CELLULAR BIOLOGY OF MURINE MAST CELL DEVELOPMENT

小鼠肥大细胞发育的细胞生物学

• 批准号: 6353057
• 负责人: KARL FRANK AUSTEN
• 金额: $ 32.71万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6353057
• 关键词: Trichinella; cell differentiation; chymase; cytokine; helminthiasis; hematopoietic growth factor; immunocytochemistry; immunoglobulin E; inflammation; interleukin 10; interleukin 3; interleukin 6; laboratory mouse; leukocyte activation /transformation; leukopoiesis; lipoxygenase; mast cell; northern blottings; nuclear runoff assay; prostaglandins; respiratory hypersensitivity; tryptase; western blottings

Mast cells are key initiators of inflammatory reactions in allergic diseases and normal hot defense. Circulating progenitor mast cells (PrMC) migrate to tissues where they mature into potent effector cells with considerable inter tissue heterogeneity. This heterogeneity is likely determined both by cognitive and soluble microenvironmental factors, and by developmentally regulated PrMC characteristics, which are not well understood. This project compares PrMC developed (PrMC/Triad), with replicates grown in IL-3 (PrMC/IL-3). Based on a relative ack of granule associated neural proteases and requirements for both SCF and IL-3 for maximal thymidine uptake, PrMC/Triad represent a more primitive PrMC than previously recognized in vitro. Because granule protease content partly determines mast cell effector capability. Specific Aim 1 focuses on the mechanistic basis for the differences in granule protease content between the two PrMC, and explores the basis for cytokine-induced maturation and protease acquisition as determined by steady-state RNA expression for murine mast cell proteases 1,2,4,5,6,7, and 9 *by RNA blot analysis), measurement of the corresponding proteins (by SDS-PAGE immunoblot), and nuclear run-on with measurement of the half-life of each RNA species in each respective PrMC population before and after week-long changes in cytokine provision, including culture in the presence of SCF alone, SCF + IL-10, and the triad of SCF/6/10. Preliminary studies also suggest a novel inductive effect of IL-6 on 5-LO pathway activity, another key mast cell effector system with special relevance to asthma, and suggest a concomitant IL-6 induced suppression of PGD2 generation. Specific Aim 2 therefore aims to establish the basis for these IL-6-mediated effects by comparing PrMC/Triad with PrMC/IL-3 for their IgE-dependent generation of 5-LO pathway products and PGD2, comparing the two PrMC for their content of each constituent 5- LO or PGHS/PGD2S pathway protein by SDS-PAGE immunoblot, RNA blot, and cell-free enzymatic assays, and exploring the mechanistic basis for any alterations in either pathway occurring in response to the above noted changes in cytokine supplementation by the same analyses. Finally, because PrMC characteristics may influence their tissue homing properties in vivo. Specific Aim 3 compares intravenous infusion of PrMC/triad with PrMC/IL-3 for reconstruction of the tissue mast cells mast cell-deficient c-kit w/wv mice, and examines the restoration of jejunal reactive mast cell hyperplasia in response to infection with Trichinella spiralis and the changes in pulmonary function immediately following inhalation challenge of ovalbumin-sensitized reconstituted mice.

肥大细胞是过敏性疾病和正常热防御中炎症反应的关键启动者。循环祖肥大细胞（PrMC）迁移到组织中，在那里它们成熟为具有相当大的组织间异质性的有效效应细胞。这种异质性可能是由认知和可溶性微环境因素，并通过发育调节PrMC的特点，这是不太清楚。该项目比较了开发的PrMC（PrMC/Triad）与在IL-3中生长的重复样品（PrMC/IL-3）。基于相对缺乏颗粒相关的神经蛋白酶和SCF和IL-3的最大胸苷摄取的要求，PrMC/Triad代表了比以前在体外认识到的更原始的PrMC。因为颗粒蛋白酶的含量部分决定了肥大细胞的效应能力。具体目标1集中于两种PrMC之间颗粒蛋白酶含量差异的机制基础，并探索如通过鼠肥大细胞蛋白酶1、2、4、5、6、7和9的稳态RNA表达（通过RNA印迹分析）、相应蛋白质的测量所确定的精氨酸诱导的成熟和蛋白酶获得的基础。（通过SDS-PAGE免疫印迹），和核运行，在细胞因子供应的长达一周的变化之前和之后测量每个相应PrMC群体中每个RNA种类的半衰期，包括在单独SCF、SCF + IL-10和SCF/6/10三联体存在下的培养。初步研究还表明，IL-6对5-LO途径活性（与哮喘特别相关的另一个关键肥大细胞效应系统）具有新的诱导作用，并表明伴随IL-6诱导的PGD 2生成抑制。因此，具体目的2旨在通过比较PrMC/Triad与PrMC/IL-3的IgE依赖性5-LO途径产物和PGD 2的产生，通过SDS-PAGE免疫印迹、RNA印迹和无细胞酶测定比较两种PrMC的每种组分5- LO或PGHS/PGD 2S途径蛋白的含量，并通过相同的分析探索响应于细胞因子补充的上述变化而发生的任一途径中的任何改变的机制基础。最后，因为PrMC的特性可能影响其在体内的组织归巢特性。具体目标3比较了静脉输注PrMC/triad与PrMC/IL-3重建组织肥大细胞肥大细胞缺陷型c-kit w/wv小鼠，并检查了响应于旋毛虫感染的空肠反应性肥大细胞增生的恢复以及卵清蛋白致敏重建小鼠吸入激发后立即发生的肺功能变化。