|Budget Amount *help
¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
Lipopolysaccharide (LPS), a membrane constituent of Gram-negative bacteria, is the best-studied ligand that potently activates the immune system and induces endotoxin shock. Recently we discovered MD-2 molecule, which is associated with TLR4 extracellular domain, and we showed that TLR4/MD-2 complex is indispensable for LPS recognition and response, but it is not clear how TLR4/MD-2 recognizes LPS. Firstly to address the role played by MD-2 in vivo, we generated MD-2-/- mice. B cells, Macrophages, and Dendritic cells of MD-2-/- mice did not respond to LPS. And We showed that MD-2-/- mice did survive endotoxic shock but were susceptible to Salmonella typhimurium infection. We found that in MD-2-/- embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Next we showed that MD-2 directly regulates LPS recognition by TLR4. To address the issue, we took advantage of species-specific pharmacology of lipidIVa, an analogue of lipidA. LipidIVa acted agonistically on mouse(m)TLR4/MD-2 but not on human(h)TLR4/MD-2. LipidIVa antagonized the agonistic effect of lipidA on hTLR4/MD-2. We examined the chimeric complex consisting of mTLR4 and hMD-2 to ask whether species specificity is coinferred by TLR4 or MD-2. hMD-2 conferred on mTLR4 responsiveness to lipidA but not to lipidIVa. Moreover, lipidIVa acted as a lipidA antagonist on mTLR4 that is associated with hMD-2. Collectively, MD-2 directly influences the fine specificity of TLR4.