Pseudomonas aeruginosa alkaline protease and elastase are thought to contribute to bacterial invasiveness, tissue damage, and immune suppression in animals and patients infected with the bacterium. This study examined the ability of the two proteases to inactivate a number of cytokines that mediate immune and inflammatory responses. Human recombinant gamma interferon (rIFN-y) and human recombinant tumor necrosis factor alpha were inactivated by both proteases. Murine rIFN-,y was relatively resistant to alkaline protease but was inactivated by elastase, and human recombinant interleukin-la and recombinant interleukin-lo were resistant to the effects of both proteases. Western immunoblots suggested that cytokine inactivation by these proteases, where it occurred, required only limited proteolysis of the polypeptides. The ability of different P. aeruginosa strains to inactivate IFN--y appeared to require the production of both proteases for optimum activity. These results indicate that in vitro cytokine inactivation by Pseudomonas proteases is selective, requires only limited proteolysis, and in certain instances reflects the cooperative effects of both proteases.Pseudomonas aeruginosa is an important pulmonary pathogen in conditions like cystic fibrosis (15,42). It has been suggested that the ability of P. aeruginosa to establish itself in the respiratory tract may be promoted by its suppressive effects on pulmonary immune responses (2,16,40). The mechanisms of this immunosuppression are not entirely understood, but proteolytic enzymes secreted by the bacterium have been shown to degrade surface receptors on hematogenous cells (36, 43) and inactivate interleukin-2 (IL-2) and gamma interferon (IFN--y) (16,17,41).In the case of human IFN--y, cytokine inactivation was caused by either Pseudomonas alkaline protease (AP) (17) or elastase (E) (16). Significant reductions in antiviral and immunomodulatory activities were associated with limited proteolysis of IFN--y. Of particular interest were the synergistic effects on IFN--y seen when both purified proteases were added to reaction mixtures (16). These results would predict that Pseudomonas strains that produce both enzymes should be particularly immunosuppressive, a property that may aid the bacterium in establishing initial colonization by significantly altering immune and inflammatory responses in infections like those seen in cystic fibrosis.This study was undertaken to address three questions relating to the effects of Pseudomonas protease on cytokines. First, what are the specificities of these proteases relative to the inactivation of cytokines that might be involved in Pseudomonas infections? Second, is limited proteolysis of the type seen with human IFN--y sufficient for the inactivation of other cytokines? Third, is the ability to inactivate cytokines a common property of Pseudomonas strains, and how does this property relate to their production of the two proteases? MATERIALS AND METHODSHuman subjects. The studies reported here were approved by the Human Subj...

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.