Mechanism of Action of Interferon-gamma

这个实验室分离出了表达突变形式的人类干扰素-伽马基因的小鼠细胞系，这些细胞缺乏信号序列。这些细胞合成人干扰素-γ，但不能分泌，并在细胞内积累蛋白质。虽然使用的小鼠细胞对添加到培养基中的人干扰素-γ的影响完全耐受，但在细胞内表达这种异种干扰素的细胞对水泡性口炎病毒和蒙戈病毒的感染具有抵抗力，并表现出较高的寡聚2，5A合成酶和eIF-2激酶水平，这些酶是通过干扰素治疗而诱导的。这些细胞还显示编码主要组织相容性复合体II类抗原的mRNA水平增强。这些结果提示了一个新的概念，即干扰素伽马可以跨越物种障碍在细胞内发挥作用，导致特定的基因激活。拟议的研究将详细分析这一现象，以确保观察到的结果直接归因于细胞内的干扰素-伽马，而不是由同源抗病毒药物的细胞产生。这些观察的一般性将通过确定在人类细胞内表达的小鼠干扰素-伽马是否产生类似的效果来检验。由于这些结果可以通过干扰素或其他具有抗病毒特性的药物的分泌来解释，因此将通过Northern印迹和聚合酶链式反应技术检测同源干扰素α、β和伽马以及肿瘤坏死因子的mRNA水平的增加。特定的抗体将用于测试培养上清液中具有抗病毒特性的制剂的分泌情况，并通过共培养实验进行测试。如果细胞内的干扰素-γ被发现能直接激活特定基因的表达，就会寻找导致这种效应的机制。如果细胞内干扰素-γ引起具有干扰素样属性的同源物质的产生，重要的是确定这种物质，并确定干扰素-γ如何诱导其产生，以及这种效应是否在细胞对干扰素-伽马的正常反应中发挥作用。干扰素-γ是一种生物反应调节剂，由于其抗病毒作用和对哺乳动物免疫系统细胞的刺激作用，近年来一直是人们感兴趣的主题。目前的研究是基于这样的假设，即这种激素样物质的作用与许多肽激素一样，通过与表膜受体结合，随后触发一系列细胞内生化事件。刘易斯博士的初步结果表明，干扰素-伽马有一个新的细胞内作用部位。如果是这样的话，它可能代表着一个非常重要的新行动机制，但必须做大量关键工作，以明确确立初步意见的基础。这笔赠款将支持对这些潜在重要观察结果的关键测试。

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.