Toll-like Receptor-4 Mediates Lipopolysaccharide-induced Signal Transduction

Abstract

TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-κB (NF-κB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-κB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-κB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50∼30 nm). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-κB activation and subsequent cellular events. TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-κB (NF-κB) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-κB-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-κB-mediated gene expression in cells transfected with the TLR4 gene in a dose- and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dose-dependent manner (IC50∼30 nm). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-κB activation and subsequent cellular events. Lipopolysaccharide (LPS), 1The abbreviations used are: LPS, lipopolysaccharide; TLR, toll-like receptor; IL, interleukin; NF-κB, nuclear factor-κB; sCD14, soluble CD14; CHO, Chinese hamster ovary; PMA, phorbol 12-myristate 13-acetate; pELAM-luc, ELAM-1-luciferase reporter plasmid. a component of the outer membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell types (1Schletter J. Heine H. Ulmer A.J. Rietschel E.T. Arch. Microbiol. 1995; 164: 383-389Crossref PubMed Scopus (270) Google Scholar, 2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in approximately 20,000 annual deaths in the United States (3Pinner R.W. Teutsch S.M. Simonsen L. Klug L.A. Graber J.M. Clarke M.J. Berkelman R.L. J. Am. Med. Assoc. 1996; 275: 189-193Crossref PubMed Google Scholar). Activation of LPS-responsive cells, such as monocytes and macrophages, occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein necessary for sensitive responses to LPS (1Schletter J. Heine H. Ulmer A.J. Rietschel E.T. Arch. Microbiol. 1995; 164: 383-389Crossref PubMed Scopus (270) Google Scholar, 2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar). LPS has been shown to initiate multiple intracellular signaling events (4Sweet M.J. Hume D.A. J. Leukoc. Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar), including the activation of NF-κB, which ultimately leads to the synthesis and release of a number of proinflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (1Schletter J. Heine H. Ulmer A.J. Rietschel E.T. Arch. Microbiol. 1995; 164: 383-389Crossref PubMed Scopus (270) Google Scholar). However, since CD14 is not a transmembrane protein, it lacks the ability to transduce cytoplasmic signals (2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar), and before the recent discovery of Toll-like receptors (TLRs), the identity of a transmembrane protein that could relay LPS-induced signals across the cell-surface membrane remained elusive. Toll is a transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5Morisato D. Anderson K.V. Cell. 1994; 76: 677-688Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 6Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2998) Google Scholar). Activation of the Toll receptor by its ligand Spätzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-κB activation by the IL-1 receptor in mammalian cells (7Belvin M.P. Anderson K.V. Annu. Rev. Cell. Dev. Biol. 1996; 12: 393-416Crossref PubMed Scopus (686) Google Scholar, 8Gay N.J. Keith F.J. Nature. 1991; 351: 355-356Crossref PubMed Scopus (453) Google Scholar). The cloning of a family of human receptors structurally related toDrosophila Toll revealed five proteins that have extracellular domains that contain multiple leucine-rich repeats and cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (9Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar). Furthermore, constitutively active mutants of TLR2, TLR4, and TLR5 can induce the activation of NF-κB (10Chaudhary P.M. Ferguson C. Nguyen V. Nguyen O. Massa H.F. Eby M. Jasmin A. Trask B.J. Hood L. Nelson P.S. Blood. 1998; 91: 4020-4027Crossref PubMed Google Scholar, 11Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar), and the active form of TLR4 increases the expression of NF-κB-regulated genes for the inflammatory cytokines IL-1, IL-6, and IL-8 (11Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar). Several lines of evidence suggest that one or more members of the TLR family is the cell-surface receptor for LPS, the prototypical activator of NF-κB and other proinflammatory responses. TLR2 and TLR4 are highly expressed in cells that respond to LPS, such as peripheral blood leukocytes, macrophages, and monocytes (11Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar, 12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1100) Google Scholar). Also, heterologously expressed TLR2 mediates LPS-induced NF-κB activation and IL-8 mRNA expression in HEK 293 cells (12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1100) Google Scholar, 13Kirschning C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar). However, TLR2 is not the only potential LPS signal transducer. The C3H/HeJ mouse is a spontaneous LPS resistant mutant. Poltorak et al. (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar) mapped the Lps gene, which has been shown previously to be necessary for LPS responses in LPS nonresponder C3H/HeJ mice, to TLR4. TLR4 from the C3H/HeJ mouse has a single point mutation at amino acid 712 (Pro to His) that changes the function of the receptor dramatically (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar); furthermore, the LPS-resistant C57/10ScCr mouse appears to be null for the TLR4 locus. These observations strongly support the concept that TLR4, and not TLR2, is the dominant LPS receptor in mammals and the hypothesis that TLR4 is a cell-surface component of the LPS signaling pathway. Thus, the present study was conducted to investigate whether TLR4 is involved in mediating the actions of LPS. Human TLR4 cDNA was provided by Dr. Charles A. Janeway, Jr. (Yale University). The ELAM-1-luciferase reporter plasmid, pELAM-luc, was generated by cloning a fragment (−241 to −54 base pairs) of the human E-selectin promoter into the pGL3 reporter plasmid (Promega, Inc.). All plasmid constructs were confirmed by automated sequencing analysis. Lipopolysaccharide was purchased from Sigma. The human embryonic kidney cell line HEK 293 (CRL-1573) was from American Type Culture Collection (Rockville, MD). The Chinese hamster ovary (CHO) cell line expressing CD14 was engineered and maintained as described (15Golenbock D.T. Liu Y. Millham F.H. Freeman M.W. Zoeller R. J. Biol. Chem. 1993; 268: 22055-22059Abstract Full Text PDF PubMed Google Scholar). The LPS antagonist (E5531) was synthesized as described previously (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar). Plasmid DNA was isolated with Qiagen Endo-freeTM Maxi-prep columns (Chatsworth, CA). MEM-18 anti-CD14 antibody was purchased from Accurate Chemicals & Scientific Corp. (Westbury, NY). Phorbol 12-myristate 13-acetate (PMA) and IL-1β were purchased from Calbiochem and Endogen (Woburn, MA), respectively. Luciferase activity was assayed using a commercial luciferase assay kit (Stratagene, La Jolla, CA). HEK 293 cells (ATCC, Rockville, MD) were cultured in Dulbecco's modified Eagle's medium (ATCC, Rockville, MD) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells were plated in 12-well tissue culture plates (3 × 105 cells/well) and maintained in the above medium for 24 h. Cells were transfected using the CalPhos Maximizer protocol (CLONTECH) with 250 ng of TLR4 cDNA or vector DNA (pcDNA3, Invitrogen, Inc.) and 100 ng of pELAM-luc. All cells were also transfected with a β-galactosidase control plasmid for normalizing transfection efficiencies. After transfection, cells were maintained in Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum overnight (18 h). The following day, cells were either left untreated or incubated with the indicated amount of ligand and/or compound E5531. After the indicated treatment period, cells were harvested in lysis buffer and assayed for luciferase activity per the manufacturer's protocol. The amount of luciferase activity in each sample was quantified by a Wallac 1450 MicroBetaTrilux counter. CHO cells expressing CD14 were cultured in suspension under serum-free conditions (EX-CELL 301 medium supplemented withl-glutamine). The culture supernatant was collected, filtered through a 0.22-μm nitrocellulose filter, and concentrated 5-fold in a protein concentrator (Amicon Diaflo, PM30) with a 30-kDa cut-off filter under pressure at 4 °C. This concentrate was then loaded onto an anti-CD14 affinity column. The column was washed twice with wash buffer before eluting with 0.1 m glycine, pH 2.8. The fractions were immediately neutralized with 1 mTris-HCl, pH 9.5, and an aliquot of each fraction was mixed with 2 × SDS loading buffer (Novex, San Diego, CA) and heated for 5 min at 95 °C. Expression and purification of sCD14 was verified by SDS-polyacrylamide gel electrophoresis followed by silver stain and immunoblotting with MEM-18 anti-CD14 antibody. Immunoreactive proteins were visualized using enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech). Quantitative data are presented as mean ± S.E. and analyzed using a statistical model based on a one-way classification analysis of variance. Tests of significance for all possible comparisons were determined by Student Newman-Keuls test or unpaired t test (GraphPad Prism, version 2.0a). To determine whether TLR4 mediates LPS-induced activation of NF-κB, HEK 293 cells were transiently transfected with TLR4 cDNA or empty vector control (pcDNA3) and an NF-κB-dependent ELAM-1-luciferase reporter plasmid (pELAM-luc). Twenty-four hours post-transfection, cells were left untreated or incubated with either LPS (1 μg/ml or indicated concentrations), sCD14 (10 nm) or both for an additional 6 h. Cells were then lysed and assayed for luciferase activity. Expression of TLR4 in HEK 293 cells induced activation of the NF-κB reporter gene 2.5-fold above controls (cells transfected with empty vector and the pELAM-luc reporter gene) in the absence of stimuli (Fig.1). Soluble CD14 alone did not have a significant effect on NF-κB activity in the presence or absence of TLR4. LPS treatment alone (1 μg/ml) was sufficient to elicit an increase (1.6-fold) in luciferase activity after incubation with TLR4 transfected cells, and this increase was not observed in vector controls (Fig. 1). However, cells were with LPS in the presence of sCD14, was a increase in TLR4-mediated activation of NF-κB that was not observed in cells with LPS or CD14 alone (Fig. 1). of TLR4-mediated NF-κB activation by LPS CD14 was 5-fold by TLR4 expression in controls or with CD14 LPS-induced reporter activity in a dose-dependent above controls at LPS and at 1 μg/ml LPS Furthermore, stimulation of NF-κB activity by LPS was time-dependent and at after LPS (Fig. 2 increases TLR4-mediated NF-κB activation in a dose- and time-dependent HEK 293 cells were transiently transfected with TLR4 cDNA or vector DNA and pELAM-luc. Cells were either left untreated or to sCD14 (10 nm) and the indicated of LPS for 6 or 1 of for the indicated amount of as described in the to After cell the amount of luciferase activity in each sample was These data the mean ± S.E. from with are from one In order to whether TLR4 is involved in LPS signaling at the cell HEK 293 cells were transfected with TLR4 or empty vector and the NF-κB reporter plasmid. cells were with LPS and of E5531, an LPS antagonist that has been shown to LPS-induced synthesis in and in (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar). of cellular luciferase activity after indicated that TLR4-mediated NF-κB activation in a dose-dependent manner NF-κB-dependent gene activation by LPS, and at (1 and NF-κB reporter in TLR4 expressing cells were to In to cells, 1 did not or NF-κB gene activation in cells that the TLR4-mediated NF-κB activation in response to LPS. it has been previously that LPS interacts with a transmembrane receptor or molecules on the surface of of cell types (2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar), evidence for this hypothesis has only recently al. (11Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar) identified a human TLR4 (9Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar), with signaling to observed for the IL-1 These the activation of the NF-κB and induction of mRNA for several proinflammatory cytokines (11Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar), both of which were also observed cells are with LPS (1Schletter J. Heine H. Ulmer A.J. Rietschel E.T. Arch. Microbiol. 1995; 164: 383-389Crossref PubMed Scopus (270) Google Scholar). by et al. (12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1100) Google Scholar) and et al. C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar) demonstrate the ability of TLR2 to signal in the presence of LPS and CD14, strongly a for this protein in LPS et al. C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar) also that and TLR4 to increase NF-κB reporter activity in the presence of LPS, the that TLR2 is a component of the cellular receptor for LPS. However, the recent by Poltorak et al. (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar), which the in the LPS-resistant C3H/HeJ as a mutation in the gene (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar), to the of TLR4 in LPS In the presented transfection of HEK 293 cells using a the cDNA for TLR4 was sufficient to elicit a significant response with LPS in the presence of soluble The reporter in a of the promoter for the E-selectin gene, which is NF-κB activation for activity U. Cell. Biol. 1994; PubMed Google Scholar). Thus, LPS-induced stimulation of reporter gene in TLR4 expressing cells is the NF-κB signaling pathway. have shown that constitutively active TLR4 constructs can IL-1 tumor necrosis and NF-κB of the IL-1 signaling that lead to NF-κB activation R. Preston-Hurlburt P. E. A. C. S. Janeway Jr., C.A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. G. S. M. A. J. Exp. Med. 1998; PubMed Scopus Google Scholar). demonstrate a LPS signaling and TLR4 expression in this it to be determined whether the of LPS the subsequent signaling proteins as the IL-1 receptor (4Sweet M.J. Hume D.A. J. Leukoc. Biol. 1996; 60: 8-26Crossref PubMed Scopus (710) Google Scholar). et al. C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar) to activation in HEK 293 cells transfected with human TLR4. The for this are the in HEK 293 cell have that one of HEK 293 cells to LPS, as by NF-κB in the absence of In order to were to an of HEK 293 cells, as this from the (ATCC, Rockville, expressed of TLR2 and D. T. the IL-1 the of signaling with highly homologous proteins as ligand J. X. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). hypothesis that the in is that the expression of which can be after gene and that all of HEK 293 cells are not in this also results in types of such as the of the TLR4 the amount of DNA in or other conditions that TLR4 have shown that the amount of TLR4 expression vector transfected into cells the of pELAM-luc activity by LPS. C. and This was to activity of pELAM-luc that was of TLR4 were effect has been in HEK 293 cells expressing E. M. M. M. Cell. 1997; 91: Full Text Full Text PDF PubMed Scopus Google Scholar). In support of et al. (12Yang R.B. Mark M.R. Gray A. Huang A. Xie M.H. Zhang M. Goddard A. Wood W.I. Gurney A.L. Godowski P.J. Nature. 1998; 395: 284-288Crossref PubMed Scopus (1100) Google Scholar) and et al. C.J. Wesche H. Merrill Ayres T. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (655) Google Scholar), have shown that HEK 293 cells transfected with a TLR2 are also to LPS is a potent that as an antagonist of LPS-induced activation (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar). The compound the of LPS in macrophages, of and (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar), as as the of of LPS to D.P. Hawkins L.D. Kishi Y. W.J. Kobayashi S. Kawata T. M. Yamatsu I. H. D. S. S. in and Scholar). on LPS it is believed that LPS activity at its cell-surface leading to of transmembrane signal (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar). with this the stimulation of TLR4 transfected cells by LPS CD14 in a dose-dependent manner with an of with its in other cell based T. Bristol J.R. Rose J.R. Rossignol D.P. W.J. Asano O. Dubuc G.R. Gavin W.E. Hawkins L.D. W.J. McGuinness P.D. Mullarkey M.A. Perez M. Robidoux A.L. Wang Y. Kobayashi S. Kimura A. Katayama K. Yamatsu I. J. H. from to Scholar). did not activation of the NF-κB reporter gene induced by IL-1β or the actions of in (16Christ W.J. Asano O. Robidoux A.L. Perez M. Wang Y. Dubuc G.R. Gavin W.E. Hawkins L.D. McGuinness P.D. Mullarkey M.A. Lewis M.D. Kishi Y. Kawata T. Bristol J.R. Rose J.R. Rossignol D.P. Kobayashi S. Hishinuma I. Kimura A. Asakawa N. Katayama K. Yamatsu I. Science. 1995; 268: 80-83Crossref PubMed Scopus (309) Google Scholar), this that the activity of is to and to cell-surface by LPS. In TLR4 is the only protein expression can for the LPS observed in transfected based on it is that TLR4 is a receptor for LPS and that as an antagonist of this In of the recent of the in C3H/HeJ and LPS-resistant as a mutation in the (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar), the results presented are These data demonstrate that TLR4 as a LPS receptor transfected into cells that are This is with the by al. (14Poltorak A. He X. Smirnova I. Liu M.Y. Huffel C.V. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar) in C3H/HeJ These data not a for TLR2 in LPS signal under conditions or in cell support the hypothesis that TLR4 is in LPS signaling in and in the of TLR4 as a receptor for the LPS antagonist is an for the and in the discovery of and for and for in the of the