RESUMEN
As they mature into erythrocytes during normal erythropoiesis, reticulocytes lose surface transferrin receptors before or concurrently with reticulin. Exosome release accounts for most of the loss of transferrin receptors from reticulocytes. During erythropoietic stress, reticulocytes are released early from hematopoietic tissues and have increased reticulin staining and transferrin receptors. Flow cytometry of dually stained erythrocytes of mice recovering from phlebotomy demonstrated delayed loss of reticulin and transferrin receptors during in vitro maturation compared to in vivo maturation, indicating that an in vivo process extrinsic to the reticulocytes facilitates their maturation. Splenectomy or macrophage depletion by liposomal clodronate inhibited in vivo maturation of reticulocytes and increased the numbers of reticulin-negative, transferrin receptor-positive cells during and after recovery from phlebotomy. This reticulin-negative, transferrin receptor-positive population was rarely found in normal mice. Transmission electron microscopy demonstrated that the reticulin-negative, transferrin receptor-positive cells were elongated and discoid erythrocytes, but they had intracellular and surface structures that appeared to be partially degraded organelles. The results indicate that maturation of circulating stress reticulocytes is enhanced by an extrinsic process that occurs in the spleen and involves macrophage activity. Complete loss of reticulin with incomplete loss of surface transferrin receptors in this process produces a reticulin-negative, transferrin receptor-positive erythrocyte population that has potential utility for detecting prior erythropoietic stresses including bleeding, hemolysis and erythropoietin administration, even after recovery has been completed. Am. J. Hematol. 91:875-882, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Macrófagos/fisiología , Receptores de Transferrina/análisis , Reticulocitos/patología , Bazo/fisiología , Animales , Membrana Eritrocítica/metabolismo , Eritropoyesis , Femenino , Ratones , Flebotomía , Reticulina/análisis , Reticulocitos/metabolismoRESUMEN
INTRODUCTION: Transforming growth factor (TGF)-beta1 is proposed to inhibit the growth of epithelial cells in early tumorigenesis, and to promote tumor cell motility and invasion in the later stages of carcinogenesis through the induction of an epithelial to mesenchymal transition (EMT). EMT is a multistep process that is characterized by changes in cell morphology and dissociation of cell-cell contacts. Although there is growing interest in TGF-beta1-mediated EMT, the phenotype is limited to only a few murine cell lines and mouse models. METHODS: To identify alternative cell systems in which to study TGF-beta1-induced EMT, 18 human and mouse established cell lines and cultures of two human primary epithelial cell types were screened for TGF-beta1-induced EMT by analysis of cell morphology, and localization of zonula occludens-1, E-cadherin, and F-actin. Sensitivity to TGF-beta1 was also determined by [3H]thymidine incorporation, flow cytometry, phosphorylation of Smad2, and total levels of Smad2 and Smad3 in these cell lines and in six additional cancer cell lines. RESULTS: TGF-beta1 inhibited the growth of most nontransformed cells screened, but many of the cancer cell lines were insensitive to the growth inhibitory effects of TGF-beta1. In contrast, TGF-beta1 induced Smad2 phosphorylation in the majority of cell lines, including cell lines resistant to TGF-beta1-mediated cell cycle arrest. Of the cell lines screened only two underwent TGF-beta1-induced EMT. CONCLUSION: The results presented herein show that, although many cancer cell lines have lost sensitivity to the growth inhibitory effect of TGF-beta1, most show evidence of TGF-beta1 signal transduction, but only a few cell lines undergo TGF-beta1-mediated EMT.
Asunto(s)
Células Epiteliales/citología , Invasividad Neoplásica/fisiopatología , Factor de Crecimiento Transformador beta/fisiología , Actinas/análisis , Animales , Cadherinas/análisis , Adhesión Celular/fisiología , Ciclo Celular/fisiología , Línea Celular Transformada/citología , Línea Celular Tumoral/citología , Movimiento Celular/fisiología , Células Cultivadas/citología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Humanos , Técnicas In Vitro , Proteínas de la Membrana/análisis , Mesodermo , Ratones , Proteínas de Neoplasias/fisiología , Fenotipo , Fosfoproteínas/análisis , Fosforilación , Procesamiento Proteico-Postraduccional , Proteína Smad2 , Proteína smad3 , Transactivadores/análisis , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta1 , Proteína de la Zonula Occludens-1RESUMEN
INTRODUCTION: Macaque species offer a valuable model for translational allo-transplantation and tolerance studies. Cardiac allograft vasculopathy in Macaca fascicularis is associated with elaboration of anti-donor antibodies. Since T-independent pathways of B cell activation have been described, and anti-B cell strategies have proven to be a fruitful tolerogenic adjunct in rodent and xenogenic models, here we investigate whether an anti-CD20 antibody (rituximab) would be useful to deplete B-cells in a pre-clinical allo-transplantation setting in macaques. METHODS: Three cynomolgus macaques which had previously rejected a cardiac allograft and one with concurrent subacute vascular rejection were treated weekly with rituximab 20 mg/kg i.v. for 4 and 2 weeks, respectively. B-cell levels (CD19+ cells) were measured by flow cytometry in peripheral blood, spleen, lymph node and bone marrow cells at various intervals after initiation of treatment. B-cells and plasma cells were also analyzed by immunohistochemistry at necropsy in spleen, lymph node, tonsil and thymus tissue sections. Anti-donor antibody titers were measured by flow cytometry. RESULTS: B-cells expressing CD19 were not detectable in the peripheral blood in any animal within 24 h after initial treatment, or over the ensuing month. At necropsy, the germinal centers in spleen and lymph node were completely depleted of CD20+ B-cells in 2 animals, leaving a hypocellular trabecular pattern around preserved plasma cell follicles. Substantial but incomplete depletion of B-cells was demonstrated in the other 2 animals, in each instance immunohistochemical findings in spleen and lymph node exhibiting higher sensitivity for residual B-cells compared to FACS. Anti-donor antibody titers exhibited kinetics similar to untreated animals over this short follow-up. COMMENT: Treatment with anti-CD20 very efficiently depletes peripheral and tissue B-cells but not plasma cells in this macaque species. Biopsy of lymph node is necessary and may be sufficient to assess B-cell clearance in secondary lymphoid organs in this model.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD20/inmunología , Linfocitos B/inmunología , Tejido Linfoide/efectos de los fármacos , Animales , Anticuerpos/sangre , Anticuerpos/farmacología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales de Origen Murino , Antígenos CD19/análisis , Antígenos CD19/sangre , Linfocitos B/química , Células de la Médula Ósea/química , Ligando de CD40/inmunología , Ciclosporina/farmacología , Citometría de Flujo , Reacción Injerto-Huésped , Trasplante de Corazón , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Inmunoglobulina M/análisis , Inmunoglobulina M/inmunología , Inmunohistoquímica , Ganglios Linfáticos/citología , Ganglios Linfáticos/efectos de los fármacos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Macaca fascicularis , Masculino , Tonsila Palatina/citología , Tonsila Palatina/efectos de los fármacos , Células Plasmáticas/citología , Rituximab , Bazo/citología , Bazo/efectos de los fármacos , Linfocitos T/inmunología , Timo/citología , Timo/efectos de los fármacos , Trasplante HeterotópicoRESUMEN
BACKGROUND: In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli. CONCLUSIONS/SIGNIFICANCE: We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCG's ability to protect against pulmonary TB.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antioxidantes/metabolismo , Vacuna BCG/inmunología , Tuberculosis Pulmonar/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacuna BCG/genética , Vacuna BCG/farmacología , Linfocitos T CD4-Positivos/inmunología , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Inmunización Secundaria , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/metabolismo , Interleucina-2/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Estrés Oxidativo , ARN Mensajero/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Tuberculosis Pulmonar/inmunologíaRESUMEN
Erythroblasts adhere to central macrophages forming erythroblastic islands in hematopoietic tissues, but the function of these islands is not understood. Murine erythroblastic islands were reconstituted in vitro with macrophages and developmentally synchronous proerythroblasts. Erythroblasts cocultured with macrophages proliferated 3-fold greater than erythroblasts cultured alone. Direct contact with the macrophages was necessary for this enhanced erythroblast proliferation, which resulted from decreased transit time in the G(0)/G(1) phase of cell cycle. Increased erythroblast proliferation in erythroblastic islands occurred over a wide range of erythropoietin concentrations and was the result of a mechanism different from the antiapoptotic effect of erythropoietin. Erythroblasts adherent to macrophages had slightly delayed enucleation, but otherwise differentiation was similar to erythroblasts cultured alone or those that became nonadherent in cocultures. These results suggest a mechanism for the development of anemias associated with abnormal macrophage function and for reduced responsiveness of those anemias to erythropoietin therapy.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Eritroblastos/citología , Eritroblastos/efectos de los fármacos , Eritropoyetina/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Femenino , Virus de la Leucemia Murina de Friend/fisiología , Ratones , Bazo/citologíaRESUMEN
The long form of B-cell lymphoma-x (Bcl-x(L)), an outer mitochondrial membrane protein, has been proposed to mediate the antiapoptotic action of erythropoietin on erythroid progenitor cells and to be necessary for heme synthesis in erythroblasts. Mice with conditional knockout of Bcl-x(L) (conditional bcl-x(-/-) mice) develop severe anemia that has been attributed to hemolysis and is accompanied by splenomegaly. We characterized further the anemia of conditional bcl-x(-/-) mice and investigated the role of Bcl-x(L) in the action of erythropoietin and in heme synthesis. We analyzed peripheral blood cells and cultured splenic erythroblasts of conditional bcl-x(-/-) mice and littermates that were rendered anemic by bleeding. Although they had massive splenic erythroblastosis, conditional bcl-x(-/-) mice had decreased circulating reticulocytes compared to littermates even prior to bleeding the littermates. Compared to erythroblasts of bled littermates, bcl-x(-/-) erythroblasts cultured with erythropoietin underwent apoptosis during the later, hemoglobin-synthesizing stages of differentiation. The bcl-x(-/-) erythroblasts synthesized heme, but at reduced rates compared to bled littermate erythroblasts. When cultured without erythropoietin, bcl-x(-/-) erythroblasts underwent apoptosis at early stages of differentiation, prior to hemoglobin synthesis. Bcl-x(L) is not required for heme synthesis and does not mediate the antiapoptotic effects of erythropoietin, but it prevents ineffective erythropoiesis due to apoptosis in late-stage, hemoglobin-synthesizing erythroblasts.
Asunto(s)
Apoptosis/efectos de los fármacos , Eritroblastos/efectos de los fármacos , Eritropoyetina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Eritroblastos/citología , Eritropoyesis/efectos de los fármacos , Hemo/biosíntesis , Ratones , Ratones Transgénicos , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Proteína bcl-XRESUMEN
Tumor necrosis factor alpha (TNFalpha) potently inhibits the in vitro growth of highly purified human d-6 erythroid colony forming cells (ECFC). Unlike the inhibitory effect of TNFalpha on other cells, including more immature ECFC, this antiproliferative effect of TNFalpha is not related to apoptosis because the d-6 cell descendants were morphologically normal, without apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling assay and without caspase activation by Western blots after TNFalpha treatment. TNFalpha did not appear to affect the cell cycle distribution, but the cell cycle duration was significantly longer in TNFalpha-treated cells. DNA synthesis was also significantly reduced by TNFalpha. Studies of various proteins that regulate the cell cycle showed that cyclin-dependent kinase 6 (CDK6) protein and mRNA levels were concomitantly decreased in the presence of TNFalpha, suggesting that inhibition of cell growth was related to reduced CDK6. To evaluate this, the CDK6 gene was transferred into ECFC using green fluorescence protein-retrovirus-mediated gene transfer. The results showed that the level of cell growth produced by TNFalpha was increased by 30% when the cells were transfected with CDK6. Therefore, the modification of cell cycle progression in the presence of TNFalpha through a reduction of CDK6 is an important mechanism in the TNFalpha inhibition of human ECFC expansion.