ALVEOLO-CAPILLARY SIGNALING IN LUNG IMMUNOLOGY

描述（由申请人提供）：目的：我们的总体目标是了解肺泡在肺部炎症起始中的作用。先前，我们报道了肺泡TNFalpha诱导完整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细胞质Ca2+ (cytCa2+)的增加驱动串扰。在这里，我们将验证串扰信使是来自线粒体或AEC的NAD(P)H氧化酶的H2O2的假设。我们的特定目标是通过受体介导（Specific Aims 1和2）和受体独立（Specific Aim 3）机制，在AEC中调节cytCa2+和H2O2产生的背景下，定义肺泡毛细血管信号。

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.