Proteases and oxidants in experimental pulmonary inflammatory injury

We have examined various biochemical parameters of pulmonary inflammation in experimental animals. Intrabronchial instillation of glucose oxidase-glucose (GO/G) to produce oxidants or formylated norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA) as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits. PMA also induced inflammation when administered intravenously. Each stimulus induced transudation of protein from the vascular space into the pulmonary tissues, and an influx of leukocytes during the 4-6 h period of the experiment. Pathophysiologic changes were measured by edema formation (transudation of 125I-bovine serum albumin), and histologic examination. Biochemical analysis was performed by measuring concentrations of potentially injurious agents in bronchoalveolar lavage (BAL) fluid. Increased acid protease and myeloperoxidase levels were found in the BAL fluid after administration of either of the stimuli. Evidence of oxidant generation in vivo was obtained in two different ways. In the first, specific activities for catalase were measured in the BAL fluid in the presence or absence of 3-amino, 1,2,4 triazole (AT), injected at intervals before obtaining BAL fluid. In the presence of AT, specific activities for catalase dropped to 0.22 after a double instillation of FNLP and to 0.15 in the presence of GO/G. In neutrophil-depleted FNLP animals, catalase was not greatly inhibited by AT (sp act 0.90). In the second, intracellular levels of total glutathione (GSH + GSSG) in whole lung tissue and alveolar macrophages decreased when stimuli of neutrophils were administered. Intrabronchially instilled PMA, e.g., caused a drop of glutathione in whole lung tissue from the control value of 2.3 mumol GSH equivalent/100 mg dry wt to 0.54 mumol GSH equivalent/100 mg dry wt at 4 h. Neutrophil depletion and superoxide dismutase protected from this effect. From these results, we conclude that O-2 or its metabolites can initiate severe pulmonary injury as shown by the effect of GO/G and that, during development of pulmonary injury, stimulated neutrophils generate oxidants and release proteolytic enzymes into the surrounding tissues.