Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway
Tomohiko Ogawa1,
Yasuyuki Asai1,
Masahito Hashimoto1,
Osamu Takeuchi2,
Tomoko Kurita3,
Yasunobu Yoshikai4,
Kensuke Miyake5 and
Shizuo Akira2
1 Department of Oral Microbiology, Asahi University School of Dentistry, 1851 Hozumi, Hozumi-Cho, Motosu-Gun, Gifu 501-0296, Japan 2 Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan 3 Department of Microbiology, Nihon University School of Dentistry at Matsudo, Chiba 271-8587, Japan 4 Division of Host Defense, Research Center for Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan 5 Division of Infectious Genetics, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Correspondence to: T. Ogawa; E-mail: tomo527{at}dent.asahi-u.ac.jp
Transmitting editor: K. Sugamura
 |
Abstract
|
---|
Porphyromonas gingivalis lipopolysaccharide (LPS) and its bioactive center, lipid A, are known to exhibit very low endotoxic activities and activate LPS-hyporesponsive C3H/HeJ mice that have a point mutation in the cytoplasmic portion of Toll-like receptor (TLR) 4, in contrast to classical enterobacterial LPS and their lipid A. In the present study, we attempted to determine which TLR mediates the response to lipid A from P. gingivalis strain 381. P. gingivalis LPS and its natural lipid A fraction induced NF-
B activation primarily in Ba/F3 cells expressing mouse TLR 2 (Ba/mTLR2), rather than in those expressing mouse TLR4 and its accessory protein MD2 (Ba/mTLR4/mMD2). Further purification of the natural lipid A fraction resulted in a significant decrease of NF-
B activation in Ba/mTLR2, although not in Ba/mTLR4/mMD2. The synthetic counterpart of P. gingivalis strain 381-lipid A (compound PG-381) also elicited NF-
B activation in Ba/mTLR4/mMD2, but not Ba/mTLR2. Furthermore, P. gingivalis purified natural lipid A and compound PG-381 lacked the ability to activate gingival fibroblasts from C3H/HeJ, TLR4 knockout (KO) and myeloid differentiation factor 88 (MyD88) KO mice. These findings demonstrate that the P. gingivalis lipid A molecule induces cell activation via a TLR4/MD2-MyD88-dependent pathway, and suggest the possibility that unknown bacterial components in P. gingivalis LPS and its lipid A may induce cell activation via TLR2.
Keywords: lipid A, lipopolysaccharide, periodontal disease, Porphyromonas gingivalis, Toll-like receptor
 |
Introduction
|
---|
Porphyromonas gingivalis is a Gram-negative, anaerobic oral black-pigmented rod with lipopolysaccharide (LPS) located on the cell surface that is frequently isolated from the periodontal pockets of patients with chronic periodontal diseases (1). We previously demonstrated that the chemical and biological properties of P. gingivalis LPS and its active center, lipid A, have been reported to be different from those of classical enterobacterial LPS and their lipid A (28). These unique properties of LPS have been also reported in Bacteroides fragilis and Prevotella intermedia, which formerly belonged to Bacteroides species as well as P. gingivalis (911). P. gingivalis lipid A exhibited a quite different phosphorylation and acylation pattern when compared with enterobacterial lipid A (2). Further, a remarkable property of P. gingivalis lipid A is its ability to activate cells from C3H/HeJ mice (5), which is thought to be due to its unique structure. To confirm this structureactivity relationship, we recently synthesized a counterpart of P. gingivalis strain 381-type lipid A, compound PG-381, which induced IL-6 and tumor necrosis factor (TNF)-
production in peritoneal macrophages from C3H/HeN, but not C3H/HeJ mice (12). The lack of activity observed in the synthetic lipid A compound may be attributable to either of the following two possibilities: (i) some of the structural differences between the natural product and the synthetic compound are essential for the activity or (ii) the natural lipid A fraction had not been fully purified and unknown components present in the fraction were responsible for the observed activity. However, we previously found that the natural lipid A fraction contains a trace amount of amino acids (4), thus the latter possibility is more favorable.
Mammalian Toll-like receptors (TLR) comprise a large family with extracellular leucine-rich repeats and a cytoplasmic Toll/IL-1R homology domain, and are implicated in the recognition of pathogen-associated microbial products (13,14). So far, 10 members (TLR110) have been reported (1519), among which TLR4 has been shown to be a critical receptor and signal transducer for LPS, because LPS-mediated cytokine release is abrogated in LPS-hyporesponsive C3H/HeJ mice with a natural TLR4 mutation as well as in TLR4 knockout (KO) mice (20,21). To respond efficiently to LPS, TLR4 requires an accessory protein, MD2 (22). Although TLR2 has also been implicated in LPS recognition, the responses to enterobacterial LPS in TLR2 KO mice are comparable to those of wild-type mice (23). However, a recent study suggested that overexpressed TLR2 is extremely sensitive to minor contamination in commercial LPS preparations and that neither human nor murine TLR2 plays a role in LPS signaling in the absence of contaminants (24). On the other hand, TLR2 is known to be essential for the signaling of some bacterial components other than LPS, such as Staphylococcus aureus peptidoglycan (23,25), muramyl dipeptide (26), bacterial lipoprotein (2730), zymosan (31), and bacterial fimbriae and their peptides (26). Further, both TLR2 and TLR4 require an adaptor protein, myeloid differentiation factor 88 (MyD88), to initiate an intracellular signaling cascade leading to the activation of NF-
B. MyD88 KO mice do not respond to ligands for either TLR2 or TLR4 (28,29), and it was previously demonstrated that P. gingivalis LPS activates murine macrophages through TLR2 and/or TLR4 (32,33). Therefore, we attempted to determine which TLR is used by P. gingivalis lipid A for signaling.
In the present study, we purified the P. gingivalis natural lipid A fraction on the basis of immunobiological activity, and then compared the activity between natural and synthetic lipid A in order to confirm the receptor utilized by P. gingivalis lipid A for cell activation.
 |
Methods
|
---|
Bacterial compounds
The natural lipid A fraction was separated from P. gingivalis strain 381 bacterial cells according to a previous method (2). Briefly, LPS was extracted by a hot phenolwater method and hydrolyzed by acetic acid, after which the hydrophobic products were subjected to silica gel column chromatography to give the natural lipid A fraction. Further purification of the natural lipid A fraction was performed by TLC on a silica gel 60 plate (5721; Merck, Darmstadt, Germany) using solvent system A, which consisted of a combination of chloroformmethanolwater (65/24/4, v/v/v). Spots on the TLC were visualized using anisaldehyde-sulfuric acid and ninhydrin reagents for analysis, and ethanol for separation. Silica gel on the spot (Rf = 0.3) was scraped, and the materials were extracted using chloroformmethanolwatertriethylamine (50/50/15/1, v/v/v/v) and dried. The extract was then separated by TLC using solvent system B, which consisted of a chloroformmethanol25% ammonium solution (65/25/5, v/v/v). Components corresponding to the major (Rf = 0.03) and minor (Rf = 0.2) spots were then extracted, and those from the major spot were used as purified natural lipid A.
P. gingivalis strain 381-type lipid A (compound PG-381) (Fig. 1) and Escherichia coli-type lipid A (compound 506) were chemically synthesized as described previously (12). Both bacterial and synthetic compounds were dissolved at a concentration of 2 mg/ml in a 0.1% triethylamine aqueous solution and a cell wall peptidoglycan specimen of Staphylococcus aureus was prepared in our laboratory, as described previously (34).
Luciferase assay
An IL-3-dependent line, Ba/F3 (35), was transfected with p55Ig
Luc, an NF-
B-dependent luciferase reporter construct, for measuring NF-
B activity with a luciferase assay (36). Cell lines stably expressing the reporter construct (Ba/
B cells) were screened by measuring spontaneous luciferase activity. Ba/
B cells were then transfected with the pEFBOS expression vector encoding mouse TLR2. The expression of TLR2 was confirmed by intracellular staining for the flag epitope, which was attached to the C-terminus of TLR2. The established cell line expressing mTLR2 and the p55Ig
Luc reporter construct (Ba/mTLR2) was used in this study. Ba/F3 cells stably expressing mouse TLR4/MD2 and the p55Ig
Luc reporter construct (Ba/mTLR4/mMD2) were established as described previously (22). These cells were maintained in RPMI 1640 (Sigma, St Louis, MO) supplemented with IL-3, 50 µM 2-mercaptoethanol and 10% FBS (Sigma) at 37°C in a 5% (v/v) CO2 atmosphere. They were inoculated onto 96-well plates at 1 x 105/100 µl of RPMI 1640 supplemented with 10% FBS, and stimulated with the indicated doses of each test specimen such as bacterial compounds and TNF-
(Dainippon Pharmaceutical, Osaka, Japan) as a control. After 4 h at 37°C, 100 µl of the Bright-Glo luciferase assay reagent (Promega, Madison, WI) was added to each well and then luminescence was quantified with a luminometer (Turner Designs luminometer model TD-20/20; Promega).
Preparation of murine gingival fibroblasts
TLR2 KO, TLR4 KO and MyD88 KO mice were generated by gene targeting and maintained as described previously (20,23,37). Gingival tissues (25 mg) were then obtained from the mice and the explants were cultured in
-MEM (Nikken Biomedical Kyoto, Japan) supplemented with 50 µg/ml gentamicin, 50 ng/ml amphotericin B (Sigma) and 10% FBS (12). The cells were grown and maintained in
-MEM containing 10% FBS at 37°C in a 5% (v/v) CO2 atmosphere, and were used for the assay at the fifth and 13th passages.
Cytokine assay
Murine gingival fibroblasts were suspended at 2 x 105 cells/ml of
-MEM supplemented with 10% FCS and then the indicated doses of the test specimens were added to the cell cultures (2 x 104 cells/well). Cells were incubated at 37°C for 18 h, and then KC and IL-6 production was measured in the culture supernatants by means of a commercial ELISA kit system (Genzyme, Cambridge, MA). The assay was performed according to the manufacturers instructions and the results were determined using a standard curve prepared for each assay.
Statistical analysis
Data were analyzed by a one-way ANOVA, using the Bonferroni or Dunn method, and the results are presented as the mean ± SEM. When an individual result is presented, it is representative of at least three independent experiments.
 |
Results
|
---|
Purification of P. gingivalis natural lipid A
Natural lipid A was purified by TLC from the P. gingivalis natural lipid A fraction prepared previously (2). In the TLC analysis of the natural lipid A fraction using solvent system A, we observed one anisaldehyde-sulfuric acid reagent-positive spot (Rf = 0.3) and traces of ninhydrin reagent-positive spots (Fig. 2B). An extract from the spot (Rf = 0.3) containing lipid A was then subjected to a second TLC procedure. Two spots, a major spot (Rf = 0.03), visualized by anisaldehyde-sulfuric acid reagent, and a minor spot (Rf = 0.2), visualized slightly by sulfuric acid, were observed using solvent system B (Fig. 2C). The approximate ratio of the major and minor components were 20:1 (w/w). Since the major one was confirmed to be lipid A by NMR (data not shown), it was used as purified natural lipid A.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2. TLC profiles of hydrophobic products from the weak acid hydrolysate of LPS using solvent system A (chloroformmethanolwater, 65/25/4) (A), the natural lipid A fraction using solvent system A (B) and the extract from the spot (Rf = 0.3) of lane B using solvent system B (chloroformmethanol25% ammonia solution, 65/25/5) (C). The spots were visualized with an anisaldehyde-sulfuric acid reagent, and are represented by arrows and the Rf value.
|
|
NF-
B activation of Ba/F3 cells stimulated with P. gingivalis lipid A
We examined the TLR2- and TLR4-dependent activation of P. gingivalis LPS and lipid A compounds in Ba/F3 cells using a luciferase assay. Compound 506 as a TLR4 ligand (Fig. 3F) and S. aureus peptidoglycan as a TLR2 ligand (Fig. 3G) were used as control compounds. P. gingivalis LPS (Fig. 3A) and its natural lipid A fraction (Fig. 3B) induced NF-
B activation in Ba/mTLR2 cells, while NF-
B activation in Ba/mTLR4/mMD2 cells was very weak. As a result of further purification of the natural lipid A fraction, the purified natural lipid A (Fig. 3C) caused a significant decrease of NF-
B activation in Ba/mTLR2 cells, though its activity in Ba/mTLR4/mMD2 cells remained. Furthermore, the P. gingivalis synthetic lipid A, compound PG-381, clearly induced NF-
B activation in only Ba/mTLR4/mMD2 cells (Fig. 3D); however, the activity was very weak as compared to compound 506 (Fig. 3F). On the other hand, the P. gingivalis lipid A minor component(s) clearly induced NF-
B activation in Ba/mTLR2 cells (Fig. 3E). Using RT-PCR, we previously found that Ba/F3 cells constitutively express TLR2 mRNA; however, TLR3, TLR4 and TLR5 mRNA were not detected by RT-PCR or Northern hybridization (22). For this reason, Ba/
B cells were thought to respond to P. gingivalis LPS, the natural lipid A fraction, the minor component(s) and S. aureus peptidoglycan in the present assay (Fig. 3A, B, E and G). Further, stimulation with TNF-
resulted in almost similar NF-
B activation in these three Ba/F3 cells (Fig. 3H).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3. NF- B activation in Ba/F3 cells stimulated with P. gingivalis LPS (A), P. gingivalis natural lipid A fraction (B), P. gingivalis purified natural lipid A (C), compound PG-381 (D), P. gingivalis lipid A minor component(s) (E), compound 506 (F), S. aureus peptidoglycan (G) and TNF- (H). Ba/ B, Ba/mTLR2 and Ba/mTLR4/mMD2 cells were stimulated with the indicated doses of each test specimen for 4 h, and luciferase activities were measured. Results are shown as relative luciferase activity, which is a ratio of stimulated activity to non-stimulated activity, in each cell line. Each assay was done in triplicate and the data are expressed as the means ± SEM. Significant differences were seen compared to Ba/ B cells in each dose of test specimen (P < 0.01).
|
|
Cytokine production from murine gingival fibroblasts in response to lipid A specimens
We established fibroblasts from gingival tissues derived from C3H/HeN and C3H/HeJ mice, and examined the production of KC, a chemokine, and IL-6 using murine gingival fibroblasts after stimulation with P. gingivalis purified natural lipid A, and synthetic compounds PG-381 and 506. P. gingivalis purified natural lipid A (Fig. 4A) and compound PG-381 (Fig. 4B) induced a weaker KC production in C3H/HeN gingival fibroblasts as compared with compound 506 (Fig. 4C). Among these three lipid A, P. gingivalis purified natural lipid A alone resulted in no or only marginal KC production in C3H/HeJ gingival fibroblasts (Fig. 4A). S. aureus peptidoglycan exhibited KC-producing activities in both C3H/HeN and C3H/HeJ gingival fibroblasts (Fig. 4D). Moreover, IL-6 production in gingival fibroblasts from C3H/HeN and C3H/HeJ mice coincided with KC production (Fig. 4EH) (12). We next examined KC production in gingival fibroblasts from wild-type, TLR2 KO, TLR4 KO and MyD88 KO mice. P. gingivalis purified natural lipid A induced KC production in gingival fibroblasts from wild-type and TLR2 KO mice, while KC production in those from TLR4 KO mice was seen scarcely (Fig. 5A). Compounds PG-381 as well as 506 definitely exhibited KC-producing activities in wild-type and TLR2 KO gingival fibroblasts, although not in those from TLR4 KO mice (Fig. 5B and C). On the other hand, S. aureus peptidoglycan induced KC production in gingival fibroblasts from TLR4 KO but not TLR2 KO mice (Fig. 5D). However, there was no KC production by any of these specimens in MyD88 KO gingival fibroblasts (Fig. 5AD). Further, the production of IL-6 was similar to KC in the murine gingival fibroblasts stimulated with these test specimens (Fig. 5EH). These results demonstrated that P. gingivalis synthetic lipid A activates gingival fibroblasts through a TLR4- and MyD88-dependent pathway.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4. KC and IL-6 production in gingival fibroblasts from C3H/HeN and C3H/HeJ mice in response to stimulation by P. gingivalis purified natural lipid A (A and E), compound PG-381 (B and F), compound 506 (C and G) and S. aureus peptidoglycan (D and H). Cells were cultured at 37°C for 18 h in -MEM with the indicated doses of each test specimen. After incubation, the supernatants were collected, and KC (AD) and IL-6 (EH) production determined by ELISA. Experiments were performed at least 3 times and representative results are presented. Each assay was done in triplicate, and the data are expressed as the means ± SEM. Significant differences were seen between groups with and without the test specimens (P < 0.01).
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5. KC and IL-6 production in gingival fibroblasts from wild-type, TLR2 KO, TLR4 KO and MyD88 KO mice in response to stimulation by P. gingivalis purified natural lipid A (A and E), compound PG-381 (B and F), compound 506 (C and G) and S. aureus peptidoglycan (D and H). Cells were cultured at 37°C for 18 h in -MEM with the indicated doses of each test specimen. After incubation, the supernatants were collected, and KC (AD) and IL-6 (EH) production determined by ELISA. Experiments were performed at least 3 times and representative results are presented. Each assay was done in triplicate and the data are expressed as the means ± SEM. Significant differences were seen between groups with and without the test specimens (P < 0.01).
|
|
 |
Discussion
|
---|
P. gingivalis LPS and its lipid A have been reported to exhibit very low endotoxic activities, and activate LPS-hyporesponsive C3H/HeJ mice in a manner different from other LPS and their lipid A (3,5,6,38,39). It was also shown that further purification removed the contaminating proteins from P. gingivalis 381 LPS preparations, which was different from those derived from Enterobacteriaceae, while the potency to induce cytokine production in C3H/HeJ peritoneal macrophages remained (11). Recently, Hirschfeld et al. showed that a purified preparation of P. gingivalis LPS could activate murine macrophages through TLR2, but not TLR4 (32). We also demonstrated previously that TNF-
-producing activity induced by P. gingivalis LPS remained in TLR4 KO mice (33). In the present study, P. gingivalis LPS induced NF-
B activation in Ba/mTLR2 cells and the same weakly in Ba/mTLR4/mMD2 cells (Fig. 3A). Lipid A is known to be responsible for the activity of LPS and it has been demonstrated that the natural lipid A derived from P. gingivalis activates C3H/HeJ peritoneal macrophages (5,39). These activities of P. gingivalis LPS and its lipid A have been assumed to be attributable to the unusual structure of P. gingivalis lipid A (2,40). To confirm this assumption, we recently compared cell-stimulating activities between a P. gingivalis natural lipid A fraction and its synthetic counterpart, compound PG-381 (12). The natural lipid A fraction demonstrated activity in peritoneal macrophages from both C3H/HeN and C3H/HeJ mice, whereas compound PG-381 only showed activity in the C3H/HeN mice. In the present study, we also found that the natural lipid A fraction induced NF-
B activation in Ba/mTLR2 cells and the same weakly in Ba/mTLR4/mMD2 cells (Fig. 3B), whereas the synthetic compound induced NF-
B activation in only Ba/mTLR4/mMD2 cells (Fig. 3D). We previously found that an amino acid analysis of P. gingivalis natural lipid A fraction resulted in contamination, which was calculated to be 0.32% (w/w) protein (7), and that the contaminant(s) may have an association with the activation of C3H/HeJ mice. Manthey and Vogel also noted that LPS-associated proteins were necessary for activation in C3H/HeJ mice (41). Thus, the discrepancy may have occurred because the P. gingivalis natural lipid A fraction had not been fully purified and unknown components present in the fraction could have been responsible for the observed activity.
In this study, we further purified the natural lipid A fraction based on immunobiological activity. After the first TLC separation (Fig. 2B), the extract from the spot (Rf = 0.3) still induced NF-
B activation in Ba/mTLR2 cells (data not shown), indicating that several compounds may have been present at the same Rf value in this solvent system. We then separated the extract using a basic solvent system to give purified natural lipid A. In the solvent system, an acidic component moved slowly rather than a non-acidic component. Since P. gingivalis lipid A was acidic, it was able to be separated from non-acidic contaminants (Fig. 2C). The purified natural lipid A exhibited a significant decrease of NF-
B activation in Ba/mTLR2 cell, although its activity in Ba/mTLR4/mMD2 cells remained (Fig. 3C). Although a total exclusion of Ba/mTLR2 cell activation could not be attained, the ability of Ba/mTLR2 cell activation by a P. gingivalis natural lipid A fraction was shown to be separable. Furthermore, the purified natural lipid A induced cytokine production primarily in gingival fibroblasts from C3H/HeN, wild-type and TLR2 KO mice, while cytokine production in those from C3H/HeJ and TLR4 KO mice was very weak (Figs 4A and E, and 5A and E). The remaining activity in Ba/mTLR2 cells and gingival fibroblasts from C3H/HeJ and TLR4 KO mice by the purified natural lipid A may have been caused by a trace amount of impurities that could not be removed by the system of purification used. Our results proved that P. gingivalis lipid A, with an unusual structure, possesses weak but similar immunobiological activities to those of classical enterococcal lipid A. Furthermore, since the magnitude of NF-
B activation in Ba/mTLR4/mMD2 cells was unchanged through all of the purification processes employed (Fig. 3AC), the lipid A part was assumed to be responsible for all the activities related to TLR4-MD2 in P. gingivalis LPS.
The activity related to TLR2 in the natural lipid A fraction was concentrated in the minor component(s) (Fig. 3E). Thus all the TLR2 ligand components in the natural lipid A fraction may be non-acidic judging from the mobility on TLC in the basic solvent. The natural lipid A fraction contained the purified lipid A and the minor component(s) in the approximate ratio of 20:1. The TLR2 ligand activity of the minor component(s) was theoretically expected to be
20 times higher than that of the natural lipid A fraction. Although the exact value of the activity coefficient was difficult to compare, it seemed reasonable that the minor components exerted stronger TLR2 ligand activities than did the natural lipid A fraction and the maximum stimulation was seen at concentrations ranging from 10 to 100 µg/ml (Fig. 3E). In addition to the component(s), some minor spots (Fig. 2A) that were observed in the TLC profile of the hydrophobic products from the weak acid hydrolysate of P. gingivalis LPS induced NF-
B activation in Ba/mTLR2 cells (data not shown). Since their chemical structure has not yet been determined, it is possible that the trace spots were minor lipid A with a heterogeneous number of fatty acids, phosphate, and ethanolamine (40). However, the conclusion seems to be unlikely, since there is no known evidence that a structurally defined synthetic lipid A induced cell activation through TLR2. Thus it is necessary to define the chemical structure of the minor components and confirm their activity by analysis of their chemically synthetic compounds. These results suggest that cell surface molecules from P. gingivalis contain TLR2- and TLR4/MD2-binding components.
We next demonstrated that compound PG-381, similar to compound 506, elicited cytokine production in gingival fibroblasts from C3H/HeN but not C3H/HeJ mice (Fig. 4B, C, F and G). TLR4 KO gingival fibroblasts also showed no response to these compounds (Fig. 5B, C, F and G). These findings indicate that TLR4 has a role in the recognition and signaling of the lipid A portion of LPS from various Gram-negative bacteria, including P. gingivalis. Furthermore, neither KC nor IL-6 production was induced in MyD88 KO gingival fibroblasts stimulated with P. gingivalis synthetic lipid A (Fig. 5B and F), demonstrating that P. gingivalis synthetic lipid A-induced cytokine production is mediated through a TLR4- and MyD88-dependent pathway, as in the case of E. coli-type synthetic lipid A. In contrast, stimulation with S. aureus peptidoglycan resulted in cytokine production in TLR4 KO as well as C3H/HeJ gingival fibroblasts (Fig. 5D and H).
The natural lipid A of P. gingivalis, different from enterobacterial and other types of lipid A, has been shown to stimulate LPS-hyporesponsive mice. In the present study, we purified P. gingivalis natural lipid A by further separation and the synthetic compound, similar to other bacterial lipid A, revealed cell responsiveness via the TLR4MD2 complex. The present results with the P. gingivalis natural lipid A fraction further strengthens the possibility that contamination by only a trace amount of molecule(s), such as lipoprotein, lipopeptide and other unknown components, in P. gingivalis natural lipid A may induce cell activation via TLR2.
 |
Acknowledgements
|
---|
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (11671829, 13470390 and 13771291), and Frontier Science from the Ministry of Education, Science, Sports and Culture of Japan (Nihon University School of Dentistry at Matsudo). We thank Mr M. Benton for his critical reading of the manuscript.
 |
Abbreviations
|
---|
KOknockout
LPSlipopolysaccharide
MyD88myeloid differentiation factor 88
TLRToll-like receptor
TNFtumor necrosis factor
 |
References
|
---|
- Slots, J. and Listgarten, M. A. 1988. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetem comitans in human periodontal diseases. J. Clin. Periodontol. 15:85.[ISI][Medline]
- Ogawa, T. 1993. Chemical structure of lipid A from Porphyromonas (Bacteroides) gingivalis lipopolysaccharide. FEBS Lett. 332:197.[ISI][Medline]
- Ogawa, T. 1994. Immunobiological properties of chemically defined lipid A from lipopolysaccharide of Porphyromonas (Bacteroides) gingivalis. Eur. J. Biochem. 219:737.[Abstract]
- Ogawa, T., Uchida, H. and Amino, K. 1994. Immunobiological activities of chemically defined lipid A from lipopolysaccharides of Porphyromonas gingivalis. Microbiology 140:1209.[Abstract]
- Ogawa, T., Shimauchi, H., Uchida, H. and Mori, Y. 1996. Stimulation of splenocytes in C3H/HeJ mice with Porphyromonas gingivalis lipid A in comparison with enterobacterial lipid A. Immunobiology 196:399.[ISI][Medline]
- Shimauchi, H., Ogawa, T., Uchida, H., Yoshida, J., Ogoh, H., Nozaki, T. and Okada, H. 1996. Splenic B-cell activation in lipopolysaccharide-non-responsive C3H/HeJ mice by lipopoly saccharide of Porphyromonas gingivalis. Experientia 52:909.[ISI][Medline]
- Ogawa, T., Nakazawa, M. and Masui, K. 1996. Immuno pharmacological activities of the nontoxic monophosphoryl lipid A of Porphyromonas gingivalis. Vaccine 14:70.[ISI][Medline]
- Ogawa, T. and Uchida, H. 1996. Differential induction of IL-1 beta and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in human peripheral blood mononuclear cells. FEMS Immunol. Med. Microbiol. 14:1.
- Lindberg, A. A., Weintraub, A., Zahringer, U. and Rietschel, E. T. 1990. Structureactivity relationships in lipopolysaccharides of Bacteroides fragilis. Rev. Infect. Dis. 12:S133.
- Iki, K., Kawahara, K., Sawamura, S., Arakaki, R., Sakuta, T., Sugiyama, A., Tamura, H., Sueda, T., Hamada, S. and Takada, H. 1997. A novel component different from endotoxin extracted from Prevotella intermedia ATCC 25611 activates lymphoid cells from C3H/HeJ mice and gingival fibroblasts from humans. Infect. Immun. 65:4531.[Abstract]
- Kirikae, T., Nitta, T., Kirikae, F., Suda, Y., Kusumoto, S., Qureshi, N. and Nakano, M. 1999. Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS. Infect. Immun. 67:1736.[Abstract/Free Full Text]
- Ogawa, T., Asai, Y., Yamamoto, H., Taiji, Y., Jinno, T., Kodama, T., Niwata, S., Shimauchi, H. and Ochiai, K. 2000. Immunobiological activities of a chemically synthesized lipid A of Porphyromonas gingivalis. FEMS Immunol. Med, Microbiol. 28:273.
- Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1:135.[Medline]
- Akira, S., Hoshino, K. and Kaisho, T. 2000. The role of Toll-like receptors and MyD88 in innate immune responses. J. Endotoxin Res. 6:383.
- Rock, F. L., Hardiman, G., Timans, J. C., Kastelein, R. A. and Bazan, J. F. 1998. A family of human receptors structurally related to Drosophila Toll. Proc. Natl Acad. Sci. USA 95:588.[Abstract/Free Full Text]
- Takeuchi, O., Kawai, T., Sanjo, H., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Takeda, K. and Akira, S. 1999. TLR6: a novel member of an expanding toll-like receptor family. Gene 231:59.[ISI][Medline]
- Du, X., Poltrak, Y., Wei, Y. and Beutler, B. 2000. Three novel mammalian Toll-like receptors: gene structure, expression, and evolution. Eur. Cytokine Netw. 11:362.[ISI][Medline]
- Chuang, T. H. and Ulevitch, R. J. 2000. Cloning and characterization of a subfamily of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur. Cytokine Netw. 11:372.[ISI][Medline]
- Chuang, T. H. and Ulevitch, R. J. 2001. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. Biochim. Biophys. Acta 1518:157.[ISI][Medline]
- Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. and Akira, S. 1999. Toll-Like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162:3749.[Abstract/Free Full Text]
- Poltorak, A., He, X., Smirnova, I., Liu, M. -Y., Huffel, C. V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. and Beutler, B. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085.[Abstract/Free Full Text]
- Shimazu, R., Akashi, S., Ogata, H., Nagai, Y., Fukudome, K., Miyake, K. and Kimoto, M. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189:1777.[Abstract/Free Full Text]
- Takeuchi, O., Hoshino, K., Kawai, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K. and Akira, S. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443.[ISI][Medline]
- Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N. and Weis, J. J. 2000. Repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165:618.[Abstract/Free Full Text]
- Yoshimura, A., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R. and Golenbock, D. 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.[Abstract/Free Full Text]
- Asai, Y., Ohyama, Y., Gen, K. and Ogawa, T. 2001. Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2. Infect. Immun. 69:7387.[Abstract/Free Full Text]
- Aliprantis, A. O., Yang, R. B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R., Godowski, P. and Zychlinsky, A. 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285:736.[Abstract/Free Full Text]
- Kawai, T., Adachi, O., Ogawa, T., Takeda, K. and Akira, S. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115.[ISI][Medline]
- Takeuchi, O., Kaufmann, A., Grote, K., Kawai, T., Hoshino, K., Morr, M., Muhlradt, P. F. and Akira, S. 2000. Preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J. Immunol. 164:554.[Abstract/Free Full Text]
- Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J. and Modlin, R. L. 1999. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285:732.[Abstract/Free Full Text]
- Underhill, D. M., Ozinsky, A., Hajjar, A. M., Stevens, A., Wilson, C. B., Bassetti, M. and Aderem, A. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811.[ISI][Medline]
- Hirschfeld, M., Weis, J. J., Toshchakov, V., Salkowski, C. A., Cody, M. J., Ward, D. C., Qureshi, N., Michalek, S. M. and Vogel, S. N. 2001. Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477.[Abstract/Free Full Text]
- Takeuchi, O., Takeda, K., Hoshino, K., Adachi, O., Ogawa, T. and Akira, S. 2000. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int. Immunol. 12:113.[Abstract/Free Full Text]
- Ogawa, T., Kotani, S., Tsujimoto, M., Kusumoto, S., Shiba, T., Kawata, S. and Yokogawa, K. 1982. Contractile effects of bacterial cell walls, their enzymatic digests, and muramyl dipeptides on ileal strips from guinea pigs. Infect. Immun. 35:612.[ISI][Medline]
- Palacios, R. and Steinmetz, M. 1985. IL-3 dependent mouse clones that express B220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell 41:727.[ISI][Medline]
- Fujita, T., Nolan, G. P., Liou, H.-C., Scott, M. L. and Baltimore, D. 1993. The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-
B p50 homodimers. Genes Dev. 7:1354.[Abstract]
- Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K. and Akira, S. 1998. Target disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9:143.[ISI][Medline]
- Tanamoto, K. 1999. Induction of lethal shock and tolerance by Porphyromonas gingivalis lipopolysaccharide in D-galactosamine-sensitized C3H/HeJ mice. Infect. Immun. 67:3399.[Abstract/Free Full Text]
- Tanamoto, K., Azumi, S., Haishima, Y., Kumada, H. and Umemoto, T. 1997. The lipid A moiety of Porphyromonas gingivalis lipopolysaccharide specifically mediates the activation of C3H/HeJ mice. J. Immunol. 158:4430.[Abstract]
- Kumada, H., Haishima, Y., Umemoto, T. and Tanamoto, K. 1995. Structural study on the free lipid A isolated from lipopolysaccharide of Porphyromonas gingivalis. J. Bacteriol. 177:2098.[Abstract]
- Manthey, C. L. and Vogel, S. N. 1994. Elimination of trace endotoxin protein from rough chemotype LPS. J. Endotoxin Res. 1:84.