RESUMO
Undifferentiated spermatogonia (Aund) or spermatogonial stem cells (SSCs) are committed to the establishment and maintenance of spermatogenesis and fertility throughout a male's life and are located in a highly specialized microenvironment called niche that regulates their fate. Although several studies have been developed on SSCs in mammalian testis, little is known about other vertebrate classes. The present study is the first to perform a more detailed investigation on the spermatogonial cells and their niche in a reptilian species. Thus, we characterized Aund/SSCs and evaluated the existence of SSCs niche in the Kinosternon scorpioides, a freshwater turtle found from Mexico to northern and central South America. Our results showed that, in this species, Aund/SSCs exhibited a nuclear morphological pattern similar to those described for other mammalian species already investigated. However, in comparison to other spermatogonial cell types, Aund/SSCs presented the largest nuclear volume in this turtle. Similar to some mammalian and fish species investigated, both GFRA1 and CSF1 receptors were expressed in Aund/SSCs in K. scorpioides. Also, as K. scorpioides Aund/SSCs were preferentially located near blood vessels, it can be suggested that this niche characteristic is a well conserved feature during evolution. Besides being valuable for comparative reproductive biology, our findings represent an important step towards the understanding of SSCs biology and the development of valuable systems/tools for SSCs culture and cryopreservation in turtles. Moreover, we expect that the above-mentioned results will be useful for reproductive biotechnologies as well as for governmental programs aiming at reptilian species conservation.
Assuntos
Escorpiões/citologia , Espermatogônias/citologia , Nicho de Células-Tronco , Tartarugas/metabolismo , Animais , Biomarcadores/metabolismo , Forma Celular , Tamanho Celular , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Escorpiões/metabolismo , América do Sul , Espermatogônias/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Lipopolissacarídeos/imunologia , Linfócitos/imunologia , Monócitos/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/imunologia , Quimiocinas/metabolismo , Criopreservação/métodos , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-4/imunologia , Interleucina-4/farmacologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Teste de Cultura Mista de Linfócitos/métodos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/imunologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-4/imunologia , Receptores de Interleucina-4/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
Gangliosides have been extensively described to be involved in the proliferation and differentiation of various cell types, such including hematopoietic cells. Our previous studies on murine models of stroma-mediated myelopoiesis have shown that gangliosides are required for optimal capacity of stromal cells to support proliferation of myeloid precursor cells, being shed to the supernatant and selectively incorporated into myeloid cell membranes. Here we describe the effect of gangliosides on the specific granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation. For that, we used the monocytic FDC-P1 cell line, which is dependent upon GM-CSF for survival and proliferation. Cells were cultured in the presence of GM-CSF and exogenous gangliosides (GM3, GD1a or GM1) or in the absence of endogenous ganglioside synthesis by the use of a ceramide-synthase inhibitor, D-PDMP. We observed that exogenous addition of GD1a enhanced the GM-CSF-induced proliferation of the FDC-P1 cells. Also, we detected an increase in the expression of the α isoform of the GM-CSF receptor (GMRα) as well as of the transcription factor C/EBPα. On the contrary, inhibition of glucosylceramide synthesis was accompanied by a decrease in cell proliferation, which was restored upon the addition of exogenous GD1a. We also show a co-localization of GD1a and GMR by immunocytochemistry. Taken together, our results suggest for the first time that ganglioside GD1a play a role on the modulation of GM-CSF-mediated proliferative response, which might be of great interest not only in hematopoiesis, but also in other immunological processes, Alzheimer disease, alveolar proteinosis and wherever GM-CSF exerts its effects.
Assuntos
Gangliosídeos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Densitometria , Imunofluorescência , Gangliosídeo G(M3)/farmacologia , Gangliosídeos/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Microscopia Confocal , Morfolinas/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Solubilidade/efeitos dos fármacosRESUMO
Interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are two of the best-characterized cell survival factors in hematopoietic cells; these factors induce an increase in Akt activity in multiple cell lines, a process thought to be involved in cellular survival. It is known that growth factors require sustained glucose metabolism to promote cell survival. It has been determined that IL-3 and GM-CSF signal for increased glucose uptake in hematopoietic cells. Interestingly, receptors for IL-3 and GM-CSF are present in several non-hematopoietic cell types but their roles in these cells have been poorly described. In this study, we demonstrated the expression of IL-3 and GM-CSF receptors in HEK293 cells and analyzed their effect on glucose uptake. In these cells, both IL-3 and GM-CSF, increased glucose uptake. The results indicated that this increase involves the subcellular redistribution of GLUT1, affecting glucose transporter levels at the cell surface in HEK293 cells. Also the data directly demonstrates that the PI 3-kinase/Akt pathway is an important mediator of this process. Altogether these results show a role for non-insulin growth factors in the regulation of GLUT1 trafficking that has not yet been directly determined in non-hematopoietic cells.
Assuntos
Transportador de Glucose Tipo 1/metabolismo , Glucose/farmacocinética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-3/farmacologia , Transporte Biológico/efeitos dos fármacos , Western Blotting , Linhagem Celular , Glucose/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
It is well established that granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-alpha) are involved in Langerhans' cell (LC) development and dendritic cell traffic. However, little is known about the pattern of cytokine receptors on human LC and their modulation during different stages of maturation. The expression of cytokine receptors was studied by flow cytometry on both freshly isolated LC (fLC) and 72-hr cultured LC (cLC). Epidermal cell suspensions enriched in LC were obtained after skin trypsinization and Ficoll-Hypaque gradient. LC were identified by their CD1a positivity. Although the majority of fLC were positive for the alpha chain of GM-CSF receptor (GM-CSFR), the beta chain of GM-CSFR was detected only on 15% of CD1a+ cells. fLC were also positive for IL-1 receptor (IL-1R) type 1, IL-1R type 2, 75,000 molecular weight TNF receptor (TNFR) and interferon-gamma receptor (IFN-gamma R). IL-6R and its transducing signal gp130 were present in a subset of fLC. Granulocyte colony-stimulating factor receptor (G-CSFR), macrophage colony-stimulating factor receptor (M-CSFR), the alpha and beta chain of IL-2R, IL-4R, IL-7R, IL-8R and 55,000 molecular weight TNFR were not detected on fLC. After culture, LC up-regulated the expression of both the alpha and beta chains of GM-CSFR, IL-1R type 2, alpha and beta chains of IL-2R, IL-6R and gp130. In contrast, IL-1R type 1 and 75,000 molecular weight TNFR were down-modulated and the expression of IFN-gamma R was not affected by culture. These results suggest that LC undergo changes in the cytokine receptor repertory during in vitro maturation.