Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease

Abstract

Human neutrophilic polymorphonuclear leukocyte (PMN) elastase was purified by affinity chromatography to greater than 95% homogeneity as judged by disc-gel electrophoresis. Dog lung elastin was prepared from alveolar-enriched tissue by prior extraction of soluble and collagenous lung proteins with 0.1 M NaOH at 98 degrees C. Digestion of the remaining insoluble residue by the purified PMN enzyme was monitored by Lowry assay of acid-soluble peptides released. The PMN enzyme possessed 60% of the digestive activity of crystallized porcine pancreatic elastase (weight:weight comparison) when tested in vitro against this substrate in phosphate-NaCl buffer at pH 7.5. Whole tissue studies were then performed in lungs of laboratory animals. One-ml samples containing purified PMN elastase were instilled into lavaged and saline-perfused isolated dog lung at the level of the sixth to seventh generation bronchus. Treatment with 384 mug of the PMN enzyme produced anatomic emphysema after a 90-min incubation at room temperature, which was comparable to that produced by 100 mug of porcine pancreatic elastase. Frozen sections of treated and control lungs were examined for the presence of PMN elastase by the indirect immunoperoxidase method using a monospecific rabbit antiserum against PMN elastase as the primary stain. Light microscopy revealed elastase bound to connective tissue in the treated lungs, in close proximity to aldehyde-fuchsin-counterstained elastic fibers. A similar experiment was tn of enzyme solutions containing 1;0 mg of elastase per ml produced discrete lesions within 90 min, as before. Light microscopic studies in conjunction with the indirect immunoperoxidase staining method again demonstrated elastase in association with connective tissue elements in the lesion area. In addition, part of the instilled protease could be demonstrated within alveolar macrophages. Electron microscopy combined with immunoperoxidase staining revealed direct attachment of th einstilled enzyme to elastic fibers within alveolar septa. In enzyme-treated tissue, some septa showed severe depletion of intercellular structures with the exception of colalgen, which was generally preserved. These results show that human leukocyte elastase penetrated dog alveolar septal connective tissue after airway instillation and that the enzyme attaches to elastic fibers, inducing histologic changes comparable to thos seen in human emphysema.