the LPS receptor CD14. The present study investigated
whether circulating LPS has the ability to modulate the cerebral innate
immune response caused by an intrastriatal (IS) injection of the
endotoxin. We also tested the possibility that CD14 plays a role in
these effects and male rats received an intracerebroventricular
injection with an anti-CD14 before the IS LPS administration. The
single LPS bolus into the striatum caused a strong and time-dependent
transcriptional activation of TNF-, IB, CD14, and monocyte
chemoattractant protein-1 mRNA in microglial cells ipsilateral to the
site of injection. Surprisingly, this wave of induced transcripts was
essentially abolished by the systemic endotoxin pretreatment. Such
anti-inflammatory properties of circulating LPS are mediated via
plasma corticosterone, because exogenous corticoids mimicked while
glucocorticoid receptor antagonist RU486 prevented the effects of
systemic endotoxin challenge. Of interest is the partial involvement of
CD14 in LPS-induced neuroinflammation; the anti-CD14 significantly
abolished the microglial activity at day 3, but not at times earlier.
The inflammatory response provoked by an acute intraparenchymal LPS
bolus was not associated with convincing neurodegenerative processes.
These data provide compelling evidence that systemic inflammation,
through the increase in circulating glucocorticoids, has the ability to
prevent the cerebral innate immune reaction triggered by an IS
endotoxin injection. This study also further consolidates the existence
of such system in the brain, which is finely regulated and its
transient activation is not harmful for the neuronal
elements. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B transduction pathway (3). For a long time, the brain was considered to be a privileged organ from an immunological point of view, owing to its inability to mount an immune response and process Ags. Although this is partly true, the CNS shows a well-organized innate immune reaction in response to systemic bacterial infection and cerebral injury. The CD14 and TLR4 receptors are constitutively expressed in the circumventricular organs (CVOs), choroid plexus, and leptomeninges (4, 5). Circulating LPS also causes a rapid increase in CD14 in these brain regions, and a delayed response takes place in cells located at boundaries of the CVOs and in microglia across the brain parenchyma (4, 5). The role of CD14 within the brain microglia remains unknown. Because these cells are the counterparts of macrophages, microglial-derived CD14 may be a sensor for the PAMPs produced by Gram-negative bacteria and opsonize LPS when present into the cerebral tissue. Such beneficial mechanism would be helpful for eliminating the endotoxin swiftly to prevent a sustained inflammatory response. On the other hand, microglial activation is becoming a hallmark of several neurodegenerative diseases associated with production of inflammatory molecules (6, 7). In this regard, CD14 expression may contribute to prime and/or maintain microglial activation within the brain parenchyma and yield to an exaggerated immune response that could be potentially detrimental for the neuronal elements.
Therefore, the purpose of this study was to determine the impact of intraparenchymal LPS infusion on the proinflammatory signal transduction pathway and gene transcription of molecules involved in the innate immune response. We also compared these effects in animals pretreated systemically with a single bolus of endotoxin to preinduce CD14 in the CNS and determine whether these inflammatory events were associated with neurodegenerative processes. The contribution of microglial-derived CD14 was assessed by blocking the biological activity of the LPS receptor with a neutralizing Ab that was injected 10 h before the CNS insult. Cerebral LPS administration caused a robust and transient innate immune response, which was deeply altered by circulating levels of the endotoxin. Although CD14 did not contribute to the early events triggered by the intrastriatal (IS) endotoxin treatment, it modulated the duration of the inflammation within the CNS. Such robust innate immune reaction was not associated with neurodegeneration, and in contrast, is likely to be a crucial player for restoring the homeostatic balance in presence of bacterial cell wall components within the cerebral tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult male Sprague-Dawley rats (
200 g; Charles River Canada,
St-Constant, Quebec, Canada) were acclimated to standard laboratory
conditions (14-h light, 10-h dark cycle; lights on at 06:00 and off at
20:00 h) with free access to rat chow and water. Animal breeding and
experiments were conducted according to Canadian Council on Animal Care
guidelines, as administered by the Laval University Animal Care
Committee. A total of 162 rats were assigned to four different
protocols divided among the treatment and route of administration.
Surgeries and treatments
Animals were anesthetized with an i.p. injection of a mixture (1 ml/kg body weight (b.w.)) of ketamine hydrochloride (91 mg/kg) and xylazine (9 mg/kg). The right lateral ventricle was reached stereotaxically (David Kopf Instruments, Tujunga, CA). With the incisor bar placed at 3.3 mm below the interaural line (horizontal zero), the coordinates from bregma for the guide cannula were -0.6 mm anteroposterior, -1.4 mm lateral, and -3.0 mm dorsoventral. Another guide cannula was positioned just below the corpus callosum and both guide cannulas were secured with screws and cranioplastic cement (cranioplastic powder; Plastic One, Roanoke, VA; Dentsply repair material; Dentsply International, York, PA). The rats were then housed individually for a 10-day recuperation period. During the first 3 days after the surgery, rats received once daily an s.c. injection of 8 ml of Ringer lactate (no. 7953(NOREF>150) lot no. 48-142-NA at 37°C; Abbott Laboratories, Saint-Laurent, Canada), and 150 µl ketoprofen (Rhône Mérieux Canada, Victoriaville, Canada).
On the day of the experiment (08:30 h), an internal cannula
(23-gauge, 14-mm long from the pedestal (C235I; Plastic One)) was
connected to the guide cannula implanted within the lateral ventricle.
Thereafter, either 2 µg of anti-CD14 (no. cat M305, no. lot B280;
Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 10 µl of pyrogen
free sterile saline or the vehicle solution only was injected into the
right lateral ventricle over 2 min by means of a microinjection pump
(Razel model A-99; Razel Scientific Instruments, Stanford, CT). Four
hours after the intracerebroventricular injection, animals
received an i.p. injection of either LPS (1 mg/kg b.w.; L-2880, lot no.
127H4097; Sigma-Aldrich, St. Louis, MO) or vehicle. Six hours later, an
internal cannula was inserted within the chronic indwelling cannula
placed just below the corpus callosum and rats were infused within the
dorsal striatum with a solution containing either LPS (5 µg/2 µl/2
min) or only the vehicle solution (sterile saline). The animals were
conscious and freely moving at all times throughout the procedure and
killed 12 h, 3 days, and 7 days after the intraparenchymal
administration of the endotoxin or the vehicle. Three to four rats were
used for each group and time postinjection for a total of 66 animals
for this first set of experiments. Four additional rats were killed 21
days after the intracerebral LPS administration to determine the
potential long-term consequences of the insult on the neuronal
integrity.
A second protocol consisted to determine the plasma levels of glucocorticoids after a single injection of LPS. The animals were killed 1, 3, 6, and 12 h after a single i.p. bolus of LPS (1 mg/kg) or vehicle (sterile saline) and the blood collected in cold Vacutainer tubes (5.4 mg EDTA-K2; BD Biosciences, Franklin Lakes, NJ). The blood samples were centrifuged (4°C, 20 min, 3000 rpm) and the plasma was separated and stored at -20°C until the assay. Plasma levels of corticosterone (Cort) were measured via an RIA kit (catalog no. 07120102, lot no. RCBK0108, Immunochem Double Ab RIA kit; ICN Biomedical, Costa Mesa, CA). Four rats were used for each group and time postinjection for a total of 32 animals for this assay.
A third set of experiments was performed to verify the role of circulating Cort levels on intraparenchymal LPS-induced innate immune response in the brain. A chronic indwelling cannula was implanted just below the corpus callosum as described. Rats received two i.p. injections of Cort (60 mg/kg/300 µl diluted in DMSO; C-2505, lot no. 28 H0805; Sigma- Aldrich) or vehicle (DMSO) 60 and 30 min before the IS infusion of either LPS (5 µg/2 µl/2 min) or sterile saline and were killed 12 h and 3 days afterward. Three or four animals were used in each time and group for a total 28 rats in this protocol.
To ascertain the role of endogenous Cort in mediating the effects of circulating LPS on the brain, rats received an i.p. injection with either the glucocorticoid receptor antagonist RU486 (50 mg/kg/200 µl diluted in DMSO; Sigma-Aldrich) or vehicle (DMSO) 1 h before being challenged with LPS i.p. (1 mg/kg b.w.). Rats were then infused with either the endotoxin or vehicle into the striatal region as previously described and killed 12 h following the cerebral insult. Three to five animals were used in each group for a total 32 rats in this protocol.
Brain preparation and in situ hybridization histochemistry
Animals were deeply anesthetized following the different treatments with an i.p. injection (500 µl) of a mixture of ketamine hydrochloride and xylazine and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4 at 4°C). Brains were removed from the skull, postfixed for 2 h, and then placed in 20% sucrose diluted in 4% paraformaldehyde-sodium phosphate buffer for 12–15 h. The brains were mounted on a microtome (Reichert-Jung; Cambridge Instruments, Deerfield, IL), frozen with dry ice, 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 (0.05 M sodium phosphate buffer (pH 7.3), 30% ethylene glycol, 20% glycerol) and stored at -20°C.
The riboprobes used in this study are described in Table I
and in situ hybridization using
35S-labeled cRNA probes was accomplished as described
previously (8).
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Immunohistochemistry was used to determine the extend of the endotoxin spreading after the IS infusion using an antilipid A mAb. Brain sections were washed in sterile diethylpyrocarbonate-treated 50 mM potassium PBS (KPBS) and incubated 48 h at 4°C with antilipid A Ab (clone 43, lot no. HM 2046-3422 M11; Cell Sciences, Norwood, MA), which was diluted in sterile KPBS (1/1000) + 0.4% Triton X-100 + 1% BSA (fraction V; Sigma-Aldrich). After incubation with the primary Ab, brain slices were rinsed in sterile KPBS and incubated with a mixture of KPBS + 0.02% Triton-X + 1% BSA + Cy3-conjugated anti-mouse IgG Ab (1/1500; catalog no. 515-165-003, lot no. 4947; Jackson ImmunoResearch Laboratories, West Grove, PA) for 3 h in a dark room at 20°C. Tissues were thereafter rinsed in sterile KPBS, mounted onto poly-L-lysine slides and coverslipped with VectaMount (catalog no. H-5000, lot no. L0510; Vector Laboratories, Burlingame, CA).
Staining of infiltrating cells
The Wright stain was used to visualize infiltrating cells within brain parenchyma and their identifications were based on the morphology and the color of the cytoplasm, nucleus, and granulations. Every sixth section of the whole rostrocaudal extent of each brain was mounted onto poly-L-lysine-coated slides, dried under vacuum for 1 h, and covered with 2 ml of Wright staining solution (catalog no. 4481AL; Bayer Corporation Diagnostics Division, Elkhart, IN) for 90 s. They were then covered with an additional 2 ml of buffer solution for 3 min and washed with the rinse solution provided by the company. Sections were immediately dipped in xylene and coverslipped with distrene plasticizer xylene mounting medium.
Detection of apoptosis, neuronal death, and morphological changes
Cell death induced by apoptosis was detected via a TdT-FragEL DNA Fragmentation Detection kit (catalog no. QIA39-1EA, lot no. D14545; Oncogene Research Products, San Diego, CA). Positive controls were generated from brain sections of animals that received only sham treatments. The sections were mounted on the slides and covered with 1 µg/µl of DNase I in 1x TBS/1 mM MgSO4 for 20 min at room temperature.
Neuronal death induced by necrosis or neurotoxicity was detected with the Fluoro-Jade B (FJB) method. Briefly, every sixth section of the whole rostrocaudal extent of each brain was mounted onto poly-L-lysine-coated slides, dried under vacuum 2 h, dehydrated through graded concentrations of alcohol (50, 70, and 100%; 1 min), rehydrated through graded concentrations of alcohol (100, 70, and 50%; 1 min), and 1 min in distilled water. They were then dipped and shacked into potassium permanganate (0.06%) for 10 min, rinsed 1 min in distillated water, and dipped and shacked in a solution containing FJB 0.0004% (Histochem, Jefferson, AR) + acetic acid 0.1% (catalog no. A-6404; Sigma-Aldrich) + 4',6'-diamidino-2-phenylindole 0.0002% (catalog no. D-1306; Molecular Probes, Eugene, OR) for 20 min. The slides were thereafter rinsed three times in distillated water (1 min each), dried, dipped in xylene three times (2 min each), and coverslipped with distrene plasticizer xylene.
Nissl stain was also used as a general index of cellular morphology that may be altered in response to the different treatments.
Quantitative analysis
Hybridization signals were quantified on x-ray films (Biomax; Kodak, Rochester, NY) over numerous brain sections ipsilateral to the site of LPS injection. OD and extent of positive hybridization signals were measured as previously described (8).
The number of parenchymal neutrophils for each rat was calculated at three different levels (-0.6 mm anteroposterior, 3.5 mm lateral, -3.0 mm dorsoventral; -0.6 mm anteroposterior, 4.5 mm lateral, -3.0 mm dorsoventral; -0.6 mm anteroposterior, 5.0 mm lateral, -5.0 mm dorsoventral) using a digital camera (SPOT RT Slider; Diagnostic Instruments, Sterling Heights, MI) mounted directly on a microscope (BX-60; Olympus, Tokyo, Japan) and connected to a Macintosh computer (Power Macintosh G3; Apple Computers, Cupertino, CA). An area of 250 x 250 µm2 was delimited on the computer monitor using an Objective Micrometer (Olympus B-0550; Olympus), and the number of neutrophils was counted manually and multiplied by 16 to provide the number of neutrophils per mm2. The image of three different brain regions ipsilateral to the injection site was digitalized and the data reported as mean number of neutrophils per mm2. The statistical analysis was performed by a three-way ANOVA followed by a Bonferroni/Dunn test procedure as post-hoc comparisons.
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Results |
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A single bolus of LPS injected into the dorsal basal ganglia
caused a robust and transient expression of numerous transcripts in the
cerebral tissue ipsilateral to the injection site. Fig. 1 depicts a representative example of
such induction pattern for TNF- mRNA that followed the diffusion of
the endotoxin within the tissue. The signal for the cytokine transcript
was low to undetectable in the brain of vehicle-injected rats; few
positive cells were found only in regions adjacent to the tract.
The signal was very intense and spread across the brain parenchyma
12 h after the LPS injection (Fig. 1, middle column).
Despite such profound induction of the gene encoding the
proinflammatory cytokine, the hybridization signal remained localized
to the injection site and did not extent to the hindbrain and the
rostral brain. This may be explained by the fact that the endotoxin
failed to diffuse throughout the brain and remained quite localized to
the dorsal basal ganglia, hippocampal formation, cerebral cortex, and
their adjacent structures. The extent of the endotoxin spreading was
verified by means of immunofluorescence using an mAb directed against
lipid A and the immunoreactive signal was strong at 12 h, largely
diminished 3 days after the single injection, and vanished at day 7
post-LPS infusion (Fig. 2). This
time-related clearance of the endotoxin within the cerebral tissue
paralleled the hybridization signal for TNF- that was maximal at
time 12 h and slowly returned to the background level
thereafter.
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B (as revealed by the do
novo expression of IB), expression of the LPS receptor CD14
and the chemokine monocyte chemoattractant protein-1 (MCP-1; Fig. 3, middle column). In
contrast to these NF-B-inducible genes, cerebral LPS
injection failed to trigger the transcription of the newly
characterized receptor TLR4. This receptor that recognizes
Gram-negative cell wall components is constitutively expressed in the
leptomeninges and is down-regulated by the endotoxin (Fig. 3, middle column). This contrasts with the mRNA encoding the
other LPS receptor CD14, which is strongly activated by the exogenous
ligand injected directly into the brain parenchyma.
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A single systemic injection of LPS was performed to increase CD14
and preactivate microglial cells before the central treatment with the
endotoxin. This preinduction of the endotoxin receptor was expected to
intensify the effects of LPS once present in the CNS. Surprisingly
however, circulating LPS clearly abolished the effects of IS LPS on the
proinflammatory signal transduction pathways and gene expression. Both
the intensity of the signal and brain areas expressing the different
transcripts in response to a single bolus of LPS into the caudate
putamen significantly decreased in animals that were pretreated with
the endotoxin systemically (Table II).
Representative examples of this phenomenon are depicted by Figs. 1
and 3 (V-L-L; right column). Pretreatment with the endotoxin
i.p. also significantly reduced the time in which the signal remained
positive in response to the cerebral endotoxin injection. Except for
the TNF- signal that was still positive but low at day 3
postinjection, expression levels for all the other induced transcripts
returned to background levels at that time while the mRNAs were still
expressed in animals that received the i.p. vehicle and cerebral LPS
treatment (V-V-L; Table II). Of interest is the strong and localized
hybridization signal for CD14 transcript in cells adjacent to the
microvasculature of the V-L-L-treated animal (Fig. 3, right
column).
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Systemic LPS administration is a powerful stimulus to increase
plasma levels in glucocorticoids, the most potent endogenous
anti-inflammatory molecule known (9). A single i.p.
endotoxin bolus caused a rapid increase in circulating corticosterone
levels that peaked between 3 and 6 h and declined slowly
thereafter (Fig. 4). This peak of plasma
Cort taking place at 6 h is quite interesting, because this was
actually the delay between the first systemic LPS injection and the
subsequent intracerebral treatment. Therefore, it was hypothesized that
the high plasma glucocorticoid levels occurring in animals pretreated
with the endotoxin i.p. were responsible for preventing the effects of
centrally injected LPS on the proinflammatory signal
transduction pathways and transcription of the genes assessed in this
study. Animals that received a systemic bolus of glucocorticoids 60 and
30 min before being injected into the caudate putamen with the
endotoxin responded quite similarly to the rats that were pretreated
with the endotoxin in the peritoneal cavity. Indeed, the strong
transcriptional activation of TNF- gene was essentially abolished in
animals that received i.p. Cort before the cerebral insult (Fig. 5A). Both the intensity and
extent of the positive signals were lower in animals that received
glucocorticoids before the LPS insult (Fig. 5A). This was
also the case for the mRNA encoding CD14 and IB (data not
shown).
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B). Therefore,
circulating LPS-induced elevation in Cort levels is the mechanism that
prevented microglial activity to cerebral endotoxin administration. Effects of the anti-CD14 antisera on the cerebral innate immunity
To block the biological activity of the LPS receptor CD14 within
the cerebral tissue, an anti-rat CD14-neutralizing Ab was injected
in the lateral ventricle 10 h before the LPS infusion within the
dorsal basal ganglia. Animals were equipped with two chronic indwelling
cannulas, one just above the lateral ventricle and the other at the
level of the corpus callosum. The neutralizing antisera failed to
prevent expression of the TNF- mRNA 12 h after the LPS infusion
into the brain parenchyma. The intensity and extend of the hybridized
signal were similar in the brain of animals that were pretreated or not
with the anti-CD14 into the lateral ventricle before the cerebral
LPS insult (Fig. 6). The same phenomenon
occurred for the other inflammatory transcripts, which remained
expressed at a similar level between both groups of
endotoxin-challenged rats at time 12 h (data not shown). In spite
of this, the duration of the inflammatory response was reduced by
preventing the biological activity of the LPS receptor CD14. Indeed,
the neutralizing CD14 Ab abolished the maintained expression of the
gene encoding TNF- that took place 3 days after the intraparenchymal
infusion with the Gram-negative cell wall component. This suggests that
although CD14 may not be essential for triggering the cerebral innate
immune response, it plays a key role in the duration of these
events.
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Wright staining allowed us to detect the presence of leukocytes
within the cerebral tissue and a large number of polymorphonuclear
cells (PMNs) were found within the regions that exhibited a positive
signal for the different transcripts in response to the IS endotoxin
injection (Fig. 7). These neutrophils
were detected only in the area where the inflammation occurred and
never in the contralateral site or in the brain of vehicle-administered
rats. However, it is possible that these cells did not actually
emigrate within the brain parenchyma and remained attached to the
vasculature, which was difficult to evaluate in the present case. Of
interest is that pretreatment with the endotoxin i.p. significantly
prevented accumulation of PMNs within the brain at 12 h
postintraparenchymal endotoxin infusion. Although these cells were
still found in regions ipsilateral to the injection site, circulating
endotoxin decreased the number of neutrophils by mm2 of
tissue and this effect was dependent on plasma Cort levels (data not
shown). The rat anti-CD14 neutralizing Ab failed to modulate the
accumulation of PMNs at time 12 h, but fewer cells were generally
detected in the brain of rats killed 3 days after double cerebral
treatments (A-V-L).
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The question of whether LPS-induced cerebral inflammation was
associated with neurodegenerative processes was investigated via a
number of approaches. Cell death induced by apoptosis was detected with
the fluorescein-FragEL DNA fragmentation detection kit for tissue
cryosections. Positive controls were generated by covering the entire
specimen with 1 µg/µl DNase I in 1x TBS/1 mM MgSO4
following proteinase K treatment, which caused a strong labeling over
neurons across the brain of a vehicle-treated animal (Fig. 8A). Such positive cells were
never observed in the brain of LPS-treated animals pretreated or not
with the endotoxin i.p. (Fig. 8B). The fluorochrome FJB is a
sensitive and reliable marker for the histochemical localization of
neuronal degeneration (10). Bilateral adrenalectomy is
known to provoke neurodegeneration of the hippocampal
dentate granule cells. These neurons displayed strong and selective
fluorescent FJB signal, indicating degeneration of a subpopulation of
cells within the dentate gyrus granulal layer of a rat killed 6 days
after being adrenalectomized (Fig. 8C). Surprisingly
however, no positive signal was observed across the CNS of
endotoxin-challenged animals. Indeed, only background levels were
detected for the fluorescent marker over the tissues that exhibited the
robust induction of inflammatory molecules in response to an
intraparenchymal LPS infusion (Fig. 8D). FJB was also
undetectable in the CNS of animals that were pretreated with the
anti-CD14 and the endotoxin i.p. before the CNS LPS insult and this
from 12 h to 21 days posttreatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported that circulating LPS has the ability to
trigger CD14 within the CVOs and thereafter across the brain parenchyma
(8). However, this parenchymal induction of the LPS
receptor depends on microglial-derived TNF- that acts as a paracrine
factor to trigger adjacent cells of myeloid origin (8).
Although such paracrine effect of the proinflammatory cytokine may also
take place in the brain of centrally injected LPS, the endotoxin seems
directly responsible of the inflammatory reaction across the cerebral
tissue ipsilateral to the injection site. Indeed, the pattern of LPS
immunoreactive areas is essentially the same as the one found for
CD14-, TNF--, IB-, and MCP-1-expressing structures. This
suggests that LPS is present in the regions that exhibited
transcriptional activation of these genes and the spreading of the
inflammatory response seems to depend on the diffusion of the endotoxin
after the infusion within the brain parenchyma. A paracrine influence
of TNF and other molecules may also take place between adjacent cells,
but not across the brain.
This contrasts with the effects of systemic LPS injection that cause transcriptional activation of CD14 (5) and TNF (11) first within the CVOs and thereafter throughout the brain microglial cells. However, this depends on the dose of the endotoxin, because the signal for these proinflammatory molecules remains quite localized to the regions that can be reached by the bloodstream and their adjacent structures after a single bolus of LPS injected at low to moderate doses (5, 8, 11). On the other hand, high circulating levels of the endotoxin are associated with an increased expression of a number of inflammatory genes across the brain parenchyma (8, 11, 12, 13). Such widespread induction that was not observed in response to an IS LPS infusion is in agreement with another recent study (14). This rather localized hybridization signal ipsilateral to the site of infusion may also be dependent on the dose of LPS, which was not verified in the present study.
Distinct TLRs have now been proposed as the key molecules to
selectively recognize one of the major PAMPs produced by either
Gram-negative or Gram-positive bacteria (3). The data that
mutation of the mouse Lps locus abolishes the LPS response
and that Lps encodes TLR4 provided the first evidence that
this particular receptor may play a key role in the innate immune
response to Gram-negative bacteria (reviewed in Ref. 15).
Therefore, the lack of signal for the gene encoding TLR4 in the brain
parenchyma is quite intriguing, because this receptor is now recognized
to be responsible for LPS-induced NF-B signaling, and TLR4-deficient
mice are resistant to cell wall components of Gram-negative bacteria
(3). Therefore, how LPS can trigger such profound
inflammatory response within microglial cells across the brain
parenchyma without TLR4? TLR4 transcript levels were low along the
meninges, choroid plexus, and the CVOs under basal conditions
(4). Such levels fit 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 (15, 16).
Nevertheless, it is remarkable that so few TLR4 receptors (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 (15). The technique
used in the present study is probably not sensitive enough to detect
TLR4 within microglial cells.
LPS-induced proinflammatory signal transduction pathway and gene transcription in the brain was not associated with neurodegeneration. Various approaches were used to reach these conclusions, which are in disagreement with few other studies that provided evidence of LPS-induced neurotoxicity (17, 18). Such discrepancies may be explained by the techniques used to assay neurodegeneration, which was determined by the decrease in tyrosine hydroxylase activity and the loss of tyrosine hydroxylase-positive neuronal bodies in the substantia nigra (17). The target region seems also of great importance, because injection of the endotoxin in the hippocampus, cortex, or substantia nigra of adult rats produced neurodegeneration only in the substantia nigra (18). These authors suggested that the region-specific differential susceptibility of neurons to LPS is attributable to differences in the number of microglia present within the system and may reflect levels of inflammation-related factors produced by these cells (18). In the present case, microglial activation was robust but transient and it is tempting to propose that such response is not detrimental for the brain and in contrast may act as a protective mechanism during cerebral infection. It is also possible that specific populations of neurons are more susceptible than others to inflammatory molecules and despite eliminating pathogens from the CNS, microglia, and their secreted products may be harmful for some neurons.
In contrast to glucocorticoids, CD14 does not seem to be involved in the early inflammatory events triggered by the cerebral injection of the endotoxin. The neutralizing antisera failed to prevent the inflammatory response in animals that were killed 12 h after the LPS infusion within the basal ganglia. These data are in agreement with the CD14-deficient mice that are highly resistant to shock induced by either live Gram-negative bacteria or LPS, but at very high concentrations of LPS or bacteria, responses through non-CD14 receptors were found (19). These animals show also no alteration in LPS-induced acute-phase protein, including serum amyloid A, LPS-binding protein, fibrinogen, and ceruloplasmin (20). Although the neutralizing CD14 Ab did not prevent the inflammatory reaction at times early post-LPS administration, it attenuated the duration of this response. These data support the concept of CD14-dependent and -independent pathways that also seem to occur in the CNS. However, the lack of effects at 12 h may be explained by the inability of the neutralizing antisera to completely block the biological activity of CD14 and the dose of LPS that was injected directly within the cerebral tissue.
Intraparenchymal LPS administration caused emigration of PMNs within the regions that exhibited positive signal for the proinflammatory genes. This phenomenon was significantly attenuated in animals that were pretreated with the endotoxin i.p., which suggests that the intensity of the inflammatory process is directly responsible to engage the chemoattraction. Nevertheless, these data are quite surprising, because leukocyte emigration is unlikely to occur during nonpathological conditions and during endotoxemia. Although systemic LPS treatment is generally not associated with leukocyte emigration within the brain parenchyma, intracerebral LPS infusion and the robust expression and cytokines/chemokines are likely mechanisms to allow diapedesis. Direct administration of LPS onto the BBB elicits gaps between endothelial cells and provokes leukocyte infiltration (21). Emigrating cells have also been observed in mice that received different doses of LPS within the brain parenchyma (22). This emigration was specific to the regions that exhibited expression of the different transcripts and was very dynamic, because the number of PMNs decreased at day 3 and these cells were no longer detected in the brain of animals killed 7 days after the endotoxin infusion within the basal ganglia. Therefore, leukocytes are recruited in response to a cerebral innate immune reaction and this transient phenomenon is not detrimental for the neuronal elements. Neutrophils contain acyloxyacyl hydrolase, the enzyme responsible to reduce LPS toxicity in deacylating its lipid A portion (23). These cells could then play a key role in detoxifying the endotoxin and eliminate it swiftly from the cerebral tissue.
Taken together, these data suggest that there is an innate immune response that is rapidly triggered within the brain microglia, which is associated with an emigration process. These events are regulated by circulating levels of glucocorticoids and LPS receptor CD14, at least for the duration of the inflammatory response. The endogenous expression of CD14 and specific TLRs may engage proinflammatory signal transduction pathways and production of cytokines by microglia. One of the beneficial consequences of such microglial reactivity is the release of neurotrophic factors and other molecules that have important roles in brain homeostasis, neuroprotection, and repair in case of injury (reviewed in Ref. 24). However, once engaged in severe infections, sustained microglial reactivity can overproduce inflammatory molecules and alter the BBB, a mechanism that seems to be central to several neurodegenerative disorders and demyelinating diseases. As microglia are the APCs of the brain, they are probably crucial for cell-specific immunity against neuronal elements. A better understanding of the innate immune response in cerebral tissue could lead us to the fundamental mechanisms that underlie the capability of the brain to mount an inflammatory response that either protects against or contributes to neuronal damage.
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Acknowledgments |
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B cDNA, CD14 cDNA, and MCP-1
cDNA, respectively. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Serge Rivest, Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center, 2705 Boulevard Laurier, Québec City, Québec, Canada, G1V 4G2. E-mail address: Serge.Rivest{at}crchul.ulaval.ca
3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; Cort, corticosterone; CVO, circumventricular organ; FJB, Fluoro-Jade B; IS, intrastriatal; KPBS, potassium PBS; MCP-1, monocyte chemoattractant protein-1; PMN, polymorphonuclear cell; b.w., body weight; TLR, Toll-like receptor.
Received for publication May 31, 2002. Accepted for publication July 15, 2002.
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References |
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B activity in the brain during endotoxemia. J. Neurosci. 20:3456. in the rat brain and pituitary in response to different models of systemic immune challenge. J. Neuropathol. Exp. Neurol. 58:61.[Medline]
expression in the mouse brain after systemic lipopolysaccharide administration. Proc. Natl. Acad. Sci. USA 22:11393.
mRNA expression in the brain and pituitary after peripheral administration of lipopolysaccharide. Neuroscience 83:281.
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, p
< 0.05, from their respective Cort-treated groups. B, Rats
received an i.p. injection with either the glucocorticoid receptor
antagonist RU486 (50 mg/kg/200 µl diluted in DMSO) or vehicle (DMSO)
1 h before being challenged with LPS i.p. (1 mg/kg b.w.). They
were then infused with the endotoxin or vehicle into the striatal
region and killed 12 h following the cerebral insult. A
significant interaction occurred between the two main factors for all
the dependent variables (d and e). *,
p < 0.05, from their corresponding control groups.
