Antibodies against CD99L2 and CD99 inhibit neutrophil extravasation in vivo by blocking diapedesis. (A) Peritonitis assay: mice were intravenously injected with 50 μg antibody: preimmune control IgG (co-IgG) for negative controls, or with monoclonal antibodies against P-selectin and L-selectin (anti-P-/L-selectin) for positive controls, or with affinity purified anti-CD99L2 IgG (anti-CD99L2) or F(ab′)2 fragments of anti-CD99L2 IgG (anti-CD99L2 F(ab′)2) (upper panel) or with affinity purified anti-CD99 IgG (anti-CD99) or F(ab′)2 fragments of anti-CD99 IgG (anti-CD99 F(ab′)2) (bottom panel) in PBS immediately followed by intraperitoneal administration of thioglycollate. Peritoneal leukocytes were removed at 4 hours after stimulation, and neutrophil counts were determined. Results represent 3 independent experiments, for each determination of each experiment 5 mice were analyzed. Left panel, ***, P < .001 anti-P-/L-selectin versus co-IgG; ***, P < .001 anti-CD99L2 versus co-IgG; **, P < .01 anti-CD99L2 F(ab′)2 versus co-IgG. Right panel: ***, P < .001 anti-P-/L-selectin versus co-IgG; ***, P < .001 anti-CD99L2 versus co-IgG; *, P < .05 anti-CD99L2 F(ab′)2 versus co-IgG. Statistical analysis was done by Student t test. (B) Intravital microscopy of cremaster muscle venules: mice were intrascrotally stimulated with IL-1β and intravenously injected with 50 μg antibody of preimmune control IgG (co-IgG), or affinity-purified anti-CD99L2 IgG (anti-CD99L2) or affinity-purified anti-CD99 IgG (anti-CD99) (left panels) or with 75 μg of control-IgG-F(ab′)2-fragments (co-IgG-F(ab′)2), or anti-CD99L2-F(ab′)2 or anti-CD99-F(ab′)2 (as indicated) (right panels). Four hours later, the cremaster muscle was surgically prepared, and the number of rolling leukocytes per second per millimeter of vessel diameter, the number of adherent leukocytes per 104 μm2 of venule surface area, and the number of extravasated leukocytes from cremasteric venules per 104 μm2 tissue area were determined by intravital near-infrared reflected light oblique transillumination microscopy. *, P < .05 versus co-IgG, statistical analysis was done by ANOVA. (C) Proportion of neutrophils trapped between endothelial cells and the basement membrane/pericyte layer in IL-1β treated cremaster muscle. Analysis was done by transmission electron microscopy. Animals were treated with preimmune control antibodies (co-IgG), anti-CD99L2 antibodies (anti-CD99L2), or with a mixture of 2 monoclonal antibodies against mouse PECAM-1 (anti-PECAM-1). The graph shows the number of neutrophils found between the endothelium and the underlying layer of basement membranes and pericytes, given as percentage of leukocytes that had passed the endothelial cell contacts. For each determination 30 to 52 randomly selected vessel segments were analyzed. (D) Electron micrographs of vessel segments of IL-1β activated cremaster muscle of mice treated with control antibody (ctrl: a), anti-CD99L2 antibodies (anti-CD99L2: b), or anti-PECAM-1 mAbs (anti-PECAM-1: c). E, endothelium; L, leukocyte; P, pericyte. The lumen of the vessel is indicated. Bar: 2 μm.
