Concomitant elevations of MMP-9, NGAL, proMMP-9/NGAL and neutrophil elastase in serum of smokers with chronic obstructive pulmonary disease

Abstract

A growing body of evidence points towards smoking-related phenotypic differences in chronic obstructive pulmonary disease (COPD). As COPD is associated with systemic inflammation, we determined whether smoking status is related to serum levels of matrix metalloproteinase-9 (pro- and active MMP-9), neutrophil gelatinase-associated lipocalin (NGAL) and the proMMP-9/NGAL complex in patients with COPD. Serum samples were collected in 100 stable-phase COPD patients (82 smokers, 18 never-smokers) and 28 healthy adults (21 smokers, 7 never-smokers). Serum levels of studied factors were measured in ELISA. Our data provide the first evidence of simultaneously elevated serum levels of MMP-9, NGAL and proMMP-9/NGAL in COPD smokers. While the triad discriminated between smokers and non-smokers in the COPD group, MMP-9 and proMMP-9/NGAL (but not NGAL) discriminated between smokers with and without COPD. Adjustment for age and smoking pack-years did not alter the findings. Serum MMP-9, NGAL and proMMP-9/NGAL levels were not correlated with the GOLD stage or FEV1 decline. Furthermore, serum levels of neutrophil elastase (NE) and MMP-3 (but not of IL-6 and MMP-12) were also higher in COPD smokers than in healthy smokers before and after adjustment for age and pack-years. Among COPD smokers, levels of MMP-9, NGAL and proMMP-9/NGAL were positively correlated with NE (P < 0.0001) but not with the remaining factors. Gelatin zymography detected proMMP-9 in serum samples of healthy and COPD smoking groups. Our results suggest that associated serum levels of proMMP-9, NGAL, proMMP-9/NGAL and NE may reflect the state of systemic inflammation in COPD related to cigarette smoking.

Keywords: GOLD; chronic obstructive pulmonary disease; cigarette smoke; matrix metalloproteinase; neutrophil elastase; neutrophil gelatinase-associated lipocalin; serum; systemic inflammation.

Figure 1

Serum concentrations of (pro and active) MMP‐9, NGAL and the proMMP‐9/NGAL complex in healthy individuals and patients with COPD. ELISAs were performed on seven healthy non‐smokers, 21 healthy smokers, 18 COPD non‐smokers and 82 COPD smokers. (A) Pro‐ and active MMP‐9, (B) proMMP‐9/NGAL and (C) NGAL protein levels were determined. P‐values were calculated using a Mann–Whitney U‐test. *P < 0.05, **P < 0.01. NS: non‐smoker; S: smoker; COPD: chronic obstructive pulmonary disease.

Figure 2

Correlations between serum (pro and active) MMP‐9, NGAL and proMMP‐9/NGAL levels in healthy smokers (n = 21) (A‐C) and COPD smokers (n = 82) (D‐F). Spearman's correlation coefficient (r) and the P‐value are shown. Spots are superposed in the COPD group.

Figure 3

Intercorrelations between serum (pro and active) MMP‐9, NGAL and proMMP‐9/NGAL levels in healthy smokers (n = 15) (A‐C) and smokers with COPD (n = 46) (D‐F). Spearman's correlation coefficient (r) and the P‐value are shown. Spots are superposed in the COPD group.

Figure 4

Levels of proMMP‐2 and proMMP‐9 in serum samples from healthy and COPD smokers. Representative gelatin zymograms of two samples from each group are shown. Controls include (a) proMMP‐9 and proMMP‐9/NGAL, and (b) proMMP‐2/MMP‐9 and active MMP‐2/MMP‐9. Expression profiles of proMMP‐2/MMP‐9 and the proMMP‐9/NGAL complex in sera (5 μl) for healthy smokers and COPD smokers (GOLD I‐IV).

Figure 5

RT‐PCR analysis of MMP‐2/ MMP‐9/MMP‐12, NGAL and IL‐6 transcripts in PBMCs from healthy and COPD smokers. (A) Representative RT‐PCR results for five samples from healthy and COPD smokers. The sample's cDNA content was normalized against the value for β2‐microglobulin. (B & C) Data are expressed as the ratio between the analyte transcript and the β2‐microglobulin transcript. Values are reported as the median (IQR). (B) Levels of MMP‐9 and NGAL transcripts in samples from healthy‐S (n = 12) and COPD‐S (n = 19) samples. (C) Levels of MMP‐9 and NGAL transcripts by GOLD stage groups (GOLD I/II, n = 14; GOLD III/IV, n = 5).