Cytokine secretion in macrophages and other cells: Pathways and mediators

ARTICLE IN PRESS

Immunobiology 214 (2009) 601–612 www.elsevier.de/imbio

REVIEW

Cytokine secretion in macrophages and other cells: Pathways and mediators Jennifer L. Stow, Pei Ching Low, Carolin Offenha¨user, Daniele Sangermani Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia Received 14 November 2008; accepted 14 November 2008

Abstract Cytokines and other immune mediators are secreted by cells of the immune system during immune responses and as a means of communication. While the functions of these cytokines, chemokines and mediators are well known, the intracellular pathways that lead to their secretion by different cells are only now being fully documented. Cytokines in some cells are released from secretory granules while in other cells they are released via constitutive secretory pathways that instead have more dynamic vesicular carriers. Recent studies have revealed that newly synthesized cytokines can be routed via compartments such as recycling endosomes prior to their secretion. Here we describe and show examples of some of the pathways used for cytokine trafficking and release in macrophages, including some of the cellular machinery required for this transport. Increasingly, these trafficking pathways are revealed as having important regulatory roles in the execution of immune responses. Crown Copyright r 2008 Published by Elsevier GmbH. All rights reserved. Keywords: Cytokines; Endosomes; SNAREs; Granules

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Trafficking through secretory pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Different pathways for cytokine release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Constitutive secretion of cytokines in macrophages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The recycling endosome and selective cytokine secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) as regulators in secretory pathways. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

602 602 603 604 606 607 609 609 610

Abbreviations: CTL, cytotoxic T lymphocytes; GFP, green fluorescent protein; IFN-g, interferon gamma; Ig, immunoglobulin; IL, interleukin; IL-6, interleukin 6; LPS, lipopolysaccharide; MIF, macrophage migration inhibitory factor; RANTES, regulated upon activation, normal T cell expressed and secreted; SNAP, soluble N-ethylmaleimide sensitive factor accessory protein; SNARE, soluble N-ethylmaleimide sensitive factor accessory protein receptor; TACE, tumor necrosis factor alpha converting enzyme; TGF, transforming growth factor; TGN, trans Golgi network; TLR4, toll-like receptor 4; TNF, tumour necrosis factor alpha; VAMP, vesicle associated membrane protein. Corresponding author. Tel.: +61 7 3346 2034; fax: +61 7 3346 2101. E-mail address: [email protected] (J.L. Stow). 0171-2985/$ - see front matter Crown Copyright r 2008 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2008.11.005

ARTICLE IN PRESS 602

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

Introduction The release of cytokines as soluble messengers is a fundamental mechanism for cell–cell communication and regulation within the immune system. Macrophages and granulocytes release cytokines to activate and recruit other cells during inflammation, or as direct killing agents (Holgate, 2000; Hume, 2006). Signalling from pattern recognition motif receptors or other surface receptors triggers cytokine synthesis and release by host cells responding to microbial invasion or other stimuli. Cytokines also link cells of the immune system to those in surrounding tissues. During development, after injury or in tumour growth cytokines can convey destructive or reparative signals to other cells (Salamonsen et al., 2007; Yan and Hansson, 2007). The subsequent downstream effects of most cytokines after binding to their cognate receptors on target cells are to activate or inhibit cellular functions in paracrine and autocrine fashions (Lazar-Molnar et al., 2000). Nevertheless, we have only a rudimentary understanding of how newly synthesized cytokines are transported within cells and secreted, and moreover, of how cells orchestrate the release of multiple cytokines with high fidelity. The secretory pathways, routes, organelles and molecular machinery that control cytokine secretion must ensure the regulated, timed and often directional release of cytokines from cells (Huse et al., 2006; Murray et al., 2005a). This is a complex task, especially given that many cells, including macrophages, secrete not one but many cytokines, and this secretion must occur alongside other cellular functions occurring as part of a cell’s immune response (Murray et al., 2005a). Current knowledge increasingly shows that many cells of the immune system have devised clever routes and regulatory systems to control the targeting and timing of cytokine release. Understanding how cytokines are trafficked and how their secretion is managed provides vital insight into basic immunity and into the many diseases associated with aberrant or excessive cytokine release (McInnes and Schett, 2007).

Trafficking through secretory pathways The early steps of secretory pathways are common to all eukaryotic cells. Newly synthesized protein precursors translated into the endoplasmic reticulum (ER) are folded, quality-checked and have the beginnings of glycosylation before being loaded into vesicular carriers for transport to the Golgi complex where post-translational processing and glycosylation continue (Brown and Stow, 1996; Farquhar and Palade, 1998). The trans Golgi network (TGN) is the last station in the Golgi complex (Farquhar and Palade, 1998) and beyond the TGN, secretory pathways diverge and differ in the

routes, carriers and other organelles they use to transport cytokines, other mediators, transmembrane proteins and membranes themselves, to the cell surface. Two major categories of secretory or exocytic pathways are currently known. Firstly, all cells have constitutive secretory pathways in which continuous streams of small, pleiomorphic vesicles or dynamic tubulovesicular carriers, transport newly synthesized protein precursors from the TGN to the cell surface or to intervening endosomes (Bard and Malhotra, 2006). Constitutive pathways typically result in the continuous release of small quantities of cytokines or other products. However, in some cells, such as activated macrophages, the molecules and carriers serving this pathway can be upregulated to increase trafficking for more abundant release of cytokines in response to cell activation (Lieu et al., 2008; Pagan et al., 2003). Secondly, professional secretory cells such as those with endocrine and exocrine functions, have additional, ‘regulated’ or granule-mediated secretory pathways (Arvan and Castle, 1998). After processing in the Golgi complex, selected mediators, hormones, enzymes and other specialized cargo, are redirected for packaging into secretory granules where these molecules remain stored until their release from the granules is triggered by specific signals (Arvan and Castle, 1998). Some cells in the haematopoietic system and melanocytes have granules that are specialized secretory lysosomes (Blott and Griffiths, 2002). Secretory lysosomes in granulocytes are most often preformed structures storing secretory products while awaiting stimulation, while in CTLs the protein components of secretory lysosomes and the structures themselves are produced only upon cell activation (Blott and Griffiths, 2002; Griffiths and Argon, 1995; Olsen et al., 1990). Ultimate release of the contents of granules or secretory lysosomes can occur via full fusion of the granules with the plasma membrane, by piecemeal degranulation wherein granule content is transported to the cell surface in small vesicles, or by a process known as compound exocytosis where granules fuse with each other at the cell surface (reviewed by Logan et al., 2003). Cytokines, it appears, can use any of these pathways for secretion and do so across a variety of cell types. Finally, cytokines such as IL-1a and b are released by a non-classical or unconventional secretory route in which a number of cytokines and growth factors synthesized in the cytoplasm are released through the plasma membrane without passing through the traditional organelles such as the ER and Golgi complex (reviewed in Nickel, 2003). IL-1b, for instance, lacks a signal peptide, is produced on free ribosomes in the cytoplasm, and is cleaved by caspase-1 of the inflammasome prior to its release (Dinarello, 1997; Ogura et al., 2006). How these cytokines escape from cells is still largely a mystery. Multiple pathways and mediators

ARTICLE IN PRESS J.L. Stow et al. / Immunobiology 214 (2009) 601–612

have been invoked, including ABC transporters, secretory lysosomes, endosomes, membrane blebbing (Andrei et al., 1999; Carta et al., 2006; Hamon et al., 1997) and most recently, transport via multivesicular body-derived exosomes (Qu et al., 2007). Understanding the release of these cytokines now awaits further support for any of these models.

Different pathways for cytokine release Granulocytes release cytokines from secretory granules or small vesicles and do so in ways that augment or regulate this release to coincide with a cellular immune response. Mast cells are heterogeneous granule-containing haematopoietic cells with primary roles in responding to parasitic infections and in mediating allergic responses (Metcalfe et al., 1997). Aggregation of the IgE receptor FceRI by binding to multivalent IgE-coated antigens causes rapid degranulation of mast cells, resulting in the release of various mediators such as histamine, prostaglandins and leukotrienes (Kinet, 1999). But, mast cells also express pattern recognition motif receptors such as toll like receptor 4 (TLR4) to detect bacterial infections as well as having complement, cytokine and growth factor receptors (Varadaradjalou et al., 2003). Stimulation of mast cells through these receptors causes differential mediator release (McCurdy et al., 2003; Theoharides et al., 2007; Varadaradjalou et al., 2003). Piecemeal degranulation allows the selective secretion of particular mediators, as does the stimulusdependent de novo synthesis of cytokines and their secretion via the mast cell constitutive secretory pathway (Dvorak, 1992; Theoharides et al., 2007). Mast cells secrete pre-formed chemokines IL-8 and RANTES from granules, as well as releasing de novo synthesized cytokines such as interferon gamma (IFN-g), macrophage migration inhibitory factor (MIF) and various interleukins (Theoharides et al., 2007). Interestingly, the cytokine tumour necrosis factor alpha (TNF) is secreted via both pathways (Nigrovic and Lee, 2005). Notably, mast cells are the only cells known to date to presynthesize TNF and store it in granules where it is held ready for sudden release upon degranulation (Gordon and Galli, 1990; Nigrovic and Lee, 2005; Olszewski et al., 2006, 2007; Walsh et al., 1991). Like mast cells, eosinophils are part of the innate immune system. These cells are involved in antihelminthic immune responses and in the pathogenesis of asthma and allergies (Adamko et al., 2005; Anthony et al., 2007). Eosinophils store a variety of cytokines, chemokines and growth factors in their secretory lysosomes, termed crystalloid granules (Moqbel and Coughlin, 2006). Piecemeal degranulation also seems to be an important mechanism for the differential secretion of cytokines from eosinophils (Lacy et al., 1999; Spencer

603

et al., 2006). This piecemeal mode of release is important for staging the release of cytokines which have opposing effects, such as IL-4 and IL-12 (Moqbel and Coughlin, 2006). The mechanisms that sort specific cytokines into the vesicles for transport from the granule to the cell surface are largely unknown but may include a role for intracellular pools of cytokine receptors (Spencer et al., 2006). Neutrophils have three different types of granules: primary (azurophilic), secondary (specific) and tertiary (gelatinase) granules as well as secretory vesicles (Mollinedo et al., 1999). The granules are used for the secretion of preformed, stored antimicrobial peptides and proteolytic enzymes (Borregaard et al., 2007). Additional mechanisms are needed in these cells to differentially load and secrete the different types of granules (Le Cabec et al., 1996). To accomplish this, granules are formed and loaded at different stages of neutrophil development in the bone marrow resulting in granules with distinct cargo and trafficking machinery (Borregaard et al., 2007; Gullberg et al., 1999). Cytokines, including IL-1a and b, TNF and CCchemokine ligand 3 (CCL3) are produced at later stages in the life cycle of neutrophils, namely, during the migration of the neutrophils into tissue (Borregaard et al., 2007; Kuhns and Gallin, 1995). Little is known about the secretion pathways responsible for their release. The storage and release of IL-8 from secretory vesicles, which are loaded by endocytosis during late stages of neutrophil development in the bone marrow, is controversial (Kuhns and Gallin, 1995; Pellme et al., 2006). T lymphocytes also secrete cytokines. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells release cytolytic granules to kill virally infected or transformed cells (reviewed in Hamerman et al., 2005; Joshi and Kaech, 2008). They also secrete a range of cytokines with immunostimulatory effects, antimicrobial activities or cytokines such as IFN-g can block viral replication in infected cells (Boehm et al., 1997; Copeland, 2002). Granule-mediated secretory pathways directed towards target cells at the immunological synapse are a wellknown feature of CTLs and NK cells. T helper cells (CD4+ T cells) also secrete cytokines although much less is known in general about secretory pathways or organelles in these cells. Earlier studies indicated that cytokines can be directed towards immunological synapses formed with APC (antigen presenting cells) with accumulation of cytokines in the synapses (reviewed in Jolly and Sattentau, 2007). More recently however, it has been shown that CD4+ cells have distinct pathways for polarized release of different cytokines, releasing them either at the synapse or elsewhere on the surface (Huse et al., 2006). Thus, IL-2 and IFN-g are secreted into the synapse to communicate with antigen presenting cells (APC),

ARTICLE IN PRESS 604

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

whereas proinflammatory cytokines such as TNF, IL-3 and CCL3 are secreted from other points on the cell surface and in a multi-directional manner for dispersal to promote a localized inflammatory response (Huse et al., 2006).

proinflammatory cytokines and to generally delimit the inflammatory response (Elenkov and Chrousos, 2002). TNF is a potent, multifunctional cytokine, and its rapid synthesis (Raabe et al., 1998) and early release serve to activate and recruit other cells to sites of microbial invasion and infection. TNF released by macrophages has many roles in sepsis, and while its secretion is fundamental to the immune response, excessive secretion of TNF in acute or chronic inflammatory disease is a significant clinical problem (Beutler, 1995). In lipopolysaccharide (LPS)-activated macrophages, newly synthesized TNF precursors can be detected by immunostaining in the Golgi complex, where these transiently accumulate (Shurety et al., 2000; Fig. 1). TNF is ultimately delivered to the cell surface, where it is normally rapidly cleaved by TNF converting enzyme (TACE or ADAM17) in order to release its ectodomain as a soluble cytokine (Black et al., 1997). Detection of TNF on the cell surface can be enhanced experimentally by incubating cells with a TACE inhibitor (Glaser et al., 1999; Pagan et al., 2003; Shurety et al., 2000). Immunostaining then shows TNF decorating the surface of activated cells (Fig. 1A and B).

Constitutive secretion of cytokines in macrophages Macrophages are protagonists of innate immunity which offer surveillance of tissues and phagocytose and degrade invading microbes and dying cells (Hume, 2006). Activated macrophages secrete an array of cytokines to recruit and activate other cells to initiate adaptive immune responses (Aderem and Ulevitch, 2000). Upon activation by LPS, macrophages first synthesize and secrete a temporal sequence of proinflammatory cytokines, including TNF as an early response cytokine, and IL-6, which is secreted in an overlapping time frame (Bopst et al., 1998). This is followed some time later by secretion of anti-inflammatory mediators IL-10, IL-4 and TGF-b, which are intended to shut off release of −TAPI

+TAPI II

Merge

I

G G

Golgi TNF

G TNF 10nm III

GFP 15nm IV

Surface TNF

PM

PM TNF 15nm Clathrin 5nm

PM GFP 15nm

Fig. 1. Newly synthesized TNF is located in the Golgi complex and on the cell surface. (A) Shortly after LPS activation, TNF can be immunostained with specific antibodies in the perinuclear Golgi complex of fixed, permeabilized RAW267.4 macrophages. Although TNF is then transported to the cell surface, it is cleaved by TACE and released and is therefore not usually stained at the surface ( TAPI); incubation of cells with a TACE inhibitor (+TAPI) prevents release of TNF which is now stained strongly on the surface and can also still be found at the Golgi. Immunostaining and TACE inhibition performed as previously described (Manderson et al., 2007; Pagan et al., 2003; Shurety et al., 2000). Green: immunostaining of TNF after cell permeabilization; red: surface immunostaining of TNF on unpermeabilized cells; blue: DAPI staining of nuclei. (B) Immunogold labelling of endogenous TNF with TNF antibodies or of recombinant GFP-TNF with GFP antibodies on ultrathin cryosections of fixed RAW267.4 cells. Different size gold particles were used for single or double labelling of sections as indicated. Panels I and III show endogenous newly-synthesized TNF in the Golgi complex and then appearing on the cell surface where the plasma membrane is denoted by labelling with clathrin antibodies. Panels II and IV show GFP-TNF on the Golgi complex and then decorating the surface of cells. Immunogold labelling and electron microscopy were performed as previously described (Manderson et al., 2007). G: Golgi complex; PM: plasma membrane; N: nucleus. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ARTICLE IN PRESS J.L. Stow et al. / Immunobiology 214 (2009) 601–612

track by conventional microscopy due to the fleeting existence of the carriers and the relatively dilute cargo of TNF. Indeed, these same issues have long clouded our ability to visualize the constitutive trafficking of any endogenous cargo proteins inside cells. So, for TNF, information detailing its processing and secretion was previously garnered only from biochemical experiments (Black et al., 1997; Decoster et al., 1995; Glaser et al., 1999; Jue et al., 1990; Shurety et al., 2000; Solomon et al., 1997; Watanabe et al., 1998).

GFP-TNF

Rab11

Merge

BMM

RAW 264.7

How is TNF transported from the Golgi complex to the cell surface? Not by a direct or simple route, as it turns out, but by one that is adapted to the multiple functions of macrophages engaged in microbial defence. Newly synthesized TNF is loaded into a specific subset of LPS-regulated tubulovesicular carriers (Lieu et al., 2008), which bud off the TGN to transport TNF towards the cell surface. Until recently, the constitutive trafficking of TNF between the Golgi complex and the cell surface had been an invisible process, one difficult to

605

Fig. 2. GFP-TNF trafficking via the recycling endosome in live cells. (A) RAW267.4 cells or primary bone marrow macrophages (BMM) were transiently transfected with GFP-TNF and viewed by confocal imaging 4 h later. GFP-TNF is seen accumulated in the perinuclear Golgi complex and on some more peripheral compartments and light staining is visible on the cell surface. (B) A RAW267.4 cell was allowed to ingest dyed transferrin and it was transfected to express GFP-TNF. The large panel shows a single frame of the whole cell (N: nucleus) while the series of smaller panels show successive frames in a movie. GFP-TNF (green) is seen colocalized on several recycling endosomes (REs) with transferrin (red). GFP-TNF is segregated for trafficking in a carrier (arrowhead) that is seen leaving one RE and travelling to another and then towards the cell surface. (C) GFP-TNF (green) in a transfected cell is shown colocalizing with immunostaining of the RE marker Rab11 (red) in peripheral compartments of a macrophage. The inset shows at higher magnification a single RE with both labels. (D) A macrophage cotransfected with m cherryIL-6 (red), GFP-TNF (green) and immunostained for transferrin (tfn; blue). The inset shows an area with REs that have all three proteins as cargo colocalized (white). The constructs used here, confocal imaging and immunostaining are as previously described (Manderson et al., 2007). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ARTICLE IN PRESS 606

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

IL-6 Early endosome

Late endosome

TNF

IL-6 TNF

Recycling endosome

Phagocytic cup

Golgi complex ER

Fig. 3. Cytokine secretion and trafficking pathways in macrophages. The constitutive secretory route followed by the inflammatory cytokines IL-6 and TNF in macrophages is shown (solid red lines). This pathway diverges at the recycling endosome for separate delivery of cytokines to the cell surface. Dotted lines indicate other major exocytic (red) and endocytic (blue) routes in cells connecting organelles, some of which intersect at the recycling endosome with cytokine trafficking. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In recent years however, the ability to fluorescently tag and overexpress proteins destined for secretion and the ability to track them inside living cells by imaging have produced dramatic new insights into constitutive secretion (Lippincott-Schwartz et al., 1998). Imaging of green fluorescent protein (GFP)-tagged TNF (GFPTNF) expressed in live RAW267.4 cells or primary bone marrow macrophages confirms that it initially accumulates in the Golgi complex (Fig. 2A) and can then be found on the surface of cells (Fig. 1B). The trafficking of GFP-TNF thus replicates that of endogenous TNF (see Fig. 1). Constitutive carriers or ‘secretory vesicles’, such as those leaving the TGN, are pleiomorphic but often tubular and very dynamic and some of these carriers can be seen transporting GFP-TNF in and out of macrophages (Lock et al., 2005; Lock and Stow, 2005; Murray et al., 2005a). Detailed analysis of movies of GFP-TNF moving out of the Golgi complex provided the first clues that TNF was delivered from the Golgi to other organelles in the cell periphery rather than going directly to the cell membrane (Murray et al., 2005a). Costaining or coexpression of various marker proteins revealed that these intermediate compartments are recycling endosomes (Murray et al.,

2005a; Figs. 2C and 3). Thus, for the first time, the recycling endosomes have to be considered in the context of cytokine secretion.

The recycling endosome and selective cytokine secretion The recycling endosome was first characterized as a key compartment for the surface recycling of transferrin and its receptor back to the cell surface after their internalization for the uptake of iron (see reviewed by Maxfield and McGraw, 2004; Rodriguez-Boulan et al., 2005). This is a particularly active and important pathway in macrophages and the propensity of these cells for recycling through this compartment can be demonstrated by allowing cells to internalize fluorescently labelled transferrin, which then transiently accumulates in recycling endosomes in live cells (Fig. 2B and D). The recycling endosome is now recognized as a key central compartment for endocytic, recycling and exocytic pathways in many cell types (van Ijzendoorn, 2006). Studies in epithelial cells for instance, have confirmed that constitutive secretory

ARTICLE IN PRESS J.L. Stow et al. / Immunobiology 214 (2009) 601–612

pathways intersect with recycling endosomes as the penultimate destination for cargo being transported to either the apical or basolateral cell surface domains (Ang et al., 2004; Lock and Stow, 2005). How the recycling endosomes handle the volume and diversity of cargo moving through them is not yet understood. The polarized and selective exit of cargo moving out of the recycling endosome certainly suggests that some protein sorting occurs within this compartment, a function thought previously to take place only at the TGN (Farquhar and Palade, 1998). Such mechanisms have direct relevance to the selective secretion of cytokines and the coincident capacity to recycle transferrin in inflammatory macrophages. The convergence of secretory cargo and recycling cargo such as GFP-TNF and transferrin, respectively, can be seen in recycling endosomes (Fig. 2B and D). TNF has a special fate upon leaving the recycling endosomes in macrophages engaged in anti-microbial activity. This fate is inextricably tied to another function of the recycling endosome, which is to contribute extra membrane for the formation of phagocytic cups (Huynh et al., 2007). The transmembrane precursor of TNF is efficiently packaged with this membrane and delivered to the cell surface at the site of phagocytic cup formation (Murray et al., 2005a). TACE is positioned at the phagocytic cups ready to cleave and release TNF before closure of the cup. By combining cup formation and TNF surface delivery, the macrophage uses a single transport step from the recycling endosome for polarized delivery of membrane for phagocytosis and cytokine release, ensuring TNF is abundantly and promptly secreted upon a microbial challenge where it is necessary for containment and clearance of local infection (Aderem and Ulevitch, 2000). However, this exit route is not used for all cytokines. IL-6 is a soluble protein made for some time alongside TNF; both cytokines accumulate in the Golgi together and are transported to recycling endosomes where they are subsequently colocalized (Manderson et al., 2007; Fig. 3). IL-6 is not delivered to phagocytic cups in a polarized fashion like TNF. Instead it appears to be loaded into carriers for delivery elsewhere on the cell surface. Additionally the release of IL-6 occurs in a different temporal pattern to other cytokines (Jones, 2005; Manderson et al., 2007). These observations suggest that the recycling endosome actively sorts cytokines for their individual release, perhaps by segregating them into different sub-domains within recycling endosomes (Manderson et al., 2007). Thus the recycling endosome in macrophages is preferentially the compartment orchestrating cytokine release in a spatial and temporal fashion during the immune response. It will be interesting to see whether these endosomes function similarly for granule-mediated release in other cell types.

607

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) as regulators in secretory pathways Members of many different protein families, such as small GTPases, golgins, coat proteins, adaptors, and lipids are required for intracellular trafficking (Gleeson et al., 2004). Members of the SNARE family of membrane fusion proteins are located on all organelles in trafficking pathways as key components of their machinery. Cognate pairing of a Q-SNARE complex with an R-SNARE protein on the apposing membrane forms a four helix bundle (Qa,b,c helices plus an R-SNARE helix), a trans-SNARE complex, which is essential for fusion of vesicles or granules with target membranes or for inter-organelle fusion (see review by Jahn and Scheller, 2006). The syntaxin molecules of Q-SNARE complexes and the VAMPs as R-SNAREs are generally transmembrane proteins and their steady state locations (and thus sites of function) help to denote key steps in trafficking pathways. SNARE proteins have emerged as functional mediators and as particularly useful markers of cytokine secretory pathways (Huse et al., 2006; Moqbel and Coughlin, 2006; Murray et al., 2005b; Pagan et al., 2003; Stow et al., 2006). The Q-SNARE components syntaxin4 (Qa SNARE) and SNAP23 (Qb,c SNARE) are found on the surfaces of a number of cell types where they control the fusion of incoming constitutive vesicles or secretory granules at the cell surface as the final step in secreting cytokines and other cargo (Stow et al., 2006). In cells of the immune system, syntaxin4 and SNAP23 often form trans-SNARE complexes by pairing with R-SNAREs such as VAMP8, VAMP7 or VAMP2 on secretory granules. For instance, in eosinophils, VAMP7 and VAMP8 both localize to the crystalloid granules (Moqbel and Coughlin, 2006) and VAMP7 has recently been shown to be required for mediator release (Logan et al., 2006). Piecemeal degranulation in these cells is mediated by VAMP2 on the secretory vesicles (Lacy et al., 2001) that selectively transport granule contents to the cell surface where SNAP23–syntaxin4 acts as the likely Q-SNARE partner complex (Logan et al., 2002). Syntaxin4 and SNAP23 on the surface of mast cells mediate fusion of incoming granules with the plasma membrane during degranulation, most likely by pairing with VAMP8 as a granule-associated R-SNARE (Lippert et al., 2007; Paumet et al., 2000; Puri and Roche, 2008). In neutrophils, the coupling of VAMP2 on secretory vesicles and secondary and tertiary granules with SNAP23 and syntaxin4 is responsible for secretion (Mollinedo et al., 2006). The fusion of azurophilic granules with the membrane at the phagocytic cup involves VAMP7 and syntaxin4 and possibly SNAP23 (Logan et al., 2006; Mollinedo et al., 2006). Taken

ARTICLE IN PRESS 608

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

together, these results demonstrate a high redundancy between the SNARE proteins used for the exocytosis of secretory granules from granulocytes. This redundancy is not limited to granulocytes but also includes other haematopoietic cells. Platelets also require SNAP23, syntaxin4 and VAMP8 for the exocytosis of their granules (Chen et al., 2000; Flaumenhaft et al., 1999;

Control

Ren et al., 2007). Moreover, cells outside the immune system such as adipocytes also utilize the syntaxin4–SNAP23 Q-SNARE for cell surface delivery of insulin regulated glucose transporters (Spurlin and Thurmond, 2006). In macrophages, the constitutive secretion of cytokines is mediated as a two-step, post-Golgi process

stx4 siRNA 34kDa stx4 23kDa SNAP23

Western Blot

siRNA

stx4 siRNA

*

Surface TNF

Intracellular TNF

Phalloidin/ Merge

Control

Fig. 4. Targeted siRNA knockdown of syntaxin4 and TNF cell surface delivery. (A) RAW267.4 cells were transfected with siRNA specific for syntaxin4 (stx4) or control cells transfected with no siRNA (shown) or cells transfected with scrambled siRNA (not shown). Western blotting shows knockdown of the stx4 protein. SNAP23, another SNARE protein and binding partner of stx4 is not affected by the stx4 knockdown. (B) ELISA assays were used to measure TNF secreted into the medium. The specific siRNA for stx4 reduced TNF secretion. ***Po0.001 by unpaired two-tailed t-test. (C) Cells transiently transfected with stx4 siRNA were fixed and immunostained for surface or Golgi TNF. Confocal image showing the absence of cell surface staining of TNF (red) in a cell expressing stx4 siRNA (*; panel II), as compared to control (I) and an adjacent untransfected cell. Staining of intracellular TNF (green) as a deliberate marker for stimulated, TNF producing cells. Phalloidin staining of cell actin cytoskeleton is shown in blue. siRNA knockdown and staining performed as in Murray et al., 2005a. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

ARTICLE IN PRESS J.L. Stow et al. / Immunobiology 214 (2009) 601–612

defined at each step by specific SNAREs (Stow et al., 2006). Cytokines are first transported out of the Golgi complex to the recycling endosome via pairing of the TGN Q-SNARE complex of syntaxin6-syntaxin7-Vti1b with the R-SNARE, VAMP3 (Murray et al., 2005b). VAMP3 on the recycling endosome is then the R-SNARE for transport to the cell surface by pairing – again – with the syntaxin4–SNAP23 Q-SNARE complex on the plasma membrane (Murray et al., 2005a). Some of these SNARE proteins and complexes also act in other cell types. Vti1b is associated with postGolgi and late endosome complexes in other cells and in T cells it is associated with cytokine secretion (Huse et al., 2006). Syntaxin6 is also associated with the multidirectional release of TNF and proinflammatory cytokines in T cells (Huse et al., 2006). The levels and concentrations of SNARE proteins can modulate the efficiency and volume of trafficking and secretion. Thus in neutrophils, different types of granules and vesicles express different densities of the R-SNARE VAMP2, which dictates their propensity for secretion. Both the propensity for secretion and VAMP2 density are highest in secretory vesicles, less so in gelatinase granules and less still in the earliest formed granules (Borregaard et al., 2007). This allows neutrophils to transport cytokine and chemokine receptors to the cell surface and to release antimicrobial peptides and matrix metalloproteinases without necessarily triggering the release of tissue-destructive serin proteases (Borregaard et al., 2007). In macrophages, constitutive trafficking occurs at relatively low levels in unactivated cells. The preactivation expression levels of individual SNARE proteins are rate-limiting for trafficking of cytokines after cell activation. Thus, LPS up-regulates the expression of relevant SNAREs, via regulation at gene and/or protein levels, to provide more SNAREs for more membrane fusion in activated cells (Murray et al., 2005b; Pagan et al., 2003; Stow et al., 2006). This coincides with increased numbers of carriers (Lieu et al., 2008) to support secretion of TNF and other cytokines. Experimentally altering levels of specific SNAREs also manipulates cytokine trafficking in macrophages. Transient overexpression of the TGN SNARE proteins syntaxin6 or Vti1b increases trafficking and secretion of TNF or IL-6 (Manderson et al., 2007; Murray et al., 2005b) whereas decreasing levels of VAMP3 by siRNA knockdown reduces secretion of these cytokines (Manderson et al., 2007; Murray et al., 2005a). Levels of syntaxin4 and SNAP23 as the surface Q-SNARE proteins are up-regulated by LPS (Pagan et al., 2003) and competitive inhibition of syntaxin4, by overexpression of non-membrane bound mutant forms, also blocks TNF surface delivery and secretion (Pagan et al., 2003). We now also demonstrate the importance of syntaxin4 expression for TNF secretion using an siRNA knockdown approach (Fig. 4). Introduction of specific siRNA

609

oligonucleotides reduced protein levels of syntaxin4 in macrophage cells (Fig. 4A) and this was accompanied by a significant, 4three-fold reduction in TNF secretion (Fig. 4B) and ablation of TNF surface delivery in affected cells (Fig. 4C). This result mirrors findings in other cells where siRNA knockdown of syntaxin4 in pancreatic beta cells, or in mice blocks glucosestimulated insulin secretion, but not basal insulin secretion (Spurlin and Thurmond, 2006). These findings confirm that expression levels of SNAREs, such as syntaxin4, are tightly coupled to the need for induced or regulated secretion across multiple pathways and in different cells.

Concluding remarks The examples reviewed here begin to show how cytokine secretion can be tailored to the needs of different cells in the immune system through a variety of secretory pathways and by the actions of specific regulatory molecules. The timing of cytokine release can be dictated by routing them through different granule populations or potentially through organelles such as recycling endosomes. Similarly, cytokines can be directed to specific release sites such as synapses or phagocytic cups by polarized or directional secretion (Huse et al., 2006; Murray et al., 2005a). Indeed secretory pathways must simultaneously orchestrate the spatial and temporal release of many different cytokines made by a single cell. Inevitably these secretory pathways intersect with other trafficking events and pathways in the cell for internalization, recycling, phagocytosis, antigen presentation and processing, and release of lytic molecules (Fig. 3). Further insights into the routes, compartments and molecules involved in regulating cytokine release pathways will be invaluable for a more detailed understanding of immune responses in homeostasis and disease. Large scale screening for regulated expression of trafficking proteins (Ravasi et al., 2007), coupled with siRNA knockdowns and live cell imaging of cargo transport, as exemplified here, can be employed to rapidly dissect trafficking pathways. The information gathered from such forays is potentially useful also for understanding various portals of pathogen entry and sequestration – which often involve subversion of endocytic and secretory pathways.

Acknowledgements The authors wish to thank Jason Kay and other members of the Stow laboratory for help in preparing the manuscript. This work was supported by a fellowship and Grants to JLS from the National Health & Medical Research Council of Australia.

ARTICLE IN PRESS 610

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

References Adamko, D.J., Odemuyiwa, S.O., Vethanayagam, D., Moqbel, R., 2005. The rise of the phoenix: the expanding role of the eosinophil in health and disease. Allergy 60, 13–22. Aderem, A., Ulevitch, R.J., 2000. Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787. Andrei, C., Dazzi, C., Lotti, L., Torrisi, M.R., Chimini, G., Rubartelli, A., 1999. The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol. Biol. Cell 10, 1463–1475. Ang, A.L., Taguchi, T., Francis, S., Folsch, H., Murrells, L.J., Pypaert, M., Warren, G., Mellman, I., 2004. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 167, 531–543. Anthony, R.M., Rutitzky, L.I., Urban Jr., J.F., Stadecker, M.J., Gause, W.C., 2007. Protective immune mechanisms in helminth infection. Nat. Rev. Immunol. 7, 975–987. Arvan, P., Castle, D., 1998. Sorting and storage during secretory granule biogenesis: looking backward and looking forward. Biochem. J. 332, 593–610. Bard, F., Malhotra, V., 2006. The formation of TGN-to-plasmamembrane transport carriers. Annu. Rev. Cell Dev. Biol. 22, 439–455. Beutler, B., 1995. TNF, immunity and inflammatory disease: lessons of the past decade. J. Invest. Med. 43, 227–235. Black, R.A., Rauch, C.T., Kozlosky, C.J., Peschon, J.J., Slack, J.L., Wolfson, M.F., Castner, B.J., Stocking, K.L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K.A., Gerhart, M., Davis, R., Fitzner, J.N., Johnson, R.S., Paxton, R.J., March, C.J., Cerretti, D.P., 1997. A metalloproteinase disintegrin that releases tumournecrosis factor-alpha from cells. Nature 385, 729–733. Blott, E.J., Griffiths, G.M., 2002. Secretory lysosomes. Nat. Rev. Mol. Cell Biol. 3, 122–131. Boehm, U., Klamp, T., Groot, M., Howard, J.C., 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15, 749–795. Bopst, M., Haas, C., Car, B., Eugster, H.P., 1998. The combined inactivation of tumor necrosis factor and interleukin-6 prevents induction of the major acute phase proteins by endotoxin. Eur. J. Immunol. 28, 4130–4137. Borregaard, N., Sorensen, O.E., Theilgaard-Monch, K., 2007. Neutrophil granules: a library of innate immunity proteins. Trends Immunol. 28, 340–345. Brown, D., Stow, J.L., 1996. Protein trafficking and polarity in kidney epithelium: from cell biology to physiology. Physiol. Rev. 76, 245–297. Carta, S., Tassi, S., Semino, C., Fossati, G., Mascagni, P., Dinarello, C.A., Rubartelli, A., 2006. Histone deacetylase inhibitors prevent exocytosis of interleukin-1beta-containing secretory lysosomes: role of microtubules. Blood 108, 1618–1626. Chen, D., Lemons, P.P., Schraw, T., Whiteheart, S.W., 2000. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 and 4 in lysosome release. Blood 96, 1782–1788.

Copeland, K.F., 2002. The role of CD8+ T cell soluble factors in human immunodeficiency virus infection. Curr. Med. Chem. 9, 1781–1790. Decoster, E., Vanhaesebroeck, B., Vandenabeele, P., Grooten, J., Fiers, W., 1995. Generation and biological characterization of membrane-bound, uncleavable murine tumor necrosis factor. J. Biol. Chem. 270, 18473–18478. Dinarello, C.A., 1997. Interleukin-1. Cytokine Growth Factor Rev. 8, 253–265. Dvorak, A.M., 1992. Basophils and mast cells: piecemeal degranulation in situ and ex vivo: a possible mechanism for cytokine-induced function in disease. Immunology 57, 169–271. Elenkov, I.J., Chrousos, G.P., 2002. Stress hormones, proinflammatory and antiinflammatory cytokines, and autoimmunity. Ann. N. Y. Acad. Sci. 966, 290–303. Farquhar, M.G., Palade, G.E., 1998. The Golgi apparatus: 100 years of progress and controversy. Trends Cell Biol. 8, 2–10. Flaumenhaft, R., Croce, K., Chen, E., Furie, B., Furie, B.C., 1999. Proteins of the exocytotic core complex mediate platelet alpha-granule secretion. Roles of vesicle-associated membrane protein, SNAP-23, and syntaxin 4. J. Biol. Chem. 274, 2492–2501. Glaser, K.B., Pease, L., Li, J., Morgan, D.W., 1999. Enhancement of the surface expression of tumor necrosis factor alpha (TNFalpha) but not the p55 TNFalpha receptor in the THP-1 monocytic cell line by matrix metalloprotease inhibitors. Biochem. Pharmacol. 57, 291–302. Gleeson, P.A., Lock, J.G., Luke, M.R., Stow, J.L., 2004. Domains of the TGN: coats, tethers and G proteins. Traffic (Copenhagen, Denmark) 5, 315–326. Gordon, J.R., Galli, S.J., 1990. Mast cells as a source of both preformed and immunologically inducible TNF-alpha/ cachectin. Nature 346, 274–276. Griffiths, G.M., Argon, Y., 1995. Structure and biogenesis of lytic granules. Curr. Top. Microbiol. Immunol. 198, 39–58. Gullberg, U., Bengtsson, N., Bulow, E., Garwicz, D., Lindmark, A., Olsson, I., 1999. Processing and targeting of granule proteins in human neutrophils. J. Immunol. Methods 232, 201–210. Hamerman, J.A., Ogasawara, K., Lanier, L.L., 2005. NK cells in innate immunity. Curr. Opin. Immunol. 17, 29–35. Hamon, Y., Luciani, M.F., Becq, F., Verrier, B., Rubartelli, A., Chimini, G., 1997. Interleukin-1beta secretion is impaired by inhibitors of the ATP binding cassette transporter, ABC1. Blood 90, 2911–2915. Holgate, S.T., 2000. The role of mast cells and basophils in inflammation. Clin. Exp. Allergy 30 (suppl. 1), 28–32. Hume, D.A., 2006. The mononuclear phagocyte system. Curr. Opin. Immunol. 18, 49–53. Huse, M., Lillemeier, B.F., Kuhns, M.S., Chen, D.S., Davis, M.M., 2006. T cells use two directionally distinct pathways for cytokine secretion. Nat. Immunol. 7, 247–255. Huynh, K.K., Kay, J.G., Stow, J.L., Grinstein, S., 2007. Fusion, fission, and secretion during phagocytosis. Physiology (Bethesda) 22, 366–372. Jahn, R., Scheller, R.H., 2006. SNAREs—engines for membrane fusion. Nat. Rev. 7, 631–643.

ARTICLE IN PRESS J.L. Stow et al. / Immunobiology 214 (2009) 601–612

Jolly, C., Sattentau, Q.J., 2007. Regulated secretion from CD4+ T cells. Trends Immunol. 28, 474–481. Jones, S.A., 2005. Directing transition from innate to acquired immunity: defining a role for IL-6. J. Immunol. 175, 3463–3468. Joshi, N.S., Kaech, S.M., 2008. Effector CD8 T cell development: a balancing act between memory cell potential and terminal differentiation. J. Immunol. 180, 1309–1315. Jue, D.M., Sherry, B., Luedke, C., Manogue, K.R., Cerami, A., 1990. Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulated macrophages. Biochemistry 29, 8371–8377. Kinet, J.P., 1999. The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology. Annu. Rev. Immunol. 17, 931–972. Kuhns, D.B., Gallin, J.I., 1995. Increased cell-associated IL-8 in human exudative and A23187-treated peripheral blood neutrophils. J. Immunol. 154, 6556–6562. Lacy, P., Mahmudi-Azer, S., Bablitz, B., Hagen, S.C., Velazquez, J.R., Man, S.F., Moqbel, R., 1999. Rapid mobilization of intracellularly stored RANTES in response to interferon-gamma in human eosinophils. Blood 94, 23–32. Lacy, P., Logan, M.R., Bablitz, B., Moqbel, R., 2001. Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-induced piecemeal degranulation in human eosinophils from atopic individuals. J. Allergy Clin. Immunol. 107, 671–678. Lazar-Molnar, E., Hegyesi, H., Toth, S., Falus, A., 2000. Autocrine and paracrine regulation by cytokines and growth factors in melanoma. Cytokine 12, 547–554. Le Cabec, V., Cowland, J.B., Calafat, J., Borregaard, N., 1996. Targeting of proteins to granule subsets is determined by timing and not by sorting: the specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells. Proc. Natl. Acad. Sci. USA 93, 6454–6457. Lieu, Z.Z., Lock, J.G., Hammond, L.A., La Gruta, N.L., Stow, J.L., Gleeson, P.A., 2008. A trans-golgi network golgin is required for the regulated secretion of TNFa in activated macrophages in vivo. Proc. Natl. Acad. Sci. USA 105, 3351–3356. Lippert, U., Ferrari, D.M., Jahn, R., 2007. Endobrevin/ VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells. FEBS Lett. 581, 3479–3484. Lippincott-Schwartz, J., Cole, N., Presley, J., 1998. Unravelling Golgi membrane traffic with green fluorescent protein chimeras. Trends Cell Biol. 8, 16–20. Lock, J.G., Stow, J.L., 2005. Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Mol. Biol. Cell 16, 1744–1755. Lock, J.G., Hammond, L.A., Houghton, F., Gleeson, P.A., Stow, J.L., 2005. E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97. Traffic 6, 1142–1156. Logan, M.R., Lacy, P., Bablitz, B., Moqbel, R., 2002. Expression of eosinophil target SNAREs as potential cognate receptors for vesicle-associated membrane protein-2 in exocytosis. J. Allergy Clin. Immunol. 109, 299–306.

611

Logan, M.R., Odemuyiwa, S.O., Moqbel, R., 2003. Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion. J. Allergy Clin. Immunol. 111, 923–932. Logan, M.R., Lacy, P., Odemuyiwa, S.O., Steward, M., Davoine, F., Kita, H., Moqbel, R., 2006. A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and neutrophils. Allergy 61, 777–784. Manderson, A.P., Kay, J.G., Hammond, L.A., Brown, D.L., Stow, J.L., 2007. Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNF{alpha}. J. Cell Biol. 178, 57–69. Maxfield, F.R., McGraw, T.E., 2004. Endocytic recycling. Nat. Rev. 5, 121–132. McCurdy, J.D., Olynych, T.J., Maher, L.H., Marshall, J.S., 2003. Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J. Immunol. 170, 1625–1629. McInnes, I.B., Schett, G., 2007. Cytokines in the pathogenesis of rheumatoid arthritis. Nat. Rev. Immunol. 7, 429–442. Metcalfe, D.D., Baram, D., Mekori, Y.A., 1997. Mast cells. Physiol. Rev. 77, 1033–1079. Mollinedo, F., Borregaard, N., Boxer, L.A., 1999. Novel trends in neutrophil structure, function and development. Immunol. Today 20, 535–537. Mollinedo, F., Calafat, J., Janssen, H., Martin-Martin, B., Canchado, J., Nabokina, S.M., Gajate, C., 2006. Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils. J. Immunol. 177, 2831–2841. Moqbel, R., Coughlin, J.J., 2006. Differential secretion of cytokines. Sci. STKE 2006, pe26. Murray, R.Z., Kay, J.G., Sangermani, D.G., Stow, J.L., 2005a. A role for the phagosome in cytokine secretion. Science 310, 1492–1495. Murray, R.Z., Wylie, F.G., Khromykh, T., Hume, D.A., Stow, J.L., 2005b. Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis factor-alpha. J. Biol. Chem. 280, 10478–10483. Nickel, W., 2003. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Eur. J. Biochem. 270, 2109–2119. Nigrovic, P.A., Lee, D.M., 2005. Mast cells in inflammatory arthritis. Arthritis Res. Ther. 7, 1–11. Ogura, Y., Sutterwala, F.S., Flavell, R.A., 2006. The inflammasome: first line of the immune response to cell stress. Cell 126, 659–662. Olsen, I., Bou-Gharios, G., Abraham, D., 1990. The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles. Eur. J. Immunol. 20, 2161–2170. Olszewski, M.B., Trzaska, D., Knol, E.F., Adamczewska, V., Dastych, J., 2006. Efficient sorting of TNF-alpha to rodent mast cell granules is dependent on N-linked glycosylation. Eur. J. Immunol. 36, 997–1008. Olszewski, M.B., Groot, A.J., Dastych, J., Knol, E.F., 2007. TNF trafficking to human mast cell granules: mature chaindependent endocytosis. J. Immunol. 178, 5701–5709. Pagan, J.K., Wylie, F.G., Joseph, S., Widberg, C., Bryant, N.J., James, D.E., Stow, J.L., 2003. The t-SNARE syntaxin 4 is

ARTICLE IN PRESS 612

J.L. Stow et al. / Immunobiology 214 (2009) 601–612

regulated during macrophage activation to function in membrane traffic and cytokine secretion. Curr. Biol. 13, 156–160. Paumet, F., Le Mao, J., Martin, S., Galli, T., David, B., Blank, U., Roa, M., 2000. Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment. J. Immunol. 164, 5850–5857. Pellme, S., Morgelin, M., Tapper, H., Mellqvist, U.H., Dahlgren, C., Karlsson, A., 2006. Localization of human neutrophil interleukin-8 (CXCL-8) to organelle(s) distinct from the classical granules and secretory vesicles. J. Leukoc. Biol. 79, 564–573. Puri, N., Roche, P.A., 2008. Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms. Proc. Natl. Acad. Sci. USA 105, 2580–2585. Qu, Y., Franchi, L., Nunez, G., Dubyak, G.R., 2007. Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 179, 1913–1925. Raabe, T., Bukrinsky, M., Currie, R.A., 1998. Relative contribution of transcription and translation to the induction of tumor necrosis factor-alpha by lipopolysaccharide. J. Biol. Chem. 273, 974–980. Ravasi, T., Wells, C.A., Hume, D.A., 2007. Systems biology of transcription control in macrophages. Bioessays 29, 1215–1226. Ren, Q., Barber, H.K., Crawford, G.L., Karim, Z.A., Zhao, C., Choi, W., Wang, C.C., Hong, W., Whiteheart, S.W., 2007. Endobrevin/VAMP-8 is the primary v-SNARE for the platelet release reaction. Mol. Biol. Cell 18, 24–33. Rodriguez-Boulan, E., Kreitzer, G., Musch, A., 2005. Organization of vesicular trafficking in epithelia. Nat. Rev. 6, 233–247. Salamonsen, L.A., Hannan, N.J., Dimitriadis, E., 2007. Cytokines and chemokines during human embryo implantation: roles in implantation and early placentation. Semin. Reprod. Med. 25, 437–444.

Shurety, W., Merino-Trigo, A., Brown, D., Hume, D.A., Stow, J.L., 2000. Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages. J. Interferon Cytokine Res. 20, 427–438. Solomon, K.A., Covington, M.B., DeCicco, C.P., Newton, R.C., 1997. The fate of pro-TNF-alpha following inhibition of metalloprotease-dependent processing to soluble TNFalpha in human monocytes. J. Immunol. 159, 4524–4531. Spencer, L.A., Melo, R.C., Perez, S.A., Bafford, S.P., Dvorak, A.M., Weller, P.F., 2006. Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion. Proc. Natl. Acad. Sci. USA 103, 3333–3338. Spurlin, B.A., Thurmond, D.C., 2006. Syntaxin 4 facilitates biphasic glucose-stimulated insulin secretion from pancreatic beta-cells. Mol. Endocrinol. 20, 183–193. Stow, J.L., Manderson, A.P., Murray, R.Z., 2006. SNAREing immunity: the role of SNAREs in the immune system. Nat. Rev. Immunol. 6, 919–929. Theoharides, T.C., Kempuraj, D., Tagen, M., Conti, P., Kalogeromitros, D., 2007. Differential release of mast cell mediators and the pathogenesis of inflammation. Immunol. Rev. 217, 65–78. van Ijzendoorn, S.C., 2006. Recycling endosomes. J. Cell Sci. 119, 1679–1681. Varadaradjalou, S., Feger, F., Thieblemont, N., Hamouda, N.B., Pleau, J.M., Dy, M., Arock, M., 2003. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur. J. Immunol. 33, 899–906. Walsh, L.J., Trinchieri, G., Waldorf, H.A., Whitaker, D., Murphy, G.F., 1991. Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1. Proc. Natl. Acad. Sci. USA 88, 4220–4224. Watanabe, N., Tsuji, N., Akiyama, S., Sasaki, H., Okamoto, T., Kobayashi, D., Sato, T., Hagino, T., Yamauchi, N., Niitsu, Y., 1998. Endogenous tumour necrosis factor regulates heat-inducible heat shock protein 72 synthesis. Int. J. Hyperthermia 14, 309–317. Yan, Z.Q., Hansson, G.K., 2007. Innate immunity, macrophage activation, and atherosclerosis. Immunol. Rev. 219, 187–203.