Cytokine secretion is a widely studied process, although little is known regarding the specific mechanisms that regulate cytokine release. Recent findings have shed light on some of the precise molecular pathways that regulate the packaging of newly synthesized cytokines from immune cells. These findings begin to elucidate pathways and mechanisms that underpin cytokine release in all cells. In this article, we review the highlights of some of these novel discoveries.
The secretion of cytokines and chemokines from cells is a fundamental response to injury and infection in the body. Cytokines profoundly alter the body's response to cellular damage or invasive pathogens and are secreted by a wide range of cell types. Among the first cells to secrete cytokines in response to pathogenic or harmful signals are epithelial and endothelial cells, which initiate potent immune and physiological responses (101, 121). These cells signal to a variety of innate immune cells and attract these to sites of injury or infection (5, 19). Epithelial cells work in concert with the innate immune system to mount appropriate inflammatory or adaptive immune responses (101). Innate immune cells generate a substantial range of cytokines and regulate the immune response to injury or infection, and these include macrophages/monocytes, dendritic cells (DCs), natural killer (NK) cells, mast cells, eosinophils, and neutrophils. These cells collectively fulfill an essential role in immunity by controlling the opportunistic invasion of a substantial range of viral, bacterial, and parasitic pathogens and can directly recognize pathogens, or their products, through a diverse array of receptors, including pattern-recognition receptors (PRRs), such as the Toll-like receptors (TLRs) (52). Innate immune cells can combat pathogens directly via the production of cytotoxic mediators, and in many cases this is combined with phagocytosis and intracellular killing of pathogens.
Epithelial and innate immune cells possess the ability to alert or disarm the rest of the immune system through the release of a large number of pro-inflammatory and immunoregulatory cytokines and chemokines. Some of the most potent pro-inflammatory cytokines and chemokines released by innate immune cells include tumor necrosis factor (TNF), interleukin (IL)-6, IL-1β, and CCL5, and collectively they have been reviewed previously (37, 47, 112). Cytokine secretion from these cells serves as a bridge for cross-communication with other innate immune cells and, with the adaptive immune system, to regulate the amplification of inflammation and the expansion of T cells and B cells with the associated production of antibodies and cytotoxic responses. The present understanding of cytokine secretion from lymphocytes has been reviewed elsewhere (51, 54).
Recently, more information has come to light from innate immune cells regarding the distal steps of cytokine secretion from the Golgi complex through membrane-bound organelles for classical secretion involving membrane fusion and exocytosis. Cytokines may also be released through alternative pathways, such as molecular transporters, in nonclassical secretion. Understanding these intracellular pathways is important for advancing our knowledge of cellular function in innate immunity and in disease. In this review, we focus on the latest advances describing mechanisms of cytokine and chemokine release.
The Release of Cytokines Through Diverse Trafficking Pathways
Multiple secretory pathways for cytokines have been characterized in individual innate immune cells. A key function for these distinct pathways is to confer selective control over the release of cytokines into the tissue microenvironment, both spatially and temporally, and therefore to enable the development of a controlled and appropriate immune or physiological response.
Until recently, very little was known about how cytokines are secreted. New findings have shown that most cytokines are released through classical secretion. In this form of secretion, cytokines may be packaged in the Golgi for storage in secretory vesicles or granules and then secreted only during receptor-mediated release in a form of “regulated exocytosis” (54, 79, 111), or they may be released rapidly upon their synthesis through recycling endosomes (REs) and small secretory vesicles through “constitutive exocytosis” (112, 113) (FIGURE 1). Constitutive exocytosis involves trafficking of newly synthesized protein cargo that may or may not be initiated by receptor stimulation of nuclear DNA transcription and RNA translation. A secretory granule is defined as a membrane-bound intracellular organelle, found in varying abundance depending on the cell type, that retains secretory proteins for release upon stimulation by receptors in the cell membrane. A special subset of secretory granules is the lysosome-related organelle (LRO), also known as the secretory lysosome, which is found in many secretory cells of the immune system (3, 8). LROs are similar in many regards to lysosomes in that they overlap in their physiological and structural characteristics, although they additionally possess the ability to fuse with the plasma membrane to release their contents into the extracellular environment (3, 111). Cells such as lymphocytes, DCs, and NK cells contain secretory lysosomes that have the ability to fuse with phagosomes or the cell membrane and to release lysosomal enzymes (111). The crystalloid granules in eosinophils may also be similar to secretory lysosomes, since they are considered to be specialized primary lysosomes due to their expression of lysosomal enzymes and markers (90). In some cases, the release of cytokines is polarized toward immunological synapses or phagocytic cups by constitutive exocytosis (50, 82). Cytokine release occurs either constitutively following continuous transcript expression or in response to receptor signaling from TLRs, Fcγ receptors, cytokine receptors, and complement receptors, among others.
Most cytokines possess a signal sequence allowing them to be synthesized and packaged in the endoplasmic reticulum (ER), subsequently directing their trafficking through the Golgi complex via small, membrane-bound vesicles for transport to specific sites in cells for their storage or immediate release. For example, IL-2, IL-3, and IL-7 carry a classical signal peptide and contain glycosylation sequences, implying trafficking through the ER and Golgi complex (18, 35, 126). Several other cytokines such as IL-1β, IL-15, IL-18, and macrophage migration inhibitory factor (MIF) do not have a signal sequence, as discussed later. These leaderless secretory proteins do not follow the classic exocytotic route to exit the cell and are secreted by different mechanisms (84).
Granulocytes, such as mast cells, eosinophils, and neutrophils, have the ability to release preformed cytokines from secretory vesicles or granules. These cells continuously synthesize cytokines from early stages of cell development, and then package these together with other inflammatory mediators into secretory granules or small vesicles for later release upon appropriate stimulation (14, 47, 69).
In contrast, macrophages do not contain granules. Studies based on morphological and biochemical analysis of their secretory organelles have shown that macrophages do have LROs that do not store granule proteins but instead continually export their contents to the cell surface (82, 113). These organelles have not been implicated in cytokine secretion in macrophages, and their function has never been fully addressed. The trafficking and secretion of cytokines by constitutive pathways in macrophages has now been comparatively well elucidated (67, 82, 113). Macrophages temporally regulate the secretion of an array of cytokines to initiate immune responses, as discussed below.
Protein trafficking through vesicles in almost all secretory cells ranging from yeast to mammalian cells is dependent on a range of molecular families, including members of the ras-related superfamily of guanosine triphosphatases (GTPases), the Rab and Rho proteins, actin microtubule motors, lysosomal trafficking molecules such as Lyst, and a family of intracellular membrane receptors known as SNAP (soluble NSF attachment protein) receptors (SNAREs) (8, 15, 34, 53, 57, 110). Rab GTPases are membrane-bound, whereas Rho GTPases are predominantly cytoplasmic in their inactive GDP-bound form, and these cycle between active (GTP-bound) and inactive (GDP-bound) forms to mediate vesicular trafficking (12, 110).
A number of these molecules have been described in the regulation of granule exocytosis from innate and adaptive immune cells (57, 111). Rab27a is activated in eosinophils (20) and is involved in exocytosis of azurophilic granules from neutrophils (42, 81), whereas a double knockout mouse model of Rab27a and Rab27b showed deficient mast cell degranulation (76). A deficiency in Rab27a is also associated with the human disease phenotype seen in the autosomal recessive immunodeficiency Griscelli syndrome 2, and NK cells from patients with this disorder fail to secrete their lytic granules (125). Rac1, Rac2, and Cdc42 have been implicated in granule exocytosis in mast cells and neutrophils through modulation of the actin cytoskeleton (1, 48) and have been associated with cytokine secretion in other cells, such as IL-8 from polarized epithelial cells (Table 1) (45). However, no specific role for Rab or Rho GTPases in cytokine secretion from innate immune cells has yet been established. Although some studies indicate a role for the actin cytoskeleton and microtubules in cytokine secretion, there is little known regarding the specific intracellular regulatory molecules required for cytoskeletal remodeling in cytokine trafficking and release in innate immune cells.
SNAREs are intracellular proteins that interact with each other to allow fusion of adjacent membranes (106, 115) and have proven to be the most informative group of proteins in defining pathways for the release of cytokine and granules from innate immune cells (Table 1). The SNARE hypothesis maintains that a single SNARE protein on a donor membrane binds to two cognate SNAREs on target membranes to form a four-helix complex called the trans-SNARE complex (FIGURE 2) (115). A central coiled-coil domain termed the “SNARE motif” serves as the binding region for SNARE-SNARE protein interaction between members of the SNARE family. The SNARE family is divided into R-SNAREs (usually on vesicles) and Q-SNAREs (usually at the target membrane) on the basis of the central functional residue in their SNARE motif being either arginine (R) or glutamine (Q) (31). When membrane fusion occurs, the R-SNAREs and Q-SNAREs bind together to allow the vesicle and target membranes to be brought together in close proximity (29, 115). After fusion, the trans-SNARE complex is rapidly disassembled to the cis-SNARE complex to allow continued cycling of the components for further fusion events. Members of R-SNAREs include vesicle-associated membrane proteins (VAMPs), whereas Q-SNAREs are associated with syntaxins and the SNAP-25 family of proteins. Several other families of proteins, including the Sec/Munc and Rab proteins, regulate SNARE binding and disassembly (62, 114).
In exocytosis, R-SNAREs are usually associated with the vesicle or granule membrane, whereas their cognate Q-SNAREs are commonly situated on the inner leaflet of the plasma membrane. It is the fusion of the vesicular membrane with the target plasma membrane in exocytosis that serves a fundamental role in the release of many of the cell's vesicular and granular contents to the extracellular milieu (15).
Cytokine Secretion and Trafficking in Epithelial Cells
Epithelial cells have a profound effect on shaping the immune responses in favor of Th1, Th2, or T regulatory cells following activation of TLRs by viral, bacterial, and fungal ligands (123). These cells secrete a considerable range of cytokines and chemokines including TNF and IL-1β (109), and TNF has been detected in lipid bodies of colonic epithelial cells (7). More recently, thymic stromal lymphopoietin, IL-25, and IL-33 were found to be key cytokines generated from airway epithelial cells, which promote allergic responses (101, 123). The mechanisms of trafficking of cytokines from epithelial cells is not yet understood, although this is likely to employ constitutive exocytotic pathways, and polarized protein trafficking in epithelial cells has been shown to require different sets of SNAREs. A specific pathway for apical delivery of secretory vesicles involves VAMP-7 (TI-VAMP) (61), whereas VAMP-3 (cellubrevin) localizes with the basolateral membrane (32). VAMP-3 was shown to cooperate with adaptor protein-1B to mediate basolateral membrane trafficking in MDCK cells (32). The apical transcytotic pathway may require VAMP-8 (endobrevin) (91). Although syntaxin-3 is required for the apical pathway, syntaxin-4 is primarily found in the basolateral membrane (17). A recent study showed that VAMP-7 is important in Golgi to plasma membrane trafficking and mediates the transport of CD82 as well as affecting cell surface diffusion of the EGF receptor in HeLa cells (23). All these results indicate that apical, basolateral, and transcytotic pathways in epithelial cells use distinct sets of SNAREs (17). Whether all of these pathways are utilized in cytokine secretion is yet to be determined.
Exocytosis of Packaged Cytokines
Several types of innate immune cells generate cytokines that are released upon stimulation of specific receptors (14, 69, 73, 79). These include eosinophils, mast cells, and neutrophils, which possess a diversity of granule types that release their contents by regulated exocytosis upon stimulation by PRRs and chemotactic ligands. Thus cytokines may be released immediately within minutes of receptor stimulation, whereas lymphocytes can take several hours or days to make and release cytokines (6, 59, 97, 105). Thus innate immune cells are uniquely poised to initiate a potent and immediate pro-inflammatory response upon contact with pathogens and/or inflammatory stimuli.
Eosinophils are normally rare white blood cells but dramatically increase in both the blood and tissues during allergy and helminth infection (47, 99). They are capable of synthesizing, storing, and secreting up to 35 different cytokines (47). Many of these cytokines are potent inducers of immune and inflammatory responses in asthma, eczema, rhinitis, and other inflammatory diseases. These unique cells contain large crystalloid granules that are enriched in crystallized major basic protein, as well as an extensive tubulovesicular network that is involved in rapid trafficking of small secretory vesicles (57, 73). Eosinophils release mediators and cytokines in a differential manner via piecemeal degranulation, a mode of exocytosis characterized by small vesicles budding from crystalloid granules that is frequently observed in tissue eosinophils from allergic subjects (26, 28). Piecemeal degranulation is thought to be a general secretory method used by immune cells and other cell types in the body, including enteroendocrine cells of the gastrointestinal tract, chromaffin cells of the adrenal medulla, and chief cells of the parathyroid gland (22). Eosinophil-derived cytokines are stored in crystalloid granules and small secretory vesicles, which are rapidly released in response to interferon-γ (IFNγ) and other agonists (47, 60). For example, the release of the chemokine CCL5, packaged primarily in the crystalloid granule, occurs within several minutes of stimulation by IFNγ (59, 72). Recently, several Th1 cytokines (IFNγ, IL-12) and Th2 cytokines (IL-4, IL-13), as well as TNF, IL-6, and IL-10, were shown to be transported through the tubulovesicular system and small secretory vesicles from their sites of storage in the crystalloid granules (73, 74, 108). A mechanism for CCL11-mediated IL-4 secretion was suggested by its colocalization with the IL-4 receptor α chain, thereby anchoring IL-4 to the secretory vesicle membrane for selective packaging, trafficking, and release (107). This receptor-mediated trafficking mechanism has also been described for IL-15 secretion from DCs (80) and may be important for the release of other cytokines from a variety of cells. In stimulated DCs, IL-15 and IL-15Rα are preassembled in complexes within the ER and Golgi. In this way, IL-15Rα chaperones IL-15 to the cell surface for stable IL-15/IL-15Rα complex expression on the cell membrane (80).
Cytokine release from eosinophils may be dependent on SNARE molecules for transport and exocytosis. Eosinophils express VAMP-2 (synaptobrevin-2), VAMP-7, VAMP-8, syntaxin-4, and SNAP-23 (46, 58, 65, 66). VAMP-2 is present in small secretory vesicles, containing the chemokine CCL5, that translocate to the cell membrane upon IFNγ stimulation (FIGURE 3A) (58). In crystalloid granules, VAMP-7 and VAMP-8 are strongly expressed; however, only VAMP-7 was required for the secretion of granule proteins eosinophil peroxidase and eosinophil-derived neurotoxin (66). Syntaxin-4 and SNAP-23 were localized to the cell membrane in eosinophils, where they function as cognate intracellular receptors for VAMP-2 (65). The precise functions of R- and Q-SNAREs in eosinophil cytokine release, however, have not yet been determined.
Taken together, eosinophils store the majority of their cytokines and chemokines in crystalloid granules and may rapidly release these in a SNARE-dependent manner by regulated exocytosis following receptor stimulation.
Mast cells are tissue-resident granulocytic cells that possess large secretory granules and smaller secretory vesicles. Mast cells can synthesize and release up to 33 different cytokines, chemokines, and growth factors (69) and store preformed cytokines in lipid bodies and granules (7). Mast cell-derived TNF and IL-4 are stored in secretory granules and are rapidly released upon stimulation by cross-linking cell surface complexes of IgE and antigen (36, 85, 86, 124). These studies suggest that TNF and IL-4 are released through FcεRI-mediated degranulation, although no evidence has been obtained for the mode of cytokine release. Trafficking outside of mast cell secretory granules occurs for IL-6, since IL-6 was shown to be released from a distinct population of secretory vesicles in the absence of degranulation (55). Stimulation of cytokine release from mast cells occurs in a differential manner in response to TLR activation (71). This differential release is suggested to take place through piecemeal degranulation (25), similarly to eosinophils, allowing selective secretion (117).
Mast cells express many SNARE molecules, which are required for degranulation in response to cross linking of the allergy-related IgE receptor, FcεRI (10, 88, 102). These include the R-SNAREs VAMP-2, VAMP-3, VAMP-7, and VAMP-8 (43, 88, 93, 119, 120). VAMP-8 was shown to be associated with secretory granules and translocated with VAMP-7 to the cell membrane during degranulation. These observations were recently confirmed in human mast cells isolated from intestinal samples, which showed expression of VAMP-3, VAMP-7, and VAMP-8 but only low levels of VAMP-2 (102). In addition, VAMP-7 and -8 were required for FcεRI-mediated degranulation responses in human mast cells (102).
Mast cell degranulation occurs through distinct subsets of secretory granules that are differentially regulated (93). Mast cells from the bone marrow of VAMP-8-deficient mice release TNF, IL-6, and CCL3 normally, whereas release of granule contents was deficient in response to IgE-dependent receptor stimulation (119). This was similar to another study looking at VAMP-8 knockout mast cells in which VAMP-8 was found on serotonin-containing lysosome-like secretory granules and was required for granule protein release in response to FcεRI-mediated stimulation but had no role in TNF or histamine secretion (93). These findings suggest that cytokine secretion occurs in a distinct pathway from that of VAMP-8-dependent release of granule contents in mast cells.
The Q-SNAREs SNAP-23 and syntaxin-4 are also involved as cognate partners for R-SNAREs in mast cells, mediating the secretion of mast cell granules (38, 88). Phosphorylation of SNAP-23 by protein kinase C promotes degranulation from mast cells (41). Mast cell cytokine release is dependent on phosphorylation of SNAP-23, as seen in a recent study showing a new role for IκB kinase-2 (IKK2), a kinase that usually activates the transcription factor NF-κB, in phosphorylation of SNAP-23 and promoting release of TNF from mast cells (116).
In summary, mast cells are able to transport their cytokines through a distinct compartment from secretory granules and release these in a kinase- and SNARE-dependent manner by regulated exocytosis in response to receptor stimulation. The identity of the cytokine-carrying compartment is not yet known but may be similar to REs, which are involved in transporting cytokines in macrophages.
Neutrophils constitute the majority of circulating white blood cells, and can rapidly transmigrate into tissues when inflammatory responses or infections occur. The ability of neutrophils to synthesize and secrete a range of cytokines and chemokines, including TNF and IL-8, is well established (24). These cells have four different types of granules and vesicles: primary (azurophilic), secondary (specific), and tertiary granules, along with a rapidly mobilizable pool of small secretory vesicles (13, 14, 57). At least one cytokine, transforming growth factor-α (TGF-α), is stored as a preformed mediator in peroxidase-negative secretory granules in neutrophils (16), whereas TNF was localized to cytoplasmic vesicles by immunogold labeling (7). These organelles could not be further classified into the four granule types typically described in neutrophils owing to the electron microscopy analysis approach used in these studies, and they could be secondary or tertiary granules, or secretory vesicles. The precise intracellular localization of other neutrophil-derived cytokines and chemokines is not known, although histological examination of mature peripheral blood neutrophils suggests that IL-6, IL-12, and CXCL2 may be stored within secretory vesicles or tertiary granules (24).
The specific mechanisms associated with neutrophil cytokine release have not been reported, although neutrophils express many SNARE isoforms required for granule exocytosis, including VAMP-1 (synaptobrevin-1), VAMP-2, VAMP-7, and VAMP-8 (14, 57, 66, 70, 77, 78). Consequently, SNARE-mediated cytokine release from neutrophils has been implied but not directly demonstrated (14). The R-SNARE VAMP-2 localizes to secondary and tertiary granules and CD35+ secretory vesicles, with the cognate Q-SNAREs SNAP-23 and syntaxin-4 mediating vesicle fusion and release of granule contents (70, 78). Two R-SNAREs, VAMP-7 and VAMP-8, are highly expressed in all neutrophil granule populations. However, similar to eosinophils, only VAMP-7 was essential for SNARE-mediated exocytotic release of azurophilic, specific, and tertiary granules (66). Another study showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediate rapid release of secondary and tertiary granules, whereas VAMP-1 and VAMP-7 are mainly involved in azurophilic granule release (77). These findings indicate that VAMP-7 may play a promiscuous role in controlling exocytosis of numerous granule populations in both eosinophils and neutrophils. This is compatible with the concept that SNARE molecules are capable of binding multiple cognate and non-cognate partners (30, 113). Taken together, neutrophils are not well characterized for their ability to release cytokines and chemokines, although it is clear that SNAREs and other trafficking machinery are required for release of granule contents.
Modes of Vesicular Release of Cytokines
Conventional exocytosis, involving the fusion of complete preformed vesicles or granules with the plasma membrane, has been well characterized in the cell types discussed here. However, piecemeal degranulation is likely to be the predominant form of cytokine release in eosinophils, since several studies indicate that IL-4 and CCL5 are released through piecemeal degranulation of small secretory vesicles based on electron and light microscopy, as well as subcellular fractionation (59, 107). Conventional exocytosis of complete granule release from eosinophils has only been determined using in vitro techniques and has never been detected in vivo, at least in tissues obtained from nasal polyps of allergic subjects (28). Instead, tissue eosinophils in allergic subjects largely undergo piecemeal degranulation (67%) and cytolysis (necrosis in the remainder of tissue cells) (28). Therefore, it is unlikely that cytokine release occurs through conventional granule exocytosis from eosinophils in allergic diseases. In the case of mast cells, it is abundantly clear that serotonin and histamine release are associated with conventional granule exocytosis. However, cytokine release from mast cells may occur through a distinct pathway from conventional granule exocytosis, as discussed above. This suggests that cytokines are released through a vesicular pathway that is distinct from conventional granule exocytosis in mast cells.
Constitutive Release of Cytokines
Constitutive cytokine release describes the packaging of cytokines through the ER and Golgi into a continuously trafficking pathway of carrier vesicles that transport cargo to the plasma membrane for release within minutes or hours of protein synthesis. In some innate immune cells, post-Golgi cargo to the plasma membrane has been shown to go via REs. REs are tubulovesicular structures that are responsible for transporting recycling membranes and proteins to and from the plasma membrane (122) and are present in nearly all cells, including epithelial cells, skeletal muscle cells, neuronal cells, NK cells, mast cells, and macrophages, although they have not yet been characterized in neutrophils or eosinophils. The RE has a slightly higher luminal pH (∼6.4) than peripheral early endosomes and is often located deeper in the cell near the microtubule-organizing center (89, 122). In most cells, REs have diverse roles in the delivery of membranes, receptors, and luminal contents to various parts of cells for secretion, cytokinesis, cell fate, transporter upregulation, and polarization of cells. In some innate immune cells, REs perform many of these functions along with cytokine secretion (67, 82). Factors that regulate RE trafficking include VAMP-3 (82) and Rab11a (21). REs can be detected by measurement of transferrin uptake (4), and transferrin receptor immunoreactivity (67, 82). Pathways engaged in constitutive cytokine release through REs have been best defined in macrophages for their ability to package and secrete TNF and IL-6 (67, 82). NK cells also possess REs that are involved in transporting cytokines constitutively to the cell surface, independently of their granules (98).
Macrophages are tissue-resident cells that derive from maturation of extravasated peripheral blood monocytes that are abundant on mucosal surfaces such as the airways. These cells offer surveillance of tissues and initiate multiple actions, including robust pro-inflammatory cytokine release, when they encounter pathogens and dying cells (49). Macrophages contain constitutively trafficked carrier vesicles that transport cytokines, including TNF and IL-6, between intracellular compartments, as well as to the cell surface for release (105). These carrier vesicles include REs, which are distinct from LROs or secretory lysosomes. When bacterially derived lipopolysaccharide (LPS) stimulates macrophages via TLR4, the type II transmembrane precursor pro-TNF is rapidly synthesized and accumulates in the Golgi within 20 min, and from there it is delivered to the cell surface (FIGURE 3B) (63, 82, 95, 105). Once TNF reaches the cell surface, it is cleaved by TNF converting enzyme (TACE, ADAM17) to release its ectodomain as a soluble cytokine (9). Secreted soluble TNF is detected <40 min after stimulation (105). Both microtubules and actin filaments are involved in post-Golgi-vesicular trafficking of TNF (105).
The anterograde transport of TNF from the trans-Golgi network (TGN) in macrophages was recently shown to require the TGN golgin, p230/golgin-245 (p230) (63). Golgins are long coiled-coil proteins, characterized by a COOH-terminal GRIP domain, that are specifically recruited to subdomains of the TGN from which dynamic tubular precursors arise (104). These coiled-coil membrane-bound proteins participate in membrane tethering that can lead to membrane fusion or can allow stable linking of two membranes (96). Golgins are thought to have a function in selective budding of Golgi-derived vesicles, and distinct spatial segregation allows association of different golgins, such as p230 and golgin-97, with TGN tubules bearing different cargo molecules (64). Depletion of p230 in macrophages using a micro-RNA approach blocks post-Golgi transport of TNF (63). Importantly, reduced TNF secretion was also demonstrated in vivo in mice where p230 was silenced by retroviral transduction (63). Thus p230 has an essential role in cytokine export from the Golgi and secretion from activated macrophages.
Similarly, LPS stimulation leads to the accumulation of newly synthesized IL-6 in the Golgi complex in macrophages within an hour of LPS stimulation, which is then loaded into tubovesicular carriers that bud off the TGN either as the sole labeled cargo or together with TNF (67). Transfection of exogenous IL-6 fused to a GFP tag confirmed that soluble cargo accumulated in the dilated ends of Golgi cisternae (67). Since IL-6 is a soluble cytokine rather than a transmembrane cytokine like TNF, it utilizes multiple carriers (including p230 and golgin-97) for its export from the Golgi.
Using markers of RE in confocal and epifluorescence imaging, it was shown that, following export from the Golgi, both TNF and IL-6 colocalize with REs in macrophages during their transport to the cell surface following LPS stimulation (82). Thus REs act as an intermediate step for cytokine release before secretion at the cell surface (67, 82). Furthermore, it was shown that IL-6 and TNF localize to different subcompartments of REs (67), suggesting that REs may additionally function to sort these two cytokines for release at different points on the cell surface.
A number of other molecules and pathways have been implicated in Golgi-derived cytokine release in macrophages. Choline cytidyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine (PtdCho) biosynthetic pathway required for membrane biogenesis. CCT-α is the major CCT isoform required to mediate PtdCho synthesis for maintaining normal Golgi structure and function. CCT-α expression in the trans-Golgi region is increased in LPS-stimulated macrophages, and CCT-α has been shown to be required for both TNF and IL-6 export from the Golgi complex and secretion (118). Another family of regulatory molecules known as secretory carrier membrane proteins (SCAMPs) are widely distributed integral membrane proteins that have been implicated in regulating vesicular transport in numerous neuronal and nonneuronal tissues. SCAMP5 has been shown to be involved in the secretion of signal peptide-containing cytokines including TNF and CCL5 from the Golgi complex in monocytes and macrophages (40). SCAMP5 is mainly localized in Golgi-associated compartments, and ionomycin triggers rapid translocation of SCAMP5 to the plasma membrane along the classical exocytosis pathway. SCAMP5 is thought to mediate exocytosis of cytokines in cooperation with SNARE machinery (SNAP-23 and syntaxin-4) by binding to calcium-sensitive synaptotagmins (40).
In activated macrophages, increased vesicular trafficking of cytokines occurs concomitantly with the upregulation in expression of relevant SNAREs and other components of trafficking machinery (2, 82, 83, 87), implicating SNARE molecules in RE function. Membrane fusion of TNF-containing vesicles from the TGN to REs, and subsequently from REs to the plasma membrane, is mediated by SNAREs. A novel Q-SNARE complex of syntaxin-6-syntaxin-7-Vti1b is upregulated in LPS-stimulated macrophages and has been shown to regulate transport of TNF out of the TGN (82). This Q-SNARE complex binds the R-SNARE VAMP-3 on REs, necessitating TNF delivery to the cell surface via REs (82).
Another molecule involved in the trafficking of TNF-containing vesicles to the plasma membrane in LPS-stimulated macrophages is the lysosomal cysteine protease, cathepsin B (39). In addition, evidence for a role for VAMP-8 in complement-induced TNF secretion from macrophages has been shown using VAMP-8 knockout mice (94). However, it is unclear how VAMP-3 and VAMP-8 coordinate to mediate the secretion of TNF, and why cathepsin B should be important in transport of TNF to the plasma membrane, since VAMP-8 and cathepsin B are both lysosome-associated proteins. It is possible that lysosomes and REs exchange their contents during secretion and that some form of cross-regulation between these compartments occurs.
In summary, the macrophage is currently the most well characterized innate immune cell type for its ability to traffic and secrete cytokines. Further studies should shed light on the interrelationships of these regulatory pathways in the transport of pro-inflammatory and immunoregulatory mediators. SNARE-dependent cytokine secretion from other innate immune cells, including NK cells and DCs, is not well characterized. NK target cell killing mediated by granzyme B secretion is dependent on VAMP-7 (68), and VAMP-8 is involved in phagosomal trafficking in DCs (44). NK cells also rely on REs to traffic cytokines to the cell surface (98). Understanding the regulatory mechanisms in these and other innate immune cells will be important, since these may be distinct from those of granulocytes and macrophages.
Polarity of Cytokine Release
Cytoskeleton-dependent polarized release of cytokines is an essential mechanism for receptor cross-linking in cell-cell communication and killing, and was first identified in T cells (92). Trafficking and secretion of cytokines can be customized for their cell-specific roles in immunity. Cytokines can be directed toward the immunological synapse arising from cell-cell contact between T cells or NK cells and their target cells, or with antigen-presenting cells (51, 111). Classically, an immunological synapse is a stable region of contact that forms between a central cluster of T-cell receptors (TCRs) on a T cell [supramolecular activation cluster (cSMAC)] and a surrounding ring of adhesion molecules [peripheral supramolecular activation cluster (pSMAC)] on an antigen-presenting cell (111). The release of cytokines can also occur in a polarized manner toward phagocytic cups and filipodia in macrophages (56, 67, 82, 113). In phagocytosing macrophages, TNF secretion was shown to be concurrent with the supply of VAMP-3-containing RE membranes polarized toward the expanding actin-rich phagocytic cup for SNARE-mediated fusion during the initial stages of phagocytosis (67, 82). Syntaxin-4 and TACE are also concentrated in phagocytic cups to allow fusion of REs and cleavage of soluble TNF. However, whereas TNF and IL-6 are both transported through REs, IL-6 is released circumferentially from the peripheral membrane (67, 82). One possible mechanism for the different pathways of TNF and IL-6 secretion is that they may be separated at the Golgi in subcompartments of REs, since TNF is synthesized as a membrane-bound precursor, whereas IL-6 is generated as a soluble cytokine (67).
Interestingly, it has now been demonstrated that cytokines can be released by vesicles in both polarized and non-polarized pathways in T-helper cells (50, 51, 54). Some cytokines such as IFNγ and IL-2 are delivered in a polarized fashion to the immunological synapse with antigen-presenting cells to impart specific communication, whereas others such as TNF and CCL3 are secreted separately and released multi-directionally to promote inflammation and to establish chemokine gradients. Therefore, secretion of different cytokines is not only polarized but is dependent on different trafficking proteins and molecularly distinct processes, some of which are associated with Rab GTPases and SNARE proteins syntaxin-6 and Vti1b (50).
Like T-helper cells, NK cells can also form an immunological synapse with a target cell to deliver cytotoxic signals or with a DC to impart communication. However, recent evidence suggests that although polarity is established in NK cells to ensure directional delivery of perforin in lytic granules toward the immunological synapse, cytokines are released multi-directionally (98). It is not clear how TNF and IFNγ are trafficked and released simultaneously to other sites on the cell surface for largely non-polarized secretion in a pathway dependent on REs, despite the alignment of REs with synapses. Inactivation of REs in NK cells with endocytosed activated horseradish peroxidase, or mutation of RE proteins Rab11 or VAMP-3, abrogates TNF and IFNγ secretion, yet the secretion of cytotoxic granules remains unaffected (98). Therefore, in NK cells, secretory pathways for cytokines and cytolytic agents diverge at the level of REs. This divergence in the polarity of cytotoxic molecules and cytokines allows NK cells to simultaneously kill target cells at the synapse and to recruit other immune cells with different sets of secreted mediators.
In turn, DCs can polarize the release of cytokines toward the immunological synapse during interaction with NK cells (103), a crucial step in the initiation/amplification of early phases of an immune response that favors the onset of an adaptive immune response. NK cells trigger immature DCs to polarize and secrete IL-18 toward the synapse. IL-18 is an inflammatory cytokine that lacks a secretory leader sequence and is translocated from the cytosol into secretory lysosomes for release (11). DC IL-18 is trafficked in secretory lysosomes that are recruited toward the adhering NK cell and are restricted to the synaptic cleft in a calcium-dependent and tubulin-mediated manner (103). This polarization of IL-18 activates interacting NK cells to upregulate cytotoxicity without spreading of cytokine. There is no evidence at this time to suggest that polarized cytokine secretion may occur in other innate immune cells, such as mast cells, eosinophils, and neutrophils.
Non-Classical Secretion of Cytokines
Several cytokines are released using non-classical pathways, independent of ER/Golgi trafficking. The mechanisms involved are still poorly understood. IL-1β has emerged as one of the most important mediators of inflammation and host responses to infection (27). IL-1β is a leaderless protein first generated as biologically inactive pro-IL-1β, which is synthesized directly into the cytoplasm of cells because it lacks a conventional hydrophobic signal sequence (100). Pro-IL-1β is then processed into mature, biologically active IL-1β by caspase-1 cleavage at intracellular sites and is subsequently released into the extracellular milieu. Different models of non-classical release have been proposed for IL-1β (27). These include classical exocytosis of IL-1β-containing secretory lysosomes, as well as non-classical secretion pathways of IL-1β release from shed plasma membrane microvesicles, fusion of multivesicular bodies with the plasma membrane and subsequent release of IL-1β-containing exosomes, and export of IL-1β through the plasma membrane using specific membrane transporters (27). There is also some evidence that a significant proportion of IL-1β release occurs upon cell lysis. MIF is another leaderless cytokine that does not enter the ER/Golgi secretory pathway and instead appears to be secreted from the cytoplasm, possibly through an ABC transporter (33). Secretion of MIF requires p115, a vesicle-docking protein that is localized predominantly to the cytosolic side of vesiculotubular intermediate clusters and to the cis-Golgi, to act as an intracellular binding partner (75). These different potential mechanisms of non-classical release remain controversial, and further work is required to elucidate these pathways.
In this review, we have seen that there are multiple and complex pathways for the release of cytokines by cells of the innate immune system. Increasingly, and unsurprisingly, it emerges that cells use different pathways, organelles, carriers, and molecules to exert an exquisite control of release of these important immunoregulatory messengers. This is essential for the development of appropriate innate and adaptive immune responses toward inflammatory or infectious agents. Regulation of exocytosis of cytokine-containing vesicles or granules appears to be specific to the cell type, such that it is not possible to fully extrapolate findings from a single cell type. In addition, each cytokine must be individually studied for its unique trafficking pathway, since even within a single cell type the pathway of cytokine secretion is dictated by the presence or absence of a signal peptide sequence as well as numerous other packaging processes. Since many of the components of trafficking machinery were first identified in neuronal cells, it is anticipated that further insights gained from neuronal and neuroendocrine cells will increasingly instruct us about molecules and mechanisms active in other cell types. Therefore, much remains to be determined regarding the precise mechanisms that control cytokine packaging and trafficking for release in innate immune cells and indeed in other cells of the body.
Data reported here was produced by funding from the National Health and Medical Research Council of Australia (A. C. Stanley), the Alberta Heritage Foundation for Medical Research (Visiting Scientist from Alberta Award; P. Lacy), and the Canadian Institutes of Health Research (P. Lacy).
No conflicts of interest, financial or otherwise, are declared by the author(s).
- Copyright © 2010 the American Physiological Society