FUNCTIONAL CHARACTERIZATION OF MONONUCLEAR HYPODENSE EOSINOPHILS

区别于更常见的形态嗜酸性粒细胞 与嗜酸性粒细胞相关的核呈节段性的表型 疾病状态，我们最近观察到嗜酸性粒细胞较少 离心密度(低密度嗜酸性粒细胞)也可以是单核细胞。 之前，我们观察到新鲜分离的双叶外周血 治疗健康人的嗜酸性粒细胞(正常嗜酸性粒细胞) 与嗜酸性细胞因子、白介素5、白介素3或 粒细胞巨噬细胞集落刺激因子(GM-CSF)受到保护 从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 转化为低密度的表型，为配体做好准备- 5-脂氧合酶(5-LO)途径和超氧化物歧化的启动 产生，以及抗体依赖和独立的细胞毒性。这个 项目4的重点是定义 单核嗜酸性粒细胞，一种未成熟的表型，在 多能粒细胞前体细胞培养28天后出现 皮损部位的单核嗜酸性粒细胞在物理和病理上与单核嗜酸粒细胞相当 形态标准。单核嗜酸性粒细胞培养体系的建立 利用新鲜分离的脐血单个核细胞进行体外培养 IL-3和IL-5在重组基底膜(Matrigel-TM)上。 培养14天后，混合的单个核细胞既有 嗜酸性和嗜碱性颗粒为主。这种杂交细胞 与之前描述的一种形态表型相似 具有某些白血病状态，但没有进一步的特征。这个 中间杂交细胞表型，尤其是单核细胞 培养28天后产生的嗜酸性粒细胞将被鉴定为 在没有和使用糖皮质激素治疗的情况下对细胞凋亡的易感性， 配基引发和(或)钙离子载体刺激的超氧化物歧化 产生和产生白三烯，抗体介导的靶细胞 细胞毒性，细胞相关蛋白多糖的类型和大小(S)，存在 选定的嗜酸性粒细胞标记和超微结构形态。 将特别关注5-LO途径的发展 通过监测稳定状态的转录，免疫印迹检测 蛋白质和胞浆磷脂酶A2，5-LO，5-LO，5-LO的个别测定 脂氧合酶激活肽和LTC4合成酶。最新的克隆 项目3中描述的LTC4合成酶的cDNA现在允许 嗜酸性粒细胞生成过程中完整的5-LO途径的特征 Normodense嗜酸性粒细胞的单核表型和转化 到细胞核呈节段性的低密度表型。

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.