MOLECULAR MECHANISMS OF GM-CSF PRODUCTION BY HUMAN EOSINOPHILS

外周血嗜酸粒细胞(PBE)分化的机制 以呼吸道为基础的效应细胞负责其病理生理学 人们对哮喘知之甚少。这一进程的候选调解人是 GM-CSF，一种由激活的嗜酸性粒细胞和其他细胞产生的强大细胞因子 炎性细胞。BAL液中GM-CSF水平升高 活动性哮喘患者虽然在体外这种细胞因子对 嗜酸性粒细胞功能与存活率。嗜酸性粒细胞在体外被激活 离子载体、纤维连接蛋白或VCAM-1分泌和积聚GM-CSF 并积聚GM-CSF mRNA。因此，现有数据有力地表明， GM-CSF是PBE重要的自分泌生长和生存因子 在体外和体内都有作用。尽管很可能是功能性的 GM-CSF的意义，对其分子知之甚少 作用机理)S)，控制其产生和释放的嗜酸性粒细胞。 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 产量既取决于转录增加，也取决于转录减少 M RNA降解。因此，我们假设类似的双重控制 这些机制也可以调节活化的嗜酸性粒细胞产生GM-CSF。 这样的基因调控研究还没有在嗜酸性粒细胞中进行， 它们很难转染或分离出完整的mRNA.利用 粒子介导的基因转移(PMGT)，我们已经成功地转导了 用荧光素酶报告蛋白静息外周血嗜酸粒细胞 构造。其次，我们证明了完整的mrna是可以分离出来的。 从嗜酸性粒细胞中提取并成功地与GM-CSF特异性基因杂交 探测器。因此，我们处于一个独特的位置来调查 巨噬细胞集落刺激因子产生的分子机制 激活的嗜酸性粒细胞。对此，我们的具体目标是：1)。优化 颗粒介导基因转移法转染嗜酸粒细胞。2)。 确定转录稳定性对 葛根素对嗜酸性粒细胞GM-CSF基因表达的影响 离子载体、纤维连接蛋白和血管细胞黏附分子-1。3)。确定GM-CSF的作用机制 活化的嗜酸性粒细胞中mRNA的稳定性。4)。识别顺式-反式 活化细胞中控制GM-CSF启动子上调的相互作用 嗜酸性粒细胞。累积起来，这些研究将与 并提供了有关以下方面的新信息 活化的嗜酸性粒细胞产生GM-CSF的分子调控。

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.