of March 13, 2018.This information is current as

Injection: Role of Glucocorticoids and CD14Intraparenchymal LipopolysaccharideInflammatory Wave Induced by Endotoxemia Prevents the Cerebral

Sylvain Nadeau and Serge Rivest

http://www.jimmunol.org/content/169/6/3370doi: 10.4049/jimmunol.169.6.3370

2002; 169:3370-3381; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/169/6/3370.full#ref-list-1

, 7 of which you can access for free at: cites 24 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2002 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Endotoxemia Prevents the Cerebral Inflammatory WaveInduced by Intraparenchymal Lipopolysaccharide Injection:Role of Glucocorticoids and CD141

Sylvain Nadeau and Serge Rivest2

There is a robust and transient innate immune response in the brain during endotoxemia, which is associated with a cascade ofNF-�B signaling events and transcriptional activation of genes that encode TNF-� and the LPS receptor CD14. The present studyinvestigated 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 anintracerebroventricular injection with an anti-CD14 before the IS LPS administration. The single LPS bolus into the striatumcaused a strong and time-dependent transcriptional activation of TNF-�, I�B�, CD14, and monocyte chemoattractant protein-1mRNA in microglial cells ipsilateral to the site of injection. Surprisingly, this wave of induced transcripts was essentially abolishedby the systemic endotoxin pretreatment. Such anti-inflammatory properties of circulating LPS are mediated via plasma cortico-sterone, because exogenous corticoids mimicked while glucocorticoid receptor antagonist RU486 prevented the effects of systemicendotoxin challenge. Of interest is the partial involvement of CD14 in LPS-induced neuroinflammation; the anti-CD14 signifi-cantly abolished the microglial activity at day 3, but not at times earlier. The inflammatory response provoked by an acuteintraparenchymal LPS bolus was not associated with convincing neurodegenerative processes. These data provide compellingevidence that systemic inflammation, through the increase in circulating glucocorticoids, has the ability to prevent the cerebralinnate immune reaction triggered by an IS endotoxin injection. This study also further consolidates the existence of such systemin the brain, which is finely regulated and its transient activation is not harmful for the neuronal elements. The Journal ofImmunology, 2002, 169: 3370–3381.

I nnate immunity is the early response of a host to infectionthat is characterized by a cascade of proinflammatory signal-ing events and transcriptional activation of several immune-

related genes. Host organisms detect the presence of infection byrecognizing specific elements produced by microorganisms (1).These elements—the so-called pathogen-associated molecular pat-terns (PAMPs)3—are recognized by specific cells of the immunesystem as inducers of innate responses to bacterial infection. Thereaction to endotoxin LPS, an important component of the outermembranes of Gram-negative bacteria, is the best-characterizedexample of innate recognition that leads to a robust inflammatoryresponse by the APCs—macrophages and dendritic cells, and inthe brain, microglial cells (2). The mechanisms involved in thesecretion of cytokines by the APCs in response to LPS requires a

series of signaling events, the details of which have been clarifiedin recent years. Indeed, the binding of LPS to its cognate trans-membrane receptor CD14 and Toll-like receptor 4 (TLR4) triggersthe activity of NF-�B transduction pathway (3).

For a long time, the brain was considered to be a privilegedorgan from an immunological point of view, owing to its inabilityto mount an immune response and process Ags. Although this ispartly true, the CNS shows a well-organized innate immune reac-tion in response to systemic bacterial infection and cerebral injury.The CD14 and TLR4 receptors are constitutively expressed in thecircumventricular organs (CVOs), choroid plexus, and leptome-ninges (4, 5). Circulating LPS also causes a rapid increase in CD14in these brain regions, and a delayed response takes place in cellslocated at boundaries of the CVOs and in microglia across thebrain parenchyma (4, 5). The role of CD14 within the brain mi-croglia remains unknown. Because these cells are the counterpartsof macrophages, microglial-derived CD14 may be a sensor for thePAMPs produced by Gram-negative bacteria and opsonize LPSwhen present into the cerebral tissue. Such beneficial mechanismwould be helpful for eliminating the endotoxin swiftly to preventa sustained inflammatory response. On the other hand, microglialactivation is becoming a hallmark of several neurodegenerativediseases associated with production of inflammatory molecules (6,7). In this regard, CD14 expression may contribute to prime and/ormaintain microglial activation within the brain parenchyma andyield to an exaggerated immune response that could be potentiallydetrimental for the neuronal elements.

Therefore, the purpose of this study was to determine the impactof intraparenchymal LPS infusion on the proinflammatory signaltransduction pathway and gene transcription of molecules involvedin the innate immune response. We also compared these effects inanimals pretreated systemically with a single bolus of endotoxin to

Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Universite LavalResearch Center, and Department of Anatomy and Physiology, Laval University,Quebec City, Quebec, Canada.

Received for publication May 31, 2002. Accepted for publication July 15, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by the Canadian Institutes of Health Research. S.N.holds a Studentship from the Canadian Institutes of Health Research, and S.R. is aCanadian Institutes of Health Research scientist and holds a Canadian Research Chairin Neuroimmunology.2 Address correspondence and reprint requests to Dr. Serge Rivest, Laboratory ofMolecular Endocrinology, Centre Hospitalier de l’Universite Laval Research Center,2705 Boulevard Laurier, Quebec City, Quebec, Canada, G1V 4G2. E-mail address:Serge.Rivest@crchul.ulaval.ca3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern;Cort, corticosterone; CVO, circumventricular organ; FJB, Fluoro-Jade B; IS, intra-striatal; KPBS, potassium PBS; MCP-1, monocyte chemoattractant protein-1; PMN,polymorphonuclear cell; b.w., body weight; TLR, Toll-like receptor.

The Journal of Immunology

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

preinduce CD14 in the CNS and determine whether these inflam-matory events were associated with neurodegenerative processes.The contribution of microglial-derived CD14 was assessed byblocking the biological activity of the LPS receptor with a neu-tralizing Ab that was injected 10 h before the CNS insult. CerebralLPS administration caused a robust and transient innate immuneresponse, which was deeply altered by circulating levels of theendotoxin. Although CD14 did not contribute to the early eventstriggered by the intrastriatal (IS) endotoxin treatment, it modulatedthe duration of the inflammation within the CNS. Such robust in-nate immune reaction was not associated with neurodegeneration,and in contrast, is likely to be a crucial player for restoring thehomeostatic balance in presence of bacterial cell wall componentswithin the cerebral tissue.

Materials and MethodsAnimals

Adult male Sprague-Dawley rats (�200 g; Charles River Canada, St-Con-stant, 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 freeaccess to rat chow and water. Animal breeding and experiments were con-ducted according to Canadian Council on Animal Care guidelines, as ad-ministered by the Laval University Animal Care Committee. A total of 162rats were assigned to four different protocols divided among the treatmentand route of administration.

Surgeries and treatments

Animals were anesthetized with an i.p. injection of a mixture (1 ml/kg bodyweight (b.w.)) of ketamine hydrochloride (91 mg/kg) and xylazine (9 mg/kg). The right lateral ventricle was reached stereotaxically (David KopfInstruments, Tujunga, CA). With the incisor bar placed at 3.3 mm belowthe interaural line (horizontal zero), the coordinates from bregma for theguide cannula were �0.6 mm anteroposterior, �1.4 mm lateral, and �3.0mm dorsoventral. Another guide cannula was positioned just below thecorpus callosum and both guide cannulas were secured with screws andcranioplastic cement (cranioplastic powder; Plastic One, Roanoke, VA;Dentsply repair material; Dentsply International, York, PA). The rats werethen housed individually for a 10-day recuperation period. During the first3 days after the surgery, rats received once daily an s.c. injection of 8 mlof Ringer lactate (no. 7953(150) lot no. 48-142-NA at 37°C; Abbott Lab-oratories, Saint-Laurent, Canada), and 150 �l ketoprofen (Rhone MerieuxCanada, 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 theguide cannula implanted within the lateral ventricle. Thereafter, either 2 �gof anti-CD14 (no. cat M305, no. lot B280; Santa Cruz Biotechnology,Santa Cruz, CA) diluted in 10 �l of pyrogen free sterile saline or thevehicle solution only was injected into the right lateral ventricle over 2 minby means of a microinjection pump (Razel model A-99; Razel ScientificInstruments, Stanford, CT). Four hours after the intracerebroventricularinjection, 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. Sixhours later, an internal cannula was inserted within the chronic indwellingcannula placed just below the corpus callosum and rats were infused withinthe dorsal striatum with a solution containing either LPS (5 �g/2 �l/2 min)or only the vehicle solution (sterile saline). The animals were consciousand freely moving at all times throughout the procedure and killed 12 h, 3days, and 7 days after the intraparenchymal administration of the endotoxinor the vehicle. Three to four rats were used for each group and time postin-

jection for a total of 66 animals for this first set of experiments. Fouradditional rats were killed 21 days after the intracerebral LPS administra-tion to determine the potential long-term consequences of the insult on theneuronal integrity.

A second protocol consisted to determine the plasma levels of glucocor-ticoids after a single injection of LPS. The animals were killed 1, 3, 6, and12 h after a single i.p. bolus of LPS (1 mg/kg) or vehicle (sterile saline) andthe blood collected in cold Vacutainer tubes (5.4 mg EDTA-K2; BD Bio-sciences, Franklin Lakes, NJ). The blood samples were centrifuged (4°C,20 min, 3000 rpm) and the plasma was separated and stored at �20°C untilthe assay. Plasma levels of corticosterone (Cort) were measured via an RIAkit (catalog no. 07120102, lot no. RCBK0108, Immunochem Double AbRIA kit; ICN Biomedical, Costa Mesa, CA). Four rats were used for eachgroup and time postinjection for a total of 32 animals for this assay.

A third set of experiments was performed to verify the role of circulatingCort levels on intraparenchymal LPS-induced innate immune response inthe brain. A chronic indwelling cannula was implanted just below the cor-pus callosum as described. Rats received two i.p. injections of Cort (60mg/kg/300 �l diluted in DMSO; C-2505, lot no. 28 H0805; Sigma-Aldrich) or vehicle (DMSO) 60 and 30 min before the IS infusion of eitherLPS (5 �g/2 �l/2 min) or sterile saline and were killed 12 h and 3 daysafterward. Three or four animals were used in each time and group for atotal 28 rats in this protocol.

To ascertain the role of endogenous Cort in mediating the effects ofcirculating LPS on the brain, rats received an i.p. injection with either theglucocorticoid receptor antagonist RU486 (50 mg/kg/200 �l diluted inDMSO; Sigma-Aldrich) or vehicle (DMSO) 1 h before being challengedwith LPS i.p. (1 mg/kg b.w.). Rats were then infused with either the en-dotoxin or vehicle into the striatal region as previously described and killed12 h following the cerebral insult. Three to five animals were used in eachgroup for a total 32 rats in this protocol.

Brain preparation and in situ hybridization histochemistry

Animals were deeply anesthetized following the different treatments withan i.p. injection (500 �l) of a mixture of ketamine hydrochloride and xy-lazine and then rapidly perfused transcardially with 0.9% saline, followedby 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4 at4°C). Brains were removed from the skull, postfixed for 2 h, and thenplaced in 20% sucrose diluted in 4% paraformaldehyde-sodium phosphatebuffer for 12–15 h. The brains were mounted on a microtome (Reichert-Jung; Cambridge Instruments, Deerfield, IL), frozen with dry ice, and cutinto 30-�m coronal sections from the olfactory bulb to the end of themedulla. 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.

The riboprobes used in this study are described in Table I and in situhybridization using 35S-labeled cRNA probes was accomplished as de-scribed previously (8).

Immunohistochemistry

Immunohistochemistry was used to determine the extend of the endotoxinspreading after the IS infusion using an antilipid A mAb. Brain sectionswere 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 insterile KPBS (1/1000) � 0.4% Triton X-100 � 1% BSA (fraction V;Sigma-Aldrich). After incubation with the primary Ab, brain slices wererinsed in sterile KPBS and incubated with a mixture of KPBS � 0.02%Triton-X � 1% BSA � Cy3-conjugated anti-mouse IgG Ab (1/1500; cat-alog no. 515-165-003, lot no. 4947; Jackson ImmunoResearch Laborato-ries, West Grove, PA) for 3 h in a dark room at 20°C. Tissues were there-after rinsed in sterile KPBS, mounted onto poly-L-lysine slides and

Table I. Plasmids and enzymes used for probe synthesis

Probe Vector cDNA Length (bp)

Enzyme for Linearization RNA Polymerase

Antisense Sense Antisense Sense

TNF-� pBluescript SK� 716 EcoRI BamHI T3 T7CD14 pBluescript SK� 1528 SacI KpnI T7 T3I�B� pBluescript SK� 1050 BamHI HindIII T7 T3TLR4 PCA II 2746 PST KpnI Sp6 T7MCP-1 pGEM-1 578 BamHI SacI T7 Sp6

3371The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

coverslipped with VectaMount (catalog no. H-5000, lot no. L0510; VectorLaboratories, Burlingame, CA).

Staining of infiltrating cells

The Wright stain was used to visualize infiltrating cells within brain pa-renchyma and their identifications were based on the morphology and thecolor of the cytoplasm, nucleus, and granulations. Every sixth section ofthe 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 ofWright staining solution (catalog no. 4481AL; Bayer Corporation Diag-nostics Division, Elkhart, IN) for 90 s. They were then covered with anadditional 2 ml of buffer solution for 3 min and washed with the rinsesolution provided by the company. Sections were immediately dipped inxylene and coverslipped with distrene plasticizer xylene mountingmedium.

Detection of apoptosis, neuronal death, and morphologicalchanges

Cell death induced by apoptosis was detected via a TdT-FragEL DNAFragmentation Detection kit (catalog no. QIA39-1EA, lot no. D14545; On-cogene Research Products, San Diego, CA). Positive controls were gener-ated from brain sections of animals that received only sham treatments. Thesections were mounted on the slides and covered with 1 �g/�l of DNase Iin 1� TBS/1 mM MgSO4 for 20 min at room temperature.

Neuronal death induced by necrosis or neurotoxicity was detected withthe Fluoro-Jade B (FJB) method. Briefly, every sixth section of the wholerostrocaudal extent of each brain was mounted onto poly-L-lysine-coatedslides, dried under vacuum 2 h, dehydrated through graded concentrationsof alcohol (50, 70, and 100%; 1 min), rehydrated through graded concen-trations 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 asolution containing FJB 0.0004% (Histochem, Jefferson, AR) � acetic acid0.1% (catalog no. A-6404; Sigma-Aldrich) � 4�,6�-diamidino-2-phenylin-dole 0.0002% (catalog no. D-1306; Molecular Probes, Eugene, OR) for 20min. The slides were thereafter rinsed three times in distillated water (1 mineach), dried, dipped in xylene three times (2 min each), and coverslippedwith distrene plasticizer xylene.

Nissl stain was also used as a general index of cellular morphology thatmay 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 LPSinjection. OD and extent of positive hybridization signals were measured aspreviously described (8).

The number of parenchymal neutrophils for each rat was calculated atthree different levels (�0.6 mm anteroposterior, 3.5 mm lateral, �3.0 mmdorsoventral; �0.6 mm anteroposterior, 4.5 mm lateral, �3.0 mm dorso-ventral; �0.6 mm anteroposterior, 5.0 mm lateral, �5.0 mm dorsoventral)using a digital camera (SPOT RT Slider; Diagnostic Instruments, SterlingHeights, 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 � 250 �m2 was de-limited on the computer monitor using an Objective Micrometer (OlympusB-0550; Olympus), and the number of neutrophils was counted manuallyand multiplied by 16 to provide the number of neutrophils per mm2. Theimage of three different brain regions ipsilateral to the injection site wasdigitalized and the data reported as mean number of neutrophils per mm2.The statistical analysis was performed by a three-way ANOVA followedby a Bonferroni/Dunn test procedure as post-hoc comparisons.

ResultsInduction of genes encoding proinflammatory molecules byintraparenchymal LPS infusion

A single bolus of LPS injected into the dorsal basal ganglia causeda robust and transient expression of numerous transcripts in thecerebral tissue ipsilateral to the injection site. Fig. 1 depicts a rep-resentative example of such induction pattern for TNF-� mRNAthat followed the diffusion of the endotoxin within the tissue. The

signal for the cytokine transcript was low to undetectable in thebrain of vehicle-injected rats; few positive cells were found only inregions adjacent to the tract. The signal was very intense andspread across the brain parenchyma 12 h after the LPS injection(Fig. 1, middle column). Despite such profound induction of thegene encoding the proinflammatory cytokine, the hybridizationsignal remained localized to the injection site and did not extent tothe hindbrain and the rostral brain. This may be explained by thefact that the endotoxin failed to diffuse throughout the brain andremained quite localized to the dorsal basal ganglia, hippocampalformation, cerebral cortex, and their adjacent structures. The extentof the endotoxin spreading was verified by means of immunoflu-orescence using an mAb directed against lipid A and the immu-noreactive signal was strong at 12 h, largely diminished 3 daysafter the single injection, and vanished at day 7 post-LPS infusion(Fig. 2). This time-related clearance of the endotoxin within thecerebral tissue paralleled the hybridization signal for TNF-� thatwas maximal at time 12 h and slowly returned to the backgroundlevel thereafter.

This robust and transient transcriptional activation of the geneencoding the proinflammatory cytokine was associated with a par-allel increase in the activity of NF-�B (as revealed by the do novo

FIGURE 1. Representative examples of TNF-� gene expression in re-sponse to different treatments with the endotoxin LPS. Left column, Brainsections of rats that received only vehicle solution in the lateral ventricle,dorsal striatum, and the peritoneal cavity (V-V-V). Middle and right col-umns, The hybridization signal in the brain of animals that received a singleIS endotoxin injection (V-V-L) or a systemic LPS injection before thecerebral treatment (V-L-L), respectively. Animals were killed 12 h after theintraparenchymal injection of the endotoxin or the vehicle solution. V,vehicle; L, LPS. The first letter stands for the infusion within the lateralventricle, the second/middle one is for the i.p. treatment, and the last letteris for the IS infusion. Please note that systemic LPS administration largelyabolished the cerebral expression of TNF-� in response to intraparenchy-mal endotoxin. For more details, please see Materials and Methods.

3372 INNATE IMMUNE RESPONSE IN THE BRAIN

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

expression of I�B�), expression of the LPS receptor CD14 and thechemokine 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 thenewly characterized receptor TLR4. This receptor that recognizes

Gram-negative cell wall components is constitutively expressed inthe leptomeninges and is down-regulated by the endotoxin (Fig. 3,middle column). This contrasts with the mRNA encoding the otherLPS receptor CD14, which is strongly activated by the exogenousligand injected directly into the brain parenchyma.

FIGURE 2. Time-related LPS immunoreactive regions after a single injection of the endotoxin into the dorsal striatum. All photomicrographs were takenat the level of bregma �0.6 with the same exposure time. Rats were sacrificed 12 h, (A–D), 3 days (E and F), and 7 days (G and H) after the single cerebralbolus with the Gram-negative cell wall component. Left column, The immunoreactive signal in the brain of animals that received injections with the vehiclesolutions (V-V-V); right column, the brain sections after an intraparenchymal injection of LPS (V-V-L). Magnification: A–B �3.125, C–H, �10; scale bar:A–B, 1625 �m, C–H, 500 �m.

3373The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Endotoxemia prevents the effects of centrally injected LPS

A single systemic injection of LPS was performed to increaseCD14 and preactivate microglial cells before the central treatmentwith the endotoxin. This preinduction of the endotoxin receptorwas expected to intensify the effects of LPS once present in theCNS. Surprisingly however, circulating LPS clearly abolished theeffects of IS LPS on the proinflammatory signal transduction path-ways and gene expression. Both the intensity of the signal andbrain areas expressing the different transcripts in response to asingle bolus of LPS into the caudate putamen significantly de-creased in animals that were pretreated with the endotoxin sys-temically (Table 2). Representative examples of this phenomenonare depicted by Figs. 1 and 3 (V-L-L; right column). Pretreatmentwith the endotoxin i.p. also significantly reduced the time in whichthe signal remained positive in response to the cerebral endotoxininjection. Except for the TNF-� signal that was still positive butlow at day 3 postinjection, expression levels for all the other in-duced transcripts returned to background levels at that time whilethe mRNAs were still expressed in animals that received the i.p.vehicle and cerebral LPS treatment (V-V-L; Table 2). Of interestis the strong and localized hybridization signal for CD14 transcriptin cells adjacent to the microvasculature of the V-L-L-treated an-imal (Fig. 3, right column).

Role of glucocorticoids in LPS-induced cerebral inflammation

Systemic LPS administration is a powerful stimulus to increaseplasma levels in glucocorticoids, the most potent endogenous anti-

inflammatory molecule known (9). A single i.p. endotoxin boluscaused a rapid increase in circulating corticosterone levels thatpeaked 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 LPSinjection and the subsequent intracerebral treatment. Therefore, itwas hypothesized that the high plasma glucocorticoid levels oc-curring in animals pretreated with the endotoxin i.p. were respon-sible for preventing the effects of centrally injected LPS on theproinflammatory signal transduction pathways and transcriptionof the genes assessed in this study. Animals that received asystemic bolus of glucocorticoids 60 and 30 min before beinginjected into the caudate putamen with the endotoxin respondedquite similarly to the rats that were pretreated with the endo-toxin in the peritoneal cavity. Indeed, the strong transcriptionalactivation of TNF-� gene was essentially abolished in animalsthat received i.p. Cort before the cerebral insult (Fig. 5A). Boththe intensity and extent of the positive signals were lower inanimals that received glucocorticoids before the LPS insult(Fig. 5A). This was also the case for the mRNA encoding CD14and I�B� (data not shown).

In an attempt to determine the exact contribution of endogenousglucocorticoids on LPS-induced gene expression in the CNS, anothergroup of rats were treated with RU486 before the different treatments.The glucocorticoid receptor antagonist RU486 restored the effects ofIS LPS administration in the animals that were pretreated with theendotoxin i.p. (Fig. 5B). Therefore, circulating LPS-induced elevation

FIGURE 3. Darkfield photomicro-graphs showing the expression patternof TNF-�, I�B�, CD14, TLR4, andMCP-1 mRNA in the rat brain (bregma�0.6) 12 h after a cerebral LPS admin-istration. Left column, The hybridiza-tion signals in the brain of an animalthat received vehicle solution in thelateral ventricle, peritoneal cavity, andthe dorsal striatum (V-V-V). Middlecolumn, The expression patterns in re-sponse to a single LPS bolus within thebrain parenchyma (V-V-L); right col-umn, the transcript signals in the CNSof a rat that received a systemic endo-toxin challenge before the cerebral in-sult (V-L-L). Magnification: �3.125;scale bar, 1000 �m.

3374 INNATE IMMUNE RESPONSE IN THE BRAIN

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

in Cort levels is the mechanism that prevented microglial activity tocerebral endotoxin administration.

Effects of the anti-CD14 antisera on the cerebral innateimmunity

To block the biological activity of the LPS receptor CD14 withinthe cerebral tissue, an anti-rat CD14-neutralizing Ab was injectedin the lateral ventricle 10 h before the LPS infusion within thedorsal basal ganglia. Animals were equipped with two chronicindwelling cannulas, one just above the lateral ventricle and theother at the level of the corpus callosum. The neutralizing antiserafailed to prevent expression of the TNF-� mRNA 12 h after theLPS infusion into the brain parenchyma. The intensity and extendof the hybridized signal were similar in the brain of animals thatwere pretreated or not with the anti-CD14 into the lateral ventriclebefore the cerebral LPS insult (Fig. 6). The same phenomenonoccurred for the other inflammatory transcripts, which remainedexpressed at a similar level between both groups of endotoxin-challenged rats at time 12 h (data not shown). In spite of this, theduration of the inflammatory response was reduced by preventingthe biological activity of the LPS receptor CD14. Indeed, the neu-tralizing CD14 Ab abolished the maintained expression of the geneencoding TNF-� that took place 3 days after the intraparenchymalinfusion with the Gram-negative cell wall component. This sug-gests that although CD14 may not be essential for triggering thecerebral innate immune response, it plays a key role in the durationof these events.

Leukocyte infiltration within the brain parenchyma

Wright staining allowed us to detect the presence of leukocyteswithin the cerebral tissue and a large number of polymorphonu-clear cells (PMNs) were found within the regions that exhibited apositive signal for the different transcripts in response to the ISendotoxin injection (Fig. 7). These neutrophils were detected onlyin the area where the inflammation occurred and never in the con-tralateral site or in the brain of vehicle-administered rats. However,it is possible that these cells did not actually emigrate within theT

able

II.

OD

and

area

depi

ctin

gpo

siti

vehy

brid

izat

ion

sign

als

for

the

diffe

rent

infla

mm

ator

ytr

ansc

ript

sin

resp

onse

tosy

stem

ican

d/or

cere

bral

LP

Sin

ject

ions

Gen

e

12h

3D

ays

7D

ays

V-V

-VV

-V-L

V-L

-LV

-V-V

V-V

-LV

-L-L

V-V

-VV

-V-L

V-L

-L

TN

F-�

OD

0.00

5�

0.00

30.

276*

†�

0.02

40.

085*

�0.

008

0.00

5�

0.00

30.

045*

†�

0.00

20.

003

�0.

001

0.00

3�

0.00

10.

004

�0.

002

0.00

2�

0.00

1A

rea

1.88

4�

0.50

159

.49*

†�

4.61

24.2

8*�

4.04

1.56

2�

0.51

648

.75*

†�

4.97

4.55

*�

0.23

0.57

4�

0.34

70.

739

�0.

411

0.62

4�

0.30

9I�

B�

OD

0.01

3�

0.00

30.

081*

�0.

009

0.06

6*�

0.00

70.

007

�0.

005

0.04

4*†

�0.

010

0.00

8�

0.00

40.

004

�0.

002

0.01

9*�

0.00

90.

009

�0.

001

Are

a3.

216

�0.

495

73.5

55*†

�7.

374

26.8

39*

�5.

566

0.43

4�

0.18

728

.491

*†�

3.95

11.

225

�0.

105

0.12

8�

0.03

04.

147*

�1.

507

1.20

6�

0.68

3C

D14

OD

0.01

0�

0.00

60.

398*

�0.

038

0.19

7*�

0.02

00.

007

�0.

005

0.28

7*†

�0.

026

0.02

1�

0.00

30.

009

�0.

003

0.02

4*�

0.00

40.

026

�0.

007

Are

a4.

011

�0.

051

73.5

71*†

�9.

326

23.1

61*

�3.

057

2.46

0�

0.34

045

.757

*†�

2.72

41.

784

�1.

448

1.98

1�

0.03

04.

587*

�1.

479

1.46

8�

0.60

4M

CP-

1O

D0.

010

�0.

004

0.18

7*†

�0.

020

0.08

5*�

0.01

10.

005

�0.

003

0.05

2*†

�0.

009

0.00

7�

0.00

20.

005

�0.

003

0.00

5�

0.00

30.

011

�0.

003

Are

a4.

220

�0.

360

62.3

16*†

�4.

357

27.8

05*

�5.

528

0.15

5�

0.04

93.

021*

†�

0.84

70.

271

�0.

119

0.00

2�

0.00

10.

003

�0.

002

0.00

3�

0.00

1

aV

-V-V

,in

trac

ereb

rove

ntri

cula

r,i.p

.,an

dIS

inje

ctio

nsof

ave

hicl

eso

lutio

n;V

-V-L

,in

trac

ereb

rove

ntri

cula

ran

di.p

.in

ject

ion

ofve

hicl

ean

dIS

LPS

inje

ctio

n;V

-L-L

,in

trac

ereb

rove

ntri

cula

rve

hicl

ein

ject

ion

and

i.p.

(1m

g/kg

b.w

.)an

dIS

(5�

g/2

�l/2

min

)L

PSin

ject

ions

.�

p�

0.05

vsV

-V-V

.†

p�

0.05

vsV

-L-L

.

FIGURE 4. Circulating Cort levels after a single i.p. LPS administra-tion. The endotoxin was injected at a dose of 1 mg/kg of b.w. and rats killed1, 3, 6, and 12 h afterward. Data were analyzed using a 2 � 3 ANOVA,followed by a Bonferroni-Dunn test procedure as post-hoc comparisons(Statview 4.01). �, p � 0.05, from their corresponding vehicle-treatedgroups. Results represent means � SEM for four animals per group.

3375The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

FIGURE 5. Role of glucocorticoids on LPS-induced TNF-� gene expression in the brain parenchyma. A, The animals received a double i.p. injection of eithervehicle (Veh) or Cort (60 mg/kg/300 �l) 60 and 30 min before an intraparenchymal injection of vehicle or LPS (5 �g/2 �l/2 min). Top panels, The positive signalon X-ray film (Biomax); middle panels, darkfield photomicrographs of the same 30-�m coronal sections dipped into nuclear type � 2 emulsion. Bottom panels,The quantitative analyses after the different treatments: a, OD for the TNF-� signal, b, surface/area of positive hybridization signal, and c, plasma corticosteronelevels. OD for TNF-� mRNA signal at the level of bregma �0.6 was digitized and subjected to densitometric analysis using a logarithmic specter of standardizedOD adapted from Bioimage Visage 110s (Millipore, Bedford, MA), yielding measurements of mean density per area. The average area (surface) of TNF-�mRNA-expressing regions for each animal was calculated from digitized sections at the level of bregma 0.0, �0.6, and �3.0. Plasma Cort concentrations weredetermined by RIA. Data were analyzed using a two-way ANOVA for each time, followed by a Bonferroni-Dunn test procedure as post-hoc comparisons (Statview4.01). A significant interaction occurred between the two main factors for all the dependent variables. �, p � 0.05, from their corresponding vehicle-treated groups.†, 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�l 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 intothe striatal region and killed 12 h following the cerebral insult. A significant interaction occurred between the two main factors for all the dependent variables (dand e). �, p � 0.05, from their corresponding control groups. †, p � 0.05, from all the other DMSO-treated groups. Results represent means � SEM for three tofive animals per group. Magnifications for the darkfield photomicrographs, �3.125; scale bar: 1000 �m.

3376 INNATE IMMUNE RESPONSE IN THE BRAIN

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

FIGURE 6. Effect of anti-CD14 Ab on IS LPS-induced expression of the proinflammatory cytokine TNF-�. Rats were sacrificed 12 h (A and B), 3 days(C and D), and 7 days (E and F) after a single bolus of the bacterial cell wall component in the dorsal basal ganglia. Animals received in the lateral ventriclea sham solution (left column, V-V-L) or the rat anti-CD14 (2 �g/10 �l/2 min) (right column, A-V-L) 10 h before the cerebral endotoxin insult (5 �g/2 �l/2min). The darkfield photomicrographs of 30-�m coronal section dipped into nuclear type �2 emulsion milk depict the strong hybridization signal for thegene encoding the cytokine that was essentially the same in both groups at time 12 h postinjection (A vs B), but the neutralizing CD14 Ab prevented TNFexpression at 3 days (C vs D). The histograms represent the OD (G) and extent/area (H) of the positive signal for TNF mRNA. �, p � 0.05, from theircorresponding vehicle-treated groups. †, p � 0.05, from their corresponding time-treated groups at 3 days. Results represent means � SEM for three tofour animals per group. Magnification, �3.125; scale bar, 1000 �m.

3377The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

brain parenchyma and remained attached to the vasculature, whichwas difficult to evaluate in the present case. Of interest is thatpretreatment with the endotoxin i.p. significantly prevented accu-mulation of PMNs within the brain at 12 h postintraparenchymalendotoxin infusion. Although these cells were still found in regionsipsilateral to the injection site, circulating endotoxin decreased thenumber of neutrophils by mm2 of tissue and this effect was de-pendent on plasma Cort levels (data not shown). The rat anti-CD14neutralizing Ab failed to modulate the accumulation of PMNs attime 12 h, but fewer cells were generally detected in the brain ofrats killed 3 days after double cerebral treatments (A-V-L).

Consequences on the neuronal integrity

The question of whether LPS-induced cerebral inflammation wasassociated with neurodegenerative processes was investigated viaa number of approaches. Cell death induced by apoptosis was de-tected with the fluorescein-FragEL DNA fragmentation detectionkit for tissue cryosections. Positive controls were generated bycovering the entire specimen with 1 �g/�l DNase I in 1� TBS/1mM MgSO4 following proteinase K treatment, which caused astrong labeling over neurons across the brain of a vehicle-treated

animal (Fig. 8A). Such positive cells were never observed in thebrain of LPS-treated animals pretreated or not with the endotoxini.p. (Fig. 8B). The fluorochrome FJB is a sensitive and reliablemarker for the histochemical localization of neuronal degeneration(10). Bilateral adrenalectomy is known to provoke neurodegenera-tion of the hippocampal dentate granule cells. These neurons dis-played strong and selective fluorescent FJB signal, indicating degen-eration of a subpopulation of cells within the dentate gyrus granulallayer of a rat killed 6 days after being adrenalectomized (Fig. 8C).Surprisingly however, no positive signal was observed across theCNS of endotoxin-challenged animals. Indeed, only background lev-els were detected for the fluorescent marker over the tissues thatexhibited the robust induction of inflammatory molecules in responseto an intraparenchymal LPS infusion (Fig. 8D). FJB was also unde-tectable in the CNS of animals that were pretreated with the anti-CD14 and the endotoxin i.p. before the CNS LPS insult and this from12 h to 21 days posttreatment.

To ascertain that the robust innate immune response was notassociated with alteration of neuronal elements, other histologicalstraining procedures were performed. Wright- and Nissl-stained sec-tions failed to provide convincing evidence of neurodegeneration. The

FIGURE 7. IS endotoxin challenge causes emigration of neutrophils within the brain parenchyma. Wright stain was used to label neutrophils in the brainof rats that received a cerebral injection of either vehicle solution (V-V-V, A) or LPS (5 �g/2 �l/2 min, V-V-L, B). C, A brain section of a rat treated withthe endotoxin i.p. (1 mg/kg of b.w.) before the parenchymal LPS infusion (V-L-L). Rats were killed 12 h after the cerebral insult with the Gram-negativecompound. D, The number of neutrophils per mm2 that was quantified as described in Materials and Methods. �, p � 0.05, from their correspondingvehicle-treated groups (V-V-V and A-V-V). †, p � 0.05, from their corresponding groups of rats that were challenged with an i.p. LPS administration(V-L-L and A-L-L). Results represent means � SEM for three to four per group. Filled and open arrowheads depict neutrophils and neurons, respectively.Magnification, �150; scale bar, 50 �m.

3378 INNATE IMMUNE RESPONSE IN THE BRAIN

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

cell bodies, dendrites, and fibers were quite comparable among allthe groups and the regions that exhibited the induction of the proin-flammatory molecules, including the basal ganglia, hippocampalformation, cerebral cortex, and the fibers of the corpus callosum (Fig.8, E–J).

DiscussionAn acute infusion of LPS into the brain parenchyma caused aprofound transcriptional activation of the genes encoding inflam-

matory molecules, which peaked at 12 h and slowly decreasedthereafter. Pretreatment with the endotoxin i.p. essentially abol-ished this cerebral innate immune response due to the increase inplasma glucocorticoid levels. The anti-inflammatory properties ofthese hormones that also take place in the cerebral tissue may playa determinant role in controlling the proinflammatory signal trans-duction pathways and gene transcription within myeloid cells ofthe CNS. The LPS receptor CD14 does not seem essential forengaging this immune reaction, although it contributes to the

FIGURE 8. The robust inflammatoryresponse provoked by an IS endotoxinchallenge is not associated with neurode-generation. Cell death induced by apopto-sis was detected with the fluorescein-FragEL DNA fragmentation detection kitfor tissue cryosections. A, Positive controlswere generated by covering the entirespecimen with 1 �g/�l DNase I in 1�TBS/1 mM MgSO4 following proteinase Ktreatment, which caused a strong labelingover neurons across the brain of a vehicle-treated animal. B, Such positive cells werenever observed in the brain of LPS-treatedanimals pretreated or not with the endo-toxin i.p. The fluorochrome FJB was usedas a marker for the histochemical localiza-tion of degenerative neurons. C, Degener-ation of a subpopulation of cells within thedentate gyrus granulal layer was found in arat killed 6 days after being adrenalecto-mized. D, No positive signal was observedacross the CNS of all the groups includedin this study. The cell bodies, dendrites,and fibers were quite comparable amongall the groups and the regions that exhib-ited the induction of the proinflammatorymolecules, including the basal ganglia,hippocampal formation, cerebral cortex,and the fibers of the corpus callosum (E–J).E and F, Wright stain in the striatum; Gand H, Nissl-stained sections at the level ofthe dorsal basal ganglia; I and J, Nissl-stained corpus callosum. Magnification,�100; scale bar, 100 �m.

3379The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

duration of the cerebral inflammatory response induced by an in-traparenchymal infusion with the cell wall component of Gram-negative bacteria. Despite the fact that LPS has the ability to in-duce a robust inflammatory reaction and neutrophil emigrationwithin the CNS, this innate and transient immune response is notassociated with neurodegenerative processes. As with the periph-ery, circulating glucocorticoids and CD14 are involved in the finecontrol of the cerebral innate immunity, which is not by itselfdetrimental for the brain. In contrast, it may be essential for elim-inating pathogens that can be harmful for the neuronal elements incase of cerebral infection.

We have previously reported that circulating LPS has the abilityto trigger CD14 within the CVOs and thereafter across the brainparenchyma (8). However, this parenchymal induction of the LPSreceptor depends on microglial-derived TNF-� that acts as a para-crine factor to trigger adjacent cells of myeloid origin (8). Al-though such paracrine effect of the proinflammatory cytokine mayalso take place in the brain of centrally injected LPS, the endotoxinseems directly responsible of the inflammatory reaction across thecerebral tissue ipsilateral to the injection site. Indeed, the pattern ofLPS immunoreactive areas is essentially the same as the one foundfor CD14-, TNF-�-, I�B�-, and MCP-1-expressing structures.This suggests that LPS is present in the regions that exhibitedtranscriptional activation of these genes and the spreading of theinflammatory response seems to depend on the diffusion of theendotoxin after the infusion within the brain parenchyma. A para-crine influence of TNF and other molecules may also take placebetween adjacent cells, but not across the brain.

This contrasts with the effects of systemic LPS injection thatcause transcriptional activation of CD14 (5) and TNF (11) firstwithin the CVOs and thereafter throughout the brain microglialcells. However, this depends on the dose of the endotoxin, becausethe signal for these proinflammatory molecules remains quite lo-calized to the regions that can be reached by the bloodstream andtheir adjacent structures after a single bolus of LPS injected at lowto moderate doses (5, 8, 11). On the other hand, high circulatinglevels of the endotoxin are associated with an increased expressionof a number of inflammatory genes across the brain parenchyma(8, 11–13). Such widespread induction that was not observed inresponse to an IS LPS infusion is in agreement with another recentstudy (14). This rather localized hybridization signal ipsilateral tothe 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 toselectively recognize one of the major PAMPs produced by eitherGram-negative or Gram-positive bacteria (3). The data that muta-tion of the mouse Lps locus abolishes the LPS response and thatLps encodes TLR4 provided the first evidence that this particularreceptor may play a key role in the innate immune response toGram-negative bacteria (reviewed in Ref. 15). Therefore, the lackof signal for the gene encoding TLR4 in the brain parenchyma isquite intriguing, because this receptor is now recognized to beresponsible for LPS-induced NF-�B signaling, and TLR4-deficientmice are resistant to cell wall components of Gram-negative bac-teria (3). Therefore, how LPS can trigger such profound inflam-matory response within microglial cells across the brain paren-chyma without TLR4? TLR4 transcript levels were low along themeninges, choroid plexus, and the CVOs under basal conditions(4). Such levels fit quite well with the fact that the copy number ofTLR4 is extremely low in systemic phagocytes compared to themore abundant membrane protein CD14 (15, 16). Nevertheless, itis remarkable that so few TLR4 receptors (�1000 or fewer percell), residing on macrophages alone, have such an important in-fluence in the LPS signaling and the coordination of the biological

responses to Gram-negative infections (15). The technique used inthe present study is probably not sensitive enough to detect TLR4within microglial cells.

LPS-induced proinflammatory signal transduction pathway andgene transcription in the brain was not associated with neurode-generation. Various approaches were used to reach these conclu-sions, which are in disagreement with few other studies that pro-vided evidence of LPS-induced neurotoxicity (17, 18). Suchdiscrepancies may be explained by the techniques used to assayneurodegeneration, which was determined by the decrease in ty-rosine hydroxylase activity and the loss of tyrosine hydroxylase-positive neuronal bodies in the substantia nigra (17). The targetregion seems also of great importance, because injection of theendotoxin in the hippocampus, cortex, or substantia nigra of adultrats produced neurodegeneration only in the substantia nigra (18).These authors suggested that the region-specific differential sus-ceptibility of neurons to LPS is attributable to differences in thenumber of microglia present within the system and may reflectlevels of inflammation-related factors produced by these cells (18).In the present case, microglial activation was robust but transientand it is tempting to propose that such response is not detrimentalfor the brain and in contrast may act as a protective mechanismduring cerebral infection. It is also possible that specific popula-tions of neurons are more susceptible than others to inflammatorymolecules and despite eliminating pathogens from the CNS, mi-croglia, and their secreted products may be harmful for someneurons.

In contrast to glucocorticoids, CD14 does not seem to be in-volved in the early inflammatory events triggered by the cerebralinjection of the endotoxin. The neutralizing antisera failed to pre-vent the inflammatory response in animals that were killed 12 hafter the LPS infusion within the basal ganglia. These data are inagreement with the CD14-deficient mice that are highly resistant toshock induced by either live Gram-negative bacteria or LPS, but atvery high concentrations of LPS or bacteria, responses throughnon-CD14 receptors were found (19). These animals show also noalteration in LPS-induced acute-phase protein, including serumamyloid A, LPS-binding protein, fibrinogen, and ceruloplasmin(20). Although the neutralizing CD14 Ab did not prevent the in-flammatory reaction at times early post-LPS administration, it at-tenuated the duration of this response. These data support the con-cept of CD14-dependent and -independent pathways that alsoseem to occur in the CNS. However, the lack of effects at 12 h maybe explained by the inability of the neutralizing antisera to com-pletely block the biological activity of CD14 and the dose of LPSthat was injected directly within the cerebral tissue.

Intraparenchymal LPS administration caused emigration ofPMNs within the regions that exhibited positive signal for theproinflammatory genes. This phenomenon was significantly atten-uated in animals that were pretreated with the endotoxin i.p., whichsuggests that the intensity of the inflammatory process is directlyresponsible to engage the chemoattraction. Nevertheless, thesedata are quite surprising, because leukocyte emigration is unlikelyto occur during nonpathological conditions and during endotox-emia. Although systemic LPS treatment is generally not associatedwith leukocyte emigration within the brain parenchyma, intrace-rebral LPS infusion and the robust expression and cytokines/che-mokines are likely mechanisms to allow diapedesis. Direct admin-istration of LPS onto the BBB elicits gaps between endothelialcells and provokes leukocyte infiltration (21). Emigrating cellshave also been observed in mice that received different doses ofLPS within the brain parenchyma (22). This emigration wasspecific to the regions that exhibited expression of the differenttranscripts and was very dynamic, because the number of PMNs

3380 INNATE IMMUNE RESPONSE IN THE BRAIN

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

decreased at day 3 and these cells were no longer detected in thebrain of animals killed 7 days after the endotoxin infusion withinthe basal ganglia. Therefore, leukocytes are recruited in responseto a cerebral innate immune reaction and this transient phenome-non is not detrimental for the neuronal elements. Neutrophils con-tain acyloxyacyl hydrolase, the enzyme responsible to reduce LPStoxicity in deacylating its lipid A portion (23). These cells couldthen play a key role in detoxifying the endotoxin and eliminate itswiftly from the cerebral tissue.

Taken together, these data suggest that there is an innate im-mune response that is rapidly triggered within the brain microglia,which is associated with an emigration process. These events areregulated by circulating levels of glucocorticoids and LPS receptorCD14, at least for the duration of the inflammatory response. Theendogenous expression of CD14 and specific TLRs may engageproinflammatory signal transduction pathways and production ofcytokines by microglia. One of the beneficial consequences of suchmicroglial reactivity is the release of neurotrophic factors andother 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 microglialreactivity can overproduce inflammatory molecules and alter theBBB, a mechanism that seems to be central to several neurode-generative disorders and demyelinating diseases. As microglia arethe APCs of the brain, they are probably crucial for cell-specificimmunity against neuronal elements. A better understanding of theinnate immune response in cerebral tissue could lead us to thefundamental mechanisms that underlie the capability of the brainto mount an inflammatory response that either protects against orcontributes to neuronal damage.

AcknowledgmentsWe thank Dr. Alain Israel (Institut Pasteur, Paris, France), Dr. Doug Fein-stein (University of Illinois, Chicago, IL), and Dr. S. C. Williams (TexasTechnical University, Lubbock, TX) for the gift of the plasmid containingI�B� cDNA, CD14 cDNA, and MCP-1 cDNA, respectively.

References1. Anderson, K. V. 2000. Toll signaling pathways in the innate immune response.

Curr. Opin. Immunol. 12:13.2. Wright, S. D. 1999. Toll, a new piece in the puzzle of innate immunity. J. Exp.

Med. 189:605.3. Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins

linking innate and acquired immunity. Nat. Immunol. 2:675.

4. Laflamme, N., and S. Rivest. 2001. Toll-like receptor 4: the missing link of thecerebral innate immune response triggered by circulating gram-negative bacterialcell wall components. FASEB J. 15:155.

5. Lacroix, S., D. Feinstein, and S. Rivest. 1998. The bacterial endotoxin lipopoly-saccharide has the ability to target the brain in upregulating its membrane CD14receptor within specific cellular populations. Brain Pathol. 8:625.

6. Antel, J. P., and T. Owens. 1999. Immune regulation and CNS autoimmunedisease. J. Neuroimmunol. 100:181.

7. Stoll, G., and S. Jander. 1999. The role of microglia and macrophages in thepathophysiology of the CNS. Prog. Neurobiol. 58:233.

8. Nadeau, S., and S. Rivest. 2000. Role of microglial-derived tumor necrosis factorin mediating CD14 transcription and NF-�B activity in the brain during endo-toxemia. J. Neurosci. 20:3456.

9. Webster, J. I., L. Tonelli, and E. M. Sternberg. 2002. Neuroendocrine regulationof immunity. Annu. Rev. Immunol. 20:125.

10. Schmued, L. C., C. Albertson, and W. Slikker, Jr. 1997. Fluoro-Jade: a novelfluorochrome for the sensitive and reliable histochemical localization of neuronaldegeneration. Brain Res. 751:37.

11. Nadeau, S., and S. Rivest. 1999. Regulation of the gene encoding tumor necrosisfactor � in the rat brain and pituitary in response to different models of systemicimmune challenge. J. Neuropathol. Exp. Neurol. 58:61.

12. Breder, C. D., C. Hazuka, T. Ghayur, C. Klug, M. Huginin, K. Yasuda, M. Teng,and C. B. Saper. 1994. Regional induction of tumor-necrosis factor-� expressionin the mouse brain after systemic lipopolysaccharide administration. Proc. Natl.Acad. Sci. USA 22:11393.

13. Quan, N., M. Whiteside, and M. Herkenham. 1997. Time course and localizationpatterns of interleukin-1� mRNA expression in the brain and pituitary after pe-ripheral administration of lipopolysaccharide. Neuroscience 83:281.

14. Stern, E. L., N. Quan, M. G. Proescholdt, and M. Herkenham. 2000. Spatiotem-poral induction patterns of cytokine and related immune signal molecule mRNAsin response to intrastriatal injection of lipopolysaccharide. J. Neuroimmunol.106:114.

15. Beutler, B. 2000. Tlr4: central component of the sole mammalian LPS sensor.Curr. Opin. Immunol. 12:20.

16. Ulevitch, R. J. 1999. Toll gates for pathogen selection. Nature 401:755.17. Castano, A., A. J. Herrera, J. Cano, and A. Machado. 1998. Lipopolysaccharide

intranigral injection induces inflammatory reaction and damage in nigrostriataldopaminergic system. J. Neurochem. 70:1584.

18. Kim, W. G., R. P. Mohney, B. Wilson, G. H. Jeohn, B. Liu, and J. S. Hong. 2000.Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicityin the rat brain: role of microglia. J. Neurosci. 20:6309.

19. Haziot, A., E. Ferrero, F. Kontgen, N. Hijiya, S. Yamamoto, J. Silver,C. L. Stewart, and S. M. Goyert. 1996. Resistance to endotoxin shock and re-duced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity4:407.

20. Haziot, A., X. Y. Lin, F. Zhang, and S. M. Goyert. 1998. The induction of acutephase proteins by lipopolysaccharide uses a novel pathway that is CD14-inde-pendent. J. Immunol. 160:2570.

21. Persidsky, Y., M. Stins, D. Way, M. H. Witte, M. Weinand, K. S. Kim, P. Bock,H. E. Gendelman, and M. Fiala. 1997. A model for monocyte migration throughthe blood-brain barrier during HIV-1 encephalitis. J. Immunol. 158:3499.

22. Andersson, P. B., V. H. Perry, and S. Gordon. 1992. The acute inflammatoryresponse to lipopolysaccharide in CNS parenchyma differs from that in otherbody tissues. Neuroscience 48:169.

23. Kitchens, R. L., and R. S. Munford. 1995. Enzymatically deacylated lipopoly-saccharide (LPS) can antagonize LPS at multiple sites in the LPS recognitionpathway. J. Biol. Chem. 270:9904.

24. Nguyen, M. D., J. P. Julien, and S. Rivest. 2002. Innate immunity: the missinglink in neuroprotection and neurodegeneration. Nat. Rev. Neurosci. 3:216.

3381The Journal of Immunology

by guest on March 13, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from