Biochemistry, Pathology and Genetics of Pulmonary Emphysema: Proceedings of an International Symposium Held in Sassari, Italy, 27-30 April 1980

Biochemistry, Pathology and Genetics of Pulmonary Emphysema documents the proceedings of an international symposium held in Sassari, Italy, 27-30 April 1980. Research on the origins of emphysema has acquired more importance than functional diagnostic studies. There are various hypotheses concerning the development of emphysema. Some cases of emphysema are linked to defects in metabolic functions of the vessels while others are linked to a disturbance in repair processes. The papers in this volume are organized into four sections. Section 1 contains studies on the pathology and biochemistry of lung connective tissue. Section 2 deals with animal models. Section 3 on proteases and antiproteases includes studies on the characteristics and identification of biological specimens, and alpha1-proteinase inhibitor. Section 4 takes up the risk factors and therapeutic approaches for lung disease. Other papers in the volume were presented during two roundtable discussions on the biochemistry of connective tissue components in emphysema and therapeutic approaches.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483157955

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SECTION 1

PATHOLOGY AND BIOCHEMISTRY OF LUNG CONNECTIVE TISSUE

PATHOLOGICAL AND PATHOGENETIC ASPECTS OF CHRONIC OBSTRUCTIVE LUNG DISEASE

ASPECTS PATHOLOGIQUES ET PATHOGÉNÉTIQUES DES MALADIES PULMONAIRES OBSTRUCTIVES CHRONIQUES

J. Bignon and H. de Grémoux*

ABSTRACT

Elastase-induced emphysema mimics the histological and biochemical aspects of panlobular emphysema in humans. Experimental animal data such as these, and the association between α1-antiproteinase deficiency and emphysema, have led many investigators to postulate that this acquired, progressive disorder is the result of a chronic elastase-antielastase imbalance. Most of the recent studies on this problem have investigated the role played by cigarette smoke and other pollutants, since 1) those agents increase the recruitment of macrophages and neutrophils and the release of elastase within bronchiolar and alveolar structures, and 2) pollutants may inactivate α1-antiproteinase by an oxidative pathway. This elastinolytic injury does not result in a decrease in the elastin content of the lung because active resynthesis leads to the formation of a disorganized network of elastic fibres.

Animal models

antiprotease

chronic obstructive lung disease

emphysema

protease

During the past decade, it was demonstrated that two major conditions are responsible for the limitation of airflow in chronic obstructive lung disease : emphysema and small airway disease. In structural terms, emphysema is characterized by a permanent increase in the size of the alveolar spaces of the acinus, beyond the terminal bronchiole, which may or may not be accompanied by rupture of the alveolar wall [2, 14, 72]; small airway disease is characterized by narrowing or tortuosity of the peripheral bronchioles in relation to inflammatory hypertrophic or atrophic changes in the airway wall [5, 33]. These two lesions are usually associated, so that it is practically impossible to make an accurate diagnosis in living persons [72].

Of the exogenous agents (pollutants, microorganisms, allergens) that have been significantly associated with the development of airflow limitation (fig 1), those which are linked most closely to the pathogenesis of emphysema and small airway disease are inhaled pollutants, particularly tobacco smoke, and exacerbations of acute infectious bronchitis. There is, however, wide variations in individual response to these etiological agents, since only a small proportion of subjects develop the disease [20]. The source of these differences is probably the peripheral lung, the « black box », where airborne pollutants challenge the cells and macromolecules of the alveolar membrane, bronchiolar wall and circulating blood or interstitial fluid.

Fig. 1 Diagram summarizing the pathogenesis of chronic obstructive lung disease (COLD). CRI : childhood respiratory infection; ANTI P : antiproteinase; aat : α1-antiproteinase; a2m : α2-macroglobulin; b. inh. : low molecular weight bronchial inhibitor; p : proteinase.

Two biochemical events that may be associated with this condition have so far been investigated : 1) qualitative defects (congenital or acquired) in connective tissue synthesis, and 2) the protease-antiprotease imbalance. We will review briefly what is known and what still remains in the « black box » concerning how these molecular systems are involved in the pathogenesis of emphysema and small airway disease.

On the basis of experimental and human data, a number of pathogenetic hypotheses for pulmonary emphysema have been formulated [6]; only a few of them will be described, and it should be kept in mind that several factors can act together synergistically.

ANIMAL DATA

1. Emphysema has been induced in various species after intratracheal instillation or inhalation of various proteolytic enzymes from plants (papain) [27], bacteria (brinase, pronase) and mammalian cells (pancreatic and leucocytic elastases) [13, 29, 38]. Only elastolytic enzymes, mostly when instillated in the airways, are able to induce emphysema [74]; collagenase does not induce emphysema [44].

A typical picture of panacinar emphysema is produced consistently in hamsters by a single injection of porcine pancreatic elastase [29]. The pathological events of the first few days are characterized by diffuse, acute, inflammatory intra-alveolar exudation of red blood cells and particularly of leukocytes, which is associated with disruption of the alveolar wall (fig. 2). This intra-alveolar attraction of leukocytes during the first days was confirmed by a study of bronchoalveolar lavage after inhalation of papain; this showed a marked rise in polymorphonuclear (PMN) leukocytes, peaking at 24 h, followed four days later by a progressive increase in the alveolar macrophage count [55]. The inflammatory changes diminish regularly over two to three weeks, leaving behind diffuse panacinar emphysema without cell infiltrate; pulmonary capacity and static compliance have been shown to increase up to the 26th week, indicating a slow progression of the emphysema [69].

Fig. 2 Early inflammatory reaction during the three days following an intratracheal instillation of porcine pancreatic elastase (0.2 µg ml) in the hamster.

These pathological observations and the fact that emphysema can be produced in dogs with an aerosol of leukocyte homogenates [52] suggest that elastase- or papain-induced emphysema might develop in two steps : 1) there is an early inflammatory lung injury which is associated with the release of chemotactic factors; 2) subsequently, during the next few days, the leukocytes and macrophages attracted into the alveolar spaces might be responsible for an endogenous elastase release associated with the progressive development of emphysema. This hypothesis is still under controversy (see Ch. KUHN, this meeting, p. 127).

The elastase-induced emphysema is, however, too aggressive a model : there is a need to develop animal models that mimic the human elastase-α1-antiproteinase (AP) imbalance, which associates an α1-AP defect (as obtained recently with chloramine-T [18] or D-galactosamine [8]) with a continuous alveolar release of elastase by leukocytes or alveolar macrophages.

There is both structural and biochemical evidence that changes take place in elastic lung tissue in experimental emphysema induced by elastolytic enzymes : use of special stains for elastic tissue and light microscopy revealed disorganization or disappearance of elastic fibres in alveolar walls [29, 43], and electron microscopy showed destruction of the amorphous core of elastic fibres although the surrounding microfibrils were intact [39]. However, these early lesions appeared to have been repaired after two to four weeks; and, at this time, biochemical analysis showed no quantitative changes in the elastin content of the lung [41, 46] and a relative decrease only in non-polar amino acids of the amorphous core of elastin [51]. During the healing phase, after acute elastase-induced injury, active resynthesis of elastin has been demonstrated [25, 46].

2. Another model has been designed to investigate the role of genetic or acquired defects in elastin and collagen anabolism in emphysema. Administration of various drugs (D-penicillamine, β-lathyrogen) or a copper-deficient diet to experimental animals can induce defects in the cross-linkage of elastin and collagen fibres (fig. 3). In young rats, such treatments can induce emphysema [32, 61]. This model requires further development, since it could help in understanding the cellular and molecular mechanisms involved in the synthesis of lung connective tissue and their interaction with genetic (blotchy or skin-tight mouse) and/or toxic factors.

Fig. 3 Schematic representation of the synthesis of elastic fibers in order to indicate how lathyrogens and D-penicillamine can work to induce emphysema.

3. Many exogenous irritants have been used to induce pulmonary emphysema in animals [13]. Nitrogen oxide- [22] and cadmium- [68] induced emphysema are useful models for understanding the mechanisms leading to various inflammatory reactions, the release of protease and to emphysematous changes, which are not yet clearly understood.

4. There is no strong evidence that an acute, systemic lung injury occurring, for example, after intravenous injection of an endotoxin can lead to pulmonary emphysema; however, in one experiment, repetitive insults with intravenously administered endotoxin for nine consecutive weeks induced a marked sequestration of pulmonary leukocytes and a slight increase in the alveolar linear intercept [76].

HUMAN DATA

Many hypotheses have been suggested to explain the pathogenesis of human emphysema [6]. In this chapter, we will review the clinical, pathological, biochemical and epidemiological bases for only two of them : 1) a genetic or acquired defect in elastic tissue synthesis, and 2) an imbalance in the protease-antiprotease system.

1. Defects in the synthesis of lung connective tissue

Three human diseases involving lysyl oxidase — cutis laxa (dermatolysis), type V Ehlers-Danlos syndrome and, possibly, Marfan syndrome — are due to failure to form stable collagen or elastin cross-links in connective tissue throughout the body [63]. Some patients with these diseases have clinical pulmonary emphysema, with abnormal elastic fibres in the lung [21]. However, these syndromes are highly exceptional sources of pulmonary emphysema.

2. Imbalance in the elastase-antielastase system

2.1. The similarity between some widespread types of human emphysema and the enzyme-induced animal model led to investigation of the different elastases and their inhibitors which may be involved.

a) Elastases. Elastase is one of several proteases isolated from the cytoplasmic granules of human neutrophil leukocytes [3, 35]. Purified elastase can induce emphysema in dogs after intracheal instillation [38]. The proteolytic damage is inhibited by human α1AP [38, 55].

Alveolar macrophages also secrete an elastolytic enzyme, which is a metallo-protease, calcium-dependent and only poorly inhibited by α1AP; these characteristics are very different from those of PMN elastase [17, 28, 30, 66]. The amount of elastase secreted is small or nil when the cells are not stimulated; however, it is very much increased in smokers [66] and during phagocytosis [17]. Moreover the number of macrophages increases three to five fold in smokers, and perhaps also under other environmental conditions. The intratracheal administration of alveolar macrophage proteases to experimental animals produces emphysematous lesions [56]. These cells are probably implicated in the pathogenesis of emphysema in human lungs.

b) Elastase inhibitors. Of the serum protease inhibitors so far identified, α1AP and α2-macroglobulin (α2M) appear to be most important. α2M is a large protein, of 800,000 daltons, which is restricted almost entirely to the vascular compartment and does not normally reach the alveolar spaces. No α2M deficiency has been observed in humans; however, the complexity of the interaction between elastase and α2M and α1AP may result in an increase in human leukocyte elastase activity in the presence of α2M when there is a deficiency of α1AP [24, 73].

α1AP is a glycoprotein of 54,000 daltons which can cross the alveolar capillary membrane to reach the interstitial and alveolar spaces [4]. It is a potent inhibitor of leukocyte elastase, but only partially inhibits alveolar macrophage elastase. Immunochemical quantification and phenotyping of serum α1AP have yielded useful information regarding the characterization of the phenotypes associated with low (Pi Z) or intermediary (SZ, MZ, SS) levels of α1AP [15], but provide no information to assess the functional activity of α1AP.

Apart from these serum inhibitors, a low molecular weight inhibitor is synthesized by the airway cells [31, 62].

2.2. Epidemiological evidence of the role of protease-antiprotease imbalance in the pathogenesis of chronic obstructive lung disease. Studies have been designed from two different points of view : 1) since the blood passes throughout the pulmonary circulation, it would seem logical to expect that there is some link between the symptoms and functional test impairment related to chronic obstructive lung disease and biochemical changes in the serum; 2) in chronic insults to the lung by airborne pollutants and by the cells that are subsequently recruited locally, the effects are concentrated in a critical area represented by the intermediate zone at the junction between the conducting airways and the lung parenchyma. Pollutants all become concentrated in that area since it is there that the air velocity becomes nil, thus facilitating the sedimentation of particles, and since it is there that the clearance mechanisms concentrate pollutants.

a) Peripheral blood as a reflection of chronic lung proteolysis. The significant association between α1AP deficiency and pulmonary emphysema found by LAURELL and ERIKSSON in 1963 [481 has been extensively documented. Sixty to eighty percent of individuals with severe Pi Z α1AP deficiency have clinical, radiological or functional symptoms of pulmonary emphysema [6]; however, the homozygote Pi Z phenotype is rare, being observed in the general population at a frequency of 0.04 or 0.06%. In contrast, other phenotypes (SZ, MZ, SS) associated with intermediate levels of α1AP (120-150 mg/100 ml) occur more frequently in up to 4 % [19, 53]. There is still some controversy concerning the role of this intermediate deficiency in the development of chronic obstructive lung disease : recent transversal epidemiological studies showed no differences in subjects with Pi M phenotypes [58, 59, 60], and this problem probably requires a longitudinal study.

Functional assays of α1AP activity were originally carried out using trypsin as the proteolytic enzyme. Since there is some evidence that trypsin and pancreatic elastase are not inhibited at the same site on the α1AP molecule [16], recent reports pointed out that an assay of elastase inhibition was more appropriate for testing the inhibitory capacity of whole serum or of its α1AP fraction [67]. It was thought that this method could be used to detect any lack of correlation between the serum concentration of α1AP and elastase inhibitory capacity. In fact, recent work with a gel plate assay did not confirm that hypothesis, and showed that there was no evidence of a functional deficiency of α1AP in the patients with chronic obstructive lung disease studied [7]. However, MARTIN and TAYLOR [54], using an agarose-elastin gel, found that PMN lysosomal extract retained residual elastolytic activity more often when incubated with sera of patients with chronic obstructive lung disease that when incubated with that from controls. The reasons for this residual elastolysis remain unclear; the hypothesis suggested by JANOFF and CARP [36] that smoking might denature the antielastase activity of α1AP is not supported by the work of MARTIN and TAYLOR since no correlation was found with smoking history.

Data concerning the elastase activity of blood neutrophils from patients with chronic obstructive lung disease are no clearer, and become even more confusing when Pi phenotype is taken into account [1, 45, 47, 64, 65]. These discrepancies can be related to differences in methods or in the criteria used to select cases [45].

b) Sputum and broncho-alveolar lavage fluid as a reflection of local parenchymal digestion. During the acute exacerbations of chronic bronchitis, the number of leukocytes in bronchial secretions increases dramatically. In sputum, pulmonary inflammation is associated with an increase in leukocyte elastase activity which overwhelms the elastase inhibitory activity [50, 71]. These findings contrast surprisingly with the lack of correlation between bronchial infection and decline in FEV1 reported by FLETCHER et al. [20] in a longitudinal survey of industrial workers. This paradox may be explained by the fact that α1AP increases dramatically during acute infections and that there is inflammatory transudation of other inhibitors, such as α2M, which can compensate for the release of leukocyte elastase.

Recent investigations have shown in vitro that several oxidizing agents (chloramine-T, ozone and the gaseous phase of cigarette smoke) block the elastase inhibitory capacity of human α1AP and that methionyl residues may play an important role in the activity of α1AP [10, 36, 40, 49]. Partial inactivation of lung and serum α1AP in vivo after inhalation of several puffs of cigarette smoke has been demonstrated in rats and in humans [16, 23, 37]. This finding supports the possible role of a defect in the elastase inhibitory capacity of α1AP due to the effect of oxidants on α1AP in the pathogenesis of chronic obstructive lung disease. However, as mentioned above, there is no epidemiological evidence that a chronic functional deficiency of serum α1AP is related to the smoking habit [7, 54]. The oxidizing agents in cigarette smoke may become localized in the lung and may not occur in significant concentrations in the peripheral blood. Thus, in conducting airways and in the alveolar spaces, inactivation of α1AP might induce repeated protease-antiprotease imbalance and thus cause small airway disease and emphysema in smokers, particularly if the cigarette smoke is accompanied by other oxidants of industrial origin and by bacterial infection. It has been shown that human granulocytes can generate hydroxyl radicals, particularly when stimulated by phagocytosis or by immune complexes [11, 57, 75] which can inactivate the elastase inhibitory capacity of α1AP. This would explain why STOCKLEY and BURNETT [71] found a decrease of this capacity in infected sputum from chronic bronchitis patients.

These findings emphasize the complexity of the elastase-α1-antiproteinase balance (fig. 4). Thus, the alveolar macrophages can operate in two directions : by contributing to elastase secretion and by releasing a chemotactic factor for leukocytes [34]; they may also function as a negative feedback system by secreting lysosymes that can inhibit the chemotaxis of PMN leukocytes [26] and by selectively binding and internalizing PMN elastase [9]. This might explain why leukocyte elastase has been found inside human alveolar macrophages [30].

Fig. 4 The protease/α1-antiproteinase balance, with the different regulatory mechanisms.

CONCLUSIONS

Present knowledge about the protease-antiprotease balance suggests that there are two pathogenetic types of emphysema [9] (fig. 5).

Fig. 5 Schematic representation of the pathogenesis of panlobular emphysema (PLE) and centrilobular emphysema (CLE). PMN : polymorphonuclear leukocytes; AM : alveolar macrophages; OH : hydroxyl.

The panlobular type of emphysema might be caused by errors in the remodelling of lung elastin and related proteins after a diffuse, chronic, low-grade elastase injury. The proteolytic enzymes are probably released by leukocytes, monocytes and platelets that are sequestered in the pulmonary capillary bed. The elastolytic activity is particularly significant in the presence of Pi Z α1AP deficiency and perhaps also in smokers, in whom elastase inhibitory capacity is reduced. In this type of emphysema, the lesions occur primarily in the lower lobes where pulmonary circulation is greatest and where cells are sequestered to the greatest extent.

The centrilobular or centriacinar type of emphysema might be caused by a selective destruction of lung structures in the centriacinar area, leading to fenestrations in the alveolar and respiratory bronchiolar walls. Proteolysis is probably due to a release of elastase from leukocytes, and primarily from alveolar macrophages which occur in large quantities in the centriacinar areas in smokers; inactivation of α1AP present in the alveolar lining fluid by oxidants from tobacco smoke, by airborne pollutants or from stimulated leukocytes may initiate this local proteolysis.

It is possible that the protease-antiprotease system also plays a role in the pathogenesis of small airway disease. In elastase-induced emphysema in hamsters, severe inflammatory lesions of the small airways can be observed; and after the initial injury, chronic goblet-cell metaplasia has been seen [12]. In some injuries induced by airborne pollutants, leukocytes or macrophages may be recruited across the bronchiolar epithelium [42] (fig. 6). It is important to determine what happens to the connective tissue components of the bronchiolar wall during this injury, what are the respective roles of elastase, collagenase and other proteases in inducing pathological changes in the small airways, and how the bronchial inhibitor acts to maintain the integrity of the airways.

Fig. 6 Pathogenesis of small airway disease induced by the release of polymorphonuclear (PMN) leukocytes and alveolar macrophages (AM) elastase. The two inhibitors working at this level, α1-antiproteinase (α1AP) and bronchial inhibitor (BI), are indicated.

RÉSUMÉ

Les emphysèmes induits par l’élastase ressemblent à l’emphysème panlobulaire humain tant au plan histologique que biochimique. Ces observations, jointes à l’association de déficit en alpha1-antiprotéase et d’emphysème, suggèrent que cette maladie acquise est la conséquence d’un déséquilibre chronique de la balance protéase-antiprotéase. De nombreux travaux récents ont étudié le rôle de la fumée de cigarette et d’autres polluants. De tels agents sont en effet susceptibles de déséquilibrer cette balance, d’une part en augmentant le recrutement de macrophages et de neutrophiles au niveau des structures bronchiolaires et alvéolaires, avec augmentation de la sécrétion d’élastase, d’autre part en inactivant l’alpha1-antiprotéase par un mécanisme oxydatif. L’élastinolyse qui résulte de ce déséquilibre ne se traduit pas par une baisse quantitative de l’élastine, qui est resynthétisée, mais conduit à la formation d’un réseau de fibres élastiques pulmonaires dystrophiques.

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