Bucher Kirsten, Schmitt Fee, Autenrieth Stella E, Dillmann Inken, Nürnberg Bernd, Schenke-Layland Katja, Beer-Hammer Sandra
*Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, and University Women's Hospital, Eberhard-Karls-University Tübingen, Germany; Department of Hematology, Oncology, Immunology, Rheumatology and Pulmology, University Hospital Tübingen, Germany; Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany; and Department of Medicine/Cardiology, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles, California, USA.
*Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, and University Women's Hospital, Eberhard-Karls-University Tübingen, Germany; Department of Hematology, Oncology, Immunology, Rheumatology and Pulmology, University Hospital Tübingen, Germany; Department of Cell and Tissue Engineering, Fraunhofer Institute for Interfacial Engineering and Biotechnology, Stuttgart, Germany; and Department of Medicine/Cardiology, Cardiovascular Research Laboratories, David Geffen School of Medicine at University of California Los Angeles, California, USA
J Leukoc Biol. 2015 Sep;98(3):365-72. doi: 10.1189/jlb.1AB1014-488RR. Epub 2015 May 27.
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.
荧光标记的Ly6G抗体能够追踪小鼠体内的中性粒细胞，而纯化的抗Ly6G抗体则能迅速使循环中的中性粒细胞减少。中性粒细胞减少的潜在机制仍存在争议。在此，我们研究了与不同荧光染料偶联的相同Ly6G抗体如何影响体内中性粒细胞的命运。将用Ly6G抗体染色的骨髓细胞注射到小鼠体内。与抗Ly6G-APC(+)或抗Ly6G-PE(+)细胞相比，回收的抗Ly6G-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显示，抗Ly6G-FITC(+)中性粒细胞在体外优先被骨髓巨噬细胞吞噬，在体内则被脾脏、肝脏和骨髓巨噬细胞吞噬。直接注射抗Ly6G-FITC而非抗Ly6G-PE抗体，可使中性粒细胞减少至与纯化抗Ly6G相同的程度，这表明FITC偶联抗体通过与未偶联抗体相似的机制清除中性粒细胞。通过蛋白G结合试验，我们证明APC和PE偶联而非FITC偶联会抑制与抗Ly6G抗体上相互作用位点的结合。我们得出以下结论：1）巨噬细胞对中性粒细胞的吞噬作用是抗Ly6G诱导的中性粒细胞减少的核心机制；2）荧光染料偶联可影响抗Ly6G抗体的功能特性，从而改变巨噬细胞对Ly6G标记的中性粒细胞的摄取以及过继性细胞转移或注射荧光抗Ly6G后中性粒细胞的回收情况。
