CYTOKINE REGULATION OF EOSINOPHIL GENES

嗜酸性粒细胞是被认为在骨髓细胞中起关键作用的细胞， 在多种疾病中的促炎或细胞毒性效应细胞 过敏性、寄生虫性、肿瘤性和特发性来源。 的 骨髓中这些粒细胞的增殖和分化， 以及它们在组织中的有丝分裂后功能成熟和活化 发生在响应于多种生长因子和细胞因子，包括 GM-CSF、IL-3和EL-5。 虽然GM-CSF和IL-3部分负责 粒细胞祖细胞的谱系定型，包括 某些嗜酸性粒细胞反应，如氧化爆发、脂质和蛋白质 介质释放，IL-5特异性参与分化， 嗜酸性粒细胞的功能性活化;所有三种细胞因子都支持延长 嗜酸性粒细胞存活和体外活化表型的发展。 我们的具体目标是研究分子机制， 嗜酸性粒细胞基因对细胞因子（IL-3，IL-5）的应答调节， 生长因子（GM-CSF）介导的对正常嗜酸性粒细胞的刺激 分化和有丝分裂后功能激活。 使用cDNA和 我们现有的嗜酸性粒细胞颗粒蛋白基因组克隆 包括嗜酸性粒细胞阳离子蛋白（ECP）、嗜酸性粒细胞衍生的神经毒素 (EDN)、主要碱性蛋白（MBP）、嗜酸性粒细胞过氧化物酶（EPO）和 Charcot-Leyden晶体（CLC）蛋白，我们将初步定义最 用于研究的适当嗜酸性粒细胞基因（即最嗜酸性粒细胞特异性的 和/或转录上调）。 我们将重点监管 诱导型嗜酸性粒细胞白血病细胞中只有一个或两个基因 细胞系，正常骨髓或脐带的精氨酸驱动分化 脐带血来源的祖细胞和细胞因子引发/激活 来自正常供体或患有以下疾病的患者的外周血的嗜酸性粒细胞： 嗜酸性粒细胞增多症 这项建议的目的是利用对下列问题的研究： 嗜酸性粒细胞基因调控，以了解参与的过程， 嗜酸性粒细胞谱系定型、分化和启动/激活 对特定细胞因子和生长因子的反应，并最终 鉴定嗜酸性粒细胞特异性转录因子。 拟议研究 将讨论以下问题：（1） 嗜酸性粒细胞颗粒蛋白基因的上调2)有哪些 嗜酸性粒细胞颗粒蛋白基因的启动子和调控元件？第三章 在这些基因中观察到的DNA酶超敏性的变化是什么？ 髓样（嗜酸性粒细胞）和非髓样细胞以及这些变化是如何发生的 与病程中mRNA表达的变化相关， 嗜酸性粒细胞分化或有丝分裂后激活 特异性细胞因子4)什么是顺式作用的DNA转录控制 负责嗜酸性粒细胞特异性基因高水平表达的元件 在苦参碱诱导的分化或细胞活化过程中？5)有哪些 组成型或精氨酸诱导的转录因子，其可与 这些控制元件来调节嗜酸性粒细胞基因的表达？拟议 研究旨在提供对分子生物学的更全面的了解， 复杂的细胞因子和生长因子介导的过程的基础， 发生在正常嗜酸性粒细胞分化和功能性 人类疾病组织中嗜酸性粒细胞的活化。

Eosinophils are myeloid cells recognized to play a pivotal role as pro-inflammatory or cytotoxic effector cells in a wide variety of diseases of allergic, parasitic, neoplastic, and idiopathic origins. The proliferation and differentiation of these granulocytes in the bone marrow, and their post-mitotic functional maturation and activation in tissues occurs in response to a variety of growth factors and cytokines including GM-CSF, IL-3 and EL-5. While GM-CSF and IL-3 are responsible in part for the lineage commitment of granulocyte progenitors, including priming for certain eosinophil responses such as oxidative burst, lipid and protein mediator release, IL-5 is specifically involved in the differentiation and functional activation of eosinophils; all three cytokines support prolonged eosinophil survival and the development of an activated phenotype in vitro. Our specific aims are to investigate the mechanisms for molecular regulation of eosinophil genes in response to cytokine (IL3, IL-5) and growth factor (GM-CSF) mediated stimulation of normal eosinophil differentiation and post-mitotic functional activation. Using cDNA and genomic clones we have available for the eosinophil granule proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), major basic protein (MBP), eosinophil peroxidase (EPO) and Charcot-Leyden crystal (CLC) protein, we will initially define the most appropriate eosinophil gene for study (i.e. the most eosinophil-specific and/or transcriptionally upregulated). We will focus on the regulation of only one or two genes in inducible eosinophil-committed leukemic cell lines, cytokine-driven differentiation of normal bone marrow or umbilical cord blood-derived progenitors, and cytokine priming/activation of eosinophils from the peripheral blood of normal donors or patients with eosinophilia. The objectives of this proposal are to use studies of eosinophil gene regulation to understand the processes involved in eosinophil lineage commitment, differentiation and priming/activation in response to specific cytokines and growth factors, and ultimately to identify eosinophil-specific transcription factors. The proposed research will address the following questions: 1) What are the mechanisms for the upregulation of the eosinophil granule protein genes? 2) What are the promoters and regulatory elements of eosinophil granule protein genes? 3) What changes in DNAse hypersensitivity are observed in these genes in myeloid (eosinophil) and non-myeloid cells and how are these changes cor-related with alterations in the expression of mRNA during the course of eosinophil differentiation or post-mitotic activation in response to specific cytokines? 4) What are the cis-acting DNA transcriptional control elements responsible for high level expression of eosinophil-specific genes during cytokine-induced differentiation or cell activation? 5) What are the constitutive or cytokine-induced transcription factors which may react with these control elements to regulate eosinophil gene expression? The proposed studies aim to provide a more integrated understanding of the molecular basis for the complex cytokine and growth factor-mediated processes that occur both during normal eosinophil differentiation and the functional activation of eosinophils in tissues in human disease.