Role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. We first determined the location of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. We then asked the involvement of phagocytosis in the prevention of neoplasia examining animals where the expression of engulfment receptors instead of apoptosis was retarded. We found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila.

Signaling pathway for phagocyte priming upon encounter with apoptotic cells.

The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity.

Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death.

Cells that have become unwanted by the body need to be selectively, rapidly, and safely removed. The removal of these cells is achieved by apoptosis-dependent phagocytosis: unwanted cells are induced to undergo apoptosis and given susceptibility to phagocytosis. Phagocytes recognize these cells using engulfment receptors that bind substances expressed on the surface of target cells during the apoptotic process. The phagocytic elimination of cells undergoing apoptosis is a mechanism that is conserved among multicellular organisms. Malfunctions in this process may lead to structural and functional defects in morphogenesis and tissue homeostasis. Therefore, molecules involved in this phenomenon may be targeted in medical treatments. The mechanisms responsible for the apoptosis-dependent phagocytosis of unwanted cells as well as its physiological and pathological consequences are described herein.

Integrin αPS3/ßν-mediated phagocytosis of apoptotic cells and bacteria in Drosophila.

Integrins exert a variety of cellular functions as heterodimers of two transmembrane subunits named α and ß. Integrin ßν, a ß-subunit of Drosophila integrin, is involved in the phagocytosis of apoptotic cells and bacteria. Here, we searched for an α-subunit that forms a complex and cooperates with ßν. Examinations of RNAi-treated animals suggested that αPS3, but not any of four other α-subunits, is required for the effective phagocytosis of apoptotic cells in Drosophila embryos. The mutation of αPS3-encoding scb, deficiency, insertion of P-element, or alteration of nucleotide sequences, brought about a reduction in the level of phagocytosis. The defect in phagocytosis by deficiency was reverted by the forced expression of scb. Furthermore, flies in which the expression of both αPS3 and ßν was inhibited by RNAi showed a level of phagocytosis almost equal to that observed in flies with RNAi for either subunit alone. A loss of αPS3 also decreased the activity of larval hemocytes in the phagocytosis of Staphylococcus aureus. Finally, a co-immunoprecipitation analysis using a Drosophila cell line treated with a chemical cross-linker suggested a physical association between αPS3 and ßν. These results collectively indicated that integrin αPS3/ßν serves as a receptor in the phagocytosis of apoptotic cells and bacteria by Drosophila phagocytes.

Independent recognition of Staphylococcus aureus by two receptors for phagocytosis in Drosophila.

Integrin ßν, one of two ß subunits of Drosophila integrin, acts as a receptor in the phagocytosis of apoptotic cells. We here examined the involvement of this receptor in defense against infection by Staphylococcus aureus. Flies lacking integrin ßν died earlier than control flies upon a septic but not oral infection with this bacterium. A loss of integrin ßν reduced the phagocytosis of S. aureus and increased bacterial growth in flies. In contrast, the level of mRNA of an antimicrobial peptide produced upon infection was unchanged in integrin ßν-lacking flies. The simultaneous loss of integrin ßν and Draper, another receptor involved in the phagocytosis of S. aureus, brought about a further decrease in the level of phagocytosis and accelerated death of flies compared with the loss of either receptor alone. A strain of S. aureus lacking lipoteichoic acid, a cell wall component serving as a ligand for Draper, was susceptible to integrin ßν-mediated phagocytosis. In contrast, a S. aureus mutant strain that produces small amounts of peptidoglycan was less efficiently phagocytosed by larval hemocytes, and a loss of integrin ßν in hemocytes reduced a difference in the susceptibility to phagocytosis between parental and mutant strains. Furthermore, a series of experiments revealed the binding of integrin ßν to peptidoglycan of S. aureus. Taken together, these results suggested that Draper and integrin ßν cooperate in the phagocytic elimination of S. aureus by recognizing distinct cell wall components, and that this dual recognition system is necessary for the host organism to survive infection.

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila.

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur.

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the Caenorhabditis elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which we name Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells.

Integrin ßν-mediated phagocytosis of apoptotic cells in Drosophila embryos.

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin ßν, one of the two ß subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin ßν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin ßν in hemocytes of an integrin ßν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin ßν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin ßν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.

Phagocytic removal of cells that have become unwanted: implications for animal development and tissue homeostasis.

Cells that have become unwanted need to be promptly, selectively, and safely removed. This is made possible by apoptosis-dependent phagocytosis, in which cells unnecessary, obstructive, or dangerous to organisms are induced to undergo apoptosis so that they are earmarked for phagocytosis. The phagocytic elimination occurs so quickly that cells with hallmarks of apoptosis are barely detectable in vivo. The removal of particular types of cells at appropriate stages of development not only contributes to the disposal of spent cells, the creation of space for morphogenesis, and the exclusion of pathogenic or noxious cells, but seems to actively control tissue renewal, tissue remodeling, tissue function, and pathogenic state. This event thus plays an indispensable role in the maintenance of animal development and tissue homeostasis.

Immune response to bacteria in seminiferous epithelium.

The luminal part of the seminiferous epithelium, a tissue compartment protected by the blood-testis barrier, has been considered a site of immune privilege. However, there are reports describing the production of anti-microbial peptides and the expression of Toll-like receptors in cells present in the seminiferous epithelium, evoking the possibility that this tissue compartment is immunologically active at least with regard to the innate immune response. To test this, we injected Escherichia coli into seminiferous tubules of live mice and examined the fate of bacteria, the production of chemokines and inflammatory cytokines, and the infiltration of neutrophils. The bacteria actively propagated and reached a maximal level in a day, but started to decrease after 5 days and completely disappeared in 2 months. The expression of macrophage inflammatory protein-2 and tumor necrosis factor-alpha became evident in macrophages present in the interstitial compartment of testes as early as 1-3 h after the inoculation of bacteria. Neutrophils first accumulated in the interstitial space at 9-12 h and entered the tubules after a day. On the other hand, impairment of spermatogenesis was observed a day after bacteria injection and seemed unrecoverable even after the bacteria were eliminated. By contrast, bacteria injected into the interstitial compartment were more rapidly cleared with no damage in the seminiferous epithelium. These results suggest the existence of immunity against invading microbes in the seminiferous epithelium although its effectiveness in maintaining tissue homeostasis remains equivocal.

Inhibitory effects of viral infection on cancer development.

Immune responses evoked on viral infections prevent the dissemination of infection that otherwise leads to the development of diseases in host organisms. In the present study, we investigated whether viral infection influences tumorigenesis in cancer-bearing animals using a Drosophila model of cancer. Cancer was induced in the posterior part of wing imaginal discs through the simultaneous inhibition of apoptosis and cell-cycle checkpoints. The larvae and embryos of cancer-induced flies were infected with Drosophila C virus, a natural pathogen to Drosophila, and larval wing discs and adult wings were morphologically examined for cancer characteristics relative to uninfected controls. We found that viral infections brought about an approximately 30% reduction in the rate of cancer development in both wing discs and wings. These inhibitory effects were not observed when growth-defective virus was used to infect animals. These results indicate that productive viral infections repress tumorigenesis in Drosophila.

Activator protein 1-mediated expression of monocyte chemoattractant protein 1 in cultured rat luteal cells.

We have proposed that luteal cells undergo apoptosis-dependent phagocytosis by invading monocyte-derived macrophages in regressive corpora lutea of the rat. Accumulation of monocytes/macrophages seems to be mediated by monocyte chemoattractant protein 1 (MCP-1) or CCL2, because apoptosis and the production of MCP-1 mRNA occur simultaneously, but in different luteal cells, in a manner dependent on nuclear factor kappaB (NF-kappaB). In this study, we determined the mechanisms underlying the induction of these two events using primary cultures of rat luteal cells. We found that the activity of the transcription factor activator protein 1 (AP-1) increased during culturing concomitantly with an increase of MCP-1 mRNA. The increase of MCP-1 mRNA was abolished when cultures were maintained in the presence of an inhibitor of either AP-1 or c-Jun amino-terminal kinase (JNK) that phosphorylates and activates c-Jun, a subunit of AP-1. Furthermore, the presence of an inhibitor of NF-kappaB abrogated an increase in the activity of both AP-1 and JNK. In contrast, the induction of apoptosis in cultured luteal cells required the action of JNK but appeared to be independent of AP-1. This may explain why apoptosis and MCP-1 mRNA production are concomitantly but differentially induced in distinct luteal cells. We therefore suggest the following signaling pathways for the induction of apoptosis and mcp-1 gene expression during involution of the corpus luteum; NF-kappaB's actions lead to the activation of JNK, and the active JNK, at one side, stimulates mcp-1 gene transcription by activating AP-1 and, at the other side, induces apoptosis.

Oral Administration of Edible Seaweed Undaria Pinnatifida (Wakame) Modifies Glucose and Lipid Metabolism in Rats: A DNA Microarray Analysis.

SCOPE: Wakame is an edible seaweed that is a common constituent in the Japanese diet. Previous studies showed that wakame consumption is associated with the prevention of metabolic syndrome, but the molecular mechanisms underlying the protective effects are poorly understood. METHODS AND RESULTS: To determine if the expression of hepatic genes is affected by ingestion of the brown seaweed Undaria pinnatifida (wakame), rats were fed a diet containing 0, 0.1, or 1.0 g per 100 g dried wakame powder for 28 days. Administration of 1% wakame significantly decreased serum total cholesterol levels. Hepatic gene expression was investigated using DNA microarray analysis, and the results showed that wakame suppresses the lipogenic pathway by downregulating SREBF-1. Moreover, bile acid biosynthesis and gluconeogenesis were promoted by upregulation of the PPAR signaling pathway, which leads to a reduction in the accumulation of cholesterol and promotion of ß-oxidation. CONCLUSIONS: These results suggest that wakame ingestion affects glucose and lipid metabolism by altering the expression of SREBF-1 and PPAR signal-related genes.

Peptidoglycan recognition protein-triggered induction of Escherichia coli gene in Drosophila melanogaster.

Interaction between the host and pathogen determines the fate of both organisms during the infectious state. The host is equipped with a battery of immune reactions, while the pathogen displays a variety of mechanisms to compromise host immunity. Although bacteria alter their pattern of gene expression in host organisms, studies to elucidate the mechanism behind this are only in their infancy. We here examined the possibility that host immune proteins directly participate in the change of gene expression in bacteria. Escherichia coli was treated with a mixture of the extracellular region of peptidoglycan recognition protein (PGRP)-LC and the antimicrobial peptide attacin of Drosophila, and subjected to DNA microarray analysis for mRNA repertoire. We identified 133 annotated genes whose mRNA increased after the treatment, and at least four of them were induced in response to PGRP-LC. One such gene, lipoprotein-encoding nlpI, showed a transient increase of mRNA in adult flies depending on PGRP-LC but not PGRP-LE. NlpI-lacking E. coli had a lowered growth rate and/or viability in flies than the parental strain. These results suggest that a host immune receptor triggers a change of gene expression in bacteria simultaneously with their recognition and induction of immune responses.

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila.

Draper, a receptor responsible for the phagocytosis of apoptotic cells in Drosophila, possesses atypical epidermal growth factor (EGF)-like sequences in the extracellular region and the two phosphorylatable motifs NPxY and YxxL in the intracellular portion. We previously suggested that Pretaporter, a ligand for Draper, binds to the EGF-like repeat and augments the tyrosine phosphorylation of Draper. In this study, we first tested the binding of Pretaporter to various parts of the extracellular region of Draper and found that a single EGF-like sequence is sufficient for the binding. We next determined roles of the two intracellular motifs by forcedly expressing Draper proteins, in which tyrosine residues within the motifs had been substituted with phenylalanine, in hemocytes of Draper-lacking flies. We found that Draper proteins with Y-to-F substitution in either motif still underwent tyrosine phosphorylation, suggesting the occurrence of phosphorylation at both motifs. The Draper protein with substitution in the YxxL motif rescued a defect of phagocytosis, as did intact Draper, but the Draper protein with substitution in the NPxY motif did not, indicating a role of the motif NPxY, but not YxxL, in Draper-mediated phagocytosis. This coincides with our previous finding that Ced-6, an NPxY-binding signaling adaptor, is required for Draper's actions in apoptotic cell clearance. In summary, we demonstrated that Draper binds to its ligand Pretaporter using EGF-like sequences, and that the NPxY motif in the intracellular region of Draper plays an essential role in its actions as an engulfment receptor.

Perturbation of spermatogenesis by androgen antagonists directly injected into seminiferous tubules of live mice.

Natural and artificial substances present in the environment can affect our health. Testicular toxicants in particular are troublesome, because they disturb gonadal function of males. Translocation of substances into the seminiferous epithelium where sperm production proceeds is restricted due to the blood-testis barrier, but this permeability barrier temporarily disappears under physiological and sub-physiological conditions. This means that any substance could enter the seminiferous epithelium and disturb sperm production. To reduce the risk posed by such toxins, it is important to accurately determine which substances possess the toxicity. However, existing assay systems are not satisfactory in terms of both accuracy and sensitivity. Here, we report the establishment of such a system. We injected the androgen antagonists, flutamide and vinclozolin, directly into seminiferous tubules of live mice, which had been treated with busulfan for a temporal arrest of spermatogenesis, and the testes were histologically examined to see the effect of the injected materials on spermatogenesis that was in the process of recovery. The injection of either substance brought about a severe impairment of spermatogenesis at an amount over a million times smaller than that used in the previous assay systems where animals are administered with test substances outside of the testis. In contrast, these androgen antagonists at the same doses showed lesser effects when intratubularly or intraperitoneally administered into mice that had not been pretreated with busulfan. We propose that the method adopted in this study is a novel assay system to identify potential testicular toxicants.

Phosphatidylserine- and integrin-mediated phagocytosis of apoptotic luteal cells by macrophages of the rat.

Corpora lutea disappear from ovaries in the absence of conception. The present study was undertaken to examine the hypothesis that disappearance of corpora lutea is accomplished through apoptosis-dependent phagocytosis of luteal cells. When bone marrow cells expressing green fluorescence protein were transplanted into X-ray-irradiated mice, macrophages derived from donor mice were detected within corpora lutea, suggesting macrophage infiltration into the tissue. Dispersed rat luteal cells underwent spontaneous apoptosis during culture and were phagocytosed by luteal macrophages. Treatment with doxorubicin increased the extent of apoptosis in luteal cells, and those cells were more efficiently phagocytosed than cells left untreated. The phagocytosis was inhibited by liposomes containing phosphatidylserine or a peptide containing the integrin-targeted sequence, and was stimulated by milk fat globule epidermal growth factor 8. These results collectively indicate that apoptotic luteal cells are phagocytosed by macrophages in a manner mediated by phosphatidylserine and integrin.

Determination of cell type specificity and estrous cycle dependency of monocyte chemoattractant protein-1 expression in corpora lutea of normally cycling rats in relation to apoptosis and monocyte/macrophage accumulation.

In regressive corpora lutea, apoptosis of luteal cells, expression of monocyte chemoattractant protein-1 (MCP-1), and accumulation of monocytes/macrophages occur. However, whether these three events are correlated and what cell type expresses MCP-1 have yet to be determined. To clarify these issues, we performed histochemical examinations to determine the localization and the numbers of MCP-1 mRNA-containing cells, apoptotic cells, and monocytes/macrophages in corpora lutea of normally cycling rats. We found that the Mcp-1 gene is expressed in nonapoptotic steroidogenic luteal cells. Corpora lutea that contained MCP-1 mRNA-expressing cells increased in number at estrus together with those containing apoptotic luteal cells. When individual corpora lutea at estrus were analyzed, those with many MCP-1-expressing cells contained few apoptotic cells, and vice versa. These results collectively suggest the following pathway for apoptosis- and MCP-1-dependent regression of the corpus luteum: 1) luteal cells are induced to undergo apoptosis at estrus, and the activation of Mcp-1 gene expression follows in nonapoptotic luteal cells; 2) monocytes/macrophages are chemoattracted by MCP-1 toward corpora lutea containing apoptotic luteal cells; and 3) monocytes/macrophages invade corpora lutea and eliminate apoptotic luteal cells by phagocytosis.

Concomitant induction of apoptosis and expression of monocyte chemoattractant protein-1 in cultured rat luteal cells by nuclear factor-kappaB and oxidative stress.

It has previously been shown that expression of monocyte chemoattractant protein (mcp)-1 and apoptosis of luteal cells occur concomitantly during the estrous cycle in the rat corpus luteum; however, luteal cells containing mcp-1 mRNA did not seem to be apoptotic. In the present study, the relationship between the induction of apoptosis and mcp-1 expression in cultures of dispersed rat luteal cells was examined. Both apoptosis and mcp-1 expression were spontaneously induced in cultured luteal cells in a manner inhibitable by antioxidative reagents or an inhibitor of nuclear translocation of nuclear factor-kB. However, the cells containing mcp-1 mRNA were distinct from those undergoing apoptosis, and the inhibition of apoptosis by the pan-caspase inhibitor z-VAD-fmk did not influence the induction of mcp-1 expression. These results collectively indicate that oxidative stress simultaneously, but independently, induces apoptosis and mcp-1 expression in luteal cells through the activation of nuclear factor-kB. This phenomenon might help to explain how monocytes/macrophages accumulate in regressive corpora lutea where their target apoptotic cells exist.