The human LT system. VIII. A target cell-dependent enzymatic activation step required for the expression of the cytotoxic activity of human lymphotoxin.
- Author(s): Weitzen, M;
- Granger, GA
- et al.
We have used various protease inhibitors to examine the role of esterase in α class human lymphotoxin (α-LT) mediated lysis of mitomycin C-treated murine L-929 cells in vitro. Seven protease inhibitors were shown to inhibit lysis by α-LT when present throughout the reaction in vitro. These inhibitors were of two types, the reversible substrate analogues (PME, TME, LME, TAME, and BAEE) and the irreversible protease inactivators (TPCK and TLCK). The concentrations needed for optimum inhibition by the seven inhibitors ranged from 2.5 x 10-5 M to 2.5 x 10-2 M. There was enhanced lysis with EACA, and no measurable effect on lysis when Trasylol and soybean trypsin inhibitor were tested. Since TLCK irreversibly inactivates enzymes, it was used to determine whether the esterase necessary for lysis was associated with the target cell or the α-LT molecule. The α-LT was shown to be fully active after treatment with TLCK and subsequent dialysis. Similar experiments with DFP gave the same results. However, pretreatment of the L-929 cells with TLCK partially inhibited α-LT-mediated lysis. To determine whether intact protease substrates were required for inhibition, the products of hydrolysis of PME, LME, TAME, and BAEE were tested for their ability to inhibit lysis and were shown to be ineffective. The results of these studies indicate than an esterase(s), probably derived from the L-929 cell, is required for α class human LT-mediated cytolysis in vitro. These studies therefore suggest that α-LT is carried by the lymphocyte in an inactive form and requires an esterase or possibly protease-mediated activation step to enable it to lyse the L-929 target cell.