Involvement of lipid mediators on cytokine signaling and induction of secretory phospholipase A(2) in immortalized astrocytes (DITNC)

Our previous studies demonstrated the ability of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL -1 beta), to stimulate NF kappa B/DNA binding and synthesis of secretory ph ospholipase A(2) (sPLA(2)) in immortalized astrocytes (DITNC). In this stud y, we examined possible involvement of lipid mediators in the cytokine acti on. Using [C-14]serine to label sphingomyelin and ceramide in these cells, subsequent exposure of cells to cytokines did not result in alteration of s phingomyelin/ceramide ratio. Furthermore, neither exogenous sphingomyelinas e nor cell-permeable ceramides could stimulate NF kappa B/DNA binding. On the other hand, C-2 ceramide (0.3 mu M) as well as other lipid mediators , such as lysophosphatidylcholine and arachidonic acid, were able to elicit a small increase in sPLA(2) and potentiate the induction of sPLA(2) by TNF -alpha. When DITNC cells were prelabeled with [P-32]Pi, an increase in labe led phosphatidic acid (PA) was observed on treatment of cells with IL-1 bet a (200 U/mL). However, despite the ability of phorbol myristate acetate (PM A) to stimulate phospholipase D (PLD) and synthesis of phosphatidylethanol (PEt) in these cells, PLD activity was not affected by IL-1 beta. With the [P-32]labeled cells, however, PA-phosphohydrolase inhibitors, such as chlor promazine and propranolol, could elicit large increases in labeled PA, indi cating active PA metabolism in these cells. Cytokines also caused an increa se in levels of diacylglycerol (DG) in these cells, although the source of this lipid pool is presently not understood. Taken together, these results provide evidence for the participation of PA and DG in cytokine signaling a ctivity. Furthermore, although cytokines did not cause the release of ceram ide, lipid mediators, such as lysophospholipids, and AA could modulate cyto kine-mediated induction of sPLA(2) in astrocytes.