Innate immune host defense mediated by pulmonary sutfactant proteins and Toll-like receptors, and its clinical application.

本项目旨在探讨hg的分子机制和肺集合素、SP-A、SP-D和toll样受体（TLR）的识别，以及集合素介导的炎症功能和巨噬细胞功能的调节。由Met1-Arg587组成的可溶性重组TLR2 （sTLR2）通过杆状病毒-昆虫细胞表达系统进行了表达。sTLR2被发现直接结合从金黄色葡萄球菌和酵素衍生的肽聚糖2。SP-A与sTLR2结合，SP-A与sTLR2结合显著减弱sTLR2与肽聚糖和酶酶的结合。SP-D和SP-A与sTLR2.4交互。SP-A以依赖SP-A浓度和Ca2+的方式与肺炎链球菌和鸟分枝杆菌结合。SP-A通过与肺泡巨噬细胞的直接相互作用增强了肺炎链球菌和鸟分枝杆菌的吞噬作用，而不依赖于与细菌的结合。此外，SP-A刺激了清道夫受体a介导的肺炎链球菌和甘露糖受体介导的鸟芽胞杆菌的吞噬。虽然SP-D增强了M.avium的吞噬，但该蛋白并没有增强肺炎链球菌的吞噬，提示肺收集物刺激细菌吞噬的不同机制。制备了重组TLR4胞外结构域（sTLR4）和重组MD-2，并在杆状病毒-昆虫细胞表达系统中进行了表达。MD-2被证明直接与sTLR4结合，而不与sTLR2结合。sTLR4和MD-2的复合体能够与脂多糖共价结合，而sTLR4单独不能与脂多糖结合。这些结果表明，肺集合素、SP-A和SP-D、sTLR2和sTLR4可以作为tlr介导的炎症和巨噬细胞吞噬的调节剂。

The purpose of this project was to investigate the molecular mechanisms of hg and recognition by lung collectins, SP-A and SP-D, and Toll-like receptor(TLR) and of the colletin-mediated and-inflammatory function and modulation of macrophage functions.1.A soluble form of recombinant TLR2 consisting of Met1-Arg587(sTLR2) was generated and expressed by baculovirus-insect cell expression system. sTLR2 was found to bind directly to peptidoglycn derived from Staphylococcus aureus and zymosan.2.SP-A bound to sTLR2 and the binding of SP-A to sTLR2 significantly attenuated the binding of sTLR2 to peptidoglycan and zymosan.3.SP-D as well as SP-A interacted with sTLR2.4.SP-A bound to Streptococcus pneumoniae and Mycobacterium avium in a manner dependent upon the SP-A concentrations and Ca2+. SP-A augemented the phagocytosis of S.pneumoniae and M.avium by its direct interaction with alveolar macrophages independent of its binding to the bacteria. In addition, SP-A stimualted the scavenger receptor A-mediated phagocytosis of S.pneumoniae and the mannose receptor-mediated phagocytosis of M.avium. Although SP-D augmented the M.avium phagocytosis, this protein did not enhance the phagocytosis of S.pneumoniae, indicating the different mechanisms of the bacterial phagocytosis stimulated by lung collectins.5.A soluble form of recombinant TLR4 extracellular domain(sTLR4) and recombinant MD-2 were generated and expressed in bacluovirus-insect cell expression system. MD-2 was demonstrated to bind directly to sTLR4 but not to sTLR2. A complex of sTLR4 and MD-2 was able to bind to LPS covalenrly linked to sepharose beads although sTLR4 alone failed to bind LPS.6.These results suggest that lung collectins, SP-A and SP-D, sTLR2 and sTLR4 can function as mudulators for TLR-mediated inflammation and for macrophage phagocytosis.