Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema

Abstract

The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme with epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing proline-glycine-proline (PGP), a triamino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild-type mice to CS over 5 mo while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bioproduction of leukotriene B4. Emphysematous phenotypes were assessed by premortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the bronchoalveolar lavage fluid and infiltration of neutrophils into the lungs were significantly reduced without affecting the levels of leukotriene B4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the leukotriene A4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema.

Figure 1

Assessment of the emphysema phenotype in 129sv wild type mice post exposure to cigarette smoke by Teague TE-2 smoking apparatus over 5 months. Panel A: Pre mortem total lung volume measured by Sireq Flexivent. Panel B: Pre mortem quasi-static lung compliance measured by Sireq Flexivent. Panel C: Post mortem alveolar L m. Panel D: Representative H&E histology of the lungs at low power (2.5× magnification). Time = duration of exposure to cigarette smoke. Cigarette dose = 3 cigarettes per 9 minutes, 5 hours a day, 5 days a week. * represents analysis by ANOVA. N = 9–13 animals per groups in panels A and B. N = 5 animals per group in panel C.

Figure 2

Assessment of the LTA₄H protein expression in 129sv wild type mice post exposure to cigarette smoke over 5 months. Panel A: The levels of LTA₄H protein in the whole lung BALF assessed by ELISA. Panel B: The levels of LTA₄H protein in the whole lung protein soup assessed by ELISA. Panel C: Flow cytometry of the whole lung single cell suspension. Air = mice exposed to ambient air for 20 weeks. SM = mice exposed to cigarette smoke for 20 weeks. Panel D: Immunohistochemistry with antibody specific to murine LTA₄H protein in lung tissues from mice exposed to cigarette smoke for 20 weeks (63× oil magnification). Positive signals appear brown and counterstained with blue. Arrowheads show nuclei with positive counterstain (blue) indicating absence of LTA₄H protein. Arrows show nuclei with positive stain (grown) indicating presence of LTA₄H protein. Gray bars in Panels A & B = ambient-air exposed mice with ages 26 – 28 weeks. * represents analysis by ANOVA, ** by Bonferroni subgroup comparison, and *** by nonparametric t-Test. N = 5 per group.

Figure 3

Assessment of 129sv wild type mice post exposure to cigarette smoke by Teague TE-2 smoking apparatus for 5 months. Panel A: Levels of LTB₄ in BALF. Panel B: Levels of PGP in BALF. Panel C: Levels of CD45⁺CD11b⁺Ly6G⁺ cells in whole lung single cell suspension. Antibodies are with PerCP-labeled CD45, ACP-labeled CD11b, and PE-labeled Ly5G. Panel D: pH of BALF over 20-week cigarette smoke exposure. Panel E: In vitro aminopeptidase activity assay using human recombinant LTA₄H at pHs 6.7 and 7.2. * represents analysis by nonparametric t-Test. ** represents analysis by ANOVA.*** represents two-way ANOVA with AP activity and time as two factors. N = 5 per group. Air = ambient air exposure. SM = cigarette smoke exposure.

Figure 4

Panel A: Diagram of CDX encapsulating 4MDM. 4MDM is in the inner pocket of CDX and highlighted with an oval circle. Panel B: Levels of 4MDM in BALF 24, 48, and 72 hours after CDX-4MDM was started to be administered as drinking water in mice. * represents column statistics comparing the levels of 4MDM to the hypothetical value of untreated mice, in this case, 0 ng/mL. N = 5 animals per group.

Figure 5

Effects of CDX-4MDM treatment on the levels of LTB₄, PGP, and neutrophils in mice exposed to cigarette smoke for 20 weeks. Panel A: In silico modeling of the LTA₄H binding pocket occupied by 4MDM and PGP overlaid with the predicted binding model of the LTA₄. 4MDM = orange. PGP = yellow. LTA₄ = purple. Panel B: Levels of LTB₄ in BALF with oral CDX-4MDM or vehicle treatment post exposure to cigarette smoke for 20 weeks. Panel C: Levels of PGP in BALF with oral CDX-4MDM or vehicle treatment post exposure to cigarette smoke for 20 weeks. Panel D: Levels of CD45⁺CD11b⁺Ly6G⁺ cells in whole lung single cell suspension with oral CDX-4MDM or vehicle treatment post exposure to cigarette smoke for 20 weeks. Panel E: Levels of CD45⁺CD11b⁺Ly6G⁺ cells in whole lung single cell suspension of 129sv WT mice (black bars) and 129sv mice with null mutation at the LTA₄H loci (gray bars) with oral CDX-4MDM or vehicle treatment post exposure to cigarette smoke for 7 days. * represents analysis by ANOVA, ** by Bonferroni subgroup comparison, and *** by nonparametric t-Test. N = 5 – 6 per group.

Figure 6

Effects of CDX-4MDM treatment on pulmonary emphysema in WT mice exposed to cigarette smoke or ambient air for 20 weeks. Panel A: Pre mortem total lung volume measured by Sireq Flexivent. Total lung volume was measured after inflating the lungs with 30 cm H₂O pressure. Panel B: Pre mortem quasi-static lung compliance measured by Sireq Flexivent. Panel C: Post mortemL m. Panel D: Representative H&E histology of the lungs (10× magnification). + in the box and whisker plot represents mean while horizontal bar represents median. * represents analysis by ANOVA, and ** by Bonferroni subgroup comparison. N = 9–13 animals per groups in panels A and B. N = 5 animals per group in panels C.

Figure 7

LTA₄H aminopeptidase activity assay. Panel A. In vitro LTA₄H aminopeptidase activity assay with human recombinant LTA₄H in the presence of increasing doses of 4MDM at culture medium pH 6.7. AP activity is assessed by UV light absorption at λ = 405. Panel B. LTA₄H aminopeptidase activity assay with BAL fluid collected from mice with wild type (WT) or null mutation (LTA₄H KO) at the LTA₄H loci. N = 5 per group. Panel C. LTA₄H aminopeptidase activity assay with BALF fluid collected from mice treated with vehicle or 4MDM after exposure to cigarette smoke for 20 weeks. N = 6 per group. All data points are mean ± SEM. * represents two-way ANOVA with AP activity as the first factor and 4MDM dose (Panel A) or time (Panels B & C) as the second factor.

Figure 8

Effects of CDX-4MDM treatment on apoptosis in WT mice exposed to cigarette smoke or ambient air for 20 weeks. Counting of TUNEL positive cells and calculating Apoptosis Index (percentage of cells positively stained in TUNEL assay). Ten random pictures of each animal were examined. N = 5 per group. Air = ambient air exposure. SM = cigarette smoke exposure. Veh = CDX containing vehicle. Tx = CDX-4MDM. * represents analysis by ANOVA, and ** by Bonferroni subgroup comparison.

Figure 9

Effects of CDX-4MDM treatment on WT mice exposed to cigarette smoke or ambient air for 20 weeks. Panel A: Levels of KC in the BALF from WT mice exposure to cigarette smoke for 20 weeks. Panel B: Levels of MIP2 in the BALF from WT mice exposure to cigarette smoke for 20 weeks. Panel C: Levels of RNA transcribing for MMP8 in whole lung RNA isolate normalized by β-actin. Panel D: Levels of RNA transcribing for MMP9 in whole lung RNA isolate normalized by β-actin. * represents analysis by ANOVA and ** by Bonferroni subgroup comparison. N = 5–7 animals per groups. Air = ambient air exposure. SM = cigarette smoke exposure. Veh = CDX containing vehicle. Tx = CDX-4MDM.