title: Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice.
abstract: In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-kappaB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [(3)H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Co-instillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-alpha levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.
authors.affiliation: Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuzawa
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nishitani
authors.forename: Naoki
authors.initials: N
authors.lastname: Hyakushima
authors.forename: Takeyuki
authors.initials: T
authors.lastname: Shimizu
authors.forename: Hitomi
authors.initials: H
authors.lastname: Sano
authors.forename: Norio
authors.initials: N
authors.lastname: Matsushima
authors.forename: Koichi
authors.initials: K
authors.lastname: Fukase
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kuroki
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D045604:Extracellular Fluid / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D005260:Female; D016209:Interleukin-8 / Q000378:metabolism; D008050:Lipid A / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology* / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D011014:Pneumonia / Q000139:chemically induced / Q000276:immunology* / Q000517:prevention & control; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000276:immunology / Q000378:metabolism*
chemical_list: D016209:Interleukin-8; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D011994:Recombinant Proteins; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4