MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

巨噬细胞激活

• 批准号: 6373276
• 负责人: KENNETH L BOST
• 金额: $ 20.01万
• 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/6373276
• 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. 巨噬细胞是控制许多免疫系统的核心。 反应，特别是在粘膜表面。 作者证明了 巨噬细胞表达一种受体，对P物质相对特异 （NK1）。 表达随着激活而增加。 P物质诱导单核因子 IL-1、IL-6、IL-12、IL-15、IL-18和TNF α。 所有这些 增强Th1应答。 IL-10也由P物质诱导，并增强 Th2细胞 另一种产品TGF β是免疫抑制剂。 虽然 TGF-β仅由P物质诱导，TGF-β被P物质抑制。 P在LPS诱导期间。 P物质诱导的单核因子分泌水平 与其它活化剂的比较没有详细讨论。 IL-12水平 发表在一份报纸上。 在一个细胞中诱导其他单核因子的mRNA 动物展示。 作者计划描述物质的动力学 P诱导的单核因子蛋白和mRNA的更详细的。 体外活化 细胞因子mRNA和蛋白的动态分析 激活，通过肠和淋巴结细胞的mRNA含量。 A2. P物质在24小时适度诱导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。 诱导的单核因子的混合物还包括IL-10，一种有效的单核因子的下调因子。 MHC II和B7。 将在体外或体内试验后定量抑制 (by口服saltrin或基于毒素的方法）活化。 A3. NK 1受体由巨噬细胞基于受体结合来表达 测定和用对NK1受体具有特异性的抗血清识别 （在作者的实验室中生成）。 NK2受体的mRNA存在于 24-1h LPS刺激。 它的水平低于NK1受体， 具有不同的诱导动力学。 这些数据将被认为是 很快出版。 实验计划更仔细地定义速激肽 巨噬细胞的受体表达。 NK1和NK2，在用 P物质加上或减去辅活化剂，或在体内，将被检测， RT-PCR、竞争性RT-PCR和流式细胞术。 A4. 巨噬细胞+LPS 活化，不像单核细胞系，不表现出Ca ++通量的物质 P刺激。 这些实验将被复制， 对照 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，但仅在被口腔病原体激活后 （Salmonella）和免疫原。 作者将定量细胞类型与 用流式细胞术测定离体细胞因子含量。 这是在早些时候扩展的。 证明P物质mRNA在不同的细胞类型，是一个在体内 速激肽产生量对细胞类型的快照。