Increased matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1 ratio in smokers with airway hyperresponsiveness and accelerated lung function decline

Figures & data

Table 1 Patient demographics and lung function

Characteristics	AHR − NS (n=24)	AHR − S (n=13)	AHR + S (n=20)	P-value
Age (years)	42.0±3.2	49.8±4.5	36.0±3.4	0.107
Male/female	6/18	7/6	11/9	0.083a
Body mass index (kg/m^2)	23.2±0.9	26.1±1.5	24.2±1.2	0.324
Blood eosinophil (%)	2.5±0.8	1.5±0.5	5.5±2.2	0.374
Total IgE (IU/mL)	104.1±25.1	255.3±78.4	356.7±124.8	0.136
Smoking history (pack-years)	–	31.1±2.7	27.6±2.3	0.215b
Remain smoking at year 5	–	10	16	0.833a
Spirometry
FEV1/FVC (%)
Year 0	87.8±1.1	86.1±0.8	83.7±1.4*	0.034
Year 5	86.5±1.0	83.0±1.3$	75.1±2.2***,$$$	0.0002
FEV1%pred
Year 0	98.9±2.3	96.8±3.4	87.7±2.5*	0.013
Year 5	98.6±2.7	98.1±3.8	72.7±4.8****,##,$$	<0.0001
FVC%pred
Year 0	98.5±2.1	94.9±2.9	93.9±2.0	0.356
Year 5	98.0±2.4	97.1±4.0	83.9±4.8*,$	0.0111
MMEF%pred
Year 0	92.1±5.9	90.5±5.1	66.8±7.5#,*	0.004
Year 5	97.9±5.5	91.9±4.1	59.8±7.8**,#,$	0.0011

Notes: Data are presented as mean ± standard error of the mean. AHR−NS, nonsmokers without AHR; AHR−S, smokers without AHR; AHR+S, smokers with AHR.

* P<0.05 versus AHR−NS.

** P<0.01 versus AHR−NS.

*** P<0.001 versus AHR−NS.

**** P<0.0001 versus AHR−NS.

# P<0.05 versus AHR−S.

## P<0.01 versus AHR−S.

$ P<0.05 versus lung function at Year 0.

$$ P<0.01 versus lung function at Year 0.

$$$ P<0.0001 versus lung function at Year 0;

a Determined by Mann-Whitney test.

b Determined by Chi-Square test.

Abbreviations: AHR, airway hyperresponsiveness; IgE, immunoglobulin E; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; MMEF, maximal mid-expiratory flow; %pred, percentage of the predicted value.

Figure 1 More rapid decline in airflow in AHR + S.

Notes: Spirometry was performed during the recruitment of the AHR − NS (n=24), AHR − S (n=13) and AHR + S (n=20) and was repeated 5 years later. Annualized reduction from baseline in FEV₁/FVC (A), FEV₁%pred (B), FVC%pred (C) and MMEF%pred (D) is presented as individual data point, mean and standard error of mean. *P<0.05, **P<0.01 versus AHR−NS; ^#P<0.05 versus AHR−S. AHR−NS, nonsmokers without AHR; AHR−S, smokes without AHR; AHR + S, smokers with AHR.
Abbreviations: AHR, airway hyperresponsiveness; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; MMEF, maximal mid-expiratory flow; %pred, as a percentage of the predicted value.

Table 2 Bronchoalveolar lavage fluid findings from nonsmokers and smokers

	AHR − NS (n=10)	AHR − S (n=13)	AHR + S (n=8)
Total number of cells, 10^4	508.0±43.6	2,125.4±104.6***	1,179.8±74.3#
Recovered volume (mL)	62.4±3.1	57.4±2.4	56.3±3.6
Cellularity, 10^4 cells/mL	8.2±0.7	37.0±0.7***	21.0±0.3#
Cell viability (%)	92.7±0.5	92.5±0.9	92.3±2.4
AM (%)	96.1±1.0	98.5±2.5	82.8±1.5####
10^4 cells/mL	7.9±0.62	36.4±0.9***	17.4±0.4#
Lymphocytes (%)	2.6±1.0	0.7±0.1	0.6±0.1
10^4 cells/mL	0.2±0.1	0.3±0.1	0.11±0.1
Neutrophils (%)	1.0±0.3	0.7±0.1	15.5±5.6**,###
10^4 cells/mL	0.1±0.0	0.3±0.1	3.3±1.2***,##

Notes: Data are presented as mean ± standard error. AHR − NS, nonsmokers without AHR; AHR − S, smokers without AHR; AHR + S, smokers with AHR.

** P<0.01 versus AHR − NS.

*** P<0.0001 versus AHR − NS.

# P<0.05 versus AHR − NS.

## P<0.01 versus AHR − NS.

### P<0.001 versus AHR − NS.

#### P<0.0001 versus AHR − NS.

Abbreviations: AHR, airway hyperresponsiveness; AM, alveolar macrophage.

Figure 2 Greater release of MMP-9 (A) but lesser release of TIMP-1 (B) by cultured AMs from AHR + S.

Notes: AMs from AHR−NS (n=10), AHR−S (n=13) or AHR+S (n=8) were incubated for 24 hr. The level of MMP-9 (A) and TIMP-1 (B) in the culture supernatants was determined by enzyme-linked immunosorbent assays. The molar ratio between MMP-9 and TIMP-1 was also calculated (C). Values are shown as individual data point, mean, and standard error of mean. *P<0.05 compared to AHR − NS. AHR − NS, nonsmokers without AHR; AHR − S, smokers without AHR; AHR + S, smokers with AHR.

Abbreviations: AHR, airway hyperresponsiveness; AMs, alveolar macrophages; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of metalloproteinase-1.

Figure 3 Association between MMP-9/TIMP-1 ratio and the development of airflow limitation.

Notes: Spirometry and bronchoscopy were performed during the recruitment, and spirometry was repeated 5 years later. AMs from 21 smokers, including 13 AHR − S and 8 AHR + S, were incubated for 24 h. The level of MMP-9 and TIMP-1 in the culture supernatants was determined by enzyme-linked immunosorbent assays. The MMP-9/TIMP-1 ratio was less related to the reduction in FEV₁/FVC (A) but was positively associated with the annualized reduction in FEV₁%pred (B), FVC%pred (C), and MMEF%pred (D). AHR − S, smokers without AHR; AHR + S, smokers with AHR.
Abbreviations: AHR, airway hyperresponsiveness; AMs, alveolar macrophages; FEV₁, forced expiratory volume in 1 s; FVC, forced vital capacity; MMEF, maximal mid-expiratory flow; MMP-9, matrix metalloproteinase-9; %pred, percentage of the predicted value; TIMP-1, tissue inhibitor of metalloproteinase-1.

Figure 4 Inhibition of p38 MAPK and ERK reduced the release of MMP-9, but not TIMP-1, from AMs from smokers.

Notes: AMs from AHR − NS (n=8) or AHR + NS (n=8) were incubated in the presence of SB203580 10 μM (p38 MAPK inhibitor) (A) or PD98059 30 μM (MEK1/2 inhibitor) (B) for 24 h. The release of MMP-9 and TIMP-1 in the culture supernatants was determined by enzyme-linked immunosorbent assays. *P<0.05 compared to untreated controls. AHR − S, smokers without AHR; AHR + S, smokers with AHR.
Abbreviations: AHR, airway hyperresponsiveness; AMs, alveolar macrophages; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of metalloproteinase-1.