Characterising the role of ICAM-3 and apoptotic cell-derived extracellular vesicles in the clearance of apoptotic cells

Abstract

Apoptotic cells (AC) are removed by macrophages (MØ) quickly to maintain healthy tissues. Apoptotic cell-derived extracellular vesicles (ACdEV) and ICAM-3 are shed during apoptosis and aid in AC removal by attracting macrophages (MØ). ICAM-3 on AC also mediates tethering to MØ. However the mechanism of ICAM-3 action is not known. This project aims to characterise the role of ICAM-3 and ACdEVs in the clearance of apoptotic cells. In agreement with previous studies, human lymphocytes were induced to apoptosis by UV irradiation and ACdEV were isolated and characterised. Using qNano, this work reveals, for the first time, release of ACdEV (~200nm) from early (6h) to late (18h) stages of apoptosis. Furthermore, the ability of ICAM-3 on ACdEV to promote phagocyte recruitment was confirmed and extended by using both vertical and horizontal migration assays. Also, ICAM-3 is confirmed as an apoptotic cell ligand that promotes interaction of AC with MØ. How ICAM-3 on ACdEV promotes MØ migration is not clear but novel data here suggests that it may be acting as an adhesion molecule to improve EV-MØ binding. Using in vitro assays of inflammation, AC and their EV were assessed for anti-inflammatory effects though any anti-inflammatory effect were unexpectedly small. In novel in vivo studies using a xenograft model, data show that anti-ICAM-3 mAb is capable of causing a significant reduction in growth of a transplanted ICAM-3+ tumour. The mAb also reduced MØ number within the tumours suggesting ICAM-3 can function in vivo for MØ recruitment to sites of cell death. Analyses of partner proteins for ICAM-3 was done in further novel studies. These were problematic as CO-IP approaches to the isolation of ICAM-3 were inefficient. However, mass spec analysis of crude membrane preparations revealed two proteins upregulated in apoptotic HeLa-ICAM-3 cells that may function to reorganize cytoskeleton. Future work is required to test if these observations are significant for the function of ICAM-3 in AC clearance.