TUMOR-NECROSIS-FACTOR-ALPHA INCREASES ANTIFIBRINOLYTIC ACTIVITY OF CULTURED HUMAN MESANGIAL CELLS

Tumor necrosis factor alpha (TNF-alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties, TNF-alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF-alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF-alpha binds specifically to a single class of high affinity receptors (K(d) 5.10(-11) M; 1500 receptors/cell). TNF-alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by H-3-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated Cr-51 was not increased by 100 ng/ml TNF-alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF-alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF-alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours. Finally, in contrast to what was reported for rat mesangial cells, we failed to demonstrate a production of TNF-alpha in human mesangial cells themselves by immunoradiometric assay, immunocytochemistry and Northern blot analysis using a specific TNF-alpha cDNA probe. We conclude that TNF-alpha has an antimitogenic activity on human mesangial cells in culture and enhances the synthesis of PAI-1. Whether TNF-alpha plays a role in glomerular PAI-1 deposition and persistency of fibrin in vivo remains to be determined.