The role of matrix metalloproteinase-9 in cigarette smoke-induced emphysema

Abstract

Rationale: Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction.

Objectives: To determine if MMP-9 causes cigarette smoke-induced emphysema using MMP-9 knockout mice and human samples.

Methods: Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury.

Measurements and main results: Mice deficient in MMP-9 develop the same degree of cigarette smoke-induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage.

Conclusions: These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke-induced emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).

Trial registration: ClinicalTrials.gov NCT00757120.

Figures

Figure 1.

Airspace size after 6 months of cigarette smoke exposure. Age-matched nonsmoked matrix metalloproteinase (MMP)-9 wild-type (WT) (A) and knockout (KO) (C) and MMP-9 WT (B) and KO (D) mice after 6 months of cigarette smoke exposure. Inflation-fixed lungs demonstrate similar airspace size in nonsmoked animals (A and C) with similar airspace enlargement in smoked animals (B and D) comparable with mild emphysematous changes in humans. An increase in alveolar macrophages (arrows, B and D) but not neutrophils is seen in both smoke-exposed WT and KO animals.

Figure 2.

Mouse lung lavage inflammatory cell profile after smoking. Chronic smoke exposure induces a modest but significant increase in alveolar macrophages at 6 months (*P < 0.05 in both wild-type and knockout (KO) compared with nonsmoked). Matrix metalloproteinase (MMP)-9 KO mice demonstrated no significant difference in number or type of inflammatory cells after smoking compared with wild-type mice.

Figure 3.

Matrix metalloproteinase (MMP)-9 gene expression in emphysematous lung. (A) MMP-9 expression in whole-lung compared with CD68+ macrophages. Using mRNA from single slices of lung cores or laser capture isolated alveolar macrophages quantitative polymerase chain reaction was performed to determine mRNA quantity after adjustment for loading by normalizing to the housekeeping gene GAPDH. Ten samples each from five subjects with GOLD 4 chronic obstructive pulmonary disease were evaluated. Gene expression is in relative units based on a simultaneous standard curve. MMP-9 is much more abundant in alveolar macrophages and demonstrates good correlation with whole-lung MMP-9 quantity (ρ = 0.7; P < 0.01). (B) MMP-9 expression in CD68+ alveolar macrophages compared with emphysema severity of the lung core. Emphysema severity is expressed as the mean density of the region of the lung core on computed tomography. MMP-9 mRNA quantity from alveolar macrophages plotted on a log scale (values of 0 converted to 1) demonstrates no correlation with emphysema severity (ρ = 0.14; P = 0.33).

Figure 4.

Monocyte matrix metalloproteinase (MMP)-9 expression relative to emphysema index. Monocytes from former and current heavy smokers were evaluated for MMP-9 mRNA by quantitative polymerase chain reaction. MMP-9 quantity is expressed as relative units after normalization to the housekeeping gene L32. Emphysema index is calculated from low-dose computed tomography scan whole-lung density.

Figure 5.

Monocyte matrix metalloproteinase (MMP)-9 expression in relation to plasma Clara cell secretory protein (CCSP) and surfactant protein-D (SP-D). (A) Plasma CCSP in current smokers (black circles) and former smokers (gray circles) compared with monocyte MMP-9 mRNA levels (log scale). Current smoking causes a significant decrement in CCSP (t test vs. former smokers, P < 0.05) but no relationship with monocyte MMP-9 mRNA is seen (ρ = 0.17; P = 0.2). (B) Plasma SP-D in current smokers (black circles) and former smokers (gray circles) compared with monocyte MMP-9 mRNA graphed on a log scale. Current smoking causes a significant increase in SP-D (t test vs. former smokers, P = 0.02) but no relationship with monocyte MMP-9 mRNA is seen (ρ = 0.05; P = 0.6).