Detection of extracellular neutrophil elastase in hamster lungs after intratracheal instillation of E. coli lipopolysaccharide using a fluorogenic, elastase-specific, synthetic substrate.
Repeated intratracheal instillations of E. coli lipopolysaccharide (LPS) in hamster lungs cause an influx of polymorphonuclear leukocytes (PMNs) into the alveolar walls, with concomitant development of severe emphysema. It has been suggested that elastase, released by these PMNs, is involved in the development of emphysema. This study demonstrates the release of elastase from recruited PMNs in cryostat sections of hamster lungs, after being treated once, twice, or thrice with LPS, intratracheally. Elastase activity was visualized using two elastase-specific synthetic substrates, to which a methoxynaphthylamine (MNA) group had been bound covalently. Liberated MNA, when made insoluble by coupling with 5-nitrosalicylaldehyde, fluoresces strongly. The authors observed that the interval between start of incubation and appearance of fluorescence and the intensity of fluorescence correlated with the number of LPS administrations. Fluorescence was observed to be located in or in close vicinity to alveolar walls. No fluorescence was observed in sections of untreated hamsters. Liberation of MNA from synthetic substrates was delayed strongly by the addition of a recombinant secretory leukocyte proteinase inhibitor or a substituted cephalosporin neutrophil elastase inhibitor. The authors conclude that LPS-mediated PMN influx into the lung is accompanied by release of elastase from these cells and speculate that this PMN-elastase is involved in the development of LPS-mediated emphysema.
Rudolphus
A
A
Department of Pulmonology, University Hospital, Leiden, The Netherlands.
Stolk
J
J
van Twisk
C
C
van Noorden
C J
CJ
Dijkman
J H
JH
Kramps
J A
JA
D000818:Animals; D006224:Cricetinae; D004926:Escherichia coli / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology* / Q000648:ultrastructure; D006651:Histocytochemistry; D008070:Lipopolysaccharides / Q000378:metabolism / Q000502:physiology*; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D008647:Mesocricetus; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000097:blood*; D013379:Substrate Specificity
D008070:Lipopolysaccharides; D010196:Pancreatic Elastase