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.

Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

“…This outcome is in conflict with findings of others who observed that following co-injection of Ly6G (Gr1)-APC-labeled “aged” neutrophils and Ly6G (Gr1)-FITC-labeled “young” neutrophils, the APC-labeled “aged” neutrophil population dominated in the BM of the recipients [26]. We have previously shown that coupled fluorochromes can alter the functional properties of neutrophil-targeting Ly6G antibodies [25] and assume that the conflicting results might be explained by the cell-depleting effect of the FITC-coupled Ly6G antibody.…”

“…Single cell suspensions from blood, spleen, lungs and BM were isolated as described previously [22, 24, 25, 26]. Leukocytes from single cell suspensions were prepared by lysis of red blood cells in ACK buffer (0.155 M NH 4 Cl, 0.01 M KHCO 3 , 0.1 mM EDTA).…”

“…Then, BM cells were stained with anti-Ly6G (1A8)-APC (6 h neutrophils) or anti-Ly6G (1A8)-PE (0 h neutrophils). APC- and PE-coupled Ly6G antibodies were chosen since neutrophils labeled with these antibodies showed comparable retrieval rates following adoptive transfer into mice [25]. After washing twice with PBS, the separately stained anti-Ly6G (1A8)-APC + and -PE + BM-leukocytes were pooled and 2.5 × 10 7 cells in 150 μL PBS were injected into the tail vein of each recipient mouse.…”

Macrophages: Key regulators of steady-state and demand-adapted hematopoiesis

“…This is thought to occur through a density feedback mechanism that detects cell loss, yet the sensors of this cell loss have not been elucidated [81]. Notably, antibody-mediated neutrophil depletion, which enhances emergency granulopoiesis, requires Mϕ-dependent efferocytosis [82]. Thus, while efferocytosis suppresses granulopoiesis at steady state [24, 25, 83], this process may enhance granulopoiesis under stress conditions where rapid neutrophil turnover and clearance occur.…”

In vivo imaging of the pathophysiological changes and neutrophil dynamics in influenza virus-infected mouse lungs

“…into the mice. We used 5 μg of antibody because administration of a low dose (1 to 40 μg) of fluorochromecoupled anti-Ly-6G antibody to mice does not affect neutrophil recruitment (56), and injection with PE-conjugated antibodies does not induce antibodydependent neutrophil depletion compared with other fluorescent dyes (such as FITC) in vivo (57).…”