TNF-α pretreatment prevents subsequent activation of cultured brain cells with TNF-α and hypoxia via ceramide

We have developed a cellular model in which cultured astrocytes and brain capillary endothelial cells preconditioned with tumor necrosis factor-alpha (TNF-alpha) fail to upregulate intercellular adhesion molecule-1 (ICAM-1) protein (80%, inhibition) and mRNA (30% inhibition) when challenged with TNF-alpha or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-alpha, its levels were measured at various times after the TNF-alpha preconditioning. We present evidence for the first time that, in normal brain cells, TNF-alpha pretreatment causes a biphasic increase of ceramide levels: an early peak at 15-20 min, when ceramide levels increased 1.9-fold in astrocytes and 2.7-fold in rat brain capillary endothelial cells, and a delayed 2- to 3-fold ceramide increase that occurs 18-24 h after addition of TNF-alpha. The following findings indicate that the delayed ceramide accumulation results in cell unresponsiveness to TNF-alpha: 1) coincident timing of the ceramide peak and the tolerance period, 2) mimicking of preconditioning by addition of exogenous ceramide, and 3) attenuation of preconditioning by fumonisin B-1, an inhibitor of ceramide synthesis. In contrast to observations in transformed cell lines, the delayed ceramide increase was transient and did not induce apoptosis in brain cells.