Activation Mechanisms of Defense Systems by Substances on the Surface of Pathogens

尽管遗传学研究表明toll样受体（TLR） 4在脂多糖（LPS）的生物学反应中起着关键作用，但异位表达的TLR4和TLR2的活性仍存在争议。我们发现在适当的转染条件下，TLR2和TLR4均介导lps诱导的人胚胎肾293细胞NF-κB活化。我们在本研究中建立的重组系统允许直接的生化表征和比较每个受体的激活。在细胞质区域有点突变的突变TLRs在刺激时对信号的转导不活跃，并且作为显性负突变体特异性地抑制LPS介导的受体类型的激活，而不抑制其他类型的受体，这表明每个TLR独立地转导LPS信号。TLR4对商业LPS制剂的敏感性是TLR2的10-100倍。与对商业LPS制剂的反应相反，TLR2对再纯化的LPS或合成脂质a无反应。然而，两种TLRs的反应都强烈依赖于血清和CD14，而LPS结合蛋白对于激活是必不可少的。此外，脂质a中和试剂多粘菌素B阻断了LPS刺激TLR2和TLR4的能力。因此，尽管有不同的和独立的激活，强烈建议CD14和脂质A之间的相互作用的关键参与，不仅对TLR4的激活，而且对TLR2的激活。在上述研究的同时，我们筛选了lps刺激下上调的分子，发现了一个新的负调节炎症反应的分子。

Although genetic studies have revealed a critical role for the toll-like receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPS-induced NF-κB activation in human embryonic kidney 293 cells. The reconstitution systems we established in this study allowed direct biochemical characterization and comparison of activation of each receptor. Mutant TLRs harboring a point mutation in the cytoplasmic domain was inactive in transducing the signal upon stimulation, and acted as dominant-negative mutants specifically inhibiting the LPS-mediated activation by corresponding type of the receptor but not the othertype, indicating that each TLR independently transduces the LPS signal. TLR4 was 10-100-fold more sensitive to a commercial LPS preparation than TLR2. In contrast to the response to the commercial LPS preparations, TLR2 was unresponsive to re-purified LPS or synthetic lipid A.Nevertheless, the responses of both TLRs were strongly dependent on serum and CD14 and LPS-binding protein were essential for the activation. Furthermore, a lipid A-neutralizing reagent, polymyxin B, blocked the ability of LPS to stimulate TLR2 as well as TLR4. Therefore, despite the distinct and independent activation, critical involvement of an interaction between CD14 and lipid A was strongly suggested not only for the activation of TLR4 but also for that of TLR2.In parallel with the above study, we screened molecules that was up-regulated upon LPS-stimulation and found a new molecule that would negatively regulate inflammation reactions.