CYTOKINE-INDUCTION OF EOSINOPHILS IN AIRWAYS

实验拟检查细胞因子白细胞介素-3的影响， （IL-3）、IL-5和粒细胞-巨噬细胞集落刺激因子（GM-CSF） 对造血过程中生物活性嗜酸性粒细胞蛋白的合成诱导作用。 编码核心主要碱性蛋白mRNA的表达研究 (MBP)和基质蛋白[嗜酸性粒细胞阳离子蛋白（ECP），嗜酸性粒细胞- 来源的神经毒素（EDN）和嗜酸性粒细胞过氧化物酶（EPO）] 将使用新开发的制剂进行嗜酸性粒细胞生成， 来源于人脐带血的培养的嗜酸性粒细胞。 数据从 将完全成熟的细胞与从外周血中提取的细胞进行比较 正常志愿者的血液。 研究将在一个新的 开发了豚鼠“活外植体”制备和隔离灌注 大鼠体内支气管制备，以评估 成熟的嗜酸性粒细胞对应于苦参碱诱导的 同样的细胞。 来自成熟分离人外周血的数据 嗜酸性粒细胞将在每个步骤中使用，以验证 培养细胞的生物活性。 这些理论的核心假设 IL-5诱导颗粒蛋白mRNA的合成， 由IL-3和GM-CSF引起的增量合成依赖于共刺激 IL-5 2)嗜酸性粒细胞的生物活性将增加， 甜菜碱刺激超过了单独成熟所需的刺激。 细胞 产后立即从新生儿脐带血中分离培养 并通过用以下物质刺激诱导分化为嗜酸性粒细胞祖细胞： IL-3、IL-5和GM-CSF单独和组合。 mrna表达 对于MBP、ECP、EDN和EPO，最初将在培养的细胞中进行评估， 在第1周（< 5%明确分化）、第2周、第3周、第4周和第5周（~ 98% 纯度）。 使用新开发的原位方法评估力 由炎性刺激激活的呼吸道中的生物生成 来自每个阶段的培养细胞的活性将被评估为 等长力产生（豚鼠外植体制备）和 内皮激活的肺阻力增加（大鼠支气管 制备）。 将评估这些研究中嗜酸性粒细胞的活化 通过新开发的EPO动力学测定和通过测量 LTC 4分泌。 所有必要的可行性研究已经完成， 提案的两个阶段。 这些研究将提供一个直接 肾上腺素刺激的作用与发展之间的相关性 生物活性蛋白质在造血中的作用 数据来源于这些 研究应该提供对嗜酸性粒细胞 在诸如人类过敏的情况下引起气道高反应性， 哮喘

Experiments are proposed to examine the effects of cytokines interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the synthetic-induction of bioactive eosinophil proteins during poiesis. Studies of the expression of m-RNA encoding the core major basic protein (MBP) and matrix proteins [eosinophil cationic protein (ECP), eosinophil- derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] during eosinopoiesis will be performed using a newly developed preparation of cultured eosinophils derived from human umbilical cord blood. Data from fully mature cells will be compared to cells extracted from the peripheral blood of normal human volunteers. Studies will be performed in a newly developed guinea pig "living explant" preparation and an isolated-perfused rat bronchial preparation in vivo to assess the biological activity of maturing eosinophils corresponding to cytokine-induced mRNA expression of the same cells in vitro. Data from mature isolated human peripheral eosinophils will be used at each step to validate the relevance of the biological activity of cultured cells. The central hypotheses of these studies are 1) IL-5 induces synthesis of granular protein mRNA and that incremental synthesis caused by IL-3 and GM-CSF depends upon co-stimulation with Il-5. 2) The biological activity of eosinophils will be increased if cytokine-stimulation exceeds that required for maturation alone. Cells isolated immediately postpartum from neonatal cord blood will be cultured and induced to differentiate as eosinophil progenitors by stimulation with IL-3, IL-5 and GM-CSF both singly and in combination. The mRNA expression for MBP, ECP, EDN, and EPO will be assessed in cultured cells initially and during weeks 1 (< 5% explicit differentiation), 2, 3, 4, and 5 (~ 98% purity). Using newly developed in situ methods for assessing force generation in airways activated by inflammatory stimulation, biological activity of cultured cells from each stage will be assessed as change in isometric force generation (guinea pig explant preparation) and endothelially activated increase in lung resistance (rat bronchial preparation). Activation of eosinophils in these studies will be assessed in vitro by a newly developed kinetic assay for EPO and by measurement of LTC4 secretion. All essential feasibility studies have been completed for both stages of the proposal. These studies will provide a direct correlation between the effects of cytokine-stimulation and development of biologically active proteins in eosinopoiesis. Data derived from these studies should provide insight into the mechanism by which eosinophils elicit airway hyperresponsiveness in conditions such as human allergy and asthma.