A novel flow cytometric method for quantifying phagocytosis of apoptotic cells

Apoptosis is a form of cell death induced by various external and internal cellular signals during development, cell differentiation, and normal tissue homeostasis (14). Induction of the cell death program intiates diverse cellular changes, including membrane blebbing, condensation of chromatin along the nuclear membrane, activation of calcium sensitive endonucleases resulting in DNA fragmentation, and the generation of membrane-enclosed apoptotic bodies (6). Apoptotic cells are specifically recognized and efficiently removed by various phagocytes before their continued degeneration results in a loss of apoptotic cell membrane integrity. Thus phagocytosis of apoptotic cells plays an important role in the clearance of nonfunctional dying cells, thereby preventing the release of toxic cellular enzymes and other by-products which would stimulate a potent inflammatory response and consequently contribute to tissue damage. Multiple types of somatic cells are capable of recognizing apoptotic cells. Examples include macrophage engulfment of apoptotic lymphocytes (1, 11), fibroblast phagocytosis of apoptotic neurotrophils (12), Langerhans’ cell engulfment of apoptotic vaginal epithelial cells during the murine estrous cycle (16), and liver endothelial cell phagocytosis of apoptotic liver cells (8). A number of methods have been used to quantify this process in vitro. Most current procedures quantify the number of apoptotic cells (internalized and surface bound) by light microscopy. These assays are based on the assumption that bound targets are destined for internalization (23), which is often an invalid assumption in cell-cell recognition systems where the target cell tightly adheres to the phagocytic cell regardless of the apoptotic state. An example of this is the specific interaction between endothelial cells and lymphocytes where endothelial cells regulate the transendothelial migration of lymphocytes as well as phagocytose apoptotic lymphocytes (4, 8, Cook-Mills, unpublished observation). Since endothelial cells bind leukocytes during both processes, binding of leukocytes must be distinguished from leukocyte internalization to specifically quantify phagocytosis. Other phagocytosis assays distinguish internalized targets from bound targets, but these assays require manual counting, which is cumbersome and time consuming. For example, Fadok et al. (10) described an assay whereby macrophages are treated with trypsin to dissociate bound cells and then the internalized cells are counted by light microscopy. In another assay, where the apoptotic target cell is a red blood cell, the bound but not internalized red blood cells are removed by hypotonic lysis and the remaining red blood cells are counted (19). This is not universally applicable, since apoptotic lymphocytes are not differentially susceptible to hypotonic lysis. Another current quantitative assay for phagocytosis utilizes the quenching of external but not phagocytosed FITC labels with trypan blue (18, 22). However, the FITC label itself is partially quenched by the acidic environment of the phagolysosome, thereby reducing assay sensitivity (18). This report describes a novel rapid method to quantify the percentage of endothelial cells which have phagocytosed apoptotic lymphocytes by distinguishing the following 3 populations: 1) endothelial cells that have phagocytosed apoptotic leukocytes, 2) endothelial cells that have surface-bound leukocytes, and 3) endothelial cells not associated with leukocytes. This method has broad applications as a technique to study phagocytic recognition of multiple apoptotic cell types.