Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phase I/II/III clinical trials. Erythrocytes do not have a nucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin­V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Geraniin inhibits whole blood IFN-γ and IL-6 and promotes IL-1ß and IL-8, and stimulates calcium-dependent and sucrose-sensitive erythrocyte death.

Correlations between circulating cytokine levels and disease states are well established, and pharmacological modulation of the immune response is thus an important aspect of the assessment of investigational new drugs. Moreover, chemotherapy-related anemia is a major obstacle in cancer treatment. Geraniin (GRN), a tannin extracted from Geranium and other plants, possesses promising antitumor potential. However, the effect of GRN on whole blood (WB) cytokine response and RBC physiology remains unexplored. Heparinized blood from consented, healthy adults was challenged with 100 ng/mL of lipopolysaccharide (LPS) with and without pretreatment with 10 µM of GRN for 24 h at 37 °C, and tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, IL-8, and IL-10 were assayed by ELISA. Moreover, single-cell RBC suspensions were treated with 5-100 µM of GRN for 24 or 48 h at 37 °C and cytotoxicity and canonical eryptotic markers were examined by flow cytometry. It was revealed that GRN significantly attenuated LPS-induced IFN-γ levels, increased IL-1ß, decreased IL-6 only in absence of LPS, and aggravated LPS-induced IL-8 while together with LPS significantly diminished IL-10. Furthermore, GRN induced dose-responsive, Ca2+-dependent, and sucrose-sensitive hemolysis, along with phosphatidylserine exposure and Ca2+ accumulation with no appreciable cell shrinkage or oxidative damage. GRN was also selectively toxic to platelets, significantly delayed reticulocyte maturation, and significantly disrupted leukocyte proportions. In conclusion, GRN regulates the WB cytokine response and promotes premature hemolysis and eryptosis. This study provides insights into the therapeutic utility of GRN in a highly relevant cellular model system.

Lauric Acid, a Dietary Saturated Medium-Chain Fatty Acid, Elicits Calcium-Dependent Eryptosis.

Cardiovascular diseases (CVD) are a leading cause of mortality worldwide, and dietary habits represent a major risk factor for dyslipidemia; a hallmark of CVD. Saturated fatty acids contribute to CVD by aggravating dyslipidemia, and, in particular, lauric acid (LA) raises circulating cholesterol levels. The role of red blood cells (RBCs) in CVD is increasingly being appreciated, and eryptosis has recently been identified as a novel mechanism in CVD. However, the effect of LA on RBC physiology has not been thoroughly investigated. RBCs were isolated from heparin-anticoagulated whole blood (WB) and exposed to 50-250 µM of LA for 24 h at 37 °C. Hemoglobin was photometrically examined as an indicator of hemolysis, whereas eryptosis was assessed by Annexin V-FITC for phosphatidylserine (PS) exposure, Fluo4/AM for Ca2+, light scatter for cellular morphology, H2DCFDA for oxidative stress, and BODIPY 581/591 C11 for lipid peroxidation. WB was also examined for RBC, leukocyte, and platelet viability and indices. LA caused dose-responsive hemolysis, and Ca2+-dependent PS exposure, elevated erythrocyte sedimentation rate (ESR), cytosolic Ca2+ overload, cell shrinkage and granularity, oxidative stress, accumulation of lipid peroxides, and stimulation of casein kinase 1α (CK1α). In WB, LA disrupted leukocyte distribution with elevated neutrophil-lymphocyte ratio (NLR) due to selective toxicity to lymphocytes. In conclusion, this report provides the first evidence of the pro-eryptotic potential of LA and associated mechanisms, which informs dietary interventions aimed at CVD prevention and management.

Bioymifi, a novel mimetic of TNF-related apoptosis-induced ligand (TRAIL), stimulates eryptosis.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a cytokine that initiates apoptosis upon binding to death receptor 5 (DR5) on cancer cells. Small molecule TRAIL mimetics have therefore been investigated as promising chemotherapeutic agents. Since anemia of chemotherapy is common, our goal is to investigate the hemolytic and eryptotic properties of novel DR5 agonist bioymifi (BMF) and identify the underlying molecular mechanisms. Whole blood (WB) was stimulated with 100 µM of BMF, whereas red blood cells (RBCs) were treated with 10-100 µM of BMF for 24 h at 37 °C. WB was analyzed for RBC, leukocyte, and platelet indices, while RBCs were examined for hemolysis by light absorbance of free hemoglobin, membrane scrambling by Annexin V-FITC, calcium by Fluo4/AM, cellular morphology by light scatter, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) using flow cytometry. Caspase inhibitor Z-VAD-FMK, p38 inhibitor SB203580, casein kinase 1α inhibitor D4476, receptor-interacting protein 1 inhibitor necrostatin-2, reduced glutathione, or cyclooxygenase (COX) inhibitor aspirin were added accordingly. BMF exerted dose-responsive, calcium-independent hemolysis, reduced RBC hemoglobin, significantly increased Annexin V-, Fluo4-, and DCF-positive cells, along with a dual effect on forward and side light scatter. Notably, the cytotoxic potential of BMF was significantly mitigated upon pharmacological inhibition of p38. Furthermore, BMF exhibited selective toxicity to eosinophils and significantly diminished reticulocyte hemoglobin content. Altogether, these novel findings highlight the adverse outcomes of BMF exposure on RBC physiology and provide the first toxicological assessment of BMF as an antitumor agent.

Flow Cytofluorometric Analysis of Molecular Mechanisms of Premature Red Blood Cell Death.

This chapter describes, in detail, the operational principles and experimental design to analyze the premature death of human red blood cells (RBCs; erythrocytes). Necrosis (i.e., hemolysis), eryptosis, and necroptosis are the three types of cell death thus far known to exist in RBCs, and distinctive markers of each are well established. Here, methods based on flow cytometry are presented in an easily reproducible form. Moreover, manipulation of incubation medium to promote or inhibit certain physiological phenomena, along with a step-by-step approach to examine membrane scrambling, cell volume, surface complexity, calcium activity, oxidative stress, and signal transduction pathways are also discussed.

Erythrocytes as model cells for biocompatibility assessment, cytotoxicity screening of xenobiotics and drug delivery.

Erythrocytes (RBCs) represent the main cell component in circulation and recently have become a topic of intensive scientific interest. The relevance of erythrocytes as a model for cytotoxicity screening of xenobiotics is under the spotlight of this review. Erythrocytes constitute a fundamental cellular model to study potential interactions with blood components of manifold novel polymer or biomaterials. Morphological changes, subsequent disruption of RBC membrane integrity, and hemolysis could be used to determine the cytotoxicity of various compounds. Erythrocytes undergo a programmed death (eryptosis) which could serve as a good model for evaluating certain mechanisms which correspond to apoptosis taking place in nucleated cells. Importantly, erythrocytes can be successfully used as a valuable cellular model in examination of oxidative stress generated by certain diseases or multiple xenobiotics since red cells are subjected to permanent oxidative stress. Additionally, the antioxidant capacity of erythrocytes, and the activity of anti-oxidative enzymes could reflect reactive oxygen species (ROS) generating properties of various substances and allow to determine their effects on tissues. The last part of this review presents the latest findings on the possible application of RBCs as drug delivery systems (DDS). In conclusion, all these findings make erythrocytes highly valuable cells for in vitro biocompatibility assessment, cytotoxicity screening of a wide variety of substances as well as drug delivery.

Modulation of Redox Signaling and Thiol Homeostasis in Red Blood Cells by Peroxiredoxin Mimetics.

Red blood cell death or erythrocyte apoptosis (eryptosis) is generally mediated by oxidative stress, energy depletion, heavy metals exposure, or xenobiotics. As erythrocytes are a major target for oxidative stress due to their primary function as O2-carrying cells, they possess an efficient antioxidant defense system consisting of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin 2 (Prx2). The oxidative stress-mediated activation of the Ca2+-permeable cation channel results in Ca2+ entry into the cells and subsequent cell death. Herein, we describe for the first time that selenium compounds having intramolecular diselenide or selenenyl sulfide moieties can prevent the oxidative stress-induced eryptosis by exhibiting an unusual Prx2-like redox activity under conditions when the cellular Prx2 and CAT enzymes are inhibited.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Loss of membrane asymmetry alters the interactions of erythrocytes with engineered silica nanoparticles.

Disruption of plasma membrane integrity is a primary mechanism of nanoparticle toxicity in cells. Mechanistic studies on nanoparticle-induced membrane damage have been commonly performed using model membranes with a focus on symmetric bilayers, overlooking the fact that the membrane has an asymmetric phospholipid composition. In this study, erythrocytes with normal and scrambled membrane asymmetry were utilized to examine how the loss of membrane asymmetry and the resulting alterations in the outer leaflet lipid composition affect nanoparticle-membrane interactions. Unmodified, amine-modified, and carboxyl-modified silica (30 nm) were used as nanoparticle models. Loss of membrane asymmetry was achieved by induction of eryptosis, using a calcium ionophore. Erythrocyte membrane disruption (hemolysis) by unmodified silica nanoparticles was significantly reduced in eryptotic compared to healthy cells. Amine- and carboxyl-modified particles did not cause hemolysis in either cell. In agreement, a significant reduction in the binding of unmodified silica nanoparticles to the membrane was observed upon loss of membrane asymmetry. Unmodified silica particles also caused significant cell deformation, changing healthy erythrocytes into a spheroid shape. In agreement with findings in the cells, unmodified particles disrupted vesicles mimicking the erythrocyte outer leaflet lipid composition. The degree of disruption and nanoparticle binding to the membrane was reduced in vesicles mimicking the composition of scrambled membranes. Cryo-electron microscopy revealed the presence of lipid layers on particle surfaces, pointing to lipid adsorption as the mechanism for vesicle damage. Together, findings indicate an important role for the lipid composition of the membrane outer leaflet in nanoparticle-induced membrane damage in both vesicles and erythrocytes.

Inhibition of suicidal erythrocyte death by pyrogallol.

Pyrogallol, a polyphenolic component of Acacia nilotica has previously been reported to induce apoptosis of diverse cell types. Pyrogallol is in part effective by influencing gene expression and by interference with mitochondrial function. Despite lack of nuclei and mitochondria, erythrocytes may undergo eryptosis, a suicidal death apparent from phosphatidylserine translocation to the erythrocyte surface and cell shrinkage. Eryptosis is triggered by glucose depletion, by oxidation, by hyperosmotic cell shrinkage and by excessive Ca2+ entry. As enhanced eryptosis is a common cause of anemia, uncovering inhibitors and stimulators of eryptosis may, both, be of clinical interest. Here we tested, whether eryptosis of human erythrocytes is modified by pyrogallol. Utilizing flow cytometry, phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding and cell volume from forward scatter. Prior to determinations erythrocytes were incubated with or without glucose, without or with added oxidant tert-butyl-hydroperoxide (t-BOOH, 0.5 mM), without or with added hyperosmotic sucrose (550 mM) or without or with added Ca2+ ionophore ionomycin (1 µM). Treatment of erythrocytes with pyrogallol (2-8 µM) was without significant effect on annexin-V-binding and forward scatter. Glucose deprivation, t-BOOH, sucrose and ionomycin, each, triggered annexin-V-binding and decreased forward scatter. Pyrogallol significantly blunted the effects on annexin-V-binding but not on forward scatter. Pyrogallol thus blunts phosphatidylserine translocation in erythrocytes exposed to glucose depletion, oxidative stress, hyperosmotic shock and excessive Ca2+ entry.

Protective Effect of Tamarind Seed Coat Ethanol Extract on Eryptosis Induced by Oxidative Stress.

Suicidal erythrocyte death, or eryptosis, is the key event in eliciting anemia in numerous pathological conditions, including diabetes, chronic kidney disease, cancer, sepsis, etc. Oxidative stress is an important trigger in the acceleration of erythrocyte loss via eryptosis and an underlying mechanism of anemia emergence in the above pathologies. Therefore, there is an increasing demand for identification of antioxidants and anti-eryptotic agents for the management of stress-related ailments. Here, we demonstrated the antioxidant and anti-eryptotic properties of the tamarind seed coat ethanol extract (TSCEE) against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress and eryptosis. The presence of probable secondary metabolites in the TSCEE extract was investigated by RP-HPLC. Active groups present in the TSCEE were studied by the Fourier-transform infrared spectroscopy. Cyclic voltammetric studies confirmed the antioxidant potential of TSCEE. The protective effect of TSCEE on red blood cells was confirmed by assessing various eryptotic markers, such as reactive oxygen species generation, intracellular calcium levels, and phosphatidylserine exposure. TSCEE reduced lipid peroxidation and protein carbonyl content and restored the levels of glutathione, antioxidant enzymes, and enzymes involved in glutathione replenishment. In conclusion, TSCEE was found to exhibit multiple therapeutic properties, which makes it a promising agent for treating oxidative stress-induced eryptosis and subsequent anemia in various pathologies.

Genotoxicity induced by hexavalent chromium leading to eryptosis in Ctenopharyngodon idellus.

The initiation of eryptosis as a result of genotoxic action of Cr(VI), seen through micronucleus and comet assay in the peripheral erythrocytes of Ctenopharyngodon idellus was evaluated through RT-qPCR. For this, fish was exposed to sublethal concentration of hexavalent chromium (5.30 and 10.63 mg/L), and the blood was sampled on different endpoints (15, 30 and 45 days). Accumulation of chromium in the erythrocytes was also studied, which depicted a significant increase in toxicant concentration and time dependent manner. Both concentrations of hexavalent chromium induced DNA damage, visible in the form of comet tails. The presence of micronuclei in the erythrocytes was accompanied with occurrence of nuclear bud (NBu), lobed nucleus (Lb), notched nucleus (Nt), vacuolated nucleus (Vn), binucleated cell (Bn) as nuclear abnormalities; and acanthocytes (Ac), echinocytes (Ec), notched cells (Nc), microcytes (Mc) and vacuolated cytoplasm (Vc) as cytoplasmic abnormalities. The expression of genes related to intrinsic apoptotic pathway induced by Cr(VI) presented significant (p < 0.05) upregulation in the expression of p53, Bax, Apaf-1, caspase9 and caspase3, and downregulation of Bcl2; inferring the initiation of apoptotic pathway. The ration of Bax and Bcl2 also appended the apoptotic state of the erythrocytes. From the present investigation, it can be concluded that genotoxicity induced by hexavalent chromium lead to eryptosis in C. idellus.

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

7-Keto-Cholesterol and Cholestan-3beta, 5alpha, 6beta-Triol Induce Eryptosis through Distinct Pathways Leading to NADPH Oxidase and Nitric Oxide Synthase Activation.

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 µM) and TRIOL (2, µM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect.

Hydroxytyrosol Decreases Phosphatidylserine Exposure and Inhibits Suicidal Death Induced by Lysophosphatidic Acid in Human Erythrocytes.

BACKGROUND/AIMS: Lysophosphatidic acid (LPA) is a phospholipid signal molecule that regulates many cellular processes both physiological and pathological. Moreover, its high plasma concentrations are toxic for several cellular types, including erythrocytes (RBC), as it acts as a pro-thrombotic and pro-atherogenic agent. It is therefore essential to explore the potential protective role of nutrition in protecting cells from the possible toxic effects of high plasma concentrations of LPA by testing bioactive nutrients. In particular, our focus was on hydroxytyrosol (HT), a phenolic antioxidant occurring naturally in virgin olive oil, investigating its possible protective effect in preventing LPA-induced programmed cell death (eryptosis) in human RBC. METHODS: Intact RBC were incubated in the presence of 2.5 µM LPA and increasing concentrations of HT. Phosphatidylserine (PS) exposure with cell shrinkage, influx of extracellular calcium (Ca2+), adenosine triphosphate (ATP) and glutathione levels were measured by FACS analysis. In addition, confocal laser scanning microscopy was used to determine RBC morphological alterations, as well as microvesicle formation. RESULTS: Our study confirms that LPA-induced eryptosis is characterized by PS exposure at the cell surface, with cell shrinkage and ATP and glutathione depletion; (Ca2+) influx is also a key event that triggers eryptosis. Here we report for the first time that cell co-incubation with LPA and in quantities as low as 0.1 µM HT causes a significant decrease in PS-exposing RBC, in addition to providing significant protection from the decrease in cell volume. Moreover, treatment of RBC with HT counters the influx of extracellular Ca2+ and completely restores ATP and glutathione content at 1 µM. Finally, under the same experimental conditions, HT exerts a protective effect on RBC morphological changes and microvescicle release, completely restoring the typical biconcave shape at 1 µM. CONCLUSION: Taken together, the findings reported in this paper point to a novel biological effect for HT in preventing programmed suicidal death in anucleated cells and indicate that prevention from LPA toxic effects may represent an additional mechanism responsible for the health-promoting effect of this dietary phenol which has been claimed, particularly related to cardiovascular diseases.

Uremia and Hypoxia Independently Induce Eryptosis and Erythrocyte Redox Imbalance.

BACKGROUND/AIMS: Red blood cell (RBC) death could contribute to anemia in chronic kidney disease (CKD) patients. Recent observational research has suggested a relationship between RBC death (eryptosis) and hypoxemia in hemodialysis patients. Thus, we studied the isolated and joint effects of a uremic toxin (indoxyl sulfate; IS) and hypoxia on RBC biology. METHODS: We incubated RBC from healthy donors with IS at concentrations of 0.01mM, 0.09mM and 0.17mM under both normoxic (21% O2) and hypoxic (5% O2) conditions for 24 hours. Eryptosis was evaluated by RBC phosphatidylserine (PS) exposure, cell volume, and cytosolic calcium which were quantified by Annexin-V+, forward scatter, and Fluo-3AM+ binding, respectively. RBC redox balance was reported by reactive oxygen species (ROS) production and intracellular reduced glutathione (GSH). Analyses were performed by flow cytometry. RESULTS: Hypoxia induced a 2-fold ROS production compared to normoxia. PS exposure and cytosolic calcium increased, while cell volume decreased by hypoxia and likewise by IS. IS increased ROS production in a dose-dependent manner under conditions of both normoxia and hypoxia. The same conditions promoted a GSH decrease with IS intensifying the hypoxia-induced effects. CONCLUSION: In summary, our results indicate that the concurrent presence of hypoxia and uremia augments RBC death and may therefore, contribute to the genesis of anemia in CKD.

Sublytic Terminal Complement Components Induce Eryptosis in Autoimmune Haemolytic Anaemia Related to IgM Autoantibodies.

BACKGROUND/AIMS: Eryptosis, the suicidal death of red blood cells (RBCs), is characterized by phosphatidylserine (PS) exposure at the cell surface. It can be catalysed by a variety of abnormal conditions and diseases. Until now, the many questions surrounding the physiology and pathophysiology of eryptosis have not been sufficiently answered. Recently, we demonstrated IgM and IgA autoantibodies (aab) to induce PS exposure on circulating RBCs of patients with autoimmune haemolytic anaemia (AIHA). However, it remained unclear how these aab lead to eryptosis. METHODS: Serum and plasma samples from patients with clinically relevant AIHA of cold type were used to induce eryptosis in O RBCs. Serum containing fresh complement from healthy donors, antibodies to complement component, and complement factor depleted sera were added to examine the influence of the complement on PS-exposure. RBC bound annexin V PE were analysed by flow cytometry. RESULTS: Eryptosis related to IgM aab was found to be dependent on complement activation and could be effectively inhibited by EDTA, serum heat inactivation and anti-C5. PS exposure increased with sequential activation of the sublytic terminal complement components C5b6, C5b-7 and was most significant at the C5b-8 stage. A decrease was observed following the formation of the lytic membrane attack complex C5b-9, either because of lysis of eryptotic RBCs or because of inhibition of eryptosis by C9. CONCLUSION: Our findings reflect new aspects on RBC destruction in AIHA as well the impact of the terminal complement complexes on the RBC membrane. The striking differences to nucleated cell apoptosis may even have physiological meaning of RBC acting as a buffer of the complement system.

Triggering of eryptosis, the suicidal erythrocyte death, by phenoxodiol.

Phenoxodiol is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. At least in theory, phenoxodiol could similarly stimulate suicidal erythrocyte death or eryptosis. Eryptosis is characterized by cell shrinkage and breakdown of cell membrane asymmetry with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), formation of reactive oxygen species (ROS), and increase of ceramide abundance at the cell surface. The present study explored whether phenoxodiol induces eryptosis and whether it modifies Ca2+ entry, ROS, and ceramide. Using flow cytometry, phosphatidylserine exposure at the cell surface was quantified from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, ROS from DCFDA-dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48-h exposure of human erythrocytes to phenoxodiol (100 µg/ml [416 µM]) significantly increased the percentage of annexin V binding cells, significantly decreased average forward scatter and Fluo3 fluorescence and significantly increased ceramide abundance, but did not significantly modify DCFDA fluorescence. The effect of phenoxodiol on annexin V binding tended to decrease following removal of extracellular Ca2+, an effect, however, not reaching statistical significance. In conclusion, phenoxodiol triggers eryptosis, an effect paralleled by increase of ceramide abundance.

Extracellular Histones Induced Eryptotic Death in Human Erythrocytes.

BACKGROUND/AIMS: Circulating or extracellular histones (EHs) in the bloodstream act as a damage-associated-molecular-pattern (DAMP) agent that plays a critical role in the pathogenesis of many diseases such as sepsis and sterile inflammation. To date, not much information is available to describe the mechanistic relationship between human erythrocytes and the cytotoxicity of EHs, the protein members from a highly conserved histone family across species. The present study explored this key question with a hypothesis that EHs induce eryptosis. METHODS: Freshly isolated human red blood cells (RBCs) from healthy donors were treated with EHs or agents for positive controls in a physiological buffer for 3 or 24 h. After treatments, flow cytometry was employed to quantify surface phosphatidylserine (PS) exposure from annexin-V-RFP binding, cell shrinkage from flow cytometric forward scatter (FSC) analysis, Ca2+ rise by fluo-4, reactive oxygen species (ROS) production by H2DCFDA, and caspase-3 activation by FAM-DEVD-FMK measurement. Hemolysis and membarne permeabilization were estimated respectively from hemoglobin release into supernatant and calcein leakage from RBC ghosts. RESULTS: With positive controls for validation, EHs in the pathophsyiological range were found to accumulate annexin-V binding on cell surface, decrease FSC, upregulate ROS production, elevate Ca2+ influx and increase caspase-3 activity in a 3-h incubation. Of note, no RBC hemolysis and no calcein release from ghosts were obtained after EHs treatment for 24 h. Interestingly, external Ca2+ was not a prerequisite for the EHs-mediated ROS production and PS externalization. Also, the eryptotic hallmarks in the apoptotic RBCs were partially blocked by heparin and antibody (Ab) against Toll-like receptor 2 (TLR2). CONCLUSION: EHs act as a DAMP agent in the human RBCs that induces eryptosis. The cytotoxic effect is rapid as the hallmarks of eryptosis such as cell shrinkage, surface PS exposure, [Ca2+]i rise, ROS production and caspase-3 activation can be seen 3 h after treatment in a dose-dependent manner. The EHs' cytotoxic effects could be blocked by heparin and the Ab against TLR2.