MD-2 Binds to Bacterial Lipopolysaccharide

Abstract

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesotaand Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability. The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesotaand Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability. lipopolysaccharide bovine serum albumin toll-like receptor recombinant soluble CD14 radioiodinated sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Salmonella minnesota Re595 lipopolysaccharide fluorescein isothiocyanate phosphate-buffered saline enzyme-linked immunosorbent assay Chinese hamster ovary monoclonal antibody biotinylated Re595 LPS polyacrylamide gel electrophoresis hemagglutinin A nuclear factor κB fluorescence-activated cell-sorting interleukin Mammals respond to microbial invasion with both innate and adaptive immune responses. The LPS1 of Gram-negative bacteria is a potent initiator of the innate inflammatory response that has an important antimicrobial role. For that reason, and because Gram-negative bacteria are common initiators of septic shock, the receptors for LPS have received much scrutiny in recent years. Recent work has shown that myeloid cells use at least four proteins to mount a sensitive cellular response to LPS: LBP, CD14, TLR4, and MD-2 (1Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1378) Google Scholar, 2Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3399) Google Scholar, 3Poltorak 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 (6422) Google Scholar, 4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar). CD14 is a critical receptor for LPS because anti-CD14 monoclonal antibody (Mab) can inhibit LPS-induced effects both in vitroand in vivo (5Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar, 6Leturcq D.J. Moriarty A.M. Talbott G. Winn R.K. Martin T.R. Ulevitch R.J. J. Clin. Invest. 1996; 98: 1533-1538Crossref PubMed Scopus (140) Google Scholar). However, because CD14 has no transmembrane domain, it cannot transmit a transmembrane signal. The type 1 transmembrane protein TLR4 is probably the signal-transducing component of the LPS receptor, based on genetic studies and a variety of other studies (3Poltorak 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 (6422) Google Scholar, 7Heine H. Kirschning C.J. Lien E. Monks B.G. Rothe M. Golenbock D.T. J. Immunol. 1999; 162: 6971-6975PubMed Google Scholar, 8Lien E. Means T.K. Heine H. Yoshimura A. Kusumoto S. Fukase K. Fenton M.J. Oikawa M. Qureshi N. Monks B. Finberg R.W. Ingalls R.R. Golenbock D.T. J. Clin. Invest. 2000; 105: 497-504Crossref PubMed Scopus (685) Google Scholar). Comparative studies of TLR4 from mice and humans suggest that it interacts directly with LPS (9Poltorak A. Ricciardi-Castagnoli P. Citterio S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2163-2167Crossref PubMed Scopus (395) Google Scholar), as do cross-linking studies with a photoactivable derivative of LPS (10da silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). MD-2 was identified as a TLR4-binding protein that is required for the function of TLR4 (4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar, 11Akashi S. Shimazu R. Ogata H. Nagai Y. Takeda K. Kimoto M. Miyake K. J. Immunol. 2000; 164: 3471-3475Crossref PubMed Scopus (423) Google Scholar). Like CD14, it has no transmembrane domain and apparently remains cell-associated because it binds to TLR4 (4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar). In this work, we describe the use of several biochemical techniques to address the question of whether LPS interacts directly with MD-2 in the absence of other LPS-binding proteins and investigate the role of this interaction in cellular activation by LPS. Re595 LPS and its FITC and125I-sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivatives (125I-ASD-LPS) were prepared as described previously (12Kirkland T.N. Virca G.D. Kuus-Reichel T. Multer F.K. Kim S.Y. Ulevitch R.J. Tobias P.S. J. Biol. Chem. 1990; 265: 9520-9525Abstract Full Text PDF PubMed Google Scholar, 13Viriyakosol S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Labeling results obtained with this material were unchanged (10da silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar) by repurification according to Hirschfeld et al. (14Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (969) Google Scholar). Biotinylated Re595 LPS (biotin-LPS) was a gift from R. Dziarski and had no TLR2 agonist activity. 2R. Dziarski, personal communication. Tritiated Ra LPS ([3H]LPS) was obtained from List Biochemicals. Partially disaggregated LPS from Escherichia coli K-12 strain LCD25 was prepared as described previously (15Kitchens R.L. Munford R.S. J. Immunol. 1998; 160: 1920-1928PubMed Google Scholar). Recombinant soluble CD14 and LBP with C-terminal His tags were prepared as described previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Control protein was a 62-kDa heat shock protein of Coccidioides immitis with a C-terminal His tag (a gift from A. Kanauchi). Polyclonal rabbit anti-MD-2 IgG was purified using a protein A column (Amersham Pharmacia Biotech) from rabbit sera immunized with purified recombinant MD-2 by QED Biosciences Inc. Mab to MD-2, 5H10, was generated as described previously (17Stanley H.A. Reese R.T. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6272-6275Crossref PubMed Scopus (13) Google Scholar) using recombinant MD-2. The IgG1 fraction was purified from the culture supernatant by protein G (Amersham Pharmacia Biotech) affinity chromatography. Mabs to TLR4, HTA 405, HTA 414, HTA 125, and HTA 1216 were generous gifts from K. Miyake. Control mouse IgG1 and rabbit IgG were obtained from Caltag. All protein biotinylations were done using the EZ-Link Sulfo-NHS-LC Biotinylation Kit (Pierce). All reagents were tested for LPS contamination with Limulus Amoebocyte Lysate (BioWhittaker). When necessary, LPS was removed from the reagents using END-X (Associates of Cape Cod, Inc.). The human MD-2 cDNA clone (accession number AA099571) was obtained from Genome System Inc. The coding sequence, excluding the signal sequence, was amplified from plasmid DNA by polymerase chain reaction using Takara LA ta-DNA polymerase (Pan Vera). The fragments were cloned into baculovirus transfer vector pBAC11 (Novagen) for of protein using the signal The MD-2 that the C-terminal tag was by the polymerase chain reaction The use of this vector of the His tag from the protein using recombinant the recombinant pBAC11 the were with DNA (Novagen) into cells using (Novagen) as described by the The recombinant generated from the cells were by the assay The purified were amplified to a and to cells for protein The cells and supernatant were of the at of by the The protein was purified from the culture supernatant by affinity as described (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The purified protein was on with and The His tag protein was identified with antibody and using the (Amersham Pharmacia The protein was as by and gel on chromatography. The His tag was removed from MD-2 by with the C-terminal of MD-2 and the His 1 of the of was of protein in at The reaction was to at for The was removed by with protein and His tag were removed in using The was for the absence of His tag by with Mab to His tag binding were LPS MD-2. In the MD-2 a was with MD-2, CD14, in at The of MD-2 was the of CD14 and was The were and as described previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). was (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) at and for at In was to the to the was and the reaction was and as described previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The assay for MD-2 binding using LPS was done to the described previously P.S. Soldau K. Ulevitch R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Re595 LPS to was to a The was for at with and with no LPS were with in and MD-2 protein with the His in 1 in was in a of MD-2 was by biotinylated monoclonal antibody at in 1 in was using and its in in and the reaction was with the of was at nm. of MD-2 binding was by LPS with MD-2 for at the to the The of MD-2 and LPS was based on the that the of LPS is and the of MD-2 is of to MD-2 was done as described previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). was with MD-2 in an and the was as a function of The apparent using a variety of of MD-2, the in signal at and the was with of proteins in and at for by on for The were on an gel as described previously (12Kirkland T.N. Virca G.D. Kuus-Reichel T. Multer F.K. Kim S.Y. Ulevitch R.J. Tobias P.S. J. Biol. Chem. 1990; 265: 9520-9525Abstract Full Text PDF PubMed Google Scholar). The were done by with MD-2. The were on a on a of as described previously P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The were at in a for were and the of was by was according to the previously E. M. Wright S.D. J. Exp. Med. PubMed Scopus Google Scholar). MD-2 was with Re595 LPS in with 1 in a of at for The was and the was using The gel was using according to the The coding of TLR2 and TLR4 the signal were polymerase chain using Takara polymerase (Pan from TLR4 from P. and C. The fragments were into the of The protein a and an hemagglutinin A were in the as described previously S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The with the of a human binding was a gift from Golenbock R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; Google Scholar). DNA was prepared using an were generated using according to the The cells proteins were by using Mab The were generated by with Inc.). cells were for of the by using Mab by the of cells with the were with 1 MD-2 in with serum for at the protein with the the cells were with Mab to MD-2 to TLR4 by rabbit and by as described previously S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). cells to were to in with serum and in a at 1 the The cells were in of and The cells were using and 1 with Mab and by as described previously S. Kirkland T.N. J. Biol. Chem. 1995; 270: 361-368Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). cells were from and in with and The cells were with for The cells were in culture at and with LPS with for The supernatant was and for by In the was with antibody and by rabbit by and the The was at and the of was using a derived from purified The cells were in a and with reagents in with human serum The supernatant was of activation and for by as described previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). from a was in and recombinant human MD-2 was to a of whole the blood. The was to of LPS coli K-12 strain was and the were for at in a The were by of the cells were removed by at and were in the by factor and were by using reagents according to the The ability of MD-2, and CD14 to was tested in on a MD-2 and CD14 much The binding of MD-2 and CD14 is LPS the is with binding to MD-2 and CD14 is that MD-2 is binding to the A of LPS. that the protein is with rabbit the binding can MD-2 binding to LPS could LPS was on the The protein His tag binds to LPS as as the protein with His and binding of MD-2 was no LPS on the His tag at the of the protein is not in LPS that the binding of MD-2 to LPS can inhibited by MD-2 with LPS in a that the binding is The LPS to the binding of MD-2 to the by is a in the complex and an apparent KD nm. MD-2 binds to LPS in the as shown in four different Re595 LPS with a photoactivable (125I-ASD-LPS) can to LPS-binding proteins by the of the to the protein work has shown the of this (12Kirkland T.N. Virca G.D. Kuus-Reichel T. Multer F.K. Kim S.Y. Ulevitch R.J. Tobias P.S. J. Biol. Chem. 1990; 265: 9520-9525Abstract Full Text PDF PubMed Google Scholar, P.S. Soldau K. K. Martin Ulevitch R.J. J. Immunol. Google Scholar). the results of a that LBP and CD14 were strongly the 62-kDa His tag protein no Labeling of MD-2 is shown in MD-2 interaction in the absence of LBP and The that and 1 of LBP with of MD-2 for the of CD14 not The for LPS binding to LBP is and the for LPS binding to CD14 is and (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). When derivatives of LPS are by LBP CD14, an in the signal is (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). We found that MD-2 enhanced the of not to as an as LBP CD14 has in previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the of as a function of MD-2 an of 65 was is to the of for CD14 (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). We to the of LPS binding to MD-2. In previously (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, P.S. Soldau K. D. Ulevitch R.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), we have found that LBP and CD14 a of in the as shown in the the of of in the absence and of of MD-2. In the absence of MD-2 A of the LPS the of the to its highly a in the of the In the of MD-2 at and the of in In not are MD-2 was found in we have to the interaction LPS and a protein is was previously shown that the binding of LPS to a in the of the protein on E. M. Wright S.D. J. Exp. Med. PubMed Scopus Google Scholar). that at of the MD-2 in the of LPS. that the of LPS binding to MD-2 is and is in with the obtained by assay The MD-2 to with to the binding of LPS with E. M. Wright S.D. J. Exp. Med. PubMed Scopus Google Scholar). The that of the protein that MD-2 binds to LPS binding LPS The results of the and to the that MD-2 is a genuine LPS-binding In previously al. (4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar) have shown that MD-2 with TLR4 by of MD-2 and TLR4 into we that MD-2 binds to cells by the protein to the that the cells TLR4 as by but no MD-2 was with the When MD-2 was the cells with the that MD-2 to The was obtained using cells the plasmid not binding of MD-2 was to cells with CD14, vector not We tested the for MD-2 on LPS activation of a is with an that can by as an of activation R.L. Yoshimura A. Ingalls R.R. Golenbock D.T. J. Immunol. 1998; Google Scholar). the of as the number of in and vector cells the of Re595 LPS in the of MD-2, CD14, His tag The of in a in cells and MD-2 was CD14 protein had no on LPS activation When MD-2 with no His tag was an was obtained not We the role of MD-2 in LPS activation of the human The cells of CD14 with P.S. Soldau K. K. Martin Ulevitch R.J. J. Immunol. Google Scholar). cells TLR4 and MD-2 as by using Mabs and rabbit anti-MD-2 not that the response of cells to Re595 as by an can inhibited by rabbit anti-MD-2 as as by the HTA and results indicate that not TLR4 and CD14, as previously Akashi S. Miyake K. Tobias P.S. J. Immunol. 2000; 165: PubMed Scopus Google Scholar), but MD-2 play roles in LPS activation in rabbit anti-MD-2 LPS binding to MD-2 results suggest that MD-2 to LPS for cellular In to the cells that a of TLR4, we an of MD-2 LPS activation in cells that of The U373 cells for TLR4 and MD-2 but not for TLR2 as by chain reaction not of TLR4 MD-2 cannot by with to TLR4 MD-2 not LPS U373 cells in the of CD14 to (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). that MD-2 by the U373 cells with CD14 and LPS in a Re595 LPS and 1 CD14, was from the U373 When MD-2 was from the response was inhibited by at a of as as of LPS can In the we that LPS with MD-2 at this of that MD-2 inhibit activation of U373 cells by with for binding to LPS. We tested whether MD-2 binding to LPS to its role in LPS activation of U373 cells by using an antibody to MD-2 that MD-2 binding to LPS to this that the response in U373 cells in the of and LPS was by MD-2. the that the MD-2 is LPS. Mab to CD14 inhibited the response The of MD-2 on LPS-induced activation in whole was We whole with a variety of LPS in the absence of MD-2. We factor and in response to LPS. shown in MD-2 at factor and from whole with LPS in from to The of MD-2 did not inhibit the ability of recombinant human to in whole not is that LPS to MD-2 from LBP of the were done with MD-2, were done with MD-2 from the His tag had that as we have shown previously for CD14 (16Viriyakosol S. Mathison J.C. Tobias P.S. Kirkland T.N. J. Biol. Chem. 2000; 275: 3144-3149Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), the His tag is to the binding of LPS. In of the we whether a 62-kDa heat shock protein from C. immitis LPS with data (10da silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar) using MD-2 with TLR4 on cells that CD14, TLR4, and MD-2 to for of MD-2 by In (10da silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar), cell-associated components were for of MD-2 not have because MD-2 not have cell-associated in the absence of TLR4 (4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar). The affinity of MD-2 for LPS to that of CD14, with an apparent KD of 65 as shown by using The that LPS binding to MD-2 is by LPS The are with this The of to as shown by and with the binding of MD-2 to cells TLR4 suggest that MD-2 can with TLR4 for of the as we are studies with MD-2 have MD-2 in the cells as the of the MD-2 (4Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1746) Google Scholar, 11Akashi S. Shimazu R. Ogata H. Nagai Y. Takeda K. Kimoto M. Miyake K. J. Immunol. 2000; 164: 3471-3475Crossref PubMed Scopus (423) Google Scholar). The effects of MD-2 were In the the of MD-2 enhanced to in other cells and whole the of MD-2 inhibited to LPS. could to the of of TLR4 and the of MD-2. the cells do not MD-2 to with the TLR4, MD-2 that TLR4 to and the cells to enhanced to LPS. the U373 cells and monocytes in activation is TLR4, and it is to that the cells are MD-2 to with In this as we MD-2 to for the LPS and inhibit cellular is based on that the cells derived from Chinese are hamster TLR4, hamster MD-2, and human TLR4, the U373 cells and the whole were derived from humans that the MD-2 is et al. K. Akashi S. Shimazu R. T. Miyake K. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) have at to LPS in four of mouse TLR4, mouse MD-2, human TLR4, and human MD-2. data that the TLR4 and MD-2 to from the to in an because the cells in human TLR4 cannot human MD-2, require human MD-2 for function of the human is of the effects of recombinant MD-2 on the of results that MD-2 can LPS in and that the MD-2 has biological with the in different assay However, we were to a of MD-2 on LPS in human whole blood. The to for LPS because the response to was not is that MD-2 MD-2 for LPS These data suggest that MD-2 can LPS in whole in a to of can to it can inhibit LPS in whole and in cells that CD14 the is at found in A. G.W. J. J. Immunol. Google Scholar). can the of LPS that has to the R.L. G. J.J. Munford R.S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). data not suggest that MD-2 the of LPS from In human MD-2 at the P. S. R. and T. N. in this we MD-2 at is whether of MD-2 could play a role in the LPS response in vivo are in its However, the ability of MD-2 to inhibit LPS activation in human whole that it as a for LPS in We Golenbock for the and Soldau for and and the for of baculovirus