EXPERIMENTAL EMPHYSEMA INDUCED WITH PURIFIED HUMAN NEUTROPHIL ELASTASE - TISSUE LOCALIZATION OF INSTILLED PROTEASE

Publisher Website
Google Scholar
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.degree. 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 6th-7th generation bronchus. Treatment with 384 .mu.g of the PMN enzyme produced anatomic emphysema after a 90-min incubation at room temperature, which was comparable to that produced by 100 .mu.g 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 then performed using dog lungs in vivo. Localized instillation of enzyme solutions containing 1.0 mg of elastase/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. EM combined with immunoperoxidase staining revealed direct attachment of the instilled enzyme to elastic fibers within alveolar septa. In enzyme-treated tissue, some septa showed severe depletion of intercellular structures with the exception of collagen, which was generally preserved. Human leukocyte elastase penetrated dog alveolar septal connective tissue after airway instillation and the enzyme attached to elastic fibers, inducing histologic changes comparable to those seen in human emphysema.