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Apoptotic cell-derived extracellular vesicles as attractants for phagocytes
Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of ‘flags’ at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. We have shown previously that dying lymphocytes release apoptotic cell-derived extracellular vesicles (ACDEV) and these are strongly attractive to phagocytes in vitro. These ACDEV carry molecules including ICAM-3 that we have shown plays an important role in promoting phagocyte migration. Here we extend our previous studies to address EV from other apoptotic cells. Methods:
Supernatants from serum-free cultures, induced to apoptosis by UV, were centrifuged (1000xg) to remove cell debris and large apoptotic bodies. Dihydroxyvitamin D3-stimulated THP-1 cells, that efficiently remove apoptotic cells, were used as a model monocytic human phagocyte for these studies. Phagocyte migration to EV-replete supernatants was assayed (a) in a horizontal Dunn chemotaxis chamber where cell migration was mapped using Image J after time-lapse microscopy over 4 hours; or (b) using Cell-IQ live cell imaging to assess cell migration across a barrier of endothelial cells. Involvement of specific molecules was assessed using monoclonal antibodies or ICAM-3-deficient cells
Our data reveal that ACDEV promote strong directional migration of phagocytes in a manner dependent upon ICAM-3 released in ACDEV from the surface of dying leukocytes. Monoclonal antibody studies demonstrate that blockade of ICAM-3 can reduce phagocyte migration. Using transwell studies we further demonstrate that THP-monocytes are attracted across endothelial cell barriers to dying cells, also in an ICAM-3-dependent manner. Finally, our preliminary data indicate that ICAM-3 may be important in recruiting phagocytes to apoptotic lipid-laden macrophages (foam cells) but not control macrophages.
Taken together our data suggest ICAM-3 may play an important role in the recruitment of macrophages to sites of cell death. Such sites may include tumours and atherosclerotic plaques. Furthermore the inhibition of monocyte migration in the presence of anti- ICAM-3 mAbs suggests ICAM-3 may be a useful target for modulation of monocyte recruitment for therapeutic gain.