Host defense system in the lung using pulmonary surfactant proteins and Toll-like reosptors

本课题的目的是研究肺表面活性蛋白A和D(SP-A和SP-D)以及Toll样受体(Tiso)在抗感染和炎症中的作用机制，并为这些蛋白的临床应用奠定分子基础。研究成果总结如下：1.研究成果。我们发现SP-A和SP-D与TLR2、TLR4和MD-2结合。SP-A抑制光滑脂多糖与表达TLR4和MD-2的细胞的结合，抑制光滑脂多糖诱导的炎症反应。功能结构域是SP-A的糖识别结构域(CRD)，但去除胶原区的SP-A胶原酶抗性片段(CRF)与TLR4结合很弱，不能抑制内毒素诱导的炎症反应，表明SP-A十八聚体的寡聚结构对其功能的表达具有重要意义。与SP-A不同，SP-D抑制不同血清型的内毒素、粗面内毒素和光面内毒素引起的炎症反应。对SP-A/SP-D嵌合蛋白的实验表明，SP-D由长胶原区组成的十字形结构对抑制炎症至关重要。我们还发现TLR4的氨基末端区域Glu24-Lys47是MD-2结合的位点。重组可溶性TLR4胞外结构域和MD-2可抑制内毒素诱导的小鼠肺部炎症。与TLR4突变体TLR4C88A的实验表明，MD-2具有表达TLR4及其N-连接复合型糖基化的关键能力。SP-A和SP-D与禽分枝杆菌结合并诱导细菌聚集。与SP-A相比，SP-D具有更强的聚集禽类支原体的能力，在禽类支原体上，SP-D的配体是脂阿拉伯甘露聚糖(LAM)，而SP-A的配体存在于禽类支原体的脂类部位。

The purposes of this project were to investigate the mechanisms by which pulmonary collections, surfactant proteins A and D (SP-A and SP-D), and Toll-like receptor (TISO function against infection and inflammation, and to establish the molecular basis for the clinical application of these proteins. The research results are summarized below.1. We have found that SP-A and SP-D bind to TLR2, TLR4 and MD-2. SP-A inhibited the binding of smooth LPS on the cells expressing TLR4 and MD-2 and suppresses inflammation elicited by smooth LPS. The functional domain was the carbohydrate recognition domain (CRD) of SP-A, but the collagenase-resistant fragment of SP-A (CRF) in which the collagenous domain was removed bound to TLR4 very weakly and did not inhibit LPS-induced inflammation, indicating that the oligometric structure composed of SP-A octadecamers is important for expressing its function. Unlike SP-A SP-D inhibits inflammation elicited by different serotypes of LPS, rough LPS and smooth LPS. The experiments with SP-A/SP-D chimeric proteins revealed that the cruciform structure of SP-D composed of long collagenous domain was crucial for suppressing inflammation.2. We have also found that the amino-terminal region of TLR4, Glu24-Lys47, is a site for MD-2 binding. Recombinant soluble forms of the extracellular TLR4 domain and MD-2 were demonstrated to dampen endotoxin-induced pulmonary inflammation in mice.3. The experiments with TLR4 mutant, TLR4C88A, revealed that MD-2 possesses crucial abilities to express cell surface expression of TLR4 and its N-linked complex type glyoosylation.4. SP-A and SP-D bound to Mycobacterium avium and induced bacrerial aggregation. SP-D possesses a potent ability to aggregate M. avium compared with SP-A The ligand for SP-D on M. avium was lipoarabinomannan (LAM) and that for SP-Aexisted in the lipid fraction extracted M. avium.