title: Isolation of an endotoxin-MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations.
abstract: Host proinflammatory responses to minute amounts of endotoxins derived from many Gram-negative bacteria require the interaction of lipopolysaccharide-binding protein (LBP), CD14, Toll-like receptor 4 (TLR4) and MD-2. Optimal sensitivity to endotoxin requires an ordered series of endotoxin-protein and protein-protein interactions. At substoichiometric concentrations, LBP facilitates delivery of endotoxin aggregates to soluble CD14 (sCD14) to form monomeric endotoxin-sCD14 complexes. Subsequent interactions of endotoxin-sCD14 with TLR4 and/or MD-2 have not been specifically defined. This study reports the purification of a stable, monomeric, bioactive endotoxin-MD-2 complex generated by treatment of endotoxin-sCD14 with recombinant MD-2. Efficient generation of this complex occurred at picomolar concentrations of endotoxin and nanogram per milliliter doses of MD-2 and required presentation of endotoxin to MD-2 as a monomeric endotoxin-CD14 complex. TLR4-dependent delivery of endotoxin to human embryonic kidney (HEK) cells and cell activation at picomolar concentrations of endotoxin occurred with the purified endotoxin-MD-2 complex, but not with purified endotoxin aggregates with or without LBP and/or sCD14. The presence of excess MD-2 inhibited delivery of endotoxin-MD-2 to HEK/TLR4 cells and cell activation. These findings demonstrate that TLR4-dependent activation of host cells by picomolar concentrations of endotoxin occurs by sequential interaction and transfer of endotoxin to LBP, CD14, and MD-2 and simultaneous engagement of endotoxin and TLR4 by MD-2.
authors.affiliation: Inflammation Program, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. theresa.gioannini@uiowa.edu
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
authors.forename: Athmane
authors.initials: A
authors.lastname: Teghanemt
authors.forename: DeSheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Coussens
authors.forename: Wendie
authors.initials: W
authors.lastname: Dockstader
authors.forename: S
authors.initials: S
authors.lastname: Ramaswamy
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000302:isolation & purification* / Q000378:metabolism; D004731:Endotoxins / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007107:Immune System / Q000276:immunology / Q000378:metabolism; D007313:Insecta / Q000276:immunology / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000276:immunology / Q000378:metabolism; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors
chemical_list: D000954:Antigens, Surface; D004731:Endotoxins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors