Inflammation is a response of the immune and vascular systems to the stimuli perceived as harmful to the host. Inflammation can be acute or chronic, and the recruitment of leukocytes to the affected tissues is a fundamental process during the course of both these events. Leukocytes are recruited in a process known as the multi-step adhesion cascade, which is a highly co-ordinated series of adhesive events mediated by the cell adhesion molecules (CAMs) on both leukocytes and endothelial cells. L-selectin is a CAM involved in the initial stages of the cascade, i.e. tethering and rolling, although there is a mounting body of evidence that points towards its role at later stages of the cascade, such as chemotaxis beyond transendothelial migration (TEM). The work outlined in this thesis explores the possibility that L-selectin actively contributes to TEM in monocytes. There are two important and measurable properties of L-selectin: (i) its rapid proteolysis (or “shedding”) upon cell activation, and (ii) its transition from being monomeric in the plasma membrane to being clustered, following ligand binding, which is a hallmark of downstream signalling. By expressing C-terminally green fluorescent protein (GFP)-tagged L-selectin in THP-1 monocytes, it was possible for the first time to monitor and analyse the spatio-temporal distribution of L-selectin and its shedding during TEM. In addition, co-expressing L-selectin-GFP with L-selectin tagged to red fluorescent protein (RFP) enabled measurement of L-selectin clustering during TEM via the use of fluorescence lifetime imaging microscopy (FLIM) to monitor Forster resonance energy transfer (FRET) between the GFP and RFP-tags. Interestingly, the majority of L-selectin was found to be clustered exclusively in pseudopods protruding beneath the endothelial monolayer, where L-selectin ligands are known to exist. L-selectin clustering was also found to occur following cross-linking of either CD43 or PECAM-1, suggesting inside-out signalling is an important factor in modulating L-selectin function. Moreover, both the extracellular cleavage domain and two cytoplasmic tail serine residues were involved in fundamentally regulating L-selectin clustering. Finally, the sub-cellular distribution of L-selectin clustering correlated tightly with the dynamics of the pseudopods that protruded beneath the endothelial monolayer during TEM. This thesis aims to understand the contribution that L-selectin has during the later stages of the adhesion cascade, and will re-shape the currently held perspective that this cell adhesion molecule’s role is solely restricted to just tethering and rolling.
Understanding the biological significance of L-selectin shedding during leukocyte transendothelial migration
Rzeniewicz, K. A. (Author). 2014
Student thesis: Doctoral Thesis › Doctor of Philosophy