A probable role for trail-induced apoptosis in the pathogenesis of marrow failure. Implications from an in vitro model and from marrow of aplastic anemia patients.

Aplastic anemia (AA) is a syndrome of hematopoietic failure involving increased apoptosis of stem cells. In order to investigate the molecular mechanisms participated in the process of marrow failure, we created an in vitro model of hematopoietic cell suppression, by continuous addition of TNF-alpha and IFN-gamma in an vitro long-term bone marrow culture system. An up-regulation of Fas expression was observed in CD34+ cells in cytokine treated cultures, compared to controls. This was accompanied by significant TRAIL and decreased caspase 3 mRNA expression, whereas the expression of Bcl-2 family members was low (Bcl-xl) or absent (Bcl-2, Bax). The expression of these apoptotic genes was also investigated in aplastic anemia patients. Apart from Fas mRNA expression in total marrow and/or CD34+ cells, TRAIL mRNA expression was found only in CD34+ cells in active disease while in total marrow cell compartment this remains a constant finding even in patients in remission. The above data are in agreement with previous studies proposing a major role for the extrinsic apoptosis pathway in the pathogenesis of aplastic anemia and additionally introduce TRAIL as a probable important molecule in the process.

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.

Impaired clonogenic growth of myelodysplastic bone marrow progenitors in vitro is irrelevant to their apoptotic state.

Excessive intramedullary apoptosis has been considered to account for the paradox of hypercellular marrow and refractory cytopenias in myelodysplastic syndrome (MDS). However, a causative relationship of apoptosis to the progenitor's defective clonogenic growth has not been sufficiently demonstrated. We investigated the degree of apoptosis and its contribution to ineffective hematopoiesis in MDS, by assessing the differential clonogenic capacity of purified "apoptotic" and "non-apoptotic" bone marrow progenitors in a short-term semisolid culture system. Although increased apoptosis was indeed detected in MDS bone marrow progenitors, there was no correlation between the existence of apoptosis and culture performance. Non-apoptotic as well as apoptotic CD34+ cells gave similar patterns of growth, both defective compared to normal. The ability of "apoptotic" CD34+ cells to proceed in colony formation as well as the abnormal growth of "non-apoptotic" progenitors are probably pointing towards the need to reconsider the role of apoptosis in the defective clonogenicity of MDS.