MACROPHAGE ACTIVATION & SUBSTANCE P RECEPTOR EXPRESSION

描述：（改编自申请人的摘要）：宏观经济学 对吞噬作用和杀死微生物至关重要， 分泌细胞因子以激活白细胞，并用于呈递抗原 与MHC相容的T淋巴细胞 由于活化的巨噬细胞 这些功能更有效率，了解事件， 激活这些细胞所需要的是相当重要的。 而 细胞因子，如干扰素-γ，具有重要的巨噬细胞 激活因子，很少关注肽， 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。 并明确 巨噬细胞表达P物质受体。 这个项目的总体目标是 项目的目的是确定P物质生产的重要性， 巨噬细胞P物质受体表达的体外和体内研究 巨噬细胞活化的体内模型。 具体来说，核酸酶保护 将进行测定和放射免疫测定， 前速激肽原mRNA表达和P物质分泌。 这些研究具有极端重要性，因为它们挑战了当前的 认为P物质只是神经细胞的产物。 的 巨噬细胞分泌P物质和表达 这种肽表明可以发生自分泌机制。 不幸的是，不可能明确地讨论 试剂活化巨噬细胞P物质受体表达 来检测这种受体。 为此，鼠 巨噬细胞P物质受体将使用双脱氧 链终止方法，以及用于开发 灵敏的定量竞争性逆转录PCR（QC-RT-PCR）。 这些测定将定量巨噬细胞表达的受体mRNA， 在体外或体内活化。 使用蛋白质表达系统， 将有可能表达重组受体蛋白， 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 在体外和体内活化的巨噬细胞上表达。 的能力 激活的巨噬细胞上调P物质受体， 表明在巨噬细胞活化中的作用。 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将是 测定 这些功能包括：1）吞噬和杀死 鼠伤寒沙门氏菌; 2）单核因子、II类MHC和 通过QC-RT-PCR定量的P物质受体mRNA; 通过ELISA和生物测定定量的单核因子;和4）细胞表面 表达II类MHC和P物质受体蛋白， 免疫荧光 为了证明其特殊性和重要性， P物质诱导的效应，将使用该反应的拮抗剂。 这些拮抗剂将包括：1）肽拮抗剂（Spantide II）; 2)抗P物质受体抗体;和3）反义 寡核苷酸最终，P物质拮抗剂能够 增加胞内病原体沙门氏菌的致病性 鼠伤寒沙门氏菌，将使用鼠模型测定。 综合这些 研究很可能表明P物质-P物质 受体相互作用是一个相当重要的 激活巨噬细胞。

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.