title: A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4.
abstract: MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2.LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2.LPS complex. Finally we show that the MD-2.LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2.LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4(+)/MD-2(-) cells in the inflammatory microenvironment.
authors.affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Margaret N
authors.initials: MN
authors.lastname: Kennedy
authors.forename: Gregory E D
authors.initials: GE
authors.lastname: Mullen
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Leifer
authors.forename: ChangWoo
authors.initials: C
authors.lastname: Lee
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mazzoni
authors.forename: Kottarappat N
authors.initials: KN
authors.lastname: Dileepan
authors.forename: David M
authors.initials: DM
authors.lastname: Segal
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007249:Inflammation; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
chemical_list: D000954:Antigens, Surface; D015850:Interleukin-6; D016209:Interleukin-8; C495377:LY96 protein, human; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors