Mitochondrial disruption and limited apoptosis of erythroblasts are associated with high risk myelodysplasia. An ultrastructural analysis.

AIMS: To investigate the ultrastructural characteristics of erythroblasts in myelodysplasia (MDS) which might be of additional importance in understanding its pathogenesis. METHODS AND RESULTS: 22 patients were classified according to FAB (French-American-British classification), IPSS (international prognostic score system), cytogenetic risk factors and transfusion dependency. Using electron microscopy, in 77% of the cases, nuclear abnormalities consisting of disrupted membranes and cystic/dilated perinuclear spaces were noted. In a limited number of patients (n=7), a low percentage of apoptosis in the erythroid lineage (mean 3.1+/-1.6%; median 3%: range 1-6) (normal controls: <0.5%) could be noted, primarily in mature erythroblasts and significantly associated with spongiform nuclear features. In all patients extensive cytoplasmic vacuolization and myelin figures in erythroblasts were demonstrated. In 55% of the cases, enlarged and abnormal mitochondria were observed, significantly associated with iron-accumulation. A significant inverse relation existed between the absence of apoptosis and more advanced, or high risk disease and cytogenetic risk factors. Mitochondrial abnormalities were significantly correlated with high risk disease as well with an increase in transfusion dependency. CONCLUSIONS: These data indicate that in MDS apoptosis may play a role in early stages of disease. The overall prominent defects in mitochondria might be an additional defect that is involved in ineffective erythropoiesis.

Excessive apoptosis of bone marrow erythroblasts in a patient with autoimmune haemolytic anaemia with reticulocytopenia.

We report a patient with autoimmune haemolytic anaemia (AIHA) with reticulocytopenia, who showed excessive apoptosis of erythroblasts. Ultrastructural analysis of bone marrow cells showed that 50% of erythroblasts had characteristic features of apoptosis, which was confirmed by staining with Annexin-V. In addition, in contrast to normal erythroblasts, Ig staining of the entire erythroblast population could be shown. These data show that apoptosis may contribute to the mechanism of reticulocytopenia in AIHA.

CD34+/CD36- cells from myelodysplasia patients have a limited capacity to proliferate but can differentiate in response to Epo and MGF stimulation.

Myelodysplasia (MDS) is mostly characterized by a normal or increased number of normoblasts in the bone marrow and an impaired in vitro colony formation. In the present study we analyzed whether this might be due to a disconnection between proliferation and differentiation. CD34+/CD36- sorted bone marrow cells of 18 MDS patients were cultured in a clonogenic and suspension culture assay in the presence of erythropoietin (Epo) and mast cell growth factor (MGF). Burst-forming units erythroid (BFU-E, 75 +/- 88/10(4) CD34+ cells, X +/- s.d.) and colony-forming units E (CFU-E) were observed in eight of the 13 cases (62%) with refractory anemia with or without ring sideroblasts (RA and RARS) and one of the five cases with RA with excess of blasts or in transformation (RAEB and RAEB-T). Suspension cultures with CD34+/CD36- sorted cells with Epo plus MGF demonstrated an 8.9 +/- 6.5-fold expansion after 7 days in cases with >10 BFU-E/10(4) CD34+/CD36- cells while cases with <10 BFU-E/10(4) CD34+/CD36- cells demonstrated 1.0 +/- 0.8-fold expansion especially in cases with RAEB/RAEB-T. FACS and morphology analysis after 7 days of suspension culture demonstrated partial differentiation along the erythroid lineage in cases with RA/RARS (75%) and RAEB/RAEB-T (66%) reflected by the presence of erythroblasts and normoblasts with variable expression of CD34, CD36 and Glycophorin A. In cases with erythroid colony formation 69 +/- 24% of the cells were CD34-/CD36+ and in cases with <10 BFU-E/10(4) CD34+ cells 18 +/- 16% of cells were CD34-/CD36+. Iron staining showed the presence of ring sideroblasts in two cases with RARS indicating that the cells originate from the abnormal erythroid clone. Finally, it was shown that cases with an impaired proliferative response demonstrate an enhanced binding of Annexin-V on CD34+ cells during the first days of the cell suspension culture phase. These results suggest that a defect in the proliferative response is most pronouncedly expressed in MDS whereas a subpopulation of cells retain the capacity to differentiate between transition to a terminated stage.

The effects of IL-1 and IL-4 on the Epo-independent erythroid progenitor in polycythaemia vera.

Human recombinant interleukin-1 (IL-1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL-1 was noticed on the normal, erythropoietin (Epo) dependent, erythroid burst-forming unit (BFU-E) using peripheral blood or bone marrow. In contrast, the Epo-independent BFU-E from peripheral blood of PV patients could be stimulated significantly. This enhancing effect of IL-1 was not only observed with unsorted but also with sorted CD34+ cells. In addition, it was shown that IL-1 indirectly stimulated the Epo-independent BFU-E because anti-GM-CSF could abrogate the supportive effects of IL-1. In contrast to the Epo-independent BFU-E, the Epo-dependent erythroid colony formation from PV patients could not be augmented by IL-1. Finally, we studied the effects of IL-4 on the Epo-independent BFU-E, because IL-4 can affect the erythroid colony formation and modulate the effects of IL-1. IL-4 suppressed the Epo-independent BFU-E. This effect could be counteracted by the addition of IL-1 to the culture medium. However, the suppressive effect of IL-4 was not related to a decline in spontaneous release of IL-1, because an anti-IL-1 antibody did not modify the spontaneous erythroid colony formation. These data indicate that IL-1 and IL-4 exert separate influences on the Epo-independent erythroid colony formation in PV.

Interleukin-4 receptor regulation in human monocytic cells.

The regulation of the interleukin-4 receptor (IL-4R) was studied at mRNA and protein level in monocytic cells on stimulation with activators of different intracellular signaling pathways and IL-4. Activation of protein kinase C-dependent pathways with phorbol myristate acetate (PMA) or activation of protein kinase A-dependent pathways with DBcAMP and prostaglandin E2 resulted in an augmented IL-4R expression at mRNA and protein level. Transcriptional and posttranscriptional mechanisms seemed to be involved in the promotive effect of DBcAMP because the transcription rate increased 1.8-fold, and the half-life of IL-4R mRNA was prolonged to 150 minutes compared with 120 minutes in unstimulated cells. In contrast, the effect of PMA could only be ascribed to changes at transcriptional level. However, activation of Ca(2+)-dependent pathways with A23187 or stimulation with IL-4 had no effect on the IL-4R expression. The unresponsiveness to IL-4 could not be ascribed to a nonfunctional receptor because IL-4 did modulate the CD14, CD23, and HLA-DR antigen expression. These results are in contrast with IL-4R regulation in T cells, which is affected by IL-4- and Ca(2+)-dependent pathways. The discrepancy might be caused by the presence of the common IL-2 receptor gamma chain (gamma c) in T cells and the absence of the gamma c in monocytic cells, as has been shown by polymerase chain reaction. These data indicate that IL-4Rs are differentially regulated, depending on the cell type studied.

Recombinant human mast cell growth factor supports erythroid colony formation in polycythemia vera in the presence and absence of erythropoietin and serum.

The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.

Erythroid progenitors in polycythemia vera demonstrate a different response pattern to IL-4 compared to the normal BFU-E from peripheral blood.

Human recombinant interleukin 4 (IL-4) was studied for its effects on erythroid burst-forming units (BFU-E) from normal peripheral blood and from patients with polycythemia vera (PV). IL-4 enhanced the proliferation of normal peripheral blood BFU-E (183% +/- 20% enhancement), whereas in the presence of interleukin 3 (IL-3) no further augmentation was noticed. The IL-4-mediated effects were independent of the absence or presence of adherent cells, B cells, or T cells. These data are in contrast with results obtained from normal human bone marrow cells, in which IL-4 antagonized the enhancing effects of IL-3. In PV a different response pattern was observed. The effects of IL-4 on the erythropoietin (Epo)-independent BFU-E were variable. In five PV patients no suppressive or enhancing effects of IL-4 were observed, whereas in two additional patients a significant decline in the Epo-independent BFU-E was noted. In the presence of IL-3, IL-4 significantly antagonized the IL-3-supported Epo-independent BFU-E in all patients (272% +/- 57% vs 187% +/- 49% enhancement, p less than 0.05). In contrast, IL-4 did not modify the IL-3-supported Epo-dependent BFU-E. In summary, these data suggest a difference between the normal and PV peripheral blood BFU-E. The Epo-dependent erythroid progenitors in PV patients showed a response pattern with IL-3 and IL-4 comparable to that of normal peripheral blood BFU-E, whereas the Epo-independent erythroid progenitors behaved like normal human bone marrow BFU-E, suggesting a shift in the stem cell compartment in PV. This is further supported by the finding that erythroid colony-forming units (CFU-E), normally only present in the bone marrow, could be cultured from the peripheral blood of PV patients in the presence or absence of Epo.