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
Source: PubMed


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.

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Available from: Rachael Zoe Murray, Jan 05, 2016
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    • "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). "
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    • "Lipopolysaccharide (LPS) up-regulates the expression of specific membrane fusion proteins [5], [6], [7] and increases the budding of TGN-derived membrane carriers to facilitate TNF trafficking and secretion [8], [9]. 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 [10], [11]. "
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    • "A number of different compartments, namely recycling endosomes, late endosomes/lysosomes and secretory granules, have been shown to fuse with plasma membrane during phagocytosis [36]. In macrophages, recycling endosomes fuse with the plasma membrane at the level of the phagocytic cup in a SNAP23-dependent manner [36], [37]. 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. "
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