CD177 ligation blocks migration in a PECAM-1-independent manner. Peripheral blood neutrophils were allowed to migrate through 3-µm pores toward LTB₄ (B-D, G-H) or supernatants from resting or TNF/IL-17-activated FLS (E-F). (A) Representative histograms of CD177 expression by flow cytometry from a donor with both CD177^pos and CD177^neg cells (CD177^mixed) or CD177^neg cells only (CD177^null). (B) Time course of migration of CD177^pos and CD177^neg neutrophils from donors with both populations, demonstrating equal intrinsic migratory efficiency. (C) Addition of blocking antibodies (10 µg/mL) to cells prior to introduction into the transwell demonstrates dependence of migration on CD18 but not PECAM-1. (D) CD177 ligation with MEM166 (10 µg/mL, termed αCD177) impedes neutrophil migration. (E) αCD177 attenuates neutrophil migration toward supernatants from activated FLS. TNF/IL-17-supplemented medium alone does not induce migration (data not shown). (F) Anti-IL-8 blocks the chemotactic effect of stimulated FLS. Data in panels B-F are normalized to no-chemoattractant migration to enable pooling of data from multiple donors. (G) αCD177 decreased the proportion of CD177^pos neutrophils in the bottom chamber, indicating selective migratory blockade of CD177^pos cells. (H) Migration of neutrophils obtained from a CD177^null donor was unaffected by αCD177. All experiments reflect duplicate or triplicate wells per condition and are representative of 3 to 5 similar experiments. Means ± SEMs. *P < .05; **P < .01; ****P < .0001; by 2-way analysis of variance (ANOVA) (B-G) or 1-way ANOVA (C,D), followed by Tukey's multiple comparisons test. Ab, antibody; activ, activated; ctrl, control; iso, isotype; ns, not significant; unstim, unstimulated.