CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation

Abstract

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177^pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177^pos and CD177^neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177^pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration.

Graphical abstract

Figure 1.

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.

Figure 2.

Migratory blockade by CD177 ligation reflects enhanced adherence. (A) The proportion of neutrophils adherent to the 3-µm transwell filters that were CD177^pos was quantitated by confocal microscopy in the presence of isotype control or αCD177. Anti-CD177 increased the proportion of adherent CD177^pos cells. (B) Neutrophils were exposed to a migratory stimulus (LTB₄ 100 nM) across a barrier with pores too small to permit migration (0.4 µm), and the proportion of adherent neutrophils was assessed in the presence of isotype or αCD177. Anti-CD177 increased adherence to the membrane. Data for panels A and B represent triplicate wells per condition, representative of at least 2 to 3 similar experiments. (C) Neutrophils were incubated on glass coverslips in the presence of LTB₄ 100 nM for 15 min and washed, and CD177^pos cells were characterized by paired differential interference contrast and immunofluorescence microscopy as attached (rounded shape with or without a trailing tail) or spreading (“halo” staining of CD177, lamellar protrusions, or both). Scale bars measure 10 µm. αCD177 markedly induced spreading morphology (D) and increased the cellular area of CD177^pos neutrophils (E). (F) Treatment with anti-CD177 increases the fraction of shear stress resistant neutrophils. Data reflect 2 replicates enumerating 90 to 110 cells each (C-D), 25 to 30 cells each (E), and 3 independent experiments performed in duplicate, each replicate analyzing 10 to 50 cells per field of view (F). Means ± SEMs. *P < .05; **P < .01; ****P < .0001; by 2-way ANOVA (A,D) or unpaired t test (B,E,F). DIC, differential interference contrast. Ctl, control.

Figure 3.

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.

Figure 4.

CD177 ligation activates neutrophils in a β2 integrin-dependent manner. (A) Neutrophils were incubated with αCD177 or isotype (10 µg/mL × 30 min), with or without LTB₄ 100 nM, followed by assessment of surface activation by flow cytometry. (B) Quantitation of change in surface activation markers in CD177^pos versus CD177^neg cells. Panels A and B reflect pooled data from 3 individual donors. (C) Activation of neutrophils from a CD177^null donor was not affected by αCD177. Data are pooled from 2 replicate experiments. (D) Peripheral blood neutrophils were incubated with αCD177 or isotype 10 µg/mL ± LTB₄ 100 nM at 37°C for times as are indicated, detecting Src and ERK activation by western blot. (E) Phosphorylation of Src and ERK by αCD177 is inhibited by contemporaneous addition of blocking antibody against CD18 (MEM148, 10 µg/mL). Panels D and E are representative of 3 experiments in neutrophils from 2 donors. Means ± SEMs. **P < .01; ***P < .001; ****P < .0001; by 2-way ANOVA (A-B). Max, maximum; Min, minimum.

Figure 5.

CD177 ligation impairs integrin internalization. (A) Peripheral blood neutrophils were incubated with Filipin III (inhibitor of calveolin-mediated endocytosis, 3 µg/mL), Dynasore (inhibitor of dynamin, required for clathrin or calveolin-mediated endocytosis, 80 µM), AEBSF (pan-serine protease inhibitor, 10 µM), or corresponding vehicles and allowed to migrate across a 3-µm pore transwell toward LTB₄ 100 nM × 2 h. Effect on migration is expressed as a proportion of corresponding vehicle control. Inhibition of endocytosis but not proteolytic cleavage impairs migration. (B) Neutrophils were preincubated with αCD177 or isotype 10 µg/mL RT for 10 min and then added to a transwell apparatus with a 0.4-µm pore membrane that was impermeable to neutrophil transit. After 2 h of attraction toward LTB₄ 100nM in the bottom chamber, attached neutrophils were liberated from the membrane using EDTA and assessed for CD11b expression. (C) Neutrophils exposed to isotype immunoglobulin G (IgG) or αCD177 10 µg/mL for 15 min at RT were allowed to adhere to 6-well plates and then incubated for with anti-CD11b-phycoerythrin (1 µg/mL, 30 min) at 4°C. After washing, cells were incubated at 37°C in prewarmed media containing 100 nM LTB₄, as is indicated, acid-washed to strip off the surface signal, and then detached using EDTA for analysis of internalized fluorescence by flow cytometry. (D) Neutrophils exposed to isotype IgG, αCD177 10 µg/mL, or MEM148 for 10 min at RT were incubated with complement-opsonized 2-µm fluorescent latex beads for 1 h at 37°C, followed by washing in PBS 5-mM EDTA to remove adherent beads. Internalized fluorescence was then analyzed by flow cytometry. Each experiment is representative of at least 3 replicates. Means ± SEMs. *P < .05; **P < .01; ***P < .001; ****P < .0001; by unpaired t test (A-B), 2-way ANOVA (C) or 1-way ANOVA (D).

Figure 6.

CD177-mediated ERK activation impairs chemoreceptor signaling. (A) Neutrophils were pretreated with MEK inhibitor U0126 (1 µM, 15 min), exposed to αCD177, and then allowed to migrate through a 3-µm pore transwell toward LTB₄ for 2 h. ERK inhibition partially rescued migration from CD177 ligation-mediated blockade. (B) Neutrophils were preincubated with αCD177 or isotype (10 µg/mL, 10 min RT) and allowed to migrate across 3- µm transwells for 2 h in response to increasing doses of LTB₄. (C) Neutrophils were loaded with the Ca²⁺-sensitive dye Fluo4 and stimulated with LTB₄ ± preincubation with U0126 (1 µM, 15 min), demonstrating suppression of Ca²⁺ flux by CD177 ligation. Each experiment is representative of at least 2 replicates. Means ± SEMs. *P < .05; **P < .01; ***P < .001; ****P < .0001; by 1-way ANOVA (A) or 2-way ANOVA (B-C).