| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Toll-like receptor 4(TLR4) and MD-2 complex recognizes lipidA, the active moiety of microbial Lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2.
Firstly we found the physical interaction between LPS and TLR4-MD-2. 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. 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.
Secondly we found the ligand-induced TLR4 oligomerization, homotypic interaction of TLR4, which directly leads to TLR4 signaling. LipidIVa, a lipidA precursor, is agonistic on mouse TLR4/MD-2 but turns antagonistic on chimeric mouse TLR4/ human MD-2, demonstrating that the antagonistic activity of lipidIVa is determined by human MD-2. Binding studies with radioactive lipidA and lipidIVa revealed that lipidIVa binds mouse TLR4/ human MD-2 but does not trigger TLR4 oligomerization. Given that the antagonistic activity of lipidIVa is determined by MD-2, MD-2 has an important role in a link between ligand interaction and TLR4 oligomerization.
Finally we found that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS. The protective effect of the mAb was not due to inhibition of LPS response, since serum TNF, which was induced by LPS and caused lethal hepatitis, was highly upregulated by this mAb pretreatment. The unique agonistic mAb to TLR4/MD-2 may be useful for therapeutic intervention in tissue damages due to excessive inflammation.
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