RESUMEN
Schistosoma mansoni egg-induced pulmonary granuloma formation is a cell-mediated inflammatory response associated with dominant Th2-type cytokine expression, tissue eosinophilia, and high levels of serum IgE. In the present study, we show that in vivo blockade of the Th2 cytokine IL-13, using soluble IL-13R alpha2-Fc fusion protein, significantly reduced the size of pulmonary granulomas in unsensitized as well as egg-sensitized mice. Blocking IL-13 also significantly reduced total serum IgE levels. Interestingly, however, IL-13 blockade did not affect the evolving egg-induced Th2-type cytokine response. IL-4, IL-5, as well as IL-13 responses were indistinguishable in control-Fc- and soluble IL-13R alpha2-Fc fusion protein-treated animals. The smaller granulomas were also phenotypically like the control Fc-treated mice, displaying a similar eosinophil content. Additional studies in IL-4-deficient mice demonstrated that IL-13 was produced, but at much lower levels than in wild-type mice, while IL-4 expression was completely independent of IL-13. Moreover, while granuloma formation was partially reduced in IL-4-deficient mice, blocking IL-13 in these animals almost completely abrogated granuloma development and the pulmonary eosinophilia, while it simultaneously increased IFN-gamma production. Together, these data demonstrate that IL-13 serves as an important mediator of Th2-mediated inflammation and plays a role in eliciting IgE responses triggered by schistosome eggs.
Asunto(s)
Granuloma del Sistema Respiratorio/inmunología , Inmunoglobulina E/biosíntesis , Interleucina-13/inmunología , Enfermedades Pulmonares Parasitarias/inmunología , Óvulo/inmunología , Esquistosomiasis mansoni/inmunología , Células Th2/inmunología , Animales , Granuloma Eosinófilo/inmunología , Granuloma Eosinófilo/prevención & control , Femenino , Granuloma del Sistema Respiratorio/patología , Granuloma del Sistema Respiratorio/prevención & control , Inmunoglobulina E/sangre , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunofenotipificación , Interferón gamma/biosíntesis , Interleucina-13/antagonistas & inhibidores , Interleucina-13/biosíntesis , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/deficiencia , Interleucina-4/genética , Cinética , Enfermedades Pulmonares Parasitarias/patología , Enfermedades Pulmonares Parasitarias/prevención & control , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina/genética , Receptores de Interleucina/fisiología , Receptores de Interleucina-13 , Proteínas Recombinantes de Fusión/farmacología , Esquistosomiasis mansoni/patología , Esquistosomiasis mansoni/prevención & control , Solubilidad , Células Th2/metabolismo , Células Th2/parasitología , Regulación hacia Arriba/inmunologíaRESUMEN
The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. The pathophysiological features of allergic asthma are thought to result from the aberrant expansion of CD4(+) T cells producing the type 2 cytokines interleukin-4 (IL-4) and IL-5, although a necessary role for these cytokines in allergic asthma has not been demonstrable. The type 2 cytokine IL-13, which shares a receptor component and signaling pathways with IL-4, was found to be necessary and sufficient for the expression of allergic asthma. IL-13 induces the pathophysiological features of asthma in a manner that is independent of immunoglobulin E and eosinophils. Thus, IL-13 is critical to allergen-induced asthma but operates through mechanisms other than those that are classically implicated in allergic responses.
Asunto(s)
Asma/inmunología , Interleucina-13/fisiología , Alérgenos/inmunología , Animales , Asma/patología , Asma/fisiopatología , Hiperreactividad Bronquial , Líquido del Lavado Bronquioalveolar/citología , Eosinófilos , Células Caliciformes/patología , Humanos , Inmunoglobulina E/sangre , Interleucina-13/antagonistas & inhibidores , Interleucina-13/farmacología , Subunidad alfa1 del Receptor de Interleucina-13 , Interleucina-4/fisiología , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos A , Ovalbúmina/inmunología , Receptores de Interleucina/fisiología , Receptores de Interleucina-13 , Receptores de Interleucina-4/antagonistas & inhibidores , Receptores de Interleucina-4/fisiología , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13R alpha 1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13R alpha 2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13R alpha 2. The predicted protein sequence of murine IL-13R alpha 2 (mIL-13R alpha 2) has 59% overall identity to human IL-13R alpha 2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13R alpha 1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13R alpha 2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13R alpha 2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13R alpha 1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13R alpha 1.Fc was 100-fold less effective than IL-13R alpha 2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13R alpha 2 and mIL-13R alpha 1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.
Asunto(s)
Interleucina-13/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/genética , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Línea Celular , Mapeo Cromosómico , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Interleucina-13/antagonistas & inhibidores , Interleucina-13/farmacología , Subunidad alfa1 del Receptor de Interleucina-13 , Activación de Linfocitos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Polimorfismo Conformacional Retorcido-Simple , Unión Proteica/inmunología , ARN Mensajero/biosíntesis , Receptores de Interleucina/aislamiento & purificación , Receptores de Interleucina/fisiología , Receptores de Interleucina-13 , Transfección/inmunologíaRESUMEN
We have investigated the mechanism of action of the thrombopoietic cytokine, recombinant human interleukin-11 (rhIL-11), on megakaryocytopoiesis in vitro. We have shown that rhIL-11-induced murine and human megakaryocytopoiesis are not mediated by thrombopoietin (Tpo). Murine megakaryocytes (MKs) were produced from bone marrow (BM) mononuclear cells cultured with rhIL-11, IL-3, and a combination of the two cytokines. Conditioned media (CM) were collected and assayed for the presence of biologically active Tpo. Tpo activity was not detected in any of the CMs tested. Next, human BM CD34+ cells were cultured in serum-free fibrin clot medium with rhIL-11, IL-3, or rhIL-11 plus IL-3 and an antibody that neutralizes human Tpo activity. No inhibition of either burst-forming unit-MK- or colony-forming unit-MK-derived colony formation was observed. The antibody did partially inhibit steel factor-induced MK-colony formation, suggesting that the actions of this cytokine are mediated, in part, by Tpo. We determined that MKs can be direct targets of rhIL-11 by showing the expression of functional IL-11 receptor on these cells. Total RNA was prepared from cultured human BM CD41+CD14- cells (MKs) and IL-11 receptor alpha chain mRNA was detected in the MKs by reverse transcription-polymerase chain reaction. Analysis of single-sorted CD41+CD14- cells confirmed that the observed IL-11 receptor expression was not due to contaminating CD41- cells in the pool. The presence of rhIL-11 receptor alpha chain protein in the cells was established by Western blot analysis. After a short exposure of purified BM MKs to rhIL-11, enhanced phosphorylation of both its signal transduction subunit, gp130, and the transcription factor, STAT3 was detected, showing a direct activation of receptor signaling by the cytokine. Consistent with the lack of effect of rhIL-11 on platelets in vivo, IL-11 receptor alpha chain mRNA and protein were not detected in isolated human platelets. These data indicate that rhIL-11 acts directly on MKs and MK progenitors but not on platelets.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Interleucina-11/farmacología , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Interleucina-11/metabolismo , Subunidad alfa del Receptor de Interleucina-11 , Megacariocitos/metabolismo , Ratones , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-11 , Proteínas Recombinantes/farmacologíaRESUMEN
Previous studies have shown that subcutaneous (SC) bolus administration of recombinant human interleukin-11 (rhIL-11) stimulates megakaryocytopoiesis and increases peripheral platelet counts in naive mice. This study was designed to determine whether administration of rhIL-11 by constant SC infusion altered either the magnitude of the nature of the hematologic response. Female C57BL/6 mice were implanted subcutaneously with 7-day Alzet mini-osmotic pumps containing either rhIL-11 with 0.5% homologous mouse serum (delivery rate of 250 microg/kg/d) or vehicle alone. Mice were sacrificed on days 3, 7, 10, and 13 after pump implantation, and the hematopoietic response was compared to mice receiving an equivalent dose of rhIL-11 administered by SC injection (250 microg/kg/d, 7 days) or vehicle controls. Subcutaneous injection of rhIL-11 resulted in a significant increase in peripheral platelet counts with a maximum platelet increase of 44% over controls observed on day 7 of the study. Platelet counts subsequently declined (24% by day 10) returning to control values by day 13. The increase in peripheral platelet counts was accompanied by an increase in reticulated platelets on day 7 and a shift to higher ploidy bone marrow megakaryocytes on days 3 and 7. Compared to SC injection, both the magnitude and duration of the platelet increase were significantly enhanced following continuous SC infusion of rhIL-11. Maximum platelet counts were detected on day 10 (115% above vehicle controls), and platelets remained significantly elevated on day 13 (84%), 6 days after rhIL-11 administration had stopped. Consistent with the platelet response, the modal ploidy of bone marrow megakaryocytes was shifted from 16N to 32N on days 3 and 7, with increases in 32N megakaryocytes still apparent on days 10 and 13. There was also a significant increase in reticulated platelets detected in the peripheral blood on days 3, 7, and 10 compared to mice administered rhIL-11 by SC injection, The changes in reticulated platelets and bone marrow megakaryocyte ploidy are consistent with the increased and prolonged platelet response following SC infusion of rhIL-11. In addition to the effects observed on peripheral platelet counts, constant SC infusion of rhIL-11 dramatically enhanced splenic hematopoietic activity, increasing spleen weight and cellularity as well as splenic megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared to mice receiving rhIL-11 by SC injection.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Interleucina-11/administración & dosificación , Animales , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Citocinas/fisiología , Femenino , Bombas de Infusión , Inyecciones Subcutáneas , Interleucina-11/farmacología , Megacariocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas/efectos de los fármacos , Ploidias , Ratas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacologíaRESUMEN
Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony-forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU-S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine-induced pathways for mobilization of primitive stem cells.
Asunto(s)
Recuento de Células Sanguíneas/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-11/farmacología , Factor de Células Madre/farmacología , Animales , Células de la Médula Ósea , Ensayo de Unidades Formadoras de Colonias , Sinergismo Farmacológico , Humanos , Subunidad alfa del Receptor de Interleucina-11 , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Proteínas Proto-Oncogénicas c-kit/sangre , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/fisiología , Ratas , Receptores de Interleucina/clasificación , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/fisiología , Receptores de Interleucina-11 , Proteínas Recombinantes/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Esplenectomía , Estimulación QuímicaRESUMEN
Interleukin-11 (IL-11) is a novel multifunctional hematopoietic cytokine capable of stimulating cells of the myeloid, lymphoid, erythroid, and megakaryocytic lineages in vitro. We have tested the pleiotropic properties of this cytokine on the hematopoietic recovery of mice after a combined regimen of sublethal irradiation and carboplatin administration. This regimen results in severe myelosuppression, characterized by a prolonged period of thrombocytopenia and severe anemia. Administration of recombinant human IL-11 (rhIL-11; 250 micrograms/kg/d) had multilineage effects on bone marrow and spleen hematopoietic activity, increasing the number of megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared with the vehicle-treated controls. This was reflected in the peripheral circulation by a reduction of both the platelet and hematocrit nadirs and a significantly reduced period of thrombocytopenia and anemia in the rhIL-11-treated mice. The results from this study support the broad spectrum of biologic activities that have been attributed to rhIL-11 in vitro and suggest that this cytokine may be an effective agent in the treatment of myelosuppression associated with cancer chemotherapy and bone marrow transplantation.
Asunto(s)
Médula Ósea/efectos de la radiación , Carboplatino/toxicidad , Hematopoyesis/efectos de los fármacos , Interleucina-11/farmacología , Animales , Médula Ósea/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacologíaRESUMEN
Lethally irradiated mice were transplanted with syngeneic bone marrow cells infected with a recombinant retrovirus vector containing the human interleukin-11 (hIL-11) cDNA under the control of the human cytomegalovirus (CMV) immediate early promoter. The hIL-11 RNA transcript from the vector was detected in the spleen and bone marrow of the recipient mice, and hIL-11 protein accumulated in their serum. The hematological reconstitution of these mice was compared with recipient mice rescued with bone marrow infected with the parental retrovirus vector not containing the hIL-11 cDNA. The hIL-11-expressing mice had an accelerated recovery of circulating platelets and red and white blood cells. Three months after the transplantation, bone marrow was harvested from the mice and used to rescue other lethally irradiated recipients. The hIL-11 mRNA and protein were also detected in these secondary recipients, and the mice showed improved hematological reconstitution relative to a control group. No abnormal cell proliferation or other histopathology was observed in the hIL-11-expressing mice.
Asunto(s)
Trasplante de Médula Ósea/patología , Médula Ósea/química , Médula Ósea/patología , Médula Ósea/efectos de la radiación , Hematopoyesis/fisiología , Interleucina-11/análisis , Interleucina-11/fisiología , Animales , Línea Celular , ADN Viral/análisis , ADN Viral/genética , Relación Dosis-Respuesta a Droga , Femenino , Vectores Genéticos , Hematopoyesis/efectos de los fármacos , Hematopoyesis/efectos de la radiación , Interleucina-11/genética , Ratones , Ratones Endogámicos C3H , ARN Mensajero/análisis , ARN Mensajero/genética , Retroviridae/genética , Bazo/química , Bazo/citología , Donantes de Tejidos , TransfecciónRESUMEN
The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11-treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.
Asunto(s)
Plaquetas/citología , Hematopoyesis , Interleucina-11/farmacología , Megacariocitos/citología , Esplenectomía , Animales , Células de la Médula Ósea , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas , PloidiasRESUMEN
Etanidazole was developed as an oxygen-mimetic radiosensitizer less lipophilic than misonidazole. Sensitization depends on an adequate concentration of drug in the tumor at the time of irradiation. Therefore, due to the presence of the blood-brain barrier, brain tumors may theoretically be difficult to radiosensitize due to the hydrophilic characteristics of etanidazole. Based on previous reports of loss of BBB integrity in brain tumors, we investigated the ability of etanidazole to penetrate into malignant gliomas of patients receiving etanidazole as part of a Phase I continuous infusion protocol. The patients had completed previous external beam irradiation. Twenty-two patients were studied and their etanidazole plasma and biopsy data were compared to the 2-compartment model derived from a second group of 19 patients with bolus etanidazole. Etanidazole concentration in brain tumor biopsies varied widely and appeared to be clustered into a higher and a lower pharmacokinetic group having mean tumor to well-perfused second compartment ratios of 1 and 0.25, respectively. Both high and low etanidazole concentrations were evident in different biopsies obtained from the same patient. Correlations between histology and tissue concentrations suggested that the higher level correspond to malignant tissue. These data indicate that the blood brain barrier is disrupted to varying degrees by the brain tumor and/or prior irradiation and that etanidazole penetrates into brain tumors.