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).     

Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation

Cells that undergo apoptosis or necrosis are promptly removed by phagocytes. Soluble opsonins such as complement can opsonize dying cells, thereby promoting their removal by phagocytes and modulating the immune response. The pivotal role of the complement system in the handling of dying cells has been demonstrated for the classical pathway (via C1q) and lectin pathway (via mannose-binding lectin and ficolin). Herein we report that the only known naturally occurring positive regulator of complement, properdin, binds predominantly to late apoptotic and necrotic cells, but not to early apoptotic cells. This binding occurs independently of C3b, which is additional to the standard model wherein properdin binds to preexisting clusters of C3b on targets and stabilizes the convertase C3bBb. By binding to late apoptotic or necrotic cells, properdin serves as a focal point for local amplification of alternative pathway complement activation. Furthermore, properdin exhibits a strong interaction with DNA that is exposed on the late stage of dying cells. Our data indicate that direct recognition of dying cells by properdin is essential to drive alternative pathway complement activation.     

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.     

细胞死亡清除的趋化和危险信号分子

细胞死亡是机体组成发育及其平衡的重要组成部分,细胞死亡后能够迅速被邻近细胞或巨噬细胞识别吞噬及消化.死细胞自身或细胞死亡时释放的物质有利于死亡细胞的清除及免疫学转归.研究发现凋亡细胞主要通过释放dRPS19、LPC、EMAPⅡ、TSP-1、核苷酸、FKN等趋化信号分子招来吞噬细胞,启动清除过程;坏死细胞则主要通过危险信号分子HSP、S100蛋白、HMGB-1、ATP、尿酸等物质启动和介导炎性反应.然而任何一个信号都不能独立执行功能,多种信号间往往相互联系与制约,共同构成了死细胞清除的信号网络.     

Apoptosis: the importance of being eaten

In vivo, cells undergoing apoptosis are usually recognised and swiftly ingested by macrophages or neighbouring cells acting as semi-professional phagocytes. This review debates evidence that the contents of apoptotic cells represent a danger to the organism, being capable of injuring tissue directly or triggering autoimmune responses, concluding that phagocytic clearance of intact apoptotic cells is a safe disposal route. Indeed, new data suggest that, in certain circumstances, phagocytes ingesting apoptotic cells may actively downregulate inflammatory and immune responses. Consequently, increasing evidence that there may be factors capable of perturbing safe clearance of apoptotic cells in vivo suggests that failure of this process may be a hitherto unrecognised pathogenetic factor in inflammatory and autoimmune diseases. New treatments designed to promote safe phagocytic clearance of dying cells can be anticipated, and it may even prove possible to eliminate unwanted cells by inducing appearance of cell surface 'eat me' signals.     

Serum-dependent processing of late apoptotic cells for enhanced efferocytosis

Binding of the serum protein complement component C1q to the surface of dying cells facilitates their clearance by phagocytes in a process termed efferocytosis. Here, we investigate during which phase of apoptotic cell death progression C1q binding takes place. Purified C1q was found to bind to all dying cells and, albeit weaker, also to viable cells. The presence of serum abrogated completely the binding to viable cells. In addition, C1q binding to dying cells was limited to a specific subpopulation of late apoptotic/secondary necrotic cells. Co-culturing serum-treated apoptotic cells with human monocytes revealed a much higher phagocytosis of C1q-positive than of C1q-negative late apoptotic/secondary necrotic cells. But this phagocytosis-promoting activity could not be observed with purified C1q. Serum-treated C1q-positive late apoptotic/secondary necrotic cells exhibited a similar volume, a similar degraded protein composition, but a much lower DNA content in comparison with the remaining late apoptotic/secondary necrotic cells. This was mediated by a serum-bound nuclease activity that could be abrogated by G-actin, which is a specific inhibitor of serum DNase I. These results show that serum factors are involved in the prevention of C1q binding to viable cells and in the processing of late apoptotic/secondary necrotic cells promoting cell death progression toward apoptotic bodies. This process leads to the exposure of C1q-binding structures and facilitates efferocytosis.     

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.     

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.     

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.     

Role of surfactant proteins A,D, and C1q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and CD91 as a common collectin receptor complex

Removal of cells dying by apoptosis is essential to normal development, maintenance of tissue homeostasis, and resolution of inflammation. Surfactant protein A (SP-A) and surfactant protein D (SP-D) are high abundance pulmonary collectins recently implicated in apoptotic cell clearance in vitro. Other collectins, such as mannose-binding lectin and the collectin-like C1q, have been shown to bind to apoptotic cells and drive ingestion through interaction with calreticulin and CD91 on the phagocyte in vitro. However, only C1q has been shown to enhance apoptotic cell uptake in vivo. We sought to determine the relative importance of SP-A, SP-D, and C1q in pulmonary clearance of apoptotic cells using knockout and overexpressing mice, and to determine the role of calreticulin and CD91 in this process. SP-A, SP-D, and C1q all enhanced apoptotic cell ingestion by resident murine and human alveolar macrophages in vitro. However, only SP-D altered apoptotic cell clearance from the naive murine lung, suggesting that SP-D plays a particularly important role in vivo. Similar to C1q and mannose-binding lectin, SP-A and SP-D bound to apoptotic cells in a localized, patchy pattern and drove apoptotic cell ingestion by phagocytes through a mechanism dependent on calreticulin and CD91. These results suggest that the entire collectin family of innate immune proteins (including C1q) works through a common receptor complex to enhance removal of apoptotic cells, and that collectins are integral, organ-specific components of the clearance machinery.     

Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c⁺ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets.     

Loss of the RhoGAP SRGP-1 promotes the clearance of dead and injured cells in Caenorhabditis elegans

Multicellular animals rapidly clear dying cells from their bodies. Many of the pathways that mediate this cell removal are conserved through evolution. Here, we identify srgp-1 as a negative regulator of cell clearance in both Caenorhabditis elegans and mammalian cells. Loss of srgp-1 function results in improved engulfment of apoptotic cells, whereas srgp-1 overexpression inhibits apoptotic cell corpse removal. We show that SRGP-1 functions in engulfing cells and functions as a GTPase activating protein (GAP) for CED-10 (Rac1). Interestingly, loss of srgp-1 function promotes not only the clearance of already dead cells, but also the removal of cells that have been brought to the verge of death through sublethal apoptotic, necrotic or cytotoxic insults. In contrast, impaired engulfment allows damaged cells to escape clearance, which results in increased long-term survival. We propose that C. elegans uses the engulfment machinery as part of a primitive, but evolutionarily conserved, survey mechanism that identifies and removes unfit cells within a tissue.     

Differentiation of apoptotic and necrotic cells in suspension cultures of Taxus cuspidata by the combined use of fluorescent dying and histochemical staining methods

A method to differentiate apoptotic and necrotic cells in suspension cultures of Taxus cuspidata was developed by the combined use of the double-fluorescence dying and histochemical staining methods. The cultured cells were directly detected without the operations such as the removal of cell wall, fixing and section of cells as required by other conventional methods. Besides, the living cells, apoptotic and necrotic cells were simultaneously differentiated under a fluorescence microscope and the typical apoptotic morphological features could be observed throughout the whole process. The advantages of this method include simple operation, quick response and high accuracy.     

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.     

Migration to apoptotic "find-me" signals is mediated via the phagocyte receptor G2A

Phagocytosis of apoptotic cells is fundamentally important throughout life, because non-cleared cells become secondarily necrotic and release intracellular contents, thus instigating inflammatory and autoimmune responses. Secreted "find-me" and exposed "eat-me" signals displayed by the dying cell in concert with the phagocyte receptors comprise the phagocytic synapse of apoptotic cell clearance. In this scenario, lysophospholipids (lysoPLs) are assumed to act as find-me signals for the attraction of phagocytes. However, both the identity of the lyso-PLs released from apoptotic cells and the nature of the phagocyte receptor are largely unknown. By a detailed analysis of the structural requirements we show here that lysophosphatidylcholine (lysoPC), but none of the lysoPC metabolites or other lysoPLs, represents the essential apoptotic attraction signal able to trigger a phagocyte chemotactic response. Furthermore, using RNA interference and expression studies, we demonstrate that the G-protein-coupled receptor G2A, unlike its relative GPR4, is involved in the chemotaxis of monocytic cells. Thus, our study identifies lysoPC and G2A as the crucial receptor/ligand system for the attraction of phagocytes to apoptotic cells and the prevention of autoimmunity.     

The disposal of dying cells in living tissues

Cells continuously die and disappear from the midst of living tissues. However, some of their constituents survive. DNA is horizontally transferred to phagocytic cells, and apoptotic cell antigens shape the immune repertoire. When massive apoptosis occurs, which overwhelms tissue scavenger cells, or when the function of phagocytes abates, dying cells escape clearance in vivo. Remnant dying cells come to phagocytes disguised: factors capable to envelop their membranes pervade the entire organism, or are generated in given tissues. Some are constitutively present, while other are generated during early or late phases of the inflammatory response, possibly to face the further burden of the dead inflammatory cells. This camouflage influences the disposal of the corpses: decoying molecules either bridge the corpse to the phagocyte or hide it. Furthermore, factors associated to the plasma membrane of the apoptotic cell shape the signals the phagocyte releases in situ. Finally, molecules contained or released by the dying cell alter the apprehension by the phagocyte of its prey, influencing its immunogenicity.     

Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice

Exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane is an early event during apoptotic cell death and serves as a recognition signal for phagocytes. Usually the clearance of apoptotic cells does not initiate inflammation or immune response. We investigated the immune response in Balb/c mice towards apoptotic human T-cells. Animals injected with apoptotic cells showed significantly reduced humoral immune responses, especially Th1-dependent IgG2a titres, compared to controls immunised with viable cells. However, treatment of apoptotic cells with annexin V (AxV) significantly increased the humoral immune response. AxV binds with high affinity to anionic phospholipids and as a result interferes with the phosphatidylserine recognition by phagocytes. Our results indicate that AxV treatment may be used to increase the efficiency of apoptotic cell-based vaccines, e.g. some tumour vaccines.     

Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages

Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy.