Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Dev Comp Immunol. 2010 Nov;34(11):1144-59. doi: 10.1016/j.dci.2010.06.006. Epub 2010 Jun 25.
Phagocytosis provides a critical first line of defense against invading pathogens. Engagement of particles through receptor-mediated binding precedes internalization and induction of cellular antimicrobial responses. Phagocytes have the capacity to differentially regulate binding and internalization processes through changes in their receptor profile and modulation of downstream events. This is necessary for the intricate control of phagocytic antimicrobial responses. Several methods are available for evaluation of phagocytosis. Unfortunately, none allow for accurate quantitation of both binding and internalization events. To overcome these limitations, we have developed a novel phagocytosis assay based on a multi-spectral imaging flow cytometry platform. This assay discriminates between internalized and surface-bound particles in a statistically robust manner and allows multi-parametric analysis of phagocytosis and downstream anti-microbial responses. We also devised a novel approach for examination of phagolysosome fusion, which provides an improved capacity for quantitative assessment of phagolysosome fusion in mixed populations of intact cells. Importantly, our approaches are likely amenable to a broad range of comparative model systems based on our examination of murine RAW 264.7 cells and a goldfish primary kidney macrophage (PKM) model system. The latter allowed us to examine the evolutionary conservation of phagocytic antimicrobial responses in a lower vertebrate model. While it has been previously reported that mixed populations of these macrophage cultures are phagocytic, it remained unclear if sub-populations within them contributed differentially to this activity. In accordance with higher vertebrate models, we found that differentiation along the macrophage pathway leads to an increased capacity for phagocytosis in goldfish PKM. Interestingly, cellular activation differentially regulated particle internalization in PKM monocyte and mature macrophage subsets. We also found differential regulation of phagolysosome fusion and downstream production of reactive oxygen intermediates (ROI). The temporal activation of specific phagocytic antimicrobial responses at distinct stages of PKM differentiation suggests specialization within the macrophage compartment early in evolution, geared to meet specific host immunity requirements within specialized niches.
吞噬作用为抵御入侵病原体提供了第一道重要防线。通过受体介导的结合来结合颗粒，随后是内化和诱导细胞抗菌反应。吞噬细胞通过改变受体谱和调节下游事件的方式，具有差异化调节结合和内化过程的能力。这对于精细控制吞噬抗菌反应是必要的。有几种方法可用于评估吞噬作用。不幸的是，没有一种方法可以准确定量结合和内化事件。为了克服这些限制，我们开发了一种基于多谱成像流式细胞术平台的新型吞噬作用测定法。该测定法以统计学上稳健的方式区分内化和表面结合的颗粒，并允许对吞噬作用和下游抗菌反应进行多参数分析。我们还设计了一种新的方法来检查吞噬溶酶体融合，这为定量评估混合完整细胞群体中的吞噬溶酶体融合提供了更好的能力。重要的是，我们的方法可能适用于广泛的比较模型系统，这是基于我们对鼠 RAW 264.7 细胞和金鱼原代肾巨噬细胞（PKM）模型系统的检查。后者使我们能够在较低的脊椎动物模型中检查吞噬抗菌反应的进化保守性。虽然之前已经报道过这些巨噬细胞培养物的混合群体具有吞噬作用，但仍不清楚它们中的亚群是否对此活性有不同的贡献。与高等脊椎动物模型一致，我们发现沿着巨噬细胞途径的分化导致金鱼 PKM 的吞噬能力增加。有趣的是，细胞激活差异化调节了 PKM 单核细胞和成熟巨噬细胞亚群中的颗粒内化。我们还发现吞噬溶酶体融合和下游活性氧中间体（ROI）产生的差异化调节。在 PKM 分化的不同阶段特异性吞噬抗菌反应的时间激活表明，在进化早期，巨噬细胞隔间内的专业化，旨在满足专门小生境中的特定宿主免疫需求。
