Fig. 2
Treatment with anti-M7 prevents inflammatory leukocyte recruitment in vitro and in vivo. a Murine peritoneal macrophages were incubated on dishes coated with mouse CD40L in the presence of 10μg/ml of IgG, anti-M7, or anti-Mac-1 (clone M1/70). Adhering cells were normalized to % of the IgG-treatment. b Murine RAW-cell adhesion on TNFα-primed murine endothelial cells under physiological flow in the presence of IgG or anti-M7 antibodies (10 μg/ml). c–f C57Bl/6 mice were injected i.p. with 200 ng TNFα and 15 min before microscopy with Fab fragments of an IgG control antibody, or antibodies against RAGE, ICAM-1, LFA-1, Mac-1 (clone M1/70), or anti-M7 (100 μg i.p.). Leukocyte recruitment was monitored by intravital microscopy 4 h later: Adhering (d) and rolling leukocytes (e) were quantified as % of IgG. (f) Cumulative frequency of leukocyte rolling velocity. g Leukocyte adhesion in intravital microscopy in IgG or anti-M7 treated WT and Mac-1−/− mice. h, i Number of CX3CR1-GFP+ monocytes (white arrows) in the para-vascular space 4 h after treatment with IgG or anti-M7 and 200ng TNFα. j Confocal in vivo imaging of the greater omentum after local stimulation with TNFα and i.v. injection of the indicated antibodies (100 μg) in LysM-GFP reporter mice. LysM-GFP+ myeloid cells were tracked over time. Time lapse and cell tracking is shown over 15 min (right panel). k Plasma cytokine levels of mice subjected to intravital microscopy after IgG or anti-M7 Fab treatment. l Peritoneal exudate cells (PECs) 72 h after induction of a sterile peritonitis and the indicated antibody treatment. Scale bars represent 100μm (c, h, j). Error bars indicate mean ± SEM. Statistical significance was assessed by a two-sided, unpaired Student’s T-test between the indicated groups (a, b, g, i, k, l) or in comparison to IgG-treatment (d, e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N ≥ 3 independent experiments (a, b). N ≥ 10 mice per group (d, e, f, g), N ≥ 6 mice per group (l, h)
