MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

巨噬细胞激活

• 批准号: 2886762
• 负责人: KENNETH L BOST
• 金额: $ 18.86万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2886762
• 关键词: Salmonella typhimurium; bactericidal immunity; chemical kinetics; flow cytometry; laboratory mouse; leukocyte activation /transformation; macrophage; monokines; neurotransmitter receptor; phagocytosis; receptor expression; substance P; tachykinin; tissue /cell culture

DESCRIPTION: A1. Macrophages are central to the control of many immune responses, particularly at mucosal surfaces. The authors have demonstrated that macrophages express a receptor, relatively specific for substance P (NK1). Expression increases with activation. Substance P induces monokine production -- IL-1, IL-6, IL-12, IL-15, IL-18, and TNFalpha. All of these enhance Th1 responses. IL-10 is also induced by substance P and enhances Th2 cells. Another product, TGFbeta, is immunosuppressive. Although TGFbeta is induced by substance P alone, TGF-beta is inhibited by substance P during LPS induction. Levels of monokine secretion induced by substance P versus other activators are not discussed in detail. IL-12 levels are discussed in a paper in press. Induction of mRNA for other monokines in one animal is shown. The authors plan to describe the kinetics of substance P-induced monokine protein and mRNA in more detail. in vitro activation is followed by kinetic analysis ofcytokine mRNA and protein; in vitro activation , by mRNA content of gut and lymph node cells. A2. Substance P modestly induces MHC class II and B7-2 at 24 hours. This is not necessarily a direct effect, and could be from induced monokines. The mix of induced monokines also includes IL-10, a potent downregulator of MHC II and B7. Suppression will be quantitated after in vitro or in-vovo (by oral salmonella or a toxin based method) activation. A3. NK1 receptors are expressed by macrophages based on receptor binding assays, and recognition with an antiserum specific for NK1 receptors (generated in the author's lab). mRNA for the NK2 receptor is present after 24-hour LPS stimulation. It is at lower levels than the NK1 receptor and has different induction kinetics. These data will presumably will be published soon. Experiments are planned to more carefully define tachykinin receptor expression by macrophages. NK1 and NK2, after activation with substance P plus or minus co-activators, or in vivo, will be detected with RT-PCR, competitive RT-PCR, and by flow cytometry. A4. Macrophages + LPS activation, unlike monocytic cell lines, exhibit no Ca++ flux upon substance P stimulation. These experiments will be replicated with more extensive controls. This is an important observation/discrepancy, for the NK1 receptor is linked to G proteins in monocyte lines, and this suggests and alternate signalling pathway for substance P in macrophages. A5. In vivo, macrophages from gut-associated lymphoid tissue appear to secrete substance P, but only after activation with oral pathogens (salmonella) and immunogens. The authors will quantitate cell type versus cytokine content ex vivo with flow cytometry. This expands upon earlier demonstration of substance P mRNA in different cell types, and is an in vivo snapshot of amount of tachykinin production versus cell type.

描述：A1。 巨噬细胞是许多免疫系统控制的核心 反应，特别是在粘膜表面。 作者已经证明 巨噬细胞表达一种对 P 物质相对特异的受体 (NK1)。 表达随着激活而增加。 P物质诱导单核因子 生产——IL-1、IL-6、IL-12、IL-15、IL-18 和 TNFα。 所有这些 增强Th1反应。 IL-10 也由 P 物质诱导并增强 Th2细胞。 另一种产品 TGFbeta 具有免疫抑制作用。 虽然 TGFβ仅由P物质诱导，TGF-β受物质抑制 LPS诱导期间的P。 P 物质诱导的单核因子分泌水平 与其他激活剂的比较没有详细讨论。 IL-12 水平为 在一篇已发表的论文中进行了讨论。 在一种细胞中诱导其他单核因子的 mRNA 显示动物。 作者计划描述物质的动力学 P 诱导的单核因子蛋白和 mRNA 更详细。 体外激活是 随后进行细胞因子mRNA和蛋白的动力学分析；体外 激活，通过肠道和淋巴结细胞的 mRNA 含量。 A2。 P 物质在 24 小时内适度诱导 MHC II 类和 B7-2。 这 不一定是直接作用，可能来自诱导的单核因子。 诱导单核因子的混合物还包括 IL-10，一种有效的下调因子 MHC II 和 B7。 抑制将在体外或体内进行定量 （通过口服沙门氏菌或基于毒素的方法）激活。 A3。 NK1 受体由巨噬细胞根据受体结合表达 检测和识别 NK1 受体特异性抗血清 （在作者实验室生成）。 NK2 受体的 mRNA 存在于 24小时LPS刺激。 它的水平低于 NK1 受体，并且 具有不同的诱导动力学。 这些数据大概会是 即将发布。 计划进行实验以更仔细地定义速激肽 巨噬细胞表达受体。 NK1 和 NK2，激活后 物质 P 加或减共激活剂，或在体内，将用以下方法检测 RT-PCR、竞争性 RT-PCR 和流式细胞术。 A4。 巨噬细胞+脂多糖 与单核细胞系不同，激活在物质上不表现出 Ca++ 通量 P刺激。 这些实验将被更广泛地重复 控制。 对于 NK1 来说，这是一个重要的观察/差异 受体与单核细胞系中的 G 蛋白相关，这表明 巨噬细胞中 P 物质的替代信号通路。 A5。 在体内，来自肠道相关淋巴组织的巨噬细胞似乎 分泌 P 物质，但仅在被口腔病原体激活后 （沙门氏菌）和免疫原。 作者将定量细胞类型与 使用流式细胞术离体测定细胞因子含量。 这扩展了之前的 展示不同细胞类型中 P 物质 mRNA，并且是体内 速激肽产生量与细胞类型的快照。