A Role for the Phagosome in Cytokine Secretion

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Science  02 Dec 2005:
Vol. 310, Issue 5753, pp. 1492-1495
DOI: 10.1126/science.1120225


Membrane traffic in activated macrophages is required for two critical events in innate immunity: proinflammatory cytokine secretion and phagocytosis of pathogens. We found a joint trafficking pathway linking both actions, which may economize membrane transport and augment the immune response. Tumor necrosis factor α (TNFα) is trafficked from the Golgi to the recycling endosome (RE), where vesicle-associated membrane protein 3 mediates its delivery to the cell surface at the site of phagocytic cup formation. Fusion of the RE at the cup simultaneously allows rapid release of TNFα and expands the membrane for phagocytosis.

In response to a microbial challenge, activated macrophages initiate multiple actions, including the secretion of proinflammatory cytokines and the phagocytosis of microorganisms (1). Both of these actions require substantial up-regulation of protein trafficking and deployment of membrane to the cell surface. TNFα, the earliest and most potent proinflammatory cytokine released, has an essential role in immunity but also plays a causative role in inflammatory disease (2). The secretory pathway for the trafficking of the newly synthesized transmembrane form of TNFα to the cell surface is unknown. Our approach has been to identify specific vesicular machinery that functions in this pathway (3, 4). Soluble N-ethylmaleimide–sensitive factor (NSF) attachment receptor (SNARE)–mediated fusion of vesicular carriers is a requirement of this and other trafficking pathways (5). Q-SNARE complexes on the Golgi (syntaxin 6–syntaxin 7–Vti1b) and at the cell surface [syntaxin 4–SNAP23 (synaptosomal-associated protein of 23 kD)] function in and are rate-limiting for TNFα trafficking and secretion (3, 4). A microarray screen identified vesicle-associated membrane protein 3 (VAMP3) as a lipopolysaccharide (LPS)-regulated SNARE protein (table S1). Interferon-γ (IFN-γ)-primed, LPS-stimulated macrophages synthesize more TNFα and secrete it faster than cells activated with LPS alone (6). VAMP3 is also up-regulated by IFN-γ priming, and its expression is modulated in temporal and quantitative fashions to match relevant Q-SNARE expression and TNFα trafficking (Fig. 1A).

Fig. 1.

VAMP3 up-regulation and function as the R-SNARE for TNFα secretion. (A) VAMP3 protein expression: up-regulation by LPS compared with other relevant SNAREs and in IFN-primed cells. WB, Western blotting. (B) SNARE partners coimmunoprecipitated (IP) with VAMP3 or VAMP8 are detected by immunoblotting. (C) TNFα secreted into media of transfected cells (GFP alone or GFP-VAMP3) measured by enzyme-linked immunosorbent assay (ELISA). Error bar indicates SEM. (D) Surface TNFα stained on unpermeabilized, TACE inhibitor–treated cells. Bar graph shows proportion of cells expressing GFP alone or GFP-VAMP3(1-81) mutant with >2-fold reduction in surface TNFα-staining expression. (E) Targeted knockdown of VAMP3 protein using three different specific siRNAs (left), and surface staining of TNFα in control (no siRNA) or siRNA-transfected cells (right) (23).

By coimmunoprecipitation, VAMP3 interacted with syntaxin 4, syntaxin 6, and Vti1b (Fig. 1B), indicating its potential as the cognate R-SNARE for the Golgi-associated and cell-surface Q-SNARE complexes (3). Trafficking in a variety of cells, including macrophages, can be enhanced by overexpression of full-length SNAREs, whereas overexpression of the SNARE cytoplasmic domains competitively inhibits and blocks vesicle docking and fusion (3, 4, 7). Overexpression of green fluorescent protein (GFP)–tagged VAMP3 in macrophages resulted in a fourfold increase in TNFα secretion (Fig. 1C). In contrast, expression of the inhibitory cytoplasmic domain of VAMP3 (GFP-VAMP3 amino acids 1 to 81) [but not of syntaxin 2, an irrelevant SNARE (3, 4)] blocked the delivery of TNFα to the cell surface (Fig. 1D) without compromising newly synthesized TNFα at the level of the Golgi (8). Short interfering RNA (siRNA) knockdown of VAMP3 expression (by >80%) also inhibited delivery and reduced surface staining of TNFα without affecting its synthesis (Fig. 1E). Thus, VAMP3 acts as the functional and rate-limiting R-SNARE for the known Golgi and cell surface Q-SNAREs required for post-Golgi transport of TNFα.

In macrophages, endogenous VAMP3 is located on vesicular membranes and plasma membrane ruffles (Fig. 2A). GFP-VAMP3 is similarly distributed, and at higher expression amounts it is also on enlarged cytoplasmic organelles and is more widespread on the cell surface (Fig. 2A). The majority of vesicular VAMP3 or GFP-VAMP3 colocalized with internalized transferrin (Tfn), Tfn receptor (TfnR), or Rab11 as markers of the recycling endosome (RE) (9, 10) (Fig. 2A and fig. S1). Notably, a proportion of TNFα (30%) in peripheral structures colocalized with GFP-VAMP3 (Fig. 2B) and with Tfn on the RE, suggesting an unexpected route for TNFα exocytosis. In live macrophages, GFP-TNFα was transported from the Golgi to peripheral structures identified as Tfn-positive REs en route to the cell surface (Fig. 2, D and E, and movies S1 to S3). This transport occurred in pleiomorphic carriers whose trajectories and behavior are consistent with post-Golgi carriers (11, 12) (Fig. 2, D and E, and movies S1 to S3). Close analysis confirmed that carriers and the RE fuse (Fig. 2D), and that at least 80% of the TNFα carriers exiting the Golgi fused with REs, verifying the requirement for two SNARE-mediated fusion events in the post-Golgi trafficking of TNFα. Golgi-to-RE transport involves pairing of the syntaxin 6–syntaxin 7–Vti1b–Q-SNARE with the R-SNARE–VAMP3 on the RE, then VAMP3 pairs with the Q-SNARE (syntaxin 4–SNAP23) on the plasma membrane.

Fig. 2.

TNFα trafficking to the cell surface via the RE. (A) In fixed cells, immunostaining of VAMP3 and GFP-VAMP3 colocalized with staining of Tfn (enlarged from box) and with Tfn receptor (TfnR). (B) Immunostaining and colocalization of TNFα with GFP-VAMP3 in REs (enlarged from box) and with Tfn. (C) Proportion of Tfn or TNFα immunostaining colocalized with GFP-VAMP3. Error bars indicate SEM. (D) GFP-TNFα imaged in live cells is concentrated in the Golgi (G) region and in carriers (∼250-nm diameter; arrow) moving along a path (sequential fixed points enlarged; arrowheads) toward a RE (1.5-μm diameter) in the cell periphery. (Right) Resulting fusion (γ) of a GFP-TNFα carrier (β) with the RE (α) is shown and quantified by fluorescence intensity of each compartment. Middle bar graph shows GFP-TNFα carriers (%) imaged leaving the Golgi region in live cells delivered to a RE compared with other destinations. (E) Live imaging of a cell expressing GFP-TNFα (green) after uptake of labeleled Tfn (red). Sequence of images showing movement of a Golgi-derived GFP-TNFα carrier (arrowheads) moving to and fusing with a RE (at 4.4 min) followed by exit of another carrier (at 11 min) from this RE.

The LPS- and IFN-γ-induced up-regulation of another RE protein, Rab11 (Fig. 3A), mirrored the expression of other machinery required for TNFα trafficking (3). A constitutively active mutant of Rab11 expressed in macrophages increased cell surface delivery of TNFα, whereas dominant-negative Rab11 blocked TNFα cell surface delivery without affecting newly synthesized TNFα at the Golgi complex (Fig. 3B). The functional requirement for the RE as a transit station in TNFα exocytosis was further demonstrated by the use of a horseradish peroxidase (HRP) inactivation assay (13). Cell surface delivery of TNFα was effectively blocked by inactivation of REs, accumulating TNFα at the Golgi and en route (Fig. 3C). The RE in activated macrophages thus assumes a pivotal role as a hub for exocytic trafficking. Correspondingly, the RE is integral to polarized exocytosis in epithelial cells (11, 13). How does this route serve to expedite the release of TNFα as an early response proinflammatory cytokine?

Fig. 3.

Recycling endosomes are required for TNFα trafficking. (A) LPS- and IFN-regulated expression of the RE protein Rab11. (B) Surface delivery and staining of TNFα in cells treated with LPS and TACE inhibitor and expressing Rab11 mutants. TNFα staining in the Golgi after cell permeabilization. (C) Surface delivery and staining of TNFα in cells after uptake of HRP-Tfn for enzymatic inactivation of the RE (13) and treatment with LPS and TACE inhibitor.

Activated macrophages increase their cell surface area for phagocytosis (14) by using extra membrane from endoplasmic reticulum (ER), lysosomes, and endosomes in SNARE-mediated fusion events (10, 1518), although the contribution from the ER is now controversial (19). We confirmed a role for VAMP3 in surface membrane expansion by fluorescence-activated cell sorter (FACS) analysis where overexpression of GFP-VAMP3 maximally increased the surface area of fluorescently labeled macrophages (fig. S2). Because VAMP3 function is also required for fully efficient phagocytosis of yeast (16, 20), we next investigated the possible convergence of VAMP3 roles in phagocytosis and surface delivery of TNFα. Primed macrophages incubated with the yeast Candida albicans for 20 to 40 min were imaged live or after fixation. In the initial stages of phagocytosis (before closure), actin-rich phagocytic cups (21) were labeled for VAMP3 (10) and for TNFα (Fig. 4A). TNFα staining was not seen after internalization or in more mature phagosomes labeled with LAMP1 (21) (Fig. 4, A and B). Addition of a TNFα converting enzyme (TACE) inhibitor to block proteolytic release of surface TNFα revealed that newly delivered surface TNFα was highly concentrated in the phagocytic cups compared with the surrounding plasma membrane (Fig. 4C). This was not the case for an unrelated and evenly distributed plasma membrane marker, GFP-tagged K-ras C-terminal targeting motif (GFP-tK) (Fig. 4C), or a secreted protein apolipoprotein E (movie S6). TNFα was not delivered randomly to the cell surface but was specifically delivered to sites of phagocytic cup formation. The absence of surface and soluble forms of TNFα from mature phagolysosomes suggests that soluble TNFα is cleaved and released rapidly, before closure of the cup. VAMP3 and other relevant machinery were clustered at the delivery site, including syntaxin 4, part of the cell surface Q-SNARE complex required for TNFα delivery (Figs. 4D and 1B), and TACE, the enzyme responsible for cleavage and release of soluble TNFα (22) (Fig. 4D). Lastly, we observed the movement of GFP-TNFα to the phagocytic cup during the internalization of C. albicans in live cells (Fig. 4E and movies S4 and S5). Membranes containing GFP-TNFα or GFP-VAMP3 were seen forming the initial phagocytic cup. As membrane moved around the yeast, engulfing and internalizing it, TNFα was concentrated in a patch on the outermost point of the cell surface from where it was cleaved and released (Fig. 4E, fig. S3, and movies S4 and S5). Thus, TNFα- and VAMP3-containing RE membranes translocate to the nascent phagocytic cup for SNARE-mediated fusion during the initial stages of phagocytosis. This presents both an unexpected site for cytokine secretion and a rapid and efficient mechanism for release of an early response inflammatory mediator. Having a single route for membrane flow to the cell surface via the RE that both delivers TNFα to the plasma membrane and expands the plasma membrane for phagocytic cup formation neatly combines two early actions of activated macrophages mounting an innate immune response.

Fig. 4.

RE delivers TNFα to newly forming phagocytic cups. (A) Primed macrophages were incubated with C. albicans (autofluorescing) and then fixed, permeabilized, and stained for VAMP3, TNFα, and F-actin. Boxed area enlarged to show staining at an actin-rich phagocytic cup. (B) Proportion of TNFα staining on phagocytic cups (actin-rich) compared with internalized phagosomes. Internalized mature phagosomes stain for LAMP1 but not F-actin. (C) Staining of TNFα at the actin-rich phagocytic cup enhanced in the presence of TACE inhibitor. Bar graph shows TNFα immunofluorescence in cups compared with rest of the plasma membrane. There is even distribution of another marker, GFP-tK, in transfected cells across all of the plasma membrane. Error bars indicate SEM. (D) Immunostaining for syntaxin 4 and TACE in actin-rich phagoctyic cups and phagososmes. (E) Live imaging of a primed macrophage expressing GFP-TNFα during phagocytosis (movie S4). Enlarged images show GFP-TNFα as it moves around the yeast (asterisk indicates phase contrast at 0 min) and culminates at the outermost point (arrowheads) after engulfment.

Supporting Online Material

Materials and Methods

Figs S1 to S3

Table S1


Movies S1 to S7

References and Notes

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