James D. Calore

Role of alpha-macroglobulin-elastase complexes in the pathogenesis of elastase-induced emphysema in hamsters.

Radiolabeled, enzymatically active or chloromethyl ketone-inactivated porcine pancreatic elastase was endotracheally instilled into hamsters. Gel filtration of the bronchopulmonary lavage fluid revealed two major radioactive fractions: one, eluting at 780,000 daltons, corresponding to an alpha-macroglobulin-pancreatic elastase complex, and another, at 68,000 daltons, corresponding to an alpha-1-protease inhibitor-pancreatic elastase complex. Elastolytic activity was recovered in the bronchopulmonary lavage fluid up to 4 d after elastase instillation and was associated with the alpha-macroglobulin-pancreatic elastase complex. Small amounts of this complex were recovered 14 d after instillation. When less than 1% (1.5--1.7 micrograms) of the usual dose of elastase was instilled into hamsters, the major radioactive complex was alpha-1-protease inhibitor-pancreatic elastase complex, and little or no elastolytic activity was found in the lavage fluid. In contrast to the instillation of 220 micrograms of elastase, no disease or hemorrhagic reaction was detected with this low dose, and without hemorrhage only insignificant amounts of alpha-macroglobulin-pancreatic elastase complexes were recovered from the lungs. To study the interaction of circulating antiproteases with elastase, hamster plasma was allowed to interact directly with the radiolabeled elastase; alpha-macroglobulin bound much more of the elastase than alpha-1-protease inhibitor, confirming the findings in the lung lavage experiments. The hamsters susceptibility to pancreatic elastase-induced emphysema may depend on the preferential binding of elastase to alpha-macroglobulin, which protects the elastolytic potential, rather than to alpha-1-protease inhibitor, which inactivates elastase. We speculate that if even a fraction of the residual radioactivity found in the hamster lungs as long as 144 d after instillation of elastase represents enzymatically active alpha-macroglobulin-pancreatic elastase complex, this could serve as a source of persistent elastolytic activity, which might explain the progressive nature of the pulmonary lesion.

Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury.

SummaryA neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema.

The Association of an Elastase with Amyloid Fibrils

Abstract The fibrils of all systemic forms of amyloid (primary, AL; secondary, AA; and hereditary, AF) that had been isolated by the water extraction procedure demonstrated elastolytic enzyme activity when examined in a specific assay using tritiated elastin. The source of this fibril-bound enzyme activity was consistent with human neutrophil elastase (HNE), since it was readily extracted by high salt solutions and inhibited by an elastase-specific chloromethyl ketone inhibitor, human α-1-protease inhibitor or by an antibody specific for HNE. The presence of an elastase on the amyloid fibril may suggest physiologic mechanisms of amyloid precursor protein degradation.

Serum Antielastase and Neutrophil Elastase Levels in PiM Phenotype Cigarette Smokers with Airflow Obstruction

In order to assess blood factors which might explain why some cigarette smokers develop airflow obstruction while others do not, we compared two groups of PiM phenotype volunteers matched for age, sex and total pack-years of cigarette smoking; one group had airflow obstruction and the other did not. Functional levels of alpha-2-macroglobulin (alpha-2-M) and alpha-1-protease inhibitor (alpha-1-PI) were separately assessed by a protease binding procedure. Neutrophils were isolated from blood by counterflow centrifugation, and their elastase content was assayed with 3H-elastin-SDS (sodium dodecyl sulfate). The obstructed and nonobstructed groups were not different with respect to functional or immunoreactive levels of alpha-1-PI and alpha-2-M or elastase levels in their neutrophils. We do not find imbalances of circulating elastase or antielastase levels in PiM phenotype smokers with airflow obstruction.

Amyloid-Agarose Plate Test: Ultrastructural Changes in the Fibril and its Association with Human Neutrophil Elastase

In the amyloid agarose plate test we examined the ultrastructure of amyloid fibrils after the clearing phenomenon caused by ethylenediamine tetraacetic acid (EDTA) and by serum. We quantitated total fibril length in the cleared and noncleared (control) areas by digital image analysis. EDTA resulted in shortening of the AA fibril by 31% in 3 h and 40% in 24 h. Serum resulted in shortening only 24% of the fibril in 3 h, but shortening to less than one half its original length in 24 h. Further examination of the fibrils from each of the systemic amyloid types (AL, AA, and AF) demonstrated similar clearing in agar plates. In addition, all had large amounts of elastolytic enzyme activity in a specific assay using tritiated elastin. The source of this fibril-bound enzyme activity was consistent with human neutrophil elastase (HNE), since it was inhibited by an elastase specific chloromethyl ketone inhibitor, as well as by an antibody specific for human neutrophil elastase.