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* Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan;
Division of Biophysics, Sapporo Medical University School of Health Sciences, Sapporo, Japan;
Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan; and
Core Research for Engineering, Science, and Technology, Japan Science and Technology, Kawaguchi, Japan
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-
B 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 [3H]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. Coinstillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-
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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.
2 Address correspondence and reprint requests to Dr. Yoshio Kuroki, Department of Biochemistry, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address: kurokiy{at}sapmed.ac.jp
3 Abbreviations used in this paper: sTLR, soluble form of rTLR; HEK, human embryonic kidney; sCD14, soluble CD14; sMD-2, soluble form of rMD-2; wt, wild type.
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  S. Divanovic, A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, R. Madan, J. J. Haky, and C. L. Karp Regulation of TLR4 signaling and the host interface with pathogens and danger: the role of RP105 J. Leukoc. Biol., August 1, 2007; 82(2): 265 - 271. [Abstract] [Full Text] [PDF]
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