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.     

Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D

BACKGROUND: Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay. METHODS: Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells. RESULTS: The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method. CONCLUSIONS: We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.     

Phagocytosis of formol treated red blood cells

Formol treated red blood cells have been established to be phagocytized by human leukocytes in the presence of compatible AB0 standard test sera in case such red blood cells are stored continuously in water or in case they are incubated in saliva in advance for one hour at least at 37 degrees C. If formol treated red blood cells are continuously stored in 0.9% NaCl solution or if they are lyophilized in addition they will not be engulfed by human leukocytes in presence of compatible standard test sera of the AB0 system. The additional treatment with papain of formol treated red blood cells will cause a moderate phagocytosis.     

Engulfment mechanism of apoptotic cells

Apoptotic cells are engulfed and removed by phagocytes. This ensures proper development of the organism and can modulate immune responses. Recent studies have examined molecules on apoptotic cells, such as phosphatidylserine, which may signal for engulfment through multiple receptors. Apoptotic recognition mechanisms may vary with the apoptotic and engulfing cell type, and even with the age of the corpse.     

Phagocytosis in rat Kupffer cells in vitro

Phagocytosis was studied in rat Kupffer cells in vitro by using opsonized sheep red cells as objects and inducing attachment and ingestion through the Fc and C₃ receptors. The Fc receptors functioned by and large in the same manner as in the peritoneal macrophages. When the red cells were opsonized with IgM and complement, there was attachment but little ingestion in a serum-free medium. Newborn calf serum was found to trigger ingestion. Our experiments provided no conclusive evidence as to the nature of this triggering mechanism. The limiting factor in phagocytosis was the cytoplasmic volume of the phagocyte rather than the availability of surface receptors. The expression of surface receptors on cells in culture depended on length of culture and degree of spreading. We confirmed the available information on the energy requirements of phagocytosis as studied in peritoneal macrophages. As judged by isotope release, digestion of the red cells was in process shortly after ingestion. However, morphological examination failed to detect any changes in appearance prior to 4 h. After a blocking dose of sheep red cells, a rather long period (40 h) was required before cells fully recovered their phagocytic capacity.     

Two-step engulfment of apoptotic cells

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.     

Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms

Caspase activation, exposure of phosphatidylserine (PS) on the outer surface of the plasma membrane, and rapid phagocytic removal of dying cells are key features of apoptosis. Nonapoptotic/necrotic modes of death occur independent of caspase activation, but the role of phagocytosis is largely unknown. To address this issue, we studied phagocytosis by human monocyte-derived macrophages (HMDM) and rat microglial cells. Target cells (Jurkat) were stimulated by several different methods that all caused caspase-independent death. First, we induced necrosis by combining toxins with ATP-depleting agents. Under these conditions, neither PS was exposed nor were such cells phagocytosed before their death. However, once the plasma membrane integrity was lost, the dead cells were rapidly and efficiently engulfed by HMDM. Next, we triggered Jurkat cell death with staurosporine in the presence of the pan-caspase inhibitor zVAD-fmk. Under these conditions, death occurred by delayed necrosis and without exposure of PS. Nevertheless, such lethally challenged cells were phagocytosed before the loss of membrane integrity. Finally, we triggered Ca2+ influx in Jurkat cells with an ionophore, or in neurons by glutamate receptor stimulation, respectively. In both models, PS was exposed on the cell surface. Ca2+-stressed cells were phagocytosed starting at 30 min after stimulation. Protein kinase C inhibitors prevented Ca2+-mediated PS exposure and phagocytosis. Essentially, similar phagocytosis data were obtained for all models with HMDM and microglia. We conclude that also cells dying nonapoptotically and independent of caspase activation may be recognized and removed before, or very quickly after, membrane lysis.     

Homing of annexin-labeled stem cells to apoptotic cells

Ischemic diseases are characterized by the presence of pro-apoptotic stimuli, which initiate a cascade of processes that lead to cell injury and death. Several molecules and events represent detectable indicators of the different stages of apoptosis. Among these indicators is phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane, which can be detected by annexinV (ANXA5) conjugation. This is a widely used in vivo and in vitro assay marking the early stages of apoptosis. We report here on an original method that employs PS-ANXA5 conjugation to target stem cells to apoptotic cells. Mesenchymal stem cells (MSCs) from GFP-positive transgenic rats were biotinylated on membrane surfaces with sulfosuccinimidyl-6-(biotinamido) hexanoate (sulfo-NHS-LC-biot) and then bound to avidin. The avidin-biotinylated MSCs were labeled with biotin conjugated ANXA5. Bovine aortic endothelial cells (BAE-1 cells) were exposed to UVC to induce caspasedependent apoptosis. Finally, we tested the ability of ANXA5-labeled MSCs to bind BAE-1 apoptotic cells: suspended ANXA5-labeled MSCs were seeded for 1 hour on a monolayer of UV-treated or control BAE-1 cells. After washing, the number of MSCs bound to BAE-1 cells was evaluated by confocal microscopy. Statistical analysis demonstrated a significant increase in the number of MSCs tagged to apoptotic BAE-1 cells. Therefore, stem cell ANXA5 tagging via biotin-avidin bridges could be a straightforward method of improving homing to apoptotic tissues. A. Gerasimou, R. Ramella and A. Brero contributed equally to this paper.     

Phagocytosis by somatic cells from dry cow secretion

Independently of medium in which the process occurred, serum or PBS, phagocytosis and killing of Staphylococcus aureus by somatic cells from dry cow secretion were significantly higher at the early dry period than at the steady state period. Total bacterial survival was highly correlated with phagocytosis and with intracellular survival. Correlations between phagocytosis and intracellular survival were much lower. Percentage of S. aureus phagocytosed after incubation in bovine blood serum showed highly significant variation among samples of cells isolated from secretion of different cows at the early dry period and significant variation among samples of cells isolated from different cows at the steady state period.     

TUNEL vision spots apoptotic cells

RanGTP is known to regulate the spindle assembly checkpoint (SAC), but the underlying molecular mechanism is unclear. BuGZ stabilizes SAC protein Bub3 through direct interaction and facilitates its mitotic function. Here we show that RanGTP promotes the turnover of BuGZ and Bub3 in metaphase, which in turn facilitates metaphase-to-anaphase transition. BuGZ and Bub3 interact with either importin-β or an E3 ubiquitin ligase, Ubr5. RanGTP promotes the dissociation of importin-β from BuGZ and Bub3 in metaphase. This results in increased binding of BuGZ and Bub3 to Ubr5, leading to ubiquitination and subsequent turnover of both proteins. We propose that elevated metaphase RanGTP levels use Ubr5 to couple overall chromosome congression to SAC silencing.     

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.     

Degradation of chromatin in apoptotic cells

We present here a model for the degradation of chromatin in cells undergoing apoptosis. This model rationalises all aspects of the fragmentation process that have been described to date, explaining not only the patterns of degradation seen within individual cells, but also the variability in extent of degradation seen in different cells. Although DNA fragmentation in apoptosis was initially considered to be solely internucleosomal, it is now apparent that the process is much more complex and most, if not all, cells also produce much larger DNA fragments. However, in the same way that internucleosomal DNA fragmentation is a reflection of chromatin structure, the generation of these larger fragments is a reflection of chromatin structure, too. By comparing the ionic requirements for the complete pattern of chromatin degradation in nuclei with those required for apoptosis, it is apparent that the whole process may be catalysed by two pools of Mg-activated\Ca-modulated DNase I-like enzyme activities.     

Coccidioides immitis endospores: Phagocytosis by human cells

Phagocytosis of killed endospores by glass adherent peripheral human mononuclear cells was studied. Phagocytosis continued through 30 minutes of incubation. No difference in rates of ingestion could be detected when cells from coccidioidin-reactive and nonreactive subjects were compared although both groups ingested endospores more avidly than latex particles.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Stabilization of apoptotic cells: generation of zombie cells

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn²⁺ (a caspase inhibitor) and coenzyme Q₁₀ (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.Apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in multicellular organisms, including humans.¹ The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (increased apoptosis) or cancer (reduced apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of apoptosis by radiation, chemotherapy or both. In particular, many antineoplasic drugs such as campothecin, a topoisomerase I inhibitor, kill tumor cells by inducing apoptosis.Apoptosis is thought to be physiologically advantageous because apoptotic cells are removed by phagocytosis before they lose their permeability barrier, thus preventing induction of an inflammatory response to the dying cells and potential harmful secondary effects. However, when massive cell death overwhelms macrophage clearance, as for example in early postchemotherapy or viral infection,² apoptotic cells may progress to secondary necrosis characterized by cell membrane degradation with spillage of intracellular contents to the extracellular milieu.³ Similarly, cells undergoing apoptosis in vitro cannot usually be cleared by phagocytes and undergo a late process of secondary necrosis.⁴In the execution phase of apoptosis, effector caspases cleave vital cellular proteins, leading to the morphological changes that characterize apoptosis. These changes include destruction of the nucleus and other organelles, DNA fragmentation, chromatin condensation, cell shrinkage, cell detachment and membrane blebbing.⁵ In apoptosis, all the degradative processes are isolated from the extracellular space by the plasma membrane that remains impermeable. However, the mechanisms involved in plasma membrane and associated protein protection from the action of caspases are not completely understood. In contrast, necrosis is accompanied by disruption of plasma membrane integrity with the subsequent release of all intracellular compounds to the intercellular space, thus inducing inflammation and more toxic effects to adjacent cells.^{6, 7}To allow the dramatic morphological changes that accompany the execution phase, an apoptotic cell undergoes a series of profound cytoskeletal breakdowns/rearrangements. Previous evidence suggests that the actomyosin cytoskeleton plays an essential role in apoptotic cell remodeling during the early events of the execution phase, whereas all other cytoskeleton elements (microtubules and intermediate filaments) are dismantled.⁸ However, during the course of the execution phase and after actininomyosin ring contraction, the actomyosin filaments are also depolymerized by a caspase-dependent mechanism. In this situation, the apoptotic cell forms a network of apoptotic microtubules that becomes the main cytoskeleton element of the apoptotic cell. The presence of microtubules in apoptotic cells has previously been reported.^{9, 10} Moreover, more recent results indicate that microtubules during apoptosis assist in the dispersal of nuclear and cellular fragments,^{11, 12} and may help to preserve the integrity of plasma membrane of the dying cell.¹³Reactive oxygen species (ROS) are also important mediators of apoptosis. ROS have been shown to play a major role in apoptosis signaling.^{14, 15, 16} Electron leak in the presence of oxygen during the process of oxidative phosphorylation make mitochondria the major endogenous source of ROS in the cell. Although mitochondria have been identified as a key player, the mechanism connecting ROS and apoptosis remains unclear.¹⁷ It has been debated whether increased ROS during apoptosis is a cause or a consequence of impaired mitochondrial function, and whether ROS are a death signal to the mitochondria or are produced as effector molecules by the mitochondria in response to apoptosis signal.^{18, 19} Hyperproduction of ROS in execution stages of apoptosis is thought to be caused by the disruption of the mitochondrial respiratory chain after release of cytochrome c into the cytosol.²⁰The main objective of this work was to develop a method for the stabilization of apoptotic cells for proper apoptosis detection or safer potential therapeutic applications. Our results show that apoptotic cells can be stabilized by a cocktail of a microtubule stabilizer (taxol), a caspase inhibitor such (Zn²⁺) and an antioxidant (coenzyme Q₁₀ (CoQ)).     

Phagocytosis

Phagocytosis is the process of recognition and engulfment of microorganisms or tissue debris that accumulate during infection, inflammation or wound repair. This ingestion, which is performed most efficiently by migrating, bone marrow-derived cells called ‘professional phagocytes’, is essential for successful host defense. Ingestion results when an invading microorganism is recognized by specific receptors on the phagocyte surface and requires multiple, successive interactions between the phagocyte and the target. Each of these interactions results in a signal transduction event, which is confined to the membrane and cytoskeleton around the ligated receptor and which is required for successful phagocytosis. Many molecules found at sites of inflammation or infection stimulate phagocytosis, so that efficient ingestion is confined to the site of infection or inflammation, which in turn limits the proinflammatory and tissue-destructive processes that accompany phagocytosis. This review summarizes current understanding of this critical component of host defense and of its regulation.