Effect of Tumor Necrosis Factor-Alpha on Erythropoietin and Erythropoietin Receptor-Induced Erythroid Progenitor Cell Proliferation in ß-Thalassemia/Hemoglobin E Patients.

OBJECTIVE: Thalassemia is one of the genetic diseases that cause anemia and ineffective erythropoiesis. Increased levels of several inflammatory cytokines have been reported in ß-thalassemia and might contribute to ineffective erythropoiesis. However, the mechanism by which tumor necrosis factor-alpha (TNF-α) is involved in ineffective erythropoiesis in thalassemic patients remains unclear. The objective of this study is to investigate the effect of TNF-α on the erythropoietin (EPO) and erythropoietin receptor (EPOR) expression involved in proliferation of ß-thalassemia/hemoglobin (Hb) E erythroid progenitor cells compared with cells from healthy subjects. MATERIALS AND METHODS: CD34-positive cells were isolated from heparinized blood by using the EasySep® CD34 selection kit. Cells were then cultured with suitable culture medium in various concentrations of EPO for 14 days. The effect of TNF-α on percent cell viability was analyzed by trypan blue staining. In addition, the percentage of apoptosis and levels of EPOR protein were measured by flow cytometry. RESULTS: Upon EPO treatment, a higher cell number was observed for erythroid progenitor cells from both healthy participants and ß-thalassemia/Hb E patients. However, a reduction of apoptosis was found in EPO-treated cells especially for ß-thalassemia/Hb E patients. Interestingly, TNF-α caused higher levels of cell apoptosis and lower levels of EPOR protein in thalassemic erythroid progenitor cells. CONCLUSION: TNF-α caused a reduction in the level of EPOR protein and EPO-induced erythroid progenitor cell proliferation. It is possible that TNF-α could be involved in the mechanism of ineffective erythropoiesis in ß-thalassemia/Hb E patients.

Cytokine-induced apoptosis of beta-thalassemia/hemoglobin E erythroid progenitor cells via nitric oxide-mediated process in vitro.

BACKGROUND/AIM: ß-Thalassemia/hemoglobin E (ß-thal/HbE) is a common hereditary anemia in Thailand. Ineffective erythropoiesis due to apoptosis and decreased lifespan of circulating thalassemic red blood cells are the major causes of anemia. Changes to bone marrow microenvironment could contribute to apoptotic events. This study examined the effects of cytokines interleukin-1ß, tumor necrosis factor-α and interferon-γ on apoptosis of ß-thal/HbE erythroid progenitor cells in vitro, including nitric oxide-mediated apoptotic processes. METHODS: Percent apoptosis of erythroid progenitor cells from 5 ß-thal/HbE patients and 5 normal control subjects was examined using flow cytometry. In addition, the inducible nitric oxide synthase (iNOS) mRNA level and nitrite production were measured using quantitative PCR and the Griess method, respectively. RESULTS: Upon cytokine treatment, a higher percent apoptosis was obtained with ß-thal/HbE erythroid progenitor cells compared with control, and the maximum effect was observed using 20 ng/ml interferon-γ on day 14 of culture. There was an increase in iNOS mRNA level and a concomitant elevation of nitrite concentration in culture medium. Apoptosis and nitrite level were abrogated when ß-thal/HbE and control cells were treated with S-methylisothiourea sulfate, an iNOS inhibitor. CONCLUSION: The marked sensitivity of erythroid progenitor cells from ß-thal/HbE patients to cytokine-induced apoptosis via an NO-mediated process reflects a proapoptotic status of such thalassemic red blood cells.