The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitsuzawa, H.
Right arrow Articles by Kuroki, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitsuzawa, H.
Right arrow Articles by Kuroki, Y.
The Journal of Immunology, 2006, 177: 8133-8139.
Copyright © 2006 by The American Association of Immunologists, Inc.

Recombinant Soluble Forms of Extracellular TLR4 Domain and MD-2 Inhibit Lipopolysaccharide Binding on Cell Surface and Dampen Lipopolysaccharide-Induced Pulmonary Inflammation in Mice1

Hiroaki Mitsuzawa*,§, Chiaki Nishitani*,§, Naoki Hyakushima*,§, Takeyuki Shimizu*,§, Hitomi Sano*,§, Norio Matsushima{dagger}, Koichi Fukase{ddagger} and Yoshio Kuroki2,*,§

* Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan; {dagger} Division of Biophysics, Sapporo Medical University School of Health Sciences, Sapporo, Japan; {ddagger} Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan; and § Core Research for Engineering, Science, and Technology, Japan Science and Technology, Kawaguchi, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-{kappa}B activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [3H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Coinstillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-{alpha} levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The innate immune system plays central roles in the first line of defense against microbial pathogens and in stimulating the clonal responses of adaptive immunity (1). LPS is a principal component of the outer membrane of Gram-negative bacteria that activates the innate immune system and elicits excessive release of proinflammatory cytokines from immune cells. TLRs have been implicated in recognition and signaling of pathogen-associated molecular patterns (2).

TLR4 has been shown to play a critical role in recognition and signaling of LPS (3). TLR4 is a type I membrane protein consisting of the extracellular domain that possesses a characteristic leucine-rich motif structure and an intracellular signaling domain. MD-2 (4) and CD14 (5) are pivotal components in LPS-induced TLR4 signaling. TLR4 does not respond efficiently to LPS without MD-2 (4). Secreted MD-2 confers LPS sensitivity to TLR4 (6). Soluble MD-2 has been demonstrated to bind bacterial LPS (7). CD14 and LPS-binding protein also bind LPS and play important roles in LPS-induced cellular activation (8). CD14 augments LPS responsiveness, which is thought to be an event occurring upstream of TLR4-MD-2 interaction.

We have previously generated a soluble form of rTLR (sTLR)34 lacking the putative intracellular and transmembrane domains and a soluble form of rMD-2 (sMD-2) (9). sTLR4 directly binds sMD-2, and LPS-conjugated beads precipitate sMD-2, but do not sediment sTLR4. However, LPS beads coprecipitate sTLR4 and MD-2 when both proteins are coincubated, suggesting that the extracellular TLR4 domain-MD-2 complex, but not TLR4 alone, is capable of binding LPS. Addition of sTLR4 and sMD-2 attenuates LPS-elicited NF-{kappa}B activation and IL-8 release in TLR4-expressing cells (9). One study has reported a 20-kDa naturally occurring soluble form of mouse TLR4 that is expressed by alternatively spliced TLR4 mRNA (10). The alternative spliced cDNA encodes 86 aa of the extracellular TLR4 domain and an additional 36 aa. Overexpression of this mutant partially inhibits TNF-{alpha} production and NF-{kappa}B activation in mouse macrophage cell line, suggesting critical roles of the extracellular TLR4 domain in interaction with LPS and in regulation of LPS responsiveness. Natural sTLR2 have been shown to exist in human milk and plasma and to modulate TLR2 signaling (11). However, the mechanisms by which these natural soluble forms of TLRs alter microbe-induced cell responses remain unknown.

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using sTLR4 and sMD-2. We have found that the extracellular TLR4 domain does not directly bind LPS by itself, but that it augments lipid A binding to sMD-2 and increases the affinity of lipid A for the binding. Coadministration of sTLR4 and sMD-2 inhibits the binding of LPS to the TLR4- and MD-2-expressing cells, and dampens pulmonary inflammation caused by LPS in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

Macrophage-like cell line U937 (JCRB9021; obtained from Health Science Research Resource Bank, Osaka, Japan) and RAW264.7 (TIB-71; obtained from American Type Culture Collection) were maintained in RPMI 1640 (Sigma-Aldrich) containing 10% FCS. Human embryonic kidney (HEK) 293 cells (CRL-1573; obtained from American Type Culture Collection) were maintained in DMEM (Invitrogen Life Technologies) containing 10% FCS. LPS from Salmonella minnesota Re595 was purchased from Sigma-Aldrich. [3H]Lipid A was prepared, as described previously (12, 13).

Recombinant proteins

sTLR4 consisting of the putative extracellular domain (Met1-Lys631) and a 6xHis tag at the C-terminal end, sTLR2 consisting of the putative extracellular domain (Met1-Arg587) and a 6xHis tag at the C-terminal end, and sMD-2 containing V5 tag and 6xHis tag were generated by the baculovirus-insect cell expression system, and the recombinant proteins were purified, as described previously (9, 14). sTLR4 and sTLR2 start at Glu24 and Glu21, respectively.

Antibodies

For preparation of anti-sTLR4 polyclonal Ab, 100 µg of sTLR4 protein was emulsified in TiterMax Gold (CytRx) and given intradermally to rabbit. After 3 wk, a booster with 80 µg of sTLR4 in the same adjuvant was injected. The blood samples were collected 2 wk later, and IgG was isolated from the sera using a protein G-Sepharose 4FF column (Amersham Biosciences). Anti-V5 mAb-conjugated agarose was obtained from Sigma-Aldrich. Anti-V5 polyclonal Ab was purchased from Medical & Biological Laboratories.

Binding of biotinylated sTLR4 to MD-2

sTLR4 was biotinylated using sulfo-N-hydroxysuccinimide-biotin (Pierce), according to the manufacturer’s instructions. Biotinylated sTLR4 (1.5 µg) was mixed with or without sMD-2 (1 µg) in PBS containing 10% FCS and incubated at 37°C for 2 h. Streptavidin-agarose (50 µl, 50% suspension in PBS; Invitrogen Life Technologies) was then added into the reaction mixture and incubated at 4°C for 2 h. After the incubation, the agarose beads were washed with PBS containing 0.1% Triton X-100. The final pellets were subjected to SDS-PAGE, and the proteins on gel were transferred onto polyvinylidene difluoride membrane. Western blot analysis was performed to detect sMD-2 and sTLR4 by using anti-V5 polyclonal Ab and anti-sTLR4 polyclonal Ab, respectively.

Binding of biotinylated sTLR4 to [3H]lipid A

Biotinylated sTLR4 (750 ng) or BSA (750 ng) was incubated with the indicated amounts of [3H]lipid A in the presence or the absence of sMD-2 (500 ng) in PBS containing 10% FCS for 2 h at 37°C. Streptavidin-agarose beads were then added into the mixture, and the suspension (500 µl) was further incubated for 6 h. After the incubation, the agarose beads were washed with PBS containing 0.1% Triton X-100. The radioactivities of the final pellets were determined by a liquid scintillation counter.

Binding of sMD-2 to sTLR4

V5 tag-sMD-2 (100 ng) was mixed with or without sTLR4 (2.5 µg) in PBS containing 10% FCS and incubated at 37°C for 2 h. Anti-V5 mAb-agarose (10 µl, 50% suspension in PBS) was then added into the suspension, and total volume of the suspension was adjusted to 500 µl by the addition of PBS containing 3% BSA. The suspension was incubated at 4°C for 6 h with gentle rocking. After the incubation, the agarose beads were washed with PBS containing 0.1% Triton X-100. The final pellets were subjected to SDS-PAGE, and the proteins on gel were transferred onto polyvinylidene difluoride membrane. Western blot analysis was performed to detect sMD-2 and sTLR4 by using anti-V5 polyclonal Ab and anti-sTLR4 polyclonal Ab, respectively.

Binding of sMD-2 to [3H]lipid A

sMD-2 (100 ng) or BSA (100 ng) was incubated at 37°C for 2 h with the indicated amounts of [3H]lipid A in the presence or the absence of sTLR4 in PBS containing 10% FCS. Anti-V5 mAb-conjugated agarose (10 µl, 50% suspension in PBS) was then added into the suspension, and total volume of the suspension was adjusted to 500 µl by the addition of PBS containing 3% BSA. The suspension was incubated at 4°C for 6 h with gentle rocking. After the incubation, the agarose beads were washed with PBS containing 0.1% Triton X-100. The radioactivities of the final pellets containing agarose beads were measured by a liquid scintillation counter.

Effects of sTLR4 and sMD-2 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 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 (5 µg/ml) and/or sMD-2 (0.25 µg/ml) that had been preincubated for 1 h with the indicated concentrations of LPS. After the LPS stimulation, the concentrations of IL-8 secreted from U937 cells were determined by ELISA using an OptEIA human IL-8 set (BD Pharmingen) according to the manufacturer’s instructions.

Effects of sTLR4 and sMD-2 on LPS-induced NF-{kappa}B activation in HEK293 cells cotransfected with TLR4 and MD-2

Activation of NF-{kappa}B was measured, as previously described (14, 15). HEK293 cells were plated at 0.15 x 105 cells/well in 96-well plates on the day before transfection. The cells were transiently transfected by FuGENE 6 transfection reagent (Roche Molecular Biochemicals) with 7.5 ng of an NF-{kappa}B reporter construct (pNF-{kappa}B-Luc; Stratagene) and 3 ng of a construct directing expression of Renilla luciferase (pRL-TK; Promega), together with 10 ng of each cDNA for wild-type (wt) TLR4 and wt MD-2. Twenty-four hours after transfection, the cells were stimulated for 6 h with 4 ng/ml LPS that had been preincubated with sMD-2 (0.25 µg/ml) and/or sTLR4 (5 µg/ml) for 1 h, and luciferase activity was measured by the dual luciferase reporter assay system (Promega), according to the manufacturer’s instructions.

Cell surface binding of Alexa-conjugated LPS

RAW264.7 cell and HEK293 cell line expressing TLR4, MD-2, and CD14 (InvivoGen) (1 x 107 cells/ml) were incubated at 4°C for 45 min with Alexa Fluor 488-conjugated LPS (0.5 µg/ml, LPS from Escherichia coli 055:B5; Molecular Probes) that had been preincubated with sTLR4 (10 µg/ml) and sMD-2 (10 µg/ml) at 37°C for 30 min in RPMI 1640 or DMEM containing 10% FCS. After the incubation, the cells were washed with PBS containing 0.5% BSA. The binding of LPS to cell surface was analyzed using FACSCalibur and CellQuest software (BD Biosciences).

Expression of cell surface TLR4 in HEK293 cells used in the experiments was confirmed by using PE-conjugated anti-TLR4 Ab (eBioscience).

Murine model of LPS-induced pulmonary inflammation

Female BALB/c mice from 7 to 8 wk of age were anesthetized by i.p. injection of 90 mg/kg ketamine HCl and 20 µg/kg xylazine hydrochloride. LPS (50 µg/kg in 20 µl of PBS) was instilled intratracheally with sTLR2 (150 µg/kg), sTLR4 (150 µg/kg), sMD-2 (125 µg/kg), or sTLR4 plus sMD-2, after tracheostomy. Sixteen hours after LPS instillation, mice were euthanized and a stainless 18G tube was inserted as a tracheostomy cannula. The lungs were lavaged via the tracheal tube with Hanks’ balanced solution (1 x 1 ml; Sigma-Aldrich). The volume of collected bronchoalveolar lavage fluids was measured in each sample, and the number of leukocytes was counted. Differential cell counts were performed by counting at least 300 cells on cytocentrifuged preparations (Autosmear; Sakura) after May-Giemsa staining. The concentrations of TNF-{alpha} in the lavage fluids were determined by ELISA using an OptEIA mouse TNF-{alpha} set (BD Pharmingen), according to the manufacturer’s instructions. To assess histological change of LPS-induced lung injury, lungs were inflated through the trachea with 1 ml of 10% formalin and fixed in 10% formalin by immersion for 24 h. The lung lobes were then washed for 8 h with tap water to remove formaldehyde. For light microscopy, the lung tissue was dehydrated with graded ethanol, put into xylene for 20 min, and then embedded in paraffin at 60°C. A series of 4.0-µm sections were cut and stained with H&E.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
sTLR4 plus sMD-2 is effective as an LPS antagonist

A previous study (9, 16) from this laboratory has revealed that the exogenously added TLR4 protein down-regulates LPS-induced signaling and cytokine release in TLR4-expressing cells. Thus, we first examined the efficiency of sTLR4 and/or sMD-2 in attenuating LPS-induced cell responses. Addition of sTLR4 alone into the culture medium did not affect LPS-induced IL-8 production (Fig. 1A). sMD-2 tended to decrease IL-8 secretion from U937 cells slightly. When both sTLR4 and sMD-2 were added, IL-8 secretion was significantly inhibited at all concentrations of LPS tested.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. Addition of sTLR4 with sMD-2 down-regulates LPS-induced cell responses in TLR4- and MD-2-expressing cells. A, IL-8 secretion from differentiated U937 cells. Differentiated U937 cells (1 x 105) were incubated for 6 h with or without sMD-2 (0.25 µg/ml) and/or sTLR4 (5 µg/ml) that had been preincubated with the indicated concentrations of LPS at 37°C for 1 h. After the LPS stimulation, the concentrations of IL-8 secreted into the medium were determined by ELISA. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with the experiments without sTLR4 or sMD-2 (none). B, NF-{kappa}B activation in HEK293 cells cotransfected with wt TLR4 and wt MD-2. HEK293 cells (0.15 x 105) were transfected with TLR4 cDNA and MD-2 cDNA together with an NF-{kappa}B reporter plasmid (pNF-{kappa}B-Luc) and Renilla luciferase control reporter plasmid (pRL-TK). Twenty-four hours after transfection, the cells were incubated for 6 h with or without sMD-2 (0.25 µg/ml) and/or sTLR4 (5 µg/ml) that had been preincubated with LPS (4 ng/ml) at 37°C for 1 h. NF-{kappa}B activities were then determined. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with the experiments with LPS alone.

 
We next examined the effects of sTLR4 and/or sMD-2 on LPS-induced NF-{kappa}B activation in HEK293 cells cotransfected with wt TLR4 and wt MD-2. LPS elicited NF-{kappa}B activation in these cells. Addition of sTLR4 alone or sMD-2 alone did not significantly alter LPS-induced NF-{kappa}B activation (Fig. 1B). In contrast, LPS-elicited NF-{kappa}B activation was significantly inhibited when both sTLR4 and sMD-2 were added.

Taken together, these results clearly demonstrate that the exogenously added sTLR4 and sMD-2 significantly attenuate LPS-induced cell responses in vitro in TLR4- and MD-2-expressing cells, and indicate that addition of sTLR4 plus sMD-2 is effective as an LPS antagonist.

The role of the extracellular TLR4 domain in interaction of lipid A with MD-2

We next pursued the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. sTLR4 and sMD-2 were expressed by baculovirus-insect cell system, and recombinant proteins were purified from the medium by a column of nickelnitrilotriacetic acid beads. The purified proteins of sTLR4 and sMD-2 exhibited bands with apparent molecular masses of 80 and 23–30 kDa, respectively, under reducing conditions when analyzed by electrophoresis, as described previously (9). We first examined whether the extracellular TLR4 domain directly binds lipid A using biotinylated sTLR4 and streptavidin-agarose. Streptavidin-agarose coprecipitated the biotinylated sTLR4 protein (Fig. 2A). When biotinylated sTLR4 was incubated with increasing amounts of [3H]lipid A and was coprecipitated with streptavidin-agarose, almost no lipid A was associated with the precipitates of sTLR4 and the beads (Fig. 2B). The results clearly demonstrate that the extracellular TLR4 domain does not directly bind lipid A by itself. In contrast, a complex of sTLR4 and sMD-2, which was coprecipitated with the streptavidin-agarose (Fig. 2A), precipitated significant amounts of [3H]lipid A in a manner dependent upon lipid A concentration (Fig. 2B), indicating that the sTLR4-sMD-2 complex can bind lipid A.


Figure 2
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 2. sTLR4 does not directly bind lipid A by itself. A, sMD-2 is coprecipitated with biotinylated sTLR4. Biotinylated sTLR4 (b-sTLR4, 1.5 µg) was mixed with or without sMD-2 (1 µg) and incubated at 37°C for 2 h. Streptavidin-agarose was then added into the reaction mixture and incubated at 4°C for 2 h. After the incubation, the agarose beads were washed and the final pellets obtained were subjected to SDS-PAGE, and Western blot (WB) analysis was performed to detect sMD-2 and sTLR4 by using anti-V5 Ab and anti-sTLR4 Ab, respectively. B, Biotinylated sTLR4 (750 ng) or BSA (750 ng) was incubated with the indicated amounts of [3H]lipid A in the presence or the absence of sMD-2 (500 ng) at 37°C for 2 h. Streptavidin-agarose beads were then added into the mixture, and the suspension was further incubated at 4°C for 6 h. After the incubation, the agarose beads were washed and the radioactivities of the final pellets were determined by a liquid scintillation counter. The data shown are the means ± SE from three separate experiments. {diamond}, sTLR4; {diamondsuit}, BSA; •, sTLR4 plus sMD-2.

 
We next examined the binding of sMD-2 to lipid A using anti-V5 mAb-conjugated agarose beads. When sMD-2 and sTLR4 were coincubated and sMD-2 possessing V5 tag was immunoprecipitated, sTLR4 was coprecipitated with sMD-2 (Fig. 3A). The result confirms the direct binding of the extracellular TLR4 domain to MD-2, and indicates the formation of TLR4-MD-2 complex.


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 3. sTLR4 enhances the binding of [3H]lipid A to sMD-2. A, sTLR4 is coprecipitated with sMD-2. sMD-2 (100 ng) possessing V5 tag was mixed with or without sTLR4 (2.5 µg) and incubated at 37°C for 2 h. Anti-V5 mAb-conjugated agarose beads were then added into the reaction mixture, and the suspension was further incubated at 4°C for 6 h with gentle rocking. After the incubation, the agarose beads were washed and the final pellets obtained were subjected to SDS-PAGE. Western blot (WB) analysis was performed to detect sMD-2 and sTLR4 by using anti-V5 Ab and anti-sTLR4 Ab, respectively. B, The binding of [3H]lipid A to sMD-2 in the presence or the absence of sTLR4. sMD-2 (100 ng) was incubated with 250 ng of [3H]lipid A in the absence or the presence of increasing amounts (0.31, 0.62, 1.25, 2.5, and 5 µg) of sTLR4 at 37°C for 2 h. Anti-V5 mAb-conjugated agarose beads were then added into the reaction mixture. After the suspension was incubated at 4°C for 6 h with gentle rocking, the agarose beads were washed and the radioactivities of the final pellets were determined by a liquid scintillation counter. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with the experiments in the absence of sTLR4. C, Concentration-dependent binding of lipid A to sMD-2. sMD-2 (100 ng) or BSA (100 ng) was incubated with the indicated amounts of [3H]lipid A in the presence or the absence of sTLR4 (2.5 µg) at 37°C for 2 h. Anti-V5 mAb-conjugated agarose beads were then added, and the amounts of lipid A binding to the immunoprecipitated sMD-2 were determined, as described above. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with the experiments with sMD-2 alone. {square}, sMD-2; {diamond}, BSA; •, sTLR4 plus sMD-2. D, Scatchard plot analysis of the data in C. •, sTLR4 plus sMD-2; {square}, sMD-2. E and F, Electrophoretic analysis of sMD-2. E, Five micrograms of rsMD-2 was resolved by 13% SDS-PAGE under reducing (R) and nonreducing (NR) conditions, and the proteins were visualized by Coomassie brilliant blue staining. F, For native PAGE analysis, 15 µg of sMD-2 was subjected to a NativePAGETM Novex 4–16% bis-Tris gel (Invitrogen Life Technologies). St, Molecular mass standards.

 
When sMD-2 was incubated with [3H]lipid A in the absence of sTLR4 and [3H]lipid A binding to the immunoprecipitated sMD-2 was determined, sMD-2 coprecipitated significant amounts of [3H]lipid A (Fig. 3B), indicating direct binding of lipid A to MD-2. Addition of sTLR4 significantly increased the amounts of [3H]lipid A binding to the immunoprecipitated sMD-2, which was considered as a complex with sTLR4. The increase in the amounts of [3H]lipid A binding to the immunoprecipitated sMD-2 depended on the amount of sTLR4 added. When 2.5 µg of sTLR4 was incubated, the amount of lipid A associated with sMD-2 increased by a factor of 2.1 compared with that in the absence of sTLR4. These results clearly demonstrate that the extracellular TLR4 domain augments the binding of [3H]lipid A to a complex with sMD-2.

We next determined whether sTLR4 alters the properties of [3H]lipid A binding to sMD-2. Various concentrations of [3H]lipid A were incubated with sMD-2 in the presence or the absence of sTLR4, and [3H]lipid A binding to the immunoprecipitated sMD-2 was determined. When BSA was used instead of sMD-2, no [3H]lipid A was precipitated. Lipid A bound to sMD-2 in a concentration-dependent manner, and its binding reached saturation at ~250 ng of [3H]lipid A (Fig. 3C). Consistent with the results obtained (Fig. 2B), addition of sTLR4 into the reaction mixture containing sMD-2 significantly increased the binding of lipid A to the immunoprecipitated sMD-2 at all concentrations of lipid A tested (Fig. 3C). The slope of the lipid A binding to sMD-2 at low concentrations of lipid A in the presence of sTLR4 is clearly steeper than that in the absence of sTLR4, suggesting that sTLR4 increases the affinity of lipid A for the binding. Thus, the binding of lipid A to sMD-2 was further analyzed by Scatchard plot (17) (Fig. 3D). When sTLR4 was absent, the maximum amount of lipid A binding to sMD-2 was ~0.75 ng, and the plot gave an apparent dissociation constant, KD = 71.4 nM. This was calculated from the slope of the Scatchard plot and the molecular mass of [3H]lipid A (1842 Da). Addition of sTLR4 apparently increased the maximum lipid A binding and its affinity. The maximum binding of lipid A to the immunoprecipitated sMD-2 in the presence of sTLR4 increased ~2-fold. The KD in the presence of sTLR4 was calculated as 30.8 nM. These results demonstrate that sTLR4 increases the amount of lipid A binding and the affinity of lipid A for the binding, indicating an important role of the extracellular TLR4 domain in LPS-MD-2 interaction.

When analyzed by SDS-PAGE, sMD-2 migrated at 23–25 kDa under reducing conditions (Fig. 3E, R), and showed various degrees of oligomers under nonreducing conditions (Fig. 3E, NR). A monomeric sMD-2 appeared as a major band. However, native PAGE analysis (Fig. 3F) revealed that most of the sMD-2 existed in aggregated forms and that the monomer population was very small (arrowhead).

Soluble CD14 (sCD14) is required for sTLR4-stimulated lipid A binding to sMD-2, but does not form a complex with sMD-2 and sTLR4

The experiments shown above were performed in the presence of FCS. Thus, we examined whether serum factors including sCD14 and LBP are required for the sTLR4-mediated increase of lipid A binding to sMD-2. When sMD-2 was incubated with [3H]lipid A in the presence of sTLR4 in PBS containing 10% FCS or PBS containing 3% BSA, the enhanced binding of lipid A to the immunoprecipitated sMD-2 was not observed under serum-free condition (Fig. 4A). These results indicate that FCS is critical for sTLR4-mediated increase of lipid A to sMD-2.


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 4. sCD14 is required for the sTLR4-stimulated binding of lipid A to sMD-2. A, FCS is required for the sTLR4-stimulated lipid A binding to sMD-2. sMD-2 (100 ng) and sTLR4 (2.5 µg) were incubated with [3H]lipid A (125 ng) in the presence of 10% FCS (FCS(–)) or 3% BSA (FCS–) for 2 h at 37°C. Anti-V5 mAb-conjugated agarose beads were then added, and the amounts of lipid A binding to the immunoprecipitated sMD-2 were determined, as described in Fig. 3B. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with FCS(+). B, Effect of sCD14 on the lipid A binding to sMD-2. sMD-2 (100 ng) was incubated with [3H]lipid A (125 ng) and the indicated amounts of sCD14 in the presence (•) or the absence ({square}) of sTLR4 (2.5 µg) in PBS containing 3% BSA at 37°C for 2 h. The amounts of lipid A binding to the immunoprecipitated sMD-2 were determined, as described in Fig. 3B. The data shown are the means ± SE from three separate experiments. *, p < 0.05 when compared with sMD-2. C, Concentration-dependent binding of lipid A to sMD-2 in the presence of sTLR4. sMD-2 (100 ng) and sTLR4 (2.5 µg) were incubated with the indicated amounts of [3H]lipid A in the presence (•) or the absence ({square}) of sCD14 (2.5 µg) in PBS containing 3% BSA at 37°C for 2 h. The amounts of lipid A binding to the immunoprecipitated sMD-2 were determined as described in Fig. 3B. The data shown are the means ± SE from three separate experiments. All values in the experiments with sCD14(+) are significantly (p < 0.05) higher than those with sCD14(–). D, sCD14 is not coprecipitated with sTLR4-sMD-2 complex regardless of the presence of LPS. V5-tagged sMD-2 (100 ng) and sCD14 (2.5 µg) were mixed with or without sTLR4 (2.5 µg) or LPS (1 µg) in PBS containing 3% BSA and incubated at 37°C for 2 h. Anti-V5-agarose beads (10 µl, 50% suspension in PBS) were then added into the mixture. After the incubation, the agarose beads were washed; the final pellets obtained were subjected to SDS-PAGE; and Western blot analysis was performed.

 
Because FCS contains sCD14 and LBP, we examined whether addition of sCD14 or LBP restored the sTLR4-enhanced lipid A binding to sMD-2 in the absence of FCS. Addition of sCD14 significantly increased the binding of [3H]lipid A to sMD-2 in the presence of sTLR4 in a manner dependent upon sCD14 concentration, although sCD14 did not affect the lipid A binding to sMD-2 in the absence of sTLR4 (Fig. 4B). When the binding study with various concentrations of lipid A was performed in the presence of 2.5 µg of sTLR4, the binding of lipid A to sMD-2 was concentration dependent and saturable regardless of the presence of sCD14 (Fig. 4C). However, the amount of lipid A binding to sMD-2 in the presence of sCD14 was significantly greater than that in the absence of sCD14 at all concentrations of lipid A tested. In contrast, LBP did not significantly alter the binding of [3H]lipid A to sMD-2 in the presence or the absence of sTLR4 (data not shown). Taken together, these results demonstrate that sCD14, but not LBP, is required for sTLR4-stimulated lipid A binding to sMD-2.

We next investigated whether sCD14 is coprecipitated with sMD-2 or a complex of sMD-2 and sTLR4. sMD-2 was incubated with or without sTLR4 and/or LPS, and was immunoprecipitated, and it was examined whether sTLR4 and sCD14 were coprecipitated (Fig. 4D). Although sTLR4 was coprecipitated with sMD-2 in the presence or the absence of LPS, sCD14 was not detected in the immunoprecipitate containing sMD-2. These results indicate that sMD-2 can form a complex with sTLR4, but not with sCD14.

Addition of sTLR4 and sMD-2 inhibits LPS binding on cell surface

Because addition of sTLR4 plus sMD-2 was the most effective in inhibiting LPS-elicited signaling and cytokine release, as shown in Fig. 1, we next examined whether addition of sTLR4 plus sMD-2 affects cell surface binding of Alexa-conjugated LPS on RAW264.7 cell (Fig. 5A) and HEK293 cell line expressing TLR4, MD-2, and CD14 (Fig. 5B). When 0.5 µg/ml Alexa-conjugated LPS was incubated with the cells at 4°C, a significant labeling of LPS was observed on the surface of these cell lines (Fig. 5, solid line). Inclusion of sTLR4 and sMD-2 in LPS-containing medium inhibited cell surface labeling of Alexa-conjugated LPS in these cells (Fig. 5, dotted line). These results clearly demonstrate that the sTLR4-sMD-2 complex inhibits cell surface binding of LPS, and suggest that the exogenously added sTLR4-sMD-2 complex competes with membrane-bound receptor complex of TLR4 and MD-2 for LPS binding.


Figure 5
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 5. Addition of sTLR4 and sMD-2 inhibits the binding of Alexa-conjugated LPS on cell surface. RAW264.7 cell (A) and HEK293 cell line (B) expressing TLR4, MD-2, and CD14 (1 x 107 cells/ml) were incubated at 4°C for 45 min with Alexa Fluor 488-conjugated LPS (0.5 µg/ml, LPS from E. coli 055:B5) that had been preincubated with sTLR4 (10 µg/ml) and sMD-2 (10 µg/ml) at 37°C for 30 min. After the incubation, the cells were washed with PBS containing 0.5% BSA. The binding of LPS on cell surface was analyzed by using FACSCalibur and CellQuest software (both BD Biosciences). The histograms shown are representatives from three experiments. Gray shadow, control without Alexa-LPS; solid line, Alexa-LPS; dotted line, sTLR4 plus sMD-2 plus Alexa-LPS.

 
Intratracheal instillation of sTLR4 and sMD-2 dampens LPS-induced pulmonary inflammation in vivo

Intratracheal instillation of LPS causes pulmonary inflammation. Thus, we examined whether coadministration of sTLR4 and sMD-2 with LPS protects the lungs against LPS-induced pulmonary inflammation. LPS instillation induced interstitial edema, neutrophil influx, and hemorrhage in the lung tissue (Fig. 6B), whereas PBS-treated murine lungs did not show any findings indicative of pulmonary inflammation (Fig. 6A). When sTLR4 and sMD-2 were coadministered with LPS, the histological findings indicative of LPS-induced inflammation were not observed (Fig. 6C).


Figure 6
View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 6. Intratracheal instillation of sTLR4 and sMD-2 improves LPS-induced pulmonary inflammation in mice. Female BALB/c mice at 7–8 wk of age were anesthetized, and LPS (50 µg/kg) was instilled intratracheally with (C) or without (B) sTLR4 (150 µg/kg) plus sMD-2 (125 µg/kg) after tracheostomy. PBS was also instilled instead of LPS as control (A). Sixteen hours after LPS instillation, lungs were fixed by intratracheal instillation of 1 ml of 10% formalin and then embedded in paraffin. Sections were stained with H&E, and viewed by light microscopy at x200 magnification. D and E, Sixteen hours after LPS instillation with or without sTLR2, sTLR4, sMD-2, or sTLR4 plus sMD-2, bronchoalveolar lavage was performed with 1 ml of HBSS. The number of neutrophil (D) in the lavage fluids was counted after May-Giemsa staining. The concentrations of TNF-{alpha} (E) in the lavage fluids were determined by ELISA. The data shown are the means ± SE from 10–12 experiments. *, p < 0.05 when compared with the experiments with LPS alone.

 
We also analyzed inflammatory indicators obtained from bronchoalveolar lavage fluids after LPS challenge. Intratracheal LPS instillation induced neutrophil infiltration and increased TNF-{alpha} in the lavage fluids (Fig. 6, D and E). When sTLR2, sTLR4, or sMD-2 was separately administered intratracheally, no significant reduction of neutrophil infiltration or TNF-{alpha} level was observed. In contrast, coadministration of sTLR4 and sMD-2 resulted in significant decreases of neutrophil infiltration and TNF-{alpha} concentration in the lavage fluids. These results indicate that a complex of sTLR4 and sMD-2 modulates TLR4-mediated signaling to minimize acute lung inflammation caused by LPS challenge to murine lung.

Taken together, the results indicate that intratracheal instillation of sTLR4 with sMD-2 dampens LPS-induced pulmonary inflammation in vivo, and suggest that the exogenous administration of sTLR4 and sMD-2 can be used as a new therapeutic strategy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we pursued the possibility that our recombinant soluble form of the extracellular TLR4 domain can modulate LPS-induced inflammation in cooperation with sMD-2. We have shown that the complex of sTLR4 and sMD-2 is the most potent inhibitor for LPS-induced IL-8 release and NF-{kappa}B activation in U937 cells and HEK293 cells cotransfected with TLR4 and MD-2 when compared with sTLR4 or sMD-2 alone (see Fig. 1). A recent study (16) has shown that TLR4-Fc fusion protein down-regulates LPS-induced cellular responses in TLR4-expressing cells. Streptavidin fails to pull down wt TLR4 in the presence of TLR4-Fc when the binding of biotinylated LPS to MD-2 is used as a means to precipitate wt TLR4, suggesting that the TLR4-Fc fusion protein inhibits the ability of MD-2 to bind TLR4. In this study, we have shown that the presence of sTLR4 plus sMD-2 inhibits LPS binding to the cells expressing TLR4 and MD-2 (see Fig. 5). Thus, the mechanism of down-regulation by sTLR4 plus sMD-2 is suggested to be competition for LPS binding between sTLR4-sMD-2 complex and membrane-bound receptor complex of wt TLR4 and MD-2. In addition, the in vivo experiments demonstrate that intratracheal instillation of sTLR4 with sMD-2 dampens LPS-induced pulmonary inflammation (see Fig. 6). Although sMD-2 alone can bind LPS by itself, it does not exhibit strong inhibition of LPS-induced inflammation in vitro and in vivo. Because the affinity of lipid A for sMD-2 binding and the amount of lipid A binding to MD-2 in the absence of sTLR4 are less than those in the presence of sTLR4 (see Fig. 3), sMD-2 by itself may not be enough to compete with the receptor complex of wt TLR4 and wt MD-2 for LPS recognition. Addition of both sTLR4 and sMD-2 results in strong inhibition of LPS-induced inflammation, presumably because sTLR4 dramatically increases the affinity and the amount of LPS binding, which becomes sufficient to compete with wt TLR4-MD-2 complex for LPS binding.

This study clearly demonstrates that the extracellular TLR4 domain does not directly bind lipid A by itself. This is consistent with previous studies showing that TLR4 cannot transmit LPS signaling without MD-2 (4) and that the extracellular TLR4 domain alone is not coprecipitated with LPS-conjugated beads (9). However, this is in stark contrast to TLR2. Transfection of TLR2 cDNA into HEK293 cells confers responsiveness to peptidoglycan and zymosan (18, 19), and the extracellular TLR2 domain directly binds to these ligands (14, 20). Although it is now well recognized that interaction of TLR4 with MD-2 is critical for recognition and signaling of LPS, little is known about the role of the extracellular TLR4 domain. Thus, in this study, we focused on the extracellular TLR4 domain in interaction with MD-2 and lipid A. We have demonstrated that the extracellular TLR4 domain increases lipid A binding to MD-2 in terms of the affinity and the amount of binding, implicating the extracellular TLR4 domain in LPS-MD-2 interaction. One recent study (21) has shown that lipid A interacts with cell surface TLR4-MD-2 with higher affinity than MD-2 or CD14 expressed on the cell surface. The binding of MD-2 to TLR4 is dependent upon Cys95 and Cys105 of MD-2 that form a interchain disulfide bond (22, 23). The MD-2 region of amino acid residues 119–132, which is rich in basic and aromatic residues, has been found to be responsible for LPS responsiveness (23, 24), indicating that separate functional domains of MD-2 mediate TLR4 binding and LPS responsiveness. Thus, the binding of TLR4 to MD-2 may cause a conformational change that brings greatly increased lipid A binding into the MD-2 molecule. In contrast, there is also a possibility that TLR4 acquires a lipid A-binding property after a complex of TLR4 and MD-2 is formed, although we have demonstrated that the extracellular TLR4 domain does not directly bind lipid A by itself, at least not in the absence of MD-2. The region of leucine-rich repeats in the extracellular TLR4 domain may still be available for ligand binding even after MD-2 binds to TLR4 because the N-terminal region of TLR4 containing no leucine-rich repeats has been shown to be responsible for MD-2 binding (25, 26).

Plasma from patients with severe sepsis and septic shock and even normal plasma appears to contain soluble MD-2 that elicits LPS activation in TLR4-transfected HEK293 cells and HUVECs (16, 27), whereas the patients’ plasma tends to somewhat decrease, although not significantly, LPS responsiveness in leukocytes and differentiated THP-1 cells expressing both TLR4 and MD-2 on cell surface. Previous studies (6, 28) have shown that soluble MD-2 interacts with the extracellular TLR4 domain and confers LPS responsiveness on TLR4-transfected cells. In contrast, this study shows that addition of soluble MD-2 significantly down-regulates LPS-elicited responses in vitro and in vivo when it is coadministered with sTLR4. Taken together, these studies indicate that the opposing effects of the activities of soluble MD-2 depend on the extracellular TLR4 domain.

Only a small amount of lipid A bound to sMD-2 and a complex of sMD-2 plus sTLR4 when 500-2000 ng of lipid A was added (see Figs. 2B and 3, B and C). Native PAGE analysis indicated that most of the sMD-2 existed as noncovalent aggregates and that there was a very small population of monomeric sMD-2 (see Fig. 3F). Because only the monomeric form of MD-2 can interact with LPS (16), the small amount of lipid A binding to sMD-2 may have been due to the fact that there was little monomer in our sMD-2 preparation.

sTLR4 (5 µg/ml) plus sMD-2 (0.25 µg/ml) significantly down-regulates IL-8 secretion from differentiated U937 cells stimulated with low concentrations (~25 ng/ml) of LPS (see Fig. 1A). When LPS concentrations were increased up to 0.1 and 1 µg/ml, sTLR4 plus sMD-2 failed to inhibit IL-8 secretion. Much higher concentrations of sTLR4 and sMD-2 may be required to inhibit the cytokine production elicited with high doses of LPS. If the proportion of sMD-2 monomer in our preparation increases, it may be possible to efficiently down-regulate the cytokine production induced by high doses of LPS, because our preparation contains only a small population of sMD-2 monomer (see Fig. 3F) and only the MD-2 monomer can interact with LPS (16). There is also a possibility that high concentrations of LPS transmit signaling through other mechanisms besides the TLR4-MD-2-mediated mechanism.

We also tested whether sTLR4 plus sMD-2 modulates serum TNF-{alpha} concentration after i.p. LPS injection. Peritoneal injection of LPS (250 µg/kg) into mice increased serum TNF-{alpha} concentration up to 68.7 ± 15.1 pg/ml (means ± SE; n = 19), resulting in systemic inflammation. When sTLR4 and sMD-2 were coadministered with LPS, serum TNF-{alpha} levels were decreased to 40.1 ± 16.3 pg/ml (n = 11), suggesting the possibility that sTLR4 plus sMD-2 could become a target for the development of a novel therapy against systemic inflammation caused by a TLR4-mediated mechanism. Further studies to establish appropriate doses and ratios of the recombinant proteins to be used and to determine the increase of sMD-2 monomer population in the recombinant protein preparation are required for efficient inhibition of systemic inflammation.

This study demonstrates that sTLR4 enhances the binding of lipid A to sMD-2 and increases the affinity of lipid A for the binding, although sTLR4 does not directly bind lipid A by itself. Coadministration of sTLR4 and sMD-2 inhibits the binding of LPS to the TLR4-expressing cells and down-regulates LPS-induced inflammation in vitro and in vivo, raising the possibility of a new therapeutic strategy for dampening endotoxin-induced pulmonary inflammation.


   Acknowledgment
 
We thank Dr. Kensuke Miyake (University of Tokyo, Tokyo, Japan) for providing us with cDNAs for human TLR4 and human MD-2.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. Back

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 Back

3 Abbreviations used in this paper: sTLR, soluble form of rTLR; HEK, human embryonic kidney; sCD14, soluble CD14; sMD-2, soluble form of rMD-2; wt, wild type. Back

Received for publication May 11, 2006. Accepted for publication August 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, R. A. B. Ezekowitz. 1999. Phylogenetic perspectives in innate immunity. Science 284: 1313-1318. [Abstract/Free Full Text]
  2. Aderem, A., R. J. Ulevitch. 2000. Toll-like receptors in the induction of the innate-immune response. Nature 406: 782-787. [Medline]
  3. Chow, J. C., D. W. Young, D. T. Golenbock, W. J. Christ, F. Gusovsky. 1999. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 274: 10689-10692. [Abstract/Free Full Text]
  4. Shimazu, R., S. Akahsi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, M. Kimoto. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189: 1777-1782. [Abstract/Free Full Text]
  5. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, J. C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 1431-1433. [Abstract/Free Full Text]
  6. Visintin, A., A. Mazzoni, J. A. Spitzer, D. M. Segal. 2001. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc. Natl. Acad. Sci. USA 98: 12156-12161. [Abstract/Free Full Text]
  7. Viriyakosol, S., P. S. Tobias, R. L. Kitchens, T. N. Kirkland. 2001. MD-2 binds to bacterial lipopolysaccharide. J. Biol. Chem. 276: 38044-38051. [Abstract/Free Full Text]
  8. Ulevitch, R. J., P. S. Tobias. 1995. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu. Rev. Immunol. 13: 437-457. [Medline]
  9. Hyakushima, N., H. Mitsuzawa, C. Nishitani, H. Sano, K. Kuronuma, M. Konishi, T. Himi, K. Miyake, Y. Kuroki. 2004. Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. J. Immunol. 173: 6949-6954. [Abstract/Free Full Text]
  10. Iwami, K., T. Matsuguchi, A. Masuda, T. Kikuchi, T. Musikacharoen, Y. Yoshikai. 2001. Naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling. J. Immunol. 165: 6682-6686.
  11. LeBouder, E., J. E. Rey-Nores, N. K. Rushmere, M. Grigorov, S. D. Lawn, M. Affolter, G. E. Griffin, P. Ferrara, E. J. Schiffrin, B. P. Morgan, M. O. Labeta. 2003. Soluble forms of Toll-like receptor (TLR) 2 capable of modulating TLR2 signaling are present in human plasma and breast milk. J. Immunol. 171: 6680-6689. [Abstract/Free Full Text]
  12. Fukase, K., T. Kirikae, F. Kirikae, W.-C. Liu, M. Oikawa, Y. Suda, M. Kurosawa, Y. Fukase, H. Yoshizaki, S. Kusumoto. 2001. Synthesis of 3H-labeled bioactive lipid A analogs and their use for detection of lipid A-binding proteins on murine macrophages. Bull. Chem. Soc. Jpn. 74: 2189-2197.
  13. Saitoh, S., S. Akahsi, T. Yamada, N. Tanimura, M. Kobayashi, K. Knno, F. Matsumoto, K. Fukase, S. Kusumoto, Y. Nagai, et al 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. [Abstract/Free Full Text]
  14. Iwaki, D., H. Mitsuzawa, S. Murakami, H. Sano, M. Konishi, T. Akino, Y. Kuroki. 2002. The extracellular Toll-like receptor 2 domain directly binds peptidoglycan derived from Staphylococcus aureus. J. Biol. Chem. 277: 24315-24320. [Abstract/Free Full Text]
  15. Mitsuzawa, H., I. Wada, H. Sano, D. Iwaki, S. Murakami, T. Himi, N. Matsushima, Y. Kuroki. 2001. Extracellular Toll-like receptor 2 region containing Ser40-Ile64 but not Cys30-Ser39 is critical for the recognition of Staphylococcus aureus peptidoglycan. J. Biol. Chem. 276: 41350-41356. [Abstract/Free Full Text]
  16. Visintin, A., K. A. Halmen, E. Latz, B. G. Monke, D. T. Golenbock. 2005. Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2. J. Immunol. 175: 6465-6472. [Abstract/Free Full Text]
  17. Scatchard, G.. 1949. The attractions of proteins for small molecules and ions. Ann. NY Acad. Sci. 51: 660-672.
  18. Brightbill, H. D., D. H. Libraty, S. R. Krutzik, R.-B. Ynag, J. T. Belisle, J. R. Bleharski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, et al 1999. Host defense mechanism triggered by microbial lipoproteins through Toll-like receptors. Science 285: 732-736. [Abstract/Free Full Text]
  19. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, C. J. Kirschning. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J. Biol. Chem. 274: 17406-17409. [Abstract/Free Full Text]
  20. Sato, M., H. Sano, D. Iwaki, K. Kudo, M. Konishi, H. Takahashi, T. Takahashi, H. Imaizumi, Y. Asai, Y. Kuroki. 2003. Direct binding of Toll-like receptor 2 to zymosan, and zymosan-induced NF-{kappa}B activation and TNF-{alpha} secretion are down-regulated by lung collectin surfactant protein A. J. Immunol. 171: 417-425. [Abstract/Free Full Text]
  21. Akashi, S., S. Saitoh, Y. Wakabayashi, T. Kikuchi, N. Takamura, Y. Nagai, Y. Kusumoto, K. Fukase, S. Kusumoto, Y. Adachi, et al 2003. Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14. J. Exp. Med. 198: 1035-1042. [Abstract/Free Full Text]
  22. Mullen, G. E. D., M. N. Kennedy, A. Visintin, A. Mazzoni, C. A. Leifer, D. R. Davies, D. M. Segal. 2003. The role of disulfide binds in the assembly and function of MD-2. Proc. Natl. Acad. Sci. USA 100: 3929-3924.
  23. Re, F., J. L. Strominger. 2003. Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness. J. Immunol. 171: 5272-5276. [Abstract/Free Full Text]
  24. Mancek, M., P. Pristovsek, R. Jerala. 2002. Identification of LPS-binding peptide fragment of MD-2, a Toll-receptor accessory protein. Biochem. Biophys. Res. Commun. 292: 880-885. [Medline]
  25. Fujimoto, T., S. Yamazaki, A. Eto-Kimura, K. Takeshige, T. Muta. 2004. The amino-terminal region of Toll-like receptor 4 is essential for binding to MD-2 and receptor translocation to the cell surface. J. Biol. Chem. 279: 47431-47437. [Abstract/Free Full Text]
  26. Nishitani, C., H. Mitsuzawa, N. Hyakushima, H. Sano, N. Matsushima, Y. Kuroki. 2005. The Toll-like receptor 4 region Glu24-Pro34 is critical for interaction with MD-2. Biochem. Biophys. Res. Commun. 328: 586-590. [Medline]
  27. Pugin, J., S. Stem-Voeffray, B. Daubeuf, M. A. Matthay, G. Elson, I. Dunn-Siegrist. 2004. Soluble MD-2 activity in plasma from patients with severe sepsis and septic shock. Blood 104: 4071-4079. [Abstract/Free Full Text]
  28. Re, F., J. L. Strominger. 2002. Monomeric recombinant MD-2 binds Toll-like receptor 4 tightly and confers lipopolysaccharide responsiveness. J. Biol. Chem. 277: 23427-23432. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
S. Divanovic, A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, R. Madan, J. J. Haky, and C. L. Karp
Regulation of TLR4 signaling and the host interface with pathogens and danger: the role of RP105
J. Leukoc. Biol., August 1, 2007; 82(2): 265 - 271.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mitsuzawa, H.
Right arrow Articles by Kuroki, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mitsuzawa, H.
Right arrow Articles by Kuroki, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS