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1 Department of Oromaxillofacial Infection and Immunity, School of Dentistry, Seoul National University, Seoul, Republic of Korea
2 Department of Periodontology, School of Dentistry, Seoul National University, Seoul, Republic of Korea
3 Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea
4 Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
Correspondence
Bong-Kyu Choi
bongchoi{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1
. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4–MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coli LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Moter et al., 1998; Wyss et al., 1999, 2004).
Treponema socranskii is one of the most frequently found oral spirochaetes in endodontic infections as well as in chronic and aggressive periodontitis (Baumgartner et al., 2003; Ellen & Galimanas, 2005; Moter et al., 1998). Its presence is correlated with clinical parameters such as pocket depth and attachment loss, and thus it is associated with the severity of periodontal tissue destruction (Socransky et al., 1991; Takeuchi et al., 2001). Riviere et al. (2002) detected several species of oral spirochaetes including T. socranskii in the brain cortex of patients with Alzheimer's disease by PCR and immunohistochemical analyses. A protease showing chymotrypsin-like activity was identified in this bacterium (Correia et al., 2003; Heuner et al., 2001). More recently we observed that T. socranskii induced osteoclastogenesis by upregulating prostaglandin E2, which stimulated the receptor activator of nuclear factor-
B ligand (Choi et al., 2005).
LPS localized in the outer membrane of Gram-negative bacteria is a representative pathogen-associated molecular pattern that exerts biologically diverse activities and is one of the most powerful stimulators of innate immune and inflammatory responses (Miller et al., 2005). Biological activities of LPS or LPS-like materials from the outer membrane of oral spirochaetes, also named lipooligosaccharides, glycolipids or glycoconjugates, have been reported in different species. The Treponema denticola lipooligosaccharide induced TNF- and nitric oxide in macrophages prepared from the peritoneal cavity of endotoxin-responsive and unresponsive mice (Rosen et al., 1999). It also induced osteoclastogenesis by upregulating the osteoclast differentiation factor in a co-culture system using mouse calvaria and bone marrow cells, and matrix metalloproteinase-9 expression in mouse calvaria-derived osteoblastic cells (Choi et al., 2003). Glycolipids from Treponema maltophilum induced IL-6 and TNF- in human monocytes and a murine macrophage cell line, and NF-B translocation in Chinese hamster ovary cells transfected with human CD14 (Opitz et al., 2001). LPS from Treponema pectinovorum induced IL-6, IL-8 and monocyte chemoattractant protein 1 in human gingival fibroblasts (HGFs) (Kesavalu et al., 2002).
LPS signalling is initiated by the combined actions of LPS-binding protein (LBP), the membrane-bound (mCD14) or soluble forms (sCD14) of CD14 and the Toll-like receptor 4 (TLR4)–MD-2 complex (Akashi et al., 2003; Mancek et al., 2002; Miyake 2003; Re & Strominger 2003; Palsson-McDermott & O'Neill, 2004). LBP, a serum protein, enhances the binding of LPS to CD14, and CD14 facilitates the recognition of LPS by the TLR4–MD-2 complex. MD-2, a glycoprotein, is an adaptor molecule that is associated with the extracellular leucine-rich repeats of TLR4 (Mancek et al., 2002; Miyake, 2003). LPS, presented by CD14, binds to TLR4–MD-2, and the LPS interaction with TLR4–MD-2 induces the TLR4 oligomerization on which TLR4 signalling is dependent (Saitoh et al., 2004). The intracellular signalling of TLR4 is homologous to the signalling systems of IL-1, as they possess a common cytoplasmic Toll-interleukin-1 receptor (TIR) domain (Martin & Wesche, 2002). Although TLR4 is known to be a predominant receptor mediating LPS activation, recent studies have suggested a role for TLR2 as a receptor for some LPS (Darveau et al., 2004; Hirschfeld et al., 2001; Opitz et al., 2001; Werts et al., 2001). Furthermore, the LPS of some other bacteria show antagonism against the activity induced by typical LPS of Gram-negative bacteria (Coats et al., 2003; Jarvis et al., 1997; Lepper et al., 2005; Yoshimura et al., 2002). The agonistic and antagonistic activities of LPS are known to be correlated with the chemical structure of lipid A in the LPS molecule (Brandenburg et al., 2003). Glycolipids of small-sized oral spirochaetes like T. denticola and T. maltophilum have been suggested to be structurally similar to lipoteichoic acid (LTA; Schröder et al., 2000; Schultz et al., 1998). In T. maltophilum, LTA-like glycolipids are the major membrane components responsible for various biological effects, and these glycolipids use TLR2 rather than TLR4 (Opitz et al., 2001). Recently, the glycolipids of the medium-sized oral spirochaete T. medium showed antagonized cell activation induced by Escherichia coli LPS as well as by peptidoglycan (Asai et al., 2003a). Their structure is also similar to LTA (Hashimoto et al., 2003).
In our preliminary study comparing the effect of glycolipids of several oral spirochaetes on the host innate immune response, a phenol/water extract of T. socranskii subsp. socranskii (TSS-P) revealed antagonism against E. coli LPS activity. In this study we examined TSS-P antagonism in more detail, including the antagonistic mechanism, mainly using the human monocytic THP-1 cell line but also using primary cultured HGFs in some experiments.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) or NOS broth (Chan et al., 1993) for 3 to 5 days. Actinobacillus actinomycetemcomitans ATCC 33384 and Escherichia coli DH5 were also included for LPS isolation. A. actinomycetemcomitans was grown anaerobically in brain heart infusion broth for 2 days. E. coli was grown aerobically in Luria–Bertani broth.
Bacterial phenol/water extraction.
The bacterial cells (50 ml cultures for Treponema species and 5 ml cultures for A. actinomycetemcomitans and E. coli) were harvested by centrifugation at 5000 g for 10 min at 4 °C. The cells were then washed with phosphate-buffered saline (PBS). Bacterial phenol/water extracts were prepared by using an LPS extraction kit (iNtRON Biotechnology) according to the manufacturer's protocol with a slight modification. Briefly, cells were mixed with 1 ml lysis buffer containing phenol from the kit and vortexed vigorously until the cell clump disappeared. After adding 200 µl chloroform, the mixture was vortexed vigously for 30 s and incubated at room temperature for 5 min. The mixture was centrifuged at 13 200 g at 4 °C for 10 min and the upper phase (400 µl) was transferred to a new tube. This crude preparation was incubated with endonucleases (100 µg DNase I ml–1 and 100 µg RNase A ml–1, 1 h at 37 °C) and subsequently proteinase K (250 µg ml–1, 1 h at 50 °C). After adding 800 µl lysis buffer, the same procedure as described above was repeated. The second preparation was mixed well with the purification buffer (800 µl) from the kit, incubated at –20 °C for 10 min, and centrifuged at 13 200 g at 4 °C for 15 min. The pellets were washed with 1 ml 70 % ethanol, air-dried, and dissolved in endotoxin-free distilled H2O. The phenol/water extracts were quantified by lyophilization and dry weight measurement. They were dissolved in endotoxin-free distilled H2O at the concentration of 1 mg ml–1; dissolution was ensured by sonication. To visualize and compare the patterns, the phenol/water extracts (10 µg each) were subjected to SDS-PAGE (15 % polyacrylamide gel) and silver stained. The phenol/water extracts of E. coli and A. actinomycetemcomitans showed typical ladder-like LPS patterns, whereas those of T. socranskii and T. lecithinolyticum did not. In this study, we called the phenol/water extracts of E. coli and A. actinomycetemcomitans LPS, and those of T. socranskii and T. lecithinolyticum phenol/water extracts TSS-P (for the phenol/water extract of T. socranskii) and TL-P (for the phenol/water extract of T. lecithinolyticum).
E. coli O127 : B8 LPS was purchased from Sigma; possible contaminants were removed by treatment with endonucleases and proteinase K and subsequent phenol/water extraction with the LPS extraction kit, as described above. This repurified E. coli LPS was used throughout this study.
Endotoxin assay.
Endotoxin activity of the LPS or phenol/water extracts was determined by the Limulus amoebocyte lysate (LAL) assay using an LAL Endochrome kit (Charles River Endosafe) according to the manufacturer's protocol. In principle, endotoxin activated a proenzyme in LAL. In the presence of a colourless substrate, the activated enzyme rapidly catalysed the cleavage of the chromophore p-nitroaniline, which produced a yellow colour and was measured at 405 nm. A standard curve was run with E. coli control standard endotoxin from the kit.
Culture and treatment of cells.
The human monocytic THP-1 cell line was purchased from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented with L-glutamine, 10 % heat-inactivated fetal bovine serum, and antibiotics (100 U penicillin ml–1 and 100 µg streptomycin sulfate ml–1). The cells were cultured overnight, plated in 35 mm-diameter cell culture dishes at a concentration of 1x106 cells ml–1, and treated with LPS (0·01–10 µg ml–1), phenol/water extracts (TSS-P or TL-P, 0·01–50 µg ml–1), peptidoglycan from Staphylococcus aureus (0·1–10 µg ml–1, Sigma) or human recombinant IL-1 (300 pg ml–1; R&D Systems) in the presence or absence of 2 % heat-inactivated human serum (HS, Sigma) for 12–24 h. In some experiments, THP-1 cells were treated with TSS-P or TL-P in the presence of LPS, peptidoglycan or IL-1 at the indicated concentrations. In another set of experiments, the 2 % HS in the cell culture medium was replaced by recombinant human LBP (rhLBP, 50 ng ml–1, R&D Systems) and recombinant human CD14 (rhCD14, 200 ng ml–1, R&D Systems). Cells were harvested for flow cytometry analysis and RNA isolation for RT-PCR. The culture supernatants were collected and stored at –70 °C for cytokine measurement by ELISA. To examine the tolerance induction by TSS-P, THP-1 cells (1x106 cells ml–1) cultured in 2 % HS were pretreated with TSS-P (10 µg ml–1) for 12 h. The cells were then washed three times with RPMI medium and treated with E. coli LPS (10 ng ml–1) for 12 h. ICAM-1 expression was measured by flow cytometry.
To ensure that the cell-stimulating activity of the E. coli LPS used in this study was not caused by contaminants, THP-1 cells were treated with E. coli LPS (10 ng ml–1) in the presence of polymyxin B (50 µg ml–1) and assessed for ICAM-1 expression. Polymyxin B completely inhibited ICAM-1 expression induced by the E. coli LPS.
To confirm the LPS signalling via TLR4, THP-1 cells were preincubated with mouse anti-human TLR4 mAb HTA125 (5 µg ml–1), a TLR4-blocking antibody, or an isotype-matched antibody for 30 min and were then stimulated with E. coli LPS (10 ng ml–1) or A. actinomycetemcomitans LPS (10 ng ml–1) for 12 h. IL-8 levels in the culture supernatants were determined by ELISA.
In order to determine the effect of TSS-P on primary cultured cells, HGFs were obtained from explants of a healthy donor, cultured in alpha-minimal essential medium (-MEM) as described previously (Choi et al., 2001), and treated with TSS-P (10 µg ml–1) or TL-P (10 µg ml–1) in the presence of E. coli LPS (10 ng ml–1).
Flow cytometry analysis.
The expression of mCD14, TLR2, TLR4 and ICAM-1 by THP-1 cells, unstimulated or stimulated as described above, was analysed by flow cytometry. THP-1 cells were collected by centrifuging and washed with PBS. The cells (1x106 cells in 100 µl) were reacted with mouse anti-human CD14 mAb, mouse anti-human TLR2 mAb, mouse anti-human TLR4 mAb or mouse anti-human ICAM-1 mAb at 4 °C for 20 min. After washing, the cells were stained with FITC-labelled goat anti-mouse IgG at 4 °C for 20 min. The cells were washed with PBS, and expression of the proteins was analysed using a fluorescence-activated cell sorter (FACS). The data were obtained by counting 15 000 cells. As a control for nonspecific binding, the cells were stained with isotype-matched IgG; no nonspecific reactivity was observed.
RT-PCR.
MD-2 expression in the THP-1 cells was analysed by RT-PCR. RNA was isolated from the THP-1 cells using TRIzol reagent (Invitrogen Life Technology) according to the manufacturer's protocol. RT-PCR was performed as described previously (Lee et al., 2005) using the following primers: 5'-AGG GGC ACG AGG TAA ATC TT-3' for the sense primer and 5'-GGC TCC CAG AAA TAG CTT CA-3' for the anti-sense primer (annealing temperature 52 °C). PCR without reverse transcription was performed as a negative control. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for gene expression.
ELISA for cytokines (TNF- and IL-8).
The culture supernatants of the cells treated with LPS and other stimuli were assayed to determine TNF- and IL-8 levels using ELISA kits from R&D Systems.
Inhibition assay for LBP and CD14 binding.
In order to analyse the antagonistic mechanism, we evaluated whether the binding of biotinylated E. coli LPS to CD14 or LBP was inhibited by TSS-P. Biotinylation was performed using EZ-Link Biotin-LC-Hydrazide (Pierce Biotechnology). E. coli LPS (500 µl of 2 mg ml–1) or TSS-P (500 µl of 2 mg ml–1) was incubated with 20 mM cold sodium metaperiodate at 4 °C for 30 min and then dialysed overnight in coupling buffer (0·1 M sodium acetate, pH 5·5). The mixture was transferred to a new tube and biotin hydrazide solution was added to a final concentration of 5 mM. After agitation at room temperature for 2 h, the mixture was dialysed three times in distilled H2O for 24 h.
For LBP binding, anti-human LBP mAb (50 ng per well in 50 µl) was coated on EIA plates (Corning) at 4 °C overnight. The plates were washed with PBS and blocked with 2 % BSA in PBS at room temperature for 1 h. The plates were washed five times with PBS/0·1 % Tween 20 (PBST), then rhLBP (62·5 ng per well in 50 µl) was added and incubated at room temperature for 1 h. After washing the plates five times with PBST, the biotinylated LPS, alone or with nonbiotinylated E. coli LPS, TSS-P or TL-P, was added at the indicated dose and incubated at room temperature for 1 h. The plates were washed five times with PBST and biotinylated LPS captured by rhLBP was reacted with 50 µl horseradish peroxidase (HRP)-labelled streptavidin (1 µg ml–1) in PBST containing 2 % BSA for 1 h. The plates were washed five times with PBST, and then 50 µl 3,3',5,5'-tetramethylbenzidine (TMB) solution was added as a substrate for HRP. The enzyme reaction was stopped after 20 min by adding 50 µl 0·5 M sulfuric acid, and the absorbance at 450 nm was measured using an ELISA reader. For sCD14 binding, anti-human CD14 mAb (125 ng per well in 50 µl) was coated on EIA plates at 4 °C overnight. The plates were washed with PBS, blocked with 2 % BSA in PBS at room temperature for 1 h, and washed five times with PBST. In parallel, 500 ng of the biotinylated E. coli LPS and rhCD14 (100 ng) were incubated with or without the indicated dose of nonbiotinylated E. coli LPS, TSS-P or TL-P at room temperature for 1 h in a total volume of 50 µl. This mixture was then added to each well of the anti-human CD14 mAb-coated plates and incubated at room temperature for 1 h. The plates were washed five times with PBST and binding of biotinylated E. coli LPS to CD14 captured by anti-CD14 mAb was detected as described above. To perform a binding assay to immobilized CD14, EIA plates were directly coated with rhCD14 (100 ng per well in 50 µl) at 4 °C overnight, and subsequent procedures were carried out as described above. Biotinylated E. coli LPS was used at a concentration of 500 ng per well in 50 µl, which was saturated by rhLBP (62·5 ng per well in 50 µl) or rhCD14 (100 ng per well in 50 µl).
LPS binding assay to MD-2 and THP-1 cells.
Since MD-2 physically associated with TLR4 on cells is known to interact with LPS, we analysed whether E. coli LPS and TSS-P bound to MD-2 and THP-1 cells. The E. coli LPS or TSS-P was biotinylated as described above. EIA plates were coated with recombinant human MD-2 (rhMD-2, 50 ng per well in 50 µl, R&D Systems) at 4 °C overnight. The plates were washed with PBS and blocked with 2 % BSA in PBS at room temperature for 1 h. After washing five times with PBST, the plates were incubated with the biotinylated E. coli LPS (0·01–1 µg per well) or TSS-P (0·01–1 µg per well) in the presence of rhLBP (62·5 ng per well in 50 µl) and rhCD14 (100 ng per well in 50 µl). The plates were washed five times with PBST; the bound LPS or TSS-P was detected with HRP-labelled streptavidin as described above. For the binding inhibition assay, biotinylated E. coli LPS (500 ng per well) was incubated with nonbiotinylated E. coli LPS, TSS-P or TL-P at the indicated doses in the presence or absence of rhLBP and rhCD14.
For the cell binding assay by E. coli LPS and TSS-P, THP-1 cells (1x106 cells ml–1) cultured in RPMI 1640 medium with or without 2 % HS were incubated with the biotinylated E. coli LPS or TSS-P at various concentrations at 37 °C for 1 h in a CO2 incubator. The cells were collected by centrifugation at 360 g for 3 min, washed three times with PBS, resuspended in 100 µl DPBS, and incubated with FITC-labelled streptavidin (1 µg) for 30 min. After three washes, the cells were analysed by FACS. For the binding inhibition assay, biotinylated E. coli LPS (0·01–1 µg ml–1) was incubated with nonbiotinylated E. coli LPS (10 µg ml–1) or TSS-P (10 µg ml–1).
Statistical analysis.
The statistical significance of the differences between nontreated and LPS- or phenol/water extract-treated cells was evaluated by a one-way analysis of variance (ANOVA). The statistical significance of the differences between agonist (LPS)-treated and inhibitor-treated cells was evaluated using Student's t-test. A P-value of less than 0·05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Expression of mCD14, TLR2, TLR4 and MD-2 on THP-1 cells
In this study, we mainly used THP-1 cells to investigate cell response to LPS. In order to ensure that the molecules necessary for LPS response in the cell were expressed, the expression of mCD14, TLR2, TLR4, and MD-2 was analysed by flow cytometry or RT-PCR. As shown in Fig. 1, THP-1 cells expressed all of these molecules.
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and IL-8 production from THP-1 cells and HGFs after stimulation with these molecules. As shown in Fig. 2, TSS-P (10 µg ml–1) and TL-P (10 µg ml–1) did not induce TNF- or IL-8 production in THP-1 cells, whereas the LPS from E. coli (10 ng ml–1) and A. actinomycetemcomitans (10 ng ml–1) significantly induced the production of these cytokines. The induction of IL-8 was not observed in HGFs stimulated with TSS-P or TL-P either (data not shown).
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a). Second, in serum-free medium, the THP-1 cell response to E. coli LPS was drastically reduced (Fig. 3b). ICAM-1 expression was not induced in the cells with 10 ng ml–1 of E. coli LPS, was slightly induced with 100 ng ml–1, and was significantly induced with 1 µg ml–1, although the response was less than that observed with the same concentration in the presence of 2 % HS. A high concentration of E. coli LPS (10 µg ml–1) showed the same cell-stimulating activity regardless of the presence of serum. Significant inhibition by TSS-P was not observed under the serum-free condition. Third, in the presence of rhLBP and rhCD14 instead of 2 % HS in the cell culture medium, E. coli LPS showed the same stimulatory effect as in the presence of serum and the inhibitory effect by TSS-P was also the same (data not shown). Fig. 3(c) shows the inhibitory effect of TSS-P on the ICAM-1 expression induced by 10 ng ml–1 of E. coli LPS under three conditions. TL-P showed neither an agonistic nor an antagonistic effect. The addition of either rhLBP or rhCD14 alone instead of human serum showed only limited stimulation by E. coli LPS (data not shown).
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As E. coli LPS is known to transduce signals via TLR4, we tested whether TSS-P inhibits the ICAM-1 induced by another known TLR4 ligand, A. actinomycetemcomitans LPS. This molecule stimulated ICAM-1 in the same concentration-dependent manner as found with the E. coli LPS, and TSS-P (10 µg ml–1) completely inhibited the ICAM-1 expression induced by 10 ng ml–1 of A. actinomycetemcomitans LPS (Fig. 3d
). The inhibitory effect of 10 µg ml–1 of TSS-P was not observed at the highest concentration (10 µg ml–1) of either E. coli or A. actinomycetemcomitans LPS.
Endotoxin tolerance is the phenomenon that prior exposure of the cells to LPS induces a transient state of reduced ability to respond to second LPS exposure. In experiments to test whether TSS-P made the cells tolerant to LPS by treating THP-1 cells with TSS-P and subsequently with E. coli LPS, we observed that TSS-P pretreatment did not affect the ICAM-1 expression induced by E. coli LPS, whereas TSS-P significantly inhibited E. coli LPS-induced ICAM-1 expression in the cells treated with TSS-P and E. coli LPS simultaneously (data not shown).
Effects of anti-TLR4 mAb and TSS-P on IL-8 production
To confirm the functional role of TLR4 in IL-8 secretion induced by LPS, THP-1 cells were pre-incubated with a TLR4-blocking antibody, HTA125. This antibody significantly blocked the LPS-induced production of IL-8, while an isotype-matched control antibody did not (Fig. 4a). In addition to the inhibitory effects on ICAM-1 expression, TSS-P significantly inhibited IL-8 production in THP-1 cells and HGFs stimulated with E. coli LPS, whereas TL-P did not affect the activity (Fig. 4b).
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a) or absence (data not shown) of serum. This stimulation was not affected by TSS-P (10 µg ml–1). In order to differentiate the target for antagonism by TSS-P, we analysed its effect on IL-1-induced ICAM-1 expression, as the IL-1 receptor (IL-1R) and TLR4 have a common intracellular TIR domain. As shown in Fig. 5(b), TSS-P (10 µg ml–1) did not affect the ICAM-1 expression stimulated by IL-1 (300 pg ml–1). This result indicates that TSS-P antagonism is specific to the TLR4 pathway.
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. No significant TSS-P antagonism was observed under the serum-free condition. These results indicated that the antagonistic target may be LBP and/or CD14, which are components of serum. To address this issue, we performed an experiment to determine whether TSS-P was in competition with the E. coli LPS for binding to LBP and CD14. Biotinylated E. coli or TSS-P bound to LBP and to both forms of CD14 (immobilized and soluble) in a concentration-dependent manner (data not shown). Since the binding saturation to rhLBP and rhCD14 was reached at 500 ng per well of biotinylated E. coli LPS, this concentration was used for the inhibition study. As shown in Fig. 6, TSS-P inhibited the binding of biotinylated E. coli LPS to both forms of CD14 as well as to LBP, in a dose-dependent manner. TL-P, which showed no antagonistic effect, did not inhibit the E. coli LPS binding to LBP and CD14. Nonbiotinylated E. coli LPS also inhibited the binding of biotinylated LPS to LBP and CD14. This inhibition was more efficient than that by TSS-P.
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a and 8a). In contrast, TSS-P bound neither to MD-2 nor to the THP-1 cells, regardless of the presence of serum (data not shown). However, TSS-P inhibited the binding of the E. coli LPS to MD-2 and THP-1 cells in the presence of rhLBP/rhCD14 or serum (Figs 7b and 8a). Nevertheless, under the serum-free condition, TSS-P did not affect the binding of LPS to MD-2 or THP-1 cells (Figs 7c and 8b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and many studies have reported its association with periodontitis and endodontic infections (Baumgartner et al., 2003; Ellen & Galimanas, 2005; Moter et al., 1998; Socransky et al., 1991; Takeuchi et al., 2001). However, its virulence has not been throughly investigated. Here, we studied the biological activity of a phenol/water extract of T. socranskii, by analysing its effect on the expression of proinflammatory cytokines and ICAM-1 in THP-1 cells and HGFs. The molecules of E. coli and A. actinomycetemcomitans prepared by phenol/water extraction revealed a typical LPS pattern on silver-stained SDS-PAGE gels, and the endotoxicity of both commercial and the extracted E. coli LPS was nearly identical. From these results, we concluded that the phenol/water extracts of oral spirochaetes are also LPS-like molecules. However, the chemical composition of these molecules remains to be characterized. The LPS-like materials in oral spirochaetes prepared by phenol/water extraction are termed glycolipids (Schröder et al., 2000; Schultz et al., 1998), lipooligosaccharides (Choi et al., 2003; Rosen et al., 1999) and glycoconjugates (Asai et al., 2003a) in various reports.
Although the cell-stimulating activities of glycolipids in a few oral spirochaetes have been reported (Choi et al., 2003, Kesavalu et al., 2002; Opitz et al., 2001; Rosen et al., 1999), TSS-P showed minimal endotoxicity (about 10 000-fold less endotoxicity than E. coli LPS) and no cell-stimulating activity. Rather, it exhibited antagonistic activity against the TLR4-mediated signalling of LPS from E. coli and A. actinomycetemcomitans. The ICAM-1 expression stimulated by the LPS of these bacteria was significantly inhibited by a 10- to 1000-fold mass excess of TSS-P in the presence of serum. In contrast, TSS-P did not affect the ICAM-1 expression induced by peptidoglycan or IL-1 in THP-1 cells. TSS-P pretreatment did not affect the cellular response to subsequent E. coli LPS treatment. This suggests that TSS-P-dependent antagonism of E. coli LPS in THP-1 cells did not occur via an indirect mechanism like endotoxin tolerance.
An antagonistic effect of LPS has been reported in other bacteria such as Rhodobacter sphaeroides (Jarvis et al., 1997), Rhodobacter capsulatus (Loppnow et al., 1990), Helicobacter pylori (Lepper et al., 2005) and some periodontal pathogens. Among periodontal pathogens, Porphyromonas gingivalis LPS antagonized the response to LPS of TLR4 ligands in human endothelial cells (Coats et al., 2003) and HGFs (Yoshimura et al., 2002). Capnocytophaga ochracea LPS showed the same antagonistic effect in HGFs as did the P. gingivalis LPS (Yoshimura et al., 2002). T. medium glycolipid has been reported to show antagonism against a TLR4 ligand, and the antagonistic mechanism has been suggested to be competitive binding to LBP and CD14 (Asai et al., 2003a). In our study, TSS-P exhibited similar antagonistic effects, showing competitive binding to LBP and CD14. Since TSS-P did not inhibit the intracellular signalling of IL-1, and TLR4 and IL-1R have a common intracellular TIR domain, the inhibition targets of TSS-P should be upstream of the convergence point of TLR4 and IL-1 signalling. LBP monomerizes LPS molecules to prevent them from forming aggregates and transfers them to CD14, which in turn presents LPS to TLR4–MD-2. In the presence of LBP, the presentation of LPS by CD14 is facilitated. In our experimental system, despite expression of mCD14 on the THP-1 cells, the addition of sCD14 along with LBP was necessary for the cellular response to a low dose of LPS and for the antagonistic effect of TSS-P. It seems that a low dose of LPS forms a complex with sCD14 more efficiently than with mCD14. Thomas et al. (2002) also observed that sCD14 and LBP enhanced the sensitivity of human monocytes to minute amounts of LPS. They suggested that minute concentrations of LPS are better delivered to the TLR4–MD-2 complex by sCD14 than by mCD14. In the binding inhibition assay, we used soluble and immobilized CD14; the latter was intended to mimic mCD14. Although TSS-P inhibited LPS binding to immobilized CD14, it did not significantly antagonize the induction of ICAM-1 expression by LPS under the serum-free condition, in which mC14 was the only CD14 available. Furthermore, TSS-P did not bind to THP-1 cells independent of serum and inhibited E. coli LPS binding to the cells only in the presence of sCD14 and LBP. These results suggest that TSS-P antagonized LPS signalling by interfering with the function of sCD14 rather than that of mCD14, and matrix-immobilized molecules could not represent membrane-bound forms. Further studies are necessary to address the difference between sCD14 and mD14 in LPS binding.
Although peptidoglycan was dependent on sCD14 for its cellular signalling (Asai et al., 2003a), we did not observe an enhanced cellular response by the addition of rhCD14 and the inhibition of peptidoglycan-induced activity by TSS-P. This discrepancy could be due to the peptidoglycan used. Asai et al. (2003a) used peptidoglycan that was extracted from S. aureus in their laboratory, whereas we used commercial S. aureus peptidoglycan. The molecules involved in the cellular response by peptidoglycan were believed to be TLR2 and possibly CD14. However, the concept of TLR2-mediated peptidoglycan signalling is challenged by the fact that this activity did not originate from peptidoglycan but from contaminating cell wall components like lipopeptides and lipoteichoic acid (Travassos et al., 2004). The different response to sCD14 observed by us and by Asai et al. (2003a) could be due to a difference in the contamination degree and molecules in the peptidoglycan used by the two groups. Shimizu et al. (2004) showed that cells expressing mCD14 did not need exogenous sCD14 for TLR2-mediated peptidiglycan signalling. This result is consistent with our observation that peptidoglycan stimulated cells in an sCD14-independent manner. Shimizu et al. (2004) also suggested the presence of CD14-independent peptidoglycan signalling by observing that mCD14-deficient epithelial cells responded to peptidoglycan to some extent under serum-free conditions. Recent investigations revealed that peptidoglycan recognition proteins (PGRP) and nucleotide-binding oligomerization domain (NOD) proteins play a key role in peptidoglycan-induced cell signalling (Girardin & Philpott, 2004).
It is known that LPS presented by CD14 binds to cells via the receptor TLR4–MD-2, forming LPS–TLR4–MD-2 complexes (Mancek et al., 2002; Miyake 2003; Palsson-McDermott & O'Neill, 2004). MD-2 is a molecule that is associated with the extracellular domain of TLR4. Therefore, TLR4–MD-2 could be another target of TSS-P antagonism, in addition to LBP and CD14. TSS-P bound to neither immobilized rhMD-2 nor THP-1 cells, whereas the E. coli LPS bound to both of them in a dose-dependent manner. However, TSS-P interfered with the binding of E. coli LPS to immobilized MD-2 and to THP-1 cells in the presence of 2 % HS or rhCD14/rhLBP. This binding inhibition was not observed in the absence of serum. These results confirmed that TSS-P antagonized LPS signalling by interfering with the interaction of LPS with CD14 and LBP.
The agonistic and antagonistic effects of various LPS molecules appear to be due to their structural differences. The presence and the structure of fatty acids in lipid A play a key role in the biological activities of LPS. Lipid A with less than six acyl chains showed little or no cell-stimulating activity in human cells or antagonistic activity against active LPS (Schromm et al., 2000). In addition to the number, length and position of the acyl chains, the saturation degree and the charge density determine the intramolecular conformation of amphiphilic LPS molecules, which affects their biological activity. Lipid A from R. sphaeroides and lipid IVa, a lipid A precursor with a tetra-acyl group, are antagonistic, but are concurrently agonistic to TLR4, although the differential response is species-specific (Hajjar et al., 2002, Lohmann et al., 2003; Saitoh et al., 2004). The characteristics of lipid A from R. sphaeroides are penta-acyl chains with shorter chain lengths, including one unsaturated chain (Brandenburg et al., 2003). P. gingivalis LPS showed an antagonistic effect to TLR4 and an agonistic effect to TLR2 (Coats et al., 2003; Hirschfeld et al., 2001). Also, a single strain of P. gingivalis showed multiple lipid A forms that activate cells via both TLR2 and TLR4 (Darveau et al., 2004).
The structures of glycolipids in oral spirochaetes like T. medium, T. maltophilum and T. denticola have been suggested to resemble lipoteichoic acid (Hashimoto et al., 2003; Schröder et al., 2000; Schultz et al., 1998). The structural components essential for typical LPS like heptose, 2-keto-3-deoxyoctonate (KDO) and -hydroxy fatty acids were absent in T. denticola (Schultz et al., 1998) and T. medium (Schröder et al., 2000). In T. pectinovorum LPS, KDO was found, whereas heptose and -hydroxy fatty acids were absent (Walker et al., 1999). All these observations indicate structural differences between glycolipids of oral spirochaetes and typical LPS, suggesting the possible use of different receptors and possibly different functions. T. maltophilum glycolipids used TLR2 as a main receptor to induce TNF- in a murine macrophage cell line and in a human embryonic kidney cell line, HEK293, transfected with TLR2 (Opitz et al., 2001). Asai et al. (2003b) reported that outer-membrane extracts of Treponema vincentii, T. medium and T. denticola induced IL-8 in human gingival epithelial cells, and this induction was inhibited by anti-human TLR2 monoclonal antibody. However, the single active component in the outer-membrane extracts in this organism was not elucidated. In other spirochaetes, LPS from Leptospira interrogans has been reported as a TLR2 agonist (Werts et al., 2001). TSS-P did not show cell-stimulating activity in THP-1 cells and HGFs that expressed TLR2.
Growth conditions are known to influence the biochemical and antigenic properties of the bacterial outer membrane. LPS of P. gingivalis grown in haemin-containing medium showed an additional antigenic determinant (Cutler et al., 1996) as compared with that of cells grown in haemin-depleted medium. Different medium compositions resulted in heterogeneous LPS types (Darveau et al., 2004). To address this problem, we used two different media (OMIZ-Pat and NOS) for T. socranskii culture and found no difference in the antagonistic effects of TSS-P extracted from the two cultures.
In summary, TSS-P exhibited no cell-stimulating activity in cells expressing TLR4 and TLR2. By blocking the function of LBP/CD14, it potentially antagonized LPS activity, which normally would stimulate proinflammatory cytokines and ICAM-1 in THP-1 cells and HGFs. These properties indicate that TSS-P may allow the bacteria to escape the innate host defence system during the initiation and progression of periodontitis. The antagonistic property may be a great advantage for the bacteria, enabling them to modulate the innate immune system and therefore to facilitate host tissue colonization and disease progression. This may contribute to prolonged persistent chronic periodontitis. The elucidation of the structure of TSS-P in the future may provide more insight into the correlation between the structure and function of glycolipids of oral spirochaetes.
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Asai, Y., Hashimoto, M. & Ogawa, T. (2003a). Treponemal glycoconjugate inhibits Toll-like receptor ligand-induced cell activation by blocking LPS-binding protein and CD14 functions. Eur J Immunol 33, 3196–3204.[CrossRef][Medline]
Asai, Y., Jinno, T. & Ogawa, T. (2003b). Oral treponemes and their outer membrane extracts activate human gingival epithelial cells through Toll-like receptor 2. Infect Immun 71, 717–725.
Baumgartner, J. C., Khemaleelakul, S. U. & Xia, T. (2003). Identification of spirochetes (treponemes) in endodontic infections. J Endod 29, 794–797.[Medline]
Brandenburg, K., Andra, J., Muller, M., Koch, M. H. & Garidel, P. (2003). Physicochemical properties of bacterial glycopolymers in relation to bioactivity. Carbohydr Res 338, 2477–2489.[CrossRef][Medline]
Chan, E. C., Siboo, R., Touyz, L. Z., Qui, Y. S. & Klitorinos, A. (1993). A successful method for quantifying viable oral anaerobic spirochetes. Oral Microbiol Immunol 8, 80–83.[Medline]
Choi, B. K., Jung, J. H., Suh, H. Y., Yoo, Y. J., Cho, K. S., Chai, J. K. & Kim, C. K. (2001). Activation of matrix metalloproteinase-2 by a novel oral spirochetal species Treponema lecithinolyticum. J Periodontol 72, 1594–1600.[CrossRef][Medline]
Choi, B. K., Lee, H. J., Kang, J. H., Jeong, G. J., Min, C. K. & Yoo, Y. J. (2003). Induction of osteoclastogenesis and matrix metalloproteinase expression by the lipooligosaccharide of Treponema denticola. Infect Immun 71, 226–233.
Choi, B. K., Moon, S. Y., Cha, J. H., Kim, K. W. & Yoo, Y. J. (2005). Prostaglandin E2 is a main mediator in receptor activator of nuclear factor-kappaB ligand-dependent osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii. J Periodontol 76, 813–820.[CrossRef][Medline]
Coats, S. R., Reife, R. A., Bainbridge, B. W., Pham, T. T. & Darveau, R. P. (2003). Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at Toll-like receptor 4 in human endothelial cells. Infect Immun 71, 6799–6807.
Correia, F. F., Plummer, A. R., Ellen, R. P., Wyss, C., Boches, S. K., Galvin, J. L., Paster, B. J. & Dewhirst, F. E. (2003). Two paralogous families of a two-gene subtilisin operon are widely distributed in oral treponemes. J Bacteriol 185, 6860–6869.
Cutler, C. W., Eke, P. I., Genco, C. A., Van Dyke, T. E. & Arnold, R. R. (1996). Hemin-induced modifications of the antigenicity and hemin-binding capacity of Porphyromonas gingivalis lipopolysaccharide. Infect Immun 64, 2282–2287.[Abstract]
Darveau, R. P., Pham, T. T., Lemley, K., Reife, R. A., Bainbridge, B. W., Coats, S. R., Howald, W. N., Way, S. S. & Hajjar, A. M. (2004). Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both Toll-like receptors 2 and 4. Infect Immun 72, 5041–5051.
Ellen, R. P. & Galimanas, V. B. (2005). Spirochetes at the forefront of periodontal infections. Periodontol 2000 38, 13–32.[CrossRef]
Girardin, S. E. & Philpott, D. J. (2004). Mini-review: the role of peptidoglycan recognition in innate immunity. Eur J Immunol 34, 1777–1782.[CrossRef][Medline]
Hajjar, A. M., Ernst, R. K., Tsai, J. H., Wilson, C. B. & Miller, S. I. (2002). Human Toll-like receptor 4 recognizes host-specific LPS modifications. Nat Immunol 3, 354–359.[CrossRef][Medline]
Hashimoto, M., Asai, Y., Jinno, T., Adachi, S., Kusumoto, S. & Ogawa, T. (2003). Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293. Eur J Biochem 270, 2671–2679.[Medline]
Heuner, K., Bergmann, I., Heckenbach, K. & Göbel, U. B. (2001). Proteolytic activity among various oral Treponema species and cloning of a prtP-like gene of Treponema socranskii subsp. socranskii. FEMS Microbiol Lett 201, 169–176.[Medline]
Hirschfeld, M., Weis, J. J., Toshchakov, V., Salkowski, C. A., Cody, M. J., Ward, D. C., Qureshi, N., Michalek, S. M. & Vogel, S. N. (2001). Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun 69, 1477–1482.
Jarvis, B. W., Lichenstein, H. & Qureshi, N. (1997). Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS. Infect Immun 65, 3011–3016.[Abstract]
Kesavalu, L., Falk, C. W., Davis, K. J., Steffen, M. J., Xu, X., Holt, S. C. & Ebersole, J. L. (2002). Biological characterization of lipopolysaccharide from Treponema pectinovorum. Infect Immun 70, 211–217.
Lee, S. H., Kim, K. K. & Choi, B. K. (2005). Upregulation of intercellular adhesion molecule 1 and proinflammatory cytokines by the major surface proteins of Treponema maltophilum and Treponema lecithinolyticum, the phylogenetic group IV oral spirochetes associated with periodontitis and endodontic infections. Infect Immun 73, 268–276.
Lepper, P. M., Triantafilou, M., Schumann, C., Schneider, E. M. & Triantafilou, K. (2005). Lipopolysaccharides from Helicobacter pylori can act as antagonists for Toll-like receptor 4. Cell Microbiol 7, 519–528.[CrossRef][Medline]
Lohmann, K. L., Vandenplas, M., Barton, M. H. & Moore, J. N. (2003). Lipopolysaccharide from Rhodobacter sphaeroides is an agonist in equine cells. J Endotoxin Res 9, 33–37.[Medline]
Loppnow, H., Libby, P., Freudenberg, M. A., Kraus, J. H., Weckesser, J. & Mayer, H. (1990). Cytokine induction by lipopolysaccharide (LPS) corresponds to the lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS. Infect Immun 58, 3743–3750.
Mancek, M., Pristovsek, P. & Jerala, R. (2002). Identification of LPS-binding peptide fragment of MD-2, a Toll-receptor accessory protein. Biochem Biophys Res Commun 292, 880–885.[CrossRef][Medline]
Martin, M. U. & Wesche, H. (2002). Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family. Biochim Biophys Acta 1592, 265–280.[Medline]
Miller, S. I., Ernst, R. K. & Bader, M. W. (2005). LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 3, 36–46.[CrossRef][Medline]
Miyake, K. (2003). Innate recognition of lipopolysaccharide by CD14 and Toll-like receptor 4-MD-2: unique roles for MD-2. Int Immunopharmacol 3, 119–128.[CrossRef][Medline]
Moter, A., Hoenig, C., Choi, B. K., Riep, B. & Göbel, U. B. (1998). Molecular epidemiology of oral treponemes associated with periodontal disease. J Clin Microbiol 36, 1399–1403.
Opitz, B., Schröder, N. W., Spreitzer, I. & 7 other authors (2001). Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-kappaB translocation. J Biol Chem 276, 22041–22047.
Palsson-McDermott, E. M. & O'Neill, L. A. (2004). Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunol 113, 153–162.[CrossRef][Medline]
Re, F. & Strominger, J. L. (2003). Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness. J Immunol 171, 5272–5276.
Riviere, G. R., Riviere, K. H. & Smith, K. S. (2002). Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer's disease. Oral Microbiol Immunol 17, 113–118.[CrossRef][Medline]
Rosen, G., Sela, M. N., Naor, R., Halabi, A., Barak, V. & Shapira, L. (1999). Activation of murine macrophages by lipoprotein and lipooligosaccharide of Treponema denticola. Infect Immun 67, 1180–1186.
Saitoh, S., Akashi, S., Yamada, T. & 10 other authors (2004). Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization. Int Immunol 16, 961–969.
Schröder, N. W., Opitz, B., Lamping, N., Michelsen, K. S., Zahringer, U., Göbel, U. B. & Schumann, R. R. (2000). Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids. J Immunol 165, 2683–2693.
Schromm, A. B., Brandenburg, K., Loppnow, H., Moran, A. P., Koch, M. H., Rietschel, E. T. & Seydel, U. (2000). Biological activities of lipopolysaccharides are determined by the shape of their lipid A portion. Eur J Biochem 267, 2008–2013.[Medline]
Schultz, C. P., Wolf, V., Lange, R., Mertens, E., Wecke, J., Naumann, D. & Zahringer, U. (1998). Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola. Functionin
