The antioxidant effect of erythropoietin on thalassemic blood cells

Abstract

Because of its stimulating effect on RBC production, erythropoietin (Epo) is used to treat anemia, for example, in patients on dialysis or on chemotherapy. In β-thalassemia, where Epo levels are low relative to the degree of anemia, Epo treatment improves the anemia state. Since RBC and platelets of these patients are under oxidative stress, which may be involved in anemia and thromboembolic complications, we investigated Epo as an antioxidant. Using flow-cytometry technology, we found that in vitro treatment with Epo of blood cells from these patients increased their glutathione content and reduced their reactive oxygen species, membrane lipid peroxides, and external phosphatidylserine. This resulted in reduced susceptibility of RBC to undergo hemolysis and phagocytosis. Injection of Epo into heterozygous (Hbb(th3/+)) β-thalassemic mice reduced the oxidative markers within 3 hours. Our results suggest that, in addition to stimulating RBC and fetal hemoglobin production, Epo might alleviate symptoms of hemolytic anemias as an antioxidant.

Figure 1

Flow cytometry analysis of the Epo effect on ROS generation by RBC and platelets. A diluted blood sample obtained from a thalassemia patient was treated for 2 hrs with Epo (1 U/ml) at 37°C, stained with DCF and then stimulated with 1 mM H₂O₂ for 15 min. (a) FCS versus SSC dot plot. The gates indicate the position of platelets (R1) and RBC (R2). (b-c) Distribution histograms showing DCF-derived fluorescence (FL-1) of untreated (grey) and Epo-treated (white) RBC (b) and platelets (c). The mean fluorescent intensity (MFI) of each population is indicated.

Figure 2

The kinetics of EPO effect on ROS generation by RBC and platelets. A diluted blood sample obtained from a thalassemia patient was stained with DCF, washed, and then incubated at room temperature with Epo (1 U/ml). Fluorescence was measured by flow cytometry at the indicated time points. The time-related changes in the mean fluorescent intensity (MFI) of each population are indicated.

Figure 3

Effect of Epo on the oxidative stress of RBC and platelets from thalassemic patients. Diluted blood samples were untreated with or without 1 U/ml Epo for 3 hrs at 37°C, then stimulated (a, b) or not (c, d) with 1 mM H₂O₂ for 15 min, and assayed for ROS. RBC (e) and platelets (f) treated with or without Epo were also assayed for GSH. The results, presented as the average (N = 11) of the mean fluorescence index (MFI) ± SD, show a decrease in ROS (P < .05) and an increase in GSH (P < .05) following Epo treatment.

Figure 4

The Epo dose-effect relationship on ROS and GSH of thalassemic RBC and platelets. Diluted blood samples were exposed to different concentrations of Epo for 2 hrs at 37°C and then stimulated with 1 mM H₂O₂ for 15 min. The results show the average (N = 4) mean fluorescence index (MFI) ± SD of ROS and GSH in RBC and platelets.

Figure 5

Effect of Epo on ROS production by H₂O₂-stimulated normal RBC and platelets. Diluted blood samples were treated with the indicated concentrations of H₂O₂ for 30 min, then incubated with or without Epo (2 U/ml) for additional 2 hrs, washed and assayed for ROS. The results show the average (N = 3) mean fluorescence index (MFI) ± SD.

Figure 6

The long-term effects of Epo on RBC oxidative markers, ageing, and susceptibility to hemolysis, and phagocytosis. Thalassemic RBC were diluted to 4 × 10⁶/ml with their plasma and incubated with or without Epo (2 U/ml) for 3 days. The cells were then harvested and assayed for ROS (a), GSH (b), calcein (c), external phosphatidylserine (PS) (d), hemolysis (e) and phagocytosis (f) as detailed in Section 2. The results in (a), (b) and (c) are presented the mean fluorescence index (MFI), in (d) as the percentage of PS positive RBC, and in (e) and (f) as the percentage of hemolysed and phagocytosed RBC, respectively, compared to the RBC input. The data are the average ± SD of 4 experiments performed with blood samples derived from different patients.

Figure 7

Effect of Epo on thalassemic mice. Heterozygous β-thalassemic intermedia (Hbb^th3/+) mice (N = 4) with low hemoglobin levels (7–9 g/dL) were inoculated (i.p) with Epo (5000 U/kg). Before (Without Epo) and 2 hrs after (With Epo) injection, blood was drawn and RBC were assayed. The changes in the indicated parameters are shown. Values of untreated mice (Without Epo) were taken as 100%.