MOLECULAR MECHANISMS OF GM-CSF PRODUCTION BY HUMAN EOSINOPHILS

外周血嗜酸性粒细胞（PBE）分化的机制 转化为基于气道的效应细胞， 哮喘知之甚少。 这一进程的候选调解人是 GM-CSF是一种由活化的嗜酸性粒细胞和其他细胞因子产生的强效细胞因子， 炎症细胞 GM-CSF水平在BALF中升高， 而在体外，这种细胞因子对哮喘患者有深远的影响， 嗜酸性粒细胞功能和存活率。 体外激活的嗜酸性粒细胞 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，现有数据有力地表明， GM-CSF是PBE重要的自分泌生长和存活因子 在体外和体内都起作用。 尽管可能的功能 GM-CSF的重要性，对GM-CSF的分子机制知之甚少。 其控制嗜酸性粒细胞产生和释放。 在T淋巴细胞、成纤维细胞和肥大细胞中，丝裂原诱导GM-CSF 生产依赖于增加的转录和减少的转录。 mRNA降解。 因此，我们假设类似的双重控制 这些机制还调节从活化的嗜酸性粒细胞产生GM-CSF。 此类基因调控研究尚未在嗜酸性粒细胞中进行， 很难从它们中分离或分离出完整的mRNA。 利用 颗粒介导的基因转移（PMGT），我们已经成功地转染 静息外周血嗜酸性粒细胞与荧光素酶报告基因 结构。 其次，我们证明了完整的mRNA可以被分离出来， 并成功地与GM-CSF特异性cDNA杂交 probes. 因此，我们处于一个独特的位置来调查 负责GM-CSF的制作的分子机制， 活化的嗜酸性粒细胞 这一点，我们的具体目标是：1）。优化 通过颗粒介导的基因转移转染嗜酸性粒细胞。 2）。 确定转录稳定性对 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的作用机制 活化的嗜酸性粒细胞中的mRNA稳定。 4）。识别顺反式 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 SCOR的其他组成部分，并提供新的信息， 活化的嗜酸性粒细胞对GM-CSF产生的分子控制。

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.