TNF-ALPHA INDUCES APOPTOSIS IN CELLS UNDER ERYTHROID DIFFERENTIATION

VITTORI DANIELA; PÉREZ GLADYS; NESSE ALCIRA

Amsterdam

Congreso; 11th Congress of the European Hematology Association.; 2006

European Hematology Association

&amp;lt;!-- /* Font Definitions */ @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-1610611985 1107304683 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman","serif"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt;} @page Section1 {size:595.3pt 841.9pt; margin:42.55pt 3.0cm 85.9pt 70.9pt; mso-header-margin:35.45pt; mso-footer-margin:35.45pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&amp;gt; BACKGROUND: Inflammatory cytokines inhibit the proliferation of erythroid progenitor cells, among other effects upon iron homeostasis and erythropoietin synthesis, all of which contribute to the pathogenesis of anaemia, a common complication of chronic diseases. Recent studies suggest that the increased release of TNF-α could be responsible for the development of anaemia through the induction of an apoptotic mechanism mediated by death receptors. It has been suggested that this cytokine effect is dependent on the stage of erythroid differentiation. AIM: The effect of TNF-α upon differentiation, proliferation and apoptosis was investigated in cells subjected to erythroid differentiation. METHODS: K562 (erythropoietin-independent) and UT-7 (erythropoietin-dependent) cells were cultured in the presence of haemin (H) for 48 h to study proliferation (Trypan blue test), differentiation (DAF staining) and apoptosis evaluated by apoptotic cells (Hoechst fluorescent nuclear stain) and caspase-3 activity (proteolitic cleavage of chromogenic substrate). mRNA analysis was performed by RT-PCR. RESULTS: After 48 h with H, high levels of haemoglobinized cells were observed (K562: 85%, UT-7: 78%). On the other hand, 30 ng/ml TNF-α treatment did not induce significant changes in the development, maturation and viability of both cell lines. Non-differentiated K562 cells were not affected by TNF-α (T) whereas haemin-treated cells were sensitive to the TNF-α proapoptotic effect (Fig. A: H-T vs. H, P<0.002). This apoptotic action was enhanced by PI3Kinase inhibition with Ly294002 (Fig. A. H-Ly-T vs. H-Ly, P<0.05). The negative effects observed in the presence of TNF-α were dramatically decreased by a previous treatment with anti-TNF neutralizing antibody (Fig. A). Only in simultaneous experiments with TNF-α and Ly, UT-7 cells cultured in the presence of erythropoietin and induced to differentiation by haemin were induced to apoptosis. This effect resulted significantly higher than that due to signalling inactivation of the growth factor erythropoietin via PI3Kinase (Apoptotic cells: H-Ly-T 92.2±2.4% vs. H-Ly 68.9±5.3%, P<0.01). Results of caspase-3 activity measured at 6 h-incubation of cell lysates with chromogenic substrate parallel those of apoptotic K562 cells (Fig. B). mRNA levels of Bcl-x, the Bcl-2 related protein that acts as important regulator of cell death, were not modified under the experimental conditions mentioned above. The mRNA of c-FLIP, the suppressor protein of apoptotic signals induced by death receptors, was found diminished in K562 induced to erythroid differentiation but not in UT-7 cells grown under similar conditions. CONCLUSIONS: During the process of differentiation, cells become sensitive to proapoptotic action of TNF-α. A decrease in c-FLIP expression would explain the apoptosis produced by TNF-α in K562 cells induced to differentiation since this cytokine effect was not observed in differentiated UT-7 cells with non-altered mRNA c-FLIP levels.        Besides, cells with different dependence on the growth factor erythropoietin, analysed under  similar conditions of erythroid differentiation, show different sensitivity to proinflammatory cytokines. Protective mechanisms against cellular apoptosis caused by TNF-α seem to be mediated by PI3Kinase signalling and proved to be independent from Bcl-x. These findings may have potential implications in the understanding of the mechanisms underlying anaemia in chronic inflammatory diseases.