SNAP‐23 and VAMP‐3 contribute to the release of IL‐6 and TNFα from a human synovial sarcoma cell line

Abstract

Fibroblast-like synoviocytes are important mediators of inflammatory joint damage in arthritis through the release of cytokines, but it is unknown whether their exocytosis from these particular cells is SNARE-dependent. Here, the complement of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in human synovial sarcoma cells (SW982) was examined with respect to the secretion of interleukin-6 (IL-6) and tumour necrosis factor a (TNFa), before and after knockdown of a synaptosome-associated protein of molecular mass 23 kDa (SNAP-23) or the vesicle-associated membrane protein 3 (VAMP-3). Wild-type SW982 cells expressed SNAP-23, VAMP-3, syntaxin isoforms 2-4 and synaptic vesicle protein 2C (SV2C). These cells showed Ca 2+ -dependent secretion of IL-6 and TNFa when stimulated by interleukin-1b (IL-1b) or in combination with K + depolarization. Specific knockdown of SNAP-23 or VAMP-3 decreased the exocytosis of IL-6 and TNFa; the reduced expression of SNAP-23 caused accumulation of SV2 in the peri-nuclear area. A monoclonal antibody specific for VAMP-3 precipitated SNAP-23 and syntaxin-2 (and syntaxin-3 to a lesser extent). The formation of SDS-resistant complexes by SNAP-23 and VAMP-3 was reduced upon knockdown of SNAP-23. Although the syntaxin isoforms 2, 3 and 4 are expressed in SW982 cells, knockdown of each did not affect the release of cytokines. Collectively, these results show that SNAP-23 and VAMP-3 participate in IL-1b-induced Ca 2+ -dependent release of IL-6 and TNFa from SW982 cells.

Fluorescence Lifetime Imaging Microscopy reveals rerouting of SNARE trafficking driving dendritic cell activation

“…Indeed, our data show that siRNA knockdown of VAMP3 impairs IL-6 secretion by monocyte-derived dendritic cells as previously observed for RAW264.7 murine macrophages (Manderson et al, 2007) and SW982 human synovial sarcoma cells (Boddul et al, 2014). As supported by our FRET-FLIM data the increased complexing of VAMP3 with Stx4 is thus likely responsible for the increased secretion of IL-6 and other cytokines by activated dendritic cells.…”

“…Because of the photobleaching we were unable to measure FLIM of the same cell pre- and post-LPS treatment. VAMP3 is known to mediate release of inflammatory cytokines by the murine macrophage cell line RAW264.7 (Manderson et al, 2007) and the human synovial sarcoma cell line SW982 (Boddul et al, 2014). VAMP3 is also required for the secretion of cytokines by LPS activated monocyte-derived dendritic cells, as siRNA knockdown of this SNARE impaired release of the cytokine IL-6, as shown by ELISA (Figure 5C–D; Figure 5—figure supplement 1E).…”

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules

“…Because of the limited imaging depth of TIRF microscopy, we could only measure exocytic events at the ventral membrane and not at the dorsal membrane. We recently showed that a post-fusion complex of the SNARE VAMP3 (responsible for IL-6 release [3,6,23]) is present at the dorsal membrane of dendritic cells, suggesting that exocytosis does take place at this side [3]. This is in line with findings in macrophages where no evidence was found for polarized secretion of IL-6, whereas tumor necrosis factor a (TNFa) was secreted predominantly at the phagocytic cup [6].…”

“…The lower estimate is assuming completely nonpolarized secretion, as suggested by a macrophage study where there was no evidence found for polarized secretion of IL‐6 in macrophages, whereas TNFα was predominantly secreted at the nascent cup of phagosomes . The higher estimate is for the case that IL‐6 is exclusively released at the ventral membrane, which we consider unlikely given that we recently showed the presence of VAMP3 in postfusion cis ‐SNARE complexes (i.e., the SNARE for IL‐6 release ) at the dorsal membrane. In the case where release of IL‐6 would be higher at the dorsal than the ventral membranes, we underestimate the rate of exocytic events and the average number of IL‐6 molecules per vesicle will be even lower.…”

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion

“…VAMP3 and SNAP-23 contribute to IL-6 and TNF secretion stimulated by IL-1b (61), and recent evidence supports the idea that VAMP3 mediates the secretion of components specifically from the recycling endosomes (62). In macrophages, for example, VAMP3 was found to be a marker of recycling endosomes and was involved in LPS-induced TNF secretion (63).…”