DEFINING A ROLE FOR L-SELECTIN IN REGULATING TRANSENDOTHELIAL MIGRATION

The passage of white blood cells (leukocytes) from the circulation in to the surrounding tissue is absolutely critical for a healthy immune system. Most animals have evolved to have a fast-moving circulation to rapidly deploy oxygen to every cell in the body, and at the same time the leukocytes within the same bloodstream have evolved a method to gradually slow down against the force of blood flow in order to successfully exit the vasculature - e.g. during inflammation. Immediate arrest of leukocytes against the blood flow is virtually impossible. Scaled-up, if a leukocyte is the size of an average family car, it would move at half a million miles per hour through the largest vessel and would have to slow down to a just few car lengths per second. Just imagine what kind of breaks would need to be designed to slow that car down?! Biology has come up with a different answer for leukocytes......
Leukocytes interact with endothelial cells (EC), which are broad flat cells that line the entire inner surface of the vasculature. EC provide a water-tight barrier between the blood and the surrounding tissue. They act as gate-keepers, selectively allowing the passage of leukocytes and macromolecules (e.g. hormones, proteins etc) from the blood and into the surrounding tissue. It is therefore not surprising that EC are also the first cell type to come in contact with leukocytes during an inflammatory response. When stimulated with a "pro-inflammatory signal", EC bring to their surface molecules that are adhesive to leukocytes. The leukocytes are able to bind immediately to EC under the immense flow of blood. The first contact bonds that are generated between EC and leukocytes are relatively weak and are broken by the immense force of the blood flow. As the leukocyte breaks free from its interaction with EC, it is re-captured almost immediately by another part of the leukocyte. The continued capture, breakage and re-capture under the force of blood flow results in "leukocyte rolling", which, under the microscope, looks like the leukocytes adopts a "tumbleweed" motion along the inner vessel wall. L-selectin is a protein that is found on the surface of nearly all circulating leukocytes, and has been shown to mediate leukocyte rolling. L-selectin spans the outer membrane of the cell so that it has an outside (extracellular) domain and an inside (intracellular) domain. The extracellular domain of L-selectin binds to sugars that are presented by EC. The most compelling evidence to suggest that L-selectin has another role outside leukocyte rolling comes from studies where the L-selectin gene has been deleted. In these studies, leukocytes can still undergo rolling, but cannot migrate through in to the surrounding tissue - leukocytes tend to stay very close to the vessel once transmigrated. So far, there have been no follow-up studies to explore this observation at the molecular level. L-selectin can be cleaved at a site in the extracellular domain, close to the plasma membrane. Intriguingly, mice that have been genetically engineered to possess a form of L-selectin that cannot be cleaved (in other words it is continually present at the cell surface) display the same migratory behaviour as those cells without L-selectin. This paradoxical observation suggests that L-selectin may have to be cleaved and then re-presented on its surface for correct cell migration across the endothelium and then in to the surrounding tissue. Using cell-based approaches that include state-of-the-art microscopy, we aim to investigate how the intracellular and extracellular domains of L-selectin are involved in regulating leukocyte migration across the endothelium and into the surrounding tissue. This will be the first ever attempt to address this at the molecular level and will identify an important role for L-selectin beyond the process of leukocyte rolling.

L-selectin is a cell adhesion molecule that is involved in the initial stages of leukocyte capture (tethering) and rolling. We have previously shown that ligand binding of L-selectin promotes its clustering, and furthermore clustering of its intracellular binding partners, Calmodulin (CaM) and Ezrin-Radixin-Moesin (ERM). We found biochemically that L-selectin, CaM and ERM could form a heterotrimeric complex, which was confirmed in cells using state-of-the-art fluorescence resonance energy transfer (FRET) microscopy techniques. We now wish to extend our studies to GFP-tag L-selectin at the C-terminus and monitor its fate during transendothelial migration. We have used the THP-1 monocytic cell line to generate stable cells line expressing WT and mutant forms of L-selectin. Thorough characterisation of this cell line confirmed that the L-selectin-GFP chimera functions like WT L-selectin in that: (i) cells can tether and roll, (ii) the L-selectin can be shed in response to PMA and TNF-alpha , (iii) the L-selectin chimera is present on microvilli. We have performed anti-GFP Western blots in an attempt to pin-point when L-selectin is proteolytically cleaved upon THP-1 interaction with activated endothelial cells. We found that shedding occurs much later (15 min) than it takes for the THP-1 cell to protrude its pseudopods beneath the endothelium (3-5 min). This presents L-selectin with a "window of opportunity" to signal during transmigration. Using a combination of cell/molecular biological approaches, including FRET microscopy techniques, we will determine where and when L-selectin exerts its signalling during TEM under flow conditions. Most excitingly, we have generated a sheddase-resistant form of L-selectin that do not appear to protrude at all, suggesting that shedding is required for monocyte transmigration. The work outlined in this proposal will use mutagenesis studies to dissect out how L-selectin contributes to the latter stages of the adhesion cascade.

Who will benefit from the research?

The immediate impacts will be in scientific advancement and new knowledge in fundamental aspects of inflammation. Much of the impact will therefore be targeted basic cellular/molecular scientist, but will also impact clinical fields.

The outcome of the proposed research will touch on many disparate areas of clinical and non-clinical research that embody processes occurring in human health, but also disease. Examples include research in to:
(i) The inflammatory response
(ii) Human embryo implantation
(iii) Metastatic spread of cancers
(iv) Muscle stem cell trafficking

This research aims to provide enhanced understanding of how L-selectin shedding impacts leukocyte recruitment, remodelling of the cortical actin cytoskeleton, cell adhesion and migration. These basic cellular processes may be the very mechanisms that contribute to some of the physiological processes that are thought to involve L-selectin in non-leukocytic cells, such as those listed above.

How will they benefit from this research?
(i) Understanding conceptually how the cytoplasmic tail of L-selectin regulates shedding can provide useful information for researchers in related fields, where cell adhesion molecules and other transmembrane proteins are proteolytically cleaved from the cell surface. Examples include: TNF-alpha, CD44, TGF-alpha, Interleukin-6 receptor, TNF receptor etc. This will certainly benefit translational research and anti-inflammatory-based therapeutics.

(ii) A great deal of interest comes from understanding how leukocytes undergo changes in cell shape during recruitment. Most circulating cells adopt a spherical morphology, which then changes dramatically upon recruitment. Understanding the molecular basis for this change, particularly in the context of blocking L-selectin shedding could have potential commercial interest.

What will be done to ensure that they have the opportunity to benefit from this research?
(i)Much of the communication of the results gained from this work will be disseminated in the form of primary research papers and reviews in scientific journals. Other methods of communication will occur through invited seminars at other higher education and research institutional bodies, as well as through invitations to present posters/talks at national and international meetings/conferences/symposia.
(ii) Public engagements and outreach activities with the public and schools will be continued and introduced into existing frameworks within the Department (see pathways to impact).