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Infection and Immunity, June 2003, p. 3221-3226, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3221-3226.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations

Masashi Muroi, Takahiro Ohnishi, Satoko Azumi-Mayuzumi, and Ken-ichi Tanamoto*

Division of Microbiology, National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan

Received 4 November 2002/ Returned for modification 18 February 2003/ Accepted 12 March 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lipopolysaccharide (LPS) preparations are known to often contain substances which activate cells through Toll-like receptor 2 (TLR2), and it is suspected that bacterial lipoproteins are responsible for this activation. We compared the mode of action of the TLR2-stimulatory substances with that of a synthetic bacterial lipopeptide (tripalmitoyl-Cys-Ser-Ser-Asn-Ala [Pam3CSSNA]), as well as with that of peptidoglycan. Six out of eight LPS preparations tested induced NF-{kappa}B-dependent reporter activity in 293 cells expressing CD14 and TLR2. Phenol extract (PEX) prepared from Escherichia coli LPS by modified phenol extraction induced reporter activity in 293 cells expressing TLR2, and this activity was enhanced by coexpression of CD14, whereas the activity of Pam3CSSNA was not dependent on CD14. The activity of PEX, but not that of Pam3CSSNA or peptidoglycan, was also enhanced by LPS binding protein or serum and blocked by polymyxin B. In addition, the activity of PEX was inhibited by a lipid A precursor (compound 406) in 293 cells expressing CD14 and TLR2. These results indicate that E. coli LPS preparations contain LPS-mimetic TLR2-stimulatory substances which differ from bacterial lipopeptides or peptidoglycan.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bacterial lipopolysaccharide (LPS) is a constituent of the outer membrane of the cell wall in gram-negative bacteria and plays a major role in septic shock in humans (32, 41). Exposure of macrophages to nanogram quantities of LPS results in the rapid activation of a number of transcription factors including NF-{kappa}B, leading to the synthesis of inflammatory cytokines (10). Cell surface molecules which bind to LPS have been extensively studied, and CD14 has long been known to be the major receptor (9, 44). Now, Toll-like receptors (TLRs) are widely accepted as central molecules which transmit extracellular signals induced by LPS as well as other bacterial components into intracellular components (4, 34).

TLRs are mammalian homologues of the Drosophila melanogaster Toll protein. Among TLRs identified to date, TLR4 is currently considered to be the predominant receptor mediating LPS activation (3). TLR4 was initially recognized as a molecule that increases constitutive but not LPS-inducible NF-{kappa}B activity. However, the finding of a novel accessory molecule, MD-2 (34), which confers LPS responsiveness on TLR4 and the analyses of TLR4-deficient (15, 28, 30, 35, 42) mice have provided strong evidence for the involvement of TLR4 in LPS signaling. TLR2 was also initially reported to confer LPS responsiveness on LPS-unresponsive cells (17, 45). However, Takeuchi et al. (35) and Heine et al. (11) found that TLR2-deficient mice responded normally to LPS and indicated that TLR2 is not essential for mammalian responses to LPS. In addition, Hirschfeld et al. (13) and Tapping et al. (39) reported that repurification of LPS by a modified phenol extraction method eliminated signaling through TLR2, although they did not characterize the contaminants. They concluded that contaminants other than LPS are responsible for TLR2-mediated signaling. The TLR2-stimulatory substances in Escherichia coli K-12 LPS were recently examined, and it was concluded that two lipoproteins are the major components responsible for TLR2-mediated signaling (18). TLR2 was also reported to respond to various bacterial cell wall components (2, 20, 21, 33, 46) and was recognized as a pattern recognition receptor (20). Interestingly, recent studies have also shown that leptospiral LPS (43) and Porphyromonas gingivalis LPS (14, 16, 38) activate cells through a TLR2-dependent or a non-TLR4-dependent mechanism, indicating that certain types of LPS are capable of activating TLR2.

In the present study, we attempted to characterize substances responsible for the TLR2-mediated activity of LPS preparations of bacterial origin and found that these LPS preparations contain TLR2-stimulatory LPS-mimetic substances which differ from bacterial lipopeptide or peptidoglycan in their modes of action.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. The human embryonic 293 cell line (obtained from the Human Science Research Resources Bank, Tokyo, Japan) was grown in Dulbecco's modified Eagle medium (Gibco BRL, Rockville, Md.) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS; Gibco BRL), penicillin (100 U/ml), and streptomycin (100 µg/ml). E. coli O111 LPS purified by phenol extraction followed by gel filtration (Ec O111a), E. coli O111 LPS purified by phenol extraction (Ec O111b), and E. coli F583 Rd LPS (Ec F583 Rd) purified by phenol-chloroform-petroleum ether extraction were obtained from Sigma (St. Louis, Mo.). E. coli J5 Rc LPS (Ec J5 Rc) purified by the method of Galanos et al. (8) was obtained from List Biological (Campbell, Calif.). E. coli R3 F653 R-type LPS (Ec R3 F653) and Salmonella enterica serovar Minnesota Re595 LPS (Sm Re595) were purified by the method of Galanos et al. (8), and S. enterica serovar Minnesota S-type LPS (Sm S-type) and S. enterica serovar Abortusequi LPS (Sab) were purified by phenol extraction in our laboratory. LPS preparations were subjected to sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis, and LPS was visualized with a silver staining kit (Bio-Rad, Hercules, Calif.). In addition, electrophoretic transfer to polyvinylidene difluoride membranes was performed, and protein was detected by using a colloidal gold reagent with a gold enhancement kit (Bio-Rad), which allows detection of as little as 10 to 100 pg of protein. Peptidoglycan from Staphylococcus aureus and polymyxin B were obtained from Sigma. The E. coli murein lipoprotein-derived synthetic lipopeptide tripalmitoyl-Cys-Ser-Ser-Asn-Ala (Pam3CSSNA) was obtained from Bachem (Bubendorf, Switzerland) and suspended in 25 mM octyl glucoside. Synthetic E. coli-type lipid A (compound 506), Salmonella-type lipid A (compound 516), and a lipid A precursor (compound 406, also known as compound IVa) were kindly provided by Daiichi Kagaku (Tokyo, Japan). An antibody (no. 1060) against the equine infectious anemia virus tag epitope (amino acid sequence: ADRRIPGTAEE) was a generous gift from Nancy Rice (NCI-Frederick Cancer Research and Development Center). The anti-mouse CD14 antibody M-20 was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Recombinant human LPS-binding protein (LBP) was prepared as previously described (26).

Phenol fractionation. LPS preparations were fractionated according to the modified phenol extraction method described by Hirschfeld et al. (13) with a slight modification, as follows. LPS preparations were dissolved in purified water containing 0.2% triethylamine and 0.5% deoxycholate and further extracted twice with purified water containing 0.2% triethylamine and 0.5% deoxycholate. The final phenol phase was dialyzed against methanol and air dried. The residue was dissolved in purified water by adding a trace amount of triethylamine and then used as the phenol extract (PEX; yield: 15 to 20%). The water phase obtained at the first phenol extraction was further extracted with phenol, and the final water phase was extensively dialyzed against purified water. To the dialysate, a 1/10 volume of 3 M sodium acetate (pH 4) and 3 volumes of cold ethanol were added. The mixture was incubated overnight at -20°C and then centrifuged at 4°C and at 10,000 x g for 15 min. The precipitate was washed with cold 70% ethanol and then air dried. The dried precipitate was dissolved in purified water by adding a trace amount of triethylamine and was used as the water fraction (WEX; yield: 40 to 60%).

Expression plasmids. Mammalian expression plasmids for human CD14, TLR4, and MD-2 were described previously (22-24). The region encoding human TLR2 was amplified by reverse transcription-PCR from total RNA prepared from THP-1 cells and cloned into mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, Calif.). The region encoding TLR2 minus its signal peptide sequence of the construct described above was subcloned into the downstream portion of a modified pcDNA3 vector; in the product of this vector the coding sequence for the preprotrypsin signal peptide sequence precedes that for the NH2-terminal equine infectious anemia virus tag epitope. The NF-{kappa}B-dependent luciferase reporter plasmid pELAM-L was described previously (26).

NF-{kappa}B reporter assay. The NF-{kappa}B-dependent luciferase reporter assay was performed as described elsewhere (23, 26). Briefly, human embryonic kidney 293 cells (3 x 105 to 5 x 105/well) were plated in six-well dishes and on the following day were transfected by the calcium phosphate precipitation method with the indicated amounts of the respective expression plasmids along with 0.2 µg of pELAM-L described above and 0.05 µg of reporter plasmid pRL-TK (Promega, Madison, Wis.) for normalization. At 24 h after transfection, cells were stimulated for 6 h, and the reporter gene activity was measured according to the manufacturer's (Promega) instructions.

Limulus amoebocyte gelation activity. Activation of the proclotting enzyme of the horseshoe crab was tested by using a quantitative Limulus assay reagent (Endospecy; Seikagaku Kogyo, Tokyo, Japan) as described previously (37). Briefly, each sample (50 µl) was incubated with the same volume of the assay reagent for 30 min at 37°C and optical density at 405 nm was measured.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TLR2-stimulatory activities of various LPS preparations. We first tested the TLR2-stimulatory activities of commercial and our own LPS preparations obtained from E. coli and Salmonella in 293 cells expressing human CD14 and TLR2 (Fig. 1). Among eight LPS preparations tested, six induced NF-{kappa}B-dependent reporter activity in a concentration-dependent manner. Sab and Sm Re595, up to 10 µg/ml, did not induce this activity. Only J5 Rc showed a detectable amount of protein contamination with silver-enhanced colloidal gold staining (data not shown). Expression of human MD-2 in addition to CD14 and TLR2 did not affect these activities (data not shown).



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  		FIG. 1. Activation of NF-{kappa}B in response to LPS preparations in 293 cells expressing CD14 and TLR2. 293 cells were transiently transfected with CD14 (0.1 µg) and TLR2 (0.1 µg) plasmids, together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were either left unstimulated (open bars) or stimulated for 6 h with LPS preparations in the presence of 10% (vol/vol) FCS, and luciferase activity was then measured. Values are means ± standard errors from two to four independent experiments. LPS preparations used were Ec O111a, Ec O111b, Ec R3 F653, Ec J5 Rc, Ec F583 Rd, Sab, Sm S-type, and Sm Re595.

 
To characterize the TLR2-stimulatory substances contained in LPS preparations, the E. coli O111a LPS preparation, which showed no detectable protein contamination, was subjected to modified phenol fractionation. The preparation was extracted with phenol in the presence of deoxycholate, and the resulting phenol fraction (PEX) and water fraction (WEX) were analyzed for TLR2-stimulatory activity (Fig. 2). PEX induced NF-{kappa}B-dependent reporter activity in 293 cells expressing CD14 and TLR2, and this effect was TLR2 dependent because no activation was observed in 293 cells transfected with a control vector or cells expressing only CD14 (data not shown). PEX induced stronger activity than the original LPS preparation, whereas WEX showed no TLR2-stimulatory activity (Fig. 2A). On the other hand, WEX activated NF-{kappa}B-dependent reporter activity in 293 cells expressing CD14, TLR4, and MD-2, and this activity was equivalent to that of the original LPS preparation (Fig. 2B). PEX also possessed TLR4-stimulatory activity, although the activity was approximately 100-fold weaker than that of the original LPS preparation (Fig. 2B). The activities of these preparations in 293 cells expressing CD14, TLR4, and MD-2 paralleled their Limulus activities (Fig. 2C).



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  		FIG. 2. TLR2- and TLR4-mediated activation of NF-{kappa}B in response to phenol and water extracts prepared from an E. coli O111 LPS preparation and their Limulus amoebocyte gelation activities. 293 cells were transiently transfected with plasmids encoding either CD14 (0.1 µg) and TLR2 (0.1 µg) (A) or CD14 (0.1 µg), TLR4 (2 ng), and MD-2 (2 ng) (B), together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were either left unstimulated ({circ}) or stimulated for 6 h with E. coli O111 LPS, WEX, or PEX obtained from E. coli O111 LPS in the presence of 10% (vol/vol) FCS, and luciferase activity was then measured. Values are means ± standard errors from one to four independent experiments. (C) Limulus amoebocyte gelation activities of E. coli O111 LPS, WEX, and PEX were measured by using a quantitative Limulus assay reagent, and the chromogen released was measured at an optical density (O.D.) of 405 nm.

 
CD14 and LBP are required for the TLR2-stimulatory activity of PEX. We next asked whether the TLR2-stimulatory activity of PEX was attributable to bacterial lipoprotein or peptidoglycan contaminations. To answer this question, we compared their modes of action in TLR2-mediated activation of NF-{kappa}B. In 293 cells expressing human CD14 and TLR2, PEX induced NF-{kappa}B-dependent reporter activity. This activity was CD14 dependent because expression of only TLR2 significantly reduced the activity (Fig. 3A), although the expression levels of TLR2 in cells transfected with TLR2 and in cells transfected with CD14 plus TLR2 were equivalent (Fig. 3A, inset). The phenol extract prepared from Sm S-type also activated NF-{kappa}B in a CD14- and TLR2-dependent manner (data not shown). On the other hand, the activation of NF-{kappa}B in response to the synthetic bacterial lipopeptide Pam3CSSNA was not CD14 dependent because comparable activation was seen in 293 cells expressing TLR2 alone and CD14 plus TLR2 (Fig. 3B). The TLR2 ligand peptidoglycan also activated NF-{kappa}B in a CD14-dependent manner (Fig. 3C). In 293 cells transfected with a control vector or cells expressing only human CD14, no stimulation of reporter activity was observed with PEX, Pam3CSSNA, or peptidoglycan at concentrations up to 10 µg/ml (data not shown).



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  		FIG. 3. CD14-dependent activation of NF-{kappa}B by PEX. 293 cells were transiently transfected with either an empty vector (0.1 µg) plus a TLR2 plasmid (0.1 µg; {circ}) or plasmids encoding CD14 (0.1 µg) and TLR2 (0.1 µg) (•), together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were stimulated for 6 h with PEX (A), a synthetic murein lipopeptide (Pam3CSSNA; B), or peptidoglycan (PG; C) in the presence of 10% (vol/vol) FCS, and luciferase activity was then measured. Values are means ± standard errors from at least three independent experiments. A part of the cellular extract prepared for the measurement of the luciferase activities was analyzed for the expression of TLR2 and CD14 by Western blotting (A, inset).

 
The activity of PEX was also enhanced by the presence of serum (Fig. 4). In the presence of 10% serum, 0.1 µg of PEX/ml induced NF-{kappa}B-dependent reporter activity comparable to that observed with 1 µg of PEX/ml in the absence of serum. When human LBP was used instead of serum, the reporter activity in response to PEX was also increased, and only 1/10 the amount of PEX was sufficient to produce activity levels similar to those observed in the absence of LBP or serum (Fig. 4A). On the other hand, reporter activities in response to peptidoglycan and Pam3CSSNA were not affected by the presence of serum (Fig. 4B).



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  		FIG. 4. Serum-dependent activation of NF-{kappa}B by PEX. 293 cells were transiently transfected with plasmids encoding CD14 (0.1 µg) and TLR2 (0.1 µg), together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were washed with phosphate-buffered saline twice and stimulated for 6 h with PEX (A), a synthetic murein lipopeptide (Pam3CSSNA; B), or peptidoglycan (PG; B) in Dulbecco's modified Eagle medium in the absence (open bars) or presence (hatched bars) of LBP (100 ng/ml) or FCS (10% [vol/vol]) (solid bars), and luciferase activity was then measured. Values are means ± standard errors from at least four independent experiments. Responses were compared with responses in the absence of LBP or serum by a two-tailed Student t test (*, P < 0.05; **, P < 0.01).

 
LPS-mimetic activity of PEX. The TLR2-stimulatory activity of PEX was CD14 and LBP dependent, thus closely resembling the effect of LPS on TLR4-mediated activity. Therefore, we next asked whether the TLR2-stimulatory activity in response to PEX is inhibited by polymyxin B. Polymyxin B at 10 to 50 µg/ml inhibited LPS-induced activation of NF-{kappa}B in 293 cells expressing CD14, TLR4, and MD-2. A similar extent of inhibition was observed in PEX-induced activation of NF-{kappa}B in 293 cells expressing CD14 and TLR2, and 50 µg of polymyxin B/ml inhibited activation almost completely (Fig. 5A). On the other hand, peptidoglycan-induced activation of NF-{kappa}B was not affected by polymyxin B, and there was only a slight inhibition in Pam3CSSNA-induced activation of NF-{kappa}B in 293 cells expressing CD14 and TLR2 (Fig. 5B).



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  		FIG. 5. Polymyxin B and lipid A precursor inhibit PEX-induced activation of NF-{kappa}B. (A and B) 293 cells were transiently transfected with plasmids encoding either CD14 (0.1 µg) and TLR2 (0.1 µg) (A, left five bars, and B) or CD14 (0.1 µg), TLR4 (2 ng), and MD-2 (2 ng) (A, right five bars), together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were either left untreated (open bars) or treated with the indicated concentrations of polymyxin B followed by either PEX (0.1 µg/ml; A), LPS (10 ng/ml; A), peptidoglycan (PG; 10 µg/ml; B), or Pam3CSSNA (0.1 µg/ml; B) for 6 h in the presence of 100 ng of LBP/ml, and luciferase activity was then measured. Luciferase activities were expressed relative to the activity in response to PEX, LPS, PG, or Pam3CSSNA in the absence of polymyxin B. Values are means ± standard errors (SE) from at least three independent experiments. (C and D) 293 cells were transiently transfected with plasmids encoding either CD14 (0.1 µg) and TLR2 (0.1 µg) (C and D, left four bars) or TLR2 (0.1 µg; D, right four bars), together with an NF-{kappa}B-dependent luciferase reporter plasmid. After 24 h, cells were either left untreated (open bars) or treated with the indicated concentrations of 506, 516, or 406 followed by either PEX (0.1 µg/ml; C), PG (10 µg/ml; D), or Pam3CSSNA (0.1 µg/ml; D) for 6 h in the presence of 100 ng of LBP/ml, and luciferase activity was then measured. Luciferase activities were expressed relative to the activity in response to PEX, PG, or Pam3CSSNA in the absence of lipid A compounds. Values are means ± SE from at least three independent experiments. Responses were compared with responses in the absence of any lipid A compounds by a two-tailed Student t test (*, P < 0.05; **, P < 0.01.

 
The observation that polymyxin B inhibited PEX-induced activation of NF-{kappa}B strongly suggested the involvement of an LPS-like substance(s) in this activation. We thus examined the effect of synthetic lipid A compounds on PEX-induced activation in 293 cells expressing CD14 and TLR2. The activity was not significantly affected by either synthetic E. coli-type lipid A (compound 506) or Salmonella-type lipid A (compound 516), whereas a lipid A precursor (compound 406) significantly inhibited activation, and complete inhibition was observed at 10 µg of 406/ml (Fig. 5C). On the other hand, 406 inhibited neither peptidoglycan- nor Pam3CSSNA-induced activation of NF-{kappa}B (Fig. 5D). These synthetic lipid A compounds by themselves did not activate NF-{kappa}B (data not shown) in 293 cells expressing CD14 and TLR2.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Some of LPS preparations of bacterial origin have been shown to activate NF-{kappa}B through TLR2 and TLR4 (13, 39). In the present study, we also found that several LPS preparations from E. coli and Salmonella activated NF-{kappa}B through TLR2 and that this activity was transferred into PEX after modified phenol extraction. Hirschfeld et al. (13) and Tapping et al. (39) found that modified phenol extraction of LPS eliminated the activity mediated through TLR2 and concluded that contaminants other than LPS are responsible for TLR2-mediated signaling. Recently, lipoproteins Lip19 and Lip12 were isolated from E. coli K-12 LPS and were shown to be substances that stimulated the TLR2-dependent pathway (18). The activation in response to these lipoproteins was not inhibited by polymyxin B treatment (18), and we also confirmed that activation in response to a synthetic bacterial lipopeptide (Pam3CSSNA) was not inhibited by polymyxin B. On the contrary, TLR2-mediated activation in response to PEX was blocked by polymyxin B. In addition, the activation of NF-{kappa}B in response to PEX was CD14 and serum (or LBP) dependent and was inhibited by a lipid A precursor (compound 406), whereas that in response to Pam3CSSNA was not dependent on CD14 or serum and was not inhibited by 406. Therefore, the TLR2-stimulatory substance(s) contained in PEX clearly differs from bacterial lipoproteins. Furthermore, this substance(s) also clearly differs from peptidoglycan because the activity of peptidoglycan was serum independent and was not inhibited by either polymyxin B or 406.

Besides peptidoglycan (33, 35, 46) and lipoprotein/lipopeptide (2, 12, 20, 25, 36), lipoarabinomannan (21) and lipoteichoic acid (27, 33) have been reported to activate cells via a TLR2-dependent mechanism. It has been reported that both lipoarabinomannan (31) and lipoteichoic acid (5, 7) require CD14 and LBP for their activities. NF-{kappa}B activation and interleukin-12 p40 gene expression in response to lipoarabinomannan (31) and lipoteichoic acid (5) in THP-1 cells were inhibited by Rhodobacter sphaeroides LPS or lipid A and by compound 406. These modes of action resemble those of PEX. However, there is also a contradictory report (33) indicating that lipoteichoic acid-induced activation of NF-{kappa}B is independent of CD14 and serum (as a source of LBP). In addition, it is reported that NF-{kappa}B activation in response to lipoteichoic acid (33) and tumor necrosis factor alpha production by macrophages in response to lipoarabinomannan (1) were not inhibited by polymyxin B. Critically, it is not conceivable that these compounds contaminate LPS preparations from gram-negative bacteria. Therefore, lipoarabinomannan and lipoteichoic acid are also unlikely to be involved in the effect of PEX.

In the present study, PEX-induced activation of NF-{kappa}B was inhibited by 406 but not by 506 or 516. Although the signals of all chemically synthesized compounds (506, 516, and 406) are considered to be mediated through TLR4, these compounds differ in their actions despite their structural similarity. All compounds activate NF-{kappa}B in murine macrophages, but only 506 is active in human macrophages (6, 19, 29, 37). Poltorak et al. (29) and Lien et al. (19) found that expression of only mouse TLR4 in THP-1 cells or in immortalized C3H/HeJ macrophages was sufficient to confer responsiveness to 406 and concluded that TLR4 was responsible for its species specificity. On the other hand, we previously demonstrated that expression of mouse TLR4 in THP-1 cells did not confer responsiveness to 516 and that expression of both mouse TLR4 and MD-2 was needed to confer responsiveness (22). These and our present results suggest that the sites of action of these compounds differ slightly. Triantafilou and Triantafilou (40) proposed a new model of LPS recognition in which the innate recognition of diverse bacterial products involves an activation cluster consisting of multiple molecules such as TLRs, CD55, CD11b/CD18, and so on. They also suggested that the combinational diversity of these molecules could provide flexibility and specificity in the recognition. Thus, it may be possible that a slight difference in LPS structure can be discriminated by using a particular combination of many membrane receptors.

At present, we do not know the biochemical nature of the TLR2-stimulatory substance(s) contained in PEX. The mode of activation of PEX closely resembled that of LPS in terms of CD14 or LBP dependency, polymyxin B blockade, and inhibition by 406. LPS preparations obtained from bacteria contain LPS molecules with a variety of structures. These include premature and degraded structures in addition to the complete LPS structure, and the TLR2-stimulatory activity of these partial LPS molecules awaits study. It is, therefore, reasonable to expect that an LPS molecule with a certain structure, found in LPS preparations, is responsible for the TLR2-stimulatory activity of PEX. It has been reported that LPS or lipid A obtained from P. gingivalis stimulates C3H/HeJ macrophages even after modified phenol extraction (14, 16, 38) and that LPS from Leptospira interrogans stimulates cells through a TLR2-dependent mechanism (43). These findings also support the concept that LPS-like substances are responsible for the TLR2-mediated activity of PEX. We are currently attempting to identify the substance(s) responsible for the activity of PEX.


   ACKNOWLEDGMENTS
 
This work was supported in part by grants from the Japan Health Sciences Foundation and the Ministry of the Environment.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbiology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. Phone: 81-3-3700-1141, ext. 272. Fax: 81-3-3707-6950. E-mail: tanamoto{at}nihs.go.jp. Back

Editor: A. D. O'Brien


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, June 2003, p. 3221-3226, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3221-3226.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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