CD99 on EC represents the ligand for PILRs on neutrophils. (A) bEnd.5 cells were either untreated or pretreated with antibodies from preserum or anti-CD99 (as indicated) and subsequently allowed to adhere onto a surface immobilized with E-selectin Fc (control; red bar), PILR-α Fc (blue bars), or PILR-β Fc (light green bars) constructs for 60 minutes at 37°C (n > 17 from 3 independent experiments). (B) bEnd5 cells were pretreated either with control small-interfering RNA (siRNA) or with CD99 siRNA (as indicated), and adhesion assays were performed as described in panel A (n > 16 from 3 independent experiments). (C) Endothelial cells used in panel B were immunoblotted for CD99 or for tubulin (as indicated) after treatment with either control or CD99 siRNA. (D) Polymorphonuclear neutrophils (PMNs) were allowed to adhere to TNF-α–treated primary lung endothelial cells isolated from WT mice or from CD99 knockout mice (as indicated) for 30 minutes at 37°C. The cell mix was then subjected to immunoprecipitation with anti-PILR antibodies or control IgG (as indicated). Immunoprecipitates (left panel) or cell lysates (right panel) were immunoblotted for CD99 and PILR (as indicated). (E) Lysates of mouse primary endothelial cells and PMNs were immunoblotted for CD99. Note the difference in electrophoretic mobility. (F) CD99 Fc or huIgG were labeled with sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-[p-azido benzamido]-hexanoamido) ethyl-1,3′-dithioproprionate biotin and separately incubated with PMNs for 1 hour at 37°C, followed by photoactivation (312 nm ultraviolet light) to crosslink with the interacting binding partners for 10 minutes. Lysates were prepared from washed cells and crosslinked biotinylated proteins were immunoprecipitated (IP) by streptavidin and immunoblotted for PILR. Samples were reduced by 100 mM dithiothreitol to complete the biotin transfer. Data in panels D and F are representative of 2 (D) and 3 (F) independent experiments. *P < .05, ***P < .001. Error bars show standard error of the mean. One-way analysis of variance was followed by the Holm-Šídák test for panels A and B.