Lavie Lena, Dyugovskaya Larissa, Polyakov Andrey, Rogovoy Oksana, Leder Eva
The Lloyd Rigler Sleep Apnea Research Laboratory, Unit of Anatomy and Cell Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Insitute of Technology;
The Lloyd Rigler Sleep Apnea Research Laboratory, Unit of Anatomy and Cell Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Insitute of Technology.
J Vis Exp. 2017 Jan 25(119):54826. doi: 10.3791/54826.
Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (Gϕ). Unlike neutrophils, Gϕ are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. Gϕ also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing Gϕ in order to further investigate their significance and functions.
中性粒细胞（PMN）以其针对入侵病原体和微生物的吞噬功能而最为人所知。它们在白细胞中半衰期最短，在未激活状态下会自然发生凋亡。当被招募到炎症部位以消除炎症时，它们会产生一系列具有强大抗菌杀伤力的细胞毒性分子。然而，当这些强大的细胞毒性分子以不受控制的方式释放时，它们会损害周围组织。然而近年来，中性粒细胞的多功能性越来越多地得到证实，表现为可塑性和免疫调节功能。我们最近鉴定出一种新的源自中性粒细胞的亚群，它在标准培养条件下自发形成，无需添加细胞因子/生长因子，如粒细胞集落刺激因子（GM-CSF）/白细胞介素（IL）-4。它们对中性粒细胞残余物的吞噬能力在很大程度上极大地增加了它们的大小；因此它们被称为巨型吞噬细胞（Gϕ）。与中性粒细胞不同，Gϕ在培养中寿命较长。它们表达分化簇（CD）中性粒细胞标志物CD66b/CD63/CD15/CD11b/髓过氧化物酶（MPO）/中性粒细胞弹性蛋白酶（NE），并且缺乏单核细胞谱系标志物CD14/CD16/CD163以及树突状细胞CD1c/CD141标志物。它们还摄取乳胶和酵母聚糖，并通过氧化爆发对调理酵母聚糖和佛波酯的刺激做出反应。Gϕ还表达清道夫受体CD68/CD36，与中性粒细胞不同，它们会内化氧化型低密度脂蛋白（oxLDL）。此外，与新鲜中性粒细胞或培养的单核细胞不同，它们对oxLDL摄取的反应是活性氧（ROS）产生增加。此外，这些吞噬细胞含有微管相关蛋白1轻链3B（LC3B）包被的液泡，表明自噬被激活。使用特异性抑制剂很明显，吞噬作用和自噬都是它们发育的先决条件，并且可能依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶产生ROS。我们在此描述一种在培养中制备这种源自中性粒细胞的长寿吞噬细胞新亚群的方法、它们的鉴定以及它们目前已知的特征。该方案对于获得和表征Gϕ以进一步研究它们的意义和功能至关重要。
