By examining endogenous IL-6 and fluorescently tagged IL-6 in live and fixed macrophages, we describe here, for the first time, the secretory pathway for the soluble cytokine IL-6. Fluorescent IL-6 was observed exiting the Golgi complex in tubulovesicular carriers, where it appeared as labeled cargo alone or in conjunction with TNFα. Overall, our results are in agreement with the limited observations of intracellular IL-6 in the literature, including an early study showing the costaining of TNFα and IL-6 in the Golgi complex of activated monocytes (Andersson and Matsuda, 1989
) and electron microscopic labeling showing that, as a constitutive secretory product in mast cells, IL-6 was excluded from entry into secretory granules and was instead found clustered in small constitutive vesicles after leaving the Golgi complex (Kandere-Grzybowska et al., 2003
). Now, a major revelation in this study is that IL-6, upon leaving the Golgi complex, is trafficked to the recycling endosome before it is delivered to the cell surface. Moreover, we show that the recycling endosome represents a critical point of divergence for the cytokines IL-6 and TNFα, with TNFα but not IL-6 delivered to phagocytic cups. Compartmentalization of cargo within the recycling endosome underpins the individual exit and release of these cytokines, revealing new capacities and an important role for this organelle in orchestrating the macrophage immune response.
Fluorescent IL-6 is loaded into tubulovesicular structures budding from the TGN in live macrophages. The size, appearance, and kinetics of these carriers are consistent with carriers seen previously in macrophages labeled with TNFα as cargo, and they are also similar to post-Golgi carriers visualized in other cell types (Polishchuk et al., 2000
; Lock et al., 2005
; Murray et al., 2005a
). By immunofluorescence, EM, and biochemical analyses, we show that IL-6 is the lone labeled cargo in some carriers, whereas in other cases, it is in carriers that also contain TNFα. Although TGN-derived carriers containing fluorescent IL-6 were abundant (for example, see Video 3), those containing TNFα alone or IL-6 were seen less frequently. Thus, there is little apparent sorting of soluble cargo like IL-6 at the TGN, whereas membrane-bound TNFα must be actively sorted for more selective loading into carriers. This is also borne out by previous data from HeLa cells, in which we showed that TNFα is selectively loaded into only a subset of golgin-labeled carriers budding off the TGN (Lock et al., 2005
). In general, there is little understanding of how soluble cargo in constitutive secretory pathways is handled at the TGN. Other soluble cargo such as the lysosomal enzyme is sorted in the TGN by binding to M6PR for trafficking to endosomes (Ghosh et al., 2003
), and, in cells with regulated secretion, secretory products are clustered by binding to chromogranins in the TGN for loading into secretory granules (Kim et al., 2001
; Arvan et al., 2002
). An earlier study suggests that in epithelial cells, soluble proteins are sorted in a pH-dependent compartment for polarized delivery to the cell surface (Caplan et al., 1987
). Our current findings now highlight the fact that the TGN may not be the only place for the sorting of biosynthetic cargo such as cytokines. The recycling endosome, which is appropriately slightly acidic (Teter et al., 1998
; Gagescu et al., 2000
), must now also be considered as a possible sorting site for membrane-bound (Rodriguez-Boulan and Musch, 2005
) and soluble cargoes.
Several lines of evidence show that the post-Golgi carriers containing IL-6 are indeed delivered to recycling endosomes. First, overexpression or knockdown of the SNARE proteins Vti1b, Stx6, and VAMP3 affected the secretion of IL-6. We have previously shown that these proteins are part of the Q-SNARE complex (Stx6–Stx7–Vti1b) on the TGN with the cognate R-SNARE VAMP3 on recycling endosome membranes and that this complex regulates TNFα trafficking in macrophages (Murray et al., 2005b
). Implicating these SNAREs also in IL-6 trafficking is consistent with both cytokines sharing some carriers and/or being trafficked in separate carriers regulated by the same SNAREs, although it is possible that additional SNARE complexes could be involved. VAMP3 function in IL-6 secretion confirms the recycling endosome as a post-Golgi destination for this cytokine. Second, by live cell imaging, fluorescently tagged IL-6 in tubular and vesicular carriers budding off the TGN was frequently observed fusing with preexisting recycling endosomes in the cell periphery. In these same structures, IL-6 was colocalized with recycling endosome markers, including VAMP3 and endocytosed Tfn. Finally, inactivation of the recycling endosome completely ablated the secretion of IL-6, showing that this is a requisite compartment en route to the cell surface for newly synthesized IL-6. The direct delivery of all or most of IL-6 to the recycling endosome is in agreement with the transport of other biosynthetic cargo from the TGN to this compartment, including TNFα in macrophages and endothelial cadherin and vesicular stomatitis virus-G in epithelial cells (Ang et al., 2004
; Lock et al., 2005
). IL-6 is the first example of a constitutively transported soluble cargo trafficking to the cell surface via the recycling endosome, and, as such, it underscores the diverse roles played by the recycling endosome in exocytosis.
The recycling endosome handles cargo from both endocytic/recycling routes as well as exocytic cargo (for review see van Ijzendoorn, 2006
). In the present study, we present evidence that IL-6 and TNFα converge with recycling Tfn and with the resident SNARE VAMP3 within recycling endosomes. Importantly, however, all three of these cargo proteins appear to be partially segregated within this compartment based on colocalization and image reconstruction in fixed and live cells. This compartmentalization differentiates not only recycling and exocytic cargo but even segregates different exocytic cargo within the same structure. Live imaging and image reconstructions demonstrate that such a compartmentalization of cargo is an extremely dynamic process, with cargo-rich domains that continuously merge and then segregate (Video 3). Mechanisms for sorting and compartmentalizing the membrane-bound cargoes such as TNFα and Tfn/TfnR can be envisioned, but very little is known about how soluble proteins are or could be sequestered (Rodriguez-Boulan et al., 2005
; for review see Ellis et al., 2006
). It is possible that surface receptors for IL-6, such as IL6R or gp130 (Kishimoto et al., 1995
), could be involved, but this awaits further investigation. In addition, it is notable that IL-GFP switched from accumulating in the lumens of Golgi cisternae to being on the luminal face of recycling endosome membranes in our immuno-EM images. This is suggestive of membrane association that could provide a mechanism for IL-6 in sorting at the level of the recycling endosome.
The concept of having compartmentalized recycling endosomes is already soundly established within the literature, particularly with reference to the membrane-associated trafficking machinery. Light, fluorescence, and EM studies show this to be a highly reticulated and tubular compartment (Ullrich et al., 1996
; Cox et al., 2000
; Ang et al., 2004
; Lock et al., 2005
). It has a complex and segmented molecular landscape with multiple resident GTPases such as Arf6 and the Rab11 subfamily proteins (Casanova et al., 1999
; Schlierf et al., 2000
; Powelka et al., 2004
). Members of the Rab11 family demark different compartments of the recycling endosome in MDCK cells with the locations of Rab11a and Rab25 distinct from that of Rab11b (Lapierre et al., 2003
). Similarly, the large Rab11-FIP family of Rab11a effectors offers many opportunities for further spatial and functional complexity, as does another Rab11 effector, myosinVb, which has also been shown to differentially regulate different cargo trafficking through the recycling endosome (Meyers and Prekeris, 2002
; Lapierre and Goldenring, 2005
; Jin and Goldenring, 2006
). Resident recycling endosome markers such as Rab11 and the SNARE VAMP3 only partially colocalize with Tfn as cargo (Ullrich et al., 1996
; Teter et al., 1998
). A targeted fluorescence method used by Teter et al. (1998)
showed only a 20–30% overlap between Tfn and VAMP3 on pericentriolar recycling endosomes. There is also a dynamic overlap or continuity of recycling endosomes with early endosomes, as demonstrated by the sequential but overlapping distributions of Rabs 11, 4, and 5, portending the possibility of recycling endosomes being part of a continuous tubular network (Daro et al., 1996
; Ullrich et al., 1996
; Teter et al., 1998
; Sonnichsen et al., 2000
; Bonifacino and Rojas, 2006
). Thus, the complex handling and segregation of cargoes within the recycling endosome that we show here now suggests a functional consequence for this array of machinery found associated with these membranes.
TNFα and IL-6 are both proinflammatory cytokines that are secreted temporally in overlapping profiles from macrophages, but, because both cytokines have different immune functions, it is important for their secretion to be tightly coordinated throughout the inflammatory response. In this study, we show for the first time that the secretion of these cytokines can be differentially regulated at the level of intracellular trafficking in addition to their known regulation at the transcriptional and translational levels (Kracht and Saklatvala, 2002
). Similarly, Huse et al. (2006)
recently identified the existence of two separate exocytic pathways for the targeted or polarized trafficking of cytokines to the immunological synapse or to the entire plasma membrane in activated T cells. This bears analogy to the targeted delivery of TNFα directly to the phagocytic cup and the more generalized secretion of IL-6, which is shown here in macrophages. It is not known whether the recycling endosome is involved in the differential trafficking of cytokines in T cells, as we show here for macrophages. Our results may also be comparable with the differential release of IL-4 and -12 from eosinophil crystalloid granules (Moqbel and Coughlin, 2006
; Spencer et al., 2006
). In these granules, IL-4 is selectively mobilized to move from the granules into secretory vesicles by IL-4Rα after the stimulation of eosinophils (Spencer et al., 2006
). Such examples highlight the importance for immune cells to independently regulate and release cytokines. Indeed, by analogy, we propose that this is a function assigned to recycling endosomes in macrophages, which, unlike granulocytes, do not have granules for packaging and selective release of cytokines.
Unlike TNFα, the secretion of IL-6 was not linked to the formation of phagocytic cups. This possibly reflects the temporal nature of cytokine secretion, with an essential role for TNFα in very early inflammation, whereas IL-6 is important during the transition from innate to adaptive immunity (for review see Jones, 2005
). Our studies to date do not help define the location or nature of the cell surface sites to which IL-6 is delivered for release. Tracking fluorescently tagged IL-6 during its delivery to the plasma membrane has not yet revealed any particular feature or special sites for plasma membrane fusion. There may well be organization of IL-6 secretion sites at the level of membrane lipid microdomains, as there is for TNFα (Kay et al., 2006
), or exocyst-demarked delivery sites, as seen on the apico-lateral membranes of epithelial cells (Yeaman et al., 2004
; Rodriguez-Boulan et al., 2005
). Interestingly, we also found that TfnR in recycling endosomes is not delivered to the actin-rich phagocytic cups. The cognate delivery of VAMP3 and TNFα to the cups and the concomitant exclusion of IL-6 and TfnR is evidence once again that sorting occurs in the recycling endosomes. Importantly, it shows that the recruitment of recycling endosome membrane for the formation of phagocytic cups at the cell surface occurs in a highly selective fashion, perhaps being designed to ensure that other trafficking and other cell functions can proceed unfettered by the onset of phagocytosis. It also highlights the temporal nature of phagosome maturation, a process that requires the sequential recruitment of different organelles in a SNARE-mediated fashion (Bajno et al., 2000
; Collins et al., 2002
; Braun et al., 2004
). Although TfnR is excluded from early stage phagocytic cups, it can appear in more mature phagosomes (Clemens and Horwitz, 1995
), suggesting that the recycling endosome plays a dynamic and evolving role throughout the phagocytic process.
In conclusion, the results shown in this study demonstrate the differential trafficking and secretion of cytokines from activated macrophages. Aspects of the post-Golgi trafficking that are shared or not shared by IL-6 and TNFα may suggest strategies for the joint or separate therapeutic targeting of these cytokines in inflammatory disease. A new route is demonstrated for IL-6 trafficking, one including recycling endosomes as a way station and possible sorting site. Our studies suggest that mechanistically, sorting and compartmentalization of cargo within the macrophage recycling endosome enables the differential secretion of these proinflammatory cytokines and helps orchestrate the immune response.