Phagocytosis of cells dying through autophagy evokes a pro-inflammatory response in macrophages

Autophagy as a natural part of cellular homeostasis usually takes place unnoticed by neighboring cells. However, its co-occurrence with cell death may contribute to the clearance of these dying cells by recruited phagocytes. Autophagy associated with programmed cell death has recently been reported to be essential for presentation of phoshatidylserine (PS) on the cell surface (Qu et al. 2007) that has a key role in the clearance of apoptotic cells. Recently, we have demonstrated that upon triggering cell death by autophagy in MCF-7 cells, the corpses were efficiently phagocytosed by both human macrophages and non-dying MCF-7 cells. Death as well as engulfment could be prevented by inhibiting autophagy. Based on our data, two molecular mechanisms have been proposed for the uptake of cells which die through autophagy: a PS-dependent pathway which was exclusively used by the living MCF-7 cells acting as non-professional phagocytes, and a PS-independent uptake mechanism that was active in macrophages acting as professional phagocytes. Several lines of evidence suggest that macrophages utilize calreticulin-mediated recognition, tethering, tickling and engulfment processes. Phagocytic uptake of cells dying through autophagy by macrophages leads to a pro-inflammatory response characterized by the induction and secretion of IL-6, TNFalpha, IL-8 and IL-10.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Clearance of apoptotic and necrotic cells and its immunological consequences

The ultimate and most favorable fate of almost all dying cells is engulfment by neighboring or specialized cells. Efficient clearance of cells undergoing apoptotic death is crucial for normal tissue homeostasis and for the modulation of immune responses. Engulfment of apoptotic cells is finely regulated by a highly redundant system of receptors and bridging molecules on phagocytic cells that detect molecules specific for dying cells. Recognition of necrotic cells by phagocytes is less well understood than recognition of apoptotic cells, but an increasing number of recent studies, which are discussed here, are highlighting its importance. New observations indicate that the interaction of macrophages with dying cells initiates internalization of the apoptotic or necrotic targets, and that internalization can be preceded by “zipper”-like and macropinocytotic mechanisms, respectively. We emphasize that clearance of dying cells is an important fundamental process serving multiple functions in the regulation of normal tissue turnover and homeostasis, and is not just simple anti- or pro-inflammatory responses. Here we review recent findings on genetic pathways participating in apoptotic cell clearance, mechanisms of internalization, and molecules involved in engulfment of apoptotic versus necrotic cells, as well as their immunological consequences and relationships to disease pathogenesis. Katharina D’Herde and Peter Vandenabeele share senior authorship. This study was supported by Ghent University GOA grant No. 12050502, IUAP-V/12-12.0C14.02, FWO-Vlaanderen 3G.0218.06, and Flanders Interuniversity Institute for Biotechnology (VIB).     

Phagocytosis of Cells Dying through Autophagy Evokes a Pro-Inflammatory Response in Macrophages

Autophagy as a natural part of cellular homeostasis usually takes place unnoticed by neighboring cells. However, its co-occurrence with cell death may contribute to the clearance of these dying cells by recruited phagocytes. Autophagy associated with programmed cell death has recently been reported to be essential for presentation of phoshatidylserine (PS) on the cell surface (Qu et al. 2007) that has a key role in the clearance of apoptotic cells. Recently, we have demonstrated that upon triggering cell death by autophagy in MCF-7 cells, the corpses were efficiently phagocytosed by both human macrophages and non-dying MCF-7 cells. Death as well as engulfment could be prevented by inhibiting autophagy. Based on our data, two molecular mechanisms have been proposed for the uptake of cells which die through autophagy: a PS-dependent pathway which was exclusively used by the living MCF-7 cells acting as non-professional phagocytes, and a PS-independent uptake mechanism that was active in macrophages acting as professional phagocytes. Several lines of evidence suggest that macrophages utilize calreticulin-mediated recognition, tethering, tickling and engulfment processes. Phagocytic uptake of cells dying through autophagy by macrophages leads to a pro-inflammatory response characterized by the induction and secretion of IL-6, TNFα, IL-8 and IL-10.

Addendum to:Clearance of Dying Autophagic Cells of Different Origin by Professional and Non-Professional Phagocytes

G. Petrovski, G. Zahuczky, K. Katona, G. Vereb, W. Martinet, Z. Nemes, W. Bursch and L. Fésüs

Cell Death Differ 2007;14:1117-28     

From regulation of dying cell engulfment to development of anti-cancer therapy

Cell death and efficient engulfment of dying cells ensure tissue homeostasis and is involved in pathogenesis. Clearance of dying cells is a complex and dynamic process coordinated by interplay between ligands on dying cell, bridging molecules, and receptors on engulfing cells. In this review, we will discuss recent advances and significance of molecular changes on the surface of dying cells implicated in their recognition and clearance as well as factors released by dying cells that attract macrophages to the site of cell death. It is now becoming apparent that phagocytes use a specific set of mechanisms to discriminate between live and dead cells, and this phenomenon will be illustrated here. Next, we will discuss potential mechanisms by which removal of dying cells could modulate immune responses of phagocytes, in particular of macrophages. Finally, we will address possible strategies for manipulating the immunogenicity of dying cells in experimental cancer therapies.     

Clearance of apoptotic cells by phagocytes

Phagocytic clearance of apoptotic cells may be considered to consist of four distinct steps: accumulation of phagocytes at the site where apoptotic cells are located; recognition of dying cells through a number of bridge molecules and receptors; engulfment by a unique uptake process; and processing of engulfed cells within phagocytes. Here, we will discuss these individual steps that collectively are essential for the effective removal of apoptotic cells. This will illustrate our relative lack of knowledge about the initial attraction signals, the specific mechanisms of engulfment and processing in comparison to the extensive literature on recognition mechanisms. There is now mounting evidence that clearance defects are responsible for chronic inflammatory disease and contribute to autoimmunity. Therefore, a better understanding of all aspects of the clearance process is required before it can truly be manipulated for therapeutic gain.     

Engulfment and clearance of apoptotic cells based on a GlcNAc-binding lectin-like property of surface vimentin

The clearance of apoptotic cells is important to maintain tissue homeostasis. The engulfment of apoptotic cells is performed by professional phagocytes, such as macrophages, and also by non-professional phagocytes, such as mesenchymal cells. Here, we show that vimentin, a cytoskeletal protein, functions as an engulfment receptor on neighboring phagocytes, which recognize O-linked β-N-acetylglucosamine (O-GlcNAc)-modified proteins from apoptotic cells as "eat me" ligands. Previously, we reported that vimentin possesses a GlcNAc-binding lectin-like property on cell surface. However, the physiological relevance of the surface localization and GlcNAc-binding property of vimentin remained unclear. In the present study, we observed that O-GlcNAc proteins from apoptotic cells interacted with the surface vimentin of neighboring phagocytes and that this interaction induced serine 71-phosphorylation and recruitment of vimentin to the cell surface of the neighboring phagocytes. Moreover, tetrameric vimentin that was disassembled by serine 71-phosphorylation possessed a GlcNAc-binding activity and was localized to the cell surface. We demonstrated our findings in vimentin-expressing common cell lines such as HeLa cells. Furthermore, during normal developmental processes, the phagocytic engulfment and clearance of apoptotic footplate cells in mouse embryos was mediated by the interaction of surface vimentin with O-GlcNAc proteins. Our results suggest a common mechanism for the clearance of apoptotic cells, through the interaction of surface vimentin with O-GlcNAc-modified proteins.     

Phagocytosis of apoptotic cells and the resolution of inflammation

Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.     

The N-Terminus of CD14 Acts to Bind Apoptotic Cells and Confers Rapid-Tethering Capabilities on Non-Myeloid Cells

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called ‘non-professional’ phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material.     

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.     

Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of "eat-me" signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A(2). These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.     

Programmed cell clearance: Molecular regulation of the elimination of apoptotic cell corpses and its role in the resolution of inflammation

Programmed cell clearance is a physiological process of elimination of apoptotic cell corpses. Recent studies have disclosed several ligand-receptor interactions that dictate the recognition or non-recognition of cells by macrophages and other phagocytes. The externalization of the anionic phospholipid, phosphatidylserine is effectively recognized by specific receptors on professional phagocytes and facilitates the clearance of apoptotic cells. Macrophage disposal of cells at sites of inflammation is believed to play an important role in the resolution of the inflammatory process, and recent studies have suggested a role for the NADPH oxidase in the process of macrophage elimination of activated neutrophils. The present review will focus on the molecular regulation of programmed cell clearance, and discuss the role of cell elimination in the resolution of inflammation.     

Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans

The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the “eat-me” signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.     

Clearing the Dead: Apoptotic Cell Sensing,Recognition, Engulfment,and Digestion

Clearance of apoptotic cells is the final stage of programmed cell death. Uncleared corpses can become secondarily necrotic, promoting inflammation and autoimmunity. Remarkably, even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. Recently, significant progress has been made in understanding the steps involved in prompt cell clearance in vivo. These include the sensing of corpses via “find me” signals, the recognition of corpses via “eat me” signals and their cognate receptors, the signaling pathways that regulate cytoskeletal rearrangement necessary for engulfment, and the responses of the phagocyte that keep cell clearance events “immunologically silent.” This study focuses on our understanding of these steps.Multicellular organisms execute the majority of unwanted cell populations in a regulated fashion via the process of apoptosis (Henson and Hume 2006; Nagata et al. 2010). Examples of unwanted cells include excess cells generated during development, cells infected with intracellular bacteria or viruses, transformed or malignant cells capable of tumorigenesis, and cells irreparably damaged by cytotoxic agents. Swift removal of these cells is necessary for maintenance of overall health and homeostasis and prevention of autoimmunity, pathogen burden, or cancer. Quick removal of dying cells is a key final step, if not the ultimate goal of the apoptotic program.The term “phagocytosis” refers to an internalization process by which larger particles, such as bacteria and dead/dying cells, are engulfed and processed within a membrane-bound vesicle called the phagosome (Ravichandran and Lorenz 2007). A phagocyte is any cell that is capable of engulfment, including “professional” phagocytes such as macrophages, immature dendritic cells, and neutrophils. Metazoa have multiple mechanisms for clearing apoptotic cells, often depending on the tissue and apoptotic cell type (Gregory 2009). Macrophages and immature dendritic cells readily engulf dead or dying cells in tissues such as bone marrow (where a large number of new hematopoietic cells are generated), spleen (during or after an immune response), and the thymus (in young animals during T-lymphocyte development). In other tissues, neighboring “nonprofessional” phagocytes can also mediate the clearance of apoptotic targets. For example, in the mammary epithelium, viable mammary epithelial cells engulf apoptotic mammary epithelial cells after cessation of lactation (Monks et al. 2005, 2008). What distinguishes the phagocytosis of apoptotic cells from the phagocytosis of most bacteria or necrotic cells is the lack of a pro-inflammatory immune response (Henson 2005). This article discusses apoptotic cell engulfment, specifically the recruitment of phagocytes, through “find me” signals, the recognition of apoptotic cells by phagocytes via “eat me” signals, the internalization process and signaling pathways used for cytoskeletal rearrangement, and finally the digestion of apoptotic cells and phagocytic response to this process (Fig. 1).Open in a separate windowFigure 1.The steps of efficient apoptotic cell clearance. First, “find me” signals released by apoptotic cells are recognized via their cognate receptors on the surface of phagocytes. This is the sensing stage and stimulates phagocyte migration to the location of apoptotic cells. Second, phagocytes recognize exposed “eat me” signals on the surface of apoptotic cells via their phagocytic receptors, which leads to downstream signaling events culminating in Rac activation. Finally, further signaling events within the phagocyte regulate the digestion and processing of the apoptotic cell meal and the secretion of anti-inflammatory cytokines.     

Apoptotic cells are cleared by directional migration and elmo1- dependent macrophage engulfment

Apoptotic cell death is essential for development and tissue homeostasis. Failure to clear apoptotic cells can ultimately cause inflammation and autoimmunity. Apoptosis has primarily been studied by staining of fixed tissue sections, and a clear understanding of the behavior of apoptotic cells in living tissue has been elusive. Here, we use a newly developed technique to track apoptotic cells in real time as they emerge and are cleared from the zebrafish brain. We find that apoptotic cells are remarkably motile, frequently migrating several cell diameters to the periphery of living tissues. F-actin remodeling occurs in surrounding cells, but also within the apoptotic cells themselves, suggesting a cell-autonomous component of motility. During the first 2 days of development, engulfment is rare, and most apoptotic cells lyse at the brain periphery. By 3 days postfertilization, most cell corpses are rapidly engulfed by macrophages. This engulfment requires the guanine nucleotide exchange factor elmo1. In elmo1-deficient macrophages, engulfment is rare and may occur through macropinocytosis rather than directed engulfment. These findings suggest that clearance of apoptotic cells in living vertebrates is accomplished by the combined actions of apoptotic cell migration and elmo1-dependent macrophage engulfment.     

A secreted factor NimrodB4 promotes the elimination of apoptotic corpses by phagocytes in Drosophila

Programmed cell death plays a fundamental role in development and tissue homeostasis. Professional and non‐professional phagocytes achieve the proper recognition, uptake, and degradation of apoptotic cells, a process called efferocytosis. Failure in efferocytosis leads to autoimmune and neurodegenerative diseases. In Drosophila, two transmembrane proteins of the Nimrod family, Draper and SIMU, mediate the recognition and internalization of apoptotic corpses. Beyond this early step, little is known about how apoptotic cell degradation is regulated. Here, we study the function of a secreted member of the Nimrod family, NimB4, and reveal its crucial role in the clearance of apoptotic cells. We show that NimB4 is expressed by macrophages and glial cells, the two main types of phagocytes in Drosophila. Similar to draper mutants, NimB4 mutants accumulate apoptotic corpses during embryogenesis and in the larval brain. Our study points to the role of NimB4 in phagosome maturation, more specifically in the fusion between the phagosome and lysosomes. We propose that similar to bridging molecules, NimB4 binds to apoptotic corpses to engage a phagosome maturation program dedicated to efferocytosis.     

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.     

Exposure of a specific pleioform of multifunctional glyceraldehyde 3-phosphate dehydrogenase initiates CD14-dependent clearance of apoptotic cells

Rapid clearance of apoptotic cells by phagocytes is crucial for organogenesis, tissue homeostasis, and resolution of inflammation. This process is initiated by surface exposure of various ‘eat me’ ligands. Though phosphatidylserine (PS) is the best recognized general recognition ligand till date, recent studies have shown that PS by itself is not sufficient for clearance of apoptotic cells. In this study, we have identified a specific pleioform of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) that functions as an ‘eat me’ signal on apoptotic cell surface. This specific form of GAPDH which is exposed on surface of apoptotic cells was found to interact with CD14 present on plasma membrane of phagocytes leading to their engulfment. This is the first study demonstrating the novel interaction between multifunctional GAPDH and the phagocytic receptor CD14 resulting in apoptotic cell clearance (efferocytosis).Subject terms: Cell biology, Apoptosis