Intact Apoptotic Cells Research Articles

Logic-Gated Proximity Aptasensing for Cell-Surface Real-Time Monitoring of Apoptosis.

In nature, intact apoptotic cells release ATP as a signaling molecule to trigger prompt phagocytic clearance, even at the earliest stage of apoptosis. Inspired by this, here we introduce a straightforward strategy for real-time monitoring ATP exocytosis and drug-stimulated apoptosis in the cancer cell surroundings. Triplex-boosted G-quadruplexes (tb-G4s) responding to cell environmental factors (H+ and K+ ) are engineered to construct a DNA logic-gated nanoplatform for proximity ATP aptasensing on the cell surface. It enables the real-time monitoring of cell apoptosis by capturing released endogenous ATP during chemotherapy drug stimulation, providing a sensitive approach for dynamically evaluating drug-induced apoptosis and therapeutic efficacy.

Logic‐Gated Proximity Aptasensing for Cell‐Surface Real‐Time Monitoring of Apoptosis

AbstractIn nature, intact apoptotic cells release ATP as a signaling molecule to trigger prompt phagocytic clearance, even at the earliest stage of apoptosis. Inspired by this, here we introduce a straightforward strategy for real‐time monitoring ATP exocytosis and drug‐stimulated apoptosis in the cancer cell surroundings. Triplex‐boosted G‐quadruplexes (tb‐G4s) responding to cell environmental factors (H+ and K+) are engineered to construct a DNA logic‐gated nanoplatform for proximity ATP aptasensing on the cell surface. It enables the real‐time monitoring of cell apoptosis by capturing released endogenous ATP during chemotherapy drug stimulation, providing a sensitive approach for dynamically evaluating drug‐induced apoptosis and therapeutic efficacy.

Innate and adaptive immune responses to dead and dying cells

Under steady state conditions, billions of dead and dying cells are removed by extrusion from epithelial surfaces as well as by phagocytosis. Cells such as macrophages and dendritic cells have specialized receptors that directly recognize altered protein or lipids on apoptotic cells or opsonins that bind to the dying cell. Once engulfed, phagosomes containing apoptotic cells are rapidly acidified and the contents degraded by proteases and nucleases in lysozymes. During necrosis, cellular material is released prior to engulfment and extracellular nucleases as well as intracellular sensors dictate the inflammatory potential of the cellular debris. The outcome may be release of TNF-a, IL-1-b or interferon (IFN)-a depending upon the type of phagocyte, molecular nature of the cellular particle and the intracellular sensor engaged. In addition to responses by cells of the innate immune system, we have recently defined a link between processing of apoptotic cells and their debris to T cell activation [1]. MFG-E8 is an opsonin (bridging protein) that binds to phosphatidylserine on apoptotic cells and facilitates their removal through interaction with integrins on phagocytes. Mice deficient in MFG-E8 develop lupus like autoimmunity associated with accumulation of apoptotic cells in vivo. We observed that older MFG-8-/- mice spontaneously developed a dermatitis associated with CD8 T cell infiltration and striking activation of effector memory CD8 T cells. T cell responses to both exogenous and endogenous apoptotic cell associated antigens were enhanced in MFG-E8 deficient mice and transfer of ovalbumin (OVA) reactive OT-I CD8 T cells caused accelerated diabetes in MFG-E8-/- RIP-mOVA mice and skin disease in kmOVA transgenic mice. The enhanced CD8 T cell response was attributed to increased cross-presentation by dendritic cells (DCs) associated with increased detection of antigen peptide MHCI complexes. Investigation of intracellular trafficking revealed that, whereas intact apoptotic cells ingested by wild type DC rapidly fused with lysosomes, in the absence of MFG-E8, smaller apoptotic cell fragments persisted in endosomal compartments and failed to fuse with lysosomes. These observations suggest that in addition to altering the rate of clearance of apoptotic cells, MFG-E8 deficiency promotes immune responses to self antigens by altered intracellular processing leading to enhanced antigen presentation. Thus, handling of dead and dying cells impacts both innate and adaptive immune responses to self antigens.

Autoimmunity in MFG-E8–deficient mice is associated with altered trafficking and enhanced cross-presentation of apoptotic cell antigens

Apoptotic cells must be rapidly cleared, as defects in this process can lead to autoimmunity. Milk fat globule EGF factor 8 (MFG-E8) binds to apoptotic cells and facilitates their removal through interaction with phagocytes. Mice deficient in MFG-E8 develop lupus-like autoimmunity associated with accumulation of apoptotic cells in vivo. Here, we have shown that MFG-E8 controls phagocytic ingestion of cell fragments as well as their intracellular processing into MHC-antigen complexes. Older Mfge8-/- mice spontaneously developed dermatitis associated with CD8+ T cell infiltration and striking activation of effector memory CD8+ T cells. CD8+ T cell responses to both exogenous and endogenous apoptotic cell-associated antigens were enhanced in Mfge8-/- mice. MFG-E8 deficiency accelerated the onset of disease in a mouse model of autoimmune diabetes. Enhanced CD8+ T cell responses were attributed to increased cross-presentation by DCs along with increased detection of antigen-MHCI complexes. Intracellular trafficking analysis revealed that intact apoptotic cells ingested by wild-type DCs rapidly fused with lysosomes, whereas smaller fragments persisted in Mfge8-/- DC endosomal compartments for 24 hours. These observations suggest that MFG-E8 deficiency promotes immune responses to self antigens not only by delaying the clearance of dying cells but also by altering intracellular processing, leading to enhanced self-antigen presentation.

Toll-like Receptor 2 and Type 1 Diabetes

Clearance of apoptotic cells by phagocytosis helps prevent the leakage of cellular contents that may stimulate inflammation or autoimmunity. Nonobese diabetic (NOD) mice develop type I diabetes (T1D) because of the impaired phagocytosis of apoptotic pancreatic β cells by macrophages; however the mechanism involved is unclear. Kim et al . (see commentary by Filippi and von Herrath) examined the responses of mouse macrophages [which enter the pancreas at the onset of T1D and later act as antigen-presenting cells (APCs) for T cells] to exposure to MIN6N8 insulinoma cells in various stages of radiation-induced cell death. Whereas incubation of macrophages with apoptotic MIN6N8 cells did not stimulate production of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), as measured by enzyme-linked immunosorbant assay (ELISA), secondary necrotic (SN) cells resulted in substantial TNF-α production. SN cells are late-stage, intact apoptotic cells that can be distinguished from apoptotic cells by flow cytometry on the basis of cell size and staining for markers of cell death and are characteristic of apoptotic cells that escaped phagocytosis. Gel-shift experiments showed that incubation of macrophages with SN MIN6N8 cells resulted in activation of the transcription factor nuclear factor-κB (NF-κB). Exposure of macrophages from Tlr2 –/– mice to SN cells resulted in a lower amount of TNF-α production than did exposure of wild-type macrophages to the cells. Tlr2 –/– mice were more resistant to T1D induced by pharmacological killing of β cells than were wild-type mice (as assessed by histological analysis of islets). Coculture and adoptive transfer experiments in mice showed that TLR2 was necessary for the maturation of APCs and the subsequent activation and proliferation of T cells that mediate T1D. These results link the accumulation of apoptotic cells with TLR2-dependent initiation of autoimmunity, raising the possibility of targeting TLR2 signaling in apoptosis-related diseases and autoimmune disorders. H. S. Kim, M. S. Han, K. W. Chung, S. Kim, E. Kim, M. J. Kim, E. Jang, H. A. Lee, J. Youn, S. Akira, M.-S. Lee, Toll-like receptor 2 senses β-cell death and contributes to the initiation of autoimmune diabetes. Immunity 27 , 321-333 (2007). [PubMed] C. M. Filippi, M. G. von Herrath, Islet β-cell death: Fuel to sustain autoimmunity? Immunity 27 , 183-185 (2007). [Online Journal]

Toll-like Receptor 2 Senses β-Cell Death and Contributes to the Initiation of Autoimmune Diabetes

Although it is established that defective clearance and, hence, increased accumulation of apoptotic cells can lead to autoimmunity, the mechanism by which this occurs remains elusive. Here, we observed that apoptotic cells undergoing secondary necrosis but not intact apoptotic cells provoked substantial immune responses, which were mediated through the toll-like receptor 2 (TLR2) pathway. The development of autoimmune diabetes was markedly inhibited in Tlr2(-/-) mice but not in Tlr4(-/-) mice, showing that TLR2 plays an important role in the initiation of the disease. Apoptotic beta-cell injury could stimulate the priming of diabetogenic T cells through a TLR2-dependent, but TLR4-independent, activation of antigen-presenting cells. These findings suggest that beta-cell death and its sensing via TLR2 may be an initial event for the stimulation of antigen-presenting cells and development of autoimmune diabetes.

Relative timing of redistribution of cytochrome c and Smac/DIABLO from mitochondria during apoptosis assessed by double immunocytochemistry on mammalian cells

Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (≤15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.

Oxidised lipoproteins may promote inflammation through the selective delay of engulfment but not binding of apoptotic cells by macrophages

During normal tissue homeostasis apoptotic cells (AC) are rapidly recognised and engulfed by neighbouring cells or macrophages (Mφ), thus preventing an inflammatory response. Conversely, in chronically inflamed tissues, including the atherosclerotic artery and rheumatoid joint, removal of AC is defective despite the co-localisation of seemingly adequate numbers of Mφ. Mechanisms preventing removal of AC in vivo remain obscure, but might include oxidised low-density lipoprotein (ox-LDL), which is abundant in chronic inflammatory lesions. Although implicated in their pathogenesis, defining the role of ox-LDL on inflammatory processes has proved complicated. In fact, seemingly contradictory results have previously been described, though these may in part reflect the heterogeneous nature of ox-LDL applied in these studies. We wished to investigate the effect of physiologically representative ox-LDL on the binding and engulfment of apoptotic vascular smooth muscle cells (VSMC) and fibroblasts, as these have previously been shown to co-localise with Mφ in chronically inflamed tissues in vivo. We show that Mφ recognition of AC in vitro is not affected at physiological levels of ox-LDL. However, engulfment of intact AC is dramatically reduced/delayed. Importantly, in the absence of ox-LDL rapid phagocytosis of intact AC suppresses Mφ inflammatory cytokine release. In striking contrast, in the presence of ox-LDL, despite binding of AC to Mφ, release of IL-6 and MCP-1 is no longer suppressed. We propose that ox-LDL could maintain an inflammatory response by inhibiting the engulfment of AC, required for Mφ de-activation. This mechanism may contribute to chronic persisting inflammation in the atherosclerotic artery and rheumatoid joint.

Controlling phagocytosis: brief encounter or death embrace?

Phagocytosis defines apoptotic cell death: in its absence, secondary necrosis and eventual cytolysis would release histotoxic mediators. The engulfment of intact apoptotic cells by phagocytes, the removal specialists of the immune system, is crucial for limiting inflammatory and immune responses. The complex mechanisms by which phagocytic cells, such as macrophages, distinguish between viable and dying cells are the subject of intense interest. Previously, recognition of apoptotic cells was thought to be mediated by the presentation of ‘eat me’ signals on the surface of the dying cell. However, Brown et al. [1xApoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Brown, S. et al. Nature. 2002; 418: 200–203Crossref | PubMed | Scopus (215)See all References[1] describe the potential discrimination of cell fate on the basis of generation of detachment signals by viable cells.Brown et al. investigated cellular interactions under shear stress to reproduce conditions in vivo more faithfully. Under these conditions, they observed temperature-dependent tethering of peripheral blood leukocytes to macrophages. A temperature shift from 20°C to 37°C caused disruption of the interaction between macrophages and viable cells, indicating active detachment; by contrast, apoptotic cells remained tethered. Using plasma membrane-enriched fractions of leukocytes to isolate macrophage-bound proteins, the cell surface adhesion molecule CD31 was purified. A member of the immunoglobulin superfamily, CD31 is expressed on leukocytes and phagocytes, and was identified as a potential mediator of tethering. Viable CD31-negative T-cell lines fail to bind to macrophages under low shear stress, even at previously permissive temperatures, and transfection of functional CD31 reintroduces the temperature-sensitive binding phenotype. Expression of a ‘signalling disabled’ CD31 mutant inhibits detachment of viable cells at 37oC, indicating that detachment requires intracellular signalling downstream of CD31 binding. Moreover, disruption of CD31-derived signalling was observed following induction of apoptosis, as shown by impaired recruitment of secondary signalling molecules. In a static assay to assess engulfment, inhibition of CD31 with blocking antibody, on either macrophage or leukocyte subset, inhibits ingestion, consistent with homophilic interaction of CD31 molecules on the phagocyte and target cell. Apoptotic CD31-negative cells are still ingested by phagocytes, albeit at a reduced rate compared with parental CD31-positive cells, implicating CD31-dependent interactions contribute to the efficiency of phagocytosis. However, this also indicates the importance of recognition mediated by alternative engulfment signals, such as externalization of phosphatidylserine on the apoptotic cell.The authors propose that, under conditions of flow, initial interaction between phagocytes and dying cells occurs through CD31. Such molecular interaction determines whether the encounter between phagocyte and prey will be fleeting or result in the ‘death embrace’ of phagocytosis. In healthy cells a signalling pathway, activated by binding of CD31, results in the generation of a repulsion signal and subsequent detachment. In dying cells, no such signal is generated, thereby facilitating prolonged interaction and engagement of various ‘eat me’ signals presented on the surface of the doomed cell. The intriguing nature of the repulsion signal and the mechanism responsible for apoptosis-associated dysfunction of CD31 signalling are eagerly anticipated.

C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells.

Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the alpha-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates.

To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(beta-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

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.

Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.

Proteolysis mediated by the interleukin 1 beta-converting enzyme (ICE) homologues is an important mechanism of the apoptotic process. The ICE homologue apopain/CPP-32/Yama (subsequently referred to as apopain) cleaves poly(ADP-ribose)polymerase (PARP) early during apoptosis. Additional apoptosis-specific protein cleavages have been observed in which the direct involvement of ICE-like proteases has been postulated. These substrates include the 70-kD protein component of the U1-ribonucleoprotein (U1-70kD), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The present studies demonstrate that U1-70kD and DNA-PKcs are excellent substrates for apopain, with cleavage occurring at sites that are highly similar to the cleavage site within PARP. The fragments generated from isolated protein substrates by apopain are identical to those observed in intact apoptotic cells, in apoptotic cell extracts, and in normal cell extracts to which apopain has been added. Like PARP, cleavage of these substrates in apoptotic cell extracts is abolished by nanomolar concentrations of Ac-DEVD-CHO and micromolar amounts of Ac-YVAD-CHO, confirming the involvement of apopain or an apopain-like activity. We propose that a central function of apopain or similar homologues in apoptosis is the cleavage of nuclear repair proteins, thereby abolishing their critical homeostatic functions.

A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation

Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies relatively small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342 bright lymphocytes by conventional immunofluoresce techniques and to evaluate membrane integrity of Hoechst 33342 bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact cells containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death.