UC San Diego

Neutrophil extravasation, a critical component in 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 migration in 3-dimensional (3D) matrices is currently unclear. To address this question, we employ a novel transwell (TW)-based leukocyte treatment assay in which human umbilical vascular endothelial cells (HUVEC) are cultured on a fibronectin-treated TW 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 for further experimental use. TW treated cells are subsequently seeded in a well-characterized, custom-built chemotaxis device containing a collagen gel matrix to observe 3D migration in the presence or absence of an externally imposed chemotactic gradient. Analysis of nearly 50,000 dHL-60 and primary human neutrophil (PMN) trajectories, leveraging sequential and variational Bayesian inference algorithms, demonstrate that increases in neutrophil activity and concomitant loss of migratory persistence after TEM are modulated respectively and quasi-independently by HUVEC interaction and junctional trafficking. Moreover, neutrophils exhibit two distinct migratory subpopulations: one characterized by a fast, directed, chemotactically efficient high motility phenotype, and one characterized by a slower, tortuous, almost chemotactically agnostic low motility phenotype. These migratory subpopulations are conserved across treatment conditions and recapitulate the behavior of PMNs. The relative prevalence of these subpopulations controls the population level changes in neutrophil migration observed following extravasation and are modulated by TEM-mediated changes in neutrophil expression of GRK2, a kinase closely associated with chemokine receptor trafficking, migrational persistence, effector functionality, and collective neutrophil response to invading pathogens. In conjunction with observed population level increases in neutrophil effector functionality after TEM, these results suggest an important role for the low motility migration phenotype in promoting efficient pathogen clearance and motivate future investigation into the connection between neutrophil motility phenotype and effector functionality.