The Journal of Experimental Medicine
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Published online 29 September 2003 doi:10.1084/jem.20031076
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© Rockefeller University Press, 0022-1007/2003/10/1035 $5.00
The Journal of Experimental Medicine, Volume 198, Number 7, 1035-1042

Lipopolysaccharide Interaction with Cell Surface Toll-like Receptor 4-MD-2 : Higher Affinity than That with MD-2 or CD14



Sachiko Akashi1, Shin-ichiroh Saitoh1, Yasutaka Wakabayashi1, Takane Kikuchi1, Noriaki Takamura1, Yoshinori Nagai1, Yutaka Kusumoto1, Koichi Fukase2, Shoichi Kusumoto2, Yoshiyuki Adachi4, Atsushi Kosugi3,5 and Kensuke Miyake1,5

1 Division of Infectious Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan
2 Department of Chemistry, Graduate School of Science
3 Department of Medical Technology and Science, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan
4 Laboratory of Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo 192-0392, Japan
5 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo 101-0062, Japan

Address correspondence to Kensuke Miyake, Division of Infectious Genetics, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5290; Fax: 81-3-5449-5410; email: kmiyake{at}ims.u-tokyo.ac.jp

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS–TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS–TLR4-MD-2 complexes, but is not coprecipitated with LPS–TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS–TLR4-MD-2 complexes was ~3 nM, which is ~10–20 times lower than the reported Kd for LPS–MD-2 or LPS–CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Key Words: innate immunity • cell surface molecule • activation • macrophage


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