RESUMEN
Transforming growth factor (TGF)-beta1 exerts autocrine and paracrine effects on hematopoiesis. Here, we have attempted to evaluate the effect of endogenous TGF-beta1 on early erythroid development from primitive human hematopoietic stem cells (HSCs) and to assess the effects of TGF-beta1 on different phases of erythropoiesis. Cord blood CD34(+)CD38(-) lineage-marker-negative (Lin(-)) cells were cultured in serum-free conditions using various combinations of stem cell factor (SCF), erythropoietin (Epo), and TGF-beta-neutralizing antibody. Generation of erythroid progenitors was assessed using colony assay and flow cytometry. Terminal erythroid differentiation was examined when SCF/Epo-stimulated cells were recultured in the presence of Epo with and without TGF-beta1. Anti-TGF-beta augmented the proliferation of CD34(+)CD38(-)Lin(-) cells (day 21) in SCF-stimulated (6.4-fold +/- 1.5-fold) and SCF/Epo-stimulated (2.9-fold +/- 1.2-fold) cultures. Cells stimulated by SCF/Epo underwent similar levels of erythroid differentiation with and without anti-TGF-beta. While SCF alone stimulated the production of tryptase-positive mast cells, cells stimulated by SCF/anti-TGF-beta were predominantly erythroid (CD36(+)CD14(-) and glycophorin A positive). A distinct expansion of erythroid progenitors (CD34(+)CD36(+)CD14(-)) with the potential to form erythroid colonies was seen, revealing early Epo-independent erythroid development. In contrast, the kinetics of erythroid progenitor generation from primitive HSCs indicate that TGF-beta1 is not inhibitory in late erythropoiesis, but it accelerated the conversion of large BFU-E into colony-forming units-erythroid. Finally, TGF-beta1 accelerated Epo-induced terminal erythroid differentiation and resulted in a greater level of enucleation (22% +/- 6% versus 7% +/- 3%) in serum-free conditions. Serum addition stimulated enucleation (54% +/- 18%), which was lower (26% +/- 14%) with anti-TGF-beta, suggesting that optimal erythroid enucleation is Epo dependent, requiring serum factors including TGF-beta1.
Asunto(s)
ADP-Ribosil Ciclasa/análisis , Antígenos CD34/análisis , Antígenos CD/análisis , Células Precursoras Eritroides/citología , Eritropoyesis , Sangre Fetal/citología , Factor de Células Madre/farmacología , Factor de Crecimiento Transformador beta/fisiología , ADP-Ribosil Ciclasa/inmunología , ADP-Ribosil Ciclasa 1 , Antígenos CD/inmunología , Antígenos CD34/inmunología , Antígenos de Diferenciación/análisis , Comunicación Autocrina , Diferenciación Celular , Linaje de la Célula , Células Precursoras Eritroides/química , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Sangre Fetal/metabolismo , Hematopoyesis , Factores de Crecimiento de Célula Hematopoyética/efectos de los fármacos , Humanos , Cinética , Glicoproteínas de Membrana , Pruebas de Neutralización , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
Because interleukin-18 (IL-18) is similar to IL-1 and is known to be involved in the hematopoietic progenitor cell growth, the effect of IL-18 on circulating cell populations was examined. Repeated administration of IL-18 induced significant amounts of neutrophilia in mice. In parallel, high levels of interferon-gamma (IFN-gamma), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in the serum of these mice. Interestingly, the cytokine profiles as well as the cell populations in circulation altered around 2 weeks after the beginning of IL-18 administration. A weak but definite eosinophilia was observed concurrently with the appearance of serum IL-5. Consistent with these observations, IL-18 induced secretion of IFN-gamma, GM-CSF, and IL-6 from splenocytes in culture. IL-18 also induced low levels of IL-5 in the splenocyte culture, which was inhibited by IL-12. However, markedly high levels of IL-5 were secreted into the culture medium when splenocytes from IFN-gamma-deficient mice were stimulated by IL-18. CD4(+) T cells strongly responded to IL-18 to secrete IL-5 and GM-CSF. IL-18 stimulated secretion of IL-6 and expression of G-CSF mRNA in splenic adherent cells. Expression of IL-18 receptors was detected in CD4(+) T cells and splenic adherent cells (macrophages). These results show that IL-18 stimulates CD4(+) T cells and macrophages to secrete IL-5, GM-CSF, IL-6, and granulocyte-colony stimulating factor in the absence of IL-12, which in turn induces hematopoietic cell proliferation causing neutrophilia and eosinophilia in mice.
Asunto(s)
Citocinas/biosíntesis , Granulocitos/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/biosíntesis , Factores de Crecimiento de Célula Hematopoyética/efectos de los fármacos , Interleucina-18/farmacología , Animales , Citocinas/sangre , Citocinas/efectos de los fármacos , Interacciones Farmacológicas , Femenino , Granulocitos/citología , Interferón gamma/genética , Interferón gamma/farmacología , Interleucina-18/administración & dosificación , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Mannuronan (poly-beta-(1-->4)-D-mannuronate or poly-M), produced by Pseudomonas aeruginosa as a mucoid exopolysaccharide, has previously been shown to exhibit immunostimulating activity. The authors investigated the in vivo and in vitro effects of mannuronan on murine haematopoiesis. In vivo, prophylactic (-24 h, intraperitoneal) administration of mannuronan enhanced survival of lethally irradiated mice from zero day 40 survivors (NaCl) to 20, 80 and 70% survival at 0.5, 1 and 2 mg/kg bw mannuronan, respectively. In vitro, primary stromal cultures stimulated with mannuronan produced high levels of interleukin(IL)-1, IL-6 and colony stimulating activity. Mannuronan alone did not have any colony stimulating activity on GM-CFC, BFU-E, Mix-CFC or HPP-CFC progenitors in clonogenic assays, but acted synergistically with suboptimal amounts of growth factors on GM-CFC, Mix-CFC and HPP-CFC colony formation. Limiting dilution analysis showed that 1 of 423 bone marrow cells formed colonies in response to suboptimal GM-CSF plus mannuronan compared to 1 of 592 for suboptimal GM-CSF alone. The primitive Lin-Sca-1+ haematopoietic progenitors showed increased day 10 colony size in the presence of mannuronan in single cells assays. These stimulating effects of mannuronan on haematopoiesis may prove to have clinical importance.