Receptors Involved in the Oxidized 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine-mediated Synthesis of Interleukin-8

We demonstrated previously that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and, specifically, the component lipid 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine increase interleukin-8 (IL-8) synthesis in aortic endothelial cells. The goal of the current studies was to characterize the receptor complex mediating the increased transcription of IL-8. We demonstrate that scavenger receptor class A, types I and II, lectin-like ox-LDL receptor-1, macrophage receptor with collagenous structure, and CD36 are not responsible for the increase in IL-8. Using dominant-negative constructs and antisense oligonucleotides, we demonstrate a role for Toll-like receptor 4 (TLR4) as the ox-PAPC receptor mediating IL-8 transcription. We demonstrate that a glycosylphosphatidylinositol (GPI)-anchored protein is also necessary because phosphatidylinositol-specific phospholipase C pretreatment inhibited the effect of ox-PAPC. CD14, a GPI-anchored protein that associates with TLR4 in mediating lipopolysaccharide action, did not appear to mediate ox-PAPC action because ox-PAPC-induced IL-8 transcription was not blocked by anti-CD14 neutralizing antibodies nor was it augmented by the addition of soluble CD14 or overexpression of membrane CD14. Instead, anti-TLR4 antibodies immunoprecipitated a 37-kDa protein that also bound ox-PAPC. A protein of this same size was found in aerolysin overlays used to detect GPI-anchored proteins. Therefore, these studies suggest that ox-PAPC may initially bind to a 37-kDa GPI-anchored protein, which interacts with TLR4 to induce IL-8 transcription. We demonstrated previously that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and, specifically, the component lipid 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine increase interleukin-8 (IL-8) synthesis in aortic endothelial cells. The goal of the current studies was to characterize the receptor complex mediating the increased transcription of IL-8. We demonstrate that scavenger receptor class A, types I and II, lectin-like ox-LDL receptor-1, macrophage receptor with collagenous structure, and CD36 are not responsible for the increase in IL-8. Using dominant-negative constructs and antisense oligonucleotides, we demonstrate a role for Toll-like receptor 4 (TLR4) as the ox-PAPC receptor mediating IL-8 transcription. We demonstrate that a glycosylphosphatidylinositol (GPI)-anchored protein is also necessary because phosphatidylinositol-specific phospholipase C pretreatment inhibited the effect of ox-PAPC. CD14, a GPI-anchored protein that associates with TLR4 in mediating lipopolysaccharide action, did not appear to mediate ox-PAPC action because ox-PAPC-induced IL-8 transcription was not blocked by anti-CD14 neutralizing antibodies nor was it augmented by the addition of soluble CD14 or overexpression of membrane CD14. Instead, anti-TLR4 antibodies immunoprecipitated a 37-kDa protein that also bound ox-PAPC. A protein of this same size was found in aerolysin overlays used to detect GPI-anchored proteins. Therefore, these studies suggest that ox-PAPC may initially bind to a 37-kDa GPI-anchored protein, which interacts with TLR4 to induce IL-8 transcription. Monocyte/endothelial interactions have been shown to play an important role in all stages of atherosclerosis (1Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. Edwards P.A. Fogelman A.M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 831-842Crossref PubMed Scopus (612) Google Scholar). Our laboratory has demonstrated that phospholipid oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) 1The abbreviations used are: PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; ox-PAPC, oxidized PAPC; PEIPC, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine; POVPC, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine; MM-LDL, minimally modified low density lipoprotein; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; PMA, phorbol 12-myristate 13-acetate; IL-8, interleukin-8; TLR4, Toll-like receptor 4; GPI, glycosylphosphatidylinositol; HAEC, human aortic endothelial cells; MAEC, murine aortic endothelial cells; CHO, Chinese hamster ovary; MARCO, macrophage receptor with collagenous structure; SRA, scavenger receptor class A; ELISA, enzyme-linked immunosorbent assay; PI-PLC, phosphatidylinositol-specific phospholipase C; RT, reverse transcription; MCP-1, monocyte chemotactic protein-1; ANOVA, analysis of variance; LOX-1, lectin-like ox-LDL receptor-1. found in minimally modified low density lipoprotein (MM-LDL) activate this interaction (2Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 3Leitinger N. Watson A.D. Hama S.Y. Ivandic B. Qiao J.H. Huber J. Faull K.F. Grass D.S. Navab M. Fogelman A.M. de Beer F.C. Lusis A.J. Berliner J.A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1291-1298Crossref PubMed Scopus (144) Google Scholar). These phospholipids have been shown to accumulate in atherosclerotic lesions of mice and rabbits. Antibodies that recognize these lipids demonstrate their presence in human lesions. Furthermore, PAPC oxidation products are increased in apoptotic cells (4Huber J. Vales A. Mitulovic G. Blumer M. Schmid R. Witztum J.L. Binder B.R. Leitinger N. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 101-107Crossref PubMed Scopus (247) Google Scholar) and cells exposed to oxidative stress (5Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). We have determined that treatment of human aortic endothelial cells (HAEC) and HeLa cells with oxidized PAPC (ox-PAPC) increased the synthesis of interleukin-8 (IL-8), a chemokine involved in monocyte transmigration and retention in the vessel wall. We have also determined that isomers of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC) and to a lesser extent 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) are responsible for most of the effect of ox-PAPC to induce IL-8 (5Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The increase in IL-8 was shown to be mediated by increased transcription; importantly, native PAPC was not found to have an effect (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). The goal of these studies was to characterize the receptor responsible for the ability of ox-PAPC to increase IL-8 transcription. Several lines of evidence suggest that the induction of IL-8 by ox-PAPC is receptor-mediated. The increase in message is rapid with accumulation being observed as early as 15 min after treatment with ox-PAPC (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). PEIPC, the major bioactive lipid in ox-PAPC, is active at concentrations as low as 100 nm, and its effect is saturable and maximal at 1 μg/ml. We first examined the possibility that ox-PAPC and its derivatives act on a receptor belonging to the scavenger receptor family. Recent studies demonstrated that autoantibodies from apoE-deficient mice that bound to oxidized phospholipids (e.g. EO6 antibody) inhibited 60–80% of the binding of copper-oxidized LDL to scavenger receptors on mouse peritoneal macrophages (7Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. J. Clin. Invest. 1999; 103: 117-128Crossref PubMed Scopus (474) Google Scholar). Although these particular studies were performed using macrophages, they suggested a possible role for scavenger receptors in endothelial cells mediating the effect of ox-PAPC. The expression of several known scavenger receptors was examined in the current study. Scavenger receptor class A, types I and II (SRA I/II), is a multifunctional receptor that binds to a broad variety of ligands including oxidized LDL (8de Winther M.P. van Dijk K.W. Havekes L.M. Hofker M.H. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 290-297Crossref PubMed Scopus (197) Google Scholar). Another class A receptor, macrophage receptor with collagenous structure (MARCO), binds to modified LDL as well (9Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (411) Google Scholar). CD36, a class B scavenger receptor, binds to the lipid moiety of oxidized LDL (10Nicholson A.C. Frieda S. Pearce A. Silverstein R.L. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 269-275Crossref PubMed Scopus (223) Google Scholar). LOX-1, an oxidized LDL receptor belonging structurally to the C-type lectin family, was initially identified in vascular endothelial cells (11Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1175) Google Scholar). We present evidence that these receptors are not responsible for ox-PAPC-induced IL-8 synthesis. We also examined the role of Toll-like receptor 4 (TLR4) and associated proteins in mediating the action of ox-PAPC. C3H/HeJ mice have a missense mutation in the Tlr4 gene resulting in nonfunctional TLR4 (12Poltorak 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 (6471) Google Scholar). These mice are resistant to atherosclerosis, and aortic endothelial cells from these mice are unresponsive to MM-LDL (13Shi W. Haberland M.E. Jien M.L. Shih D.M. Lusis A.J. Circulation. 2000; 102: 75-81Crossref PubMed Scopus (162) Google Scholar). We hypothesized a role for TLR4 additionally because of the epitope similarity of bacterial lipids, known regulators of TLR4 (14Medzhitov R. Nat. Rev. Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3279) Google Scholar), and oxidized phospholipids (recognized by the antibody EO6) (7Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. J. Clin. Invest. 1999; 103: 117-128Crossref PubMed Scopus (474) Google Scholar). We further hypothesized that, like lipopolysaccharide (LPS), the interaction of ox-PAPC with TLR4 may be enhanced by binding to a glycosylphosphatidylinositol (GPI)-anchored protein. These studies present evidence for a role of both TLR4 and a GPI-anchored protein in mediating ox-PAPC induction of IL-8 synthesis in endothelial cells. Reagents—Tissue culture media and reagents were obtained from Irvine Scientific, Inc. unless otherwise stated. Fetal bovine serum was obtained from Hyclone. PAPC was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) or Sigma. PAPC was oxidized as described previously (2Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). PEIPC was isolated as described previously (5Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). POVPC was prepared as described previously (2Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 15Leitinger N. Tyner T.R. Oslund L. Rizza C. Subbanagounder G. Lee H. Shih P.T. Mackman N. Tigyi G. Territo M.C. Berliner J.A. Vora D.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12010-12015Crossref PubMed Scopus (229) Google Scholar). Mass analysis, liquid chromatography/mass spectrometry, tandem mass spectrometric analysis, and quantitation of oxidized phospholipids were performed as described previously (5Subbanagounder G. Wong J.W. Lee H. Faull K.F. Miller E. Witztum J.L. Berliner J.A. J. Biol. Chem. 2002; 277: 7271-7281Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). LPS from Escherichia coli O111:B4 (a natural, smooth strain) was obtained from List Biological Laboratories, Inc. Phorbol 12-myristate 13-acetate (PMA) and anti-β-coatomer protein monoclonal antibodies (catalog number G6160) were obtained from Sigma. Tumor necrosis factor-α (TNF-α), anti-human CD14 polyclonal antibodies (catalog number AB383), anti-human CD14 monoclonal antibodies (catalog number MAB3831), and a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit were obtained from R & D Systems. Anti-CD36 monoclonal antibodies (clone FA6-152) were obtained from Immunotech (Westbrook, ME) (16Finnemann S.C. Silverstein R.L. J. Exp. Med. 2001; 194: 1289-1298Crossref PubMed Scopus (112) Google Scholar). CD36 peptides were obtained from Dr. S. Frieda A. Pearce (Cornell University) (17Pearce S.F. Roy P. Nicholson A.C. Hajjar D.P. Febbraio M. Silverstein R.L. J. Biol. Chem. 1998; 273: 34875-34881Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Anti-human TLR4 monoclonal antibodies were obtained from eBioscience (catalog number 14-9917). Anti-Rab8 monoclonal antibodies were obtained from BD Transduction Laboratories (catalog number 610844). Anti-histone monoclonal antibodies were obtained from Chemicon International (catalog number MAB052). A murine monocyte chemotactic protein-1/JE (MCP-1/JE) ELISA kit was obtained from Pharmingen. EO6 antibodies were obtained from Dr. L. Witztum of Anti-human TLR4 polyclonal antibodies (catalog number anti-human monoclonal antibodies (catalog number anti-human polyclonal antibodies (catalog number antibodies (catalog number antibodies (catalog number and antibodies (catalog number were obtained from Inc. human CD14 was obtained from Dr. and antibodies were obtained from phospholipase C was obtained from (catalog number The IL-8 was obtained from Dr. Leitinger of (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). and antisense to TLR4 were obtained from These the to the of The dominant-negative TLR4 a mutation to that found in C3H/HeJ mice and the human CD14 were obtained from Dr. L. assay and assay were obtained from MM-LDL was obtained by treatment of LDL with and phospholipase or by oxidation Fogelman A.M. A. Territo M.C. Berliner J.A. J. Clin. Invest. PubMed Scopus Google Scholar). J.A. Territo M.C. E. Haberland M.E. Fogelman A.M. Berliner J.A. Arterioscler. Thromb. PubMed Google Scholar), murine aortic endothelial cells W. Shih D.M. X. Lusis A.J. 2000; PubMed Scopus Google Scholar), and G. A. A. J. Immunol. Google Scholar) were isolated and as described HeLa cells and cells were obtained from the endothelial cells were obtained from Dr. T. Reddy aortic endothelial cells were obtained from Inc. was isolated using transcription was performed after was performed on the resulting using for human TLR4 human CD14 human human CD36 human human human 1 human human or human and in a were with were or were with were performed using anti-TLR4 monoclonal antibodies anti-CD14 monoclonal antibodies & D monoclonal antibodies or antibodies to with was to the which was in a at were performed using anti-β-coatomer protein, and and IL-8 cells were with in bovine serum for at after for in bovine were performed on the using were in culture were performed with of TLR4 or or antisense TLR4 oligonucleotides, human CD14 or with the IL-8 or was used for to the after cells were with ox-PAPC, LPS, TNF-α, or for were and for were with or were with ox-PAPC LPS or for 4 the treatment were and for IL-8. were to with and with were and in a of A was to a at for and in A and 1 was for to a and at for The were used for with anti-human TLR4 polyclonal antibodies using the protein of was determined by and of protein were by and in by in The protein was to and antibodies to were used for using and proteins were using the assay as described previously B. G. W. M. Lee H. C. M.P. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar). using the are as The are of at of Scavenger in of IL-8 Although studies have been performed scavenger receptors in macrophages, has been to scavenger receptor expression in human aortic endothelial cells. Therefore, to the role of endothelial scavenger receptors in ox-PAPC action, we determined which of the known scavenger receptors are present in and oxidized phospholipids their was performed to the presence of CD36, LOX-1, and on were found to low of CD36 and of or not to be low or not present with being with of the Our were by a that expression was by in atherosclerosis H. Kume N. S. M. Moriwaki H. T. T. Masaki T. N. Kita T. Circulation. 1999; PubMed Scopus Google Scholar). of with ox-PAPC was found to increase the for CD36, for MARCO, and not we to CD36 is necessary for from CD36 mice of Dr. University) and mice were with LPS MM-LDL or native LDL in to both LPS and MM-LDL not in to native from CD36 mice in to MM-LDL at with from cells with antibodies and CD36 peptides effect on with ox-PAPC and PEIPC not These that CD36 is not for and ox-PAPC-induced and of Toll-like 4 studies by R. Y. E. A. M. M. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated that human endothelial cells of TLR4 as determined by and protein, as determined by and S. L. R. R. Lee J. Clin. Invest. 1999; PubMed Scopus Google Scholar) found that endothelial cells of TLR4 as determined by studies have been performed that vessel endothelial expression of TLR4 were to detect and to detect protein was determined by from isolated from that they TLR4 HeLa cells and human endothelial were also found to TLR4 from the that TLR4 protein on the we determined TLR4 is involved in ox-PAPC-induced We previously demonstrated that ox-PAPC IL-8 transcription in HeLa cells and an IL-8 (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). We used this to the role of TLR4 in the ox-PAPC induction of IL-8. HeLa cells were with a dominant-negative of an IL-8 was to detect ox-PAPC-induced The dominant-negative of TLR4 effect on IL-8 transcription; the effect of on transcription was in the effect of ox-PAPC was inhibited in cells dominant-negative TLR4 with with that TLR4 a role in ox-PAPC-induced HeLa cells were with antisense TLR4 with the was a in the of cells with the antisense with with the effect on IL-8 synthesis was of GPI-anchored in the of the LPS receptor complex GPI-anchored CD14 and we hypothesized that ox-PAPC may also bind to a GPI-anchored protein with this we with bacterial PI-PLC, which GPI-anchored proteins from the and the cells with ox-PAPC. were and for IL-8. with was found to IL-8 in to ox-PAPC and LPS that a GPI-anchored protein a role in ox-PAPC-induced did not the induction of IL-8 by or We the role of CD14 in ox-PAPC action because CD14 is known to complex with TLR4 J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). were found to CD14 from the that CD14 on the We that CD14 was to the because the for the protein, and increased with the for CD14 did not not the role of CD14 in ox-PAPC-induced IL-8 neutralizing anti-CD14 antibodies were Although these antibodies blocked IL-8 synthesis in HAEC, they did not the effect of ox-PAPC the role of CD14, were with ox-PAPC or LPS in the presence of soluble CD14. shown in the induction of IL-8 by ox-PAPC was in the presence of soluble CD14 with the effect of LPS was which TLR4 E. H. A. S. K. M. N. B. J. Clin. Invest. 2000; PubMed Scopus Google Scholar), were with a human CD14 and an IL-8 to membrane CD14 the to ox-PAPC. ox-PAPC expression of cells did not from cells with LPS expression of cells were that of soluble nor membrane CD14 an ox-PAPC the expression of of the LPS receptor was Although HeLa cells are to ox-PAPC, they not that is not for ox-PAPC-induced IL-8 synthesis in HeLa cells. that CD14 and were not necessary for ox-PAPC induction we examined the binding of ox-PAPC to proteins. an antibody which binds to oxidized phospholipids in the or bound to protein (7Horkko S. Bird D.A. Miller E. Itabe H. Leitinger N. Subbanagounder G. Berliner J.A. Friedman P. Dennis E.A. Curtiss L.K. Palinski W. Witztum J.L. J. Clin. Invest. 1999; 103: 117-128Crossref PubMed Scopus (474) Google Scholar), bound to a protein of that was immunoprecipitated from of cells with anti-TLR4 antibodies binding of EO6 to a extent in cells cells. protein was observed in the A protein of this same size was also using an aerolysin to detect GPI-anchored proteins These suggest that ox-PAPC may initially bind to a 37-kDa GPI-anchored protein which interacts with TLR4 to induce Our studies demonstrated that ox-PAPC IL-8 transcription in and HeLa cells (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar). The current studies characterize the receptor responsible for this effect of ox-PAPC. We first examined the that scavenger receptors mediate the effect of ox-PAPC. Scavenger receptors have been in macrophages in the of atherosclerosis A. Bird D.A. Dennis E.A. Friedman P. K. Horkko S. Palinski W. O. P. D. Witztum J.L. N. Y. Acad. Sci. 2001; PubMed Scopus Google Scholar, Clin. 1999; PubMed Scopus Google Scholar, S. 2001; PubMed Scopus Google Scholar), their role in endothelial cells is We demonstrate that not MARCO, or expression of these did not increase ox-PAPC low of CD36 which is by ox-PAPC treatment not to was observed in we present evidence that CD36 are to MM-LDL, of which ox-PAPC is a major bioactive component (2Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar) and that with antibodies or CD36 peptides are to ox-PAPC not that CD36, important in macrophage M. E.A. Hajjar D.P. K. Silverstein R.L. J. Clin. Invest. 2000; PubMed Scopus Google Scholar), is not necessary for endothelial by MM-LDL or ox-PAPC. we examined the presence and of TLR4, the component of the LPS receptor in ox-PAPC-induced IL-8 HAEC, HeLa and human endothelial all of which to ox-PAPC, TLR4 A and Furthermore, ox-PAPC TLR4 synthesis dominant-negative constructs and antisense for TLR4 inhibited IL-8 by ox-PAPC, that TLR4 an important role in ox-PAPC-induced IL-8 synthesis. studies demonstrate that TLR4 is by endothelial cells in atherosclerotic K. J. Circulation. 2002; PubMed Scopus Google Scholar) and an TLR4 which receptor is associated with a of atherosclerosis S. E. M. E. J. D.A. N. J. Med. 2002; PubMed Scopus Google Scholar). We also present evidence that GPI-anchored proteins are necessary for ox-PAPC-induced IL-8 synthesis. pretreatment ox-PAPC-induced IL-8 synthesis not be by the of the bioactive lipids in ox-PAPC because the and the lipids were not present and demonstrate the presence of CD14 in A and early of human endothelial cells not were previously shown to CD14 A. 2001; PubMed Scopus Google Scholar). we demonstrate that CD14 is not involved in ox-PAPC-induced IL-8 synthesis Furthermore, we demonstrate that is not necessary for ox-PAPC induction of IL-8 transcription in HeLa as these cells not this The role of in ox-PAPC induction of IL-8 in endothelial cells is not antibodies a 37-kDa protein that also binds ox-PAPC a 37-kDa protein was also found in aerolysin overlays used to detect GPI-anchored proteins may the GPI-anchored protein with TLR4 that is responsible for ox-PAPC-induced IL-8 expression is not CD14, which has a mass of studies by Miller S. Binder Witztum J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have that MM-LDL and of macrophages by binding to CD14 and the LPS receptor complex of TLR4 and Miller found that MM-LDL binding was in macrophages of the and a in cells. were in cells with human CD14. also demonstrated that cells with human TLR4 and a cells with TLR4 that is for maximal to demonstrate that CD14 is not for ox-PAPC-induced IL-8 synthesis and that is not necessary for ox-PAPC-induced IL-8 transcription in HeLa cells. These may be to the that MM-LDL is a of possible including and oxidized ox-PAPC oxidized may to an or IL-8 transcription. are several important the of the Miller studies S. Binder Witztum J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and studies suggest an important role for TLR4 in the of MM-LDL and ox-PAPC (a major bioactive component of in macrophages and endothelial cells. like on ox-PAPC induction of IL-8 (6Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (102) Google Scholar), Miller determined that of the most well of TLR4, is not because an of did not the to both studies suggest a of TLR4 that may be important in