PHAGOCYOTSIS OF APOPTOTIC CELLS IN INVERTEBRATES ISJ 3: 89-96, 2006 ISSN 1824-307X Minireview Mechanisms and roles of phagocytosis in Drosophila and Caenorhabditis elegans Y Nakanishi, A Shiratsuchi Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan Accepted September 19, 2006 Abstract Our understanding of the humoral immune response in both vertebrates and invertebrates has dramatically deepened in the past decade. In contrast, many of the mechanisms and roles of the cellular immune response remain to be elucidated. Phagocytosis is at the center of the cellular responses in both innate and adaptive immunity. Targets of phagocytosis are either invading microbes or altered self, that is, own cells that have become dispensable or harmful. The selective recognition and engulfment of target cells by phagocytes are achieved through the specific binding of receptors of phagocytes to ligands present on the surface of the target cells. However, these phagocytosis receptors and ligands are still being identified. The fundamental mechanism of phagocytosis appears to be the same in vertebrates and invertebrates, but whether or not genes are evolutionally conserved has yet to be determined. Key words: apoptosis; Caenorhabditis elegans; Drosophila; innate immunity; microbial pathogen; phagocyte; phagocytosis Introduction Innate immunity is defined as a type of immune responses in which only products of genes already present in germ lines play roles, in contrast to adaptive immunity that uses both germ line genes and genes acquired by rearrangement of existing genes during development (Janeway and Medzhitov, 2002). Simpler organisms like invertebrate animals have only innate immunity, while organisms more complex than jawed fish possess both innate and adaptive immune systems. Innate immunity was once thought to be a prototype of the “more sophisticated” adaptive immunity, but it is now apparent that the two systems play individual roles and cooperate to protect against invaders and endogenous insults (Hoebe et al., 2004). Furthermore, the boundary between the two immune systems is becoming obscure (Flajnik and Pasquier, 2004). Given that either form of immunity consists of humoral and cellular responses, there are two types of humoral and cellular reactions in vertebrates and only one type in invertebrates. ___________________________________________________________________________ Corresponding author: Yoshinobu Nakanishi Graduate School of Medical Science, Kanazawa University, Shizenken, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan E-mail: nakanaka@kenroku.kanazawa-u.ac.jp This raises the possibility that the roles of innate immunity in vertebrates and invertebrates are somewhat distinct. If so, a better way to fully understand innate immunity would be to compare the mechanisms and roles of immune responses between vertebrates and invertebrates. Previous studies have revealed that at least the mechanistic part of the innate immune response is well conserved between vertebrates and invertebrates although the players are not exactly the same (Iwanaga, 2002; Brennan and Anderson, 2004). While the mechanisms and consequences of the humoral innate immune response have been intensively investigated particularly in insects and mammals (Janeway and Medzhitov, 2002), many issues still remain to be solved before we can gain a good understanding of the cellular response. Provided that the fundamental mechanisms of the cellular innate immune response are almost the same in vertebrates and invertebrates, a smarter way to address these issues is to begin with the analysis of genetically tractable invertebrate animals such as Drosophila and Caenorhabditis elegans (Mylonakis and Aballay, 2005). In this minireview, we summarize what is known of phagocytosis, an event at the center of cellular responses, in Drosophila and C. elegans, in an attempt to gain a deeper understanding of this phenomenon. 89 mailto:nakanaka@kenroku.kanazawa-u.ac.jp Phagocytosis at a glance Phagocytosis, a phenomenon whereby cells are engulfed and digested by other cells (Aderem and Underhill, 1999), is at the center of the cellular immune response in both innate and adaptive immunity; other cellular responses include cell-mediated killing in vertebrates, and cell-mediated killing and encapsulation in invertebrate animals. The cells in charge of phagocytosis are called phagocytes and consist of various types. Phagocytes are classified into two groups, professional and amateur cells. Professional phagocytes, macrophages as a representative, are full-time executors. In contrast, amateur phagocytes, which exert functions other than phagocytosis most of the time, exhibit phagocytic activity only when it is needed. There is another way to classify phagocytes; that is, phagocytes that circulate through the body and are responsible for phagocytosis in various places, and others that are localized to certain places and engaged in phagocytosis only there. The phagocytes of invertebrate animals have not been intensively characterized compared with those of vertebrates. In many invertebrates circulatory cells (either coelomocytes or hemocytes) exist, several types of which act as professional phagocytes. In the fruit fly Drosophila, plasmatocytes are such phagocytes and responsible for the phagocytic elimination of invaders and altered self in many areas of the body (Meister and Lagueux, 2003). Glia and some ectodermal cells also act as phagocytes in Drosophila. In contrast, there are no mobile phagocytes in C. elegans; the cells that simply neighbor target cells seem to carry out phagocytosis (Gravato-Nobre and Hodgkin, 2005). Phagocytes selectively recognize and engulf cells that are foreign to our body or own cells that have become dispensable. The foreign cells are invading microbes, and the altered self includes cells that have become structurally and/or functionally spent, unwanted, aged, or harmful. Removal of the former targets is accomplished to eliminate microbial pathogens that may cause infectious diseases, while that of the latter is necessary for morphogenesis, establishment of tissue functions, and maintenance of tissue homeostasis, i.e. tissue renewal, avoidance of excessive cellular actions, and extermination of pathogenic or noxious materials (Stuart and Ezekowitz, 2005). Failure in the expeditious removal of such “unwanted” cells impairs normal development as well as increases the risk of infectious diseases, inflammation, or autoimmunity. Phagocytosis is induced when receptors present on the surface of phagocytes are activated by target cells (Aderem and Underhill, 1999) (Fig. 1). Upon the binding of target cells, the intracellular portion of phagocytosis receptors activates a signaling pathway, which in most cases leads to rearrangement of the actin cytoskeleton. As a result, the plasma membrane of phagocytes locally extends and surrounds the target, which is then ingested as membrane vesicles called phagosomes. There is another mode of phagocytosis whereby target cells appear to “sink” into phagocytes without any extension of the plasma membrane. It is presumed Fig. 1 Recognition and engulfment of target cells by phagocytes. Phagocytosis receptors of phagocytes bind to phagocytosis markers present at the surface of target cells. Marker-bound receptors activate a signaling pathway that leads to reorganization of the actin cytoskeleton. Portions of the plasma membrane of phagocytes then extend and surround targets. Finally, target cells are incorporated into phagocytes as phagosomes. that the mode of engulfment varies depending on which receptors are responsible for the induction of phagocytosis as well as the size and shape of target cells (Champion and Mitragotri, 2006). Target selectivity in phagocytosis is achieved through specific molecular recognition between phagocytosis receptors residing at the surface of phagocytes and their ligands or phagocytosis markers on the surface of target cells. The phagocytosis markers are either constituents of the surface of target cells or soluble molecules in body fluid that bind to the targets. The latter are called opsonins, being represented by immunoglobulin and complement of vertebrates that presumably do not exist in invertebrates: it is unclear if opsonin-dependent phagocytosis occurs in invertebrate animals (see below). Cell surface constituents specific for microbes are exemplified by a group of molecules called the pathogen-associated molecular patterns that are recognized by a group of receptors of immune cells, the pattern recognition receptor (Janeway, 2001). However, it is presumed that pattern recognition receptors and pathogen-associated molecular patterns, which are responsible for the induction of humoral innate immune responses, do not serve, at least in a direct way, as receptors and ligands, respectively, in phagocytosis. Mammalian receptors responsible for the phagocytosis of microbes have been characterized, including mannose receptors, Fc receptors, and complement receptors (Aderem and Underhill, 1999; Taylor et al., 2005). Mannose receptors directly recognize components of the bacterial cell wall, but Fc receptors and complement receptors bind to the opsonins immunoglobulin and complement, respectively. Several membrane proteins have recently been proposed to be receptors responsible for the phagocytosis of bacteria by phagocytes of Drosophila, but the corresponding bacterial ligands have yet to be identified (see below). Altered own cells are often induced to undergo apoptosis, a physiologic mode of cell death (Wyllie et al., 1980; Ellis et al., 1991), and become susceptible to phagocytosis (Savill et al., 1993; Savill and Fadok, 2000). Apoptotic cells 90 express phagocytosis ligands that do not exist at the surface of viable cells. These ligands are either endogenous molecules that move to the cell surface or pre-existing molecules at the surface whose structure or distribution changes during apoptosis (Lauber et al., 2004). The apoptosis-dependent structural reorganization of the cell surface has been well studied with mammalian cells, and the membrane phospholipid phosphatidylserine is the best-characterized ligand for phagocytosis receptors (Fadok et al., 1998; Schlegel and Williamson, 2001). Apoptosis-dependent expression of phosphatidylserine at the cell surface is also observed in cells of Drosophila and C. elegans. However, it is unclear whether phosphatidylserine serves as a phagocytosis ligand for apoptotic cells to be recognized by phagocytes of Drosophila and C. elegans. To date, no molecule has been identified as a marker for phagocytosis of altered self in Drosophila and C. elegans Targets are incorporated into phagocytes as membrane vesicles called phagosomes, which are surrounded by the plasma membrane of phagocytes (Aderem and Underhill, 1999). The main fate of engulfed target cells is decomposition and digestion. Phagosomes are processed so that the engulfed targets are killed and degraded mainly by reactive species such as reactive oxygen species and reactive nitrogen species (Halliwell, 2006) and lysosomal enzymes, respectively (Fig. 2). A key step in the production of reactive oxygen species is the activation of an enzyme called NADPH oxidase (Underhill and Ozinsky, 2002; Geiszt and Leto, 2004). A prerequisite of the lysosomal degradation of engulfed target cells is the fusion of phagosomes with lysosomes that provide various enzymes for degrading components of engulfed cells. These processes are collectively called phagosome maturation and seem to be under a strict regulation. Fig. 2 Fate of engulfed cells in phagocytes. Engulfed cells are killed and digested by a reactive oxygen-mediated mechanism and lysosomal enzymes, respectively. Both reactions occur during structural and functional changes of phagosomes. Contents of engulfed cells are sometimes used as antigens for the activation of T lymphocytes and thus for the induction of adaptive immunity. MHC, major histocompatibility complex. On the other hand, engulfed targets are sometimes not completely degraded but subjected to partial digestion. This occurs in a particular type of phagocyte, i.e. antigen-presenting cells such as dendritic cells in vertebrate animals, and processed components of engulfed cells are expressed at the surface together with the major histocompatibility complex for the activation of T lymphocytes (Ackerman and Cresswell, 2004). Microbes, some types of bacteria in particular, possess the ability to resist the actions of phagocytes at various steps (Ernst, 2000; Coombes et al., 2004). Yersinia inhibits phagocytosis itself through the actions of their own proteins that are delivered to phagocytes via the type III secretion system. Salmonella produce, after engulfment, proteins that inhibit the activation of NADPH oxidase in phagosomes. Listeria sneaks out of phagosomes by disrupting phagosome membranes using their own proteins. Other bacteria including Leginonella and Chlamydia, and the protozoa Leishmania also inhibit phagosome maturation. Phagocytes of the host organisms counterattack such microbes, in at least two ways as follows. Bacteria that have come out of phagosomes are surrounded again by membranes through a process called autophagy (Shintani and Klionsky, 2004), and phagocytes invaded by long-living microbes are often induced to undergo apoptosis and engulfed together with the microbes by other phagocytes. All these phenomena have been observed with mammalian phagocytes, and whether or not the same is true for phagocytes of invertebrate animals remains to be determined. Phagocytosis of microbes in Drosophila and C. elegans Phagocytosis of bacteria by Drosophila phagocytes In Drosophila, most humoral immune responses are accomplished by cells of the fat body, a tissue equivalent to the mammalian liver, while blood cells called hemocytes are responsible for most cellular events (Hoffmann and Reichhart, 2002; Mylonakis and Aballay, 2005). There exist three cell lineages of Drosophila hemocytes, which emerge at different stages of development and participate in particular types of cellular immune responses (Meister and Lagueux, 2003). Two lineages, the plasmatocyte and the crystal cell, differentiate during the second half of embryogenesis. Plasmatocytes account for no less than 90 % of circulating hemocytes and act primarily as phagocytes. Therefore, these cells are considered to be responsible for the phagocytic elimination of invading microbes and altered self in Drosophila. Crystal cells are seemingly involved in humoral melanization mediated by phenoloxidase, but their role is not fully understood. The third hemocyte lineage emerges at the larval stage, and these cells called lamellocytes are responsible for the encapsulation of microbes, a cellular response often followed by phagocytosis. Differently from mammalian phagocytes such as neutrophils and macrophages, how plasmatocytes act to phagocytose target cells has not been intensively studied. Even the identity of phagocytosis receptors and corresponding microbial ligands has been elusive. It is presumed that most phagocytosis 91 receptors of Drosophila phagocytes directly recognize molecules present at the surface of target microbes and apoptotic cells, because no molecules present in the hemolymph have been shown in vivo to serve as opsonin. Several proteins have been proposed to be phagocytosis receptors (Table 1) (Cherry and Silverman, 2006), although their ligands presumably present at the surface of microbes remain to be identified. The first receptor reported is known as a pattern recognition receptor. In Drosophila, a family of proteins called peptidoglycan recognition proteins (PGRPs) serves as pattern recognition receptors (Brennan and Anderson, 2004), as do Toll-like receptors in mammals (Akira et al., 2006). PGRP-LC, not PGRP-LA or -LD, has been identified as a protein, a decrease in the expression of which leads to a decrease in the level of phagocytosis of Gram-negative Escherichia coli, but not Gram-positive Staphylococcus aureus, by S2 cells (Rämet et al., 2002), a cell line established from hemocytes of Drosophila embryos. This approach, namely, a genome-wide screen with RNA interference-mediated inhibition of gene expression in phagocytes, was adopted by other investigators, and a group of proteins resembling a human protein called CD36 have been spotlighted. CD36 belongs to the class B scavenger receptor family (SR-B) that is responsible for the control of serum cholesterol levels as well as the removal of denatured serum proteins in mammals (Peiser and Gordon, 2001). Besides these actions, two SR-B proteins, SR-BI of mammals (Shiratsuchi et al., 1999) and Croquemort of Drosophila (Franc et al., 1996), have been shown to be involved in the phagocytosis of apoptotic cells. The genome-wide screen revealed that the Drosophila SR-B proteins Peste and Croquemort serve as receptors for the phagocytosis of bacteria by S2 cells. Peste targets Mycobacteria and Lysteria but not E. coli and S. aureus (Agaisse et al., 2005), but this specificity seems to change when Peste is expressed in mammalian cells (Philips et al., 2005). On the other hand, S. aureus is a preferred target for Croquemort in phagocytosis by S2 cells (Stuart et al., 2005). In addition, SR-CI, a class C scavenger receptor, of Drosophila seems to have some role in the phagocytosis of bacteria (Rämet et al., 2001; Philips et al., 2005). All the aforementioned proteins remain as candidate phagocytosis receptors at present, because their role in vivo in the phagocytic removal of bacteria is yet to be shown. Ezekowitz and colleagues extended their RNA interference screen with S2 cells, in which the transcription factor Serpent was found to be important for the phagocytosis of bacteria (Rämet et al., 2002). They searched for gene products whose expression is regulated by Serpent and examined their role in the phagocytosis of bacteria by S2 cells. Eventually one protein named Eater was found to bind to and engulf both E. coli and S. aureus (Kocks et al., 2005). Eater is a single-path membrane protein with epidermal growth factor (EGF)-like repeats in its extracellular portion, and is expressed primarily in plasmatocytes. Hemocytes prepared from mutant flies lacking the expression of eater showed a decreased level of the phagocytosis of both E. coli and S. aureus. Furthermore, the phagocytosis of those bacteria injected into the adult mutant flies was significantly impaired. The final candidate for a phagocytosis receptor of Drosophila is quite unique in that it possesses an immunoglobulin-like structure and is expressed as over one thousand isoforms through alternative splicing in hemocytes and the fat body (Watson et al., 2005). This family of proteins, named Dscam for immunoglobulin-superfamily receptor Down syndrome cell adhesion molecule, bind to E. coli, and larval hemocytes prepared from mutant flies with a reduced level of the expression of dscam showed less activity to phagocytose E. coli than Table 1 Candidate receptors for the phagocytosis of bacteria by Drosophila phagocytes Receptor name Domains E. coli S. aureus Mycobacterium Other targets In vivo evidence References___ PGRP-LC peptidoglycan binding yes no nd1 M.luteus2 nd2 Rämet et al., 2002 Peste scavenger receptor3 no no yes Listeria nd Philips et al., 2005 Croquemort scavenger receptor3 no yes nd nd Stuart et al., 2005 SR-CI scavenger receptor4 yes5 nd yes5 nd Philips et al., 2005 Eater EGF-like repeat yes yes nd yes6 Kocks et al., 2005 Dscam Immunoglobulin-like yes nd nd yes7 Watson et al., 2005 1. Not determined; 2. Only viability of flies lacking PGRP-LC expression upon infection with bacteria was examined; 3. Class B scavenger receptor family; 4. Class C scavenger receptor family; 5. Extent of contribution is small; 6. Levels of phagocytosis of bacteria by larval hemocytes of flies lacking Eater expression are reduced. Levels of phagocytosis of bacteria injected into adult mutant flies are reduced; 7. Levels of phagocytosis of bacteria by larval hemocytes of flies lacking Dscam expression are reduced. 92 those from wild-type flies. In addition, its soluble form is present in the hemolymph. These findings evoke the possibility that Dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. More recently, a family of secreted proteins, called Teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by S2 cells, was reported (Stroschein-Stevenson et al., 2006). Of 6 Teps TepII, TepIII, and TepVI have been suggested to be involved in the phagocytosis of E. coli, S. aureus, and Candida albicans, respectively, though in vivo confirmation is required. Teps resemble the complement C3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (Lagueux et al., 2000; Kocks et al., 2003). Phagocytosis of bacteria by C. elegans phagocytes The nematode C. elegans maintained in laboratories propagates when fed with E. coli, and the lifespan of this worm is altered when the food source is changed to other bacteria. This suggests that C. elegans is immune to microbial pathogens. The study of innate immunity in C. elegans has begun with the analysis of the humoral immune response, and its mechanism and role have been shown to be basically the same as those in Drosophila and mammals (Gravato-Nobre and Hodgkin, 2005; Kim and Ausubel, 2005; Mylonakis and Aballay, 2005). Infection with some types of bacteria causes apoptosis in germ lines, and a mutant line of the worm lacking the expression of ced-3 and ced-4 is more sensitive to infection. This means that C. elegans uses the apoptotic pathway for innate immune responses against invading microbes. In contrast, the role of cellular responses in the protection of the worm from infectious diseases has not yet been settled. There exists a type of cell that serves as a phagocyte in C. elegans, but the importance of the phagocytic elimination of pathogenic microbes is expected to be small. The phagocytosis of invading microbes by C. elegans phagocytes is inefficient, simply because those phagocytes are not mobile. Further studies are needed to clarify the mechanisms and roles of the phagocytosis of microbes by C. elegans phagocytes. Phagocytosis of apoptotic cells in Drosophila and C. elegans Phagocytosis of apoptotic cells by C. elegans phagocytes Although the contribution of phagocytosis to defense against the invasion of pathogenic microbes is unclear, apoptotic cells are definitely eliminated by phagocytosis in C. elegans. There are no circulating “professional” phagocytes in this worm, and cells that neighbor dying cells are in charge of phagocytosis. The pioneer work done by Horvitz and coworkers has revealed the existence of a set of genes responsible for the induction, execution, and regulation of programmed cell death or apoptosis in C. elegans (Ellis et al., 1991; Lettre and Hengartner, 2006). Such genes include those that play roles at the final stage of apoptosis, i.e. the engulfment and degradation of apoptotic cells (Gumienny and Hengartner, 2001; Fig. 3 Signaling pathways for the induction of phagocytosis of apoptotic cells. Two partly overlapping signaling pathways for the induction of phagocytosis of apoptotic cells, which were genetically identified in C. elegans and are considered to be conserved beyond species, are schematically presented. Shown in the parentheses are names of the Drosophila counterparts of the C. elegans proteins. Reddien and Horvitz, 2004; Mangahas and Zhou, 2005). Seemingly there are two partly overlapping signaling pathways, which involve signal mediators conserved beyond species, for the induction of the phagocytosis of apoptotic cells by C. elegans phagocytes (Lettre and Hengartner, 2006) (Fig. 3), although a different opinion was recently provided (Yu et al., 2006). The onset of engulfment should be the activation of receptors residing at the surface of phagocytes. This occurs most likely by the binding of marker molecules of phagocytosis present on the surface of target apoptotic cells. Presumably there are two sets of phagocytosis receptor and ligand, but only one receptor has been identified to date. A single-path membrane protein named CED-1 has been genetically discovered and shown to serve as a phagocytosis receptor (Zhou et al., 2001). There are counterparts of CED-1 in Drosophila and human, which are respectively called Draper (Freeman et al., 2003) and MEGF10 (Callebaut et al., 2003). Draper seems to be responsible for the phagocytic removal of apoptotic cells by Drosophila phagocytes (Freeman et al., 2003; Manaka et al., 2004) (see below), but whether or not MEGF10 plays roles in the clearance of apoptotic cells by mammalian phagocytes remains to be determined. CED-1 contains several structural domains, including EGF-like repeats, in the extracellular region and two segments containing tyrosine residues, which are candidate domains for protein-protein interaction, in the intracellular region. The former domain could serve as a site for the binding of an as-yet unidentified phagocytosis ligand, and the latter could be, most likely after phosphorylation of the tyrosine residues, a site for the assembly of downstream signal mediators such as CED-6 (Mangahas and Zhou, 2005). There has been no information regarding the identity of the other phagocytosis receptor. In contrast to the fact that many molecules have been proposed to be phagocytosis markers in mammals (Lauber et al., 2004), no molecules have 93 been shown to be ligands for phagocytosis receptors of C. elegans. C. elegans cells seem to express phosphatidylserine at their surface during apoptosis, but it is not known if externalized phosphatidylserine serves as a phagocytosis marker. The involvement of the C. elegans homologue of the mammalian phosphatidylserine receptor in the phagocytosis of apoptotic cells was reported, but the role for the mammalian protein itself as a phosphatidylserine-recognizing phagocytosis receptor is now doubted. It will be necessary to adopt an experimental strategy other than genetics for the identification of the other phagocytosis receptor and a couple of ligands, but the C. elegans system where mobile phagocytes and suitable cell lines are unavailable does not appear to be suitable for a rapid solution of these issues. Phagocytosis of apoptotic cells by Drosophila phagocytes In contrast to studies with C. elegans (see above) and mammals (Lauber et al., 2004), mechanisms of the phagocytosis of apoptotic cells in Drosophila have not been intensely investigated. Three membrane proteins have so far been proposed as receptors responsible for the phagocytosis of apoptotic cells by Drosophila phagocytes, but no molecules have been identified as phagocytosis markers presumably present on the surface of apoptotic cells. Franc and coworkers were the first to identify a receptor responsible for the phagocytosis of apoptotic cells by Drosophila phagocytes (Franc et al., 1996, 1999). They searched for members of the C-type lectin family in larvae at the third instar stage and found a protein that belongs not to the C-type lectin family but to the SR-B family. This protein, named Croquemort, standing for “catcher of death”, is a membrane protein (single- or double-path) and expressed in hemocytes (plasmatocytes/lamellocytes) of embryos and larvae. Analyses of flies with a chromosomal deletion including the croquemort locus revealed that Croquemort is responsible at least in part for the phagocytosis of apoptotic cells, but not of bacteria, in Drosophila embryos, though the latter conclusion recently became controversial (Stuart et al., 2005). It is still unknown what molecule at the surface of apoptotic cells Croquemort binds to, and as to whether this receptor is contained in either one of the two signaling pathways (see Fig. 3) is not clear. Another molecule that has been shown to act as a receptor for the phagocytosis of apoptotic cells is Draper, the Drosophila homologue of the C. elegans phagocytosis receptor CED-1. Draper, a single-path membrane protein with EGF-like repeats, appears to serve as a receptor for the phagocytic elimination of apoptotic cells by both hemocytes and glia (Freeman et al., 2003; Manaka et al., 2004). Moreover, Draper is involved in the removal of axons by glia during metamorphosis for remodeling of the neural network and recovery from injury (Awasaki et al., 2006; Hoopfer et al., 2006; MacDonald et al., 2006). A ligand(s) for Draper remains to be identified for both apoptotic cells and degenerating axons, but phosphatidylserine is not likely to be the one (Manaka et al., 2004). The externalization of phosphatidylserine occurs also in Drosophila cells during apoptosis, but whether or not phosphatidylserine serves as a phagocytosis marker remains to be determined. The third candidate for a Drosophila phagocytosis receptor is a protein named Six microns under (Simu) that is expressed in hemocytes and glia (Kurant et al., 2006). The overall structure of Simu resembles that of Draper; both are single-path membrane proteins containing EGF-like repeats in the extracellular region. A decrease in the expression level of Simu in a hemocyte-derived cell line as well as in embryos leads to an increase in the number of apoptotic cells. Two structurally similar phagocytosis receptors, Draper and Simu, are co-expressed in hemocytes and glia, and how they cooperate with each other needs to be investigated. Perspectives In animals having both innate and adaptive immunity, cooperation between the two systems is necessary to maximize immune responses. For invertebrate animals lacking adaptive immunity, innate immune responses, either humoral or cellular, are more important than those in animals with both types of immunity. It is thus speculated that the role and mode of action of innate immune responses in invertebrate animals are somewhat different from those in vertebrate animals. Phagocytosis is at the center of cellular immune responses, and thus clarification of its mechanisms and consequences in invertebrate animals should lead to a better understanding of immunity in general. There are many issues to be solved in order to achieve a full understanding of innate immunity in invertebrate animals. First, it is not known how these animals die upon being infected with some types of microbes. In mammals, septic shock is considered a consequence of excessive host responses to invading microbes, which are mostly mediated by proteins called cytokines secreted from immune cells. Is this also true for invertebrates? Probably the answer is yes, because cytokine-like proteins are secreted from Drosophila hemocytes when insults such as infections with microbes occur (Agaisse et al., 2003). The secreted proteins move via the hemolymph and stimulate the fat body, a Drosophila tissue equivalent to the mammalian liver, to produce stress proteins. There could thus be septic shock in invertebrate animals, at least in Drosophila. Taking into consideration that Drosophila produce immunoglobulin-like proteins (Watson et al., 2005) and complement-like proteins (Lagueux et al., 2000; Kocks et al., 2003; Stroschein-Stevenson et al., 2006), the architecture and operation of immunity do not seem to differ between invertebrate and vertebrate animals. It needs to be confirmed in vivo if these proteins act as opsonins to mediate the phagocytosis of microbes and altered self by Drosophila phagocytes. Conversely, it is necessary to determine to what extent the opsonin-independent phagocytosis of microbes and microbe-infected cells contributes to the immune response against pathogenic microbes in vertebrate animals. Another question to be answered is how invertebrates protect themselves against microbes other than bacteria, such as viruses and protozoa. This issue is also important for humans, because arthropods such as 94 insects can act as a vector for parasitic protozoa that cause severe infectious diseases. It is totally unclear how immune surveillance against invading Plasmodia, the parasite responsible for malaria, is accomplished in mosquitoes. A study on innate immune responses to viral infections has just started with Drosophila (Cherry and Silverman, 2006). The final general question is whether or not the contents of engulfed cells, microbes or altered self, are used to evoke further immune reactions in invertebrate animals. In mammals, cell contents are sometimes processed and presented as a complex with the major histocompatibility complex at the surface of specialized immune cells, antigen-presenting cells, for the activation of T lymphocytes (Ackerman and Cresswell, 2004). This seems unlikely to occur in invertebrates lacking adaptive immunity, but the presence of immunoglobulin-like molecules in Drosophila catches our imagination. Leaving the above-mentioned questions for a future task, issues to be immediately addressed are: 1) to identify marker molecules that exist at the surface of bacteria and are bound by phagocytosis receptors of Drosophila phagocytes; 2) to identify a molecule that exists at the surface of apoptotic cells and is bound by C. elegans CED-1/Drosophila Draper; and 3) to identify the second phagocytosis receptor, after CED-1/Draper, and its ligand for the phagocytosis of apoptotic cells by phagocytes of C. elegans and Drosophila. Acknowledgements We thank an anonymous reviewer for helping us to improve the manuscript. Our studies cited in this paper were supported by the Grant-in-Aid for Scientific Research from JSPS and MEXT, and also by institutional research grants from Kanazawa University. References Ackerman AL, Cresswell P. Cellular mechanisms governing cross-presentation of exogenous antigens. Nat. Immunol. 5: 678–684, 2004. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: 593–623, 1999. Agaisse H, Burrack LS, Philips JA, Rubin EJ, Perrimon N, Higgins DE. Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309: 1248–1251, 2005. Agaisse H, Petersen U-M, Boutros M, Mathey-Prevot B, Perrimon N. Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev. Cell 5: 441–450, 2003. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 124: 783–801, 2006. Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, et al. Essential role of the apoptotic cell engulfment genes draper and ced-6 in developmentally programmed axon pruning during Drosophila metamorphosis. Neuron 50: 855–867, 2006. Brennan CA, Anderson KV. Drosophila: the genetics of innate immune recognition and response. Annu. Rev. Immunol. 22: 457–483, 2004. Callebaut I, Mignotte V, Souchet M, Mornon J-P. EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem. Biophys. Res. Commun. 300: 619–623, 2003. Champion JA, Mitragotri S. Role of target geometry in phagocytosis. Proc. Natl. Acad. Sci. USA 103: 4930–4934, 2006. Cherry S, Silverman N. Host-pathogen interactions in drosophila: new tricks from an old friend. Nat. Immunol. 7: 911–917, 2006. Coombes BK, Valdez Y, Finlay BB. Evasive maneuvers by secreted bacterial proteins to avoid innate immune responses. Curr. Biol. 14: R856–R867, 2004. Ellis RE, Yuan J, Horvitz HR. Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7: 663–698, 1991. Ernst JD. Bacterial inhibition of phagocytosis. Cell. Microbiol. 2: 379–386, 2000. Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death Differ. 5: 551–562, 1998. Flajnik MF, Pasquier LD. Evolution of innate and adaptive immunity: can we draw a line? Trends Immunol. 25: 640–644, 2004. Franc NC, Dimarcq J-L, Lagueux M, Hoffmann J, Ezekowitz RAB. Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4:431–443, 1996. Franc NC, Heitzler P, Ezekowitz RAB, White K. Requirement for Croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284: 1991–1994, 1999. Freeman MR, Delrow J, Kim J, Johnson E, Doe CQ. Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38: 567–580, 2003. Geiszt M, Leto TL. The Nox family of NAD(P)H oxidases: host defense and beyond. J. Biol. Chem. 279: 51715–51718, 2004. Gravato-Nobre MJ, Hodgkin J. Caenorhabditis elegans as a model for innate immunity to pathogens. Cell. Microbiol. 7: 741–751, 2005. Gumienny TL, Hengartner MO. How the worm removes corpses: the nematode C. elegans as a model system to study engulfment. Cell Death Differ. 8: 564–568, 2001. Halliwell B. Phagocyte-derived reactive species: salvation or suicide? Trends Biochem. Sci. 31: 509–515, 2006. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat. Immunol. 5: 971–974, 2004. Hoffmann JA, Reichhart J-M. Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3: 121–126, 2002. Hoopfer ED, McLaughlin T, Watts RJ, Schuldiner O, O’Leary DDM, Luo L. Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning. Neuron 50: 883–895, 2006. Iwanaga S. The molecular basis of innate immunity in the horseshoe crab. Curr. Opin. Immunol. 14: 87–95, 2002. Janeway Jr CA. How the immune system works to 95 protect the host from infection: a personal view. Proc. Natl. Acad. Sci. USA 98: 7461–7468, 2001. Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu. Rev. Immunol. 20: 197–216, 2002. Kim DH, Ausubel FM. Evolutionary perspectives on innate immunity from the study of Caenorhabditis elegans. Curr. Opin. Immunol. 17: 4–10, 2005. Kocks C, Cho JH, Nehme N, Ulvila J, Pearson AM, Meister M, et al. Eater, a transmembrane protein mediating phagocytosis of bacterial pathogens in Drosophila. Cell 123: 335–346, 2005. Kocks C, Maehr R, Overkleeft HS, Wang EW, Iyer LK, Lennon-Duménil A-M, et al. Functional proteomics of the active cysteine protease content in Drosophila S2 cells. Mol. Cell. Proteom. 2: 1188–1197, 2003. Kurant E, Leaman D, Gaul U. The novel glial factor, Six microns under (Simu), functions in the phagocytosis of apoptotic particles. Abstract in the 47th Annual Drosophila Research Conference, Houston, TX, USA, 2006. Lagueux M, Perrodou E, Levashina EA, Capovilla M, Hoffmann JA. Constitutive expression of a complement-like protein Toll and JAK gain-of-function mutants of Drosophila. Proc. Natl. Acad. Sci. USA 97: 11427–11432, 2000. Lauber K, Blumenthal SG, Waibel M, Wesselborg S. Clearance of apoptotic cells: getting rid of the corpses. Mol. Cell 14: 277–287, 2004. Lettre G, Hengartner MO. Developmental apoptosis in C. elegans: a complex CEDnario. Nat. Rev. Mol. Cell Biol. 7: 97–108, 2006. MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR. The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 50: 869–881, 2006. Manaka J, Kuraishi T, Shiratsuchi A, Nakai Y, Higashida H, Henson P, et al. Draper-mediated a n d p h o s p h a t i d y l s e r i n e - i n d e p e n d e n t phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages. J. Biol. Chem. 279: 48466–48476, 2004. Mangahas PM, Zhou Z. Clearance of apoptotic cells in Caenorhabditis elegans. Sem. Cell Dev. Biol. 16: 295–306, 2005. Meister M, Lagueux M. Drosophila blood cells. Cell. Microbiol. 5: 573–580, 2003. Mylonakis E, Aballay A. Worms and flies as genetically tractable animal models to study host-pathogen interactions. Infect. Immun. 73: 3833–3841, 2005. Peiser L, Godon S. The function of scavenger receptors expressed by macrophages and their role in the regulation of inflammation. Microb. Infect. 3: 149–159, 2001. Philips JA, Rubin EJ, Perrimon N. Drosophila RNAi screen reveals CD36 family member required for Mycobacterial infection. Science 309: 1251–1253. Rämet M, Pearson A, Manfruelli P, Li X, Koziel H, Gobel V, et al. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria. Immunity 15: 1027–1038, 2001. Rämet M, Manfruelli P, Pearson A, Mathey-Prevot B, Ezekowitz RAB. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644–648, 2002. Reddien PW, Horvitz HR. The engulfment process of programmed cell death in Caenorhabditis elegans. Annu. Rev. Cell Dev. Biol. 20: 193–221, 2004. Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature 407: 784–788, 2000. Savill J, Fadok V, Henson P, Haslett C. Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14: 131–136, 1993. Schlegel RA, Williamson P. Phosphatidylserine, a death knell. Cell Death Differ. 8: 551–563, 2001. Shintani T, Klionsky DJ. Autophagy in health and disease: a double-edged sword. Science 306: 990–995, 2004. Shiratsuchi A, Kawasaki Y, Ikemoto M, Arai H, Nakanishi Y. Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells. J. Biol. Chem. 274: 5901–5908, 1999. Stroschein-Stevenson S, Foley E, O’Farrell PH, Johnson AD. Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol. 4: e4, 2006. Stuart LM, Deng J, Silver JM, Takahashi K, Tseng AA, Hennessy EJ, et al. Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J. Cell Biol. 170: 477–485, 2005. Stuart LM, Ezekowitz RAB. Phagocytosis: elegant complexity. Immunity 22 : 539–550, 2005. Taylor PR, Martinez-Pomares L, Stacey M, Lin H-H, Brown GD, Gordon S. Macrophage receptors and immune recognition. Annu. Rev. Immunol. 23: 901–944, 2005. Underhill DM, Ozinsky A. Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20: 825–852, 2002. Watson FL, Püttmann-Holgado R, Thomas F, Lamar DL, Hughes M, et al. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309: 1874–1878, 2005. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68: 251–306, 1980. Yu X, Odera S, Chuang C-H, Lu N, Zhou Z. C. elegans dynamin mediates the signaling of phagocytic receptor CED-1 for the engulfment and degradation of apoptotic cells. Dev. Cell 10: 743–757, 2006. Zhou Z, Hartwieg E, Horvitz HR. CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104: 43–56, 2001. 96 Mechanisms and roles of phagocytosis in Drosophila and Caenorhabditis elegans Y Nakanishi, A Shiratsuchi Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan Abstract Introduction Phagocytosis at a glance Phagocytosis of microbes in Drosophila and C. elegans Phagocytosis of bacteria by Drosophila phagocytes those from wild-type flies. In addition, its soluble form is present in the hemolymph. These findings evoke the possibility that Dscam serves not only as a receptor but also as an immunoglobulin-like opsonin in the phagocytosis of bacteria. More recently, a family of secreted proteins, called Teps for thioester-containing proteins, which serve as opsonins to mediate phagocytosis of microbes by S2 cells, was reported (Stroschein-Stevenson et al., 2006). Of 6 Teps TepII, TepIII, and TepVI have been suggested to be involved in the phagocytosis of E. coli, S. aureus, and Candida albicans, respectively, though in vivo confirmation is required. Teps resemble the complement C3, and the presence of other complement-like proteins has also been noted though their action as opsonins remains to be shown (Lagueux et al., 2000; Kocks et al., 2003). Phagocytosis of bacteria by C. elegans phagocytes Phagocytosis of apoptotic cells in Drosophila and C. elegans Phagocytosis of apoptotic cells by C. elegans phagocytes Phagocytosis of apoptotic cells by Drosophila phagocytes Perspectives References