Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha.

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.