Involvement of a serine protease in tumour‐necrosis‐factor‐mediated cytotoxicity

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Abstract
We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1 The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, Nα-p-tosyl-L-lysine chloromethane, Nα-p-tosyl-L-phenylalanylchloromethane, Nα-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-β-naphthyl ester, p-nitrophenyl p′-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2 Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3 TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1, 3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with Nα-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4 The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5 Protease inhibitors did not influence TNF binding, internalization or metabolization. 6 No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth-inhibiting processes induced by TNF.