The passage of leukocytes across blood vessel walls plays a key role in the immune response to infection in inflammatory conditions. ADAM10 is a ubiquitously expressed molecular scissor that proteolytically cleaves key cell surface proteins including vascular endothelial (VE)-cadherin and transmembrane chemokines. Their shedding by ADAM10 promotes leukocyte transmigration in cell line models, however the precise mechanism behind ADAM I O's involvement is unknown. ADAM10 associates with six different membrane organising tetraspanins (Tspan5/10/14/15/17/33) termed the TspanC8s. These tetraspanins regulate ADAM10 enzymatic maturation and trafficking to the cell surface and emerging evidence indicates that different TspanC8s can promote ADAM10 cleavage of specific substrates. It was hypothesised that ADAM10 promotes leukocyte transmigration by cleaving one of its endothelial substrates and one or more of the TspanC8s could facilitate this process. The aim of this thesis was to test this hypothesis using in vitro leukocyte adhesion assays with primary human leukocytes and human umbilical vein endothelial cells (HUVECs). siRNA knockdown or pharmacological inhibition of ADAM10 on HUVECs impaired the transmigration of lymphocytes, but not neutrophils or monocytes. ADAM10 knockdown/inhibition caused a reduction in VE-cadherin shedding and an increase in VE-cadherin surface expression. Partial knockdown of VE-cadherin, in the presence of ADAM10 knockdown/inhibition, reduced VE-cadherin levels to normal and restored basal lymphocyte transmigration. Systematic knockdown of TspanC8s in HUVECs revealed that the presence of either Tspan5 or Tspan17 was sufficient to maintain basal lymphocyte transmigration and reduced VE-cadherin surface levels. Tspan5 and Tspan17 are functionally uncharacterised, but they are the most highly related TspanC8s by sequence (78% amino acid identity) and may share a common role in lymphocyte transmigration by regulation of ADAM10 and VE-cadherin.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:685376 |
| Date | January 2016 |
| Creators | Reyat, Jasmeet Singh |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/6650/ |
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