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Clearance of apoptotic cells: implications in health and disease

Clearance of apoptotic cells: implications in health and disease JCB: Review Clearance of apoptotic cells: implications in health and disease Michael R. Elliott and Kodi S. Ravichandran Center for Cell Clearance and the Department of Microbiology, University of Virginia, Charlottesville, VA 22908 macrophages and dendritic cells) and by nonprofessional Recent advances in defining the molecular signaling path - “neighboring” phagocytes (such as epithelial cells, endothelial ways that regulate the phagocytosis of apoptotic cells cells, and fibroblasts). Current evidence suggests that the steps have improved our understanding of this complex and involved in the phagocytic clearance of apoptotic cells are similar evolutionarily conserved process. Studies in mice and between professional and nonprofessional phagocytes (Fig. 1), humans suggest that the prompt removal of dying cells is although the kinetics may differ, with professional phagocytes crucial for immune tolerance and tissue homeostasis. exhibiting higher rates and capacity for phagocytosis (Parnaik et al., 2000). Failed or defective clearance has emerged as an impor- Based on work from many laboratories over the past decade, tant contributing factor to a range of disease processes. several broadly den fi ed steps have been identie fi d in the recogni - This review addresses how specific molecular alterations tion and removal of apoptotic cells by phagocytes. Each step ap- of engulfment pathways are linked to pathogenic states. pears to be tightly regulated by signaling events to ensure swift A better understanding of the apoptotic cell clearance and efc fi ient clearance (Fig. 1). At the early stage of apoptosis, process in healthy and diseased states could offer new the dying cells release “n fi d-me” signals that are sensed by motile phagocytes, which help attract these phagocytes to the proximity therapeutic strategies. of the dying cell. Several soluble chemoattractant n fi d-me signals released during apoptosis have been recently den fi ed, including triphosphate nucleotides (ATP/UTP), lysophosphatidylchloline Introduction (lysoPC), and the chemokine CX CL1 (Lauber et al., 2003; Apoptosis plays an essential role in the development and mainte- Truman et al., 2008; Elliott et al., 2009; Muñoz et al., 2010). Once nance of all mammalian tissues. The apoptotic program ensures in the proximity of the dying cell, the physical contact between that damaged, aged, or excess cells are deleted in a regulated the apoptotic cell and the phagocyte is mediated via ligands on manner that is not harmful to the host. Beyond the cell intrinsic apoptotic cells (referred to as “eat-me” signals) and engulfment apoptotic program initiated after a variety of insults, an integral receptors on phagocytes that can recognize these eat-me markers. second step in apoptosis is the removal of the cell corpse (Kerr Among the array of identie fi d eat-me molecules (Ravichandran et al., 1972). Indeed, the physical removal and subsequent deg- and Lorenz, 2007), the exposure of phosphatidylserine (PtdSer) on radation of the corpse via phagocytosis represents the n fi al act the outer leae fl t of the apoptotic cell plasma membrane appears to necessary for the successful removal of a cell fated to die. Recent be a key eat-me marker (Fadok et al., 1992; Vandivier et al., 2006). advances in our understanding of apoptotic cell clearance have Phagocyte recognition of PtdSer is mediated directly via one or led to the identic fi ation of molecules and signaling pathways that more PtdSer recognition receptors, including Bai1, Tim-4, and orchestrate this process (Lauber et al., 2004; Ravichandran and Stabilin-2 (Kobayashi et al., 2007; Park et al., 2007, 2008, 2009; Lorenz, 2007; Erwig and Henson, 2008). Miyanishi et al., 2007; Nakayama et al., 2009), or by soluble The efficiency of the phagocytic clearance of apoptotic bridging molecules that bind PtdSer on the apoptotic cell and a cells appears enormous when one considers that despite the receptor on the phagocyte (MFG-E8/v , Gas6/MER; Savill 3/5 loss of >10 cells per day, the incidence of histologically de- et al., 1990; Scott et al., 2001; Hanayama et al., 2004). Engagement tectable apoptotic cells is rare in normal tissues (Mochizuki of the PtdSer receptors initiates signaling events within the phago- et al., 1996; Scott et al., 2001; Schrijvers et al., 2005; Yang cytes that lead to activation of the small GTPase Rac, and subse- et al., 2006; Elliott et al., 2009). The engulfment of apoptotic quent cytoskeletal reorganization of the phagocyte membrane to cells is performed by both professional phagocytes (such as allow corpse internalization (Albert et al., 2000; Gumienny et al., 2001). From studies in Caenorhabditis elegans and Drosophila Correspondence to Kodi S. Ravichandran: Ravi@virginia.edu; or Michael © 2010 Elliott and Ravichandran This article is distributed under the terms of an Attribution– R. Elliott: elliott@virginia.edu Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - Abbreviations used in this paper: CF, cystic fibrosis; CFTR, cystic fibrosis trans - lication date (see http://www.rupress.org/terms). After six months it is available under a membrane conductance regulator; COPD, chronic obstructive pulmonary dis- Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, ease; LDL, low-density lipoprotein; PtdSer, phosphatidylserine. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 189 No. 7 1059–1070 www.jcb.org/cgi/doi/10.1083/jcb.201004096 JCB 1059 THE JOURNAL OF CELL BIOLOGY Figure 1. Stages of apoptotic cell engulfment and associated cell signaling events that regulate each stage. The four stages of apoptotic cell clearance are shown, with some of the specific key signaling players identified. The “find-me” step occurs when apoptotic cells release soluble chemoattractants that promote chemotaxis of phagocytes via corresponding receptors on the phagocyte. The broken line from LPC to G2A indicates uncertainty of direct ligand– receptor interaction. The “eat-me” stage is characterized by the appearance of ligands on the surface of the dying cell that mark it as a target to be engulfed by phagocytes bearing appropriate DAMP or PtdSer recognition receptors. The “engulfment” stage occurs when signaling downstream of the apoptotic cell recognition receptors stimulates Rac-dependent cytoskeletal rearrangement and formation of the phagocytic cup around the target and subsequent internal- ization. Once fully internalized, the cell corpse undergoes “processing” through the phagolysosomal pathway that results in the degradation and reprocessing of the dead cell material. DAMP, damage-associated molecular patterns; LPC, lysophosphatidylcholine; MBL, mannose-binding lectin; PS, phosphatidylserine. melanogaster, and in vitro mammalian cell experiments, two key ELMO, and Dock180 appear to be widely expressed (Hasegawa evolutionarily conserved Rac-dependent apoptotic cell engulf- et al., 1996; Gumienny et al., 2001), whereas the expression ment pathways have been identie fi d (Fig. 1; Reddien and Horvitz, of many of the surface molecules responsible for recognition 2004; Kinchen, 2010). In addition to receptors that can directly of apoptotic cells varies widely among different tissues and cell signal after engaging eat-me signals, there are also contributions types (Ferrero et al., 1990; Graham et al., 1994; Falkowski et al., from other “tethering” receptors (e.g., CD14 and CD31) that help 2003; Miyanishi et al., 2007; Park et al., 2007). Thus, because of the binding/specic fi recognition between the apoptotic cell and the redundancy in the engulfment machinery among cell types, it the phagocyte (Brown et al., 2002; Devitt et al., 2003). Once is critical to know the expression pattern of identie fi d phagocytic inside the phagosome, the ingested apoptotic cargo is processed receptors when considering apoptotic cell clearance in a specic fi via a phagolysosomal pathway that shares both overlapping and tissue or by a particular cell type. Interestingly, many of the disease unique features with the endocytic machinery (Erwig et al., 2006; states linked to failed clearance have been associated with aberra- Kinchen et al., 2008; Yu et al., 2008; Kinchen and Ravichandran, tions in the recognition or eat-me step of clearance (Table I). This 2010; Bohdanowicz and Grinstein, 2010). Because of this over- observation might reflect an investigator-induced bias toward lap, it is difc fi ult to distinguish disease states related specic fi ally phagocyte–corpse interactions, or it may be the result of selective to aberrant signaling in the phagosomal pathways from those in- expression of phagocytic receptors that reduces the redundancy volving endocytosis dysfunction. Indeed, a role for endocytosis in of uptake mechanisms, and thus is more likely to reveal failures human disease has been well established (Mosesson et al., 2008; in clearance. Ballabio and Gieselmann, 2009). Thus, we will focus on diseases Regardless of the specific molecules mediating uptake, related to engulfment signaling upstream of corpse degradation. the ability to efficiently clear apoptotic cells is strongly linked Although the clearance of apoptotic cells occurs throughout to the homeostatic maintenance of healthy tissues in mammals. the body, the specic fi molecular pathways can vary by tissue. For This is thought to be the result of two key features of the clear- example the intracellular engulfment signaling molecules Rac, ance process. The first is the obvious function of phagocytes 1060 JCB • VOLUME 189 • NUMBER 7 • 2010 Table I. A survey of disease states associated with defects in engulfment-related genes Gene Disease relationship Human/mouse References Find me G2A AI M Le et al., 2001 CX CR1 Neuropathy M Cardona et al., 2006 CX CL1 Atherosclerosis M Combadière et al., 2003 Eat-me/tickling MER AI, cancer, neuropathy, atherosclerosis H/M Gal et al., 2000; Scott et al., 2001; Cohen et al., 2002; Keating et al., 2006; Nandrot et al., 2007; Ait-Oufella et al., 2008; Thorp et al., 2008 MFG-E8 AI, atherosclerosis, neuropathy M Hanayama et al., 2004; Ait-Oufella et al., 2007; Nandrot et al., 2007 C1q AI, atherosclerosis, neuropathy M Botto et al., 1998; Fonseca et al., 2004; Bhatia et al., 2007 v AI, atherosclerosis M Weng et al., 2003; Lacy-Hulbert et al., 2007 3/5 TIM-4 AI M Rodriguez-Manzanet et al., 2010 Gas6 Atherosclerosis M Lutgens et al., 2008 Engulfment ELMO1 Diabetic nephropathy H Shimazaki et al., 2005; Leak et al., 2009; Pezzolesi et al., 2009a a a GULP1 Arthritis , schizophrenia H Qingchun et al., 2008; Chen et al., 2009 MEGF10 Schizophrenia H Chen et al., 2009 Post-engulfment LXR/ AI M A-Gonzalez et al., 2009 PPAR AI M Mukundan et al., 2009 DNase II AI M Kawane et al., 2003 Genes are grouped by known roles in engulfment (find-me, eat-me, engulfment, and post-engulfment). AI, autoimmune phenotype; H, human; M, mouse. There is evidence of genetic linkage but no direct causal relationship was established. as “garbage collectors,” mediating the physical removal of The release of intracellular contents from necrotic cells is thought the dying cells. Such clearance sequesters the dying cell and to provoke an inflammatory response, particularly toward intra - prevents the release of potentially toxic or immunogenic intra- cellular antigens and DNA released from the dying cells. This cellular contents from the dying cell into the local environment. may provide the immunogenic impetus for the onset of some This is a key distinction from necrotic cell death, where the un- autoimmune disorders in humans, including systemic lupus regulated release of dead cell material can cause very strong erythematosus and rheumatoid arthritis (Gaipl et al., 2004). inflammatory responses (such as ischemic injury). The second Early experiments in mice showed that the administration of homeostatic function of the clearance process is the production excess syngeneic apoptotic cells or the masking of PtdSer on of anti-inflammatory mediators by phagocytes that suppress in - apoptotic cells via annexin V (to block PtdSer-mediated uptake) ammation fl and facilitate the “immunologically silent” clear - produces hallmarks of autoimmunity, such as autoantibody ance of apoptotic cells. production and IgG deposition in the glomeruli (Mevorach The purpose of this review is to examine the current body et al., 1998; Asano et al., 2004). More recently, several genetic of knowledge linking apoptotic cell clearance to disease patho- mouse models bearing defects in PtdSer-mediated recognition genesis. We will discuss several families of disease states that have further confirmed that the failure to efficiently clear apop - appear to have as a contributing factor some level of impaired totic cells can result in autoimmunity (Botto et al., 1998; Scott cell clearance. We will also attempt to highlight how components et al., 2001; Cohen et al., 2002; Hanayama et al., 2004; Lacy- of the engulfment signaling pathways may function in myriad Hulbert et al., 2007; Rodriguez-Manzanet et al., 2010). Nuclear disease processes. antigens, particularly DNA and DNA–protein complexes (e.g., high mobility group box 1–containing nucleosomes), appear Failed clearance, altered immune tolerance, especially crucial in human systemic lupus erythematosus and and autoimmunity rheumatoid arthritis (Taniguchi et al., 2003). Studies in knock- Autoimmune disorders represent the best-characterized rela- out mice demonstrated that to maintain self-tolerance, DNase- tionship between apoptotic cell clearance and disease pathogen- mediated degradation of apoptotic cell-derived DNA in the esis (Table I; Savill et al., 2002; Gaipl et al., 2004; Erwig and phagosome is necessary (Napirei et al., 2000; Krieser et al., Henson, 2007; Nagata et al., 2010). The self-contained, regulated 2002; Kawane et al., 2003). There is now a solid link between nature of apoptotic cell death preserves membrane integrity and the inefficient engulfment of apoptotic cells and autoimmunity prevents the release of potentially inflammatory and immuno- in humans (Ren et al., 2003; Gaipl et al., 2004). genic intracellular contents. However, if the apoptotic cells An additional means for controlling the immune response are not promptly cleared, the membrane integrity is lost over to apoptotic cells is through the active production of anti- time, and apoptotic cells can progress to secondary necrosis. inflammatory mediators by phagocytes. The PtdSer-dependent Apoptotic cell clearance and disease • Elliott and Ravichandran 1061 recognition of apoptotic cells by a phagocyte elicits the re- At least two factors in CF sputum have been shown to disrupt lease of anti-inflammatory mediators such as IL-10, TGF , and apoptotic cell engulfment, including elevated levels of neutrophil- prostaglandins in vitro (Voll et al., 1997; Fadok et al., 1998; derived elastase, which may cleave eat-me signals (Vandivier et al., McDonald et al., 1999; Ogden et al., 2005). Moreover, this 2002), and pyocyanin, a toxic by-product of Pseudomonas recognition actively suppresses inflammatory cytokine release aeruginosa, a common infectious pathogen found in the lungs in vitro, particularly those elicited via Toll-like receptors (TLRs; of about half of all CF patients (Bianchi et al., 2008). Finally, Voll et al., 1997; Fadok et al., 1998). This immunosuppressive the inflammation associated with lung disease appears to create response extends in vivo, as studies in mice have shown that a cytokine milieu (notably increased TNF) that may suppress the systemic administration of apoptotic cells induces a toler- apoptotic cell engulfment (Borges et al., 2009), perhaps by hin- izing effect on the immune response in rodent allograft models dering the differentiation of monocytes to macrophages, thus (Sun et al., 2004; Wang et al., 2009). Recently, key insights into exacerbating these clearance defects. the signaling events that regulate the release of these immune Intrinsic defects in macrophages in the context of the dis- modulators have been gained. PtdSer-dependent engagement of eased lung also appear to contribute to the reduced clearance apoptotic cells induces in phagocytes the p38 MAPK-dependent seen in these respiratory diseases. Alveolar macrophages from transcriptional regulation of IL-10, as well as translational con- COPD, CF, and asthma patients show a decreased ability to en- trol of TGF in the phagocyte (Chung et al., 2007; Xiao et al., gulf apoptotic cells in vitro (Hodge et al., 2003, 2007; Huynh 2008). The ability of apoptotic cells to suppress TLR-dependent et al., 2005; Vandivier et al., 2009). To date, there are no re- release of IL-6, IL-8, and TNF has also been shown to be regu- ported links to specic fi engulfment pathways that are defective lated at the transcript level (Cvetanovic and Ucker, 2004). Thus, in these lung diseases, although decreased expression of at least in addition to the physical removal of dying cells, the “tickling” two collectins (mannose-binding lectin and surfactant protein-D) of phagocytic receptors generates signals that lead to regulation in COPD patients suggests a possible role for decreased pat- of anti-inflammatory mediators and in turn, the elicitation of tern recognition receptor (PRR)/C1q receptor–mediated up- an immunosuppressive environment during removal of apoptotic take (Hodge et al., 2008). Intriguingly, Vandivier et al. (2009) cells. Even under normal healthy conditions, there is a turnover recently found that cystic b fi rosis transmembrane conductance of >200 billion cells per day in many tissues throughout our regulator (CFTR)-dec fi ient epithelial cells are defective in the body, and therefore interruptions to the finely tuned clearance phagocytosis of apoptotic cells, whereas CFTR-deficient al - system can lead to inflammation, tissue destruction, and the veolar macrophages show no engulfment defect. These n fi dings onset of disease. suggest that a persistent disease state in the lung (i.e., COPD) and/or genetic anomalies may drive engulfment defects, and Respiratory diseases and impaired thus point to a prominent role for engulfment in the establish- cell clearance ment and progression of disease. Moreover, the relative contri- Intriguingly, increased levels of apoptotic cells are seen in the butions of macrophages and the epithelial cells for apoptotic cell sputum and lung tissue of several serious respiratory diseases, clearance, as well as the anti-ina fl mmatory cytokines generated including chronic obstructive pulmonary disease (COPD), cystic (or lack thereof), need to be determined in the context of lung in- b fi rosis (CF), and asthma (Henson and Tuder, 2008). Because a fl mmation. Future genetic studies that target engulfment genes aberrant lung ina fl mmation is a common feature of these dis - in particular phagocyte populations may reveal some important eases, one possibility is that uncleared apoptotic cells progress- information on the onset and progression of lung ina fl mmation. ing to secondary necrosis may contribute to lung ina fl mmation. An interesting feature of defective apoptotic cell clearance But a common underlying question is whether or not these “un- in the diseased lung is the potential role of the small GTPase cleared” apoptotic cells represent increased rates of apoptosis RhoA. During engulfment, activation of the small GTPase Rac in or defects in apoptotic cell clearance. In the past few years, the phagocyte is crucial for actin rearrangement during corpse several studies have established considerable links between re- internalization (Fig. 1). In contrast, RhoA antagonizes Rac in spiratory disease and inefc fi ient apoptotic cell clearance in the this process, and increased levels of RhoA-GTP potently im- lung (Vandivier et al., 2002; Hodge et al., 2003; Huynh et al., pair engulfment (Leverrier and Ridley, 2001; Tosello-Trampont 2005). Although the focus of these studies has primarily been et al., 2003; Nakaya et al., 2006). Independent studies have on the phagocytic activity of lung resident macrophages (alveo- shown that CFTR deficiency in lung epithelial cells results in lar macrophages), it will be interesting to determine the relative higher basal levels of activated RhoA (Kreiselmeier et al., 2003; contribution of healthy lung epithelial cells in the clearance of Vandivier et al., 2009). Studies using in vitro treated lung epi- neighboring apoptotic cells. thelial cells similarly show increased basal levels of RhoA-GTP The environment of the diseased lung contributes to poor in response to cigarette smoke (Richens et al., 2009). Pharma- apoptotic cell clearance. Cigarette smoking, the leading cause cological inhibitors of RhoA activity, particularly statins, en- of COPD, is correlated with increased apoptotic cell debris in hance apoptotic cell engulfment in vitro and in vivo, and thus the lung (Hodge et al., 2005), and cigarette smoke impairs the suggest that elevated RhoA-GTP levels may play a signifi - uptake of apoptotic cells by alveolar macrophages in vitro cant role in the impaired clearance observed in diseased lungs (Kirkham et al., 2004; Hodge et al., 2007). Sputum from CF pa- (Morimoto et al., 2006). Although the molecular events leading tients, when added to normal alveolar macrophages, inhibits their to increased levels of RhoA-GTP levels are poorly understood, ability to engulf apoptotic targets in vitro (Vandivier et al., 2002). cigarette smoke exerts a similar effect (activation of RhoA) and 1062 JCB • VOLUME 189 • NUMBER 7 • 2010 may in part explain the defective engulfment seen in COPD and apoptotic cell engulfment. We and others have found that (Richens et al., 2009). There is currently no definite linkage be - macrophages engulfing apoptotic cells up-regulate the key lipid tween lung disease and specific engulfment receptors, and the transporter ABCA1, and this leads to enhanced cholesterol high rate of cell death in the lung due to inhaled toxins could efflux from the phagocytes (Gerbod-Giannone et al., 2006; provide valuable insights into clearance mechanisms through the Kiss et al., 2006a). This cholesterol efflux requires PtdSer- use of genetically modified mice. dependent recognition and signaling within the phagocytes (Kiss et al., 2006a). These findings reveal that a phagocyte taking up Atherosclerosis and an apoptotic cell has the ability to regulate and normalize the engulfment-related consequences level of cellular material. Another intracellular engulfment sig- Macrophages play a prominent role in the development of naling protein, GULP1, has been shown to promote cholesterol atherosclerotic plaques, and their function in clearing apoptotic efflux, and GULP1 functions downstream of the LDL-receptor cells appears to be a key to the pathogenesis of this widespread related protein 1 (LRP1), which is also linked to engulfment of and life-threatening disease. At the onset of plaque formation, apoptotic cells (Su et al., 2002; Gardai et al., 2005; Kiss et al., monocytes in the blood adhere to intimal smooth muscle cells and 2006b). Nuclear receptors, a family of transcriptional regulators differentiate almost exclusively to macrophages. These macro- that control the response to cellular lipids (Hong and Tontonoz, phages then take up low-density lipoprotein (LDL) via scaven- 2008), have been implicated in this response, as antagonists ger receptors and, once they are cholesterol-laden, are known as blocked this efflux (Gerbod-Giannone et al., 2006; Kiss et al., “foam cells.” These foam cells eventually undergo apoptosis, yet 2006a). As further evidence of the interplay between engulfment early atherosclerotic lesions display few uncleared apoptotic and lipid metabolism, mice deficient in the LXR / or PPAR cells, which suggests efc fi ient clearance (Tabas, 2005). As leuko - nuclear receptors showed decreased expression of engulfment cytes continue to inl fi trate the lesion and release ina fl mmatory genes, with impaired engulfment of apoptotic cells by macro- mediators, cell death increases (Schrijvers et al., 2005). Indeed, phages in vitro and in vivo (A-Gonzalez et al., 2009; Mukundan late plaques feature much higher levels of free, uncleared apop- et al., 2009). These mice also showed aberrant expression of totic cells, and eventually a necrotic core forms and becomes un- inflammatory mediators and eventually develop hallmarks of stable, leading to possible lesions that can cause thrombosis autoimmunity. Because uncleared dead cells are a fundamental (Tabas, 2005). issue in atherogenesis, it would seem that the ability to modu- In recent years, the role of apoptotic cell clearance has late apoptotic cell clearance in this environment could serve as begun to be appreciated in atherogenesis. Through the use of a useful and novel tool to prevent or treat disease. / / atherosclerosis mouse models—ApoE and Ldlr —genetic studies of engulfment molecules have demonstrated the role of Cell clearance defects in cell clearance in atherosclerosis (Table I). Mice deficient in the neurological diseases apoptotic cell-bridging molecules MFG-E8 (Ait-Oufella et al., Over a decade ago, several studies identie fi d excess apoptotic 2007) and C1q (Bhatia et al., 2007) develop accelerated athero- cells associated with chronic neurodegenerative diseases, includ- genesis and display increased plaque-bound apoptotic cells on ing in patients with Parkinson’s, Alzheimer’s, and Huntington’s / / ApoE and Ldlr genetic backgrounds, respectively. Like- disease, and in aging brains (Su et al., 1994; Thomas et al., 1995; wise, mice deficient in transglutamase 2 (TG2), a cross-linking Zhang et al., 1995; Mochizuki et al., 1996). Microglia are one of enzyme that promotes engulfment via v (Lorand and the primary phagocytes for apoptotic cells and debris in the brain 3/5 Graham, 2003; Szondy et al., 2003), also enhances atheroscle- (Witting et al., 2000; Magnus et al., 2002; Stolzing and Grune, / rotic plaque formation in Ldlr -deficient mice (Boisvert et al., 2004; Garden and Möller, 2006). Considered to be of myeloid 2006), but not in ApoE-deficient mice (Williams et al., 2010). lineage, these highly motile cells provide necessary surveillance In addition, the receptor tyrosine kinase MER, which recog- to respond to cell death associated with acute injury and stroke nizes apoptotic cells via the PtdSer-binding Gas6 bridging mol- (Davalos et al., 2005; Garden and Möller, 2006). Upon the initia- ecule, functions in vivo to inhibit plaque formation and can tion of neuronal cell death, microglia migrate to the site of injury promote apoptotic cell clearance in atherosclerosis models and mediate the ina fl mmatory response (Davalos et al., 2005; (Ait-Oufella et al., 2008; Thorp et al., 2008). Paradoxically, Koizumi et al., 2007). Recently, engulfment signaling pathways / Gas6 deficiency on the ApoE background leads to the forma- have been implicated in glial function during chronic neurologi- tion of more stable plaques with smaller necrotic cores, fewer cal diseases. Although the discussion in the following paragraph macrophages, and increased TGF levels (Lutgens et al., 2008), focuses on microglial cells, it is important to keep in mind that which suggests possible additional nonengulfment related anti- other cell types in the brain such as astrocytes can also engulf atherogenic roles for MER. These studies suggest divergent apoptotic cells (Chang et al., 2000; Magnus et al., 2002; Park roles for the receptor–ligand interactions in atherogenesis, et al., 2007) and thus may play a role in clearance and disease which may be due to nonengulfment functions of both proteins in the brain. or the lack of our full understanding of cell death/cell clearance To date, MFG-E8 is the engulfment-related molecule in an atherosclerotic plaque. best linked to clearance of apoptotic cells in the brain. Cultured Lipid handling by macrophages plays an important role in astrocytes and microglia produce MFG-E8, and MFG-E8 can atherosclerosis, and so it is interesting that there is considerable promote the phagocytosis of apoptotic neurons by microglia overlap in the cellular mechanisms that regulate lipid metabolism in vitro (Boddaert et al., 2007; Fuller and Van Eldik, 2008). Apoptotic cell clearance and disease • Elliott and Ravichandran 1063 There is also a correlative relationship between MFG-E8 and MFG-E8 in mouse models of solid tumors also enhances anti- Alzheimer’s disease, as suppressed levels of MFG-E8 are as- tumor activity (Jinushi et al., 2008; Jinushi et al., 2009). These sociated with the disease in humans and mice (Boddaert et al., findings suggest that interfering with PtdSer uptake promotes 2007; Fuller and Van Eldik, 2008). Additional evidence of en- dendritic cell-mediated antitumor activity by favoring ina fl m - gulfment signaling in the brain comes from studies of microglial matory uptake mechanisms. Still, despite what appears to be a chemoattractants. Dying neurons release find-me cues, namely plausible scenario wherein apoptotic cell clearance could have a extracellular nucleotides as well as CX CL1 (fractalkine or neuro- profound impact on carcinogenesis, there is only limited genetic tactin) that promote chemotaxis of microglia via the P2Y and evidence to implicate specic fi engulfment signaling pathways in CX CR1 receptors, respectively (Harrison et al., 1998; Koizumi this process. Indeed, the expression of several key engulfment et al., 2007). Interestingly, both fractalkine and UDP appear players, including MER (Linger et al., 2008) and v (Burvenich to enhance glial cell engulfment: fractalkine by enhancing et al., 2008), is up-regulated in neoplastic cells, but the impor- microglial secretion of MFG-E8, and UDP through an as yet un- tance of this observation is unclear. known mechanism (Koizumi et al., 2007; Fuller and Van Eldik, With the recent discovery of several “find-me” factors 2008). The role of fractalkine signaling has been studied in the released by apoptotic cells that act to promote recruitment of context of amyotrophic lateral sclerosis and Parkinson’s disease phagocytes to apoptotic cells, new insights have been gained in using CX CR1-deficient mice. In these disease models, loss of our understanding of connections between cell clearance and fractalkine signaling resulted in increased numbers of dying tumorigenesis. Several insightful studies from the laboratory of neurons, which suggests a potential role for fractalkine as an C.D. Gregory (Ogden et al., 2005; Truman et al., 2008) have fo- important find-me signal in the maintenance of brain homeosta - cused on how macrophages sense and subsequently engulf apop- sis (Cardona et al., 2006). A key unexplored area of clearance totic Burkitt lymphoma cells and how these signaling events may in the central nervous system is the immune response generated impact disease progression. These neoplastic B cells express high by microglial cells or astrocytes during engulfment (i.e., the levels of fractalkine on their surface that is cleaved during apop- release of anti-inflammatory mediators) and how that impacts tosis and subsequently functions as a potent chemoattractant for homeostasis and disease. Finally, in the developed brain, cell macrophages (Truman et al., 2008). Recruitment of macrophages turnover is thought to be quite low with the exception of restricted to splenic follicles is impaired in fractalkine receptor-dec fi ient regions where adult neurogenesis takes place (Kempermann mice, an observation consistent with a role for fractalkine as a key et al., 2004; Zhao et al., 2008; Taupin, 2009). Defining how mediator of macrophage recruitment to germinal centers (Truman apoptotic cell clearance impacts other developmental processes et al., 2008). Within the germinal center environment, high levels in the brain related to cell turnover, including adult neuro- of IL-10 (likely produced by the enguln fi g macrophages) appear genesis, will require additional studies with appropriate neuro- to suppress tumor immunity, whereas the release of B cell sur- logical models. vival factors by enguln fi g macrophages is thought to promote tumor growth (Ogden et al., 2005). Tumorigenesis and cell clearance Additionally, we have recently found that apoptotic cells Because apoptotic cell clearance typically generates an immuno- release nucleotide triphosphates (ATP/UTP) early during the suppressive environment, its role in the development and pro- apoptotic process (within 2–4 h), and that these nucleotides act as gression of cancer is enigmatic. As has been reviewed elsewhere chemoattractants for monocytes and macrophages in vitro and (Coussens and Werb, 2002; Condeelis and Pollard, 2006; Solinas in vivo (Elliott et al., 2009). The amount of ATP released by apop- et al., 2009), chronic ina fl mmation is a key factor in tumorigene - totic cells under these conditions, which promotes silent clear- sis. Thus, the efc fi ient clearance of dying cells, and the associated ance, represents a very small percentage of the total intracellular production of anti-ina fl mmatory mediators, would be predicted pool of nucleotides (<2%; Elliott et al., 2009). In contrast, a few to be beneficial in limiting tumorigenesis. However, within a other recent studies have demonstrated that ATP is released tumor environment where rapid cell proliferation and apoptosis are by tumor cells undergoing apoptosis in response to chemo- ongoing, phagocyte-mediated clearance can exert an unwanted therapeutics, with considerably higher amounts of ATP release immunosuppressive effect. This is particularly the case upon the (10–100 fold greater) seen at later times after induction (12–24 h; administration of antitumor chemotherapeutics, most of which Ghiringhelli et al., 2009; Martins et al., 2009; Aymeric et al., 2010). act by inducing apoptosis of tumor cells. In this setting, efc fi ient This apoptotic cell-derived ATP stimulates activation of the engulfment and the characteristic release of anti-ina fl mmatory NLRP3 ina fl mmasome in dendritic cells via the P2X7 receptor mediators, particularly TGF, upon encounter with eat-me sig- (Ghiringhelli et al., 2009). This heightened activation state appears nals during this process appear to suppress the antitumor immune necessary to drive IL-1 secretion and subsequent priming of response. Indeed, in several rodent tumor models, treatment with CD8+ T cells for IFN production and antitumor responses. These monoclonal antibodies to block PtdSer-mediated uptake retards studies highlight an emerging role for factors released by apoptotic the growth of tumors (Huang et al., 2005; Ran et al., 2005; He cells in shaping the immune response in normal and tumor environ- et al., 2009). Similarly, vaccination of mice with UV-irradiated ments. This has led to the concept of “immunogenic” versus “non- lymphoma cells coated with annexin V to mask PtdSer provides immunogenic” cell death, and the idea that immunogenic cell signic fi ant tumor protection against subsequent challenge with death may be benec fi ial in antitumor therapies (Green et al., 2009; living tumor cells, presumably by initiating an antitumor ina fl m - Locher et al., 2009). Thus, whether apoptotic cell clearance has a matory response (Bondanza et al., 2004). Antibody depletion of benec fi ial or detrimental effect in the context of tumor progression 1064 JCB • VOLUME 189 • NUMBER 7 • 2010 Figure 2. Pathogens usurp the ELMO–Dock–Rac engulfment module. Examples of mechanisms whereby microbial pathogens use the ELMO–Dock–Rac module to alter the host cellular response. The area above the broken line shows mechanism of enhanced S. flexneri invasion via IPGB1 interaction with ELMO, leading to enhanced Rac activation and membrane ruffles that serve as entry points for the bacteria. The area below the broken line shows that HIV-1 uses Nef interaction with the ELMO–Dock2 complex to disrupt CXCR4-dependent chemotaxis in CD4+ T cells. or anticancer therapies will depend on gaining a better under- The small GTPase RhoG acts upstream of ELMO1, and active standing of the role of factors released by apoptotic tumor cells. RhoG-GTP interacts with ELMO1, and thereby recruits the ELMO–Dock180 complex to the membrane to promote Rac acti- Engulfment molecules vation, membrane rufifl ng, and engulfment (Katoh and Negishi, in microbial pathogenesis 2003; deBakker et al., 2004). IPGB1 mimics the activity of RhoG- An emerging facet of engulfment signaling is how these path- GTP, and the Rac-generated ruffles serve as a site of entry for ways can be usurped by microbial pathogens. It has been known S. e fl xneri (Handa et al., 2007). Similarly, Yersinia enterocolitica for some time that bacteria can hijack or mimic host signaling virulence factors Invasin and YopE also modulate Rac1 activity pathways to aid in pathogenic steps, including cell entry and at the level of RhoG, and appear to do so in an ELMO–Dock180- immune evasion (Stebbins and Galán, 2001). This is achieved by dependent manner in cultured cells (Roppenser et al., 2009). delivery of bacterial effector proteins into the host cell that mimic However, neither of these Y. enterocolitica virulence factors have a range of cellular activities. As key regulators of the cytoskeleton been reported to directly interact with ELMO–Dock180, and the and numerous other cellular processes, small G proteins, particu- role of this module was inferred by expression of a dominant- larly the Rho family (e.g., RhoA, Rac, and Cdc42), are frequent negative mutant of ELMO1 that did not further alter Rac activa- targets for these clever effector mechanisms (Mattoo et al., 2007). tion in the presence of YopE (Roppenser et al., 2009). The signaling machinery that controls phagocyte morphology Usurping the engulfment machinery is not exclusive to during apoptotic cell engulfment relies on these GTPases as well, bacteria, and in fact can be used by viruses to promote patho- and thus it is not surprising that several bacteria target these path- genesis. Janardhan et al. (2004) found that the Nef gene product ways. In particular, the RhoG–ELMO–Dock–Rac pathway has of HIV-1 is able to complex with the ELMO2–Dock2 module in been found to be such a target (Fig. 2). The invasive pathogen T cells to promote Rac activation. Further, we have found that Shigella e fl xneri utilizes a type III secretion system to inject ef- Nef interacts with Dock2 in Jurkat T cells and promotes the fectors to promote entry into epithelial cells, including IPGB1 activation of a key cytoskeletal Rac effector, p21-activated (Handa et al., 2007). IPGB1 promotes membrane rufi fl ng via kinase (PAK; unpublished data). The outcome of this inter- Rac activation in a mechanism that requires binding to ELMO1. action appears to be dysregulated Rac activation, which is Apoptotic cell clearance and disease • Elliott and Ravichandran 1065 associated with enhanced activation through the T cell receptor topic (using in vivo models) portend potentially therapeutic ben- and improper CXCR4-dependent chemotaxis. However, the hi- et fi s by targeting the components of the engulfment machinery. jacking of the engulfment signaling machinery has only been We thank members of the Ravichandran laboratory for helpful comments shown using cultured cells, and it will be important to determine during preparation of this manuscript. This work was supported by a post-doctoral fellowship from the Ameri- if in vivo pathogenesis is dependent on these activities as well. can Cancer Society (to M.R. Elliott), and grants from the National Institute of General Medical Sciences/National Institutes of Health (to K.S. Ravichandran). Engulfment genes and other types of K.S. Ravichandran is a William Benter Senior Fellow of the American Asthma Foundation. disease associations Several recent studies have discovered associations with human Submitted: 20 April 2010 disease and genetic mutations of components of the engulfment Accepted: 7 June 2010 signaling machinery. 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Clearance of apoptotic cells: implications in health and disease

The Journal of Cell Biology , Volume 189 (7) – Jun 28, 2010

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© 2010 Elliott and Ravichandran
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0021-9525
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10.1083/jcb.201004096
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Abstract

JCB: Review Clearance of apoptotic cells: implications in health and disease Michael R. Elliott and Kodi S. Ravichandran Center for Cell Clearance and the Department of Microbiology, University of Virginia, Charlottesville, VA 22908 macrophages and dendritic cells) and by nonprofessional Recent advances in defining the molecular signaling path - “neighboring” phagocytes (such as epithelial cells, endothelial ways that regulate the phagocytosis of apoptotic cells cells, and fibroblasts). Current evidence suggests that the steps have improved our understanding of this complex and involved in the phagocytic clearance of apoptotic cells are similar evolutionarily conserved process. Studies in mice and between professional and nonprofessional phagocytes (Fig. 1), humans suggest that the prompt removal of dying cells is although the kinetics may differ, with professional phagocytes crucial for immune tolerance and tissue homeostasis. exhibiting higher rates and capacity for phagocytosis (Parnaik et al., 2000). Failed or defective clearance has emerged as an impor- Based on work from many laboratories over the past decade, tant contributing factor to a range of disease processes. several broadly den fi ed steps have been identie fi d in the recogni - This review addresses how specific molecular alterations tion and removal of apoptotic cells by phagocytes. Each step ap- of engulfment pathways are linked to pathogenic states. pears to be tightly regulated by signaling events to ensure swift A better understanding of the apoptotic cell clearance and efc fi ient clearance (Fig. 1). At the early stage of apoptosis, process in healthy and diseased states could offer new the dying cells release “n fi d-me” signals that are sensed by motile phagocytes, which help attract these phagocytes to the proximity therapeutic strategies. of the dying cell. Several soluble chemoattractant n fi d-me signals released during apoptosis have been recently den fi ed, including triphosphate nucleotides (ATP/UTP), lysophosphatidylchloline Introduction (lysoPC), and the chemokine CX CL1 (Lauber et al., 2003; Apoptosis plays an essential role in the development and mainte- Truman et al., 2008; Elliott et al., 2009; Muñoz et al., 2010). Once nance of all mammalian tissues. The apoptotic program ensures in the proximity of the dying cell, the physical contact between that damaged, aged, or excess cells are deleted in a regulated the apoptotic cell and the phagocyte is mediated via ligands on manner that is not harmful to the host. Beyond the cell intrinsic apoptotic cells (referred to as “eat-me” signals) and engulfment apoptotic program initiated after a variety of insults, an integral receptors on phagocytes that can recognize these eat-me markers. second step in apoptosis is the removal of the cell corpse (Kerr Among the array of identie fi d eat-me molecules (Ravichandran et al., 1972). Indeed, the physical removal and subsequent deg- and Lorenz, 2007), the exposure of phosphatidylserine (PtdSer) on radation of the corpse via phagocytosis represents the n fi al act the outer leae fl t of the apoptotic cell plasma membrane appears to necessary for the successful removal of a cell fated to die. Recent be a key eat-me marker (Fadok et al., 1992; Vandivier et al., 2006). advances in our understanding of apoptotic cell clearance have Phagocyte recognition of PtdSer is mediated directly via one or led to the identic fi ation of molecules and signaling pathways that more PtdSer recognition receptors, including Bai1, Tim-4, and orchestrate this process (Lauber et al., 2004; Ravichandran and Stabilin-2 (Kobayashi et al., 2007; Park et al., 2007, 2008, 2009; Lorenz, 2007; Erwig and Henson, 2008). Miyanishi et al., 2007; Nakayama et al., 2009), or by soluble The efficiency of the phagocytic clearance of apoptotic bridging molecules that bind PtdSer on the apoptotic cell and a cells appears enormous when one considers that despite the receptor on the phagocyte (MFG-E8/v , Gas6/MER; Savill 3/5 loss of >10 cells per day, the incidence of histologically de- et al., 1990; Scott et al., 2001; Hanayama et al., 2004). Engagement tectable apoptotic cells is rare in normal tissues (Mochizuki of the PtdSer receptors initiates signaling events within the phago- et al., 1996; Scott et al., 2001; Schrijvers et al., 2005; Yang cytes that lead to activation of the small GTPase Rac, and subse- et al., 2006; Elliott et al., 2009). The engulfment of apoptotic quent cytoskeletal reorganization of the phagocyte membrane to cells is performed by both professional phagocytes (such as allow corpse internalization (Albert et al., 2000; Gumienny et al., 2001). From studies in Caenorhabditis elegans and Drosophila Correspondence to Kodi S. Ravichandran: Ravi@virginia.edu; or Michael © 2010 Elliott and Ravichandran This article is distributed under the terms of an Attribution– R. Elliott: elliott@virginia.edu Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub - Abbreviations used in this paper: CF, cystic fibrosis; CFTR, cystic fibrosis trans - lication date (see http://www.rupress.org/terms). After six months it is available under a membrane conductance regulator; COPD, chronic obstructive pulmonary dis- Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, ease; LDL, low-density lipoprotein; PtdSer, phosphatidylserine. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 189 No. 7 1059–1070 www.jcb.org/cgi/doi/10.1083/jcb.201004096 JCB 1059 THE JOURNAL OF CELL BIOLOGY Figure 1. Stages of apoptotic cell engulfment and associated cell signaling events that regulate each stage. The four stages of apoptotic cell clearance are shown, with some of the specific key signaling players identified. The “find-me” step occurs when apoptotic cells release soluble chemoattractants that promote chemotaxis of phagocytes via corresponding receptors on the phagocyte. The broken line from LPC to G2A indicates uncertainty of direct ligand– receptor interaction. The “eat-me” stage is characterized by the appearance of ligands on the surface of the dying cell that mark it as a target to be engulfed by phagocytes bearing appropriate DAMP or PtdSer recognition receptors. The “engulfment” stage occurs when signaling downstream of the apoptotic cell recognition receptors stimulates Rac-dependent cytoskeletal rearrangement and formation of the phagocytic cup around the target and subsequent internal- ization. Once fully internalized, the cell corpse undergoes “processing” through the phagolysosomal pathway that results in the degradation and reprocessing of the dead cell material. DAMP, damage-associated molecular patterns; LPC, lysophosphatidylcholine; MBL, mannose-binding lectin; PS, phosphatidylserine. melanogaster, and in vitro mammalian cell experiments, two key ELMO, and Dock180 appear to be widely expressed (Hasegawa evolutionarily conserved Rac-dependent apoptotic cell engulf- et al., 1996; Gumienny et al., 2001), whereas the expression ment pathways have been identie fi d (Fig. 1; Reddien and Horvitz, of many of the surface molecules responsible for recognition 2004; Kinchen, 2010). In addition to receptors that can directly of apoptotic cells varies widely among different tissues and cell signal after engaging eat-me signals, there are also contributions types (Ferrero et al., 1990; Graham et al., 1994; Falkowski et al., from other “tethering” receptors (e.g., CD14 and CD31) that help 2003; Miyanishi et al., 2007; Park et al., 2007). Thus, because of the binding/specic fi recognition between the apoptotic cell and the redundancy in the engulfment machinery among cell types, it the phagocyte (Brown et al., 2002; Devitt et al., 2003). Once is critical to know the expression pattern of identie fi d phagocytic inside the phagosome, the ingested apoptotic cargo is processed receptors when considering apoptotic cell clearance in a specic fi via a phagolysosomal pathway that shares both overlapping and tissue or by a particular cell type. Interestingly, many of the disease unique features with the endocytic machinery (Erwig et al., 2006; states linked to failed clearance have been associated with aberra- Kinchen et al., 2008; Yu et al., 2008; Kinchen and Ravichandran, tions in the recognition or eat-me step of clearance (Table I). This 2010; Bohdanowicz and Grinstein, 2010). Because of this over- observation might reflect an investigator-induced bias toward lap, it is difc fi ult to distinguish disease states related specic fi ally phagocyte–corpse interactions, or it may be the result of selective to aberrant signaling in the phagosomal pathways from those in- expression of phagocytic receptors that reduces the redundancy volving endocytosis dysfunction. Indeed, a role for endocytosis in of uptake mechanisms, and thus is more likely to reveal failures human disease has been well established (Mosesson et al., 2008; in clearance. Ballabio and Gieselmann, 2009). Thus, we will focus on diseases Regardless of the specific molecules mediating uptake, related to engulfment signaling upstream of corpse degradation. the ability to efficiently clear apoptotic cells is strongly linked Although the clearance of apoptotic cells occurs throughout to the homeostatic maintenance of healthy tissues in mammals. the body, the specic fi molecular pathways can vary by tissue. For This is thought to be the result of two key features of the clear- example the intracellular engulfment signaling molecules Rac, ance process. The first is the obvious function of phagocytes 1060 JCB • VOLUME 189 • NUMBER 7 • 2010 Table I. A survey of disease states associated with defects in engulfment-related genes Gene Disease relationship Human/mouse References Find me G2A AI M Le et al., 2001 CX CR1 Neuropathy M Cardona et al., 2006 CX CL1 Atherosclerosis M Combadière et al., 2003 Eat-me/tickling MER AI, cancer, neuropathy, atherosclerosis H/M Gal et al., 2000; Scott et al., 2001; Cohen et al., 2002; Keating et al., 2006; Nandrot et al., 2007; Ait-Oufella et al., 2008; Thorp et al., 2008 MFG-E8 AI, atherosclerosis, neuropathy M Hanayama et al., 2004; Ait-Oufella et al., 2007; Nandrot et al., 2007 C1q AI, atherosclerosis, neuropathy M Botto et al., 1998; Fonseca et al., 2004; Bhatia et al., 2007 v AI, atherosclerosis M Weng et al., 2003; Lacy-Hulbert et al., 2007 3/5 TIM-4 AI M Rodriguez-Manzanet et al., 2010 Gas6 Atherosclerosis M Lutgens et al., 2008 Engulfment ELMO1 Diabetic nephropathy H Shimazaki et al., 2005; Leak et al., 2009; Pezzolesi et al., 2009a a a GULP1 Arthritis , schizophrenia H Qingchun et al., 2008; Chen et al., 2009 MEGF10 Schizophrenia H Chen et al., 2009 Post-engulfment LXR/ AI M A-Gonzalez et al., 2009 PPAR AI M Mukundan et al., 2009 DNase II AI M Kawane et al., 2003 Genes are grouped by known roles in engulfment (find-me, eat-me, engulfment, and post-engulfment). AI, autoimmune phenotype; H, human; M, mouse. There is evidence of genetic linkage but no direct causal relationship was established. as “garbage collectors,” mediating the physical removal of The release of intracellular contents from necrotic cells is thought the dying cells. Such clearance sequesters the dying cell and to provoke an inflammatory response, particularly toward intra - prevents the release of potentially toxic or immunogenic intra- cellular antigens and DNA released from the dying cells. This cellular contents from the dying cell into the local environment. may provide the immunogenic impetus for the onset of some This is a key distinction from necrotic cell death, where the un- autoimmune disorders in humans, including systemic lupus regulated release of dead cell material can cause very strong erythematosus and rheumatoid arthritis (Gaipl et al., 2004). inflammatory responses (such as ischemic injury). The second Early experiments in mice showed that the administration of homeostatic function of the clearance process is the production excess syngeneic apoptotic cells or the masking of PtdSer on of anti-inflammatory mediators by phagocytes that suppress in - apoptotic cells via annexin V (to block PtdSer-mediated uptake) ammation fl and facilitate the “immunologically silent” clear - produces hallmarks of autoimmunity, such as autoantibody ance of apoptotic cells. production and IgG deposition in the glomeruli (Mevorach The purpose of this review is to examine the current body et al., 1998; Asano et al., 2004). More recently, several genetic of knowledge linking apoptotic cell clearance to disease patho- mouse models bearing defects in PtdSer-mediated recognition genesis. We will discuss several families of disease states that have further confirmed that the failure to efficiently clear apop - appear to have as a contributing factor some level of impaired totic cells can result in autoimmunity (Botto et al., 1998; Scott cell clearance. We will also attempt to highlight how components et al., 2001; Cohen et al., 2002; Hanayama et al., 2004; Lacy- of the engulfment signaling pathways may function in myriad Hulbert et al., 2007; Rodriguez-Manzanet et al., 2010). Nuclear disease processes. antigens, particularly DNA and DNA–protein complexes (e.g., high mobility group box 1–containing nucleosomes), appear Failed clearance, altered immune tolerance, especially crucial in human systemic lupus erythematosus and and autoimmunity rheumatoid arthritis (Taniguchi et al., 2003). Studies in knock- Autoimmune disorders represent the best-characterized rela- out mice demonstrated that to maintain self-tolerance, DNase- tionship between apoptotic cell clearance and disease pathogen- mediated degradation of apoptotic cell-derived DNA in the esis (Table I; Savill et al., 2002; Gaipl et al., 2004; Erwig and phagosome is necessary (Napirei et al., 2000; Krieser et al., Henson, 2007; Nagata et al., 2010). The self-contained, regulated 2002; Kawane et al., 2003). There is now a solid link between nature of apoptotic cell death preserves membrane integrity and the inefficient engulfment of apoptotic cells and autoimmunity prevents the release of potentially inflammatory and immuno- in humans (Ren et al., 2003; Gaipl et al., 2004). genic intracellular contents. However, if the apoptotic cells An additional means for controlling the immune response are not promptly cleared, the membrane integrity is lost over to apoptotic cells is through the active production of anti- time, and apoptotic cells can progress to secondary necrosis. inflammatory mediators by phagocytes. The PtdSer-dependent Apoptotic cell clearance and disease • Elliott and Ravichandran 1061 recognition of apoptotic cells by a phagocyte elicits the re- At least two factors in CF sputum have been shown to disrupt lease of anti-inflammatory mediators such as IL-10, TGF , and apoptotic cell engulfment, including elevated levels of neutrophil- prostaglandins in vitro (Voll et al., 1997; Fadok et al., 1998; derived elastase, which may cleave eat-me signals (Vandivier et al., McDonald et al., 1999; Ogden et al., 2005). Moreover, this 2002), and pyocyanin, a toxic by-product of Pseudomonas recognition actively suppresses inflammatory cytokine release aeruginosa, a common infectious pathogen found in the lungs in vitro, particularly those elicited via Toll-like receptors (TLRs; of about half of all CF patients (Bianchi et al., 2008). Finally, Voll et al., 1997; Fadok et al., 1998). This immunosuppressive the inflammation associated with lung disease appears to create response extends in vivo, as studies in mice have shown that a cytokine milieu (notably increased TNF) that may suppress the systemic administration of apoptotic cells induces a toler- apoptotic cell engulfment (Borges et al., 2009), perhaps by hin- izing effect on the immune response in rodent allograft models dering the differentiation of monocytes to macrophages, thus (Sun et al., 2004; Wang et al., 2009). Recently, key insights into exacerbating these clearance defects. the signaling events that regulate the release of these immune Intrinsic defects in macrophages in the context of the dis- modulators have been gained. PtdSer-dependent engagement of eased lung also appear to contribute to the reduced clearance apoptotic cells induces in phagocytes the p38 MAPK-dependent seen in these respiratory diseases. Alveolar macrophages from transcriptional regulation of IL-10, as well as translational con- COPD, CF, and asthma patients show a decreased ability to en- trol of TGF in the phagocyte (Chung et al., 2007; Xiao et al., gulf apoptotic cells in vitro (Hodge et al., 2003, 2007; Huynh 2008). The ability of apoptotic cells to suppress TLR-dependent et al., 2005; Vandivier et al., 2009). To date, there are no re- release of IL-6, IL-8, and TNF has also been shown to be regu- ported links to specic fi engulfment pathways that are defective lated at the transcript level (Cvetanovic and Ucker, 2004). Thus, in these lung diseases, although decreased expression of at least in addition to the physical removal of dying cells, the “tickling” two collectins (mannose-binding lectin and surfactant protein-D) of phagocytic receptors generates signals that lead to regulation in COPD patients suggests a possible role for decreased pat- of anti-inflammatory mediators and in turn, the elicitation of tern recognition receptor (PRR)/C1q receptor–mediated up- an immunosuppressive environment during removal of apoptotic take (Hodge et al., 2008). Intriguingly, Vandivier et al. (2009) cells. Even under normal healthy conditions, there is a turnover recently found that cystic b fi rosis transmembrane conductance of >200 billion cells per day in many tissues throughout our regulator (CFTR)-dec fi ient epithelial cells are defective in the body, and therefore interruptions to the finely tuned clearance phagocytosis of apoptotic cells, whereas CFTR-deficient al - system can lead to inflammation, tissue destruction, and the veolar macrophages show no engulfment defect. These n fi dings onset of disease. suggest that a persistent disease state in the lung (i.e., COPD) and/or genetic anomalies may drive engulfment defects, and Respiratory diseases and impaired thus point to a prominent role for engulfment in the establish- cell clearance ment and progression of disease. Moreover, the relative contri- Intriguingly, increased levels of apoptotic cells are seen in the butions of macrophages and the epithelial cells for apoptotic cell sputum and lung tissue of several serious respiratory diseases, clearance, as well as the anti-ina fl mmatory cytokines generated including chronic obstructive pulmonary disease (COPD), cystic (or lack thereof), need to be determined in the context of lung in- b fi rosis (CF), and asthma (Henson and Tuder, 2008). Because a fl mmation. Future genetic studies that target engulfment genes aberrant lung ina fl mmation is a common feature of these dis - in particular phagocyte populations may reveal some important eases, one possibility is that uncleared apoptotic cells progress- information on the onset and progression of lung ina fl mmation. ing to secondary necrosis may contribute to lung ina fl mmation. An interesting feature of defective apoptotic cell clearance But a common underlying question is whether or not these “un- in the diseased lung is the potential role of the small GTPase cleared” apoptotic cells represent increased rates of apoptosis RhoA. During engulfment, activation of the small GTPase Rac in or defects in apoptotic cell clearance. In the past few years, the phagocyte is crucial for actin rearrangement during corpse several studies have established considerable links between re- internalization (Fig. 1). In contrast, RhoA antagonizes Rac in spiratory disease and inefc fi ient apoptotic cell clearance in the this process, and increased levels of RhoA-GTP potently im- lung (Vandivier et al., 2002; Hodge et al., 2003; Huynh et al., pair engulfment (Leverrier and Ridley, 2001; Tosello-Trampont 2005). Although the focus of these studies has primarily been et al., 2003; Nakaya et al., 2006). Independent studies have on the phagocytic activity of lung resident macrophages (alveo- shown that CFTR deficiency in lung epithelial cells results in lar macrophages), it will be interesting to determine the relative higher basal levels of activated RhoA (Kreiselmeier et al., 2003; contribution of healthy lung epithelial cells in the clearance of Vandivier et al., 2009). Studies using in vitro treated lung epi- neighboring apoptotic cells. thelial cells similarly show increased basal levels of RhoA-GTP The environment of the diseased lung contributes to poor in response to cigarette smoke (Richens et al., 2009). Pharma- apoptotic cell clearance. Cigarette smoking, the leading cause cological inhibitors of RhoA activity, particularly statins, en- of COPD, is correlated with increased apoptotic cell debris in hance apoptotic cell engulfment in vitro and in vivo, and thus the lung (Hodge et al., 2005), and cigarette smoke impairs the suggest that elevated RhoA-GTP levels may play a signifi - uptake of apoptotic cells by alveolar macrophages in vitro cant role in the impaired clearance observed in diseased lungs (Kirkham et al., 2004; Hodge et al., 2007). Sputum from CF pa- (Morimoto et al., 2006). Although the molecular events leading tients, when added to normal alveolar macrophages, inhibits their to increased levels of RhoA-GTP levels are poorly understood, ability to engulf apoptotic targets in vitro (Vandivier et al., 2002). cigarette smoke exerts a similar effect (activation of RhoA) and 1062 JCB • VOLUME 189 • NUMBER 7 • 2010 may in part explain the defective engulfment seen in COPD and apoptotic cell engulfment. We and others have found that (Richens et al., 2009). There is currently no definite linkage be - macrophages engulfing apoptotic cells up-regulate the key lipid tween lung disease and specific engulfment receptors, and the transporter ABCA1, and this leads to enhanced cholesterol high rate of cell death in the lung due to inhaled toxins could efflux from the phagocytes (Gerbod-Giannone et al., 2006; provide valuable insights into clearance mechanisms through the Kiss et al., 2006a). This cholesterol efflux requires PtdSer- use of genetically modified mice. dependent recognition and signaling within the phagocytes (Kiss et al., 2006a). These findings reveal that a phagocyte taking up Atherosclerosis and an apoptotic cell has the ability to regulate and normalize the engulfment-related consequences level of cellular material. Another intracellular engulfment sig- Macrophages play a prominent role in the development of naling protein, GULP1, has been shown to promote cholesterol atherosclerotic plaques, and their function in clearing apoptotic efflux, and GULP1 functions downstream of the LDL-receptor cells appears to be a key to the pathogenesis of this widespread related protein 1 (LRP1), which is also linked to engulfment of and life-threatening disease. At the onset of plaque formation, apoptotic cells (Su et al., 2002; Gardai et al., 2005; Kiss et al., monocytes in the blood adhere to intimal smooth muscle cells and 2006b). Nuclear receptors, a family of transcriptional regulators differentiate almost exclusively to macrophages. These macro- that control the response to cellular lipids (Hong and Tontonoz, phages then take up low-density lipoprotein (LDL) via scaven- 2008), have been implicated in this response, as antagonists ger receptors and, once they are cholesterol-laden, are known as blocked this efflux (Gerbod-Giannone et al., 2006; Kiss et al., “foam cells.” These foam cells eventually undergo apoptosis, yet 2006a). As further evidence of the interplay between engulfment early atherosclerotic lesions display few uncleared apoptotic and lipid metabolism, mice deficient in the LXR / or PPAR cells, which suggests efc fi ient clearance (Tabas, 2005). As leuko - nuclear receptors showed decreased expression of engulfment cytes continue to inl fi trate the lesion and release ina fl mmatory genes, with impaired engulfment of apoptotic cells by macro- mediators, cell death increases (Schrijvers et al., 2005). Indeed, phages in vitro and in vivo (A-Gonzalez et al., 2009; Mukundan late plaques feature much higher levels of free, uncleared apop- et al., 2009). These mice also showed aberrant expression of totic cells, and eventually a necrotic core forms and becomes un- inflammatory mediators and eventually develop hallmarks of stable, leading to possible lesions that can cause thrombosis autoimmunity. Because uncleared dead cells are a fundamental (Tabas, 2005). issue in atherogenesis, it would seem that the ability to modu- In recent years, the role of apoptotic cell clearance has late apoptotic cell clearance in this environment could serve as begun to be appreciated in atherogenesis. Through the use of a useful and novel tool to prevent or treat disease. / / atherosclerosis mouse models—ApoE and Ldlr —genetic studies of engulfment molecules have demonstrated the role of Cell clearance defects in cell clearance in atherosclerosis (Table I). Mice deficient in the neurological diseases apoptotic cell-bridging molecules MFG-E8 (Ait-Oufella et al., Over a decade ago, several studies identie fi d excess apoptotic 2007) and C1q (Bhatia et al., 2007) develop accelerated athero- cells associated with chronic neurodegenerative diseases, includ- genesis and display increased plaque-bound apoptotic cells on ing in patients with Parkinson’s, Alzheimer’s, and Huntington’s / / ApoE and Ldlr genetic backgrounds, respectively. Like- disease, and in aging brains (Su et al., 1994; Thomas et al., 1995; wise, mice deficient in transglutamase 2 (TG2), a cross-linking Zhang et al., 1995; Mochizuki et al., 1996). Microglia are one of enzyme that promotes engulfment via v (Lorand and the primary phagocytes for apoptotic cells and debris in the brain 3/5 Graham, 2003; Szondy et al., 2003), also enhances atheroscle- (Witting et al., 2000; Magnus et al., 2002; Stolzing and Grune, / rotic plaque formation in Ldlr -deficient mice (Boisvert et al., 2004; Garden and Möller, 2006). Considered to be of myeloid 2006), but not in ApoE-deficient mice (Williams et al., 2010). lineage, these highly motile cells provide necessary surveillance In addition, the receptor tyrosine kinase MER, which recog- to respond to cell death associated with acute injury and stroke nizes apoptotic cells via the PtdSer-binding Gas6 bridging mol- (Davalos et al., 2005; Garden and Möller, 2006). Upon the initia- ecule, functions in vivo to inhibit plaque formation and can tion of neuronal cell death, microglia migrate to the site of injury promote apoptotic cell clearance in atherosclerosis models and mediate the ina fl mmatory response (Davalos et al., 2005; (Ait-Oufella et al., 2008; Thorp et al., 2008). Paradoxically, Koizumi et al., 2007). Recently, engulfment signaling pathways / Gas6 deficiency on the ApoE background leads to the forma- have been implicated in glial function during chronic neurologi- tion of more stable plaques with smaller necrotic cores, fewer cal diseases. Although the discussion in the following paragraph macrophages, and increased TGF levels (Lutgens et al., 2008), focuses on microglial cells, it is important to keep in mind that which suggests possible additional nonengulfment related anti- other cell types in the brain such as astrocytes can also engulf atherogenic roles for MER. These studies suggest divergent apoptotic cells (Chang et al., 2000; Magnus et al., 2002; Park roles for the receptor–ligand interactions in atherogenesis, et al., 2007) and thus may play a role in clearance and disease which may be due to nonengulfment functions of both proteins in the brain. or the lack of our full understanding of cell death/cell clearance To date, MFG-E8 is the engulfment-related molecule in an atherosclerotic plaque. best linked to clearance of apoptotic cells in the brain. Cultured Lipid handling by macrophages plays an important role in astrocytes and microglia produce MFG-E8, and MFG-E8 can atherosclerosis, and so it is interesting that there is considerable promote the phagocytosis of apoptotic neurons by microglia overlap in the cellular mechanisms that regulate lipid metabolism in vitro (Boddaert et al., 2007; Fuller and Van Eldik, 2008). Apoptotic cell clearance and disease • Elliott and Ravichandran 1063 There is also a correlative relationship between MFG-E8 and MFG-E8 in mouse models of solid tumors also enhances anti- Alzheimer’s disease, as suppressed levels of MFG-E8 are as- tumor activity (Jinushi et al., 2008; Jinushi et al., 2009). These sociated with the disease in humans and mice (Boddaert et al., findings suggest that interfering with PtdSer uptake promotes 2007; Fuller and Van Eldik, 2008). Additional evidence of en- dendritic cell-mediated antitumor activity by favoring ina fl m - gulfment signaling in the brain comes from studies of microglial matory uptake mechanisms. Still, despite what appears to be a chemoattractants. Dying neurons release find-me cues, namely plausible scenario wherein apoptotic cell clearance could have a extracellular nucleotides as well as CX CL1 (fractalkine or neuro- profound impact on carcinogenesis, there is only limited genetic tactin) that promote chemotaxis of microglia via the P2Y and evidence to implicate specic fi engulfment signaling pathways in CX CR1 receptors, respectively (Harrison et al., 1998; Koizumi this process. Indeed, the expression of several key engulfment et al., 2007). Interestingly, both fractalkine and UDP appear players, including MER (Linger et al., 2008) and v (Burvenich to enhance glial cell engulfment: fractalkine by enhancing et al., 2008), is up-regulated in neoplastic cells, but the impor- microglial secretion of MFG-E8, and UDP through an as yet un- tance of this observation is unclear. known mechanism (Koizumi et al., 2007; Fuller and Van Eldik, With the recent discovery of several “find-me” factors 2008). The role of fractalkine signaling has been studied in the released by apoptotic cells that act to promote recruitment of context of amyotrophic lateral sclerosis and Parkinson’s disease phagocytes to apoptotic cells, new insights have been gained in using CX CR1-deficient mice. In these disease models, loss of our understanding of connections between cell clearance and fractalkine signaling resulted in increased numbers of dying tumorigenesis. Several insightful studies from the laboratory of neurons, which suggests a potential role for fractalkine as an C.D. Gregory (Ogden et al., 2005; Truman et al., 2008) have fo- important find-me signal in the maintenance of brain homeosta - cused on how macrophages sense and subsequently engulf apop- sis (Cardona et al., 2006). A key unexplored area of clearance totic Burkitt lymphoma cells and how these signaling events may in the central nervous system is the immune response generated impact disease progression. These neoplastic B cells express high by microglial cells or astrocytes during engulfment (i.e., the levels of fractalkine on their surface that is cleaved during apop- release of anti-inflammatory mediators) and how that impacts tosis and subsequently functions as a potent chemoattractant for homeostasis and disease. Finally, in the developed brain, cell macrophages (Truman et al., 2008). Recruitment of macrophages turnover is thought to be quite low with the exception of restricted to splenic follicles is impaired in fractalkine receptor-dec fi ient regions where adult neurogenesis takes place (Kempermann mice, an observation consistent with a role for fractalkine as a key et al., 2004; Zhao et al., 2008; Taupin, 2009). Defining how mediator of macrophage recruitment to germinal centers (Truman apoptotic cell clearance impacts other developmental processes et al., 2008). Within the germinal center environment, high levels in the brain related to cell turnover, including adult neuro- of IL-10 (likely produced by the enguln fi g macrophages) appear genesis, will require additional studies with appropriate neuro- to suppress tumor immunity, whereas the release of B cell sur- logical models. vival factors by enguln fi g macrophages is thought to promote tumor growth (Ogden et al., 2005). Tumorigenesis and cell clearance Additionally, we have recently found that apoptotic cells Because apoptotic cell clearance typically generates an immuno- release nucleotide triphosphates (ATP/UTP) early during the suppressive environment, its role in the development and pro- apoptotic process (within 2–4 h), and that these nucleotides act as gression of cancer is enigmatic. As has been reviewed elsewhere chemoattractants for monocytes and macrophages in vitro and (Coussens and Werb, 2002; Condeelis and Pollard, 2006; Solinas in vivo (Elliott et al., 2009). The amount of ATP released by apop- et al., 2009), chronic ina fl mmation is a key factor in tumorigene - totic cells under these conditions, which promotes silent clear- sis. Thus, the efc fi ient clearance of dying cells, and the associated ance, represents a very small percentage of the total intracellular production of anti-ina fl mmatory mediators, would be predicted pool of nucleotides (<2%; Elliott et al., 2009). In contrast, a few to be beneficial in limiting tumorigenesis. However, within a other recent studies have demonstrated that ATP is released tumor environment where rapid cell proliferation and apoptosis are by tumor cells undergoing apoptosis in response to chemo- ongoing, phagocyte-mediated clearance can exert an unwanted therapeutics, with considerably higher amounts of ATP release immunosuppressive effect. This is particularly the case upon the (10–100 fold greater) seen at later times after induction (12–24 h; administration of antitumor chemotherapeutics, most of which Ghiringhelli et al., 2009; Martins et al., 2009; Aymeric et al., 2010). act by inducing apoptosis of tumor cells. In this setting, efc fi ient This apoptotic cell-derived ATP stimulates activation of the engulfment and the characteristic release of anti-ina fl mmatory NLRP3 ina fl mmasome in dendritic cells via the P2X7 receptor mediators, particularly TGF, upon encounter with eat-me sig- (Ghiringhelli et al., 2009). This heightened activation state appears nals during this process appear to suppress the antitumor immune necessary to drive IL-1 secretion and subsequent priming of response. Indeed, in several rodent tumor models, treatment with CD8+ T cells for IFN production and antitumor responses. These monoclonal antibodies to block PtdSer-mediated uptake retards studies highlight an emerging role for factors released by apoptotic the growth of tumors (Huang et al., 2005; Ran et al., 2005; He cells in shaping the immune response in normal and tumor environ- et al., 2009). Similarly, vaccination of mice with UV-irradiated ments. This has led to the concept of “immunogenic” versus “non- lymphoma cells coated with annexin V to mask PtdSer provides immunogenic” cell death, and the idea that immunogenic cell signic fi ant tumor protection against subsequent challenge with death may be benec fi ial in antitumor therapies (Green et al., 2009; living tumor cells, presumably by initiating an antitumor ina fl m - Locher et al., 2009). Thus, whether apoptotic cell clearance has a matory response (Bondanza et al., 2004). Antibody depletion of benec fi ial or detrimental effect in the context of tumor progression 1064 JCB • VOLUME 189 • NUMBER 7 • 2010 Figure 2. Pathogens usurp the ELMO–Dock–Rac engulfment module. Examples of mechanisms whereby microbial pathogens use the ELMO–Dock–Rac module to alter the host cellular response. The area above the broken line shows mechanism of enhanced S. flexneri invasion via IPGB1 interaction with ELMO, leading to enhanced Rac activation and membrane ruffles that serve as entry points for the bacteria. The area below the broken line shows that HIV-1 uses Nef interaction with the ELMO–Dock2 complex to disrupt CXCR4-dependent chemotaxis in CD4+ T cells. or anticancer therapies will depend on gaining a better under- The small GTPase RhoG acts upstream of ELMO1, and active standing of the role of factors released by apoptotic tumor cells. RhoG-GTP interacts with ELMO1, and thereby recruits the ELMO–Dock180 complex to the membrane to promote Rac acti- Engulfment molecules vation, membrane rufifl ng, and engulfment (Katoh and Negishi, in microbial pathogenesis 2003; deBakker et al., 2004). IPGB1 mimics the activity of RhoG- An emerging facet of engulfment signaling is how these path- GTP, and the Rac-generated ruffles serve as a site of entry for ways can be usurped by microbial pathogens. It has been known S. e fl xneri (Handa et al., 2007). Similarly, Yersinia enterocolitica for some time that bacteria can hijack or mimic host signaling virulence factors Invasin and YopE also modulate Rac1 activity pathways to aid in pathogenic steps, including cell entry and at the level of RhoG, and appear to do so in an ELMO–Dock180- immune evasion (Stebbins and Galán, 2001). This is achieved by dependent manner in cultured cells (Roppenser et al., 2009). delivery of bacterial effector proteins into the host cell that mimic However, neither of these Y. enterocolitica virulence factors have a range of cellular activities. As key regulators of the cytoskeleton been reported to directly interact with ELMO–Dock180, and the and numerous other cellular processes, small G proteins, particu- role of this module was inferred by expression of a dominant- larly the Rho family (e.g., RhoA, Rac, and Cdc42), are frequent negative mutant of ELMO1 that did not further alter Rac activa- targets for these clever effector mechanisms (Mattoo et al., 2007). tion in the presence of YopE (Roppenser et al., 2009). The signaling machinery that controls phagocyte morphology Usurping the engulfment machinery is not exclusive to during apoptotic cell engulfment relies on these GTPases as well, bacteria, and in fact can be used by viruses to promote patho- and thus it is not surprising that several bacteria target these path- genesis. Janardhan et al. (2004) found that the Nef gene product ways. In particular, the RhoG–ELMO–Dock–Rac pathway has of HIV-1 is able to complex with the ELMO2–Dock2 module in been found to be such a target (Fig. 2). The invasive pathogen T cells to promote Rac activation. Further, we have found that Shigella e fl xneri utilizes a type III secretion system to inject ef- Nef interacts with Dock2 in Jurkat T cells and promotes the fectors to promote entry into epithelial cells, including IPGB1 activation of a key cytoskeletal Rac effector, p21-activated (Handa et al., 2007). IPGB1 promotes membrane rufi fl ng via kinase (PAK; unpublished data). The outcome of this inter- Rac activation in a mechanism that requires binding to ELMO1. action appears to be dysregulated Rac activation, which is Apoptotic cell clearance and disease • Elliott and Ravichandran 1065 associated with enhanced activation through the T cell receptor topic (using in vivo models) portend potentially therapeutic ben- and improper CXCR4-dependent chemotaxis. However, the hi- et fi s by targeting the components of the engulfment machinery. jacking of the engulfment signaling machinery has only been We thank members of the Ravichandran laboratory for helpful comments shown using cultured cells, and it will be important to determine during preparation of this manuscript. This work was supported by a post-doctoral fellowship from the Ameri- if in vivo pathogenesis is dependent on these activities as well. can Cancer Society (to M.R. Elliott), and grants from the National Institute of General Medical Sciences/National Institutes of Health (to K.S. Ravichandran). Engulfment genes and other types of K.S. Ravichandran is a William Benter Senior Fellow of the American Asthma Foundation. disease associations Several recent studies have discovered associations with human Submitted: 20 April 2010 disease and genetic mutations of components of the engulfment Accepted: 7 June 2010 signaling machinery. 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Journal

The Journal of Cell BiologyPubmed Central

Published: Jun 28, 2010

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