LPS-MEDIATED REGULATORY EVENTS DURING MACROPHAGE ACTIVATION

拟议研究的总体目标是阐明内毒素- 脂多糖触发杀瘤活性过程中的调节事件 巨噬细胞。这些研究的基本原理是，尽管 众多实验室的广泛努力，巨噬细胞的机制 激活情况仍不清楚。根据我们的初步结果，我们建议 检验假设，在内毒素触发期间的事件序列 杀瘤活性开始于几种不同的 蛋白激酶。这会导致转录因子的激活 然后转移到细胞核并激活各种细胞因子基因 诱导型一氧化氮合酶基因。几种细胞因子，如干扰素-β，由内毒素产生- 活化的巨噬细胞参与血管内皮细胞的进一步发展 巨噬细胞以自动/旁分泌方式激活。这 假设将在以下具体目标下进行检验：1) 研究内毒素触发的信号转导机制 核因子-kappa-B的激活；以及2)研究 或者巨噬细胞在体外和体内都可以被干扰素-β激活， 干扰素-γ和/或单核细胞趋化肽-1(MCP-1，又称乙脑) 由稳定转染的各种肿瘤细胞分泌或 插入干扰素-β、干扰素-γ或单核细胞趋化蛋白-1基因的真核表达载体 表达载体。我们实现第一个具体目标的方法是 研究：1)I-kappa-B相关蛋白的类型和性质 核因子-kappaB蛋白在J774细胞中的表达；2)蛋白激酶的作用 和可能参与内毒素触发的磷酸化和蛋白水解酶 I-kappa-B蛋白的降解；3)内毒素介导的转录和 Kappa-B生物合成的转录后调控；4)潜能 多种内源性细胞因子在内毒素触发的核因子-kappaB中的作用 5)H-89和/或H-7介导的抑制作用 脂多糖触发的核因子-kappaB和AP-1对细胞因子基因的激活。我们的 实现具体目标2的方法是：1)稳定地生产肿瘤细胞-- 转染或转导小鼠干扰素-β、干扰素-γ基因， 和/或单核细胞趋化蛋白-1；2)检测干扰素-β和/或干扰素-γ 基因修饰的肿瘤细胞分泌的物质将激活J774细胞 体外培养；3)检测肿瘤细胞是否分泌MCP-1 将单核/巨噬细胞吸引到肿瘤部位，可被激活 对于由基因-β和/或干扰素-γ分泌的杀瘤活性- 改良的肿瘤细胞。拟议研究的意义在于 这些具体目标的完成有望为我们提供更好的 对脂多糖诱导的肿瘤细胞杀伤活性机制的认识 巨噬细胞，这可能是针对癌症的免疫治疗。

The overall objective of the proposed research is to elucidate LPS- mediated regulatory events during LPS-triggered tumoricidal activation of macrophages. The rationale for these studies is that despite extensive efforts by numerous laboratories, the mechanisms of macrophage activation remain unclear. Based on our initial results, we propose to examine the hypothesis that the sequence of events during LPS-triggered tumoricidal activation begins with the activation of several different protein kinases. This leads to the activation of transcription factors which then translocate to the nucleus and activate various cytokine genes and iNOS gene. Several cytokines, such as IFN-beta, produced by LPS- activated macrophages participate in further development of the macrophage activation process in an auto/paracrine fashion. This hypothesis will be examined under the following specific aims: 1) to investigate LPS-triggered signal transduction mechanisms which lead to the activation of NF-kappa-B; and 2) to investigate the question whether or not macrophages can be activated, in vitro and in vivo, by IFN-beta, IFN-gamma and/or monocyte chemotactic peptide-1 (MCP-1, also known as JE) secreted from various tumor cells which are stably transfected or transduced with IFN-beta, IFN-gamma or MCP-1 genes inserted in eukaryotic expression vectors. Our approaches to Specific Aim #1 are to investigate: 1) the types and properties of I-kappa-B proteins associated with NF-kappa-B proteins in J774 cells; 2) the roles of protein kinases and proteases potentially involved in LPS-triggered phosphorylation and degradation of I-kappa-B proteins; 3) LPS-mediated transcriptional and post-transcriptional regulation of kappa-B biosynthesis; 4) potential role of various endogenous cytokines in LPS-triggered NF-kappa-B activation; 5) the effects of the H-89 and/or H-7 mediated inhibition of LPS-triggered NF-kappa-B and AP-1 on cytokine gene activation. Our approaches to Specific Aim #2 are: 1) to produce tumor cells stably- transfected or transduced with mouse genes for IFN-beta, IFN-gamma, and/or MCP-1; 2) to test whether or not IFN-beta and/or IFN-gamma secreted from gene modified tumor cells will activate J774 cells in vitro; 3) to examine whether or not MCP-1 secreted from tumor cells attracts monocytes/macrophages into tumor site, which can be activated for tumoricidal activity by IFN-beta and/or IFN-gamma secreted from gene- modified tumor cells. The significance of the proposed research is that completion of these specific aims is expected to provide us with better understanding of mechanisms of LPS-triggered tumoricidal activation of macrophages, which may be directed to cancer immunotherapy.