RESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Mielopoese/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Técnicas de Cocultura , Células do Tecido Conjuntivo/fisiologia , Ferritinas/análise , Fibroblastos/citologia , Fibroblastos/fisiologia , Glicocálix/química , Glicocálix/ultraestrutura , Glicosaminoglicanos/análise , Glicosaminoglicanos/isolamento & purificação , Glicosaminoglicanos/fisiologia , Concentração de Íons de Hidrogênio , Indóis/análise , Camundongos , Camundongos Endogâmicos C3H , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , Células Progenitoras Mieloides/química , Células Progenitoras Mieloides/fisiologia , Células Progenitoras Mieloides/ultraestrutura , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Compostos Organometálicos/análise , Ligação Proteica , Proteoglicanas/análise , Proteoglicanas/fisiologia , Pseudópodes/ultraestrutura , Rutênio Vermelho/análise , Rutênio Vermelho/farmacologia , Espalhamento de Radiação , Células Estromais/química , Células Estromais/fisiologia , Células Estromais/ultraestruturaRESUMO
Staining by ruthenium red (0.5 mg/ml in borate buffer at pH = 9.2) has been used for light and electron microscopic visualization of polyanion containing structures in sections from glutaraldehyde-fixed, epoxy-embedded tissues. This staining technique can be applied in a simple and rapid way, showing the reactive cell components with suitable resolution and contrast. Preliminary spectrophotometric studies show the correspondence in absorption characteristics of the dye which is bound to polyanions in situ or in vitro.