Daniela Vittori

MORPHOLOGIC AND FUNCTIONAL ALTERATIONS OF ERYTHROID CELLS INDUCED BY LONG-TERM INGESTION OF ALUMINIUM

Anaemia has been associated with aluminium (Al) accumulation in plasma and/or bone tissue in patients with chronic renal insufficiency. Nevertheless, in previous works, we have found shortened red-cell life span, increased osmotic resistance and inhibition of colony-forming units-erythroid (CFU-E) development in Al-overloaded rats with normal renal function. To elucidate further the action of Al on in vivo erythropoiesis, aluminium citrate was provided to Sprague Dawley rats (n = 18) in the drinking water for 8 months. Significant decreases in haematocrit (38.8 +/- 4.29 versus 43.1 +/- 3.58%, p < 0.05) and blood haemoglobin concentration (137 +/- 10.1 versus 148 +/- 8.5 g/l, p < 0.05), reticulocytosis (1.8/1.3-4.2 versus 1.2/0.4-3.7%, p < 0.05), and severe inhibition of CFU-E growth (670/120-950 versus 1530/810-2440 CFU-E/2 x 10(5) cells, p < 0.005) were found. Anysocytosis, poikilocytosis and schistocytosis were detected in peripheral blood stained films. Scanning electron microscopy revealed the presence of erythrocytes with abnormal shape, including crenated and target cells. Aluminium was localised specially inside the schistocytes by EDAX analysis. Decreased haptoglobin concentration (107/83-127 versus 139/89-169 mg/l, p < 0.05) supports the assumption of haemolytic nature of the anaemia. Rats were not iron depleted, as plasma iron concentration and total iron binding capacity were found in the range of control values, and sideroblasts and haemosiderin deposits were observed in bone marrow smears. Total 59Fe uptake and 59Fe incorporated to haem by the bone marrow cells were found decreased. In conclusion, the erythropoiesis impairment induced by Al may be a combined effect of direct action on circulating erythrocytes and interference with the cellular iron metabolism in erythroid progenitors.

TNF-alpha-induced apoptosis is prevented by erythropoietin treatment on SH-SY5Y cells

The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-alpha. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-alpha or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-alpha. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-kappaB nuclear translocation, TNF-alpha induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-kappaB, through mechanisms involving Jak/STAT and PI3K signalling pathways.

Human erythroid cells are affected by aluminium. Alteration of membrane band 3 protein.

There is evidence that anaemia is associated with aluminium (Al). We have already reported on the sensitivity to Al, showed by erythroid cell populations of animals chronically exposed to the metal. In order to investigate whether Al could also affect human cells, experiments were carried out both on immature and mature human erythroid cells. Erythroid progenitors (CFU-E, colony-forming units-erythroid) concentrated from human peripheral blood were cultured in an Al-rich medium under erythropoietin stimulation and their development analysed. Human peripheral erythrocytes were aged in the presence of Al. Cells were examined using scanning electron microscopy, and membrane proteins analysed by polyacrylamide gel electrophoresis with sodium dodecyl sulphate and immunoblotting. The development of the Al-treated progenitors was 8750/6600-9200 CFU-E/10(6) cells, a significantly lower median value (P<0.05) than that showed by non-treated cells (12300/11200-20700 CFU-E/10(6) cells). Erythrocyte morphological changes were induced by Al during the in vitro ageing. The cells lost their typical biconcave shape, turning into acanthocytes and stomatocytes. Simultaneously, an increased membrane protein breakdown compatible with band 3 degradation was detected. Besides, Al was found within the cells and attached to the membrane. The present in vitro results suggest that Al may disturb human erythropoiesis through combined effects on mature erythrocytes and cellular metabolism in late erythroid progenitors.

Signaling pathways of cell proliferation are involved in the differential effect of erythropoietin and its carbamylated derivative.

It is now recognized that in addition to its activity upon erythroid progenitor cells, erythropoietin (Epo) is capable of stimulating survival of different non-erythroid cells. Since stimulation of erythropoiesis is unwanted for neuroprotection, Epo-like compounds with a more selective action are under investigation. Although the carbamylated derivative of erythropoietin (cEpo) has demonstrated non-hematopoietic tissue protection without erythropoietic effect, little is known about differential mechanisms between Epo and cEpo. Therefore, we investigated signaling pathways which play a key role in Epo-induced proliferation. Here we show that cEpo blocked FOXO3a phosphorylation, allowing expression of downstream target p27(kip1) in UT-7 and TF-1 cells capable of erythroid differentiation. This is consistent with the involvement of cEpo in slowing down G1-to-S-phase progression compared with the effect of Epo upon cell cycle. In contrast, similar antiapoptotic actions of cEpo and Epo were observed in neuronal SH-SY5Y cells. Inhibition and competition assays suggest that Epo may act through both, the homodimeric (EpoR/EpoR) and the heterodimeric (EpoR/βcR) receptors in neuronal SH-SY5Y cells and probably in the TF-1 cell type as well. Results also indicate that cEpo needs both the EpoR and βcR subunits to prevent apoptosis of neuronal cells. Based on evidence suggesting that cell proliferation pathways were involved in the differential effect of Epo and cEpo, we went forward to studying downstream signals. Here we provide the first evidence that unlike Epo, cEpo failed to induce FOXO3a inactivation and subsequent p27(kip1) downregulation, which is clearly shown in the incapacity of cEpo to induce erythroid cell growth.

Disturbance of cellular iron uptake and utilisation by aluminium.

Aluminium (Al) affects erythropoiesis but the real mechanism of action is still unknown. Transferrin receptors (TfR) in K562 cells are able to bind Tf, when carrying either iron (Fe) or Al, with similar affinity. Then, the aim of this work was to determine whether Al could interfere with the cellular Fe uptake and utilisation. K562 cells were induced to erythroid differentiation by either haemin (H) or sodium butyrate (B) and cultured with and without Al. The effect of Al on cellular Fe uptake, Fe incorporation to haem and cell differentiation was studied. H- and B-stimulated cells grown in the presence of 10 microM Al showed a reduction in the number of haemoglobinised cells (by 18% and 56%, respectively) and high amounts of Al content. Al(2)Tf inhibited both the (59)Fe cellular uptake and its utilisation for haem synthesis. The removal of Al during the (59)Fe pulse, after a previous incubation with the metal, allowed the cells to acquire Fe quantities in the normal range or even exceeding the amounts incorporated by the respective control cells. However, the Fe incorporated to haem could not reach control values in B-stimulated cells despite enough Fe acquisition was observed after removing Al. Present results suggest that Al might exert either reversible or irreversible effects on the haemoglobin synthesis depending on cellular conditions.

Protective action of erythropoietin on neuronal damage induced by activated microglia

Inflammation is a physiological defense response, but may also represent a potential pathological process in neurological diseases. In this regard, microglia have a crucial role in either progression or amelioration of degenerative neuronal damage. Because of the role of hypoxia in pro‐inflammatory mechanisms in the nervous system, and the potential anti‐inflammatory protective effect of erythropoietin (Epo), we focused our investigation on the role of this factor on activation of microglia and neuroprotection. Activation of microglial cells (EOC‐2) was achieved by chemical hypoxia induced by cobalt chloride (CoCl2) and characterized by increased levels of nitrite, tumor necrosis factor‐α and reactive oxygen species production, as well as up‐regulation of inducible nitric oxide synthase expression. Under these conditions, cell proliferation data and proliferating cell nuclear antigen (PCNA) staining demonstrated a mitogenic effect of chemical hypoxia. Even though pre‐treatment with Epo did not prevent nitrite production, inducible nitric oxide synthase protein expression or tumor necrosis factor‐α secretion, it prevented the oxidative stress induced by CoCl2 as well as cell proliferation. Neuronal cells (SH‐SY5Y) cultured in the presence of conditioned medium from activated EOC‐2 cells or macrophages (RAW 264.7) developed significant apoptosis, an effect that was abolished by Epo via Epo/Epo receptor activation. The results show that even though Epo did not exert a direct anti‐inflammatory effect on microglia activation, it did increase the resistance of neurons to subsequent damage from pro‐inflammatory agents. In addition to its anti‐apoptotic ability, the Epo antioxidant effect may have an indirect influence on neuronal survival by modulation of the pro‐inflammatory environment.

Modulation of protein tyrosine phosphatase 1B by erythropoietin in UT-7 cell line.

Background/ Aims: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. Methods: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. Results: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). Conclusion: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.

Eryptosis is induced by hyperthermia in hereditary spherocytosis red blood cells.

Fil: Crisp, Renee L.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Quimica Biologica; Argentina. Hospital Nacional Alejandro Posadas; Argentina

Erythrocyte: Programmed Cell Death

Erythrocytes are produced by a complex and finely regulated process of erythropoiesis. It starts with a pluripotential stem cell that, in addition of its self replication capacity, can give rise to separate cell lineages. Erythropoiesis passes from the stem cell through the multipotent progenitor CFU-GEMM (colony-forming unit granulocyte erythroid monocyte and megakaryocyte), and then BFU-E (burst-forming unit erythroid) and CFU-E (colonyforming unit eryhtroid), to the first recognizable erythrocyte precursor in the bone marrow, the pronormoblast. This cell gives rise to a series of progressively smaller normoblasts with increasing content of hemoglobin. The nucleus is finally extruded from the late normoblast leading to mature red blood cell through the reticulocyte stage. Erythropoiesis ends with the mature circulating red cell, which is a non-nucleated biconcave disc, performing its function of oxygen delivery. In this process, the glycoprotein hormone erythropoietin has been known as the major humoral regulator of red cell production. It is now well established that erythropoietin stimulates erythropoiesis, at least in part, by protecting erythroblasts from apoptosis. Human mature erythrocytes are terminally differentiated cells that are devoid of mitochondria, as well as of nucleus and other organelles. In circulation, the red cell is constantly tested for its capacity to undergo marked cellular deformation. This ability to change its shape is essential for optimal cell function, since the resting diameter of the human red cell far exceeds that of the capillaries and splenic endothelial slits through which it must pass (Mohandas & Groner, 1989). A two dimensional network of proteins interacting between transmembrane location and cytoplasmic surface of the plasma membrane gives the red blood cell its properties of elasticity and flexibility that allows the success of this journey. The mature erythrocyte is unable to self-repair and has no capacity to synthesize proteins. Therefore, its lifespan is finite and is shortened further when the cell’s environment becomes hostile or when the erythrocyte’s ability to cope with damaging extracellular influences becomes impaired. The erythrocyte limited lifespan implies that, as in other cells, life and death are well regulated for erythrocytes, in spite of their lack of capacity for protein synthesis (Bosman et al., 2005). In the present review, we aim to show updated information concerning erythrocyte death in order to contribute to the understanding of the physiopathological relationship of this process with the development of anemia.

Protein tyrosine phosphatase 1B (PTP1B) is involved in the defective erythropoietic function of carbamylated erythropoietin.

Fil: Chamorro, Maria Eugenia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Quimica Biologica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Quimica Biologica de la Facultad de Ciencias Exactas y Naturales; Argentina