Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death

Abstract

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.

1. INTRODUCTION

Cells that have become unwanted emerge in the body throughout our life. These cells include those that are obstacles to morphogenesis, disturb the establishment of tissue functions, have become spent and aged, have accomplished their task, or have become pathogenic. The removal of these cells is achieved by apoptosis-dependent phagocytosis, a mechanism evolutionarily conserved among multicellular organisms.^1–3) This mechanism does not involve lymphocytes or antibodies, and, thus, is considered to be an innate immune response. The phagocytic removal of apoptotic cells appears to be accomplished so quickly and effectively that cells undergoing apoptosis are rarely detected in vivo. The malfunction of this mechanism leads not only to the retardation in morphogenesis and tissue function establishment as well as the severity of some infectious diseases, but to defects in tissue homeostasis: apoptotic cells left unengulfed eventually lyze and damage surrounding tissues and organs, sometimes causing diseases such as inflammation and autoimmunity.^4,5)

The first step of phagocytosis is the recognition of apoptotic cells by phagocytic cells, and this is executed through the action of engulfment receptors that bind to substances specifically expressed on the surface of target cells during the apoptotic process.^{1–3,6–14)} Activated receptors transmit signals to intracellular signal mediators, resulting in the onset of signal-transduction pathways for the induction of phagocytosis. These pathways culminate in the reorganization of the actin cytoskeleton, allowing phagocytes to engulf target cells.^1–3) Apoptotic cells incorporated into phagocytes eventually lyze and are digested by lysosomal enzymes. This entire process is called ‘apoptotic cell clearance’ or ‘efferocytosis’ and contributes not only to the disappearance of unwanted cells, but also to the accomplishment of phagocyte actions for the maintenance of tissue homeostasis.

This review describes the mechanisms underlying and significance of apoptosis-dependent phagocytosis as well as subsequent alterations in the functions of phagocytes.

2. MECHANISMS OF APOPTOSIS-DEPENDENT PHAGOCYTOSIS OF UNWANTED CELLS

Phagocytosis is a biological event in which a particular type of cell with phagocytic activity, called a phagocyte, engulfs relatively large substances and digests them with the aid of lysosomal enzymes (Fig. 1). Targets include invading bacteria and altered cells in the host organism after they become aged, spent, cancerous, or infected with a virus. The engulfment of large substances is, in many cases, initiated in a receptor-mediated manner, and an event subsequent to the molecular interaction between an engulfment receptor and a ligand is the protrusion of portions of a phagocyte, the pseudopodium, which surrounds the target and incorporates it into the cell. Engulfed targets exist in phagocytes as a membrane vesicle called the phagosome, which undergoes maturation from an early to late endosome and eventually becomes a phagolysosome after fusion with the lysosome. Targets are lysed in phagolysosomes by digestive enzymes contained in lysosomes.

Fig. 1. Mechanisms of Phagocytosis

Phagocytosis is initiated in phagocytes through a molecular interaction between ligands and engulfment receptors, which exist on the surface of target cells and phagocytes, respectively. Once bound by ligands, engulfment receptors activate signal transduction pathways to induce the formation of pseudopodia. Target cells are incorporated into phagocytes as phagosomes that subsequently fuse with lysosomes, giving rise to phagolysosomes. Phagocytosis is complete when target cells are digested by lysosomal enzymes.

There are two types of phagocytes, professional and amateur (Fig. 2). The professional phagocyte plays its role primarily in phagocytosis, while the amateur phagocyte exerts the phagocytic activity only when needed. In mammals, the former type of phagocytes includes macrophages, neutrophils, dendritic cells, alveolar macrophages in the lung, and Kupffer cells in the liver, while the latter includes the endothelial cells of blood vessels, epithelial cells of the airway and digestive tract, microglia of the brain, and Sertoli cells of the testis. Among the phagocytes mentioned above, macrophages circulate throughout the body to locate target substances, neutrophils and dendritic cells migrate towards targets, and others are localized and function only in particular tissues or organs (Fig. 2).

Fig. 2. Category of Phagocytes

There are two categories for phagocytes. One category is whether phagocytes are professional or amateur, the latter of which exert phagocytic activity only when needed. The names of professional and amateur phagocytes are circled and squared, respectively. The other category is whether phagocytes are migratory in the body or localized in particular tissues or organs. Refer to the text for further information.

2.1. General Mechanisms of Apoptotic Cell Clearance

Among the various types of cell death identified to date, apoptosis is considered to be a physiological, silent mode of cell death.¹⁵⁾ This is because the integrity of plasma membranes is maintained in cells undergoing apoptosis until they are eliminated, thereby protecting the surrounding tissues from being exposed to the noxious contents of dying cells. Apoptosis is executed through the actions of cysteine proteases named caspases that cleave a peptide bond located at the carboxyl side of an aspartic acid residue of target proteins.¹⁶⁾ There are two types of caspases, the initiator and effector (Fig. 3). The initiator caspase, which becomes functional at the onset of apoptosis, is responsible for the activation of the effector caspase by partial cleavage. Activated effector caspases digest a number of proteins, causing the apoptotic characteristics of cells such as the structural change of cell surface for the generation of markers for phagocytosis, and the fragmentation of DNA and nuclei to destroy a genetic material. Two pathways exist for the activation of initiator caspases: the extrinsic pathway involves a death receptor and runs independent of a mitochondrion, while the intrinsic pathway does not involve a death receptor being initiated by mitochondrial proteins¹⁷⁾ (Fig. 3).

Fig. 3. Mechanisms of Apoptosis

When cells are triggered, either extrinsically or intrinsically, for the induction of apoptosis, initiator caspases are activated and partially cleave effector caspases for their activation, which in turn degrade a number of proteins. These target proteins include those responsible for structural changes at the surface of apoptosing cells, the cleavage of DNA, and the fragmentation of nuclei.

During the process of apoptosis, the structure of cell surface is altered in order for these cells to be earmarked for recognition by phagocytes. This occurs through chemical modifications to pre-existing molecules or the surface exposure of molecules that are hindered intracellularly (Fig. 4). A variety of molecules, including proteins, sugars, and lipids, are known to receive such apoptosis-specific changes in structure or localization and called ‘markers for phagocytosis’ or ‘eat-me signals’. Phagocytes recognize apoptotic cells using engulfment receptors that bind eat-me signals either directly or indirectly with the aid of molecules bridging the receptor and ligand (Fig. 4). Several types of engulfment receptors and bridging molecules have been identified; however, the mechanisms by which they are differentially used are not completely understood. Ligand-bound engulfment receptors transmit a stimulus to intracellular molecules to activate signal transduction pathways for the induction of phagocytosis.

Fig. 4. Structural Changes of the Cell Surface during Apoptosis

The structure of the cell surface changes in the apoptotic process, and this is achieved by the translocation of intracellular molecules or the chemical modification of pre-existing molecules. These altered structures serve as ligands for engulfment receptors to induce phagocytosis.

2.2. Signal Transduction Pathway for the Induction of Phagocytosis

Previous studies using the nematode Caenorhabditis elegans showed the existence of two signaling pathways for phagocytosis that converge on a small G-protein responsible for remodeling the actin cytoskeleton (Fig. 5A). Pseudopodia are then formed, surround target cells, and incorporate them into phagocytes as phagosomes. The engulfed apoptotic cells are ultimately digested through the actions of lysosomal enzymes. This process allows for the elimination of apoptotic cells before their noxious contents leak out, and is, thus, regarded as the ‘silent removal’ of unwanted cells. The molecules involved in this process in C. elegans mostly possess counterparts in mammals, suggesting the evolutionary conservation of this biological event. In order to reach a conclusion on this aspect, we recently examined signaling pathways for the induction of apoptotic cell clearance in the fruit fly Drosophila melanogaster. Data from a series of biochemical and genetic experiments revealed that pathways in Drosophila closely resemble those in nematodes and mammals (Fig. 5B), indicating that a similar mechanism for the safe removal of unwanted cells functions among multicellular organisms.¹⁸⁾ The engulfment receptors CED-1¹⁹⁾ (C. elegans)/Draper^20,21) (Drosophila)/Jedi-1²²⁾ (mouse), MEGF10²³⁾ (human) are single-path membrane proteins that belong to the Nimrod family of proteins, with atypical epidermal growth factor-like repeats in the extracellular region. The receptor in the other pathway is integrin; α and β subunits are INA-1 and PAT-3 in C. elegans,²⁴⁾ αPS3 and βν in Drosophila,^25,26) and α_v and β₃, β₅ in mammals.²⁷⁾ These receptors, once bound by eat-me signals or bridging molecules, activate the intracellular molecules constituting two signal transduction pathways that converge at the small G proteins CED-10 (C. elegans)/Rac1, Rac2 (Drosophila)/Rac1 (mammals). Re-organization of the actin cytoskeleton then occurs for the initiation of engulfment.

Fig. 5. Signaling Pathways for Apoptotic Cell Clearance

(A) Signaling pathways in the phagocyte of C. elegans. Two engulfment receptors, CED-1 and integrin, are shown in green, and intracellular signaling molecules are squared. Two pathways converge on the small G protein CED-10 that induces the rearrangement of actin cytoskeleton for the occurrence of phagocytosis. (B) Conservation of signaling pathways among C. elegans, Drosophila, and humans. Eat-me signals, bridging molecules (in parentheses), engulfment receptors, and intracellular signaling molecules of C. elegans/Drosophila/humans are illustrated. Note that dElmo appears to be dispensable in Drosophila phagocytes. PS, phosphatidylserine; MFG-E8, milk fat globule-EGF-factor 8.

2.3. Phosphatidylserine as an Eat-Me Signal

Among the several eat-me signals identified to date, phosphatidylserine, a glycerophospholipid constituting bio-membranes, has been most intensively characterized and shown to play a role in nematodes, insects, and mammals. This phospholipid mainly exists in the inner leaflet of the plasma membrane bilayer due to the actions of proteins responsible for the trans-layer movement of phospholipids. When apoptosis is initiated, the function of these proteins is altered by the action of effector caspases, resulting in the cancellation of the asymmetrical distribution of phospholipids between two layers^28,29) (Fig. 6). As a result, phosphatidylserine, which is otherwise restricted to the inner layer, is relocated to the outer layer and exposed to the surface of apoptotic cells. Many engulfment receptors have been reported to bind phosphatidylserine in a direct or indirect manner. The receptors that directly bind this phospholipid include CED-1 of C. elegans,³⁰⁾ Draper³¹⁾ and Six-microns-under³²⁾ of Drosophila, and lectin-like oxidized low-density lipoprotein receptor 1,³³⁾ class B scavenger receptor type I,³⁴⁾ stabilin-2,³⁵⁾ brain-specific angiogenesis inhibitor 1,³⁶⁾ T-cell immunoglobulin- and mucin-domain-containing molecule 4,³⁷⁾ kidney injury molecule-1,³⁸⁾ receptor for advanced glycation end products,^39,40) CD300f,⁴¹⁾ and triggering receptor expressed on myeloid cells-like protein 2⁴²⁾ in mammals. Bridging molecules that link phosphatidylserine and engulfment receptors are TTR-52 of C. elegans⁴³⁾ and product of growth arrest-specific gene 6⁴⁴⁾ and milk fat globule-EGF-factor 8⁴⁵⁾ of mammals. TTR-52 is bound by CED-1, while product of growth arrest-specific gene 6 and milk fat globule-EGF-factor 8 are bound by the engulfment receptors Axl, Sky, and Mer, and the integrins α_v-β₃ and α_v-β₅, respectively. However, which of the two signal transduction pathways are activated by the above-described receptors largely remains to be determined.

Fig. 6. Surface Exposure of Phosphatidylserine during Apoptosis

Phospholipids are located in the two layers of the plasma membrane in certain proportions, due to the actions of proteins responsible for their trans-layer movement (lipid transporter). Upon induction of apoptosis, the activities of these proteins are altered so that the asymmetrical distribution of phospholipids in the bilayer is cancelled. As a result, phosphatidylserine that is localized in the inner layer of normal cells moves to the outer layer, being exposed on the cell surface. The cell surface phosphatidylserine serves as an eat-me signal that is recognized, either directly or indirectly with bridging molecules, by engulfment receptors of phagocytes for the induction of phagocytosis.

3. CONSEQUENCES OF APOPTOSIS-DEPENDENT PHAGOCYTOSIS OF UNWANTED CELLS

The primary outcome of apoptotic cell clearance is the removal of cells unnecessary for organisms without causing damage to surrounding tissues. However, this event induces secondary effects under specific conditions, based on the creation of empty spaces, removal of cells that impede tissue functions, elimination of pathogens, utilization of the components of engulfed cells, and changes in the characteristics of phagocytes (Fig. 7). Some examples of the consequences of apoptotic cell clearance are described below.

Fig. 7. Secondary Effects of Apoptotic Cell Clearance

The phagocytic elimination of unwanted cells provokes secondary outcomes that are important for morphogenesis, the renewal of tissues and organs, the establishment of tissue functions, and the maintenance of tissue homeostasis. Refer to the text for further information.

3.1. Role in Morphogenesis

Apoptotic cell clearance plays a role in the early development of multicellular organisms. During the generation of fingers and toes, cells occupying the space between bones are removed by apoptosis-dependent phagocytosis (Fig. 8). The mouth and anus have been suggested to be created by the phagocytic removal of cells that otherwise fill these areas. In the metamorphosis of frogs, the removal of a tadpole’s tail is partly executed by phagocytosis.

Fig. 8. Examples of Secondary Effects

Three biological events are shown as the secondary effects of apoptotic cell clearance. Refer to the text for further information.

3.2. Role in the Establishment and Maintenance of Tissue Functions

When a neural network is established, mistakenly formed connections, either neuron–neuron or neuron–peripheral tissue, are removed by the phagocytosis of those neurons. During the establishment of acquired immunity, a set of lymphocytes is eliminated by the mechanism of apoptosis-dependent phagocytosis: lymphocytes that react against self-antigens are induced to undergo apoptosis and subsequently removed by phagocytosis.

Apoptotic cell clearance also participates in the generation of gametes. In the production of sperm, a large portion of differentiating spermatogenic cells become apoptotic and are engulfed by Sertoli cells⁴⁶⁾ (Fig. 8), a testicular somatic cell with phagocytic activity. Although the physiological meaning of this selection of spermatogenic cells remains unclear, this event is necessary for the effective production of sperm.⁴⁷⁾ Ovarian follicles after ovulation form the corpus luteum, which serves as an endocrine tissue to maintain the development of fertilized eggs; however, this tissue needs to be removed in the absence of conception in order for new ovulation to occur. These corpora lutea are subjected to apoptosis-dependent phagocytosis by infiltrated macrophages.^48,49)

3.3. Role in the Renewal and Remodeling of Tissues

When cells that have become unwanted are removed, the same cells need to be replenished in order to maintain the structure and function of the corresponding organs and tissues. Apoptosis and subsequent phagocytosis appear to actively induce this replenishment, an event often called compensatory cell proliferation.⁵⁰⁾ Although the precise mechanisms involved remain to be elucidated, cytokine-like soluble substances, which are secreted from apoptotic cells and phagocytes, have been suggested to play a role. Axons and dendrites are selectively eliminated, without damage to the parent neurons, to shape and organize neural networks.⁵¹⁾ This phenomenon, called pruning, is observed during the development of the nervous system as well as in response to the injury or disease of neurons. Phagocytosis plays a role, at least partly, in the pruning of axons and dendrites: during metamorphosis in Drosophila, the axons of larval neurons are phagocytosed by glia, and new axons are generated to form a network in adult flies⁵²⁾ (Fig. 8). Although this event involves the engulfment of a part of a cell, not an entire cell, the participating players are considered to be the same as in apoptotic cell clearance.⁵³⁾ Another example is the diurnal renewal of photoreceptor cells in the retina: retinal pigment epithelial cells remove outer segments of photoreceptor cells by phagocytosis every morning, and this is necessary for the maintenance of vision.⁵⁴⁾

3.4. Role in Disease Prevention

The first study to describe the active involvement of phagocytes that have engulfed apoptotic cells in the mitigation of diseases was published nearly 20 years ago.⁵⁵⁾ In this study, mouse macrophages engulfing apoptotic neutrophils were shown to reduce the production of the pro-inflammatory cytokines interleukin-8 and tumor necrosis factor-α, and increase that of the anti-inflammatory transforming growth factor-β. This change in macrophages was interpreted as macrophages acting to resolve inflammation by directly eliminating neutrophils as well as altering the cytokine repertoire from inflammation-prone to normal. A subsequent study demonstrated that a change in the production of cytokines was achieved through alterations in gene expression patterns.⁵⁶⁾ The control of the inflammatory state by phagocytes that have accomplished the engulfment of apoptotic cells has been investigated in recent studies, and the importance of an interaction between professional and amateur or tissue-restricted phagocytes has been noted.^57,58)

Cells that have become pathogenic to organisms are eliminated by apoptosis-dependent phagocytosis. Examples of these targets are cancer cells and virus-infected cells. However, it remains controversial whether the phagocytosis of cancer cells depends on apoptosis in the target.⁵⁹⁾ The induction of apoptosis and subsequent phagocytosis of cells infected with a virus were initially reported to occur in rodents,⁶⁰⁾ and this mechanism was later shown to exist also in insects.⁶¹⁾ The phagocytic elimination of virus-infected cells is directly involved in decreasing the level of a viral load, resulting in the mitigation of pathogenicity (Fig. 9). A recent study showed that Pseudomonas aeruginosa are passively engulfed and killed by phagocytes when apoptotic epithelial cells of the digestive tract that this bacterium specifically binds undergo phagocytosis.⁶²⁾

Fig. 9. Total Prevention of Virus-Induced Diseases by Apoptotic Cell Clearance

Virus-infected cells are engulfed and digested by phagocytes in an apoptosis-dependent manner. This contributes to the reduction of viral load (Virus Removal). In addition, phagocytes change the pattern of gene expression, thereby increasing the levels of anti-inflammatory substances (Resolution of Inflammation) and engulfment receptors (Phagocyte Priming). Phagocytes also utilize the components of engulfed cells for antigen presentation towards T lymphocytes (Antigen Presentation) as well as the systemic induction of virus-specific RNA interference, including cells not infected with a virus (Systemic RNAi). These have been observed in mice and humans (mammal) or Drosophila (fly). Refer to the text for further information.

3.5. Role in Antigen Presentation

The first step towards the induction of acquired immunity is to alert CD4-positive helper and CD8-positive cytotoxic T lymphocytes of the presence of foreign substances. Stimulated helper T lymphocytes produce cytokines that make B lymphocytes prepare for antibody production, while cytotoxic T lymphocytes undergo maturation into killer T cells. Particular types of immune cells including dendritic cells and macrophages, which are called antigen-presenting cells, process proteins of foreign substances and express them on their cell surface using major histocompatibility complex to trigger T lymphocytes. In order for antigen-presenting cells to exert their task, antigens need to be produced in these cells. However, the generation of an antibody specific to a viral protein was evident in animals in which antigen-presenting cells were apparently not infected with the virus. This phenomenon, called cross-presentation, appears to involve apoptotic cell clearance, namely, antigen-presenting cells engulf virus-infected cells, which are induced to undergo apoptosis after infection, and process and present engulfed viral antigens to T lymphocytes⁶³⁾ (Fig. 9).

3.6. Role in Phagocyte Priming

Previous studies reported that the level of engulfment receptors increased in mouse macrophages after the engulfment of apoptotic cells^64,65) A similar event was more clearly observed with Drosophila phagocytes, and its role was demonstrated in vivo.^18,66) Furthermore, a transcription factor responsible for the augmented expression of engulfment receptors was identified.¹⁸⁾ This phenomenon has been interpreted as a mechanism for the priming of phagocytes; the activity of phagocytes is enhanced when they first meet target cells through an increase in the level of engulfment receptors, and this ensures the elimination of target cells that the same phagocytes subsequently encounter⁶⁷⁾ (Fig. 10). More recently, Drosophila phagocytes were shown to capture viral RNA to produce and secrete short interfering RNA for the RNA interference of viral genomes or transcripts.⁶⁸⁾ This mechanism explains the occurrence of anti-viral RNA interference in cells that are not infected with a virus. The production of virus-specific short interfering RNA appeared to last for a long time, and, thus, was regarded as immunological memory resembling the production of antibodies in evolutionarily higher organisms.⁶⁹⁾ We speculate that phagocytes can gain viral materials for the production of short interfering RNA through the apoptosis-dependent phagocytosis of virus-infected cells (Fig. 9).

Fig. 10. Phagocyte Priming by Apoptotic Cell Clearance in Drosophila

After the engulfment of apoptotic cells, Drosophila phagocytes augment the transcription of genes coding for engulfment receptors, with the aid of the transcription factor Tailless that is activated in phagocytes after the engulfment of apoptotic cells. This leads to the enhancement of phagocytic activity, which ensures the removal of apoptotic cells that phagocytes subsequently encounter. drpr and scb code for Draper and integrin αPS3, respectively.

4. CONCLUSION AND PERSPECTIVES

Cells of particular types and those at certain developmental stages are destined to die, and this is often called programmed cell death. In addition, cells of many organs and tissues are periodically renewed through the death of old cells and the subsequent differentiation of corresponding stem cells for replenishment. Cells that have become spent, non-functional, or pathogenic are also induced to undergo cell death. Apoptosis is a modality of cell death that explains the death of cells in the above-described phenomena. However, cells that have become apoptotic do not autonomously disappear, but need to be phagocytosed for removal. Thus, it is appropriate to interpret apoptotic cell death as a process that earmarks unwanted cells for phagocytosis. Apoptosis and subsequent phagocytosis are evident in different animal species from nematodes and insects through to mammals, including humans, and are executed under fundamentally conserved mechanisms. Recent studies revealed that apoptosis-dependent phagocytosis serves not merely as a mechanism to eliminate unwanted cells, but is more actively involved in the maintenance of tissue homeostasis: morphogenesis during early development, the establishment and maintenance of tissue functions, and the protection of organisms from diseases. Therefore, molecules involved in the process of apoptosis and subsequent phagocytosis as well as those controlling these processes may be targeted in novel medical treatments for intractable diseases.

Apoptotic cell clearance helps organisms keep homeostasis, but, at the same time, it becomes causative of diseases under certain circumstances. The elimination of apoptotic neurons in the brain and human immunodeficiency virus-infected immune cells might lead, if not solely, to the development of neurodegenerative diseases and AIDS, respectively. Therefore, medical treatments in both directions, enhancing and suppressing phagocytosis, are likely to be effective. Many attempts have been made to create medical treatments that modify the level of apoptosis in patients since the mechanisms underlying apoptotic cell death were mostly solved. However, not much success has so far been achieved. A difficulty in apoptosis-controlling medical treatments is that their effect must be local, and the same condition needs to be applied to the control of phagocytosis; otherwise adverse side effects are anticipated. On this assumption, pharmacological targets in apoptotic cell clearance could be engulfment receptors that participate in locally occurring phagocytosis or are expressed in a particular type of phagocytes. Alternatively, it should be feasible to change the number of phagocytes in patients: to reduce phagocytes has been successful in experimental animals where phagocytes are killed in a spatiotemporal manner. Injection of apoptosing cells into patients is likely to mitigate inflammation, and that of apoptotic cancer cells could induce acquired immunity against the same type of cancer as well. Priming or training of phagocytes in vitro might also be useful, where phagocytes are isolated from a patient, incubated with apoptotic cells, and returned to the same patient, expecting a rise in the level of phagocytic activity.

Acknowledgment

The authors’ studies referred to in this article have been supported in part by Grant-in-Aid for the Japan Society for the Promotion of Science Fellows (to S. N.) and the Japan Society for the Promotion of Science KAKENHI Grants (to A. S., K. N., and Y. N.).

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Lettre G, Hengartner MO. Developmental apoptosis in C. elegans: a complex CEDnario. Nat. Rev. Mol. Cell Biol., 7, 97–108 (2006).
• 2) Kinchen JM, Ravichandran KS. Journey to the grave: signaling events regulating removal of apoptotic cells. J. Cell Sci., 120, 2143–2149 (2007).
• 3) Nakanishi Y, Nagaosa K, Shiratsuchi A. Phagocytic removal of cells that have become unwanted: Implications for animal development and tissue homeostasis. Dev. Growth Differ., 53, 149–160 (2011).
• 4) Elliott MR, Ravichandran KS. Clearance of apoptotic cells: implications in health and disease. J. Cell Biol., 189, 1059–1070 (2010).
• 5) Nagata S, Hanayama R, Kawane K. Autoimmunity and the clearance of dead cells. Cell, 140, 619–630 (2010).
• 6) Savill J. Recognition and phagocytosis of cells undergoing apoptosis. Br. Med. Bull., 53, 491–508 (1997).
• 7) Ren Y, Savill J. Apoptosis: the importance of being eaten. Cell Death Differ., 5, 563–568 (1998).
• 8) Savill J, Fadok V. Corpse clearance defines the meaning of cell death. Nature, 407, 784–788 (2000).
• 9) Henson PM, Bratton DL, Fadok VA. Apoptotic cell removal. Curr. Biol., 11, R795–R805 (2001).
• 10) Lauber K, Blumenthal SG, Waibel M, Wesselborg S. Clearance of apoptotic cells: getting rid of the corpses. Mol. Cell, 14, 277–287 (2004).
• 11) Ravichandran KS, Lorenz U. Engulfment of apoptotic cells: signals for a good meal. Nat. Rev. Immunol., 7, 964–974 (2007).
• 12) Ravichandran KS. Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity, 35, 445–455 (2011).
• 13) Hochreiter-Hufford A, Ravichandran KS. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. Perspect. Biol., 5, a008748 (2013).
• 14) Penberthy KK, Ravichandran KS. Apoptotic cell recognition receptors and scavenger receptors. Immunol. Rev., 269, 44–59 (2016).
• 15) Wallach D, Kang T-B, Dillon CP, Green DR. Programmed necrosis in inflammation: Toward identification of the effector molecules. Science, 352, aaf2154 (2016).
• 16) Man SM, Kanneganti T-D. Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat. Rev. Immunol., 16, 7–21 (2016).
• 17) Riedl SJ, Salvesen GS. The apoptosome: signalling platform of cell death. Nat. Rev. Mol. Cell Biol., 8, 405–413 (2007).
• 18) Nonaka S, Ando Y, Kanetani T, Hoshi C, Nakai Y, Nainu F, Nagaosa K, Shiratsuchi A, Nakanishi Y. Signaling pathway for phagocyte priming upon encounter with apoptotic cells. J. Biol. Chem., 292, 8059–8072 (2017).
• 19) 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).
• 20) Manaka J, Kuraishi T, Shiratsuchi A, Nakai Y, Higashida H, Henson P, Nakanishi Y. Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages. J. Biol. Chem., 279, 48466–48476 (2004).
• 21) Kuraishi T, Nakagawa Y, Nagaosa K, Hashimoto Y, Ishimoto T, Moki T, Fujita Y, Nakayama H, Dohmae N, Shiratsuchi A, Yamamoto N, Ueda K, Yamaguchi M, Awasaki T, Nakanishi Y. Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells. EMBO J., 28, 3868–3878 (2009).
• 22) Wu H-H, Bellmunt E, Scheib JL, Venegas V, Burkert C, Reichardt LF, Zhou Z, Fariñas I, Carter BD. Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor. Nat. Neurosci., 12, 1534–1541 (2009).
• 23) Hamon Y, Trompier D, Ma Z, Venegas V, Pophillat M, Mignotte V, Zhou Z, Chimini G. Cooperation between engulfment receptors: the case of ABCA1 and MEGF10. PLoS ONE, 1, e120 (2006).
• 24) Hsu T-Y, Wu Y-C. Engulfment of apoptotic cells in C. elegans is mediated by integrin α/SRC signaling. Curr. Biol., 20, 477–486 (2010).
• 25) Nagaosa K, Okada R, Nonaka S, Takeuchi K, Fujita Y, Miyasaka T, Manaka J, Ando I, Nakanishi Y. Integrin βν-mediated phagocytosis of apoptotic cells in Drosophila embryos. J. Biol. Chem., 286, 25770–25777 (2011).
• 26) Nonaka S, Nagaosa K, Mori T, Shiratsuchi A, Nakanishi Y. Integrin αPS3/βν-mediated phagocytosis of apoptotic cells and bacteria in Drosophila. J. Biol. Chem., 288, 10374–10380 (2013).
• 27) Wu Y, Tibrewal N, Birge RB. Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol., 16, 189–197 (2006).
• 28) Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science, 341, 403–406 (2013).
• 29) Segawa K, Kurata S, Yanagihashi Y, Brummelkamp TR, Matsuda F, Nagata S. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science, 344, 1164–1168 (2014).
• 30) Li Z, Venegas V, Nagaoka Y, Morino E, Raghavan P, Audhya A, Nakanishi Y, Zhou Z. Necrotic cells actively attract phagocytes through the collaborative action of two distinct PS-exposure mechanisms. PLOS Genet., 11, e1005285 (2015).
• 31) Tung TT, Nagaosa K, Fujita Y, Kita A, Mori H, Okada R, Nonaka S, Nakanishi Y. Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper. J. Biochem., 153, 483–491 (2013).
• 32) Shklyar B, Levy-Adam F, Mishnaevski K, Kurant E. Caspase activity is required for engulfment of apoptotic cells. Mol. Cell. Biol., 33, 3191–3201 (2013).
• 33) Oka K, Sawamura T, Kikuta K, Itokawa S, Kume N, Kita T, Masaki T. Lectin-like oxidized low-density lipoprotein receptor 1 mediates phagocytosis of aged/apoptotic cells in endothelial cells. Proc. Natl. Acad. Sci. U.S.A., 95, 9535–9540 (1998).
• 34) Kawasaki Y, Nakagawa A, Nagaosa K, Shiratsuchi A, Nakanishi Y. Phosphatidylserine binding of class B scavenger receptor type I, a phagocytosis receptor of testicular Sertoli cells. J. Biol. Chem., 277, 27559–27566 (2002).
• 35) Park S-Y, Jung M-Y, Kim H-J, Lee S-J, Kim S-Y, Lee B-H, Kwon T-H, Park R-W, Kim I-S. Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ., 15, 192–201 (2008).
• 36) Park D, Tosello-Trampont A-C, Elliott MR, Lu M, Haney LB, Ma Z, Klibanov AL, Mandell JW, Ravichandran KS. BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature, 450, 430–434 (2007).
• 37) Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S. Identification of Tim4 as a phosphatidylserine receptor. Nature, 450, 435–439 (2007).
• 38) Ichimura T, Asseldonk EJ, Humphreys BD, Gunaratnam L, Duffield JS, Bonventre JV. Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells. J. Clin. Invest., 118, 1657–1668 (2008).
• 39) He M, Kubo H, Morimoto K, Fujino N, Suzuki T, Takahashi T, Yamada M, Yamaya M, Maekawa T, Yamamoto Y, Yamamoto H. Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells. EMBO Rep., 12, 358–364 (2011).
• 40) Friggeri A, Banerjee S, Biswas S, de Freitas A, Liu G, Bierhaus A, Abraham E. Participation of the receptor for advanced glycation end products in efferocytosis. J. Immunol., 186, 6191–6198 (2011).
• 41) Choi S-C, Simhadri VR, Tian L, Gil-Krzewska A, Krzewski K, Borrego F, Coligan JE. Cutting Edge: Mouse CD300f (CMRF-35-like molecule-1) recognizes outer membrane-exposed phosphatidylserine and can promote phagocytosis. J. Immunol., 187, 3483–3487 (2011).
• 42) de Freitas A, Banerjee S, Xie N, Cui H, Davis KI, Friggeri A, Fu M, Abraham E, Liu G. Identification of TLT2 as an engulfment receptor for apoptotic cells. J. Immunol., 188, 6381–6388 (2012).
• 43) Wang X, Li W, Zhao D, Liu B, Shi Y, Chen B, Yang H, Guo P, Geng X, Shang Z, Peden E, Kage-Nakadai E, Mitani S, Xue D. Caenorhabditis elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor. Nat. Cell Biol., 12, 655–664 (2010).
• 44) Ishimoto Y, Ohashi K, Mizuno K, Nakano T. Promotion of the uptake of PS liposomes and apoptotic cells by a product of growth arrest-specific gene, gas6. J. Biochem., 127, 411–417 (2000).
• 45) Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature, 417, 182–187 (2002).
• 46) Nakanishi Y, Shiratsuchi A. Phagocytic removal of apoptotic spermatogenic cells by Sertoli cells: mechanisms and consequences. Biol. Pharm. Bull., 27, 13–16 (2004).
• 47) Maeda Y, Shiratsuchi A, Namiki M, Nakanishi Y. Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility. Cell Death Differ., 9, 742–749 (2002).
• 48) Nagaosa K, Shiratsuchi A, Nakanishi Y. 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. Biol. Reprod., 67, 1502–1508 (2002).
• 49) Kato S, Shiratsuchi A, Nagaosa K, Nakanishi Y. Phosphatidylserine- and integrin-mediated phagocytosis of apoptotic luteal cells by macrophages of the rat. Dev. Growth Differ., 47, 153–161 (2005).
• 50) Fan Y, Bergmann A. Apoptosis-induced compensatory proliferation. The cell is dead. Long live the cell! Trends Cell Biol., 18, 467–473 (2008).
• 51) Luo L, O’Leary DDM. Axon retraction and degeneration in development and disease. Annu. Rev. Neurosci., 28, 127–156 (2005).
• 52) Yu F, Schuldiner O. Axon and dendrite pruning in Drosophila. Curr. Opin. Neurobiol., 27, 192–198 (2014).
• 53) Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K. Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron, 50, 855–867 (2006).
• 54) Nandrot EF, Kim Y, Brodie SE, Huang X, Sheppard D, Finnemann SC. Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking α_vβ₅ integrin. J. Exp. Med., 200, 1539–1545 (2004).
• 55) Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-β, PGE2, and PAF. J. Clin. Invest., 101, 890–898 (1998).
• 56) McDonald PP, Fadok VA, Bratton D, Henson PM. Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-β in macrophages that have ingested apoptotic cells. J. Immunol., 163, 6164–6172 (1999).
• 57) Cummings RJ, Barbet G, Bongers G, Hartmann BM, Gettler K, Muniz L, Furtado GC, Cho J, Lira SA, Blander JM. Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs. Nature, 539, 565–569 (2016).
• 58) Han CZ, Juncadella IJ, Kinchen JM, Buckley MW, Klibanov AL, Dryden K, Onengut-Gumuscu S, Erdbrügger U, Turner SD, Shim YM, Tung KS, Ravichandran KS. Macrophages redirect phagocytosis by non-professional phagocytes and influence inflammation. Nature, 539, 570–574 (2016).
• 59) Chao MP, Majeti R, Weissman IL. Programmed cell removal: a new obstacle in the road to developing cancer. Nat. Rev. Cancer, 12, 58–67 (2011).
• 60) Hashimoto Y, Moki T, Takizawa T, Shiratsuchi A, Nakanishi Y. Evidence for phagocytosis of influenza virus-infected, apoptotic cells by neutrophils and macrophages in mice. J. Immunol., 178, 2448–2457 (2007).
• 61) Nainu F, Tanaka Y, Shiratsuchi A, Nakanishi Y. Protection of insects against viral infection by apoptosis-dependent phagocytosis. J. Immunol., 195, 5696–5706 (2015).
• 62) Capasso D, Pepe MV, Rossello J, Lepanto P, Arias P, Salzman V, Kierbel A. Elimination of Pseudomonas aeruginosa through efferocytosis upon binding to apoptotic cells. PLOS Pathog., 12, e1006068 (2016).
• 63) Joffre OP, Segura E, Savina A, Amigorena S. Cross-presentation by dendritic cells. Nat. Rev. Immunol., 12, 557–569 (2012).
• 64) A-Gonzalez N, Bensinger SJ, Hong C, Beceiro S, Bradley MN, Zelcer N, Deniz J, Ramirez C, Díaz M, Gallardo G, de Galarreta CR, Salazar J, Lopez F, Edwards P, Parks J, Andujar M, Tontonoz P, Castrillo A. Apoptotic cells promote their own clearance and immune tolerance through activation of the nucelar receptor LXR. Immunity, 31, 245–258 (2009).
• 65) Sarang Z, Joós G, Garabuczi È, Rühl R, Gregory CD, Szondy Z. Macrophages engulfing apoptotic cells produce nonclassical retinoids to enhance their phagocytic capacity. J. Immunol., 192, 5730–5738 (2014).
• 66) Weavers H, Evans IR, Martin P, Wood W. Corpse engulfment generates a molecular memory that primes the macrophage inflammatory response. Cell, 165, 1658–1671 (2016).
• 67) Schneider D, Tate AT. Innate immune memory: activation of macrophage killing ability by developmental duties. Curr. Biol., 26, R503–R505 (2016).
• 68) Tassetto M, Kunitomi M, Andino R. Circulating immune cells mediate a systematic RNAi-based adaptive antiviral response in Drosophila. Cell, 169, 314–325.e13 (2017).
• 69) West C, Silverman N. Drosophilosophical: re-thinking adaptive immunity in the fly. Cell, 169, 188–190 (2017).