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: 6099539
• 负责人: William F Owen
• 金额: $ 16.87万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6099539
• 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 具有与嗜酸性粒细胞相关的分节核的表型 在疾病状态下，我们最近观察到较小的嗜酸性粒细胞 离心密度（低密度嗜酸性粒细胞）也可为单核。 之前，我们观察到新鲜分离的双叶外周血 来自接受治疗的健康个体的嗜酸性粒细胞（正态嗜酸性粒细胞） 与嗜酸性粒细胞细胞因子、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) 受到保护 来自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，为配体做好准备 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。这 项目 4 的重点是定义促炎特征 单核嗜酸性粒细胞，一种不成熟的表型，是在 培养28天多能粒细胞前体并出现 通过物理和方法与病变部位的单核嗜酸性粒细胞相当 形态学标准。单核嗜酸性粒细胞培养系统 利用体外培养的新鲜分离的脐带血单核细胞 重组基底膜 (Matrigel-Tm) 上含有 IL-3 和 IL-5。 培养 14 天后，获得了具有这两种细胞的杂交单核细胞 以嗜酸性和嗜碱性颗粒为主。这种混合电池 类似于先前描述的关联的形态学表型 具有某些白血病状态，但未进一步表征。这 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被表征为 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 产生和白三烯产生，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、存在 选定的嗜酸性粒细胞颗粒标记物和超微结构形态。 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 蛋白质和胞质磷脂酶 A2、5-LO、5- 的单独测定 脂氧合酶激活肽和 LTC4 合酶。最近的克隆 项目 3 中描述的 LTC4 合酶 cDNA 现在允许 嗜酸性粒细胞生成过程中完整 5-LO 途径的表征 单核表型和正常密度嗜酸性粒细胞的转化 到具有分节核的低密度表型。