RESUMEN
Airway inflammation is characterized by an accumulation of activated leukocytes. Bronchial epithelial cells may contribute to this process by releasing chemokines and by expressing surface membrane molecules involved in the adhesion and activation of the recruited leukocytes. In this study, we analyzed the effects of cytokines and glucocorticoids on the release of monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for predominantly monocytes and lymphocytes, by human bronchial epithelial cells and compared this with the release of interleukin-8 (IL-8), which potently attracts neutrophils. In addition, we analyzed the effects of cytokines and glucocorticoids on the epithelial expression of intercellular adhesion molecule (ICAM)-1, CD40, and human leukocyte antigen (HLA) class II molecules. Primary cultures of human bronchial epithelial cells constitutively released MCP-1 and IL-8. IFN-gamma greatly increased MCP-1 release, which was accompanied by increased expression of MCP-1 mRNA and an increased monocyte chemotactic potential. In contrast, IFN-gamma had no effect on the release of IL-8, but it did increase the epithelial expression of ICAM-1, CD40, and HLA class II molecules. IL-1beta increased both MCP-1 and IL-8 release, and increased the expression of ICAM-1 and CD40, but not HLA class II molecules. Dexamethasone partially inhibited the cytokine-induced release of MCP-1 and IL-8 and the expression of ICAM-1, CD40, and HLA class II molecules by human bronchial epithelial cells. Our results indicate that IFN-gamma and IL-1beta differentially regulate the MCP-1 and IL-8 release by human bronchial epithelial cells. In addition, IL-1beta and particularly IFN-gamma increase the expression of ICAM-1, HLA class II and/or CD40 molecules, which are involved in the adhesion and possibly activation of the recruited leukocytes. Finally, the beneficial effect of glucocorticoid therapy in airway inflammatory diseases may be mediated in part by inhibition of chemokine release and ICAM-1, CD40, and HLA class II expression by bronchial epithelial cells.
Asunto(s)
Bronquios/inmunología , Quimiocina CCL2/genética , Células Epiteliales/inmunología , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-8/genética , Bronquios/efectos de los fármacos , Antígenos CD40/genética , Células Cultivadas , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Dexametasona/farmacología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Antígenos HLA-D/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Molécula 1 de Adhesión Intercelular/genética , Interleucina-8/metabolismo , Monocitos/efectos de los fármacos , Monocitos/fisiología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Reacción en Cadena de la PolimerasaRESUMEN
Platelet-derived growth factor (PDGF) B-chain mRNA is readily detectable in malignant mesothelioma (MM) cell lines, but not in normal mesothelial (NM) cell lines. The high affinity receptor for PDGF B-chain dimers, the PDGF beta-receptor, is expressed in MM cell lines. NM cell lines predominantly express the PDGF alpha-receptor. Coexpression of the PDGF beta-receptor and its ligand may lead to an autocrine growth stimulating loop in the malignant cell type. In nuclear run off experiments, PDGF B-chain mRNA was detectable in MM cells only, indicating an increased level of transcription in this cell type. The proximal promoter of the PDGF B-chain gene contains DNaseI hypersensitive (DH) sites and mediates reporter gene activation in both normal and malignant cells. Nuclear proteins, extracted from both cell types, interact with DNA sequences within the proximal promoter around bp-64 to -61 relative to the transcription start site. Electrophoretic mobility shift assays (EMSAs) indicate that these factors are more abundantly present in the malignant than in the normal cell type. A DH site around -9.9 kb was found in both cell types. When tested in CAT assays, this region exerted a stimulatory effect on transcription in malignant cells. The elevated level of transcription of the PDGF B-chain gene in malignant cells may well be the result of interaction of regulatory sites in the proximal promoter and an enhancing element located at -9.9 kb from the transcription start site.
Asunto(s)
Mesotelioma/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción Genética , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The expression of platelet-derived growth factor (PDGF) and PDGF receptors was studied in human normal and malignant mesothelial cells in vitro and in vivo. Staining with anti-cytokeratin and ME1 antibodies and ultrastructural analysis confirmed the mesothelial nature of the cell lines used to study PDGF and PDGF receptor expression in vitro. Using antibodies, mesothelioma cell lines were found to express PDGF and both the PDGF alpha- and the PDGF beta-receptor, whereas cultured normal mesothelial cells expressed PDGF and PDGF alpha-receptor. This PDGF and PDGF receptor staining pattern largely reflects the earlier described mRNA expression in these cell lines. The only exception was the immunocytochemical detection of PDGF alpha-receptors in the mesothelioma cell lines, which is different from the inability to detect alpha-receptor transcripts on Northern blots. Expression was also investigated in mesothelial cells in vivo. Expression of PDGF was observed in malignant mesothelioma cells on frozen tissue sections. In pleural effusions, a double immunofluorescence staining procedure for PDGF and epithelial membrane antigen (EMA) revealed PDGF expression by EMA-positive malignant mesothelioma cells. PDGF beta-receptors and occasionally PDGF alpha-receptors were detected in frozen tissue sections of malignant mesotheliomas, whereas mesothelioma cells in effusions showed faint expression of only the PDGF beta-receptor. In contrast, in effusions containing non-malignant mesothelial cells, only a very low level of PDGF alpha-receptor could be detected. Taken together, these results indicate that the pattern of PDGF and PDGF receptor expression in mesothelial cells in vivo largely corresponds to expression of PDGF and its receptors in vitro. Malignant mesothelioma cell lines thus constitute a good model system for studies on the role of PDGF in this malignancy. Furthermore, the data reported in this paper are consistent with the idea that an autocrine growth stimulatory effect of PDGF via PDGF receptors may play a role in the pathogenesis of malignant mesothelioma.
Asunto(s)
Mesotelioma/metabolismo , Proteínas de Neoplasias/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Criopreservación , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Derrame Pleural Maligno/metabolismo , Células Tumorales CultivadasRESUMEN
In earlier studies we showed that the expression of patterns of platelet-derived growth factor (PDGF) alpha- and beta-receptors differ between normal and malignant mesothelial cell lines. Normal mesothelial cells predominantly express PDGF alpha-receptor mRNA and protein, whereas most malignant mesothelioma cell lines produce PDGF beta-receptor mRNA and protein. In this paper we studied regulation of this differential PDGF receptor mRNA expression. Such an analysis is of importance in view of the suggested PDGF autocrine activity involving the PDGF beta-receptor mesothelioma cells. The results obtained in this study demonstrate that malignant mesothelioma cell lines are not only capable of PDGF beta-receptor transcription but of alpha-receptor transcription as well, as evidenced from run off analysis and RT-PCR using alpha-receptor specific primers. However, the fact that PDGF alpha-receptor mRNA could not be detected by Northern blot analysis, even after cycloheximide treatment, suggests a difference in steady-state PDGF alpha-receptor mRNA expression levels between normal and malignant mesothelial cell lines, which is likely to be caused by a post-transcriptional mechanism. In normal mesothelial cells a half-life of more than 6 h was observed for PDGF alpha-receptor mRNA. In the majority of malignant mesothelioma cell lines clear PDGF beta-receptor mRNA expression was seen. The half-life of the PDGF beta-receptor transcript was at least 6 h in these cells. In contrast, hardly any PDGF beta-receptor transcription was observed in run off assays in normal mesothelial cells, suggesting that differences in beta-receptor transcriptional initiation most probably account for the inability to clearly detect PDGF beta-receptor transcripts in these cells. Transforming growth factor beta-1 (TGF-beta 1), which is being produced in active form by mesothelial cells was evaluated for its potential role in regulation of the differential PDGF receptor expression in these cells. Stimulation with TGF-beta 1 revealed decreased PDGF alpha-receptor mRNA expression in normal mesothelial cells. The effect on PDGF beta-receptor mRNA in the malignant mesothelioma cell lines was variable. Although the TGF-beta 1 effect cannot entirely explain the differential PDGF receptor expression pattern, TGF-beta 1 may nevertheless play a role in downregulation of an (already) low PDGF alpha-receptor mRNA level in malignant mesothelioma cell lines.
Asunto(s)
ARN Mensajero/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , Células Epiteliales , Epitelio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesotelioma/genética , Mesotelioma/metabolismo , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
We have studied the maintenance of stem cells with long-term multilineage repopulating ability from murine bone marrow, cultured on a pre-established bone marrow-derived stromal cell layer, both in a qualitative and quantitative way. Female bone marrow cells were cultured for a period of 1-4 weeks and compared with uncultured cells for their ability to establish and maintain a level of 50% chimerism in a sex-mismatched bone marrow transplantation model. Chimerism was determined in nucleated cells using fluorescence in situ hybridization with a murine Y-chromosome-specific probe. We observed a rapid decline in the ability of cultured marrow cells to repopulate the blood, bone marrow, spleen, and thymus of sublethally irradiated male recipients. After 4 weeks of culture only 5% of the long-term repopulating ability of the inoculated bone marrow cells remained. The remaining long-term repopulating cells, however, had similar qualities to establish and maintain long-term engraftment compared to fresh bone marrow, as judged from their ability to give stable chimerism over a period of 6 months. These observations are relevant for the therapeutic applications of long-term bone marrow cultures in purging protocols prior to autologous bone marrow transplantation of acute and chronic myeloid leukemic patients, and for the use of long-term marrow cultures when introducing foreign genetic material in hematopoietic stem cells.
Asunto(s)
Células de la Médula Ósea , Trasplante de Médula Ósea/patología , Células Madre Hematopoyéticas/citología , Animales , Células Cultivadas , Quimera , Femenino , Técnicas In Vitro , Masculino , Ratones , Factores de TiempoRESUMEN
Hemopoietic stem cells show extensive heterogeneity with respect to their proliferative potential and activity. We have recently reported that the accepted technique for sorting stem cells on the basis of high affinity for the lectin wheat germ agglutinin (WGA) did not select for cells initiating long-term production of new stem cells on a stromal layer in vitro. We have therefore reinvestigated the expression of cell surface sialic acid residues in the hemopoietic stem cell compartment by sorting murine bone marrow cells on the basis of affinity for WGA. Frequency analysis of long-term bone marrow culture initiating stem cells was done using the cobblestone-area-forming cell (CAFC) assay with limiting dilution set-up. In vivo stem cell quality was determined by spleen colony formation, marrow-repopulating ability (MRA) and long-term repopulating ability (LTRA) using sex-mismatched hemopoietic chimerism. The data indicate that MRA and LTRA in vivo and in vitro are among the most WGA-dim cells. In contrast, the enrichment factors for splenic colony-forming units (CFU-S) at day 12 and transient CAFC increase with increasing WGA affinity. These characteristics allowed us to concentrate LTRA cells 590- to 850-fold over their activity in normal bone marrow without significant enrichment of day-12 CFU-S. The data reveal that WGA affinity is an inverse function of the primitiveness of murine hemopoietic stem cells and that long-term production of blood cells in vivo and in vitro is provided for by primitive cells that are physically separable from the vast majority of day-12 CFU-S. In addition the data reveal, that the CAFC frequency at day 28-35 of a graft strongly correlates with the number of cells required to induce 40% donor-type chimerism at 15 months post-transplantation and thus predicts the in vivo LTRA of a graft.
Asunto(s)
Células de la Médula Ósea , Células Madre Hematopoyéticas/citología , Aglutininas del Germen de Trigo/metabolismo , Animales , División Celular , Separación Celular , Femenino , Citometría de Flujo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Magnetismo , Masculino , RatonesRESUMEN
The radiation sensitivity of various subsets in the haemopoietic stem cell hierarchy was defined using a limiting dilution type long-term bone marrow culture technique that was previously shown to allow quantification of cells with spleen colony-forming potential (day-12 CFU-S) and in vivo marrow repopulating ability (MRA). Primitive stem cells that generate new in vitro clonable colony-forming cells (CFU-C) in the irradiated marrow (MRA) and have long-term repopulation ability (LTRA) in vitro (cobblestone area forming cell, CAFC day-28) had D0 values of 1.25 and 1.38 Gy, respectively. A lower D0 was found for the less primitive CFU-S day-12, CAFC day-12 and cells with erythroid repopulating ability (0.91, 1.08 and 0.97 Gy, respectively). CFU-S day-7 were the most radiosensitive (D0 equalling 0.79 Gy), while CFU-C and CAFC day-5 were relatively resistant to irradiation (D0 1.33 and 1.77 Gy). Split-dose irradiation with a 6 h interval gave dose sparing for stem cells with MRA and even more with in vitro LTRA, less for CFU-S day-12 and CAFC day-10 and none for CFU-S day-7. The cell survival data of the specified stem cell populations were compared with the ability of a fixed number of B6-Gpi-1a donor bone marrow cells to provide for short- and long-term engraftment in single- and split-dose irradiated congenic B6-Gpi-1b mice. Serial blood glucose phosphate isomerase (Gpi) phenotyping showed less chimerism in the split as compared to the single radiation dose groups beyond 4 weeks after transplant. Radiation dose-response curves corresponding to stable chimerism at 12 weeks for single and fractionated doses revealed appreciable split-dose recovery (D2-D1) in the order of 2 Gy. This was comparable to D2-D1 estimates for MRA and late-developing CAFC (1.27 and 1.43 Gy, respectively), but differed from the poor dose recovery in cells corresponding to the committed CFU-S day-7/12 and CAFC day-10 population (0.14-0.33 Gy). These data are together consistent with differential radiosensitivity and repair in the haemopoietic stem cell hierarchy, and provide a cellular basis for explaining the dose-sparing effect of fractionated total-body irradiation conditioning on long-term host marrow repopulation.
Asunto(s)
Células Madre Hematopoyéticas/diagnóstico por imagen , Tolerancia a Radiación/fisiología , Bazo/citología , Células Madre/efectos de la radiación , Animales , Supervivencia Celular/efectos de la radiación , Radioisótopos de Cesio , Rayos gamma , Células Madre Hematopoyéticas/fisiología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Radiografía , Células Madre/fisiologíaRESUMEN
We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal "feeders" depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.