MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

巨噬细胞激活

• 批准号: 2067925
• 负责人: KENNETH L BOST
• 金额: $ 13.65万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 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 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067925
• 关键词: MHC class II antigen; RNase protection assay; Salmonella typhimurium; antireceptor antibody; antisense nucleic acid; bactericidal immunity; bioassay; enzyme linked immunosorbent assay; gene expression; immunofluorescence technique; inhibitor /antagonist; laboratory mouse; leukocyte activation /transformation; macrophage; monokines; neurotransmitter receptor; nucleic acid sequence; phagocytosis; polymerase chain reaction; radioimmunoassay; receptor expression; recombinant proteins; substance P; tachykinin; tissue /cell culture

DESCRIPTION: (Adapted from the Applicant's abstract): Macrophages are critically important for phagocytosis and killing of microbes, for secreting cytokines to activate leukocytes, and for presenting antigen to MHC compatible T lymphocytes. Since activated macrophages perform these functions with much greater efficiency, understanding the events required to activate these cells is of considerable importance. While cytokines, such as interferon-gamma, have important macrophage activating factors, little attention has been given to the peptide, substance P. This peptide is found in tissues undergoing inflammatory processes and in normal lymphoid tissues. It is also clear that macrophages express substance P receptors. The overall goal of this project is to determine the significance of substance P production and substance P receptor expression by macrophages using in vitro and in vivo models of macrophage activation. Specifically, nuclease protection assays and radioimmunoassays will be performed to quantify preprotachykinin mRNA expression and substance P secretion, respectively. These studies are of extreme importance since they challenge the current view that substance P is solely a product of neuronal cells. The ability of macrophages to secrete substance P and express receptors for this peptide suggest that autocrine mechanisms can occur. Unfortunately, it is not possible to definitively address the role of substance P receptor expression in macrophage activation since reagents to detect this receptor are not available. To this end, the murine macrophage substance P receptor will be sequenced using the dideoxy chain termination method, and cloned receptor fragments used to develop sensitive quantitative competitive-reverse transcribed PCR (QC-RT-PCR). These assays will quantify receptor mRNA expressed by macrophages activated in vitro or in vivo. Using a protein expression system, it will be possible to express recombinant receptor protein for use as an immunogen. Antibodies specific for the macrophage substance P receptor and I-125 labelled substance P will be used to quantify receptor expression on macrophages activated in vitro and in vivo. The ability of activated macrophages to up regulate substance P receptors, strongly suggests a role in macrophage activation. To prove this hypothesis, the effect substance P has on several critical macrophage functions will be determined. These functions include: 1) phagocytosis and killing of Salmonella typhimurium; 2) expression of monokine, class II MHC, and substance P receptor mRNAs as quantified by QC-RT-PCR; 3) secretion of monokines quantified by ELISA and bioassays; and 4) cell surface expression of class II MHC and substance P receptor proteins using immunofluorescence. To prove specificity and significance of the substance P induced effects, antagonists of the response will be used. These antagonists will include: 1) a peptide antagonist (Spantide II); 2) anti-substance P receptor antibodies; and 3) anti-sense oligonucleotides. Ultimately the ability of substance P antagonists to increase the pathogenicity of the intracellular pathogen, Salmonella typhimurium, will be determined using a murine model. Together these studies are likely to demonstrate that the substance P- substance P receptor interaction is one of considerable importance for the activation of macrophages.

描述：（改编自申请人的摘要）：巨噬细胞是 对于微生物的吞噬作用和杀灭作用至关重要 分泌细胞因子激活白细胞并呈递抗原 MHC 相容性 T 淋巴细胞。 由于激活的巨噬细胞执行 这些功能的效率更高，可以理解事件 激活这些细胞所需的物质非常重要。 尽管 细胞因子，例如干扰素-γ，对巨噬细胞具有重要作用 激活因子，但很少有人关注肽， P 物质。这种肽存在于发生炎症的组织中 过程和正常淋巴组织中。 还清楚的是 巨噬细胞表达 P 物质受体。 本次活动的总体目标 项目的目的是确定 P 物质生产的重要性和 巨噬细胞在体外和体内表达 P 物质受体 巨噬细胞激活的体内模型。 具体来说，核酸酶保护 将进行化验和放射免疫测定来量化 分别是前原速激肽 mRNA 表达和 P 物质分泌。 这些研究极其重要，因为它们挑战了当前的 认为 P 物质只是神经元细胞的产物。 这 巨噬细胞分泌 P 物质和表达 P 物质受体的能力 该肽表明可以发生自分泌机制。 不幸的是，不可能明确地说明 P物质受体表达在巨噬细胞激活自试剂中的应用 无法检测到该受体。 为此，小鼠 巨噬细胞 P 物质受体将使用双脱氧酶进行测序 链终止方法，以及用于开发的克隆受体片段 敏感定量竞争性逆转录 PCR (QC-RT-PCR)。 这些测定将量化巨噬细胞表达的受体 mRNA 在体外或体内激活。 使用蛋白质表达系统， 将有可能表达重组受体蛋白用作 免疫原。 巨噬细胞 P 物质受体特异性抗体 I-125标记的P物质将用于定量受体 在体外和体内激活的巨噬细胞上表达。 能力 激活的巨噬细胞上调 P 物质受体，强烈 表明在巨噬细胞激活中发挥作用。 为了证明这个假设， P 物质对巨噬细胞几个关键功能的影响 决定。 这些功能包括：1）吞噬和杀伤 鼠伤寒沙门氏菌； 2) 单核因子、II类MHC的表达，以及 通过 QC-RT-PCR 定量的 P 物质受体 mRNA； 3）分泌 通过 ELISA 和生物测定法定量单核因子； 4) 细胞表面 II 类 MHC 和 P 物质受体蛋白的表达 免疫荧光。 证明其特殊性和意义 P物质引起的效应，会使用该反应的拮抗剂。 这些拮抗剂包括： 1) 肽拮抗剂 (Spantide II)； 2)抗P物质受体抗体； 3）反义 寡核苷酸。最终 P 物质拮抗剂的能力 增加细胞内病原体沙门氏菌的致病性 鼠伤寒杆菌，将使用小鼠模型进行测定。 一起这些 研究很可能证明 P 物质- P 物质 受体相互作用对于 巨噬细胞的激活。