MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

巨噬细胞激活

• 批准号: 2672140
• 负责人: KENNETH L BOST
• 金额: $ 18.31万
• 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/2672140
• 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调节的中心细胞。 反应，特别是在粘膜表面。作者们已经证明 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物质诱导单核细胞因子 产生--IL-1、IL-6、IL-12、IL-15、IL-18和肿瘤坏死因子α。所有这些都是 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 转化生长因子β由P物质单独诱导，转化生长因子-β受物质抑制 P在内毒素诱导过程中。P物质诱导的单核细胞分泌水平 与其他激活剂的对比没有详细讨论。IL-12水平为 在一篇报纸上讨论过。一株单核细胞中其他单核细胞的mRNA诱导 图中显示了动物。作者计划描述物质的动力学 更详细地介绍了P诱导的单核细胞蛋白和mRNA。体外激活是 细胞因子mRNA和蛋白的动态分析；体外 激活，由肠道和淋巴结细胞的mRNA含量决定。 A2.P物质在24小时适度诱导MHC-II和B7-2。这 不一定是直接的影响，可能来自诱导的单核细胞。 诱导单核细胞因子的混合物还包括IL-10，一种有效的下调因子 MHC II和B7。体外或体内的抑制作用将被量化 (通过口服沙门氏菌或基于毒素的方法)激活。 A3.巨噬细胞通过受体结合表达NK1受体 NK1受体特异性抗血清的检测和识别 (在作者的实验室生成)。NK2受体的信使核糖核酸在 24小时内毒素刺激。它的水平低于NK1受体，而且 具有不同的诱导动力学。这些数据大概会是 很快就会出版。计划进行实验，以更仔细地定义速激肽 巨噬细胞的受体表达。NK1和NK2在使用激活后 P物质加或减共激活剂，或在体内，将检测到 RT-PCR法、竞争性RT-PCR法和流式细胞术。A4.巨噬细胞+内毒素 与单核细胞系不同，激活在物质上不显示钙离子通量 P刺激。这些实验将以更广泛的方式复制 控制。对于NK1来说，这是一个重要的观察/差异 在单核细胞系中，受体与G蛋白相连，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，但仅在与口腔病原体激活后 (沙门氏菌)和免疫原。作者将对细胞类型与 流式细胞仪测定体外细胞因子含量。这是在前面的基础上扩展的 P物质在不同细胞类型中的表达，是体内的一种 速激肽产生量与细胞类型的快照。