An aberrant leukotriene A4 hydrolase-proline-glycine-proline pathway in the pathogenesis of chronic obstructive pulmonary disease

Abstract

Rationale: Chronic neutrophilic inflammation is a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory response have not been delineated.

Objectives: We investigated changes to the leukotriene A4 hydrolase (LTA4H)-proline-glycine-proline (PGP) pathway and chronic inflammation in the development of COPD.

Methods: A/J mice were exposed to air or cigarette smoke for 22 weeks followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human cohorts were used to analyze changes to the LTA4H-PGP pathway in never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and LTA4H aminopeptidase activity were detected by mass spectroscopy, LTA4H amounts were detected by ELISA, and acrolein was detected by Western blot.

Measurements and main results: Mice exposed to cigarette smoke developed emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of LTA4H aminopeptidase, which ordinarily degrades PGP. We recapitulated these findings in smokers with and without COPD. PGP and AcPGP are closely associated with cigarette smoke use. Once chronic inflammation is established, changes to LTA4H aminopeptidase remain, even in the absence of ongoing cigarette use. Acrolein modifies LTA4H and inhibits aminopeptidase activity to the same extent as cigarette smoke.

Conclusions: These results demonstrate a novel pathway of aberrant regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately involved in disease progression in the absence of ongoing cigarette smoke exposure. We highlight a mechanism by which acrolein potentiates neutrophilic inflammation through selective inhibition of LTA4H aminopeptidase activity. Clinical trial registered with www.clinicaltrials.gov (NCT 00292552).

Trial registration: ClinicalTrials.gov NCT00292552.

Keywords: COPD; PGP; acrolein; inflammation; leukotriene A4 hydrolase.

Figure 1.

A/J mice exposed to once-daily whole-body cigarette smoke for 22 weeks develop an emphysematous phenotype and alterations to the leukotriene A₄ hydrolase (LTA₄H)–proline-glycine-proline (PGP) pathway. Representative hematoxilin and eosin stain of air control (n = 11) (a) and cigarette smoke–exposed (n = 6) (b) mouse and resultant increase in mean linear intercept (Lm) (c) in the lungs of cigarette smoke–exposed mice, and development of right ventricular (RV) hypertrophy measured by increased RV mass and increased RV/LV ratio as seen in d. Neutrophil burden and PGP amounts are increased in bronchoalveolar lavage fluid (BALF) of cigarette smoke–exposed mice as seen in e. Additionally, LTA₄H mRNA amounts are increased as is the amount of enzyme detected by ELISA, depicted in f. Despite these increases in enzyme amount, the aminopeptidase function is inactivated by more than 95% in cigarette smoke–exposed mice as seen in g, preventing the degradation of PGP in cigarette smoke–exposed mice. *P < 0.05, **P < 0.01 by two-sided t test, with error bars representing SEM.

Figure 2.

Changes in sputum leukotriene A₄ hydrolase (LTA₄H) amount and activity affect proline-glycine-proline (PGP) in smokers and never smokers. Sputum samples from never smokers (n = 18) and control smokers (n = 25) underwent immunoprecipitation and Western blot analysis demonstrating increased amounts of LTA₄H in smokers compared with never smokers (a, representative blot). (b) The increase was quantified and confirmed by ELISA. The amount of leukotriene B₄ (LTB₄) in the sputum of smokers was increased in smokers to the same extent that was seen in LTA₄H (b), suggesting that the epoxy-hydrolase activity is intact. Aminopeptidase activity is significantly inhibited in the sputum of smokers compared with never smokers as seen in c, suggesting that the aminopeptidase function is selectively inactivated. Levels of PGP are threefold elevated in the sputum of smokers, similar to changes seen in levels of the neutrophil marker myeloperoxidase (MPO) as depicted in d. *P < 0.05, **P ≤ 0.01, with error bars representing SEM.

Figure 3.

Selective aminopeptidase inhibition leads to proline-glycine-proline (PGP) accumulation in sputum of smokers and in patients with chronic obstructive pulmonary disease (COPD) independent of smoking status. (a) Leukotriene A₄ hydrolase (LTA₄H) amounts are increased in subjects with COPD (n = 23) compared with never smokers (n = 18) and control smokers (n = 25). (b) In the pilot cohort, PGP levels are elevated in COPD subjects compared with never smokers and elevated to a similar extent as that seen in control smokers. (c) Aminopeptidase activity is decreased by more than 80% in COPD subjects, independent of ongoing cigarette use. Acetylated PGP (AcPGP) is elevated in both current and former smokers in the pilot cohort, with a trend toward lower AcPGP in former smokers. In the ECLIPSE group, AcPGP amounts are elevated in current COPD smokers (n = 107) compared with former smokers with COPD (n = 107; P = 0.05). (d) In the pilot cohort, sputum myeloperoxidase (MPO) and leukotriene B₄ (LTB₄) levels are increased to similar extents in healthy smokers and COPD subjects compared with never smokers. *P ≤ 0.05, **P < 0.01 by two-sided t test, ^#P = 0.015 for AcPGP by one-way analysis of variance, ^¥P < 0.001 for aminopeptidase activity by one-way analysis of variance. Error bars represent SEM.

Figure 4.

Differences in acetylated proline-glycine-proline (AcPGP) between current and former smokers with chronic obstructive pulmonary disease (COPD) according to disease severity and phenotype. In the ECLIPSE cohort, AcPGP levels are elevated in both current and former smokers with COPD. (a) In severe COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 3 and 4), AcPGP is higher in current smokers (P = 0.045 for GOLD 3 and P = 0.035 for GOLD 4) compared with former smokers with similar airflow obstruction. (b) Sputum AcPGP is higher in COPD smokers with chronic bronchitis compared with former smokers (P = 0.06). Sputum AcPGP levels are similar between current and former smokers with either significant emphysema alone or in emphysema in combination with chronic bronchitis.

Figure 5.

Acrolein inhibits leukotriene A₄ hydrolase (LTA₄H) aminopeptidase activity and is detectable in the sputum of patients with chronic obstructive pulmonary disease (COPD), even in the absence of ongoing cigarette use. In an in vitro setting, cigarette smoke and acrolein significantly inhibit LTA₄H aminopeptidase activity when measured over time by Ala-pNA assay (a) or at 24 hours when measured by proline-glycine-proline (PGP) degradation (b). Acroleinated LTA₄H is present in the sputum of patients with COPD. (c) Representative Western blots (n = 4–5 per group) of LTA₄H probed for acrolein. LTA₄H was isolated from the sputum of smokers and former smokers with COPD by immunoprecipitation. The band densities shown were standardized to the amount of LTA₄H enzyme concentration as detected by ELISA and then standardized to the band density of the LTA₄H + acrolein control. (d) Relative acrolein absorbance units (AU) per nanogram of LTA₄H was significantly higher in both COPD smokers (n = 4) and COPD former smokers (n = 4) compared with never smokers (n = 5). *P < 0.05, **P < 0.01, error bars represent SEM.