Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.

The current concepts of the pathogenesis of emphysema hold that progressive, chronic destruction of the alveolar structures occurs because there was in imbalance between the proteases and antiproteases in the lower respiratory tract. In this context, proteases, particularly neutrophil elastase, work unimpeded to destroy the alveolar structures. This concept has evolved from consideration of patients with alpha 1-antitrypsin deficiency, who have decreased levels of serum alpha 1-antitrypsin and who have progressive panacinar emphysema. To directly assess the antiprotease side of this equation, the lower respiratory tract of non-smoking individuals with normal serum antiproteases and individuals with PiZ homozygous alpha 1-antitrypsin deficiency underwent bronchoalveolar lavage to evaluate the antiprotease screen of their lower respiratory tract. These studies demonstrated that: (a) alpha 1-antitrypsin is the major antielastase of the normal human lower respiratory tract; (b) alpha 2-macroglobulin, a large serum antielastase, and the bronchial mucous inhibitor, an antielastase of the central airways, do not contribute to the antielastase protection of the human alveolar structures; (c) individuals with PiZ alpha 1-antitrypsin deficiency have little or no alpha 1-antitrypsin in their lower respiratory tract and have no alternative antiprotease protection against neutrophil elastase; and (d) the lack of antiprotease protection of the lower respiratory tract of PiZ individuals is a chronic process, suggesting their vulnerability to neutrophil elastase is always present.     

Alpha 1-PI for emphysema due to alpha 1-antitrypsin deficiency

Alpha 1-antitrypsin deficiency is a genetic disorder that may result in premature pulmonary emphysema. Affected persons are deficient in the protective protein, alpha 1-proteinase inhibitor (alpha 1-PI). The diagnosis should be considered in patients with signs of emphysema by age 40 (especially nonsmokers), those with predominantly lower lobe disease and those with a family history of premature emphysema. Recently, human alpha 1-PI has been purified and is available to prevent the development of disabling emphysema in affected individuals. Weekly infusion of the concentrate is effective in raising serum levels and is very safe, with only rare minor adverse effects.     

Recent progress of pathogenesis and treatment in alpha 1-antitrypsin deficiency]

alpha 1-Antitrypsin (alpha 1-AT) deficiency, one of the most common hereditary disorders that mainly affects the lung and liver in Caucasian people, is caused by mutations of alpha 1-AT gene. Decrease of serum alpha 1-AT concentration is directly responsible for lung emphysema, replacement of plasma-derived alpha 1-AT concentrate is administered to patients of alpha 1-AT deficiency with lung emphysema, and lung transplantation is employed for end-stage lung emphysema. Augmentation therapy is not adequate for alpha 1-AT deficiency with liver disease, because liver injury is caused by accumulation of mutated alpha 1-AT protein in hepatocytes. Currently liver transplantation is the only treatment for severe liver cirrhosis. To correct the genetic defects of alpha 1-AT gene, several approaches for gene therapy are under investigation in vitro and in vivo animal models.     

Z-type alpha 1-antitrypsin is less competent than M1-type alpha 1-antitrypsin as an inhibitor of neutrophil elastase.

Alpha 1-antitrypsin (alpha 1AT) deficiency resulting from homozygous inheritance of the Z-type alpha 1AT gene is associated with serum alpha 1AT levels of less than 50 mg/dl and the development of emphysema in the third to fourth decades. Despite the overwhelming evidence that the emphysema of PiZZ individuals develops because of a "deficiency" of alpha 1AT and hence an insufficient antineutrophil elastase defense of the lung, epidemiologic evidence has shown that levels of alpha 1AT of only 80 mg/dl protect the lung from an increased risk of emphysema. With this background, we hypothesized that homozygous inheritance of the Z-type may confer an added risk beyond a simple deficiency of alpha 1AT by virtue of an inability of the Z-type alpha 1AT molecule to inhibit neutrophil elastase as effectively as the common M1-type molecule. To evaluate this hypothesis, the functional status of alpha 1AT from PiZZ individuals (n = 10) was compared with that of alpha 1AT from PiM1M1 individuals (n = 7) for its ability to inhibit neutrophil elastase (percent inhibition) as well as its association rate constant for neutrophil elastase (K association). Plasma alpha 1AT concentration, measured by radial immunodiffusion, was 34 +/- 1 mg/dl in PiZZ patients vs. 237 +/- 14 mg/dl for PiM1M1 plasma, a sevenfold difference. When titrated against neutrophil elastase, the present inhibition of PiZZ plasma was significantly less than Pi M1M1 plasma (ZZ 78 +/- 1% vs. M1M1 95 +/- 1%, P less than 0.001) as was purified Z type alpha 1AT (ZZ, 63 +/- 2% vs. M1M1 86 +/- 2%, P less than 0.001). Sodium dodecyl sulfate (SDS) gel comparisons of the complexes formed with M1-type alpha 1AT and Z-type alpha 1AT with elastase demonstrated the Z alpha 1AT-elastase complexes were less stable than the M1 alpha 1AT-elastase complexes, thus releasing some of the enzyme to continue to function as a protease. Consistent with these observations, the K association of purified Z-type alpha 1AT for neutrophil elastase was lower than that of M1-type alpha 1AT (ZZ 4.5 +/- 0.3 X 10(6) M-1s-1 vs. M1M1 9.7 +/- 0.4 X 10(6) M-1s-1, P less than 0.001), suggesting that for the population of alpha 1AT molecules, the active Z-type molecules take more than twice as long as the active M1-type alpha 1AT to inhibit neutrophil elastase. Consequently, not only is there less alpha1AT in PiZZ individuals, but the population of Z-type alpha1AT molecules is less competent as an inhibitor of neutrophil elastase than M1-type alpha1AT molecules. This combination of defects suggests that PiZZ individuals have far less functional antielastase protection than suggested by the reduced concentrations of alpha1AT alone, further explaining their profound risk for development of emphysema.     

Danazol-induced Augmentation of Serum α1-Antitrypsin Levels in Individuals with Marked Deficiency of this Antiprotease

Individuals with serum α1-antitrypsin levels below 80 mg/dl are clearly at risk for the development of accelerated panacinar emphysema. One possible approach to the therapy of this disorder would be to raise serum levels of this major antiprotease to establish protease-antiprotease homeostasis within the lung parenchyma. Because danazol, an impeded androgen, elevates levels of C1 inhibitor in patients deficient of that serum antiprotease, we hypothesized that this agent might also increase α1-antitrypsin levels in patients with α1-antitrypsin deficiency. To evaluate this concept, seven patients with severe emphysema associated with α1-antitrypsin deficiency (six PiZ and 1 M_DuarteZ) and one asymptomatic individual (PiSZ) received 600 mg of danazol daily for 30 d. Five of the six PiZ patients responded to danazol therapy with significant increases in serum α1-antitrypsin levels (mean increase of 37%; P < 0.03). The two individuals who were heterozygous for the Z protein increased their serum levels by 85% (PiM_DuarteZ) and 87% (PiSZ), respectively. These increases in serum α1-antitrypsin antigen were accompanied by commensurate increases in serum trypsin inhibition. Crossed immunoelectrophoresis showed no alterations of the microheterogeneity of the α1-antitrypsin or the presence of protease-antiprotease complexes in serum during danazol therapy. These data demonstrate that serum α1-antitrypsin levels can be augmented by danazol therapy in PiZ individuals as well as those heterozygotes with severe deficiency of α1-antitrypsin. The clinical relevance of these increases in serum α1-antitrypsin remains speculative, but these findings suggest that danazol may provide a means of improving the protease-antiprotease balance in these individuals and thus impede the progression of their lung disease.     

Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. Spontaneous release of leukotriene B4 by alveolar macrophages.

The emphysema of alpha 1-antitrypsin (alpha 1AT) deficiency is conceptualized to result from insufficient alpha 1AT allowing neutrophil elastase to destroy lung parenchyma. In addition to the deficiency of alpha 1AT in these individuals resulting from mutations in the alpha 1AT gene, it is recognized that, for unknown reasons, there are also increased numbers of neutrophils in their lungs compared with normal individuals. With the knowledge that alveolar macrophages have surface receptors for neutrophil elastase, we hypothesized that the neutrophil accumulation in the lower respiratory tract in alpha 1AT deficiency may result, in part, from release of neutrophil chemotactic activity by alveolar macrophages as they bind uninhibited neutrophil elastase. Consistent with this hypothesis, alpha 1AT-deficient alveolar macrophages spontaneously released nearly threefold more neutrophil chemotactic activity than normal alveolar macrophages. Analysis of alpha 1AT-deficient macrophage supernates by reverse-phase HPLC, molecular sieve chromatography, radioimmunoassay, and absorption with anti-LTB4 antibody revealed that the majority of the chemotactic activity was leukotriene B4 (LTB4), a mediator absent from normal macrophage supernates. Consistent with this hypothesis, incubation of normal macrophages with human neutrophil elastase resulted in the release of the same neutrophil chemotactic mediator. Furthermore, purified human alpha 1AT was able to prevent the neutrophil elastase from stimulating the macrophages to release the chemotactic factor. Together, these findings suggest that the absence of a normal antineutrophil elastase screen in the lower respiratory tract permits free neutrophil elastase to bind to alveolar macrophages, resulting in the release of LTB4, a process which attracts neutrophils to the alveoli of alpha 1AT deficient individuals, thus accelerating the lung destruction that characterizes this disorder.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.     

Recombinant DNA-produced alpha 1-antitrypsin administered by aerosol augments lower respiratory tract antineutrophil elastase defenses in individuals with alpha 1-antitrypsin deficiency.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is characterized by insufficient amounts of alpha 1AT to protect the lower respiratory tract from neutrophil elastase, resulting in emphysema. Yeast-produced recombinant alpha 1AT (rAAT) has normal antielastase function but is associated with high renal clearance, thus obviating chronic intravenous administration. As an alternative, we evaluated aerosol administration of rAAT to alpha 1AT-deficient individuals. After aerosol administration of single doses of 10-200 mg of rAAT, epithelial lining fluid (ELF) alpha 1AT antineutrophil elastase defenses were augmented in proportion to the dose of rAAT administered. ELF alpha 1AT levels and antineutrophil elastase capacity 4 h after 200 mg rAAT aerosol were increased 40-fold over preaerosol levels, and were fivefold increased over baseline at 24 h after aerosol administration. rAAT was detectable in serum after aerosol, indicating that the lower respiratory tract epithelium may be permeable to rAAT, and that aerosolized rAAT is capable of gaining access to lung interstitium. No adverse clinical effects were noted. These observations demonstrate that aerosol administration of rAAT is safe and results in significant augmentation of lung antineutrophil elastase defenses, suggesting this method is a feasible approach to therapy. Because this approach is clinically unproven, further studies will be necessary to establish the long-term clinical efficacy of aerosol therapy in alpha 1AT deficiency.     

Alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin (AAT) is a polymorphic protein with many variants collectively known as the Pi system. The most common alleles are the M, S and Z, which are co-dominantly inherited. Infants with PiZZ have approximately 16% of the normal AAT serum concentration. alpha 1-Antitrypsin deficiency (AATD) is an inborn error of metabolism which is principally associated with liver disease in children and emphysema in young adulthood. Individuals with AATD produce an abnormal protein which accumulates in the liver, resulting in decreased serum levels. Affected individuals cannot protect their lungs from digestion by elastase. Smoking is a significant risk factor for the early development of emphysema. Prolastin, human alpha 1-protease inhibitor, is now available as replacement therapy. Weekly intravenous administration, with the goal of maintaining the serum AAT greater than 80 mg/dl, appears to arrest pulmonary damage. Its effect on liver disease is unknown at this time. A recombinant alpha 1-protease inhibitor is being tested in aerosol form with promising early results.     

Inhibition of intracellular degradation increases secretion of a mutant form of alpha1-antitrypsin associated with profound deficiency.

The mutant Z form of alpha1-antitrypsin (alpha1AT) is responsible for > 95% of all individuals with alpha1AT deficiency, an important inherited cause of emphysema and liver disease. Since secreted Z alpha1AT is a functional antiprotease, we hypothesized that interrupting catabolism of retained Z alpha1AT might increase its transport out of cells, causing an increase in extracellular protease protection. Both the protein translation inhibitor cycloheximide and the specific inhibitor of proteasome function, lactacystin, prevented intracellular degradation of Z alpha1AT. Moreover, this inhibition of degradation was associated with partial restoration of Z alpha1AT vesicular transport. This effect was observed in a model system of transfected CHO cells as well as in human alveolar macrophages synthesizing Z alpha1AT. This study supports the hypothesis that altering the intracellular fate of a mutant protein may be an option in the treatment of diseases associated with misfolded but potentially functional proteins.     

Synthesis of stress proteins is increased in individuals with homozygous PiZZ alpha 1-antitrypsin deficiency and liver disease.

Individuals who are homozygous for the protease inhibitor phenotype Z (PiZ) genetic variant of alpha 1-antitrypsin (alpha 1-AT) have reduced plasma concentrations of alpha 1-AT, and are susceptible to premature development of pulmonary emphysema. A subset of this population develops chronic liver disease. The reduction in plasma concentrations of alpha 1-AT results from a selective defect in secretion as the abnormal PiZ alpha 1-AT protein accumulates within the cell. It has recently been shown in several experimental systems that the heat shock/stress response, a response characterized by the synthesis of a family of highly evolutionarily conserved proteins during thermal or chemical stress, may also be activated by the presence of abnormal proteins within the cell. Therefore, we predicted that the heat shock/stress response would be induced in the absence of thermal or chemical stress in alpha 1-AT-synthesizing cells of PiZZ individuals. In the following study, however, we show that net synthesis of proteins in the heat shock/stress gene family (SP90, SP70, ubiquitin) is increased only in a subset of the population, PiZZ individuals with liver disease. It is not significantly increased in PiZZ individuals with emphysema or in those without apparent tissue injury. Net synthesis of stress proteins is not increased in individuals with another variant of the alpha 1-AT gene (PiS alpha 1-AT) and is not increased in individuals with severe liver disease but a normal alpha 1-AT haplotype (PiM alpha 1-AT). These results demonstrate that the synthesis of stress proteins is increased in a subset of individuals with homozygous PiZZ alpha 1-AT deficiency, those also having liver disease.     

Principles and clinical management of alpha 1-antitrypsin deficiency

Alpha 1-antitrypsin (alpha 1-AT) deficiency can cause a lung disease that, until a few years ago, has proved fatal to all patients diagnosed with this disease. This deficiency can now be treated with alpha 1-proteinase inhibitor, a protein derived from human plasma and delivered via intravenous infusion. Infusion therapy for alpha 1-AT deficiency can be delivered in the hospital or home environments. This article discusses the pathophysiology of alpha 1-AT deficiency and outlines nursing considerations for the intravenous clinician who is administering this therapy.     

Rapid screening for deficiency of alpha 1-proteinase inhibitor

This rapid screening procedure for detection of low but functional elastase-inhibitory activity in human plasma is based on the fact that incubation of excess porcine pancreatic elastase (EC 3.4.21.36) with plasma results in formation of a complex with active alpha 1-proteinase inhibitor (alpha 1PI, also called alpha 1-antitrypsin). In normal individuals all of the elastase is complexed, leaving no free enzyme to hydrolyze the elastase substrate, and the reaction mixture remains clear. Because individuals homozygous for the Z allele have relatively low concentrations of alpha 1PI, their plasma cannot complex all of the elastase in the assay. The uncomplexed enzyme hydrolyzes the elastase-specific p-nitroanilide substrate, producing a yellow reaction mixture. Use of this simple assay for early screening of individuals for alpha 1PI deficiency may substantially decrease the number of untreated cases of familial emphysema, a disorder that develops as a result of a genetically derived proteinase-proteinase inhibitor imbalance.     

Interrelationships between the Human Alveolar Macrophage and Alpha-1-Antitrypsin

Alveolar macrophages lavaged from human lungs contain protease activity at an optimum pH of 3.0 and possibly a lesser peak of activity at pH 5.5. Protease activity measured at pH 4.1 is inhibited by purified alpha-1-antitrypsin.Fluorescent antibody studies of human alveolar macrophages showed that alpha-1-antitrypsin is present in normal alveolar macrophages. In addition, macrophages from a patient with a homozygous deficiency of alpha-1-antitrypsin exhibited less fluorescence when incubated in autologous serum than the same macrophages incubated in normal serum. Macrophages from normal subjects showed maximal fluorescence when removed from the lung and additional incubation with serum did not increase fluorescence.These results implicate the human alveolar macrophage as a possible source of an enzyme that may cause emphysema in patients deficient in alpha-1-antitrypsin. They also show that alpha-1-antitrypsin has access to the alveolus in normal subjects.     

alpha 1-Antitrypsin phenotypes in cor pulmonale due to chronic obstructive airways disease.

The clinical and radiological features of 23 patients with cor pulmonale due to chronic obstructive airways disease were reviewed. Twenty-two patients had evidence of pulmonary vascular disease and 52 per cent had secondary polycythaemia. Eighteen (78 per cent) had radiological evidence of emphysema. Thirteen of the patients (56.5 per cent) had alpha 1-antitrypsin phenotypes associated with serum deficiency and of these the largest single group was the MZ phenotype (34.8 per cent). No difference was found between the clinical features of patients with the MM phenotype or those associated with alpha 1-antitrypsin deficiency, although radiological emphysema was more common in the latter group (92 per cent compared to 60 per cent).     

Deficiency of the Chemotactic Factor Inactivator in Human Sera with α1-Antitrypsin Deficiency

As revealed by appropriate fractionation procedures, human serum deficient in alpha(1)-antitrypsin (alpha(1)-AT) is also deficient in the naturally occurring chemotactic factor inactivator. These serum donors had severe pulmonary emphysema. Serum from patients with clinically similar pulmonary disease, but with presence of alpha(1)-AT in the serum, showed no such deficiency of the chemotactic factor inactivator. When normal human serum and alpha(1)-AT-deficient human sera are chemotactically activated by incubation with immune precipitates, substantially more chemotactic activity is generated in alpha(1)-AT-deficient serum. These data indicate that in alpha(1)-AT-deficient serum there is an imbalance in the generation and control of chemotactic factors. It is suggested that the theory regarding development of pulmonary emphysema in patients lacking the alpha(1)-antitrypsin in their serum should be modified to take into account a deficiency of the chemotactic factor inactivator.     

The effect of tamoxifen in intermediate alpha 1-antitrypsin deficiency associated with the phenotype PiSZ

Three patients, one with lung disease and two with liver disease, with intermediate alpha 1-antitrypsin (AAT) deficiency phenotype PiSZ, (plasma AAT concentration approximately 1/3 of normal) were treated with the anti-oestrogen drug, tamoxifen, for 2-24 months. Patients responded with 50-70% increases in plasma AAT concentrations. Progress of emphysema appeared to be retarded in one case but no improvement in liver function was recorded in the other two patients.     

Plasma levels of elastase-specific fibrinopeptides correlate with proteinase inhibitor phenotype. Evidence for increased elastase activity in subjects with homozygous and heterozygous deficiency of alpha 1-proteinase inhibitor.

There is indirect evidence that unopposed human neutrophil elastase (HNE) is responsible for emphysema in patients with alpha 1-proteinase inhibitor (Pi) deficiency. To directly explore this possibility, we developed an assay for fibrinopeptide A alpha 1-21 and its degradation products and used it to measure HNE activity in 128 subjects of known Pi phenotype. The mean elastase-specific fibrinopeptide (ESF) level in 49 deficient PiZ individuals is significantly higher than that in 56 PiMZ heterozygotes (4.5 and 1.5 nM, respectively; P less than 0.01), while the mean ESF value in heterozygotes is significantly elevated over that in 23 normal PiM subjects (1.5 and 0.6 nM, respectively; P less than 0.01), consistent with increased HNE activity in those deficient in the major regulator of the enzyme. These results are not due to differences in smoking history because after correction for pack-years of smoking, ESF values in PiZ subjects are fourfold higher than those in PiMZ individuals (P = 0.005), while the ESF levels in heterozygotes are threefold higher than those in PiM subjects (P = 0.02). In addition, this analysis suggests that cigarette smoking and alpha 1-proteinase inhibitor deficiency have additive effects on ESF levels thereby explaining why PiZ and some PiMZ individuals are at especially high risk for the development of lung disease if they smoke. Finally, the observation that ESF levels in nonsmoking PiZ subjects are inversely related to the percent of predicted forced expiratory volume in 1 s (FEV 1%) provides direct support for the concept that unregulated HNE activity causes alveolar septal destruction in patients with alpha 1-proteinase inhibitor deficiency.     

Alpha 1-antiproteinase deficiency

The dysbalance between proteolytic neutrophil elastase and its endogeneous inhibitor seems to be a pathogenetic key mechanism in the origin of pulmonary emphysema (elastase-antielastase hypothesis). This hypothesis is supported by the observation, that low serum levels of alpha 1-antitrypsin can be observed in smokers with premature pulmonary emphysema. alpha 1-proteinase inhibitor is an acute phase protein with known structural and moleculargenetic aspects, which is synthesized by the liver and reaches the lung by the circulation. Its role is the inactivation of excessive neutrophil elastase in the pulmonary parenchyma, which is liberated during inflammation and destroys elastin and other components of extra-cellular connective tissue matrix. This is an overview on epidemiology, clinical aspects, genetics and molecular biology of this particular disease which was described in 1963.     

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.