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

本研究的目的是探讨汞、肺凝集素（SP-A、SP-D）和Toll样受体（TLR）识别汞的分子机制，以及肺凝集素介导的抗炎和调节巨噬细胞功能的分子机制。SP-A与sTLR 2的结合能显著减弱sTLR 2与肽聚糖和酵母聚糖的结合; SP-D和SP-A与sTLR 2的结合能显著减弱sTLR 2与肽聚糖和酵母聚糖的结合; SP-A通过其与肺泡巨噬细胞的直接相互作用增强肺炎链球菌和鸟分枝杆菌的吞噬作用，而不依赖于其与细菌的结合。此外，SP-A刺激清道夫受体A介导的肺炎链球菌和甘露糖受体介导的鸟分枝杆菌的吞噬作用。SP-D能增强鸟支原体的吞噬功能，但不能增强肺炎链球菌的吞噬功能，说明肺凝集素刺激细菌吞噬功能的机制不同。5.构建了可溶性重组TLR 4胞外区（sTLR 4）和重组MD-2，并在杆状病毒-昆虫细胞表达系统中表达。MD-2被证明直接结合sTLR 4，但不结合sTLR 2。结果表明，肺凝集素SP-A和SP-D、sTLR 2和sTLR 4可作为TLR介导的炎症和巨噬细胞吞噬功能的调节剂。

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.