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Wall Componentsand Gram-Negative Bacteria and Their CellTLR2-Mediated Responses to Gram-Positive Lipopolysaccharide and Enhances(TLR2)-Mediated Responses to MD-2 Enables Toll-Like Receptor 2

Kirschning and Dipika GuptaRoman Dziarski, Qiuling Wang, Kensuke Miyake, Carsten J.

http://www.jimmunol.org/content/166/3/1938doi: 10.4049/jimmunol.166.3.1938

2001; 166:1938-1944; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/166/3/1938.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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MD-2 Enables Toll-Like Receptor 2 (TLR2)-MediatedResponses to Lipopolysaccharide and EnhancesTLR2-Mediated Responses to Gram-Positive andGram-Negative Bacteria and Their Cell Wall Components1

Roman Dziarski,2* Qiuling Wang, 3* Kensuke Miyake,† Carsten J. Kirschning,‡ andDipika Gupta*

MD-2 is associated with Toll-like receptor 4 (TLR4) on the cell surface and enables TLR4 to respond to LPS. We tested whetherMD-2 enhances or enables the responses of both TLR2 and TLR4 to Gram-negative and Gram-positive bacteria and theircomponents. TLR2 without MD-2 did not efficiently respond to highly purified LPS and LPS partial structures. MD-2 enabledTLR2 to respond to nonactivating protein-free LPS, LPS mutants, or lipid A and enhanced TLR2-mediated responses to bothGram-negative and Gram-positive bacteria and their LPS, peptidoglycan, and lipoteichoic acid components. MD-2 enabled TLR4to respond to a wide variety of LPS partial structures, Gram-negative bacteria, and Gram-positive lipoteichoic acid, but not toGram-positive bacteria, peptidoglycan, and lipopeptide. MD-2 physically associated with TLR2, but this association was weakerthan with TLR4. MD-2 enhanced expression of both TLR2 and TLR4, and TLR2 and TLR4 enhanced expression of MD-2. Thus,MD-2 enables both TLR4 and TLR2 to respond with high sensitivity to a broad range of LPS structures and to lipoteichoic acid,and, moreover, MD-2 enhances the responses of TLR2 to Gram-positive bacteria and peptidoglycan, to which the TLR4-MD-2complex is unresponsive. The Journal of Immunology,2001, 166: 1938–1944.

M ammalian innate immune system recognizes bacteriaand their cell wall components through two pattern-recognition receptors, CD14 (1, 2) and Toll-like re-

ceptors (TLR)4 2 and 4. TLR2, first identified as the cell-activatingreceptor for Gram-negative LPS (3, 4), also serves as the receptorfor both Gram-positive and Gram-negative bacteria, mycobacteria,Mycoplasma, and spirochetes, and their peptidoglycan (PGN), li-poteichoic acid (LTA), lipoarabinomannan, and lipoprotein cellwall or cell membrane components (5–11). TLR2 seems to serveas the primary receptor for Gram-positive bacteria and their cellwall components, whereas TLR4 (but not TLR2) serves as theprimary LPS receptor, because TLR2 (but not TLR4) knockoutmice are unresponsive to Gram-positive bacteria (12), whereasTLR4 (but not TLR2) knockout mice are unresponsive to Gram-negative bacteria and LPS (12, 13). This notion is consistent withthe hyporesponsiveness to LPS, but not to Gram-positive bacteria,of C3H/HeJ and in C57BL/10ScCr mice, which have a mutation in

the Tlr4 gene (14, 15), and with the responsiveness of TLR2-de-ficient cells to LPS, but not to Gram-positive bacteria (16). More-over, in the initial studies the responses in TLR2-transfected non-macrophage cells required much higher concentrations of LPSthan the concentrations needed to activate macrophages (1–6, 17),and most recently highly purified LPS was shown not to stimulatecells through TLR2 (18). Therefore, these results suggested that byitself TLR2 does not function as an LPS receptor.

However, TLR4 by itself does not function as an LPS receptoreither. To function as an LPS receptor, TLR4 requires a helpermolecule, MD-2 (19). MD-2 is a 160-amino acid protein that isassociated with TLR4 on the cell surface and enables TLR4 torespond to LPS (19, 20). The discovery of MD-2 explained whyTLR4-transfected cells (in which MD-2 was not expressed) wereunresponsive to LPS (4–6, 8) and supported the proposed functionof the TLR4-MD-2 complex as the LPS receptor. However, it isnot known whether MD-2 enables TLR4 to respond to Gram-pos-itive bacterial components and also what effect MD-2 has onTLR2-mediated responses. Therefore, the aim of this study was todetermine whether MD-2 functions as a helper molecule for bothTLR2 and TLR4 for the responses to both Gram-positive andGram-negative bacterial components.

Materials and MethodsMaterials

All materials were purchased from Sigma (St. Louis, MO), unless other-wise indicated. Smooth LPS fromEscherichia coliK12 LCD25 (List,Campbell, CA),E. coli O127:B8,E. coli O113 (refined endotoxin standard,Ribi, Hamilton, MT),Salmonella minnesota,Salmonella typhimurium, andShigella flexneri1A were obtained by phenol-water extraction. Rough LPSmutants fromE. coli EH100 (Ra),S. typhimuriumTV119 (Ra),E. coli J5(Rc),S. minnesotaR5 (Rc),S. typhimuriumSL684 (Rc),E. coli F583 (Rd),S. minnesotaR7 (Rd),E. coli K12 D31 m4 (Re) (List),S. minnesotaRe595(Re),S. typhimuriumSL1181 (Re), andS. flexneri(Re) were obtained byphenol-chloroform-petroleum ether extraction. Ra mutants have the entire

*Northwest Center for Medical Education, Indiana University School of Medicine,Gary, IN 46408;†Department of Immunology, Saga Medical School, Saga, Japan;and‡Institute for Medical Microbiology, Immunology, and Hygiene, Technical Uni-versity of Munich, Munich, Germany

Received for publication July 26, 2000. Accepted for publication November 13, 2000.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the U.S. Public Health Service Grant AI2879 from theNational Institutes of Health.2 Address correspondence and reprint requests to Dr. Roman Dziarski, Indiana Uni-versity School of Medicine, 3400 Broadway, Gary, IN 46408. E-mail address:rdziar@iun.edu3 Current address: Washington University School of Medicine, St. Louis, MO 63110.4 Abbreviations used in this paper: TLR, Toll-like receptor; LTA, lipoteichoic acid;PGN, peptidoglycan; sPGN, soluble PGN; JNK-1, c-Jun N-terminal kinase; PC pro-tein C.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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lipid A and core polysaccharide, but are devoid of theO-polysaccharide,Rc mutants are devoid of theO-polysaccharide and four terminal sugars inthe core polysaccharide, Rd mutants are devoid of theO-polysaccharideand five or six terminal sugars in the core polysaccharide, and Re mutantsare devoid of theO-polysaccharide and seven terminal sugars of the corepolysaccharide, with only three KDO units of the core polysaccharide thatremain bound to lipid A. Diphosphoryl lipid A were fromE. coli F-583 andS. minnesotaRe595. Detoxified (delipidized) LPS fromE. coli O127:B8was chromatographically purified. The purity of LPS was analyzed by pe-riodate-modified silver staining of 12% SDS-polyacrylamide gels (17, 21)and by silver-enhanced colloidal gold staining (22) using a Bio-Rad kit(Hercules, CA) as recommended by the manufacturer.

Soluble PGN (sPGN) fromStaphylococcus aureuswas purified by af-finity chromatography and contained,24 pg endotoxin/mg (23). LTAfrom S. aureus,Streptococcus pyogenes, andStreptococcus mutans, pre-pared by phenolic extraction and purified by hydrophobic interaction chro-matography on octyl-Sepharose (17), contained,500 pg endotoxin/mg.Synthetic lipopeptide (Pam3Cys-Ser-Lys4OH) was obtained from Boehr-inger Mannheim (Indianapolis, IN).Micrococcus luteusATCC 4698,Ba-cillus subtilis ATCC 6633,S. aureus845 (all contained,500 pg endo-toxin/mg), E. coli K12, Enterobacter cloaceATCC 13047, SerratiamarcescensATCC 13880,S. typhimuriumATCC 10289, andProteus vul-garis ATCC 13315 were heat killed (70°C, 30 min).

Cell cultures

Human HEK 293 cells were cultured in DMEM with 10% endotoxin-free(,6 pg/ml) FCS as before (5). Stable transfectants of B lymphocytic cellline Ba/F3, expressing human TLR4 and human MD-2, were generated asbefore (19). RAW264.7 cells were stimulated, and supernatants were as-sayed for TNF-a and IL-6 and cell lysates for NF-kB (2, 17).

Transfection and luciferase assay

For cell activation, 293 cells were cultured at 0.3–0.353 106/ml in 48-wellplates (0.25 ml/well) for 16–20 h and transfected with Lipofectamine in aserum-free medium with optimal concentrations of the following plasmids:0.22 mg/ml NF-kB reporter endothelial leukocyte adhesion molecule 1luciferase plasmid (5); 0.027mg/ml human CD14 (5); 0.22mg/ml of Flag-tagged human TLR1, TLR2, or TLR4 (5); and 0.22mg/ml of 6His andFlag-tagged human MD-2 (19). When TLRs, MD-2, or CD14 were notused, equivalent amounts of appropriate control vectors were included. Inaddition, the total DNA concentration was brought up to 1.25mg/ml withsalmon sperm DNA. After 5 h, 10% FCS was added, and, after an addi-tional 16–18 h, fresh medium with 10% FCS was added and cells werestimulated for 6 h as indicated inResults. The lysates were assayed forluciferase activity (5), and the data were expressed as normalized relativeluciferase units.

Immunoprecipitation

Human MD-2 (19) was tagged with protein C epitope followed by the 6Hisepitope and subcloned into the pEFBOS expression vector. For coimmu-noprecipitation experiments, 293 cells were cultured in 10-cm plates (sixper group) until 60–70% confluent and transfected with Lipofectaminewith 0.22–0.44mg/ml of Flag-tagged human TLR2 or TLR4 (5), 0.22mg/ml human Flag-tagged c-Jun N-terminal kinase (JNK)-1 (24), and0.22–0.6mg/ml 6His and protein C (PC)-tagged human MD-2. WhenTLRs or MD-2 were not used, equivalent amounts of appropriate control

vectors were included. In addition, the total DNA concentration wasbrought up to 1.25mg/ml with salmon sperm DNA. Thirty-three hours aftertransfection, the cells were lysed in 1 ml of lysis buffer per plate (0.05 MTris-HCl, pH 8.0, with 0.3 M NaCl, 1% Triton X-100, 10 mM imidazole,and EDTA-free protease inhibitor mixture) at 4°C. Insoluble cell debriswere removed by centrifugation (10,0003 g) and 5 ml of each supernatantwas rotated for 5–10 h with 5ml of anti-Flag (M-2)-agarose (Sigma) foranti-PC Western blot to detect coprecipitated MD-2, 0.5 ml of each super-natant was rotated with 2.5ml of Ni-NTA-agarose (Qiagen, Chatsworth,CA) for anti-PC Western blot to detect the expression of MD-2, and an-other 0.5 ml of each supernatant was rotated with 1.25ml of anti-Flag(M-2)-agarose for anti-Flag Western blot to detect the expression of TLRs.For other immunoprecipitation experiments, done to detect the expressionof each construct, lysates from cells from one 10-cm plate/group wereused. The agarose immunoprecipitates were washed in the lysis buffer andsubjected to SDS-PAGE (12%) and Western blot analysis with anti-FlagM2 (Sigma) or anti-PC (Boehringer Mannheim) primary Abs, peroxidase-labeled anti-mouseg-chain secondary Abs, and ECL-Plus ECL reagent(Amersham Pharmacia Biotech, Piscataway, NJ).

ResultsTLR2 does not efficiently recognize highly purified endotoxicLPS and LPS partial structures

We tested the responsiveness of 293 cells transfected with TLR2 to20 different LPS and LPS partial structures, including a number ofsmooth LPS from various bacterial species, as well as a series ofRa, Rc, Rd, and Re LPS mutants (which have progressively trun-cated polysaccharide portion), lipid A (the minimum endotoxicstructure of LPS), and detoxified LPS. In all experiments, the cellswere also cotransfected with CD14 to mimic the natural expressionof CD14 in monocytic cells. Several LPS preparations inducedvery high responses, whereas other LPS preparations induced in-termediate or very low responses, or did not stimulate the cells atall (Fig. 1). However, there was no correlation between the LPSstructure and the ability to activate cells, e.g., some smooth LPS(E. coli LCD25) and some ReLPS (E. coli) were highly stimula-tory, whereas other smooth LPS (E. coli O113 orS. flexneri), someReLPS (S. minnesotaor S. typhimurium), or lipid A were non-stimulatory or very weakly stimulatory (Fig. 1). All of these prep-arations induced very high secretion of cytokines in mouse mac-rophages or human monocytes (Refs. 1, 2, and 17 and data notshown).

This lack of correlation between the LPS structure and activat-ing capacity suggested that possibly some contaminants couldhave been responsible for the stimulatory activity of these LPSpreparations. Indeed, it was previously reported that three of sixcommercial LPS preparations were contaminated with endotoxinprotein (22), and very recently it was shown that highly purifiedLPS did not stimulate cells through TLR2 (18). We tested thepurity of our LPS preparations with a periodate-silver stain that

FIGURE 1. TLR2 efficiently recog-nizes some, but not all endotoxic LPSand LPS partial structures. 293 cells,transfected with TLR2, CD14, and NF-kB-luciferase reporter plasmids, werestimulated with the indicated LPS prep-arations, and cell lysates were assayedfor luciferase activity. The results aremeans6 SE of three to five experiments.Ec, E. coli; Sm,S. minnesota; St,S. ty-phimurium; Sf,S. flexneri.

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stains both LPS and proteins (with the sensitivity of 10 ng protein/lane) and with a silver-enhanced colloidal gold that stains proteinsonly, but not LPS (with the sensitivity of 0.4 ng protein/lane). Themost stimulatory LPS preparations (ReLPS fromE. coli, LPS fromE. coli LCD25, RaLPS fromE. coli, LPS fromE. coli 0127) hadthe highest protein contamination (Fig. 2), and the extent of con-tamination (Fig. 2) correlated with the stimulating capacity ofthese LPS preparations (Fig. 1). On the other hand, LPS prepara-tions that were not contaminated with proteins (lipid A fromS.minnesota, ReLPS fromS. minnesotaand S. typhimurium, andLPS fromE. coli 0113, which were.99.99% protein free) (Fig. 2)did not stimulate cells through TLR2 (Fig. 1).

These results demonstrate that TLR2 does not efficiently recog-nize highly purified LPS endotoxic structures and are consistent

with the recent report (18) that contaminating endotoxin proteinsmight have been responsible for the previously observed TLR2-activating capacity of LPS.

MD-2 enables TLR2-mediated responses to LPS and LPS partialstructures and enhances TLR2-mediated responses to sPGN,LTA, and both Gram-positive and Gram-negative bacteria

Because activation of cells by LPS through TLR4 requires MD-2(19), we next tested the hypothesis that MD-2 may also enableTLR2 and CD14 to respond to LPS. When 293 cells were cotrans-fected with TLR2, CD14, and MD-2, they became highly respon-sive to all LPS preparations and LPS partial structures (exceptdetoxified LPS), including highly purified protein-free LPS,ReLPS, and lipid A preparations that did not activate or verypoorly activated TLR2/CD14-transfected cells (Fig. 3 and data notshown). In the presence of MD-2, LPS preparations efficiently ac-tivated TLR2/CD14-transfected cells even at 0.1–1 ng/ml (Fig. 4).For example, 0.1 ng/mlS. minnesotaReLPS and 1 ng/mlE. coli0113 LPS yielded about 20% of maximum responses, which ishigher than the response to 10mg/ml of these ReLPS or LPSwithout MD-2 (Fig. 4). Thus, MD-2 enhanced 104–105 times theresponses of TLR2/CD14-transfected cells to LPS and ReLPS.These results demonstrate that MD-2 enables very sensitive TLR2/CD14-mediated recognition of protein-free LPS and LPS partialstructures and enhances responses to all endotoxic LPS.

We next tested the hypothesis that MD-2 may also enhanceTLR2/CD14-mediated responses to sPGN and LTA from Gram-positive bacteria, a synthetic lipopeptide (an analogue of bacteriallipoproteins), as well as intact bacteria. MD-2 highly enhanced theresponses to sPGN, LTA (Fig. 3), and both Gram-positive andGram-negative bacteria (Fig. 5), but not to lipopeptide. Cell acti-vation by Gram-positive bacteria or their cell wall components wasnot due to endotoxin contamination, because these preparationswere endotoxin free and because cells transfected with TLR4,CD14, and MD-2 were unresponsive to these stimuli, whereas theywere highly responsive to endotoxin and Gram-negative bacteria(see next section below).

Cells transfected with TLR2 or TLR2 and MD-2 without CD14showed lower responses to all of the stimulants than cells trans-fected with all three plasmids (data not shown), which confirmsthat CD14 is not indispensable, but that it enhances TLR2-medi-ated responses (5, 6). Cells transfected with MD-2 alone, CD14alone, or with MD-2 and CD14 were unresponsive to all of thestimulants (data not shown), thus confirming the requirement forTLR2 for the above responses and for the enhancing effect ofMD-2. Cells transfected with TLR1, CD14, and MD-2 were alsounresponsive to all of the stimuli tested (data not shown).

MD-2 enables TLR4-mediated responses to LPS, LPS partialstructures, Gram-negative bacteria, and LTA, but not to sPGN,lipopeptide, and Gram-positive bacteria

We next tested whether MD-2 could enable TLR4 to recognize thesame large variety of LPS, LPS partial structures, and other cellwall components, as well as intact Gram-positive and Gram-neg-ative bacteria. None of these stimulants activated cells transfectedwith TLR4 and CD14 without MD-2. However, cells transfectedwith TLR4, CD14, and MD-2 were highly responsive to all LPSand LPS partial structures (except detoxified LPS) (Fig. 6). Effec-tive cell activation could be even achieved with 0.1 ng/ml endo-toxin (data not shown). These cells were also responsive to LTAfrom Gram-positive bacteria, but not to sPGN and lipopeptide(Fig. 6), and were highly responsive to Gram-negative, but not toGram-positive bacteria (Fig. 7). Similarly, a B lymphocytic cell

FIGURE 2. Purity of LPS and LPS partial structures. The indicated LPSpreparations (25mg/lane inA and 15mg/lane inB) were subjected to 12%SDS-PAGE and staining with periodate-sliver stain, which stains LPS andproteins (A), or blotting and staining with enhanced colloidal gold, whichstains proteins only (B). For abbreviations, see the legend to Fig. 1.

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line Ba/F3, stably expressing TLR4 and MD-2 (19), was also un-responsive to sPGN andS. aureus, whereas, as expected (19), itwas highly responsive toE. coli LPS andS. minnesotaReLPS(Fig. 8).

MD-2 associates with both TLR2 and TLR4 and enhancesexpression of TLR2 and TLR4

To begin to understand the mechanism of MD-2 enhancement ofTLR2-mediated responses, we first tested whether MD-2 physi-cally associates with TLR2, because it was earlier shown thatMD-2 physically associates with TLR4 (19, 20). Indeed, in cellstransiently cotransfected with PC-tagged MD-2 and Flag-taggedTLR2 or TLR4, MD-2 could be detected with anti-PC Abs onWestern blots of Flag immunoprecipitates (Fig. 9), thus indicatingphysical association of MD-2 with TLR2 and TLR4. MD-2 wasnot detectable in Flag immunoprecipitates from cells transfectedwith MD-2 alone, control vectors alone, TLR2 or TLR4 alone, orMD-2 and Flag-tagged unrelated molecule, JNK-1 (Fig. 9), thusdemonstrating the specificity of MD-2-TLR association, as well asthe specificity of immunoprecipitation and anti-Flag and anti-PCAbs. However, the association of MD-2 with TLR2 was weakerthan with TLR4, as judged by a much lower amount of MD-2 inthe TLR2 than in the TLR4 coprecipitations, despite equivalentamounts of MD-2, TLR2, and TLR4 expressed in both groups andloaded on the gel (Fig. 9).

FIGURE 4. MD-2 enables TLR2-mediated responses to low concentra-tions of LPS. 293 cells, transfected with TLR2, with or without MD-2, andwith CD14 and NF-kB-luciferase reporter plasmids, were stimulated withthe indicated concentrations of protein-free ReLPS or LPS, and cell lysateswere assayed for luciferase activity. The results are from one of two similarexperiments. For abbreviations, see the legend to Fig. 1.

FIGURE 5. MD-2 enhancesTLR2-mediated responses toboth Gram-positive and Gram-negative bacteria. 293 cellswere transfected as in Fig. 3and stimulated with the indi-cated bacteria. The results aremeans6 SE of three to fiveexperiments.

FIGURE 3. MD-2 enables or enhances TLR2-mediated responses to LPS, LPS partial structures, sPGN, and LTA. 293 cells, transfected with TLR2,CD14, and NF-kB-luciferase reporter plasmids, with (1) or without (2) MD-2 plasmid, were stimulated with the indicated preparations, and cell lysateswere assayed for luciferase activity. The results are means6 SE of three to five experiments. Sa,S. aureus; Sp,S. pyogenes; others as in the legend toFig. 1.

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To look for other possible mechanisms of MD-2 enhancementof TLR2-mediated responses, we also studied the effect of MD-2on the expression of TLR2 and TLR4. Cotransfection of cells withMD-2 and TLR2 or TLR4 greatly enhanced the expression ofTLR2, TLR4, and MD-2 (Fig. 10). This effect was specific forMD-2, TLR2, and TLR4, because cotransfection of cells with an-other unrelated Flag-tagged molecule (JNK-1) or control vectorsdid not enhance the expression of MD-2 (Figs. 9 and 10) or TLR2or TLR4 (data not shown). The enhancement of MD-2, TLR2, andTLR4 expression was observed with both Flag-tagged MD-2 (Fig.10) and PC-tagged MD-2 (Fig. 9 and data not shown).

These results suggest that MD-2 enables or enhances TLR2- andTLR4-mediated responses by physically associating with TLR2and TLR4 and by increasing the amount of expressed TLR2 andTLR4 protein.

DiscussionOur results demonstrate that MD-2 can work not only with TLR4,as discovered earlier (19, 20), but also with TLR2 to enable TLR2to respond to nonactivating or low-activating stimuli, such as pro-tein-free LPS, LPS mutants, or lipid A. MD-2 can also enhance theresponses of TLR2 to both Gram-positive and Gram-negative bac-teria, as well as their sPGN, LTA, and LPS-associated cell wallcomponents that can activate cells through TLR2 without MD-2.Thus, TLR2 is able to respond to several (but not all) stimuli with-out MD-2, and MD-2 enhances its responses to these stimuli,

whereas TLR4 cannot be activated by any of these stimuli withoutMD-2 and, therefore, TLR4 has an absolute requirement for MD-2for cell activation. However, for some ligands (e.g., protein-freeLPS), TLR2 behaves like TLR4, i.e., it also requires MD-2 for cellactivation by these ligands. Therefore, MD-2 both broadens thespectrum of stimulants that can activate cells through TLR2 andenhances the responsiveness of TLR2 to ligands that do not abso-lutely require MD-2 for TLR2-mediated cell activation.

The mechanism of the enhancement of TLR2-mediated re-sponses could be based on both the physical association of MD-2with TLR2 (which, however is much weaker than with TLR4) andon the enhancement by MD-2 of TLR2 protein expression (or itcould be based on either of these mechanisms alone). However, theability of MD-2 to enable the responses of TLR2 and TLR4 to thestimulants that are not active in the absence of MD-2 is unlikely tobe solely due to the enhancement of TLR expression, because cellsoverexpressing TLR2 or TLR4 without MD-2 are unresponsive tothese stimulants (4–6, 18, 19). Therefore, the enabling function ofMD-2 for TLR2- and TLR4-mediated responses is likely to bebased on both the physical association of MD-2 with TLRs and theenhancement of their expression. The exact mechanism of thisenhanced expression is unknown, but, because of the physical as-sociation of TLRs with MD-2, it may rely on a greater stability ofMD-2-TLR complexes, compared with the stability of TLR andMD-2 alone.

FIGURE 6. MD-2 enables TLR4-mediated responses to LPS, LPS partial structures, and LTA, but not to sPGN and lipopeptide. 293 cells, transfectedwith TLR4, CD14, and NF-kB-luciferase reporter plasmids, with (1) or without (2) MD-2 plasmid, were stimulated with the indicated preparations, andcell lysates were assayed for luciferase activity. The results are means6 SE of three to five experiments. Abbreviations: see Figs. 1 and 3.

FIGURE 7. MD-2 enables TLR4-mediated responses to Gram-negative,but not to Gram-positive bacteria. 293cells were transfected as in Fig. 6 andstimulated with the indicated bacteria.The results are means6 SE of three tofive experiments.

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Our data confirm previous results that TLR4 associates withMD-2 and requires MD-2 for the responses to LPS (19, 20) anddemonstrate that MD-2 makes TLR4 highly responsive to a widevariety of endotoxic LPS and LPS partial structures, and also tointact Gram-negative bacteria and isolated LTA from Gram-posi-tive bacteria. They also demonstrate that the TLR4-MD-2 complexdoes not respond to Gram-positive bacteria, their sPGN compo-nent, and lipopeptide. Thus, the TLR4-MD-2 complex has a nar-rower specificity than the TLR2-MD-2 complex. Our data on thespecificity of TLR4 responses are consistent with the experimentson TLR4 knockout mice (12, 13), which showed that TLR4 re-sponds to Gram-negative LPS and Gram-positive LTA. Our dataon the specificity of TLR2 responses are consistent with the pre-vious reports showing TLR2 responsiveness to both Gram-nega-tive LPS and Gram-positive bacteria and their PGN and LTA com-

ponents, as well as lipopeptide (5, 6, 8, 25), and with the recentreport showing unresponsiveness of TLR2 (without MD-2) tohighly purified protein-free LPS (18).

Our results also explain the variable and often low responses orthe lack of responses through TLR2 (in TLR2-transfected cells) tovarious endotoxic LPS preparations (that are much more active inmonocytic cells; Refs. 1, 2, and 17) by showing that TLR2 aloneis unresponsive to highly purified protein-free LPS, LPS mutants,and lipid A, and that the responses to “active” LPS preparationscorrelate with the level of their protein contamination. Our resultsalso extend these observations by showing that in the presence ofMD-2 both TLR2 and TLR4 are highly and equally sensitive to allendotoxic LPS, including protein-free LPS.

However, our results in 293 cells are different from the initialresults obtained in TLR2 and TLR4 knockout mice and inTlr4mutant mice, which showed that TLR4 knockout andTlr4 mutant

FIGURE 8. Ba/F3 cells expressing TLR4 and MD-2 are re-sponsive to LPS, but not to sPGN andS. aureus. Stable Ba/F3transfectants expressing NF-kB reporter luciferase plasmid andTLR4 alone or TLR4 and MD-2 were stimulated with low, me-dium, or high concentrations of sPGN (0.4, 2, or 10mg/ml), S.aureus(0.83 108, 4 3 108, or 20 3108 cells/ml), or LPS (0.1,1, or 10 mg/ml), and cell lysates were assayed for luciferaseactivity. The results are from one of two similar experiments.

FIGURE 9. MD-2 coprecipitates with both TLR2 and TLR4. 293 cellswere transfected with the indicated Flag-tagged TLRs or JNK-1 and/orPC-tagged MD-2, and cell lysates were subjected to immunoprecipitation.MD-2 was detected in anti-Flag TLR2 and TLR4 immunoprecipitates byWestern blotting with anti-PC Abs. The expression of MD-2 was demon-strated in Ni-NTA-agarose precipitates by Western blotting with anti-PCAbs and of TLRs and JNK-1 in anti-Flag immunoprecipitates by Westernblotting with anti-Flag Abs. The results are from one of three similarexperiments.

FIGURE 10. MD-2 enhances expression of TLR2 and TLR4, and TLR2and TLR4 enhance expression of MD-2. 293 cells were transfected withFlag-tagged MD-2, TLR2, TLR4, or JNK-1 alone or in combination, andthe expression of each protein was detected in anti-Flag immunoprecipi-tates by Western blotting with anti-Flag Abs. The results are from one ofthree similar experiments.

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mice, which had an intactTlr2 gene, were unresponsive to LPSand LTA, thus indicating that in these mice TLR2 could not func-tion as an effective LPS and LTA receptor (12–15). This differencemay be due to the inducible expression of TLR2 (26) or due to theinsufficient expression or function of MD-2 in TLR4 knockout andTlr4 mutant mice. This possibility is consistent with much strongerassociation of MD-2 with TLR4 than with TLR2 observed by us.Therefore, the amount of MD-2 in TLR2 knockout mice may besufficient to support the function of TLR4, but in TLR4 knockoutor Tlr4 mutant mice may not be sufficient to support the functionof TLR2. Moreover, in most primary cells, the expression of TLR2is much lower than TLR4 (26, 27) and the expression of TLR2 isinducible by cell stimulation (26). Because the expression of MD-2is enhanced by the expression of TLRs, it is possible that TLR4knockout and mutant mice have insufficient expression of MD-2,because of the lack of the enhancing effect of TLR4 on the ex-pression of MD-2 and because of the low expression of TLR2.Insufficient expression of MD-2 and low expression of TLR2would then explain the unresponsiveness of TLR4 knockout andmutant mice to pure LPS, which requires MD-2, but not to Gram-positive bacteria, which do not require MD-2.

Moreover, our results are consistent with two recent studies.The first one (28) demonstrated that unresponsiveness of TLR4knockout mice was only limited to some LPS preparations;however, because there is no data on the purity of the LPSpreparations used, these results need to be interpreted with cau-tion. The second one indicated thatgd T cells respond to LPSand lipid A through TLR2 (29).

Our results confirm the specificity of the TLR4-MD-2 complexfor glycolipids, which is a structural feature shared by both LPSand LTA, and suggest the requirement for both the glycan and thelipid components of LPS and LTA, because neither detoxified (de-lipidated) LPS nor the lipid-containing lipopeptide (that lacks theglycan component) could stimulate cells through the TLR4-MD-2complex. Our results also suggest that LTA does not contribute tothe responses to intact Gram-positive bacteria, because cells trans-fected with TLR4, CD14, and MD-2 were responsive to isolatedLTA, but were unresponsive to LTA-containing Gram-positivebacteria. This lack of cell activation by intact bacteria may be dueto the anchoring of the lipid part of LTA in the bacterial cellmembrane, which is located underneath the thick PGN layer.

In summary, our results demonstrate that MD-2 enhancesTLR2-mediated responsiveness to both Gram-negative and Gram-positive bacteria and their LPS, PGN, and LTA components andthat it enables TLR2 to respond with high sensitivity to a muchbroader variety of stimulants, including protein-free LPS.

AcknowledgmentsWe thank Dr. Roger J. Davis for providing the JNK-1 plasmid.

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