MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

巨噬细胞激活

• 批准号: 2067926
• 负责人: KENNETH L BOST
• 金额: $ 13.98万
• 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/2067926
• 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 对于吞噬和杀死微生物至关重要，因为 分泌细胞因子以激活白细胞，并呈递抗原 到MHC相容的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，了解事件 激活这些细胞所需的物质是相当重要的。而当 细胞因子，如干扰素-γ，有重要的巨噬细胞 激活因子，很少有人注意到这种多肽， P物质。这种多肽存在于炎症组织中。 在突起和正常淋巴组织中。同样明显的是， 巨噬细胞表达P物质受体。这个项目的总体目标是 该项目是为了确定P物质生产的意义和 巨噬细胞在体外和体内P物质受体的表达 巨噬细胞激活的活体模型。具体地说，核酸酶保护 将进行化验和放射免疫分析以量化 原速激肽原mRNA表达和P物质分泌。 这些研究具有极其重要的意义，因为它们挑战了当前 认为P物质只是神经细胞的产物。这个 巨噬细胞分泌P物质和表达P物质受体的能力 这种多肽提示自分泌机制是可以发生的。 不幸的是，不可能明确地解决 P物质受体在巨噬细胞活化过程中的表达 检测这种受体是不可用的。为此，小鼠 巨噬细胞P物质受体将使用双脱氧测序 链终止法和用于开发的克隆受体片段 敏感的定量竞争-逆转录聚合酶链式反应(QC-RT-PCR)。 这些检测将量化巨噬细胞表达的受体mrna。 在体外或体内被激活。使用蛋白质表达系统，它 将有可能表达重组受体蛋白用作 免疫基因。巨噬细胞P物质受体特异性抗体 和I-125标记的P物质将被用来定量受体 在体外和体内激活的巨噬细胞上表达。一种能力 激活的巨噬细胞强烈上调P物质受体 提示在巨噬细胞激活中起作用。为了证明这一假设， P物质对几种关键的巨噬细胞功能的影响 下定决心。这些功能包括：1)吞噬和杀死 鼠伤寒沙门氏菌；2)单核细胞因子II类MHC的表达； QC-RT-PCR定量P物质受体mRNAs；3)分泌 通过酶联免疫吸附试验和生物测定定量的单因子；4)细胞表面 用免疫印迹技术表达第二类MHC和P物质受体蛋白 免疫荧光。以证明其特殊性和重要性。 P物质诱导效应时，将使用拮抗剂的反应。 这些拮抗剂包括：1)多肽拮抗剂(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 增强细胞内病原体沙门氏菌的致病性 鼠伤寒沙门氏菌，将使用小鼠模型进行测定。把这些放在一起 研究很可能证明P物质-P物质 受体的相互作用是一个相当重要的 巨噬细胞的激活。