Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice
Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice
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DOI：
10.1189/jlb.1ab1014-488rr
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发表时间：
2015-09-01
影响因子：
5.5
通讯作者：
Beer-Hammer, Sandra
中科院分区：
文献类型：
--
作者：
Bucher, Kirsten;Schmitt, Fee;Beer-Hammer, Sandra
荧光标记的Ly 6 G抗体能够追踪小鼠中的嗜中性粒细胞，而纯化的抗Ly 6 G抗体迅速耗尽循环中的嗜中性粒细胞。中性粒细胞耗竭的机制仍在争论中。在这里，我们研究了相同的Ly 6 G抗体与不同的荧光染料偶联如何影响体内中性粒细胞的命运。将用Ly 6 G抗体染色的BM细胞注射到小鼠中。与抗Ly 6 G-APC（+）或抗Ly 6 G-PE+细胞相比，回收的抗Ly 6 G-FITC+细胞数量显著减少。流式细胞术和多光谱成像流式细胞术分析表明，抗Ly 6 G-FITC+中性粒细胞优先吞噬的Bcyline在体外和脾，肝，和BM巨噬细胞在体内。直接注射抗Ly 6 G-FITC抗体，但不注射抗Ly 6 G-PE抗体，中性粒细胞消耗程度与纯化的抗Ly 6 G抗体相同，表明FITC偶联抗体通过与未偶联抗体相似的机制消除中性粒细胞。与使用的蛋白G结合试验，我们证明，APC和PE，但不是FITC偶联抑制访问的抗Ly 6 G抗体上的相互作用位点。我们得出以下结论：1）巨噬细胞的嗜中性粒细胞吞噬作用是抗Ly 6 G诱导的嗜中性粒细胞耗竭的中心机制，2）荧光染料偶联可影响抗Ly 6 G抗体的功能特性，从而在过继细胞转移或注射荧光抗Ly 6 G后改变Ly 6 G标记的嗜中性粒细胞的巨噬细胞摄取和嗜中性粒细胞回收。
Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC+ cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE+ cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC+ neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central-mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.