Kindlin‐3 organizes a ring of clustered high affinity β₂ integrins during human neutrophil spreading under flow

Abstract

Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high‐resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM‐1 and P‐selectin under flow. A homogenous binding assay using the conformation‐reporter antibodies mAb24 (reporting high‐affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk‐mediated integrin outside‐in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin‐3‐GFP fusion proteins, a ring of kindlin‐3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin‐3. Support or Funding Information This research was supported by funding from the National Institutes of Health, USA (NIH, HL078784 and R01HL145454), WSA postdoctoral fellowships and a Career Development Award from the American Heart Association, USA (AHA, 16POST31160014, 19POST34450228, and 18CDA34110426). Integrin activation on a spread neutrophil footprint. Red is the mAb KIM127, which reports integrin extension; Green is the mAb24, which reports integrin high‐affinity; Blue is conformational non‐specific aLb2 integrins. Kindlin‐3‐GFP molecules formed a ring after the spreading of a neutrophil‐like HL60 cell.