Figure 2.

Anti-Ly6G ligation enhances TNFα-induced neutrophil oxidative burst

(a-b). Five mice were treated with anti-Ly6G or isotype CTR for 5 d. Bone marrow (BM) and blood cells were collected and incubated with or without TNFα for 1 hour at 37°C. Cells were subsequently stained with the pauci-competitive anti-Gr1at the surface a and Ly6C (b). Note that the surface levels for Gr1 in the blood neutrophils are lower than levels in the BM, while the neutrophil exit in physiological conditions normally goes with an increase of surface Ly6G, suggesting an active loss of the Ly6G antigen upon in vivo anti-Ly6G treatment p < .05, ** p < .01, *** p < 0,001, **** p < 0,0001 from Mann-Whitney test; error bars represent s.d. (c). Schematic representation of the Ly6G membrane availability upon anti-Ly6G after treatment induction and maintenance. (d). 200000 BM cells were incubated with anti-Ly6G for 15 minutes at 4°C (or isotype CTR, 10 µg/mL), extensively washed, pulsed with TNFα (5 ng/mL) for 30 min at 37°C, and incubated with a ROS detecting probe for 0–15-30 or 60 minutes (n = 6 per group per endpoint). **** p < 0,0001 from 2way ANOVA test; error bars represent s.d e. The experimental setting from d was reproduced for the endpoint 15 min, in the presence of PBS, FcBlock or Cytochalasin D to evaluate the role of residual surface Fc-FcGr interactions as well as the internalization of the Ly6G/anti-Ly6G complex on the enhanced ROS production. Six bone marrows coming from six untreated mice were used. Each bone marrow is divided to test all conditions. treatment p < .05, ** p < .01, *** p < 0,001, **** p < 0,0001 from ANOVA test with multiple comparison; error bars represent s.d