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Interaction of Soluble Form of Recombinant Extracellular TLR4 Domain with MD-2 Enables Lipopolysaccharide Binding and Attenuates TLR4-Mediated Signaling

2004; American Association of Immunologists; Volume: 173; Issue: 11 Linguagem: Inglês

10.4049/jimmunol.173.11.6949

1550-6606

Naoki Hyakushima, Hiroaki Mitsuzawa, Chiaki Nishitani, Hitomi Sano, Koji Kuronuma, Masanori Konishi, Tetsuo Himi, Kensuke Miyake, Yoshio Kuroki,

NF-κB Signaling Pathways

Abstract TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu24-Lys631. MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of KD = 6.29 × 10−8 M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-κB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu24-Lys631 enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.