Tumor necrosis factor (formerly known as TNF-α) is a 185-aminoacid glycoprotein that was initially described for its ability to induce necrosis in certain tumors (13). It stimulates the acute phase of the immune response. This potent pyrogenic cytokine is one of the first to be released in response to a pathogen, and is able to exert its effects in many organs (12). As such, TNF is one of the main cytokines responsible for septic shock. In the hypothalamus, TNF stimulates the release of corticotropic releasing hormone, suppresses appetite, and induces fever. In liver, it stimulates the acute inflammatory response by elevating the synthesis of C-reactive protein and other mediators. TNF induces vasodilation and loss of vascular permeability, which is propitious for lymphocyte, neutrophil, and monocyte infiltration. It helps recruit these cells to the inflammation site by regulating chemokine release. TNF, in concert with IL-17, triggers the expression of neutrophil-attracting chemokines CXCL1, CXCL2, and CXCL5 (14) and can also augment the expression of cell adhesion molecules (15) that facilitate diapedisis. This in turn increases CXCR2-dependent neutrophil migration to the inflammation site. Being an inducer of the inflammatory response, excess amounts of TNF have been found to play pathological roles in ailments such as inflammatory bowel disease, psoriasis, rheumatoid arthritis, asthma, cancer, infectious diseases, and other auto-immune pathologies. Some of these conditions are currently co-treated with monoclonal antibodies that neutralize this cytokine (16).

In macrophages, TNF is released to the extracellular milieu via the constitutive secretion pathway, and its trafficking is the best understood of all cytokines (9, 17, 18). Details on TNF trafficking will be discussed in another article of this issue. After synthesis in the ER, the SNARE proteins Stx6, Stx7, Vtib mediate the fusion of TNF-containing vesicles from the Golgi complex with VAMP3⁺-recycling endosomes (17, 19). Thence, the Stx4/SNAP23/VAMP3 complex facilitates the passage of TNF from recycling endosomes to the cell membrane (17, 18). Rho1 and Cdc42, two proteins that govern cell shape via actin remodeling, also regulate the post-recycling endosome trafficking of TNF to the plasmalemma (20). Moreover, LPS was found to increase the expression of vesicle trafficking proteins that regulate TNF trafficking (17, 18). Finally, release of mature TNF from the plasmalemma requires cleavage of the membrane-bound precursor by the TNF-α-converting enzyme (TACE) (21). The process of phagocytosis requires extensive membrane exocytosis from several organelles that also partake in TNF secretion (7). Interestingly, it was found that TNF is not only secreted to the extracellular milieu at the plasma membrane, but also in a polarized manner at the phagocytic cup (17). This highlights an efficient and elegant strategy where macrophages can promptly release cytokines at the same time that they phagocytose microbial invaders. The importance of regulating TNF secretion implies that there exist negative regulators for its secretion. One such regulator is the recently characterized protein Syt XI, which associates to recycling endosomes and lysosomes in macrophages (11, 22). Syts constitute a group of membrane proteins that regulate vesicle docking and fusion in processes such as exocytosis (11, 23) and phagocytosis (11, 24, 25). Syts control vesicle fusion by virtue of their Ca²⁺-binding C2 domains (26). However, Syt XI cannot bind calcium and inhibits vesicle fusion (27). Upon LPS stimulation of macrophages treated with siRNA to Syt XI, more TNF and IL-6 are released. The inverse is true when Syt XI is overexpressed (11). Though the mechanism for this finding is not yet known, Syt XI likely regulates cytokine release by interacting with members of the SNARE complex. Indeed, Syt XI was found to interact with the Golgi SNARE Vti1a (28), raising the question of whether Syt XI regulates SNARE complex formation at the Golgi.

Three forms of IL-1 are known: IL-1α, IL-1β and IL-1Ra. Although both IL-1α and IL-1β are strongly proinflammatory, perform many of the same functions and bind to the IL-1 receptor (IL-1R), there is only 25% aminoacid homology between them. Similarly to TNF, IL-1β is also an endogenous pyrogen that is produced and released at the early stages of the immune response to infections, lesions, and stress. Although monocytes and macrophages are the main sources of IL-1β, it is also released by NK cells, B cells, dendritic cells, fibroblasts, and epithelial cells. During inflammation, IL-1β stimulates the production of acute phase proteins from the liver and acts on the central nervous system to induce fever and prostaglandin secretion. In mast cells, IL-1β induces the release of histamine, which in turn elicits vasodilation and localized inflammation. It is also a chemoattractant for granulocytes, enhances the expansion and differentiation of CD4 T cells (29), and increases the expression of cell adhesion molecules on leukocytes and endothelial cells. Additionally, IL-1β augments the expression of genes that produce it (30). To quell the proinflammatory action of IL-1α and IL-1β, IL-1Ra competes for the same receptor. IL-1Ra is secreted via the classical secretory, though the exact mechanism is not well known. Its binding to the IL-1R does not induce the proinflammatory signaling program induced by IL-1α and IL-1β.

In stimulated macrophages, IL-1α is synthesized de novo and can be actively secreted (31) or passively released from apoptotic cells (32). It can also exert its effects in an intracrine fashion and act as a transcription factor (29, 30). IL-1β is synthesized as a leaderless precursor that must be cleaved by inflammasome-activated caspase-1. After activation, autophagy plays a major role in the release of this cytokine. Autophagy is a highly conserved process in eukaryotes in which the cytoplasm, aberrant, or damaged organelles are sequestered in double-membrane vesicles and released into the lysosome for breakdown and eventual recycling of resulting macromolecules (33). This process plays a crucial role in adaptation to changing environmental conditions, starvation, cellular remodeling during development, and senescence. Autophagy is characterized by the formation of double-membrane vesicles, called autophagosomes, which capture and transport cytoplasmic material to acidic compartments where material is degraded by hydrolytic enzymes (33). Autophagy has also been recognized to mediate the secretion of proteins (34) – such as IL-1β and IL-18 (35, 36) – that would otherwise not enter the classical secretory pathway due to lack of a leader peptide. In the case of IL-1β, the autophagic protein Atg5, the Golgi protein GRASP55, and Rab8a are essential for translocating IL-1β-containing cargo to the outside of the cell. In peritoneal macrophages, it has been shown that IL-1β is transported to the extracellular milieu via membrane transporters (37); knockdown of ABC transporters inhibits IL-1β secretion (38). Additionally, exocytosis of P2X7R-positive multivesicular bodies containing exosomes has also been reported to play an important role in the release of this cytokine (39). The various modes of IL-1 secretion highlight the exquisite machinery that macrophages have evolved as a means for rapidly responding to inflammatory stimuli.

IL-6 is a pleiotropic cytokine that has both proinflammatory and anti-inflammatory functions that affect processes ranging from immunity to tissue repair and metabolism. It promotes differentiation of B cells into plasma cells, activates cytotoxic T cells, and regulates bone homeostasis. As with other proinflammatory cytokines, IL-6 is has been implicated in Crohn’s disease and rheumatoid arthritis (40). Similar to TNF and IL-1β, IL-6 is an endogenous pyrogen that promotes fever and the production of acute phase proteins from liver. Proinflammatory properties are elicited when IL-6 signals in trans via soluble IL-6 receptors binding to gp130, which is ubiquitous in all cells. Inhibition of trans signaling via gp130 blockade in murine sepsis models rescues mice from widespread inflammation and death (41). IL-6 trans signaling also leads to recruitment of monocytes to the inflammation site (42), promotes the maintenance of Th17 cells, and inhibits T cell apoptosis and development of Tregs (43). In contrast, anti-inflammatory properties are elicited when IL-6 signals through the classical pathway, which occurs via the IL-6 receptor that only few cells express. The anti-inflammatory properties of IL-6 are illustrated by IL-6^−/− mice, which exhibit hepatosteatosis, insulin resistance, and liver inflammation (44). IL-6 classic signaling also mediates apoptosis inhibition and the regeneration of intestinal epithelial cells (43).

IL-6 is a soluble cytokine that is synthesized in the ER and, unlike TNF, is not processed as a membrane-bound precursor. Upon stimulation of macrophages with LPS, IL-6 starts accumulating in the Golgi after 4 h of stimulation (45). From the Golgi, IL-6 exits in tubulovesicular carriers that may also contain TNF. Golgi-derived vesicles then fuse with VAMP3-positive recycling endosomes. Three-dimensional reconstruction of fluorescence images showed that recycling endosomes can harbor both TNF and IL-6, albeit both occupy different subcompartments (45). The post-Golgi trafficking of IL-6 follows a route that is also dependent on Stx6 and Vti1b, which form a complex with cognate SNARE VAMP3 at recycling endosomes (17, 18). Knockdown and overexpression of these SNAREs decreases and augments IL-6 release, respectively (45). Syt XI may be negatively modulating the secretion of this cytokine by regulating the formation of these SNARE complexes (11, 28). Unlike TNF, IL-6 is not secreted at the phagocytic cup (45).

IL-12 is produced primarily by monocytes, macrophages, and other antigen-presenting cells; it is essential for fighting infectious diseases and cancer. IL-12 is a heterodimeric cytokine comprised of the p35 and p40 subunits, which come together after their synthesis. Deletions within the p40 gene have been observed in patients suffering from concurrent multiple bacterial infections (46, 47). IL-12 promotes cell-mediated immunity via stimulation of Th1 cells. It synergizes with TNF and other proinflammatory cytokines in stimulating IFN-γ production, as well as the cytotoxicity of NK and CD8 T cells (48). IL-12 can also inhibit angiogenesis through IFN-γ-mediated upregulation of the anti-angiogenic chemokine CXCL10. The involvement of this cytokine in these processes has made it a target in both auto-immune pathologies and cancer (46, 47). After protein synthesis, both p40 and p35 subunits associate at the ER, where they undergo subsequent glycosylation steps prior to being released at the cell membrane (49). Although the precise post-Golgi trafficking mechanisms in macrophages are not known, the release route is likely to resemble that of TNF and IL-6 (9). Data from neutrophils localized the SNAREs VAMP2, VAMP7, Stx2, Stx6, and SNAP23 in the granules that contain and secrete IL-12 (50, 51). Although macrophages do not possess secretory granules, IL-12 release from these cells may involve some of the same SNARE complexes. Furthermore, IL-12 is secreted in a polarized manner from lymphocytes; this process is dependent on Cdc42 (52), which also regulates release of TNF to the plasma membrane. This raises the interesting prospect that IL-12 may be released in a polarized fashion, along with TNF (17), at nascent macrophage phagosomes.

IL-18 is a member of the IL-1 family and also an inducer of IFN-γ production. It synergizes with IL-12 to activate T cells and NK cells. Albeit the fact that IL-18 signals similarly to IL-1β, IL-18 is not a pyrogen, and can even attenuate IL-1β-induced fever (53). Lack of fever induction may be explained by the fact that IL-18 signals through the MAPK p38 pathway instead of the NF-κB pathway, which is used by IL-1β (54). IL-18 trafficking is similar to that of IL-1β, with secretory autophagy also playing a major role in its release (35, 36).

IL-23 is also an IFN-γ inducer and T cell activator that is involved in a variety of diseases ranging from psoriasis to schizophrenia (47). It is similar to IL-12 in that both induce inflammation. Moreover, both IL-12 and IL-23 share the IL-12p40 subunit and thus have similar signaling pathways. In contrast to IL-12, IL-23 augments IL-10 release and induces IL-17 synthesis by activated naïve T cells (55).

IL-27 is a member of the IL-12 family, and is composed of subunits p28 and Epstein–Barr virus-induced gene 3. Similar to TNF, it is produced early in monocytes and macrophages stimulated with LPS and IFN-γ. Knockout of its receptor ensues in increased susceptibility of mice to bacterial and parasitic infections due to impaired IFN-γ production (56). In addition to favoring the differentiation of naïve T cells to Th1 cells via IFN-γ induction, IL-27 can also inhibit the differentiation of Th17 cells (57). IL-27 also has anti-inflammatory properties, which are exemplified by the fact that IL-27 receptor-deficient mice are more susceptible to auto-immune encephalomyelitis, which correlates with increased levels of Th17 cells (55). The fact that this cytokine has selective inflammatory and anti-inflammatory properties supports the concept that the inflammatory response is prompt, but also carefully calibrated to avoid damage to the host.

Inflammation is tightly regulated by multiple inhibitors and antagonists. IL-10 is a 35 kD cytokine identified in 1989, and is produced by activated macrophages, B cells, and T cells (58). Its main activities concern the suppression of macrophage activation and production of TNF, IL-1β, IL-6, IL-8, IL-12, and GM-CSF (59). IL-10 suppresses MHC-II expression in activated macrophages and is thus a potent inhibitor of antigen presentation (60). Of particular interest is that IL-10 inhibits the production of IFN-γ by Th1 and NK cells, and induces the growth, differentiation, and secretion of IgGs by B cells (61, 62). Macrophages themselves are affected by IL-10 in that exposure to this cytokine lowers their microbicidal activity and diminishes their capacity to respond to IFN-γ (63, 64). Experiments in murine models have shown that blocking or neutralizing IL-10 leads to increased levels of TNF and IL-6; on the contrary, exogenous IL-10 improves survival and reduces the levels of inflammatory cytokines (65). It has been observed that reduced levels of IL-10 favor the development of gastrointestinal pathologies such as inflammatory bowel disease (65). Recombinant IL-10 has indeed been effective in the treatment of some of these diseases.

The mechanism of IL-10 trafficking and release resembles that of TNF and IL-6 (66). IL-10 traffics from Golgi tubular carriers associated with p230/golgin-245 along with TNF-containing vesicles, or in golgin-97-associated tubules. The Golgi-associated p110δ isoform of PI3K was also found to be a positive regulator of IL-10 release. From the Golgi, IL-10-containing vesicles move to recycling endosomes, where VAMP3 and Rab11 then modulate the transit of this cytokine – and of TNF and IL-6 – to the cell surface. Independent of recycling endosomes, IL-10 was also observed to exit directly from the Golgi to the cell surface in apoE-labeled vesicles (66).

Together with IL-10, TGF-β is another powerful anti-inflammatory cytokine that acts on many target cells and tones down the inflammatory effects of TNF, IL-1β, IL-2, and IL-12, etc. (61, 67, 68). TGF-β is a potent suppressor of both Th1 and Th2 cells, but foments the maintenance and function of Tregs (67, 69). The importance of TGF-β in the immune system is highlighted by the fact that mice lacking the TGF-β1 isoform, which is predominant in cells of the immune system, develop severe multi-organ inflammation and die by week 4 (70). TGF-β is also implicated in hematopoiesis and has a crucial role in embryogenesis, tissue regeneration, and cell proliferation and differentiation.

Transforming growth factor beta is synthesized as a precursor and is directed to the ER by virtue of its signal peptide. Cleavage by the endoprotease furin, which can happen at the ER or in the extracellular environment, is required for activation of this cytokine (70). Although the secretory mechanism of this cytokine has not been explored, it is possible that it follows a post-Golgi pathway similar to that of TNF, IL-6, or IL-10.

CXCL1 and CXCL2 (also known as macrophage inflammatory protein 2-α, MIP) share 90% amino acid similarity and are secreted by monocytes and macrophages to recruit neutrophils and hematopoietic stem cells (72, 73). Both chemokines are angiogenic and may promote the development of tumors such as melanomas (74).

CCL5, or the regulated upon activation normal T cell expressed and secreted (RANTES), is an inflammatory chemoattractant for T cells, basophils, eosinophils, and dendritic cells to the site of inflammation (75). Aside from this role, it can also mediate the activation of NK cells into chemokine-activated killers (CHAK) (76). Similar to CXCL1 and 2, it promotes tumorigenesis and metastasis (74). CCL5 is synthesized in the ER and traffics to the Golgi complex before being exported outside of the cell. The secretory carrier membrane protein (SCAMP)5, a recycling endosome-associated protein, governs post-Golgi trafficking of CCL5 to the plasmalemma. Stimulation of macrophages with ionomycin induces SCAMP5 translocation to the plasma membrane, where it colocalizes and interacts with Syt I and II, which in turn mediate interactions with various SNAREs (77).

CXCL8 is a potent chemoattractant for neutrophils, in which it also induces degranulation and morphological changes (78, 79). Since macrophages are some of the first cells to respond to an antigen, they are likely the first cells to release CXCL8. Other cells such as keratinocytes, endothelial cells, eosinophils, and basophils also respond to this chemokine. The importance of IL-8 has made this chemokine important in inflammatory diseases such as psoriasis, Crohn’s disease, and cancer (80, 81).

CXCL9, also known as monokine induced by gamma interferon (MIG), is a strong T cell chemoattractant to the site of inflammation (71, 82). It mediates cell recruitment necessary for inflammation and repair of tissue damage. CXCL9 also inhibits neovascularization (83) and has anti-tumor and anti-metastatic effects (74).

CXCL10, or interferon gamma-induced protein 10, is secreted not only by monocytes and macrophages, but also by fibroblasts and endothelial cells (83). It serves to attract T cells, NK cells, dendritic cells (84), and also has potent anti-cancer activity.

Similar to CXCL9 and CXCL10, CXCL11 is interferon-inducible and also mediates T cell recruitment, although more potently than CXCL9 and CXCL10 (85). It also inhibits angiogenesis and tumor formation (74).