L-Selectin Shedding Does Not Regulate Constitutive T Cell Trafficking but Controls the Migration Pathways of Antigen-activated T Lymphocytes

L-Selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LΔP-selectin) was engineered. Transgenic mice expressing either LΔP or wild-type (WT) L-selectin on T cells were crossed with L-selectin knockout (KO) mice. The cellularity and subset composition of secondary lymphoid organs did not differ between LΔP and WT mice, however, they were different from C57BL/6. Plasma levels of soluble L-selectin in LΔP mice were reduced to <5% of WT and C57BL/6 mice. The rolling properties of T lymphocytes from LΔP and WT mice on immobilized L-selectin ligands were similar. Furthermore, similar numbers of LΔP and WT T lymphocytes were recruited from the bloodstream into PLNs in mice, although LΔP T cells transmigrated HEVs more slowly. WT, but not LΔP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LΔP T cells retained the capacity to enter PLNs from the bloodstream. These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs. However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs

The role of L-selectin shedding in regulating lymphocyte migration

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