Abstract
Destruction of lung elastin is critical for development of emphysema associated with chronic obstructive pulmonary disease (COPD). Lung macrophages release elastolytic enzymes, including matrix metalloproteinase (MMP)-9, along with tissue inhibitors of MMP (TIMP). We examined the production and activity of macrophage-derived MMP-9 and TIMP-1 from alveolar macrophages (AM) from smokers with COPD, healthy smokers (HS), and nonsmokers (NS). AM were stimulated with either lipopolysaccharide (LPS), interleukin (IL)-1 β , or cigarette smoke–conditioned culture medium (CSM). AM from patients with COPD released greater amounts of MMP-9 with greater enzymatic activity than HS and NS. In contrast, AM from NS released more TIMP-1 than cells from HS and subjects with COPD. LPS and IL-1 β caused a dose-dependent increase in MMP-9 release and activity, together with increased levels of TIMP-1. Dexamethasone prevented the increase in MMP-9 release, and increased TIMP-1 release. CSM increased MMP-9 and TIMP-1 release from AM of all groups. Dexamethasone decreased CSM-stimulated MMP-9 release, but had no effect on MMP-9 activity This study suggests that macrophages might be important in the development of COPD because these cells exhibit increased levels of elastolytic activity.
Chronic obstructive pulmonary disease (COPD) is a major cause of respiratory morbidity and mortality in the world. The incidence of the disease is increasing and it has been estimated that by 2010 COPD will be the fourth largest cause of death in the world (1). COPD affects more than 16 million people and is the fourth leading cause of death in the USA (2). Cigarette smoking is the major cause of COPD in developed countries (2); this may possibly arise from the fact that cigarette smoking induces airway inflammation and increases alveolar macrophage number, both of which may lead to the development of emphysema.
Elastin comprises ∼ 2.5% (wt/wt) of the dry weight of the lung (3) and is vital to the normal structure and function of the lung (4). In the small airways elastin maintains patency and alveolar wall resilience. Elastin is distributed widely throughout the lungs and is thought to be conserved throughout a lifetime (5). Elastin replacement may occur but newly synthesized elastin monomers may fail to form chains (fibrils) and are thus unable to play a structural role in the lung. Elastin degradation products, such as desmosine, are increased in the urine of subjects with COPD (6) and correlate with the rate of decline of FEV1 (7).
COPD is a disease of the small airways and lung parenchyma, and the relatively low numbers of neutrophils in the distal airways, as sampled by bronchoalveolar lavage (BAL), questions the hypothesis that neutrophils are the pre-eminent cell in emphysema. There is increasing evidence that macrophages play a role in the pathogenesis of COPD and particularly emphysema (8, 9) because they are localized to the areas of the lungs where cigarette- induced emphysema usually occurs. Furthermore, the numbers of macrophages in the small airways correlate with parenchymal damage (10).
Chapman and colleagues demonstrated that macrophages secrete two elastolytic cysteine proteinases (cathepsin L and cathepsin S) (11) and also several matrix metalloproteinases (MMPs). MMPs are a family of structurally related enzymes that are capable of degrading all components of the extracellular matrix (12), and over twenty members of this family have been identified. Enzymes in the MMP family share 32–49% amino acid similarity and have similar structural domains. MMPs are inhibited by specific inhibitors, namely, tissue inhibitors of metalloproteinases (TIMP), of which four members have been identified.
Macrophages are derived from circulating monocytes. During the differentiation process the pattern of MMP expression changes from a predominantly MMP-7 phenotype toward expression of MMP-2 and MMP-9. These MMPs are both capable of elastolysis as well as the degradation of collagen. MMP-9 is predominantly produced by macrophages but is also found in neutrophil granules, whereas MMP-2 is produced in smaller quantities by macrophages but is also released by fibroblasts (13). MMP-2 is involved in the processing of cell receptors and TIMP release, whereas MMP-9 is a major elastolytic MMP (13). The mechanism of MMP activation is essentially the same for each family member via the cleavage of a 10-kD proenzyme peptide. In vivo activation of MMP may occur through several mechanisms. MMP can autocatalyze the cleavage of the propeptide (14), and other proteases, such as NE, MMP-7, and cathepsins, will also activate MMP (12, 15). Recent studies have investigated the role of reactive oxygen species in MMP activation (16). Peroxynitrite and hydrogen peroxide have both been shown to activate MMP-2 and MMP-9 in human smooth muscle cell culture systems. These findings may be significant as cigarette smoking increases oxidative stress and therefore may induce activation of the MMP. MMP release from alveolar macrophages can be stimulated by lipopolysaccharide (LPS), phorbol esters, interleukin (IL)-1β, platelet derived growth factor (PDGF), and tumor necrosis factor (TNF)-α (17), thus suggesting that proinflammatory stimuli might regulate MMP activity in the COPD airway.
TIMPs are produced by macrophages and epithelial cells and have two structural and functional domains. The N-terminal domains of TIMPs are inhibitors of all MMPs and mediate their effect by binding to the catalytic domain of MMPs. The C-terminal domain of TIMPs has two separate enzyme binding sites (18). There appears to be a lack of specificity, because all four TIMPs are capable of inhibiting all MMPs (19).
Thus far no effective therapy has been found for COPD (20). Corticosteroids are widely prescribed to patients with COPD in spite of the fact that well-conducted, long-term trials of inhaled corticosteroids have not shown changes in rate of decline of lung function (30). However, changes in rate of change of quality of life have been demonstrated. There is in vitro data that suggests that steroids can decrease inflammatory mediator and enzyme release, and thus a rationale for the continued investigation into their usefulness in COPD (21, 22).
This study investigated the release and activity of MMP-9 and of TIMP-1 from alveolar macrophages obtained from patients with COPD, and compared these with age- and smoking status–matched healthy smokers. The AM were stimulated with IL-1β, LPS, and cigarette smoke–conditioned media to mimic pathologic conditions. The effect of corticosteroids on the release and activity of MMP-9 was investigated to clarify the role corticosteroids may play in the treatment of COPD.
Subjects were recruited from a general practice database by searching for smokers over the age of 40. Each contact attended for screening and spirometry. Subjects were excluded if they had > 15% improvement in forced expiratory volume in 1 s (FEV1) or forced vital capacity (FVC) with either β-agonist or corticosteroids (2 wk 40 mg/d) and if they had any history of asthma, atopy, or allergy or were taking any respiratory medication. All were current smokers. Nonsmokers were recruited from patients having investigations for other reasons. COPD was defined as an FEV1/FVC ratio < 0.7 and FEV1% predicted < 70%. For the purposes of this study eight subjects were recruited in each group. The East Berkshire Ethics Committee approved this study and all patients gave written informed consent.
Bronchoscopy, BAL collection, and processing were performed as previously described (23). Subjects underwent bronchoscopy at between 6 and 12 wk after steroid challenge, and all were free from exacerbation and upper respiratory tract infection.
BAL cells were resuspended in RPMI-1640 medium containing 2 mM glutamine, 10% (vol/vol) fetal calf serum at a concentration of 1 × 106 macrophages/ml and seeded onto 24-well Primaria cell culture plates (250 μl/well). The cells were allowed to adhere for 4 h in a humidified incubator (95% air, 5% CO2 vol/vol) at 37°C. Nonadherent cells were removed by washing three times with Hanks' balanced salt solution. Cells were incubated overnight and washed again before exposure to experimental conditions. Cells were then stimulated overnight and the cell supernatant was collected and stored at −70°C until analyzed.
Cigarette smoke–conditioned medium (CSM) was produced following the method of Wirtz and colleagues (24). Briefly, cigarette smoke was bubbled through sterile cell culture media. The media thus produced was standardized to a standard curve of CSM concentration against absorbance at 320 nm. Passing the smoke from four cigarettes (“Players Navy Cut;” John Player and Sons, Nottingham, UK) through 100 ml of cell culture media set an arbitrary value of 1 for the concentration of CSM. All CSM produced was referred back to the original standard curve and a dilution factor calculated such that on any day the concentration of CSM used was the same. CSM is toxic to AM in high concentrations. Preliminary dose–response studies demonstrated that relative concentrations of CSM above 0.2 were deleterious as measured by Trypan Blue exclusion as a test of cell viability. Thus 0.1 was selected as the most concentrated CSM in these experiments.
MMP-2, MMP-9, and TIMP-1 in the samples were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D systems, Abingdon, UK and Amersham Pharmacia Biotech, Little Chalfont, UK) according to the manufacturers' directions. The kits measured total MMP released (pro and active MMP protein together with that complexed to TIMP-1+2). TIMP-1 ELISA measured both bound and free TIMP-1.
Semiquantitative measurement of MMP activity was performed using zymography (25). Samples (15 μl) and standards (1 μg; Amersham Pharmacia Biotech) were loaded into 10% polyacrylamide gels (wt/vol) incorporating 0.1% (wt/vol) gelatin substrate. Proteins were subjected to electrophoresis at 125 V for 60 min and the gels then washed three times in 20 mM Tris/HCl, pH 7.8, 2.5% (vol/vol) Triton X-100 for 15 min. The gels were then washed twice in 1% Triton X-100 (vol/vol) containing 10 mM CaCl2, 5 μM ZnCl2 pH 7.8 in Tris/HCl and incubated for 18 h at 37°C. Gels were also incubated in the presence of phenylmethylsulfonyl fluoride (PMSF; 5 mM) or ethylenediaminetetraacetic acid (EDTA; 5 mM) in experiments to inhibit serine proteases and MMPs.
After incubation gels were stained with 1% (wt/vol) Coomasie blue in 45% methanol (vol/vol). Bands of lysis (enzyme activity) were visualized by washing in 25% (vol/vol) methanol, 7.5% acetic acid solution (vol/vol). The zones of lysis in the gels were analyzed using Gelworks (UVP Ltd, Cambridge, UK) software system. Images were taken and bandwidth and density measured. Only the bands that corresponded to active MMP-2+9 were analyzed. A sample zymogram is shown Figure 1. Standard curves were generated using known amounts of MMP-2 and MMP-9 protein and were linear at the concentrations measured; results are therefore expressed as %control (1 μg of control MMP run in each gel) activity for each gel. The assay was found to be linear for MMP activity at the concentrations of MMP-9 found in this study (data not shown). Confirmation that the activity seen was due to MMP-2 and MMP-9 was obtained using immunoprecipitation of the samples and standards before being run in the zymography gels. Antibodies to MMP-2 and MMP-9 (Oncogene Research Products, Cambridge, MA) were incubated with samples and standards and separated using Protein A-Sepharose beads. The resulting supernatants were run in zymography gels together with supernatants obtained following separation of the antibodies from the bound MMP-2 and MMP-9. Gels were analyzed as described above.
Fig. 1. Sample zymogram. 1 μg of MMP-9 and MMP-2 loaded to control lanes and loaded by volume (15 μl). Lane 1: MMP-2 (latent and active) control; lane 2: MMP-9 (active) control; lanes 3–5: COPD; lanes 6–8: healthy smokers; lanes 9–10: nonsmokers. Pro–MMP-9 is shown at 92 kD, activated MMP-9 at 82 kD, and MMP-2 at 72 kD.
[More] [Minimize]Supernatant MMP-9 protein levels were also measured using Western blotting. Electrophoresis was preformed using 3–8% polyacrylamide gel electrophoresis Tris acetate gels, followed by translation onto Hybond-ECL nitrocellulose membrane. After transfer, the membranes were blocked in a 10% (wt/vol) nonfat milk solution in PBS/T (PBS, 0.05% [vol/vol] Tween 20) overnight at 4°C. Mouse anti-human antibodies (1:200 wt/vol, PBS, 0.1% BSA) raised against active MMP-9 were added and incubated with the membranes for 1 h at room temperature. Membranes were then washed 5 × 5 min in PBS/T and incubated for 1 h in 2° antibody (1:5,000 goat anti-mouse horseradish peroxidase [HRP]-linked IgG, PBS, 0.05% [vol/vol]Tween 20). After a further five washes in PBS/T the proteins were visualized using enhanced chemiluminescence solution (ECL). The images were developed on X-ray film and band density measured using a Gelworks image analysis system. Antibodies against active MMP-9 were purchased form Oncogene Research Products (Cambridge, MA).
Results were analyzed using one-way analysis of variance (ANOVA). Significant differences (P < 0.05) were confirmed using the Mann-Whitney test for nonparametric data. Eight subjects were studied in each group. All other data are presented as means ± SE.
The characteristics of the three subject groups are shown in Table 1. Groups were similar with respect to age and sex distribution. There was no difference between the healthy smokers and the subjects with COPD in smoking status, absolute FEV1, or FVC. FEV1% predicted and FEV1/FVC ratio were significantly lower in the patients with COPD than the healthy smokers or nonsmokers. Significant differences were found in the subjects' residual volume and transfer factor measurements, suggesting significant levels of gas trapping.
| Nonsmokers | Healthy Smokers | Patients with COPD | ||||
|---|---|---|---|---|---|---|
| Age, yr | 58 ± 7.2 | 63.6 ± 4.1 | 70.4 ± 4.9 | |||
| Sex (M:F) | 3:5 | 4:4 | 5:3 | |||
| Smoking history, pack-years | — | 44.8 ± 3.0 | 51.4 ± 3.2 | |||
| FEV1/FVC ratio | 82.6 ± 2.7* | 81.3 ± 2.3† | 59.1 ± 3.5 | |||
| FEV1, L | 2.8 ± 0.3* | 2.52 ± 0.2 | 1.9 ± 0.2 | |||
| FEV1, %predicted | 98.1 ± 4.8*,‡ | 89.5 ± 1.8† | 56.3 ± 3.0 | |||
| FVC, L | 3.46 ± 0.4 | 3.1 ± 0.2 | 3.1 ± 0.2 | |||
| RV, %predicted | 97 ± 3.8*,‡ | 124.7 ± 5.7† | 172.7 ± 14.5 | |||
| KCO, %predicted | 105 ± 7.2*,‡ | 85.5 ± 3.3 | 80.5 ± 4.7 |
BAL cytospin absolute cell counts and differentials are shown in Table 2. There was a significantly greater cell number recovered from the BAL of the subjects with COPD compared with nonsmokers. There was no statistical difference in the differential cell counts between the three groups.
| Nonsmokers | Healthy smokers | Patients with COPD | ||||
|---|---|---|---|---|---|---|
| Total Cell Count (×106) | 5.5 ± 0.8 | 6.5 ± 1.2 | 8.9 ± 1.2* | |||
| Macrophages (%) | 86.3 ± 2.4 | 89.8 ± 2.9 | 91.4 ± 1.6 | |||
| Neutrophils (%) | 10.6 ± 2.5 | 7.9 ± 2.9 | 6.2 ± 1.8 | |||
| Eosinophils (%) | 0.5 ± 0.13 | 0.7 ± 0.2 | 0.8 ± 0.14 | |||
| Bronchial Epithelial Cells (%) | 2.6 ± 0.6 | 1.6 ± 0.4 | 1.6 ± 0.4 |
AM from subjects with COPD released more MMP-9, as measured by ELISA, without stimulation than from AM of the healthy smokers or nonsmokers (7.2 versus 5.2 versus 3.9 ng/ml, P < 0.05; Figure 2A). MMP-2 was only detectable using the ELISA in a minority of samples (6/24) and so was excluded from further analysis.
Fig. 2. MMP-9 release from alveolar macrophages (AM). AM from patients with COPD (black bars), healthy smokers (gray bars), and nonsmokers (open bars) were incubated overnight in the presence or absence of interleukin-1β (IL-1β, A), lipopolysaccharide (LPS, B), or cigarette smoke–conditioned media (CSM, C). Media was harvested and release of MMP-9 measured using ELISA. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
[More] [Minimize]IL-1β dose-dependently increased the release of MMP-9 from AM (Figure 2A). AM from COPD subjects released more MMP-9 at all concentrations of IL-1β when compared with both healthy smokers and healthy normal subjects (P < 0.01). AM from healthy smokers released more MMP-9 than AM from nonsmokers when stimulated with 10 ng/ml IL-1β (P < 0.05), but there was no statistical difference between the two groups at lower concentrations of IL-1β.
LPS had an effect similar to that of IL-1β on MMP-9 release (Figure 2B). AM from COPD subjects released more MMP-9 protein in response to stimulation than those from either of the two normal subject groups (P < 0.01 for all comparisons). AM from healthy smokers released more MMP-9 following LPS stimulation than those from nonsmokers, but there was no difference between the two groups under nonstimulated conditions (P < 0.01 for all comparisons).
MMP-9 production from all subject groups was increased in response to CSM in a dose-dependent manner (Figure 2C). CSM caused death of AM within the 24-h culture period at concentrations greater than 0.1 (data not shown). There was an increased release of MMP-9 from AM from COPD subjects when compared with cells from healthy smokers and nonsmokers (P < 0.05 and 0.01, respectively). Furthermore, AM from healthy smokers released more MMP-9 than nonsmokers (P < 0.01 at the higher CSM concentrations).
Both IL-1β and LPS increased release of TIMP-1 from AM from all groups above control levels (P < 0.01) (Figures 3A and 3B). The AM from nonsmokers released more TIMP-1 at baseline than cells from either healthy smokers or COPD patients (P < 0.01)(Figure 3). There was no difference between the release of TIMP-1 from the AM of healthy smokers and subjects with COPD.
Fig. 3. TIMP-1 release from AM. AM from patients with COPD (black bars), healthy smokers (gray bars), and nonsmokers (open bars) were incubated overnight in the presence or absence of IL-1β (A), LPS (B), or CSM (C). Media was harvested and release of TIMP-1 measured using ELISA. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
[More] [Minimize]CSM increased TIMP-1 release when stimulated with relative concentrations of 0.01 and above (P < 0.05 for all three subject groups). AM from nonsmokers and patients with COPD produced more TIMP-1 in response to CSM stimulation than AM from healthy smokers (P < 0.01).
Dexamethasone (1 μM) decreased the production of MMP-9 protein by AM of all subjects groups (Figure 4) following LPS, IL-1β, and CSM stimulation (P < 0.01 for all comparisons). TIMP-1 production was increased by 1 μM dexamethasone alone, but dexamethasone had no effect on release of LPS-, IL-1β-, and CSM-stimulated TIMP-1 production (Figure 5).
Fig. 4. Effect of dexamethasone on MMP-9 release from AM. AM from patients with COPD, healthy smokers, and nonsmokers were incubated overnight in the presence or absence of dexamethasone (10−6 M) with IL-1β (1 ng/ml, A), LPS (10 ng/ml, B) or CSM (0.1, C). Media was harvested and release of MMP-9 measured using ELISA. Open bars, control; light gray bars, dexamethasone alone; dark gray bars, stimulation alone; black bars, stimulation + dexamethasone. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
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Fig. 5. Effect of dexamethasone on TIMP-1 release from AM. AM from patients with COPD, healthy smokers, and nonsmokers were incubated overnight in the presence or absence of dexamethasone (10−6 M) with IL-1β (1 ng/ml, A), LPS (10 ng/ml, B) or CSM (0.1, C). Media was harvested and release of TIMP-1 measured using ELISA. Open bars, control; light gray bars, dexamethasone alone; dark gray bars, stimulation alone; black bars, stimulation + dexamethasone. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
[More] [Minimize]AM from subjects with COPD demonstrated a greater level of MMP-9 activity than those of the healthy smokers and nonsmokers (P < 0.01, Figure 6). AM from healthy smokers released more active MMP-9 when compared with AM from nonsmokers (34.1% versus 16.0 versus 9.3, P < 0.01). LPS, IL-1β, and CSM all increased MMP-9 activity in a dose-dependent manner (Figure 6). This finding was confirmed by Western blotting (Figure 7).
Fig. 6. Release of MMP-9 activity from AM measured by zymography. AM from patients with COPD (black bars), healthy smokers (gray bars), and nonsmokers (open bars) were incubated overnight in the presence or absence of IL-1β (A), LPS (B), or CSM (C). Media was harvested and activity of MMP-9 measured using zymography. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
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Fig. 7. Western blot showing active MMP-9 protein release from AM of patients with COPD incubated with CSM and the effect of dexamethasone. Five micrograms of protein were loaded to each lane. Lane 1: active MMP-9 control; lane 2: control; lane 3: dexamethasone (10−6 M) alone; lane 4: CSM (0.1); lane 5: CSM (0.1) plus dexamethasone (10−6 M).
[More] [Minimize]Dexamethasone treatment of AM decreased MMP-9 activity in cell culture supernatants from AM stimulated with IL-1β and LPS (Figures 8A and 8B) for all subject groups to baseline (P < 0.01). However, dexamethasone did not decrease the MMP-9 activity in the AM supernatant treated with CSM (Figures 8C and 9). Gelatinolytic activity was confirmed as being MMP in origin by suppression with EDTA (5 mM), an MMP inhibitor, and the lack of effect of PMSF (5 mM), a specific serine protease inhibitor, added to the zymogram incubation media (Figure 9). Immunoprecipitation confirmed that the activity seen was mainly due to MMP-9; incubation of the samples with MMP-9 antibody resulted in a 64.7% (±9.6) reduction in activity at 82 and 92 kD.
Fig. 8. Effect of dexamethasone on MMP-9 activity from AM. AM from patients with COPD, healthy smokers, and nonsmokers were incubated overnight in the presence or absence dexamethasone (10−6 M) with IL-1β (1 ng/ml, A), LPS (10 ng/ml, B) or CSM (0.1, C). Media were harvested and release of TIMP-1 was measured using ELISA. Open bars, control; light gray bars, dexamethasone alone; dark gray bars, stimulation alone; black bars, stimulation + dexamethasone. Data are means ± SEM. * P < 0.05, **P < 0.01 (ANOVA) for comparisons shown.
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Fig. 9. Sample zymograms showing MMP activity of AM from patients with COPD incubated with CSM and dexamethasone. One microgram of MMP-9 and MMP-2 was loaded to control lanes, and sample lanes were loaded by volume (15 μl). Lane 1: MMP-2 (latent and active) control; lane 2: MMP-9 (latent and active) control; lane 3: CSM (0.1) + dexamethasone (10−6 M); lane 4: CSM (0.1); lane 5: dexamethasone (10−6 M) alone; lane 6: Control. A, control gel; B, gel incubated in serine protease inhibitor PMSF (5 mM); C, gel incubated in MMP inhibitor EDTA (5 mM).
[More] [Minimize]This study demonstrates that AM are a significant source of MMP-9 in the airways of subjects with COPD, in agreement with a previous study (26). This study examined the nature of different stimuli on MMP-9 release, as release of MMP from AM has been previously described (23). AM from COPD patients release more MMP-9, and it is more active, than that released from AM of healthy smokers and nonsmoking subjects. The increased level of MMP-9 might be due to the relative decrease in release of the specific endogenous inhibitor of MMP-9, TIMP-1. However, this cannot explain the increase in the absolute levels of MMP-9 released from AM derived from COPD patients. The releases of MMP-9 and of TIMP-1 need to be considered together, as it is the balance of enzyme to inhibitor which may be critical in determining elastolytic activity in vivo (27).
Few studies have addressed the effects of cigarette smoke on AM. Cigarette smoke is a complex mixture of more than 4,700 chemical compounds and contains a high concentration of free radicals and other oxidants in both the gas and tar phase (28). There are estimated to be 1015 free radicals per puff (29) and the concentration of nitric oxide, a free radical, is 500–1,000 ppm. Cigarette tar also contains oxidants, which can generate hydrogen peroxide, so that CSM could potentially provide a source of oxidant stress. This potent mixture might therefore exhibit major effects on the airway and may act in part by altering the MMP/TIMP balance.
The major novel finding in this study is that corticosteroids fail to decrease MMP-9 activity induced by CSM, in spite of decreasing MMP-9 protein release, which was confirmed using two methods. This lack of effect of corticosteroids may be one of the reasons for the lack of efficacy of corticosteroids seen clinically in COPD treatment (30). There are several mechanisms that may explain this finding. It has previously been shown that an increase in oxidant stress may increase MMP-9 activation and decrease TIMP-1 activity (16, 31), leading to an imbalance in favor of increased elastolysis. Our study has only examined a high concentration of corticosteroids, but it is possible that there are differences in responsiveness to corticosteroids between individuals that may account for the susceptibility to the effects of cigarette smoke and the failure of inhaled corticosteroids to be effective. The molecular basis for the impaired response to CSM-induced MMP-9 release compared with other inflammatory stimuli remains to be determined, but may be linked to oxidative stress.
Macrophages respond in many ways to pro-inflammatory stimuli such as LPS and IL-1β. The finding that one such response is an increase in MMP-9 production is relevant in the context of COPD. It has been hypothesized that acute exacerbations of COPD, either virally or bacterially induced, are significant events leading to a progressive decline in lung function. Levels of pro-inflammatory cytokines and airway cells have been shown to be increased in COPD and these may contribute to macrophage-mediated elastolysis (32). MMP-9 may significantly increase the elastolytic load in the lungs in such circumstances and therefore accelerate the loss of lung function. The increase in MMP production is offset, in part, by an increase in TIMP-1 release. However, our study has shown an increase in MMP-9 activity in COPD, along with an increase in MMP-9 and TIMP-1 protein release. The molar ratio of MMP: TIMP is critical, as an imbalance may result in destruction of elastin fibers in the lung parenchyma (33).
Our study has demonstrated that macrophages have a potential role in emphysema through the increased production of MMP-9 and that COPD subjects produce more MMP-9 than healthy smokers and nonsmokers in response to inflammatory stimuli, including cigarette smoke. Other investigators have demonstrated increases in MMP-9 from BAL, not only in subjects with emphysema but also in those with preclinical disease, although not from cultured alveolar macrophages (34). The present study further supports the idea that macrophages are likely to be the critical cell type in the pathogenesis of COPD.
This work was supported by a research grant from the British Lung Foundation (R.R.) and the Medical Research Council (S.V.C.).
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Abbreviations: alveolar macrophages, AM; bronchoalveolar lavage, BAL; chronic obstructive pulmonary disease, COPD; cigarette smoke–conditioned medium, CSM; enhanced chemiluminescence, ECL; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immunosorbent assay, ELISA; forced expiratory volume in 1 s, FEV1; forced vital capacity, FVC; healthy smokers, HS; interleukin, IL; lipopolysaccharide, LPS; matrix metalloproteinase, MMP; nonsmokers, NS; polyacrylamide gel electrophoresis, PAGE; platelet-derived growth factor, PDGF; polymethyl sulfonyl fluoride, PMSF; tissue inhibitor of MMP; TIMP; tumor necrosis factor, TNF.
