Pentoxifylline inhibits tumor necrosis factor-α-mediated cytotoxicity and cytostasis in L929 murine fibrosarcoma cells

Tumor necrosis factor-α (TNFα) is recognized as a principal mediator of a variety of inflammatory conditions. In animal models, pentoxifylline attenuates the morbidity and mortality of bacterial sepsis, an effect which has been attributed to its ability to suppress the induction of TNFα. To determine whether pentoxifylline also directly inhibits the effects of TNFα, the ability to inhibit cytotoxicity on the TNFα-sensitive murine fibrosarcoma cell line, L929, was examined. Cell viability was assessed by crystal violet staining and cell proliferation was assessed by [ 3H]-thymidine uptake assay. TNFα induced dose-dependent cytotoxicity. At concentrations of TNFα of 1000 U/ml, viability at 3 days was approximately 35% of control. When L929 cells were co-incubated with TNFα (1000 U/ml) and pentoxifylline (1 mM), cell viability increased to approximately 75% of control ( P = 0.001). At concentrations of TNFα of 10,000 U/ml, cell viability which was 11% of control with TNFα alone increased to 53% in the presence of pentoxifylline ( P = 0.002). TNFα at 1000 and 10,000 U/ml concentrations decreased [ 3H]-thymidine uptake to approximately 5% of control values. Co-incubation with pentoxifylline significantly increased uptake to 13% of control at both TNFα concentrations ( P = 0.002). Pentoxifylline did not affect the level of type I TNFα receptor-ligand cross-link product. However, in TNFα receptor binding assays, incubation with pentoxifylline 1 mM for 4 h was associated with an increase in the receptor affinity (control: K D = 0.42 nM vs pentoxifylline-treated: K D = 0.21 nM, P = 0.006), without significant change in number of type I TNFα receptors, suggesting that pentoxifylline affects post-receptor signalling events. We have observed that pentoxifylline prevents the TNFα-mediated activation of sn-2 arachidonic acid-specific cytosolic phospholipase A 2, an important component of the signal transduction pathway of TNFα cytotoxicity. Because pentoxifylline does not inhibit all activities mediated by the type I TNFα receptor, its selective inhibition of post-receptor signalling may facilitate further study into the mechanisms underlying the diverse effects of TNFα.