Rab27a and Rab27b regulate neutrophil azurophilic granule exocytosis and NADPH oxidase activity by independent mechanisms

Abstract

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Figure 1. Rab27b expression is upregulated in Rab27a deficient neutrophils

A, Western blot analysis of the expression of Rab27a and Rab27b in human neutrophils. B, Immunofluorescence analysis of the subcellular distribution of Rab27b in wild type murine neutrophils. Scale bar = 5 μm. The data depicted in A and B are representative of at least three similar experiments. C, Western blot analysis of the expression of Rab27b in ashen (Rab27a^ash/ash) or wild type (WT) neutrophils from the BL6 mouse strain. Thirty five μg of protein from the indicated neutrophil lysates were loaded in each lane. C′, The bands were quantified by densitometry analysis. The data represent the mean ± SD from two independent experiments. D, Western blot analysis of the expression of Rab27a in Rab27b KO or wild type (WT) neutrophils from the BL6 mouse strain was performed as described above. In this assay, we used the monoclonal antibody 4B12 that specifically detects Rab27a but does not react with Rab27b (30). Quantification by densitometry is shown in D′.

Figure 2. Subcellular distribution of Rab27a and Rab27b in neutrophils

A, Western blot analysis of the distribution of Rab27b in human neutrophils. The cells were lysed by nitrogen cavitation and organelles were separated using a two-layer Percoll gradient. Granule markers in the various fractions were identified by Western blot and quantified by densitometry. B, Immunofluorescence staining of endogenous Rab27b (green) and myeloperoxidase (red) in murine neutrophils. B′ shows a different layer from the z-series obtained from the upper right cell showed in B and the magnification of the selected area indicating partial colocalization of Rab27b and MPO (arrow). Scale bar = 2.5 μm. C, Immunofluorescence analysis of the distribution of Rab27a in wild type (upper panels) or Rab27b KO neutrophils (lower panels). Arrows indicate areas of colocalization of Rab27a with MPO. Arrowheads indicate granules that were positive for one marker but not for the other. Scale bar = 5 μm.

Figure 3. Neutrophils deficient in GTPases from the Rab27 family have impaired azurophilic granule secretion

A and B, Peripheral neutrophils from Rab27a^ash/ash, Rab27b knockout (KO), Rab27a/b double knockout (DoKo) or wild type (WT) control mice were resuspended in serum-free RPMI, stimulated with 0.1 μg/ml PMA (+) or left untreated (−) and incubated for 1h at 37 °C. MPO release was measured using a murine MPO-specific enzyme-linked immunosorbent assay. Results are mean ± SEM of data from 6 to 10 mice.*, p<0.03. #, statistical outliers.

Figure 4. No gross morphological abnormalities are present in the granules from Rab27a^ash/ash, Rab27b knockout or Rab27a/b double knockout neutrophils

A, Peripheral neutrophils from Rab27a^ash/ash, Rab27b knockout (KO) and Rab27a/b double knockout (DoKo) or wild type (WT) mice were seeded on poly-L-lysine-coated 1.5 coverglass, fixed, stained for the visualization of endogenous myeloperoxidase and analyzed by immunofluorescence confocal microscopy. A representative image of each type is shown in the upper panels. Magnifications of the indicated areas are shown in the lower panels. At least 15 cells of each type were analyzed. Scale bar = 5 μm. B, Transmission electron microscopy analysis of Rab27a^ash/ash, Rab27b KO and Rab27a/b double knockout neutrophils. The panels on the right are magnifications of the corresponding areas selected in the left panels. Similar results were observed in Rab27a^ash/ash neutrophils from the C3H strain (Supplementary Fig. S4).

Figure 5. Rab27-deficient neutrophils have a decreased number of azurophilic granules in proximity to the plasma membrane

Neutrophils from Rab27a^ash/ash (A) or Rab27b knockout (KO) and Rab27a/b double knockout (DoKo) (B) mice and their appropriate wild type (WT) controls were seeded on coverglass and the distribution of endogenous myeloperoxidase-containing granules was analyzed by total internal reflection fluorescence microscopy (TIRFM). Representative images of each type are shown in the upper panels. The number of azurophilic granules identified in the TIRFM zone (calibrated at 108 nm from the coverslip) per adherent membrane area unit for wild type or Rab27-deficient neutrophils is shown in the lower panels. The data depicted in A′ and B′ are represented as the mean ± SEM. Statistical analysis was performed using the non-parametric Mann-Whitney test.***, p<0.01; **, p<0.02*, p<0.05. Twenty to 40 cells from each group were analyzed. Rab27a^ash/ash and wild type neutrophils from the C3H mouse strain were used in A and neutrophils from BL6 mice were used in B. Scale bar = 5 μm

Figure 6. Rab27a and Rab27b are not essential components of the secretory machinery that regulates mobilization of CD11b or LAMP-2-containing organelles in neutrophils

Exocytosis of readily mobilizable organelles was analyzed by following the surface expression of CD11b in neutrophils from wild type (WT) and ashen mice (Rab27a^ash/ash) (A) or Rab27b knockout (KO) and Rab27a/b double knockout (DoKo) mice (B). Neutrophils were stimulated with 0.1 μg/ml PMA (+) or left untreated (−), double labeled for the granulocyte marker Gr-1 and for the surface expression of the β2 integrin subunit CD11b. The samples were analyzed by flow cytometry. Each symbol represents the result obtained using an individual mouse. No significant differences were detected between the populations analyzed (unpaired Student’s t-test). C and D, Wild type (WT), Rab27a ^ash/ash, Rab27b KO or Rab27a/b double KO neutrophils were stimulated with 0.1 μg/ml PMA (+) or left untreated (−), double labeled for the granulocyte marker Gr-1 and for the surface expression of the membrane protein LAMP-2. The samples were analyzed by flow cytometry. Each symbol represents the value obtained using an individual mouse. No significant differences were observed between stimulated Rab27-deficient neutrophils and wild type cells (unpaired Student’s t-test). Neutrophils from the C3H and the BL6 mouse strains were used in A and C, and B and D, respectively.

Figure 7. Global ROS production is impaired in Rab27a^ash/ash and Rab27a/b double knockout mice

A, Neutrophils isolated from ashen (Rab27a^ash/ash) or wild type mice (C57BL/6 background) were stimulated with PMA (0.1μg/ml) and the luminol-dependent chemiluminescence was continuously monitored for 25 min at 37°C. The results show the mean ± SEM of samples from three individual mice. Black and grey squares correspond to stimulated WT or Rab27a^ash/ash neutrophils, respectively. Circles correspond to unstimulated cells. B, The integrated chemiluminescence response of WT and Rab27a^ash/ash neutrophils is expressed as relative light units (RLU). C, The results show the chemiluminescence response of neutrophils from WT or Rab27a^ash/ash (C3H) mice. D, Chemiluminescence response of cells from WT, Rab27b knockout (KO) or Rab27a/b double knockout (DoKo) from C57BL/6 mice. Results are the mean ± SEM from data obtained using samples from three individual mice for each colony. *, p< 0.01. E, Luminol-dependent chemiluminescence of wild type or ashen neutrophils after incubation with LPS (10 ng/ml) for 30 min and activation with 1μM fMLP (▲ and +) or untreated cells (● and ▼) is shown.

Figure 8. Plasma membrane-associated NADPH oxidase activity is impaired in neutrophils from Rab27-deficient mice

Peripheral neutrophils from ashen (Rab27a^ash/ash), Rab27b knockout (KO), Rab27a/b double knockout (DoKo) or wild type (WT) mice were incubated in the presence of 10 μg/ml cytochalasin D for 10 min and stimulated with 10 μM fMLP or left untreated. NADPH oxidase-dependent extracellular chemiluminescence was measured by inhibiting endogenous MPO (sodium azide) and using cell-impermeant isoluminol. Wild type neutrophil kinetics are shown with black squares (■) and those from Rab27-deficient cells using triangles (▲). Inverted triangles in the left panel (▼) indicate the negligible chemiluminescence response obtained from wild type or Rab27a-deficient neutrophils in the presence of superoxide dismutase. Data is presented as mean ± SEM.

Figure 9. Rab27-deficient neutrophils are characterized by normal intraphagosomal ROS production

A, Murine neutrophils were isolated from peripheral blood obtained by cardiac puncture. The cells were stimulated with opsonized heat-killed Listeria monocytogenes (HKLM) and luminol-dependent chemiluminescence was continuously monitored at 37°C. Results represent the mean ± SEM (n=3). B, Measurement of ROS release in the phagovacuoles of immune complex-stimulated wild type or Rab27a^ash/ash neutrophils using Fc OxyBurst. The base line represents the dichlorodihydrofluorescein (DCF) fluorescence obtained in the absences of neutrophils (◆). The inset shows the mean ± SEM of three experiments performed with cells from three individual mice.