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

尽管遗传学研究揭示了Toll样受体(TLR4)在脂多糖(LPS)生物反应中的重要作用，但异位表达的TLR4和TLR2的活性存在争议。我们发现，在适当的转导条件下，TLR2和TLR4均可介导脂多糖诱导的人胚胎肾293细胞中NF-κB的活化。我们在这项研究中建立的重组系统可以直接进行生化表征和比较每个受体的激活情况。在胞浆区存在点突变的突变体TLRs在刺激时信号传导不活跃，并且以显性-负性突变体的形式特异性地抑制相应类型的受体而不是另一种类型的内毒素介导的激活，表明每个TLR独立地转导内毒素信号。TLR4对商业内毒素制剂的敏感性是TLR2的10-100倍。与商业脂多糖制剂的反应不同，TLR2对重新纯化的脂多糖或合成脂蛋白A无反应。然而，这两种TLR的反应强烈依赖于血清和CD14和内毒素结合蛋白。此外，脂类A中和剂多粘菌素B阻断了内毒素刺激TLR2和TLR4的能力。因此，尽管CD14和脂质A之间的相互作用是不同的和独立的激活，但强烈建议不仅对TLR4的激活，而且对TLR2的激活，我们还筛选了对内毒素刺激上调的分子，并发现了一个新的负调控炎症反应的分子。

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.