Rab27a is a key component of the secretory machinery of azurophilic granules in granulocytes

Abstract

Neutrophils kill micro-organisms using microbicidal products that they release into the phagosome or into the extracellular space. The secretory machinery utilized by neutrophils is poorly characterized. We show that the small GTPase Rab27a is an essential component of the secretory machinery of azurophilic granules in granulocytes. Rab27a-deficient mice have impaired secretion of MPO (myeloperoxidase) into the plasma in response to lipopolysaccharide. Cell fractionation analysis revealed that Rab27a and the Rab27a effector protein JFC1/Slp1 (synaptotagmin-like protein 1) are distributed principally in the low-density fraction containing a minor population of MPO-containing granules. By immunofluorescence microscopy, we detected Rab27a and JFC1/Slp1 in a minor subpopulation of MPO-containing granules. Interference with the JFC1/Slp1-Rab27a secretory machinery impaired secretion of MPO in permeabilized neutrophils. The expression of Rab27a was dramatically increased when promyelocytic HL-60 cells were differentiated into granulocytes but not when they were differentiated into monocytes. Down-regulation of Rab27a in HL-60 cells by RNA interference did not affect JFC1/Slp1 expression but significantly decreased the secretion of MPO. Neither Rab27a nor JFC1/Slp1 was integrated into the phagolysosome membrane during phagocytosis. Neutrophils from Rab27a-deficient mice efficiently phagocytose zymosan opsonized particles and deliver MPO to the phagosome. We conclude that Rab27a and JFC1/Slp1 permit MPO release into the surrounding milieu and constitute key components of the secretory machinery of azurophilic granules in granulocytes. Our results suggest that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

“…Intracellular ROS Production and Phagosomal Maturation Are Normal in RAB27A KO Neutrophils-RAB27A and its effector MUNC13-4 are master regulators of exocytosis in neutrophils (26,27,29,30). Furthermore, similar to that shown in this work for MUNC13-4, RAB27A also plays an important role in the regulation of the neutrophil extracellular oxidative response (28).…”

“…Human neutrophils were isolated from normal donor's blood by Ficoll density centrifugation as described previously (40). Cell fractionation assays using sucrose gradients were performed exactly as depicted before (26).…”

“…with the phagosomal membrane in neutrophils (26,28). Here, we investigated a possible role of RAB27A in the phagosomal oxidative response and in the regulation of phagosomal maturation induced by P. aeruginosa.…”

Munc13-4 Restricts Motility of Rab27a-expressing Vesicles to Facilitate Lipopolysaccharide-induced Priming of Exocytosis in Neutrophils

“…Other studies suggest a possible combined role for Munc13-4 and Rab27a during lytic granule maturation (16). In neutrophils, both Rab27a and Munc13-4 regulate the exocytosis of secretory organelles (3,7,17), although whether they jointly or independently regulate transport toward the plasma membrane (PM), tethering, docking, or fusion is currently unknown and needs further characterization.…”

“…The amplification of the neutrophil secretory process by LPS plays a very important role during the neutrophil response to Gram-negative bacterial infections, in which neutrophils are exposed to multiple pathogen-associated molecular patterns and to endogenous inflammatory cytokines (5,6). Although the molecular mechanisms that mediate LPS-induced neutrophil priming and the molecular components involved in LPS-induced exocytosis of azurophilic granules are currently unknown, our previous studies showing that mice deficient for the small GTPase Rab27a have impaired secretion of myeloperoxidase (MPO) 2 in response to LPS in vivo suggest that Rab27a plays a significant role in this process (7).…”