Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.595090
Title: SNARE proteins in human mast cells
Author: Friend, Reuben
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2013
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Abstract:
Mast cells form an integral part of both innate and adaptive immunity; they help to orchestrate the inflammatory immune response through the release of a variety of inflammatory mediators. Adverse reaction to allergens can lead to activation of mast cells, causing degranulation and release of a range of pro-inflammatory mediators contributing to the onset of allergy. The most studied activation pathway in the adaptive immune response of mast cells is through the Immunoglobulin E (IgE) cell surface receptor FceRI. Crosslinking of FceRI leads to degranulation and de novo synthesis of mediators. Every eukaryotic cell undergoes constitutive secretion. Alongside this general process, cells such as neuronal endocrine and immune cells, including mast cells, perform regulated secretion. This enables the cell to rapidly release mediators stored in secretory granules upon stimulation by a particular extracellular ligand. Mediators released fall into two categories; pre-formed, contained within these secretory granules; monoamines such as histamine as well as many proteases, and de novo synthesized that are released through the constitutive secretory pathway, including prostaglandins, leukotrienes, cytokines and chemokines. Elucidating the mechanisms of mast cell mediator release is imperative for understanding many disease processes; however, knowledge of the precise mechanisms by which mast cell exocytosis is controlled remains elusive. The aim of this study was to identify and characterise Soluble NSF attachment protein receptor (SNARE) proteins involved in the release of inflammatory mediators in human mast cells. Using LAD 2 human mast cells and primary human lung mast cells (HLMCS), expression of a variety of syntaxins and Vesicle associated membrane proteins (VAMPs), as well as the ubiquitously expressed SNAP-23 were found. To study the roles of individual VAMPs in exocytosis a novel technique utilising pH sensitive pHluorins was developed. Using VAMPs tagged with pHluorins, the cellular distribution of VAMP-3 and VAMP-8 containing vesicles and their behaviour upon IgE stimulation in live cells was monitored. In unstimulated cells, VAMP- 3 and 8 were found to have distinct cellular distributions. Upon IgE stimulation both VAMP-3 and VAMP-8 containing vesicles translocated to the membrane and underwent membrane fusion, consistent with roles in exocytosis. However, their responses showed distinct time courses and calcium dependences. Importantly the VAMP-3 vesicle pool could be selectively targeted with a botulinum neurotoxin serotype B (BoNT)/B LC construct and in doing so inhibited the release of IL-6. The findings in this study support the notion that distinct vesicle pools, defined in part by expression of VAMP-3 and VAMP-8, regulate the release of inflammatory mediators from mast cells and that BoNTs might provide a novel means of targeting the release of chronic inflammatory mediators from mast cells for treatment of chronic inflammatory diseases.
Supervisor: Seward, Elizabeth Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.595090  DOI: Not available
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