MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation
© Mercer et al. 2005
Received: 27 July 2005
Accepted: 22 December 2005
Published: 22 December 2005
Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.
This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.
Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.
MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).
During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.
Chronic obstructive pulmonary disease (COPD) is a classic disease of airway damage and remodelling, characterised by slowly progressive airflow obstruction, resulting in increasing dyspnoea and exercise limitation. It is a widely accepted theory that an important causative factor is extracellular matrix (ECM) remodelling, resulting from aberrant inflammation and disruption of the proteinase – antiproteinase balance; reviewed . The identity of candidate proteinases has been a subject of much debate, however the balance between matrix metalloproteinases (MMPs) and their inhibitors; the tissue inhibitors of metalloproteinase (TIMPs), is thought to be important in pathogenesis [1–3].
MMPs are a family of up to 26 homologous endopeptidases, which can collectively degrade all of the protein components of the extracellular matrix . They are produced by a range of stromal cells and by two of the major inflammatory cells implicated in COPD – neutrophils and alveolar macrophages [5, 6]. Recent studies have shown that levels of MMPs, especially MMP-9, are elevated in the bronchial alveolar lavage (BAL) fluid from patients with COPD, compared to normal controls [5, 7], and high levels of both MMP-9 and its cognate inhibitor TIMP-1 have been found in sputum from chronic bronchitics  and correlated with decrease in lung function [9, 10]. While these studies focused on stable COPD, there has been no analysis to date of MMP-9 and TIMP-1 variation during exacerbations.
Patients with moderate to very severe COPD are prone to frequent exacerbations, which are associated with a poorer health-related quality of life . Exacerbations can be triggered by viral or bacterial infection of the lower respiratory tract [12, 13] both of which increase airway and systemic inflammatory response [14–16]. Exacerbations also contribute to the accelerated decline in FEV1 that is characteristic of COPD [17, 18].
Using a well monitored group of COPD patients, this study has prospectively investigated changes in sputum levels of MMP-9 and TIMP-1 during exacerbations in COPD and compared these to samples taken from the same individuals prior to exacerbation. The aim of the study was to see whether the MMP/TIMP balance was upset during exacerbation as this might suggest a pathway between exacerbations, ECM remodelling and lung function decline.
Nineteen patients were recruited from a cohort of patients followed since October 1995 by the East London COPD study [14, 19]. Inclusion criteria and enrolment for the study have been previously published [14, 19].
The patients were recording on diary cards each morning any increase over their normal, stable condition of (i) dyspnoea, sputum purulence or sputum volume (major symptoms) and (ii) colds (nasal discharge/congestion), wheeze, sore throat, cough and fever (minor symptoms). This was a yes/no answer. Exacerbation onset was identified as the first of two or more consecutive days with increase in either two or more major symptoms, or any one major symptom plus any minor symptom. The prospective data collected on the diary cards before and at onset of the exacerbation showed that peak expiratory flow fell from 246 l/min (223 to 313) averaged over days 14-8 preceding exacerbation by 7.1 l/min (-22 to 3) on the day of onset. Similarly, the number of symptoms rose from zero to 2 (2 to 3) on the day of onset. Patients with any deterioration of symptoms were encouraged to contact the clinical team by telephone, and were seen in clinic for sputum sampling, generally within 48 hours. Patients also attended 3 monthly clinics at which samples were obtained when the patient was exacerbation free (clinically stable).
Characteristics of the 19 patients in the study
63 to 74
FEV1, L (stable)
0.9 to 1.2
27.3 to 46.2
25.9 to 54.8
8.45 to 9.51
5.49 to 6.36
O2 saturation breathing room air (%)
92 to 95
Reversibility to salbutamol (%)
5.0 to 13.5
Daily taking oral steroids
Daily taking inhaled steroids
Sputum samples were processed as per Bhowmik et al 2000 . Briefly, the sputum was incubated with four times its weight of 0.01 M DTT in HBSS, at 4°C for 15 minutes. Sputum dilution was in proportion to the weight collected thus standardizing between time points and patients the supernatants used for assay of proteinases and other markers. The volume of HBSS was then doubled (ten fold dilution of original sputum sample) and incubated for a further 5 minutes. The suspension was then filtered through 50 μm nylon gauze to remove mucus and debris without removing any of the cells and centrifuged at 790 g for 10 minutes. The cell free supernatant was removed and stored at -70°C. The cell pellet was resuspended. The total cell count was determined with a Neubauer haemocytometer using the trypan blue exclusion method to determine cell viability, blue cells were counted as non viable. Cytospins were made from the cell suspension and stained with Diff-Quik to obtain a differential cell count.
Band intensity was captured and digitised by scanning densitometry (using scan analysis). The optical optical density for each band of interest was calculated as area under curve. This value was converted to total MMP-9 for each patient by combining the values for each band (220, 125 and 92 kDa) and normalizing to MMP-9 standard (1 ng/lane, Biogenesis. Dorset, UK) (see Figure 1). Samples outside of the accurate limit of detection were diluted up to a hundred fold and rerun.
Analysis of TIMP-1
Samples were analysed using a commercial Biotrak® TIMP-1 ELISA (Amersham Biosciences. Bucks. UK.) specific for both total free TIMP-1 and TIMP-1 complexed to MMPs. ELISAs were run according to the manufacturer's instructions.
All differences within sets of paired data were analysed using the non-parametric Wilcoxon signed rank test. Correlations were calculated using the non-parametric Spearman correlation. P value < 0.05 was considered significant.
Levels of MMP-9 and TIMP-1 in paired samples
MMP-9 and cellular inflammation
Total and differential leukocyte cell counts in COPD sputum sampled prior to and during exacerbation† (n = 12)
Total cell (106 cells/g)
Neutrophil (106 cells/g)
Macrophage (106 cells/g)
Eosinophil (106 cells/g)
Lymphocyte (106 cells/g)
Percentage squamous cell contamination of sputum varied from 5.14% (IQR 1.81–6.20) during stable COPD, to 1.6% (0.43–4.58) during exacerbation (data not shown).
Spearman's correlation between MMP-9 level and inflammatory cell counts during exacerbation† (n = 19)
This is the first pilot study to look at how levels of the inflammatory proteinase, MMP-9 and its cognate inhibitor, TIMP-1 change in the airway during an exacerbation. The results indicate that exacerbations are associated with an increase in MMP-9 burden in the airways. This increase could result in ECM destruction in the airways and contributing to airway remodelling and the decline in lung function seen in COPD patients. Previous studies have related MMP-9 to parenchymal destruction and lung function decline, in sub clinical  and established emphysema . Studies with induced sputum from stable COPD patients have found that levels of MMP-9 and TIMP-1 are elevated in COPD patients compared to controls [9, 10]. Levels were found to correlate with both increased neutrophilia and decrease in lung function. These studies suggest that an underlying proteolytic environment exists in the airways in COPD, however studies where molar ratio of MMP-9 to TIMP-1 was analysed found that TIMP-1 levels outweighed MMP-9 [8, 21]. Our current study indicates that prior to exacerbation MMP-9/TIMP-1 balance favours the inhibitor. During exacerbation however MMP-9 levels significantly increase, tipping the balance in favour of MMP-9. A similar finding has been reported in asthmatic exacerbations which found that circulating levels of MMP-9 were increased in patients during exacerbation compared with convalescence . The frequency of shift between a "proteolytic" phenotype during exacerbation and "fibrotic" phenotype outside exacerbation, may be an important driving factor in airway remodelling, and deserves further research.
Our data show that number of neutrophils, eosinophils, and lymphocytes all increase in the airways during an exacerbation, whilst levels of macrophages remained constant. Recent studies have shown that alveolar macrophages from smokers increased production of MMP-9 in response to cigarette smoke conditioned medium . Pro-inflammatory stimuli such as IL-1β and LPS also increased MMP-9 production, leading the authors to suggest that Alveolar Macrophage release of MMP-9 could have relevance in COPD exacerbations. In this present study, a strong correlation was observed during exacerbation between increased neutrophil number and MMP-9 while a weaker association existed between macrophages and MMP-9. While the contribution of macrophage derived MMP-9 cannot be ruled out, it is tempting to speculate from these data that neutrophil influx is the primary source of increased MMP-9 during exacerbation. The stimulus for increased neutrophil influx was not assessed in this study, however it seems likely that neutrophil specific chemokines such as IL8 are involved this response. Recently, Bhowmik et al, using sputa from the East London COPD study, reported that levels of IL8 during exacerbation were related to neutrophil number .
Additionally, it is of note that the increase in MMP-9 during exacerbation also shows a very close association with increase in lymphocyte number. It is unclear whether these events are related; however it is pertinent to suggest that the increase in neutrophil and lymphocyte numbers is linked. In support of this concept, a recent study in mice has shown that intranasal stimulation by the cytokine IL17, produced by CD4+ T-lymphocytes induced a pronounouned accumulation in both pro and active MMP-9 associated with local accumulation of neutrophils . A recent study by Zhu et al reported an increase in the number of CD4+ lymphocytes in the airways during exacerbation in COPD . The authors suggested that virally induced upregulation of the chemokine RANTES during exacerbation could contribute to this. In addition an increased airway eosinophilia, also attributed to RANTES upregulation, was observed.
While eosinophils do not have a clearly defined role in COPD, an increased airway eosinophilia during exacerbation has now been observed in a range of studies including our own [16, 25]. The role of eosinophils in disease progression may therefore deserve further investigation. Previous studies using asthmatic BAL identified eosinophils as important sources of MMP-9 . However a comparatively weak, albeit significant association between MMP-9 and eosinophils in our study, suggests eosinophils are not the primary source of MMP-9 during COPD exacerbation.
We report here that during an exacerbation in COPD there is an increased MMP-9 burden in the airways, which combined with unchanging TIMP-1 leads to MMP-9/TIMP-1 imbalance in favour of MMP-9. Increase in MMP-9 correlates with influx in both neutrophils and lymphocytes and to a lesser extent eosinophil and macrophage numbers. The cross talk between these cells during exacerbation and their contribution to airway remodelling requires further detailed investigation.
The authors wish to thank the MRC (UK) and Pfizer for the funding of this work. The authors are particularly grateful to Dr Mike Yeadon (Pfizer) for his helpful discussions.
- Hogg JC, Senior RM: Chronic obstructive pulmonary disease - part 2: pathology and biochemistry of emphysema. Thorax 2002, 57:830–834.View ArticlePubMedPubMed CentralGoogle Scholar
- Atkinson JJ, Senior RM: Matrix metalloproteinase-9 in lung remodeling. Am J Respir Cell Mol Biol 2003, 28:12–24.View ArticlePubMedGoogle Scholar
- Barnes PJ: New treatments for COPD. Nat Rev Drug Discov 2002, 1:437–446.View ArticlePubMedGoogle Scholar
- Visse R, Nagase H: Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003, 92:827–839.View ArticlePubMedGoogle Scholar
- Betsuyaku T, Nishimura M, Takeyabu K, Tanino M, Venge P, Xu S, Kawakami Y: Neutrophil granule proteins in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Am J Respir Crit Care Med 1999, 159:1985–1991.View ArticlePubMedGoogle Scholar
- Finlay GA, O'Driscoll LR, Russell KJ, D'Arcy EM, Masterson JB, FitzGerald MX, O'Connor CM: Matrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am J Respir Crit Care Med 1997, 156:240–247.View ArticlePubMedGoogle Scholar
- Finlay GA, Russell KJ, McMahon KJ, D'Arcy EM, Masterson JB, FitzGerald MX, O'Connor CM: Elevated levels of matrix metalloproteinases in bronchoalveolar lavage fluid of emphysematous patients. Thorax 1997, 52:502–506.View ArticlePubMedPubMed CentralGoogle Scholar
- Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D'accardi P, Bousquet J, Bonsignore G: Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998, 158:1945–1950.View ArticlePubMedGoogle Scholar
- Culpitt SV, Rogers DF, Traves SL, Barnes PJ, Donnelly LE: Sputum matrix metalloproteases: comparison between chronic obstructive pulmonary disease and asthma. Respir Med 2005, 99:703–710.View ArticlePubMedGoogle Scholar
- Vernooy JH, Lindeman JH, Jacobs JA, Hanemaaijer R, Wouters EF: Increased activity of matrix metalloproteinase-8 and matrix metalloproteinase-9 in induced sputum from patients with COPD. Chest 2004, 126:1802–1810.View ArticlePubMedGoogle Scholar
- Seemungal TA, Donaldson GC, Paul EA, Bestall JC, Jeffries DJ, Wedzicha JA: Effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998, 157:1418–1422.View ArticlePubMedGoogle Scholar
- Patel IS, Seemungal TA, Wilks M, Lloyd-Owen SJ, Donaldson GC, Wedzicha JA: Relationship between bacterial colonisation and the frequency, character, and severity of COPD exacerbations. Thorax 2002, 57:759–764.View ArticlePubMedPubMed CentralGoogle Scholar
- Seemungal T, Harper-Owen R, Bhowmik A, Moric I, Sanderson G, Message S, Maccallum P, Meade TW, Jeffries DJ, Johnston SL, Wedzicha JA: Respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001, 164:1618–1623.View ArticlePubMedGoogle Scholar
- Bhowmik A, Seemungal TA, Sapsford RJ, Wedzicha JA: Relation of sputum inflammatory markers to symptoms and lung function changes in COPD exacerbations. Thorax 2000, 55:114–120.View ArticlePubMedPubMed CentralGoogle Scholar
- Crooks SW, Bayley DL, Hill SL, Stockley RA: Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4. Eur Respir J 2000, 15:274–280.View ArticlePubMedGoogle Scholar
- Saetta M, Di Stefano A, Maestrelli P, Turato G, Ruggieri MP, Roggeri A, Calcagni P, Mapp CE, Ciaccia A, Fabbri LM: Airway eosinophilia in chronic bronchitis during exacerbations. Am J Respir Crit Care Med 1994, 150:1646–1652.View ArticlePubMedGoogle Scholar
- Donaldson GC, Seemungal TA, Bhowmik A, Wedzicha JA: Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease. Thorax 2002, 57:847–852.View ArticlePubMedPubMed CentralGoogle Scholar
- Kanner RE, Anthonisen NR, Connett JE: Lower respiratory illnesses promote FEV(1) decline in current smokers but not ex-smokers with mild chronic obstructive pulmonary disease: results from the lung health study. Am J Respir Crit Care Med 2001, 164:358–364.View ArticlePubMedGoogle Scholar
- Seemungal TA, Donaldson GC, Bhowmik A, Jeffries DJ, Wedzicha JA: Time course and recovery of exacerbations in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000, 161:1608–1613.View ArticlePubMedGoogle Scholar
- Kleiner DE, Stetler-Stevenson WG: Quantitative zymography: detection of picogram quantities of gelatinases. Anal Biochem 1994, 218:325–329.View ArticlePubMedGoogle Scholar
- Beeh KM, Beier J, Kornmann O, Buhl R: Sputum matrix metalloproteinase-9, tissue inhibitor of metalloprotinease-1, and their molar ratio in patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and healthy subjects. Respir Med 2003, 97:634–639.View ArticlePubMedGoogle Scholar
- Oshita Y, Koga T, Kamimura T, Matsuo K, Rikimaru T, Aizawa H: Increased circulating 92 kDa matrix metalloproteinase (MMP-9) activity in exacerbations of asthma. Thorax 2003, 58:757–760.View ArticlePubMedPubMed CentralGoogle Scholar
- Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ: Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2002, 26:602–609.View ArticlePubMedGoogle Scholar
- Prause O, Bozinovski S, Anderson GP, Linden A: Increased matrix metalloproteinase-9 concentration and activity after stimulation with interleukin-17 in mouse airways. Thorax 2004, 59:313–317.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhu J, Qiu YS, Majumdar S, Gamble E, Matin D, Turato G, Fabbri LM, Barnes N, Saetta M, Jeffery PK: Exacerbations of Bronchitis: bronchial eosinophilia and gene expression for interleukin-4, interleukin-5, and eosinophil chemoattractants. Am J Respir Crit Care Med 2001, 164:109–116.View ArticlePubMedGoogle Scholar
- Warner JA, Julius P, Luttmann W, Kroegel C: Matrix metalloproteinases in bronchoalveolar lavage fluid following antigen challenge. Int Arch Allergy Immunol 1997, 113:318–320.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.