Report

Requirement for Croquemort in Phagocytosis of Apoptotic Cells in Drosophila

See allHide authors and affiliations

Science  18 Jun 1999:
Vol. 284, Issue 5422, pp. 1991-1994
DOI: 10.1126/science.284.5422.1991

Abstract

Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed thatcrq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria in Drosophila.

Phagocytosis is the terminal event of the apoptotic process (1, 2) and is also critical for the engulfment of microorganisms (3). It has been proposed that the recognition of both nonself (microorganisms) and effete self (corpses) may share common receptors (4). Blocking experiments have implicated a number of receptors as important for target recognition (2–4). Genetic studies indicate that some of these receptors participate in phagocytosis of pathogens in vivo (5, 6). However, the multiplicity and redundancy of recognition mechanisms in mammalian systems have made it difficult to evaluate the relative roles of these receptors in the phagocytosis of corpses. Although several genes of Caenorhabditis elegans are involved in the phagocytosis of corpses (7–9), none of these molecules seem to act directly as a receptor in the recognition of the corpse.

In Drosophila embryos, like in mammals and in contrast to worms, the clearance of apoptotic cells is primarily mediated by macrophages, hemocytes that become phagocytic at the initiation of developmentally regulated apoptosis (10). Croquemort (CRQ), a Drosophila CD36-related receptor, is specifically expressed on all embryonic macrophages (11). Human CD36 acts as a scavenger receptor (12–14) and also binds apoptotic cells in combination with the macrophage vitronectin receptor and thrombospondin (15,16). CD36 has the ability to confer phagocytic activity on nonphagocytic cells on transfection (17,18). CRQ expression in nonphagocytic Cos7 cells allows these cells to recognize and engulf apoptotic thymocytes (11). Thus, CRQ may participate in the removal of apoptotic cells duringDrosophila embryogenesis. We genetically evaluated the relative role of this receptor in phagocytosis of apoptotic cells and in other macrophage functions in vivo.

To look at the crq-null phenotype, we used two overlapping deletions of the 21C region, Df(2L)al(19) and Df(2L)TE99(Z)XW88 (W88) (20). The Drosophila genome project sequence indicates that crq is at position 21C4 betweenexpanded (ex) (21) andu-shaped (ush) (22, 23).Df(2L)al removes about 180 kb from the aristalessgene (al) (19) to ush, whereasW88 uncovers about 100 kb from ex toush (20). Homozygous embryos for either of these deficiencies can be distinguished by morphological defects (19, 22). Both polymerase chain reaction (PCR) on single embryos (24) and CRQ immunostaining (11) confirmed that these homozygous embryos arecrq null.

We assayed phagocytosis of apoptotic corpses in Df(2L)al andW88 homozygous embryos with a double fluorescent immunolabeling for CRQ and peroxidasin, a hemocyte marker (10), and a nuclear dye, 7-amino actinomycin D (7-AAD) (25). Macrophages in wild-type embryos and embryos homozygous for the balancer chromosome were phagocytic for apoptotic corpses (Fig. 1, A and D) with a mean phagocytic index (P.I.) of 3.96 corpses per macrophage (Fig. 1G) (26). Although they accumulated at the site of cell death, macrophages within Df(2L)al and W88homozygous embryos remained very small and round (Fig. 1, B, C, E, and F), with P.I.s of 0.26 and 0.21, respectively (Fig. 1G).

Figure 1

Macrophages in crq-deficient embryos have very poor phagocytic activity for apoptotic cells. (A to F) In confocal micrographs, peroxidasin-stained hemocytes appear green, CRQ staining appears blue, 7-AAD–stained apoptotic corpses appear as bright red round particles, and the nuclei of viable cells appear as large red diffused components. All images are the sum of eight focal planes. (A) to (C) show a ×40 magnified lateral view of the head region of (A) a In(2L)Cyhomozygous embryo, (B) a Df(2L)al homozygous embryo, and (C) a W88 homozygous embryo. (D) to (F) show high-magnification views (×400) of their respective macrophages. As compared with the wild-type distribution (A) and phagocytic activity (D) of macrophages withinIn(2L)Cy homozygous embryos, macrophages inDf(2L)al (B) and W88 (C) homozygous embryos accumulate in the head and around the amnioserosa and show very poor phagocytic activity despite their recruitment at sites of abundant apoptosis (E and F). Asterisks indicate the nucleus of each macrophage seen in these fields. (G) A chart summarizes the efficiency of phagocytosis of apoptotic corpses observed within each genotyped embryos assayed. Results shown are the mean P.I. ± SE;n is the total number of macrophages scored for each genotype. Dark blue, w; In(2L)Cy/In(2L)Cy; red, w; In(2L)Cy/Df(2L)al; yellow, w; Df(2L)al/Df(2L)al; and light blue, w; W88/W88.

In the absence of crq single mutants, we could not definitively conclude that the phenotype observed inDf(2L)al or W88homozygous embryos resulted solely from the deletion ofcrq. Therefore, we generated a UAS-crq transgene (27) and tested the ability of ubiquitously expressed CRQ to rescue the engulfment defect in Df(2L)al homozygous embryos, using a hsGal4 transgene to drive expression. In heat-shocked mutant embryos that carried both the hsGal4 and UAS-crq transgenes, macrophages showed substantial CRQ expression and phagocytic activity for apoptotic cells, with a P.I. of 2.20 (Fig. 2, C, F, and G). This indicates that crq is sufficient to rescue the phagocytosis defect in Df(2L)al homozygous embryos.

Figure 2

Expression of a croquemort transgene reinstates the ability of macrophages in Df(2L)alhomozygotes to recognize and engulf apoptotic corpses. A UAS-crq transgene was expressed under the control of a hsGal4 driver in all cells of Df(2L)al embryos, and phagocytosis of apoptotic corpses was assessed as in Fig. 1 (25). (A toC) Single focal planes. (D to F) Images are the sum of eight focal planes. (A) to (C) show a ×100 view of macrophages within the head of embryos with the following genotypes: (A) control: w; CyO,S/CyO,S; hsGal4/+; (B) homozygous mutant, as recognized by aberrant morphology: w; Df(2L)al/Df(2L)al; hsGal4/hsGal4; and (C) transgene rescue of homozygous mutant: w, UAS-crq; Df(2L)al/Df(2L)al; hsGal4/+. All embryos were heat-shocked for 1 hour at 39°C and assayed 2 hours later. (D) to (F) show high-magnification views (×400) of macrophages within the embryos shown in (A) to (C), respectively. Asterisks indicate the nucleus of each macrophage seen in these fields. Arrows indicate free apoptotic corpses. (G) A chart summarizes the efficiency of phagocytosis of apoptotic corpses observed within each category of embryos assayed. Results shown are the mean P.I. ± SE; n is the total number of macrophages scored for each genotype. Blue, w; CyO,S/CyO,S; hsGal4/hsGal4; red,w; Df(2L)al/Df(2L)al; hsGal4/hsGal4; and yellow, w, UAS-crq; Df(2L)al/Df(2L)al; hsGal4/+.

In serial sections of embryos that ubiquitously expressed CRQ, we observed that apoptotic corpses were not engulfed by cells other than macrophages (28, 29). Thus, ectopic expression of CRQ is not sufficient to confer phagocytic ability on other cells in the embryo. This finding is in contrast with our previous observation that CRQ expression was sufficient to confer phagocytic activity on Cos7 cells (13). However, in UAS-crq; hsGal4embryos, CRQ was found at only low levels in cells other than macrophages, suggesting that CRQ might be unstable in other cells.

A human macrophage receptor, CD14, participates in both recognition and engulfment of pathogens as well as of apoptotic cells (30, 31). We tested whether crqparticipates in phagocytosis of pathogens by Drosophilaembryonic macrophages. We injected fluorescently labeled bacteria into living stage 11 wild-type and W88 embryos and monitored their fate by confocal microscopy (32). Wild-type embryonic macrophages engulfed both Gram-negative (Escherichia coli) (Fig. 3, A to C) and Gram-positive bacteria (Staphylococcus aureus) (28). InW88 homozygous embryos identified by their u-shaped phenotype (Fig. 3D), macrophages also engulfed bacteria (Fig. 3, D and E). Although these assays are not quantitative, we conclude thatcrq is specifically required for the phagocytosis of apoptotic corpses and is not essential for the engulfment of bacteria.crq is also not necessary for endocytosis of acetylated low density lipoproteins (LDLs) (33) (Fig. 3F) or for the production of the extracellular matrix components peroxidasin and MDP-1 (Fig. 1, B and C) (28).

Figure 3

Wild-type and crq-deficientDrosophila embryonic macrophages are competent to recognize and engulf bacteria and perform endocytosis. Stage 11 yw andW88 homozygous embryos were injected with TRITC-labeled bacteria (32). The fate of the bacteria was monitored by confocal microscopy. (A to C) High-magnification images (×100) of a macrophage in a yw injected embryo taken at three successive focal planes. The overlay of Nomarski and fluorescent images shows a macrophage that has engulfed several labeled bacteria. (D) A Nomarski image of a magnified view (×40) of the head region of a W88 embryo injected with TRITC-labeled bacteria. In this panel, a very large cell can be seen that has the typical morphology of a macrophage (arrowhead). (E) As seen at high magnification (×400), macrophages in this mutant can engulf bacteria. (F) A high-magnification image (×100) of a macrophage (arrowhead) in a W88 embryo that had been injected with dioctadecyl tetramethyl indocarbocyanine–labeled acetylated LDL (DiI AcLDL) (33). Nomarski and fluorescent images of a single focal plane were merged so that the morphology of a macrophage that had taken up DiI AcLDL (red stain) can be seen.

The onset of CRQ expression corresponds to the time at which developmentally regulated apoptosis begins. We tested whether the presence of apoptotic cells might regulate CRQ expression by examining CRQ protein levels in embryos with altered amounts of apoptosis. InDf(3L)H99 (H99) homozygous embryos, apoptosis does not occur, as a result of the deletion of the cell death regulators reaper (rpr), grim, andhead involution defective (hid) (34–36). However, macrophages in H99homozygotes engulf corpses when apoptosis is induced by high levels of x-ray irradiation (34). When quantified by confocal microscopy, CRQ expression was decreased by 74% in H99embryos (Fig. 4, B and E) as compared with wild-type embryos (Fig. 4, A and D) (37). Some hemocytes in these embryos do not express detectable levels of CRQ (Fig. 4H). However, after x-ray irradiation, apoptosis is induced inH99 embryos (34), and CRQ expression increases (28). This suggests that rpr,grim, and hid themselves do not regulate CRQ expression but that the absence of apoptotic corpses results in CRQ down-regulation. MDP-1 expression is also down-regulated inH99 embryos (38), suggesting that multiple macrophage functions might be activated in the presence of apoptotic cells.

Figure 4

Croquemort expression is regulated by the amount of apoptosis. Projected confocal images of a CRQ immunostaining in the head of a wild-type embryo (A,D, and G), an H99 homozygous embryo (B, E, and H), and an irradiated wild-type embryo (34) (C, F, andI). (A) to (C) are at ×40; (D) to (I) are isolated macrophages at ×400. Images shown in (A) to (F) were taken with constant excitation and detection settings to show relative levels of CRQ staining. (G) to (I) show overlays of (D) to (F) with corresponding Nomarski images. CRQ expression is considerably down-regulated inH99 homozygous embryos (B and E). In x-ray–irradiated wild-type embryos, the amount of apoptosis is considerably increased. Macrophages become greatly enlarged as they engulf numerous apoptotic corpses, and the level of CRQ expression in each macrophage is remarkably up-regulated (C and F). (E) and (H) show three small macrophages side by side, one of which does not appear to express CRQ (arrowhead). (D), (G), (F), and (I) show single macrophages.

We tested whether increased apoptosis resulted in increased CRQ expression by subjecting wild-type embryos to x-ray irradiation (34). In such embryos, giant macrophages were seen that had engulfed many apoptotic corpses. In these embryos, macrophages showed a 3.3-fold increase in CRQ expression as compared with wild-type embryos (Fig. 3, C and F) (37). CRQ expression was similarly up-regulated after treatment of l(2)mbn cells with ecdysone (39), which induces increased apoptosis and increases the phagocytic activity in these cells (40). Thus, signals generated by dying cells cause increased expression of CRQ, which could facilitate the clearance of the cell corpses. The expression of the related protein CD36 in human monocytes is also increased by binding to one of its ligands, oxidized LDL (41).

This work characterizes a phagocytosis mutant inDrosophila and indicates that the CRQ protein is necessary, but probably not sufficient, for efficient phagocytosis of apoptotic cells in the embryo. Blocking studies on mammalian macrophages predicted a role for CD36 in the engulfment of apoptotic cells, and our in vivo data support this model. Because phagocytosis of apoptotic cells was not completely abolished in crq-deficient embryos (Fig. 1F), other receptors are probably involved in this process. Two other Drosophila macrophage receptors, the Scavenger Receptor dSR-C1 and Malvolio (42, 43), may share overlapping functions with CRQ in the engulfment of apoptotic corpses. However, the rather low efficiency of the residual phagocytic activity in crq-deficient embryos implicates the CRQ pathway as a major participant in the phagocytosis of apoptotic cells. CRQ is not required for the phagocytosis of bacteria by embryonic macrophages, but because dSR-C1 and Malvolio are similar to molecules involved in mammalian immune responses (42, 43), they may be specific pattern-recognition receptors for pathogens.

A genetic dissection of phagocytosis in Drosophila should further elucidate the phagocytic pathways for apoptotic corpses during development and for the engulfment of pathogens during an immune response. A greater understanding of the molecular mechanisms of both these processes in the fly, as well as of the macrophage responses they trigger, is likely to provide insights relevant to mammalian systems.

  • * To whom correspondence should be addressed. E-mail: kristin.white{at}CBRC2.MGH.Harvard.edu

REFERENCES AND NOTES

View Abstract

Stay Connected to Science

Navigate This Article