Mechanism of Action of Interferon-gamma

该实验室分离出表达一种 一种缺乏信号的人类干扰素-γ基因的突变形式 顺序 这些细胞合成人类干扰素-γ， 分泌并在细胞内积累蛋白质。 尽管所用的鼠细胞对免疫抑制剂完全耐受， 加入到培养基中的人干扰素-γ的作用， 细胞内表达这种异源干扰素的细胞 对水泡性口炎病毒的感染具有抗性， 门戈病毒，并表现出高水平的寡2，5 A合成酶 和eIF-2激酶，这些酶已知通过用 干扰素 这些细胞还显示出mRNA水平的增强 编码主要组织相容性复合体II类抗原。 这些结果提出了新的概念，干扰素γ可以 在细胞内跨越物种屏障， 特异性基因激活 本研究旨在分析 这一现象的细节，以确保观察是由于 直接作用于细胞内干扰素-γ， 由细胞产生同源的抗病毒剂。 的 这些观察结果的一般性将通过确定 鼠干扰素-γ是否在细胞内表达， 人类细胞也会产生类似的效果。 因为结果可能是 解释为分泌干扰素或其他药物， 抗病毒特性，测定增加的mRNA水平， 同种干扰素α、β和γ与肿瘤坏死 因子将通过北方印迹和聚合酶进行 链式反应技术 特异性抗体将用于 抗病毒分泌试验 培养物上清液和通过共培养实验。 如果 发现细胞内干扰素-γ激活 特定的基因直接，负责这一机制 将寻求效果。 如果细胞内的干扰素γ 具有干扰素样同源试剂的生产 重要的是要识别这种物质， 确定干扰素γ如何诱导其产生， 这种效应是否在正常反应中起作用， 干扰素-γ。 生物反应调节剂，干扰素-γ一直是 由于其抗病毒作用， 及其对哺乳动物免疫细胞的刺激作用 系统 目前的研究是基于这样的假设， 类激素物质的作用与许多肽类激素一样， 与表面膜受体结合，随后 引发一系列细胞内生化反应 博士 刘易斯的初步结果表明， 干扰素-γ的作用部位。 如果是这样的话， 可能是一种非常重要的新的作用机制， 必须做大量重要工作， 初步意见的基础。 这笔资金将支持 对这些潜在的重要观测结果的关键检验。

This laboratory has isolated murine cell lines which express a mutated form of the human interferon-gamma gene lacking a signal sequence. These cells synthesize human interferon-gamma but fail to secrete it and accumulate the protein intracellularly. Although the murine cells used are completely resistant to the effects of human interferon-gamma added to the culture medium, the cells expressing this heterologous interferon intracellularly are resistant to infection with vesicular stomatitis virus and Mengo virus and exhibit elevated levels of oligo 2,5 A synthetase and eIF-2 kinase, enzymes known to be induced by treatment with interferons. The cells also show enhanced levels of mRNA encoding major histocompatibility complex class II antigen. These results suggest the novel concept that interferon gamma can function intracellularly across a species barrier to cause specific gene activation. The proposed research is to analyze this phenomenon in detail to ensure that the observations are due directly to the intracellular interferon-gamma and not to production by the cells of a homologous antiviral agent. The generality of these observations will be tested by determining whether murine interferon-gamma expressed intracellularly in human cells produces similar effects. Since the results could be explained by the secretion of interferons or other agents with antiviral properties, assays for increased levels of mRNA for homologous interferons alpha, beta and gamma and tumor necrosis factor will be carried out by Northern blotting and polymerase chain reaction techniques. Specific antibodies will be used to test for the secretion of agents with antiviral properties in culture supernatants and by co-culture experiments. If intracellular interferon-gamma is found to activate expression of specific genes directly, the mechanism responsible for this effect will be sought. If intracellular interferon-gamma causes the production of a homologous agent with interferon-like properties it will be important to identify this substance and determine how interferon gamma can induce its production and whether such an effect plays a part in the normal response of cells to interferon-gamma. The biological response modifier, interferon-gamma has been the subject of much recent interest because of its antiviral action and its stimulatory effects on cells of the mammalian immune system. Current research is based on the assumption that this hormone-like substance acts as do many peptide hormones, by binding to a surface membrane receptor and, subsequently triggering a cascade of intracellular biochemical events. Dr. Lewis has preliminary results suggesting a novel, intracellular site of action for interferon-gamma. If this is the case it could represent a very important new mechanism of action, but much critical work must be done to establish unequivocally the basis for the preliminary observations. This grant will support the critical test of these potentially important observations.