An in vitro investigation of the responses of C6 rat astroglioma cells to interleukin-1(beta) and tumor necrosis factor alpha
Description
Because of its similarity to normal, mature astrocytes, the C6 astrocytoma cell line was chosen as an in vitro system to model mechanisms involved in gliosis. Gliosis, the astrocytic response to central nervous system injury, is believed to be triggered by the cytokines interleukin-1$\beta$ (IL-1$\beta$) and tumor necrosis factor-$\alpha$ (TNF$\alpha$). The cellular responses of C6 cells to IL-1$\beta$ and TNF$\alpha$ were found to include alterations in morphology and increases in the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and $\alpha$-tubulin The similarity in C6 cell responses to IL-1$\beta$ and TNF$\alpha$ suggested that some aspects of the intracellular signal transduction pathways for those two cytokines might be shared. Phospholipid metabolites involved in signal transduction were identified by chromatographic methods. Both IL-1$\beta$ and TNF$\alpha$ stimulated a biphasic increase in intracellular diacylglycerol, predominantly from phosphatidylcholine. There was also evidence for activation of phospholipase A$\sb2$ and a phosphatidylinositol phospholipase C. Results of enzymatic assays and inhibition experiments suggested that the phosphatidylcholine-derived diacyiglycerol was produced by the phospholipase D/phosphatidic acid phosphatase pathway Since protein kinase C (PKC) activity is modulated by various phospholipase-catalyzed hydrolysis products and long-term activation of phospholipase D has been attributed to a positive feedback loop with PKC, PKC involvement was addressed. Phospholipase D was found to be activated by PKC-dependent and PKC-independent mechanisms. Also, IL-1$\beta$- and TNF$\alpha$-induced $\alpha$-tubulin increases were blocked by PKC inhibitors. The GFAP response to IL-1$\beta$ and TNF$\alpha$ was sensitive to the PKC inhibitors calphostin C and H7. However, another PKC inhibitor, staurosporine, was found to increase GFAP, an effect that was synergistic with that of IL-1$\beta$ and TNF$\alpha$. Elucidation of these signaling pathways is important in that intracellular mediators represent potential therapeutic targets for pharmacological intervention