Augmentation of neutrophil-mediated erythrocyte lysis by cells derived in vitro from human monocytes.

Neutrophilic polymorphonuclear leukocytes (PMNs) were incubated with opsonized zymosan and lysed human erythrocytes (RBCs) as measured by a 51Cr release method. Conversely, myeloperoxidase (MPO)-negative hydrogen peroxide (H2O2)-generating cells, derived in vitro from human monocytes (monocyte-derived cells (MDCs), were ineffective per se but capable of augmenting the lysis by PMNs. The lysis by PMNs and PMNs plus MDCs was inhibited by catalase, azide, taurine, and alanine, consistent with the requirement for hypochlorous acid (HOCl). As detected under conditions similar to those used for lytic assays, MDCs failed to produce HOCl but augmented the HOCl recovery from the PMN-RBC system. Moreover, when the extent of the lysis was plotted as a function of the HOCl recovery, a positive linear relationship was found. Although the actual size of the H2O2 extracellular pool could not be measured because of the inexistence of a reliable assay to probe our cytolytic model without perturbing the equilibrium of the system, the results presented suggest that MDCs enhance the PMN-mediated lysis by improving the HOCl production, presumably by supplying extra amounts of H2O2 to be handled by PMN MPO. In fact, the events mediated by MDCs could be reproduced by using an appropriate H2O2-generating enzymatic system (glucose-glucose oxidase). The present study provides direct evidence for the possibility of cooperation between MPO-positive and MPO-negative phagocytes in exerting functions (HOCl production and, in turn, cytolysis) possibly relevant to the outcome of inflammatory processes.

Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils.

Triggered human neutrophils degraded denatured type I collagen (gelatin) by releasing and activating the latent metalloenzyme, gelatinase. The ability of the neutrophil to activate this enzyme was significantly, but not completely, inhibited by agents known to inhibit or scavenge chlorinated oxidants generated by the H2O2/myeloperoxidase/chloride system. A direct role for chlorinated oxidants in this process was confirmed by the ability of reagent HOCl to activate the latent enzyme in either the cell-free supernatant or in a highly purified state. Gelatinase activity was also expressed by triggered neutrophils isolated from patients with chronic granulomatous disease. The amount of gelatinolytic activity expressed by the patients' cells was similar to that released by normal neutrophils that were triggered in the presence of antioxidants. Thus, human neutrophils have the ability to activate gelatinase by either an HOCl-dependent process or an uncharacterized oxygen-independent process. The ability of the neutrophil to directly regulate this enzyme suggests an important role for the metalloproteinase in physiologic and pathophysiologic connective tissue metabolism.

Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis.