Draper-mediated and Phosphatidylserine-independent Phagocytosis of Apoptotic Cells by Drosophila Hemocytes/Macrophages

Abstract

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.

Apoptosis-dependent Externalization and Involvement in Apoptotic Cell Clearance of DmCaBP1, an Endoplasmic Reticulum Protein of Drosophila

“…Antibody and Immunochemistry-The generation and use of anti-Draper (14), anti-Croquemort (14), anti-focal adhesion kinase (21), anti-Pretaporter (17), and anti-Calreticulin (22,23) antibodies were reported previously. Anti-Drosophila DmCaBP1 antibody was raised by immunizing rats with recombinant DmCaBP1 expressed in E. coli as a protein fused to GST.…”

“…l(2)mbn cells were incubated with 20-hydroxyecdysone (Sigma-Aldrich) (1 M) for 48 h before being used in an assay for phagocytosis. To induce apoptosis, S2 cells were incubated in the presence of cycloheximide (Sigma-Aldrich) (2 g/ml) for 24 h, as described previously (14). Apoptotic S2 cells were stained with the DNA-binding fluorochrome Hoechst 33342 (1 M) and by TUNEL (Millipore) for the examination of chromatin condensation and DNA cleavage, respectively, according to standard protocols.…”

“…Cell Culture-The cell lines l(2)mbn, established from larval hemocytes, and embryonic-cell derived S2 were maintained at 25°C with Schneider's Drosophila medium (Invitrogen) supplemented with 10% (v/v) heat-inactivated FBS, as described previously (14). l(2)mbn cells were incubated with 20-hydroxyecdysone (Sigma-Aldrich) (1 M) for 48 h before being used in an assay for phagocytosis.…”

“…To fractionate externalized DmCaBP1, culture media of apoptotic S2 cells were centrifuged at varying levels of forces according to published procedures (20), and the resulting pellets and supernatants at each centrifugation were analyzed by Western blotting. RNAi using double-stranded RNA synthesized in vitro was done as described previously (14).…”

“…CED-1 is a single-path membrane protein containing atypical EGF-like repeats in its extracellular region (11). There are counterparts of CED-1 in other species (12), namely Draper in Drosophila (13,14), Jedi in mice (15), and MEGF10 in humans (16), and their involvement in the phagocytosis of apoptotic cells has been shown. However, it is almost obscure how CED-1 and its counterparts are activated for the induction of engulfment in phagocytes.…”