title: MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.
abstract: MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.
authors.affiliation: Laboratory of Oncology, Pharmacy Practice and Sciences, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Sendai 980-8578, Japan; Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan. Electronic address: tsukamoh@m.tohoku.ac.jp.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tsukamoto
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Ihara
authors.forename: Ritsu
authors.initials: R
authors.lastname: Ito
authors.forename: Ippo
authors.initials: I
authors.lastname: Ukai
authors.forename: Naoto
authors.initials: N
authors.lastname: Suzuki
authors.forename: Masao
authors.initials: M
authors.lastname: Kimoto
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Tomioka
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D051218:Lymphocyte Antigen 96 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4
keywords: Bio-; C-terminally FLAG-tagged MD-2; CM; FCS; LBP; LBP-N; LPS; LPS-binding protein; Lipopolysaccharide; MD-2; MD-2F; Monoclonal antibody; PE; TLR; Toll-like receptor; Toll-like receptor 4; biotinylated; conditioned medium; fetal calf serum; lipopolysaccharide; mAb; monoclonal antibody; phycoerythrin; sCD14; sMD-2; soluble MD-2; soluble form of CD14; streptavidin; stv; truncated N-terminal form of LBP