RESUMEN
ABSTRACT Macrophage inflammatory protein-1alpha (MIP-1alpha) is a C-C chemokine which has antiproliferative effects on early hematopoietic progenitors and stimulatory effects on later progenitors. It also possesses chemotactic and activating properties for monocytes, macrophages, and T-cells. CD34+ progenitors isolated utilizing an avidin-biotin immunoadsorption column produced significant amounts of MIP-1alpha from 24 h onward when cultured in medium with 10% fetal calf serum (>200 pg/ml). Such production persisted through 96 h of culture and was greater when such progenitors were cocultured with a preformed marrow stromal layer (4000 pg/ml at 24 h). The production of MIP-1alpha declined over time of coculture with stromal layers, and stromal layers themselves produced minimal MIP-1alpha as detected by ELISA: <100 pg/ml. In contrast, CD34+ cells isolated by flow cytometry or by magnetic bead adsorption produced minimal MIP-1alpha (0-30 pg/ml). MIP-1alpha production also increased when cells isolated by these two methods were cocultured with stromal layers. The difference in MIP-1alpha production could not be accounted for by differences in purity of the CD34+ population between isolation methods nor on the basis of monocytic or lymphocytic contamination as assessed by the presence of CD14 or CD3 positive cells. CD34+ cells isolated by immune adsorption had increased expression of endothelial and mesenchymal associated antigens, however, suggesting that this subpopulation might account for the MIP-1alpha production observed. Freshly isolated CD34+ cells expressed MIP-1alpha message as assessed by RT-PCR and by in situ hybridization. Coculture of CD34+ cells isolated by any means with stromal cells increased transforming growth factor-beta (TGF-beta) production, in this case by the stromal layer itself. Both MIP-1alpha and TGF-beta have been found to influence cell cycle status and proliferation status of early hematopoietic progenitors, and both have potential effects on accessory cell function. These studies indicate that progenitor-stromal cell interactions may influence local cytokine output, thus potentially influencing progenitor cycling status and accessory cell activation. The method of isolation of CD34+ progenitors may influence secretion of certain cytokines and chemokines.
Asunto(s)
Antígenos CD34/metabolismo , Proteínas Inflamatorias de Macrófagos/biosíntesis , Células Madre/metabolismo , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Comunicación Celular , Separación Celular/métodos , Células Cultivadas , Quimiocina CCL3 , Quimiocina CCL4 , Técnicas de Cocultivo , Regulación de la Expresión Génica , Humanos , Leucemia/patología , Proteínas Inflamatorias de Macrófagos/farmacología , ARN/genética , ARN/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células del Estroma/citología , Factor de Crecimiento Transformador beta/genética , Células Tumorales CultivadasRESUMEN
Stimulation of CD34(+)-enriched marrow or light density marrow with various growth factor combinations can generate granulocyte progenitors and mature neutrophils in vitro. In this work, we have examined the influence of irradiated marrow stromal layers on growth factor-induced myeloid and early multipotential progenitor expansion from enriched marrow CD34+ progenitors. We have also explored whether the addition of early-acting growth factors known to enhance myelopoiesis in long-term culture, such as fibroblast growth factor (b-FGF), insulin growth factor (IGF-1), c-kit ligand or stem cell factor (SCF), and flk-2flt-3 ligand (FL), can lengthen survival of CD34+ progenitors in these cultures. Stromal cell coculture resulted in greater numbers of total cells and CFU-GM at day 7 and day 14, but with the addition of multiple growth factors, these effects of stromal cell coculture were diminished. At day 14, generally < 1% of the expanded cells over stromal coculture conditions were CD34+, with up to 90% demonstrating CD15 positivity. Culture of CD34+ cells in the presence of early-acting growth factors did not cause significant expansion of CD34+ cells over a 14-day life span, even in the presence of marrow stromal cells. These data suggest that although stromal cell coculture for a period up to 14 days can enhance expansion of total cell numbers and CFU-GMs, stromal cell presence does not lead to expansion of CD34+ cells in these cultures and may diminish the number of clonogenic cells present when growth factors with differentiating capacity are present. Mature neutrophils harvested from such cultures are capable of chemotaxis, actin polymerization, and migration, suggesting a replete functional status.
Asunto(s)
Antígenos CD34 , Células Madre Hematopoyéticas/fisiología , Neutrófilos/fisiología , Células del Estroma/fisiología , Actinas , Quimiotaxis , Técnicas de Cocultivo , Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Neutrófilos/efectos de los fármacos , FagocitosisRESUMEN
The megakaryoblastic cell line, UT-7, is dependent for its growth upon interleukin-3 (IL-3), erythropoietin, or granulocyte-macrophage colony stimulating factor (GM-CSF). A subculture of this line can be maintained in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this subculture from SCF results in rapid loss of viability and decreased proliferation. Cells grown in SCF also can be maintained in GM-CSF but not vice versa. In this work, we have characterized the SCF dependence of this UT-7 subculture. Stem cell factor removal results in apoptosis and a decline in viability which can be restored partially by re-addition of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented by light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quantitate the degree of apoptosis in the cell populations, and to assess whether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exposure of previously factor-starved cells to stroma decreased the percentage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggesting contribution of other stromal cell factors. Experiments performed using trans-well inserts which do not allow cell passage, showed greatest viability of cells in contact with stroma, but viability was also improved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a role for soluble stroma-produced substances. These data demonstrate that SCF alone can prevent apoptosis in cells dependent upon its presence for proliferation. Also, marrow stromal cells can serve as a partial substitute for growth factor in the prevention of apoptosis in these cells, probably due to constitutive presentation of SCF and other hematopoietic growth factors in both soluble and surface-bound forms.
Asunto(s)
Apoptosis , Células de la Médula Ósea , Leucemia Megacarioblástica Aguda/patología , Factor de Células Madre/farmacología , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Bucladesina/farmacología , División Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular , Técnicas de Cocultivo , ADN de Neoplasias/análisis , Eritropoyetina/farmacología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Sustancias de Crecimiento/inmunología , Sustancias de Crecimiento/farmacología , Sustancias de Crecimiento/fisiología , Humanos , Interleucina-3/farmacología , Cinética , Microscopía Electrónica , Proteínas Recombinantes/farmacología , Células del Estroma/fisiología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Hematopoiesis is influenced by the presence of the hematopoietic microenvironment, and Dexter-type liquid culture systems represent an in vitro representation of some aspects of the microenvironment that are optimal for the propagation of myeloid progenitors. Marrow stromal layers, which constitute part of these culture systems, produce growth factors, including stem cell factor (SCF), a ligand for the c-kit proto-oncogene that has been found to increase detection of myeloid, erythroid, and megakaryocytic progenitors in short-term marrow colony assays. In this work, the role of SCF in Dexter-type culture systems was examined to better define its contribution to steady-state myelopoiesis. When cultured in the continued presence of 100 ng/mL SCF, both primary and recharged cultures demonstrated significantly greater CFU-GM output, with quantitative differences noted throughout culture duration (up to 6 weeks). This increase in CFU-GM could be inhibited specifically with the addition of 1:1500 SR-1, a neutralizing anti-c-kit monoclonal antibody (MAb) that neutralizes the biological effects of SCF, and the increase was noted both with recharged light-density marrow cells and purified CD34+ progenitor cells. On the other hand, when primary or recharged marrow cultures were established in the absence of exogenous SCF, but in the continuous presence of SR-1, no inhibition of CFU-GM output was observed. When light-density marrow cells were purged of pre-existing CFU-GM by 4-hydroperoxycyclophosphamide (4-HC) and were seeded over irradiated stromal layers, exogenous SCF resulted in detection of CFU-GM from 4-HC-treated cells as early as 1 week of culture, as compared to the lack of significant emergence of CFU-GMs at 4 weeks in the control cultures. This SCF effect was also inhibited by SR-1. Purified CD34+ progenitor cells did not adhere to SCF immobilized to tissue culture plates, and the adhesion of such progenitors to murine Steel lines transfected with membrane-bound SCF was not greater than to the parent nontransfected Steel line, suggesting that the effect of SCF was not on CD34+ cell adhesion. These studies confirm the action of SCF at a pre-CFU level, and they demonstrate the ability of SCF to stimulate increased production of myeloid progenitors in long-term liquid culture systems.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Anticuerpos Monoclonales/farmacología , Antígenos CD/análisis , Antígenos CD34 , Células de la Médula Ósea , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Factores de Crecimiento de Célula Hematopoyética/análisis , Humanos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-kit , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores del Factor Estimulante de Colonias/inmunología , Factor de Células MadreRESUMEN
Human immunodeficiency virus (HIV) infection often has effects on the hematopoietic system which can be distinguished from the concurrent effects of medications or opportunistic infections. Exactly how the virus mediates these effects remains uncertain, but both in vivo and in vitro studies have pointed up possible direct and indirect modes of hematopoietic suppression. Whether a significant fraction of CD34+ cells in vivo are infected with HIV remains controversial, but most studies using in situ polymerase chain reaction techniques would suggest not. Other more indirect modes of hematopoietic cell suppression such as production of autoantibodies, production of other humoral inhibitory factors, T-cell mediated suppression of hematopoiesis, or production of inhibitory or stimulatory cytokines may also be contributory. It is probable that several of these mechanisms may occur simultaneously, and an increased understanding of their role may lead to improved strategies to correct the cytopenias which often accompany HIV disease.
Asunto(s)
Anemia/fisiopatología , Infecciones por VIH/sangre , Leucopenia/fisiopatología , Trombocitopenia/fisiopatología , Anemia/terapia , Antígenos CD , Antígenos CD34 , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Médula Ósea/patología , Citocinas/biosíntesis , Inhibidores de Crecimiento/biosíntesis , VIH/patogenicidad , Hematopoyesis , Factores de Crecimiento de Célula Hematopoyética/uso terapéutico , Células Madre Hematopoyéticas/patología , Células Madre Hematopoyéticas/virología , Humanos , Leucopenia/terapia , Modelos Biológicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Trombocitopenia/terapiaRESUMEN
The capacity of normal CD34+ marrow cells and CD34+ leukemic cell lines to adhere to human umbilical vein endothelial cells has been examined. Such interactions have importance since the processes of homing and egress within the marrow microenvironment involve the traverse of sinusoidal endothelium. Umbilical vein endothelial monolayers expressed CD44 and CD54 constitutively, and expression of both E-selectin (ELAM) and vascular cell adhesion molecule-1 (VCAM-1) were inducible with interleukin-1 (IL-1) alpha and beta and tumor necrosis factor (TNF). CD34+ marrow cells bound to unstimulated endothelial layers (33 vs. 16% to plastic), and their adhesion was significantly increased in the presence of IL-1 or TNF. This increased adhesion was not inhibited by functionally blocking antibodies to E-selectin or to CD54 but was partially inhibited by antibodies to VCAM. CD34+ KG1a cells also bound to endothelial monolayers (33 vs. 8% to plastic), and such adhesion was also upregulated by pretreatment of the endothelial cells with IL-1 or TNF. In contrast to normal CD34+ cells, this increased adhesion was inhibited by antibodies to E-selectin but not to VCAM. These findings indicate that adhesion of both normal CD34+ cells and leukemic blasts to endothelial cells can be upregulated by inflammatory mediators such as TNF and IL-1.