Tanousis, Kyriakos Michael; (2000) The role of L-selectin shedding in regulating lymphocyte migration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The recirculation of lymphocytes between blood and lymphoid organs is fundamental to the function of a healthy immune system. In lymph nodes (LN) lymphocytes exit the blood at specialised post capillary vessels known as high endothelial venules (HEVs), attaching to and migrating between the high endothelial cells (HECs) of which HEVs are comprised. The L-selectin cell adhesion molecule plays an essential role in this "homing" to LN. The extracellular portion of L-selectin is rapidly cleaved in response to a number of physiological and non-physiological stimuli, although the role of this "shedding" in lymphocyte migration is not well understood. To study this role, "non shedding" mutations were employed starting with an eight residue membrane proximal deletion (M-N) in human L-selectin. This mutation has been shown to completely abrogate phorbol ester-induced L-selectin shedding when expressed in the mouse pre-B cell line 300.19. Mouse EL4 T lymphoma cells, were stably transfected with either wildtype or M-N human L-selectin cDNA. Adhesion and transendothelial migration events were modelled using a cultured HEC monolayer system which allows lymphocyte interactions with HEV to be mimicked in vitro. The equivalent eight residue deletion (K-N) was created for mouse L-selectin and a further panel of stable EL4 transfectants was generated and tested. To address conflicting data on the ability of such deletion mutants to abolish shedding, as well as anomalies observed within this study, another "non shedding" mouse L-selectin mutation (LP) was engineered. Finally, mice transgenic for either K-N or LP or wildtype L-selectin were created. Transgene expression was directed towards the T lymphocyte compartment. Preliminary characterisation of transgenic mice indicated no gross effects of either mutation on lymphoid organ cellularity or short term lymphocyte trafficking. A number of important experiments will be possible when the transgenic mice are crossed to L-selectin knockout mice, removing expression of endogenous L-selectin.

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