Neutrophils exhibit distinct migration phenotypes that are modulated by transendothelial migration

Neutrophil extravasation, a critical component of the innate immune response, comprises two sequential processes: transendothelial migration (TEM) and interstitial migration. TEM requires intimate physical contact between leukocytes and vascular endothelial cells, triggering a cascade of biochemical and biomechanical interactions whose effect on leukocyte interstitial migration is currently unclear. To address this question, we culture human umbilical vascular endothelial cells on a fibronectin-treated transwell insert, mimicking the endothelium and basement membrane in vitro. Neutrophil-like differentiated HL60 (dHL-60) cells are loaded in the upper chamber of the insert, and cells that cross the membrane-supported monolayer are collected from the lower chamber. These cells are then seeded in a custom-built chemotaxis device containing a collagen gel matrix to study their 3D migration in the presence or absence of an fMLP gradient. dHL-60 cells that do not pass through the transwell insert serve as controls. Data collected from over 37,000 dHL-60 trajectories show two distinct migratory phenotypes conserved across treatment conditions: the high motility subpopulation is characterized by faster, more persistent cells undergoing fewer turning events; while the mid-motility subpopulation demonstrates lower speeds, increased incidence of large angle and 180o turns, and loitering periods in localized regions of the matrix. Changes in the relative proportion of subpopulations between conditions are correlated with changes in GRK2 expression levels. Our results suggest TEM may modulate GRK2 expression to prime neutrophils for a migration phenotype better suited for spatial exploration than highly persistent chemotaxis. In conjunction with previously presented TEM-induced changes in neutrophil ICAM-1 expression, associated with increased phagocytosis and neutrophil effector functions, this suggests a functional purpose underlying the shift in primary migration phenotype after TEM. These observations provide novel insight into the biophysical impacts of TEM that prime neutrophils to conduct sentinel functions.