ALVEOLO-CAPILLARY SIGNALING IN LUNG IMMUNOLOGY

描述(由申请者提供)：目的：我们的总体目标是了解肺泡在肺部炎症发生中的作用。以前，我们报道了肺泡肿瘤坏死因子α诱导完整的肺泡毛细血管膜上皮细胞和内皮细胞之间的矢量串扰，并且AEC胞浆钙离子(细胞内钙离子)的增加驱动串扰。在这里，我们将检验一个假设，即串扰信使是源于线粒体或AEC的NAD(P)H氧化酶的H_2O_2。我们的具体目的是通过受体介导的(特异性目标1和2)和受体非依赖性的(特异性目标3)机制来定义肺泡毛细血管信号转导机制对AEC中细胞钙和过氧化氢产生的调节。 程序：我们将使用分离的、血液灌流的小鼠和大鼠肺标本。肺泡将被装载特定的荧光团，用于检测胞浆、线粒体和内体存储的钙离子，以及ROS的产生。随之而来的是，毛细管将装载类似的染料以确定串扰响应。单个肺泡毛细血管内皮细胞和肺泡毛细血管内皮细胞将通过广角和共聚焦显微镜实时光学成像。对于促炎症激活，肺泡毛细血管将受到肿瘤坏死因子α、脂多糖、花生四烯酸、浓盐酸和光解去钙的刺激。EC的反应将取决于细胞内钙离子和ROS的产生以及白细胞的募集。这些假说也将在含有NAD(P)H氧化酶(gp91Phox(-/-))和cPLA2基因缺陷的小鼠身上进行测试。 意义：虽然肺泡内的病理刺激可引起肺部炎症，但肺泡上皮屏障在产生这种反应中的作用尚不清楚。我们的研究将揭示AEC中不同的钙动员模式，即受体介导的与受体非依赖的模式，在建立将肺泡反应转化为毛细血管内皮细胞促炎症激活的信使方面的重要性。这项研究是新颖的，它将促进我们对调节肺部炎症的基本机制的理解。

DESCRIPTION (provided by applicant): Aims: Our overall aim is to understand the role of alveoli in the initiation of lung inflammation. Previously, we reported that alveolar TNFalpha induces vectorial crosstalk between epithelial and endothelial cells of intact alveolo-capillary membranes, and that increases in the AEC cytosolic Ca2+ (cytCa2+) drive the crosstalk. Here, we will test the hypothesis that the crosstalk messenger is H2O2 originating from mitochondria or NAD(P)H oxidase of AEC. Our Specific Aims are to define alveolo-capillary signaling in the context of the regulation of cytCa2+ and H2O2 production in AEC by receptor-mediated (Specific Aims 1 and 2) and receptor-independent (Specific Aim 3) mechanisms. Procedures: We will use the isolated, blood-perfused lung preparation for mouse and rat. Alveoli will be loaded with specific fluorophores for the detection of cytosolic, mitochondrial and endosomal store Ca2+, and for ROS production. Concomitantly, capillaries will be loaded with similar dyes to determine crosstalk responses. Single AEC and EC in the alveolo-capillary region will be optically imaged in real-time by of wide-angle and confocal microscopy. For proinflammatory activation, alveoli capillaries will be challenged with TNFalpha, LPS, arachidonate, concentrated HCI and photolytic uncaging of Ca2+. EC responses will be determined in terms of cytCa2+ and ROS production and the recruitment of leukocytes. The hypotheses will also be tested in mice containing genetically defective in NAD(P)H oxidase (gp91phox(-/-)), and in cPLA2. Significance: Although pathological stimuli in the alveolus induce lung inflammation, the role of the alveolar epithelial barrier in generating this response is not understood. Our research will reveal the importance of different modes of Ca2+ mobilization in AEC, namely those that are receptor-mediated versus receptor-independent, in establishing the messenger that translates the alveolar response to proinflammatory activation of EC in capillaries. This research is novel and it will advance our understanding of fundamental mechanisms regulating lung inflammation.