Coupled nucleotide and mucin hypersecretion from goblet-cell metaplastic human airway epithelium

Abstract

Adenosine triphosphate (ATP) and its metabolite adenosine regulate airway mucociliary clearance via activation of purinoceptors. In this study, we investigated the contribution of goblet cells to airway epithelial ATP release. Primary human bronchial epithelial (HBE) cultures, typically dominated by ciliated cells, were induced to develop goblet cell metaplasia by infection with respiratory syncytial virus (RSV) or treatment with IL-13. Under resting conditions, goblet-cell metaplastic cultures displayed enhanced mucin secretion accompanied by increased rates of ATP release and mucosal surface adenosine accumulation as compared with nonmetaplastic control HBE cultures. Intracellular calcium chelation [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester] or disruption of the secretory pathways (nocodazole, brefeldin A, and N-ethylmaleimide) decreased mucin secretion and ATP release in goblet-cell metaplastic HBE cultures. Conversely, stimuli that triggered calcium-regulated mucin secretion (e.g., ionomycin or UTP) increased luminal ATP release and adenyl purine accumulation in control and goblet-cell metaplastic HBE cultures. Goblet cell-associated ATP release was not blocked by the connexin/pannexin hemichannel inhibitor carbenoxolone, suggesting direct nucleotide release from goblet cell vesicles rather than the hemichannel insertion. Collectively, our data demonstrate that nucleotide release is increased by goblet cell metaplasia, reflecting, at least in part, a mechanism tightly associated with goblet cell mucin secretion. Increased goblet cell nucleotide release and resultant adenosine accumulation provide compensatory mechanisms to hydrate mucins by paracrine stimulation of ciliated cell ion and water secretion and maintain mucociliary clearance, and to modulate inflammatory responses.

Figure 1.

Time course of respiratory syncytial virus (RSV) infection, RSV-induced goblet cell metaplasia, and extracellular adenyl purine profiles. The data were obtained at 3, 14, and 42 days after RSV (or mock) infection. (A) Top: Live intracellular RSV intensity visualized by green fluorescent protein fluorescence of the recombinant virus (rgRSV). Bar, 30 μm. Bottom: Quantitation of green fluorescent protein fluorescence intensity (by Image J). (B) IL-8 levels in serosal medium. (C) Representative x-z sectioned histological pictures (Richardson staining) of primary human bronchial epithelial cultures at 14 days after mock (left) or RSV (right) infection. Bar, 10 μm. (D) Percentages of mucus-producing cells (goblet cells) quantitated in laser confocal microscopy images of differential immunostaining for ciliated cells (red; tubulin) and goblet cells (green; periodic acid-Schiff) overlayed with differential interference contrast images. (E) Mucin secretion rates in control and RSV-induced goblet-cell metaplastic cultures as measured by ELISA over 12 hours. Mucosal surfaces were washed, and cultures were rested for 24 hours. At t = 0, 3, 6, 9, and 12 hours, 75 μl Dulbecco's modified Eagle's medium was added on cultures mucosally. Cultures were incubated for 10 minutes at 37°C and sampled for ELISA. Mucin secretion rates over 12 hours were calculated from the amount of mucins in samples at each time point. (F) Concentrations of adenyl purines in airway surface liquid sampled from resting cultures at 42 days after mock (left blocks) or RSV (right blocks) infection, as analyzed by etheno-derivatization. *Significant difference (P < 0.05) over mock-infected control cultures. Values are mean ± SE of four Transwells/subject established from three different subjects.

Figure 2.

ATP release rates from cultures after RSV infection. (A) ATP accumulation in airway surface liquid when epithelial cell surface-mediated ATP hydrolysis was inhibited by the addition of ecto-ATPase inhibitors (β, γ-methylene-ATP [300 μM], ebselen [30 μM], and levamisole [10 mM]) to airway surface liquid at t = 0. Open circles and solid circles indicate mock- and RSV-infected cultures, respectively, at 42 days after infection. (B) ATP release rates in resting cultures at 3, 14, and 42 days after RSV (or mock) infection, as measured from ATP accumulation rates by using the method depicted in Fig. 2A and Ref. 12. (C) Correlation between goblet cell percentages (data imported from Fig. 1D) and ATP release rates (data imported from Fig. 2B). *Significant difference (P < 0.05) over mock-infected control cultures. Values are mean ± SE of four Transwells/subject established from three different subjects.

Figure 3.

Goblet cell metaplasia after IL-13 treatment. (A) Representative images of differential immunohistochemistry for ciliated cells (red; tubulin) and goblet cells (green; periodic acid-Schiff) after 5 days of treatment with vehicle (left) or IL-13 (right). Bar, 30 μm. (B) Goblet cell percentages in vehicle- or IL-13–treated cultures quantitated from the confocal images as represented in A. (C) Mucin secretion rates in control and IL-13–treated goblet-cell metaplastic cultures as measured by ELISA over 12 hours. (D) Concentrations of adenyl purines in airway surface liquid sampled from vehicle- or IL-13–treated cultures under resting condition, as measured by etheno-derivatization. (E) ATP release rates in vehicle- or IL-13–treated cultures. (F) Correlation between goblet cell percentages (data imported from B) and ATP release rates (data imported from E). *Significant difference (P < 0.05) over vehicle-treated control cultures. Values are mean ± SE of three Transwells/subject established from three different subjects.

Figure 4.

ATP and mucin secretion from goblet-cell metaplastic cultures. (A and B) Amounts of secreted mucins as measured by slot blot (A) and ATP release rates (B) in control, RSV-infected (42 d), or IL-13–treated cultures. These cultures were treated with BAPTA-AM (100 μM, 30 min) (black bars) or vehicle (white bars) immediately before the mucin or ATP measurement assays. (C and D) Amounts of secreted mucins as measured by slot blot (C) and ATP release rates (D) in control, RSV-infected, or IL-13–treated cultures. These cultures were treated with secretory pathway inhibitors: nocodazole (Noco; 20 μM, 4 h), brefeldin A (BFA) (40 μM, 2.5 h), and N-ethylmaleimide (NEM) (1 mM, 15 min) or vehicle immediately before the mucin or ATP measurement assays. For slot blots (A and C), 300 μl Hanks' balanced salt solution buffered with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid was added to mucosal culture surface, and 100 μl was sampled 15 minutes later. Signal intensity in slot blots was expressed as relative to that of mock-infected cultures without inhibitor treatment. *Significant difference (P < 0.05) between vehicle and the reagent treatment. ^†Significant difference (P < 0.05) over nonmetaplastic control subjects under the same reagent treatment. Values are mean ± SE of four Transwells/subject established from three different subjects.

Figure 5.

Enhanced nucleotide release associated with robust mucin secretion. (A) Mucin secretion after UTP (100 μM, 10 min) or ionomycin (5 μM, 10 min) treatment in control, RSV-infected (42 d), or IL-13–treated cultures. (B) Adenyl purine profiles at basal (left), UTP-treated (middle), or ionomycin-treated (right) conditions in control, RSV-infected (42 d), or IL-13–treated cultures. *Significant difference (P < 0.05) over nonmetaplastic control subjects. †Significant difference (P < 0.05) over basal values (i.e., without UTP or ionomycin treatment). Values are mean ± SE of three Transwells/subject established from three different subjects.

Figure 6.

UDP-glucose release from goblet-cell metaplastic cultures. UDP-glucose concentrations in the sampled airway surface liquid from control, RSV-infected (42 d), or IL-13–treated cultures. After 2 hours of incubation with mucosal assay buffer, 300 μM β, γ-methylene ATP was added to airway surface liquid to inhibit UDP-glucose hydrolysis and was incubated for additional 15 minutes. *Significant difference over mock-infected cultures (P < 0.05). Values are mean ± SE of four Transwells/subject established from two different subjects.

Figure 7.

ATP release from human bronchial epithelial cultures treated with connexin or pannexin hemichannels. (A) Control and IL-13–treated goblet-cell metaplastic cultures at resting conditions were preincubated with carbenoxolone (CBX) of indicated concentrations or vehicle for 15 minutes, and ATP release rates were measured. (B and C) After the preincubation with CBX or vehicle, cultures were stimulated with UTP (100 μM) (B) or ionomycin (5 μM) (C), and ATP release rates were measured. Values are mean ± SE of three Transwells/subject established from three different subjects.