receptor-mediated endocytosis - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10375-10379, November 1992 Cell Biology

Macrophages in Drosophila embryos and L2 cells exhibit scavenger receptor-mediated endocytosis JOHN M. ABRAMS*t, ALISON Lux*, HERMANN STELLER*t, AND MONTY

KRIEGER*f

*Department of Biology, and the tHoward Hughes Medical Institute in the Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

Communicated by Emilio Bizzi, July 24, 1992

ABSTRACT Mammalian macrophage scavenger receptors exhibit unusually broad binding specificity and are implicated in atherosclerosis and host defense. Scavenger receptorlike endocytosis was observed in Drosophila melanogaster embryos and in primary embryonic cell cultures. This receptor activity was expressed primarily by macrophages. The Drosophila Schneider L2, but not the Kc, cell line also exhibited a scavenger receptor-mediated endocytic pathway similar to Its mammalian counterpart. L2 receptors medated hg-afnity internalization and subsequent temperature- and chioroquine-

sensitive degradation of 'NI-labeled acetylated low density lipoprotein and displayed characteristic ligand specificity. These rindings suggest that scavenger receptors mediate important, well-conserved functions and raise the possibility that they may be pattern recognition receptors that arose early in the evolution of host defense mechanisms. They also establish additional systems for the investigation of endocytosis in Drosophila and scavenger receptor function in disease, host defense, and development.

Macrophages play multifaceted roles in mammalian host defense systems (1). They recognize and process antigens for T-cell presentation, release key mediators of inflammation (e.g., tumor necrosis factor), and are perhaps best characterized by their ability to endocytose and phagocytose a wide variety of molecules and cells. Macrophages recognize foreign substances identified by antibodies and complement (1). In addition, they can independently differentiate between self and nonself (2-4). This self-nonself discrimination may have predated the emergence of immunity mediated by lymphocytes and antibodies (1, 2). The polyspecificity and selfnonself discrimination required of macrophages in primitive organisms could have been achieved by receptors with multiple or broad ligand-binding specificities (2). We have suggested that macrophage scavenger receptors are attractive candidates for such pattern recognition receptors (5-7). Macrophage scavenger receptors have been implicated in the etiology of atherosclerosis (8, 9). These receptors have been characterized in detail at the molecular level and exhibit unusually broad ligand specificity (5, 6, 9-12). Their ligands are polyanions, including modified proteins such as acetylated low density lipoprotein (AcLDL) and maleylated bovine serum albumin (M-BSA), but not native LDL or native BSA, certain polyribonucleotides [poly(I) and poly(G) but not poly(C)], and certain polysaccharides and phospholipids (6, 12). Bacterial endotoxin (lipopolysaccharide) is a ligand and scavenger receptors play a important role in endotoxin clearance in vivo (6). To further explore the functions of scavenger receptors, we used fluorescently labeled AcLDL and 125I-labeled AcLDL (1251-AcLDL) to look for scavenger receptor-like activity in Drosophila melanogaster embryos and cell cultures. Using The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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these ligands, we found that macrophages in Drosophila embryos were strikingly similar to mammalian macrophages in that they express scavenger receptor activity. Furthermore, we found that the Drosophila Schneider L2 cell line also exhibits scavenger receptor-mediated endocytosis.

METHODS AND MATERIALS Drosophila Strains. The D. melanogaster embryos used were from Canton S (wild-type) and the enhancer-trap strain 197, kindly provided by J. Campos-Ortega (University of Cologne). Strain 197 contains a single copy of an enhancer-

trap P element inserted on the second chromosome. This line expresses the lacZ reporter gene specifically in macrophage nuclei, as verified by histological analysis (J. CamposOrtega, personal communication and J.M.A., H.S., and Kristin White, unpublished data). Eggs were collected on molasses/agar plates and staged according to CamposOrtega and Hartenstein (13). Analysis of Embryos. For in vivo studies, embryos were injected with -0.2-0.5 nl of 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate-labeled AcLDL [DiI-AcLDL (25 ,ug of DiI-AcLDL protein per ml in 0.1 mM phosphate buffer/5 mM KC1, pH 6.8)] per embryo following the protocol of Steller and Pirrotta (14). DiI-AcLDL, a fluorescent derivative of AcLDL, was prepared as described (15). After incubation at 18'C, the live embryos were refrigerated briefly to retard their movement and examined by confocal microscopy. Samples were viewed with an MRC 600 confocal scanning laser microscope (Bio-Rad) using a YHS color cube to detect red fluorescence. Confocal image processing was performed either with software provided by the manufacturer or with the Voxel View program (Vital Images,

Fairfield, IA). To label macrophages in situ, embryos were fixed and immunostained essentially as described in Steller et al. (16) except that the postfixation and blocking steps were done as described in Lee et aL (17). A rabbit anti-,B-galactosidase antibody (1:500 dilution, Cappel Laboratories) and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (1:100, Cappel Laboratories) were used to detect the distribution of P-galactosidase in embryos from strain 197. Primary Embryonic Cultures and Cell Lines. Primary cell cultures were prepared essentially as described by Ashburner (18). Briefly, 100-300 embryos were dechorionated in bleach, washed in water, and homogenized in 0.2 ml of Schneider's Drosophila medium (GIBCO or Sigma) supplemented with 10o fetal bovine serum, 50 units of penicillin per ml, and 50 tug of streptomycin per ml (medium A). For competition studies, the cells were incubated with DiI-AcLDL and then mounted on standard glass slides and examined. For coloAbbreviations: AcLDL, acetylated LDL; M-BSA, maleylated bovine serum albumin; DiI-AcLDL, 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate-labeled AcLDL; X-Gal, 5-bromo4chloro-3-indolyl 3-D-galactoside. tTo whom reprint requests should be addressed.

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FIG. 1. Comparison of DiI-AcLDL (A, C, and D) and macrophage (B) distributions in Drosophila embryos. (A, C, and D) Canton S strain Drosophila stage 11 embryos (5.5-6.5 hr after egg laying at 250C) were injected posteriorly with DiI-AcLDL and, after a 16-hr incubation at 180C, live embryos were examined by confocal fluorescence (A and C) and Nomarski optics (D, same field as C) microscopy. (B) Macrophages in a fixed embryo from strain 197 were visualized by staining for P-galactosidase immunoreactivity. A and B are superimpositions of several optical sections comprising -40 .m. [Bars = 100 Aum (A and B) or 10 ,m (C and D).]

calization studies, cells were mounted on poly(L-lysine)(Sigma) treated glass slides (18) and representative fields of cells were photographically recorded by combined Nomarski optics and fluorescence microscopy. These preparations were then stained with 5-bromo-4-chloro-3-indolyl fB-Dgalactoside (X-Gal) (18) to detect (3-galactosidase activity in macrophage nuclei. Each field of cells previously analyzed for DiI-AcLDL fluorescence was revisited and photographed after X-Gal staining to determine the extent ofcolocalization.

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The reported frequencies of colocalization are the averages determined by analysis of three separate experiments independently scored by two observers. Schneider L2 cells and Kc cells (19) were maintained in medium A supplemented with an additional 50 jug of streptomycin per ml (medium B) or D-22 medium (Sigma). Assays. 1251-AcLDL assays were performed with minor modifications as described (11, 20). In brief, 750,000 Schneider L2 cells in suspension were set in 0.5 ml of medium B

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FIG. 2. Uptake of DiI-AcLDL by primary cultured Drosophila embryonic cells. Cultures of stage 12-14 embryos were incubated at room temperature for 3 hr with DiI-AcLDL (5 jig of protein per ml) and the indicated additions prior to examination by confocal fluorescence (Upper) and Nomarski optics (Lower) microscopy. (Bar = 50 ,um.)


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containing 125I-AcLDL and other additions as indicated. After incubation at 250C for 5 hr or as otherwise noted, cells were harvested and washed by centrifugation (750-5000 x g) once or twice with BSA/Tris.HCl buffer (10-min incubations in the wash buffer) and once or twice rapidly with Tris HCl buffer. The amounts of trichloroacetic acid-soluble 1251_ AcLDL degradation products released into the incubation medium and cell-associated 1251-AcLDL were determined. The data are expressed as ng of 1251-AcLDL protein metabolized per mg of cell protein determined by the method of Lowry et al. (21). For chloroquine sensitivity studies, cells were preincubated for 0.5 hr and then incubated for 5 hr with the indicated concentration of chloroquine at 250C. For assays at 40C, the cells were preincubated at 40C for 0.5 hr prior to addition of reagents.

RESULTS AND DISCUSSION To determine if scavenger receptor-like activity could be detected in D. melanogaster, we injected a fluorescently labeled derivative of AcLDL (DiI-AcLDL) into live, stage 11 embryos. Sixteen hours later, scattered, punctate fluorescence (Fig. 1A) was observed throughout the interstitial spaces of the body cavity distributed in a pattern similar to that of embryonic macrophages (Fig. 1B) (14, 22). At higher magnification (Fig. 1 C and D), the DiI-AcLDL fluorescence could be seen in cells with multivesicular inclusions characteristic of macrophages (23). We also observed internalization of significant amounts of DiI-AcLDL by a subset of cells in primary embryonic cultures (Fig. 2 Left). Scavenger receptor competitors [poly(I), M-BSA, AcLDL] blocked DiI-AcLDL uptake, whereas their noncompetitive controls [poly(C), BSA, LDL] did not (Fig. 2 and data not shown). Thus, DiI-AcLDL uptake by cultured embryonic cells exhibited the ligand-binding specificity characteristic of scavenger receptors. To identify the cell type(s) that expressed scavenger receptor activity, we examined the uptake of DiI-AcLDL in primary cultures of embryonic cells from the Drosophila enhancer-trap strain 197. In this strain, bacterial f-galactosidase is specifically expressed in macrophage nuclei (see Methods and Materials). Approximately 83% of all P-galactosidase-positive macrophages (X-Gal blue staining, Fig. 3B) expressed scavenger receptor activity (DiI-AcLDL red fluorescence, Fig. 3A) and -70% of all scavenger receptorpositive cells were f8-galactosidase positive. Because these cultures were derived from heterozygous parents, only 75% of the macrophages express the P-galactosidase marker. Thus, most of the scavenger receptor-positive cells were macrophages and most, if not all, macrophages expressed scavenger receptor activity. Because of the low abundance of macrophages in primary embryonic cultures, we used 125I-AcLDL to screen for the expression of this activity in two Drosophila cultured cell lines. Kc cells expressed virtually no activity (not shown); however, L2 cells expressed considerable activity. Fig. 4A shows that the specific degradation and binding plus uptake of 1251-AcLDL by L2 cells were high affinity, essentially saturable processes with Km values of -3.5 ,ug of protein per ml. At the higher 125I-AcLDL concentrations there was a nonsaturable component of binding and uptake that was not seen in the degradation data. The 125I-AcLDL binding, uptake, and degradation exhibited the characteristic broad ligand specificity of scavenger receptors. At 400 ug/ml, scavenger receptor ligands [AcLDL, M-BSA, poly(G), fucoidan, dextran sulfate] inhibited activity but their controls [LDL, BSA, poly(C)] did not (Fig. 4B). M-BSA completely inhibited 1251-AcLDL binding, uptake, and degradation with an affinity (ID50 = 4.2 ,ug/ml) considerably higher than that for mammalian scavenger receptors (9-11, 24, 25).

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FIG. 3. Comparison of scavenger receptor and macrophage distributions in primary cultures of strain 197 Drosophila embryos. Primary cell cultures from strain 197 stage 12-16 embryos were incubated at room temperature for 24 hr with 5 gtg of Dil-AcLDL protein per ml, fixed with 1.25% glutaraldehyde, examined by combined Nomarski and fluorescence (red) optics (A), and then stained with X-Gal to detect (3-galactosidase activity (blue) in macrophage nuclei (B, Nomarski optics). (Bar = 50 g~m.)

The release of trichloroacetic acid-soluble 1251-AcLDL

degradation products into the culture medium suggested that the L2 cells exhibit a receptor-mediated endocytic pathway involving lysosomal hydrolysis of the ligand similar to that of mammalian cells. Fig. 4C shows that the binding plus uptake of 125I-AcLDL was rapid and approached a steady state after 6 hr. As is the case with mammalian macrophages (11), there was a 50-min lag before degradation at a linear rate began, and degradation was inhibited at 40C (not shown). Furthermore, chloroquine had an effect in L2 cells similar to that in mammalian macrophages; it inhibited degradation and led to a compensatory increase in binding plus uptake (Fig. 4D). Taken together, these results show that Drosophila embryonic macrophages exhibit in vivo and in vitro a scavenger receptor activity similar to that expressed by mammalian


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FIG. 4. Metabolism of 125I-AcLDL by Schneider L2 cells at 250C. (A) Saturation kinetics of the degradation and binding plus uptake of 125I-AcLDL. The indicated amounts of 125I-AcLDL were added to L2 cells in the absence (total activity, duplicate incubations) or presence (nonspecific activity, single incubations) of the competitive ligand M-BSA (400 pg/ml). The specific values (total minus nonspecific) are shown. The nonspecific values were -7% and d11% of the totals at 10 and 25 Ag of 125I-AcLDL protein per ml. (B) Specificity of receptor activity. The degradation and binding plus uptake of 125I-AcLDL (5 pg of protein per ml) were determined in the absence (quadruplicate incubations) or presence (duplicate incubations) of the indicated scavenger receptor competitors (400 Ag/ml) and controls. (C) Degradation and binding plus uptake of 125I-AcLDL (5 Ag of protein per ml) were measured at the indicated times in the absence (triplicate incubations) or presence (duplicate incubations) of M-BSA (400 ,ug/ml). The specific values are shown. (D) Specific degradation and binding plus uptake of 125I-AcLDL (5 fig of protein per ml) were determined as in A in the presence of the indicated concentrations of chloroquine. The total activities (sum of binding plus uptake and degradation) are indicated.

macrophages (9, 11). The Drosophila L2 cell line has a similar, possibly identical, activity. The scavenger receptor pathway of L2 cells was characterized by high-affinity, broad specificity ligand binding and uptake that was followed by temperature- and chloroquine-dependent degradation. Previous studies (26, 27) have shown that Drosophila as well as mammalian cells contain coated pits and associated internal organelles that participate in endocytic pathways. In contrast to mammalian systems (28), few receptor-mediated endocytic pathways in Drosophila cells have been examined in detail (29, 30). Scavenger receptor-mediated endocytosis in macrophages and L2 cells may prove useful for further analysis of the mechanisms underlying the cellular uptake of macromolecules in Drosophila. These observations also raise the possibility that L2 cells may represent a macrophage-cell line. The structure and function of the scavenger receptors in Drosophila and their relationships to mammalian scavenger receptors (5, 9) remain to be elucidated by further molecular, genetic, and physiologic studies. Application of the genetic techniques available in Drosophila will provide a powerful approach for the investigation of scavenger receptor function. Scavenger receptor expression by mammalian and Drosophila macrophages suggests that these receptors me-

diate important, well-conserved functions and raises the possibility that they may indeed be pattern recognition receptors that arose early in the evolution of host defense mechanisms (2, 5, 7). It is noteworthy that postembryonic macrophage-like hemocytes in Drosophila participate in wound healing, encapsulation of pathogens, and phagocytosis (31). Furthermore, macrophages apparently play an important role in the recognition of effete cells during the course of development and aging (1, 13). We recently have observed that a large number of cells undergo programmed cell death during Drosophila embryogenesis and that the apoptotic cell corpses are phagocytosed by macrophages (23). Additional studies will be required to determine if scavenger receptors are involved in this crucial developmental process. We thank Alan Pearson for help in developing the assays for L2 cell metabolism of 125I-AcLDL and his ongoing contributions to this study. We also thank Jose Campos-Ortega for the gift of Drosophila strain 197, R. Rosenberg and K. White for helpful comments, and Dave Smith for assistance with the confocal microscopy and image processing. This work was supported by the National Institutes of Health-National Heart, Lung, and Blood Institute. H.S. is an Assistant Investigator with the Howard Hughes Medical Institute and J.M.A. was supported by an American Cancer Society fellowship.

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