Abnormal pro-gly-pro pathway and airway neutrophilia in pediatric cystic fibrosis

Abstract

Background: Proline-glycine-proline (PGP) is a bioactive fragment of collagen generated by the action of matrix metalloproteinase-9 (MMP-9) and prolylendopeptidase (PE), and capable of eliciting neutrophil chemotaxis and epithelial remodelling. PGP is normally then degraded by leukotriene A₄ hydrolase (LTA₄H) to limit inflammation and remodelling. This study hypothesized that early and persistent airway neutrophilia in Cystic Fibrosis (CF) may relate to abnormalities in the PGP pathway and sought to understand underlying mechanisms.

Methods: Broncho-alveolar lavage (BAL) fluid was obtained from 38 CF (9 newborns and 29 older children) and 24 non-CF children. BAL cell differentials and levels of PGP, MMP-9, PE and LTA₄H were assessed.

Results: Whilst PGP was present in all but one of the older CF children tested, it was absent in non-CF controls and the vast majority of CF newborns. BAL levels of MMP-9 and PE were elevated in older children with CF relative to CF newborns and non-CF controls, correlating with airway neutrophilia and supportive of PGP generation. Furthermore, despite extracellular LTA₄H commonly being greatly elevated concomitantly with inflammation to promote PGP degradation, this was not the case in CF children, potentially owing to degradation by neutrophil elastase.

Conclusions: A striking imbalance between PGP-generating and -degrading enzymes enables PGP accumulation in CF children from early life and potentially supports airway neutrophilia.

Keywords: Cystic fibrosis; Matrikine; Neutrophil; Protease.

Graphical abstract

Fig. 1

The neutrophil matrikine PGP is elevated in the BAL fluid of older children with CF. (A) Levels of PGP peptide in the BAL fluid of non-CF controls (n = 24), RSISP-CF children (n = 9) and CC-CF children (n = 25), as determined by LC-MS/MS. (B) Correlation between levels of BAL fluid PGP and airway neutrophils (n = 37: 16 non-CF, 7 RSISP-CF, 14 CC-CF). (C) Levels of IL-8 in the BAL fluid of non-CF controls (n = 23), RSISP-CF children (n = 9) and CC-CF children (n = 28), as determined by ELISA. Closed symbols represent patients that were culture negative and open symbols patients that were culture positive. The horizontal bar depicts the median of each group. Statistical significance between groups was tested using a Kruskal-Wallis test followed by a Dunns post-test and correlation analysis was performed using Spearman rank test. * = P < .05; ** = P < .01; *** = P < .001. RSISP, routine screened infant surveillance program; CC, bronchoscopy for clinical concern; CF = Cystic Fibrosis.

Fig. 2

Elevated PGP-generating enzymes in the BAL fluid of older children with CF. Levels of total MMP-9 (A; n = 24 non-CF, 9 RSISP-CF, 29 CC-CF) and active MMP-9 (B; n = 22 non-CF, 9 RSISP-CF, 28 CC-CF) and PE (C; n = 24 non-CF, 9 RSISP-CF, 29 CC-CF) in the BAL fluid. Closed symbols represent patients that were culture negative and open symbols patients that were culture positive. The horizontal bar depicts the median of each group. Statistical significance between groups was tested using a Kruskal-Wallis test followed by a Dunns post-test. * = P < .05; ** = P < .01; *** = P < .001. RSISP, routine screened infant surveillance program; CC, bronchoscopy for clinical concern; CF = Cystic Fibrosis.

Fig. 3

Neutrophil elastase can cleave LTA₄H, abrogating its PGP-degrading activity. (A) Levels of LTA₄H in the BAL fluid of non-CF controls (n = 24), RSISP-CF patients (n = 9) and CC-CF patients (n = 29), as determined by ELISA. (B) NE activity in the BAL fluid of non-CF controls (n = 23), RSISP-CF patients (n = 7) CC-CF patients (n = 20), as determined by fluorometric assay. (C-E) Recombinant LTA₄H was co-incubated with recombinant neutrophil elastase (NE) for 2 h at 37 °C and cleavage of LTA₄H assessed by Coomassie stained gel (C) and Western blot (D). (E) Recombinant LTA₄H was co-incubated with recombinant NE for 2 h at 37 °C and capacity of LTA₄H to degrade PGP subsequently determined by incubation with the peptide for 2 h and assessing PGP degradation by LC-MS/MS. (F) Correlation between levels of active MMP-9 and NE activity in the BAL fluid (n = 46: 22 non-CF, 7 RSISP-CF, 17 CC-CF). (G) Correlation between levels of PGP and NE activity in the BAL fluid (n = 50: 23 non-CF, 7 RSISP-CF, 20 CC-CF). Closed symbols represent patients that were culture negative and open symbols patients that were culture positive. For (A and B) the horizontal bar depicts the median of each group. Statistical significance between groups was tested using a Kruskal-Wallis test followed by a Dunns post-test and correlation analysis was performed using Spearman rank test. * = P < .05; ** = P < .01; *** = P < .001. RSISP, routine screened infant surveillance program; CC, bronchoscopy for clinical concern; CF = Cystic Fibrosis.

Fig. 4

An imbalance between PGP-generating and -degrading enzymes permits PGP accumulation in CF children. Ratio of [MMP-9]/[LTA₄H] (A) or [PE]/[LTA₄H] (B) in the BAL fluid of non-CF controls (n = 24), RSISP-CF patients (n = 9) and CC-CF patients (n = 27), as determined by ELISA. (C) Correlation between the ratio of [MMP-9]/[LTA₄H] versus [PGP] or (D) between the ratio of [PE]/[LTA₄H] versus PGP (n = 56: 24 non-CF, 9 RSISP-CF, 23 CC-CF). Closed symbols represent patients that were culture negative and open symbols patients that were culture positive. For (A and B) the horizontal bar depicts the median of each group. Statistical significance between groups was tested using a Kruskal-Wallis test followed by a Dunns post-test and correlation analysis was performed using Spearman rank test. * = P < .05; ** = P < .01; *** = P < .001. RSISP, routine screened infant surveillance program; CC, bronchoscopy for clinical concern; CF = Cystic Fibrosis.