IMMUNE FUNCTIONS OF ACCESSORY CELLS IN THE LUNG

描述（由申请人提供）：本提案的长期目标是确定肺中树突状细胞（DC）的免疫功能。 DC表达紧密连接（TJ）蛋白和金属蛋白酶，以及紧密连接蛋白（TJ蛋白的一个家族）可以调节金属蛋白酶活性的观察提示了以下三个问题，这些问题对于理解肺DC生物学至关重要。 1. DC如何维持TJ鹞，因为它们穿过细胞旁途径，以采样气道中的病原体？ 为了回答这个问题，在趋化因子诱导的DC穿过鼠气道上皮细胞系单层的迁移过程中体外评估DC和上皮细胞之间形成的TJ的结构/功能，并在抗原吸入后体内评估。 2.金属蛋白酶MMP-2和/或MMP-9促进DC通过TJ迁移吗？ 这通过在体内检查MMP-2-/-或MMP-9-/-突变小鼠中的DC来确定MMP-2和/或MMP-9在促进DC通过TJ的迁移中的作用来解决。 使用短干扰RNA，体外研究将检查密蛋白-1在调节MT 1- MMP介导的MMP-2活化中的作用。 3. DCs是否通过释放MMP-2和/或MMP-9激活细胞因子并促进DC向局部淋巴结迁移，在肉芽肿性免疫应答中发挥关键作用？ 为了回答这个问题，向MMP-2-/-或MMP-9-/-突变小鼠施用抗原包被的珠以诱导肉芽肿形成。 随时间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真实的时间定量PCR用于检测肉芽肿内的细胞因子环境。 这些研究将为肺DC在肺免疫防御中的作用提供重要的新见解。 所获得的信息将有助于制定新的战略，以防止吸入病原体。

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.