Phagocytic Activity of Apoptotic Cells

ABSTRACT

Apoptosis is a distinct mechanism of cell death involved in many physiological and pathological processes. Various stimuli, including phagocytosis of bacteria, can induce apoptosis. As the cells proceed through apoptosis, functional activities decline in accord with phenotypic changes. However, decline in functional activities does not mean instantaneous shut-down of all functions, which is rather the characteristic of necrosis. Phagocytosis and oxidative burst are two of the major tasks of mloid cells for engulfment and killing of microbes. It was reported that the myeloid cells which phagocytosed bacteria underwent apoptosis, rendering resolution of acute inflammation. On the contrary, it was known that phagocytosis of latex beads did induce apoptosis. However, we found phagocytosis of latex beads within the apoptotic cell fraction. Thus we investigated whether phagocytosis of latex beads induced apoptosis or apoptotic cells phagocytosed the beads. We used human promyelocytic cell line HL-60 cultured for 4 days in RPMI1640 supplemented with 10% fetal bovine serum and 1 uM all-trans retinoic acid for phagocytic assay. Phagocytic activity was analyzed by flow cytometry after shaking incubation of HL- 60 cells (5 x 10 cells/ml) with fluorochrome-cougated latex beads for 1 hour at 37C followed by elimination of the un-phagocytosed beads by centrifugation on the density of fetal bovine serum. Apoptotic cells were identified as subdiploid fraction by staining the cells with DNA-dye. To investigate whether phagocytosis of latex beads leads to apoptosis or apoptotic cells phagocytose the beads, the cells wbich had phagocytosed the beads were sequentially analyzed before and after 1, 3, 6, 12 and 24 hours of incubation. On the other hand, the apoptotic cell fraction was sorted to be analyzed for phagocytic activity. The sorted cells were also analyzed by chemiluminescence assay for capability of oxidative burst by stimulation with PMA (5 mM). The results showed little increase in the apoptotic fraction among phagocytic cells during incubation up to 24 hours. Rather the sorted apoptotic cells did phagocytose latex beads. But the sorted cells did not show any capability of oxidative burst. Taken these results into consideration, the apoptotic cells seemed to be on the way of dying process in which oxidative burst was lost while phagocytic activity remained. Thus it was suggested that the primitive function of phagocytosis remained longer in the cells proceeding through apoptosis, while oxidative bunt, requiring mitochondrial function, was lost earlier.