CELLULAR BIOLOGY OF MURINE MAST CELL DEVELOPMENT

在过敏性疾病和正常的热防御中，肥大细胞是炎症反应的关键启动者。循环中的祖细胞肥大细胞(PRMC)迁移到组织中，在那里它们成熟为强大的效应细胞，具有相当大的组织间异质性。这种异质性很可能是由认知和可溶的微环境因素以及发育调节的PRMC特征决定的，这些特征还没有被很好地理解。本项目比较了PRMC开发的(PRMC/Triad)和在IL-3中生长的复制(PRMC/IL-3)。基于颗粒相关神经蛋白水解酶的相对ACK，以及对SCF和IL-3的最大胸苷摄取的需求，PRMC/Triad代表了比先前在体外所认识的更原始的PRMC。因为颗粒蛋白水解酶的含量部分决定了肥大细胞的效应器能力。具体目的1侧重于两种PRMC中颗粒蛋白水解酶含量差异的机制基础，并探索细胞因子诱导成熟和蛋白酶获得的基础，这是通过RNA印迹分析小鼠肥大细胞蛋白酶1、2、4、5、6、7和9*的稳定RNA表达、相应蛋白质的测量(通过SDS-PAGE免疫印迹)以及在细胞因子提供的持续一周的变化(包括单独存在SCF、SCF IL-10、初步研究还表明，IL-6对另一个与哮喘相关的肥大细胞效应系统5-LO途径的活性具有新的诱导作用，并伴随着IL-6诱导抑制PGD2的产生。因此，特定目的2旨在通过比较PRMC/Triad和PRMC/IL-3依赖于IgE的5-LO途径产物和PGD2的产生，通过SDS-PAGE免疫印迹、RNA印迹和无细胞酶分析比较两种PRMC各自5-LO或PGHS/PGD2S途径蛋白的含量，并通过相同的分析来探索这两种途径中任何一种途径发生变化的机制基础，从而为这些IL-6介导的作用奠定基础。最后，由于PRMC的特性可能会影响其在体内的组织归巢特性。具体目的3比较静脉注射PRMC/Triad和PRMC/IL-3重建c-kit w/WV小鼠组织肥大细胞缺陷的效果，并检测旋毛虫感染后空肠反应性肥大细胞增殖的恢复和卵蛋白致敏重组小鼠吸入攻击后即刻肺功能的变化。

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.