Killing me softly - suicidal erythrocyte death

Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is characterized by cell shrinkage, cell membrane blebbing and cell membrane phospholipid scrambling. Eryptotic cells are removed and thus prevented from undergoing hemolysis. Eryptosis is stimulated by Ca(2+) following Ca(2+) entry through unspecific cation channels. Ca(2+) sensitivity is enhanced by ceramide, a product of acid sphingomyelinase. Eryptosis is triggered by hyperosmolarity, oxidative stress, energy depletion, hyperthermia and a wide variety of xenobiotics and endogenous substances. Eryptosis is inhibited by nitric oxide, catecholamines and a variety of further small molecules. Erythropoietin counteracts eryptosis in part by inhibiting the Ca(2+)-permeable cation channels but by the same token may foster formation of erythrocytes, which are particularly sensitive to eryptotic stimuli. Eryptosis is triggered in several clinical conditions such as iron deficiency, diabetes, renal insufficiency, myelodysplastic syndrome, phosphate depletion, sepsis, haemolytic uremic syndrome, mycoplasma infection, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase-(G6PD)-deficiency, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, and Wilson's disease. Enhanced eryptosis is observed in mice with deficient annexin 7, cGMP-dependent protein kinase type I (cGKI), AMP-activated protein kinase AMPK, anion exchanger AE1, adenomatous polyposis coli APC and Klotho as well as in mouse models of sickle cell anemia and thalassemia. Eryptosis is decreased in mice with deficient phosphoinositide dependent kinase PDK1, platelet activating factor receptor, transient receptor potential channel TRPC6, janus kinase JAK3 or taurine transporter TAUT. If accelerated eryptosis is not compensated by enhanced erythropoiesis, clinically relevant anemia develops. Eryptotic erythrocytes may further bind to endothelial cells and thus impede microcirculation.     

Mechanisms of suicidal erythrocyte death.

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure ('eryptosis') mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in 'eryptosis' may similarly contribute to apoptosis of nucleated cells.     

Stimulation of erythrocyte cell membrane scrambling by amiodarone.

Side effects of amiodarone, an effective antiarrhythmic drug, include anemia, which may be caused by decreased formation or accelerated death of erythrocytes. Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Stimulators of erythrocyte membrane scrambling include increase of cytosolic Ca2+ concentration ([Ca2+]i) following activation of Ca2+-permeable cation channels. Moreover, eryptosis is triggered by ceramide. The present study has been performed to test for an effect of amiodarone on eryptosis. Erythrocytes from healthy volunteers were exposed to amiodarone and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), [Ca2+]i (Fluo3-dependent fluorescence), and ceramide formation (anti-ceramide-FITC antibody and radioactive labelling) determined by flow cytometry. Exposure of erythrocytes to amiodarone (1 microM) increased [Ca2+]i and triggered annexin V binding, but did not significantly decrease forward scatter and did not significantly influence ceramide formation. Amiodarone augmented the increase of annexin binding following hypertonic shock (addition of 550 mM sucrose) but did not significantly alter the enhanced annexin binding following Cl- removal (replacement with gluconate). Amiodarone did not significantly modify the decrease of forward scatter following hypertonic shock or Cl- removal. The present observations disclose a novel action of amiodarone which may contribute to the side effects of the drug.     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca²⁺-permeable cation channels with subsequent entry of Ca²⁺ and stimulation of Ca²⁺-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl^– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca²⁺ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E₂ (PGE₂) formation, cation channel activity, Ca²⁺ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl^– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE_2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca²⁺ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes. annexin V; osmotic cell shrinkage; calcium; apoptosis     

Dicoumarol activates Ca2+-permeable cation channels triggering erythrocyte cell membrane scrambling

Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether dicoumarol induces eryptosis. [Ca(2+)](i) was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 μM) significantly increased [Ca(2+)](i), enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 μM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of Ca(2+). In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca(2+) entry and subsequent triggering of Ca(2+) dependent cell membrane scrambling.     

Ceranib‐2‐induced suicidal erythrocyte death

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca²⁺‐activity ([Ca²⁺]_i) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib‐2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine‐exposure at the cell surface from annexin‐V‐binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3‐fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib‐2 significantly increased the percentage of annexin‐V‐binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib‐2 significantly increased Fluo3‐fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib‐2 on annexin‐V‐binding was not significantly blunted by removal of extracellular Ca²⁺. Ceranib‐2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca²⁺ entry. Copyright © 2016 John Wiley & Sons, Ltd.     

Studying Mechanisms of Eryptosis

Cellular stress leads to activation of erythrocyte cation channels with subsequent Ca²⁺ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Both signaling molecules then activate the death program of erythrocytes (eryptosis) which is characterized by phosphatidylserine exposure, cellular shrinkage, membrane blebbing and activation of death-inducing proteases. Some of the mediators accounting for activation of the erythrocyte death machinery, i. e. prostaglandin E₂ (PGE₂) and platelet activating factor (PAF), have been described and the respective signaling cascades disclosed. The present article outlines and discusses the methods which have been used to analyze erythrocyte death pathways. Furthermore, some of the pathophysiological implications of eryptosis signaling are delineated and the methods to screen for eryptosis defects in those conditions are presented. Needless to say that further research will be required to fully understand the mechanisms leading to suicidal red blood cell death and to elucidate the role of eryptosis during anemic complications.     

Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca²⁺ activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca²⁺-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin–luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca²⁺-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca²⁺. The present observations disclose a novel effect of chromium (VI), i.e. Ca²⁺ entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.     

Effect of anandamide on erythrocyte survival.

The endocannabinoid anandamide (Arachidonylethanolamide, AEA) is known to induce apoptosis in a wide variety of nucleated cells. The present study explored whether anandamide induces suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus cleared from circulating blood. Triggers of eryptosis include increase of cytosolic Ca2+ activity, formation of PGE(2), oxidative stress and excessive cell shrinkage. Erythrocyte Ca2+ activity was estimated from Fluo3 fluorescence, phosphatidylserine exposure from annexin V binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to anandamide (= 2.5 microM) increased cytosolic Ca2+ activity, enhanced the percentage of annexin V binding erythrocytes and decreased erythrocyte forward scatter, effects significantly blunted in the presence of cycloxygenase inhibitors acetylsalicylic acid (50 microM) or ibuprofen (100 microM) and in the nominal absence of extracellular Ca2+. Anandamide further enhanced the stimulating effects of hypertonic (addition of 550 mM sucrose) or isotonic (isosmotic replacement of Cl- with gluconate) cell shrinkage on annexin V binding. The present observations demonstrate that anandamide increases cytosolic Ca2+ activity, thus leading to cell shrinkage and cell membrane scrambling of mature erythrocytes.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

Curcumin induced suicidal erythrocyte death.

The natural nutrient component Curcumin with anti-inflammatory and antitumor activity has previously been shown to stimulate apoptosis of several nucleated cell types. The present study has been performed to explore whether Curcumin could similarly induce suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing cells are phagocytosed and thus rapidly cleared from circulating blood. Erythrocyte membrane scrambling may be triggered by increase of cytosolic Ca(2+) activity or formation of ceramide. To test for eryptosis, erythrocyte phosphatidylserine exposure has been estimated from annexin V binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to Curcumin (= 1 microM) increased annexin V binding and decreased forward scatter, pointing to phosphatidylserine exposure at the cell surface and cell shrinkage. According to Fluo3 fluorescence Curcumin increased cytosolic Ca(2+) activity and according to immunofluorescence Curcumin increased ceramide formation. As shown previously, hypertonic shock (addition of 550mM sucrose), chloride removal and glucose depletion decreased the forward scatter and increased annexin V binding. The effects on annexin binding were enhanced in the presence of Curcumin. Exposure to Curcumin did, however, not significantly enhance the shrinking effect of hypertonic shock or Cl(-) removal and reversed the shrinking effect of glucose withdrawal. The present observations disclose a proeryptotic effect of Curcumin which may affect the life span of circulating erythrocytes.     

Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin

Eryptosis, a suicidal death of mature erythrocytes, is characterized by decrease of cell volume, cell membrane blebbing, and breakdown of cell membrane asymmetry with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increased cytosolic Ca(2+) activity, which could result from activation of Ca(2+)-permeable cation channels. Ca(2+) triggers phosphatidylserine exposure and activates Ca(2+)-sensitive K(+) channels, leading to cellular K(+) loss and cell shrinkage. The cation channels and thus eryptosis are stimulated by Cl(-) removal and inhibited by erythropoietin. The present experiments explored eryptosis in transgenic mice overexpressing erythropoietin (tg6). Erythrocytes were drawn from tg6 mice and their wild-type littermates (WT). Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorting (FACS) analysis. The percentage of annexin binding was significantly larger and forward scatter significantly smaller in tg6 than in WT erythrocytes. Transgenic erythrocytes were significantly more resistant to osmotic lysis than WT erythrocytes. Cl(-) removal and exposure to the Ca(2+) ionophore ionomycin (1 microM) increased annexin binding and decreased forward scatter, effects larger in tg6 than in WT erythrocytes. The K(+) ionophore valinomycin (10 nM) triggered eryptosis in both tg6 and WT erythrocytes and abrogated differences between genotypes. An increase of extracellular K(+) concentration to 125 mM blunted the difference between tg6 and WT erythrocytes. Fluo-3 fluorescence reflecting cytosolic Ca(2+) activity was larger in tg6 than in WT erythrocytes. In conclusion, circulating erythrocytes from tg6 mice are sensitized to triggers of eryptosis but more resistant to osmotic lysis, properties at least partially due to enhanced Ca(2+) entry and increased K(+) channel activity.     

Eryptosis triggered by bismuth

Bismuth is used for multiple industrial purposes and in the treatment of several gastrointestinal diseases. Untoward effects of bismuth include anemia, which could, in theory, result from suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. Signaling leading to eryptosis includes increase in cytosolic Ca²⁺ activity and formation of ceramide. The present experiments explored whether bismuth elicits eryptosis. To this end, phosphatidylserine exposure was estimated from annexin V-binding, cell shrinkage from decrease of forward scatter in FACS analysis, cytosolic Ca²⁺ activity from Fluo3 fluorescence and ceramide abundance from binding of fluorescent antibodies. A 48 h exposure to bismuth (≥500 μg/l BiCl₃) enhanced the percentage of annexin V-binding cells and decreased forward scatter, increased cytosolic Ca²⁺ activity, and stimulated ceramide formation. In conclusion, bismuth stimulates eryptosis, the suicidal death of erythrocytes. The effect may contribute to or even account for the development of anemia during bismuth treatment. Moreover, ceramide formation in intestinal cells may participate in the therapeutic efficacy of bismuth preparations. This study was supported by the Deutsche Forschungsgemeinschaft SFB 766 and the Carl Zeiss Stiftung. The experimental work and studies on human subjects have been approved by the Ethical commission of the University of Tübingen.     

Inhibition of suicidal erythrocyte death by blebbistatin

Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.     

PGE2-induced apoptotic cell death in K562 human leukaemia cells.

Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.     

Stimulation of erythrocyte phosphatidylserine exposure by paclitaxel.

Side effects of cytostatic treatment include development of anemia resulting from either decreased generation or accelerated clearance of circulating erythrocytes. Recent experiments revealed a novel kind of stress-induced erythrocyte death, i.e. eryptosis, which is characterized by enhanced cytosolic Ca(2+) levels, increased ceramide formation and exposure of phosphatidylserine at the cell surface. The present study explored whether cytostatic treatment with paclitaxel (Taxol) triggers eryptosis. Blood was drawn from cancer patients before and after infusion of 175 mg/m2 Taxol. The treatment significantly decreased the hematocrit and significantly increased the percentage of annexin-V-binding erythrocytes in vivo (by 37%). In vitro incubation of human erythrocytes with 10 microM paclitaxel again significantly increased annexin-V-binding (by 129%) and augmented the increase of annexin-V-binding following cellular stress. The enhanced phosphatidylserine exposure was not dependent on caspase-activity but paralleled by erythrocyte shrinkage, increase of cytosolic Ca(2+) activity, ceramide formation and activation of calpain. Phosphatidylserine exposure was similarly induced by docetaxel but not by carboplatin or doxorubicin. Moreover, eryptosis was triggered by the Ca(2+) ionophore ionomycin (10 microM). In mice, ionomycin-treated eryptotic erythrocytes were rapidly cleared from circulating blood and sequestrated into the spleen. In conclusion, our data strongly suggest that paclitaxel-induced anemia is at least partially due to induction of eryptosis.     

Stimulation of suicidal erythrocyte death by lipoxygenase inhibitor Bay-Y5884.

The prostaglandin PGE(2), a metabolite of the cyclooxygenase pathway, activates Ca(2+)-permeable cation channels in erythrocyte cell membranes leading to entry of Ca(2+) with subsequent eryptosis, i.e. cell shrinkage, breakdown of phosphatidylserine (PS) asymmetry and membrane blebbing, all features typical for apoptosis in nucleated cells. PS exposing cells are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether the specific lipoxygenase inhibitor Bay-Y5884 influences eryptosis. As determined by competitive ELISA, Bay-Y5884 (20 microM) enhanced the release of PGE(2) from human erythrocytes. According to whole-cell patch-clamp, Bay-Y5884 (20 microM) activated nonselective cation channels. The effect of Bay-Y5884 on cation channels was abolished by the cyclooxygenase inhibitor diclophenac (10 microM). Bay-Y5884 (30-40 microM) significantly increased erythrocyte free Ca(2+) concentration and PS exposure as analyzed in flow cytometry by Fluo3 fluorescence and annexin-V binding, respectively. PS exposure triggered by 20 microM (but not by 40 microM) Bay-Y5884 was blunted by cyclooxygenase inhibitors acetylsalicylic acid (50 microM) and diclophenac (10 microM). In conclusion, the lipoxygenase inhibitor Bay-Y5884 enhances erythrocyte PGE(2) formation with subsequent activation of non-selective cation channels, Ca(2+) entry and phospholipid scrambling.     

Induction of suicidal erythrocyte death by listeriolysin from Listeria monocytogenes.

Listeriolysin, the secreted cytolysin of the facultative intracellular bacterium Listeria monocytogenes, is its major virulence factor. Previously, non-lytic concentrations of listeriolysin were shown to induce Ca2+-permeable nonselective cation channels in human embryonic kidney cells. In erythrocytes, Ca2+ entry is followed by activation of K+ channels resulting in K+-exit as well as by membrane scrambling resulting in phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Phosphatidylserine exposure is a key event of eryptosis, the suicidal death of erythrocytes. The present study utilized patch-clamp technique, Fluo3-fluorescence, and annexin V-binding in FACS analysis to determine the effect of listeriolysin on cell membrane conductance, cytosolic free Ca2+ concentration, and phosphatidylserine exposure, respectively. Within 30 minutes, exposure of human peripheral blood erythrocytes to low concentrations of listeriolysin (which were non-hemolytic for the majority of cells) induced a Ca2+-permeable cation conductance in the erythrocyte cell membrane, increased cytosolic Ca2+ concentration, and triggered annexin V-binding. Increase of extracellular K+ concentration blunted, but did not prevent, listeriolysin-induced annexin V-binding. In conclusion, listeriolysin triggers suicidal death of erythrocytes, an effect at least partially due to depletion of intracellular K+. Listeriolysin induced suicidal erythrocyte death could well contribute to the pathophysiology of L. monocytogenes infection.     

p38 MAPK activation and function following osmotic shock of erythrocytes

p38 protein kinase is activated by hyperosmotic shock, participates in the regulation of cell volume sensitive transport and metabolism and is involved in the regulation of various physiological functions including cell proliferation and apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include hyperosmotic shock, which increases cytosolic Ca(2+) activity and ceramide formation. The present study explored whether p38 kinase is expressed in human erythrocytes, is activated by hyperosmotic shock and participates in the regulation of eryptosis. Western blotting was utilized to determine phosphorylation of p38 kinase, forward scatter to estimate cell volume, annexin V binding to depict phosphatidylserine exposure and Fluo3 fluorescence to estimate cytosolic Ca(2+) activity. As a result, erythrocytes express p38 kinase, which is phosphorylated upon osmotic shock (+550 mM sucrose). Osmotic shock decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence, all effects significantly blunted by the p38 kinase inhibitors SB203580 (2 μM) and p38 Inh III (1 μM). In conclusion, p38 kinase is expressed in erythrocytes and participates in the machinery triggering eryptosis following hyperosmotic shock.