RELEASE & EFFECT OF MAST CELL/BASOPHIL MEDIATORS

这项建议有两个主要目标：进一步了解肥大细胞 肥大细胞的激活及其结构和功能的阐明 具有趋化活性的介体。第一个主要目标将是 通过发展中的肥大细胞群体来解决，这反映了 这种细胞类型的异质性。结缔组织肥大细胞将 从大鼠浆膜腔和粘膜型肥大细胞中获得 用IL-3富含培养液培养大鼠和小鼠骨髓 物种间和物种内肥大细胞功能和介体的分析。 这些肥大细胞群体将阐明肥大中是否存在差异 细胞功能和介体是由物种差异或真正的肥大引起的 细胞异质性。这些肥大细胞随后将被用于研究 调节介质释放，与人肺肥大细胞一起，作为一种 肥大细胞来源的趋化介质。肥大细胞的研究进展 调控将集中在胞外钙/镁ATPase和细胞表面的作用上 腺苷受体在刺激-分泌偶联中的调节作用。特定的 ATPase和腺苷受体的激动剂和拮抗剂探针将 根据介体评估，检查它们影响肥大细胞的能力 释放，钙离子动态平衡，钙离子通量，磷脂(特别是肌醇 磷酸盐)代谢、蛋白磷酸化与受体表达 短期(0-60min)体外研究和长期(1-7d)体内研究 在组织培养中也是如此。要使用的探针包括特定的ATPase DIDS(4‘，4-二异硫氰基-2’，2-二磺酸) 抑制介质释放的二苯乙烯)；导致介质释放的三磷酸腺苷 释放，(以及各种核苷酸类似物)；腺苷及其 激动剂类似物，促进预先形成的介体释放；以及 抑制腺苷结合的甲基黄嘌呤及其作用。这些 研究将有助于进一步深入了解 这些化合物的作用，从而形成肥大细胞的重要元素 激活。 对肥大细胞来源的趋化因子的评估将包括其 其靶细胞的理化特性及其鉴定 专一性。纯化后的趋化因子对 影响白细胞产生炎性介质将被测定 并评估了它们在体内诱导白细胞聚集的能力。

This proposal has two major goals: further understanding of mast cell activation and elucidating the structure and function of mast cell mediators with chemotactic activity. The first major goal will be addressed by developing populations of mast cells which reflect the heterogeneity of this cell type. Connective tissue mast cells will be obtained from rat serosal cavities and mucosal-type mast cells will be cultured from rat and mouse bone marrow utilizing IL-3 rich media to permit analysis of mast cell function and mediators across and within species. These mast cells populations will clarify whether differences noted in mast cell function and mediators are due to species differences or to true mast cell heterogeneity. These mast cells will then be utilized in studies of regulation of mediator release and, along with human lung mast cells, as a source of mast cell derived chemotactic mediators. Studies of mast cell regulation will focus upon the role of ecto Ca/Mg ATPases and cell surface adenosine receptors in modulation of stimulus-secretion coupling. Specific agonist and antagonist probes of ATPases and adenosine receptors will be examined for their ability to affect mast cells as assessed by mediator release, Ca++ homeostasis, Ca++ flux, phospholipid (esp. inositol phosphates) metabolism, protein phosphylation and receptor expression in short term (0-60 min) in vitro and in longer term (1-7d) studies in vivo and in tissue culture. Probes to be utilized include specific ATPase inhibitors such as DIDS (4',4-diisothiocyano - 2', 2 - disulfonic acid stilbene) which inhibits mediator release; ATP which causes mediator release, (as well as various nucleotide analogues); adenosine and its agonist analogues which enhance preformed mediator release; and methylxanthines which inhibit adenosine binding and its action. These studies will aid in developing further insights into the precise sites of action of these compounds and thereby into important elements of mast cell activation. Assessment of mast cell derived chemotactic factors will include their physiochemical characterizations and identification of their target cell specificity. After purification the ability of chemotactic factors to affect leukocyte production of inflammatory mediators will be determined and their ability to induce leukocyte accumulation in vivo assessed.