MOLECULAR MECHANISMS OF GM-CSF PRODUCTION BY HUMAN EOSINOPHILS

人类嗜酸性粒细胞产生 GM-CSF 的分子机制

• 批准号: 6410558
• 负责人: James S Malter
• 金额: $ 19.62万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6410558
• 关键词: asthma; cell adhesion molecules; cell differentiation; colony stimulating factor; eosinophil; fibronectins; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; human tissue; immunogenetics; ionophores; leukocyte activation /transformation; tissue /cell culture; transcription factor; transfection

The mechanisms by which peripheral blood eosinophils (PBE) differentiate into airway based, effector cells responsible for the pathophysiology of asthma are poorly understood. A candidate mediator for this process is GM-CSF, a potent cytokine produced by activated eosinophils and other inflammatory cells. GM-CSF levels are elevated in the BAL fluid of active asthmatics while in vitro this cytokine has profound effects on eosinophil function and survival. Eosinophils activated in vitro with ionophore, fibronectin or VCAM-1 secrete GM-CSF and accumulate GM-CSF and accumulate GM-CSF mRNA. Thus, available data strongly suggest that GM-CSF is an important autocrine growth and survival factor for PBE function both in vitro and in vivo. Despite the likely functional significance of GM-CSF, very little is known about the molecular mechanism)s) which control its production and release by eosinophils. In T lymphocytes, fibroblasts and mast cells, mitogen induced GM-CSF production is dependent on both increased transcription and decreased mRNA degradation. Therefore, we hypothesize that similar, dual control mechanisms also modulate GM-CSF production from activated eosinophils. Such gene regulation studies have not been performed in eosinophils as they are difficult to transfect or isolate intact mRNA from. Utilizing particle mediated gene transfer (PMGT), we have successfully transfected resting peripheral blood eosinophils with luciferase reporter constructs. Secondly, we demonstrate that intact mRNA can be isolated from eosinophils and successfully hybridized with GM-CSF specific cDNA probes. Therefore, we are in a unique position to investigate the molecular mechanisms responsible for the elaboration of GM-CSF by activated eosinophils. This, our specific aims are: 1). Optimize the transfection of eosinophils by particle mediated gene transfer. 2). Determine the relative contributions of transcription stability to accumulation of GM-CSF mRNA in eosinophils after treatment with ionophore, fibronectin and VCAM-1. 3). Determine the mechanism of GM-CSF mRNA stabilization in activated eosinophils. 4). Identify the cis-trans interactions which control GM-CSF promoter upregulation in activated eosinophils. Cumulatively, these studies will closely interact with other components of the SCOR and provide novel information concerning the molecular control of GM-CSF production by activated eosinophils.

周围血液嗜酸性粒细胞（PBE）的机制分化 进入基于气道的效应细胞负责导致的病理生理 哮喘知之甚少。 此过程的候选人是 GM-CSF，一种有效的细胞因子，由活化的嗜酸性粒细胞和其他 炎性细胞。 GM-CSF水平在BAL流体中升高 活跃的哮喘患者在体外这种细胞因子对 嗜酸性粒细胞功能和生存。 嗜酸性粒细胞在体外激活 离子载体，纤连蛋白或VCAM-1分泌GM-CSF并积累GM-CSF 并积累GM-CSF mRNA。 因此，可用的数据强烈表明 GM-CSF是PBE的重要自分泌生长和生存因子 体外和体内功能。 尽管可能有功能 GM-CSF的重要性，对分子知之甚少 机制）s）控制嗜酸性粒细胞的生产和释放。 在T淋巴细胞，成纤维细胞和肥大细胞中，有丝分裂原诱导的GM-CSF 生产取决于转录的增加和减少 mRNA降解。 因此，我们假设相似的双重控制 机制还可以调节活化嗜酸性粒细胞的GM-CSF产生。 这种基因调节研究尚未在嗜酸性粒细胞中进行 它们很难从中转染或分离完整的mRNA。 利用 粒子介导的基因转移（PMGT），我们已成功转染 与荧光素酶报告基因静止的外周血嗜酸性粒细胞 构造。 其次，我们证明可以分离完整的mRNA 来自嗜酸性粒细胞，并成功地与GM-CSF特异性cDNA杂交 探针。 因此，我们处于独特的位置来研究 负责阐述GM-CSF的分子机制 活化的嗜酸性粒细胞。 这是我们的具体目标：1）。优化 通过粒子介导的基因转移对嗜酸性粒细胞转染。 2）。 确定转录稳定性的相对贡献 治疗后，GM-CSF mRNA在嗜酸性粒细胞中的积累 离子载体，纤连蛋白和VCAM-1。 3）。确定GM-CSF的机制 激活的嗜酸性粒细胞中的mRNA稳定。 4）。识别顺式传播 控制GM-CSF启动子上调的相互作用激活 嗜酸性粒细胞。 累积地，这些研究将与 SCOR的其他组成部分，并提供有关有关的新信息 激活的嗜酸性粒细胞对GM-CSF产生的分子控制。