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Nod2 Is a General Sensor of Peptidoglycan through Muramyl Dipeptide (MDP) Detection*

Stephen E. GirardinDagger §, Ivo G. Boneca||, Jérôme Viala**, Mathias ChamaillardDagger Dagger §§, Agnès Labigne, Gilles ThomasDagger Dagger , Dana J. Philpott**¶¶||||, and Philippe J. SansonettiDagger ¶¶***

From the Dagger  Unité de Pathogénie Microbienne Moléculaire, INSERM U389,  Unité de Pathogénie Bactérienne des Muqueuses, ** Groupe d'Immunité Innée et Signalisation, Institut Pasteur, 28, Rue du Dr. Roux, 75724 Paris Cedex 15, France and the Dagger Dagger  INSERM U434, Fondation Jean Dausset/CEPH, Paris, France

Received for publication, November 21, 2002, and in revised form, January 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nod2 activates the NF-kappa B pathway following intracellular stimulation by bacterial products. Recently, mutations in Nod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of this disorder. We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria. Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP. Together, these results impact on the understanding of Crohn's disease development. Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities of this immunomodulatory compound.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Crohn's disease is a chronic inflammatory disorder of the gastrointestinal tract. The development of this disease is known to be influenced by both environmental factors and genetic predisposition. A significant advance in the understanding of this disease was achieved by the identification of Nod2/CARD15 as the first susceptibility gene for Crohn's disease in Caucasian populations (1, 2). Nod2 belongs to the recently described family of intracellular Nod proteins, which contain a nucleotide-binding site domain flanked by a leucine-rich repeat domain (3, 4). Among this family, proteins such as Nod1/CARD4 and Nod2 detect bacterial products to induce the activation of proinflammatory signaling pathways, such as the NF-kappa B pathway (2, 5, 6), while others like Nalp1 and Nalp3/cryopyrin have no known ligands but are also implicated in inflammation (7, 8). The critical importance of the Nods in inflammatory processes is further reinforced by the recent characterization of Nod2 and Nalp3 as susceptibility genes for five hereditary inflammatory disorders (1, 2, 8-10).

Since bacterial products have been shown to activate Nod2 (2, 6), the implication of Nod2 in Crohn's disease may support the hypothesis that enteric bacteria-host interactions could have an etiological role in this disorder. However, the precise characterization of the bacterial motifs detected by Nod2 remains to be addressed. In this study, we aimed to examine the sensitivity of Nod2 toward different peptidoglycans (PGNs),1 a cell wall product common to both Gram-positive and Gram-negative bacteria. Our results show that Nod2 senses muramyl dipeptide (MDP), which is the minimal PGN motif common to both Gram-positive and Gram-negative bacteria. These findings impact on our current understanding of the etiology of Crohn's disease. Additionally, these data will contribute to the elucidation of the mode of action of MDP, an immunomodulatory agent used for decades in adjuvant preparations.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Preparation of Highly Purified Peptidoglycans from Gram-negative and Gram-positive Bacteria-- Bacterial strains used to prepare PGN were as follows: Escherichia coli K12, Shigella flexneri 5a M90T (wild type), and Bacillus subtilis 168, Staphylococcus aureus COL (from Olivier Chesneau, Institut Pasteur). PGNs of E. coli and S. flexneri were purified as described by Glauner et al. (11). PGNs of B. subtilis and S. aureus were purified as described by de Jonge et al. (12). Briefly, bacteria were harvested in exponential growth phase at an optical density (600 nm) of 0.4-0.6 and quickly chilled in an ice-ethanol bath to minimize PGN hydrolysis by endogeneous autolysins. Pellets were resuspended in ice-cold water and added drop by drop to 8% SDS boiling. Samples were boiled for 30 min, allowing immediate inactivation of autolysins. Polymeric PGN, which is insoluble, was recovered by centrifugation and washed several times until no SDS could be detected. SDS assay was done as described by Hayashi (13). SDS treatment removes contaminating proteins, non-covalently bound lipoproteins and LPS. Gram-positive samples were physically broken with acid washed glass beads (<100 nm). The PGN fraction was recovered by differential centrifugation to remove cellular debris. All PGNs were further treated with alpha -amylase to remove any glycogen and with trypsin (3× crystallized trypsin, Worthington) digestions to remove covalently bound proteins (LPXTG proteins in Gram-positive bacteria) or lipoproteins (Gram-negative bacteria). Samples were further boiled in 1% SDS to inactivate trypsin and were washed to remove SDS. Gram-positive samples were treated with 49% hydrofluoridic acid during 48 h at 4 °C. This mild acid hydrolysis allows removal of secondary polysaccharides covalently bound to the PGN by phosphodiester bonds such as teichoic acid, capsules, poly(beta ,1-6-GlcNAc), etc. Further treatment of both Gram-positive and Gram-negative PGNs included washes with M LiCl, 0.1 M EDTA to remove any polypeptidic contamination and with acetone to remove lipoteichoic acids or any traces of LPS. Samples were lyophilized to measure PGN amounts. Purity of samples was assessed by HPLC amino acid and saccharide analysis after HCl hydrolysis (14).

Cells and Reagents-- HEK293T and HeLa epithelial cells, and RAW macrophage cells, were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Prior to transfection, HEK293T cells were seeded into 24-well plates at a density of 1 × 105 cells/ml as described previously (5). MDP LD and MDP LL were from Calbiochem and reported to be 98% pure by TLC. LPS was from E. coli O111:B4 (Sigma), commercial S. aureus PGN was from Fluka, and TNFalpha was from R & D Systems.

Expression Plasmids and Transient Transfections-- The expression plasmid for FLAG-tagged Nod1 was from Gabriel Nuñez (University of Michigan Medical School, Ann Arbor, MI) and has been described previously (15). For Nod2, a peripheral blood leukocyte cDNA library (lambda  Zap ExpressTM/EcoRI vector, Stratagene, catalog number 938202), kindly provided by S. Gisselbrecht (Paris), was screened for Nod2-expressing clones (GenBankTM/EBI accession number CAC42117). A full-length Nod2-expressing vector was verified by complete automatic sequencing and inserted into pBKCMV (Stratagene). The Nod2 3020InsC frameshift mutation was subsequently introduced using QuikChange XL site-Directed mutagenesis kit (Stratagene). The entire coding region was verified by sequencing and the size of the encoded product was verified by immunoblotting. Transfections were carried out in HEK293T cells as described previously (5).

NF-kappa B Activation Assays-- Studies examining the synergistic activation of NF-kappa B by peptidoglycans or MDPs in cells overexpressing Nod2 were carried out as described by Inohara et al. (6). Briefly, HEK293T cells were transfected overnight with 30 ng of Nod2 plus 75 ng of Igkappa luciferase reporter plasmid. At the same time, 1 µg of PGN preparations or MDPs were added, and the synergistic NF-kappa B-dependent luciferase activation was then measured following 24 h of co-incubation. NF-kappa B-dependent luciferase assays were performed in duplicate, and data represent at least three independent experiments. Data show mean ± S.E.

For immunofluorescence studies, NF-kappa B activation was assessed by nuclear translocation of NF-kappa B p65 in HeLa cells and RAW macrophages following microinjection of MDPs as described previously (16). At least 50 microinjected cells were examined per coverslip, and experiments were performed at least two times independently with similar results.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nod2 Senses Peptidoglycans from Gram-negative and Gram-positive Bacteria-- Previous studies demonstrated that Nod2 can be activated by commercial PGN from Staphyloccocus aureus and LPSs from various Gram-negative bacteria (2, 6). We aimed to investigate the sensing specificity of Nod2 toward PGNs from Gram-negative or Gram-positive bacteria. Indeed, even though Gram-negative and Gram-positive bacteria contain PGN, its organization and structure differ substantially between these two groups (17). Therefore, PGNs from E. coli, S. flexneri, B. subtilis, and S. aureus were purified according to experimental procedures specifically designed for Gram-positive or Gram-negative bacteria (11, 12). These harsh purification steps are well suited for the elimination of the possible contaminants, such as LPS (through SDS treatments), lipoproteins (through SDS and trypsin treatments), lipoteichoic acid (LTA), teichoic acid (TA), capsule (through HF treatment of Gram-positive PGN). Indeed, amino acid and saccharide analysis of the purified PGNs following HPLC revealed the absence of amino acids or amino sugars other than those related to PGN, thus excluding the possibility that contaminant polypeptidic residues remained at the end of our purification procedures (data not shown).

Equivalent amounts of purified PGNs (1 µg) were then added to cells transiently transfected with Nod2, and the subsequent activation of the NF-kappa B pathway was measured using a NF-kappa B-driven luciferase reporter gene assay. As demonstrated previously (6), transfection of low amounts (30 ng) of Nod2 itself results in a moderate action of NF-kappa B (~5-fold over vector-expressing cells). We observed that PGN preparations from Gram-negative and Gram-positive bacteria were similarly efficient in potentiating the Nod2-dependent activation of the NF-kappa B pathway (Fig. 1a), suggesting that Nod2 is a general sensor of PGNs. To completely eliminate the possibility that trace amounts of contaminants in the PGN preparations could be responsible for some of the Nod2-dependent activation of NF-kappa B, PGNs from E. coli and S. aureus were subjected to additional incubation with polymyxin B or proteinase K and boiling. These treatments did not lead to any change in the Nod2-dependent stimulation of the NF-kappa B pathway compared with the non-treated PGNs (Fig. 1b).


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Fig. 1.   Nod2 is a general sensor of PGNs through MDP detection. a, HEK293T cells were transfected with low amounts of Nod2 expression vector (30 ng) together with 1 µg of each purified PGN (E.c, E. coli; S.f, S. flexneri; B.s, B. subtilis; S.a, S. aureus), and NF-kappa B activity was measured after 4 h using an NF-kappa B-luciferase reporter assay, and fold activation over vector-expressing cells is shown. CTR, no PGN added. b, PGNs from Gram-negative and Gram-positive bacteria were subjected to polymyxin B or proteinase K/boiling treatments to exclude a role for trace amounts of LPS or lipoproteins in PGN-dependent stimulation of Nod2. NF-kappa B activity was measured as in a. Values represent the fold synergy in luciferase activity compared with activation induced by Nod2 alone. CTR, buffer minus or plus PGN. c, schematic representation of PGN repetitive structure, showing that MDP is the minimal signature of Gram-negative and Gram-positive bacterial PGNs. d, HEK293T cells were transfected with low amounts of Nod2 (30 ng), Nod1 (30 ng), or TLR2 (50 ng) expression vectors together with 1 µg of either MDP (MDP LD) or the biologically inactive MDP LL, and NF-kappa B activity was measured after 4 h using an NF-kappa B-luciferase reporter assay. CTR, no MDPs added.

Nod2 Mediates PGN Sensing through MDP Detection-- These observations prompted us to define what is the minimal PGN compound that would be common to both Gram-negative and Gram-positive bacteria and could activate Nod2. The sugar backbone of PGN is made of chains of repeating disaccharide units composed of N-acetylglucosamine (GlcNAc) beta -1,4 linked to N-acetylmuramic acid (MurNAc). Additionally, the MurNAc residues are substituted with short DL-amino acid peptides (Fig. 1c). Since GlcNAc is a sugar moiety also found in LPS, LTA, TA, and other complex polysaccharides, this sugar is unlikely to account for specific PGN sensing by Nod2. On the contrary, the MurNAc substituted with two amino acids, known as muramyl dipeptide (MDP LD or simply MDP), corresponds to the minimal PGN signature found in all bacteria. We took advantage of the availability of chemically synthesized MDP, thus avoiding the risk of contamination by other bacterial products. We observed that addition of MDP could strongly enhance Nod2-dependent activation of the NF-kappa B pathway (Fig. 1d). To test the specificity of the synergistic activation of Nod2 by MDP, we tested whether Nod1, another Nod molecule closely related to Nod2, could also sense MDP. Strikingly, Nod1-dependent activation of the NF-kappa B pathway was completely insensitive to MDP (Fig. 1d). Since TLR2 has been shown to be implicated in PGN detection (18), we investigated whether it could sense MDP. Extracellular stimulation of TLR2-transfected cells with MDP (1 µg) failed to potentiate the TLR2-dependent activation of the NF-kappa B pathway (Fig. 1d), while addition of commercial preparations of S. aureus PGN could do so (data not shown). This result suggests either that TLR2 might require other yet unknown structural PGN motifs to achieve recognition, or, alternatively, that co-receptors would be required for this recognition. Therefore, Nod2 is the only pathogen-recognition molecule identified so far that detects PGN through MDP, its minimal invariant motif.

MDP has been studied extensively in the past 2 decades because of its immunostimulatory properties (19). It is also well documented that the biological activity of this molecule is completely abolished if its second amino acid, D-Glx, is replaced by the L-Glx enantiomer (MDP LL). We therefore investigated whether Nod2 could detect the biologically inactive MDP LL to the same extent as MDP. Chemically synthesized MDP LL (1 µg) was then added to Nod2-transfected cells, and in these conditions we detected no synergistic activation of the Nod2-dependent NF-kappa B pathway by MDP LL (Fig. 1d). Even though we cannot exclude that pathogen-recognition molecules distinct from Nod2 could also detect MDP, these observations strongly suggest that Nod2 plays a key role in mediating the biological activities of MDP.

Macrophages but Not Epithelial Cells Can Detect MDP-- In contrast to Nod1, which is expressed in virtually all adult tissues (15, 20), the expression of Nod2 is predominantly in monocytes derived from peripheral blood leukocytes (21). In epithelial cells, Nod2 expression remains below detection limits in basal conditions (Ref. 22 and data not shown). Therefore, we took advantage of this restricted expression profile to compare the sensitivity of epithelial cells and macrophages toward MDP stimulation. To this end, macrophages or epithelial cells were stimulated with MDP LD or MDP LL, and the activation of the NF-kappa B pathway was assessed by immunostaining through the detection of nuclear translocation of the NF-kappa B p65 subunit in activated cells. Epithelial cells and macrophages were first stimulated by extracellular addition of MDP LD or MDP LL. In neither case could we observe any activation of the NF-kappa B pathway (Fig. 2a). As positive controls, epithelial cells and macrophages were stimulated by TNFalpha and LPS, respectively. Therefore, these results suggest that none of these cells possess pathogen-recognition molecules at their plasma membrane that could detect MDP and trigger downstream signaling pathways. As macrophages express most of the TLRs, including TLR2 and TLR4, this observation is consistent with our findings that TLR2 is unable to detect MDP. Since Nod2 is a cytoplasmic protein, we investigated whether MDP detection could occur inside microinjected cells. Epithelial cells and macrophages were then microinjected with MDP LD or MDP LL together with fluorescein isothiocyanate-dextran, to track microinjected cells. While neither epithelial cells nor macrophages were stimulated by intracellular presentation of MDP LL, 80% of MDP LD-microinjected macrophages displayed activation of the NF-kappa B pathway as observed by nuclear translocation of the NF-kappa B p65 subunit (Fig. 2b). In contrast, 100% of MDP LD-microinjected epithelial cells remained NF-kappa B-inactive (Fig. 2b). Together, these results are in agreement with our observation that Nod2, which is an intracellular pathogen-recognition molecule expressed in macrophages, detects MDP LD but not MDP LL. Moreover, these observations demonstrate that epithelial cells, which do not express Nod2, do not possess an additional pathogen-recognition molecule distinct from Nod2 that could either extracellularly or intracellularly detect MDP.


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Fig. 2.   Intracellular sensing of MDP in macrophages but not in epithelial cells. a, extracellular addition of MDP (MDP LD) or MDP LL in epithelial cells or macrophages fails to activate the NF-kappa B pathway, as observed in immunostaining by the lack of p65 NF-kappa B subunit nuclear translocation. Positive controls were TNFalpha (100 ng/ml) and LPS (1 µg/ml) stimulation for epithelial cells and macrophages, respectively. b, microinjection of MDP (MDP LD) and MDP LL into macrophages and epithelial cells. Immunostaining of the p65 subunit of NF-kappa B reveals nuclear translocation in macrophages but not in epithelial cells microinjected with MDP.

Defective PGN or MDP Sensing in Crohn's Disease-associated Nod2 Variant-- Following the discovery of Nod2 as a susceptibility gene for Crohn's disease, a critical issue is now to understand how mutations that affect Nod2 can account for development of the disease. As Nod2 is a pathogen-recognition molecule, it is therefore of interest to investigate whether Nod2 mutants found in Crohn's disease patients display an altered ability to detect bacterial products. Indeed, previous studies had suggested that Nod2 variants found in Crohn's disease displayed an altered sensitivity toward LPS detection (2). Our findings that Nod2 is a general sensor of PGNs through the detection of MDP prompted us to investigate whether PGN or MDP sensing was altered in a Crohn's disease-associated Nod2 mutant. To this end, we compared the ability of wild-type Nod2 and Nod2 3020InsC (Nod2fs), the most frequent Nod2 variant found associated with Crohn's disease patients, to detect PGNs from Gram-negative or Gram-positive bacteria (Fig. 3a). Strikingly, while Nod2fs could activate the NF-kappa B pathway as well as wild-type Nod2 when overexpressed, which is consistent with previous reports (2), the activation of the NF-kappa B pathway by Nod2fs was absolutely not potentiated by the addition of any PGN, thus showing that Nod2fs is unable to detect PGNs. This defect in PGN sensing was further illustrated by the fact that Nod2fs is also deficient in detecting MDP (Fig. 3b). Therefore, these results suggest that defects in MDP sensing influence the development of Crohn's disease in patients with Nod2 mutations.


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Fig. 3.   3020insC, the most frequent Nod2 mutation associated with Crohn's disease, abrogates Nod2-dependent sensing of PGNs and MDP. a, HEK293T cells were transfected with Nod2 (30 ng) or Nod2fs (20 ng) expression vectors together with 1 µg of each purified PGN (E.c, E. coli; S.f, S. flexneri; B.s, B. subtilis; S.a, S. aureus), and NF-kappa B activity was measured after 4 h using an NF-kappa B-luciferase reporter assay. CTR, no PGN added. b, HEK293T cells were transfected with Nod2 (30 ng) or Nod2fs (20 ng) expression vectors together with 1 µg of either MDP (MDP LD) or MDP LL, and NF-kappa B activity was measured after 4 h using an NF-kappa B-luciferase reporter assay. CTR, no MDPs added.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Innate immunity to microbial pathogens relies on the specific detection of pathogen-associated molecular patterns (PAMPs) by specific host receptors (3). A major difficulty concerning the identification of which PAMP is detected by a specific pathogen-recognition molecule resides in the fact that most of these products need to be purified from bacterial cell walls, and therefore contamination with other cell wall components can often occur and may lead to erroneous conclusions. As our aim was to investigate the sensing specificity of Nod2 toward PGNs, we adapted the most stringent purification protocols for PGN preparations to obtain the highest purity. Using these purified PGN preparations, we were able to show that Nod2 is a general sensor of both Gram-negative and Gram-positive PGN. Previous studies showed that Nod2 displayed varying sensitivities toward different commercial LPSs (2, 6), which, in light of our data, suggests that these preparations could be contaminated more or less with PGN, and this possibility remains to be addressed.

Over the last decades, muramyl peptides and other synthetic derivatives of this molecule have been reported to be potent immunoadjuvants that enhance protective immunity against pathogens and tumors by stimulating immune-competent cells, such as monocytes and macrophages (19, 23). Despite a long standing investigation in the field of muramyl peptides, little is known about their interaction with target cells and their mode of action. Indeed, the nature of the MDP-sensing molecule(s) has been a subject of controversy (23-25). Interestingly, it has been also reported that MDP binding sites are located within the intracellular compartment (26). Our results have defined Nod2 as an intracellular sensor of MDP, which is in agreement with many of the known biological properties of MDP. First, Nod2 expression is restricted to monocytes/macrophages in basal conditions, which is the major cell population stimulated by MDP. Second, the fact that the MDP sensor is actually intracellular correlates with the characterization of cytoplasmic MDP binding sites (26) and would explain why extracellular presentation of MDP in the absence of lipidic emulsifying compounds such as liposomes or paraffin oil (the base of Freund's preparation) is poorly immunostimulatory (27). Indeed, it can now be hypothesized that lipidic moieties might be required to allow MDP to cross the hydrophobic plasma membrane.

The homeostasis of the intestine relies on a finely tuned surveillance system mediated by mononuclear cells, including monocytes/macrophages and dendritic cells that are permanent residents of the intestinal mucosa. The constant presence of luminal antigens maintains the tissue in a state of physiological inflammation through a tightly controlled equilibrium that balances pro- versus anti-inflammatory signals. Indeed, the discovery that Nod2-dependent activation of the NF-kappa B pro-inflammatory cascade is modulated by PGN sensing in these cells highlights the essential role of Nod2 in the maintenance of mucosal homeostasis. In Crohn's disease patients carrying mutations in Nod2, the manifestation of pathology is then likely to be related to a defect in mononuclear-driven homeostasis at the level of the intestinal mucosa. Our discovery of MDP as the bacterial ligand that initiates Nod2-dependent signaling and the insensitivity of a Crohn's disease-associated Nod2 mutation to this bacterial product underscores the critical role of bacterial sensing by Nod2 in intestinal mucosal homeostasis. Therefore, it will be of interest to examine whether some of the Nod2-independent Crohn's disease susceptibility alleles are associated with defects in pathways that are involved in the processing of PGN, such as lysozyme in the endosomal compartment.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Muguette Jéhanno as well as Martine Parmier who helped with plasmid preparations. We also acknowledge Didier Blanot for the amino acid and saccharide analysis of the PGN samples. We thank those individuals that donated bacterial strains and plasmids. We also thank the "TLR Group" at the Institut Pasteur and Bill Goldman for helpful discussion.

    FOOTNOTES

* This work was supported in part by a grant from the Institut Pasteur, "Program Transversal de Recherche" (to S. E. G. and D. J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a grant from Danone Vitapole, Paris, France.

|| Supported by a postdoctoral fellowship from the Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/1567/2000).

§§ Supported by a grant from the Ministère Français de l'Education Nationale.

¶¶ These authors share senior authorship.

|||| To whom correspondence should be addressed. Tel.: 33-1-45-68-89-93; Fax: 33-1-40-61-39-02; E-mail: philpott@pasteur.fr.

*** Howard Hughes International Research Scholar.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.C200651200

    ABBREVIATIONS

The abbreviations used are: PGN, peptidoglycan; MDP, muramyl dipeptide; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; TNF, tumor necrosis factor; LTA, lipoteichoic acid; TA, teichoic acid; PAMP, pathogen-associated molecular pattern.

    REFERENCES
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ABSTRACT
INTRODUCTION
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REFERENCES

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