FUNCTIONAL CHARACTERIZATION OF MONONUCLEAR HYPODENSE EOSINOPHILS

单核低密度嗜酸性粒细胞的功能表征

• 批准号: 3727366
• 负责人: WILLIAM F OWEN
• 金额: --
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3727366
• 关键词: apoptosis; basophils; cell nucleus; cytology; enzyme activity; eosinophil; fatty acid biosynthesis; granule; hematopoiesis; interleukin 3; interleukin 5; leukotrienes; lipoxygenase; monocyte; phospholipase A2; proteoglycan; tissue /cell culture; western blottings

In distinction to the more commonly recognized morphologic eosinophil phenotype with a segmented nucleus that is associated with eosinophilic disease states, we have recently observed that eosinophils of a lesser centrifugation density (hypodense eosinophils) can also be mononuclear. Previously, we observed that freshly isolated, bilobed peripheral blood eosinophils from healthy individuals (normodense eosinophils) treated with the eosinophilopoietic cytokines, interleukin (IL)-5, IL-3, or granulocyte-macrophage colony-stimulating factor (GM-CSF), are protected from apoptosis and glucocorticoid-induced cytotoxicity. These eosinophils are converted to a hypodense phenotype that is primed for ligand- initiated activation of the 5-lipoxygen-ase (5-LO) pathway and superoxide generation, and for antibody-dependent and independent cytotoxicity. The focus of Project 4 is to define the pro-inflammatory characteristics of the mononuclear eosinophil, an immature phenotype that is achieved after 28 days of culture of pleuripotent granulocyte precursors and appears comparable to mononuclear eosinophils in lesional sites by physical and morphologic criteria. The culture system for mononuclear eosinophils utilizes freshly isolated cord blood mononuclear cells cultured in vitro with IL-3 and IL-5 on a reconstituted basement membrane (Matrigel-Tm). After 14 days of culture, a hybrid mononuclear cell with both eosinophilic and basophilic granules predominates. This hybrid cell resembles a morphologic phenotype previously described in association with certain leukemic states but not further characterized. The intermediate hybrid cell phenotype, and especially the mononuclear eosinophil generated after 28 days of culture, will be characterized for susceptibility to apoptosis without and with glucocorticoid treatment, ligand-initiated and (or) calcium ionophore-stimulated superoxide generation and leukotriene production, antibody-mediated target cell cytotoxicity, type and size of cell-associated proteoglycan(s), presence of selected eosinophil-granule markers, and ultrastructural morphology. Particular attention will be paid to the development of the 5-LO pathway by monitoring steady-state transcription, immunoblot detection of protein, and individual assay of cytosolic phospholipase A2, 5-LO, 5- lipoxygenase-activating peptide, and LTC4 synthase. The recent cloning of the cDNA for LTC4 synthase as described for Project 3 now permits the characterization of the full 5-LO pathway during eosinophilopoiesis to the mononuclear phenotype and with conversion of normodense eosinophils to the hypodense phenotype with a segmented nucleus.

与更常见的形态学嗜酸性粒细胞不同， 与嗜酸性粒细胞相关的分叶核表型 疾病状态，我们最近观察到，嗜酸性粒细胞的一个较小的 离心密度（低密度嗜酸性粒细胞）也可以是单核的。 以前，我们观察到新鲜分离的双叶外周血 来自健康个体的嗜酸性粒细胞（正常密度嗜酸性粒细胞） 与嗜酸性粒细胞生成细胞因子，白细胞介素（IL）-5，IL-3，或 粒细胞-巨噬细胞集落刺激因子（GM-CSF）， 细胞凋亡和糖皮质激素诱导的细胞毒性。这些嗜酸性粒细胞 转化为低密度表型， 启动5-脂氧合酶（5-LO）途径和超氧化物的激活 产生，以及抗体依赖性和非依赖性细胞毒性。的 项目4的重点是定义 单核嗜酸性粒细胞是一种不成熟表型， 培养28天的多能性粒细胞前体和出现 与病变部位的单核嗜酸性粒细胞相当， 形态学标准单核嗜酸性粒细胞的培养体系 利用体外培养的新鲜分离的脐带血单核细胞 用IL-3和IL-5在重建的基底膜（Matrigel-Tm）上进行。 培养14天后， 嗜酸性和嗜碱性颗粒占优势。这种混合细胞 类似于先前描述的形态学表型， 患有某些白血病但没有进一步的特征。的 中间杂交细胞表型，特别是单核细胞 培养28天后产生的嗜酸性粒细胞，将表征 在没有和有糖皮质激素治疗的情况下对细胞凋亡的易感性， 配体引发和（或）钙离子载体刺激的超氧化物 生成和白三烯产生，抗体介导的靶细胞 细胞毒性、细胞相关蛋白聚糖的类型和大小、是否存在 选择的嗜酸性粒细胞颗粒标志物，和超微结构形态。 将特别关注5-LO途径的发展 通过监测稳态转录，免疫印迹检测 蛋白质，和细胞溶质磷脂酶A2，5-LO，5-LO的单独测定。 脂氧合酶激活肽和LTC4合酶。最近的克隆 如项目3所述的LTC4合酶的cDNA现在允许 在嗜酸性粒细胞生成过程中完整的5-LO途径的表征， 单核细胞表型和正常密度嗜酸性粒细胞转化 低密度表型与核分裂。