Pseudomonas aeruginosa alkaline protease (AP) has recently been shown to produce limited proteolysis of human gamma interferon (IFN--y) and thereby destroy the antiviral and macrophage-activating activities of the lymphokine. In the present study we describe some of the characteristics of Pseudomonas elastase (E) with regard to inactivation of human IFN-,y. The inhibitory effect of E on IFN--y bioactivity differed from that of AP in that the direct effects of E were reduced in the presence of human serum. That this property of human serum was in large part attributable to the protease inhibitor a2-macroglobulin (%2-M) was suggested by the following observations: (i) methylamine treatment of serum reduced its effect on E, (ii) E interacted directly with OL2-M to induce a characteristic conformational change in the protease inhibitor, and (iii) preformed E-a2-M complexes lacked IFN-y-degrading activity. Despite these findings, anti-E antiserum partially neutralized the effect that a Pseudomonas filtrate showed on IFN--y, suggesting that E contributes to the activity of bacterial filtrates. Treatment of IFN--y with E in the presence of a suboptimal concentration of AP resulted in an E dose-dependent inactivation of the lymphokine. Preformed E-_2-M complexes, although ineffective by themselves at cleaving IFN-y, degraded the lymphokine, providing AP was also present in the reaction mixture.

Mononuclear phagocytes produce proteinases that are thought to play a role in regulating the activity of cytokines. Activated macrophages secrete urokinase-type plasminogen activator (uPA), which mediates the formation of the serine proteinase plasmin from the ubiquitous zymogen plasminogen. We previously observed a correlation between in vitro plasminogen activation by the promonocytic cell line U937 and the apparent ability of these cells to inactivate recombinant interferon-gamma (rIFN-gamma) by proteolysis. The present study was designed to test the hypothesis that plasmin, generated in U937 cell cultures, is both necessary and sufficient to inactivate rIFN-gamma by limited proteolysis. The following observations are consistent with this hypothesis: (1) inactivation of rIFN-gamma was prevented by inhibitors of serine proteinases or an antibody that specifically immunodepleted plasmin activity; (2) purified plasmin inactivated rIFN-gamma as efficiently as U937 culture supernatants and was similarly sensitive to serine proteinase inhibitors; and (3) plasmin removed an 11 amino acid carboxyl-terminal peptide from rIFN-gamma, which included a region known to be required for bioactivity. Overall, these data indicate that plasminogen activation by U937 promonocytic cells leads to the proteolytic inactivation of IFN-gamma by a process that only requires the production of plasmin.

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