OXIDATIVE INACTIVATION OF ALPHA-1-PROTEINASE INHIBITOR BY ALVEOLAR MACROPHAGES FROM HEALTHY SMOKERS REQUIRES THE PRESENCE OF MYELOPEROXIDASE

The aim of this work was to study the ability of human alveolar macrophages (AM) of 10 healthy smokers to inactivate alpha-1-proteinase inhibitor (alpha-1PI). Purified alpha-1PI was incubated for 45 min, with human alveolar macrophages before and after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. As a Positive control, the same experiments were performed in parallel with blood human neutrophils (PMN). Results are expressed as percentage of inactivation of alpha-1PI as evaluated from its inhibitory activity against porcine pancreatic elastase. A strong correlation (r = 0.99) was shown when inhibitory activity of alpha-1PI was evaluated against porcine pancreatic elastase or human neutrophil elastase. Unstimulated AM (1.57 +/- 0.9%) as well as stimulated AM (PMA: 1 +/- 0.4%; zymosan: 3 +/- 0.6%) were unable to inactivate alpha-1PI. Gel electrophoresis of alpha-1PI demonstrated that AM before or after stimulation induced a slight proteolysis of alpha-1PI, whereas both cleaved and complexed alpha-1PI were found when alpha-1PI was incubated with activated PMN. Both unstimulated (22 +/- 2.6%) and activated PMN (PMA: 91.7 +/- 4.7%; zymosan: 90 +/- 5.5%) were responsible for a significant inactivation of alpha-1PI. Catalase, in contrast to superoxide dismutase, was responsible for a near complete protection of alpha-1PI inactivation by PMN. To better determine the role of PMN secretory products, especially myeloperoxidase (MPO), we also investigated the effect of zymosan-activated PMN supernatants or of purified MPO on the alpha-1PI-AM reaction. MPO assay in PMN supernatants demonstrated that activated neutrophils released significant amounts of MPO (16.8 +/- 4.1 U/ml), whereas MPO was undetectable in activated AM supernatants. Inactivation of alpha-1PI by activated AM was dramatically increased by addition of PMN supernatant (up to 40%) or of MPO (up to 98%). Effect of MPO was dose dependent and was optimal at 25 mU/ml. Addition of NaN3, an inhibitor of MPO activity, prevented the enhancing effect of PMN supernatants on alpha-1PI inactivation by AM (from 47.5 +/- 9.7% to 13.5 +/- 4%). Addition of catalase to the reaction significantly prevented the inactivation of alpha-1PI by activated AM in the presence of MPO (25 mU/ml) (from 97 +/- 0.3% 0.3% to 1 +/- 0.3%), whereas superoxide dismutase did not, demonstrating the crucial role of hydrogen peroxide generation by AM. It is likely that culture conditions, especially duration of incubation, may explain discrepancies with previously published data. However, our results support the hypothesis that oxidative inactivation of alpha-1PI by reactive oxygen species generated by activated human AM from smokers requires the presence of the MPO release by PMN.