Investigation of molecular mechanisms underlying the role of ICAM-3 in the phagocytic clearance of apoptotic leukocytes

Damaged, infected, aged or unwanted cells in the body are induced to die via a process known as apoptosis. The final step in this process is the removal (phagocytosis) of dying cells by healthy neighbouring cells where the cell corpses are eaten and degraded in a safe and controlled environment. A range of molecules have been suggested to play a role in this process though the amount of information relating to molecules recognised on dying cells is limited. It has been suggested that changes at the surface of dying cells result in the exposure of signals or flags that identify the cell, to viable neighbours, as dying and ready for removal. Relatively little is currently known about the nature of such 'eat me' signals. Lipids at the cell surface are known to change during death with phosphatidylserine (PS) becoming exposed at the cell surface where in viable cells it is usually maintained within the inner leaflet of the cell membrane. Once exposed PS is recognised by receptors on viable cells and mediates dying cell clearance. A change in another molecule, ICAM-3 (Intercellular Adhesion Molecule-3) on the surface of dying leukocytes have been implicated in the recognition and removal of dying leukocytes by professional phagocytes, macrophages. ICAM-3 is a molecule found only on leukocytes and, when present on viable cells, is important in the initiation of immune responses by facilitating cell-cell interactions. To mediate this interaction ICAM-3 binds to counter-receptors including the integrin LFA-1 and the non-integrin DC-SIGN. Previous work has indicated that ICAM-3 on dying cells loses its ability to bind to its prototypic counter-receptor LFA-1. This apoptosis-related loss of function is associated with a gain of alternate function where ICAM-3 becomes able to bind to another, as yet unidentified, receptor - a function that is important in mediating the phagocytic clearance of apoptotic leukocytes. The removal of dying leukocytes is especially important for the resolution of inflammation and the control of immune responses. Failure to remove leukocytes at the appropriate time may lead to chronic inflammatory conditions and autoimmune disease. A better understanding of the molecules and processes involved within the clearance of dying leukocytes will be central to future strategies for improving human health and well-being. This project seeks to identify the changes that occur in ICAM-3 that underlie the loss of function (loss of LFA-1 binding) and the gain of function (ability to bind other receptor(s) and mediate clearance of dying leukocytes) that is reported during apoptosis. The change of function associated with ICAM-3 at the apoptotic cell surface may be due to changes in ICAM-3 location, partner molecules or structure (e.g. sugar content of ICAM-3) and any such changes may arise from or contribute to changes in the mobility of ICAM-3 as cells undergo apoptosis. Within this project we will identify and characterise changes in the structure and/or environment of ICAM-3 occurring during apoptosis and will relate these changes to dead cell clearance.

Unwanted cells are removed in vivo through a carefully executed programme known as apoptosis that culminates in the safe and controlled removal of dying cells in an efficient manner. This application addresses the nature of molecular changes at the surface of dying cells that are fundamental to the success of apoptotic cell clearance. ICAM-3 (the most heavily glycosylated member of the Immunoglobulin Super Family) has previously been shown to change its function during apoptosis such that it loses its ability to bind LFA-1 (its viable cell counter-receptor) and gains the ability to bind a different receptor(s). This project seeks to identify the nature of the molecular changes underlying the role of ICAM-3 in apoptotic cell clearance. Using a range of fluorescence microscopy (including real-time analyses) and biochemical techniques we will analyse the localisation of ICAM-3 in relation to other cell surface molecules during apoptosis to identify whether a change of location and/or partner molecule(s) defines 'apoptotic cell-associated ICAM-3' and how this relates to phagocytic clearance. Real time studies will provide a powerful strategy for characterising the poorly characterised dynamic processes involved in apoptotic cell clearance. Further we will assess the structural integrity of ICAM-3 on viable and apoptotic cells to identify whether glycosylation changes underlie the ability of ICAM-3 to mediate apoptotic cell clearance. Finally we will characterise the ability of other IgSF members to function as ICAM-3 in clearance. This work will help better define the molecular processes and molecules involved in the clearance of apoptotic cells - a process central to the resolution of inflammation, control of immune responses, normal tissue homeostasis and development.