Specificity and sensitivity of the assay for elastin-derived peptides in chronic obstructive pulmonary disease.

One prediction of the protease-antiprotease hypothesis of chronic obstructive pulmonary disease (COPD) pathogenesis is the appearance of elastin-derived degradation products in the plasmas of affected patients that are due to the breakdown of alveolar interstitial elastin by an excess of elastolytic activity in the lung. We previously demonstrated a significant elevation of plasma elastin-derived peptides (EDP) in subjects with COPD in comparison with asymptomatic smokers with normal spirometry or normal nonsmokers. To better determine the selectivity of the assay for EDP as a marker of COPD, we measured plasma EDP levels in different patient populations. These included subjects with COPD, subjects with diseases that may involve accelerated elastolysis (pneumonia, atherosclerotic vascular disease, and inflammatory arthritis), subjects with diseases hypothesized to involve pulmonary inflammation without elastolysis (asthma and acute tracheobronchitis), asymptomatic smokers with normal spirometry, and healthy, nonsmoking subjects. Mean plasma EDP levels in subjects with COPD were elevated above those in all other subjects (p less than 0.01). The prospective analyses of specificity and sensitivity of plasma EDP levels as markers of COPD gave values of 91 and 65%, respectively. Utilizing receiver operating characteristic curve analysis to assess the diagnostic and screening performance of plasma EDP as a test for COPD (perfect test equals an area under the curve of 1.0), the area under the curve was 0.87, which compares favorably with many widely used clinical tests. These data demonstrate that the assay for plasma EDP is a quantitative, easily measured, and highly specific marker for subjects with COPD.

Size distribution of human lung elastin-derived peptide antigens generated in vitro and in vivo.

The protease-antiprotease hypothesis of emphysema development suggests that degradation of elastin in the lung interstitium may give rise to abnormal quantities of circulating elastin-derived peptides (EDP) during periods of inflammation. Recent studies have shown a relationship between emphysema and high levels of EDP in human plasma. This report characterizes elastin digests on the basis of antigenicity, size, and method of preparation, as well as the size distribution of EDP found in the plasmas of nonsmokers, smokers, and emphysema patients. Gel filtration of elastin digests prepared by hydrolysis of human lung elastin using a low (1:500) ratio of neutrophil elastase to elastin generated a broad protein peak of approximately 70,000 daltons. In contrast, a high (1:25) ratio of neutrophil elastase to human lung elastin gave a broad protein peak, with a size distribution in the 10,000 to 30,000 dalton range. This digest showed distinct immunochemical properties. A polyclonal antibody directed against the low-ratio digest showed a minimum detection of 2 ng/ml for the homologous antigen but required 1,000 ng/ml of the high-ratio digest for detectable inhibition in an indirect ELISA assay. Gel filtration of plasmas from normal nonsmokers and the majority of normal smokers revealed a single immunoreactive EDP fraction of approximately 70,000 daltons. Plasmas from selected normal smokers and emphysema patients with high levels of circulating EDP (greater than 90 ng/ml) fractionated into a complex pattern of peptides in which the 70,000 dalton component represented 50% of the immunoreactive material and several lower molecular weight peptides represented the remaining circulating elastin antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute cigarette smoke exposure in dogs: the inflammatory response.

Acute cigarette smoke causes polymorphonuclear leukocyte (neutrophil, PMN) recruitment to the lung followed by loss of elastase from the recruited cells. Dogs were exposed to cigarette smoke with different oxidant content, bronchoalveolar lavage (BAL) was performed, and the cell distribution in the recovered alveolar lining fluid was analyzed. Exposures were 1, 3, or 6 cigarettes on one or multiple days with a maximum dose of 42 cigarettes. The mean percent PMN present in control lavage was 2.01%, while the mean percent PMN recovered in BAL after a dose of 42 1R1 cigarettes was 13.05%. Recoverable PMN, after a single exposure to three 1R1 cigarettes, also increased from 1.7 to 10.4% by 15 h after cessation of smoke exposure. The cell response for multiple (2 and 7) day exposures was similar. The elastase content per BAL neutrophil decreased relative to peripheral blood PMN from the same animals. No free elastolytic activity was found in BAL, but PMN elastase antigen was present. Increased frequency of cigarette smoke exposure delayed the return to homeostatic cell conditions. The increased PMN accumulation observed may result in an increased proteolytic load in the pulmonary interstitium and contribute to the pathogenesis of emphysema.

Immunolocalization of elastase in human emphysematous lungs.

The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema.

Utilization of a peroxidase antiperoxidase complex in an enzyme-linked immunosorbent assay of elastin-derived peptides in human plasma.

Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and significant impairment of lung function usually occurs before the disease is diagnosed. Because lung elastin degradation appears to be a prerequisite for the development of the disease, immunologic detection of elastin-derived peptides in the blood might be an effective approach to the early detection and monitoring of the disease. We here report an improved enzyme-linked immunosorbent assay for elastin peptides using a peroxidase-antiperoxidase complex as the reporter group. The assay is sensitive to 2 ng/ml elastin peptides. We show that for optimal, reproducible results the assay should be carried out at 16 degrees C rather than at room temperature and that determinations should be made on plasma containing protease inhibitors rather than on serum. The levels of elastin-derived peptides appeared to remain relatively constant when multiple samples were taken during a 5- to 10-wk period from individual subjects. In addition, patients with COPD had elevated elastin peptide levels (127 +/- 47 ng/ml) compared with levels in normal nonsmokers (58 +/- 17 ng/ml), whereas normal smokers had values intermediate between the 2 groups (mean peptide levels of 76 +/- 42 ng/ml). A small group of normal smokers (20%) had elevated elastin peptide levels similar to those in the emphysema group and may represent that group of smokers who are at risk of developing obstructive lung disease.

Immunologic measurement of elastin-derived peptides in human serum.

Chronic obstructive pulmonary disease (COPD), a major cause of morbidity and death in the smoking population, develops insidiously over many years, and usually significant impairment of lung function has occurred before the disease is diagnosed. It is likely that destruction of the elastic fiber is a prerequisite for the development of the disease, and it is possible that immunologic identification in the serum of peptides derived from lung elastin degradation might be an effective approach to the early detection and monitoring of the disease. We prepared antibodies to peptides derived from human lung parenchymal elastin and used these antibodies in an enzyme-linked immunosorbant assay to quantitate elastin-derived peptides in the serum of 39 normal control nonsmokers, 33 smokers with normal lung function, and 40 patients with COPD. On average, statistically significant higher levels of elastin-derived peptides were found in the normal smokers and COPD patients compared to the controls. Further work with larger numbers of subjects is necessary to determine whether such a test is effective in identifying those individuals who are at risk of developing COPD.

A model of decreased functional alpha-1-proteinase inhibitor. Pulmonary pathology of dogs exposed to chloramine T.

The objective of this study was to develop an animal model representative of chronic human alpha-1-proteinase inhibitor deficiency. Eight dogs were treated with a mild oxidizing agent, chloramine T, with varying regimens for 3--27 wk. The capacity of the serum to inhibit both trypsin and elastase was examined and found to respond differently. Although immunologically determined levels of protease inhibitor did not change, the ability of serum to inhibit elastase in an in vitro assay decreased in direct response to chloramine T treatment. The trypsin inhibitory capacity was less affected. Emphysemalike alterations in lung morphology were observable when histologic sections were evaluated both subjectively and objectively by mean linear intercept measurements. The data suggest that this model parallels the emphysema associated with the genetic alpha-1-proteinase inhibitor deficiency in man.

Emphysema induced in vitro and in vivo in dogs by a purified elastase from homologous leukocytes.

The protease hypothesis of emphysema development evolved from systems using intratracheal instillation or aerosols of heterologous enzymes, such as papain or porcine pancreatic elastase, which bear no relation to the animal species treated. Although these enzymes did produce experimental emphysema, their exogenous origin and superphysiological dosages limit their use in definitive model systems. The observation that dog leukocyte homogenates could induce canine emphysema led us to purify the causative agent from canine neutrophils. This report establishes that a single elastolytic enzyme from dog neutrophils is responsible for inducing experimental emphysema in the dog. Two purification methods were employed. The first used solvents of increasing ionic strength in a sequential extraction of acetone powders of purified dog neutrophils. The ability to initiate emphysema-like lesions was tested in every fraction of the purification and was localized in the extract with the highest true elastolytic activity. The second purification involved neutrophil intracytoplasmic organelle fractionation and established that only extracts of the lysosomal granules were capable of emphysema induction. Finally, the enzyme was purified to homogeneity from the granules using affinity chromatography and was shown to be a true elastase. Emphysema development was quantitated using mean linear intercept and was shown to be dependent on elastase concentration. There does not appear to be any other single enzyme in the canine neutrophil capable of inducing experimental emphysema.

Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome.

To test the hypothesis that adult respiratory-distress syndrome (ARDS) is related to increased activity of the proteolytic enzyme elastase released from neutrophils in the lung, we determined the differential white-cell count, the elastolytic activity, the source of elastase, and the concentration and activity of the endogenous protease inhibitor alpha-1-antiprotease (alpha-1-AP) in bronchoalveolar lavage fluid from 23 patients with ARDS and from 55 patients without this syndrome. Neutrophil predominance (> 80 per cent) was observed in 18 of 23 patients with ARDS. High elastolytic activity of neutrophil origin was found in 12 of 23 patients with ARDS (52 per cent), in none of 16 normal nonsmokers (P < 0.01), in two of 17 normal smokers, and in three of 22 patients with chronic obstructive pulmonary disease. Although there were no significant differences in alpha-1-AP concentrations, its activity was reduced in eight of nine patients with ARDS and high elastolytic activity. We conclude that in many patients with ARDS, high levels of neutrophil elastolytic activity in the lungs are associated with reduced alpha-1-AP function.

The effect of the oxidizing agents chloramine-T and cigarette smoke on dog serum proteinase inhibitor(s).

Dog serum treated with the oxidant chloramine-T is rapidly and selectively depleted of its ability to inhibit porcine pancreatic elastase or dog neutrophil elastase. Trypsin inhibitory capacity of serum is not affected. Purified dog alpha-1-proteinase inhibitor (alpha-1-PI) is similarly oxidized with an apparent rate constant of 1.1 x 10(3) M-1 sec-1. Reversal of the oxidative inactivation using dithiothreitol was demonstrated. Cigarette smoke also directly affects the inhibitory capacity of both serum and pure alpha-1-PI. These studies form a basis for developing a model of functionally deficient alpha-1-PI by taking advantage of oxidative inactivation of normal proteinase inhibitor levels.

Chronic obstructive pulmonary disease: rehabilitation program with continuation on an outpatient basis.

A total of 187 patients with chronic obstructive pulmonary disease (COPD) were treated in a rehabilitation program, initially as inpatients and then scheduled to continue 6 months or more in an outpatient clinic. The mean age was 61 (range, 28-76 years). Changes shown by participants in the outpatient program were compared to those shown by the nonparticipants. For both groups, the mortality rates were similar to published figures, and were strongly related to the levels of forced expiratory volume/sec (FEV 1.0). The FEV 1.0 declined significantly within one year in both groups. Psychologic test scores were unchanged. There was a sharp increase in unemployment. Although rehabilitative therapy must be continued, high priority should be given to early detection of COPD in patients who have airway obstruction but are otherwise asymptomatic. Possibly, at that stage, the elimination of cigarette smoking may slow the process.

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.