Figure 3.
GEF-H1 is specifically required for shear stress–induced neutrophil migration. (A) Fluorescently labeled GEF-H1+/+ and GEF-H1−/− neutrophils (5 × 105) were plated on HUVECs in triplicate and treated with fMLP or DMSO. Nonadhered cells were removed, and mean fluorescence intensity (MFI) per well was determined. MFI of labeled neutrophils is shown as a positive control. Shown are means ± SD. (B) Fluorescently labeled neutrophils were plated in triplicate on confluent HUVECs in transwell filters. Cells were allowed to transmigrate for 1 h toward 1 µM fMLP in the wells below each filter. Transmigrated cells were imaged at 20× by widefield microscopy, and automated cell counting was performed using CellProfiler software. Three independent experiments were performed. Results are mean ± SD from a representative experiment. FOV, field of view. (C) GEF-H1+/+ and GEF-H1−/− neutrophils were resuspended in a collagen matrix, and migration toward a gradient of C5a was monitored in triplicate wells. Representative data from one experiment are shown. Distance from the origin is indicated on x and y axes in µm. The direction of the chemotactic gradient is indicated. (D–L) GEF-H1+/+ and GEF-H1−/− neutrophils were stimulated with fMLP and plated on ICAM-1–coated surfaces (D–F), TNF-activated HUVECs (G–I), or TNF-activated C166 mouse endothelial cells (J–L). Cell migration in the presence or absence of 4 dynes/cm2 constant shear stress was determined from at least 60 cells per experiment. Mean percentage of migrating cells (E, H, and K) and the mean cell displacement (F, I, and L) ± SEM are indicated and were determined from three independent experiments. Moving cells were defined as those that migrated a minimum distance of 15 µm. Neutrophil positions relative to the origin are indicated from a representative experiment (D, G, and J). Distance from the origin is indicated on the x and y axes in micrometers. Arrows indicate direction of shear.