Metalloproteinases in Chronic Lung Injury

Lung Emphysema

Lung emphysema is a major pathological condition usually associated with cigarette smoking. The disease is characterized by abnormal and permanent enlargement of respiratory regions of the lung distal to the terminal bronchioles, accompanied by the destruction of the alveolar septa.37 The disease provokes the disappearance or severe disturbance of the orderly structure of the pulmonary acinus where gas exchange occurs, leading to progressive and irreversible respiratory insufficiency. Corresponding to their locations in the secondary pulmonary lobule, two major forms of emphysema have been described: panacinar and centriacinar. Panacinar emphysema, generally seen in patients with a1-antiprotease deficiency, is less common, and is characterized by air space enlargement throughout the acinus. Centriacinar emphysema is strongly associated with cigarette smoking, and results from destruction of alveoli around the proximal respiratory bronchiole.

The most prevalent theory about the pathogenesis of emphysema involves an abnormal balance between proteases and antiproteases in the lung, where emphysema develops as a result of excessive proteinase burden—mainly neutrophil elastase—in the lower respiratory tract.37,39 The hypothesis linking cigarette smoke, increased elastolytic activity, and the development of emphysema attributes the recruitment of elastase-producing neutrophils in smokers' lungs and the inactivation of the a1-proteinase inhibitor to tobacco compounds.

However, several crucial questions challenge this hypothesis: Are neutrophils primary participants in lung destruction? Is neutrophil elastase the only enzyme capable of degrading lung elastin? And are the elastic fibers the only extracellular matrix molecules affected during the development of the disease?

Is Neutrophil the Main Culprit Cell?

Although much attention has focused on neutrophil elastase as a mediator of the lung destruction observed in emphysema, the accumulated evidence is mostly circumstantial, and in fact, studies attempting to find increased tissue levels of this enzyme in emphysematous lesions have yielded controversial results.40,41 The concept that increased numbers of neutrophils and macrophages are present in the lungs of smokers is mainly based on findings in BALF, but few morphologic studies have attempted to describe the types of inflammatory cells present in the alveolar walls of smokers. Nevertheless, macrophages, and not neutrophils, are the most abundant inflammatory cells found in the BALF of cigarette smokers, as well as in the respiratory bronchioles where emphysematous changes first manifest themselves.42,43

But most importantly, a recent morphometric study performed by Finkelstein et al44 held that the extent of emphysema, determined by the volume density of the lung parenchyma, was directly related to the number of alveolar macrophages and T-lymphocytes present in the lesions. Moreover, a negative correlation was found between the number of neutrophils and the amount of lung destruction. Therefore, macrophages are the most abundant inflammatory cell in areas of lung destruction, and alveolar disruption is associated with the presence of this cell type.

Macrophages are a major source of proteases capable for lung destruction, and in addition to interstitial collagenase-1 (MMP-1), they produce at least four different metalloproteinases also able to degrade insoluble elastin: metalloelastase (MMP-12), matrilysin (MMP-7), and gelatinases A and B (MMP-2, MMP-9).32,45-47 Interestingly, several preliminary reports suggest that in both human emphysematous lung tissue specimens and experimentally induced emphysema, the number of cells expressing 92 kDa gelatinase increases.48-50 In these studies, the cells were located along alveolar walls, spaces, and inter-stitium, and most likely represented activated alveolar and interstitial macrophages along with some epithelial cells.

In this way, these inflammatory cells may also contribute to the disruption of elastic tissue. A complication is that alveolar macrophages in the lower respiratory tract of cigarette smokers are a potential source of oxidants capable of inactivating the active site of a1-antiproteinase and rendering it ineffective as an inhibitor of neutrophil elastase.51 Therefore, macrophages, by virtue of their increased numbers in the site of lesions and exaggerated production of metalloproteinases and oxidants, are ideal candidates as agents eroding the morphological and functional integrity of smoker's lungs.

Could the Elastic Fiber Rupture Alone Explain the Emphysematous Lesion?

Alveolar walls are constituted of a dynamic and complex connective tissue framework including interstitial collagens (the predominant component), elastic fibers, proteoglycans, fibronectin, and other glycoproteins.52

In emphysema, the terminal respiratory unit is often completely demolished and moreover, the emphysematous spaces may coalesce into larger bullae which in some cases are several centimeters in diameter. It is difficult to conceive of such damage to the alveolar septa without the action of proteases capable of degrading interstitial collagen—the main component of lung parenchyma extracellular matrix. The collagenases, a subgroup of the metalloproteinases gene family have as members with specific substrate specificity for fibrillar collagen,53-55 interstitial collagenase, neutrophil collagenase, and collagenase 3. In other words, lung destruction occurring during the development of emphysema should necessarily affect more than elastic tissue and involve the multiple action of proteolytic enzymes like interstitial collagenase released into the local milieu.

The first important contribution showing a possible role for interstitial collagenase in the pathogenesis of this disease was published by D'Armiento et al.56 They demonstrated that transgenic mice expressing a collagenase transgene in their lungs developed morphological changes strikingly similar to human emphysema. Histological analysis of the lungs revealed disruption of the alveolar walls and coalescence of the alveolar spaces with no evidence of fibrosis or inflammation. Moreover, those mice expressing the highest levels of the transgene developed the most severe emphysematous lesions. The enzyme used in these transgenic mice was interstitial collagenase (MMP-1), the collagenase present in macrophages and fibroblasts. In support of this finding, lungs showed a conspicuous decrease in collagen fibers whereas elastic fibers seemed to be normal. From this evidence, it was concluded that, the upregulation of interstitial collagenase, an enzyme that does not degrade elastin, provoked lung emphysematous changes.

This finding was later confirmed in our laboratory using an experimental model of lung disease induced by tobacco smoke in guinea pigs.57 During cigarette smoke exposure, lungs exhibited progressive inflammatory lesions of mononuclear predominance, and after 6 to 8 weeks, varied degrees of emphysematous changes were observed. Coinciding with the progression of bronchiolar and alveolar inflammation and development of emphysema-tous lesions, an increased expression and synthesis of interstitial collagenase in alveolar macrophages was identified. Furthermore, these findings were accompanied by an elevation of endogenous collagenolytic activity. The increase in collagenase activity occurred simultaneously with a higher degree of inflammation and alveolar rupture, suggesting that active collagen breakdown takes place during the progression of emphysema. In addition to alveolar macrophages, alveolar epithelial cells also expressed interstitial collagenase. Interestingly, type II pneumocyte cells seem very active in matrix remodeling after injury, complementing the observation that they also produce a number of MMPs in acute lung injury.20 Recent reports have confirmed the upregulation of MMP-1 in human emphysematous lung tissues and the presence of collagenolytic activity in the BALF of patients with pulmonary emphy-sema.50,58,59

Taken together, the findings in transgenic mice, the model of tobacco-induced damage, and in human disease strongly suggest that emphysematous lesions involve more than elastic tissue disruption, and also that the degradation of interstitial collagens contributes significantly to the pathogenesis of this disease.

An Unrestrained Positive Feedback Between Enzymes and Inhibitors?

In dealing with this system, it is important to realize that excessive collagenolytic activity may affect more extensively than normal the lung connective tissue metabolism because both macrophage/fibroblast collagenase and neutrophil collagenase are able to hydrolyze and inactivate (^-proteinase inhibitor.60,61 Evidence like this indicates that MMP-1 and MMP-8 display expanded substrate repertoires and support theories about the existence of a new interface between interstitial matrix turnover and serine proteinase inhibitors. Excessive secretion of interstitial collagenases in the lung microenvironment could conceivably perturb the serine-proteinase/a1-proteinase inhibitor balance and contribute to the recorded increase in elastolytic activity. Similarly, other metalloproteinases such as stromelysins 1 and 3 can cleave a1-antiproteinase and consequently may also potentiate the activity of neutrophil elastase.62,63

Fig. 13.3. Interstitial collagenase and neutrophil elastase may contribute to the development of emphysema both through degradation of matrix molecules as well as by degradation of specific enzymatic inhibitors.

Because of these related activities, it has also been shown that neutrophil elastase and other serine proteases degrade TIMP, thus increasing collagenolytic and other metalloproteinases activities64 (Fig. 13.3).

In this scenario, we postulate that a complex network of interrelated proteinases — serine proteases and metalloproteinases— capable of degrading different extracellular matrix molecules and proteinase inhibitors, are pivotal members in the pathogenesis of pulmonary emphysema.

Diffuse Pulmonary Fibrosis

Pulmonary fibrosis is a consequence of a large number of diseases and a variety of lung injuries.65 In general, lung injury may produce transient and mild pathological changes that are quickly repaired, allowing the lung to return to normal condition. By contrast, if the injury is severe or repetitive, or if it occurs in a "susceptible" individual, the lesion may evolve to diffuse interstitial and intra-alveolar fibrosis.

When fibrosis occurs, these diseases progress slowly into irreversible and lethal conditions and until now, there has been no therapy capable of reverting the pathological process. Idiopathic pulmonary fibrosis (IPF), a disease of unknown etiology, is the prototype of one of the most frequent and aggressive fibrotic disorders of the lung, and is usually fatal, with the survival period averaging four to five years.

Regardless of the etiology, the pathogenesis of diffuse lung fibrosis generally presents the following sequence: initial lung damage, interstitial and intra-alveolar inflammation (alveolitis), fibroblast proliferation, and finally the endstage lung, with its abnormal accumulation of interstitial collagens. In other words, these diseases evolve from an initial cellular inflammatory reaction (early phase), to extensive fibrosis (advanced phase), often ending in terminal honeycomb change (end-stage lung).

The Early Stage. Rupture of the Basement Membranes, a Role for Gelatinases?

Several studies performed in human fibrotic lung disease and in experimental models have furthered the conclusion that, if during the initial injury and subsequent inflammation there is extensive destruction of lung scaffolding and damage to the integrity of the basement membrane repair by epithelial cells becomes impossible. This situation results in a fibrotic response, and in these areas where fibroblast proliferation and fibrosis becomes prominent, hyperplastic type II pneumocytes replace type I alveolar cells and line residual air spaces. For alveolar repair and correct re-epithelialization to occur following injury,66-69 intact basal lamina is essential. Furthermore, intra-alveolar fibrosis, a frequent feature of the fibrotic lung disorders, requires migration of interstitial fibroblasts and deposition of extracellular matrix outside of the epithelial basement membrane. Since mesenchymal cells are confined to the interstitial space bounded by the alveolar basal lamina, focal disruption of this membrane should be required for airspace fibrosis.

Toward this line, Raghu et al16 demonstrated by immunohistochemical methods that early in the course of fibrotic diseases, basement membrane is disrupted by an interstitial collagen invasion of the alveolar spaces. Disruption was signified by gaps in the distribution of type IV collagen and laminin, and in these areas, there was continuity between the interstitial ECM and the ECM occupying the alveolar spaces.

Increased levels of gelatinases A and B, which degrade several components of basement membrane including type IV collagen, seem to be associated with lysis of basal lamina in lung disorders such as acute lung injury, cancer and bronchiectasis.20,70,71 However, their role in fibrotic lung disorders is presently unknown.

Recently, Hayashi and colleagues33 have approached, through immunohistochemical and confocal microscopic techniques, the localization in lung tissues from patients with diffuse alveolar damage and idiopathic pulmonary fibrosis, of gelatinases A and B, TIMP-1 and TIMP-2, and type IV collagen. In the former, the injury initiates inflammatory and fibroproliferative processes that either progress to end-stage fibrosis or resolve themselves completely, whereas IPF appears to be a continuing pathological process with progressive deposition of extracellular matrix and remodeling of lung parenchyma culminating in endstage fibrosis. However, when evolved to fibrosis, the entire process of lung injury and diffuse alveolar damage matures during a period of a few weeks while the process in IPF is a more gradual phenomenon, taking due course in the range of several months to years. The authors found that myofibroblasts and alveolar epithelial cells lining thickened septa had increased reactivity for MMPs and TIMPs in both disorders, although the expression was stronger in diffuse alveolar damage.

Myofibroblasts and type II pneumocytes seem important in the pathogenesis of pulmonary fibrosis. After lung injury, myofibroblasts proliferate and often emerge as the predominant mesenchymal component of the fibrotic tissue. In addition to their contractil capacity, myofibroblasts are active collagen producing cells and have been implicated in lung architectural distortions, contributing to the decreased lung compliance observed in pulmonary fibrosis.72 According to the study of Hayashi et al,33 myofibroblasts may also participate in the fibrous remodeling of extracellular space and basement membrane by secreting MMPs and TIMPs.

On the other hand, failure to replace damaged type I epithelium by proliferation and differentiation of type II pneumocytes appears to be an important determinant of whether or not progression to fibrosis ensues.73,74 When appropriate re-epithelialization does not occur after injury, type II pneumocytes proliferate and line the airspaces, synthesizing several cytokines, growth factors, and MMPs.4,75-77 Interestingly enough, in both diffuse alveolar damage and IPF, the expression of MMP-2 by myofibroblasts and type II pneumocytes showed focal colocalization with type IV collagen, suggesting that activation of this metalloproteinase contributes to subsequent proteolysis of basement membrane components.

In general, these findings show some similarity with those in hyperoxia-induced acute lung injury, where increased gelatinolytic activity accompanied increased expression of MMP-2 and MMP-9 in situ.20 Furthermore, it has been shown that the use of recombinant TIMP-2 significantly reduces immune complex-induced acute alveolitis in vivo, supporting the pathogenic nature of gelatinases.78 In this context, a potential hypothesis is that following lung injury, gelatinases A and B are overexpressed, provoking focal disruptions of the alveolar basement membrane and deteriorating normal re-epithelialization, enhancing fi-broblast migration to the alveolar spaces. In light of these properties, it is probable that a rapid response mediated by TIMPs would revert this process.

However, given their pleomorphic functions, MMP and TIMP function in basement membrane turnover during lung fibrosis should be considerably complex, and obviously further studies are necessary to pinpoint the enzyme-inhibitor in vivo relationship.

The Fibrotic Phase. A Role for Interstitial Collagenases?

Independently of etiology, the final common pathway in pulmonary fibrosis is the inappropriate and uncontrolled accumulation of collagenous extracellular matrix in the distal respiratory tract, which eventually leads to destruction of the lung parenchyma. An important aspect of pulmonary fibrosis development is the altered metabolism of several components of the extracellular matrix. However, the role of increased deposits of elastin, proteoglycans, and fibronectin in the pathogenesis of lung fibrosis and in the loss of lung architecture remains unclear. Nonetheless, the absolute increase of collagens, taken together with the abnormalities in their spatial distribution, seem culpable for the disorganization and distortion of the normal lung parenchyma during fibrosis progression. In this way, collagen metabolism regulation is central to the pathogenesis of fibrosis that follows injury and inflammation.

Collagen accumulation appears to be progressive because of demonstrations that the concentration of this protein is usually higher in postmortem lung tissues than in biopsy samples taken in the same patients months or years before.79,80 All interstitial collagens increase in the lung parenchyma, but the temporal patterns of deposition differ among them. Thus, a predominance of the type III and VI collagens associated with loose, reticular matrix, can be observed in sites of early or active fibrogenesis, while an abundance of dense connective tissue composed mainly fibrillar type I collagen is observed in areas of advanced fibrosis.16,81

In a normal adult, the net matrix production is zero, the concentration of collagen per unit lung mass remains constant, and matrix synthesis and degradation are balanced.82 Therefore, collagen accumulation in pulmonary fibrosis represents an imbalance in the collagen turnover rates favoring degradation, and resulting in an excessive deposit of this protein. This increase in collagen content must result from an increase in synthesis, a decrease in degradation, or the sum of both processes.

Rates of synthesis are usually increased in experimental lung fibrosis, although the two studies performed in humans have failed to provide direct evidence for increased lung collagen production.79,83 Figures obtained from animal models indicated a significant but transient increase in collagen synthesis, whereas collagen accumulation is usually constant.84,85 Since human studies have been done in advanced stages of the disease, increased collagen biosynthesis could be an early and transient stage during the development of pulmonary fibrosis, perhaps representing the activation or selective expansion of high collagen-producing fibroblasts or myofibroblasts. Indirect evidence for this hypothesis is given by an immunohistochemical study performed by Kuhn et al,86 who corroborated that in the lungs of patients with early stage fibrosis, a large population of collagen-producing cells was typical. By contrast, in biopsies of patients with chronic pulmonary fibrosis, none or only a few type I collagen-synthesizing fibroblasts were identified. Moreover, foci of highly activated fibroblasts expressing type I collagen were seen in areas of active fibrogenesis whereas no type I procollagen expression was observed in dense fibrotic areas of the lung.76

On the other hand, several lines of evidence indicate that changes in collagen degradation are an integral part of the fibrotic process. Normally, a significant amount of collagen synthesized by mesenchymal cells is intracellularly degraded. Therefore, the amount of procollagen secreted might be modulated by alterations in this intracellular degradative pattern. In this context, increased collagen secretion might be at least partially associated with a decrease in the proportion of procollagen degraded intracellularly.87

However, once the lung collagen deposit forms, it is mainly modulated by the extracellular collagenolysis, of interstitial collagenases, which are unknown entities in the patho-genesis of lung fibrosis. One possibility is that collagen degradation is crucial to in the abnormal remodeling observed during fibrosis. Evidence for this hypothesis is largely related to the discovery of comparatively higher levels of active collagenase in BALF from patients with interstitial lung diseases.88-90 However, other studies have demonstrated that collagenase and collagenolytic activity can be present or absent in BALF without any association with the activity and prognosis of the disease or with tissue collagenolytic activity.91-93

Therefore, in accordance with the results obtained in a number of studies in our laboratory, we postulate that a decrease in collagen degradation is an essential mechanism for collagen accumulation in the fibrotic lung. As an example in support, patients with IPF usually exhibit a remarkable decrease in lung collagen degradation.79 Furthermore, in chronic hypersensitivity pneumonitis, a disease in which most patients improve or heal, we have demonstrated that healing appears associated with higher levels of lung collagenolytic activity, while progression to fibrosis seems related to significantly lower collagenase activity.92 Though regulated by complex mechanisms, a decrease in collagenolytic activity, is probably related to a decreased expression and synthesis of interstitial collagenases or with an upregulation of TIMPs. In support of this point of view, we have found that the TIMP-1/ collagenase molar ratio is higher in fibroblasts derived from human fibrotic lungs than in normal ones.94 This finding makes possible the presence of lung fibroblast subpopulations with different abilities to produce collagenase and TIMP. In fibrotic lungs, low-collagenase, high TIMP producing subsets predominate. Likewise, collagenase inhibitory activity appears significantly higher in the lung parenchyma of patients with IPF and chronic hypersensitivity pneumonitis.95 Furthermore, in the fibrotic phase of both diffuse alveolar damage and IPF, TIMP-1 and TIMP-2 increase in areas of dense fibrosis, suggesting that upregulation of these inhibitors contributes to the stability of collagen and the other matrix components.

Sequential studies of experimental lung fibrosis induced in rats using paraquat and oxygen or silica, have consistently revealed the dual nature of collagenolysis. During the early inflammatory process, experimental rats generally display a clear increase in lung collagen degradation.96,97 However, in more advanced phases that coincide with active fibrogenesis, nearly all rats displayed a significantly lower rate of collagenase activity. While increased collagenolytic activity is one outcome, the initial increase of matrix degradation at sites of parenchymal inflammation could also contribute to matrix reorganization through more subtle means. For example, preliminary studies performed in our laboratory have shown increased collagenase-3 mRNA lung expression in experimental silicotic rats, principally in areas of developing silicotic granulomas.98 On the other hand, and analogous to human studies, an upregulation of TIMP-1 appears to occur in experimental lung fibrosis.99

The conclusion to be drawn from this data is that a decrease in collagenase activity is a general mechanism of tissue fibrotic processes, and has been well documented in cirrhotic livers, in the skin of patients with Progressive Systemic Sclerosis, and in keloid lesions.100-102

Regulatory Cytokines. A Key Role for Transforming Growth Factor Beta (TGF-p)?

A great variety of cytokines capable of affecting every part of the pathological processes are upregulated and released by inflammatory, connective tissue, and epithelial cells during the development of the diffuse lung fibrotic disorder. These include TNF-a, PDGF, and TGF-p, all considered profibrotic cytokines.75-77 In particular, TGF-p, secreted first by macrophages and in more advanced stages by fibroblasts and epithelial cells, maximizes collagen accumulation by getting at several points in the pathway of collagen synthesis and degradation. It stimulates procollagen gene transcription, increases a-chain mRNA stability, decreases procollagen intracellular degradation, inhibits collagenase expression, and stimulates TIMP production.103-105 Furthermore, TGF-p stimulates the synthesis of 72 kDa type IV collagenase and consequently may participate in basement-membrane restructuring.106 In addition of enhancing a fibrotic response, TGF-p may also contribute to lung damage by inhibiting both the proliferation and differentiation of alveolar epithelial cells.107 This cytokine has also been connected to the emergence of myofibroblasts in the lung microenvironment during fibrotic processes.108 Taken together, these fragments of evidence support a central role for TGF-p in the pathogenesis of diffuse lung fibrosis. However, it is important to consider that the fibrotic response represents an imbalance between the profibrotic and antifibrotic cytokines released, where profibrotic prevail and provoke exaggerated collagen accumulation. This phenomenon was recently confirmed by the finding that aFGF, as profibrotic cytolcine, and its receptors are upregulated during the entire evolution of a pulmonary fibrosis induced by paraquat plus hyperoxia.109 The expression of this factor seemed to be stronger in areas where the lung architecture was better preserved. Recent evidence suggests that aFGF appears to exert an antifibrotic role since downregulates a1 (I) collagen gene expression and upregulates interstitial collagenase expression by lung fibroblasts.110

Further research and analysis of the regulation of MMPs and TIMPs in progressive fibrosis are required for the satisfactory understanding of detailed mechanisms of excessive extracellular matrix accumulation.


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