Dalina I. Tanyong

Objective: The major pathophysiological features of β-thalassemia are anemia and ineffective erythropoiesis. Ineffective erythropoiesis has been shown by an intense marrow erythroid hyperplasia and increased apoptosis during basophilic to orthochromatic normoblast stages. Some cytokines like interferon-γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) have been found to involved in apoptosis. Although the pro-apoptotic activity of IFN-γ and TNF-α is well documented, there are only few studies on IL-1, especially on erythroid lineage. In this in vitro study, the role of cytokine IL-1α and IL-1β in apoptosis of erythroid progenitor cells from β- thalassemia/HbE patients was assessed. Methods: Erythroid progenitor cells were isolated from peripheral blood of healthy subjects and β-thalassemia/HbE patients. Cells were then cultured, with and without 20 ng/ml IL-1α and IL-1β. Total cells and percent cell viability were performed by using trypan blue staining. Percent cell apoptosis was analyzed by using flow cytometer. Results: Both IL-1α and IL-1β were significantly decreased erythroid progenitor cells. IL-1 at 20 ng/ml reduced the glycophorin A positive cells and percent cell viability of erythroid progenitor cells from β-thalassemia/HbE patients, while there was increased apoptosis in this group. The highest percent apoptosis was observed in 20 ng/ml IL-1β treated β-thalassemia/HbE erythroid progenitor cells. Conclusion: IL-1β could be involved in apoptosis of erythroid progenitor cells from β-thalassemia/HbE patients which might be related with ineffective erythropoiesis of the disease. DOI: http://dx.doi.org/10.3126/ajms.v3i4.6805 Asian Journal of Medical Science Vol.3(4) 2012 pp.8-14

Proinflammatory cytokines play a role in the pathogenesis of anemia of chronic disease (ACD), which is a common cause of anemia in rheumatoid arthritis (RA). Anemia in RA is associated with increased apoptosis of erythroid cells. However, there is unclear information on the mechanism of ACD in the disease. The purpose of this study is to investigate the role of cytokines on nitric oxide-mediated apoptosis in erythroid progenitor cells of ACD in RA patients. Erythroid progenitor cells from healthy subjects and RA patients with ACD were treated with cytokines like interleukin-1β, tumor necrosis factor-α, and interferon-γ at concentrations of 2, 20, and 40 ng/ml for 14 days. Cell viability and cell apoptosis were analyzed by trypan blue staining and flow cytometry, respectively. The results showed that the highest effect of cytokines on reduction cell viability and induction cell apoptosis was found in 20 ng/ml IFN-γ-treated cells of RA patients. In addition, IFN-γ showed significantly increased nitric oxide production and iNOS mRNA expression, which was measured by Griess assay and real-time PCR, respectively. The percentage of cell apoptosis and NO production was reduced after an inducible nitric oxide synthase inhibitor, SMT, treatment. In conclusion, IFN-γ could induce nitric oxide production–mediated apoptosis process, which might be involved in the pathogenesis of ACD in RA patients.

Background/Aim: β-Thalassemia/hemoglobin E (β-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-1β, tumor necrosis factor-α and interferon-γ on apoptosis of β-thal/HbE erythroid progenitor cells in vitro, including nitric oxide-mediated apoptotic processes. Methods: Percent apoptosis of erythroid progenitor cells from 5 β-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 β-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 β-thal/HbE and control cells were treated with S-methylisothiourea sulfate, an iNOS inhibitor. Conclusion: The marked sensitivity of erythroid progenitor cells from β-thal/HbE patients to cytokine-induced apoptosis via an NO-mediated process reflects a proapoptotic status of such thalassemic red blood cells.

Background: Leukemia is characterized by the uncontrolled accumulation of white blood cells. Recently, cytokines have been used in immunotherapy, which is a new strategy for leukemia treatment. Objective: To investigate the effect of cytokines on induction of apoptosis in acute leukemic cell lines; HL-60, MV4-11, K-562 and Molt-4 and in addition, to study the involvement of nitric oxide (NO) in apoptotic pathways. Methods: Leukemic cell lines were incubated with cytokines; interleukin-1 beta, tumor necrosis factor-a, and interferon-gamma in various concentrations and for variable periods of time. The percent apoptosis and caspase 3 activation were examined by flow cytometry. Moreover, NO production and inducible nitric oxide synthase (iNOS) mRNA were measured by using Griess method and Real-time PCR, respectively. Results: Cytokines caused a time and dose-dependent induction of apoptosis in leukemic cell lines. The highest cell apoptosis was found in K-562 treated with 40 U/ml interferon-gamma for 48 hours; this correlated with the result of cell growth inhibition and caspase 3 activation. NO and iNOS mRNA were increased in cytokines treated cells. Moreover, apoptosis was reduced by SMT, an iNOS inhibitor, which confirms the possible involvement of NO in the apoptotic pathway. Conclusion: Cytokines especially interferon-gamma induced apoptosis in acute leukemia via NO and caspase 3 pathway. (Asian Pac J Allergy Immunol 2011;29:102-11)

In order to study the role of the cytokine interleukin-3 (IL-3) and its signaling pathways in erythropoiesis of beta-thalassemia/HbE erythroid progenitor cells, CD34 positive cells were isolated from peripheral blood of patients and healthy subjects. After culturing the cells in the presence or absence of IL-3, cell viability was measured by trypan blue staining and apoptotic cells were analyzed by flow cytometry. After 7 days of culture the highest percent erythroid progenitor cell viability was obtained with cells from healthy subjects, while the lowest percentage was found in those from splenectomized beta-thalassemia/HbE. Viability of beta-thalassemia/HbE erythroid progenitor cells in the presence of IL-3 was higher than that of nonsupplemented cells. In addition, specific inhibitors of protein kinase C (Ro-318220), phospholipase C (U-73122) and Janus kinase 2 (AG-490) were used to investigate the involvement of signaling pathways in erythropoiesis. Percent apoptosis of erythroid progenitor cells from splenectomized beta-thalassemia/HbE subjects treated with RO-318220 was higher than those of nonsplenectomized beta-thalassemia/HbE and healthy subjects. Treatment with U-73122 resulted in enhanced percent apoptotic cells from normal and beta-thalassemia/HbE subjects. All these effects were independent of IL-3 treatment.