CYTOKINE-INDUCTION OF EOSINOPHILS IN AIRWAYS

有人建议进行实验，以检验细胞因子白介素3的作用。 白介素3、白介素5和粒细胞巨噬细胞集落刺激因子 嗜酸性粒细胞生物活性蛋白合成诱导的研究。 编码核心主要碱性蛋白m-RNA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- 衍生神经毒素(EDN)和嗜酸性粒细胞过氧化物酶(EPO) 将使用一种新开发的制剂进行嗜酸性粒细胞生成 培养的来源于人脐血的嗜酸性粒细胞。数据来自 将完全成熟的细胞与从外周血中提取的细胞进行比较 正常人类志愿者的血液。研究将在一个新的 豚鼠“活体外植体”制剂的研制及体外灌流 大鼠体内制备支气管液评价其生物活性 成熟的嗜酸性粒细胞与细胞因子诱导的mRNA表达 同样的细胞在体外。来自成熟的、分离的人类外周血细胞的数据 将在每个步骤中使用嗜酸性粒细胞来验证 培养细胞的生物学活性。这些理论的中心假设 研究表明：1)IL-5诱导颗粒蛋白mRNA的合成 IL-3和GM-CSF诱导的递增合成依赖于共刺激 与Il-5一起。2)嗜酸性粒细胞的生物活性在以下情况下会增加 细胞因子的刺激超过了单独成熟所需的水平。单元格 产后立即从新生儿脐带血中分离出来进行培养 并通过刺激诱导分化为嗜酸性粒细胞前体细胞 IL-3、IL-5、GM-CSF单独或联合应用。基因的表达 对于MBP、ECP、EDN和EPO，最初将在培养细胞中进行评估，并 在第1周(&lt；5%的显性分化)、第2、3、4和5周(~98% 纯度)。使用新开发的现场测力方法 在炎症刺激激活的呼吸道中产生，生物 每一阶段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 内皮细胞激活的肺阻力增加(大鼠支气管 准备)。在这些研究中将评估嗜酸性粒细胞的激活情况。 通过建立一种新的促红细胞生成素动力学测定方法和通过测定 LTC4分泌。所有必要的可行性研究均已完成 提案的两个阶段。这些研究将提供直接的 细胞因子刺激效应与糖尿病发生发展的相关性 嗜酸性粒细胞生成中的生物活性蛋白。从这些数据派生的数据 研究应该提供对嗜酸性粒细胞的机制的见解 引起呼吸道高反应性的条件，如人类过敏和 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.