The dual role of neutrophil elastase in lung destruction and repair

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The International Journal of Biochemistry & Cell Biology 40 (2008) 1287–1296

Review

The dual role of neutrophil elastase in lung destruction and repair Giuseppe Lungarella ∗ , Eleonora Cavarra, Monica Lucattelli, Piero A. Martorana Department of Physiopathology and Experimental Medicine, University of Siena, via Aldo Moro n.6, I-53100 Siena, Italy Available online 24 December 2007

Abstract The purpose of this review was to modify the prevailing view that neutrophil elastase (NE) is mainly a matrix-degrading enzyme. Recent observations indicate that the role of NE in inflammation is more complex than the simple degradation of extra-cellular matrix components. Several lines of evidence suggest that NE aims specifically at a variety of regulatory functions in local inflammatory processes. This enzyme can modulate many biological functions by promoting chemokine and cytokine activation and degradation, cytokine receptor shedding, proteolysis of cytokine binding proteins and the activation of different specific cell surface receptors. However, the current knowledge of regulatory mechanisms by which NE potentially regulates inflammatory processes is primarily derived from in vitro studies. The extent of these NE-dependent pathways and their relevance under various pathophysiological conditions remains poorly understood and a matter for further investigation. Recent studies suggest that NE not only plays a key role in lung destruction (emphysema) but can also modulate proliferative changes (fibrosis) in inflammatory processes. Thus, NE could be considered to have potential multiple roles in the pathogenesis of both emphysema and lung fibrosis. © 2007 Elsevier Ltd. All rights reserved. Keywords: Neutrophil elastase; Inflammation; Cytokine network; Lung emphysema; Lung fibrosis

Contents 1. 2. 3. 4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neutrophil elastase, more than an elastolytic enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neutrophil elastase in pulmonary emphysema and fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pulmonary emphysema and fibrosis can coexist. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neutrophil elastase as a putative link between emphysema and fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: A1 AT, alpha1 antitrypsin; BAL, bronchoalveolar lavage; BLM, bleomycin; DIP, Desquamative Interstitial Pneumonia; ECM, extracellular matrix; IPF, Idiopathic Pulmonary Fibrosis; Lm, mean linear intercept; NE, neutrophil elastase; PAR-1, proteinase activated receptor 1; PLCH, Pulmonary Langerhans’ Cell Histiocytosis; RB-ILD, Respiratory Bronchiolitis-associated Interstitial Lung Disease; UIP, usual interstitial pneumonia. ∗ Corresponding author. Tel.: +39 0 577 234 028; fax: +39 0 577 234 019. E-mail address: [email protected] (G. Lungarella). 1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.12.008

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1. Introduction Neutrophils are recruited to the lung at the site of inflammation in response to infection and inflammation. These cells are one of the first lines of host defense against infection achieving their anti-microbial function through a series of coordinated responses (intracellular sequestration of microorganisms into the phagosome, fusion of neutrophil granules with the phagolysosome, release of proteases and antimicrobial peptides into the phagolysosomes, release of large quantities of reactive oxygen species generated by the activation of the membrane bound NADPH oxidase system) that culminate in the destruction of pathogens (Nathan, 2006). Based on their protein content, their formation during different stages of cell differentiation and their tendency to undergo exocytosis following neutrophil activation by inflammatory stimuli or phagocytosis of invading pathogens, neutrophil granules are classified into four types: primary (also known as azurophil) granules, secondary (also known as specific) granules, tertiary (also known as gelatinase) granules and secretory granules (Faurschou & Borregaard, 2003). The azurophil granules, which mainly contribute to the intracellular degradation of microorganisms in phagolysosomes, contain a high amount of bactericidal protein defensins, myeloperoxidase and a family of structurally related serine proteases, such as neutrophil elastase (NE), cathepsin G and proteinase 3. The importance of these enzymes in the antimicrobial action of neutrophils in vivo has been recently demonstrated (Belaaouaj et al., 1998; Reeves et al., 2002; RibeiroGomes et al., 2007; Tkalcevic et al., 2000). Although the role in host defense has been acknowledged, neutrophil serine proteinases have also been implicated in various non-infectious, inflammatory processes. For example, during an acute inflammatory response these enzymes may be released extracellularly playing crucial roles in extracellular proteolytic processes at sites of inflammation (Antonicelli, Bellon, Debelle, & Hornebeck, 2007). NE is believed to play a particularly important pathogenic role in several lung conditions including pulmonary emphysema (Snider, 1989), acute lung injury (Lee & Downey, 2001), acute respiratory distress syndrome (Lee & Downey, 2001) and chronic inflammatory airway diseases (Fischer & Voynow, 2002). NE is able to degrade most of the components of the pulmonary extracellular matrix (ECM), including elastin, type I–IV collagens, proteoglycans fibronectin and laminin (Ginzberg et al., 2001) and to induce apoptosis of epithelial cells via activation of surface receptors

such as proteinase activated receptor 1 (PAR-1) (Suzuki et al., 2005). 2. Neutrophil elastase, more than an elastolytic enzyme Due to these capacities, until recently most studies have focused on the deleterious effects of NE on the ECM components in various pathological conditions. However, over the past few years it has become increasingly clear that various bioactive molecules (i.e. cytokines and chemokines), specific cell surface receptors and cytokine binding proteins are also natural substrates of NE (Table 1). This led to the hypothesis that extracellularly released neutrophil serine proteases, and in particular NE, have active regulatory functions during inflammation in tissue destruction and in repair. The interaction of NE with the cytokine network includes (a) the release of active cytokines from their inactive precursor molecules, (b) proteolytic cleavage, and thus inactivation of active cytokines, (c) proteolysis of cell surface-bound cytokine receptors, (d) proteolysis of the cytokine binding proteins, and finally, (e) the activation of specific cell surface receptors (such as PARs or Toll-like receptor 4). In particular, recent findings indicate that NE can modulate the activity of important regulators of (a) inflammatory processes (such as TNF-␣, IL-6, IL-8, SDF1␣, MIP1␣, chemerin, GCSF), (b) adaptive immune responses (such as IL-2, chemerin, CD2, CD4, CD8) or (c) repair processes (such as TGF-␤, TGF-␣, EGF, IGF, TNF-␣, TNF-RII, proepithelin). Unfortunately, the current knowledge of the molecular mechanisms by which NE potentially regulates these processes has been deduced primarily from in vitro studies. The net biological effects in vivo might reflect a balance between the levels of activation/inactivation of various cytokines and receptors by NE, and it may be greatly influenced by the different microenvironments at sites of inflammation. Thus, the relevance of these NE-dependent pathways for specific disease processes still remains poorly understood and a matter for further investigations. In support of the role of NE in the local inflammatory response, the inhibition of this enzyme has been shown to reduce leucocyte infiltration and neutrophil mediated injury in several in vivo models of inflammation such as collagen-induced arthritis, ischemia and reperfusion injury and endotoxin-induced acute lung injury (Carden & Korthuis, 1996; Kakimoto, Matsukawa, Yoshinaga, & Nakamura, 1995; Kawabata et al., 2000). Furthermore, there is growing evidence that NE plays a dual role in lung destruction and repair.

Table 1 Overview of different targets of neutrophil elastase Hypothetical biological function

Reference

Cytokines/chemokines Chemerin IL-8 (CXCL8) IL-6 TNF-␣ IL-2 SDF1␣ (CXCL12) MIP1␣ (CCL3) EGF GCSF

Modulation of cytokine half-life time, activation of chemotaxis Modulation of cytokine half-life time, activation of chemotaxis Modulation of cytokine half-life time Modulation of cytokine half-life time Modulation of cytokine half-life time Modulation of cytokine half-life time, inhibition of chemotaxis Modulation of cytokine half-life time, inhibition of chemotaxis Modulation of cytokine half-life time Growth inhibition

Wittamer et al. (2005) Leavell, Peterson, and Gross (1997) Bank, Kupper, Reinhold, Hoffmann, and Ansorge (1999a) van Kessel, van Strijp, and Verhoef (1991) Ariel et al. (1998) Rao et al. (2004) Ryu et al. (2005) DiCamillo et al. (2006) Hunter, Druhan, Massullo, and Avalos (2003)

Integrins/others ICAM-1 Vascular endothelium cadherin Proepithelin TGF-␤ binding protein IGF-binding proteins

Modulation of adhesion Modulation of adhesion Wound healing Enhanced growth factor availability Enhanced growth factor availability

Champagne, Tremblay, Cantin, and St Pierre (1998), Robledo et al. (2003) Hermant et al. (2003) Zhu et al. (2002) Hyytiainen, Taipale, Heldin, and Keski-Oja (1998) Gibson and Cohen (1999)

Activation, modulation of response Inactivation, modulation of response, apoptosis Inactivation, modulation of response Inactivation, modulation of response Inhibiting cellular response and prolongation of cytokine half-life time Inhibiting cellular response and prolongation of cytokine half-life time Inhibition of chemotaxis, feedback mechanism Inhibition of complement signaling Modulation of cell migration Growth inhibition Modulation of adhesion

Walsh et al. (2001), Devaney et al. (2003) Loew et al. (2000), Suzuki et al. (2005) Loew et al. (2000) Cumashi et al. (2001) Bank et al. (1999b) Porteu, Brockhaus, Wallach, Engelmann, and Nathan (1991) Tralau, Meyer-Hoffert, Schroder, and Wiedow (2004) Sadallah et al. (1999) Beaufort et al. (2004) Hunter et al. (2003) Hunter et al. (2003) Galon et al. (1998) Le Barillec, Si-Tahar, Balloy, and Chignard (1999) Doring et al. (1995) Doring et al. (1995) Doring et al. (1995)

Receptors TLR4 PAR-1 PAR-2 PAR-3 IL-2R␣ TNF-RII CD88 (C5aR) CD35 (C3b/C4b R) CD87 (urokinase R) GCSF-R CD43 (sialophorin) CD16 CD14 CD2 CD4 CD8

Inhibition of LPS-mediated cell activation Impairment of T lymphocytes Impairment of T lymphocytes Impairment of T lymphocytes

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Target

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This review focuses on this particularly intriguing aspect of NE as mediator of extracellular matrix destruction and accumulation in the lung. There is sufficient evidence to suggest that NEdependent pathways in the cytokine network are associated with the development of pulmonary emphysema and fibrosis, and consequently that NE may be a common pathogenic link between these two disorders. 3. Neutrophil elastase in pulmonary emphysema and fibrosis The dual role played by NE is highlighted by its involvement in the pathogenesis of the two lung diseases that exemplify destructive and reparative processes, emphysema and fibrosis. Pulmonary emphysema is a destructive lesion of the lung parenchyma. At a National Heart, Lung, and Blood Institute workshop on emphysema held in 1985, this disease was defined in anatomical terms as a condition of the lung characterized by abnormal, permanent enlargement of airspaces distal to the terminal bronchiole and accompanied by destruction of their walls (Snider, Kleinermann, Thurlbeck, & Bengali, 1985). It is generally accepted that cigarette smoking is the main risk factor for the development of emphysema. The pathogenesis of emphysema has been a matter of debate for long time, however the most widely accepted hypothesis is based on the interaction between proteases and antiproteases. The protease-antiprotease hypothesis for the development of emphysema originated in the early 1960s. At that time, a clinical investigation indicated that patients with a deficiency of serum antiprotease alpha1 antitrypsin (A1 AT) developed severe, early onset emphysema (Laurell & Eriksson, 1963). The protease-antiprotease hypothesis suggested that emphysema may result from an imbalance, either inborn (A1 AT deficiency) or acquired (cigarette smokeinduced oxidative inactivation of A1 AT) of the elastase/antielastase homeostasis in the lung. Since human neutrophil elastase was also found to induce emphysema when instilled in the lung of laboratory animals (Senior et al., 1977), this protease has been considered to be the main, but not the only, culprit in the pathogenesis of emphysema for more than 30 years. It has been shown that other proteinases might contribute to emphysema (Churg et al., 2003, 2004; Hautamaki, Kobayashi, Senior, & Shapiro, 1997), but it is likely that NE plays a significant role. In particular, it has been demonstrated that the lesion of the matrix

which results in emphysema is the end-result of crosstalk between macrophage metalloelastase and neutrophil elastase (Churg et al., 2002; Shapiro et al., 2003). However, the latter protease is responsible for the greater portion of the final proteolytic attack (Churg et al., 2002). On the other hand, pulmonary fibrosis and in particular the most common form idiopathic pulmonary fibrosis (IPF) also known as cryptogenic fibrosing alveolitis, is a proliferative disease of the lung defined as “a distinctive type of chronic fibrosing interstitial pneumonia of unknown cause limited to the lungs and associated with a histologic pattern of usual interstitial pneumonia (UIP)” (ATS/ERS, 2002). The main histologic features of the UIP pattern include structural destruction, fibrosis often associated with honeycombing, scattered fibroblastic foci, patchy distribution and involvement of the periphery of the lobule. IPF/UIP has a heterogeneous appearance with alternating areas of normal lung, interstitial inflammation, fibrosis and honeycomb changes. These changes most severely affect the subpleural parenchyma. Interstitial inflammation is usually mild to moderate, patchy, and consists of an alveolar septal infiltrate of lymphocytes, plasma cells, and histiocytes associated with hyperplasia of type II pneumocytes. The fibrotic areas show deposition of extracellular matrix, such as collagen and fibronectin, and scattered fibroblastic foci. Smooth muscle hyperplasia is commonly seen in areas of fibrosis and honeycomb change (ATS/ERS, 2002). Other forms of lung fibrosis, such as Desquamative Interstitial Pneumonia (DIP), Respiratory Bronchiolitisassociated Interstitial Lung Disease (RB-ILD), and Pulmonary Langerhans’ Cell Histiocytosis (PLCH) are usually associated with cigarette smoking (ATS/ERS, 2002). Several animal (Azuma et al., 1998; Gardi et al., 1994, 1990; Mitsuhashi et al., 1996; Nagai et al., 1992; Pacini et al., 1990; Taooka, Maeda, Hiyama, Ishioka, & Yamakido, 1997) and human (Cardoso, Sekhon, Hyde, & Thurlbeck, 1993; Cottin et al., 2005; Finlay, O’Donnell, O’Connor, & FitzGerald, 1996; Hiwatari, Shimura, & Takishima, 1993; Lang et al., 1994; Merritt et al., 1983; Mura et al., 2006; O’Donnel et al., 1999; Wells et al., 2003) studies have suggested an involvement of NE in the development of lung fibrosis. Some years ago, it was shown that lung collagen breakdown products, obtained by digestion with elastase, were able to induce collagen synthesis in fibroblasts (Gardi et al., 1994). A long-term treatment of rabbits with these peptides led to the induction of pulmonary fibrosis (Gardi et al., 1990; Pacini et al., 1990).

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In 1993 it was reported that the administration of A1 AT significantly reduced pulmonary lesions in a model of bleomycin-induced lung fibrosis in hamsters. However, no evidence could be found that this effect was due to altered elastase activity (Nagai et al., 1992). This evidence was found a few years later in a similar model of bleomycin-induced lung fibrosis in the hamster in which treatment with a truncated secretory leukoprotease inhibitor (SLPI) significantly ameliorated pulmonary fibrosis. This effect was associated with a dose-dependent inhibition of elastase activity in bronchoalveolar lavage (BAL) fluids, suggesting that neutrophil elastase may be implicated in the pathogenesis of BLM-induced pulmonary fibrosis (Mitsuhashi et al., 1996). Similarly, administration of the low molecular weight NE inhibitor ONO-5046 in mice challenged with bleomycin suppressed the appearance of the pulmonary lesion and significantly prevented the increase of interleukin 1-␤, macrophage inflammatory protein2, platelet-derived growth factor-A and insulin-like growth factor-1 mRNA levels in BAL cells. The authors suggested a potential role of these cytokines in the fibrogenic process and postulated that NE may be involved in the early stages of lung inflammation during the development of pulmonary fibrosis (Taooka et al., 1997). The early development of fibrosis was also investigated in a rat model of bleomycin-induced acute lung injury. In this model, the administration of erythromycin significantly prevented the appearance of the lung lesion and this effect was accompanied by a decrease in NE activity and interleukin-8 content in BAL fluids (Azuma et al., 1998). Furthermore, clinical studies have demonstrated that a lack of A1 AT activity (the major serum inhibitor of neutrophil elastase) in infants with Respiratory Distress Syndrome is associated with chronicity and the development of fibrosis (Merritt et al., 1983). 4. Pulmonary emphysema and fibrosis can coexist In a workshop report of the National Heart, Lung, and Blood Institute the definition of emphysema also included the condition “and without obvious fibrosis” (Snider et al., 1985). Thus, the generally accepted definition of emphysema excluded coexistence with lung fibrosis. However, studies then started to appear that documented the coexistence of these two diseases in humans and in experimental animals.

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4.1. Human studies Cardoso et al. examined 147 samples of human lungs removed for cancer. Elastin was decreased in all grades of panacinar air-space enlargement and also in severe centriacinar air-space enlargement. Collagen in the homogenates was increased only in irregular air-space enlargement, but collagen was consistently increased histochemically in centriacinar, distal acinar, and irregular air-space enlargement sections. Thus, these data indicate that destructive matrix changes coexist with proliferative ones. The authors concluded: “The question arises whether the current definition of emphysema is appropriate” (Cardoso et al., 1993). Similarly, in another study it was reported that there is a loss of airspace wall tissue that is accompanied by a net increase in collagen mass in smokers with emphysema. These results suggested an active alveolar wall fibrosis in emphysema as a consequence of cigarette smoking (Lang et al., 1994). In another study the extent of extracellular matrix remodeling in human emphysematous lungs was assessed by ultrastructural examination of elastin and collagen templates. In emphysematous lungs, sheets of elastin were disrupted and perforated with multiple fenestrations. This disintegration was accompanied by a pattern of thickened collagen fibrils and disorganized collagen deposition. The authors concluded that their findings supported the concept of increased collagen deposition and aberrant collagen remodeling in the pathogenesis of emphysema (Finlay et al., 1996). The coexistence of emphysema and fibrosis in man has been repeatedly reported in subsequent years (Hiwatari et al., 1993; Mura et al., 2006; Wells et al., 2003) and it is interesting that centrilobular emphysema may be associated with some subsets of idiopathic interstitial pneumonias, such as DIP, RB-ILD, PLCH and IPF in smokers and ex-smokers. This was clearly outlined in a recent ATS/ERS document defining the clinical manifestations, pathology and radiologic features of patients with Idiopathic Interstitial Pneumonias (ATS/ERS, 2002). The coexistence of emphysema and fibrosis in man has recently been characterized as a distinct entity, “Combined Pulmonary Fibrosis and Emphysema” (Cottin et al., 2005). 4.2. Animal studies The coexistence of emphysema and fibrosis has also, been observed in some animal models, when the main goal was the induction of emphysema, that was found to be accompanied by fibrotic foci, and when the induction of fibrosis was associated with emphysema.

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In models of emphysema, scanning electron microscopical examination of rat lungs treated intratracheally with porcine pancreatic elastase showed sheets of disrupted and perforated elastin with multiple fenestrations and a marked increase in the thickness of collagen fibrils (Finlay et al., 1996). Similarly, using this technique the lungs of tight-skin mice, a mutant with spontaneously occurring emphysema, showed a fragmentation and distortion of elastin surrounding enlarged alveolar sacs and ducts, associated with thickening and matting of the underlying collagen scaffold (O’Donnel et al., 1999). Recently, in a model of cigarette smoke-induced emphysema, DBA/2 mice were exposed to cigarette smoke for 6 months. After 3 months of smoke exposure these mice already showed overt emphysema characterized by significant changes in the mean linear intercept (Lm) and in the internal surface area. Interestingly, the foci of fibrosis were seen after 4 months of smoke exposure and their severity progressively increased with time. Six months after smoke exposure the fibrotic lesions mainly consisted of subpleural foci (Fig. 1). In some areas the fibrotic reaction was seen in the lung parenchyma associated or not with foci of emphysema (Bartalesi et al., 2005; Lucattelli et al., 2005). The appearance of emphysema in models of fibrosis is also well documented. It was reported in 1982 that cadmium chloride administered intratracheally to hamsters caused an acute lung injury which evolved into a lesion with functional and morphological features of diffuse fibrosis. With simultaneous feeding of a lathyrogen, beta-aminoproprionitrile, this injury evolved into functional and morphological changes of bullous emphysema. Intriguingly, these results suggested that the same lung injury might result in either fibrosis or emphysema (Niewoehener & Hoidal, 1982).

More recently, the dynamic changes in fibrosis were investigated in 3 strains of mice either sensitive (C57Bl/6J and their mutant pallid) or resistant (Balbc) to the fibrogenic agent bleomycin (BLM) (Cavarra et al., 2001). In Balbc mice following intratracheal BLM, negligible foci of cellular infiltration and fibrosis and no areas of air-space enlargement were detected during the period of the study. On the contrary, a progressive increase in areas of fibrosis and emphysema was seen in alpha1-PI deficient C57Bl/6J and pallid mice. Interestingly, at 3 and 7 days after BLM, C57Bl/6J and pallid mice showed significant morphologic emphysema with spotty areas of inflammatory cell infiltration in absence of fibrotic changes. At these time periods the anatomical emphysema was associated with a significant increase in the Lm and a significant decrease in lung desmosine content. The lungs of C57Bl/6J and pallid mice showed large areas of fibrosis only at 14 days after BLM. Both lesions (emphysema and fibrosis) were widely spread and intermixed. Nevertheless, areas of emphysema could also be detected in lung lobes without any fibrotic reaction thus distant from fibrotic foci. These findings are interesting, particularly in light of recent reports on transgenic mice overexpressing the inflammatory cytokine tumor necrosis factor-␣ (TNF␣). On one hand, mice overexpressing TNF-␣ have been reported to have fibrosis in the parenchyma, together with elevated lymphocytes, macrophages and neutrophils within the interstitium, leading to the conclusion that it could be considered a model of idiopathic pulmonary fibrosis (Miyazaki et al., 1995), On the other hand, another study (Fujita et al., 2001) found that the inflammation in this mouse is resolved by the age of 6 months, at which time the lung histology and physiologic function is more consistent with emphysema. Another

Fig. 1. DBA/2 lung 6 months after chronic cigarette smoke exposure. Areas of moderate emphysema are associated with foci of subpleural and septal fibrosis (A). (B) Shows a higher magnification of (A). Hematoxylin-eosin stain, original magnification, 40× (A) and 100× (B).

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study in a mouse with an inducible TNF-␣ gene construct suggested that TNF-␣ expression in the lung may produce emphysema in conjunction with lymphoid follicles (Vuillemenot, Rodriguez, & Hoyle, 2004). Finally, in another study pathological changes consistent with both emphysema and fibrosis were demonstrated in TNF-␣ over-expressing transgenic mice (Lundblad et al., 2005). Thus, these two disorders, emphysema and fibrosis, once believed to be basically different in downstream pathogenesis (a destructive vs. a proliferative process) have been observed to coexist in the same lung, both in humans and in animal models. This may suggest a common pathogenic mechanism particularly since both disorders are associated with an ongoing inflammatory process. 5. Neutrophil elastase as a putative link between emphysema and fibrosis A recent study has highlighted NE as a common pathogenic mechanism linking pulmonary emphysema and fibrosis (Lucattelli et al., 2005). This study was done in two animal models in which emphysema and fibrosis were induced either by bleomycin (BLM) or by chronic exposure to cigarettesmoke. In order to assess the protease-dependence of the BLM-induced lesion, a group of mice was treated with 4(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, a serine proteinase inhibitor active against neutrophil elastase. The results obtained in this study demonstrate that in BLM-treated ␣1 -proteinase inhibitor deficient mice (i) the development of elastolytic emphysema precedes that of fibrosis; (ii) a significant amount of elastase in the alveolar interstitium is associated with an increased expression of TGF-␤ and TGF-␣; and finally, (iii) emphysematous and fibrotic lesions can be significantly attenuated by using a protease inhibitor active against neutrophil elastase. In DBA/2 mice that develop both emphysema and fibrosis after chronic cigarette-smoke exposure, the presence of elastase in alveolar structures is also associated with a positive immunohistochemical reaction of both TGF-␤ and TGF-␣ (Bartalesi et al., 2005). These results strongly suggest that neutrophil elastase may be a common pathogenic link between emphysema and fibrosis, acting as a regulatory factor in the generation of soluble cytokines with mitogenic activity for mesenchymal cells resulting either in emphysema or in fibrosis or both (Lucattelli et al., 2005). In a recent study the release of fibroblast growth factor-2 (FGF-2) and TGF-␤ was investigated in mice

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after intratracheal instillation of porcine pancreatic elastase (Buczek-Thomas et al., 2004). It was found that elastase promoted a time-dependent release of FGF-2 and TGF-␤ from the lung into BALF. A large percentage of the TGF-␤ in BALF was an active form, suggesting that elastase may participate in the activation of TGF-␤ from its latent form. Analysis of the levels of FGF-2 and TGF-␤ in mouse blood indicated that the growth factors in BALF were not entirely derived from blood. Moreover, elastase treatment of pulmonary fibroblast cultures caused the release of TGF-␤, suggesting that the TGF-␤ in BAL fluids could have come from lung cells/matrix (Buczek-Thomas et al., 2004). This study has clarified the role of elastase on the release of TGF-␤, which may be important for the development of fibrosis. The potential role of NE and TGF-␤ in the lung fibrogenic response to bleomycin has been recently confirmed “in vivo” in mice lacking NE (Chua et al., 2007). This is an additional proof of concept for future studies regarding the role of NE in human IPF. It has been proposed that the levels of protease burden, and therefore the differences in cleavage and removal of collagen molecules, may affect the remodeling of ECM after alveolar injury. A recent study has provided evidence that different interstitial levels of NE burden in emphysema may be associated with different routes of collagen clearance (intracellular vs. extracellular) and different degrees of remodeling of the ECM in emphysema (Lucattelli et al., 2003). This point merits further investigation. 6. Conclusions Recent observations indicate that the role of NE in inflammation is more complex than the simple degradation of ECM components. Several lines of evidence suggest that NE aims specifically at a variety of regulatory functions in local inflammatory processes. However, the current knowledge of regulatory mechanisms by which NE potentially regulates inflammatory processes is primarily derived from in vitro studies. The extent of these NE-dependent pathways and their relevance under various pathophysiological conditions remains poorly understood and a matter for further investigation. The implication of NE in lung destruction and repair and its pathogenic role in emphysema and fibrosis could lead to a novel approach for therapeutic interventions. References American Thoracic Society. American Thoracic Society/European Respiratory Society. (2002). International multidisciplinary con-

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