Putative role of neutrophil elastase in the pathogenesis of emphysema.

Emphysema in humans takes several different forms: centrilobular, panacinar, paraseptal, and airspace enlargement with fibrosis. The varying morphologic and background features of these forms of emphysema suggest that they differ in pathogenesis. Elastic fiber rupture and fraying are a feature of emphysema. Experimental emphysema may be induced by human neutrophil elastase and other elastolytic enzymes but not by nonelastolytic proteases. Disruption of elastic fibers also appears to be the underlying feature of lathyrogen-induced airspace enlargement and of the emphysema in the blotchy mouse. However, there is no evidence of elastic fiber destruction in cadmium-induced airspace enlargement with fibrosis or in emphysema associated with hyperoxia or severe starvation. Thus, elastic fiber disruption is not common to all forms of experimental emphysema. We posit that airspace enlargement may be a stereotyped response of the lungs to different injuries. Emphysema can be induced in experimental animals by repeated induction of pulmonary neutrophilia. However, the evidence for involvement of neutrophil elastase in human emphysema is not clear: there are studies using a variety of approaches that weigh on both sides of the question. There is also in vitro evidence that alveolar macrophages can degrade elastin or elastic fibers with which they are in contact by means of a metalloelastase or the cooperative action of plasminogen activator and an acid cysteine protease. We conclude that the pathogenesis of emphysema is complex. Neutrophil elastase likely plays a major role in the development of some forms of emphysema, but our understanding of the interactions between the alveolar walls and neutrophils is still fragmentary.

Hypercapnic ventilatory response in unanesthetized normal hamsters.

In order to measure the hypercapnic ventilatory response (HCVR) in experimental animals, it is desirable that the animals be unanesthetized and unrestrained. In the present study, we used a barometric chamber to measure steady state HCVR of hamsters with chronic cannulation of their aortas. In 16 hamsters, the mean (+/- SD) of HCVR, defined as changes in ventilation divided by changes in Paco2 (range 53-73 mmHg), was 10.1 +/- 4.9 ml X min-1 X mmHg-1. The rise in ventilation during CO2 inhalation at low concentrations was due to a rise in mean inspiratory flow and tidal volume. As inhaled Pco2 increased, frequency of breathing increased because expiratory time fell progressively. Inspiratory time rose slightly with small increases in inhaled CO2, but returned to baseline values as the concentration of inhaled Pco2 increased. These changes in ventilation and its components appear to resemble those found in the unanesthetized cat and in man, but are somewhat different from those found in the rat.

Effect of chloromethyl ketone on the progression of elastase-induced emphysema in hamsters.

Intratracheal administration of an elastase inhibitor (Succinyl alanyl alanyl prolyl valine chloromethyl ketone) into hamsters 20 days after induction of emphysema by porcine pancreatic elastase did not affect the lesion seen at 120 days. This study suggests that the progressive increase in lung volume between 20 and 120 days after emphysema induction was not due to a residue of active elastase in the lungs.

Differing susceptibility of young and adult hamster lungs to injury with pancreatic elastase.

A study was undertaken to determine whether there are differences in the initial response of the lungs of young (28 days) and adult (105 days) hamsters to pancreatic elastase treatment. The elastase was given intratracheally (0.1 mg/100 g body weight), and the animals were studied 24 h later. The mean linear intercept of the lungs of young animals increased less than that of adult animals (p less than 0.05). Adult body weights decreased significantly with elastase treatment, whereas those of young elastase-treated animals did not. The lungs of young animals gained proportionately less weight than those of adult animals, suggesting they experienced less hemorrhage and edema. When the volumes, at given transpulmonary pressures, of fluid-filled lungs were expressed as percent predicted, the values for young animals were significantly less than those for adult animals. The most pronounced differences were at low transpulmonary pressures (1 to 3 cm H2O). Volume-pressure hysteresis was not altered by elastase treatment in either the young or adult animals. We conclude that the lungs of adult hamsters are more susceptible to elastase injury than the lungs of young hamsters.

Pitfalls in antiprotease therapy of emphysema.

Many individuals with emphysema are unable to stop smoking despite the best efforts of specialists in smoking cessation. Because emphysema is a slowly progressive disease, it is rational to attempt to develop drugs for it. The hope is that drug therapy will slow the rate of decline of lung function, thereby delaying the onset of disability and prolonging life. The major emphasis in drug development has been on antiproteases having the ability to inhibit neutrophil elastase. There are a number of potential pitfalls in the development of such drugs. Although there is gathering evidence that elastin degradation is a part of the development of human emphysema, it is evident from studies in experimental emphysema that protease-antiprotease imbalance is not the only pathogenetic mechanism that gives rise to emphysema. There is strong evidence that human centrilobular and panacinar emphysema are different in pathogenesis. Indeed, airspace enlargement may be considered one of the stereotyped ways that the lung heals after a variety of injuries. There is accumulating evidence that macrophages as well as neutrophils may participate in elastolysis; antiproteases designed to inhibit neutrophil elastase may not inhibit the metalloproteases produced by macrophages. Some antiproteases may serve to transport elastase into the interstitium of the lung and actually increase the risk of emphysema. A process study of antiprotease therapy, using a measure of alteration of elastase burden of the lungs and urinary elastin peptides and desmosine measurements as markers of elastin degradation is now feasible. An outcome study of antiprotease therapy of emphysema should not be undertaken unless there is evidence from a process study that an antiprotease has biochemical efficacy and no unacceptable side effects.

Pulmonary ventilation and blood gas values in emphysematous hamsters.

Emphysema is known to progress in severity during the year after its induction by pancreatic elastase. A barometric chamber and indwelling aortic cannulas were used to evaluate the effects of worsening emphysema on pulmonary ventilation and arterial blood gases. Unanesthetized, unrestrained hamsters were studied 1, 5, and 13 months after panlobular emphysema was induced by intratracheal injection of porcine pancreatic elastase (0.2 mg in 0.5 ml of 0.15 M NaCl solution/100 g body weight). Lung volumes were subsequently measured in the anesthetized animals and the lungs were examined histologically and stereologically. The pattern of breathing in the 1-month emphysematous hamsters (n = 12) wsa the same as that of untreated control animals (n = 28) but the 5-month (n = 7) and 13-month (n = 6) animals breathed more deeply and slowly; there were no changes in mean inspiratory flow rate of proportion of time per breath occupied by inspiration. The PaO2 for all elastase-treated groups was significantly lower than the control but hypoxemia did not progress significantly with advancing age of the animals. The hematocrit was elevated for the 1-month and 5-month treated animals but not for the 13-month emphysematous animals. The arterial pH and PaCO2 values were not significantly different from control values in any of the three groups of emphysematous animals. We conclude that as hamsters with emphysema age their breathing becomes slower and deeper, that hypoxemia is present from 1 month onwards and does not progress and that hypercapnia is not found at any time.

Induction and exacerbation of emphysema in hamsters with human neutrophil elastase inactivated reversibly by a peptide boronic acid.

In solution, MeO-Suc-Ala-Ala-Pro-D,L-boro-Val pinacol ester (Boroval) is a highly effective but reversible inhibitor of both porcine pancreatic elastase and human neutrophil elastase (HNE) (50% inhibition with a 1.5 M ratio of Boroval to elastase). Boroval has been shown to prevent porcine-pancreatic-elastase-induced emphysema in hamsters. But with HNE-induced emphysema in hamsters, pretreatment with as much as a 170-fold M excess of Boroval, given intratracheally 1 h before 0.3 mg HNE, did not prevent emphysema. Indeed, lung volumes were larger after Boroval pretreatment than after HNE alone. Emphysema was also induced by instilling HNE that had been mixed with and inactivated by a 41-fold M excess of Boroval (a molar ratio of 42). When 0.25 or 0.5 mg of HNE were given mixed with a 41-fold M excess of Boroval, the emphysema was much more severe with the 0.5 mg dose. Two hours after instillation of 0.3 mg HNE inactivated with a 34-fold M excess of Boroval, bronchoalveolar lavage contained elastolytic activity but no evidence of hemorrhage. In contrast, hemorrhage was severe in hamsters that had been instilled with 0.3 mg HNE alone. We conclude that Boroval can enhance HNE-induced emphysema. We postulate that Boroval suppresses HNE-induced hemorrhage and the resultant influx of plasma protease inhibitors; the HNE-Boroval complex is transported into the alveolar interstitium, followed by dissociation of the inhibitor from the active site of HNE. Because of its small size, free Boroval is rapidly cleared, and the reactivated HNE attacks elastic fibers, giving rise to emphysema.

The specific treatment of emphysema.

Cigarette smoking is the major risk factor for development of emphysema. Many people are unable to stop smoking despite skilled support. The elastase-antielastase imbalance hypothesis for the pathogenesis of emphysema suggests that treatment with a supplemental elastase inhibitor might prevent development of emphysema in susceptible people. Many elastase inhibitors have been developed. Poorly soluble inhibitors do not prevent emphysema when tested in an animal model of elastase-induced emphysema. Irreversible inhibitors are effective in a dose-response manner. Reversible but tight-binding large molecular weight inhibitors, which clear slowly from the lungs, are effective in vivo. Small molecular weight, reversible inhibitors prevent haemorrhage after human neutrophil elastase instillation into the lungs but may potentiate emphysema. Only 15% of long-term smokers are susceptible to the development of emphysema. Susceptible smokers can be identified by the development of airflow obstruction. An outcome study of efficacy of elastase inhibitor therapy would be prohibitively expensive. However, a study of the process of development of elastase-induced emphysema is feasible. Measurement of alterations in elastase load of the lungs, elastase derived fibrinopeptides, circulating elastin peptides and urinary desmosines could be used for this purpose.

Maximum expiratory flow and transpulmonary pressure in the hamster.

Maximum expiratory flow was measured in 19 normal, anesthetized, tracheostomized, supine hamsters from records of forced deflation produced by the application of varying degrees of negative pressure to the tracheostomies of animals whose lungs had been previously inflated to a transpulmonary pressure (PL) of 25 cmH2O. Flow was measured with a pneumotachograph, volume with a constant-volume pressure plethysmograph and pleural surface pressure (Ppl) with a water-filled esophageal catheter. The esophageal pressure measurement overestimated Ppl and a simple technique was based on an estimate of the resting volume of the chest wall. This volume, at which the Ppl is zero, was calculated for anesthetized supine hamsters from the measurement of respiratory-system pressure and PL made independently of esophageal pressure and was found to be about 30% of vital capacity (VC). Flow limitation was present below 70% of VC with a tracheal deflation pressure of -30cmH2O. Negative effort dependence of flow was seen in small segments of the flow-volume curves. Mean +/- SD maximum expiratory flow at 50% VC was 52 +/- 9.5 ml/s or 9.1 VC/s. Upstream resistance was 0.09 +/- 0.03 cmH2O/ml per s.

Premorbid ventilatory response to hypercapnia is not related to resting arterial carbon dioxide tension in hamsters with elastase-induced emphysema.

Marked variability in resting steady-state arterial PCO2 (PaCO2) values are observed among patients with chronic obstructive pulmonary disease (COPD), independent of severity of their obstructive airways defect. The reasons for the development of hypercapnia in some but not in the others remain unclear. One hypothesis states that the level of morbid resting PaCO2 may be related to the premorbid hypercapnic ventilatory response (HCVR); accordingly, subjects who were relatively insensitive to CO2 breathing (low responders) develop CO2 retention in the face of lung disease. The present study investigated this hypothesis in the hamster model of elastase-induced emphysema. After obtaining steady-state HCVR in 19 unanesthetized unrestrained hamsters, emphysema was induced by intratracheal instillation of pancreatic elastase. Forty-five days later, minute ventilation and PaCO2 measurements were done, and lung function tests were obtained. The slopes of HCVR and morbid PaCO2 values varied from -0.09 to 2.36 ml/min/mmHg inspired PCO2 and 48.7 to 63.1 mmHg, respectively. There were no significant correlations between morbid PaCO2 values and premorbid HCVR or lung function test abnormalities caused by emphysema. These animal model studies do not support the hypothesis that the level of PaCO2 in patients with COPD is related to their premorbid HCVR.

Effect of diet on urinary excretion of desmosine and hydroxylysyl pyridinoline.

To help validate the use of urinary desmosine (DES), isodesmosine (IDES), and hydroxylysyl pyridinoline (HP) as specific markers of host elastin and collagen degradation, respectively, a study was carried out on the effect of dietary elastin and collagen on urinary DES, IDES, and HP. Ingestion of a meal of calf ligamentum nuchae containing 33 g elastin, 500 mg DES, and 400 mg IDES produced a 10-fold increase in urinary DES and an 8-fold increase in IDES. The urinary DES values remained elevated for more than 10 days following the ingestion. We estimate that about 0.3 mg, or < 0.1%, of the ingested DES was excreted in the urine. Since ligamentum nuchae is not a usual ingredient of human diets, we also determined whether a more typical source and amount of DES, IDES, and HP might affect urinary DES, IDES, or HP values. Lean ground beef (454 g) was ingested. Our analysis showed that this meal contained 4 mg DES, 2 mg IDES, and 0.9 mg HP. The meat-rich diet caused a significant increase of 16 and 34% in the creatinine and DES content of the urine, respectively. When DES, IDES, and HP values were normalized for the urine creatinine content, diet had no effect on the measured amounts. The baseline values (mean +/- SE) for the volunteers before ingestion of the beef were 8.3 +/- 0.7 micrograms DES/24 h, 8.3 +/- 0.6 micrograms IDES/24 h, and 340 +/- 48 nmol HP/24 h; 5.7 +/- 0.5 micrograms DES/g creatinine, 5.6 +/- 0.4 micrograms IDES/g creatinine, and 26.9 +/- 2.2 nmol HP/mmol creatinine.(ABSTRACT TRUNCATED AT 250 WORDS)

The ultrastructure of hamster bronchial epithelium.

The central intrapulmonary bronchi of adult male Syrian hamsters were examined by electron microscopy to identify the principal types and proportions of epithelial cells. A differential count of cells displaying both a basal lamina and luminal border (transepithelial cells) showed that, on average, ciliated cells constituted 63% and granule-containing (granulated) secretory cells 25% of the total. Other transepithelial cells included nongranulated secretory cells (9%), preciliated cells (1.5%), and indeterminate cells (1%). The most frequent granulated secretory cell (77% of the population) was the Clara cell. It was identified by the presence of prominent apical smooth endoplasmic reticulum and secretory granules. It was subclassified into three types based on the presence or absence of rough endoplasmic reticulum and on granule morphology. Mucous cells (little or no smooth endoplasmic reticulum but with typical mucous granules) constituted approximately 20% of the granulated secretory cells. Serous cells were very infrequent. A differential count of nucleated epithelial cells demonstrated an average of 2% basal cells (hemidesmosomes present) and 20% pseudobasal cells (hemidesmosomes absent). Neuroepithelial bodies and solitary "small-granule" cells were infrequent. Brush cells and apoptotic bodies were rarely found but are noteworthy because their occurrence in hamster airways was not demonstrated previously. These results provide a foundation for subsequent analysis of alterations of epithelial homeostasis induced by injurious agents of exogenous and endogenous origin.

An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.

An ultrastructural morphometric analysis of bronchial secretory cells was carried out on hamsters treated intratracheally with 300 micrograms of human neutrophil elastase (HNE) in 0.5 ml saline, saline alone, or left untreated. Five to 6 animals were killed at 2 h and at 3, 8, and 16 days after treatment. Electron micrographs were prepared from the hilar region of the left main intrapulmonary airway; 125 +/- 16 (mean +/- SE) granulated secretory cells extending from basement membrane to lumen were analyzed for each group. The number (Ng) and area (Ag) of granular profiles per cell, the area of cell profiles (Ac), and the volume density of secretory granules per cell (Vv) were determined using an electronic image analyzer. There were significant decreases in Ng, mean Ag, and Vv in the 2-h HNE group when compared with the saline group. Values of Ng, mean Ag, and Vv were similar for HNE and saline groups at 3 days, but were significantly increased at 8 and 16 days. The Ac of HNE-treated groups was similar to their saline control groups at all time points except at 16 days when the HNE-treated group enlarged to double that of its saline control group. An ultrastructural differential cell count showed a decrease in frequency of granulated secretory cells at 2 h and an increase at 8 and 16 days; there was an inverse change in the frequency of nongranulated secretory cells at these times. The proportion of ciliated, preciliated, and indeterminate cells remained constant over time in all treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of stretch and contractile activity in the prevention of connective tissue accumulation in muscle.

The loss of serial sarcomeres which results when muscles are immobilised in a shortened position is accompanied by an increase in the proportion of collagen and an increased muscle stiffness. In order to determine whether it is lack of stretch or lack of contractile activity which is the main factor involved in these changes experiments were carried out using different combinations of immobilisation and electrical stimulation. It was found that the connective tissue accumulation that occurs in inactive muscles can be prevented either by passive stretch or by active stimulation. It was also shown that in muscle that is working over a reduced range there is, as in muscle immobilised in the shortened position, a reduction in serial sarcomeres. In this case, however, there is no concomitant increase in connective tissue, again indicating that contractile activity is important for the maintenance of normal muscle compliance.

An 18-month study of the effects on hamster lungs of intratracheally administered human neutrophil elastase.

A study was made of the evolution of emphysema and airway injury induced in the lungs of male golden Syrian hamsters by a single intratracheal injection of 350 micrograms human neutrophil elastase (HNE). Saline control and HNE-treated groups of 8 animals were studied 1, 3, 6, 12, and 18 months posttreatment. HNE treatment caused a significant increase in all lung volumes and a significant decrease in maximum expiratory flows at all study times. The mean linear intercept (MLI) values of the left lung were significantly increased over control values. There was no progression with time in MLI values, lung volumes, or lung compliance. Secretory-cell metaplasia was present at 1 month and persisted throughout the study. The HNE-treated lungs showed clusters of ferric iron-containing macrophages in the terminal airspaces. The amount of iron in the lungs, determined morphometrically, was greatest at 1 month, was decreased by 6 months, and then did not change further to 18 months. At 18 months the amount of iron was still significantly above control amounts. We conclude that the airway and parenchymal lesions induced by HNE persist without progression for 18 months. Clearance of ferric iron, which was probably a result of the hemorrhage induced by HNE treatment, continued for 6 months with no evident subsequent clearance.

Effect of the elastase inhibitor, eglin-c, on antibacterial mechanisms in experimental pneumonia. Description of a system to quantitate phagocytic and bactericidal activity of resident murine alveolar macrophages in vivo.

To evaluate the influence of the elastase inhibitor, eglin-c, on lung host defense, normal CD-1 mice were challenged intratracheally with type 3 Streptococcus pneumoniae suspended in phosphate-buffered saline alone or containing 10 mg/ml eglin-c. After infection with 5 X 10(3) colony-forming units (cfu), animals given eglin-c demonstrated a significant enhancement in their capacity to clear viable pneumococci from the lungs at 24 h after challenge; the augmented pulmonary clearance was associated with an increased influx of granulocytes at 6 and 24 h. After challenge with higher inocula (5 X 10(4) and 5 X 10(5) cfu), animals treated with eglin-c exhibited a significant impairment in pulmonary clearance at 6 h; however, in the presence of larger numbers of granulocytes within the bronchoalveolar spaces, the attenuation in pulmonary clearance resolved between 6 and 24 h. Changes in the kinetics of pulmonary clearance that were similar to those noted after infection with high pneumococcal inocula were also observed after challenge with 1 X 10(6) cfu Staphylococcus aureus. In addition, although it did not influence the in vivo phagocytic capacity of resident alveolar against S. aureus, eglin-c depressed the bactericidal activity of these cells. We conclude that in the mouse, high doses of eglin-c alter pulmonary antimicrobial mechanisms important for preventing and eradicating bacterial infection of the lower respiratory tract.