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. Author manuscript; available in PMC: 2013 Feb 26.
Published in final edited form as: J Immunol. 2011 Jun 3;187(1):391–400. doi: 10.4049/jimmunol.1003112

Granule Exocytosis Contributes to Priming and Activation of the Human Neutrophil Respiratory Burst

Silvia M Uriarte *, Madhavi J Rane *,†, Gregory C Luerman †, Michelle T Barati *, Richard A Ward *, William M Nauseef ‡,§,, Kenneth R McLeish *,†,‖
PMCID: PMC3582343  NIHMSID: NIHMS438324  PMID: 21642540

Abstract

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT–SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT–SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT–SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT–SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT–SNAP-23 inhibited the increase in plasma membrane expression of gp91phox in TNF-α–primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.

Polymorphonuclear leukocytes, or neutrophils, are professional phagocytes that play a central role in the killing of invading microorganisms. Optimal microbial killing requires the production of toxic reactive oxygen species (ROS), generated by the multicomponent enzyme NADPH oxidase, and oxygen-independent events; in particular, the release of proteolytic enzymes, defensins, myeloperoxidase, and bactericidal peptides from intracellular granules into phagosomes (1, 2). Granule contents and ROS function both independently and cooperatively within phagosomes to enhance microbicidal activity (2, 3). In the absence of microbial phagocytosis, activated neutrophils release these products into the extracellular space, resulting in damage to normal tissue (4).

Not only do granule proteins synergize with oxidants for optimal microbial killing, but also they interface with ROS in several other ways. At low concentrations, products of both systems participate in intracellular signaling (5–7). ROS inactivate protease inhibitors, such as secretory leukocyte protease inhibitor, thereby enhancing protease activity (8). Neutrophil granule contents could be involved in regulating NADPH oxidase activity. Myeloperoxidase-deficient neutrophils, as well as normal neutrophils treated with a peroxidase inhibitor, exhibit augmented NADPH oxidase activity (9–11), suggesting that termination of oxidase activity might be myeloperoxidase mediated. Degranulation has been temporally linked to the augmented oxidase activity seen in neutrophils exposed to a variety of agents in a process termed priming (12–16). Priming agents induce exocytosis of neutrophil granules leading to fusion of granule membranes with the plasma membrane, resulting in increased expression of functionally important receptors, signaling molecules, and membrane components of the NADPH oxidase (17–19). The contribution of neutrophil granule exocytosis per se to priming of the respiratory burst has not been demonstrated, as it has not been possible to selectively inhibit exocytosis experimentally.

SNARE proteins play a central role in intracellular membrane trafficking events by mediating fusion of membranes of different cellular compartments (20, 21). SNARE proteins, including synaptosome-associated protein-23 (SNAP-23), syntaxin-4, syntaxin-6, vesicle-associated membrane protein (VAMP)-1, VAMP-2, and VAMP-7, have been identified on neutrophil granules and plasma membranes and have been functionally implicated in neutrophil exocytosis (22–27). Based on the role of SNARE proteins in neutrophil granule exocytosis, we reasoned that selective inhibition of exocytosis could be accomplished by introducing the SNARE interaction domain from one or more of these SNARE proteins into neutrophils, thereby blocking the interaction among SNARE partners. To that end, we used fusion proteins containing the N-terminal SNARE domain of SNAP-23 and the HIV transactivator of transcription (TAT) protein transduction peptide (28) to block exocytosis selectively and to examine the interaction between exocytosis and priming and activation of the neutrophil respiratory burst.

Materials and Methods

Neutrophil isolation

Neutrophils were isolated from the blood of healthy donors using plasma-Percoll gradients as previously described (29, 30). Microscopic evaluation of isolated cells showed that >95% of cells were neutrophils. Trypan blue exclusion indicated that >97% of cells were viable. The Institutional Review Boards of the University of Louisville and the University of Iowa approved the use of human donors at their respective institutions.

TAT–SNAP-23 and TAT–Control fusion proteins

Two sets of primers incorporating an NcoI restriction site on the forward primer (5′-CTTGAGTTTTGATTCACCATGGATAAT-3′) and a HindIII restriction site on the reverse primer (5′-GAAGTGAATAAGCTTTAAAGAAGAACA-3′) were designed to generate the cDNA sequence for the N-terminal 78 residues of SNAP-23 from cDNA generated from neutrophil RNA. Verification of the PCR product was accomplished by DNA sequencing. The PCR product of the N terminus of SNAP-23 (TAT–SNAP-23, Mr 19 kDa) and the pTAT-vector (TAT–Control, Mr 15 kDa) were digested with NcoI and HindIII, ligated, and used for the transformation of Escherichia coli DH5α competent cells (Invitrogen, Carlsbad, CA). Colonies were selected and the DNA was extracted using a DNA Maxi Prep from Marlingen Biosciences (Rockville, MD). E. coli BL21-AI cells (Invitrogen) were transformed to overexpress the recombinant TAT fusion proteins. Purification of TAT–SNAP-23 and TAT–Control was performed by sonication and lysis of the bacterial pellet with a denaturing buffer (7 M urea, 0.5 M NaCl, 50 mM NaPO4 [pH 8], 20 mM imidazole), followed by protein separation from the supernatant by Ni-NTA beads (Invitrogen). Protein eluted from the beads was dialyzed against 10% glycerol, 0.01% Triton X-100 in PBS, pH 7.4, and stored at −80°C until use.

Exocytosis

Exocytosis was stimulated by fMLF (Sigma, St. Louis, MO), TNF-α (R&D Systems, Minneapolis, MN), or platelet-activating factor (PAF; Sigma). Exocytosis of secretory vesicles, specific granules, azurophil granules, and gp91phox was determined by measuring the increase in plasma membrane expression of FITC-conjugated monoclonal anti-CD35 (clone E11; Pharmingen, San Diego, CA), FITC-conjugated monoclonal anti-CD66b (clone CLB-B13.9; Accurate Chemical, Westbury, NY), FITC-conjugated anti-CD63 (clone AHN16.1/46-4-5; Ancell Corporation, Bayport, MN), and FITC-conjugated monoclonal anti-gp91phox (clone 7D5; MBL, Woburn, MA), respectively, on 4 × 106/ml neutrophils using flow cytometry as previously described (29, 12). Exocytosis of gelatinase granules was determined by ELISA for matrix metalloproteinase-9 (R&D Systems) as previously described (29). The data are represented as mean ± SEM increase in membrane expression. An Epics Profile II (Coulter, Hialeah, FL) flow cytometer and Expo 32 XL4 software were used to analyze the samples.

GST pulldown

Recombinant GST–VAMP-2 (2 µg), His–syntaxin-4 (2 µg), TAT–SNAP-23 (2 µg), and/or TAT–Control (2 µg) were incubated with glutathione-coupled Sepharose (2 µl) in PBS containing 0.025% (v/v) Triton X-100 for 2 h at 4°C with constant rotation. After incubation, glutathione beads were washed four times in PBS containing 0.1% (v/v) Triton X-100. Proteins bound to the beads were separated by 10% SDS-PAGE, and immunoblot analysis with anti–histidine-tag (His-tag) (1:1000; Invitrogen), anti–hemagglutinin tag (HA-tag) (1:1000; Abcam, Cambridge, MA), or anti–VAMP-2 (1:000; Synaptic Systems, Göttingen, Germany) was performed. Protein signal was visualized by chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. His–syntaxin-4 recombinant protein (12 kDa), containing the SNARE domain of mouse syntaxin-4, was a generous gift from Dr. Chuan Hu (University of Louisville, Louisville, KY). The GST–VAMP-2 construct (pGEX4T–VAMP-2) containing the mouse cDNA for VAMP-2 was a generous gift from Dr. Jeff Sands (Emory University, Atlanta, GA).

Kinase phosphorylation

At completion of the experimental protocol, neutrophils (1 × 107 cells/ml) were pelleted by centrifugation at 2500 × g for 20 s and lysed in 100 µl ice-cold lysis buffer as previously described (31). Total cell lysate was separated by 4–12% gradient SDS-PAGE, followed by immunoblot analysis for phospho-p38 MAPK (1:1000; Cell Signaling, Danvers, MA), total p38 (1:1000; Cell Signaling), phospho-ERK1/2 (1:500; Santa Cruz, Santa Cruz, CA), and total ERK1/2 (1:500; Santa Cruz). The appropriate labeled secondary Abs were used at 1:5000 (Alexa Fluor 680 and Alexa Fluor 700; Invitrogen). Protein signal was visualized by immunofluorescence using a Li-Cor scanner and Odyssey 2.0 software. Densitometry was performed on images captured from the Li-Cor scanner using ImageJ software.

Phagocytosis and respiratory burst activity

To measure H2O2 production in phagosomes, neutrophils (4 × 106 cells/ml) were incubated with 2′,7′-dichlorofluorescin diacetate (final concentration 0.5 mM; Molecular Probes/Invitrogen, Carlsbad, CA) for 10 min at 37°C. Fifty microliters of cell suspension was sampled before, and 10 min after, the addition of 50 µl of opsonized, propidium iodide-labeled Staphylococcus aureus (final concentration ~108 bacteria/ml), and samples were analyzed by flow cytometry (Epics Profile II; Coulter, Hialeah, FL), as previously described (12). Extracellular superoxide release was determined as ferricytochrome c reduction measured spectrophotometrically, as previously described (12). Briefly, neutrophils (4 × 106/ml) were suspended in KRPB containing calcium and magnesium and 1 mg/ml ferricytochrome c. After stimulation of O2− production, the reaction was stopped by placing the tubes on ice and pelleting the cells by centrifugation at 4°C. Superoxide production was quantified using the change in absorbance of the supernatant at 550 nm and expressed as nanomoles of O2− per 4 × 106 cells using an extinction coefficient of 2.1 × 104/M/cm.

Extracellular release of azurophil granule contents

Extracellular release of human neutrophil elastase (HNE) and myeloperoxidase (MPO) was measured continuously at 37°C for 30 min. Extracellular HNE was quantitated spectrofluorometrically as the conversion of nonfluorescent elastase substrate (MeO-Suc-Ala-Ala-Pro-Val-methylcoumarin) to fluorescent aminomethylcoumarin using a modification of a previously published method (32). The change in fluorescence was measured in a Spectramax 5 (Molecular Devices, Sunnyvale, CA), using λex 345 nm and λem 444 nm. MPO release was quantitated as NaN3-inhibitable isoluminol chemiluminescence in the presence of an exogenous supply of H2O2. The assay exploits the widely used assay for extracellular H2O2 release as peroxidase-dependent enhancement of isoluminol chemiluminescence (33). Neutrophils were stimulated in the presence of isoluminol (50 µM) and an H2O2-generating system (GGO; 1 µg/ml glucose oxidase in 5 mM glucose) in the absence or presence of 1 mM NaN3. Chemiluminescence was measured continuously at 37°C for 10 min, and MPO-dependent isoluminol chemiluminescence was defined as that which was azide-inhibitable. The amount of MPO released was calculated from a standard curve of the isoluminol–GGO system in the presence of known amounts of pure MPO (0–1 nM).

Intraphagosomal generation of HOCl

Fluorescence of intraphagosomal S. aureus (RN6390) expressing super-folded GFP (34) was monitored in a FACS using a modification of a previously published procedure (35). Data were derived from the mean fluorescence index of each sample.

TAT fusion protein uptake

Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or TAT–Control for 10 min. Cells were fixed in 3.7% para-formaldehyde for 15 min at room temperature, permeabilized with 2% saponin for 15 min at room temperature, followed by overnight incubation at 4°C with anti–HA-tag Ab (final concentration 2 µg/250 µl). Neutrophils were then washed twice with 0.02% saponin in Krebs+ followed by 60-min incubation at room temperature with secondary Ab Alexa Fluor 546 (final concentration 1 µg/250 µl; Invitrogen). Cells were imaged by a Zeiss Axiovert 100 M confocal microscope with a Zeiss Plan-Neofluar × 100/1.3 oil-immersion lens using LSM510 (version 3.2) software.

Statistical analysis

All data are expressed as mean ± SEM. Statistical analysis was performed using a one-way ANOVA with the Tukey–Kramer multiple-comparison test. The p value for each set of data is provided in the Results section and/or the figure legends. When required, a log transformation was performed to normalize the data.

Results

Characterization of TAT–SNAP-23 inhibition of neutrophil granule exocytosis

To study the contributions of exocytosis to neutrophil functional responses, we generated a fusion protein containing the 11-aa cell-penetrating peptide of the HIV TAT and the N-terminal SNARE domain of SNAP-23 (TAT–SNAP-23). The TAT sequence fused to hexa histidine tag (6 × His) and HA-tag sequences was generated to serve as a control (TAT–Control). Transduction of both TAT fusion proteins into neutrophils was confirmed by confocal microscopy (Fig. 1). Incubation of TAT–SNAP-23 or TAT–Control for 10, 60, or 240 min did not alter cell viability, as determined by trypan blue dye exclusion (data not shown). To determine the effect of incubation with TAT–SNAP-23 or TAT–Control for 60 min on constitutive neutrophil apoptosis, propidium iodide detection of DNA fragmentation and plasma membrane expression of annexin V were determined by flow cytometry (36). A small increase in DNA fragmentation from 14 ± 3% in untreated cells to 20 ± 3% in cells pretreated with either TAT–SNAP-23 or TAT–Control was observed (n = 4). The percentage of cells demonstrating annexin V binding was 8–9% in all three groups (n = 2).

FIGURE 1.

FIGURE 1

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Uptake of TAT fusion proteins by neutrophils. Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or TAT–Control for 10 min, followed by permeabilization, fixation, and overnight incubation with anti–HA-tag Ab to identify TAT fusion proteins. A and B, Confocal microscopy images illustrate fluorescence in the absence (Control) and presence of TAT–SNAP-23 (A) and TAT–Control (B) detected by anti–HA-tag. Cells were imaged by a Zeiss Axiovert 100 M confocal microscope with a Zeiss Plan-Neofluar × 100/1.3 oil-immersion lens using LSM510 (version 3.2) software.

To establish the ability of TAT–SNAP-23 to inhibit exocytosis, a concentration-inhibition experiment of fMLF-stimulated exocytosis of secretory vesicles (CD35 expression) and specific granules (CD66b expression) was performed. Optimal inhibition of exocytosis of both granule subsets occurred at 0.8 µg/ml (Supplemental Fig. 1). That concentration of TAT–SNAP-23 and TAT–Control was used in all subsequent experiments. To determine the degree of inhibition of exocytosis at this optimal concentration, neutrophils were pretreated with or without TAT–SNAP-23 or TAT–Control for 10 min at 37°C, followed by stimulation with fMLF (300 nM, for 5 min), and exocytosis of all four granule subsets was measured. Pretreatment with TAT–SNAP-23 inhibited fMLF-stimulated secretory vesicle exocytosis, measured as plasma membrane expression of CD35, by 74 ± 5% (p < 0.001, Fig. 2A). Similarly, pretreatment with TAT–SNAP-23 inhibited fMLF-stimulated specific granule exocytosis, measured as plasma membrane expression of CD66b, by 53 ± 5% (p < 0.001, Fig. 2B) and gelatinase granule exocytosis, measured as gelatinase release, by 78 ± 14% (p = 0.002, Fig. 2C). Basal expression of CD35 and CD66b and basal gelatinase release did not differ between untreated cells and cells treated with TAT–SNAP-23 and TAT–Control (Fig. 2A–C). In contrast, pretreatment with TAT–SNAP-23 or TAT–Control did not inhibit fMLF-stimulated azurophilic granule exocytosis, measured as plasma membrane expression of CD63 in the presence of latrunculin A (Fig. 2D).

FIGURE 2.

FIGURE 2

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Effect of TAT–SNAP-23 on exocytosis. Neutrophils (4 × 106/ml) were incubated with buffer, TAT–SNAP-23 (0.8 µg/ml, 10 min), or TAT–Control (0.8 µg/ml, 10 min), then treated with or without fMLF (300 nM, 5 min). For experiments examining CD63 expression, cells were pretreated with latrunculin A (1 µM) for 30 min. A, Exocytosis of secretory vesicles was determined using the plasma membrane expression of CD35. Results are presented as mean ± SEM of the mean channel of fluorescence (mcf) for 12 separate experiments. B, Exocytosis of specific granules was determined using the plasma membrane expression of CD66b. Results are presented as the mean ± SEM of the mcf intensity for 12 separate experiments. C, Exocytosis of gelatinase granules was measured as the extracellular release of gelatinase. Results are presented as mean ± SEM in nanograms gelatinase released per 1 × 106 cells from eight separate experiments. D, Exocytosis of azurophil granules was determined by the plasma membrane expression of CD63. Results are expressed as mean increase above basal expression ± SEM of the mcf intensity from four separate experiments.

The effect of TAT–SNAP-23 pretreatment on azurophil granule exocytosis was also assessed by quantifying the fMLF-stimulated (10−6 M) release of HNE and MPO in the absence or presence of dihydrocytochalasin B (DHCB), an actin disrupting drug that enhances exocytosis of this granule subset. Similar levels of HNE were released by fMLF-stimulated neutrophils pretreated with buffer alone (1738 ± 356 relative fluorescence units [RFU]), TAT–Control (3234 ± 551 RFU), or TAT–SNAP-23 (2736 ± 390 RFU) (n = 8). In the presence of DHCB, HNE released from fMLF-stimulated neutrophils was dramatically increased, but no statistically significant differences among cells pretreated with buffer alone (48,332 ± 2,598 RFU), TAT–Control (48,437 ± 2,541 RFU), or TAT–SNAP-23 (46,558 ± 2,316 RFU) were observed (n = 8). MPO released by fMLF-stimulated neutrophils did not differ among cells pretreated with buffer alone (48.8 ± 4.9 fmol), TAT–Control (77.3 ± 14.1 fmol), or TAT–SNAP-23 (70.0 ± 8.8 fmol) (n = 5). In the presence of DHCB, MPO release from fMLF-stimulated neutrophils was dramatically increased, but no statistically significant differences among cells pretreated with buffer alone (2.64 ± 0.80 nmol), TAT–Control (2.69 ± 0.60 nmol), or TAT–SNAP-23 (1.59 ± 0.70 nmol) (n = 5) were observed. TAT–SNAP-23 contains two methionine and two cysteine amino acids, residues that would be vulnerable to oxidation by hypochlorous acid (HOCl) (37), and thus may serve as a competing substrate in the MPO assay. However, washing away the extracellular TAT–SNAP-23 before stimulating neutrophils with fMLF did not alter detection of MPO (data not shown). Taken together, the data indicate that TAT–SNAP-23 inhibits exocytosis of secretory vesicles, gelatinase granules, and specific granules but has no effect on azurophil granule exocytosis.

Based on the current understanding of SNARE-mediated membrane fusion, we postulated that TAT–SNAP-23 inhibited exocytosis by binding to endogenous SNARE proteins and blocking the coiled-coil formation of four SNARE domains necessary for membrane fusion (38). To test that hypothesis, a GST pulldown assay using recombinant GST–VAMP-2, a recombinant SNARE domain of syntaxin-4, TAT–SNAP-23, and/or TAT–Control was performed (Fig. 3). TAT–SNAP-23 precipitated with the combination of GST–VAMP-2 and syntaxin-4 (Fig. 3A, lane 3) but did not bind to glutathione beads alone (lane 1) or to GST–VAMP-2 alone (lane 5). TAT–Control bound to glutathione beads alone (lane 2), but the amount of this nonspecific binding was not altered by the presence of VAMP-2 (lane 6) or VAMP-2 and syntaxin-4 (lane 4). Similar results were seen when the membrane was stripped and reprobed with anti–His-tag that recognizes TAT–SNAP-23, TAT–Control, and syntaxin-4 (Fig. 3B). These data confirm that syntaxin-4 and TAT–SNAP-23 precipitated with GST–VAMP-2 (Fig. 3A, 3B, lane 3) and that TAT–Control nonspecifically bound to glutathione beads (Fig. 3A, 3B, lane 2). Equivalent amounts of GST–VAMP-2 bound to glutathione beads under all conditions (Fig. 3C). These data support our hypothesis that TAT–SNAP-23 inhibited exocytosis by binding to SNARE proteins and blocking their interaction.

FIGURE 3.

FIGURE 3

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TAT–SNAP-23 interacts with SNARE proteins in vitro. TAT–SNAP-23 (lanes 1, 3, and 5) and TAT–Control (lanes 2, 4, and 6) containing both HA-tag and 6 × His sequences were incubated with glutathione-coupled Sepharose in the absence (lanes 1 and 2) or presence of recombinant GST–VAMP-2 (lanes 3–6) and/or a recombinant SNARE domain of syntaxin-4 containing 6 × His (lanes 3 and 4). Proteins adherent to the beads were separated by SDS-PAGE and subjected to immunoblot analysis with anti–HA-tag (A), anti–His-tag (B), or anti–VAMP-2 (C).

A number of signal transduction pathways containing protein kinases are activated by fMLF, some of which also regulate exocytosis (39–42). To determine if TAT–SNAP-23 altered basal or fMLF-stimulated activation of signal transduction pathways, phosphorylation of p38 MAPK and ERK1/2 was measured by immunoblot analysis in neutrophils pretreated with or without TAT–SNAP-23 or TAT–Control (Fig. 4A). Fig. 4B shows the densitometric analysis of these experiments. fMLF stimulation induced an increase in p38 MAPK and ERK1/2 phosphorylation that was not altered by pretreatment with TAT–SNAP-23 or TAT–Control. Although both TAT–SNAP-23 and TAT–Control induced a small increase in basal p38 MAPK phosphorylation, densitometric analysis showed that this difference was not statistically significant (Fig. 4B). Neither TAT–SNAP-23 nor TAT–Control had an effect on basal ERK1/2 phosphorylation. Incubation of neutrophils with a concentration of TNF-α (Fig. 4C) that primed the respiratory burst increased p38 MAPK and ERK1/2 phosphorylation. Sequential incubation with TNF-α followed by fMLF did not result in an additive activation of either p38 MAPK or ERK1/2. Similar to the results with fMLF alone, the increased p38 MAPK and ERK1/2 phosphorylation induced by TNF-α alone or in combination with fMLF was not altered by pretreatment with TAT–SNAP-23 or TAT–Control (Fig. 4C). Incubation of neutrophils with concentrations of PAF that primed the respiratory burst did not induce p38 MAPK or ERK1/2 phosphorylation (Fig. 4D). Activation of either p38 MAPK or ERK1/2 resulted only with sequential incubation with PAF followed by fMLF (Fig. 4D). Pretreatment with TAT–SNAP-23 or TAT–Control did not alter the increase in p38 MAPK and ERK1/2 phosphorylation induced by sequential treatment with PAF then fMLF. These data suggest that TAT–SNAP-23 did not alter the activation of signal transduction pathways by fMLF, and inhibition of exocytosis did not change fMLF-stimulated p38 MAPK or ERK1/2 activity in neutrophils primed with PAF or TNF-α.

FIGURE 4.

FIGURE 4

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Effect of TAT–SNAP-23 and TAT–Control on signal transduction pathways. A, Neutrophils (1 × 107/ml) were incubated for 10 min in the presence or absence of TAT–SNAP-23 (0.8 µg/ml) or TAT–Control (0.8 µg/ml), followed by incubation with or without 300 nM fMLF for 1 min. Cells were lysed and proteins separated by SDS-PAGE, followed by immunoblot analysis for phospho-p38 MAPK and phospho-ERK1/2. The blots were then stripped and reblotted for total p38 MAPK and ERK1/2. Immunoblots representative of eight separate experiments are shown. B, Densitometric analysis of the eight immunoblots for phospho-p38 MAPK and phospho-ERK1/2 was performed. Values were normalized to the total amount of p38 MAPK and ERK1/2 and expressed as mean ± SEM as the ratio of phosphorylated to total kinase. C, Neutrophils (1 × 107/ml) were incubated for 10 min in the presence or absence of TAT–SNAP-23 (0.8 µg/ml) or TAT–Control (0.8 µg/ml) followed by incubation with or without TNF-α (2 ng/ml) for 10 min, then stimulated with or without fMLF (300 nM) for 1 min. Cells were lysed and proteins separated by SDS-PAGE, followed by immunoblot analysis for phospho-p38 MAPK and phospho-ERK1/2. The blots were then stripped and reblotted for total p38 MAPK and ERK1/2. Densitometric analysis of immunoblots from three separate experiments was performed. Values were normalized to the total amount of p38 MAPK or ERK1/2 and expressed as mean ± SEM. D, Neutrophils (1 × 107/ml) were incubated for 10 min in the presence or absence of TAT–SNAP-23 (0.8 µg/ml) or TAT–Control (0.8 µg/ml) followed by incubation with or without PAF (30 nM) for 5 min, then stimulated with or without fMLF (300 nM) for 1 min. Cells were lysed and proteins separated by SDS-PAGE, followed by immunoblot analysis for phospho-p38 MAPK and phospho-ERK1/2. The blots were then stripped and reblotted for total p38 MAPK and ERK1/2. Densitometric analysis of immunoblots from six separate experiments was performed. Values were normalized to the total amount of p38 MAPK or ERK1/2 and expressed as mean ± SEM.

Exocytosis participates in phagocytosis-stimulated, but not fMLF-stimulated, respiratory burst activity

TAT–SNAP-23 was used to examine the role of exocytosis in the respiratory burst stimulated by phagocytosis or by activation of formyl peptide receptors. For plasma membrane receptor-mediated respiratory burst activity, neutrophils were pretreated with TAT–SNAP-23 or TAT–Control prior to stimulation with 300 nM fMLF for 5 min, and extracellular superoxide release was measured. The amount of superoxide release stimulated by fMLF-treated neutrophils was not altered by pretreatment with TAT–SNAP-23 or TAT–Control compared with basal conditions (Fig. 5A). Pretreatment with TAT–SNAP-23 inhibited phagocytosis-stimulated hydrogen peroxide production by 60 ± 1.6% (p < 0.001), whereas pretreatment with TAT–Control had no effect (Fig. 5B). Neither TAT–SNAP-23 nor TAT–Control significantly altered phagocytosis of S. aureus. Based on the inhibition of phagocytosis-stimulated H2O2 production, the ability of TAT–SNAP-23 to block phagosomal generation of HOCl, the product of MPO-catalyzed, H2O2-dependent oxidation of chloride was examined. HOCl production was monitored by measuring the bleaching of GFP-expressing S. aureus within neutrophil phagosomes, as previously described (35). Pretreatment with TAT–SNAP-23 did not alter the rate at which fluorescence was lost after phagocytosis of GFP-expressing S. aureus for up to 120 min (Fig. 5C). The data indicate that sufficient H2O2 was generated and MPO released into the phagosome of TAT–SNAP-23–treated cells to support the HOCl-dependent bleaching of GFP. Additionally, pretreatment with TAT–SNAP-23 did not compromise the ability of neutrophils to establish the conditions necessary to kill S. aureus (data not shown).

FIGURE 5.

FIGURE 5

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Effect of TAT–SNAP-23 on respiratory burst activity. A, Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to stimulation with 300 nM fMLF for 5 min. Superoxide production was measured as the reduction of ferricytochrome c. Data are expressed as mean ± SEM superoxide release in nmol/4 × 106 cells/5 min for seven separate experiments. B, Neutrophils (4 × 106/ml) were incubated with 0.5 mM 2′,7′-dichlorofluorescin diacetate for 10 min, then incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to addition of opsonized propidium iodide-labeled S. aureus. Uptake of labeled bacteria and oxidation of 2′,7′-dichlorofluorescin to 2′,7′-dichlorofluorescein were measured by flow cytometry. Data are expressed as mean ± SEM in mcf units for nine separate experiments. C, To determine the effect of TAT–SNAP-23 on phagosomal HOCl generation, neutrophils were incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to addition of S. aureus expressing superfolded GFP. HOCl-mediated bleaching of superfolded GFP was measured by flow cytometry. Results are expressed as mean ± SEM in percentage fluorescence intensity for four separate experiments.

Taken together, these results suggest that delivery of NADPH oxidase components by granule fusion with phagosomes was inhibited by blocking SNARE protein interaction. In contrast, fusion of azurophilic granules with phagosomes was not impaired, resulting in delivery of sufficient MPO to support HOCl production and microbicidal action.

Role of exocytosis in priming of respiratory burst activity by TNF-α and PAF

To kill microbial organisms more effectively, the neutrophil respiratory burst is augmented by a variety of biological agents, including cytokines, chemokines, and bacterial products, through a process termed “priming.” To evaluate the contribution of exocytosis to neutrophil priming, we examined the effect of TAT–SNAP-23 on priming of superoxide release by TNF-α and PAF, agents that induce priming through different signal transduction pathways (43).

To assess the ability of TNF-α and PAF to stimulate exocytosis and the capacity of TAT–SNAP-23 to inhibit the resultant granule release, neutrophils were pretreated with TAT–SNAP-23 or TAT–Control, followed by incubation with TNF-α (2 ng/ml, 10 min) or PAF (30 nM, 10 min). TNF-α and PAF stimulated exocytosis of secretory vesicles and specific granules, measured as an increase in plasma membrane expression of CD35 and CD66b, respectively (Fig. 6A, 6B). Pretreatment with TAT–SNAP-23, but not TAT–Control, inhibited the increase in CD35 and CD66b expression induced by TNF-α (82 ± 4.8% for CD35, p = 0.01, and 75 ± 6.3% for CD66b, p < 0.001). Pretreatment with TAT–SNAP-23 significantly inhibited the PAF-induced increase in CD35 by 40 ± 5.6% (p = 0.05). Although there was a numerical reduction in PAF-induced CD66b expression compared with that of untreated cells (42 ± 11%) after incubation with TAT–SNAP-23, that reduction did not achieve statistical significance. In contrast, the reduction in PAF-induced CD66b expression after incubation with TAT–SNAP-23 was significant compared with that of cells pretreated with TAT–Control (p < 0.05). In the presence of latrunculin A, PAF induced azurophil granule exocytosis, measured as an increase in CD63 expression, and pretreatment with TAT–SNAP-23 did not alter that increase (Fig. 6C). TNF-α did not stimulate an increase in CD63 expression in the presence of latrunculin A. These results indicate that both priming agents stimulated exocytosis. TAT–SNAP-23 dramatically reduced TNF-α–stimulated exocytosis of secretory vesicles and specific granules, whereas TAT–SNAP-23 was a less potent inhibitor of PAF-stimulated exocytosis.

FIGURE 6.

FIGURE 6

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TAT–SNAP-23 inhibits exocytosis induced by TNF-α and PAF. Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to addition of TNF-α (2 ng/ml) or PAF (30 nM) for 10 min. Exocytosis of secretory vesicles, specific granules, and azurophil granules was measured as the increase in plasma membrane expression of CD35 (secretory vesicles) (A), CD66b (specific granules) (B), and CD63 (azurophil granules) (C). For experiments examining CD63 expression, some groups of cells were stimulated with latrunculin A (1 µM) in combination with TNF-α or PAF. Data are expressed as the mean increase above basal expression ± SEM of the mcf intensity for six separate experiments.

The absence of an effect of TAT–SNAP-23 on fMLF-stimulated superoxide release provided the opportunity to examine directly the role of exocytosis in neutrophil priming. Neutrophils were incubated with or without TAT–SNAP-23 or TAT–Control prior to, or after, priming with TNF-α (2 ng/ml) or PAF (30 nM), then stimulated with fMLF (300 nM). Pretreatment with TAT–SNAP-23, but not TAT–Control, significantly inhibited fMLF-stimulated superoxide release from neutrophils pretreated with TNF-α or PAF (Fig. 7; p < 0.01 for TNF-α; p < 0.001 for PAF). Addition of TAT–SNAP-23 after incubation with TNF-α or PAF had no effect on priming of fMLF-stimulated superoxide release (Fig. 7B). The data indicate that exocytosis of neutrophil granules contributed significantly to TNF-α and PAF-induced priming of the respiratory burst.

FIGURE 7.

FIGURE 7

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TAT–SNAP-23 inhibits priming of respiratory burst activity. A, Neutrophils (4 × 106/ml) were pretreated with or without 0.8 µg/ml TAT–SNAP-23 or TAT–Control, incubated in the presence or absence of TNF-α (2 ng/ml, 10 min) or PAF (30 nM, 5 min), then stimulated with 300 nM fMLF. Superoxide production was measured as the reduction of ferricytochrome c. Data are expressed as mean ± SEM superoxide release in nmol/4 × 106 cells/5 min for six separate experiments. B, Neutrophils (4 × 106/ml) were pretreated with or without TNF-α (2 ng/ml, 10 min) or PAF (30 nM, 5 min), then incubated in the presence or absence of TAT–SNAP-23 or TAT–Control, followed by stimulation with 300 nM fMLF. Superoxide release was measured as the reduction in ferricytochrome c. Data are expressed as mean increase in superoxide release above basal ± SEM for six separate experiments.

Two membrane components of the NADPH oxidase, gp91phox and gp22phox, are expressed on secretory vesicles, gelatinase granules, and specific granules (17, 23, 24, 44, 45). To test the hypothesis that exocytosis contributed to priming through translocation of membrane NADPH oxidase components to the plasma membrane, membrane expression of gp91phox was measured after neutrophil priming and/or stimulation. Exposure to fMLF (p < 0.001) and TNF-α (p < 0.001) increased plasma membrane expression of gp91phox, and sequential incubation with TNF-α followed by fMLF did not further increase that expression (Fig. 8A). Pretreatment with TAT–SNAP-23 significantly inhibited the increase in expression of gp91phox stimulated by TNF-α by 55 ± 5% (p = 0.01) and reduced the increase in expression stimulated by the combination of TNF-α and fMLF by 39 ± 18% (p = 0.05). PAF, as well as the combination of PAF followed by fMLF, also significantly increased plasma membrane expression of gp91phox (Fig. 8B). Although pretreatment with TAT–SNAP-23 reduced the increase in gp91phox expression, that reduction did not achieve statistical significance. Pretreatment with TAT–SNAP-23 reduced the increase in gp91phox expression stimulated by fMLF in both sets of experiments; however, this reduction was statistically significant only in the set of experiments also examining PAF (Fig. 8B). Pretreatment with TAT–Control had no effect on gp91phox expression under any treatment condition.

FIGURE 8.

FIGURE 8

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Effect of TAT–SNAP-23 on plasma membrane expression of gp91phox. A, Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to incubation in the presence or absence of TNF-α (2 ng/ml, 10 min), followed by stimulation with or without 300 nM fMLF. Plasma membrane expression of gp91phox was measured by flow cytometry. Data are expressed as mean increase above basal expression ± SEM in mcf units for 10 separate experiments. B, Neutrophils (4 × 106/ml) were incubated with or without 0.8 µg/ml TAT–SNAP-23 or 0.8 µg/ml TAT–Control for 10 min prior to incubation in the presence or absence of PAF (30 nM, 5 min), followed by stimulation with or without 300 nM fMLF. Plasma membrane expression of gp91phox was measured by flow cytometry. Data are expressed as mean increase above basal expression ± SEM in mcf units for 16 separate experiments.

Discussion

In the current study, we show that the introduction of the N-terminal SNARE domain of SNAP-23 into human neutrophils as a TAT fusion protein significantly inhibited exocytosis of three of four granule subsets without altering signal transduction pathway activation. The ability to inhibit exocytosis selectively allowed the relationship between exocytosis and priming and activation of the respiratory burst to be examined. Our data show that exocytosis played a significant role in the respiratory burst activity induced in phagosomes, but not in chemoattractant receptor-mediated activation of the NADPH oxidase at the plasma membrane. Additionally, selective inhibition of exocytosis allowed direct confirmation, for the first time to our knowledge, that granule exocytosis was an event critical for neutrophil priming.

SNARE proteins mediate membrane trafficking events by forming a coiled-coil among four different SNARE domains contained on opposing membranes, leading to their fusion (20, 21). Disruption of SNARE complexes by the introduction of Abs to various SNARE proteins into human neutrophils inhibits exocytosis of specific, gelatinase, and azurophil granules (25–27). Based on those observations, we postulated that introduction of a single SNARE domain into neutrophils would interfere with the interaction of endogenous SNARE proteins and inhibit exocytosis. We chose to examine the SNARE domains of SNAP-23 because of the ability of anti–SNAP-23 to inhibit stimulated exocytosis of gelatinase and specific granules and the identification of SNAP-23 on neutrophil plasma membranes, secretory vesicles, gelatinase granules, and specific granules (22, 23, 25, 27). Whereas a fusion protein containing the C-terminal SNARE domain of SNAP-23 had no effect on stimulated exocytosis (S. Uriarte and K. McLeish, unpublished observations), a fusion protein containing the N-terminal SNARE domain inhibited exocytosis of secretory vesicles, specific granules, and gelatinase granules. Our results show, for the first time to our knowledge, that SNARE proteins mediate exocytosis of secretory vesicles and suggest that SNAP-23 participates in that process. TAT–SNAP-23 had no effect on azurophil granule exocytosis, measured by increased CD63 expression, HNE release, or MPO release. This finding is consistent with the report by Mollinedo et al. (27) showing that azurophil granule exocytosis is not affected by anti–SNAP-23 and with the inability to demonstrate SNAP-23 on azurophil granules by proteomic or immunoblot analyses (23). The results of the current study support the conclusion by Mollinedo et al. (27) that SNAP-23 plays a role in exocytosis of gelatinase and specific, but not azurophil, granules.

Fasshauer and Margittai (38) reported that SNARE-mediated fusion occurs in stages, with two SNARE proteins forming a loose coil structure before a third SNARE protein bound to induce a coiled-coil. Our in vitro data showing that TAT–SNAP-23 bound to the syntaxin-4 SNARE domain and VAMP-2, but not to VAMP-2 alone, suggests that TAT–SNAP-23 may prevent SNARE protein interaction by binding to an intermediate structure. Our data demonstrate that TAT–SNAP-23 selectively inhibited SNARE-mediated membrane fusion. Introduction of TAT–SNAP-23 had no effect on fMLF-stimulated activation of p38 MAPK and ERK1/2, indicating that TAT–SNAP-23 did not prevent activation of signal transduction pathways known to mediate exocytosis or respiratory burst activity (29, 43, 46). We and others have reported that bacterial LPS induces degranulation and primes the respiratory burst in human neutrophils (12, 13, 29). As the TAT fusion proteins used in this study were produced in bacteria, there was a potential for LPS contamination. All fusion proteins were tested for endotoxin using a quantitative Limulus amebocyte lysate assay. The final concentration of endotoxin in the reaction mixtures did not exceed 0.1 EU/ml for any of the fusion protein preparations. Additionally, the inability of TAT fusion proteins alone to stimulate granule exocytosis (Fig. 2), to prime the fMLF respiratory burst (Fig. 5A), or to induce ERK1/2 activation (Fig. 4) indicate that any contamination of TAT fusion proteins with bacterial products was below a level that affected neutrophil responses.

A major finding of this study is that inhibition of exocytosis reduced respiratory burst activity inside phagosomes by ~60% without altering phagocytic activity. Although the mechanism by which TAT–SNAP-23 reduced phagosomal respiratory burst activity was not clearly established, we propose that inhibition of granule fusion with phagosomes prevented delivery of cytochrome b558 to the phagosomal membrane. Neutrophil microbicidal activity requires delivery of antimicrobial proteins to the phagosome and activation of the NADPH oxidase at the phagosomal membrane, leading to the generation of HOCl by MPO-catalyzed oxidation of chloride in the presence of H2O2. To evaluate the effect of the reduction in phagosomal H2O2 production on neutrophil bactericidal activity, the bleaching of cytosolic GFP contained in ingested S. aureus was examined. Bleaching of GFP requires the complete HOCl-generating system of MPO, H2O2, and chloride, and the loss of fluorescence reflects the chlorination of GFP (35). Despite the 60% reduction in phagosomal respiratory burst activity after pretreatment with TAT–SNAP-23, no inhibition of GFP-S. aureus bleaching was observed. Our findings indicate that despite the inhibition of exocytosis, adequate H2O2 and MPO were present to support generation of amounts of HOCl sufficient to bleach GFP.

As opposed to the effect on respiratory burst activity in phagosomes, inhibition of exocytosis had no effect on extracellular superoxide release stimulated by fMLF. The basis for the differential effect of inhibition of exocytosis on respiratory burst activity in phagosomes and the plasma membrane was not examined in our study. It is possible that granule fusion with phagosomes is necessary to provide a component needed for activation of the NADPH oxidase that is normally present at the plasma membrane. The recent report of p40phox deficiency and a selective defect in oxidase activity on phagosomes (47) illustrates that there are clearly distinct and different requirements for oxidase assembly at the plasma membrane versus nascent phagosomes.

The lack of a requirement for exocytosis in respiratory burst activity at the plasma membrane allowed us to examine directly the role of exocytosis in neutrophil priming. Priming is defined as the ability of an agent incapable of independently inducing NADPH oxidase activity to enhance the production of reactive oxygen species in response to exposure to a second agent. Three mechanisms of neutrophil priming have been postulated. First, phosphorylation of cytosolic components of the NADPH oxidase (p47phox, p67phox, p40phox) leads to enhanced translocation of these components to the plasma membrane and assembly of an active NADPH oxidase complex (1, 13, 17). Second, granule exocytosis induces increased plasma membrane expression of the membrane components of the NADPH oxidase (gp91phox, p22phox) (12, 19). Third, granule exocytosis increases plasma membrane expression of receptors, G proteins, and components of signal transduction pathways that lead to enhanced NADPH oxidase activation (29, 43, 44, 46, 48–50). Our data clearly demonstrate that TNF-α stimulated neutrophil granule exocytosis and confirm that exocytosis was a critical component of priming by TNF-α. TNF-α stimulated an increase in plasma membrane expression of gp91phox that was dependent on granule exocytosis. The ability of fMLF to stimulate p38 MAPK and ERK1/2 activation after TNF-α priming was not reduced by inhibition of exocytosis (Fig. 4C), suggesting that increased plasma membrane expression of receptors and signal transduction pathway components did not contribute to priming. These results are most consistent with the hypothesis that exocytosis contributes to TNF-α priming by increasing the plasma membrane expression of NADPH oxidase components. The mechanism by which TAT–SNAP-23 inhibited PAF priming was less clear. PAF clearly induced granule exocytosis and increased the plasma membrane expression of gp91phox. Whereas TAT–SNAP-23 inhibited TNF-α and PAF priming to a similar degree, TAT–SNAP-23 was a less potent inhibitor of PAF-induced exocytosis and failed to inhibit significantly the increase in plasma membrane gp91phox expression after PAF stimulation. The absence of an effect of pretreatment with TAT–SNAP-23 on p38 MAPK and ERK1/2 phosphorylation after fMLF stimulation of PAF-primed neutrophils suggests that the mechanism of PAF priming is not through an increase in plasma membrane receptors or signal transduction components. Further studies are required to determine the mechanism by which TAT–SNAP-23 inhibits priming by PAF.

In summary, we have characterized a novel reagent that selectively inhibits stimulated exocytosis of three neutrophil granule subsets and used it to examine the relationship between granule exocytosis and neutrophil priming. We demonstrate for the first time, to our knowledge, that granule exocytosis per se contributes directly to TNF-α–mediated priming of the NADPH oxidase. In light of the recent identification of Pin1-dependent conformational changes in p47phox as essential for TNF-α priming of the oxidase (51), it is clear that the assembly and activity of the phagocyte NADPH oxidase are regulated by biologic modifiers at several distinct sites on individual components of the enzyme complex. More detailed characterization of other priming agents, including PAF, endotoxin, and GM-CSF, will demonstrate the different mechanisms underlying achievement of the primed neutrophil phenotype.

Supplementary Material

01

Acknowledgments

We thank Karen Brinkley, Junyi Le, Shweta Tandon, Noelle Lang, Terri Manning, and Kevin G. Leidal for expert technical help.

This work was supported by grants from the Department of Veterans Affairs Merit Review Board (to K.R.M. and W.M.N.), the American Heart Association (BGIA 0765387B to S.M.U.), and the National Institutes of Health (K99/R00 HL087924 to S.M.U., R01 AI075212 to M.J.R., and R01 AI07958 and PO1 AI04462 to W.M.N.).

Abbreviations used in this article

DHCB

dihydrocytochalasin B

HA-tag

hemagglutinin tag

6 × His

hexa histidine tag

His-tag

histidine tag

HNE

human neutrophil elastase

mcf

mean channel of fluorescence

MPO

myeloperoxidase

PAF

platelet-activating factor

RFU

relative fluorescence units

ROS

reactive oxygen species

SNAP-23

synaptosome-associated protein-23

TAT

HIV transactivator of transcription

VAMP

vesicle-associated membrane protein

Footnotes

The online version of this article contains supplemental material.

Disclosures

The authors have no financial conflicts of interest.

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01
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