Human neutrophils are prominent white blood cells that play an important role in defense against microbial infection. Intense activation by bacterial or inflammatory stimuli trigger neutrophil degranulation, releasing a range of antibacterial factors which can exert collateral tissue damage. Degranulation involves granule translocation to the plasma membrane followed by exocytosis, a tightly regulated process (33
). Excessive degranulation from neutrophils is a central feature in numerous inflammatory disorders such as severe asthma, emphysema, and rheumatoid arthritis (43
Neutrophils contain four different granule subtypes: primary (azurophilic), secondary (specific)and tertiary (gelatinase) granules, as well as secretory vesicles. The signaling pathways that control exocytosis of granules appear to be unique to each subtype. Granules are released in a hierarchical fashion in response to secretagogues by first releasing secretory vesicles, followed by tertiary, secondary, and finally primary granules (42
). Granule subtypes contain overlapping as well as unique luminal contents. Primary granules contain some of the most potent cytolytic enzymes that aid in digestion of pathogens, such as elastase and myeloperoxidase (MPO), which also significantly contribute to host tissue damage (23
), which emphasizes the importance of studying the regulation of their exocytosis mechanism.
Rab and Rho GTPases have been shown to regulate exocytosis in a variety of secretory cells including mast cells (5
), cytotoxic T lymphocytes (3
) and eosinophils (31
). Recent studies have defined the need for these two classes of small monomeric GTPases in regulating exocytosis of primary granules in neutrophils. Both Rab27a- and Rac2-deficient mice show impaired secretion of the primary granule enzyme MPO (1
). Rab27a, via its effector protein JFC1/Slp1, may act to discriminate between primary granules destined for exocytosis from those which preferentially fuse intracellularly with phagosomes (37
). The precise role that Rac2 plays in exocytosis remains unclear. In many cell types, Rho GTPases such as Rac are known to be key regulators of cytoskeletal remodeling. This was demonstrated in their ability to activate cytoskeletal remodeling and contribute to cell motility and chemotaxis (39
). Importantly, neutrophils deficient in Rac2 have defects in filamentous (F-) actin assembly which prevents cell migration, as distinct from Rac1 deficiency leading to inhibition of cell spreading (14
). Rac2 is also the predominant Rac protein expressed in neutrophils, and is the main GTPase required for activation of the superoxide-generating NADPH oxidase complex (14
Myeloid cells contain an F-actin-rich cortical region that is proposed to act as a barrier against granule docking and fusion at the plasma membrane. A proteomic analysis of neutrophil granule subtypes revealed that actin associates with all granule populations (28
). Indeed, exocytosis of all granules is enhanced by pre-incubation of neutrophils with the actin depolymerization drug, cytochalasin B, which favors the actin-barrier hypothesis (7
). However, other studies have shown that actin depolymerization inhibits exocytosis, suggesting that F-actin formation instead facilitates exocytosis (13
). This is plausible since neutrophil chemotaxis is triggered by polarized F-actin assembly, which could similarly drive polarized mobilization of granules on this actin network (18
). Therefore, a role for both actin depolymerization and polymerization during exocytosis is feasible.
In this study, we propose that Rac-mediated actin polymerization is necessary for directing granules in the cell cytoplasm to the plasma membrane, while actin depolymerization must occur concurrently at the cell cortex to allow exocytosis. We found that drugs which promote actin depolymerization stimulated receptor-mediated exocytosis at low dosage. Stabilization of F-actin specifically inhibited primary granule exocytosis, but had little effect on secondary granule exocytosis. Microscopic analyses of neutrophils stimulated with the actin drug, cytochalasin B together with f-Met-Leu-Phe, showed that cortical actin was remodelled to a polarized state with primary granule marker co-localization. The actin drugs, latrunculin and jasplakinolide affected both granule distribution and the polarization of actin remodelling, however, only jasplakinolide blocked exocytosis. A recently discovered small molecule inhibitor of Rac (15
) was also found to block actin remodelling and primary granule exocytosis. Surface exposure of the primary granule membrane marker CD63 was used to further quantify the effects of actin and Rac-directed drugs. Our findings suggest that Rac-mediated F-actin formation is necessary for primary granule movement to the cell membrane, while concurrent actin depolymerization at the cell cortex stimulates granule exocytosis in general.