Molecular and Cellular Targets of Adenosine in Lung

“肺中腺苷的分子和细胞靶点”将重点关注识别对介导博来霉素对肺的毒性至关重要的炎性细胞，以及A2 A腺苷受体（A2 AAR）激动剂抑制肺部炎症的机制。目的1A是表征来自在骨髓源性细胞、T细胞、粒细胞或内皮细胞（EC）中具有A2 AAR的组织特异性缺失的遗传修饰小鼠的细胞。将通过定量RT-PCR在纯化的中性粒细胞、巨噬细胞、T细胞和纯化的肺EC中测量四种腺苷受体（AR）亚型的转录物。目的1B是在源自这些小鼠的细胞中表型A2 AAR应答，是中性粒细胞（氧化爆发）、巨噬细胞（TNF α释放和整联蛋白表达）、T细胞（INF γ释放和粘附）和EC（粘附和粘附分子表达）的功能测定或细胞表面活化标志物的FACS检测。假设1是，将有可能几乎消除来自各种遗传修饰小鼠的细胞中的A2 AAR表达和功能，而对其他AR亚型的表达几乎没有影响。这些研究将有助于独特的AR亚型选择性配体的可用性。目的2是检查响应于LPS（阳性对照）和博来霉素的由巨噬细胞和其它细胞中的炎症诱导的mRNA的诱导。诱导的AR将通过mRNA、放射性配体结合（如可能）和功能性 测定。假设2是腺苷的功能性抗炎反应是由炎症刺激强烈诱导的。目的3是确定博来霉素和A2 AAR活化如何影响体内白细胞运输、肺细胞因子产生和纤维化，使用具有各种靶向A2 AAR缺失的小鼠和缺乏肺巨噬细胞、INF γ或CXCR 2受体（结合KC或MIP-1 α）的小鼠。将通过BALF、分散肺、免疫组织化学中的细胞计数测量细胞运输，并在活体动物中通过超高分辨率γ成像和MRI测量细胞运输。假设3是肺巨噬细胞和/或T细胞影响对A2 AAR活化的反应。这些结果将有助于确定A2 AAR激活在保护肺免受其他损伤（LPS和缺血-再灌注损伤）中的作用，这些损伤正在其他项目中进行研究。

"Molecular and Cellular Targets of Adenosine in Lung" will focus on identifying inflammatory cells that are important for mediating bleomycin toxicity to the lung, and the mechanisms used by agonists of the A2A adenosine receptor (A2AAR) to inhibit lung inflammation. Aim 1A is to characterize cells from genetically modified mice with tissue specific deletions of the A2AAR in bone marrow-derived cells, T cells, granulocytes or endothelial cells (EC). Transcripts for the four adenosine receptor (AR) subtypes will be measured by quantitative RT-PCR in purified neutrophils, macrophages, T cells and lung EC purified. Aim 1B is to phenotype A2AAR responses in cells derived from these mice is functional assays of neutrophils (oxidative burst), macrophages (TNFalpha release and integrin expression), T cells (INFgamma release and adherence) and EC (adherence and adhesion molecule expression) or FACS detection of cell surface activation markers. Hypothesis 1 is that it will be possible to nearly eliminate A2AAR expression and function in cells derived from various genetically modified mice with little effect on the expression other AR subtypes. These studies will be helped by the availability of unique AR subtype selective ligands. Aim 2 is to examine the induction in response to LPS (positive control) and bleomycin of mRNAs induced by inflammation in macrophages and other cells. Induced ARs will be quantified by mRNAs, radioligand binding (where possible), and functional assays. Hypothesis 2 is that functional anti-inflammatory responses to adenosine are strongly induced by inflammatory stimuli. Aim 3 is to determine how bleomycin and A2AAR activation affect leukocyte trafficking, pulmonary cytokine production and fibrosis in vivo using mice with various targeted A2AAR deletions, and mice lacking pulmonary macrophages, INFgamma or CXCR2 receptors (that bind KC or MIP-1alpha). Cell trafficking will be measured by counting cells is BALF, dispersed lung, immunohistochemistry and, in live animals by ultra-high resolution gamma-imaging and MRI. Hypothesis 3 is that the response to A2AAR activation influenced by pulmonary macrophages and/or T cells. These results will be helpful for determining the role of A2AAR activation in protecting lungs from other injuries (LPS and ischemia-reperfusion injury) that are being investigated in the other projects.