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Human Neutrophils the Secretion of Cytoplasmic Granules In The Journal of Immunology Combinatorial SNARE Complexes Modulate the Secretion of Cytoplasmic Granules in Human Neutrophils1 Faustino Mollinedo,2* Jero Calafat,† Hans Janssen,† Bele´n Martı´n-Martı´n,* Javier Canchado,* Svetlana M. Nabokina,* and Consuelo Gajate*‡ 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, coimmunopre- cipitation, 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. The Journal of Immunology, 2006, 177: 2831–2841. he innate immune response is characterized by the accu- differ in their respective contents and proneness for exocytosis. It mulation of granulocytic cells at sites of infection and is well established that major differences exist between the differ- T injury. The extracellular release of granular contents from ent granule populations, specifically regarding the extent to which these cells yields an effective barrier in combating infectious or- they are mobilized both in vitro and in vivo (5–8). Specific and ganisms, but also can cause massive organ injury during acute or tertiary granules are secreted in response to a plethora of stimuli chronic inflammatory conditions (1). Polymorphonuclear neutro- including chemoattractants, phorbol esters and calcium iono- phils (PMN),3 which are the most numerous granulocyte subpopu- phores; secretion of azurophilic granules (in addition to specific lation, comprise the first line of cellular defense against infection and tertiary granules) occurs during phagocytosis or when neutro- and constitute a key mediator in inflammation (2). Central to their phils are pretreated with the fungal metabolite cytochalasin B be- physiological role, neutrophils contain three major types of cyto- fore stimulation with a wide variety of stimuli (5–10). Specific and plasmic granules: azurophilic or primary granules, specific or sec- tertiary granules are more readily exocytosed upon cell activation ondary granules, and gelatinase-rich tertiary granules (3, 4), which than azurophilic granules (6, 7, 11), modulating crucial neutrophil functions, including adhesion to endothelium, diapedesis, and kill- ing of microorganisms. Azurophilic granules mainly fuse with *Centro de Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Consejo Superior de Investigaciones Cientificas-Universidad de Salamanca, phagosomal membranes, and contain a wide array of lytic enzymes Salamanca, Spain; †Division of Cell Biology, The Netherlands Cancer Institute, Am- and proteins with bactericidal activity that can be detrimental to ‡ sterdam, The Netherlands; and Unidad de Investigacio´n, Hospital Universitario de the surrounding tissue if secreted in an unregulated way. In con- Salamanca, Salamanca, Spain trast, specific and tertiary granules mainly fuse with the plasma Received for publication December 28, 2005. Accepted for publication June 8, 2006. membrane, and contain a number of proteins involved in adhe- 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 sion and extravasation, comprising a reservoir of plasma mem- with 18 U.S.C. Section 1734 solely to indicate this fact. brane proteins that are translocated to the cell surface upon 1 This work was supported by Grants FIS-FEDER 04/0843, 02/1199, 01/1048 from neutrophil activation (3, 4, 12–14). In addition to the granules the Fondo de Investigacio´n Sanitaria and European Commission, Grant SAF2005- discussed, human neutrophils contain yet another releasable 04293 from the Ministerio de Educacio´n y Ciencia, Fundacio´n de Investigacio´n Me´d- ica Mutua Madrilen˜a, Grant BM05-30-0 from Fundacio´n “la Caixa”, and Grant membrane-bound organelle-type named the secretory vesicle CSI04A05 from Junta de Castilla y Leo´n. C.G. was supported by the Ramo´n y Cajal that are storage organelles for membrane receptors (3, 4). An Program from the Ministerio de Educacio´n y Ciencia of Spain. inflammatory reaction always accompanies infections; its mag- 2 Address correspondence and reprint requests to Dr. Faustino Mollinedo, Centro de nitude usually depends on the extent of neutrophil infiltration Investigacio´n del Ca´ncer, Instituto de Biologı´a Molecular y Celular del Ca´ncer, Con- sejo Superior de Investigaciones Cientificas-Universidad de Salamanca, Campus and the release of neutrophil products. Thus, secretion of neu- Miguel de Unamuno, E-37007 Salamanca, Spain. E-mail address: [email protected] trophil granules must be tightly regulated to achieve an efficient 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; SNAP, syn- antimicrobial response and to prevent uncontrolled release of aptosome-associated protein; SNARE, soluble N-ethylmaleimide-sensitive factor at- tachment protein receptor; VAMP, vesicle-associated membrane protein; TeTx, tet- noxious components that could lead to tissue destruction or un- anus toxin. desirable inflammatory events. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2832 COMBINATORIAL SNARE COMPLEXES IN PMN EXOCYTOSIS The ability of intracellular secretory organelles to specifically Cell culture recognize appropriate acceptor membrane targets underlies the or- Human leukemia HL-60 cells were grown in RPMI 1640 supplemented ganization of the exocytic pathway. Soluble N-ethylmaleimide- with 10% (v/v) heat-inactivated FCS, L-glutamine, and antibiotics. Neu- sensitive factor attachment protein receptor (SNARE) proteins are trophil differentiation of HL-60 cells was induced with 1.3% (v/v) DMSO key mediators of membrane fusion (15). The neuronal SNARE as described previously (30). proteins synaptobrevin/vesicle-associated membrane protein (VAMP)-2, syntaxin 1A, and 25-kDa synaptosome-associated pro- Neutrophil isolation and activation tein (SNAP-25) share a homologous SNARE motif of ϳ60 aa Neutrophils were obtained from human peripheral blood by dextran sedi- (16–18), which mediates the association of SNARE proteins into mentation and Ficoll-Hypaque centrifugation as described previously (23). 6 a core complex composed of a tightly packed parallel four-helical Freshly isolated human neutrophils were resuspended at 3–5 ϫ 10 bundle. VAMP-2 and syntaxin 1A contribute one ␣-helix each and cells/ml in HEPES/glucose buffer (150 mM NaCl, 10 mM HEPES, 5 mM KCl, 1.2 mM MgCl , 1.3 mM CaCl , 5.5 mM glucose, (pH 7.5)). For cell ␣ 2 2 SNAP-25 contributes two -helices. All known SNARE motifs activation assays, neutrophils were incubated with 100 ng/ml PMA for 10 fall into two major subfamilies that contain either a conserved min at 37°C or preincubated with 5 ␮g/ml cytochalasin B for 5 min at Ϫ glutamine (Q) or arginine (R) at the ionic “0” layer in the middle 37°C, and then stimulated with 10 7 M FMLP for 10 min at 37°C. Release ␤ of the bundle, leading to the classification of SNARE proteins into of gelatinase, lactoferrin, -glucuronidase, and peroxidase following neu- trophil activation was determined as described elsewhere (6, 11, 31). Q-SNARE and R-SNARE. The hydrophilic 0 layer of the neuronal SNARE complex is made of three glutamine residues from the Subcellular fractionation Q-SNARE motifs (one contributed by syntaxin 1A and two by SNAP-25) and one arginine residue from the R-SNARE motif Resting neutrophils were resuspended in 50 mM Tris-HCl (pH 7.5), con- (contributed by VAMP-2) (18). The crystal structure of the endo- taining 2 mM PMSF, disrupted by repeated freeze-thaw, and soluble and membrane fractions from postnuclear extracts were prepared as described somal SNARE complex has revealed that it is also formed by a previously (24). four-helix bundle containing three Q-SNARE contributors (syn- To prepare the distinct subcellular fractions, freshly isolated resting and taxin 7, syntaxin 8, vti1b) and one R-SNARE (endobrevin/ PMA-activated neutrophils (ϳ3–5 ϫ 108) were gently
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