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Combinatorial SNARE Complexes Modulate the Secretion of Cytoplasmic Granules in Human Neutrophils¹

Faustino Mollinedo^2,*, Jero Calafat, Hans Janssen, Belén Martín-Martín^*, Javier Canchado^*, Svetlana M. Nabokina^* and Consuelo Gajate^*,

^* Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Salamanca, Spain; Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and Unidad de Investigación, Hospital Universitario de Salamanca, Salamanca, Spain

	Abstract

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Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The innate immune response is characterized by the accumulation of granulocytic cells at sites of infection and injury. The extracellular release of granular contents from these cells yields an effective barrier in combating infectious organisms, but also can cause massive organ injury during acute or chronic inflammatory conditions (1). Polymorphonuclear neutrophils (PMN),³ which are the most numerous granulocyte subpopulation, comprise the first line of cellular defense against infection and constitute a key mediator in inflammation (2). Central to their physiological role, neutrophils contain three major types of cytoplasmic granules: azurophilic or primary granules, specific or secondary granules, and gelatinase-rich tertiary granules (3, 4), which differ in their respective contents and proneness for exocytosis. It is well established that major differences exist between the different granule populations, specifically regarding the extent to which they are mobilized both in vitro and in vivo (5, 6, 7, 8). Specific and tertiary granules are secreted in response to a plethora of stimuli including chemoattractants, phorbol esters and calcium ionophores; secretion of azurophilic granules (in addition to specific and tertiary granules) occurs during phagocytosis or when neutrophils are pretreated with the fungal metabolite cytochalasin B before stimulation with a wide variety of stimuli (5, 6, 7, 8, 9, 10). Specific and tertiary granules are more readily exocytosed upon cell activation than azurophilic granules (6, 7, 11), modulating crucial neutrophil functions, including adhesion to endothelium, diapedesis, and killing of microorganisms. Azurophilic granules mainly fuse with phagosomal membranes, and contain a wide array of lytic enzymes and proteins with bactericidal activity that can be detrimental to the surrounding tissue if secreted in an unregulated way. In contrast, specific and tertiary granules mainly fuse with the plasma membrane, and contain a number of proteins involved in adhesion and extravasation, comprising a reservoir of plasma membrane proteins that are translocated to the cell surface upon neutrophil activation (3, 4, 12, 13, 14). In addition to the granules discussed, human neutrophils contain yet another releasable membrane-bound organelle-type named the secretory vesicle that are storage organelles for membrane receptors (3, 4). An inflammatory reaction always accompanies infections; its magnitude usually depends on the extent of neutrophil infiltration and the release of neutrophil products. Thus, secretion of neutrophil granules must be tightly regulated to achieve an efficient antimicrobial response and to prevent uncontrolled release of noxious components that could lead to tissue destruction or undesirable inflammatory events.

The ability of intracellular secretory organelles to specifically recognize appropriate acceptor membrane targets underlies the organization of the exocytic pathway. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key mediators of membrane fusion (15). The neuronal SNARE proteins synaptobrevin/vesicle-associated membrane protein (VAMP)-2, syntaxin 1A, and 25-kDa synaptosome-associated protein (SNAP-25) share a homologous SNARE motif of 60 aa (16, 17, 18), which mediates the association of SNARE proteins into a core complex composed of a tightly packed parallel four-helical bundle. VAMP-2 and syntaxin 1A contribute one -helix each and SNAP-25 contributes two -helices. All known SNARE motifs fall into two major subfamilies that contain either a conserved glutamine (Q) or arginine (R) at the ionic "0" layer in the middle of the bundle, leading to the classification of SNARE proteins into Q-SNARE and R-SNARE. The hydrophilic 0 layer of the neuronal SNARE complex is made of three glutamine residues from the Q-SNARE motifs (one contributed by syntaxin 1A and two by SNAP-25) and one arginine residue from the R-SNARE motif (contributed by VAMP-2) (18). The crystal structure of the endosomal SNARE complex has revealed that it is also formed by a four-helix bundle containing three Q-SNARE contributors (syntaxin 7, syntaxin 8, vti1b) and one R-SNARE (endobrevin/VAMP-8) contribution (19). This finding reveals common structural principles of SNARE core complexes consisting of four-helix bundles of the three (Q-SNARE)/one (R-SNARE) type.

A number of SNARE proteins have been identified in human neutrophils (20, 21, 22, 23, 24, 25, 26), and some of them have been recently involved in the secretion of specific and tertiary granules (24, 26). However, our knowledge on neutrophil exocytosis is far from complete. Regulation of azurophilic granule secretion is largely unknown, and a long-standing question lies on how distinct neutrophil granules are differentially mobilized upon cell activation. In this study, we show the involvement of different SNARE proteins as well as the formation of distinct SNARE complexes in the exocytosis of the major neutrophil cytoplasmic granules.

	Materials and Methods

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Antibodies

Specific rabbit anti-VAMP-1 Ab (Synaptic Systems) was raised against a synthetic peptide corresponding to residues 2–14 of human VAMP-1, a specific VAMP-1 sequence showing <23% identity with its closest relative VAMP-2. Anti-VAMP-2 mAb (clone 69.1; Synaptic Systems), raised against the N-terminal peptide (residues 2–17) of rat VAMP-2, specifically recognized VAMP-2 from different species including human, and showed no cross-reactivity with VAMP-1 (27). Specific anti-VAMP-7 mAb (clone 158.2) (28) was a gift from Dr. T. Galli (Institut National de la Santé et de la Recherche Médicale, Paris, France). Specific anti-human syntaxin 4 mAb (clone 49) was obtained from BD Transduction Laboratories. Specific anti-human SNAP-23 polyclonal Ab was prepared from recombinant human SNAP-23 as described previously (24). To avoid possible cross-reactivity with SNAP-25, the antiserum was incubated overnight at 4°C with GST-SNAP-25 coupled to glutathione-agarose beads, and after centrifugation the supernatant was immunoabsorbed to GST-SNAP-23 to obtain affinity-purified anti-SNAP-23 Ab as described elsewhere (24). Anti-CD20 and anti-CD3 mAbs were generously provided by Dr. M. Romero (Hospital Rio Hortega, Valladolid, Spain). Specific mAbs against human CD63 (CLB-gran/12,435) and CD66b (CLB-B13.9) were obtained from the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service (CLB; Amsterdam, The Netherlands). Rabbit anti-human lactoferrin Ab was purchased from Cappel Laboratories. Rabbit anti-human myeloperoxidase Ab was purchased from DakoCytomation. Rabbit anti-gelatinase Ab (29) was generously provided by Dr. N. Borregaard (National University Hospital, Copenhagen, Denmark). Biotinylated anti-mouse and anti-rabbit IgG were obtained from Amersham Biosciences. FITC-conjugated anti-mouse Ig was obtained from Dakopatts. P3X63 myeloma culture supernatant, provided by Dr. F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain), was used as a negative control.

Cell culture

Human leukemia HL-60 cells were grown in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS, L-glutamine, and antibiotics. Neutrophil differentiation of HL-60 cells was induced with 1.3% (v/v) DMSO as described previously (30).

Neutrophil isolation and activation

Neutrophils were obtained from human peripheral blood by dextran sedimentation and Ficoll-Hypaque centrifugation as described previously (23). Freshly isolated human neutrophils were resuspended at 3–5 x 10⁶ cells/ml in HEPES/glucose buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 1.2 mM MgCl₂, 1.3 mM CaCl₂, 5.5 mM glucose, (pH 7.5)). For cell activation assays, neutrophils were incubated with 100 ng/ml PMA for 10 min at 37°C or preincubated with 5 µg/ml cytochalasin B for 5 min at 37°C, and then stimulated with 10^–7 M FMLP for 10 min at 37°C. Release of gelatinase, lactoferrin, -glucuronidase, and peroxidase following neutrophil activation was determined as described elsewhere (6, 11, 31).

Subcellular fractionation

Resting neutrophils were resuspended in 50 mM Tris-HCl (pH 7.5), containing 2 mM PMSF, disrupted by repeated freeze-thaw, and soluble and membrane fractions from postnuclear extracts were prepared as described previously (24).

To prepare the distinct subcellular fractions, freshly isolated resting and PMA-activated neutrophils (3–5 x 10⁸) were gently disrupted and the postnuclear fraction was layered onto a 27-ml, 15–40% (w/w) continuous sucrose gradient, with a 1-ml cushion of 60% (w/w) sucrose, and centrifuged at 25,000 rpm in a Beckman L8-70B ultracentrifuge using a SW27 rotor (24). Subcellular fractions were assayed for marker proteins, namely lactate dehydrogenase (cytosol), HLA (plasma membrane), gelatinase (tertiary granules), lactoferrin (specific granules), and peroxidase (azurophilic granules) as described (26). Membranes from each fraction were obtained as described (26).

RT-PCR

Total RNA (10 µg) was reverse-transcribed into cDNA with Moloney murine leukemia virus reverse transcriptase (Promega). The generated cDNA was amplified by using TaqDNA polymerase (ECOGEN) and primers for human vamp-1 (5'-CCCTCCTCCTAACATGACCA-3' and 5'-CTACCACGATGATGGCACAG-3'); vamp-2 (5'-ATGTCGGCTACCGCTGCCAC-3' and 5'-TTAAGAGCTGAAGTAAACTA-3'); vamp-3 (5'-ATGTCTACAGGTCCAACTGC-3' and 5'-TCATGAAGAGACAACCCAC-3'); vamp-4 (5'-ATGCCTCCCAAGTTTAAGCG-3' and 5'-TCAAGTACGGTATTTCATGAC-3'); vamp-5 (5'-ATGGCAGGAATAGAGTTGGAG-3' and 5'-GTCAGTTCCCAGGCCCTGAG-3'); vamp-7 (5'-ATGGCGATTCTTTTTGCTGTTG-3' and 5'-CTATTTCTTCACACAGCTTGGC-3'); vamp-8 (5'-ATGGAGGAAGCCAGTGAAGG-3' and 5'-CTTAAGAGAAGGCACCAGTG-3'); and -actin (5'-AATCTGGCACCACACCTTCTACA-3' and 5'-CGACGTAGCACAGCTTCTCCTTA-3'). The PCR profiles were as follows: 1 cycle at 95°C for 5 min as an initial denaturation step, then denaturation at 95°C for 30 s, annealing for 30 s, and extension at 72°C for 90 s (16 cycles for -actin; 25 cycles for vamp-1, vamp-4, vamp-5, vamp-8; 27 cycles for vamp-7; 30 cycles for vamp-2 and vamp-3), followed by further incubation for 15 min at 72°C (1 cycle). The annealing step was conducted at 52°C (vamp-3), 54°C (vamp-7), 56°C (vamp-2), 58°C (vamp-1), or 65°C (vamp-4, vamp-5, vamp-8, and -actin). These experimental conditions were shown to be at the linear phase of amplification for each gene. The expected PCR products (263 for vamp-1, 351 for vamp-2, 303 for vamp-3, 426 for vamp-4, 352 for vamp-5, 663 for vamp-7, 304 for vamp-8, and 407 for -actin) were size fractionated onto a 2% agarose gel and stained with ethidium bromide.

Cloning and sequencing

The PCR products were cloned into the pCR 2.1 vector using the TA cloning kit (Invitrogen Life Technologies) following the manufacturer’s indications, and sequenced in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

Western blotting

Proteins were separated by SDS-PAGE and then immunoblotted as described elsewhere (26). Immunoblots were developed using the ECL detection system (Amersham Biosciences).

Coimmunoprecipitation

A total of 5 x 10⁶ cells was lysed with 200 µl of lysis buffer (20 mM Tris-HCl, 100 mM KCl, 0.9% Triton X-100, 10% glycerol, 2 mM orthovanadate, and 2 mM PMSF). Lysates were precleared with 500 µl of protein A-Sepharose at 4°C for 2 h and immunoprecipitated by incubation with Abs against the indicated SNARE proteins precoupled to protein A-Sepharose for 2 h at 4°C. After extensive washing with lysis buffer, the precipitates were subjected to SDS-PAGE and Western blot analysis. P3X63 was used as a negative control for immunoprecipitation, and no signal was detected.

Tetanus toxin (TeTx) treatment

Recombinant toxigenic L chain TeTx, provided by Dr. J. Blasi (Universidad de Barcelona, L’Hospitalet de Llobregat, Barcelona, Spain), was preactivated by incubation with 10 mM DTT at 37°C for 1 h. Neutrophil extracts or membranes (90 µg protein) were incubated in the presence of 0.5% Triton X-100 for 1 h at 37°C with or without 400 nM preactivated TeTx.

Electropermeabilization and immunofluorescence flow cytometry

Neutrophils were electropermeabilized by two discharges of 5 kV/cm, 25 µF, and 72 Ohm using a BTX electroporator (Biotechnologies & Experimental Research) as described previously (24, 32, 33). Electropermeabilized neutrophils were incubated for 5 min with different concentrations of Abs or with 400 nM TeTx for 30 min, and then incubated for 5 min with 5 µg/ml cytochalasin B at 37°C, followed by stimulation with 1 µM Ca²⁺ (0.1 mM CaCl₂, 5.37 mM MgCl₂, 5 mM hydroethyl EDTA, 10 mM glucose) and 50 µM GTP--S for 10 min at 37°C as described elsewhere (26). In some cases, cells were stimulated by incubation with 100 ng/ml PMA for 10 min at 37°C without cytochalasin B pretreatment, or pretreated with cytochalasin B and then activated with 10^–7 M FMLP for 10 min at 37°C. Cells were then fixed with 1% paraformaldehyde and processed for immunofluorescence flow cytometry. Control untreated electropermeabilized cells were run in parallel. Ag cell surface expression was measured in paraformaldehyde-fixed neutrophils as described (24) using a BD Biosciences FACSCalibur flow cytometer.

Immunoelectron microscopy

Resting human neutrophils and exudate neutrophils from skin window chambers after phagocytosis of latex beads (34), provided by Dr. N. Borregaard (National University Hospital), were fixed and processed for ultrathin cryosectioning as described previously (26, 35). For double immunolabeling, ultrathin frozen sections were incubated with the indicated Abs followed with 10- and 15-nm protein A-conjugated colloidal gold probes (Electron Microscopy Laboratory, Utrecht University, The Netherlands) (36). After immunolabeling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM10 electron microscope. Negative controls, prepared by replacing the primary Ab by a nonrelevant rabbit or mouse Ab, showed no staining.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
VAMP expression in human neutrophils and neutrophil-differentiating HL-60 cells

Because neutrophils contain a high number of granules and VAMPs constitute the major SNARE proteins located in vesicles (37), we first analyzed the expression of VAMP genes in human mature peripheral blood neutrophils and neutrophil-differentiating HL-60 cells by semiquantitative RT-PCR. The human HL-60 cell line can be induced to differentiate toward the neutrophil lineage, and it has been widely used as a cell culture model to study neutrophil gene expression (30). VAMP-1, VAMP-2, VAMP-3 (cellubrevin), VAMP-4, VAMP-5 (myobrevin), VAMP-7 (or tetanus neurotoxin-insensitive vesicle-associated membrane protein/TI-VAMP), and VAMP-8 were expressed in both HL-60 cells and human neutrophils (Fig. 1). VAMP-1, VAMP-2, VAMP-4, and VAMP-5 mRNA levels were up-regulated following HL-60 differentiation (Fig. 1), and thus their expression was under regulatory control during neutrophil maturation. These VAMP genes were up-regulated after 1-day incubation of HL-60 cells with DMSO (Fig. 1), the time required to commit HL-60 cells toward the neutrophil lineage (38). The VAMP gene expression pattern shown in mature human neutrophils was similar to that found in DMSO-differentiated HL-60 cells (after a 5-day DMSO treatment), except for a lower expression of VAMP-8 in neutrophils (Fig. 1). Each VAMP was identified by the expected amplicon size, and by subsequent cloning and sequencing of each amplified fragment.

View larger version (18K): [in this window] [in a new window]   FIGURE 1. Expression of VAMP genes in neutrophil-differentiating HL-60 cells and human peripheral blood neutrophils. Total RNA was purified from human peripheral blood mature neutrophils (PMN), untreated HL-60 cells, and HL-60 cells treated with 1.3% (v/v) DMSO for the indicated times, and subjected to semiquantitative RT-PCR analysis using specific oligonucleotide primers for each gene, under conditions shown to be at the linear phase of amplification. PCR amplification of -actin was used as an internal loading control. The PCR products were electrophoresed onto a 2% agarose gel and stained with ethidium bromide.

TeTx-sensitive and insensitive mechanisms in neutrophil exocytosis

TeTx specifically cleaves some VAMPs, and thereby constitutes an excellent tool to study functional aspects of these proteins (41, 42). The selective action of TeTx relies on the presence of a highly conserved QF motif in the amino acid sequence of both human VAMP-1 (GASQFESS) and human VAMP-2 (GASQFETS), but not in other VAMPs, such as VAMP-7 (TF) and rat VAMP-1 (VF) (41, 42). We have recently shown that TeTx degraded VAMP-2 and inhibited secretion of specific and tertiary granules in human neutrophils (26). Now, we asked whether azurophilic granules were also regulated by TeTx-sensitive proteins. To access the neutrophil cytoplasm in whole functional neutrophils with TeTx, we prepared electropermeabilized human neutrophils and treated them with 400 nM TeTx. The viability of such permeabilized cells determined by latency of lactate dehydrogenase averaged 82.3 ± 4.6% (n = 5) for at least 50 min, which is in agreement with previous estimates (32, 43). The granules and the plasma membrane of electropermeabilized human neutrophils did not show any significant difference with intact neutrophils as assessed by electron microscopy (44), and electropermeabilized neutrophils have been shown to remain functional, retaining their responsiveness to distinct stimuli (24, 26, 32, 45). The effect of TeTx on neutrophil exocytosis in electropermeabilized neutrophils was analyzed following the up-regulation of the two granule membrane markers CD63 (azurophilic membrane marker) (46) and CD66b (specific/tertiary granule membrane marker) (26, 47) at the cell surface, which constitutes a reliable method to monitor neutrophil degranulation in electropermeabilized neutrophils (24, 33). We have previously shown that most of the intracellular CD63 and CD66b pools (>80%) were shown incorporated into the cell surface of electropermeabilized neutrophils after cell activation with Ca²⁺ and GTP--S (24). TeTx practically prevented CD66b up-regulation, but only partially inhibited the CD63 up-regulation following cell activation with Ca²⁺ and GTP--S (Fig. 2A). This finding suggests that exocytosis of specific/tertiary and azurophilic granules is mainly mediated by TeTx-sensitive and insensitive proteins, respectively. Azurophilic granules are lysosome-like organelles, and TeTx-insensitive VAMP-7 has been involved in the mobilization of lysosomes in different systems (48, 49, 50, 51). Thus, we next asked for the putative involvement of VAMP-7 in neutrophil exocytosis. In addition, because VAMP-1 and VAMP-2 expression was up-regulated during neutrophil differentiation of HL-60 cells, and both proteins are cleaved by TeTx (42), we further analyzed their respective roles in neutrophil exocytosis.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Effect of TeTx on neutrophil exocytosis and identification, subcellular distribution and TeTx sensitivity of VAMPs in human neutrophils. A, Electropermeabilized human neutrophils were incubated in the absence (Control) or in the presence of TeTx (400 nM), activated with Ca2+ + GTP--S, and analyzed for CD63 and CD66b cell surface expression. Data are expressed as percentage of the cell surface Ag increase upon electropermeabilized neutrophil activation compared with the CD63 and CD66b cell surface up-regulation detected in control Ca2+ + GTP--S-stimulated electropermeabilized neutrophils in the absence of TeTx, considered as 100% increase in cell surface Ag expression. Mean values ± SD of three independent determinations are shown. B, Neutrophil extracts (90 µg protein) and rat brain homogenates (10 µg protein) were resolved by SDS-PAGE and immunoblotted for VAMP-1 and VAMP-7. The molecular mass of each band is indicated. C, Equal amounts of postnuclear extract (E), soluble (S), and membrane (M) proteins (80 µg) from resting human neutrophils were run on SDS-polyacrylamide gels and analyzed by immunoblotting using specific anti-VAMP-1 or anti-VAMP-7 Abs. Rat brain homogenates (10 µg protein) are included as positive controls. D, Extract and membrane proteins (80 µg) were incubated in the absence (–) or presence (+) of 400 nM TeTx, and then subjected to SDS-PAGE and immunoblotted for VAMP-1 or VAMP-7. Electropermeabilized human neutrophils were incubated in the absence (Control) or in the presence of TeTx (400 nM) for 30 min and then subjected to SDS-PAGE and immunoblotted for VAMP-1. E, Resting human neutrophils were gently disrupted and subjected to subcellular fractionation. Fractions were collected and analyzed for the activity of specific organelle markers, which are plotted normalized to the fraction with maximal activity. The following markers were assayed: lactate dehydrogenase for cytosol (CYT) (); HLA for plasma membrane (PM) (•); gelatinase for tertiary granules (TG) (); lactoferrin for specific granules (SG) (); peroxidase for azurophilic granules (AG) (). F, Membrane proteins (80 µg) from the subcellular fractions 2–8 of resting (Rest.) and PMA-activated (Act.) human neutrophils were assayed for VAMP-1, VAMP-2, and VAMP-7 by immunoblotting. Membranes from fractions enriched in plasma membrane (PM), tertiary granules (TG), specific granules (SG), and azurophilic granules (AG) were analyzed. Results shown are representative of three separate experiments.

Previously, we and others showed the expression of TeTx-sensitive VAMP-2 in human neutrophils (20, 25, 26). Here we found that specific anti-VAMP-1 and anti-VAMP-7 mAbs recognized bands of 18 and 25 kDa, respectively, in both neutrophil extract and brain homogenate, although greater amounts of neutrophil protein were loaded to allow protein detection (Fig. 2B). These immunoreactive bands were detected in the membrane fraction of human neutrophils, but not in the soluble fraction containing the cytosol (Fig. 2C), indicating that both proteins were membrane bound. The immunoreactive 18-kDa band was extensively degraded by treatment with TeTx, whereas the immunoreactive 25-kDa band was resistant (Fig. 2D), further confirming their identity as VAMP-1 and TeTx-insensitive VAMP-7, respectively. TeTx also degraded VAMP-1 in electropermeabilized cells (Fig. 2D), suggesting a role for this protein in TeTx-sensitive neutrophil exocytosis.

Differential subcellular localization of VAMPs in human neutrophils

To determine the subcellular localization of VAMPs in resting human neutrophils we performed subcellular fractionation assays that resolved cytosol, plasma membrane, as well as tertiary, specific, and azurophilic granules (Fig. 2E). We found that VAMP-1 was located in the membranes prepared from subcellular fractions 4–6, enriched in specific and tertiary granules, as well as from fractions 7–8, enriched in azurophilic granules (Fig. 2F). This subcellular location differed from that of VAMP-2, mainly localized in the readily mobilizable tertiary and specific granules (Fig. 2F). When human neutrophils were activated with PMA that released tertiary and specific granules, but not azurophilic granules (26), VAMP-2 and the tertiary/specific granule location of VAMP-1 were translocated to the plasma membrane, whereas the azurophilic granule-located VAMP-1 remained in the last fractions of the subcellular fractionation (Fig. 2F). Interestingly, we found that VAMP-7 was mainly localized in the membranes of the fractions enriched in azurophilic granules in resting human neutrophils (Fig. 2F), and was not mobilized upon PMA activation (Fig. 2F).

To further define the subcellular localization of VAMP-1, VAMP-2 and VAMP-7, cryosections of resting human neutrophils were double-labeled with Abs against these VAMPs and with markers for the different cytoplasmic granules, namely myeloperoxidase (azurophilic granules), lactoferrin (specific granules), or gelatinase (tertiary granules). Cryosections were analyzed by immunogold electron microscopy. VAMP-1 was localized on the membranes of specific, tertiary and azurophilic granules (Fig. 3, A–D), and on the internal vesicles of multivesicular bodies (Fig. 3B). VAMP-2-positive granules were mostly positive for specific and tertiary granule markers (data not shown), corroborating our previous findings (26). Interestingly, VAMP-7 was mainly located on the membrane of myeloperoxidase-positive granules (Figs. 3E and 4). The degree of colocalization of the VAMPs with the distinct granule markers is shown in Fig. 4, after analyzing at least 200 positive granules for VAMPs. Because labeling with anti-VAMP Abs was much weaker than labeling with the corresponding granule markers and only one section was examined for each granule, we analyzed colocalization only in VAMP-positive granules to avoid that the less abundant granule constituent, i.e., VAMP could be missed in a particular section of the same granule. VAMP-1 was evenly detected in the three cytoplasmic granules, whereas VAMP-2 was predominantly present in specific and tertiary granules and VAMP-7 in azurophilic granules (Fig. 4).

View larger version (111K): [in this window] [in a new window]   FIGURE 3. Electron microscopy characterization of VAMP-1 and VAMP-7 positive granules. Cryosections of neutrophils were double-labeled for VAMP-1 and lactoferrin (Lf) (A and B), gelatinase (Gel) (C), or myeloperoxidase (MPO) (D), respectively, using specific immunogold-labeled Abs with different gold particle sizes (10 and 15 nm) as indicated. Double-labeled granules are indicated (arrows). VAMP-1 was also seen in Golgi (G) and multivesicular bodies (mvb). E, Neutrophils were immunogold-labeled with anti-VAMP-7 and anti-myeloperoxidase specific Abs, showing colocalization of both proteins (arrows). Scale bar, 200 nm.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Differential subcellular localization of VAMP in neutrophil granules. Ultrathin cryosections were double immunogold-labeled for the corresponding VAMP and either myeloperoxidase (marker for azurophilic granules), lactoferrin (marker for specific granules), or gelatinase (marker for gelatinase-rich tertiary granules). Histograms indicate the percentage of VAMP-positive granules displaying colocalization with each granule marker. For each experiment, at least 200 positive granules were analyzed.

View larger version (114K): [in this window] [in a new window]   FIGURE 5. VAMP-1 and VAMP-7 are present in phagolysosomes. Cryosections of exudate neutrophils from skin window chambers after phagocytosis of latex beads were incubated with anti-VAMP-1 (A) and anti-VAMP-7 (B) Abs for immunogold detection. A, An area of a cell shows labeling of VAMP-1 (arrows) on the outer membrane and on vesicles in the phagolysosomes (ph) and in the endoplasmic reticulum (er). B, Three phagolysosomes are labeled (arrows) for VAMP-7. Scale bar, 200 nm.

Next we analyzed whether the VAMPs, with different subcellular locations, could play a role in neutrophil exocytosis. To this aim, we prepared electropermeabilized neutrophils that allowed rapid access of Abs into the cytoplasm and were able to undergo exocytosis of cytoplasmic granules upon cell activation with Ca²⁺ and GTP--S (24, 33). More than 95% of electropermeabilized neutrophils were permeable to either propidium iodide or FITC-conjugated anti-CD3 mAb (24, 33). Incubation of electropermeabilized neutrophils with irrelevant mouse Igs, including P3X63 myeloma culture supernatant or isotype-matched unrelated mouse mAbs anti-CD20 or anti-CD3, used as negative controls, had no effect on neutrophil degranulation (Fig. 6A). However, incubation of electropermeabilized neutrophils with specific anti-VAMP-1 Abs inhibited exocytosis of both CD63- and CD66b-rich granules although a higher inhibitory effect on the secretion of azurophilic granules was observed (Fig. 6A). Specific anti-VAMP-2 Abs inhibited CD66b up-regulation in a dose-dependent manner, but had no effect on CD63 up-regulation (Fig. 6A). In contrast, specific anti-VAMP-7 Abs showed a potent inhibitory action on the exocytosis of azurophilic granules with little effect on CD66b-positive granule secretion (Fig. 6A). The combination of anti-VAMP-1 and anti-VAMP-7 Abs highly potentiated the inhibitory effect on azurophilic granule secretion, but no further inhibitory action was observed on CD66b-rich granule exocytosis (Fig. 6A). Ab neutralization of SNAREs has been shown to impair secretory responses in a variety of cell types (24, 26, 53, 54), likely by hampering SNARE complex formation through steric hindrance or conformational change.

View larger version (49K): [in this window] [in a new window]   FIGURE 6. Effect of Abs against VAMPs and syntaxin 4 on the up-regulation of CD63 and CD66b cell surface expression in activated electropermeabilized human neutrophils. A, Electropermeabilized neutrophils (PMN) were incubated in the absence (Control) or in the presence of P3X63 (20 µg/ml), anti-CD20 mAb (20 µg/ml), anti-CD3 mAb (20 µg/ml), or of increasing concentrations of anti-VAMP-1, anti-VAMP-2, and anti-VAMP-7 Abs, and then activated with Ca2+ + GTP--S, and assayed for CD63 and CD66b Ag expression by flow cytometry. In addition, electropermeabilized cells were also incubated with anti-VAMP-1 + anti-VAMP-7 Abs at the indicated concentrations. Data are expressed as the percentage of the cell surface Ag increase upon electropermeabilized neutrophil activation compared with the CD63 and CD66b cell surface up-regulation detected in control Ca2+ + GTP--S-stimulated electropermeabilized neutrophils in the absence of any Ab (Control), considered as 100% increase in cell surface Ag expression. Mean values ± SD of three independent determinations are shown. B, Electropermeabilized neutrophils (PMNs) were incubated in the absence (Control) or in the presence of P3X63 (20 µg/ml), or of increasing concentrations of anti-syntaxin 4 (STX4) Ab, and then activated with Ca2+ + GTP--S (Ca2+ + GTP), and assayed for CD63 and CD66b Ag expression by flow cytometry. The results shown are representative of three separate experiments. C, Electropermeabilized neutrophils (PMN) were incubated in the absence (Control) or in the presence of P3X63 (20 µg/ml), anti-CD20 mAb (20 µg/ml), anti-CD3 mAb (20 µg/ml), increasing concentrations of anti-syntaxin 4 (STX4) Ab, or combinations of Abs against syntaxin 4 and different VAMPs, and then activated with Ca2+ + GTP--S, and assayed for CD63 and CD66b Ag expression by flow cytometry as described. Data are expressed as in A. Mean values ± SD of three independent determinations are shown.

The granule membrane VAMPs discussed are expected to interact with target plasma membrane proteins during exocytosis. Thus, we next analyzed the role of syntaxin 4, which is located in the plasma membrane of human neutrophils (20, 26), in the exocytosis of the distinct granule populations. Incubation of electropermeabilized neutrophils with specific anti-syntaxin 4 mAb inhibited CD63 up-regulation in a dose-response manner (Fig. 6, B and C). Higher amounts of anti-syntaxin 4 mAb were required to inhibit CD66b up-regulation (Fig. 6, B and C). Following dose-response assays, we found that exocytosis of azurophilic granules was 3.5-fold more sensitive to the effect of the anti-syntaxin 4 Ab than the corresponding exocytosis of specific/tertiary granules. Ab inhibition experiments suggested that distinct VAMP/syntaxin 4 complexes were involved in the exocytosis of neutrophil granules (Fig. 6C). Combinations of anti-syntaxin 4/anti-VAMP-1 Abs potentiated the inhibition of the secretion of azurophilic and specific/tertiary granules, whereas combinations of anti-syntaxin 4/anti-VAMP-2 Abs and anti-syntaxin 4/anti-VAMP-7 Abs further enfeebled specific/tertiary and azurophilic granule secretion, respectively (Fig. 6C). These data suggest that syntaxin 4 mediates secretion of specific/tertiary and azurophilic granules via its putative interaction with VAMP-1 or VAMP-2 and VAMP-1 or VAMP-7, respectively.

SNAP-23 is involved in VAMP-mediated exocytosis of specific and tertiary granules

The requirement for higher amounts of anti-syntaxin 4 Abs to block exocytosis of specific/tertiary granules, as compared with azurophilic granule mobilization (Fig. 6C), would be compatible with a higher number of syntaxin 4-containing SNARE complexes involved in the secretion of specific/tertiary granules. In addition, a higher diversity of SNARE proteins in specific and tertiary granules might lead to their proneness to exocytose. We have recently reported by subcellular fractionation the presence of SNAP-23 in specific and tertiary granules, with a minor location in plasma membrane (24). In this study, we have found by electron microscopy that SNAP-23 was mainly present at the membranes of specific and tertiary granules with a secondary location in the plasma membrane (Fig. 7). Labeling of SNAP-23 in docked granules in contact with the plasma membrane was also observed (Fig. 7A, inset), further supporting its role in membrane fusion processes in these cells. Incubation of electropermeabilized neutrophils with specific anti-SNAP-23 Abs blocked CD66b up-regulation following cell activation with Ca²⁺ and GTP--S, without affecting CD63 up-regulation (Fig. 8). However, incubation of electropermeabilized neutrophils with anti-SNAP-23 Ab previously preincubated with SNAP-23 recombinant protein did not affect exocytosis, further demonstrating the specific effect of the anti-SNAP-23 Ab on the exocytosis of specific/tertiary granules (data not shown). In addition, a combination of Abs against SNAP-23 with either anti-VAMP-1 or anti-VAMP-2 Abs further inhibited secretion of specific and tertiary granules, with no effect on the release of azurophilic granules (Fig. 8).

View larger version (172K): [in this window] [in a new window]   FIGURE 7. Subcellular localization of SNAP-23 in human neutrophils by electron microscopy. A, Cryosections of neutrophils were immunogold-labeled with rabbit anti-SNAP-23 Ab and 10-nm protein A-gold. SNAP-23 localized at the membrane of the granules (arrows). An area of another cell with a docked granule (inset) showing labeling on the granule in contact with labeling underneath the plasma membrane (arrows). To characterize SNAP-23-positive granules, neutrophils were double-labeled with anti-SNAP-23 Abs and anti-gelatinase (Gel) (B) or anti-lactoferrin (Lf) (C) Abs, respectively. B, After double-labeling with anti-gelatinase Ab (10-nm gold), SNAP-23 (15-nm gold) was found in gelatinase-positive granules (arrows). C, After double-labeling with anti-lactoferrin Ab (10-nm gold), SNAP-23-positive granules (g) (arrows) were also lactoferrin-positive. SNAP-23 is also shown at the plasma membrane (m) (arrows). n, Nucleus. Scale bar, 200 nm.

View larger version (35K): [in this window] [in a new window]   FIGURE 8. Involvement of SNAP-23 in the secretion of specific and tertiary granules. Electropermeabilized neutrophils (PMN) were incubated in the absence (Control) or presence of P3X63 (20 µg/ml), anti-CD20 (20 µg/ml), anti-CD3 (20 µg/ml), increasing concentrations of anti-SNAP-23 Ab, or combinations of Abs against SNAP-23 and different VAMPs, and then activated with Ca2+ + GTP--S, and assayed for CD63 and CD66b Ag expression by flow cytometry as in Fig. 6. Data are expressed as the percentage of CD63 and CD66b cell surface increase. Mean values ± SD of three independent determinations are shown.

Human neutrophils electropermeabilized under the same conditions as in this work have been previously shown to respond to different stimuli, including the phorbol ester PMA and the chemotactic factor FMLP (32). PMA induces secretion of specific and tertiary granules, whereas FMLP is a general secretagogue that releases contents of tertiary, specific, and azurophilic granules (10). We found that PMA, without cytochalasin B pretreatment, induced a potent up-regulation of CD66b, similar to that induced by Ca²⁺ and GTP--S electropermeabilized cells, but it did not prompt CD63 up-regulation. This observation is in agreement with the known ability of PMA to induce secretion of specific and tertiary granules (6, 55). The complete secretagogue FMLP promoted a weaker exocytic response, 42% of that induced by Ca²⁺ and GTP--S. Similarly to what we obtained in electropermeabilized neutrophils activated with Ca²⁺ and GTP--S, we found that Abs against VAMP-2 and SNAP-23 were potent inhibitors of CD66b up-regulation following stimulation with either PMA (Fig. 9A) or FMLP (Fig. 9B). Abs against VAMP-1 and syntaxin 4 were also able to inhibit CD66 up-regulation, although to a lesser extent, whereas anti-VAMP-7 Abs hardly affected the exocytosis of CD66b-positive granules (Fig. 9). In contrast, Abs against VAMP-7, syntaxin 4, and VAMP-1 drastically inhibited CD63 up-regulation, whereas Abs against VAMP-2 and SNAP-23 did not affect CD63 up-regulation (Fig. 9B). These results further support that exocytosis of specific and tertiary granules is mediated by VAMP-1, VAMP-2, SNAP-23, and syntaxin 4, whereas exocytosis of azurophilic granules is mediated by VAMP-1, VAMP-7, and syntaxin 4, irrespective of the stimulus used for secretion.

View larger version (37K): [in this window] [in a new window]   FIGURE 9. Involvement of VAMPs, SNAP-23, and syntaxin 4 in the secretion of neutrophil granules upon activation with PMA (A) and FMLP (B). Electropermeabilized neutrophils (PMN) were incubated in the absence (Control) or in the presence of Abs (10 µg/ml) against CD3, VAMP-1, VAMP-2, VAMP-7, SNAP-23, and syntaxin 4 (STX4), and then activated with PMA (A) or FMLP (B), and assayed for CD63 and CD66b Ag expression by flow cytometry as in Fig. 6. Data are expressed as the percentage of CD63 and CD66b cell surface increase. Mean values ± SD of three independent determinations are shown.

We next sought whether VAMPs and SNAP-23, located in granules, and syntaxin 4, located in the plasma membrane, could interact each other during neutrophil exocytosis resulting in the formation of SNARE complexes. Using similar activation conditions as those used in Fig. 9, we found that SNAP-23 coimmunoprecipitated with VAMP-1, VAMP-2, and syntaxin 4 after neutrophil activation with PMA (Fig. 10A), which induced selective secretion of specific and tertiary granules (>67% secretion of gelatinase and lactoferrin, with <5% secretion of the azurophilic granule marker -glucuronidase, following incubation with 100 ng/ml PMA for 10 min). Interestingly, VAMP-2 coimmunoprecipitated with SNAP-23 and syntaxin 4, but not with VAMP-1, after PMA stimulation (Fig. 10A), suggesting that VAMP-1 or VAMP-2 are mutually exclusive of each other in the formation of SNARE complexes. These data suggest that VAMP-1, VAMP-2, and SNAP-23, mainly found in granules, and syntaxin 4, mainly found in plasma membrane, are brought together during neutrophil activation forming two combinatorial SNARE complexes, VAMP-2/SNAP-23/syntaxin 4 and VAMP-1/SNAP-23/syntaxin 4, which eventually lead to secretion of specific and tertiary granules. VAMP-1 and syntaxin 4 coimmunoprecipitated after cell activation with the specific inducer of specific/tertiary granule exocytosis PMA (Fig. 10B). Coimmunoprecipitation of VAMP-1 and syntaxin 4 was further enhanced following cell stimulation with the complete secretagogue FMLP (Fig. 10B), which induced the discharge of specific/tertiary granules and azurophilic granules (release of 55% lactoferrin, 63% gelatinase, and 36% -glucuronidase, following stimulation with 10^–7 M FMLP for 10 min). This result suggests the involvement of VAMP-1/syntaxin 4 complexes in the secretion of specific/tertiary and azurophilic granules. We found coimmunoprecipitation of syntaxin 4 and VAMP-7 only under conditions that promoted azurophilic granule exocytosis, i.e., in FMLP-treated neutrophils, but not in untreated or PMA-treated neutrophils (Fig. 10B), suggesting that VAMP-7/syntaxin 4 complexes were specifically formed during the azurophilic granule secretion. These results further support a role for syntaxin 4 as a major target Q-SNARE in the plasma membrane of human neutrophils, acting as an anchor for the distinct granule-located SNAREs.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. Combinatorial SNARE complexes in activated human neutrophils. SNAP-23 and VAMP-2 (A), and syntaxin 4 (B) were immunoprecipitated (IP) from detergent-solubilized cell extracts of resting (Rest) and of PMA- or FMLP-activated human neutrophils. Immunoprecipitates were resolved by SDS-PAGE and subjected to an immunoblot analysis using Abs directed against the indicated SNAREs (immunoblot).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

View larger version (30K): [in this window] [in a new window]   FIGURE 11. Model for the involvement of SNARE proteins in human neutrophil exocytosis. Schematic diagram portrays a currently plausible mechanism for the involvement of SNARE proteins in granule release. Syntaxin 4 (Q-SNARE) and VAMPs (R-SNARE) contain transmembrane domains, whereas SNAP-23 (Q-SNARE) is bound to the granule membrane through a palmitoylated cysteine-rich membrane anchor domain located at the central part of the molecule connecting the two helical Q-SNARE motifs. Although not represented in the scheme for simplicity, all four SNARE motifs of the SNARE complex are arranged in parallel, with the N termini at one end of the complex and the C termini at the membrane-anchor end. Different combinations of granule SNARE proteins interact with plasma membrane syntaxin 4 to form a 3 (Q-SNARE)/1 (R-SNARE) complex in the release of specific and tertiary granules.

Additional proteins, including small GTPases, could also control the exocytic pathway in human neutrophils. Distinct subsets of low m.w. GTP-binding proteins have been associated with the membranes of the different neutrophil granule populations (59, 60, 61). Rac2 has been shown to be critical for the exocytosis of neutrophil azurophilic granules using bone marrow and peritoneal exudate neutrophils from Rac2-deficient mice (62). Thus, small GTPases and SNARE proteins seem to play a pivotal role in neutrophil exocytosis, and hence these molecules could link together to form a common signaling pathway for neutrophil degranulation (63). Elucidation of the molecular mechanisms involved in neutrophil granule secretion is of major pharmacological interest inasmuch as new anti-inflammatory targets can be unraveled.

	Acknowledgments

 
We are grateful to Nico Ong for the preparation of micrographs. We thank the Blood Bank of the Hospital Universitario de Salamanca for blood supply.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants FIS-FEDER 04/0843, 02/1199, 01/1048 from the Fondo de Investigación Sanitaria and European Commission, Grant SAF2005-04293 from the Ministerio de Educación y Ciencia, Fundación de Investigación Médica Mutua Madrileña, Grant BM05-30-0 from Fundación "la Caixa", and Grant CSI04A05 from Junta de Castilla y León. C.G. was supported by the Ramón y Cajal Program from the Ministerio de Educación y Ciencia of Spain.

² Address correspondence and reprint requests to Dr. Faustino Mollinedo, Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain. E-mail address: fmollin{at}usal.es

³ Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; SNAP, synaptosome-associated protein; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; VAMP, vesicle-associated membrane protein; TeTx, tetanus toxin.

Received for publication December 28, 2005. Accepted for publication June 8, 2006.

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