RESUMO
The quality and quantity of BCR signals impact on cell fate decisions of B lymphocytes. Here, we describe novel gene-targeted mice, which in the context of normal VDJ recombination show hypomorphic expression of immunoglobulin µ heavy chain (µHC) mRNA levels and hence lower pre-BCR and BCR levels. Hypomorphic expression of µHC leads to augmented selection processes at all stages of B-cell development, noticeably at the expansion of pre-B cells, the positive selection of immature B lymphocytes in the bone marrow and the selection of the follicular (FO), marginal zone (MZ) and B1 B-lymphocyte compartment in peripheral lymphoid organs. Immature as well as mature FO and MZ B lymphocytes in the peripheral lymphoid organs express lower levels of the receptor for B-cell activating factor (BAFF). In addition, hypomorphic expression of the BCR favours receptor editing. Together, our results highlight the critical importance of pre-BCR and BCR receptor levels for the normal development of B-lymphocyte subpopulations in the context of intact VDJ recombination and a diverse antibody repertoire.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Diferenciação Celular/imunologia , Genes de Cadeia Pesada de Imunoglobulina/fisiologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Alelos , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/fisiologia , Receptor do Fator Ativador de Células B/metabolismo , Subpopulações de Linfócitos B/metabolismo , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Genes de Cadeia Pesada de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/genética , Cadeias mu de Imunoglobulina/metabolismo , Lectinas/genética , Lectinas/imunologia , Lectinas/metabolismo , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/fisiologia , Receptores de Antígenos de Linfócitos B/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido SiálicoRESUMO
BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34+ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34+ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5-50 ng/ml had not effect on the expansion of CD34+ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34+ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proliferação de Células/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Antígenos CD34/biossíntese , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteína Morfogenética Óssea 7 , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/biossíntese , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/biossíntese , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos , Recém-Nascido , Interleucina-3/farmacologia , Proteínas de Membrana/farmacologia , Fator de Células-Tronco/farmacologia , Fatores de TempoRESUMO
Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the alpha4beta1 and alpha5beta1 integrins very-late antigen (VLA)-4 and VLA-5 on peripheral blood-derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA-4 and VLA-5 on CD34+/c-kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient-derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long-term culture-initiating cell. The improvement of the homing abilities of SCF-stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic-scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.
