RESUMO
BACKGROUND/AIMS: Disease-related anemia in chronic lymphocytic leukemia (CLL) occurs when the obvious causes are excluded while its pathogenesis is still obscure. We investigated its underlying mechanisms in 56 untreated patients with CLL. METHODS: Bone marrow (BM) lymphocytic infiltration was estimated in trephine biopsies. Serum erythropoietin (EPO) and tumor necrosis factor-alpha (TNF-alpha) levels were measured by ELISA. The potential of BM CD34+ to differentiate into erythroid cells was evaluated by methylcellulose-based assays and in liquid cultures supplemented with EPO, SCF, IL-3 +/- TNF-alpha. The response of erythroid precursors to EPO +/- TNF-alpha was assessed by detecting activated key proteins of EPO-EPO receptor signalling pathway using Western Blot and EMSA. RESULTS: Bone marrow lymphocytic infiltration was not exclusively responsible for disease-related anemia and CD34+ cells were intrinsically capable of generating erythroid precursors. Also, no deficiency of serum erythropoietin (EPO) or defective intracellular response of erythroid precursors to EPO +/- TNF-alpha stimulation was observed. Serum TNF-alpha levels were found increased in anemic CLL patients and TNF-alpha appeared to directly inhibit the erythroid development in early stages of erythropoiesis. CONCLUSION: We concluded that CLL-related anemia was not due to intrinsic defects of erythroid precursors, but might result from the direct suppressive effect of TNF-alpha on the erythroid production.
Assuntos
Anemia/etiologia , Eritropoese/fisiologia , Leucemia Linfocítica Crônica de Células B/complicações , Proteínas de Neoplasias/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/fisiopatologia , Medula Óssea/patologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/patologia , Células Precursoras Eritroides/efeitos dos fármacos , Células Precursoras Eritroides/patologia , Eritropoetina/sangue , Feminino , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Infiltração Leucêmica , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Anemia Aplástica/metabolismo , Proteínas Reguladoras de Apoptose/biossíntese , Apoptose , Células-Tronco Hematopoéticas/metabolismo , Glicoproteínas de Membrana/biossíntese , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima , Adulto , Idoso , Anemia Aplástica/patologia , Antígenos CD34/biossíntese , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteína X Associada a bcl-2/biossíntese , Proteína bcl-X/biossíntese , Receptor fas/biossínteseRESUMO
Myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. MDS patients have a defective immune response manifested by increased susceptibility to bacterial infections, autoimmune phenomena, and high incidence of lymphoid malignancies. Presently, we investigated the phenotype and function of monocyte-derived dendritic cells (MoDC) in 23 MDS patients and 15 controls at different stages of differentiation using the maturation stimuli tumor necrosis factor-alpha (TNF-alpha) and LPS. Monocytes from MDS patients showed low potential to differentiate into dendritic cells (DC), as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of mannose receptor and reduced endocytic capacity. MDS-MoDCs showed a diminished response to TNF-alpha with low CD83, CD80, and CD54 expression and allostimulatory capacity. In patients with 5q syndrome, monocytes and MoDCs were positive for the 5q deletion, suggesting their origin from the malignant clone. Our data indicate that MoDCs in MDS display quantitative and functional abnormalities that may contribute to the defective immune response of these patients.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Monócitos/efeitos dos fármacos , Monócitos/patologia , Síndromes Mielodisplásicas/patologia , Fator de Necrose Tumoral alfa/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antígenos/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Endocitose , Feminino , Citometria de Fluxo , Humanos , Lectinas Tipo C/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Pessoa de Meia-Idade , Monócitos/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismoRESUMO
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.
Assuntos
Antígenos CD34/análise , Células Dendríticas/patologia , Células-Tronco Hematopoéticas/patologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Diferenciação Celular , Divisão Celular , Células Cultivadas , Células Clonais/patologia , Células Dendríticas/imunologia , Feminino , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunofenotipagem , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/imunologiaRESUMO
The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies, but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice, whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases, the regenerated cells showed the same clonal markers (trisomy 8, n = 3; and 5q-, n = 1) as the original sample and, in one instance, regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly, the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3, granulocyte-macrophage colony-stimulating factor, and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS.