title: Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.
abstract: 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.
authors.affiliation: *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.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Bucher
authors.affiliation: *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.
authors.forename: Fee
authors.initials: F
authors.lastname: Schmitt
authors.affiliation: *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.
authors.forename: Stella E
authors.initials: SE
authors.lastname: Autenrieth
authors.affiliation: *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.
authors.forename: Inken
authors.initials: I
authors.lastname: Dillmann
authors.affiliation: *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.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nürnberg
authors.affiliation: *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.
authors.forename: Katja
authors.initials: K
authors.lastname: Schenke-Layland
authors.affiliation: *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 sandra.beer-hammer@uni-tuebingen.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Beer-Hammer
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000950:Antigens, Ly / Q000378:metabolism*; D001665:Binding Sites; D001854:Bone Marrow Cells / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000166:cytology*; D010587:Phagocytosis; D010798:Phycocyanin / Q000378:metabolism; D010799:Phycoerythrin / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000378:metabolism
chemical_list: D000906:Antibodies; D000950:Antigens, Ly; D005456:Fluorescent Dyes; C480309:Ly6G antigen, mouse; C002497:allophycocyanin; D010798:Phycocyanin; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate
keywords: APC; FITC; PE; cell tracking; granulocytes