High Endothelial Venule (HEV) cell phagocytosis of apoptotic leukocytes

HEV cells executed a novel function, the phagocytosis of apoptotic leukocytes as determined by a fluorescence-based phagocytosis assay and confocal microscopy. To examine the mechanisms for this phagocytosis, we compared the acquisition of leukocyte apoptotic characteristics with HEV cell receptor recognition of these apoptotic cells. 50% maximum acquisition of apoptotic characteristics were defined in γ-irradiated (500 rads) splenic lymphocytes, anti-Ig-treated WEHI-231 cells (B lymphoma), and IL-3-deprived 32D cells (premyeloid cell line). Apoptotic splenic lymphocytes were at 50% maximum of DNA fragmentation and propidium iodide (PI)-labeling of ethanol-fixed cells at 4 hours. Apoptotic splenic lymphocytes were defined as in a mid apoptotic stage at 10 hours as determined by PI-permeability and in a late apoptotic stage at 20 hours as determined by MC540 and annexin V-FITC labeling. Apoptotic WEHI-231 cells were defined as in an early stage of apoptosis at 24 hours as determined by DNA fragmentation, in a mid stage of apoptosis at 32 hours as determined by MC540 and annexin V-FITC labeling and by PI- labeling of ethanol-fixed cells, and in a late stage of apoptosis at 40 hours as determined by PI-labeling of non-fixed cells. In contrast, apoptotic 32D cells were defined as in an early stage of apoptosis at 10 hours as determined by DNA fragmentation, in a mid stage of apoptosis at 20 hours as determined by PI-labeling of ethanol-fixed and non-fixed cells, and in a late stage of apoptosis at 30 hours as determined by MC540 and annexin V-FITC labeling. Recognition and ingestion of early stage apoptotic WEHI-231 cells by a murine HEV cell line involved a fucose-specific receptor as determined by receptor inhibition studies. Since removal of apoptotic cells is mandatory for tissue protection and preservation, investigation of endothelial cell phagocytosis of apoptotic cells can lead to a better understanding of essential clearance mechanisms employed in this biological process.