Human neutrophil elastase-mediated goblet cell metaplasia is attenuated in TACE-deficient mice

Abstract

Neutrophilic inflammation is associated with chronic airway diseases. It has been observed that human neutrophil elastase (HNE), which is secreted by active neutrophils during inflammation, induces both mucin overproduction and goblet cell metaplasia. Several in vitro studies suggest that tumor necrosis factor-α converting enzyme (TACE) regulates the signaling axis that mediates HNE-induced mucin overproduction; however, it is unknown whether TACE performs a similar function in HNE-induced goblet cell metaplasia in vivo. We conducted this study to determine whether the inactivation of Tace gene expression attenuates HNE-induced goblet cell metaplasia in mice. Deletion of Tace is lethal shortly after birth in mice; therefore, we utilized Tace(flox/flox)R26CreER(+/-) mice and induced conditional deletion of Tace using a tamoxifen injection. Wild-type mice were given tamoxifen to control for its effect. Tace conditional deletion mice and wild-type mice were exposed to HNE via nasal instillation three times at 3-day intervals, and the lungs were harvested on day 11 after initial HNE exposure. Using periodic acid-Schiff staining and MUC5AC immunohistochemical staining to visualize goblet cells in the lungs, we found that HNE induced goblet cell metaplasia in the wild-type mice and that HNE-induced goblet cell metaplasia was significantly attenuated in the Tace conditional deletion mice. These findings suggest that TACE could be a potential target in the treatment of goblet cell metaplasia in patients with chronic airway diseases.

Keywords: airway epithelium; airway mucin; chronic airway disease; neutrophilic inflammation.

Fig. 1.

Experimental procedure for tamoxifen injection and human neutrophil elastase (HNE) exposure. Tamoxifen was injected intraperitoneally (IP) daily for 5 days. On day 7 after the last tamoxifen injection, mice were instilled intranasally with HNE (50 μg in 40 μl of PBS) or 40 μl of PBS 3 times at 3-day intervals. On day 4 after the last HNE exposure, lungs were harvested for histology and mRNA analysis of Muc5ac.

Fig. 2.

Detection of Tace mRNA and tumor necrosis factor-α converting enzyme (TACE) protein expression using real-time PCR and Western blot analysis, respectively. Intraperitoneal injection of tamoxifen (daily for 5 days, 0.04 mg/g of body wt) in Tace^flox/flox/Cre^+/− mice (Tace conditional deletion mice) reduced the levels of Tace mRNA expression (A; P < 0.01, mean ± SE, n = 3 independent experiments) and TACE protein (B) expression detected on day 7 after the last tamoxifen injection, compared with those detected in Tace^flox/flox/Cre^−/− mice (wild-type mice) injected with tamoxifen on the same dose schedule. Semiquantitative measurement of TACE protein normalized to α-tubulin from 3 independent experiments (C).

Fig. 3.

Periodic acid-Schiff (PAS) staining of the airway. A–D: PAS staining was performed to visualize goblet cells in the lungs harvested on day 11 after the first exposure to HNE. Tace^flox/flox/Cre^−/− mice (wild-type mice) that were instilled with HNE developed goblet cell metaplasia, but HNE-mediated goblet cell metaplasia was attenuated in Tace^flox/flox/Cre^+/− mice (Tace conditional deletion mice). PBS instillation was used as a negative control and did not induce goblet cell metaplasia in either genotype. E: the goblet cell index determined by PAS staining shows that HNE significantly increased the number of goblet cells in the airways of wild-type mice (n = 10) compared with Tace conditional deletion mice (n = 14).

Fig. 4.

Immunohistochemical staining of MUC5AC. Immunohistochemical staining of MUC5AC was performed to specifically immunolabel goblet cells (×200 magnification, A and B). Strong immunostaining was detected on the tissue from Tace^flox/flox/Cre^−/− mice (wild-type mice) that were exposed to HNE, whereas no staining was observed on the tissue from Tace^flox/flox/Cre^+/− mice (Tace conditional deletion mice) that were exposed to HNE. The dotted areas in A and B are magnified in C and D. Secondary antibody alone (E) was used to confirm the specificity of the MUC5AC antibody. Scale bars, 100 μm.

Fig. 5.

Muc5ac mRNA expression, tissue inflammation index, and concentration of TNF-α in bronchoalveolar lavage (BAL) fluid. A: Muc5ac mRNA expression in homogenized lung tissue was determined by real-time PCR analysis and normalized to the expression of Gapdh. B: tissue inflammation, which was determined by hematoxylin and eosin staining, was not significantly different between the genotypes. Number of mice used for each condition: Tace^flox/flox/Cre^−/− with PBS (wild-type-PBS), n = 6; Tace^flox/flox/Cre^−/− with HNE (wild-type-HNE), n = 9; Tace^flox/flox/Cre^+/− with PBS (Tace conditional deletion-PBS), n = 3; Tace^flox/flox/Cre^+/− with HNE (Tace conditional deletion-HNE), n = 14. C: concentration of TNF-α in BAL fluid. Number of mice used for each condition: Tace^flox/flox/Cre^−/− with PBS (wild-type-PBS), n = 2; Tace^flox/flox/Cre^−/− with HNE (wild-type-HNE), n = 3; Tace^flox/flox/Cre^+/− with PBS (Tace conditional deletion-PBS), n = 3; Tace^flox/flox/Cre^+/− with HNE (Tace conditional deletion-HNE), n = 5.