CELLULAR BIOLOGY OF MURINE MAST CELL DEVELOPMENT

小鼠肥大细胞发育的细胞生物学

• 批准号: 6202255
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202255
• 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因子。循环祖肥大细胞（PrMC）迁移到组织中，在那里它们成熟为有效的效应细胞，具有相当大的组织间异质性。这种异质性可能是由认知和可溶性微环境因素以及发育调节的PrMC特征决定的，这些特征尚未得到很好的理解。本项目比较了PrMC培养（PrMC/Triad）和IL-3中培养的重复（PrMC/IL-3）。基于颗粒相关神经蛋白酶的相对缺乏以及SCF和IL-3对最大胸腺嘧啶摄取的要求，PrMC/Triad代表了比以前在体外认识的更原始的PrMC。因为颗粒蛋白酶含量在一定程度上决定了肥大细胞的效应能力。特异性目标1侧重于两种PrMC颗粒蛋白酶含量差异的机制基础，并探讨细胞因子诱导成熟和蛋白酶获得的基础(通过稳态RNA表达确定小鼠肥大细胞蛋白酶1、2、4、5、6、7和9（通过RNA印迹分析），相应蛋白的测量（通过SDS-PAGE免疫印迹），在细胞因子供应变化前后，包括单独培养SCF、SCF + IL-10和SCF/6/10三联培养，测量每个PrMC群体中每种RNA物种的半衰期。初步研究还表明，IL-6对与哮喘特别相关的另一个关键肥大细胞效应系统5-LO通路活性具有新的诱导作用，并提示IL-6同时诱导抑制PGD2的产生。因此，Specific Aim 2旨在通过比较PrMC/Triad与PrMC/IL-3对5-LO通路产物和PGD2的ige依赖性产生，并通过SDS-PAGE免疫印迹、RNA印迹和无细胞酶分析比较两种PrMC中各成分5-LO或PGHS/PGD2S通路蛋白的含量，从而为这些il -6介导的效应建立基础。并通过相同的分析，探索任何途径发生变化的机制基础，以响应上述细胞因子补充的变化。最后，因为PrMC的特性可能会影响它们在体内的组织归巢特性。特异性目的3比较静脉输注PrMC/triad与PrMC/IL-3重建组织肥大细胞肥大细胞缺陷c-kit w/wv小鼠，并研究旋毛虫感染后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刺激后肺功能的变化。