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.     

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.     

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.     

Suicidal erythrocyte death following cellular K+ loss.

Hallmarks of apoptosis include cell shrinkage, which is at least partially due to cellular K(+) loss. The decline of cellular K(+) concentration has been suggested to participate in the triggering of apoptosis. Suicidal erythrocyte death or eryptosis is triggered by increased cytosolic Ca(2+) activity leading to activation of Ca(2+)-sensitive K(+) channels with subsequent cellular K(+) loss and cell shrinkage, and to Ca(2+)-sensitive scambling of the cell membrane with subsequent phosphatidylserine (PS) exposure at the cell surface. Phosphatidylserine exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether cellular loss of K(+) and/or cell shrinkage actively participate in the triggering of cell membrane phospholipid scrambling. Cellular K(+) loss was achieved by treatment of human erythrocytes with the K(+) ionophore valinomycin (1 nM) at different extracellular K(+) concentrations (5-125 mM) and osmolarities (300-550 m Osm). Cell volume was estimated from forward scatter and PS exposure from annexin V binding in FACS analysis. Treatment with 1 nM valinomycin indeed decreased forward scatter and increased annexin V binding. The effect was significantly blunted in the presence of staurosporine (1 microM). Increase of extracellular K(+) concentration gradually blunted the decrease of forward scatter but inhibited annexin V binding only at extracellular K(+) concentrations >or=75 mM. An increase of extracellular osmolarity (+150 mM or 250 mM sucrose) reversed the protective effect of 75 mM KCl during valinomycin treatment. A correlation between forward scatter and annexin binding at different osmolarities and K(+) concentrations suggests that the cellular K(+) content determines the rate of suicidal erythrocyte death primarily through its influence on cell volume.     

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.     

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.     

Retinoic acid induced suicidal erythrocyte death.

Vitamin A and retinoic acid have previously been shown to confer some protection against a severe course of malaria by fostering the phagocytosis of parasitized erythrocytes. Phagocytosis of erythrocytes is stimulated by phosphatidylserine exposure at the cell surface. The present study has thus been performed to explore the effect of retinoic acid and the specific retinoic acid receptor (RAR) agonist 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl) benzoic acid (TTNPB) on erythrocyte annexin V binding, which reflects phosphatidylserine exposure at the cell surface. A 24 hours exposure to either, retinoic acid (3 microM) or TTNPB (3 microM), indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter reflecting cell shrinkage. According to Fluo3 fluorescence, exposure to either, retinoic acid (10 microM, 24 hours) or TTNPB (10 microM, 6 hours), significantly increased cytosolic Ca(2+)-activity, a known trigger of phosphatidylserine exposure. Infection of erythrocytes with Plasmodium falciparum increased phosphatidylserine exposure, an effect increased in the presence of TTNPB. In conclusion, retinoid acid and TTNPB trigger phosphatididylserine exposure and cell shrinkage of erythrocytes, typical features of suicidal erythrocyte death or eryptosis. The eryptosis could participate in the accelerated clearance of parasitized erythrocytes from circulating blood following treatment with retinoids.     

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene

Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.     

Protein kinase CK1α regulates erythrocyte survival

Protein kinase CK1 (casein kinase 1) isoforms are involved in the regulation of various physiological functions including apoptosis. The specific CK1 inhibitor D4476 may either inhibit or foster apoptosis. Similar to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity following energy depletion (removal of glucose) or oxidative stress (exposure to the oxidant tert-butyl hydroperoxide [TBOOH]). Western blotting was utilized to verify that erythrocytes express the protein kinase CK1α, and FACS analysis to determine whether the CK1 inhibitor D4476 and CK1α activator pyrvinium pamoate modify forward scatter (reflecting cell volume), annexin V binding (reflecting phosphatidylserine exposure), and Fluo3 fluorescence (reflecting cytosolic Ca(2+) activity). As a result, both, human and murine erythrocytes express CK1 isoform α. Glucose depletion (48 hours) and exposure to 0.3 mM TBOOH (30 minutes) both decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence. CK1 inhibitor D4476 (10 μM) significantly blunted the decrease in forward scatter, the increase in annexin V binding and the increase in Fluo 3 fluorescence. (R)-DRF053, another CK1 inhibitor, similarly blunted the increase in annexin V binding upon glucose depletion. The CK1α specific activator pyrvinium pamoate (10 μM) significantly enhanced the increase in annexin V binding and Fluo3 fluorescence upon glucose depletion and TBOOH exposure. In the presence of glucose, pyrvinium pamoate slightly but significantly increased Fluo3 fluorescence. In conclusion, CK1 isoform α participates in the regulation of erythrocyte programmed cell death by modulating cytosolic Ca(2+) activity.     

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.     

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.     

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.     

Protein kinase C activation induces phosphatidylserine exposure on red blood cells

We have shown previously that red blood cells (RBCs) can be induced to influx Ca(2+) when treated with lipid mediators, such as lysophosphatidic acid and prostaglandin E(2), that are released during clot formation. Since calcium loading of RBCs can lead to both protein kinase C (PKC) activation and phosphatidylserine (PS) exposure, we decided to investigate the possible linkage between PKC activation and membrane PS scrambling using phorbol 12-myristate-13-acetate (PMA), a commonly used activator of PKC. Treatment of RBCs with PMA in a calcium-containing buffer caused immediate PS exposure in an RBC subpopulation. The size of the subpopulation did not change upon further incubation, indicating that not all RBCs are equally susceptible to this treatment. Using a fluorescent indicator, we found a subpopulation of RBCs with elevated intracellular calcium levels. In the absence of extracellular calcium, no PS exposure was found. However, we did find cells with high levels of calcium that did not expose PS, and a variable percentage of PS-exposing cells that did not show elevated calcium concentrations. Inhibition of PKC with either calphostin C, a blocker of the PMA binding site, or chelerythrine chloride, an inhibitor of the active site, diminished the level of formation of PS-exposing cells. However, the inhibitors had different effects on calcium internalization, indicating that a high calcium concentration alone was not responsible for inducing PS exposure in the absence of PKC activity. Moreover, PKC inhibition could prevent PS exposure induced by calcium and ionophore treatment of RBCs. We conclude that PKC is implicated in the mechanism of membrane phospholipid scrambling.     

PGE(2) in the regulation of programmed erythrocyte death

Hyperosmotic shock, energy depletion, or removal of extracellular Cl(-) activates Ca(2+)-permeable cation channels in erythrocyte membranes. Subsequent Ca(2+) entry induces erythrocyte shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. PS-exposing cells are engulfed by macrophages. The present study explored the signalling involved. Hyperosmotic shock and Cl(-) removal triggered the release of prostaglandin E(2) (PGE(2)). In whole-cell recording, activation of the cation channels by Cl(-) removal was abolished by the cyclooxygenase inhibitor diclophenac. In FACS analysis, phospholipase-A(2) inhibitors quinacrine and palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibitors acetylsalicylic acid and diclophenac, blunted the increase of PS exposure following Cl(-) removal. PGE(2) (but not thromboxane) induced cation channel activation, increase in cytosolic Ca(2+) concentration, cell shrinkage, PS exposure, calpain activation, and ankyrin-R degradation. The latter was attenuated by calpain inhibitors-I/II, while PGE(2)-induced PS exposure was not. In conclusion, hyperosmotic shock or Cl(-) removal stimulates erythrocyte PS exposure through PGE(2) formation and subsequent activation of Ca(2+)-permeable cation channels.     

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.     

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.     

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.     

Contributions of Ca2+ to galectin-1-induced exposure of phosphatidylserine on activated neutrophils

Apoptotic cells redistribute phosphatidylserine (PS) to the cell surface by both Ca(2+)-dependent and -independent mechanisms. Binding of dimeric galectin-1 (dGal-1) to glycoconjugates on N-formyl-Met-Leu-Phe (fMLP)-activated neutrophils exposes PS and facilitates neutrophil phagocytosis by macrophages, yet it does not initiate apoptosis. We asked whether dGal-1 initiated Ca(2+) fluxes that are required to redistribute PS to the surface of activated neutrophils. Prolonged occupancy by dGal-1 was required to maximally mobilize PS to the surfaces of fMLP-activated neutrophils. Like fMLP, dGal-1 rapidly elevated cytosolic Ca(2+) levels in Fluo-4-loaded neutrophils. An initial Ca(2+) mobilization from intracellular stores was followed by movement of extracellular Ca(2+) to the cytosolic compartment, with return to basal Ca(2+) levels within 10 min. Chelation of extracellular Ca(2+) did not prevent PS mobilization. Chelation of intracellular Ca(2+) revealed that fMLP and dGal-1 independently release Ca(2+) from intracellular stores that cooperate to induce optimal redistribution of PS. Ca(2+) mobilization by ionomycin did not permit dGal-1 to mobilize PS, indicating that fMLP initiated both Ca(2+)-dependent and -independent signals that facilitated dGal-1-induced exposure of PS. dGal-1 elevated cytosolic Ca(2+) and mobilized PS through a pathway that required action of Src kinases and phospholipase Cgamma. These results demonstrate that transient Ca(2+) fluxes contribute to a sustained redistribution of PS on neutrophils activated with fMLP and dGal-1.     

Effect of cyclosporine on parasitemia and survival of Plasmodium berghei infected mice

Cyclosporine triggers suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. The present study explored whether cyclosporine influences eryptosis of Plasmodium infected erythrocytes, development of parasitemia and thus the course of the disease. Annexin V binding was utilized to depict phosphatidylserine exposure and forward scatter in FACS analysis to estimate erythrocyte volume. In vitro infection of human erythrocytes with Plasmodium falciparum increased annexin binding and decreased forward scatter, effects potentiated by cyclosporine (> or = 0.01 microM). Cyclosporine (> or = 0.001 microM) significantly decreased intraerythrocytic DNA/RNA content and in vitro parasitemia (> or = 0.01 microM). Administration of cyclosporine (5 mg/kg b.w.) subcutaneously significantly decreased the parasitemia (from 47% to 27% of circulating erythrocytes 20 days after infection) and increased the survival of P. berghei infected mice (from 0% to 94% 30 days after infection). In conclusion, cyclosporine augments eryptosis, decreases parasitemia and enhances host survival during malaria.