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1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants. With respect to secretory cell metaplasia and haemorrhage, rALP231 tended to be a better inhibitor than recombinant antileucoprotease and rALP242. 5. We conclude that the recombinant antileucoprotease mutants are less sensitive to oxidation and consequently inhibit human-neutrophil-elastase-induced emphysema to a greater extent than recombinant antileucoprotease.

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.