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Asthma and chronic obstructive pulmonary disease (COPD) are different conditions with contrasting airway inflammation and parenchymal disease patterns. A number of matrix metalloproteases (MMPs) are implicated in the pathophysiology of COPD and asthma. Different profiles of airway MMPs may, therefore, be expected in asthma and COPD. The present study compared MMP profiles in the airways of non-smokers, non-symptomatic cigarette smokers, and patients with COPD or asthma ( subjects per group). Induced sputum was assessed for MMP-1, -2, -3, -8 and -9, and tissue inhibitor of metalloproteases (TIMP)-1 by ELISA. Gelatinase activity was determined by zymography. Sputum from COPD patients contained increased levels of MMP-1, -8 and -9 compared with the other groups (2–7-fold, depending upon group). MMP-9 activity was elevated in COPD sputum by 3–12-fold above the other groups. Sputum from COPD patients had 3-fold higher levels of TIMP-1 than samples from asthmatics or controls, but was not different to smokers. FEV1 correlated negatively with MMP-1, -8, -9, MMP-9 activity and TIMP-1, whereas percent neutrophils in sputum correlated positively with MMP-1, -8, -9, TIMP-1 and MMP-9 activity. The MMP profile in COPD differs to that in asthma and cigarette smokers. This may contribute to, or be a marker of, different pathophysiologies of asthma and COPD.
Cigarette smoking is the major risk factor for development of COPD, and smoking cessation is currently the only intervention that slows disease progression.
COPD comprises three disease components, namely chronic bronchitis (mucous hypersecretion), chronic bronchiolitis (also known as small airways disease) and emphysema (alveolar destruction).
The contribution of each component to disease status and clinical presentation varies from one patient to another. Endogenous protease activity is implicated in the pathophysiology of each of these three disease components.
In contrast to COPD, asthma is defined as a clinical entity characterised by variable airflow obstruction and increased airway responsiveness that is reversible, either spontaneously or with treatment.
A number of studies have examined the expression of individual MMPs in either asthma or COPD and compared it with healthy control subjects. For example, there is increased expression of the collagenases MMP-1 and -8 in asthmatic airways compared with non-asthmatic controls
Soluble membrane-type 1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis.
with no reports of its expression in the lung in COPD. MMP activity is regulated by endogenous inhibitors termed tissue inhibitors of metalloproteinase (TIMPs), which are also elevated in asthma
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.
However, to the knowledge of the authors, there has been no direct comparison of airway MMP and TIMP expression between patients with asthma and patients with COPD.
The aim of the present study was to compare and contrast the concentrations of MMP-1, -2, -3, -8 and 9, together with TIMP-1, in induced sputum from patients with asthma and patients with COPD. Non-smoking subjects and non-symptomatic cigarette smokers were included as relevant controls. In addition, the enzymatic activity of MMP-9 was assessed using gelatin zymography. Moreover, glucocorticosteroids down-regulate MMP expression, and upregulate TIMP expression, in asthma.
Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma.
Consequently, we recruited mild asthmatics who were not currently taking glucocorticosteroids, which might confuse interpretation of the data. In addition, they were matched for pharmacotherapy with the COPD patients (bronchodilator therapy alone).
Methods
Subjects
Four groups of subjects were recruited: 15 patients with COPD (cigarette smokers), diagnosed according to current standard criteria,
15 current smokers without airways obstruction (FEV1>80% predicted) and 15 non-smoking control subjects without lung disease (Table 1). Smokers and COPD patients had a smoking history of >20 pack years. Six of the COPD patients could be classified as stage II, eight patients as stage III and one patient as stage IV.
None of the current smokers could be classified as stage 0 COPD. The asthmatic patients had bronchial hyperreactivity as evidenced by a PC20 methacholine of <4mg/ml. Otherwise, the asthmatic patients were considered to be mild and clinically stable, with no change in treatment for at least 8 weeks prior to study and no more than four short courses of oral corticosteroids (<2 weeks) during the preceding year. Asthmatic subjects maintained their current therapy (i.e. β2-agonists alone, with no inhaled or oral glucocorticosteroids for at least 8 weeks prior to the study), as did the COPD patients (i.e. β2-agonists and/or anticholinergics only). Neither the asthmatic nor the COPD patients had suffered an exacerbation for at least 8 weeks prior to the study. Smokers were unmedicated. The study was approved by the Ethics Committee of the Royal Brompton and Harefield NHS Trust, and subjects gave informed written consent.
Table 1Clinical characteristics of control subjects, asthmatic patients, cigarette smokers and COPD patients
The concentrations of total MMP and TIMP-1 in sputum supernatant were determined using paired antibody quantitative ELISAs and appropriate blanks (Amersham Life Sciences, Little Chalfont, UK). The assay detected both active and latent forms of MMPs. The presence of dithiothreitol (DTT) (0.05% w/v) in the sputum samples reduced the sensitivity of the assays. However, this was controlled for by inclusion of DTT in the standards for each ELISA. The lower limit of detection of the assays was 1.8ng/ml for MMP-1, 0.4ng/ml for MMP-2, 2.4ng/ml for MMP-3, 32pg/ml for MMP-8, 0.6ng/ml for MMP-9 and 1.3ng/ml for TIMP-1.
Zymography
Sputum supernatant was incubated with β-galactosidase (30 i.u. per 100μl) for 2h with shaking at 37°C to partially digest mucopolysaccharides in order to improve resolution of the sputum samples on the zymographic gel. Sputum protein content was measured using the BioRad protein assay reagent according to the manufacturer's instructions (BioRad Ltd., Hemel Hempstead, UK). Two micrograms of sputum protein was diluted with Tris-Glycine sodium dodecyl sulphate (SDS) sample running buffer (0.25M Tris-HCl containing glycerol 20% v/v, 4% w/v SDS and 0.05% w/v bromophenol blue), and zymography performed as described previously.
Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease.
It should be noted that MMP-2 activity was not detected in any of the samples (see Results). Consequently, gelatinase activity in these samples was due to MMP-9. Standards of 0.1μg human recombinant active MMP-9 (Oncogene Research Products, Cambridge, MA, USA) were resolved on each gel to provide a reference for gelatinase activity (MMP-2 and MMP-9) in the samples. Gels were analysed using the Gelworks (UVP Ltd., Cambridge, UK) software system which calculated a measurement of gelatinase activity according to band thickness and density. MMP-9 activity in the samples was expressed as a percentage of the MMP-9 standard for each gel.
Statistical analysis
Comparisons between subject groups were made using the Kruskal–Wallis test followed by Dunn's multiple comparison test, or a Student's t-test where applicable. The significance of the relationship between two variables was assessed using Spearman's rank correlation coefficient. The null hypothesis was rejected at .
Results
Clinical characteristics of subjects
The current smokers and patients with COPD were older than both the asthmatic patients and the non-smoking controls (Table 1). The patients with COPD were older than the current smokers and had smoked more cigarettes. None of the current smokers had an FEV1:FVC ratio of <0.7, indicating that they were not stage 1 COPD subjects according to GOLD criteria.
We cannot anticipate whether or not these subjects would in later life develop COPD and, therefore, have specifically not referred to them herein as ‘healthy’ smokers.
Inflammatory cell counts
There were no significant differences in the total number of inflammatory cells recovered in sputum from any of the groups (Table 2). Patients with COPD had a significantly reduced percentage macrophage count and an increased percentage neutrophil count compared with any of the other groups (Table 2). Eosinophils were not detected in sputum of normal controls, smokers or COPD patients, whereas patients with asthma had a mean % eosinophil count of 2.7% (s.e.m. 0.8%).
Table 2Induced sputum total cell counts and differential cell counts
MMP-1 was significantly increased in the sputum of patients with COPD, with increases in median values of almost 4-fold above controls, 3-fold above asthmatics and ∼2-fold above smokers (Fig. 1, panel A). In contrast, there were no significant differences in concentrations of either MMP-2 or -3 between the four subject groups (Fig. 1, panels B and C). MMP-8, however, was increased in the sputum of patients with COPD, with increases in median values of ∼4-fold above controls, ∼5-fold above asthmatics and ∼3-fold above smokers (Fig. 1, panel D). Similarly, MMP-9 was increased in the sputum of patients with COPD, with increases in median values of 8.5-fold above controls, 6.5-fold above asthmatics and 4-fold above smokers (Fig. 1, panel E). In addition, there was an increase of ∼2-fold in the sputum of smokers above that of controls or asthmatics. TIMP-1 was increased in the sputum of patients with COPD above that in controls and asthmatics (∼3-fold increase for both subject groups), but with no significant change compared with smokers (Fig. 1, panel F). In addition, TIMP-1 was elevated in the sputum of smokers compared with that of controls or asthmatics (∼3-fold increase for both).
Figure 1Concentrations of MMPs and tissue inhibitor of MMP (TIMP)-1 in induced sputum from control subjects, asthmatics, cigarette smokers and patients with COPD. Panel A=MMP-1, panel B=MMP-2, panel C=MMP-3, panel D=MMP-8, panel E=MMP-9 and panel F=TIMP-1. , , .
Active MMP-9 (86kDa band) was significantly elevated in the sputum of patients with COPD (Fig. 2, panel A), with increases in median values of ∼12-fold above controls, 5-fold above asthmatics and ∼3-fold above smokers (Fig. 2, panel B). In addition, MMP-9 activity was increased in the sputum of smokers compared with controls by 4.5-fold. There was no increase in activity in the sputum from the asthmatics (Fig. 2, panel B). MMP-2 activity was not detected in any of the sputum samples (data not shown). The high molecular weight zones of lysis on the zymography gels corresponded to latent MMP-9. β-galactosidase did not lyse the gels (data not shown).
Figure 2MMP-9 activity in induced sputum from control subjects, asthmatics, cigarette smokers and patients with COPD. Panel A shows a representative gelatin zymogram of MMP-9 activity in individual subjects from the different groups: C=controls, A=asthmatic patients, S=smokers and COPD patients. MMP-9 is increased in the smokers and patients with COPD. Panel B=group data. , .
Relationship between MMP and TIMP-1 with lung function and sputum inflammatory cells
MMP-8 and -9 concentrations and activity correlated negatively with FEV1 (Table 3; Fig. 3, panels A and D). Similarly, MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, also correlated negatively with FEV1. MMP-8 concentrations and MMP-9 concentrations and activity correlated positively with both the percentage (Table 3; Fig. 3, panels B and E) and number of neutrophils (Table 3). Similarly, MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, also correlated positively with the percentage and number of neutrophils. MMP-8 concentrations and MMP-9 concentrations and activity correlated negatively with the percentage of macrophages (Table 3; Fig. 3, panels C and F). Similarly MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, correlated negatively with the percentage of macrophages. No correlations were observed with macrophage numbers. There was also no correlation between MMP-8 levels and FEV1 or neutrophil count, or between MMP-9 activity and FEV1 or neutrophil count for any of the individual groups. Similarly, there was no correlation between MMP-9 activity, % neutrophils or % macrophages and age for any of the subject groups. There was also no correlation between age and MMP-8 concentrations for normal controls and COPD patients. However, MMP-8 correlated negatively with age in the asthmatic group (, ) and positively with age in the smokers (, ).
Table 3Relationship between MMPs and TIMP-1 with lung function and sputum inflammatory cells
Figure 3Relationship between MMP-8 concentrations and MMP-9 activity with lung function and sputum inflammatory cells. Panels A–C=MMP-8 vs. FEV1, % neutrophils and % macrophages respectively; panels D–F=MMP-9 vs. FEV1, % neutrophils and % macrophages, respectively. ○=controls, ●=asthmatics, □=smokers, ■=COPD patients. r=Spearman's rank correlation coefficient.
In the present study, MMP-1, -8, and -9, were increased in sputum from patients with COPD compared with patients with asthma. To our knowledge, this is the first direct demonstration of differences in pulmonary MMP expression between asthma and COPD. The levels of these MMPs in COPD sputum were also increased compared with non-smokers and current cigarette smokers without COPD. This is consistent with observations that these MMPs are elevated in the lungs of patients with COPD.
The increase in concentration of MMP-9 in sputum from patients with COPD may be related to smoking status, since MMP-9 levels were also elevated in the sputum from smokers, or may be due to age differences within our groups. However, it is noteworthy that there was no significant correlation between, for example, smoking history (pack years) and MMP-9 activity (). Similarly, there was an overall lack of positive correlation between age and the inflammatory markers measured herein, except for MMP-8 in the smokers. Herein, there was no change in MMP-2 or MMP-3 in the sputum of COPD patients. This differs from the observation of increased MMP-2 levels in lung tissue from patients with emphysema.
The reason(s) underlying this discrepancy may relate to sampling differences. The present study used induced sputum whereas the cited study examined lung tissue. Alternatively, the discrepancy could be related to different study populations since it is reported that MMP-2 activity is detected in induced sputum in only 25% of a cohort of COPD patients.
In the present study, TIMP-1 was elevated in COPD patients compared with the other groups. This is consistent with the observation that sputum TIMP-1 levels were enhanced in COPD compared with healthy subjects.
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.
Therefore, the increase in TIMP-1 expression observed in COPD may not be sufficient to counteract the effects of the numerous MMPs that are elevated in COPD, leading to a protease–antiprotease imbalance and an enhanced proteolytic environment.
In the present study, levels of MMPs or TIMP-1 were not elevated in our asthmatic patients. This is in contrast to certain other studies.
Soluble membrane-type 1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis.
The reason(s) for these discrepancies may reflect asthma severity, with our patients being milder than in the studies cited above. Consequently, it is possible that the lungs of our asthmatic patients exhibited less remodelling than those with more severe disease. Part of the remodelling process might be due to the activity of MMPs, which would be less marked in these milder patients. Consistent with this suggestion is the observation that MMP-9 levels are greater in BAL fluid and induced sputum of patients with severe asthma compared with patients with mild or moderate asthma.
Subepithelial basement membrane immunoreactivity for matrix metalloproteinase 9 association with asthma severity, neutrophilic inflammation, and wound repair.
The increase in MMP-9 may reflect an increase in neutrophils that are found in the induced sputum of patients with severe asthma and during exacerbations.
In the present study, lung function was inversely related to MMP-1, -8,-9 and TIMP-1 levels. This is consistent with a number of other diverse observations.
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.
Subepithelial basement membrane immunoreactivity for matrix metalloproteinase 9 association with asthma severity, neutrophilic inflammation, and wound repair.
Matrix metalloproteinase-9, but not tissue inhibitor of matrix metalloproteinase-1, increases in the sputum from allergic asthmatic patients after allergen challenge.
The negative correlation between MMP-1 and FEV1 in the current study has not been reported previously, although polymorphisms in the MMP-1 and -12 genes, but not the MMP-9 gene, are associated with a rapid decline in lung function in cigarette smokers.
This relationship may be due to increased inflammatory cell infiltrate in these conditions, and consistent with the positive correlations observed between neutrophils and MMP-9 in asthmatic or COPD sputum.
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.
Matrix metalloproteinase-9, but not tissue inhibitor of matrix metalloproteinase-1, increases in the sputum from allergic asthmatic patients after allergen challenge.
The differences in MMP expression between asthmatics and patients with COPD seen herein may reflect chronicity of disease. Our asthmatic patients were relatively mild, whereas our COPD patients were moderate to severe (Table 1). However, the two groups were matched for pharmacotherapy (bronchodilator treatment alone). This is relevant because inhaled glucocorticosteroids down-regulate expression of MMP-9 in asthmatics.
Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma.
The lack of differences in any of the MMPs between the normal controls and the asthmatic patients possibly reflects the relative mildness of disease in the asthmatic group. The increased concentrations of MMP-1, -8 and -9 in the COPD patients compared with the asthmatics may reflect the destructive nature of COPD. Although airway remodelling is a feature of asthma, it is not the tissue destruction characteristic of emphysema. Therefore, the enhanced levels of MMP expression, together with MMP-9 activity, reflect the enhanced pulmonary proteolytic load in COPD.
In summary, the present study demonstrates increased concentrations of MMP-1, -8, -9 activity and TIMP-1 in induced sputum from COPD patients compared with patients with mild asthma, cigarette smokers or control subjects. Cigarette smokers have increased levels of MMP-9 and TIMP-1 compared with asthmatics and controls, and greater MMP-9 activity than controls. MMP-8 concentrations and MMP-9 activity correlate negatively with FEV1, and are positively correlated with the percentage and number of neutrophils. In conclusion, the MMP profile of COPD patients differs to that seen in mild asthmatics, and may contribute to, or be a marker of, the different pathophysiologies of asthma and COPD.
References
Barnes P.J.
The role of inflammation and anti-inflammatory medication in asthma.
Soluble membrane-type 1 matrix metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis.
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.
Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma.
Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease.
Matrix metalloproteinase-9, but not tissue inhibitor of matrix metalloproteinase-1, increases in the sputum from allergic asthmatic patients after allergen challenge.
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