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.     

How apoptotic cells aid in the removal of their own cold dead bodies

Apoptotic cell clearance facilitates the removal of aged, damaged, infected or dangerous cells although minimizing perturbation of surrounding tissues, and is a vital process in the development and homeostasis of multicellular organisms. Importantly, failure to correctly execute programmed cell death and subsequent corpse clearance is broadly associated with chronic inflammatory and/or autoimmune diseases such as systemic lupus erythematosus. Apoptotic cells develop dramatic morphological changes including contraction, membrane blebbing and apoptotic body formation, which were among the first and most readily identifiable features of cellular suicide. However, understanding the purpose of apoptotic cell morphological changes has proven to be elusive, and recent studies have made somewhat surprising, and occasionally opposing, conclusions about the contribution of blebbing to phagocytic clearance and prevention of inflammatory/autoimmune disease. We review the evidence indicating how apoptotic blebs actively promote corpse recognition, uptake, and generation of auto-reactive antibodies.     

CD38/cyclic ADP-ribose signaling: role in the regulation of calcium homeostasis in airway smooth muscle

The contractility of airway smooth muscle cells is dependent on dynamic changes in the concentration of intracellular calcium. Signaling molecules such as inositol 1,4,5-trisphosphate and cyclic ADP-ribose play pivotal roles in the control of intracellular calcium concentration. Alterations in the processes involved in the regulation of intracellular calcium concentration contribute to the pathogenesis of airway diseases such as asthma. Recent studies have identified cyclic ADP-ribose as a calcium-mobilizing second messenger in airway smooth muscle cells, and modulation of the pathway involved in its metabolism results in altered calcium homeostasis and may contribute to airway hyperresponsiveness. In this review, we describe the basic mechanisms underlying the dynamics of calcium regulation and the role of CD38/cADPR, a novel pathway, in the context of airway smooth muscle function and its contribution to airway diseases such as asthma.     

Apoptosis commitment--translating survival signals into decisions on mitochondria

Most defective and unwanted cells die by apoptosis, cells without damaging the surrounding tissue. Once a an exquisitely controlled genetic programme for removing such cell has committed to apoptosis, the process is remarkably efficient, and is completed within a few minutes of initiation. This point of no retum for an apoptotic cell is commonly held to be the point at which the outer mitochondrial membrane is permeabilised, a process regulated by the Bcl-2 family of proteins. How these proteins regulate this decision point is central to diseases such as cancer where apoptotic control is lost. In this review, we will discuss apoptotic signalling and how a cell makes the irreversible decision to die. We will focus on one set of survival signals, those derived by cell adhesion to the extracellular matrix （ECM）, and use these to highlight the complexities of apoptotic signalling. In particular, we will illustrate how multiple signalling pathways converge to determine critical cell fate decisions.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells

Chronic obstructive pulmonary disease is a highly prevalent, complex disease, usually caused by cigarette smoke. It causes serious morbidity and mortality and costs the global community billions of dollars per year. While chronic inflammation, extracellular matrix destruction and increased airway epithelial cell apoptosis are reported in chronic obstructive pulmonary disease, the understanding of the basic pathogenesis of the disease is limited and there are no effective treatments. We hypothesized that the accumulation of apoptotic airway epithelial cells chronic obstructive pulmonary disease in could be due to defective phagocytic clearance by alveolar macrophages. There have been no previous studies of the phagocytic capacity of alveolar macrophages in chronic obstructive pulmonary disease using physiologically relevant apoptotic airway epithelial cells as phagocytic targets. We developed a phagocytosis assay whereby cultured 16HBE airway epithelial cells were induced to apoptosis with ultraviolet radiation and stained with mitotracker green. Alveolar macrophages from bronchoalveolar lavage from eight control and six chronic obstructive pulmonary disease subjects were analysed following 1.5 h incubation with apoptotic airway epithelial cells, then staining with macrophage marker anti CD33. CD33+/mitotracker green + events (i.e., alveolar macrophages which had phagocytosed apoptotic airway epithelial cells) were analysed using flow cytometry. Phagocytosis of polystyrene microbeads was investigated in parallel. A significantly reduced proportion of alveolar macrophages from chronic obstructive pulmonary disease subjects ingested apoptotic airway epithelial cells compared with controls (11.6 +/- 4.1% for chronic obstructive pulmonary disease versus 25.6 +/- 9.2% for control group). Importantly, the deficiency was not observed using polystyrene beads, suggesting that the failure to resolve epithelial damage in chronic obstructive pulmonary disease may result, at least partially, from specific defects in phagocytic ability of alveolar macrophages to ingest apoptotic airway epithelial cells.     

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.     

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.     

Dangerous attraction: phagocyte recruitment and danger signals of apoptotic and necrotic cells

Tissue homeostasis in metazoa requires the rapid and efficient clearance of dying cells by professional or semi-professional phagocytes. Impairment of this finely regulated, fundamental process has been implicated in the development of autoimmune diseases, such as systemic lupus erythematosus. Various studies have provided us a detailed understanding of the interaction between dying cells and phagocytes as well as the current concept that apoptotic cell removal leads to a non- or anti-inflammatory response, whereas necrotic cell removal stimulates a pro-inflammatory reaction. In contrast, our knowledge about the soluble factors released from dying cells is rather limited, although meanwhile it is generally accepted that not only the dying cell itself but also the substances liberated during cell death contribute to the process of corpse clearance and the subsequent immune response. This review article is intended as an up-to-date survey over attraction and danger signals of apoptotic, primary and secondary necrotic cells, their function as chemoattractants in phagocyte recruitment, additional effects on the immune system, and the receptors, which are engaged in this scenario.     

Phagocytosis of apoptotic cells

Removal of apoptotic cells by phagocytes plays an important role in many biological processes, including embryological development and tissue remodelling. In addition, it has become apparent that one of the key mechanisms for the successful resolution of inflammation is the orchestrated clearance of apoptotic inflammatory cells by phagocytes (e.g., macrophages and dendritic cells) and other cells known to have phagocytic capacity (e.g., hepatocytes, endothelial cells, epithelial cells, etc.). Furthermore, phagocytosis of apoptotic cells is an active and highly regulated process that not only serves to remove potentially histotoxic cells from the inflammatory milieu, but also directs the phenotype of the phagocytic cell to be anti-inflammatory. Convincing evidence has been presented that reduced or dysregulated phagocytosis of apoptotic cells contributes to the development and propagation of inflammatory disorders. Conversely, enhanced phagocytosis of apoptotic cells may be exploited for therapeutic gain. Indeed, powerful anti-inflammatory drugs such as the glucocorticoids have been shown to augment clearance of apoptotic cells which may contribute to their therapeutic effectiveness. In this chapter, we describe methods for studying phagocytosis of apoptotic cells.     

Autoantigens are translocated into small apoptotic bodies during early stages of apoptosis

A dysregulation of apoptosis or an ineffective clearance of apoptotic material is suspected to be involved in the pathogenesis of systemic lupus erythematodes. Subcellular fragments such as apoptotic bodies (ABs) have been recognized as modulators of intercellular communication and immune function. In this context, we have been interested whether nuclear and cytoplasmic antigens are relocated into ABs. In the present study, we characterized ABs isolated from apoptozing lymphoblasts. We found an accumulation of the linker-histone (histone 1) as well as the core-histones (histone 2A, histone 2B, histone 3, histone 4) in ABs. Further, they contained DNA, RNA and the ribonuclear protein La/SSB. Proteins such as cytochrome c, HSP 70, prohibitin, p53, nuclear matrix antigen or lamin B were excluded from ABs. The content of ABs differed from that observed in membrane microparticles isolated from viable cells. Formation of ABs occurred early during apoptosis. It was observed before DNA-degradation or phosphatidylserine exposure was detected. ABs were engulfed by monocyte-derived phagocytes. These findings suggest that immunogenic molecules are actively translocated into ABs followed by a rapid engulfment of the latter by environmental phagocytes. In autoimmune diseases, a defect in the clearance of ABs or AB formation may contribute to the development of autoimmunity.     

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).     

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.     

Luminal fluid tonicity regulates airway ciliary beating by altering membrane stretch and intracellular calcium

The coordinated, directional beating of airway cilia drives airway mucociliary clearance. Here we explore the hypothesis that airway surface liquid osmolarity is a key regulator of ciliary beating. Cilia in freshly isolated human and murine airways visualized with streaming video-microscopy exhibited a reciprocal dependence on a physiological range of luminal fluid osmolarities, across the entire range of ciliary activity (0-20 beats per sec). Increasing osmolarity slowed or completely abrogated, while lower osmolarity dramatically stimulated ciliary beating. In parallel, epithelial cell height and importantly, intracellular calcium levels (as judged by fluorescence imaging) also changed. Moreover, ciliary beating was stimulated by isosmotic solutions containing membrane permeant osmolytes, suggesting that cell size and membrane stretch (governed by apical fluid tonicity), rather than osmolarity itself, contribute to the activation. These findings shed light on the pathophysiology of diseases of mucociliary clearance such as cystic fibrosis and other chronic inflammatory lung diseases.     

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.     

Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac(2-26)

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.     

Differences between the clearance of apoptotic cells by professional and non-professional phagocytes

Both professional and non-professional phagocytes [1] participate in clearing the massive numbers of cells that undergo apoptosis during animal development [2], but it is not known how they divide this task. Using time-lapse recordings of cells in culture, we show that professional phagocytes (brain macrophages or microglia) are highly motile, ingest apoptotic cells immediately, and digest them quickly. Non-professionals such as BHK and lens epithelial cells are sessile, often recognize apoptotic cells as soon as they die by showing characteristic palpating movements, but delay ingestion until several hours later. By pre-ageing apoptotic cells, we show that this delay is because the apoptotic cells must undergo further changes before non-professionals can ingest them. The difference was also apparent in vivo, using immunofluorescence and electron microscopy of the developing central nervous system. This arrangement favours prompt clearance by professionals if present in adequate numbers; if they are scarce, however, non-professional bystanders will reluctantly clear the apoptotic cells.     

Lung protease/anti-protease network and modulation of mucus production and surfactant activity

Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local ‘alarm’ anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs.