Abstract
Matrix metalloproteinases (MMPs) are elevated in the airways and blood of COPD patients, contributing to disease pathogenesis and tissue remodelling. However, it is not clear if MMP levels in airways, blood and urine are related or if MMP levels are related to disease severity or presence of exacerbations requiring hospitalisation. Seventy-two patients requiring hospitalisation for COPD exacerbations had serum, urine and sputum MMP-8, -9 and active MMP-9 measured by ELISA and gelatin zymography on day one, five and four weeks later (recovery). Clinical history, spirometry, COPD Assessment Test and MRC dyspnoea score were obtained. Twenty-two stable COPD patients had MMP measurements one week apart. During exacerbations, serum and urine MMP-9 were slightly elevated by 17% and 30% compared with recovery values respectively (p = 0.001 and p = 0.026). MMP-8 was not significantly changed. These MMP levels related to serum neutrophil numbers but not to outcome of exacerbations, disease severity measures or smoking status. In clinically stable patients, serum MMP levels did not vary significantly over 7 days, whereas urine MMPs varied by up to nine fold for MMP-8 (p = 0.003). Sputum, serum and urine contained different MMP species and complexes. Median values for sputum active MMP-9 were significantly different from serum (p = 0.035) and urine (p = 0.024). Serum and urine MMPs are only modestly elevated during exacerbations of COPD and unlikely to be useful biomarkers in this clinical setting. Airway, serum and urine MMP levels are independent of each other in COPD patients. Further, MMP levels are variable between patients and do not reflect airflow obstruction.
Introduction
Chronic obstructive pulmonary disease (COPD) is the 3rd most-frequent cause of death worldwide and affects around 3 million people in the United Kingdom (Citation1). Inhaled toxins, chiefly cigarette smoke, lead to persisting airway inflammation resulting in neutrophil chemotaxis and the release of the neutrophil derived proteases, including cathepsin G, neutrophil elastase and matrix metalloproteinases (MMP) (Citation2,Citation3). Patients with COPD experience sudden periods of deterioration in symptoms, known as exacerbations, triggered by bacteria, viral or environmental factors which are associated with increased airway neutrophil influx, protease release and an accelerated loss of lung function (Citation4,Citation5).
A number of studies have shown that MMP-8 and -9 are elevated in the airways and blood of patients with COPD compared with healthy controls (Citation6–Citation9). MMPs require activation for most functions and in the airways of those with COPD, oxidative stress, cigarette smoke, and other airway proteases activate MMPs contributing to chronic tissue remodelling (Citation10–Citation12). MMPs are involved in a number of processes of relevance to COPD including modulating growth factor, cytokine and chemokine activity, inflammatory cell migration, extracellular matrix turnover and tissue remodelling (Citation13). In mechanistic studies, deletion of MMP-9 reduces cigarette smoke induced emphysema in mice, and in humans MMP activity is linked to the specific aspects of the disease phenotype (Citation14–Citation18). Interestingly, urinary MMPs are elevated in other protease driven lung diseases (Citation19) and cancers where urinary MMPs can predict outcome (Citation20).
As a consequence of these observations, many reports describing the expression of MMPs in COPD suggest they may be useful markers of disease activity and possibly predict prognosis (Citation7,Citation21). Despite this, it is not clear if these measures could be useful clinically nor whether airway, serum and urine levels of MMPs are related and if serum or urine levels can be used as surrogates of airway MMP expression and activity. To determine if serum and urine MMPs reflect airway MMP levels and if these non-invasive measurements can be used as markers of disease severity or exacerbations we have analysed sputum, serum and urine MMPs in 72 patients with COPD who required hospital admission due to an exacerbation of COPD.
Methods
Subjects
Two groups of patients were recruited for the study. First, to determine the variability of serum and urine MMP measurements over time, outpatients with stable COPD were recruited from Nottingham University Hospitals NHS Trust. All patients were aged between 40–85 years, had a greater than 10 pack-year smoking history, symptoms compatible with COPD and a forced expiratory volume in 1 second /forced vital capacity (FEV1/FVC) ratio of < 0.7 (Citation22). Patients with a history of recent exacerbation or change in medication were excluded. The study was approved by the Nottingham research ethics committee and all patients provided written informed consent.
For the exacerbation study, patients between 40 and 85 years of age with diagnosis of COPD exacerbation made by a consultant physician were recruited within 24 hours of hospital admission (called exacerbation day 1) and a follow-up visit at 5–7 days (exacerbation day 5–7) and were seen four weeks after the initial admission when the exacerbation had resolved (recovery day 28–30).
At admission (exacerbation day 1), blood was taken for full blood count, CRP and other measurements as directed by clinical need. At all visits, venous blood, non-induced sputum and spot urine samples were taken, the COPD Assessment Test (CAT) (Citation23) and the MRC breathlessness score were determined (Citation24). Not all patients were able to provide all samples, particularly sputum, at each visit. At the final visit (recovery day 28–30), spirometry was performed according to ATS/ERS standards (Citation25). COPD was confirmed by a history of smoking greater than 10 pack-years and an FEV1/FVC ratio of < 0.7. GOLD stage was then assigned according to FEV1% predicted (Citation26). Those determined not to have COPD by lung function criteria and those with consolidation visible on chest radiograph consistent with pneumonia were not included for further analysis in the study (Figure ). The study protocol was approved by the Nottingham research ethics committee (NRES ref: 10/N0403/85) and all patients gave written informed consent.
Figure 1. Consort diagram and study protocol for the COPD exacerbation cohort. Other withdrawal includes inability to complete protocol due to illness, further disease exacerbation, inability to attend visit due to co-morbidity or frailty and withdrawal of consent.
Sample collection and storage
Blood samples were collected in serum separator tubes, allowed to clot for 30 minutes on ice and then centrifuged for 10 minutes at 4000 g. Urine samples were centrifuged for 10 minutes at 1500 g. Sputum samples were processed with PBS to obtain supernatants as described previously (Citation27). All samples were stored at –80°C prior to analysis.
MMP measurements
Total MMP-8 and -9 protein (hereafter called MMP-8 and -9) were quantitated using DuoSet Human MMP-8 and -9 immunoassays (R&D Systems) and validated as previously described (Citation28). Urine samples were corrected for dilution by measurement of creatinine concentration (Parameter assay, R&D Systems, Minneapolis, MN) and MMP concentrations in urine expressed as ng/mmol creatinine. MMP-9 species were detected and quantitated by gelatin zymography as previously described (Citation27,Citation29). Gelatin zymography also detects MMP-2, which is present in serum. Sputum samples were diluted 1:40, urine samples 1:2 and serum 1:20 prior to zymography. Dilution values were factored into comparisons between sample types.
In preliminary experiments we showed that storage under these conditions for up to 56 days and up to 4 freeze thaw cycles did not significantly affect the level of serum MMP-9 and active MMP-9 detected by zymography with coefficient of variation (CV) values of 8.1% and 15.3%, respectively. Limits of detection for ELISAs were 31.2–2000 pg/ml for MMP-9 and 62.5–4000 pg/ml for MMP-8. For gelatin zymography values were 46.9–3000 pg/ml for active MMP-9 and 156-5000 pg/ml for active MMP-2.
Analyses
Unless otherwise stated, to study the effect of exacerbations, exacerbation day 1 values were compared with recovery day 28–30. To examine the relationship between MMPs and disease severity, MMP levels from recovery day 28–30 were used when patients had returned to their baseline clinical state. Associations between variables and MMP levels were examined using Spearman's Correlation. MMPs at time of exacerbation and recovery were compared using the Mann–Whitney U-test and paired groups analysed by Wilcoxon matched pairs test. Analyses were performed using GraphPad Prism v6.02 (GraphPad Software, San Diego, California, USA) and SPSS v21 (SPSS Inc., Chicago, Illinois, USA). Statistical significance was set at p < 0.05.
Results
Stability of circulating MMPs in COPD
Twenty-two patients with COPD and no history of exacerbation for 6 weeks prior to and over the study period, had blood and urine sampled on two occasions one week apart. The patient's disease remained stable as observed clinically and by FEV1 and FEV1/FEV values (Supplemental Table ). Despite correction for dilution, urinary MMP values were markedly more variable than serum values. In serum, MMP-9, active MMP-9 and MMP-8 varied between the two time points by 16, 14 and 28%, respectively (none significantly different), whereas urinary MMP-9, active MMP-9 and MMP-8 varied by 79, 65 and 907% (Wilcoxon test p = 0.0034 for MMP-8, others non-significant) (Supplemental Table ).
Exacerbation study
A total of 153 patients were recruited at hospital admission with a working diagnosis of exacerbation of COPD. Of these, 125 patients had a follow-up visit 5–7 days later, two patients having died and 26 either having declined or being too unwell to complete a second visit. Then, 89 patients attended for the final recovery visit at 4 weeks, a further three having died. Others who had suffered a further exacerbation or were unwell due to other diseases were also excluded. 72 fitted the study criteria for COPD who comprised the final study population (Figure ). Those not completing the study tended to be slightly older, had more severe disease and a longer duration of inpatient stay than those completing the study, although these differences were not significant (Table ). Thirty-three of the final cohort had been treated with antibiotics before their first assessment.
Table 1. Characteristics of patients recruited to the study, attending follow-up visit (F/U) and confirmed to have COPD
MMP expression in the airways, serum and urine are independent
To determine whether serum and urine MMPs reflect MMP expression in the airways, we analysed the gelatinases, MMP-2 and -9, by zymography in samples of sputum, serum and urine obtained simultaneously both during exacerbations and at recovery. All three sample types had different patterns of MMP species and complexes. Sputum contained pro, active and neutrophil gelatinase associated lipocalin (NGAL) associated MMP-9, but not MMP-2. Serum contained pro and active MMP-9, MMP-9 dimers and pro MMP-2. Urine samples contained pro and active MMP-9, and MMP-9/NGAL complexes but no MMP-2 (Figure ). Fifteen patients were able to provide matched sputum, serum and urine from any visit and these samples were compared directly. The median value for active MMP-9 was 0.14 (IQR 0.009–0.88) for sputum 0.062 (0.029–0.11) for serum and 0.001 (0.0008–0.0014) for urine. Sputum active MMP-9 values were significantly different from serum (p = 0.035, n = 15) and urine (p = 0.024, n = 11) (Figure ). Prior antibiotic treatment had no significant effect upon serum or urine MMP-8, -9 or active MMP-9 levels in serum or urine (data not shown).
Figure 2. MMP expression is different in sputum, serum and urine. (a) Representative gelatin zymogram of serum, sputum and urine of a single patient showing different MMP species and complexes in separate compartments. Visit 1: admission during exacerbation, visit 2: exacerbation days 5–7, visit 3: recovery days 28–30. NGAL, neutrophil associated lipocalin. (b) Quantitation of values of active MMP-9 in sputum, serum and urine from 15 patients. (c) Relative change in MMP-8, -9 and active MMP-9 in serum and urine from exacerbation day 1 to recovery days 28–30 in 53 patients. The direction of change was not consistent for any of the MMP species tested.
Fifty-three patients from the final cohort were able to provide paired serum and urine samples from any visit which were analysed by ELISA. There was no correlation of total MMP-8, MMP-9 or active MMP-9 protein between serum and urine (Table ). Further, during exacerbations, direction of change defined by increase or decrease in serum MMP expression compared with increase or decrease in urine for individual patients; was as likely to be in different directions as the same direction. Collectively these findings suggest that serum, urine and airway MMP values are not directly related or predictive of each other (Figure ).
Table 2. Comparison of MMP measures within sputum, serum and urine obtained at the same visit
Table 3. Association between MMP values and lung function in 72 patients at recovery visit
Table 4. Median (IQR) MMP levels in COPD patient serum and urine measured during exacerbations and recovery
Supplemental Table 1 . Characteristics of stable patients measured 1 week apart
Supplemental Table 2 . MMPs values at two time points in stable patient with COPD
Serum and urine MMP expression are not associated with disease severity
The 72 patients with confirmed COPD were characterised according to disease severity at recovery visit by spirometry, GOLD stage, MRC dyspnoea score and CAT score at their recovery visit (Table ). There was no association between serum or urine MMP-8 and -9 at recovery visit with FEV1 or FEV1/FVC ratio (Table and Supplemental Figure ). Although there was a trend toward positive correlations between serum MMP-8 and GOLD stage, and serum MMP-9 and CAT score, no MMP was significantly associated with disease severity (Figure ).
Supplemental Table 3 . Baseline serum and urine levels of MMPs compared to FEV1 and FEV1/FVC
Figure 3. MMP-8 and -9 are not associated with severity of COPD. Graphs show individual MMP levels in serum and urine at different GOLD stages. (a and b) Serum MMP-8 and -9, (c and d) urine MMP-8 and -9, (e) serum MMP-9 and CAT score, (f) serum MMP-9 and MRC dyspnoea score.
The effect of exacerbations on MMP levels
Forty-one out of 72 patients had exacerbation MMP-9 levels that exceeded their baseline. For the group as a whole, MMP-9 protein was elevated over baseline by 17% (p = 0.001) in serum and by 30% (p = 0.026) in urine for the day 5 samples during exacerbations. There was no significant difference in MMP-8 or serum active MMP-9 during exacerbations. Interestingly, urine active MMP-9 expression tended to be lower during exacerbations than at baseline (p = 0.003. Figure ; Table 4).
Figure 4. Change in MMPs between exacerbations and convalescence in individual patients. (a-c) Serum MMP-9, -8 and active MMP-9, respectively, (d-f) Urine MMP-9, -8 and active MMP-9, respectively.
We then examined whether MMP levels at admission were associated with outcome from exacerbation and compared serum MMP-8 and -9 with risk of death, or length of hospital stay. There was no difference in MMP levels between patients with a hospital stay over 7 days or death compared with those able to be discharged within 7 days (Supplemental Table ).
Supplemental Table 4 . MMP levels in serum and urine at admission compared with length of hospital stay
MMP-8 and -9 levels are related to neutrophil numbers but not cigarette smoking
Of the 153 patients recruited to the study at time of exacerbation, 104 had blood neutrophil counts above the normal range. Total serum MMP-8 and MMP-9 were positively associated with neutrophil count (two-tailed Spearman's rank r = 0.17, p = 0.047 and r = 0.3, p = 0.0003 respectively) (Figure ). Blood neutrophil count was not related to either MMP-8 or -9 in urine (data not shown). Recovery visit MMP values, compared between 72 current and previous smokers were not significantly different (Supplemental Figure ).
Figure 5. Serum MMP-8 and -9 are correlated with serum neutrophils. (a) serum MMP-9, (b) serum MMP-8. Neutrophil count × 106 ml-1.
Discussion
Despite strong evidence to suggest that MMP-8 and -9 are elevated in the airways and blood of patients with COPD and that MMPs contribute to disease progression, we have shown in a real world study of patients experiencing exacerbations of COPD, that neither MMP expression nor activity reflect disease severity analysed by degree of airflow obstruction, GOLD stage or CAT score when stable. Although there was a modest increase in total serum MMP-9 in the majority of patients during COPD exacerbations, MMP-8, and active MMP-9 did not differ significantly for the group as a whole during exacerbations when compared with recovery. We also, for the first time, compared MMP expression in sputum, blood and urine: interestingly we observed that these separate compartments expressed MMP-2, -8 and -9 in different states of activation and complexed differently.
Further, during disease exacerbations, MMP levels in serum, urine and the airways varied independently of each other. Collectively, our findings suggest that in individual patients, neither MMP expression nor activity in serum or urine reflect airway MMP activity, nor are they clinically useful as markers of disease exacerbations or disease stage. The high inter-subject variation in urine MMP levels suggests they are unlikely to serve as effective biomarkers for COPD exacerbations.
The observation that proteolytic activity is regulated differently in specific body compartments is important for the measurement and understanding of proteolysis in COPD. Although previous work has shown that circulating MMP activity affects disease phenotype with respect to elastin degradation in the skin and vasculature (Citation30), to our knowledge, no studies have compared how these systemic and airway protease activities are related. Although we see a modest elevation of serum MMP-9 during exacerbations, our findings suggest, that at least for -9, protease activity in serum and urine is mostly independent of that in the lung and suggests that circulating MMP levels in COPD may be the consequence of systemic inflammation present in COPD (Citation31) rather than “spill over” from the airways.
Although our data suggest the same observation is likely to be true of MMP-8, the 28% coefficient of variation means this observation should be more guarded. Similarly in urine, it is likely that the renal tubular epithelium produces MMPs in response to systemic inflammation, rather than the renal loss of serum MMPs. In keeping with this idea, we have previously observed that the MMP inhibitor doxycycline is able to supress MMP-9 levels in urine but not the serum of patients with another protease driven destructive lung disease, lymphangioleiomyomatosis (Citation29).
The discordance of MMP expression and activity in differing body compartments is likely to reflect the complex regulatory network that has evolved to restrict proteolytic activity to specific situations. MMP expression is regulated by gene transcription, secretion and cellular localisation (Citation32). The proteolytic activity of MMPs requires co-localisation of the pro-enzyme and its activator, proteolytic cleavage of a pro-peptide and a local excess of the active protease over its inhibitor. MMP activators and inhibitors vary according to body compartment, with MMP-9 activated by plasmin/MMP-3 (Citation33), and predicted to be activated by other proteases including MMP-2 (Citation34), which are themselves independently localised and regulated.
Previous large-scale cohort studies attempting to identify serum biomarkers have shown that clinical indices including the BODE score, level of emphysema and previous hospital admissions best predict outcome in COPD with serum biomarkers including IL-6 and fibrinogen adding a small increment to these clinical measures (Citation35). Single serum factors have not been identified as potential biomarkers of COPD (Citation36). This difficulty in identifying useful protein markers is perhaps a reflection of the complexity and heterogeneity of the COPD phenotype.
Although our study did not examine the origin of MMP-8 and -9, these proteins are produced by neutrophils, to a lesser extent by lung macrophages (Citation7, Citation37, Citation38) and also lung stromal cells (Citation39–Citation41). In keeping with these findings and similar to Ilumets and colleagues we observe that expression of MMP-8 and -9 throughout exacerbations of COPD are correlated with serum neutrophil numbers which are likely to be in part the source of these proteins (Citation9). The presence of urinary MMPs in patients with brain and breast tumours (Citation42) suggest that urinary MMPs either enter the urine via the glomerulus or more likely are produced by the tubular epithelium in response to disease specific stimuli. Urinary and sputum MMP-9 was complexed with NGAL, a chaperone protein, which protects MMP-9 from degradation and is produced by neutrophils, renal and respiratory epithelia (Citation43, Citation44). The MMP-9 dimers observed in sputum, serum and urine in our study have also been suggested to protect MMP-9 from degradation (Citation45) and these complexes may prolong MMP activity or act as reservoirs of the inactive protease.
This was a “real world” observational study to determine the behaviour of neutrophil derived MMPs throughout the course of COPD exacerbations using unselected acute admissions rather than pre-recruited trial subjects and as a consequence, had some limitations. Patients had symptoms for different durations before the first study visit. Whilst this may have introduced some variation into the study measurements it does reflect the assessment of these patients in clinical practice. Similarly, not all patients were able to provide all samples at all time points, particularly sputum samples as they may have been too unwell or otherwise unable to do this. A significant number of patients did not complete all visits, due to deaths from COPD, further exacerbations, co-morbidities or general frailty.
Although patients not completing the study tended to be slightly older and had more severe disease, serum MMP levels were not significantly different from those completing the study and did not cause significant bias. We feel that this approach gives a realistic reflection of the utility of COPD measurements across the range of patients encountered in clinical practice rather than selected trial subjects which would have less application to patients with COPD generally. However, difficulties with sample collection due to frailty and other issues did reduce sample size for some elements of the study limiting the strength of the findings in some areas such as the small number of patients with GOLD stages 1 and 4.
In conclusion we found in a cohort of patients admitted to hospital with COPD that MMP levels in lung, serum and urine are independent of each other and do not reflect disease severity. While serum MMP-8 and -9 may reflect circulating neutrophil numbers, serum and urine MMPs are variable measurements only weakly associated with exacerbations of COPD and unlikely to be useful biomarkers in this clinical setting.
Declaration of Interest Statement
JLC received a Medical Research Council CASE Studentship in conjunction with Mologic Ltd and the University of Nottingham during the conduct of the study. AK reports grants from MRC, grants from Mologic, during the conduct of the study. SRJ has received lecture and advisory fees from Novartis pharmaceuticals, outside the submitted work. DLF is funded by Wellcome Fellowship WT088614. The authors alone are responsible for the content and writing of the paper.
Acknowledgments
We are grateful to Adeleine Sheehan for patient recruitment and sample collection, Dr. Gill Lowrey, Royal Derby Hospital and Dr. Andrew Molyneux Kings Mill hospital for patient recruitment and to Garry Meakin, Helen Bailey and Rebecca Simms for sample processing.
Funding
JLC received a Medical Research Council CASE Studentship in conjunction with Mologic Ltd. and the University of Nottingham. DLF is supported by Wellcome fellowship WT088614. The Nottingham NIHR Respiratory BRU supported patient recruitment and sample collection.
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