PENTOXIFYLLINE INHIBITS TUMOR NECROSIS FACTOR-ALPHA-MEDIATED CYTOTOXICITY AND CYTOSTASIS IN L929 MURINE FIBROSARCOMA CELLS

Tumor necrosis factor-alpha (TNF alpha) is recognized as a principal m ediator of a variety of inflammatory conditions. In animal models, pen toxifylline attenuates the morbidity and mortality of bacterial sepsis , an effect which has been attributed to its ability to suppress the i nduction of TNF alpha. To determine whether pentoxifylline also direct ly inhibits the effects of TNF alpha, the ability to inhibit cytotoxic ity on the TNF alpha-sensitive murine fibrosarcoma cell line, L929, wa s examined. Cell viability was assessed by crystal violet staining and cell proliferation was assessed by [H-3]-thymidine uptake assay. TNF alpha induced dose-dependent cytotoxicity. At concentrations of TNF al pha of 1000 U/ml, viability at 3 days was approximately 35% of control . When L929 cells were co-incubated with TNF alpha (1000 U/ml) and pen toxifylline (1 mM), cell viability increased to approximately 75% of c ontrol (P = 0.001). Ar concentrations of TNF alpha of 10,000 U/ml, cel l viability which was 11% of control with TNF alpha alone increased to 53% in the presence of pentoxifylline (P = 0.002). TNF alpha at 1000 and 10,000 U/ml concentrations decreased [H-3]-thymidine uptake to app roximately 5% of control values. Co-incubation with pentoxifylline sig nificantly increased uptake to 13% of control at both TNF alpha concen trations (P = 0.002). Pentoxifylline did not affect the level of type I TNF alpha receptor - ligand cross-link product. However, in TNF alph a 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), with out significant change in number of type I TNF alpha receptors, sugges ting that pentoxifylline affects post-receptor signalling events. We h ave observed that pentoxifylline prevents the TNF alpha-mediated activ ation of sn-2 arachidonic acid-specific cytosolic phospholipase A(2), an important component of the signal transduction pathway of TNF alpha cytotoxicity. Because pentoxifylline does not inhibit all activities mediated by the type I TNF alpha receptor, its selective inhibition of post-receptor signalling may facilitate further study into the mechan isms underlying the diverse effects of TNF alpha.