STIMULATION OF GROUP-II PHOSPHOLIPASE A(2) MESSENGER-RNA EXPRESSION AND RELEASE IN AN IMMORTALIZED ASTROCYTE CELL-LINE (DITNC) BY LPS, TNF-ALPHA, AND IL-1-BETA

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 A(2) In this study, we examined the interactive effects of lipopolysaccharides (LPS), interleukin 1 beta (IL-1 beta) and tumor necrosis factor (TNF alpha) to stimulate phospholipase A(2) (PLA(2)) 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 PLA(2) indicated the ability of DITNC cells to respond to all three factors in the induction of gene expression and the release of PLA(2). After an initial lag time of 2 h, PLA(2) 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 mu M completely inhibited cytokine-induced PLA(2) release. When suboptimal amounts of TNF alpha were added to the DITNC culture together with IL-1 beta or LPS, a synergistic increase in the induction of PLA(2) release could be observed. On the other hand, combination of IL-1 beta and LPS resulted only in an additive increase in PLA(2) release. Antibodies to IL-1 beta and TNF alpha completely neutralized the effects of these two agents on PLA(2) release. However, neither antibody was able to inhibit the PLA(2) release induced by LPS, suggesting that the effect of LPS was not complicated by the release of IL-1 beta or TNF alpha. 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 PLA(2) synthesis.