CD177 interacts with β2 integrins but does not alter integrin expression or affinity. (A) Immunofluorescence microscopy was performed to assess colocalization of CD177 and CD11b on human peripheral blood neutrophils. Scale bar, 10 µm. (B) Pseudocolored FLIM demonstrating molecular interaction between CD177 and CD11b, with reduction in τ1 indicative of fluorescence resonance imaging transfer quenching. (C) Quantitation of τ1 for interaction between CD11b or CD11a as donor and CD177 as acceptor. Data shown reflect at least 2 experiments each. (D) Flow cytometry was used to assess surface expression of total and active surface CD11b on freshly isolated peripheral neutrophils or after activation with LTB4 100 nM for 30 min. Similar results were obtained for CD11a, CD18, ICAM-1 binding, and integrin-dependent phagocytosis and with endogenously activated neutrophils obtained from inflamed joints (supplemental Figure 5A-D). Integrin data are pooled from 5 independent donors and at least 3 replicates. (E) CD177 ligation enhances the spatial proximity of CD177 with β2 integrins. Reduction in τ1 by FLIM upon CD177 ligation with MEM166 reflects enhanced intermolecular interaction with CD11b. Data were pooled from 3 separate experiments, including 60 to 90 pixels from the surface membrane used to calculate values. Donor-only τ1 was 1290 ± 9. Means ± SEMs. ****P < .0001, by unpaired t test (C,E) or 2-way ANOVA (D). A, acceptor; D, donor; MFI, mean fluorescence intensity; ps, picoseconds.
