Molecular and Cellular Targets of Adenosine in Lung

“肺中腺苷的分子和细胞靶点”将着重于识别介导博来霉素对肺毒性的重要炎症细胞，以及A2A腺苷受体（A2AAR）激动剂抑制肺炎症的机制。目的1A是表征来自骨髓源性细胞、T细胞、粒细胞或内皮细胞（EC）中A2AAR组织特异性缺失的转基因小鼠的细胞。四种腺苷受体（AR）亚型的转录本将通过定量RT-PCR在纯化的中性粒细胞、巨噬细胞、T细胞和肺EC中进行检测。目的1B是通过中性粒细胞（氧化爆发）、巨噬细胞（TNFalpha释放和整合素表达）、T细胞（INFgamma释放和粘附）和EC（粘附和粘附分子表达）的功能分析，或FACS检测细胞表面活化标志物，对这些小鼠细胞中的A2AAR反应进行表型分析。假设1：有可能在各种转基因小鼠衍生的细胞中几乎消除A2AAR的表达和功能，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：探讨炎症诱导巨噬细胞和其他细胞mrna对LPS（阳性对照）和博来霉素的诱导反应。诱导ar将通过mrna、放射配体结合（如果可能）和功能来量化

"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.