Carolin Offenhäuser

Cytokine secretion in macrophages and other cells: Pathways and mediators

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.

Cytokine Secretion Is Distinct from Secretion of Cytotoxic Granules in NK Cells

NK cells are renowned for their ability to kill virally infected or transformed host cells by release of cytotoxic granules containing granzymes and perforin. NK cells also have important regulatory capabilities chiefly mediated by secretion of cytokines, such as IFN-γ and TNF. The secretory pathway for the release of cytokines in NK cells is unknown. In this study, we show localization and trafficking of IFN-γ and TNF in human NK cells in compartments and vesicles that do not overlap with perforin or other late endosome granule markers. Cytokines in post-Golgi compartments colocalized with markers of the recycling endosome (RE). REs are functionally required for cytokine release because inactivation of REs or mutation of RE-associated proteins Rab11 and vesicle-associated membrane protein-3 blocked cytokine surface delivery and release. In contrast, REs are not needed for release of perforin from preformed granules but may be involved at earlier stages of granule maturation. These findings suggest a new role for REs in orchestrating secretion in NK cells. We show that the cytokines IFN-γ and TNF are trafficked and secreted via a different pathway than perforin. Although perforin granules are released in a polarized fashion at lytic synapses, distinct carriers transport both IFN-γ and TNF to points all over the cell surface, including within the synapse, for nonpolarized release.

Recycling Endosome Membrane Incorporation into the Leading Edge Regulates Lamellipodia Formation and Macrophage Migration

In comparison to our knowledge of the recycling of adhesion receptors and actin assembly, exactly how the cell controls its surface membrane to form a lamellipodium during migration is poorly understood. Here, we show the recycling endosome membrane is incorporated into the leading edge of a migrating cell to expand lamellipodia membrane. We have identified the SNARE complex that is necessary for fusion of the recycling endosome with the cell surface, as consisting of the R‐SNARE VAMP3 on the recycling endosome partnering with the surface Q‐SNARE Stx4/SNAP23, which was found to translocate and accumulate on the leading edge of migrating cells. Increasing VAMP3‐mediated fusion of the recycling endosome with the surface increased membrane ruffling, while inhibition of VAMP3‐mediated fusion showed that incorporation of the recycling endosome is necessary for efficient lamellipodia formation. At the same time, insertion of this recycling endosome membrane also delivers its cargo integrin α5β1 to the cell surface. The loss of this extra membrane for lamellipodia expansion and delivery of cargo in cells resulted in macrophages with a diminished capacity to effectively migrate. Thus, the recycling endosome membrane is incorporated into the leading edge and this aids expansion of the lamellipodia and simultaneously delivers integrins necessary for efficient cell migration.

Syntaxin 11 Binds Vti1b and Regulates Late Endosome to Lysosome Fusion in Macrophages

Syntaxin 11 (Stx11) is a SNARE protein enriched in cells of the immune system. Loss or mutation of Stx11 results in familial hemophagocytic lymphohistiocytosis type‐4 (FHL‐4), an autosomal recessive disorder of immune dysregulation characterized by high levels of inflammatory cytokines along with defects in T‐cell and natural killer cell function. We show here Stx11 is located on endosomal membranes including late endosomes and lysosomes in macrophages. While Stx11 did not form a typical trans‐SNARE complex, it did bind to the Q‐SNARE Vti1b and was able to regulate the availability of Vti1b to form the Q‐SNARE complexes Stx6/Stx7/Vtib and Stx7/Stx8/Vti1b. The mutant form of Stx11 sequestered Vti1b from forming the Q‐SNARE complex that mediates late endosome to lysosome fusion. Depletion of Stx11 in activated macrophages leads to an accumulation of enlarged late endocytic compartments, increased trafficking to the cell surface and inhibition of late endosome to lysosome fusion. These phenotypes are rescued by the expression of an siRNA‐resistant Stx11 construct in Stx11‐depleted cells. Our results suggest that by regulating the availability of Vti1b, Stx11 regulates trafficking steps between late endosomes, lysosomes and the cell surface in macrophages.

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3s surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.

Flightless, secreted through a late endosome/lysosome pathway, binds LPS and dampens cytokine secretion.

Summary Flightless (Flii) is upregulated in response to wounding and has been shown to function in wound closure and scarring. In macrophages intracellular Flii negatively modulates Toll-Like Receptor (TLR) signalling and dampens cytokine production. We now show that Flii is constitutively secreted from macrophages and fibroblasts and is present in human plasma. Secretion from fibroblasts is upregulated in response to scratch wounding and lipopolysaccharide (LPS)-activated macrophages also temporally upregulate their secretion of Flii. Using siRNA, and wild-type and mutant proteins, we show that Flii is secreted by means of a late endosomal/lysosomal pathway that is regulated by Rab7 and Stx11. Flii contains 11 leucine-rich repeat domains in its N-terminus that have nearly 50% similarity to those in the extracellular pathogen binding portion of Toll-like receptor 4 (TLR4). We show secreted Flii can also bind LPS and has the ability to alter macrophage activation. LPS activation of macrophages in Flii-depleted conditioned medium leads to enhanced macrophage activation and increased TNF secretion compared with cells activated in the presence of Flii. These results show secreted Flii binds to LPS and in doing so alters macrophage activation and cytokine secretion, suggesting that like the intracellular pool of Flii, secreted Flii also has the ability to alter inflammation.

The role of the recycling endosome in regulating lamellipodia formation and macrophage migration.

Cell migration is a highly complex process that requires the extension of cell membrane in the direction of travel. This membrane is continuously remodeled to expand the leading edge and alter its membrane properties. For a long time it has been known that there is a continual flow of polarized membrane traffic towards the leading edge during migration and that this trafficking is essential for cell migration. However, there is little information on how the cell coordinates exocytosis at the leading edge. It is also unclear whether these internal membranes are incorporated into the leading edge or are just delivering the necessary proteins for migration to occur. We have shown that recycling endosome membrane is incorporated into the plasma membrane at the leading edge to expand the membrane and at the same time delivers receptors to the leading edge to mediate migration. In order for this to happen the surface Q-SNARE complex Stx4/SNAP23 translocates to the leading edge where it binds to the R-SNARE VAMP3 on the recycling endosome allowing incorporation into the plasma membrane. Loss of any one of the components of this complex reduces efficient lamellipodia formation and restrains cell migration.

Lysosomal secretion of Flightless I upon injury has the potential to alter inflammation.

Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid.