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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 12-24, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0166TR

Translational Review

Matrix Metalloproteinase-9 in Lung Remodeling

Jeffrey J. Atkinson and Robert M. Senior

Pulmonary and Critical Care Medicine, Department of Medicine, and the Department of Cell Biology and Physiology, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri

Address correspondence to: Robert M. Senior, M.D., Barnes-Jewish Hospital (North), 216 South Kingshighway, St. Louis, MO, 63110. E-mail: seniorr{at}msnotes.wustl.edu


   Abstract
Top
 Abstract
Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
 Conclusion
 References
 
Matrix metalloproteinase (MMP)-9 (Gelatinase B, 92-kD type IV collagenase, EC 3.4.24.35) is an MMP that is present in low quantities in the healthy adult lung, but much more abundant in several lung diseases, including asthma, idiopathic pulmonary fibrosis (IPF), and chronic obstructive pulmonary disease (COPD). Despite numerous reports of MMP-9 in these and other lung diseases, whether MMP-9 is causal in lung remodeling or part of the inflammatory and reparative response remains to be determined. Many intrinsic lung cells can be stimulated to produce MMP-9, but much of the information regarding MMP-9 in the lung deals with MMP-9 from inflammatory cells. The multiple locations and cell types producing MMP-9 are consistent with multiple functions in different microenvironments. In addition to digestion of structural proteins and antiproteases, MMP-9 can modify cellular function by regulation of cytokines and matrix-bound growth factors. Determining the role of MMP-9 in health and disease will be important, because broad spectrum and specific inhibitors will soon be available to enable conversion of the bench knowledge to bedside practice. This review addresses the current understanding of MMP-9 in human asthma, IPF, and COPD, and in animal models of these conditions.

Abbreviations: bronchoalveolar lavage fluid, BALF • bronchiolitis obliterans organizing pneumonia, BOOP • chronic obstructive pulmonary disease, COPD • extracellular matrix, ECM • enzyme linked immunoabsorbant assay, ELISA • forced expiratory volume at one second, FEV1 • interleukin, IL • interstitial pulmonary fibrosis, IPF • matrix metalloproteinase, MMP • reverse transcriptase–polymerase chain reaction, RT-PCR • tissue inhibitor of matrix metalloproteinase, TIMP • transforming growth factor, TGF • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
 Conclusion
 References
 
Remodeling of the lung architecture is a hallmark of many lung diseases, for example, loss of alveolar walls in emphysema, subepithelial fibrosis in asthmatic airways, intralveolar fibrosis in idiopathic pulmonary fibrosis (IPF), cavity formation in tuberculosis, and bronchiectasis in cystic fibrosis. All of these pathologic changes involve extensive alterations of lung extracellular matrix (ECM). Matrix metalloproteinases (MMPs) have been proposed to be key in causing these changes because of their capacity to cleave structural proteins such as collagens and elastin.

MMPs are a family of neutral proteinases that are minimally composed of a prodomain that requires cleavage for activation of a zinc-binding catalytic domain. Various MMPs contain other domains that affect their activation and substrate specificity. The large amount of information about MMPs in general and specifically in relation to the lung has been the subject of several reviews (1–5). Here we focus on one member of the MMP family, MMP-9. Although many MMPs have been proposed to play a role in lung pathology, the ease of MMP-9 detection and quantification has generated an abundance of literature. The relationship of MMP-9 to the lung was the subject of more than 100 articles in the last two years (Table 1). Although diverse pathologic situations have been associated with MMP-9, much of the information pertains to asthma, IPF, and chronic obstructive pulmonary disease (COPD). This review concentrates on the role of MMP-9 in these three conditions.


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  		TABLE 1 Publications about MMP-9 in the lung

 

   MMP-9: Background
Top
 Abstract
 Introduction
 MMP-9: Background
MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
 Conclusion
 References
 
Gene and Regulation
MMP-9 cDNA was first cloned in 1989 from transformed human fibroblasts (6). Subsequently it was discovered that the so-called type IV collagenase of neutrophils is the product of the same gene (7). MMP-9 has been cloned in many mammalian species, and homology exists between the cow, rabbit, rat, mouse, and human ({cong} 80% at the mRNA level). Human MMP-9 is synthesized as a proenzyme with a molecular mass of 92 kD. The mouse mRNA has 24 additional base pairs in exon 9 and 15 in exon 13, resulting in a proenzyme with a mass of 105 kD. The MMP-9 promoter is in a 2-kb 5' flanking region that contains AP-1, AP-2, and SP-1 factor–binding sites. The human gene contains additional nuclear factor-{kappa}B– and Ets-binding sites and a transforming growth factor (TGF)-ß–inhibitory element. Proinflammatory type I cytokines like tumor necrosis factor (TNF)-{alpha} and interleukin (IL)1-ß stimulate MMP-9 production in many cell types (8, 9). CXCL8(IL-8) stimulates neutrophils to release stored MMP-9 (10). For more details about the regulation of MMP-9, see the review by Fini and coworkers (11).

The MMP-9 gene is on human chromosome 20q11.1-13.1, a position associated with bronchial hyperresponsiveness (12). There are no known occurrences of MMP-9 deficiency in humans, but a cytosine to thymidine nucleotide polymorphism (C-1562T) increases MMP-9 promoter activity (13). This polymorphism, present in one allele of 20% of sampled populations (14), may play a role in atherosclerotic disease. No association was shown with asthma in one small study of a Czechoslovakian population (15), but development of emphysema may be related, depending on the population studied (16, 17).

Structure
MMP-9 is one of two MMPs referred to as gelatinases. The other is MMP-2 (gelatinase A, 72-kD type IV collagenase). In addition to having the prototypic structure of MMPs, the gelatinases contain fibronectin type II–like repeats within their catalytic domain, resulting in a higher binding affinity to gelatin and elastin (18). Although both gelatinases readily cleave gelatin, their substrate specificity differs (19). MMP-9 contains a type V collagen-like domain that is highly glycosylated, which may effect substrate specificity and resistance to degradation. The gelatinases also differ in cellular production, transcriptional regulation, and extracellular activation.

Activation
MMP-9 is released from cells as a proenzyme. Its activation can be accomplished in vitro by the addition of organic mercurials. In vivo, MMP-9 is likely activated via a protease cascade (20). The prodomain (~ 10 kD) can be cleaved by MMP-3 (stromelysin), MMP-2, or hypochlorous acid (21), but not by plasmin, thrombin, or MMP-1 (interstitial collagenase) (22). MMP-3 may be the most efficient activator of MMP-9 (23).

Inhibitors
The predominant circulating inhibitor of MMP-9 is {alpha}2-macroglobulin. Active forms of MMP-9 are trapped by {alpha}2-macroglobulin and removed from the circulation via scavenger receptors. The tissue inhibitors of matrix metalloproteinases (TIMPs) all have affinity for MMP-9, but typically the enzyme is secreted by cells in a noncovalent complex with TIMP-1. MMP-9 is unique in that TIMP-1 complexes with the carboxy-terminal of the proenzyme as well as the catalytic domain of the active form (23). The reason for this dual association is unknown. Dimerization with another MMP-9 molecule or with MMP-1 is inhibited when MMP-9 is bound to TIMP-1 (23). Thrombospondins (24) and tissue factor protease inhibitor-2 (TFPI-2) (25) also can bind and inactivate MMP-9.

MMPs may be involved in invasion of malignant cells through intact basement membrane and/or tumor angiogenesis (26–28). The presence of MMP-2 and -9 in malignant and metastatic cells has created an interest in developing pharmaceutical inhibitors (29–34). These inhibitors can be divided into three basic categories: peptidomimetics, nonpeptidomimetics, and tetracycline derivatives. Peptidomimetics (e.g., Batimastat and Marmistat) contain a sequence that resembles MMP substrates but are relatively nonspecific. In trials as cancer chemotherapeutics they have shown limited benefit in advanced cancer with intolerance due to joint and muscle pain (35, 36). The nonpeptidomimetics (e.g., Bay 12-9566 and Prinomastat) are synthesized to resemble the catalytic pocket of MMPs. Some of these compounds are more selective for gelatinases (37) and have been tested on patients with advanced cancer. Although side effects are not an issue, phase III trials were stopped because of lack of efficacy (38). Tetracycline, doxycycline, and chemically modified tetracyclines all can decrease MMP production and activity. Doxycycline for periodontal disease is the only FDA-approved MMP inhibitor. Chemically modified tetracyclines show some selectivity for gelatinases over collagenases at low doses (39). The limited study of these compounds in asthma, IPF, or COPD likely reflects that MMPs are involved in chronic matrix remodeling, making outcomes difficult to measure. One or more of these drugs will likely be approved as anticancer agents, and clinical trials in chronic lung diseases are likely to follow.

MMP-9–Deficient Mice
Targeted deletion of MMP-9 in mice is a useful tool to study MMP-9 function in vivo. MMP-9–deficient mice have been developed by three groups (40–42) using mice of different strains. The MMP-9 null mouse has transient delayed growth plate angiogenesis (40), but survives normally into adulthood with near normal fertility (43). In models of lung injury, MMP-9 deficiency has effects that suggest the enzyme has a role in bronchiolar epithelial migration, alveolar destruction, and alveolar permeability (Table 2).


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  		TABLE 2 Lung phenotypes of MMP-9–deficient mice

 
MMP-9 and the Neutrophil
Neutrophils synthesize MMP-9 during maturation in the bone marrow and the MMP-9 is stored in specific granules from which it is readily released. Based on studies with MMP-9 null mice, neutrophils do not require MMP-9 to enter or leave the circulation (44, 45), but primates may differ (46, 47). The MMP-9 content per neutrophil is substantial ({cong} 3 µg per million neutrophils; R. Senior, unpublished data). In circumstances in which neutrophils are abundant, MMP-9 can be expected, and identifying other cellular sources of MMP-9 is difficult. The neutrophil does not secrete MMP-9 in complex with TIMP-1 as many cells do, but can secrete MMP-9 either alone or in a covalent complex with lipocalin. The role of the lipocalin is unknown. MMP-9 also binds to the surface of neutrophils where it is inaccessible to protein inhibitors (48).

Assays for MMP-9
Many approaches are used to measure MMP-9. These include zymography, in situ zymography, enzyme-linked immunoabsorbant assay (ELISA), Western blotting, immunostaining, reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blotting. The protein is present in multiple forms (e.g. TIMP-bound, latent enzyme, homodimer, active enzyme, lipocalin-bound), so that quantification of protein and enzyme activity may differ depending on the assay used. Because neutrophils do not synthesize MMP-9 outside of the bone marrow, transcript assays will not reflect the MMP-9 produced by these cells.

Zymography. One attraction of studying MMP-9 in disease or experimental models of disease is the ease and availability of sensitive MMP-9 assays, especially gelatin zymography (Figure 1) . The results of MMP-9 zymography may not correlate with assays of MMP-9 mRNA, depending on the degree of neutrophilic inflammation. Gelatin zymography will separate the multiple forms of the enzyme by molecular mass, and is sensitive to a picomolar range. The presence of latent and inactive forms of MMP-9, in zymography and other protein assays, may create a disparity between protein content and enzymatic activity. In combination with image analysis programs and a concomitant standard of known controls, zymography can provide rough estimations of MMP-9 quantity. Free gelatinolytic activity can be measured using radioactive or fluorogenic substrates. Recently, bioassays developed to localize enzymatic activity have been used on frozen tissue sections (i.e., in situ zymography). The method involves enzymatic cleavage of gelatin-based quenched fluorochromes or gelatin-containing film processing reagents that can be thinly coated on tissue sections (49–52). In situ zymography provides localization and estimation of enzyme activity, but does not differentiate other gelatinolytic enzymes from MMP-9.



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  		Figure 1. Zymogram showing MMP-9 in a murine neutrophil lysate. After electrophoresis into an 8.5% acrylamide gel containing 0.1% gelatin and overnight incubation in a zinc and calcium containing buffer, gelatinolytic activity appears as clearing on the stained gel. The bands represent (A) trimer, (B) dimer, (C) lipocalin–MMP-9 complex, and (D) latent MMP-9. Active MMP-9 is typically slightly below the latent band.

 
Immunoassays. Commercial ELISAs for human MMP-9 are sensitive to the nanomolar range, but the ELISA will not distinguish active and inactive forms of the enzyme. In some clinical studies the ratio of MMP-9 to TIMP-1 is determined as a surrogate for in vivo enzymatic activity. Because the protease and inhibitor form a 1:1 complex, the ratio should be expressed in molar quantities. An elevated ratio favors enzyme activity, whereas the opposite is inferred when the ratio is low, but global analysis of this sort could miss microenvironments in which one or the other factor predominates.

Assay for MMP-9 by Western blotting is less sensitive than zymography and generally requires concentration of the sample by gelatin affinity, but can confirm the results of zymography.

Antibodies to mouse and human MMP-9 have been used for immunohistochemistry (53–56). Permeabilized tissue samples will result in immunostaining of MMP-9 in neutrophil granules, which may overestimate extracellular enzyme. Immunostaining quantified by image analysis software can correlate with relative increases or decreases in protein level, but quantified immunostaining may not correlate with enzyme activity because most antibodies recognize both latent and active forms of MMP-9. Quantification of an MMP-9:TIMP-1 ratio by immunostaining for both proteins is a poor surrogate of enzyme activity, but does reveal co-localization and relative increments or decrements in protein quantity between samples.

Transcript assays. In situ hybridization of MMP-9 using various fragments of the human and mouse cDNA is sensitive and provides localization (57, 58). RT-PCR (real time or conventional) is also quite sensitive and reproducible, but Northern analysis and possibly gene chips may be insensitive to small, localized amounts of MMP-9 message in whole lung specimens (53, 59–61).

Lung Cells as a Source of MMP-9
In the normal lung, MMP-9 is not produced by resident cells, but under various forms of stimulation, bronchial epithelial cells (62), Clara cells (59), alveolar type II cells (63), fibroblasts (6), smooth muscle cells (64), and endothelial cells (65) produce MMP-9. Alveolar type I, neuroendocrine, and goblet cells have not yet been shown to produce MMP-9. Leukocytes in the lung can also be a source of MMP-9. Macrophages (66), eosinophils (67), mast cells (68), lymphocytes (69), NK cells (70), and dendritic cells (71) all can produce MMP-9. Lung cancer cells, both primary and metastatic, can express MMP-9 constitutively, which may correlate with metastatic potential (72, 73).

How Might MMP-9 Affect the Lung?
Cell migration. The role of MMP-9 in the lung is largely unknown, despite its presence in many situations. The only function clearly identified for MMP-9 in resident lung cells is migration of basal cells after tracheal wounding (74) and terminal airway epithelial cells after bleomycin ("alveolar bronchiolization") (53). These findings are consistent with evidence for the involvement of MMP-9 in cellular migration in tissues other than the lung (75–78). Because MMP-9 cleaves type IV collagen, it could be involved in epithelial cell movement across or through basement membranes.

Migration of human leukocytes through artificial basement membranes (Matrigel; Becton Dickinson, Bedford, MA) may require MMP-9 (79–82), but MMP-9 is not necessary for neutrophil transendothelial migration (83, 84). MMP-9 null mice show normal neutrophil emigration to stimulus (44, 85) and may even have increased leukocyte recruitment to certain stimuli (86, 87). Dendritic cells may require MMP-9 for migration before maturation, but not for migration to the lymph node (88). MMP-9 null mice have decreased T cells and dendritic cells in bronchoalveolar lavage fluid (BALF) in an antigen challenge model (89).

Processing of ECM, cytokines, and other proteins. The potential effects of MMP-9 are many as MMP-9 has many substrates (Table 3). The multiple potential substrates and the diversity of cell types expressing MMP-9 suggests the possibility of involvement in multiple events at the same time in different microenvironments. Apart from digesting components of the ECM, MMP-9 modulates the activity of other proteases and cytokines that are important in lung diseases (90). It degrades {alpha}1-antitrypsin, protecting neutrophil elastase activity (91), and potentiates the collagenolytic activity of MMP-13 (92) and fibroblasts in collagen gels (93). It cleaves a six–amino acid peptide from CXCL8, increasing its chemotactic activity for neutrophils 10-fold, but it also inactivates other neutrophil chemokines (10). MMP-9 bound to CD44 can release latent TGF-ß1 (94). MMP-9 is involved in angiogenesis through liberation of vascular endothelial growth factor (VEGF) (92, 95) and production of angiostatin (96). Whether neovascularization in lung disease is affected is not known.


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  		TABLE 3 Substrates for MMP-9

 
MMP-9 and Lung Development
In the developing rabbit lung, MMP-9 is present at low levels in epithelial cells during the glandular and canalicular stages and increases during alveolization (97). The cells that show MMP-9 synthesis are predominantly alveolar type II cells, bronchial epithelial cells, and macrophages. However, the significance of MMP-9 during development appears to be minimal, because MMP-9 null mice have normal branching morphogenesis (98) and the adult MMP-9 null mouse lung has normal histology (T. Betsuyaku and R. Senior, unpublished results).


   MMP-9: Asthma, IPF, and COPD
Top
 Abstract
 Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
IPF
 MMP-9 in COPD
 Conclusion
 References
 
Asthma
Human Studies. The literature about MMP-9 in asthma raises many issues. It is clear that MMP-9 levels have some correlation with both acute and chronic asthma, but the interpretation is uncertain with our current knowledge. Other MMPs have been studied in asthma and asthma models (55, 99–101), but MMP-9 has been the predominant MMP found in blood, BALF, sputum, and transbronchial biopsy specimens of individuals with asthma (Table 4). TIMP-1, the predominant inhibitor of MMP-9, is also elevated in asthma to molar concentrations that exceed MMP-9 and all measured MMPs combined (101). Although elevated in both chronic and acute asthma, the cellular source and function of MMP-9 may differ.


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  		TABLE 4 MMP-9 and asthma

 
Chronic Asthma.

AIRWAY REMODELING. An excess of TIMP-1 over MMP-9 has been proposed to favor airway remodeling in chronic asthma (102, 103). The thickness of subepithelial collagen in transbronchial biopsy specimens from individuals with asthma has correlated with forced expiratory volume in 1 s (FEV1) and airway hyperresponsiveness (104, 105). If low MMP-9:TIMP-1 ratios result in pathologic subepithelial collagen deposition, chronic MMP-9:TIMP-1 imbalance could lead to thickened airways with restricted airflow. Elevated serum MMP-9:TIMP-1 ratios (in ng/ml) correlate with oral corticosteroid responsiveness (106). Low sputum MMP-9:TIMP-1 ratios correlate with decreased FEV1 in outpatients with asthma (and COPD) (107), suggesting that the postulated TIMP-1 excess theory is true. However, MMP-9 levels in serum or sputum could be a surrogate of the leukocyte pool marking a subset of individuals with asthma with steroid-responsive inflammation instead of MMP-9–dependent remodeling (108).

The results of immunostains for MMP-9 in transbronchial biopsies of individuals with stable asthma are conflicting. Compared with normal control subjects, elevated MMP-9 and TIMP-1 (104) were found in one study; however, a second study did not find significant MMP-9 elevations (55). Differences in antibodies, tissue fixation, prior steroid use, and sample size make the studies difficult to compare. Tissue eosinophils and macrophages did not immunostain for MMP-9 in either of these studies, although these cells are known to make MMP-9 in asthma (67). MMP-3 was significantly elevated in individuals with asthma and may play a direct role in remodeling, but it also activates MMP-9 (55). MMP-9 activity in the tissue needs to be evaluated to determine the significance of these findings.

DISCREPANCIES IN DATA. In contradiction to the TIMP excess theory of airway remodeling in asthma, inhaled corticosteroid treatment produced a significant decrease in MMP-9 and an increase in TIMP-1 immunostaining of bronchial biopsies (54). The steroids decreased subepithelial collagen thickness, and subjects had an increased FEV1 at 6 mo. This data suggests that a decrease in the MMP-9:TIMP-1 ratio resulted in resolution of remodeling. However, because quantified immunostaining does not equate to molar ratios, the MMP-9:TIMP-1 molar ratio is unknown. Sampling at the beginning and end of a 6–mo time period may also underscore the fluctuation of both MMP-9 and TIMP-1 levels during this time, creating a false impression of a direct correlation of the two protein levels.

What does seem apparent is that sputum does not reflect airway subepithelial proteinase activity. The macrophage is the predominant cell type in the sputum of individuals with stable asthma, and neutrophils are the predominant MMP-9 immunostaining cells in bronchial biopsies (55). The different MMP-9 and TIMP-1 content of these two cell types will produce different MMP-9:TIMP-1 ratios in each respective sample. An assay of gelatinolytic activity in the tissue may need to be done to confirm the pertinence of the MMP-9 to TIMP-1 ratio in airway remodeling.

PROPOSED MECHANISMS. Whether increased MMP-9 or excess of TIMP-1 over MMP-9 in individuals with asthma relates to airway pathology is unknown. MMP-9 might modulate airway remodeling by activating TGF-ß, increasing collagen synthesis by fibroblasts, or by increasing angiogenesis and airway vascular remodeling. Conversely, a TIMP-1 excess could inhibit MMP-9 potentiation of collagenases that can cleave the increased type III collagen in asthmatic airways. Another possibility is that MMP-9 is irrelevant and unbound TIMP-1 increases the survival of fibroblasts (109). Unfortunately, levels of two proteins at brief time points in a disease process that is not static can be misleading. Is the excess of TIMP during stable time points an excessive response to brief elevations of MMPs during exacerbations? Is remodeling both a reaction to injury and overaggressive repair?

The thickened reticular basement membrane in asthmatic airways is an increase in many components in an organized manner, suggesting an aberrant coordination of many enzymes and transcription factors. Limiting the explanation of the pathology to a few factors that can be measured certainly oversimplifies the process. The proper way to assess MMPs and TIMPs in chronic asthma is not clear, as it is likely that a complex mixture of proteases and antiproteases results in remodeling. Time-specific and localized activity of MMPs in asthma increase the difficulty of interpreting assays from clinically derived samples. These studies in asthma highlight the complexity of developing a coherent picture of the role of a single enzyme (MMP-9) in a chronic disease process. Defining the role of MMP-9 in asthma likely will require a combination of tissue enzyme activity assays at multiple time points and studies of chronic airway remodeling in MMP-9–deficient mice.

Acute asthma.
EXACERBATIONS AND STATUS ASTHMATICUS. Patients with decompensated asthma have molar ratios of MMP-9 to TIMP-1 in BALF greater than that seen in patients with stable asthma, and free gelatinolytic activity is present (110, 111). Gelatin zymography of sputum and BALF in these patients typically shows a prominent MMP-9–lipocalin band, consistent with increased neutrophil counts. Intravenous corticosteroid treatment decreases the amount of MMP-9 while increasing the amount of TIMP-1 (112). In acute asthma, the level of MMP-9 in BALF correlates with numbers of neutrophils, macrophages, and bronchial epithelial cells (111). Exfoliation of airway epithelial cells, induced by MMP-9, may contribute to the pathologic airway obstruction in status asthmaticus (111), but many neutrophil- and macrophage-derived products could cause exfoliation of epithelial cells. The situation in acute asthma may bear some similarities to bullous pemphigoid, where MMP-9 is involved indirectly in the cleavage of collagen XVII (BP180) through inhibition of {alpha}1-antitrypsin (91). Some neutrophil proteases can induce mucus hypersecretion (113, 114), but whether MMP-9 has this activity is not known.

ALLERGEN CHALLENGE. In allergen challenge models of acute asthma, MMP-9 levels and neutrophil counts increase in BALF within 24 h after the challenge (115). Although this confirms neutrophils as a source of MMP-9 in acute asthma, it is not clear if MMP-9 plays a pathologic role or is simply a marker of the neutrophilic inflammatory process. Most of the MMP-9 present is in the latent form, but earlier time points might show more active forms as is seen in large animal models (116). Assessment of gelatinolytic activity from tissue samples of these patients would be useful to test the significance of the increased latent form in the BALF. If some of the MMP-9 is active and causes pathology, it may be a target worthy of inhibition during exacerbations. Likely the best way to sort out if MMP-9 has a pathologic role is to examine experimentally induced asthma in mice that lack MMP-9.

Animal models. Sensitization models. MMP-9 in mouse models of asthma is receiving increasing attention. Broad-spectrum MMP inhibitors decrease the inflammatory cell infiltrate and airway hyperresponsiveness in ovalbumin and toluene diisocyanate sensitization models of asthma (117, 118). Whether MMP-9 is one of the important MMPs inhibited is not known, but studies already report MMP-9 decrement as a secondary endpoint for inhibition of airway remodeling (119). MMP-9–deficient mice have been sensitized to ovalbumin and challenged daily for 7 d (120). These mice do have a decrease in lymphocytic emigration into the airway submucosa and BALF without an increase in airway collagen (120). However, the MMP-9–deficient mice treated with phosphate-buffered saline instead of ovalbumin have increased sensitivity to carbachol similar to the ovalbumin-treated controls, limiting the interpretation of airway hyperreactivity in this study. A more chronic model of ovalbumin or other allergen exposure may allow further assessment of airway remodeling in MMP-9–deficient mice, but the decrease in lymphocytic infiltration may reflect a difference in cellular migration or cytokine profile and make the MMP-9–deficient mice most useful for studying the role of MMP-9 in acute allergen exposure. Because the transfer of sensitized lymphocytes can recreate the phenotype of airway reactivity in mice (121), selective gelatinase inhibitors may decrease lymphocyte trafficking in asthma.

Transgenic models. A mouse that has been mutated to overexpress IL-13 develops airway inflammation and mucus cell metaplasia similar to the airway pathology of severe asthma. In these mice many protease transcripts are elevated in whole lung RNA, including MMP-9 (99). When the IL-13 transgenic mouse was bred with the MMP-9 null mouse there was a decrease in the airway and alveolar pathology, as well as the level of bioactive TGF-ß1, but mucus cell metaplasia was unchanged (122). The decrease in bioactive TGF-ß is less impressive than after neutrophil elastase inhibition, suggesting a modulatory rather than unique role of MMP-9. Further studies of MMP-9–deficient mice in sensitization and transgenic models may better define a role of MMP-9 in asthma.


   IPF
Top
 Abstract
 Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
MMP-9 in COPD
 Conclusion
 References
 
Human studies. IPF is a disorder of excessive matrix deposition from fibroblastic foci that obliterates functional alveoli. The initiating cause is unknown, but alveolar epithelial injury appears to be a critical early event (123). MMP-2, -9, -7 (matrilysin), and -12 (macrophage elastase) are elevated in IPF, but much of the recent work has examined the possibility of an excess of TIMPs to MMPs in the fibroblastic foci (60, 61, 97, 124, 125). The lesions of early fibrosis appear to have a bud of tissue intruding into the alveoli. These alveolar buds are covered by epithelial cells and filled with connective tissue and myofibroblasts. Although there is MMP staining on the epithelial surface of the alveolar buds, the fibroblasts have strong immunoreactivity to many TIMPs (125). When isolated, the fibroblasts/myofibroblasts produce an excess of TIMPs to MMPs (109).

Inflammatory cell production of MMP-9. BALF from IPF patients demonstrate elevation of MMP-9 with a prominent lipocalin-associated band, especially in patients with a more rapid course (56), consistent with the elevated neutrophil counts found in this subset of patients (126). Although neutrophilic inflammation in IPF is atypical, this subgroup of patients may represent a different process. Whether elevations of MMP-9 are a marker of activated neutrophils or involved in the alveolar damage in this subset of patients is unknown. The elevations of MMP-9 in the BALF of patients with bronchiolitis obliterans organizing pneumonia (BOOP) exceed those seen in IPF (127), suggesting an association with neutrophils rather than the lung histopathology (Table 5).


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  		TABLE 5 MMP-9 and IPF

 
Epithelial cell production of MMP-9. Disorders resembling IPF, such as nonspecific interstitial fibrosis and BOOP, can resolve without fibrosis and do not show the MMP-9 immunostaining characteristic of IPF (Figure 2) (56, 128). In IPF, MMP-9 staining is greatest in metaplastic epithelial cells and neutrophils, with minimal staining in fibroblasts. The macrophages also show MMP-9 immunostaining, both in the tissue and when isolated from BALF (129). This striking difference would point to a role for MMP-9 in IPF. Gene chip data also shows MMP-9 to be one of the elevated MMPs in IPF, but MMP-7 may play a greater modulatory role in the development of fibrosis (59).



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  		Figure 2. MMP-9 in interstitial lung disease. Immunostaining for MMP-9 in lung biopsy specimens. (A) IPF, (B) NSIP, and (C) BOOP. There is continuous MMP-9 staining of the epithelium in IPF, but not in NSIP or BOOP. Modified from Suga and coworkers with permission (56).

 
Possible functions of MMP-9 in IPF include migration of regenerating epithelial cells, activation of latent TGF-ß, and facilitation of leukocyte migration through the excessive matrix (129). Although inhibition of MMP-9 alone is unlikely to stem ongoing fibrosis in IPF, patients with rapid progression may be a subset of patients with MMP-9–related pathology.

Animal models of pulmonary fibrosis. Lack of a true IPF animal model. Although animal models of pulmonary fibrosis exist, no true model of IPF has been developed. Most animal models of fibrosis resemble diffuse alveolar damage in early stages and never develop the typical pattern of usual interstitial pneumonitis. Likely these models help in our understanding of fibrotic mediators and cellular activity, but do not further our understanding of the cause of IPF.

Bleomycin-induced fibrosis. Intratracheal or intraperitoneal administration of bleomycin produces some similarities to IPF in fibrotic protein and cytokine production, but lacks the typical histologic characteristics of temporal heterogeneity and chronicity seen in IPF (130). Not surprisingly, a single-time insult will not replicate a chronic disease. Nonspecific inhibition of MMPs before and after treatment with bleomycin results in decreased fibrosis (131, 132). However, MMP-9 null mice develop fibrosis with increases in collagen and elastin similar to wild-type littermates (53), but do not develop alveolar bronchiolization commonly seen in this model. Alveolar bronchiolization is the appearance of terminal airway-like epithelial cells in areas of injured alveoli. This phenomenon can be seen in response to many types of alveolar injury, and is believed to be a sign of terminal airway epithelial cell migration to these areas of injury and not a defect in alveolar epithelial cell differentiation. The lack of alveolar bronchiolization in MMP-9 null mice is interesting because the strongest immunostaining for MMP-9 in wild-type mice treated with bleomycin and in human IPF is in the metaplastic alveolar epithelial cells (56). Likely, these regenerating cells are part of an aberrant repair process and MMP-9 plays a role in epithelial repair and not alveolar fibrosis.

Other models of lung fibrosis in mice (e.g., silica or repeated bacterial lipopolysaccharide [LPS]) show increases in MMP-9 in BALF (133, 134), but have not been assessed for modulation by MMP-9 gene deletion. Broad-spectrum MMP inhibitors and the antifibrotic agent perfenidone did decrease both MMP-9 activity and fibrosis in an LPS-induced model of chronic fibrosis (135). More work is needed in mice with MMP-9 deletion or overexpression to evaluate the function of MMP-9 in fibrosis.


   MMP-9 in COPD
Top
 Abstract
 Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
Conclusion
 References
 
Human studies. MMPs in emphysema. The protease:antiprotease hypothesis has dominated thinking about the pathogenesis of emphysema (136). Studies of human samples show increases in many proteases, including MMP-1 (137, 138), -2 (139), -8 (140), -9 (139–142), and -14 (MT1-MMP) (139) in smoking-related emphysema. It is not clear what causes a subgroup of smokers to have progressive disease, but the ultimate event is an irreversible loss of alveolar surface area and depletion of lung elastin. Three MMPs degrade elastin: MMP-2, -9, and 12 (143, 144).

Smokers with early emphysema have increased neutrophils and activated macrophages in BALF (140, 141, 145), and the rate of decline in pulmonary function has been correlated with both of these cell types (146). As an elastolytic enzyme associated with both macrophages and neutrophils, MMP-9 has the potential to cause emphysema.

MMP-9 in airspace enlargement. Histologic samples of emphysema for analysis of MMPs generally come from lung transplant, volume reduction, or cancer resection margins. The volume reduction and transplant samples usually represent an advanced stage of emphysema and may underestimate the role of proteases or not reflect the protease profile in early disease. Homogenates of lung removed in volume reduction reveal MMP-9–related elastolytic and gelatinolytic activity and significant elevations in MMP-9, with no significant increase in neutrophil elastase by ELISA (139). However, results of immunostaining for MMP-9 are relatively unimpressive (Table 6). In situ hybridization for lung MMP-9 mRNA in a small group of patients with emphysema did show a correlation with emphysema histologic grade and an eosinophilic source of the protein (147). Analysis of inflammatory cells in the lung parenchyma of emphysematous samples reveals that macrophages, T lymphocytes, neutrophils, and eosinophils all correlate with the degree of emphysema (145). Examination of macrophages isolated from smokers suggests that cigarette smoke increases the production of MMP-9 in these cells (148). As a protease produced by all of these cell types, MMP-9 may make an excellent target for therapies aimed at decreasing the protease imbalance. The role of MMP-9 in the pathogenesis of emphysema is likely to be complex. {alpha}1-antitrypsin can be cleaved by MMP-9. Increased chemotactic activity of BALF is associated with the development of smoking-related emphysema (149), and potentiation of CXCL8 by MMP-9 could amplify the alveolar inflammation and destruction in smokers who develop emphysema (10).


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  		TABLE 6 MMP-9 and emphysema

 
MMP-9 in COPD airways. There is little information linking the airway pathology in COPD with MMPs. Neutrophil numbers are increased up to 10-fold in the sputum of subjects with COPD, and neutrophil MMP-9–associated lipocalin is elevated in the sputum of patients with COPD (150). Neutrophils have also been shown to increase in the submucosal mucus glands of patients during COPD exacerbations. Likely the elevation of mucus production in these exacerbations results from neutrophil products, but as noted before, there is no evidence that MMP-9 is involved in mucus secretion. The presence of neutrophils in endobronchial biopsies of patients with stable COPD correlated with FEV1, but MMP-9 levels in sputum did not correlate with FEV1 (107). In fact, FEV1 was inversely correlated with MMP-9:TIMP-1 ratios (107).

However, examination of small airway inflammation in patients with severe COPD does not show elevation of neutrophils, but rather T lymphocytes and macrophages (151). The fibrosis seen in the small airways of patients with COPD (152) may represent a different process than the inflammation seen in the sputum, BALF, or alveolar tissue. The role of MMPs and TIMPs in the fibrosis of the small airways in COPD needs to be examined.

MMP-9 promoter polymorphisms. Another method of assessing the role of MMP-9 in emphysema is to look at single nucleotide polymorphisms. In diseases like emphysema that develop slowly over long periods of time, minor perturbations in promoter efficiency can result in increased severity of disease. A recent study of a Japanese population reported a significant increase in allelic frequency of the C–1562T MMP-9 polymorphism in smokers with emphysema, compared with matched smokers without emphysema as defined by high-resolution chest computed tomography (17). However, in a study of smokers with a more rapid rate of decline in lung function, there was a correlation with polymorphisms of MMP-12 and MMP-1, but not MMP-9 (16). Polymorphisms in a relatively homogenous population like Japan may differ from a more ethnically heterogeneous country like the United States of America, so polymorphism studies will need to be confirmed in other populations.

Mouse models. Cigarette smoke. Chronic exposure to cigarette smoke leads to airspace enlargement in mice. This does not occur in MMP-12–deficient mice (153). Neutrophil elastase gene deletion also offers some protection (154). When exposed to cigarette smoke, MMP-9 null mice do not appear to be protected from the development of airspace enlargement (T. Betsuyaku and coworkers, unpublished results). This is intriguing because MMP-9 does appear to be involved in the airspace enlargement of genetically modified mice as discussed below (155, 156).

Transgenes and gene targeting. Inducible, airway-specific interferon-{gamma}–overproducing transgenic mice display histology compatible with emphysema. MMP-9 is increased in the BALF, but mRNA is not increased, suggesting a neutrophilic source of MMP-9 in these mice (156). The IL-13 transgenic mouse (inducible airway-specific IL-13), as discussed above (see ASTHMA), develops airspace enlargement, which is partially abrogated by MMP-9 or -12 deletion (157, 158). MMP-9 deletion in IL-13–overexpressing mice results in less airspace enlargement, despite an increase in lung lavage neutrophils, suggesting MMP-9 is more important in the development of airspace enlargement than other neutrophil-derived proteases in this model. Transgenic IL-13/MMP-9 null mice have increased neutrophil-related cytokines (KC and MIP-1) in the BALF, so that MMP-9 itself may decrease these cytokines. TIMP-3 null mice develop enlarged airspaces and in situ zymography shows an increase in lung gelatinolytic activity (159), suggesting a dysregulation of a gelatinase. Surfactant protein (SP)-D null mice also develop airspace enlargement associated with active proteases, including both MMP-12 and MMP-9 (160). The result of concomitant MMP-9 deletion in these and other mice that develop airspace enlargement is unknown, but may help to isolate MMP-9 as a modifier in the progression to airspace enlargement.

The theory that MMP-9 could be involved in emphysema requires more investigation, but likely the damage that occurs is due to a complex mix of effects, involving an imbalance between antiproteinases, cytokines, and physical forces that induce damage to the lung ECM. An important caveat in the data from mouse models is that there may be significant differences between humans and mice (as seen, for example in cystic fibrosis transmembrane receptor–deficient mice). MMP-12 appears to play a greater role in cigarette smoke–associated airspace enlargement in mice than in humans.


   Conclusion
Top
 Abstract
 Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
 Conclusion
References
 
Neutrophils contain substantial MMP-9 that can be readily released. Many lung cells can synthesize and release MMP-9. In lung diseases that include a neutrophil-predominant response, MMP-9 produced by epithelial and other structural cells may be obscured by neutrophil-produced MMP-9, but could be important in disease pathogenesis. In asthma, IPF, and emphysema, MMP-9 is elevated and likely more than a surrogate for inflammation, but localization of MMP-9 activity and the in vivo substrates need to be determined. A likely scenario is that MMP-9 modulates other enzymes and cytokines to fine-tune both destruction and repair. Whether inhibiting MMP-9 at the right place and time could improve the course of any lung disease is still unknown. It is even possible that MMP-9 produced in the lung in response to proinflammatory stimuli may have a beneficial function in a subset of cell types and should not be inhibited. In future determinations of MMP-9 related lung disease, it will be important to establish the function of MMP-9 from different cellular sources for evaluation of the effects of global inhibition of the enzyme on lung remodeling. To pursue clinical trials of gelatinase inhibitors in these and other lung diseases, better understanding of the functions of MMP-9 needs to be established. The difficulty of assessing gelatinolytic activity in clinical specimens means that animal models need to be exploited.


   Acknowledgments
 
This study was supported by NHLBI/NIH HL 29594 and HL 47328, the Alan A. and Edith L. Wolff Charitable Trust (R.M.S.), and the American Lung Association Research Training Fellowship RT-026-N (J.J.A.). The authors thank Dr. Moritaka Suga for permission to use his figure.

Received in original form August 23, 2002


   References
Top
 Abstract
 Introduction
 MMP-9: Background
 MMP-9: Asthma, IPF, and...
 IPF
 MMP-9 in COPD
 Conclusion
 References
 

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