Microglial regulation by astrocytes: effect on CD40 surface expression and Its consequences for dendritic cell maturation

Microglia are the primary immune-responsive cells in the central nervous system. Monocytically derived, they can be isolated in culture where, if stimulated by sequential treatment with granulocyte/monocyte colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS), they will upregulate the expression of major histocompatibility complex class II and surface levels of the co-stimulatory molecules CD40, CD80 and CD86 necessary for efficient antigen presentation and T cell activation. Hence, they assume a mature dendritic cell (DC) phenotype in isolation culture. In preliminary studies we have shown that the continued presence of astrocytes stunts the assumption of the microglial DC phenotype in a contact-dependent manner. The current study was designed to examine the mechanism(s) by which astrocytes prevent DC maturation in microglia. LPS signaling inhibitors SOCS1, SOCS3, TRIM30 and IRAK-M were rapidly and robustly upregulated by LPS treatment of co-cultured microglia. SOCS3 protein levels, however, were similar in both culture conditions while IRAK-M protein was elevated only in co-cultured cells. In IRAK-M-/- microglia CD11c levels were inhibited in both settings, but CD40 levels were only modestly affected. Expression of IFN, an inducer of both CD40 as well as inhibitors SOCS1 and SOCS3, was highest in isolated microglia, but neutralization of it affected only CD11c. GM-CSF pretreatment elicited a rapid and robust up-regulation of co-stimulatory molecules in isolated cells while subsequent LPS treatment increased expression primarily in co-cultured cells. CD40 and CD11c protein surface expression were maximal at the end of GM-CSF treatment in isolated cells while co-cultured cells awaited LPS signaling before placing protein on the cell surface. Differences in CD40 surface expression were not due to a more rapid mRNA or protein turnover. Rather, Western blots and flow cytometry showed that total CD40 protein levels in co-cultured microglia were high. These data suggested that CD40 protein was transcribed and translated but was retained intracellularly. Immunocytochemistry confirmed that CD40 was retained in the ER, but not in lysosomes or early endosomes. In conclusion, astrocytes affect particularly GM-CSF signaling of microglia. Future studies would be designed to address the mechanism(s) by which the extracellular environment created by astrocytes regulates the intracellular trafficking within microglia.Ph. D.Includes bibliographical referencesIncludes vitaby Giselles Maria Aceved