Stimulation of group II phospholipase A2 mRNA expression and release in an immortalized astrocyte cell line (DITNC) by LPS, TNFα, and IL-1β
Astrocytes are immunoactive cells in brain and have been implicated in the defense mechanism in response to external injury. Previous studies using cultured glial cells indicated the ability of astrocytes to respond to bacteria endotoxin and cytokines, resulting in the release of phospholipase A2. In this study, we examined the interactive effects of lipopolysaccharides (LPS), interleukin 1β (IL-1β) and tumor necrosis factor (TNFα) to stimulate phospholipase A2 (PLA2) in an immortalized astrocyte cell line (DITNC) with many properties of type I astrocytes. Northern blot analysis using oligonucleotide probes derived from the cDNA encoding the rat spleen group II PLA2 indicated the ability of DITNC cells to respond to all three factors in the induction of gene expression and the release of PLA2. After an initial lag time of 2 h, PLA2 release was proportional to time, reaching a plateau by 12 h. This event occurred at a time period preceding any signs of cell death. Cycloheximide at 1.25 μM completely inhibited cytokine-induced PLA2 release. When suboptimal amounts of TNFα were added to the DITNC culture together with IL-1β or LPS, a synergistic increase in the induction of PLA2 release could be observed. On the other hand, combination of IL-1β and LPS resulted only in an additive increase in PLA2 release. Antibodies to IL-1β and TNFα completely neutralized the effects of these two agents on PLA2 release. However, neither antibody was able to inhibit the PLA2 release induced by LPS, suggesting that the effect of LPS was not complicated by the release of IL-1β or TNFα. Taken together, results show that the immortalized astrocyte cell line (DITNC) can be used for studies to elucidate the molecular mechanism underlying the cytokine signaling cascade and subsequent induction of PLA2 synthesis.
Index EntriesAstrocytes injury glial cells endotoxin cytokines phospholipase A2 lipopolysaccharides interleukin 1β tumor necrosis factor signaling cascade
free fatty acid
fetal bovine serum
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