Rab27a-mediated protease release regulates neutrophil recruitment by allowing uropod detachment

Abstract

Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.

Fig. 1.

Rab27a promotes migration of neutrophils in vivo and in vitro. (A) Leukocytes recruited to the bronchoalveolar space of WT and Rab27a KO mice treated intranasally with MIP-2; cells were stained with antibody against Ly6G and analysed by flow cytometry. Numbers indicate Ly6G-positive cells as a percentage of the total cell number. (B) Neutrophils recruited to the bronchalveloar space of WT mice and Rab27a KO mice treated with MIP-2. Data represent the mean±s.e.m. (n=5–7 for each treatment and genotype of mice). Transwell migration of WT versus Rab27a KO BM-PMN towards (C) MIP-2 or (D) LTB4 concentrations after 30 minutes, and (E) Rab27a-knockdown (siRab27) versus non-targeting (NT) control transfected differentiated HL-60 cells towards fMLP after 60 minutes. Data represent the mean from three independent experiments (n=3 for each treatment and genotype of mice BM-PMN and knockdown HL-60 cells). *P≤0.05, ***P≤0.001 [Student's t-test (B) and two-way ANOVA (C–E)].

Fig. 2.

Rab27a regulates neutrophil migration by controlling uropod release. (A) Tracks of WT and Rab27a KO BM-PMN migration measured from stacks of images taken every 30 seconds for 30 minutes in a Zigmond chamber with medium alone added to the left well and 10 nM MIP-2 added to the right well. Black and red tracks indicate cells with net leftwards or rightwards movement respectively. Migration distance (B), velocity (C) x- and y-migration index (D) and uropod lifetime (E) of WT and Rab27a KO BM-PMN. Data represent the mean±s.e.m. from 25 random cells per experiment from four independent experiments (n=4 for each genotype of BM-PMNs). Still images of WT (F) and Rab27a KO (G) BM-PMN migration over a course of 180 seconds (from supplementary material Movies 2 and 4). Scale bars: 10 μm. *P≤0.05, ***P≤0.001 (Student's t-test).

Fig. 3.

Rab27a localises to the uropod in migrating neutrophils, and Rab27a-dependent secretion of serine proteases promotes neutrophil migration. EGFP–Rab27a BM-PMNs from transgenic EGFP–Rab27a mice were plated onto coverslips for 20 minutes at 37°C, and fixed with 4% PFA (A) or placed into a MIP-2 chemokine gradient in a Zigmond chamber for 5 minutes and then fixed (B). (C) Detail of cell rear from B. Scale bars: 5 μm. (D) Quantification of EGFP–Rab27a localisation close to (<4 μm) and far from (>4 μm) the cell rear in BM-PMNs migrating in a MIP-2 gradient. ***P≤0.001 (Student's t-test). Data are representative of three experiments (n=30 cells). (E) Flow cytometry analysis of the increase in Cd11b surface expression in WT or Rab27a KO BM-PMN after stimulation with 1 nM MIP-2 for the indicated times. Transwell chemotaxis of WT and Rab27a KO BM-PMN towards 10 nM MIP-2 for 30 minutes in the presence of indicated concentrations of (F) AEBSF and (G) elastatinal. Data represent the mean±s.e.m. from four (D) or three (F,G) independent experiments (n=4 or n=3 for each genotype of mice BM-PMN) *P≤0.05, **P≤0.01 (Student's t-test comparison of WT and Rab27a KO BM-PMN); ^#P≤0.05, ^###P≤0.001 (Student's t-test comparison of untreated and protease inhibitor treated BM-PMN). n.s., not statistically significant.