Antibodies against neutrophil LY6G do not inhibit leukocyte recruitment in mice in vivo

To the editor:

Ly6G is a marker of neutrophils, but the function of this GPI-linked receptor has remained mysterious. Wang et al provided data implicating Ly6G in regulating leukocyte migration.¹  These investigators found that low doses of antibodies (10 μg total administration) directed against Gr-1 or Ly6G reduced experimental arthritis, an effect not due to neutrophil depletion. Instead, anti–Gr-1 or anti-Ly6G antibodies were proposed to inhibit neutrophil recruitment, leading to a reduction in arthritis by acting on the β2-integrins CD11a and CD11b and on ICAM-1 binding levels.

Our laboratory uses intravital imaging to assess the entire leukocyte recruitment cascade directly in real time in vivo. Although granulocyte depletion is achieved at high anti–Gr-1 antibody doses (150-250 μg), we visualize leukocyte recruitment using lower IV doses (1-40 μg) of fluorochrome-labeled anti–Gr-1 (clone RB6-8C5) or anti-Ly6G (clone 1A8). Exploiting these antibodies as imaging tools, we have characterized rolling, adhesion, intravascular crawling, transmigration, emigration, phagocytosis, and tissue NETosis in multiple tissues including skin, liver, brain, and muscle.^2-6  Despite not observing defects in leukocyte recruitment with these antibodies, we performed new experiments to compare neutrophil recruitment directly in transgenic LysM-eGFP mice, in which peripheral blood neutrophils are visualized without antibodies to Ly6G, with LysM-eGFP mice treated with a fluorochrome-labeled anti–Gr-1 antibody (Figure 1 and supplemental Video 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Using a mouse model of Staphylococcus aureus cellulitis,⁶  we imaged neutrophil recruitment directly using spinning disk confocal intravital microscopy. LysM-eGFP mice received an intradermal injection of live S aureus and fluorochrome-conjugated anti–Gr-1 antibody (5 μg IV) or Ly6G clone 1A8 (data not shown), followed immediately by intravital imaging. Neutrophils were visualized rolling and adhering within the dermal microvasculature in both the LysM-eGFP (green) laser channel and the anti–Gr-1 (yellow) laser channel immediately after infection (Figure 1A,D and supplemental Video 1). Within 60 minutes of infection, LysM-eGFP/anti–Gr-1 double-positive peripheral blood neutrophils transmigrated into the parenchyma (Figure 1B,E) and continued to chemotax through the skin (Figure 1C,F). Therefore, we conclude that the entire recruitment cascade, from rolling to chemotaxis, occurs in anti–Gr-1 antibody-treated animals.

To ensure that the anti–Gr-1 antibody did not have subtle quantitative effects on neutrophil recruitment, we compared LysM-eGFP mice with and without anti–Gr-1 antibody (Figure 1G-L). The number of emigrated neutrophils was similar between the 2 groups (Figure 1G). Once emigrated, neutrophils from each group crawled with identical velocities (Figure 1H), meandering index (a measurement of the ability of a cell to move in a straight line; Figure 1I), and displacement (Figure 1J). Leukocyte crawling tracks are shown for tissue neutrophils in LysM-eGFP mice (Figure 1K) and LysM-eGFP mice treated with anti–Gr-1 (Figure 1L). These data demonstrated that no quantifiable difference in leukocyte recruitment and behavior occurred after anti–Gr-1 administration. A fluorochrome-conjugated anti-Ly6G antibody (1A8 clone) did not disrupt any of the leukocyte recruitment measurements (data not shown).

Our ability to visualize all aspects of the leukocyte recruitment cascade directly in vivo demonstrates that low-dose anti–Gr-1 antibodies do not interfere with neutrophil migration. Anti–Gr-1 administration in vitro inhibited stimulation-induced up-regulation of β2-integrins above baseline; however, cell-surface levels continued to be high, as were ICAM-1–binding levels. It is likely that these levels continue to mediate normal recruitment under in vivo physiologic models. The abrogation of arthritis reported by Wang et al is an interesting observation that highlights the need to better characterize Ly6G. However, given our data, we do not believe that the mechanism proposed by the authors adequately explains their observations. We demonstrate that fluorochrome-conjugated antineutrophil antibodies against Gr-1 can be used to investigate leukocyte recruitment without interfering with cell migration.