Lung chitinolytic activity and chitotriosidase are elevated in chronic obstructive pulmonary disease and contribute to lung inflammation

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers. ChTRase accounted for approximately 80% of bronchoalveolar lavage chitinolytic activity, while acidic mammalian chitinase was undetectable. Bronchoalveolar lavage chitinolytic activity and ChTRase were associated with airflow obstruction and emphysema and with the levels of interleukin (IL)-1beta, IL-8, tumor-necrosis factor (TNF)-alpha, and its type II soluble receptor. Tumor necrosis factor-alpha stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of IL-8, monocyte-chemoattractant protein-1, and metalloproteinase-9. Finally, ChTRase overexpression in the lung of normal mice promoted macrophage recruitment and the synthesis of the murine homologue of IL-8, keratinocyte-derived cytokine, and of monocyte-chemoattractant protein-1. We conclude that pulmonary ChTRase overexpression may represent a novel important mechanism involved in COPD onset and progression.

Figure 1

BAL chitinolytic activity and levels of ChTRase in subjects with COPD and asthma. A: Chitinolytic activity (in fluorescence units, FU) in BAL samples collected by fiberoptic bronchoscopy in never-smokers, in smokers, in COPD patients, and in mild, moderate and severe asthmatics (horizontal bars denote median values). B: Inhibition, by a neutralizing anti-ChTRase Ab (closed squares), but not by an anti-AMCase Ab (open squares), of chitinolytic activity induced by 25 ng/ml of recombinant human ChTRase in an enzymatic assay. C: Inhibition of chitinolytic activity in BAL samples from smokers and from patients with COPD by a neutralizing ChTRase Ab (closed squares), but not by its control rabbit IgG isotype (open squares). Columns represent means ± SEM of n = 6 to 10 independent experiments in each group. *P = 0.03, **P = 0.02. D: BAL levels of ChTRase in never-smokers, in smokers, in patients with COPD, and in mild, moderate and severe asthmatics. Horizontal bars in panels (A) and (D) denote median values.

Figure 2

Expression of ChTRase in bronchial biopsies. A: Number ChTRase-positive cells per mm² of bronchial biopsies in never-smokers, in smokers, and patients with COPD (horizontal bars denote median values). B: ChTRase (red chromogen) is undetectable in bronchial tissue sections from never-smokers, whereas faint ChTRase-positive cells are identified in the bronchial epithelium and the submucosa (arrow) of smokers. In smokers with COPD, substantially more ChTRase-positive cells infiltrate the bronchial epithelium (arrows) and the subepithelial areas. Original magnification, ×40. C and D: Double immunofluorescent labeling of bronchial tissue sections of a patient with COPD showing ChTRase- (red, right panels) expressing CD68 (green, left panel in C)-positive macrophages and elastase (green, left panel in D)-positive neutrophils. Original magnification, ×189).

Figure 3

Expression of ChTRase in lung tissues. A: Never-smokers show scant ChTRase-positive cells (red chromogen) in the alveolar septa and no immunostaining in the bronchiolar epithelium. Smokers have ChTRase-expressing macrophages in the alveoli and at the vicinity of the bronchiolar epithelium, but no immunostaining in the bronchiolar epithelium. B: Patients with COPD have prominent accumulation of ChTRase-positive macrophages in the alveoli and strong expression of ChTRase in the bronchiolar epithelium. The last right panel showing the absence of immunostaining in a control isotype (rabbit IgG)-labeled lung tissue section from a patient with COPD. Original objective ×20, except the second panel in (B, ×40). C: Double immunofluorescence labeling of lung tissue sections of a patient with COPD shows ChTRase- (red) and CD68 (green)-positive macrophages and immunolocalization of ChTRase (red) in the bronchiolar epithelium. Last right panel represents a control-isotype-labeled tissue section with no staining. Original objective ×63, except in the second panel (×189).

Figure 4

Expression and functional role of ChTRase in alveolar macrophages. A: The expression of ChTRase (red chromogen) is higher in alveolar macrophages collected by BAL from patients with COPD, as compared to smokers and to never-smokers. Original magnifications ×40. B: Mean proportions of alveolar macrophages expressing ChTRase in never-smokers (open column), in smokers (gray column) and in patients with COPD (closed column). Results are means ± SEM of n = 8 to 10 distinct cell preparations in each group. *P < 0.05, **P < 0.03 (Kruskal-Wallis and Mann-Whitney U-tests). (C, left) Levels of ChTRase mRNA (ratio over the amounts of the housekeeping gene, ubiquitin C, UBC) in freshly isolated alveolar macrophages from never-smokers (open bars), from smokers (gray bars) and from patients with COPD (closed bars). (C, right) Spontaneous and TNF-α (10 ng/ml for 24 hours)-induced ChTRase release from alveolar macrophages from never-smokers, smokers and COPD patients. Data are means ± SEM of n = 10 to 12 distinct cell preparations in each group of subjects. *P < 0.04, **P < 0.03.

Figure 5

ChTRase induces IL-8, MCP-1, and MMP-9 release by alveolar macrophages. Alveolar macrophages from never-smokers, from smokers, and from patients with COPD were stimulated over 24 hours with the medium alone (open columns), or supplemented with 10 μg/ml ChTRase (closed bars), or with 10 ng/ml TNF-α (hatched bars) and the levels of IL-8 (A), MCP-1 (B), and MMP-9 (C) were determined by ELISA. Data are means ± SEM of n = 5 to 7 independent experiments in each group. *P < 0.05 (Kruskal-Wallis and Mann-Whitney U-test).

Figure 6

Pulmonary ChTRase overexpression promotes airway inflammation in normal mice. A: Levels of ChTRase mRNA (ratio over the amounts of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase) in whole lung homogenates obtained from normal mice that had been instilled intranasally with PBS, or with empty vector adenovirus (Null Adv), or with adenovirus carrying full-length ChTRase (ChTRase Adv). Mice were sacrificed 2 or 4 days thereafter. Data are means ± SEM of n = 3 to 7 independent experiments. (**P < 0.01). B: ChTRase immunolocalization (brown deposit) in bronchial and bronchiolar epithelium and in tissue macrophages contained in lung tissue sections from ChTRase Adv-instilled mice sacrificed at 4 days. The right panel shows an exemplary isotype-stained tissue section. Original magnification, ×40. C: Levels of KC and MCP-1 (ratio over glyceraldehyde-3-phosphate dehydrogenase mRNA) in whole lung homogenates from PBS-, Null Adv-, and ChTRase Adv-instilled mice. D: Amounts of KC and MCP-1, as determined by ELISA in BAL fluid from PBS-, or Null Adv-, or ChTRase Adv-instilled mice. Data in (C) and (D) are means ± SEM of n = 3 to 7 independent experiments (*P < 0.02).