Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

Dept. of Cell Biology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7.
AJP Cell Physiology (Impact Factor: 3.78). 10/2008; 295(5):C1354-65. DOI: 10.1152/ajpcell.00239.2008
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The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

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    • "Neutrophil morphology as observed by electron microscopy. Neutrophils were isolated from human peripheral blood, and processed for electron microscopy after incubation for 15 min at 37°C with vehicle (A,B) or 10 μM cytochalasin B and 5 μM fMLF (C,D) as described in Ref. (22). (A,B) The cytosol of a resting cell is filled with vesicles, with primary granules (P) staining intensely dark with DAB, while secondary (S) and tertiary (T) granules show more translucent staining. "
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    • "Rac2 switches from a resting, inactive GDP-bound state to an active GTP-bound state in response to receptor stimulation. The GTP-bound form of Rac2 is essential for activation of the superoxide-generating NADPH oxidase complex in neutrophils [10] and is required for exocytosis of primary azurophilic granules through polymerization of F-actin filaments required for propelling granules to the cell membrane [9,12]. Macrophages utilize Rac2, in addition to the ubiquitously expressed homolog Rac1, for maximal phagocytosis and oxidant production [11]. "
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    • "The down-regulation of cytoskeleton-related proteins, including actin isoforms and coactosin-like protein, seen in the PMN from the COPD and SM-exposed hosts may reflect changes that may have occurred with regard to cell re-modeling processes in the lungs (Sheppard et al., 2005). Coactosin-like protein, which belongs to the actin-depolymerizing factor family of actin-binding proteins, interacts with 5-lipoxygenase and filamentous actin; because extensive compositional re-organization is exhibited in PMN during initial stages of phenotypic conversion (Stie & Jesaitis, 2007), upon PMN activation, the assembly of polarized F-actin promotes exocytosis (Mitchell et al., 2008). Actin (de)polymerizetion is also important for the process of transendothelial migration, the means by which leukocytes exit the blood and enter tissues – a key step in inflammatory processes in organs like the lungs. "
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