Phillips, Rhian Medi; (2001) Identification of high endothelial cell-derived chemokines that regulate T lymphocyte transendothelial migration. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

To assess the roles of chemokines in regulating adhesion and transendothelial migration of T lymphocytes across High Endothelial Venules (HEV) into lymph nodes (LN), an in vitro model of T lymphocyte binding and transendothelial migration, which employs High Endothelial Cells (HEC) cultured from rat LN HEV was used. Pertussis toxin (PTX)-treatment of T lymphocytes has minimal effects on the total binding of T lymphocytes to HEC, whereas PTX-treatment of T lymphocytes inhibited their migration across HEC in a concentration-dependent manner, with maximal inhibition of 80 %. This indicates that transendothelial migration of T lymphocytes, but not adhesion to HEC, is regulated by PTX-sensitive Gi-coupled receptors on the T lymphocytes. Expression of the chemokines MCP-1, MIP-3β and SDF-lα by HEC was demonstrated by ELISA. Two MCP-1 transcripts (approximately 1 and 4 Kb) were identified by northern blotting, and a transcript isolated and sequenced from a rat HEC cDNA library corresponded to the published rat MCP-1 sequence and 1 Kb transcript. A murine MIP-3β probe did not detect the rat homologue, and a probe for SDF-lα has not been tested. Attempts were made to establish a functional assay for effects of chemokines on rat T lymphocytes. Recombinant chemokines failed to induce mobilisation of intracellular calcium, or reproducibly polymerise actin in T lymphocytes. However MIP-3β, SLC and SDF-l? all stimulated migration of T lymphocytes in a transwell migration assay. MCP-1 did not stimulate T lymphocyte migration in this assay. Desensitisation of receptors for SLC or MIP-3β had no effect on T lymphocyte binding or transendothelial migration, however desensitisation of SDF-lα receptors significantly inhibited T lymphocyte transmigration across HEC (60 % inhibition). Desensitisation of SDF-lα, SLC and MIP-3β receptors was verified using the transwell migration assay. Although the nature of the receptors and mechanisms of receptor desensitisation are unclear, these results indicate that SDF-lα is a pivotal regulator of T lymphocyte transendothelial migration across HEC.

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