MECHANISMS INVOLVED IN PREMATURE SELF-DESTRUCTION OF RED BLOOD CELLS UNDER DIFFERENT ERYPTOSIS STIMULI

VOTA DAIANA; CALLERO MARIANA; CHAMORRO MARÍA EUGENIA; CRISP RENÉE; NESSE ALCIRA; VITTORI DANIELA

Barcelona, España

Congreso; 15th Congress of the European Hematology Association; 2010

European Hematology Association

BACKGROUND: Recently, it has been reported that under certain circumstances erythrocytes (RBCs) can suffer premature self-destruction showing cell changes that mimic features of apoptosis in nucleated cells. Although much information about this process called eryptosis is available, the mechanisms are not yet well known. AIM: Based on the hypothesis that oxidative stress and increased intracellular calcium induce eryptosis through different pathways, the aim of this work was to study the mechanisms involved in this process under these two models of eryptosis. METHODS: RBCs from healthy donors were obtained following informed consent. Cells were exposed to different eryptosis stimuli in buffer HEPES at 37°C for different periods. Morphological changes were observed by scanning electron microscopy. Flow cytometry assays were performed to detect Anexin V binding and to measure GSH, ROS, intracellular calcium, and band 3 levels. Membrane protein phosphorylation and nitrosylation were analyzed by SDS-PAGE and Western blotting. RESULTS: The presence of either H2O2+NaNO2 or Ca ionophore A23187 (CaI) induced membrane phosphatidylserine translocation (Control 1.8±0.6%; H2O2+NaNO2 71.6±27.3%; CaI 96.2±3.1%; H2O2+NaNO2 vs. C P<0.05; CaI vs. C P<0.01, n=5). Interestingly, both treatments caused different morphological changes. Spherocytes with microvesiculation or stomatocytes were the characteristic cell shapes of CaI- or H2O2+NaNO2-induced eryptosis, respectively. Significantly higher ROS and lower GSH levels with respect to untreated cells were observed in the presence of H2O2+NaNO2 (P<0.05, n=3), whereas they remained unchanged under CaI exposure. In contrast with CaI treatment, no change in intracellular calcium levels was observed in the presence of H2O2+NaNO2 (expressed as Gm of fluorescence intensity: C 53.3±6.7; H2O2+NaNO2 44.9±5.5; CaI 136.1±68.5; CaI vs. C P<0.01, n=4). Even though these results suggested that calpain activation may play an essential role to induce eryptosis, we did not detect a reduction of Anexin V positive cells in the presence of Calpain Inhibitor I under either H2O2+NaNO2 or CaI incubation. Membrane protein analysis showed a strong reduction of band 3 after exposure to CaI (Flow cytometry, Gm: C 100.0±23.0; CaI 27.4±7.1; P<0.05, n=3) whereas post-translational modifications, such as nitrosylation of membrane proteins and band 3 phosphorylation were caused by cell incubation with H2O2+NaNO2. Parallel assays to detect protein phosphorylation in cells pre-treated with staurosporine (kinase inhibitor) or sodium o-vanadate (phosphatase inhibitor) suggest higher involvement of kinase activation in eryptosis induced by H2O2+NaNO2. CONCLUSIONS: RBCs exposed to oxidative stress or CaI suffered self-destruction detected by increase of Anexin V binding, although different pathways are involved in the mechanisms of eryptosis depending on the stimuli. On the one hand, an increase in calcium influx appears to specially affect membrane proteins such as band 3, possibly due to membrane vesiculation. On the other hand, oxidative stress not only reduces the amount of cellular protective anti-oxidative compounds, but also produces membrane protein modifications probably leading to alteration of their properties. Based on the present results we can suggest that the anemia observed under several diseases associated with chronic inflammation might be produced not only by erythroid progenitor dysfunction but also by direct effects of pro-eryptotic factors on erythrocytes.