The Extracellular Toll-like Receptor 2 Domain Directly Binds Peptidoglycan Derived from Staphylococcus aureus
2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês
10.1074/jbc.m107057200
ISSN1083-351X
AutoresDaisuke Iwaki, Hiroaki Mitsuzawa, Seiji Murakami, Hitomi Sano, Masanori Konishi, Toyoaki Akino, Yoshio Kuroki,
Tópico(s)Pneumonia and Respiratory Infections
ResumoToll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-κB activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-κB activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN. Toll-like receptor 2 (TLR2) has been recognized to mediate cell signaling in response to peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria. The mechanism by which TLR2 recognizes PGN is unknown. It is not even clear whether TLR2 directly binds to PGN. In this study, we generated a soluble form of recombinant TLR2 (sTLR2) possessing only its putative extracellular domain by using the baculovirus expression system to examine the direct interaction between sTLR2 and PGN. sTLR2 bound avidly to insoluble PGN (iPGN) from Staphylococcus aureus coated onto microtiter wells in a concentration-dependent manner. In contrast, sTLR2 exhibited a very weak binding to lipopolysaccharide. iPGN cosedimented sTLR2 after the mixture of iPGN and sTLR2 had been incubated and centrifuged. sTLR2 partially attenuated the iPGN-induced NF-κB activation in TLR2-transfected HEK 293 cells and the iPGN-induced IL-8 secretion in U937 cells. One of anti-human TLR2 monoclonal antibodies, which blocked iPGN-induced NF-κB activation in TLR2-transfected cells, inhibited the binding of sTLR2 to iPGN. In addition, we found that sCD14 interacted with sTLR2 and increased the binding of sTLR2 to iPGN. From these results, we conclude that the extracellular TLR2 domain directly binds to PGN. Toll-like receptor a soluble form of extracellular TLR2 domain soluble CD14 peptidoglycan insoluble PGN lipopolysaccharide nuclear factor-κB monoclonal antibody human embryonic kidney phosphate-buffered saline interleukin The innate immune system plays key roles in the first line of host defense to limit infection after exposure to microorganisms and in stimulating the clonal response of adaptive immunity (1Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A.B. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2167) Google Scholar). Toll-like receptors (TLRs)1 function as pattern recognition receptors that recognize conserved motifs on pathogens (2Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2632) Google Scholar). To date, 10 members of the TLR family have been described (3Chaudhary P., M. Ferguson C. Nguyen V. Nguyen O. Massa H.F. Eby M. Jasmin A. Trask B.J. Hood L. Nelson P.S. Blood. 1998; 91: 4020-4027Crossref PubMed Google Scholar, 4Chuang T.H. 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Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar) has revealed that LPS is cross-linked specifically to TLR4 and MD-2 only when coexpressed with CD14, indicating that LPS directly binds to each member of the LPS receptor complex. In vitro and in vivo studies (13Hirschfeld M., Ma, Y. Weis J.H. Vogek S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (973) Google Scholar, 17Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 19Yoshimura A. Lien E. Ingalls R.R. Tuomanen E. Dziarski R. Golenbock D. J. Immunol. 1999; 163: 1-5Crossref PubMed Google Scholar, 21Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar, 23Takeuchi O. Hoshino K. Akira S. J. Immunol. 2000; 165: 5392-5396Crossref PubMed Scopus (911) Google Scholar) demonstrate that TLR2 is responsible for cell signaling in response toS. aureus and its cell wall component, peptidoglycan. PGN induces the activation of NF-κB in HEK 293 cells expressing TLR2 in the absence of CD14, although coexpression of CD14 enhances TLR2-mediated signaling (17Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). This suggests that TLR2 can interact with PGN regardless of whether CD14 is expressed. The specific objective of this study was to determine whether TLR2 directly binds PGN. In this study we generated a soluble form of recombinant TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains and conducted a binding study using sTLR2 and insoluble PGN (iPGN) derived fromS. aureus. This study clearly demonstrated that the extracellular TLR2 domain binds iPGN. iPGN derived from S. aureus was obtained from Fluka Chemical. LPS from Escherichia coliO26:B6 and from Salmonella minnesota Re595 were purchased from Sigma. Recombinant sCD14 expressed in Chinese hamster ovary cells was prepared as described previously (27Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Anti-human TLR2 monoclonal antibody (mAb 2392) was a kind gift from Genentech (San Francisco, CA). Anti-human TLR2 monoclonal antibody (mAb TL2.3) was obtained from Alexis Biochemicals (San Diego, CA). A 2.6-kb cDNA for human TLR2 was obtained by reverse transcriptase-polymerase chain reaction using RNA isolated from U937 cells. sTLR2 consists of the putative extracellular domain (Met1–Arg587) and a His6 tag at the C-terminal end and sTLR2 cDNA was constructed by using PCR. The sense and antisense primers used were 5′-GGATCCAAAGGAGACCTATAG-3′ and 5′-TTAGTGATGGTGATGGTGATGCCTGTGACATTCCGACAC-3′, respectively. The sTLR2 cDNA was subcloned into pVL1393 plasmid vector usingBamHI and NotI sites. The recombinant plasmid constructed was confirmed by a combination of restriction enzyme mapping and DNA sequencing. The sTLR2 protein was expressed in the baculovirus insect cell expression system using the methods described by O'Reilly et al. (28O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vector: A Laboratory Manual. W. H. Freeman and Company, New York1992: 139-179Google Scholar). Monolayers of Spodopera frugiperda (Sf-9) cells were cotransfected with linearized virus DNA (BaculoGold, Pharmingen) and the pVL1393 plasmid vector containing cDNA for sTLR2. Viral titers were amplified to ∼5–10 × 107 plaque-forming units/ml. The recombinant viruses were used to infect monolayers of Tni cells in serum-free medium at a multiplicity of 2. After a 3-day incubation, the sTLR2 protein was purified from the medium using a column of nickel-nitrilotriacetic acid beads (Qiagen, Valencia, CA) by the method described previously (27Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The protein concentrations were determined using a bicinchoninic assay (BCA; Pierce). The proteins were analyzed by SDS-PAGE (12.5% gel) under reducing conditions according to the method of Laemmli (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and were stained with Coomassie Brilliant Blue R-250. Antisera against sTLR2 and sCD14 were raised in rabbits. sTLR2 (100 μg) or sCD14 (100 μg) was emulsified in TiterMax® Gold (CytRx Corp.) and given intradermally. After 3 weeks, a booster with 80 μg of sTLR2 or sCD14 in the same adjuvant was injected. The blood samples were collected 2 weeks later, and IgG was isolated from the sera using a protein G-Sepharose 4FF column (Amersham Biosciences). The binding of sTLR2 and sCD14 to iPGN and LPS was performed by the method described previously for the binding of sCD14 to LPS (27Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 30Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar). The indicated amount of iPGN or LPS in 20 μl of ethanol was added onto microtiter wells (Immulon 1B, Dynex), and the solvent was evaporated in ambient air. Nonspecific binding was blocked with 10 mm HEPES buffer (pH 7.4) containing 0.15m NaCl, 5 mm CaCl2, and 5% (w/v) bovine serum albumin (buffer A). The indicated concentrations of sTLR2 or sCD14 (50 μl/well) in the buffer A were then added and incubated at 37 °C for 6 h. After the incubation, the wells were washed with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100 and were then incubated for 1 h with 30 μg/ml anti-sTLR2 IgG or anti-sCD14 IgG in the same buffer, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000) for 1 h. The peroxidase reaction was finally performed usingo-phenylenediamine as a substrate after washing the wells with PBS containing 0.1% (v/v) Triton X-100. The binding of the protein to iPGN or LPS was detected by measuring absorbance at 492 nm. Glass tubes that had been coated with silicone (SIL-COAT5, IWAKI, Japan) were used in the experiments. The preparation of 3 μg of sTLR2 or sCD14 in 100 μl of the buffer A was first centrifuged at 2,000 × g for 5 min. The supernatant was collected and mixed with 30 μg of iPGN in a fresh silicone-coated tube. The mixture of protein and iPGN was incubated at 37 °C for 3 h with gentle shaking. After the incubation, the mixture was centrifuged at 2,000 × g for 5 min, and the supernatant was removed. The pellet was suspended with 300 μl of PBS containing 0.1% (v/v) Triton X-100 and centrifuged again at 2,000 × g for 5 min, and the supernatant was removed. The washing step was repeated three times. The pellet obtained by the final centrifugation was suspended in 30 μl of the sampling buffer for SDS-PAGE and boiled for 5 min. sTLR2 or sCD14 that had cosedimented with iPGN was detected by immunoblotting analysis. The sample was electrophoresed and transferred to polyvinylidene difluoride membranes. Nonspecific binding was blocked by incubating the membrane with 20 mm Tris buffer (pH 7.4) containing 0.15 m NaCl, 3% (w/v) skim milk, and 0.05% (v/v) Tween 20 (buffer B) for 30 min. The membranes were then incubated with anti-sTLR2 or anti-sCD14 IgG (10 μg/ml) in the buffer B for 3 h at room temperature. After washing the membranes with the buffer B, they were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG. The proteins that cosedimented with iPGN were finally visualized using SuperSignal® West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions. A 2.6-kb TLR2 cDNA was subcloned into pcDNA3.1(+) (Invitrogen). Activation of NF-κB was measured as described previously (14Kirschning C.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar, 17Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar). HEK 293 cells were plated at 1 or 1.5 × 105 cells/well in 24-well plates on the day before transfection. The cells were transiently transfected by FuGENETM 6 transfection reagent (Roche Molecular Biochemicals) with 30 or 100 ng of an NF-κB reporter construct (pNF-κB-Luc; Stratagene) and 3.5 or 10 ng of a construct that directed expression of Renilla luciferase under the control of the constitutively active thymidine kinase promoter (pRL-TK; Promega), together with 150 or 200 ng of transfectant gene. Forty-eight hours after transfection, the cells were stimulated with 0.1, 1.0, or 10 μg/ml iPGN in serum-free medium for 6 h, and luciferase activity was measured by using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. In some experiments the transfected cells were preincubated with monoclonal antibody or control mouse IgG at 37 °C for 30 min. To examine the effect of sTLR2 on iPGN-induced NF-κB activation, iPGN was preincubated with sTLR2 (0–10 μg/ml) for 30 min before incubation with the cells. sTLR2 was iodinated by the method of Bolton and Hunter (31Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2403) Google Scholar) using Bolton-Hunter reagent (Amersham Biosciences). The specific radioactivity ranged from 278 to 293 cpm/ng, and more than 85% of the radioactivity was precipitated by treatment with 10% (w/v) trichloroacetic acid. The binding study with 125I-sTLR2 was performed by sedimentation assay. The indicated concentrations of125I-sTLR2 were incubated with 1 μg of iPGN (100 μl/tube) in the buffer A at 37 °C for 3 h. After the incubation, the reaction mixture was transferred into the polypropylene tube and was centrifuged at 10,000 × g for 5 min. The supernatant was removed, and the resultant pellet was washed three times with 500 μl of PBS containing 0.1% (v/v) Triton X-100. The final pellet was resuspended with PBS containing 0.1% (v/v) Triton X-100, and the suspension was transferred into a new tube. The radioactivity of 125I-sTLR2 bound to the iPGN pellet was measured using a γ-radiation counter. Specific binding was determined by subtracting the binding obtained in the absence of iPGN from that in the presence of iPGN. Fifty microliters of sTLR2 (10 μg/ml) or bovine serum albumin (10 μg/ml) was added onto microtiter wells and incubated at 4 °C overnight. After blocking the wells with the buffer A, the indicated concentrations of sCD14 (50 μl/well) in the buffer A were added and incubated at 37 °C for 3 h. After the incubation, the wells were washed with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100, and the binding of sCD14 was detected using anti-sCD14 IgG. U937 cells (5 × 105 cells) were differentiated by incubation with 10 nm phorbol 12-myristate 13-acetate in RPMI 1640 medium containing 10% fetal calf serum for 24 h. The cells were further incubated in the absence of phorbol 12-myristate 13-acetate in RPMI 1640 medium containing 10% fetal calf serum for 24 h. The cells were stimulated in serum-free RPMI medium for 6 h with 1 μg/ml iPGN that had been preincubated for 30 min with sTLR2 (0–1 μg/ml). After the incubation, concentrations of IL-8 secreted from U937 cells were determined by enzyme-linked immunosorbent assay using an OptEIATM Human IL-8 Set (Pharmingen, San Diego, CA) according to the manufacturer's instructions. Approximately 190 μg of sTLR2 was obtained from 450 ml of culture medium. The N-terminal sequence of sTLR2 determined by an Applied Biosystems Procise 492 sequencer was ESSNQASLSXDRNGIXKGSS. This result was coincident with the deduced amino acid sequence of TLR2 starting at Glu21. Electrophoretic analysis revealed that sTLR2 exhibited a single band with a molecular mass of ∼75 kDa (Fig.1 A). sCD14 migrated as broad bands with 46–56 kDa, as described previously (27Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 32Stelter F. Pfister M. Bernheiden M. Jack R.S. Bufler P. Engelmann H. Schütt C. Eur. J. Biochem. 1996; 236: 457-464Crossref PubMed Scopus (49) Google Scholar). sCD14 has been shown to bind soluble PGN as well as LPS (33Dziarski R. Tapping R.I. Tobias P.S. J. Biol. Chem. 1998; 273: 8680-8690Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). We investigated the binding of sTLR2 and sCD14 to iPGN coated onto microtiter wells. When 0–5 μg/ml of sTLR2 and sCD14 were incubated with solid phase iPGN (20 μg/well), both proteins bound to iPGN in a concentration-dependent manner (Fig. 2 A). Concentration-dependent binding of sTLR2 to iPGN increased with the increasing amounts of iPGN (Fig. 2 B). The binding of sTLR2 to O26:B6 LPS or Re LPS was also examined compared with iPGN binding (Fig. 2 C). sTLR2 avidly bound to 20 μg/well iPGN, but the binding of sTLR2 to 20 μg/well LPS was clearly weak. The amount of sTLR2 binding to O26:B6 or Re LPS was less than 13% of that to iPGN. Another binding study was also performed by sedimentation, because we used insoluble PGN. To examine whether iPGN was sedimentable by centrifugation at 2,000 × g for 5 min, we determined the abilities of the supernatant and the pellet obtained by the centrifugation to induce tumor necrosis factor-α secretion from U937 cells. Tumor necrosis factor-α secretion was measured using the L929 bioassay as described previously (30Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar). The pellet fraction contained 99.3% (mean of three experiments) of tumor necrosis factor-α-inducing activity, indicating that almost all of the iPGN was sedimentable by the centrifugation. After the mixture of sTLR2 or sCD14 and iPGN suspension was incubated and centrifuged, the proteins that had cosedimented with iPGN were analyzed by immunoblotting using anti-sTLR2 or anti-sCD14 IgG (Fig.1 B). iPGN cosedimented sTLR2 or sCD14. When sTLR2 or sCD14 was incubated without iPGN, none of them was sedimentable. When surfactant protein A, which does not bind to iPGN (34Murakami S. Iwaki D. Mitsuzawa H. Sano H. Takahashi H. Voelker D.R. Akino T. Kuroki Y. J. Biol. Chem. 2002; 277: 6830-6837Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), was used as a negative control, iPGN failed to cosediment this protein. The binding study with 125I-labeled sTLR2 was also performed by sedimentation assay (Fig.3). sTLR2 exhibited concentration-dependent and saturable binding. When the molecular mass of sTLR2 was estimated as 75 kDa (Fig. 1 A), the binding of sTLR2 to iPGN has an apparent K d of 67 × 10−9m. A maximum of ∼40 ng (0.53 pmol) of sTLR2 was assumed to bind to 1 μg of iPGN. Taken together, these data clearly demonstrate that sTLR2 directly binds to iPGN. We examined the effect of sTLR2 on iPGN-induced NF-κB activation in TLR2-transfected HEK 293 cells. Coincubation of iPGN (100 and 1000 ng/ml) and sTLR2 (0.1–10 μg/ml) attenuated the activities of NF-κB (Fig. 4 A). sTLR2 at 1 and 10 μg/ml reduced the NF-κB activity by ∼40–50%. We also examined the effect of sTLR2 on iPGN-induced IL-8 secretion in U937 cells. When differentiated U937 cells were stimulated with iPGN (1 μg/ml) that had been preincubated with sTLR2 (0–1 μg/ml), the IL-8 secretion was decreased in a manner dependent upon the sTLR2 concentrations (Fig. 4 B). sTLR2 at 1 μg/ml reduced the iPGN-induced IL-8 secretion by 36%. Anti-TLR2 monoclonal antibody (mAb 2392), whose epitope is localized at the extracellular TLR2 domain (35Aliprantis A., O. Yang R.-B. Mark M.R. Sugget S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinski A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1278) Google Scholar), has been shown to inhibit secretion of tumor necrosis factor-α and IL-8 elicited by PGN in macrophage cell line THP-CD14, a monocytic cell line that constitutively expresses CD14 (36Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (305) Google Scholar). Another mAb TL2.3 also recognizes the extracellular TLR2 domain and has been shown to immunostain peripheral blood mononuclear cells (37Flo T.H. Halaas O. Torp S. Ryan L. Lien E. Dybdahl B. Sundan A. Espevik T. J. Leukocyte Biol. 2001; 69: 474-481PubMed Google Scholar). When iPGN was incubated with TLR2-transfected HEK 293 cells in the presence of mAb 2392, the antibody inhibited the iPGN-induced NF-κB activity in a concentration-dependent manner (Fig.5 A). Coincubation of 10 μg/ml mAb 2392 with TLR2-transfected cells in the presence of 10 μg/ml iPGN significantly reduced the NF-κB activity to the level of ∼15% of that in the absence of antibody. This is essentially consistent with the results observed for cytokine secretion in THP-CD14 cells (36Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (305) Google Scholar) and supports the idea that the epitope of mAb 2392 is located at the TLR2 region that is involved in PGN recognition. However, 10 μg/ml mAb TL2.3 failed to attenuate the iPGN-induced NF-κB activation. The effects of mAbs on the binding of sTLR2 to iPGN were also examined (Fig. 5 B). mAb 2392 significantly decreased the binding of sTLR2 to iPGN in a dose-dependent manner. The binding of sTLR2 to iPGN in the presence of 10 μg/ml mAb 2392 was reduced to the level of 35% of that in the absence of the mAb. In contrast, 10 μg/ml mAb TL2.3 did not attenuate the binding of sTLR2 to iPGN. Its level was almost comparable with that obtained in the presence of control mouse IgG. The results support the conclusion obtained from the direct binding assay that the extracellular TLR2 domain directly binds iPGN. We next investigated the interaction of sCD14 with sTLR2. When 0–20 μg/ml of sCD14 were incubated with sTLR2 coated onto microtiter wells, sCD14 bound to sTLR2 in a concentration-dependent manner (Fig.6 A). When sTLR2 and sCD14 were preincubated together, the binding of sTLR2 to iPGN increased by 23% compared with that of sTLR2 alone (Fig. 6 B). Coincubation of these proteins also enhanced the binding of sCD14 to iPGN by 27% (Fig.6 C). These results indicate that sCD14 can interact with sTLR2 and that coincubation of sTLR2 and sCD14 enhances the binding of each protein to iPGN. In this report we demonstrated that the extracellular domain of TLR2 directly binds PGN. To determine the direct interaction of TLR2 with PGN, we generated the soluble form of recombinant TLR2 possessing only the putative extracellular domain. sTLR2 bound to iPGN coated onto microtiter wells in a concentration-dependent manner. Because we were unable to quantify the amount of iPGN binding to the wells, we conducted the binding assay in solution with sedimentable iPGN. Consistent with the microtiter well binding, sTLR2 cosedimented with iPGN. Taken together, these results clearly demonstrate that the extracellular domain of TLR2 directly binds to iPGN. When the binding of sTLR2 to iPGN was compared with that to LPS (Fig.2 C), sTLR2 exhibited a measurable but very weak binding to LPS. The small amount of sTLR2 binding to LPS may have been due to the contaminants contained in the LPS preparation, because one recent study (13Hirschfeld M., Ma, Y. Weis J.H. Vogek S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (973) Google Scholar) indicates that commercial preparation of LPS contained low levels of highly bioactive contaminants, endotoxin protein, which is responsible for the TLR2-mediated signaling observed upon LPS stimulation, and that repurification of LPS eliminates cell signaling mediated through TLR2. The current study indicating the avid binding of sTLR2 to iPGN is essentially consistent with the previous in vivo and in vitro studies (17Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 21Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar) showing that TLR2 functions as a physiological receptor for PGN but not for LPS. Coincubation of sTLR2 with iPGN partially neutralized both iPGN-induced NF-κB activation in TLR2-transfected HEK 293 cells and IL-8 secretion in U937 cells (Fig. 4). It is likely that the interaction of sTLR2 with iPGN causes the decreased binding of iPGN to TLR2 expressed on cell surfaces, resulting in the attenuated cell signaling. The naturally occurring soluble form of mouse TLR4 that is expressed by alternatively spliced mouse TLR4 mRNA has been shown to attenuate but not completely inhibit LPS-elicited NF-κB activities to the level of 50–60% of those observed for controls (38Iwami K. Matsuguchi T. Masuda A. Kikuchi T. Musikacharoen T. Yoshikai Y. J. Immunol. 2001; 165: 6682-6686Crossref Scopus (230) Google Scholar). These results indicate that the extracellular TLR domain alone does not explain all the activity of TLR-ligand interaction. Although it is unclear why the soluble form of TLRs did not completely inhibit cell signaling, it is possible to assume that the wild type TLRs and the soluble form of the extracellular TLR domain lacking the transmembrane and cytoplasmic domains may differ in affinities for the ligands. Actually the affinity (the apparent K d = 67 × 10−9m) of sTLR2 for iPGN was not so high as was expected. It is uncertain whether wild type TLR2 on cell surfaces interacts with PGN with the same affinity as sTLR2 constructed. Partial attenuation of iPGN-induced signaling and cytokine secretion by the soluble form of TLR may be a consequence of the binding affinity of the soluble form, which is different from membrane-bound form. Other factors including sCD14 and other classes of TLR may be required for the acquisition of high affinity binding. As shown in Fig. 6, sCD14 interacted with sTLR2 and facilitated the binding of sTLR2 to solid phase iPGN. These results are consistent to the previous studies indicating that PGN-induced NF-κB activation is increased by cotransfection of membrane bound CD14 with TLR2 in HEK 293 cells (17Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar,39Mitsuzawa H. Wada I. Sano H. Iwaki D. Murakami S. Himi T. Matsushima N. Kuroki Y. J. Biol. Chem. 2001; 276: 41350-41356Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Taken together, these results support the idea that the PGN-induced cellular response is closely related with the direct binding of PGN to the extracellular TLR2 domain. Anti-TLR2 mAb 2392 that had been generated against the extracellular TLR2 domain (35Aliprantis A., O. Yang R.-B. Mark M.R. Sugget S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinski A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1278) Google Scholar) was reported to inhibit PGN-induced cytokine release from THP1-CD14 cells (36Tapping R.I. Akashi S. Miyake K. Godowski P.J. Tobias P.S. J. Immunol. 2000; 165: 5780-5787Crossref PubMed Scopus (305) Google Scholar). Likewise, this study showed that mAb 2392 inhibited NF-κB activity induced by iPGN in TLR2-transfected 293 cells (Fig. 5 A). mAb 2392 also significantly decreased the binding of sTLR2 to solid phase iPGN (Fig. 5 B). Although the epitope for this monoclonal antibody has not been identified, the extracellular TLR2 region contiguous to the antibody epitope may be critical for ligand recognition. In contrast, another mAb TL2.3 failed to inhibit iPGN-induced NF-κB activation and sTLR2 binding to iPGN (Fig. 5). These results may give specificity and biological relevance to the experiments. The study supports the idea that the direct binding of the extracellular TLR2 domain initiates iPGN-induced cell signaling. One recent study (26da Silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar) has revealed that LPS is cross-linked specifically to TLR4 and MD-2 only in the cells expressing CD14, indicating the direct binding of LPS to TLR4. In addition, the study has shown that LPS is cross-linked to TLR2 even when CD14 and MD-2 are not transfected. This study indicates that TLR2 directly binds to PGN in the absence of CD14. These studies suggest that TLR2, unlike TLR4, does not require CD14 to bind the ligand, supporting the idea that the mechanism of TLR2-ligand interaction is different from that of TLR4. In conclusion, we demonstrated that the extracellular TLR2 domain directly binds PGN. The conclusion obtained from this study using the cell-free system is consistent with that obtained from the study using the cells with transient transfection, showing that TLR directly binds its ligand and that the TLR-ligand interaction does not require cellular energy (26da Silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar).
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