MANIPULATION OF PHAGOCYTOSIS IN MACROPHAGES

巨噬细胞吞噬作用的操纵

• 批准号: 2330417
• 负责人: JOEL A SWANSON
• 金额: $ 18.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330417
• 关键词: Salmonella typhimurium; acidity /alkalinity; drug delivery systems; electron microscopy; endocytosis; fluorescence microscopy; genetic strain; laboratory mouse; lysosomes; macrophage; membrane permeability; phagocytosis; vesicle /vacuole

Phagocytosis by macrophages is essential for immune surveillance and response. Although postphagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis by altering the course to lysosomes. These pathogens could be counteracted by therapies that redirect phagosomes toward lysosomes or that deliver drugs specifically into those phagosomes. The goal of the work described here is to design such therapies through in-proved understanding of organelle dynamics inside macrophages. We will first describe the normal course of phagosomes toward lysosomes. In our previous studies of macropinosomes, we found that their intracellular path to the lysosome includes intermediate stages. if phagosomes proceeded toward lysosomes similarly, by progressively acquiring and losing features of endosomes, then phagosome- lysosome fusion would be only the last of a series of vesicle fusion and fission events, any of which might be inhibited by a pathogen in a phagosome. We will examine phagocytosis of IgG-opsonized particles to determine if phagosomes have properties of early or late encosomes before they fuse with lysosomes. Phagosomes of different ages will be identified by pulse-labeling with fluorescein dextran during phagocytosis, then chasing without fluorophore for defined intervals before fixing and preparing cells for immunofluorescent localization of markers for early and late endosomes and for lysosomes. The rate of phagosome acidification will be measured by quantitative fluroescence microscopy and correlated with the morphology. We will then measure the effects of various factors on the course or rate of phagosomes progression. Next, we will describe the course of phagosomes containing Salmonella typhimurium, which enter mouse macrophages via macropinocytosis. We will determine where along the path phagosomes containing Salmonella slow their progression toward lysosomes. We will then examine progression of phagosomes containing mutants of S. typhimurium that fail to induce macropinocytosis. We will also ask if Salmonella -phagosomes become removed from the traffic of other endocytic organelles, and if their permeability or transport properties differ from those of other phagosomes. Finally, we will study phagocytosis and the intracellular progression of various liposomes, modifying them to optimize uptake, to delay their delivery to lysosomes, and to target them to phagosomes containing Salmonella. We will also devise strategies to deliver molecules from liposomes into macrophage cytoplasm. Although these studies aim to kill Salmonella typhimurium in its phagosome, the information gained in the process should be applicable to other organisms that evade macrophage defense machanisms. This research should therefore provide both basic cell biology and clinically relevant therapeutic strategies.

巨噬细胞的吞噬作用对于免疫监测和 回复。虽然将微生物递送到巨噬细胞中 溶酶体通常导致其降解，一些致病性 微生物通过改变溶酶体的病程来存活吞噬作用。 这些病原体可以通过重定向的疗法来抵消 吞噬对溶酶体或专门输送药物的吞噬体 吞噬体。这里描述的工作的目的是设计 通过对内部细胞器动力学的特有理解来疗法 巨噬细胞。我们将首先描述吞噬体的正常过程 朝向溶酶体。在我们先前关于大斑小体的研究中，我们发现 他们通往溶酶体的细胞内路径包括中间体 阶段。如果吞噬体类似地朝着溶酶体前进，则 逐渐获得和失去内体特征，然后是吞噬体 溶酶体融合只是一系列囊泡融合的最后一个 裂变事件，其中任何一个可能被病原体抑制 吞噬体。我们将检查IgG揭示颗粒的吞噬作用 确定吞噬体之前的早期或晚期杂种的特性 它们与溶酶体融合。将确定不同年龄的吞噬体 通过在吞噬作用过程中用荧光素葡萄糖脉冲标记，然后 在固定之前，没有荧光团的无荧光团进行定义的间隔和 制备细胞以早期标记的免疫荧光定位 和晚期内体和溶酶体。吞噬体酸化速率 将通过定量氟化显微镜测量并相关 与形态。 然后，我们将衡量各种因素的影响 关于吞噬体进展的过程或速率。接下来，我们将描述 含有鼠伤寒沙门氏菌的吞噬体的进程，进入 小鼠巨噬细胞通过大型细胞增多症。我们将确定沿哪里 含有沙门氏菌的路径吞噬体降低了它们的进展 溶酶体。然后，我们将检查包含吞噬体的进展 伤寒的突变体，无法诱导大型细胞增多症。我们将 还要询问沙门氏菌 - 斑点是否从交通中删除 其他内吞细胞器，以及它们的渗透性或运输 特性与其他吞噬体的特性不同。最后，我们将学习 吞噬作用和各种脂质体的细胞内进展， 修改它们以优化摄取，推迟溶酶体的递送， 并将其靶向含有沙门氏菌的吞噬体。 我们也会 设计将分子从脂质体传递到巨噬细胞的策略 细胞质。 尽管这些研究旨在杀死鼠伤寒沙门氏菌 它的吞噬体，该过程中获得的信息应适用 对于逃避巨噬细胞防御马奇主义的其他生物。这 因此，研究应同时提供基本细胞生物学和临床上的生物学 相关的治疗策略。