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,
Departments of
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Biochemistry and
Otolaryngology, Sapporo Medical University School of Medicine, Sapporo, Japan;
Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Core Research for Engineering, Science, and Technology, Japan Science and Technology, Kawaguchi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu24-Lys631 enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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His mutation in Tlr4 gene exhibit a defective responsiveness to LPS (14). TLR4 requires an accessory protein, MD-2, to respond efficiently to LPS (15). Secreted MD-2 confers LPS sensitivity to TLR4 (16). Immunoprecipitation analysis has revealed that MD-2 can be associated with TLR4 (17). The studies with site-directed mutagenesis of MD-2 indicate the importance of disulfide bonds and N-linked glycosylation in TLR4 signaling (18, 19). MD-2 has been demonstrated to bind bacterial LPS (20), but the region of MD-2 required for the association with TLR4 appears to be different from that for LPS responsiveness (21). The mechanisms of ligand recognition by TLRs have not been completely understood. The extracellular TLR2 domain directly binds to peptidoglycan derived from Staphylococcus aureus (22) and zymosan (23), and the extracellular TLR2 region containing Ser40-Ile64, but not Cys30-Ser39 is critical for peptidoglycan recognition (24), indicating the importance of the extracellular TLR2 domain in ligand recognition. LPS is cross-linked with TLR4 and MD-2 only when coexpressed with CD14 (25), suggesting that LPS is in close proximity to the receptor complex. TLR4 is homologous to other TLRs and CD14 in point of having leucine-rich repeats. TLR4 consists of the amino-terminal region Met1-Phe54, the region Ser55-Phe567 containing leucine-rich repeats, the region Pro568-Lys631, and the region Thr632-Ile839 containing the putative transmembrane and cytoplasmic domains. The purposes of this study were to examine the direct interactions of TLR4 with MD-2 and LPS. In this study, we generated a soluble form of rTLR4 (sTLR4)3 lacking the putative intracellular and transmembrane domains, and analyzed the direct interaction of sTLR4 protein with MD-2 protein and LPS. This study demonstrates that the extracellular TLR4 domain alone cannot bind to LPS, but that the TLR4-MD-2 complex is capable of binding LPS, and that the TLR4 region containing Glu24-Lys631 enables MD-2 binding and LPS recognition. In addition, this study suggests that sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human embryonic kidney (HEK) 293 cells were maintained in DMEM supplemented with 10% FCS. Re595 LPS was purchased from Sigma-Aldrich (St. Louis, MO). mAb to sTLR4, 4D9, was generated using the hybridoma technique based on the method described previously (26). Anti-FLAG Ab and anti-FLAG Ab-conjugated agarose beads were obtained from Sigma-Aldrich. Anti-V5 mAb was purchased from Invitrogen Life Technologies (Carlsbad, CA).
Expression vectors
The cDNAs for human TLR4 and human MD-2 were obtained, as described previously (15). TLR4-FLAG-His that contains the C-terminal fusion FLAG tag and 6 His tag was subcloned into pcDNA3.1(+) (Invitrogen Life Technologies). MD-2-V5-His that contains the C-terminal fusion V5 tag and 6 His tag was generated by using PCR and subcloned into pcDNA3.1D/V5-His-TOPO (Invitrogen Life Technologies). The mutant proteins used in this study are schematically presented in Fig. 1.
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A soluble form of extracellular rTLR4 domain (sTLR4) consists of the putative extracellular domain (Met1-Lys631) and a 6 His tag at the C-terminal end (Fig. 1). sTLR4 cDNA was constructed by using PCR. The sense primers and antisense primers used were 5'-TGCCAGGATGATGTCTGCC-3' and 5'-TAGTGATGGTGATGGTGATGCTTATTCATCTGACAGGTG-3', respectively. Constructed sTLR4 cDNA was confirmed by DNA sequencing. The sTLR4 cDNA was subcloned into pVL1392 plasmid vector using NotI and BamHI sites. The MD-2 cDNA in pcDNA3.1(+) vector was digested with PmeI and subcloned into SmaI site of pVL1392 vector. The proteins sTLR4 and MD-2 were expressed by a baculovirus-insect cell expression system using the method described by O’Reilly et al. (27). Viral titers were amplified to 
4 x 108 PFU/ml. The recombinant viruses were used to infect insect cell monolayers of Tni cells in serum-free medium at a multiplicity of 1 to 5. After 4-day incubation, the sTLR4 protein and the MD-2 protein were purified from the medium using a column of nickel-nitrilotriacetic acid beads (Qiagen, Valencia, CA) by the method described previously (22, 28). sTLR2 produced in insect cells or soluble CD14 (sCD14) produced in Chinese hamster ovary cells was prepared, as described previously (22, 28).
NF-B reporter assay
Activation of NF-B was measured, as previously described (22, 24). HEK293 cells were plated at 1 x 105 cells/well in 24-well plates on the day before transfection. The cells were transiently transfected by FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Basel, Switzerland) with 30 ng of an NF-B reporter construct (pNF-B-Luc; Stratagene, La Jolla, CA) and 10 ng of a construct directing expression of Renilla luciferase (pRL-TK; Promega, Madison, WI), together with the indicated amount of cDNA for TLR2. Twenty-four hours after transfection, the cells were stimulated with the indicated concentrations of LPS for 6 or 12 h, and luciferase activity was measured by the dual luciferase reporter assay system (Promega), according to the manufacturer’s instructions.
Biotinylation of sTLR4
sTLR4 was biotinylated with EZ-Link sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce, Rockford, IL), according to the manufacturer’s instructions.
Binding assay of TLR4 to MD-2 with microtiter wells
sTLR4, MD-2, sTLR2, sCD14, or BSA (2 µg/ml, 50 µl/well) was coated onto microtiter wells (Immulon 1B; Thermo Labsystems, Franklin, MA). After the wells were blocked with PBS containing 3% (w/v) BSA (blocking buffer), the indicated concentrations of MD-2 or biotinylated sTLR4 (50 µl/well) in the blocking buffer were added and the suspension was incubated at 37°C for 2 h. The wells were then washed with PBS containing 3% (w/v) skim milk and 0.1% (v/v) Triton X-100. MD-2 binding to the solid-phase protein was detected by anti-V5 mAb, followed by incubation with HRP-labeled anti-mouse IgG. The biotinylated sTLR4 binding to solid-phase protein was detected by HRP-conjugated streptavidin. The peroxidase reaction was conducted by using o-phenylenediamine as a substrate, and the absorbance at 492 nm was measured.
Binding assay in solution
A total of 2 µg each of sTLR4, MD-2, or sTLR4 plus MD-2 was added into 100 µl of the blocking buffer and was incubated for 30 min at 37°C. After the incubation, total volume of the mixture was adjusted to 500 µl by the addition of the blocking buffer. The mixture was precleared for 1 h at 4°C by incubation with 40 µl of protein G-Sepharose (50% suspension in PBS) and was incubated with 1 µg of anti-V5 mAb or 2 µg of anti-sTLR4 mAb 4D9 for 1 h at 4°C. Immune complexes were allowed to bind to 40 µl of protein G-Sepharose for 2 h at 4°C. The Sepharose beads were washed three times with PBS containing 0.1% (v/v) Triton X-100, and were finally resuspended in 20 µl of SDS sample buffer and boiled for 3 min under reducing conditions. The sample was subjected to SDS-PAGE (29), and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was immunoprobed with anti-sTLR4 mAb 4D9, followed by incubation with HRP-conjugated anti-mouse IgG. MD-2 was detected with HRP-conjugated anti-V5 mAb. The proteins that reacted with the Abs were visualized by using a chemiluminescence reagent (SuperSignal; Pierce), according to the manufacturer’s instructions.
Measurement of molecular interaction by the BIAcore method
The interaction between sTLR4 and MD-2 was also analyzed with BIAcore 3000 (BIAcore AB, Uppsala, Sweden). MD-2 (30 µg/ml) in 10 mM sodium acetate (pH 5.0) was immobilized on a CM5 sensor chip using the amine-coupling method. sTLR4 in 5 mM Tris buffer (pH 7.4) containing 150 mM NaCl at a concentration of 250 nM to 1.5 µM was passed over the surface of the sensor chip at a flow rate of 30 µl/min. The interaction was monitored as the changes of surface plasmon resonance response at 25°C. After 2 min of monitoring, the same buffer was introduced onto the sensor chip in place of the sTLR4 solution to start the dissociation. The sensor surface was regenerated with 10 mM HCl at the end of each experiment. Both the association rate constant (Ka) and the dissociation rate constant (Kd) were calculated according to the BIAevaluation software (version 3.1; BIAcore AB) using a program named 1:1 (Langmuir) binding model. The dissociation constant (KD) was determined by Kd/Ka.
Binding of sTLR4 and MD-2 to LPS-Sepharose beads
LPS from Escherichia coli 026:B6 (2 mg; Sigma-Aldrich) was cross-linked to epoxy-activated Sepharose 6B (3 ml of gel volume; Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer’s instructions.
A total of 2 µg each of sTLR4, MD-2, or sTLR4 plus MD-2 was added into 100 µl of the blocking buffer and preincubated at 37°C for 30 min. LPS-conjugated Sepharose beads (80 µl, 50% suspension in PBS) or control-Sepharose beads were then added into the solution containing sTLR4 and/or MD-2, and total volume of the suspension was adjusted to 500 µl by the addition of the blocking buffer. The suspension was incubated at 37°C for 2 h with gentle rocking. After the incubation, the beads were washed three times with PBS containing 0.1% (w/v) Triton X-100. The final pellet containing LPS-Sepharose beads was subjected to SDS-PAGE, and the proteins on the gel were transferred to the PVDF membrane. Western blot analysis was performed to detect sTLR4 and MD-2 that had been coprecipitated with LPS-Sepharose beads by using anti-sTLR4 mAb 4D9 and anti-V5 mAb, followed by incubation with HRP-conjugated anti-mouse IgG, as described above.
Effect of sTLR4 on LPS-induced IL-8 secretion from U937 cells
U937 cells (1 x 105) were differentiated by incubating the cells with 10 nM PMA in RPMI 1640 medium containing 10% FCS for 24 h. The cells were further incubated in the absence of PMA for 24 h. The cells were then incubated for 6 h with the medium containing sTLR4 (10 µg/ml) and MD-2 (0.25 µg/ml) that had been preincubated for 1 h with Re595 LPS. After the LPS stimulation, concentrations of IL-8 secreted from U937 cells were determined by ELISA using an OptEIA human IL-8 set (BD Pharmingen, San Diego, CA), according to the manufacturer’s instructions.
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Results |
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The N-terminal sequence of sTLR4 determined by an Applied Biosystems (Foster City, CA) Procise 492 sequencer was ESWEPCVEVVPNITYQCMEL. This was consistent with the deduced amino acid sequence of TLR4 starting at Glu24 (Fig. 1). Electrophoretic analysis revealed that MD-2 exhibited broad bands with molecular masses of 23–30 kDa (Fig. 2). sTLR4 migrated as a single band with an approximate molecular mass of 80 kDa under reducing condition. sTLR2 and sCD14 exhibited a single band with a molecular mass of 70 kDa and broad bands with 46–56 kDa, respectively, as described previously (22, 28, 30). sTLR2 starts at Glu21, as described previously (22).
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B activation
We first examined the effect of purified MD-2 protein on LPS-induced NF-B activation in TLR4-transfected HEK293 cells. LPS (100 ng/ml) failed to induce NF-B activation in the absence of MD-2 protein (Fig. 3). In contrast, the addition of MD-2 protein dramatically conferred NF-B activation to TLR4-transfected cells in response to LPS. The stimulatory effect of MD-2 protein on LPS-induced NF-B activation is concentration dependent. These results indicate that MD-2 protein exogenously added into the medium can function as an LPS receptor complex.
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When various concentrations of MD-2 were incubated with sTLR4, sTLR2, sCD14, or BSA coated onto the microtiter wells, MD-2 bound to solid-phase sTLR4 in a concentration-dependent manner (Fig. 4A). However, MD-2 did not exhibit any significant binding to sTLR2, sCD14, or BSA. When sTLR4 was incubated with MD-2 coated onto microtiter wells, sTLR4 bound to solid-phase MD-2 (Fig. 4B). The interaction of sTLR4 with MD-2 was also examined by a solution-phase assay. When both proteins were coincubated, sTLR4 was associated with MD-2 that was immunoprecipitated with anti-V5 Ab (Fig. 4C, upper panel), and likewise, MD-2 was associated with sTLR4 that was immunoprecipitated with anti-sTLR4 Ab (Fig. 4C, lower panel). These results demonstrate that the extracellular TLR4 domain containing Glu24-Lys631 can directly bind to MD-2, but that TLR2 and CD14, homologous proteins to TLR4 containing leucine-rich repeats, fail to interact with MD-2.
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The parameters of sTLR4 binding with MD-2 were determined by surface plasmon resonance analysis (Fig. 5). The passage of various concentrations of sTLR4 over MD-2 immobilized on a sensor chip yielded an association rate constant of Ka = 7.56 x 103 M–1 · s–1 and a dissociation rate constant of Kd = 4.76 x 10–4 · s–1, and a consequent dissociation constant of KD (Kd/Ka) = 6.29 x 10–8 M.
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We examined whether the sTLR4-MD-2 complex directly bound to LPS. sTLR4, MD-2, or sTLR4 plus MD-2 were incubated with LPS-Sepharose beads; LPS beads were sedimented; and the proteins binding to the beads were then analyzed by Western blotting. LPS beads coprecipitated a significant amount of MD-2 protein when MD-2 alone was incubated with LPS beads (Fig. 6), indicating that MD-2 is capable of binding LPS. LPS beads that had been incubated with sTLR4 alone failed to coprecipitate sTLR4 protein, showing that TLR4 cannot bind LPS. However, LPS beads coprecipitated a significant amount of sTLR4 protein and MD-2 protein when both sTLR4 and MD-2 were coincubated. Control beads precipitated neither sTLR4 nor MD-2. These results demonstrate that TLR4 alone cannot interact with LPS, but that the complex of the extracellular TLR4 domain and MD-2 can bind LPS.
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B activation and IL-8 secretion in TLR4-expressing cells
We next examined whether sTLR4 affected LPS-induced NF-B activation in HEK293 cells that had been transfected with wild-type (wt) TLR4. When 100 ng/ml MD-2 was exogenously added into the medium, LPS induced NF-B activation (Fig. 7). Addition of 4 µg/ml sTLR4 significantly attenuated NF-B activity elicited with 10 and 100 ng/ml LPS. These results demonstrate that sTLR4 can neutralize LPS-induced NF-B activation in cells transfected with wt TLR4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation in wt TLR4-transfected cells in the presence of MD-2.
TLR2, TLR4, and CD14 contain 19, 24, and 10 leucine-rich motifs, respectively. The proteins containing leucine-rich repeats appear to be involved in protein-protein interaction (31, 32). The structure of this motif adopts a curved shape resembling a horseshoe forming with an 
-helix lining its outer circumference and 
-strands forming a parallel -sheet along its inner circumference (31). TLR4 consists of the amino-terminal region Met1-Phe54, the region Ser55-Phe567 containing leucine-rich repeats, the region Pro568-Lys631, and the region Thr632-Ile839 containing the putative transmembrane and cytoplasmic domains. This study demonstrates that MD-2 binds to sTLR4, but not sTLR2 or CD14 (see Fig. 4). Because sTLR4 consists of Glu24-Lys631, the data indicate that the extracellular TLR4 region of Glu24-Lys631, but not the transmembrane or intracellular domains, is the functional domain for the binding to LPS and MD-2. This is supported by the data showing that sTLR4 can attenuate LPS-induced signaling and IL-8 secretion in wt TLR4-expressing cells (see Figs. 7 and 8).
Previous studies (10, 24) have shown that cells transfected with TLR2 are able to induce NF-B activation in response to peptidoglycan in the absence of CD14 or MD-2. Although cotransfection with CD14 enhances cell responsiveness, the studies indicate that an accessory protein is not required for the TLR2-mediated signaling. The previous studies (22, 23) from this laboratory consistently show that the extracellular TLR2 domain (sTLR2) directly binds to peptidoglycan and zymosan. The physiological relevance of the study is supported by the results showing that sTLR2 attenuates peptidoglycan-induced NF-B activation and IL-8 secretion in TLR2-transfected HEK293 cells (22). In addition, anti-TLR2 mAb that blocks peptidoglycan-induced signaling also inhibits sTLR2 binding to peptidoglycan. In contrast, the present study has shown that sTLR4 fails to bind LPS, but that the MD-2-TLR4 complex is capable of binding LPS (see Fig. 6). This is consistent with the previous study (15) showing that TLR4 alone cannot transmit LPS signaling and that MD-2 is requisite for LPS signaling of TLR4. Although the extracellular domains of TLR2 and TLR4 are homologous in point of having leucine-rich repeats, these studies support the idea that the extracellular domain of each TLR exhibits different ligand specificities.
Recent studies (21, 33) elucidate the MD-2 structure critical for TLR4 binding and LPS responsiveness by site-directed mutagenesis of MD-2. MD-2 binding to TLR4 is dependent on Cys95 and Cys105 (21). Although the MD-2 mutant F121G-K122A-Y131G-K132A is able to bind TLR4, this mutant is totally unresponsive to LPS. The MD-2 mutants of positively charged amino acids at Lys128 and Lys132 have also been shown to fail to interact with LPS, although these mutants can bind to TLR4 (34). These studies suggest that MD-2 interaction with TLR4 is necessary, but not sufficient for cellular response to LPS. Collectively, the MD-2 domain involved in TLR4 binding is likely to be distinct from that involved in LPS recognition. Thus, it is reasonable that MD-2 that binds to TLR4 is still able to interact with LPS. In addition, LPS can be associated with TLR4 when the cells were transfected with cDNAs for both wt TLR4 and MD-2, but not with TLR4 cDNA alone (33). Taken together, these studies are consistent with the conclusion that the extracellular TLR4 domain-MD-2 complex can bind LPS.
The addition of sTLR4 to the culture medium containing the MD-2 protein reduces the LPS-induced NF-B activation and IL-8 secretion in TLR4-expressing cells (see Figs. 7 and 8). Because the sTLR4-MD-2 complex binds LPS (see Fig. 6), it is likely that the inhibitory effect of sTLR4 on the wt TLR4-mediated LPS signaling is due to the sTLR4-MD-2 complex competing with wt TLR4-MD-2 receptor complex for LPS recognition. The previous study (23) from this laboratory has also shown that sTLR2 attenuates peptidoglycan-induced cell response. Taken together, these results raise the possibility that the extracellular TLR domain can function as a modulator of TLR-mediated inflammation. One recent study (34) supports this possibility. Blood monocytes have been shown to release natural sTLR2 constitutively. The natural soluble forms of TLR2 that exhibit the major polypeptide bands with 83, 70, and 66 kDa appear to be released from Mono Mac-6 cells and to originate from cell surface TLR2. This is likely to result from the posttranslational modification. This natural sTLR2 has been shown to inhibit IL-8 and TNF- production by Mono Mac-6 cells in response to synthetic bacterial lipopeptide Pam3Cys. The use of the soluble forms of rTLR2 and rTLR4 may lead to new therapeutic strategies for dampening TLR-mediated inflammation in infectious diseases.
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Acknowledgments |
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Footnotes |
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1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. 
2 Address correspondence and reprint requests to Dr. Yoshio Kuroki, Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: kurokiy{at}sapmed.ac.jp
3 Abbreviations used in this paper: sTLR, soluble rTLR; HEK, human embryonic kidney; PVDF, polyvinylidene difluoride; sCD14, soluble CD14; wt, wild type.
Received for publication April 16, 2004. Accepted for publication September 24, 2004.
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References |
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B activation and TNF- secretion are down-regulated by lung collectin surfactant protein A. J. Immunol. 171:417.This article has been cited by other articles:
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  K. Kuronuma, H. Mitsuzawa, K. Takeda, C. Nishitani, E. D. Chan, Y. Kuroki, M. Nakamura, and D. R. Voelker Anionic Pulmonary Surfactant Phospholipids Inhibit Inflammatory Responses from Alveolar Macrophages and U937 Cells by Binding the Lipopolysaccharide-interacting Proteins CD14 and MD-2 J. Biol. Chem., September 18, 2009; 284(38): 25488 - 25500. [Abstract] [Full Text] [PDF]
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M. Yamazoe, C. Nishitani, M. Takahashi, T. Katoh, S. Ariki, T. Shimizu, H. Mitsuzawa, K. Sawada, D. R. Voelker, H. Takahashi, et al. Pulmonary Surfactant Protein D Inhibits Lipopolysaccharide (LPS)-induced Inflammatory Cell Responses by Altering LPS Binding to Its Receptors J. Biol. Chem., December 19, 2008; 283(51): 35878 - 35888. [Abstract] [Full Text] [PDF]
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M. J. Scott and T. R. Billiar {beta}2-Integrin-induced p38 MAPK Activation Is a Key Mediator in the CD14/TLR4/MD2-dependent Uptake of Lipopolysaccharide by Hepatocytes J. Biol. Chem., October 24, 2008; 283(43): 29433 - 29446. [Abstract] [Full Text] [PDF]
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S. M. Zimmer, J. Liu, J. L. Clayton, D. S. Stephens, and J. P. Snyder Paclitaxel Binding to Human and Murine MD-2 J. Biol. Chem., October 10, 2008; 283(41): 27916 - 27926. [Abstract] [Full Text] [PDF]
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 V. Jain, A. Halle, K. A. Halmen, E. Lien, M. Charrel-Dennis, S. Ram, D. T. Golenbock, and A. Visintin Phagocytosis and intracellular killing of MD-2 opsonized Gram-negative bacteria depend on TLR4 signaling Blood, May 1, 2008; 111(9): 4637 - 4645. [Abstract] [Full Text] [PDF]
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A. Teghanemt, F. Re, P. Prohinar, R. Widstrom, T. L. Gioannini, and J. P. Weiss Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin J. Biol. Chem., January 18, 2008; 283(3): 1257 - 1266. [Abstract] [Full Text] [PDF]
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P. Prohinar, F. Re, R. Widstrom, D. Zhang, A. Teghanemt, J. P. Weiss, and T. L. Gioannini Specific High Affinity Interactions of Monomeric Endotoxin{middle dot}Protein Complexes with Toll-like Receptor 4 Ectodomain J. Biol. Chem., January 12, 2007; 282(2): 1010 - 1017. [Abstract] [Full Text] [PDF]
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C. Nishitani, H. Mitsuzawa, H. Sano, T. Shimizu, N. Matsushima, and Y. Kuroki Toll-like Receptor 4 Region Glu24-Lys47 Is a Site for MD-2 Binding: IMPORTANCE OF CYS29 AND CYS40 J. Biol. Chem., December 15, 2006; 281(50): 38322 - 38329. [Abstract] [Full Text] [PDF]
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 A. Visintin, K. A. Halmen, N. Khan, B. G. Monks, D. T. Golenbock, and E. Lien MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4) J. Leukoc. Biol., December 1, 2006; 80(6): 1584 - 1592. [Abstract] [Full Text] [PDF]
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S. K. Pathak, S. Basu, A. Bhattacharyya, S. Pathak, A. Banerjee, J. Basu, and M. Kundu TLR4-Dependent NF-{kappa}B Activation and Mitogen- and Stress-Activated Protein Kinase 1-Triggered Phosphorylation Events Are Central to Helicobacter pylori Peptidyl Prolyl cis-, trans-Isomerase (HP0175)-Mediated Induction of IL-6 Release from Macrophages J. Immunol., December 1, 2006; 177(11): 7950 - 7958. [Abstract] [Full Text] [PDF]
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H. Mitsuzawa, C. Nishitani, N. Hyakushima, T. Shimizu, H. Sano, N. Matsushima, K. Fukase, and Y. Kuroki Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice J. Immunol., December 1, 2006; 177(11): 8133 - 8139. [Abstract] [Full Text] [PDF]
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C. Yamada, H. Sano, T. Shimizu, H. Mitsuzawa, C. Nishitani, T. Himi, and Y. Kuroki Surfactant Protein A Directly Interacts with TLR4 and MD-2 and Regulates Inflammatory Cellular Response: IMPORTANCE OF SUPRATRIMERIC OLIGOMERIZATION J. Biol. Chem., August 4, 2006; 281(31): 21771 - 21780. [Abstract] [Full Text] [PDF]
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M. Kobayashi, S.-i. Saitoh, N. Tanimura, K. Takahashi, K. Kawasaki, M. Nishijima, Y. Fujimoto, K. Fukase, S. Akashi-Takamura, and K. Miyake Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering J. Immunol., May 15, 2006; 176(10): 6211 - 6218. [Abstract] [Full Text] [PDF]
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A. Visintin, K. A. Halmen, E. Latz, B. G. Monks, and D. T. Golenbock Pharmacological Inhibition of Endotoxin Responses Is Achieved by Targeting the TLR4 Coreceptor, MD-2 J. Immunol., November 15, 2005; 175(10): 6465 - 6472. [Abstract] [Full Text] [PDF]
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), CD14 (
), or BSA (
) that had been coated onto microtiter wells (2 µg/ml, 50 µl). The MD-2 protein binding to the solid-phase protein was detected with anti-V5 mAb, followed by the incubation with HRP-labeled anti-mouse IgG, as described in Materials and Methods. The data shown are the means ± SE of three experiments. B, The binding of sTLR4 to MD-2. The indicated concentrations of biotinylated sTLR4 were incubated at 37°C for 2 h with MD-2 (•) or BSA (
) LPS in medium that had been preincubated with or without 4 µg/ml sTLR4 and/or 0.1 µg/ml rMD-2 proteins. NF-