CYTOKINE REGULATION OF HUMAN EOSINOPHIL GENES

嗜酸性粒细胞是公认的发挥关键作用的髓系细胞，如 多种疾病中的促炎或细胞毒效应细胞 过敏性、寄生性、肿瘤性和特发性。这个 这些粒细胞在骨髓中的增殖和分化， 以及它们在组织中的有丝分裂后功能成熟和激活 发生于对多种生长因子和细胞因子的反应，包括 GM-CSF、IL-3、EL-5。而GM-CSF和IL-3在一定程度上是 粒系祖细胞的谱系承诺，包括启动 某些嗜酸性粒细胞反应，如氧化爆发、脂肪和蛋白质 中介物释放，IL-5特异性参与分化和 嗜酸性粒细胞的功能激活；所有三种细胞因子都支持延长 嗜酸性粒细胞在体外存活和活化表型的发展。 我们的具体目标是研究分子生物学机制。 嗜酸性粒细胞基因对细胞因子(IL-3、IL-5)和 生长因子(GM-CSF)对正常嗜酸性粒细胞的刺激 分化和有丝分裂后的功能激活。使用cdna和 我们已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 适合研究的嗜酸性粒细胞基因(即最具嗜酸性粒细胞特异性的基因 和/或转录上调)。我们将重点抓好监管 可诱导的嗜酸性粒细胞白血病细胞中只有一到两个基因 细胞因子诱导的正常骨髓或脐血分化系 脐带血来源的祖细胞和细胞因子的启动/激活 正常献血者或慢性支气管炎患者外周血中的嗜酸性粒细胞 嗜酸性粒细胞增多症。这项提案的目标是利用对 了解嗜酸性粒细胞基因调控参与的过程 嗜酸粒细胞谱系的定位、分化和启动/激活 对特定细胞因子和生长因子的反应，并最终对 鉴定嗜酸性粒细胞特异性转录因子。拟议的研究 将解决以下问题：1)有哪些机制可用于 嗜酸性粒细胞颗粒蛋白基因上调？2)什么是 嗜酸性粒细胞颗粒蛋白基因的启动子和调控元件 在这些基因中观察到了DNA酶超敏反应的哪些变化 髓系(嗜酸性粒细胞)和非髓系细胞以及这些变化是如何发生的 血管紧张素转换酶基因表达变化与血管紧张素转换酶相关性 嗜酸性粒细胞分化或有丝分裂后活化对 特定的细胞因子？4)顺式作用的DNA转录调控是什么 嗜酸性粒细胞特异性基因高水平表达的分子机制 在细胞因子诱导的分化或细胞激活过程中？5)什么是 构成或细胞因子诱导的转录因子，可能与 这些调控元件能调控嗜酸性粒细胞基因表达吗？建议数 研究的目的是提供对分子的更完整的理解 复杂的细胞因子和生长因子介导的过程的基础 发生在正常的嗜酸性粒细胞分化和功能性 人类疾病组织中嗜酸粒细胞的激活。

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.