The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 178: 6476-6481.
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoffmann, O.
Right arrow Articles by Weber, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoffmann, O.
Right arrow Articles by Weber, J. R.
Right arrowPubmed/NCBI databases
*Substance via MeSH

TLR2 Mediates Neuroinflammation and Neuronal Damage1

Olaf Hoffmann2,*, Johann S. Braun2,*, Doreen Becker*, Annett Halle*, Dorette Freyer*, Emilie Dagand*, Seija Lehnardt{dagger} and Joerg R. Weber3,*,{dagger}

* Department of Neurology and {dagger} Department of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Berlin, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Innate immunity relies on pattern recognition receptors to detect the presence of infectious pathogens. In the case of Gram-positive bacteria, binding of bacterial lipopeptides to TLR2 is currently regarded as an important mechanism. In the present study, we used the synthetic bacterial lipopeptide Pam3CysSK4, a selective TLR2 agonist, to induce meningeal inflammation in rodents. In a 6-h rat model, intrathecal application of Pam3CysSK4 caused influx of leukocytes into the cerebrospinal fluid (CSF) and induced a marked increase of regional cerebral blood flow and intracranial pressure. In wild-type mice, we observed CSF pleocytosis and an increased number of apoptotic neurons in the dentate gyrus 24 h after intrathecal challenge. Inflammation and associated neuronal loss were absent in TLR2 knockout mice. In purified neurons, cytotoxicity of Pam3CysSK4 itself was not observed. Exposure of microglia to Pam3CysSK4 induced neurotoxic properties in the supernatant of wild-type, but not TLR2-deficient microglia. We conclude that TLR2-mediated signaling is sufficient to induce the host-dependent key features of acute bacterial meningitis. Therefore, synthetic lipopeptides are a highly specific tool to study mechanisms of TLR2-driven neurodegeneration in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system recognizes bacterial molecules and generates an inflammatory response to invading pathogens. In recent years, important progress has been made in understanding how specific receptors of the immune system recognize pathogen-associated molecular patterns to induce immune response (1, 2). A highly relevant class of pattern recognition receptors is the family of TLRs, of which 12 have been found in mammals (2). In the case of Gram-positive bacteria, components of the bacterial cell wall interact with TLR2. Although the exact molecular motif required for binding has not been identified, it appears that bacterial lipopeptides and teichoicated peptidoglycans (PGs)4 are the major ligands. Controversy exists whether TLR2 is also capable of sensing lipoteichoic acid-depleted PG. However, other components of Gram-positive bacteria may be recognized by other TLRs, e.g., bacterial nucleic acids via TLR9, or they may bind to non-TLRs, e.g., the muramyl dipeptide of PGs to NOD2 receptors (3, 4). Diversity at the level of the intracellular adaptor proteins adds further complexity. Most of the evidence discussed above was gathered in vitro, mostly involving overexpressing systems, which may not truly model the in vivo situation. Indeed, experiments in TLR2 knockout mice and several infection models often failed to show a pronounced anti-inflammatory phenotype, suggesting redundancy in microbial pattern recognition (5, 6, 7, 8, 9).

Lipoproteins are present in the cell wall of all bacteria and may represent a general principle for how bacteria induce an immune response and tissue damage. Other than the complex macromolecular structure of the cell wall backbone, the basic structure of lipopeptides, an unusual S-propyl-substituted cysteine carrying three lipid residues plus a variable amino acid chain, has been chemically synthesized. Synthetic lipopeptides allow us to study TLR2-driven inflammation without the confounding effects of contaminants such as bacterial toxins, metabolic products, or other cell wall components. In previous studies, the synthetic bacterial lipopeptide Pam3CysSK4 (N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3- lysine) has been identified as a specific TLR2 agonist (10) acting mainly through TLR2/1 heterodimeric receptors (11). In the present study, we used Pam3CysSK4 to study TLR2-driven inflammation and neuronal damage in experimental meningitis as an important example of invasive bacterial infection. For comparison with a viable pathogen, meningitis was induced in further animals by intrathecal inoculation with live encapsulated Streptococcus pneumoniae, the most common microorganism in bacterial meningitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial culture

D39, an encapsulated strain of S. pneumoniae serotype 2 originally derived from a clinical isolate, was grown in standard casein plus yeast medium with 5% CO2 at 37°C. During log phase growth, bacteria were pelleted by centrifugation and resuspended in pyrogen-free 0.1 M PBS. CFU per milliliter were then determined photometrically (absorption at 620 nm) using a standard curve. Adequate dilutions in pyrogen-free PBS were used to produce defined inocula. CFU calculations were verified by plating of serial dilutions.

Rat experiments

All animal experimental procedures were reviewed by institutional and state authorities. The general experimental procedure was as described earlier (12). The experiments were performed on male Wistar rats (280–330 g) anesthetized with i.p. thiopental sodium (Byk Gulden). A dose of 100 mg/kg was used for induction of anesthesia, while 20 mg/kg was given for maintenance at ~2-h intervals (total dose per animal, 160–180 mg/kg). Adequate anesthesia was verified by absence of response to a painful stimulus. Animals were tracheotomized and mechanically ventilated (model AP-10; K. Effenberger). End-tidal CO2 was monitored continuously (Heyer Artema MM204). Body temperature was measured by a rectal probe and maintained at 37.8 ± 0.4°C using a heating pad. Mean arterial blood pressure was measured continuously by a transducer (Statham P109-EZ; Spectramed) connected to a catheter placed in the left femoral artery. From this catheter, arterial blood samples were analyzed for PaO2, PaCO2, and pH at 0, 2, 4, and 6 h. The left femoral vein was cannulated for infusion of saline as a volume replacement. A 3x3-mm area of the parietal bone lateral to the sagittal suture was thinned to allow laser Doppler flow (LDF) measurements (Periflux 4001 Master; Perimed) in cortical blood vessels. A catheter was placed into the cisterna magna through an occipital burr hole and connected to a pressure transducer (Statham P109-EZ; Spectramed) for continuous intracranial pressure (ICP) measurement. The 100 µl of cerebrospinal fluid (CSF) removed from this catheter was replaced with 100 µl of Pam3CysSK4 (1 mg/ml; EMC Microcollections) or with 100 µl of bacterial suspension containing 107 CFU. Controls received 100 µl of endotoxin-free PBS. At the end of the experiment, CSF samples were obtained to determine the CSF leukocyte count. In experiments involving live bacteria, CFU in the CSF were determined by the plating of serial dilutions on blood agar plates. Animals were then killed by i.v. injection of 3 M potassium chloride to induce cardiac arrest.

Mouse model of meningitis

Mouse experiments were conducted using a previously published model (12, 13). In brief, male C57BL/6 mice (~20 g) or TLR2–/– mice (14) were anesthetized with i.p. ketamine (100 mg/kg; DeltaSelect) and xylazine (20 mg/kg; Bayer). A skin incision was made to expose the lumbar spine. Using a 30-gauge needle and a microliter syringe, 40 µl of PBS, Pam3CysSK4 solution or bacterial suspension containing 5 x 105 CFU were slowly injected into the spinal canal at vertebrae L2 or L3. The skin incision was closed using dermal clips. Animals were then allowed to wake up and given free access to food and water. Absence of paresis and adequate waking were verified. After 24 h, the animals were again deeply anesthetized. To obtain a CSF sample, a skin incision was made over the head and neck. After dissection of the suboccipital muscles under a preparation microscope, the cisterna magna was punctured and CSF withdrawn using a 27-gauge butterfly cannula connected to a microliter syringe. White blood cells were then counted in the CSF using a Fuchs-Rosenthal chamber at x400 magnification, and CFU per milliliter were determined as discussed when appropriate. Brains were removed, snap frozen in methylbutane on dry ice, and stored at –80°C until further processing.

Histology

Cryostat sections (20 µM) were thaw-mounted on coated glass slides, including the hippocampal structures at several levels. For the evaluation of neuronal damage, we performed TdT-TUNEL using a commercially available kit (Qbiogene). Labeled sites were visualized using a FITC-conjugated Ab. Sections were counterstained using Hoechst 33258 (1/10,000; Invitrogen Life Technologies). For quantification, the TUNEL-positive nuclei in the dentate gyrus were enumerated on all sections using a fluorescent microscope at x1000 magnification. Cell count was divided by the area of the dentate gyrus as determined using Stereo Investigator software (version 4.0; MicroBrightField).

Generation of primary neuronal and glial cell cultures

Primary rat neuronal cultures were prepared from the cerebral cortex of embryonic day 18 rat embryos according to a previously published protocol (15). Following dissociation, 150,000 cells/cm2 were seeded into 24-well plates pretreated with poly-L-lysine (0.5% w/v in PBS, 1 h at room temperature) and coating medium (dissociation medium with 0.03{per thousand} w/v collagen G, 1 h at 37°C). The cultures were maintained at 36.5°C with 5% CO2 in neurobasal medium with B27 supplement (Invitrogen Life Technologies) and fed every 4 days by replacing one-third of the medium. Cultures typically consist of >90% neurons, <10% astrocytes, and <1% microglia until 14 days in vitro (16). Experiments on rat neuronal cultures were conducted after 5–8 days in vitro.

Primary cultures of purified murine cortical neurons were generated from forebrains of embryonic day 17 mice, as previously described (17). Briefly, cortices were dissociated by trituration with papain (Cell Systems) in Earle’s balanced salt solution (Invitrogen Life Technologies) for 5 min at 37°C. Subsequently, cells were resuspended in 0.25% trypsin inhibitor and 0.25% BSA (both from Sigma-Aldrich) in Earle’s balanced salt solution, and incubated at 37°C for 5 min. Cells were pelleted by centrifugation at 1000 x g for 5 min and were plated onto poly-D-lysine-coated glass slides. Neurons were maintained in DMEM with GlutaMAX medium (Invitrogen Life Technologies) supplemented with 10% FBS and penicillin/streptomycin in humidified 5% CO2/95% air at 37°C. Glial cell cultures were prepared from the brains of newborn mice (postnatal days 0–3) following previously described methods (18). After digestion with trypsin and mechanical dissociation, cells were seeded in 75 cm2 flasks in DMEM with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 0.1% glucose. After 8–10 days, microglia were detached by shaking for 2 h at 200 rpm, retrieved from the supernatant, and reseeded into 24-well plates in neurobasal medium with B27. Experiments on microglia were then conducted after 24 h in culture. The remaining astrocytes (>99% glial fibrillary acidic protein GFAP positive cells) were maintained by subcultivation in DMEM with supplements as described.

TLR2 mRNA expression. Total RNA was isolated from ~106 cells according to standard methods (19) using TRIzol reagent (Invitrogen Life Technologies). Contaminating DNA was removed by incubation with DNase (Promega), followed by phenol/chloroform extraction and ethanol precipitation. RNA was quantified by measuring the OD. Reverse transcription of 2 µg of extracted RNA was performed with random hexamer primers (Roche Applied Science) and Moloney murine leukemia virus reverse transcriptase (Promega) following the manufacturer’s instructions. Real-time PCR was performed using FastStartDNA SYBR Green I and a light cycler instrument (Roche). PCR conditions were 10 min at 95°C followed by 45 cycles at 95°C for 15 s, 64°C for 10 s, and 72°C for 15 s (amplification product data acquisition at 81°C). All reactions were performed in duplicate, and the mean threshold cycle was used for analysis. The TLR2 expression of each sample was normalized on the basis of its beta-actin and GAPDH mRNA content. The following sequence-specific primers were used: TLR2 forward, 5'-GATATTCTGAGTTCCGTGAG-3' and reverse, 5'-TGTTACCGTTTCTATTTTACC-3'; beta-actin forward, 5'-ACCCACACTGTGCCCATCTA-3' and reverse, 5'-GCCACAGGATTCCATACCCA-3'; and GAPDH forward, 5'-AGATTGTCAGCAATGCATCCTGC-3' and reverse, 5'-CCT TCTTGATGTCATCATACTTGG-3'.

Immunocytochemistry for TLR2

Cells were fixed and immunostained as previously described (17). To identify neurons and microglia, cells were stained with the Ab microtubule-associated protein-2 (Chemicon International) or with IB4-Alexa (Invitrogen Life Technologies), respectively. The mouse TLR2 Ab 6C2 was obtained from eBioscience. Specificity of the TLR2 Ab was confirmed by the absence of signal in TLR2–/– microglia.

Effects of Pam3CysSK4 in cell culture

Primary rat neuronal cultures were challenged by addition of Pam3CysSK4 to the medium at a final concentration of 0.1 µg/ml. At 24, 48, and 72 h, cell death was assessed by the measurement of lactate dehydrogenase (LDH) in the supernatant, an indicator of disintegration of the cell membrane (20). Moreover, cells were stained with the intercalating dyes acridine orange (final concentration, 2 µg/ml) and ethidium bromide (2 µg/ml), both from Sigma-Aldrich, to allow microscopic differentiation of live, apoptotic, and necrotic cells (21). Acridine orange stains all nuclei green, whereas ethidium bromide is excluded by cells with an intact cell membrane. Live cells are identified by green, regular-sized nuclei. Nuclear condensation or fragmentation identifies apoptotic cells, whereas red staining identifies late apoptotic or necrotic cells. In additional experiments, neurons were not directly challenged with Pam3CysSK4 but exposed to preconditioned medium from Pam3CysSK4-stimulated microglia instead. For this purpose, microglia was exposed to 0.1 µg/ml Pam3CysSK4 in neurobasal medium with B27 for 6, 24, or 48 h. Following centrifugation, the supernatant was filtered and then used to replace the medium of primary neuronal cultures as described. For controls, supernatant was collected from unstimulated microglia at identical times. Cell damage in the neuronal cultures was then determined by measuring the LDH concentration in the supernatant after 48 h of exposure to the preconditioned medium.

Statistics

Data are presented as mean ± SD. Two group comparisons were performed with Student’s t tests after ensuring normal distribution; otherwise, Mann-Whitney U tests were used. Multiple group comparisons on samples with normal distribution were performed with ANOVA followed by Student-Newman-Keuls post hoc testing. Otherwise, nonparametric ANOVA (Kruskal-Wallis) and Dunn’s post hoc analysis were used. The impact of TLR2 genotype on the survival rate of D39-challenged mice was examined using Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intrathecal administration of Pam3CysSK4 induces the pathophysiological hallmarks of bacterial meningitis

In anesthetized rats (n = 3), intracisternal application of pyrogen-free PBS did not induce relevant changes of regional cerebral blood flow as measured by LDF or ICP during the experimental period of 6 h (Fig. 1, A–C). At the end of the experiment, LDF was 103 ± 13% of the individual baseline (Fig. 1A), and ICP was increased by 0.5 ± 1.5 cm H2O (Fig. 1B). Leukocyte count in the CSF at 6 h was 25 ± 15 leukocytes/µl (Fig. 1C). In contrast, instillation of Pam3CysSK4 into the cisterna magna of anesthetized rats (n = 3) was followed by a gradual increase of ICP and regional cerebral blood flow. At 6 h after application, LDF was 155 ± 17% of the individual baseline (p < 0.05; Fig. 1A), whereas ICP was 11.9 ± 0.7 cm H2O above baseline (p < 0.01; Fig. 1B). By comparison, inoculation with viable pneumococci resulted in a more pronounced increase of LDF (at the end of the experiment: 216 ± 23% vs 155 ± 17% of the individual baseline; p < 0.05 vs Pam3CysSK4 and p < 0.05 vs control), whereas the increase of ICP was not different between the two challenges (at 6 h: 12.3 ± 0.3 cm H2O with D39 vs 11.9 ± 0.7 cm H2O with Pam3CysSK4, p was not significant vs Pam3CysSK4 and p < 0.01 vs control). At the end of the experiment, the CSF contained 3278 ± 355 leukocytes/µl in Pam3CysSK4-challenged rats, whereas controls had 25 ± 15 leukocytes/µl (p < 0.01; Fig. 1C). In D39 infected rats, the CSF revealed 3984 ± 1656 leukocytes/µl (p < 0.01 vs control and p is not significant vs Pam3CysSK4) and a bacterial concentration of 5.4 ± 1.6 x 108 CFU/ml. In all experimental groups, arterial blood gas analysis and invasive blood pressure monitoring revealed normal values throughout the entire experimental period (data not shown).


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		FIGURE 1. Pathophysiological effects of intrathecal challenge with the synthetic bacterial lipopeptide Pam3CysSK4 in rats (A–C) and mice (D and E). Regional cerebral blood flow, measured as LDF (A), and increase of ICP (B) were monitored during 6 h following application of Pam3CysSK4 ({blacksquare}), viable pneumococci (D39) ({blacktriangleup}), or PBS ({circ}). *, p < 0.05 vs control; #, p < 0.05 vs Pam3CysSK4 using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. C, Concentration of leukocytes in the CSF of rats 6 h after intrathecal application of PBS ({square}), Pam3CysSK4 ({blacksquare}), or D39 (Figure 1). *, p < 0.05 using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. D, Dose-response curve of the concentration of leukocytes in the CSF of mice 24 h after intrathecal challenge with PBS ({square}) or Pam3CysSK4 ({blacksquare}). Dashed line, 0. p < 0.05 using ANOVA on ranks with Dunn’s post hoc analysis. E, Concentration of CSF leukocytes after intrathecal application of Pam3CysSK4 or D39 in wild-type mice ({blacksquare}) and TLR2–/– mice ({square}). *, p < 0.05; **, p < 0.01 determined by Mann-Whitney U test.

 
Pam3CysSK4 causes leukocyte influx into the CSF in wild-type but not in TLR2-deficient mice

Wild-type mice (n = 4) receiving intrathecal pyrogen-free PBS had 11 ± 2 leukocytes/µl CSF at 24 h after surgery. Intrathecal challenge with Pam3CysSK4 caused a dose-dependent influx of leukocytes into the CSF. At 24 h after application of 2 µg of Pam3CysSK4 per animal (n = 3), 2607 ± 349 leukocytes/µl were counted in the CSF, while a higher dose of 20 µg of Pam3CysSK4 per animal (n = 6) increased pleocytosis to 6741 ± 1969 leukocytes/µl (p < 0.05; Fig. 1D). The leukocyte influx was absent in TLR2–/– mice (n = 8) 24 h after application of 20 µg of Pam3CysSK4 (24 ± 35 leukocytes/µl, p < 0.05 vs wild-type mice) (Fig. 1E). Compared with the 24-h interval, a higher number of leukocytes (20913 ± 5667/µl) were detected at 12 h after application of 20 µg of Pam3CysSK4 in wild-type mice (n = 5; p < 0.05), whereas TLR2-deficient mice (n = 4) again showed no invasion of leukocytes (15 ± 7/µl; Fig. 1E). In meningitis induced by live D39 pneumococci, two of seven wild-type mice died within the experimental period compared with no deaths in six TLR2–/– mice (p was not significant using Fisher’s exact test). CSF was not available from the two wild-type mice that died. In the remaining wild-type mice, challenge with live pneumococci resulted in a similar CSF pleocytosis at 24 h as was observed with challenge by 20 µg of Pam3CysSK4 (5103 ± 2451 leukocytes/µl, n = 5; p was not significant vs Pam3CysSK4 and p < 0.01 vs control). Unlike Pam3CysSK4, D39 also caused leukocyte influx (2428 ± 830 leukocytes/µl) in TLR2-deficient mice (n = 6; p < 0.01). D39-induced pleocytosis was less pronounced in TLR2–/– than in wild-type mice (p = 0.05).

Pam3CysSK4 induces neuronal damage in a TLR2-dependent fashion in vivo

In the dentate gyrus, TUNEL-positive cells were found in all animals 24 h after challenge (Fig. 2). In wild-type control mice (n = 3), 73 ± 18 apoptotic cells/mm2 were present following intrathecal application of pyrogen-free PBS. Meningitis induction with 20 µg of Pam3CysSK4 increased apoptotic cell number to 120 ± 18/mm2 (n = 7; p < 0.05), whereas D39-induced meningitis resulted in 146 ± 40 apoptotic cells/mm2 (n = 5; p < 0.05 vs control and p was not significant vs Pam3CysSK4). In TLR2–/– control mice (n = 3) receiving intrathecal PBS, 81 ± 3 apoptotic cells/mm2 were present (p was not significant vs wild-type control mice). No significant increase of this number was observed following challenge in TLR2–/– control mice (n = 8) with Pam3CysSK4 (74 ± 11 apoptotic cell/mm2) or with live pneumococci (92 ± 15 apoptotic cell/mm2; n = 5).


Figure 2
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 2. Studies of neurodegeneration in the murine dentate gyrus. A–D, Merged TUNEL (green) and Hoechst (chromatin, blue) staining of the dentate gyrus of wild-type mice (A and C) and TLR2–/– mice (B and D) 24 h after challenge with intrathecal Pam3CysSK4 (A and B) or live pneumococci (C and D). E, Quantitative analysis of TUNEL-positive nuclei in the dentate gyrus per area on multiple sections per animal. *, p < 0.05 determined by one-way ANOVA followed by Student-Newman-Keuls post hoc analysis.

 
Expression of TLR2 is cell type-specific

In purified primary cultures from rat cortex, real-time PCR demonstrated minor levels of TLR2 mRNA in neurons or astrocytes with a 50- to 600-fold higher presence in microglia (Fig. 3A). Accordingly, immunocytochemistry revealed the presence of TLR2 Ag on microglial cells, but not on cortical neurons (Fig. 3, B–J).


Figure 3
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 3. Cell type-specific expression of TLR2 in the CNS. A, Real-time PCR analysis of TLR2 mRNA expression (in arbitrary units) in rat neurons, astrocytes, and microglia using GAPDH (Figure 3) and beta-actin ({blacksquare}) as internal standards. Immunocytochemical localization of TLR2 (C, F, and I). Murine primary microglia from wild-type (B–D) and TLR2–/– mice (E–G) is identified by IB4 immunostaining (B and E). Murine primary neurons (H–J) are identified by microtubule-associated protein-2 immunoreactivity (H). Merged images (D, G, and J) are shown. Scale bar, 50 µm.

 
Pam3CysSK4 does not induce neuronal cell death in vitro

In unchallenged cortical rat neurons, the proportion of apoptotic cells did not increase during the experimental period. We observed 32.3 ± 8.7% apoptotic neurons at 24 h, 31.0 ± 7.4% at 48 h, and 30.7 ± 6.8% at 72 h (Fig. 4A). Accordingly, the concentration of LDH in the supernatant remained essentially unchanged with 10.1 ± 3.0 U/ml at 24 h, 9.1 ± 2.6 U/ml at 48 h, and 12.3 ± 5.0 U/ml after 72 h (Fig. 4B). Challenge with 0.1 µg/ml Pam3CysSK4 did not result in a significant increase of apoptotic neurons (35.5 ± 7.7% at 72 h; p = 0.12 vs control at 72 h; Fig. 4A) or of LDH release into the supernatant (12.0 ± 4.4 U/ml at 72 h; p = 0.86 vs control at 72 h; Fig. 4B). Conversely, exposure to 0.5 µM staurosporine resulted in apoptosis of 97.6 ± 2.1% of the neurons (p < 0.01 vs control at 72 h; Fig. 4A) and a strong increase of LDH in the supernatant (38.3 ± 10.9 U/ml; p < 0.01 vs control at 72 h; Fig. 4B). Cells exhibiting a necrotic staining pattern were not observed in any of the groups.


Figure 4
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 4. Microglia-mediated toxicity of Pam3CysSK4 in primary rat cortical neurons. A and B, Neuronal cultures were left untreated ({square}) or incubated for 24–72 h with 0.1 µg/ml Pam3CysSK4 ({blacksquare}) or with 0.5 µM staurosporine (STS) as a positive control (Figure 4). A, Proportion of apoptotic neurons after 24, 48, and 72 h. B, Concentration of LDH in the supernatant at 24, 48, and 72 h. C and D, Neuronal cultures were incubated for 48 h with supernatant that had been obtained from murine primary microglia upon stimulation with 0.1 µg/ml Pam3CysSK4 for 6–48 h. Data are the LDH concentration at 48 h in the supernatant of the neuronal cultures. Controls ({square}) were incubated with medium from unstimulated microglia collected at identical times. In these experiments, supernatant was either obtained from wild-type microglia (C) or from TLR2–/– microglia (D). *, p < 0.05; **, p < 0.01 determined by Student’s t test.

 
Next, primary mouse microglia was stimulated with 0.1 µg/ml Pam3CysSK4 (Fig. 4, C and D). Supernatant was collected at 6, 24, or 48 h and used to challenge cortical neurons as described (Fig. 4C). Supernatant from unstimulated microglia was collected at identical times to be used as a control. Supernatant from microglia after 6 h of incubation with Pam3CysSK4 did not induce an increase in LDH release from neurons compared with unstimulated microglia. LDH concentrations after incubation with "stimulated" vs "unstimulated" supernatant were 53.2 ± 17.9 U/ml vs 52.3 ± 16.2 U/ml at 24 h, p = 0.89; 63.8 ± 37.3 U/ml vs 64.1 ± 33.7 U/ml at 48 h, p = 0.98; and 80.5 ± 37.5 U/ml vs 69.1 ± 37.0 U/ml at 72 h, p = 0.46. Supernatant from microglia stimulated for 24 h with Pam3CysSK4 induced a time-dependent LDH release into the neuronal culture medium (incubation of neurons for 24 h: 64.1 ± 13.7 U/ml; 48 h: 78.4 ± 31.1 U/ml, 72 h: 89.8 ± 37.1 U/ml) that was significantly different from the effect of the supernatant collected from unstimulated microglia after 24 h (incubation of neurons for 24 h: 48.0 ± 14.1 U/ml, p < 0.01; 48 h: 53.5 ± 25.8 U/ml, p = 0.04; 72 h: 61.3 ± 30.6 U/ml, p = 0.05). Similar results were observed with the supernatant from stimulated microglia after 48 h (incubation of neurons for 24 h: 68.1 ± 19.1 U/ml vs 41.3 ± 6.4 U/ml, p < 0.01; 48 h: 85.0 ± 40.7 U/ml vs 49.5 ± 19.5 U/ml, p < 0.01; 72 h: 84.3 ± 35.8 U/ml vs 55.2 ± 23.0 U/ml, p = 0.03). Fig. 4C shows LDH release from neurons after 48 h of supernatant exposure. Pam3CysSK4 did not induce neurotoxic properties in the supernatant from TLR2–/– microglia (Fig. 4D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The major finding of our study is that the selective activation of TLR2 by a synthetic bacterial lipopeptide in vivo causes inflammatory changes typically associated with experimental bacterial meningitis. Lipopeptide-induced intracranial inflammation has all the hallmarks of bacterial meningitis, i.e., influx of leukocytes, increase of blood flow, development of intracranial hypertension as well as neuronal cell death (21, 22, 23, 24, 25, 26). In particular, the time course of the inflammatory response to the synthetic bacterial lipopeptide in the mouse experiments resembles an earlier study with purified pneumococcal cell wall components (22). In this study, challenge also resulted in a self-limiting acute influx of leukocytes into the CSF with high leukocyte concentrations at 6 h and receding numbers at 24 h. In our rat model, the maximum increases of LDF and ICP after challenge with Pam3CysSK4 were similar to previous findings in meningitis induced by purified pneumococcal cell wall components (12, 23). Although the effects of Pam3CysSK4 on leukocyte influx and ICP increase were not different from those of viable pneumococci, we observed a significantly stronger LDF increase with live D39. As the likely basis of this effect, we have previously identified release of hydrogen peroxide by S. pneumoniae as an independent vasodilator during early experimental meningitis (27). Studies of neuronal apoptosis induction in vivo by bacterial cell wall components have not previously been published. Earlier studies using viable pneumococci have argued that neuronal damage during meningitis is the combined effect of bacterial toxins and the inflammatory host response (13, 28, 29). Our present findings show that stimulation of the TLR2 pathway alone induces neuronal apoptosis in vivo.

Our data present clear evidence that TLR2 signaling is highly sufficient to trigger meningitis in vivo, including the induction of neuronal apoptosis in the dentate gyrus. The clinical relevance of this finding is underlined by the fact that pneumococcal cell wall components, a known TLR2 agonist (30, 31), are excessively released after antibiotic-induced lysis of bacteria and that patient outcome is correlated to their concentration in the CSF (32). The ubiquitous presence of lipopeptides on bacterial surfaces, including other meningitis-inducing pathogens, further broadens the clinical importance.

Conversely, the relative impact of TLR2 on inflammation and host damage in bacterial meningitis caused by a viable pathogen has not been clearly delineated. In vitro, ectopical expression of TLR2 confers responsiveness to PG and LTA of Gram-positive S. pneumoniae in Chinese hamster ovary fibroblasts (30, 33) and human embryonic kidney HEK 293 cells (8, 33, 34). In vivo, several studies report a reduced ability of TLR2–/– mice to limit the growth of Gram-positive bacteria (5, 6, 7, 35, 36). In a previous study (8), higher titers of S. pneumoniae in CSF and blood were found in TLR2–/– mice compared with wild-type mice 24 h after meningitis induction. Typical CNS complications, i.e., ICP increase and formation of brain edema, were more pronounced and clinical scores were worse in the knockout mice. However, CSF leukocyte count and tissue levels of proinflammatory cytokines were not significantly changed, and aggravation of disease in TLR2–/– mice may have resulted from pronounced cytotoxic effects of the pneumococcal metabolism. Reduced CSF pleocytosis was later observed in pneumococcal meningitis of mice lacking the adaptor protein MyD88, which is situated downstream of both TLR2 and TLR4 (37). Similarly, aggravation of the clinical course, higher bacterial titers in brain tissue, enhanced brain edema, and increased mortality were reported in TLR2–/– mice in meningitis due to S. pneumoniae or Listeria monocytogenes (7). These studies suggest that TLR2 plays an important role in the host defense during Gram-positive meningitis. However, these reports do not allow differentiation between effects of an inadequate suppression of bacterial growth and consequences of the absence of TLR2 on the inflammatory host response, nor can meningitis-associated phenomena be traced back to specific receptors. Indeed, our own findings in D39-induced meningitis do not support a more severe clinical course in TLR2-deficient mice in the presence of equivalent bacterial concentrations in the CSF. Rather, reduced CSF pleocytosis and prevention of excess neuronal damage in the knockout mice despite an identical bacterial load suggest a prominent contribution of TLR2 signaling to inflammation and host damage during meningitis.

Neuronal loss, predominantly in hippocampal structures, is a hallmark of bacterial meningitis (21, 24, 38, 39, 40). In pneumococcal meningitis, at least two distinct pathways leading to neuronal loss have been demonstrated: live pneumococci induce an early caspase-3-independent, AIF-dependent wave of apoptosis, while later, the release of bacterial cell wall components leads to a different type of cell death involving release of cytochrome c, formation of the apoptosome, and ultimately activation of caspase-3 (21, 24, 28, 29, 41). It has been argued that induction of apoptosis by bacterial cell wall components occurs secondary to the activation of immune competent host cells (28). In vitro, bacterial cell wall components induce cell death in organotypical slices and in neuronal/glial coculture, but not in primary neurons cultured alone, and NO release from activated glia has been suggested as the relevant cytotoxic principle (42). We did not observe an induction of cell death in neuronal monocultures exposed to Pam3CysSK4. These findings are conclusive and are supported by the absence of TLR2 in neurons. As demonstrated in another model, our experiments confirm that TLR2-dependent release of neurotoxins by microglia causes neuronal cell death (43). Additionally, initiation of TLR2-mediated and caspase-dependent cell death by pneumococcal cell wall has only been reported in TLR2-expressing cells such as endothelial cells or dendritic cells (41, 44). Our finding of low TLR2-mRNA expression in resting astrocytes does not argue against an additional contribution of these cells in vivo because astrocytes have been shown to up-regulate TLR2 upon activation by bacterial compounds (45).

Taken together, we have established a model of meningitis induced by a synthetic TLR2 ligand. Synthetic bacterial lipopeptides offer an opportunity to study TLR2-dependent events independently of bacterial metabolism and other bacterial cell wall components and thus enable further dissection of the complex mechanisms of intracranial immune response.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant SFB 507 B6 from the Deutsche Forschungsgemeinschaft (to O.H., J.S.B., and J.R.W.) and a Rahel Hirsch grant from the Charité-Universitätsmedizin Berlin (to S.L.). Back

2 O.H. and J.S.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Joerg R. Weber, Department of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. E-mail address: joerg.weber{at}charite.de Back

4 Abbreviations used in this paper: PG, peptidoglycan; ICP, intracranial pressure; CSF, cerebrospinal fluid; LDF, laser Doppler flow; LDH, lactate dehydrogenase. Back

Received for publication September 7, 2006. Accepted for publication February 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  2. Akira, S., S. Uematsu, O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124: 783-801. [Medline]
  3. Girardin, S. E., I. G. Boneca, J. Viala, M. Chamaillard, A. Labigne, G. Thomas, D. J. Philpott, P. J. Sansonetti. 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278: 8869-8872. [Abstract/Free Full Text]
  4. Inohara, N., G. Nuñez. 2003. NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3: 371-382. [Medline]
  5. Takeuchi, O., K. Hoshino, S. Akira. 2000. Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165: 5392-5396. [Abstract/Free Full Text]
  6. Wooten, R. M., Y. Ma, R. A. Yoder, J. P. Brown, J. H. Weis, J. F. Zachary, C. J. Kirschning, J. J. Weis. 2002. Toll-like receptor 2 is required for innate, but not acquired, host defense to Borrelia burgdorferi. J. Immunol. 168: 348-355. [Abstract/Free Full Text]
  7. Echchannaoui, H., K. Frei, C. Schnell, S. L. Leib, W. Zimmerli, R. Landmann. 2002. Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation. J. Infect. Dis. 186: 798-806. [Medline]
  8. Koedel, U., B. Angele, T. Rupprecht, H. Wagner, A. Roggenkamp, H. W. Pfister, C. J. Kirschning. 2003. Toll-like receptor 2 participates in mediation of immune response in experimental pneumococcal meningitis. J. Immunol. 170: 438-444. [Abstract/Free Full Text]
  9. Knapp, S., C. W. Wieland, C. van ’t Veer, O. Takeuchi, S. Akira, S. Florquin, T. van der Poll. 2004. Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense. J. Immunol. 172: 3132-3138. [Abstract/Free Full Text]
  10. Bessler, W. G., R. B. Johnson, K. Wiesmuller, G. Jung. 1982. B-lymphocyte mitogenicity in vitro of a synthetic lipopeptide fragment derived from bacterial lipoprotein. Hoppe Seylers Z. Physiol. Chem. 363: 767-770. [Medline]
  11. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169: 10-14. [Abstract/Free Full Text]
  12. Hoffmann, O., N. Keilwerth, M. B. Bille, U. Reuter, K. Angstwurm, R. R. Schumann, U. Dirnagl, J. R. Weber. 2002. Triptans reduce the inflammatory response in bacterial meningitis. J. Cereb. Blood Flow Metab. 22: 988-996. [Medline]
  13. Hoffmann, O., J. Zweigner, S. H. Smith, D. Freyer, C. Mahrhofer, E. Dagand, E. I. Tuomanen, J. R. Weber. 2006. Interplay of pneumococcal hydrogen peroxide and host-derived nitric oxide. Infect. Immun. 74: 5058-5066. [Abstract/Free Full Text]
  14. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11: 443-451. [Medline]
  15. Harms, C., M. Lautenschlager, A. Bergk, J. Katchanov, D. Freyer, K. Kapinya, U. Herwig, D. Megow, U. Dirnagl, J. R. Weber, H. Hörtnagl. 2001. Differential mechanisms of neuroprotection by 17beta-estradiol in apoptotic versus necrotic neurodegeneration. J. Neurosci. 21: 2600-2609. [Abstract/Free Full Text]
  16. Lautenschlager, M., M. Höltje, B. von Jagow, R. W. Veh, C. Harms, A. Bergk, U. Dirnagl, G. Ahnert-Hilger, H. Hörtnagl. 2000. Serotonin uptake and release mechanisms in developing cultures of rat embryonic raphe neurons: age- and region-specific differences. Neuroscience 99: 519-527. [Medline]
  17. Lehnardt, S., L. Massillon, P. Follett, F. E. Jensen, R. Ratan, P. A. Rosenberg, J. J. Volpe, T. Vartanian. 2003. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc. Natl. Acad. Sci. USA 100: 8514-8519. [Abstract/Free Full Text]
  18. McCarthy, K. D., J. de Vellis. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85: 890-902. [Abstract/Free Full Text]
  19. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. [Medline]
  20. Koh, J. Y., D. W. Choi. 1987. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J. Neurosci. Methods 20: 83-90. [Medline]
  21. Braun, J. S., R. Novak, P. J. Murray, C. M. Eischen, S. A. Susin, G. Kroemer, A. Halle, J. R. Weber, E. I. Tuomanen, J. L. Cleveland. 2001. Apoptosis-inducing factor mediates microglial and neuronal apoptosis caused by pneumococcus. J. Infect. Dis. 184: 1300-1309. [Medline]
  22. Tuomanen, E., H. Liu, B. Hengstler, O. Zak, A. Tomasz. 1985. The induction of meningeal inflammation by components of the pneumococcal cell wall. J. Infect. Dis. 151: 859-868. [Medline]
  23. Weber, J. R., K. Angstwurm, W. Burger, K. M. Einhaupl, U. Dirnagl. 1995. Anti ICAM-1 (CD 54) monoclonal antibody reduces inflammatory changes in experimental bacterial meningitis. J. Neuroimmunol. 63: 63-68. [Medline]
  24. Braun, J. S., R. Novak, K. H. Herzog, S. M. Bodner, J. L. Cleveland, E. I. Tuomanen. 1999. Neuroprotection by a caspase inhibitor in acute bacterial meningitis. Nat. Med. 5: 298-302. [Medline]
  25. Tauber, M. G., H. Khayam-Bashi, M. A. Sande. 1985. Effects of ampicillin and corticosteroids on brain water content, cerebrospinal fluid pressure, and cerebrospinal fluid lactate levels in experimental pneumococcal meningitis. J. Infect. Dis. 151: 528-534. [Medline]
  26. Koedel, U., A. Bernatowicz, R. Paul, K. Frei, A. Fontana, H. W. Pfister. 1995. Experimental pneumococcal meningitis: cerebrovascular alterations, brain edema, and meningeal inflammation are linked to the production of nitric oxide. Ann. Neurol. 37: 313-323. [Medline]
  27. Hoffmann, O. M., D. Becker, and J. R. Weber. 2007. Bacterial hydrogen peroxide contributes to cerebral hyperemia during early stages of experimental pneumococcal meningitis. J. Cereb. Blood Flow Metab. In press.
  28. Mitchell, L., S. H. Smith, J. S. Braun, K. H. Herzog, J. R. Weber, E. I. Tuomanen. 2004. Dual phases of apoptosis in pneumococcal meningitis. J. Infect. Dis. 190: 2039-2046. [Medline]
  29. Braun, J. S., J. E. Sublett, D. Freyer, T. J. Mitchell, J. L. Cleveland, E. I. Tuomanen, J. R. Weber. 2002. Pneumococcal pneumolysin and H2O2 mediate brain cell apoptosis during meningitis. J. Clin. Invest. 109: 19-27. [Medline]
  30. Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, D. Golenbock. 1999. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J. Immunol. 163: 1-5. [Abstract/Free Full Text]
  31. Weber, J. R., P. Moreillon, E. I. Tuomanen. 2003. Innate sensors for Gram-positive bacteria. Curr. Opin. Immunol. 15: 408-415. [Medline]
  32. Schneider, O., U. Michel, G. Zysk, O. Dubuis, R. Nau. 1999. Clinical outcome in pneumococcal meningitis correlates with CSF lipoteichoic acid concentrations. Neurology 53: 1584-1587. [Abstract/Free Full Text]
  33. Schröder, N. W., S. Morath, C. Alexander, L. Hamann, T. Hartung, U. Zähringer, U. B. Göbel, J. R. Weber, R. R. Schumann. 2003. Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J. Biol. Chem. 278: 15587-15594. [Abstract/Free Full Text]
  34. Malley, R., P. Henneke, S. C. Morse, M. J. Cieslewicz, M. Lipsitch, C. M. Thompson, E. Kurt-Jones, J. C. Paton, M. R. Wessels, D. T. Golenbock. 2003. Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection. Proc. Natl. Acad. Sci. USA 100: 1966-1971. [Abstract/Free Full Text]
  35. Mancuso, G., A. Midiri, C. Beninati, C. Biondo, R. Galbo, S. Akira, P. Henneke, D. Golenbock, G. Teti. 2004. Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease. J. Immunol. 172: 6324-6329. [Abstract/Free Full Text]
  36. Wang, G., Y. Ma, A. Buyuk, S. McClain, J. J. Weis, I. Schwartz. 2004. Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice. FEMS Microbiol. Lett. 231: 219-225. [Medline]
  37. Koedel, U., T. Rupprecht, B. Angele, J. Heesemann, H. Wagner, H.-W. Pfister, C. J. Kirschning. 2004. MyD88 is required for mounting a robust host immune response to Streptococcus pneumoniae in the CNS. Brain 127: 1437-1445. [Abstract/Free Full Text]
  38. Nau, R., A. Soto, W. Brück. 1999. Apoptosis of neurons in the dentate gyrus in humans suffering from bacterial meningitis. J. Neuropathol. Exp. Neurol. 58: 265-274. [Medline]
  39. Gerber, J., W. Brück, C. Stadelmann, S. Bunkowski, H. Lassmann, R. Nau. 2001. Expression of death-related proteins in dentate granule cells in human bacterial meningitis. Brain Pathol. 11: 422-431. [Medline]
  40. Gerber, J., G. Raivich, A. Wellmer, C. Noeske, T. Kunst, A. Werner, W. Brück, R. Nau. 2001. A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease. Acta Neuropathol. 101: 499-508. [Medline]
  41. Bermpohl, D., A. Halle, D. Freyer, E. Dagand, J. S. Braun, I. Bechmann, N. W. Schroder, J. R. Weber. 2005. Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways. J. Clin. Invest. 115: 1607-1615. [Medline]
  42. Kim, Y. S., M. G. Tauber. 1996. Neurotoxicity of glia activated by Gram-positive bacterial products depends on nitric oxide production. Infect. Immun. 64: 3148-3153. [Abstract]
  43. Lehnardt, S., P. Henneke, E. Lien, D. L. Kasper, J. J. Volpe, I. Bechmann, R. Nitsch, J. R. Weber, D. T. Golenbock, T. Vartanian. 2006. A mechanism for neurodegeneration induced by group B streptococci through activation of the TLR2/MyD88 pathway in microglia. J. Immunol. 177: 583-592. [Abstract/Free Full Text]
  44. Colino, J., C. M. Snapper. 2003. Two distinct mechanisms for induction of dendritic cell apoptosis in response to intact Streptococcus pneumoniae. J. Immunol. 171: 2354-2365. [Abstract/Free Full Text]
  45. Esen, N., F. Y. Tanga, J. A. DeLeo, T. Kielian. 2004. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J. Neurochem. 88: 746-758. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
H. J. Yoon, S.-B. Jeon, I.-H. Kim, and E. J. Park
Regulation of TLR2 Expression by Prostaglandins in Brain Glia
J. Immunol., June 15, 2008; 180(12): 8400 - 8409.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
V. B. Shah, Y. Huang, R. Keshwara, T. Ozment-Skelton, D. L. Williams, and L. Keshvara
{beta}-Glucan Activates Microglia without Inducing Cytokine Production in Dectin-1-Dependent Manner
J. Immunol., March 1, 2008; 180(5): 2777 - 2785.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. Boivin, I. Pineau, B. Barrette, M. Filali, N. Vallieres, S. Rivest, and S. Lacroix
Toll-Like Receptor Signaling Is Critical for Wallerian Degeneration and Functional Recovery after Peripheral Nerve Injury
J. Neurosci., November 14, 2007; 27(46): 12565 - 12576.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoffmann, O.
Right arrow Articles by Weber, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoffmann, O.
Right arrow Articles by Weber, J. R.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS