RESUMEN
BACKGROUND: Only a small proportion of patients respond to anti-VEGF therapy, pressing the need for a reliable biomarker that can identify patients who will benefit. We studied the biological activity of anti-VEGF antibodies in patients' blood during anti-VEGF therapy by using the Ba/F3-VEGFR2 cell line, which is dependent on VEGF for its growth. METHODS: Serum samples from 22 patients with cancer before and during treatment with bevacizumab were tested for their effect on proliferation of Ba/F3-VEGFR2 cells. Vascular endothelial growth factor as well as bevacizumab concentrations in serum samples from these patients were determined by enzyme linked immunosorbent assay (ELISA). RESULTS: The hVEGF-driven cell proliferation was effectively blocked by bevacizumab (IC50 3.7 µg ml-1; 95% CI 1.7-8.3 µg ml-1). Cell proliferation was significantly reduced when patients' serum during treatment with bevacizumab was added (22-103% inhibition compared with pre-treatment). Although bevacizumab levels were not related, on-treatment serum VEGF levels were correlated with Ba/F3-VEGFR2 cell proliferation. CONCLUSIONS: We found that the neutralising effect of anti-VEGF antibody therapy on the biological activity of circulating VEGF can be accurately determined with a Ba/F3-VEGFR2 bioassay. The value of this bioassay to predict clinical benefit of anti-VEGF antibody therapy needs further clinical evaluation in a larger randomised cohort.
Asunto(s)
Inhibidores de la Angiogénesis/sangre , Linfocitos B/efectos de los fármacos , Bevacizumab/sangre , Bioensayo , Ensayo de Inmunoadsorción Enzimática , Neoplasias/sangre , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , División Celular , Línea Celular , Interleucina-3/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Receptores de Eritropoyetina/genética , Receptores de Interleucina-3/fisiología , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
RATIONALE: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with (18)F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. OBJECTIVE: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE(-/-) mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE(-/-) hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common ß subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1(+/-), ApoE(-/-), and ApoE(-/-)Glut1(+/-) mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE(-/-) hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE(-/-) mice transplanted with ApoE(-/-) bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. CONCLUSIONS: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE(-/-) mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.
Asunto(s)
Transportador de Glucosa de Tipo 1/fisiología , Glucosa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Hipercolesterolemia/metabolismo , Mielopoyesis/fisiología , Placa Aterosclerótica/metabolismo , Animales , Aorta Torácica/metabolismo , Apolipoproteínas E/deficiencia , Trasplante de Médula Ósea , División Celular , Subunidad beta Común de los Receptores de Citocinas/fisiología , Progresión de la Enfermedad , Metabolismo Energético , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1/deficiencia , Glucólisis , Hipercolesterolemia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/deficiencia , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Metformina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Multipotentes/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Interleucina-3/antagonistas & inhibidores , Receptores de Interleucina-3/fisiología , Bazo/metabolismo , Tirfostinos/farmacologíaRESUMEN
The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common beta (betac) receptor is shared by the three cytokines and functions together with cytokine-specific alpha subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human betac (hbetac) and unidentified cysteine residues in the N-terminal domains of the alpha receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hbetac is part of the cytokine binding epitope of this receptor and that an IL-3Ralpha isoform lacking the N-terminal Ig-like domain (the "SP2" isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hbetac and the related mouse receptor, beta(IL-3), could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hbetac led to both a complete loss of high affinity binding with the human IL-3Ralpha SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, beta(IL-3), not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Ralpha SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hbetac and beta(IL-3) in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.
Asunto(s)
Interleucina-3/química , Receptores de Interleucina-3/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Proliferación Celular , Chlorocebus aethiops , Disulfuros/química , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Isoformas de Proteínas , Estructura Terciaria de Proteína , Receptores de Interleucina-3/química , Transducción de SeñalRESUMEN
Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining lymph node (LN) after allergen immunization and that the recruited basophils promote the differentiation of naive CD4 T cells into Th2 effector cells. Using IL-3(-/-) and IL-3Rbeta(-/-) mice, we report in this study that the IL-3/IL-3R system is absolutely required to recruit circulating basophils into the draining LN following helminth infection. Unexpectedly, the absence of IL-3 or of basophil LN recruitment played little role in helminth-induced Th2 immune responses. Moreover, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.
Asunto(s)
Basófilos/inmunología , Basófilos/patología , Movimiento Celular/inmunología , Interleucina-3/fisiología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Animales , Basófilos/parasitología , Movimiento Celular/genética , Técnicas de Sustitución del Gen , Interleucina-3/deficiencia , Interleucina-3/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Nippostrongylus/inmunología , Receptores de Interleucina-3/deficiencia , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Infecciones por Strongylida/parasitología , Infecciones por Strongylida/patología , Células Th2/parasitologíaRESUMEN
Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in approximately 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3R alpha-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.
Asunto(s)
Basófilos/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Precursoras de Granulocitos/inmunología , Células Progenitoras de Granulocitos y Macrófagos/inmunología , Interleucina-3/fisiología , Bazo/inmunología , Regulación hacia Arriba/inmunología , Animales , Basófilos/citología , Basófilos/metabolismo , Regulación de la Expresión Génica/inmunología , Células Precursoras de Granulocitos/citología , Células Precursoras de Granulocitos/metabolismo , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , Interleucina-3/administración & dosificación , Interleucina-3/deficiencia , Interleucina-3/genética , Recuento de Leucocitos , Mastocitos/citología , Mastocitos/inmunología , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/metabolismo , Receptores de Interleucina-3/biosíntesis , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Bazo/citología , Bazo/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
CD123 has been identified as a specific surface marker for plasmacytoid dendritic cells (PDCs). However, CD123 has recently been shown to be expressed on freshly isolated or in vitro generated myeloid dendritic cells (MDCs). In this article, we investigated whether the expression of CD123 on monocyte-derived MDCs was related to their function, especially to tumor-inhibiting potential. MDCs were induced from cord blood CD14+ monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days, and then CD123+ cells were isolated by positive immunomagnetic cell selection. We observed that CD123+ cells lost monocyte CD14 expression, acquired immature myeloid dendritic cell phenotype and morphology. They exerted more significant endocytosis and less antigen-presenting function than CD123(-)MDCs which are often referred to as typical MDCs. Meanwhile, CD123+ MDCs exhibited more significant tumor-inhibiting activity toward hematological tumor cell lines of U937 and Jurkat even at a low effector:target ratio. CD123+ MDCs expressed higher level of cytoplasmic TNF-alpha-related apoptosis-inducing ligand (TRAIL), but no detectable surface TRAIL and very little soluble TRAIL. Pretreatment with recombinant human TRAIL receptor 2:Fc fusion protein significantly reduced the tumor-inhibiting effect of CD123+ MDCs, but not of CD123(-) MDCs. Overall, our data demonstrated that CD123+ MDCs were an early-stage immature DC subset, with a significant tumor-inhibiting activity partially via involvement of enhanced cytoplasmic TRAIL.
Asunto(s)
Células Dendríticas/fisiología , Subunidad alfa del Receptor de Interleucina-3/fisiología , Células Mieloides/fisiología , Neoplasias/prevención & control , Receptores de Interleucina-3/fisiología , Presentación de Antígeno , Apoptosis , Diferenciación Celular , Células HL-60 , Humanos , Células Jurkat , Linfocitos T/inmunología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Células U937RESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5 are related cytokines that play key roles in regulating the differentiation, proliferation, survival and activation of myeloid blood cells. The cell surface receptors for these cytokines are composed of cytokine-specific alpha-subunits and a common beta-receptor (betac), a shared subunit that is essential for receptor signaling in response to GM-CSF, IL-3 and IL-5. Previous studies have reached conflicting conclusions as to whether N-glycosylation of the betac-subunit is necessary for functional GM-CSF, IL-3 and IL-5 receptors. We sought to clarify whether betac N-glycosylation plays a role in receptor function, since all structural studies of human betac to date have utilized recombinant protein lacking N-glycosylation at Asn(328). Here, by eliminating individual N-glycans in human betac and the related murine homolog, beta(IL-3), we demonstrate unequivocally that ligand-binding and receptor activation are not critically dependent on individual N-glycosylation sites within the beta-subunit although the data do not preclude the possibility that N-glycans may exert some sort of fine control. These studies support the biological relevance of the X-ray crystal structures of the human betac domain 4 and the complete ectodomain, both of which lack N-glycosylation at Asn(328).
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Polisacáridos/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina-3/fisiología , Receptores de Interleucina-5/fisiología , Animales , Células COS , Chlorocebus aethiops , Subunidad beta Común de los Receptores de Citocinas/química , Subunidad beta Común de los Receptores de Citocinas/genética , Humanos , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Polisacáridos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/químicaRESUMEN
Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL-induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor alpha and beta chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL (triple positive cells), and these cells formed colonies in soft agar, whereas BCR-ABL+ NIH 3T3 cells lacking IL-3 receptor expression did not. Signaling studies indicate that the BCR-ABL/IL-3 receptor+ NIH 3T3 cells utilize the Gab2/PI-3 kinase pathway activated by Jak2, and the Stat5 pathway activated separately by Bcr-Abl, whereas BCR-ABL+ NIH 3T3 cells lacking the IL-3 receptor do not utilize the Jak2 pathway, but still maintain activation of Stat5. The Bcr-Abl kinase inhibitor imatinib mesylate (1 microM) and two Jak2 kinase inhibitors strongly inhibited agar colony formation and the activation of Gab2 caused by Jak2. All of these findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Fusión bcr-abl/fisiología , Células 3T3 NIH , Receptores de Interleucina-3/fisiología , Animales , Fibroblastos/metabolismo , Fibroblastos/patología , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Janus Quinasa 2/metabolismo , Ratones , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interleucina-3/genética , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT5/fisiología , Transducción de Señal/genética , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
Mast cells are potent effectors of the inflammatory response, playing an important role in atopy, bacterial immunity, and animal models of arthritis, multiple sclerosis, and heart disease. Hence controlling mast cell numbers and responsiveness is essential for preventing inflammatory disease. We demonstrate that the cytokine transforming growth factor (TGF) beta1 is a potent inducer of mast cell apoptosis, a finding that was consistent in cultured mouse bone marrow-derived mast cells, peritoneal mast cells, and human mast cells. Cell death appeared to be caused by TGF-mediated repression of interleukin-3 (IL-3) receptor expression and function, leading to mitochondrial damage and activation of an apoptotic cascade acting via p53 and caspases. Although IL-3 receptor expression was reduced within 1 day of TGFbeta1 stimulation, apoptosis required at least 3 days to occur. This delay in onset is postulated to allow protective mast cell effector functions, protecting the host from infection while preventing the establishment of chronic inflammation. Our data support the theory that TGFbeta1 is an inhibitor of mast cell survival. The widespread expression of TGFbeta1 offers this cytokine as an ideal candidate for control of mast cell homeostasis.
Asunto(s)
Apoptosis/fisiología , Mastocitos/citología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Interleucina-3/antagonistas & inhibidores , Receptores de Interleucina-3/fisiología , Factor de Transcripción STAT5/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
As vascular endothelial growth factor (VEGF), interleukin-3 (IL-3), released into the tumor microenvironment stimulates motogenic and mitogenic activity of normal and transformed cells. In the present study, we investigate the effects of IL-3 and VEGF on neoplastic vascular growth. Engagement of IL-3 receptor beta common (IL-3R beta c) contributes to both IL-3- and VEGF-induced Rac1 activation, cell migration and in vitro tube-like structure formation as shown by the expression of the dominant-negative IL-3R beta c construct (Delta455). In normal and transformed endothelial cells (EC) as well as in HEK 293 cells expressing KDR and IL-3R, VEGF and IL-3 treatment induces the formation of a KDR/IL-3R beta c complex. Moreover, as shown by the IL-3R Delta455 mutant or by the kinase dead KDR, functional receptors are required for this interaction. Consistent with the contribution of IL-3R beta c in both IL-3- and VEGF-mediated angiogenic signal, a reduced number of vessels inside tumors are found in mice injected with cells expressing the IL-3R Delta455 mutant. Thus, these findings provide a novel mechanism through which IL-3 and VEGF support cell survival and tumor neovascularization.
Asunto(s)
Neoplasias/irrigación sanguínea , Neovascularización Patológica , Receptores de Interleucina-3/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN , Humanos , Inmunoprecipitación , Interleucina-3/fisiología , Invasividad Neoplásica , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells, the modulation of their functional responses, as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. In contrast, GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled, thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover, the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated, within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators. In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors, and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs, an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution.
Asunto(s)
Factores Estimulantes de Colonias/fisiología , Mielopoyesis/fisiología , Animales , Factores Estimulantes de Colonias/genética , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Humanos , Interleucina-3/genética , Interleucina-3/fisiología , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Transducción de SeñalRESUMEN
Recent studies indicate that abnormalities of the interleukin-3 receptor (IL-3R) are frequently observed in acute myeloid leukemias (AMLs) and may contribute to the proliferative advantage of leukemic blasts. This review analyzes the evidences indicating that the IL-3R represents one of the target molecules involved in the stimulation of proliferation of AMLs, and the overexpression of the IL-3Ralpha chain may represent one of the mechanisms contributing to the development of a highly malignant leukemic phenotype. Furthermore, there is evidence that the IL-3Ralpha is a marker of leukemic stem cells, at variance with normal stem cells that are IL-3Ralpha-. Finally, the IL-3R may represent an important target for the development of new antileukemic drugs.
Asunto(s)
Leucemia Mieloide/etiología , Receptores de Interleucina-3/fisiología , Enfermedad Aguda , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-3 , Leucemia Mieloide/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Interleucina-3/biosíntesis , Receptores de Interleucina-3/genéticaRESUMEN
The hematopoietic factor and inflammatory cytokine GM-CSF is involved in PNS and CNS injury and disease, and in macrophage and microglia function regulation. We presently document that injury to PNS axons induces in vivo production of GM-CSF-inhibitor and GM-CSF-augmenter activities. GM-CSF-inhibitor activity was detected in extract and conditioned medium (CM) of injured PNS but not in extract of intact PNS, and was removed from CM by GM-CSF affinity chromatography, suggesting it is carried by a secreted GM-CSF binding molecule. CM further displayed GM-CSF-augmenter activity along with GM-CSF-inhibitor activity but at contrasting concentrations; augmentation at lowest and inhibition at highest. GM-CSF activity is thus regulated during Wallerian degeneration (WD); augmenter activity characterizes the onset and inhibitor activity the later stages of WD.
Asunto(s)
Axones/patología , Proteínas Portadoras/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Superficie Celular/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Nervio Ciático/patología , Degeneración Walleriana/metabolismo , Animales , Axones/metabolismo , Axotomía , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/análisis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , ARN Mensajero/análisis , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/análisis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/deficiencia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Interleucina-3/análisis , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-3/fisiología , Nervio Ciático/metabolismo , Solubilidad , Factores de Tiempo , Regulación hacia Arriba/fisiología , Degeneración Walleriana/genética , Degeneración Walleriana/patologíaRESUMEN
Mast cells are immunoregulatory effector cells capable of releasing different mediators and cytokines implicated in inflammatory tissue processes. Previous studies suggested that IL-3 regulates growth and function of murine mast cells and human mast cell precursors, but does not affect mature human mast cells. In the present study, we found expression of IL-3 receptors (IL-3R) in freshly isolated human intestinal mast cells by reverse transcriptase (RT)-PCR and in mast cells cultured with stem cell factor (SCF) using RT-PCR and flow cytometry. IL-3R expression was enhanced when the culture medium was supplemented with IL-4 in addition to SCF. In the presence of SCF, IL-3 significantly enhanced mast cell growth in a dose-dependent fashion (179+/-51% of control, p
Asunto(s)
Interleucina-3/farmacología , Intestinos/citología , Mastocitos/efectos de los fármacos , Receptores de IgE/fisiología , Receptores de Interleucina-3/análisis , División Celular/efectos de los fármacos , Células Cultivadas , Liberación de Histamina/efectos de los fármacos , Humanos , Interleucina-4/farmacología , Leucotrieno C4/metabolismo , Mastocitos/metabolismo , Prostaglandina D2/metabolismo , ARN Mensajero/análisis , Receptores de Interleucina-3/genética , Receptores de Interleucina-3/fisiología , Factor de Células Madre/farmacologíaRESUMEN
Airway obstruction, hyperresponsiveness, and the accumulation and persistence within the airways of inflammatory cells characterize asthma. Interleukin (IL)-3, granulocyte macrophage colony- stimulating factor (GM-CSF), and IL-5 are among several cytokines that have been shown to be increased in asthma and to contribute to atopic inflammation. They mediate their effect via receptors that have a common beta subunit (beta(c)). We hypothesized that blocking of this common beta(c) would impair the airway response to antigen. We report that an antisense (AS) phosphorothioate oligodeoxynucleotide (ODN) found to specifically inhibit transcription of the beta(c) in rat bone marrow cells also caused inhibition of beta(c) mRNA expression and of immunoreactive cells within the lungs of Brown Norway (BN) rats when injected intratracheally (p < 0.01). Inhibition of beta(c) significantly reduced (p < 0.01) experimentally induced eosinophilia in vivo in ovalbumin (OVA)-sensitized BN rats after antigen challenge. Furthermore, when compared with mismatch-treated rats, beta(c) AS-ODN caused inhibition of antigen-induced airway hyperresponsiveness to leukotriene D4. Taken together, our findings demonstrate that the common beta(c) of IL-3, IL-5, and GM-CSF receptors is involved in the eosinophil influx and airway hyperresponsiveness that follow OVA challenge and underscore the potential utility of a topical antisense approach targeting beta(c) for the treatment of asthma.
Asunto(s)
Antígenos/inmunología , Asma/fisiopatología , Hiperreactividad Bronquial , Eosinófilos/patología , Pulmón/patología , Oligonucleótidos Antisentido/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina-3/fisiología , Receptores de Interleucina/fisiología , Animales , Asma/inmunología , Asma/patología , Células de la Médula Ósea/metabolismo , Recuento de Células , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inmunización , Leucotrieno D4/farmacología , Pulmón/metabolismo , Masculino , Ovalbúmina/inmunología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas BN , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-3/química , Receptores de Interleucina-3/genética , Receptores de Interleucina-5 , Transcripción GenéticaRESUMEN
OBJECTIVE: The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated. METHODS: The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses. RESULTS: Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist. CONCLUSION: PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.
Asunto(s)
Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Megacariocitos/inmunología , Proteínas de la Leche , Proteínas de Neoplasias , Transducción de Señal/inmunología , Trombopoyetina/fisiología , Secuencia de Aminoácidos , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Células de la Médula Ósea/citología , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Interleucina-3 , Janus Quinasa 2 , Macaca mulatta , Megacariocitos/efectos de los fármacos , Datos de Secuencia Molecular , Fosforilación , Fosfotirosina/análisis , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Receptores de Citocinas/metabolismo , Receptores de Interleucina-3/fisiología , Receptores de Trombopoyetina , Proteínas Recombinantes de Fusión/farmacología , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , TransfecciónAsunto(s)
Receptores de Interleucina-3/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Mapeo Cromosómico , Humanos , Subunidad alfa del Receptor de Interleucina-3 , Ratones , Datos de Secuencia Molecular , Fenotipo , Receptores de Interleucina-3/química , Receptores de Interleucina-3/fisiologíaRESUMEN
The activation of cytokine receptors is a stepwise process that depends on their specific interaction with cognate cytokines, the formation of oligomeric receptor complexes, and the initiation of cytoplasmic phosphorylation events. The recent determination of the structure of extracellular domains of several cytokine receptors allows comparison of their cytokine-binding surfaces. This comparison reveals a common structural framework that supports considerable diversity and adaptability of the binding surfaces that determine both the specificity and the orientation of subunits in the active receptor complex. These regions of the cytokine receptors have been targeted for the development of specific agonists and antagonists. The physical coupling of signaling intermediates to the intracellular domains of their receptors plays a major role in determining biological responses to cytokines. In this review, we focus principally on the receptors for cytokines of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family and, where appropriate, compare them with related cytokine receptors. Several paradigms are beginning to emerge that focus on the ability of the extracellular portion of the cytokine receptor to recognize the appropriate cytokine and on a phosphorylated motif in the intracellular region of the GM-CSF receptor that couples to a specific signaling pathway.
Asunto(s)
Receptores de Citocinas/química , Secuencias de Aminoácidos , Animales , División Celular , Citocinas/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-3/fisiología , Interleucina-5/fisiología , Ligandos , Modelos Moleculares , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Fosfoserina/química , Fosfotirosina/fisiología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Receptores de Citocinas/efectos de los fármacos , Receptores de Citocinas/fisiología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos de los fármacos , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Receptores de Interleucina/química , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/fisiología , Receptores de Interleucina-3/química , Receptores de Interleucina-3/efectos de los fármacos , Receptores de Interleucina-3/fisiología , Receptores de Interleucina-5 , Transducción de Señal , Relación Estructura-ActividadRESUMEN
OBJECTIVE: A truncated common beta chain (Deltabeta(C)) of the interleukin-3 (IL-3) receptor complex was previously identified as a key factor in inducing autonomous growth of IL-3-independent mutants. Expression of Deltabeta(C) in IL-3-dependent hematopoietic cells does not result in immediate factor-independent growth, but increases the frequency of obtaining autonomous mutants by three to four orders of magnitude. This study was designed to delineate the mechanisms by which Deltabeta(C) increases the frequency to autonomous growth. DESIGN AND METHODS: Retroviral vectors were used to express Deltabeta(C) into IL-3-dependent myeloid cells, which were then tested for factor-independent growth. To determine if secondary genetic events were required for conversion to autonomous growth, elements of the Cre-loxP recombinant system were used to excise Deltabeta(C) in factor-independent clones. RESULTS: Excision of Deltabeta(C) in factor-independent clones revealed two types of phenotypes: reversion to factor-dependent growth (1/8) or continued IL-3-dependent growth (7/8). Analysis of cells that remained factor independent revealed constitutive activation of STAT5, not observed in factor-dependent revertants. Analysis of revertant cells demonstrated the presence of interacting secondary mutations that synergize with Deltabeta(C)-induced proliferation. A cysteine residue within the truncated extracellular domain of Deltabeta(C) was also found to be required for its oncogenic potential, supporting a model of dimerization for receptor activation. CONCLUSIONS: The high incidence of obtaining factor-independent mutants from cells expressing Deltabeta(C) results from the selection of mutations that either complement Deltabeta(C) expression to promote proliferation or that singly or in synergy with other secondary mutations negate the requirement of Deltabeta(C) expression for proliferation.
Asunto(s)
División Celular/inmunología , Interleucina-3/farmacología , Proteínas de la Leche , Receptores de Interleucina-3/genética , Eliminación de Secuencia , Animales , División Celular/efectos de los fármacos , Línea Celular , Transformación Celular Neoplásica , Cisteína , Proteínas de Unión al ADN/metabolismo , Dimerización , Vectores Genéticos , Ratones , Mutagénesis Sitio-Dirigida , Plasmacitoma , Receptores de Interleucina-3/química , Receptores de Interleucina-3/fisiología , Proteínas Recombinantes/metabolismo , Retroviridae , Factor de Transcripción STAT5 , Transactivadores/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
IL-4 is a pleiotropic cytokine which plays a pivotal role in shaping immune responses. The effects of IL-4 are mediated after binding to high affinity receptor complexes present on hematopoietic as well as non-hematopoietic cells. This review will summarize the current knowledge on the molecular structure of the different types of IL-4 receptor (IL-4R) complexes as well as the signal transduction mechanisms induced by IL-4 leading to cellular proliferation and / or gene activation. IL-4 effects are modulated by soluble forms of the respective receptor molecules which are produced by several immune cells in a regulated manner. The biological impact of recently described IL-4R allotypes of mice and humans as well as the results of studies with IL-4R knockout mice will be particularly emphasized.