IMMUNE FUNCTIONS OF ACCESSORY CELLS IN THE LUNG

描述(申请人提供)：这项提案的长期目标是确定肺中树突状细胞(DC)的免疫功能。树突状细胞表达紧密连接(TJ)蛋白和金属蛋白酶，而Claudins是TJ蛋白家族中的一员，可以调节金属蛋白酶的活性，这三个观察结果提示了以下三个问题，这些问题是理解肺DC生物学的核心。1.DC如何维持TJ屏障穿过细胞旁通路以采样呼吸道中的病原体？为了回答这个问题，我们在体外评价了趋化因子诱导的DC在小鼠呼吸道上皮细胞系单层间迁移过程中，DC与上皮细胞之间形成的TJs的结构/功能。2.基质金属蛋白酶MMP2和/或MMP9是否促进DC通过TJs的迁移？这是通过检测MMP2-/-或MMP9-/-突变小鼠体内的DC来解决的，以确定MMP2和/或MMP9在促进DC通过TJ迁移中的作用。利用短干扰RNA，体外研究将检测claudin-1在调节MT1-MMP介导的MMP2激活中的作用。3.树突状细胞是否通过释放基质金属蛋白酶-2和/或基质金属蛋白酶-9而在肉芽肿性免疫反应中发挥关键作用，从而激活细胞因子并促进树突状细胞向局部淋巴结的迁移？为了回答这个问题，将包被抗原的珠子注射给突变的小鼠，以诱导肉芽肿的形成。随着时间的推移，监测肉芽肿的大小和所含DC的数量。激光捕获显微切割结合实时定量聚合酶链式反应检测肉芽肿内的细胞因子环境。这些研究将对肺树突状细胞在肺免疫防御中的作用提供重要的新见解。获得的信息将有助于制定新的战略来防御吸入性病原体。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define the immune function of dendritic cells (DC) in the lung. The observations that DC express tight junction (TJ) proteins and metalloproteinases, and that the claudins, a family of TJ proteins, can regulate metalloproteinase activity prompts the following three questions central to the understanding of lung DC biology. 1. How do DC maintain the TJ harrier as they traverse the paracellular pathway to sample pathogens in the airways? To answer this question the structure/function of TJs formed between DC and epithelial cells is assessed in vitro during chemokine induced migration of DC across murine airway epithelial cell line monolayers and in vivo following antigen inhalation. 2. Do the metalloproteinases MMP-2 and/or MMP-9 facilitate DC migration through TJs? This is addressed by examining DC in MMP2-/- or MMP-9-/- mutant mice, in vivo to determine the role of MMP-2 and/or MMP-9 in facilitating the migration of DC through TJs. Using short interfering RNAs, studies in vitro will examine the role of claudin-1 in regulating MT1- MMP mediated activation of MMP-2. 3. Do DCs play a critical role in the granulomatous immune response by releasing MMP-2 and/or MMP-9, which in turn activate cytokines and promote DC migration to local lymph nodes? To answer this question antigen-coated beads are administered to MMP-2-/- or MMP-9-/- mutant mice to induce granuloma formation. The size of the granuloma and the number of contained DC are monitored over time. Laser capture microdissection combined with real time quantitative PCR is used to examine the cytokine milieu within the granulomas. These studies will contribute important new insights into the role of pulmonary DC in the immune defense of the lung. The information obtained will assist in devising new strategies to defend against inhaled pathogens.