Molecular and Cellular Targets of Adenosine in Lung

肺中腺苷的分子和细胞靶标

• 批准号: 7232629
• 负责人: Joel M. Linden
• 金额: $ 21.7万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232629
• 关键词: T lymphocyte; adenosine; bleomycin; bone marrow; cell biology; cell migration; cell population study; cytotoxicity; fibrosis; genetically modified animals; granulocyte; inflammation; laboratory mouse; leukocyte activation /transformation; lipopolysaccharides; lung injury; macrophage; molecular biology; myocardium disorder; neurotransmitter receptor; neutrophil; pathologic process; pharmacology; protein isoforms; protein structure function; purinergic receptor; receptor expression; stimulant /agonist; vascular endothelium

"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.

“肺中腺苷的分子和细胞靶标”将着重于鉴定炎症细胞，这些细胞对于介导肺的博霉素毒性很重要，以及A2A腺苷受体（A2AAR）激动剂使用的机制来抑制肺炎症。 AIM 1a是在骨髓衍生的细胞，T细胞，粒细胞或内皮细胞（EC）中表征具有基因改性小鼠的细胞。四种腺苷受体（AR）亚型的转录本将通过纯化的嗜中性粒细胞，巨噬细胞，T细胞和肺EC纯化的定量RT-PCR测量。 AIM 1B是源自这些小鼠的细胞中的表型A2AAR反应是中性粒细胞（氧化爆发），巨噬细胞（TNFalpha释放和整联蛋白表达）的功能测定法，T细胞（Infgamma释放和依从性）和EC（粘附和粘附分子表达）或FACS FACS或FACS FACS或FACS FACS检测细胞表面表面表面激活分标。假设1是，几乎可以消除源自各种遗传修饰小鼠的细胞中的A2AAR表达和功能，而对其他AR亚型的表达几乎没有影响。这些研究将通过独特的AR亚型选择性配体的可用性来帮助。 AIM 2是检查响应于LPS（阳性对照）和巨噬细胞炎症引起的mRNA的博来霉素的诱导。诱导的ARS将通过mRNA，放射性配体结合（如有）和功能来定量 测定。假设2是炎症刺激强烈诱导了对腺苷的功能性抗炎反应。 AIM 3是确定博来霉素和A2AAR激活如何影响白细胞运输，肺细胞因子的产生和纤维化在体内使用各种靶向A2AAR缺失的小鼠，以及缺乏肺巨噬细胞的小鼠，Infgamma或cxcr2受体（结合KC或MIP-1Alpha）。细胞运输将通过计数细胞来衡量BALF，分散的肺，免疫组织化学，并通过超高分辨率γ成像和MRI在活体动物中进行测量。假设3是对A2AAR激活的反应受肺巨噬细胞和/或T细胞影响。这些结果将有助于确定A2AAR激活在保护肺部免受其他项目中正在研究的其他损伤（LPS和缺血再灌注损伤）中的作用。