LPS-MEDIATED REGULATORY EVENTS DURING MACROPHAGE ACTIVATION

这项研究的总体目标是阐明LPS- LPS触发的杀肿瘤激活过程中介导的调节事件 巨噬细胞 这些研究的基本原理是，尽管 通过众多实验室的广泛努力，巨噬细胞的机制 激活仍不清楚。 根据我们的初步结果，我们建议 检查LPS触发的事件序列的假设， 杀肿瘤的激活始于几种不同的 蛋白激酶 这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 和iNOS基因。 几种细胞因子，如IFN-β，由LPS产生， 活化的巨噬细胞参与了 巨噬细胞以自动/旁分泌方式激活过程。 这 假设将根据以下具体目标进行审查：1） 研究LPS触发的信号转导机制， NF-κ-B的活化;和2）研究是否 巨噬细胞在体外和体内都可以被IFN-β激活， IFN-γ和/或单核细胞趋化肽-1（MCP-1，也称为JE） 从稳定转染的各种肿瘤细胞分泌，或 用插入真核细胞中的IFN-β、IFN-γ或MCP-1基因转导 表达载体。 我们实现具体目标#1的方法是 研究：1）与I-κ-B蛋白相关的蛋白类型和性质 与NF-κ-B蛋白在J774细胞中的作用; 2）蛋白激酶的作用 和可能参与LPS触发的磷酸化的蛋白酶， I-κ-B蛋白的降解; 3）LPS介导的转录和 kappa-B生物合成的转录后调节; 4）潜在的 各种内源性细胞因子在LPS触发的NF-κ B B中的作用 5）H-89和/或H-7介导的抑制细胞活化的作用; LPS触发的NF-κ-B和AP-1对细胞因子基因活化的影响 我们 具体目标#2的方法是：1）稳定地产生肿瘤细胞- 用小鼠IFN-β，IFN-γ， 和/或MCP-1; 2）测试IFN-β和/或IFN-γ 从基因修饰的肿瘤细胞分泌的将激活J774细胞， 3）检测肿瘤细胞是否分泌MCP-1 吸引单核细胞/巨噬细胞进入肿瘤部位，可被激活 对于由基因-IFN-β和/或IFN-γ分泌的杀肿瘤活性， 修饰的肿瘤细胞 拟议研究的意义在于， 这些具体目标的完成，预计将为我们提供更好的 了解LPS触发的杀肿瘤激活的机制， 巨噬细胞，这可能是针对癌症免疫疗法。

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.