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(Chest. 2004;126:1802-1810.)
© 2004 American College of Chest Physicians

Increased Activity of Matrix Metalloproteinase-8 and Matrix Metalloproteinase-9 in Induced Sputum From Patients With COPD*

Juanita H. J. Vernooy, PhD; Jan H. N. Lindeman, MD, PhD; Jan A. Jacobs, MD, PhD; Roeland Hanemaaijer, PhD and Emiel F. M. Wouters, MD, PhD, FCCP

* From the Departments of Respiratory Medicine (Drs. Vernooy and Wouters) and Medical Microbiology (Dr. Jacobs), Nutrition and Toxicology Research Institute Maastricht, University Hospital Maastricht, Maastricht, the Netherlands; the Department of Vascular Surgery (Dr. Lindeman), Leiden University Medical Center, Leiden, the Netherlands; and the Department of Biomedical Research (Dr. Hanemaaijer), TNO Prevention and Health, Leiden, the Netherlands.

Correspondence to: Juanita H. J. Vernooy, PhD, Department of Respiratory Medicine, University Hospital Maastricht, PO Box 5800, 6202 AZ Maastricht, the Netherlands; e-mail: j.vernooy{at}pul.unimaas.nl


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: The increased expression of matrix metalloproteinases (MMPs) is considered to be a key factor in the development of COPD. Net MMP activity represents a tightly regulated process, which is not addressed by conventional investigation methods such as messenger RNA or protein expression. Yet, quantitative data on MMP activity in the airways of COPD patients are lacking.

Methods: We used specific immunocapture assays to quantify the activity of MMP collagenase (ie, MMP-1, MMP-8, and MMP-13) and MMP gelatinase (ie, MMP-2 and MMP-9) in the induced sputum of COPD patients (17 patients; FEV1, 56% predicted) and healthy smokers (17 subjects; FEV1, 99% predicted).

Results: Levels of total and active MMP-8 and MMP-9 were significantly increased in COPD patients vs control subjects, whereas MMP-1 activity levels were similar in both groups. The activity of MMP-2 and MMP-13 remained below the detection threshold of the assays. MMP-8 and MMP-9 activity were strongly related to neutrophilia in both groups, and the results of immunohistochemistry tests on sputum cytospins showed that MMP-9 was expressed in both alveolar macrophages and neutrophils, whereas MMP-8 expression was exclusively observed in neutrophils. A positive correlation was found between sputum MMP-8 and MMP-9 activity and the degree of airflow limitation.

Conclusion: We demonstrate increased MMP-8 and MMP-9 activity in the airway compartment of patients with mild-to-moderate COPD. This study provides further evidence of an impaired proteinase-antiproteinase balance in COPD patients.

Key Words: airway inflammation • collagenase • COPD • gelatinase • immunocapture activity assay


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
COPD is a complex heterogeneous respiratory disease that is clinically defined by the development of progressive, largely irreversible, airflow limitation, and emphysematous degeneration. The pathophysiologic basis for COPD has not been elucidated, but it is generally conceived that long-term exposure to noxious particles such as those present in cigarette smoke induces a sustained and extensive inflammatory response throughout the airways, lung parenchyma, and pulmonary vasculature.1 This view is supported by numerous studies2345678 demonstrating that the severity of COPD and the number of various inflammatory cells in the lung are strongly correlated.

The chronic inflammatory response in COPD is associated with an imbalance between proteinases and endogenous proteinase inhibitors that is held responsible for the destruction of lung parenchyma, but which may also interfere with normal tissue function and repair through the proteolysis of receptors and/or ligands.91011 The increased prevalence of early-onset emphysema in patients with a congenital deficiency of {alpha}1-antitrypsin, an endogenous inhibitor of neutrophil elastase (NE), led to the concept of NE as the proteolytic culprit in COPD, a hypothesis that has prevailed for nearly 40 years.12 The evolving concepts of the pathogenesis of COPD, however, have suggested that the involvement of other proteinases of different catalytic classes, acting on diverse substrates of the extracellular matrix components, is required to promote effective tissue destruction in COPD patients,12 which is analogous to what has been suggested about aneurysm formation.13

Evidence from animal studies1415 as well as preliminary studies in humans16171819 has suggested that members of the matrix metalloproteinase (MMP) family are critically involved in the pathogenesis of COPD. MMPs comprise a large family of structurally related zinc metalloendopeptidases with different substrate specificities that collectively are able to degrade most, if not all, protein constituents of the extracellular matrix.2021 Net MMP activity represents a tightly regulated process involving the regulation of gene expression, the activation of the secreted proenzyme, and its inhibition by specific inhibitors. MMP messenger RNA expression is increased in emphysematous lung tissue,161718 and elevated MMP levels are found in BAL fluid of emphysematous patients.1819 Yet, the answer to the critical question of how MMP messenger RNA and protein expression in COPD translate into net proteinase activity is not known. We have used in this study new specific activity assays that address the complex posttranslational regulation of proteinase activity to analyze the activity of MMP gelatinases (ie, MMP-2 and MMP-9) and MMP collagenases (ie, MMP-1, MMP-8, and MMP-13) in the induced sputum of well-characterized COPD patients with mild-to-moderate disease8 and of healthy smokers (HSs) who were matched for age and smoking history. In addition, the cellular distribution of MMP-8 and MMP-9 was assessed by immunohistochemistry on cytospins of sputum cells.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
Seventeen patients with smoking-related, clinically stable COPD (using American Thoracic Society criteria22) were included in the study. These patients had a stable airflow limitation with an FEV1 of <70% predicted, with reversibility of FEV1 of < 11% predicted (or < 200 mL) following inhaled ß-agonist administration (Spirometer Masterlab; Jaeger; Würzburg, Germany), and a smoking history of at least 20 pack-years. Five patients with COPD were ex-smokers (ie, they had quit smoking at least 1 year before the start of the study). Exclusion criteria included a history of respiratory disease other than COPD as well as increased respiratory complaints or respiratory tract infection during the 4 weeks preceding entry into the study. A control group of so-called HSs consisted of 17 subjects with a normal FEV1, no medical history of lung disease, and a smoking history of at least 15 pack-years. Seven control subjects were ex-smokers. The study was approved by the medical ethics committee of the University Hospital of Maastricht. Written informed consent was obtained from all subjects.

Sputum Induction and Processing
Sputum was induced as reported by Keatings et al.6 In short, a 3% hypertonic saline solution nebulized via an ultrasonic nebulizer (NEB2000; TEFA-Portanje; Woerden, the Netherlands) was inhaled by the subjects during three 7-min periods. To minimize saliva contamination, subjects were instructed to wash their mouths thoroughly with saline solution before expectoration. The sputum collected from each subject was pooled and kept at 4°C for not > 2 h prior to further processing. The procedure was well-tolerated, and an adequate specimen of sputum could be collected from every participant.

The volume of the pooled sputum (including sputum plugs) was recorded, and the sputum was processed with dithiothreitol (DTT) [Sputolysin; Calbiochem; La Jolla, CA] at a final concentration of 0.1%, as has been described.8 Differential cell counts (May-Grünwald Giemsa method) were carried out by one observer who was blinded to the clinical characteristics, counting 500 nucleated cells. The numbers of squamous cells were subtracted, and the differential cell counts were expressed as corrected percentages. A sample was considered to be adequate if the slides contained ≤ 15% squamous epithelial cells.

Analysis of MMP Activity in Sputum
Immunocapture Activity Assays: MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 activity assays (Amersham Biosciences; Buckinghamshire, UK) were performed according to the recommendations of the supplier. These assays measure both active (mature) MMP as well as total MMP (already active plus activatable [ie, latent] pro-MMP) activity, but are insensitive to proteinase inhibitor complexes. In brief, MMP-1, MMP-2, MMP-8, MMP-9, or MMP-13 is captured by a specific antibody that has been immobilized on a microtiter plate. The amount of active MMP is measured directly by the incubation of the captured MMP with modified prourokinase (Ukcol), and subsequent activation of Ukcol is quantified by a chromogenic substrate (S-2444). Color development is recorded at 405 nm at different time intervals. Total MMP activity (ie, the pro-MMP and active MMP forms) is assessed through the activation of pro-MMP by preincubation with 0.5 mmol/L p-aminophenylmercuric acetate for 2 h at 37°C before the addition of modified Ukcol and chromogenic substrate. Activity is expressed in recombinant enzyme equivalents (recEEs) in nanograms per milliliter. The lower detection limits were 1.4 ng/mL recEE for MMP-1, 1.3 ng/mL recEE for MMP-2, 5.0 ng/mL recEE for MMP-8, 2.7 ng/mL recEE for MMP-9, and 8.3 ng/mL recEE for MMP-13.

DTT Effect: The DTT present in Sputolysin may interfere with MMP activity. In order to study the effect of DTT on MMP activity, standard curves of appropriate recombinant proteins were incubated with 0.1% DTT (Sputolysin) under the same conditions as sputum samples during processing (20 min at room temperature). The presence of DTT did not affect MMP activities, indicating that DTT had no effect on the immunocapture activity assays used in this study (data not shown).

Immunohistochemistry for MMP-8 and MMP-9 on Sputum Cytospin
Cytospins were fixed in acetone and stored at –20°C until use. Cytospins were rehydrated in phosphate-buffered saline solution, pH 7.4, and endogenous peroxidase was quenched with 0.3% H2O2 in methanol. Cytospins were stained with rabbit antihuman MMP-8 or rabbit antihuman MMP-9 polyclonal antibodies (TNO Gaubius; Leiden, the Netherlands). After washing, biotin-conjugated donkey antirabbit IgG antibody (Amersham Biosciences) was applied as the secondary detection antibody, followed by avidin-biotinylated horseradish peroxidase complex (DAKO; Glostrup, Denmark). Enzymatic reactivity was visualized with a red stain (NovaRed; Vector Laboratories; Burlingame, CA). Cytospins were counterstained with hematoxylin and mounted. No significant staining was detected in slides incubated with a control antibody instead of the primary detecting antibody, indicating the absence of significant background staining.

Statistical Analysis
Results are presented as the mean ± SD for normally distributed variables, and as median (range) otherwise. Groups were compared by analysis of covariance adjusted for age, gender, or pack-years where appropriate. The Mann-Whitney U test was used to compare nonparametric data. Categoric variables were compared by the {chi}2 test. Correlations between parameters were evaluated using the Pearson rank correlation analysis (Statistical Package for the Social Sciences, version 10.0.7 for Windows; SPSS Inc; Chicago, IL). A p value of < 0.05 was considered to be statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subject Characteristics
Patients in the COPD group showed mild-to-moderate airflow obstruction, whereas HSs had normal FEV1 values (percent predicted) according to the selection criteria (Table 1 ). Figure 1 shows the sputum characteristics. The percentage of neutrophils was significantly higher in COPD patients than in HSs, whereas the percentage of macrophages was lower. The same was true for the absolute numbers of neutrophils and macrophages (data not shown). The degree of airflow limitation (FEV1 percent predicted) correlated strongly with the percentage of neutrophils (r = –0.599; p < 0.001) in COPD patients, but not in HSs.


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  		Table 1.. Characteristics of the Study Groups Investigated*

 


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  		Figure 1.. Differential cell counts in induced sputum from patients with COPD and HSs. AM = alveolar macrophage; PMN = polymorphonuclear neutrophil; EOS = eosinophil; LYM = lymphocytes; * = p < 0.05 vs HSs.

 
Quantitative Measurement of MMP Activity in Sputum
MMP Gelatinases: MMP-9 activities were found in both COPD patients and control subjects, whereas MMP-2 activity remained below the detection threshold of the assay (Fig 2 ). The mean total MMP-9 activity was significantly higher in the COPD group when compared to that in the control group (p < 0.05), and active MMP-9 was found in 76% of the COPD patients compared to 41% of the HSs (p < 0.05).



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  		Figure 2.. MMP gelatinase activity in induced sputum obtained from patients with COPD and HSs. Top left, A: total MMP-2; bottom left, B: total MMP-9; top right, C: active MMP-2; bottom right, D: active MMP-9. MMP activity was measured using specific immunocapture assays and was expressed in nanograms per milliliter recEEs. Dotted line = lower detection limit; solid line = mean values; * = p < 0.05 vs HSs. NS = not significant.

 
MMP Collagenases: As shown in Figure 3 , the activities of the individual MMP collagenases were highly variable. MMP-13 activities were below the detection threshold of the assay. Mean total MMP-1 activity was low, and no differences were observed between the study groups. Active MMP-1 was found in a small proportion of COPD patients (two patients) and HSs (two subjects). In both patients and control subjects, MMP-8 was the most dominant MMP collagenase. The mean total MMP-8 activity was significantly higher in the COPD group (p < 0.05), and active MMP-8 was found in 35% of the COPD patients compared to 6% of the control subjects (p < 0.05).



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  		Figure 3.. MMP collagenase activity in induced sputum obtained from patients with COPD and HSs. Top left, A: total MMP-1; middle left, B: total MMP-8; bottom left, C: total MMP-13; top right, D: active MMP-1; middle right, E: active MMP-8; bottom right, F: active MMP-13. MMP activity was measured using specific immunocapture assays and was expressed in nanograms per milliliter recEEs. Dotted line = lower detection limit; solid line = mean values; * = p < 0.05 vs HS. See Figure 2 legend for expansion of abbreviation.

 
Influence of Current Smoking Status on MMP Activity: Subjects were classified according to their current smoking behavior, and MMP activity was compared. No differences in the activity of gelatinases and collagenases were observed between individuals who were current smokers and those who were ex-smokers in both groups (data not shown).

Correlations Among MMP Activity, Inflammation, and FEV1
The active form and total activity of MMP-8 and MMP-9 were highly correlated in both study groups. In addition, MMP-8 and MMP-9 were strongly correlated with each other, but not with MMP-1 (data not shown). A significant correlation between both active and total MMP-8 and MMP-9 activity with the absolute number of neutrophils was observed in the sputum of COPD patients (Table 2 ). A similar correlation was noted for total MMP-8 activity in HSs (active MMP-8 was only detectable in one HS). We observed a positive correlation between active MMP-9 activity and total MMP-9 activity, and the absolute number of macrophages in the sputum of HSs, but not in patients with COPD (Fig 4 ).


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  		Table 2.. Correlations Between Sputum MMP Activity and Neutrophils in Patients With COPD and HSs*

 


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  		Figure 4.. Correlation between macrophage numbers (106 cells/mL sputum) and sputum levels of (top, A) total MMP-9 activity (COPD patients: r = 0.197; p = 0.448; control subjects: r = 0.711; p = 0.002) or (bottom, B) active MMP-9 (COPD patients: r = 0.373; p = 0.141; control subjects: r = 0.519; p = 0.040) in patients with COPD ({diamondsuit}) and HSs ({diamond}). MMP activity was measured using specific immunocapture assays and was expressed in nanograms per milliliter recEEs.

 
The relationship between MMP activities and the degree of airflow obstruction was explored. We found that the degree of airflow obstruction was positively related to the sputum activity of MMP-8 (10log active MMP-8: r = –0.410; p = 0.016; 10log total MMP-8: r = –0.395; p = 0.021) and MMP-9 (10log active MMP-9: r = –0.376; p = 0.028; 10log total MMP-9: r = –0.438; p = 0.010) for the complete study group. The correlations between MMP activities and airflow obstruction in the separated groups, however, failed to reach statistical significance.

MMP Immunohistochemistry on Cytospins
Immunohistochemistry on the cytospins of sputum cells was performed to determine the cellular distribution of MMP-8 and MMP-9. As shown in Figure 5 , strong positive staining for MMP-9 was observed in both alveolar macrophages and neutrophils, whereas immunoreactivity against MMP-8 was exclusively found in neutrophils. In addition, squamous epithelial cells (Fig 5), eosinophils, lymphocytes, which were recognized on basis of morphology, and bronchial epithelial cells were negative for MMP-8 and MMP-9 (not shown).



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  		Figure 5.. Immunohistochemistry for (top, A) MMP-8 and (bottom, B) MMP-9 on the cytospins of sputum cells. Note the strong positive staining for MMP-8 in neutrophils and for MMP-9 in both macrophages and neutrophils. Staining for MMP-8 and MMP-9 was absent in squamous epithelial cells (SEC). The results are representative for both COPD patients and HSs. See the legend of Figure 1 for abbreviations not used in the text.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The increased expression of the MMP is considered to be a key factor in the development and progression of COPD. MMP expression represents a tightly regulated process of proteinase expression, activation, and inhibition by specific inhibitors. In this study, we investigated MMP collagenase and gelatinase activity in the induced sputum of patients with mild-to-moderate COPD and HSs. The concept of an imbalance between the release of proteinases, in particular MMPs, and of antiproteinase enzymes in the development and progression of COPD has found wide acceptance.1223 Yet, quantitative data on the delicate balance between proteinases and antiproteinases are missing. Specific enzyme-linked immunosorbent assays have the disadvantage that information on the active/inactive state of the proteinases is lost. Alternatively, the use of zymograms is semiquantitative and, more importantly, does not differentiate between the active proteinase and the proteinase inhibitor complexes. In this study, we used specific immunocapture assays that allowed to determine the activity of both active as well as total (ie, active and latent) MMPs in human body fluids and thus to address the complex interaction between the activation and inhibition of MMP activity. In these assays, specificity is achieved through the capturing of the targeted proteinase by a specific antibody, and the activity of the immobilized proteinase is subsequently assessed by proteolytic activation of a modified urokinase. The assay is insensitive to MMP inhibitor complexes. Moreover, the activation of captured proenzyme through a mercury salt allows the determination of latent forms (potentially activatable proteinases).

MMPs constituted a large family of related proteinases with different substrate specificities. As yet, attention has been focused primarily on the MMP gelatinases MMP-2 and MMP-9.12 It has been pointed out24 that the collagenases (ie, MMP-1, MMP-8, and MMP-13) might be equally important in COPD. MMP-8 and MMP-9 appeared to be the most dominant MMP proteinases in sputum. Both activities were significantly increased in COPD patients compared to HSs, despite the overlap of values between the groups (Fig 2, 3). More importantly, net MMP-8 and MMP-9 activity was increased in a significant proportion of the COPD patients, but in a limited proportion of the "healthy" control subjects who smoked. In contrast to the activity of MMP-8 and MMP-9, the activity of MMP-1 was low in both study groups, and the activity of MMP-2 and MMP-13 was below the detection threshold of the assay.

These findings extend previous findings demonstrating increased MMP-9 concentrations (by enzyme-linked immunosorbent assay) in the induced sputum of patients with chronic bronchitis2526 and COPD,27 and nonquantitative zymographic findings demonstrating MMP-9 activity in the BAL fluid of COPD patients.181928 The presence of active MMP-8 in induced sputum supports the presupposition of Finlay et al28 that MMP-8 constitutes the prime interstitial collagenase in emphysema.

In contrast to MMP-8 and MMP-9, MMP-1 activity was low. This observation apparently contrasts with the increased expression of MMP-1 in the alveolar compartment of patients with late-stage emphysema.17 As induced sputum represents the airway compartment,29 the observed differences may relate to spatial differences in MMP expression between the alveolar compartment and the airway compartment. Alternatively, the differences in proteinase profiles may relate to differences in disease stage. In contrast to the study by Imai et al,17 patients included in the present study had mild-to-moderate COPD, and we cannot exclude that MMP-1 is expressed in later stages of the disease.

Our observation that the activity of MMP-8 and MMP-9 correlates positively with absolute neutrophil numbers in both study groups suggests that MMP-8/MMP-9 activity in sputum originates from activated neutrophils. Neutrophils are thought to play an important role in the chronic inflammatory reaction that is present in the airways of COPD patients.12 In line with previous observations, a significant increase in neutrophils was found in the induced sputum of COPD patients compared with HSs, which highly correlated with the degree of airflow limitation.463031 As sputum samples were centrifuged prior to freezing sputum to obtain cell-free supernatants, increased MMP-8 and MMP-9 activities in COPD sputum reflect enhanced MMP secretion rather than a proteinase release during the freezing and thawing of samples. This notion is supported by the finding of active forms in a higher proportion of the COPD patients. In addition, the presence of complexes of human neutrophil lipocalin/MMP-9 in the BAL fluid of subjects with subclinical emphysema also suggests that MMP-9 that is present in BAL fluid is derived from the active secretion of neutrophils19 rather than being an artifact. As several studies3233 have demonstrated that NE is able to cleave the tissue inhibitor of metalloproteinases without the significant destruction of pro-MMP forms,34 and that NE may directly cleave MMPs, resulting in their activation,33 it is tempting to speculate that increased MMP-8/MMP-9 activity may be directly related to the NE that is present in the neutrophil microenvironment in the induced sputum of patients with COPD.

Using immunohistochemical techniques on cytospins of sputum-derived cells, we found a strong positive signal for MMP-9 in both neutrophils and alveolar macrophages, whereas other cell types like eosinophils, lymphocytes, bronchial epithelial cells, and squamous epithelial cells were negative. Positive staining for MMP-8 was exclusively observed in neutrophils and not in other cell types that are present in sputum, which is consistent with other reports in the literature,2021 as MMP-8 has been labeled as neutrophil collagenase. These results confirm the prominent role of alveolar macrophages and neutrophils in COPD as major sources of MMPs in the larger airway compartment. Studies performing in vitro stimulation of alveolar macrophages isolated from BAL fluid demonstrated that these cells are a significant source of MMP-9 in the airways of subjects with COPD.2835 Alveolar macrophages from COPD patients were shown to release more MMP-9, which is more active than that released from the alveolar macrophages of HSs and nonsmoking individuals.3536

Our data clearly demonstrate active forms of MMP-8 and MMP-9 in the induced sputum samples of patients with mild-to-moderate COPD. The presence of active forms of MMPs is an uncommon finding in biological fluids. As active MMPs are commonly associated with cellular activation under pathologic circumstances, our findings provide further evidence for an impaired proteinase-antiproteinase balance in this disorder. This MMP imbalance in the airways may account for inappropriate matrix remodeling and basement membrane disruption in both the upper and lower respiratory tract. Moreover, excess MMP activity also may contribute to the chronic inflammatory response and interfere with tissue repair through its effects on a variety of nonmatrix proteins, including cytokines, chemokines, integrins, antimicrobial peptides, and other proteinases. Clearly, the precise mechanisms involving MMPs and their inhibitors in matrix remodeling and chronic inflammation in COPD patients, as well as their consequences in terms of abnormality of lung function require further investigations.

In summary, COPD patients had significantly higher levels of active as well as total (activatable) MMP-8 and MMP-9 in their sputum compared to HSs. No differences in the activity of gelatinases and collagenases were observed between individuals who were current smokers and ex-smokers in both groups. In both study groups, the activity of MMP-8 and MMP-9 (active and total) were strongly correlated with each other, and with absolute numbers of neutrophils. A positive correlation between MMP-8 and MMP-9 activity in sputum and the degree of airflow limitation was found when the values of the two study groups were combined. Immunohistochemistry demonstrated strong positive staining for MMP-9 in both alveolar macrophages and neutrophils. Specific staining for MMP-8 was only observed in neutrophils. The presence of increased MMP-8 and MMP-9 activity in the airways of patients with mild-to-moderate COPD supports the concept of an impaired proteinase-antiproteinase balance in COPD.


   Acknowledgements
 
The authors wish to thank Natascha van Lent and Adri Mulder for excellent technical help with the immunocapture activity assays and immunohistochemistry, respectively, and Dr. A.M.W.J. Schols for expert assistance with the statistical evaluation of the data.


   Footnotes
 
Abbreviations: DTT = dithiothreitol; HS = healthy smoker; MMP = matrix metalloproteinase; NE = neutrophil elastase; recEE = recombinant enzyme equivalent; Ukcol = prourokinase

This study was supported by a grant of the Development Foundation (Profileringsfonds) of University Hospital Maastricht, Maastricht, the Netherlands.

Received for publication November 21, 2003. Accepted for publication July 23, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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