SECRETION AND INACTIVATION OF MYELOPEROXIDASE BY ISOLATED NEUTROPHILS

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues , Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the m edium, Cells stimulated with phorbol myristate acetate (PMA) or opsoni zed zymosan particles secreted MPO and released super-oxide free radic als (.O2-). Dismutation of .O2- produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the tox ic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dism utase (SOD) to increase the rate of conversion of .O-2- to H2O2 had pa -dependent effects on HOCl production, From pH 6.0 to 7.4, SOD promote d HOCl production by up to 500% but SOD had no effect at pH 1.6 and in hibited by 40 +/- 10% at pH 7.8, In further experiments at pH 7.0, MPO activity in the cells decreased by 25 +/- 2 and 44 +/- 4% during 1-h incubations with PMA and zymosan, Only 1 +/- 0 and 3 +/- 1% of the tot al activity was found in the medium, indicating that most of the secre ted MPO was inactivated, Loss of activity was not accompanied by prote olytic destruction of the MPO protein, which was measured with anti-MP O antibodies, SOD raised the amount of active MPO in the medium two- t o sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect, The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but O-2- production by ionophore-stimula ted cells was less than 4% of that of PMA- or zymosan-stimulated cells and most of the secreted MPO was found active in the medium, When PMA -stimulated cells were incubated with purified MPO, the added MPO acti vity was lost from the medium, Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium, The visible absorption spectrum of MPO was lost, indicating destruction of the iron-containing prosthetic group, Loss o f activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiolog ical pH range, inactivation of MPO in the medium suppressed HOCl produ ction, Inactivation required .O-2- but not HOCl, H2O2, or free iron, I nactivation of secreted MPO may limit MPO-mediated damage to host tiss ues by stimulated neutrophils.