Chronic Obstructive Pulmonary Disease (COPD)

ROBERT M. SENIOR and NICHOLAS R. ANTHONISEN

Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; and Faculty of Medicine, The University of Manitoba, Winnepeg, Manitoba, Canada

	INTRODUCTION

TOP INTRODUCTION REFERENCES

Recognition of chronic obstructive pulmonary disease (COPD) as a major health problem is approximately 50 years old, coincident with the 50th anniversary of the National Heart, Lung, and Blood Institute (NHLBI). Research in COPD was greatly stimulated by formation of the Division of Lung Diseases (DLD) nearly 30 years ago. In this presentation we cover some clinical aspects of COPD and aspects of the pathogenesis of emphysema, with emphasis on findings attributed to support from the DLD.

	CLINICAL ASPECTS OF COPD

COPD is generally defined as slowly progressive airflow obstruction, which is only partially reversible (1). It typically occurs in individuals with substantial smoking histories (at least 20 pack-yr). It is associated with three general types of lesions: emphysema, small airways inflammation and fibrosis, and mucus gland hyperplasia, most obvious in larger airways. All of the lesions are uncommon in nonsmokers, and all may be present in patients with COPD, but this is not always the case. Smokers without dyspnea frequently have one or more of these lesions.

At present, there are major difficulties with the quantification of emphysema and small airways disease during life, so clinical investigators study COPD by measuring the degree of lung function abnormality, notably the impairment in FEV₁. This is justified on the basis that both emphysema and small airways obstruction reduce maximum expiratory flow, so that the FEV₁ represents some kind of sum of the two influences. Further, the work of the Burrows group (2) showed that in patients with COPD, the FEV₁ is, besides age, the single best predictor of mortality. This finding has been independently verified by numerous other groups. The tendency to regard COPD as best assessed by measurement of FEV₁ was powerfully supported by the work of Fletcher and colleagues (7), who studied a group of working men in London over 8 years. Fletcher and colleagues found that the average rate of decline of FEV₁ was 0.03 L/yr in nonsmokers, and that decline was twice as fast in smokers. However, given a rate of decline of FEV₁ of 0.06 L/yr, it was unlikely that the average smoker would live long enough to develop symptomatic airways obstruction as signified by an FEV₁ < 1.5 L. It followed that people who developed COPD were a subset of smokers whose decline in FEV₁ was considerably larger than the average.

The Fletcher study produced other findings of great interest. Smokers who spontaneously quit the habit had a normal rate of fall of FEV₁ thereafter, although their FEV₁ values did not increase to levels that would have existed had they never smoked. These observations have been supported and amplified recently by the DLD-sponsored Lung Health Study (8), in which smokers were randomly assigned to control or smoking cessation groups. Smoking cessation had a beneficial effect (Figure 1) that was, if anything, larger than that described by Fletcher and colleagues (7).

View larger version (21K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 1.   Rates of decline of lung function in participants in the Lung Health Study (see Reference 8). The ordinates are postbronchodilator FEV1; the abscissas are years. The top panel shows decline in the three treatment groups, with the dashed line with squares representing the usual care group, and the other symbols, the groups that received an anti-smoking interventions. The bottom panel shows results from participants in one of the treatment groups and compares individuals who stopped smoking at the onset of the study with those who continued to smoke.

Fletcher and colleagues also found that chronic cough and sputum (chronic bronchitis, chronic mucus hypersecretion) predicted the number of acute exacerbations of cough and sputum thought to represent airways infection, but did not predict rate of decline of FEV₁. Moreover, there was no discernible effect of exacerbations on long-term fall in FEV₁. These findings essentially refuted the "British hypothesis" concerning the pathogenesis of COPD, which was that COPD resulted from repetitive airways infections. This conclusion, in turn, tended to incriminate tobacco smoke as the direct cause of the lung damage of COPD.

Fletcher did not try to test the alternative "Dutch hypothesis" of the pathogenesis of COPD (9). This hypothesis was based on the observation that asthma and COPD had many common features, including airways hyperreactivity and other evidence of allergy. To oversimplify, patients with COPD were potential asthmatics who smoked. This approach involved lumping people with asthma and COPD and did not become popular in the United Kingdom or North America, where most investigators believed that asthma and COPD were different diseases that could be readily distinguished in the vast majority of cases. It is only within the last 5-10 years that some rapprochement between these views has become evident.

Risk Factors

Almost by definition, tobacco use is by far the most important risk factor for COPD, best summarized as cumulative dose or pack-years. However, as noted above, not all heavy smokers develop COPD; in fact, most do not, and there has been considerable interest in other risks. COPD is familial to a greater extent than can be accounted for by the relatively few cases of ₁-antitrypsin (₁-AT) deficiency (10). It is not known whether this familial tendency reflects genetic or environmental influences, or both. Dusty occupational environments are well established risks (11), though probably not major factors in North America. Childhood respiratory illnesses may render some people susceptible to tobacco-induced lung damage (12). All of these influences are minor compared to that of smoking, and none satisfactorily explains the differences between smokers who develop COPD and those who do not.

There was hope that susceptibility to tobacco smoke could be identified early, before permanent or major damage to the lungs occurs. This was based on the finding that young smokers had inflammatory and fibrotic lesions of the small airways (13) and the probability that the conventional lung function tests using forced expiration were relatively little influenced by such lesions. A variety of tests for small airways diseases, most notably closing volume, were developed and studied in detail with DLD support (14). However, the tests were not nearly as reproducible as the FEV₁ and were probably too sensitive, since abnormalities were often detected in the majority of otherwise healthy tobacco users. These efforts re- emphasized the value of careful repetitive spirometry in the assessment of COPD.

As noted previously, for more than 30 years Dutch clinical investigators had argued that asthma and COPD were different points in a spectrum of obstructive disease with some risk factors in common, most notably allergy and airways reactivity. Atopy (15) and eosinophilia (16) have been identified as relatively minor risk factors for COPD, but the influence of airways reactivity on the course of COPD, as differentiated from asthma, was unresolved until recently because there was ample evidence that the degree of airways reactivity was directly related to the degree of airways obstruction of whatever cause. This meant that individuals with the same level of obstruction, but differing values of airways reactivity, had to be followed for long enough to discern differences in disease progress after allowing for variation of potential confounders such as smoking.

The Lung Health Study (8) successfully accomplished this, measuring methacholine reactivity in a large number of smokers with subclinical airways obstruction at the beginning of a careful 5-yr follow-up. The initial level of airways (methacholine) reactivity was, after smoking, the most important single determinant of decline in FEV₁, and this effect was not explained by variation in the initial level of obstruction (17). Thus, it is reasonable to conclude that airways reactivity is an important risk factor for COPD. On the other hand, it is not clear what this represents in terms of biology. In particular, it is not known whether the reactivity observed in the smokers of the Lung Health Study has mechanisms similar to those in patients with asthma.

The Lung Health Study also found that airways reactivity was greater in women than men smokers, and FEV₁ may decline more rapidly in women when allowances are made for lung size and degree of smoking (18). Thus, the female gender may be a risk for COPD, an influence previously obscured by the preponderance of tobacco use by men.

Therapy

General. The DLD has been very active in supporting studies of the treatment of COPD. Indeed, most of the long-term therapy trials have been done with DLD sponsorship, and DLD-sponsored trials have established the gold standard for this kind of research.

Generally speaking, COPD therapy has two aims: ameliorating the course of the disease and/or improving the quality of life. Neither of these aims or end points is easy to assess, and in both cases statistically significant differences may be of little clinical significance, or achieved at inordinate cost. At present, changing the course of COPD implies changing the rate of decline of FEV₁ or prolonging life. Study of the first requires large patient samples with at least 3-5 yr follow-up, and study of mortality requires either a much longer follow-up or selection of end-stage patients. Both approaches are laborious and expensive. We need alterative end points that are more easily evaluable, but comparably robust, and justifiable in terms of cost.

Measures of quality of life include assessment of symptoms, exercise performance, and health care utilization. None is easy to measure in reproducible fashion, and all have subjective aspects that make things like standardization between different centers difficult. Further, quality of life measured in the short term may or may not apply in the longer term, and long-term studies are expensive and difficult. Some COPD therapies have been justified on the basis of short-term changes in lung function. Improvements in FEV₁ have been related to improvements in quality of life in the short term, so that FEV₁ can function as a surrogate for quality of life. On the other hand, the use of short-term studies as the rationale for long-term therapy carries a number of assumptions that are seldom justified. As indicated above, smoking cessation is the best way to change the course of the disease (7, 8). Nicotine substitution improves cessation success rates, but as illustrated by the Lung Health Study, most "good" cessation programs are expensive and produce long-term quit rates on the order of 25% (8).

Bronchodilators. As is perhaps best illustrated by data from the DLD-sponsored IPPB trial (19, 20), most patients with COPD have a measurable increase in FEV₁ with the inhalation of beta-agonists, and in some the change is substantial (Figure 2). Responses to anticholinergic agents are at least comparable, and these agents have been shown to improve quality of life over the short term. The method of delivery of inhaled bronchodilators has not been shown to influence their effect in a clinically significant way. Though there were suggestions that regular, inhaled bronchodilator therapy might alter the long-term course of COPD, this issue was studied in the Lung Health Study and no long-term effect was found (8).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Frequency distribution of bronchodilator response in patients with COPD enrolled in the Intermittent Positive Pressure Breathing Trial (see References 19 and 20). The ordinate is percent of patients, and the abscissa is postbronchodilator FEV1 as a percentage of the prebronchodilator value. There were 985 patients included, with a mean prebronchodilator FEV1 of 36.1% of predicted normal.

Systemically administered bronchodilators, particularly aminophylline, have been extensively studied, with differing results. In general, they add relatively little to inhaled bronchodilator therapy in the short term. On average, there is a 10% improvement in FEV₁, with some reduction by dyspnea. The size of the effect varies from patient to patient and includes some who benefit more than others. Though systemic administration is likely to affect airways not reached by inhaled agents, this is apparently of little clinical significance. The use of intravenous aminophylline in COPD exacerbations is probably not justifiable (21). Use of aminophylline for purposes other than bronchodilation in COPD has not been studied in large numbers of patients in the long term.

Corticosteroids. Numerous studies show that some patients with stable COPD have improvements in lung function when given anti-inflammatory corticosteroids. Responses are substantial in a minority of patients and are most common when steroids are given systemically in large doses (22). The long-term therapeutic implications of these findings have not been explored adequately. It is not clear how reproducible steroid responses are in a given patient with COPD nor whether steroids change the course of COPD in steroid responders or unselected patients. The advent of high-dose inhaled steroids has made steroid therapy safe and practical, and at present there are at least three major clinical trials of these agents in COPD, one of them sponsored by DLD. The results of these trials will be of great practical and theoretical interest.

Steroid responsiveness in COPD raises the issue of overlap between COPD and asthma. Some investigators have found that patients with COPD who respond to steroids have other features reminiscent of asthma, while other investigators have not. It has been argued that people who respond to steroids should be designated asthmatic, and the diagnosis of COPD reserved for those who do not. This argument assumes that steroid responsiveness is an immutable patient characteristic, which has not been demonstrated. Indeed, there is inferential evidence that this is not the case. Albert and coworkers (23) showed that systemic steroids improved lung function in unselected patients with COPD in acute exacerbations; others have confirmed these results (24). These findings suggest that all or most COPD patients are "steroid responders" during acute exacerbations, which is not the case in stable COPD. It is possible, therefore, that patients who do not respond to steroids when stable do so when in exacerbation. This hypothesis warrants further investigation.

Antibiotics for exacerbations. Acute exacerbations of symptoms of COPD are often accompanied by increased sputum volume and purulence that suggest infection of the airways. Treatment of exacerbations with broad spectrum antibiotics is common, and the balance of the evidence indicates that such treatment improves the quality of life by speeding symptomatic recovery (25). However, the effect is by no means dramatic and it is difficult to use these data to argue a purely bacterial origin of exacerbations. Most of the acceptable studies of this issue were completed more than 10 years ago and used relatively unsophisticated agents. It is not known whether the organisms involved in exacerbations have changed or whether newer antibiotics offer advantages.

It is worth noting that neither the causes nor the consequences of COPD exacerbations are known. The effects of antibiotics and of immunostimulatory agents (26) suggest that exacerbations are in part infectious, a hypothesis supported by the benefits of flu vaccine. However, steroid responses in exacerbations may imply other mechanisms. As to consequences, the studies of Fletcher and colleagues (7), mentioned previously, showed that exacerbations did not alter the long-term course of COPD in a relatively normal population. They did not study individuals with severe airways obstruction, among whom it is axiomatic that some will develop respiratory failure and die during exacerbations.

Oxygen. The first DLD-sponsored multicenter clinical trial in COPD concerned home oxygen therapy (27), comparing nocturnal treatment (about 12 h/d) with continuous treatment (about 19 h/d). This trial, known as NOTT (Nocturnal Oxygen Therapy Trial), concluded at about the same time as a British Medical Research Council (MRC)-sponsored trial (28) that compared 15 h/d of oxygen therapy with none at all. Entry criteria for the two trials were similar, involving COPD patients with chronic, stable hypoxemia, and the results were strikingly congruent. Oxygen therapy prolonged life, and the more continuous the therapy the larger the effect (Figure 3).

View larger version (20K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 3.   Results of North American and British trials of home oxygen therapy in hypoxemic chronic obstructive pulmonary disease. In both cases, survival is plotted against time of follow-up. The British trial (top panel ) (see Reference 28) compared no oxygen with 15 h of oxygen per day. The North American trial (bottom panel ) (see Reference 27) compared 12 h of nocturnal oxygen (squares) with an average of 19 h per day (circles).

These trials had a major impact upon the treatment of patients with COPD. They established oxygen therapy for advanced COPD as state of the art, and the DLD entry criteria were widely adopted as requirements by third-party payers for oxygen therapy.

The success of NOTT gave both the pulmonary community and DLD confidence to initiate other multicenter trials in COPD. Finally, it is remarkable how well the results of the MRC and National Institutes of Health (NIH) trials have stood up. Indeed, although they were accomplished nearly 20 years ago, little of importance in regard to oxygen therapy has been learned since. The role of oxygen therapy has not been well worked out in episodic hypoxemia, such as that occurring during sleep and exercise. A recent Polish trial re-examined oxygen therapy in COPD patients with less severe hypoxemia than those of the original trials, and found no survival benefit (29).

Nonpharmacologic therapy. Pulmonary rehabilitation for patients with COPD has a long and controversial history. Broadly speaking, the term refers to patient education and exercise training, and its supporters believe that it improves exercise tolerance and quality of life (30). There is little doubt that these benefits can occur, and that they can outlast the program (31). There are problems, however, in assessing the cost-effectiveness of such programs, since the benefits are modest on average and the therapeutic effort may be large (32). Further, it is not entirely clear which component(s) of a particular program are responsible for the improvement, although most believe it is the exercise training. If an inexpensive variety of pulmonary rehabilitation could be shown to be effective, it would be widely adopted.

The ground-breaking work of Macklem drew attention to the fact that COPD compromises the function of the muscles of inspiration and that the state of these muscles may determine quality of life and survival. Two therapeutic avenues were suggested: training the inspiratory muscles so that they performed better, and resting them on the assumption that they were fatigued. Many investigations of inspiratory muscle training using a wide variety of techniques showed that task-specific improvements in inspiratory muscle function were attainable, but that these did not translate well into improvements in the quality of life (33), rather as if the disease itself trained the muscles for breathing. Resting the inspiratory muscles was the subject of a DLD-sponsored clinical trial. Stable patients with COPD were given daily periods of negative pressure (tank) ventilation. Results showed that it is extremely difficult to accomplish this in the home and that it had no discernible benefit (34). Thus, it appeared that inspiratory muscle fatigue was not an important feature of stable COPD, though in other situations such fatigue might be crucial.

Two surgical procedures have been recommended for COPD: lung transplantion and volume reduction. The former is impractical in the vast majority of patients with COPD, who are elderly and infirm. Volume reduction surgery offers promise in that it appears capable of effecting improvements in ventilatory function that are not achieved by medical management (35). Patient selection and surgical technique have not been standardized, however, and results are not predictable. Much important data will doubtless emerge from the current multicenter study, National Emphysema Treatment Trial (NETT), sponsored jointly by NHLBI and Health Care Finance Administration.

	PATHOGENESIS OF EMPHYSEMA

Definition of Emphysema

A workshop of the DLD provided the generally accepted definition of emphysema as "a condition of the lung characterized by abnormal, permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of their walls, and without obvious fibrosis" (36). This definition is useful, but several aspects merit comment. First, emphysema appears to begin as an increased number and size of holes in alveolar walls so that destruction of entire alveolar septa must be a process that occurs in stages (37). Second, disruption of alveolar attachments to small airways is an important additional site of tissue destruction occurring during the loss of alveolar septal tissue, and likely important in the mechanism of reduced maximal airflow associated with emphysema (38). Third, the relationship between enlarged airspaces and lung function is not clear-cut. Increased lung compliance and decreased diffusing capacity correlate more closely with microscopic abnormalities of alveolar walls than with the presence of enlarged airspaces (39). Fourth, increased collagen in both human emphysema (40, 41) and smoke-induced experimental emphysema (42) suggests that the evolution of emphysema involves both destruction and synthesis of extracellular matrix.

Historic Note

Emphysema has been known for two centuries at least, but plausible ideas about its pathogenesis did not appear until the early 1960s, when researchers in Sweden and the United States made discoveries that have become the cornerstone of current thinking. One was discovering ₁-AT deficiency and its association with emphysema. The other was finding that lesions resembling human emphysema could be induced with proteolytic enzymes in experimental animals.

₁-Antitrypsin deficiency. While surveying serum protein electrophoresis patterns of about 1,500 clinical specimens in Malmo, Laurell and Ericksson (43) noticed five without the usual distinctive band in the ₁ zone. Because ₁-AT accounts for the sharply staining band in the ₁ zone, although it is not the only protein there, they reasoned and demonstrated that these five samples were deficient in ₁-AT. Three of the subjects had emphysema, leading them to comment, "The clinical material is too small to warrant any definite conclusions concerning possible connections between the ₁-AT deficiency and the patient's clinical pictures. It is, however, striking that three of the patients had widespread pulmonary lesions and that the sister of one had the same lung disease and obviously the same plasma protein deficiency."

Shortly after the initial report, Eriksson (44) demonstrated three groups of values of ₁-AT: values corresponding to normal; values about 60% of normal; and values less than 10% of normal in a single family. These findings pointed definitively to genetic inheritance of the deficiency and to heterozygous and homozygous states. In two of the individuals with marked deficiency, aged 38 and 48, there was COPD with hyperinflation. In a large series of deficient subjects and their families, reported in 1965, Eriksson (45) confirmed the trimodal distribution of ₁-AT and conclusively linked the deficiency with early-onset COPD.

The first five deficient subjects revealed the pulmonary spectrum of ₁-AT deficiency. Symptomatic emphysema was present in three, who were 35, 38, and 44 yr of age. Early- onset emphysema has become one of the leading clues to the presence of the deficiency. On the other hand, two subjects did not have clinical lung disease, including a woman in her seventies. Similar variability in the occurrence of COPD has been observed ever since (46). Marked ₁-AT deficiency is not necessarily associated with emphysema and a shortened life span. Because smoking is now known to accelerate COPD in ₁-AT deficiency, it seems likely that the first elderly, asymptomatic individual never smoked. The Registry for Patients with Severe Deficiency of Alpha-1-Antitrypsin, sponsored by the NHLBI, has completed its data collection and will be reporting on the clinical and laboratory course of this group of 1,129 individuals, the largest cohort with the deficiency (47).

Besides discovering ₁-AT deficiency and recognizing the clinical features, Laurell and Ericksson (43) also concluded that the deficiency is not rare, that it is probably an inherited defect, and that the ₁-AT protein in deficient subjects has a structural abnormality because it migrated slower than the normal protein upon electrophoresis. They have been proven correct in each of these conclusions.

Papain-induced emphysema. In 1964, Gross and colleagues (48) in Pittsburgh reported enzymatically produced emphysema. This result was uncovered in a project designed to test the effects of proteolytic enzymes on developing silicotic pulmonary nodules. Papain, a plant-derived proteinase, or chymotrypsin was injected intratracheally into rats exposed to quartz dust in inhalation chambers. Animals that received papain developed centriacinar emphysema; the other animals did not. Emphysema developed quickly after papain and without apparent inflammation, suggesting a direct proteolytic effect on lung tissue. Within a few years, other researchers reported marked changes in the appearance of lung elastic fibers in enzyme-induced emphysema (49).

These initial studies linking emphysema to ₁-AT deficiency and intrapulmonary proteolytic enzymes triggered a burst of research activity, and an international symposium convened on the topic of pulmonary emphysema and proteolysis in 1971. The participants accepted a connection between proteases and emphysema and agreed that ₁-AT deficiency presented an important model to dissect the pathogenesis of emphysema, even though most individuals with emphysema do not have the deficiency. Eugene Robin, the conference summarizer, noted "the growing maturity of the discipline of chest disease as one capable of assimilating and using all the basic disciplines of biology" (50). Indeed, studies into the pathogenesis of emphysema have helped with the entry of modern cell and molecular biology into lung research generally. The DLD has played a major role in these developments in many ways, including support of the first international meeting on elastin and its successor, the Gordon Research Conference on Elastin and Elastic Tissue, that has been held every 2 years over the past two decades.

Proteinase-Antiproteinase Hypothesis

The idea that emphysema results from proteolytic injury to alveolar septa has been the prevailing hypothesis about the pathogenesis of emphysema for the past three decades. According to the proteinase-antiproteinase hypothesis, there is a steady or episodic release of proteinases into the lung tissue capable of digesting structural proteins of the lung. Normally, lung tissue is protected by a shield of proteinase inhibitors, principally from the blood, but also synthesized locally. Emphysema results when the proteinase-antiproteinase balance favors proteolytic activity. The importance of elastin destruction followed recognition that elastolytic activity was required for proteolytic induction of emphysema and by the finding that the capacity of different papain preparations to cause emphysema correlated with their elastase activity (51). The fact that neutrophil elastase was shown to be the principal target of ₁-AT (52) further strengthened the connection between elastin and emphysema. Janoff (53) prepared a comprehensive review of this topic in 1985.

Lung elastin and elastic fibers. Emphysematous lung tissue has aberrant-looking elastic fibers (54) and contains less elastin than normal lung tissue (40). Elastin is the principal component of elastic fibers. Encoded by a gene on human chromosome 7, elastin is secreted from several cell types as a soluble monomer precursor of approximately 70 kilodalton (kD) called tropoelastin. In the extracellular space tropoelastin molecules align on a "scaffold" of microfibrils, which consist of a number of constituents including fibrillins and microfibril-associated proteins. Under the action of lysyl oxidase, most of the lysine residues in tropoelastin become modified, causing the tropoelastin monomers to crosslink and form elastin, a highly insoluble, rubber-like polymer. The lysine-derived crosslinks are known as desmosines.

Under normal circumstances, the synthesis of lung elastin begins late in fetal life, peaks in the early neonatal period, continues to a much lesser degree during adolescence, and stops in adult life, although the tropoelastin gene may remain transcriptionally active (55). Elastic fibers in the lung normally last a human life span (56). Elastic fibers are not distributed uniformly in the lung parenchyma. They loop around alveolar ducts, form rings at the mouths of alveoli, and penetrate as wisps into alveolar septa, where they are concentrated at bends and junctions (57). Therefore, destruction of entire alveolar septa must affect matrix components besides elastin. Important in thinking about the role of elastases in producing emphysema is that all elastases degrade multiple components of the extracellular matrix in addition to elastin.

Because they are unique to elastin, desmosines have been used to quantify elastin in tissues and as markers of elastin degradation in biologic fluids. Recently, in smokers with marked variability in annual deterioration of FEV₁, urinary excretion of desmosine was found to correlate with the rate of decline in FEV₁ (58). There was, however, no correlation between emphysema as determined by computed tomography (CT) and desmosine excretion. Although the sensitivity of CT to microscopic indices of emphysema may be a limiting factor, these data suggest that desmosines can originate from breakdown of elastin in small airways as well as lung parenchyma. Other studies, however, using plasma peptides of elastin as the marker and indices of elastic recoil, do show a relationship between increased elastin breakdown and emphysema (59).

What little is known about repair of lung elastic fibers in vivo is primarily from studies in animals given intratracheal elastases. After intratracheal instillation of elastase, much of the lung elastin is depleted within hours to a few days (60). This phase is followed by a burst of elastin synthesis so that over the next few weeks the elastin content of the lungs is restored. Yet, the lung is emphysematous and the alveolar elastic fibers look abnormal (61), resembling the aberrant alveolar elastic fibers in human emphysema (54). Accordingly, restoring the elastin content of the lung does not restore normal lung architecture in this experimental model. This result is not surprising considering that production of an elastic fiber is complex, involving temporal and physical coordination of expression of tropoelastin, microfibrillar proteins, and lysyl oxidase.

An intriguing recent finding was restoration of normal alveoli in elastase-induced emphysema by treatment with retinoic acid (62). This result was achieved in adult male rats, an animal that has continued lung growth throughout life, unlike people. Verification in other species and elucidation of the mechanisms involved in producing alveolar repair in adult lungs could prove extremely valuable.

Elastases, elastin destruction, and the absence of fibrosis dominate thinking about the pathogenesis of emphysema, but experimental studies and data from human tissue point to alveolar septal collagen destruction and aberrant collagen repair as part of the emphysematous process (63). A diet including -amino-proprionitrile (BAPN) to prevent crosslinking of newly synthesized collagen, when given to hamsters along with intratracheal elastase, resulted in worse emphysema with giant bullae than the same dose of elastase without concomitant BAPN (64). Rats given intratracheal cadmium chloride and dietary BAPN developed emphysema, but without BAPN the pulmonary lesions resembled pulmonary fibrosis (65). In guinea pigs exposed to cigarette smoke, emphysema was associated with a progressive increase in septal collagen after 6 and 12 mo. These experimental studies fit with findings of increased alveolar collagen and focally thickened alveolar walls in human emphysema (41).

Elastases and anti-elastases in the lung. Recognition that elastic fiber destruction is probably a central feature in the pathogenesis of smoking-induced emphysema has focused attention on elastolytic enzymes that might be involved. There are numerous elastolytic enzymes in lung (Table 1). Establishing their relative importance in the pathogenesis of emphysema is still not resolved. Knowing which enzyme(s) is involved is essential to develop proteinase inhibitors that may be useful clinically, because elastases of different enzyme classes require different inhibitors.

Although neutrophil elastase is almost surely pivotal in emphysema associated with ₁-AT deficiency, it is much less certain whether neutrophil elastase has a central role in emphysema that develops in smokers with normal levels of ₁-AT. Some facts support a role for it. Increased neutrophil elastase is detectable in bronchoalveolar lavage immediately after smoking (66), and neutrophil elastase has been detected in emphysematous tissue (67). The possibility that the smoker's lungs have a local deficiency of functional ₁-AT because smoke oxidizes ₁-AT in vitro was an attractive early hypothesis, but data about this subsequently have been inconclusive.

Recently, alveolar macrophages have come under increasing attention to help explain emphysema in the typical smoker who has a normal circulating level of ₁-AT. Alveolar macrophages are strong candidates because smoker's lungs contain a greatly expanded number of macrophages and because they produce several proteolytic enzymes with elastase activity, including macrophage elastase, gelatinase B, and cathepsins L and S. Correlations of alveolar wall destruction in smokers demonstrate a relationship with the number of alveolar macrophages and T lymphocytes, but not with neutrophils (68). Young adult smokers have macrophage aggregations in respiratory bronchioles, the site where emphysema typically begins (13).

One means of pinpointing the enzymes responsible for emphysema is targeting the genes that code for proteinases in experimental models (69). Using this approach, recent results show an important role for macrophage elastase, a matrix metalloproteinase, in smoke-induced emphysema in mice. Mice lacking a functional macrophage elastase gene as a result of targeted mutagenesis did not develop emphysema from cigarette smoke exposure under conditions that produced emphysema in mice and a functional macrophage elastase gene (Figure 4) (70). Macrophage elastase is not inhibited by ₁-AT.

View larger version (120K): [in this window] [in a new window]   Figure 4.   Mice with the normal expression of macrophage elastase (MME +/+), but not mice deficient in macrophage elastase (MME -/-), develop emphysema in response to cigarette smoke (two cigarettes/day, 6 d/wk for 6 mo). The lungs were inflated by intratracheal administration of 10% formalin under constant pressure, 25 cm H2O. The MM +/+ lung from a smoke-exposed mouse has centriacinar dilitation of alveolar ducts compared with the MME +/+ non-smoker mouse. In contrast, the lung of the smoke-exposed MME -/- mouse resembles the lung of the MME / non-smoker mouse. (Courtesy of Steven D. Shapiro, M.D.) (Data adapted from Reference 70.)

As noted, the history of human deficiency of ₁-AT began in 1963. Since then, the progress in understanding ₁-AT over the past three decades has been remarkable, and stands as a shining example of medical science's capacity to unravel basic aspects of human disease (71). Several rare ₁-AT phenotypes are now known to be associated with low plasma concentrations and a high risk for emphysema, but the Pi Z phenotype originally identified by Laurell and Erikkson accounts for nearly all the patients with marked deficiency. Individuals with the Pi Z phenotype have about 15% of the normal plasma ₁-AT concentration. The Pi Z ₁-AT protein has a slower association rate with neutrophil elastase than does normal ₁-AT (72), so that the Pi Z phenotype has a protein that is less effective than normal in addition to the deficiency. The threshold for the circulating level of ₁-AT above which there is little increased risk for emphysema without the aggravating effective smoking appears to be about 37% of normal (~ 88 mg/dl). This value comes from finding that Pi SZ heterozygotes who typically have about this level of ₁-AT usually have FEV₁ values above 80% of predicted normal if they have never smoked (73).

With the exception of ₂-macroglobulin, each proteinase inhibitor in the lung has activity that is restricted to one class of proteolytic enzymes (Table 2). Like ₁-AT, ₂-macroglobulin is produced primarily in the liver. The other inhibitors are produced mainly locally in the respiratory tissues. Their relative contributions to protection against alveolar septal destruction associated with smoking is not known, but the recent data incriminating macrophage elastase in mice with smoke-induced emphysema, mentioned previously, suggests that inhibitors of matrix metalloproteinases may prove to be important.

In brief, over the past 30 years a picture of the pathogenesis of emphysema in smokers has emerged that stresses proteolytic activity against extracellular matrix proteins. Damage to elastic fibers in the lung parenchyma appears to be a critical event, but the destruction of alveolar walls clearly affects other extracellular matrix components in the lung parenchyma as well. Repair, reflected in collagen deposition, also seems to occur coincident with alveolar septal destruction and may help check alveolar overdistention.

	CONCLUDING COMMENTS

Much of the progress in understanding and treating COPD during the past 30 years is ascribable to the NHLBI and DLD. It must be noted that even "negative" efforts, such as the investigation of small airways disease and the trials of artificial ventilation and intermittent positive pressure breathing, yielded large amounts of data of value to both clinicians and investigators. However, COPD is now the fourth leading cause of death in the United States and exacts an enormous toll in terms of morbidity and health care resources in industrialized countries worldwide. Accordingly, the problem is by no means solved; we still need better understanding of pathogenesis and more effective therapy.

It is important to recognize that the problem is eminently soluble simply by changing the population's smoking habits. This is, of course, easier said than done, but smoking is decreasing in North America and western Europe. Though overall mortality has not declined, the age of death from emphysema has increased steadily, as has the age of onset of clinically severe disease. People are living long enough to develop COPD who did not do so previously, and the cohorts of men who began smoking 50-75 yr ago, and who had very heavy exposure to tobacco, are working their way through the population. Thus, in North America and western Europe we may expect that COPD will be a less important problem in the future than it is at present. In contrast, in developing countries where both life expectancy and cigarette smoking are increasing, COPD will become an important problem.

A revolution in concepts about the pathogenesis of emphysema has occurred during the past 30 years. The discovery of ₁-AT deficiency led to the idea that emphysema resulted from enzymatic digestion of lung extracellular matrix. Details became available about the inhibitor profile of ₁-AT that made neutrophil elastase the principal candidate enzyme. More recent studies have revealed a higher level of complexity, with several enzymes and inhibitors present in lung tissue that might be involved in the development of emphysema. Although clinical emphysema is found almost exclusively among smokers, many smokers do not develop emphysema, and the reasons remain to be determined. Genetic factors are certain to be important, both in the development of emphysema from smoking in some individuals and the apparent resistance to this effect of smoking in others (74). The tools of cell and molecular biology will surely be used increasingly to identify factors involved in the pathogenesis of emphysema.

	Footnotes

Correspondence and requests for reprints should be addressed to Robert M. Senior, M.D., Pulmonary and Critical Care Medicine, Barnes-Jewish Hospital (North Campus), 216 South Kingshighway, St. Louis, MO 63110. E-mail: rsenior{at}imgate.wustl.edu

	References

TOP INTRODUCTION REFERENCES

1. American Thoracic Society. 1995. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med 152: S77-S120 .

2. Burrows, B., and R. H. Earle. 1969. Prediction of survival in patients with chronic airways obstruction. Am. Rev. Respir. Dis 99: 865-871 [Medline].

3. Burrows, B., and R. H. Earle. 1969. Course and prognosis of chronic obstructive lung disease. N. Engl. J. Med. 280: 397-404 .

4. Diener, C. F., and B. Burrows. 1975. Further observations on the course and prognosis of chronic obstructive lung disease. Am. Rev. Respir. Dis 111: 719-724 [Medline].

5. Traver, G. A., M. G. Cline, and B. Burrows. 1979. Predictors of mortality in chronic obstructive lung disease. Am. Rev. Respir. Dis 119: 895-902 [Medline].

6. Burrows, B., J. W. Bloom, G. A. Traver, and M. G. Cline. 1987. The course and prognosis of different forms of chronic airways obstruction in a sample from a general population. N. Engl. J. Med 317: 1309-1314 [Abstract].

7. Fletcher, C., R. Peto, C. Tinker, and F. E. Speizer. 1976. The Natural History of Chronic Bronchitis and Emphysema. Oxford University Press, London.

8. Anthonisen, N. R., J. E. Connett, J. P. Kiley, M. D. Altose, W. C. Bailey, et al . 1994. Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV₁. J.A.M.A 272: 1497-1505 [Abstract/Free Full Text].

9. Sluiter, H. J., G. H. Koeter, J. G. R. de Monchy, D. S. Postma, K. de Vries, and J. G. M. Orie. 1991. The Dutch hypothesis (chronic nonspecific lung disease) revisited. Eur. Respir. J 4: 479-489 [Abstract].

10. Larson, R. K., and M. L. Barman. 1965. The familial occurrence of chronic obstructive lung disease. Ann. Intern. Med 63: 1001-1008 .

11. Becklake, M. R.. 1985. Chronic airflow limitations: its relationship to work in dusty occupations. Chest 88: 608-617 [Abstract/Free Full Text].

12. Burrows, B., R. J. Knudson, and M. D. Lebowitz. 1977. The relationship of childhood respiratory illness to adult obstructive airways disease. Am. Rev. Respir. Dis 115: 751-760 [Medline].

13. Niewoehner, D. E., J. Kleinerman, and D. B. Rice. 1977. Pathological changes in the peripheral airways of young cigarette smokers. N. Engl. J. Med 291: 755-758 .

14. Buist, A. S., H. Ghezz, P. Renzi, T. Ducret, A. Robinson, and the PARI-IS Study Steering Committee and Research Group. 1979. Relationship between the single breath N₂ test and age, sex and smoking habit in three North American cities. Am. Rev. Respir. Dis 120: 305-318 [Medline].

15. Burrows, B., M. Halonen, M. D. Lebowitz, R. J. Knudson, and R. A. Barbee. 1982. The relationship of serum immunoglobulin E, allergy, skin tests, and smoking to respiratory disorders. J. Allergy Clin. Immunol 80: 199-204 .

16. Burrows, B., F. M. Hasan, R. A. Barbee, M. Halonen, and M. D. Lebowitz. 1980. Epidemiologic observations on eosinophilia and its relation to respiratory disorders. Am. Rev. Respir. Dis 122: 709-718 [Medline].

17. Tashkin, D. P., M. D. Altose, J. E. Connett, R. E. Kanner, W. W. Lee, and R. A. Wise. 1996. Methacholine reactivity predicts changes in lung function over time in smokers with early chronic obstructive lung disease. Am. J. Respir. Crit. Care Med 153: 1802-1811 [Abstract].

18. Xu, X., B. Li, and L. Wang. 1994. Gender difference in smoking effects on adult pulmonary function. Eur. Respir. J 7: 477-483 [Abstract].

19. Intermittent Positive Pressure Breathing Trial Group. 1983. Intermittent positive pressure breathing therapy of chronic obstructive pulmonary disease. Ann. Intern. Med 99: 612-620 .

20. Anthonisen, N. R., E. C. Wright, and the IPPB Trial Group. 1986. Bronchodilator response in chronic obstructive pulmonary disease. Am. Rev. Respir. Dis 133: 814-819 [Medline].

21. Rice, K. L., J. W. Leatherman, P. G. Duane, L. S. Snyder, K. R. Harmon, J. Abel, and D. E. Niewdehner. 1987. Aminophylline for acute exacerbations of chronic obstructive pulmonary disease. Ann. Intern. Med 107: 305-309 .

22. Wedzicha, J. A.. 1993. Inhaled corticosteroids in COPD: awaiting controlled trials. Thorax 48: 305-307 [Free Full Text].

23. Albert, R. K., T. R. Martin, and S. W. Lewis. 1980. Controlled clinical trial of methylprednisolone in patients with chronic bronchitis and acute respiratory insufficiency. Ann. Intern. Med 92: 753-758 .

24. Thompson, W. H., C. P. Nielson, P. Carvalho, N. B. Charan, and J. P. Crowley. 1996. Controlled trial of oral prednisone in outpatients with acute COPD exacerbations. Am. J. Respir. Crit. Care Med 154: 407-412 [Abstract].

25. Saint, S., S. Bent, E. Vittinghof, and D. Grady. 1995. Antibiotics in chronic obstructive pulmonary disease exacerbations: a meta-analysis. JAMA 272: 957-960 .

26. Collet, J. P., S. Shapiro, P. Ernst, N. R. Anthonisen, R. M. Cherniak, S. Ducic, P. T. Macklem, J. Manfreda, R. R. Martin, D. McCarthy, and B. B. Ross. 1997. Effects of an immunostimulating agent on acute exacerbations and hospitalizations in COPD patients. Am. J. Respir. Crit. Care Med 156: 1719-1724 [Abstract/Free Full Text].

27. Nocturnal Oxygen Therapy Trial Group. 1980. Continuous or nocturnal oxygen therapy in hypoxemic chronic obstructive pulmonary disease. Ann. Intern. Med 93: 391-398 .

28. Medical Research Council Working Party. 1981. Long term domiciliary oxygen therapy in chronic cor pulmonale complicating chronic bronchitis and emphysema. Lancet 1: 681-686 [Medline].

29. Gorecka, D., K. Gorzelak, P. Sliwinski, M. Tobiasz, and J. Zielinski. 1997. Effect of long term oxygen therapy on survival in patients with chronic obstructive pulmonary disease with moderate hypoxemia. Thorax 52: 674-679 [Abstract].

30. Pardy, R. L., M. S. Fairbarn, and S. P. Blackie. 1990. Respiratory muscle training. In M. J. Tobin, editor. Problems in Respiratory Care. Lippincott, Philadelphia.

31. Shapiro, S. H., P. Ernst, K. Gray-Donald, J. G. Martin, S. Wood-Dauphinee, A. Beaupre, W. O. Spitzer, and P. T. Macklem. 1992. Effect of negative pressure ventilation in severe chronic obstructive pulmonary disease. Lancet 340: 1425-1429 [Medline].

32. Celli, B. R.. 1997. Is pulmonary rehabilitation an effective treatment for chronic obstructive pulmonary disease? Yes. Am. J. Respir. Crit. Care Med 155: 781-783 [Medline].

33. Goldstein, R., E. Gork, D. Stubbing, M. Avendano, and G. Guyatt. 1994. Randomized controlled trial of respiratory rehabilitation. Lancet 344: 1394-1397 [Medline].

34. Albert, R. K.. 1997. Is pulmonary rehabilitation an effective treatment for chronic obstructive pulmonary disease? No. Am. J. Respir. Crit. Care Med 155: 784-785 [Medline].

35. Cooper, J. D., G. A. Patterson, R. S. Sundaresan, E. P. Trulock, R. D. Yusen, M. S. Pohl, and S. S. Lefrak. 1997. Results of 150 consecutive bilateral lung volume reduction procedures in patients with severe emphysema. J. Thoracic Cardiovasc. Surg 112: 1319-1329 [Abstract/Free Full Text].

36. Snider, G., L. J. Kleinerman, W. M. Thurlbeck, and Z. H. Bengali. 1985. The definition of emphysema: report of a National Heart, Lung and Blood Institute, Division of Lung Diseases Workshop. Am. Rev. Respir. Dis 132: 182-185 [Medline].

37. Nagai, A., H. Inano, K. Matsuba, and W. M. Thurlbeck. 1994. Scanning electronmicroscopic morphometry of emphysema in humans. Am. J. Respir. Crit. Care Med 150: 1411-1415 [Abstract].

38. Lamb, D., A. McLean, M. Gillooly, P. M. Warren, G. A. Gould, and M. MacNee. 1993. Relation between distal airspace size, bronchiolar attachments, and lung function. Thorax 48: 1012-1017 [Abstract/Free Full Text].

39. Mclean, A., P. M. Warren, M. Gillooly, W. MacNee, and D. Lamb. 1992. Microscopic and macroscopic measurements of emphysema: relation to carbon monoxide gas transfer. Thorax 47: 144-149 [Abstract/Free Full Text].

40. Cardosa, W. V., H. S. Sekhon, D. M. Hyde, and W. M. Thurlbeck. 1993. Collagen and elastin in human pulmonary emphysema. Am. Rev. Respir. Dis 147: 975-981 [Medline].

41. Lang, M. R., G. W. Fiaux, M. Gillooly, J. A. Stewart, D. J. S. Hulmes, and D. Lamb. 1994. Collagen content of alveolar wall tissue in emphysematous and non-emphysematous lungs. Thorax 49: 319-326 [Abstract/Free Full Text].

42. Wright, J. L., and A. Churg. 1995. Smoke-induced emphysema in guinea pigs is associated with morphometric evidence of collagen breakdown and repair. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 268:L17-L20.

43. Laurell, C.-B., and S. Eriksson. 1963. The electrophoretic ₁-globulin pattern of serum of ₁-antitrypsin deficiency. Scand. J. Clin. Lab. Invest 15: 132-140 .

44. Eriksson, S.. 1964. Pulmonary emphysema and alpha₁-antitrypsin deficiency. Acta Med. Scand 175: 197-205 [Medline].

45. Eriksson, S.. 1965. Studies in ₁-antitrypsin deficiency. Acta Med. Scand 432: 1-85 .

46. Silverman, E. K., J. A. Pierce, M. A. Province, D. C. Rao, and E. J. Campbell. 1989. Variability of pulmonary function in alpha-1-antitrypsin deficiency: clinical correlates. Ann. Intern. Med 111: 982-991 .

47. McElvaney, N. G., J. K. Stoller, A. S. Buist, U. B. S. Prakash, M. L. Brantly, M. D. Schluchter, and R. D. Crystal. 1997. Baseline characteristics of enrollees in the National Heart, Lung and Blood Institute Registry of ₁-Antitrypsin Deficiency. Chest 111: 394-403 [Abstract/Free Full Text].

48. Gross, P., M. A. Babyak, E. Tolker, and M. Kaschak. 1964. Enzymatically produced pulmonary emphysema: a preliminary report. J. Occup. Med 6: 481-484 [Medline].

49. Goldring, I. P., L. Greenburg, and I. M. Ratner. 1968. On the production of emphysema in Syrian hamsters by aerosol inhalation of papain. Arch. Environ. Health 16: 59-60 [Medline].

50. Robin, E. D. 1972. Summary: Symposium on Pulmonary Emphysema and Proteolysis. In C. Mittman, editor. Pulmonary Emphysema and Proteolysis. Academic Press, Inc., New York. 527-537.

51. Snider, G. L., J. A. Hayes, C. Franzblau, H. M. Kagan, P. S. Stone, and A. L. Korthy. 1974. Relationship between elastolytic activity and experimental emphysema-inducing properties of papain preparations. Am. Rev. Respir. Dis 110: 254-262 [Medline].

52. Travis, J., and G. S. Salvesen. 1983. Human plasma proteinase inhibitors. Ann. Rev. Biochem 52: 655-709 [Medline].

53. Janoff, A.. 1985. Elastases and emphysema: current assessment of the protease-antiprotease hypothesis. Am. Rev. Respir. Dis 132: 417-433 [Medline].

54. Fukuda, Y., Y. Masuda, M. Ishizaki, Y. Masugi, and V. J. Ferrans. 1989. Morphogenesis of abnormal elastic fibers in lungs of patients with panacinar and centriacinar emphysema. Hum. Pathol 20: 652-659 [Medline].

55. Swee, M., W. C. Parks, and R. A. Pierce. 1995. Developmental regulation of elastin production: expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels. J. Biol. Chem 270: 14899-14906 [Abstract/Free Full Text].

56. Shapiro, S. D., S. K. Endicott, M. A. Province, J. A. Pierce, and E. J. Campbell. 1991. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons related radiocarbon. J. Clin. Invest. 87: 1828-1834 .

57. Kuhn, C., and E. H. Oldmixon. 1990. The interstitium of the lung. In D. E. Schraufnagel, editor. Electron Microscopy of the Lung. Marcel Dekker, New York. pp. 177-214.

58. Gottlieb, D. J., P. J. Stone, D. Sparrow, M. E. Gale, S. T. Weiss, G. L. Snider, and G. T. O'Connor. 1996. Urinary desmosine excretion in smokers with and without rapid decline of lung function: the normative aging study. Am. J. Respir. Crit. Care Med 154: 1290-1295 [Abstract].

59. Dillon, T. J., R. L. Walsh, R. Scicchitano, B. Eckert, E. G. Cleary, and G. McLennan. 1992. Plasma elastin-derived peptide levels in normal adults, children, and emphysematous subjects. Am. Rev. Respir. Dis 146: 1143-1148 [Medline].

60. Goldstein, R. A., and B. C. Starcher. 1978. Urinary excretion of elastin peptides containing desmosine after intratracheal injection of elastase in hamsters. J. Clin. Invest 61: 1286-1290 .

61. Kuhn, C., S. H. Yu, M. Chraplyvy, H. E. Linder, and R. M. Senior. 1976. The induction of emphysema with elastase: II. Changes in connective tissue. Lab. Invest 34: 372-380 [Medline].

62. Massaro, G. D., and D. Massaro. 1997. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema. Nature Med 3: 675-677 [Medline].

63. Wright, J. L.. 1995. Emphysema: concepts under changea pathologist's perspective. Mod. Path 8: 873-880 . [Medline]

64. Kuhn, C., and B. C. Starcher. 1980. The effect of lathyrogens on the evolution of elastase-induced emphysema. Am. Rev. Respir. Dis 122: 453-460 [Medline].

65. Niewoehner, D. E., and J. R. Hoidal. 1982. Lung fibrosis and emphysema: divergent responses to a common injury? Science 217: 359-360 [Abstract/Free Full Text].

66. Fera, T., R. T. Abboud, A. Richter, and S. S. Johal. 1986. Acute effect of smoking on elastaselike esterase activity and immunologic neutrophil elastase levels in bronchoalveolar lavage fluid. Am. Rev. Respir. Dis 133: 568-573 [Medline].

67. Damiano, V. V., A. Tsang, U. Kucich, W. Abrams, J. Rosenbloom, P. Kimbel, M. Fallahnjad, and G. Weinbaum. 1986. Immunolocalization of elastase in human emphysematous lungs. J. Clin. Invest 78: 482-493 .

68. Finkelstein, R., R. S. Fraser, H. Ghezzo, and M. G. Cosio. 1995. Alveolar inflammation and its relation to emphysema in smokers. Am. J. Respir. Crit. Care Med 152: 1666-1672 [Abstract].

69. Shapiro, S. D.. 1997. Mighty mice: transgenic technology `knocks out' questions of matrix metalloproteinase function. Matrix Biol 15: 527-533 [Medline].

70. Hautemaki, R. D., D. K. Kobayashi, R. M. Senior, and S. D. Shapiro. 1997. Requirement for macrophage elastase for cigarette smoke- induced emphysema in mice. Science 277: 2002-2004 [Abstract/Free Full Text].

71. Crystal, R. G. 1996. Alpha-1-Antitrypsin Deficiency: Biology, Pathogenesis, Clinical Manifestations, Therapy. Marcel Dekker, Inc., New York.

72. Ogushi, F., G. A. Fells, R. C. Hubbard, S. D. Straus, and R. G. Crystal. 1987. Z-type alpha-1-antitrypsin is less competent than M1-type alpha-1-antitrypsin as an inhibitor of neutrophil elastase. J. Clin. Invest 80: 1366-1374 .

73. Turino, G. M., A. F. Barker, M. L. Brantly, A. B. Cohen, R. P. Connelly, R. G. Crystal, E. Eden, M. D. Schluchter, and J. K. Stroller. 1996. Clinical features of individuals with PI*SZ phenotype of ₁-antitrypsin deficiency. Am. J. Respir. Crit. Care Med 154: 1718-1725 [Abstract].

74. Sandford, A. J., T. D. Weir, and P. D. Pare. 1997. Genetic risk factors of chronic obstructive pulmonary disease. Eur. Respir. J 10: 1380-1391 [Abstract].