Chymotrypsin-specific protease inhibitors decrease H2O2 formation by activated human polymorphonuclear leukocytes

Stimulated phagocytic cells generate active oxygen species which are known to contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity. Until now, it was not certain whether protease inhibitors are capable of decreasing the production of those oxygen species, and if they are, what type of protease inhibitor is the most active. In this work we monitored formation of H2O2 by 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated polymorphonuclear leukocytes (PMNs) because H2O2 is the immediate precursor of the actual damaging species. These determinations were carried out in the absence or presence of protease inhibitors and/or superoxide dismutase (SOD). The protease inhibitors tested were: potato inhibitors 1 (PtI-1) and 2 (PtI-2), a chymotrypsin-inhibitory fragment of PtI-2 (PCI-2), chicken ovoinhibitor (COI), turkey ovomucoid ovoinhibitor (TOOI), Bowman-Birk inhibitor (BBI), lima bean inhibitor (LBI) and soybean (Kunitz) trypsin inhibitor (SBTI). The order of activity, as measured by inhibition of H2O2 formation by TPA-activated PMNs during incubation at 37 degrees C for 30 min, was (in descending order): PtI-1 greater than or equal to PCI-2 greater than PtI-2 greater than COI greater than BBI greater than or equal to TOOI greater than LBI greater than SBTI. Thus, the most effective were the chymotrypsin-specific inhibitors PtI-1 and PCI-2, followed by the bifunctional inhibitors recognizing both chymotrypsin and trypsin, and the least active was SBTI, a predominantly trypsin inhibitor. At the higher concentrations of protease inhibitors tested, the inhibitory activity was similar in both the absence and presence of SOD. These results show that protease inhibitors specific for chymotrypsin but not those that are trypsin-specific are capable of inhibiting formation of active oxygen species during the oxidative burst of stimulated human PMNs.