CD99 Fc supports shear resistance of the binding of neutrophils to ICAM-1 in a PILR-dependent manner. (A) Polymorphonuclear neutrophils (PMNs) were added together with CXCL1 to rotating Petri dishes (80 rpm) with immobilized ICAM-1 Fc, P-selectin (P-sel) Fc, and either CD99 Fc (blue bars) or huIgG (red bars) (as indicated) at room temperature for 30 minutes, followed by washing and fixation (left panel). PMNs attached at different distances from the center were counted by microscopy. The depicted shear resistance index represents the shear rate increase required for a set reduction of PMN binding (n = 10). Note that no increase was seen when ICAM-1 FC was omitted (n = 6) (right panel). (B) Role of PILRs for CD99-mediated shear resistance of PMN binding was investigated by repeating experiment described in panel A with PMNs pretreated with antibodies from preserum (left) (n = 16) or anti-PILR antibodies (right) (n = 9). (C) PMNs were passed through a flow chamber at 5 dyn/cm² coated with P-selectin Fc, CXCL1, ICAM-1 Fc, and either CD99 Fc (blue bars) or huIgG (red bars) for 10 minutes at room temperature followed by enumeration of adherent cells (n = 15 [with ICAM-1 Fc] or n = 6 [without ICAM-1 Fc]). (D) Cells from bone marrow were passed through a flow chamber at 5 dyn/cm² coated with P-selectin Fc, CXCL1, and either CD99 Fc (blue bars) or huIgG (red bars) for 5 minutes at room temperature followed by fluorescence-activated cell sorting analysis for binding of soluble ICAM-1 Fc. The ICAM-1-binding fraction of Gr-1⁺ cells incubated with the huIgG-coated surfaces was set as 1 (n = 6). n.s., *P < .05, and ***P < .001, as per 1-sample t test; #P < .05, as per unpaired t test; error bars show standard error of the mean.