RESUMEN
Lycopene is a hydrocarbon-carotenoid commonly found in red fruits intake with major function correlated to antioxidative capacity in several pathological conditions, including cancer and cardiovascular diseases. Recently, lycopene has been associated with hematopoiesis, although the effects on B lymphocyte differentiation and antibody production are poorly understood. In this work, the principal aim was to investigate whether lycopene affects B lymphopoiesis and terminal differentiation into plasma cells. Distinct in vivo and in vitro strategies based on lycopene supplementation were used direct in Balb/c mice or in culture systems with cells derived of these mice. In the bone marrow, lycopene expanded B220+IgM- progenitor B cells and B220+IgM+ immature B lymphocytes. In the spleen, lycopene induced terminal CD138+ plasma cell generation. In the blood, we found prominent IgA and low IgM levels after lycopene administration. Interestingly, the pattern of peritoneal IgM+ and IgA+ B cells indicated a significant IgM-to-IgA class switching after lycopene injection. These data indicated that lycopene induces B cell differentiation into IgA-producing plasma cells. Thus, a new cellular function has been attributed to lycopene for B lymphocyte biology and possibly associated with humoral responses and mucosal immunity.
Asunto(s)
Médula Ósea , Linfopoyesis , Animales , Células de la Médula Ósea , Diferenciación Celular , Inmunoglobulina A , Inmunoglobulina M , Licopeno/farmacología , Ratones , Ratones Endogámicos BALB CRESUMEN
The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.
Asunto(s)
Agammaglobulinemia/genética , Cromatina/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Adolescente , Adulto , Linfocitos B/fisiología , Diferenciación Celular/genética , Línea Celular , Niño , Preescolar , Células Dendríticas/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Lactante , Linfopoyesis/genética , Masculino , Mutación/genética , Células Precursoras de Linfocitos B/fisiología , Células Madre/fisiología , Adulto JovenRESUMEN
Glucocorticoids are produced and released by the adrenal gland and become elevated in response to stress. Although glucocorticoids are well known for their immunosuppressive effects, less is known about their effects on B cells. ABCB1 is an efflux pump expressed in both cancer and normal cells, modulating the gradient of various metabolites, including hydrocortisone. Our goal was to evaluate the effect of this glucocorticoid on murine B cell differentiation and whether sensitivity to hydrocortisone could be related to ABCB1 activity in vivo. C57BL/6 mice received one or three consecutive i.p. injections of hydrocortisone (70, 140 and 200 mg/kg/day). ABCB1 activity was evaluated via the rhodamine-123 transport and inhibited by cyclosporin A in hydrocortisone-treated and control mice. Cells from bone marrow, spleen and blood were counted, incubated with antibodies and analyzed by flow cytometry. A single hydrocortisone injection did not alter the number of bone marrow subsets. Conversely, three daily injections were able to reduce the cell number of most bone marrow subsets, excepting c-kit-sca-1+ and mature B cells. This treatment reduced marginal zone, follicular and transitional B cells, though splenic subsets were more resistant than bone marrow B cells. Recirculating follicular B cells in the blood were resistant to hydrocortisone. With the exception of follicular B cells, all subpopulations exhibited ABCB1 activity. However, hydrocortisone treatment did not affect ABCB1 activity in most subsets analyzed. Results suggest that hydrocortisone is able to regulate B cell lymphopoiesis although ABCB1 activity is not related to the susceptibility to that glucocorticoid in B cell subsets.
Asunto(s)
Subgrupos de Linfocitos B/efectos de los fármacos , Glucocorticoides/farmacología , Hidrocortisona/farmacología , Linfopoyesis/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Inmunofenotipificación , Linfopoyesis/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
Hematopoietic stem cell transplantation (HSCT) is an important therapeutic option for some hematological diseases. However, patients who undergo HSCT acquire a state of immunodeficiency that causes significant mortality. Reconstitution of thymic function is needed to support the immune system. One way to measure thymic function is through T-cell receptor excision circle (TREC) quantification. TRECs are generated by T-cell receptor gene rearrangements during T-cell maturation in the thymus and represent a reliable marker for thymic output. In this study, we aimed to assess aging and malignant hematological diseases as two important factors that may influence thymic output before HSCT. We observed that patients before HSCT presented signal joint TREC (sjTREC) numbers lower than 606.55 copies/µg DNA (low values) compared with healthy individuals, with an odds ratio (OR) of 12.88 [95% confidence interval (CI): 5.26-31.53; p < 0.001]. Our results showed that a group of older individuals (≥50 years old), comprising both healthy individuals and patients, had an OR of 10.07 (95% CI: 2.80-36.20) for low sjTREC values compared with younger individuals (≤24 years old; p < 0.001). Multiple logistic regression analysis confirmed that both older age (≥50 years old) and malignant hematological diseases and their treatments were important and independent risk factors related to thymic function impairment (p < 0.001). The median sjTREC value for patients of all ages was significantly lower than the sjTREC median for the subgroup of older healthy individuals (≥50 years old; p < 0.001). These data suggested that patients before HSCT and healthy individuals exhibited age-dependent thymic impairment, and that prior treatment for hematological diseases may exacerbate aging-related deterioration of natural thymic function. Furthermore, we analyzed these patients 9 months post-HSCT and compared patients who underwent autologous HSCT with those who underwent allogeneic HSCT. Both groups of patients achieved sjTREC copy numbers similar to those of healthy individuals. We did not find a close relationship between impaired thymic function prior to HSCT and worse thymic recovery after HSCT.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Linfopoyesis , Timo/citología , Timo/metabolismo , Adolescente , Adulto , Estudios de Casos y Controles , Femenino , Enfermedad Injerto contra Huésped/etiología , Neoplasias Hematológicas/complicaciones , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Adulto JovenRESUMEN
Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento.Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje.Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA.Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml).Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.
Asunto(s)
Linfocitos B/efectos de los fármacos , Caseínas/farmacología , Linfopoyesis/efectos de los fármacos , Animales , Médula Ósea/efectos de los fármacos , Caseínas/administración & dosificación , Caseínas/toxicidad , División Celular , Femenino , Inyecciones Intraperitoneales , Interleucina-7/biosíntesis , Interleucina-7/sangre , Interleucina-7/genética , Recuento de Linfocitos , Linfocinas/biosíntesis , Linfocinas/genética , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34+ HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3ß inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Proteínas de Homeodominio/metabolismo , Linfopoyesis , Receptores Notch/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Linaje de la Célula/genética , Factor de Transcripción GATA3/genética , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Linfopoyesis/genética , Ratones , FN-kappa B/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transducción de Señal/genética , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismo , Factores de Transcripción/genéticaRESUMEN
B lymphocytes are immune cells crucial for the maintenance and viability of the humoral response. Sleep is an essential event for the maintenance and integrity of all systems, including the immune system (IS). Thus, sleep deprivation (SD) causes problems in metabolism and homeostasis in many cell systems, including the IS. In this study, our goal was to determine changes in B lymphocytes from the bone marrow (BM) and spleen after SD. Three-month-old male Swiss mice were used. These mice were sleep deprived through the modified multiple platform method for different periods (24, 48, and 72 h), whereas another group was allowed to sleep for 24 h after 72 h of SD (rebound group) and a third group was allowed to sleep normally during the entire experiment. After this, the spleen and BM were collected, and cell analyses were performed. The numbers of B lymphocytes in the BM and spleen were reduced by SD. Additionally, reductions in the percentage of lymphocyte progenitors and their ability to form colonies were observed. Moreover, an increase in the death of B lymphocytes from the BM and spleen was associated with an increase in oxidative stress indicators, such as DCFH-DA, CAT, and mitochondrial SOD. Rebound was not able to reverse most of the alterations elicited by SD. The reduction in B lymphocytes and their progenitors by cell death, with a concomitant increase in oxidative stress, showed that SD promoted a failure in B lymphopoiesis.
Asunto(s)
Linfocitos B/inmunología , Médula Ósea/inmunología , Linfopoyesis , Células Precursoras de Linfocitos B/inmunología , Privación de Sueño/inmunología , Bazo/inmunología , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Biomarcadores/metabolismo , Médula Ósea/metabolismo , Médula Ósea/patología , Muerte Celular , Modelos Animales de Enfermedad , Recuento de Linfocitos , Masculino , Ratones , Estrés Oxidativo , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Privación de Sueño/metabolismo , Privación de Sueño/patología , Bazo/metabolismo , Bazo/patología , Factores de TiempoRESUMEN
El proceso de envejecimiento provoca cambios en el sistema inmune que afectan su funcionamiento y desarrollo. Estos cambios pueden manifestarse desde la linfopoyesis hasta la respuesta que orquesta el sistema inmune frente a determinada enfermedad o agente infeccioso. Ambas ramas de la inmunidad, innata y adaptativa, se afectan en este proceso, lo que genera un impacto negativo en la respuesta inmune de los ancianos y los predispone a padecer enfermedades infecciosas, cáncer, autoinmunidad y a desarrollar respuestas pobres tras la administración de vacunas(AU)
The aging process produces functional and developmental changes in the immune system. Those changes may appear from lymphopoiesis up to the final response of the immune system facing a certain disease. Both branches of immunity, innate and adaptive, are affected by the aging process; hence these changes can have a negative impact on the immune response of elderly patients and increase their susceptibility to infectious diseases, cancer and decreased vaccine efficacy(AU)
Asunto(s)
Humanos , Anciano , Anciano de 80 o más Años , Envejecimiento/inmunología , Linfopoyesis/inmunología , Inmunidad Adaptativa/fisiología , Inmunidad Activa/fisiología , Inmunidad Innata/fisiologíaRESUMEN
El proceso de envejecimiento provoca cambios en el sistema inmune que afectan su funcionamiento y desarrollo. Estos cambios pueden manifestarse desde la linfopoyesis hasta la respuesta que orquesta el sistema inmune frente a determinada enfermedad o agente infeccioso. Ambas ramas de la inmunidad, innata y adaptativa, se afectan en este proceso, lo que genera un impacto negativo en la respuesta inmune de los ancianos y los predispone a padecer enfermedades infecciosas, cáncer, autoinmunidad y a desarrollar respuestas pobres tras la administración de vacunas...
The aging process produces functional and developmental changes in the immune system. Those changes may appear from lymphopoiesis up to the final response of the immune system facing a certain disease. Both branches of immunity, innate and adaptive, are affected by the aging process; hence these changes can have a negative impact on the immune response of elderly patients and increase their susceptibility to infectious diseases, cancer and decreased vaccine efficacy...
Asunto(s)
Humanos , Anciano , Anciano de 80 o más Años , Envejecimiento/inmunología , Inmunidad Activa/fisiología , Inmunidad Adaptativa/fisiología , Inmunidad Innata/fisiología , Linfopoyesis/inmunologíaRESUMEN
Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL(nkt/nkt) mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSL (nkt/nkt) mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSL (nkt/nkt) mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSL (nkt/nkt) mice. Besides, BM B-cell emigration to the spleen was increased in CTSL (nkt/nkt) mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSL (nkt/nkt) mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.
Asunto(s)
Subgrupos de Linfocitos B/citología , Catepsina L/inmunología , Linfopoyesis/inmunología , Células Precursoras de Linfocitos B/citología , Animales , Apoptosis , Subgrupos de Linfocitos B/enzimología , Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Catepsina L/deficiencia , Catepsina L/genética , Proliferación Celular , Regulación de la Expresión Génica , Homeostasis , Ganglios Linfáticos/citología , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos B/enzimología , Células Precursoras de Linfocitos B/inmunología , Bazo/citología , Bazo/enzimología , Bazo/inmunología , Células Madre/citología , Células Madre/enzimología , Células Madre/inmunologíaRESUMEN
Multipotent mesenchymal stromal cells (MSCs) from the human olfactory mucosa (OM) are cells that have been proposed as a niche for neural progenitors. OM-MSCs share phenotypic and functional properties with bone marrow (BM) MSCs, which constitute fundamental components of the hematopoietic niche. In this work, we investigated whether human OM-MSCs may promote the survival, proliferation, and differentiation of human hematopoietic stem cells (HSCs). For this purpose, human bone marrow cells (BMCs) were co-cultured with OM-MSCs in the absence of exogenous cytokines. At different intervals, nonadherent cells (NACs) were harvested from BMC/OM-MSC co-cultures, and examined for the expression of blood cell markers by flow cytometry. OM-MSCs supported the survival (cell viability >90%) and proliferation of BMCs, after 54 days of co-culture. At 20 days of co-culture, flow cytometric and microscopic analyses showed a high percentage (73%) of cells expressing the pan-leukocyte marker CD45, and the presence of cells of myeloid origin, including polymorphonuclear leukocytes, monocytes, basophils, eosinophils, erythroid cells, and megakaryocytes. Likewise, T (CD3), B (CD19), and NK (CD56/CD16) cells were detected in the NAC fraction. Colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors and CD34(+) cells were found, at 43 days of co-culture. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies showed that OM-MSCs constitutively express early and late-acting hematopoietic cytokines (i.e., stem cell factor [SCF] and granulocyte- macrophage colony-stimulating factor [GM-CSF]). These results constitute the first evidence that OM-MSCs may provide an in vitro microenvironment for HSCs. The capacity of OM-MSCs to support the survival and differentiation of HSCs may be related with the capacity of OM-MSCs to produce hematopoietic cytokines.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Células Madre Hematopoyéticas/citología , Células Madre Mesenquimatosas/metabolismo , Mucosa Olfatoria/citología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígeno CD56/genética , Antígeno CD56/metabolismo , Recuento de Células , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Células Cultivadas , Microambiente Celular , Técnicas de Cocultivo/métodos , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Leucocitos Mononucleares/metabolismo , Linfopoyesis , Células Madre Mesenquimatosas/citología , Mielopoyesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Factores de TiempoRESUMEN
Ouabain (OUA) is an endogenous hormone released by the adrenal gland under stress situations. Steroid hormones and glucocorticoids have been characterized as selective inhibitors of lymphopoiesis. The present report shows in vivo modulation of mature B cells in bone marrow, spleen and peripheral blood by ouabain. Mice injected intraperitonially (i.p.) with ouabain 0.56 mg/kg for 3 consecutive days displayed, 24 h after last injection, a decreased cellularity in the bone marrow with diminution of the mature B cell subpopulation while the other B cell subpopulations were preserved. Percentually, the myeloid lineage in bone marrow was increased by ouabain. Numbers of mature B lymphocytes in spleen and peripheral blood were reduced following in vivo treatment. In vitro, the B cell populations were not affected. The effects appear to be independent of steroid hormones and strain. The presence of stable levels of glucocorticoids seems to be important because the effects could only be observed from the fourth week animal's life, when glucocorticoid levels are stable. These results open new perspectives for a potential use of ouabain as an immunomodulator.
Asunto(s)
Linfocitos B/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Linfopoyesis/efectos de los fármacos , Ouabaína/farmacología , Bazo/efectos de los fármacos , Animales , Antígenos CD/inmunología , Linfocitos B/inmunología , Médula Ósea/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Cultivadas , Femenino , Citometría de Flujo , Glucocorticoides/inmunología , Glucocorticoides/farmacología , Factores Inmunológicos/farmacología , Inyecciones Intraperitoneales , Linfopoyesis/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ouabaína/inmunología , Bazo/inmunologíaRESUMEN
Although the bone marrow (BM) microenvironment is the main inducer niche of early B lymphopoiesis during the adult life, other extramedullar microenvironments, such as the liver, may also have potential for supporting B-cell development. Previously, we reported that murine liver sinusoidal endothelial cells (LSECs) support in vitro and in vivo hematopoietic stem cell (HSC) proliferation and myeloid differentiation. In the present study, we investigated the capacity of LSEC to promote B lymphopoiesis from BM progenitor lineage-negative (Lin(-)) cells. Murine BM Lin(-) cells were co-cultured with LSEC, in the absence of exogenous cytokines. B cells were characterized by flow cytometry and cytokine expression by RT-PCR. We show that BM Lin(-) cells differentiated to early B-lymphoid progenitors (B220(+)) and subsequently to mature (CD19(+)) B cells. Functional studies showed the presence of a high number of non-adherent cells (NACs), collected from lipopolysaccharide (LPS)-treated Lin(-)/LSEC co-cultures, expressing IgM on their surface (sIgM). Colony formation from NAC was observed in the presence of IL-7 (CFU-IL-7). LSEC constitutively express IL-7, Flt-3L, and SCF at the mRNA level, and VCAM-1 on their surface, which may explain the capacity of these cells to promote B lymphopoiesis. These data demonstrate that LSEC promote all stages of B lymphopoiesis. To our knowledge, this is the first report that LSEC constitute an in vitro microenvironment for B lymphopoiesis. Further studies will establish whether LSEC can serve in vivo as a B-lymphopoietic niche under physiological or pathological condition, or when HSC are mobilized.
Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea/citología , Diferenciación Celular , Hígado/citología , Animales , Antígenos CD19/análisis , Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Endotelio/citología , Endotelio/metabolismo , Femenino , Citometría de Flujo , Expresión Génica , Interleucina-7/genética , Antígenos Comunes de Leucocito/análisis , Hígado/metabolismo , Linfopoyesis , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Kalanchoe brasiliensis (Kb) is a medicinal plant from the Crassulaceae family, used in folk medicine to treat inflammatory and infectious diseases. Here we show that short-term treatment of mice with a highly purified compound named kalanchosine dimalate (KMC), obtained from Kb, led to a strong and selective inhibition of B cell development in the bone marrow, without affecting the myeloid lineage development. Numbers of mature B lymphocytes in bone marrow or peripheral lymphoid organs were preserved in KMC treated mice. The inhibitory effect of KMC was acute and rapidly reverted with the interruption of the treatment. In vitro, KMC, inhibited the interleukin-7 dependent proliferation of B cell precursors and do not induce cell death. Also in vitro, the maturation of B cell precursors was not affected by KMC. KMC does not inhibit the proliferative response to IL-3 or IL-2. These results suggest that KMC is selectively affecting B cell lymphopoiesis, possibly acting on the IL-7 signaling pathway, opening new perspectives for a potential therapeutic usage of Kb derived drugs.
Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Interleucina-2/metabolismo , Interleucina-3/metabolismo , Interleucina-7/metabolismo , Linfopoyesis/efectos de los fármacos , Malatos/farmacología , Animales , Linfocitos B/fisiología , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Interleucina-2/inmunología , Interleucina-3/inmunología , Interleucina-7/inmunología , Kalanchoe , Linfopoyesis/inmunología , Malatos/aislamiento & purificación , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Lymphopoiesis and myelopoiesis continuously generate mature cells from hematopoietic cell progenitors during the lifetime of the organism. The identification of new endogenous or exogenous substances that can act specifically on the differentiation of distinct cell lineages is of relevance and has potential therapeutical use. Kalanchoe brasiliensis (Kb) is a medicinal plant from the Crassulaceae family, used in folk medicine to treat inflammatory and infectious diseases. Here, we show that short-term treatment of naïve mice with Kb led to a strong and selective inhibition of lymphopoiesis, affecting B and T cell lineages without reduction of the myeloid lineage development. Similar effects were observed after treatment with the highly purified compound kalanchosine dimalate (KMC), obtained from Kb. Numbers of mature lymphocytes in secondary lymphoid organs were preserved in Kb(KMC)-treated mice. The effect of Kb(KMC) was not a result of secondary augmentation of plasma levels of endogenous corticoids; neither involves TNF-alpha, type-I IFN, or TLR2/TLR4 ligands, which have all been described as selective inhibitors of lymphopoiesis. Flow cytometry analysis of the phenotypes of T and B cell precursors indicate a blockade of maturation on IL-7-dependent, proliferative stages. In vitro, Kb(KMC) inhibited the IL-7-dependent proliferation of pre-B cells and does not induce massive apoptosis of B and T cell precursors. These results suggest that Kb(KMC) is selectively blocking lymphopoiesis through a mechanism that does not involve the previously characterized substances, possibly acting on the IL-7 signaling pathway, opening new perspectives for a potential therapeutic use of Kb-derived drugs.
Asunto(s)
Interleucina-7/antagonistas & inhibidores , Linfopoyesis/fisiología , Malatos/farmacología , Animales , Antiinflamatorios/farmacología , División Celular/efectos de los fármacos , Interleucina-7/farmacología , Kalanchoe , Linfopoyesis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Extractos Vegetales , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genéticaRESUMEN
We present a cross-sectional analysis of the maturation and migratory properties of the memory CD8(+) T cell compartment, in relation to the severity of heart disease in individuals with chronic Trypanosoma cruzi infection removed from endemic areas for longer than 20 years. Subjects with none or mild heart involvement were more likely to mount T. cruzi-specific memory IFN-gamma responses than subjects with more advanced cardiac disease, and the T. cruzi-specific CD8(+) T cell population was enriched in early-differentiated (CD27(+)CD28(+)) cells in responding individuals. In contrast, the frequency of CD27(+)CD28(+)CD8(+) T cells in the total memory CD8(+) T cell population decreases, as disease becomes more severe, while the proportion of fully differentiated memory (CD27(-)CD28(-)) CD8(+) T cells increases. The analysis of CCR7 expression revealed a significant increase in total effector/memory CD8(+) T cells (CD45RA(-)CCR7(-)) in subjects with mild heart disease as compared with uninfected controls. Altogether, these results are consistent with the hypothesis of a gradual clonal exhaustion in the CD8(+) T cell population, perhaps as a result of continuous antigenic stimulation by persistent parasites.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Memoria Inmunológica/inmunología , Trypanosoma cruzi/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos de Protozoos/sangre , Apoptosis/inmunología , Enfermedad de Chagas/sangre , Enfermedad Crónica , Femenino , Humanos , Linfopoyesis , Masculino , Persona de Mediana EdadRESUMEN
The coelome-associated lympho-myeloid tissues, including the omentum, are derived from early embryo haemopoietic tissue of the splanchnopleura, and produce B lymphocytes and macrophages. They are reactive in pathologies involving coelomic cavities, in which they can expand in situ the cells of inflammatory infiltrates. We have addressed the question of the role of the adult omentum in permanent basal production of early lymphopoietic progenitors (pro-B/pre-B cells), through characterisation of omentum cells ex vivo, and study of their in vitro differentiation. We have shown that the murine omentum produces early haemopoietic progenitors throughout life, including B-cell progenitors prior to the Ig gene recombination expressing RAG-1 and lambda5, as well as macrophages. Their production is stroma-dependent. The omentum stroma can supply in vitro the cytokines (SDF-1alpha, Flt3 ligand and IL-7) and the molecular environment required for generation of these two cell lineages. Omentum haemopoietic progenitors are similar to those observed in foetal blood cell production, rather than to progenitors found in the adult haemopoietic tissue in the bone marrow--in terms of phenotype expression and differentiation capacity. We conclude that a primitive pattern of haemopoiesis observed in the early embryo is permanently preserved and functional in the adult omentum, providing production of cells engaged in nonspecific protection of abdominal intestinal tissue and of the coelomic cavity.
Asunto(s)
Linfocitos B/citología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Monocitos/citología , Epiplón/citología , Animales , Linfocitos B/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/biosíntesis , Citometría de Flujo , Células Madre Hematopoyéticas/inmunología , Linfopoyesis/fisiología , Ratones , Ratones Endogámicos C3H , Monocitos/inmunología , Epiplón/inmunología , Células del Estroma/citología , Células del Estroma/inmunologíaRESUMEN
INTRODUCTION: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation. MATERIALS AND METHODS: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies. RESULTS: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells. CONCLUSION: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Linfopoyesis , Mielopoyesis , Neoplasias/fisiopatología , Animales , Preescolar , Femenino , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/patología , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias/tratamiento farmacológicoRESUMEN
A reação inflamatória granulomatosa induzida, no fígado pelos ovos do Schistosoma mansoni é um sítio ativo de mielopoese extramedular potencialmente capaz de produzir todas as linhagens lielóides. O aumento da população de linfócitos B, ao longo da doença, associado à presença de precursores multipotentes, nos fez questionar se, em paralelo a mielopoese, haveria também linfopoese B. Foi demonstrado, por imunofenotipagem, a presença de células com característica de precursor B jovem (pro B) no granuloma. A expressão de RAG 1 e 5 foi demonstrada através da técnica de RT-PCR, confirmando a presença de precursosres B no granuloma. No entanto, colônias pré B não foram observadas, indicando um bloqueio na diferenciação de pro B para pre B. As células do estroma foram capazes de sustentar precursores B e a expressão de IL7 e SCF foi demonstrada. Porém, o meio condicionado de granuloma mostrou atividade inibidora da linfopoese B ao mesmo tempo que estimulava a mielopoese. Além disso, aproximadamente 50% dos precursores B do granuloma expressavam Mac 1. O estudo permitiu concluir que, há o desenvolvimento de um microambiente hematopoietico no granuloma, que permite a migração de progenitores e o multipotentes e o comprometimento para a linhagem B. No entanto, a produção extramedular de linfócitos B é bloqueada por fatores solúveis produzidos no granuloma