Matrix metalloproteinases -8, -9 and -12 in smokers and patients with stage 0 COPD

Abstract

COPD is underdiagnosed and its early assessment is problematic. It has been suggested that symptomatic smokers with normal FEV1/FVC (Stage 0 COPD, GOLD criteria) can develop COPD in the future. Potential early biomarkers in COPD include the matrix metalloproteinases (MMPs). It is not yet known, whether alterations in MMP expression are associated with smoking alone or with the risk of developing COPD. In this cross-sectional study MMP-8, MMP-9 and MMP-12 were determined from induced sputum and plasma by ELISA, immunocytochemistry, zymography, and/or Western blot in non-smokers (n=32), smokers with symptoms (Stage 0, GOLD criteria) (n=23) or without symptoms (n=23). Only MMP-8 differentiated Stage 0 COPD from non-symptomatic smokers (p = 0.02). MMP-9 levels were significantly elevated in the induced sputum of non-symptomatic smokers and Stage 0 COPD (p = 0.01, p < 0.001) compared to non-smokers, but did not differ between the two subgroups of smokers. MMP-12 was higher only at Stage 0 compared to non-smokers (p = 0.04). MMP-8, MMP-9 and MMP-12 immunoreactivity was localized in macrophages and neutrophils, especially in smokers. MMP-8 levels correlated significantly with the small airway flow parameters (MEF50, MEF25) (p = 0.005 and p = 0.0004) and markers of neutrophil activation (myeloperoxidase, lactoferrin). In conclusion MMP-8 may differentiate Stage 0 from healthy smokers.

Figure 1

Cell differential counts in induced sputum from non-smokers, smokers and Stage 0. Stage 0 patients had significantly higher percentage of neutrophils and eosinophils, and lower percentage of macrophages than non-smokers. There were no differences between asymptomatic smokers and Stage 0.

Figure 2

MMP-8 (A), MMP-9 (B), MMP-12 (C) and TIMP-1 (D) levels by ELISA in the induced sputum from non-smokers, asymptomatic smokers and GOLD Stage 0. Mean values are shown with horizontal bars. The levels of MMP-8 were increased in Stage 0 when compared with asymptomatic smokers (p = 0.02) and non-smokers (p < 0.0001). The levels of MMP-9 were higher in Stage 0 (p < 0.0001) and asymptomatic smokers (p = 0.01) than in non-smokers. The MMP-12 levels were higher in Stage 0 than in non-smokers (p = 0.04) and there were no significant differences in TIMP-1 levels between the groups.

Figure 3

Immunocytochemistry for MMP-8 (A), MMP-9 (B), MMP-12 (C) and TIMP-1 (D) on the cytospins of induced sputum cells from a GOLD Stage 0 smokers. Clear immunoreactivity for each MMP was detectable in macrophages and neutrophils from all patient groups, especially from smokers and those at GOLD Stage 0.

Figure 4

Zymography and Western blotting analysis of MMP-9 in the sputum specimens. Representative zymography of 11 specimens (A): Gelatinolytic activity at 92 kD (pro-MMP-9) and 77–82 kD (activated MMP-9) of non-smoking controls (lanes 1–3), cigarette smokers (lanes 4–7), and subjects at GOLD stage 0 (lanes 8–11). Individual variation occurred, but generally MMP-9 was activated in smokers and especially those at GOLD Stage 0. Representative Western blotting of 6 specimens (B): HC healthy non-smoking control (lanes 1–2), HS healthy smoker (lanes 3–4), St 0 Stage 0 (lanes 5–6). There was individual variation, when calculated from 16 specimens no significant differences between the groups could be seen. Molecular weight standards at the left.

Figure 5

Correlation between sputum MMP-8 (A) (n = 70; r = 0.70; p < 0.0001), MMP-9 (B) (n = 69; r = 0.66; p < 0.0001), MMP-12 (C) (n = 56; r = 0.50; p < 0.0001), and TIMP-1 (D) (n = 68; r = 0.36; p = 0.002) levels and total number of neutrophils.

Figure 6

Correlation between MMP-8 (A) (n = 34; r = 0.63; p < 0.0001), MMP-9 (B) (n = 35; r = 0.50; p = 0.002) and MMP-12 (C) (n = 24; r = 0.53; p = 0.008) and lactoferrin levels in induced sputum.