Cytokine Secretion via Cholesterol-rich Lipid Raft-associated SNAREs at the Phagocytic Cup
Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia. Journal of Biological Chemistry
(Impact Factor: 4.57).
05/2006; 281(17):11949-54. DOI: 10.1074/jbc.M600857200
Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha (TNFalpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFalpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNFalpha trafficking and secretion. TNFalpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNFalpha surface distribution revealed these puncta to be the initial points of TNFalpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.
Available from: Neda Moradin
- "Hence, it is conceivable that multiple aspects of phagolysosome biogenesis, crucial for the acquisition of the microbicidal properties of this organelle and its ability to properly process proteins for antigen presentation, are regulated by a series of membrane fusion regulators such as SNAREs. To determine whether Leishmania impairs the recruitment of other SNAREs, we infected bone marrow-derived macrophages (BMMs) with L. donovani promastigotes and assessed the intracellular localization of VAMP3 and VAMP8, two SNAREs known to be present on phagosomes (Furuta et al., 2010; Kay et al., 2006; Murray et al., 2005). Immunofluorescence analyses at the confocal microscope show that both VAMP3 and VAMP8 were present on zymosan (Zym)-containing phagosomes at 2 and 6 hr after the initiation of phagocytosis (Figure 1A). "
[Show abstract] [Hide abstract]
ABSTRACT: During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to T cells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced T cell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.
Cell host & microbe 07/2013; 14(1):15-25. DOI:10.1016/j.chom.2013.06.003 · 12.33 Impact Factor
Available from: Bradly J Marsh
- "Lipopolysaccharide (LPS) up-regulates the expression of specific membrane fusion proteins , ,  and increases the budding of TGN-derived membrane carriers to facilitate TNF trafficking and secretion , . Specific trafficking molecules of the SNARE and Rab families that mediate post-Golgi transport and TGN-associated golgins have been identified as regulators of TNF trafficking in macrophages , . "
[Show abstract] [Hide abstract]
ABSTRACT: Lipopolysaccharide (LPS)-activated macrophages secrete pro-inflammatory cytokines, including tumor necrosis factor (TNF) to elicit innate immune responses. Secretion of these cytokines is also a major contributing factor in chronic inflammatory disease. In previous studies we have begun to elucidate the pathways and molecules that mediate the intracellular trafficking and secretion of TNF. Rab6a and Rab6a' (collectively Rab6) are trans-Golgi-localized GTPases known for roles in maintaining Golgi structure and Golgi-associated trafficking. We found that induction of TNF secretion by LPS promoted the selective increase of Rab6 expression. Depletion of Rab6 (via siRNA and shRNA) resulted in reorganization of the Golgi ribbon into more compact structures that at the resolution of electron microcopy consisted of elongated Golgi stacks that likely arose from fusion of smaller Golgi elements. Concomitantly, the delivery of TNF to the cell surface and subsequent release into the media was reduced. Dominant negative mutants of Rab6 had similar effects in disrupting TNF secretion. In live cells, Rab6-GFP were localized on trans-Golgi network (TGN)-derived tubular carriers demarked by the golgin p230. Rab6 depletion and inactive mutants altered carrier egress and partially reduced p230 membrane association. Our results show that Rab6 acts on TNF trafficking at the level of TGN exit in tubular carriers and our findings suggest Rab6 may stabilize p230 on the tubules to facilitate TNF transport. Both Rab6 isoforms are needed in macrophages for Golgi stack organization and for the efficient post-Golgi transport of TNF. This work provides new insights into Rab6 function and into the role of the Golgi complex in cytokine secretion in inflammatory macrophages.
PLoS ONE 02/2013; 8(2):e57034. DOI:10.1371/journal.pone.0057034 · 3.23 Impact Factor
Available from: Fabienne Paumet
- "A number of different compartments, namely recycling endosomes, late endosomes/lysosomes and secretory granules, have been shown to fuse with plasma membrane during phagocytosis . In macrophages, recycling endosomes fuse with the plasma membrane at the level of the phagocytic cup in a SNAP23-dependent manner , . The fact that both overexpressed SNAP29-GFP and SNAP23-GFP affect mast cell phagocytosis suggests that both of these SNAREs may participate in fusion events occurring at the plasma membrane during internalization in mast cells. "
[Show abstract] [Hide abstract]
ABSTRACT: Mast cells play a critical role in the innate immune response to bacterial infection. They internalize and kill a variety of bacteria and process antigen for presentation to T cells via MHC molecules. Although mast cell phagocytosis appears to play a significant role during bacterial infection, little is known about the proteins involved in its regulation. In this study, we demonstrate that the SNARE protein SNAP29 is involved in mast cell phagocytosis. SNAP29 is localized in the endocytic pathway and is transiently recruited to Escherichia coli (E. coli)-containing phagosomes. Interestingly, overexpression of SNAP29 significantly increases the internalization and killing of E. coli, while it does not affect mast cell exocytosis of inflammatory mediators. To our knowledge, these data are the first to demonstrate a novel function of SNAP29 in mast cell phagocytosis and have implications in protection against bacterial infection. Citation: Wesolowski J, Caldwell V, Paumet F (2012) A Novel Function for SNAP29 (Synaptosomal-Associated Protein of 29 kDa) in Mast Cell Phagocytosis. PLoS ONE 7(11): e49886. doi:10.1371/journal.pone.0049886
PLoS ONE 11/2012; 7(11):e49886. DOI:10.1371/journal.pone.0049886 · 3.23 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.