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. 2019 Apr 1;199(7):873-881.
doi: 10.1164/rccm.201803-0442OC.

Elastase Exocytosis by Airway Neutrophils Is Associated With Early Lung Damage in Children With Cystic Fibrosis

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Free PMC article

Elastase Exocytosis by Airway Neutrophils Is Associated With Early Lung Damage in Children With Cystic Fibrosis

Camilla Margaroli et al. Am J Respir Crit Care Med. .
Free PMC article

Abstract

Rationale: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown.

Objectives: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF.

Methods: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects.

Measurements and main results: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes.

Conclusions: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Keywords: air trapping; degranulation; mucus plugging; proteolysis; scavenging.

Figures

Figure 1.
Figure 1.
Flow cytometry gating strategy and neutrophil phenotype. (A) After definition of singlets (not shown), blood (red, top left) and airway (blue, bottom left) neutrophils were gated in four sequential steps, as shown. Autofluorescence in the violet laser-excited 450/50 nm channel (Autofl.) was low in airway neutrophils and high in airway macrophages (see Figure E1). All children with cystic fibrosis (CF) showed a typical pattern in airway neutrophils with increased surface CD63 (primary granule exocytosis) and decreased surface CD16 (phagocytic receptor) expression (right panel). (B) Surface expression of CD66b (left; control cohort, n = 9; CF cohort, n = 22), CD16 (middle; control cohort n = 9; CF cohort n = 33), and CD63 (right; control cohort n = 9; CF cohort n = 33) on airway neutrophils are shown in % change compared with matched blood neutrophils. Significant differences in between-group analyses are indicated as **P < 0.01 and ***P < 0.0001. FSC = forward scatter; SSC = side scatter.
Figure 2.
Figure 2.
Correlation of neutrophil elastase (NE) exocytosis by airway neutrophils, and free extracellular NE activity with structural lung damage. (A) Correlation of the PRAGMA-%Dis with airway neutrophil CD63 levels (n = 33). (B) Correlation of the PRAGMA-%Dis score with free extracellular NE activity (interpolated to a concentration of recombinant NE standard expressed in ng/ml; n = 29). MFI = median fluorescence intensity; PRAGMA-%Dis = Perth-Rotterdam Annotated Grid Morphometric Analysis for Total Disease.
Figure 3.
Figure 3.
Neutrophil elastase (NE) exocytosis by airway neutrophils and free extracellular NE activity are not impacted by infection status. (A) Airway neutrophil CD63, as a measure of NE exocytosis (in median fluorescence intensity; n = 39) is compared between children with cystic fibrosis based on infection status (0, 1, or 2 or more proinflammatory pathogens detected in BAL fluid). (B) Free extracellular NE activity (interpolated to a concentration of recombinant NE standard expressed in ng/ml; n = 34) is also compared between children with cystic fibrosis based on infection status. MFI = median fluorescence intensity; n.s. = not significant.
Figure 4.
Figure 4.
Free extracellular neutrophil elastase (NE) activity in children with cystic fibrosis is limited by BAL fluid antiprotease activity and by compartmentalization on airway leukocytes. (A) Activity of recombinant NE was measured in vitro by Förster resonance energy transfer in the absence or presence of BAL fluid from children with cystic fibrosis with either low or high intrinsic NE activity. Results were compared with the unopposed activity of recombinant NE in the absence of BAL fluid (100%). (B) Surface NE antigen was measured by flow cytometry in cystic fibrosis blood and airway neutrophils and macrophages (n = 6). *P < 0.05. MFI = median fluorescence intensity.

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