IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells

Abstract

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

Figure 1

IL-13 modifies the proportion of differentiated phenotypes during MCD. (a) Scheme illustrating the different steps of the primary culture. Cells dissociated from human nasal polyps or turbinates are plated on thick collagen. Basal-like epithelial cells proliferate and form an epithelial monolayer. At confluence (C), collagenase removes epithelial cell sheets that are then rotary shaken to form epithelial spheroids. Epithelial cells then polarize and differentiate into secretory and ciliated cells. (b) Detection of ciliated cells (GT335) and secretory cells (Anti-M1) within epithelial spheroids during MCD in the absence (–IL-13) or in the presence (+IL-13) of the cytokine (by indirect IF). Note the absence of ciliated cells and the high number of secretory cells in the presence of the cytokine. Bar, 10 μm. (c) Quantification of IL-13 effect during MCD (flow cytometry). Cells dissociated from epithelial spheroids were analyzed using GT335 for ciliated cells and anti-M1 for secretory cells during the time course (0–30 days) of MCD. IL-13 (10 ng/ml) decreases the proportion of ciliated cells and largely increases the proportion of secretory cells. Three independent kinetics are shown. Gray symbols, –IL-13; black symbols, +IL-13.

Figure 2

Dose- and time-dependent effect of IL-13 on MCD. GT335, a mAb that detects glutamylated tubulins, gives the proportion of ciliated cells during MCD in Western blot analysis (14). The indicated numbers represent the days after confluence. (a) Dose-dependent effect of IL-13 on ciliated cell differentiation. The proportion of ciliated cells at day 15 decreases with the dose of the continuously applied cytokine during differentiation. (b) Epithelial cells were continuously treated with IL-13 during proliferation (+/–), differentiation (–/+), or both (+/+), compared with control cultures (–/–). No effect is observed on ciliated cell differentiation when cells are treated with the cytokine during proliferation (+/–). The increased GT335 staining at day 9 (–/+ compared with –/–) is not significant, varying from one experiment to another. Ciliated differentiation is strongly reduced when cells are treated during differentiation (–/+ or +/+). (c) Epithelial cells were either treated for 72 hours (between day 4 and day 7) or continuously treated with IL-13 (10 ng/ml). The maximum effect is observed for a continuous treatment. (d) Epithelial cells were continuously treated with IL-13, IL-4, or IFN-γ (10 ng/ml) during differentiation. IL-13 and IL-4 effects are identical, while IFN-γ does not alter the ciliated cell differentiation process. (e) The IL-4 antagonistic mutant protein (Y124D) antagonizes the IL-13 effect in a dose-dependent manner. The same amount of total protein extracts were deposited per lane (10 μg). Actin or tubulin were revealed as constant protein markers during the kinetics of differentiation.

Figure 3

IL-13 alters the polarization of epithelial cells. Transmission electronic microscopy analyses were performed on epithelial spheroids during MCD in the absence (a, b, c) or in the presence (d, e, f, g, h) of IL-13. Epithelial cells from untreated spheroids display a close contact of lateral membranes (a) (arrowheads) compared with IL-13–treated cells (d). The latter exhibit alterations of the lateral membranes characterized by decreased cell-cell contacts, large intercellular spaces (d) (arrowheads), and interdigitations (h). These cells also possess an apical (d) or basal (e, f) membrane surface that is lined by a condensed microvilli compared with control cells presenting a more regular membrane surface. (a) Apical; (b) basal. In control conditions (day 15), centrioles were currently detected in epithelial cells, and mature basal bodies were anchored at the apical membrane among dispersed simple microvilli (c) (arrows). In some cases, a default in centriole/basal bodies targeting is observed in IL-13–treated spheroids (arrows in g). Bars, 2 μm.

Figure 4

IL-13 affects ezrin expression and localization during mucociliary differentiation. (a) ³²P-labeled complex probes were obtained from epithelial cell RNAs at day 11 during MCD (+/– IL-13 from day 2) and hybridized on CLONTECH Laboratories Inc.’s Atlas cDNA arrays. CD9, a member of the tetraspanin protein family, is strongly expressed in both conditions. Merlin is not detectable in either case, while ezrin expression is downregulated by IL-13. (b) Ezrin protein expression was downregulated by IL-13 (+7, +12 days after confluence during MCD). At day 7, protein expression is identical in the two conditions. At day 12, the difference is significant. The Western blot analysis results shown are from one representative experiment, corresponding to a 30% decrease of ezrin expression in the presence of IL-13. Ten micrograms of total protein extract per lane. (c) Localization of ZO-1 and ezrin is affected by IL-13. Epithelial cells were labeled with GT335 mAb (red) in combination with either anti–ZO-1 Ab (green) or anti-ezrin Ab (green). In control conditions during MCD, anti–ZO-1 Ab reveals a continuous regular pattern, defining the subapical membrane domain (tight junctions) of each individual cell. IL-13-treatment strongly affects epithelial cell shape as observed with anti–ZO-1 staining. Anti-ezrin Ab decorates microvilli specifically located at the apical membrane of ciliated cells. In IL-13–treated spheroids, ezrin largely remains in the cytoplasm. Note the low number of ciliated cells (GT335 positive-cells) in the presence of the cytokine. Bar, 10 μm.

Figure 5

IL-13 effect on partially differentiated epithelial spheroids: IL-13 inhibits further ciliogenesis, increases the proportion of MUC5AC-positive cells, and decreases ciliary beat frequency. (a) Partially differentiated (ciliated) epithelial spheroids (18 days after confluence) were continuously treated with IL-13 for 11 days and processed for double IF analysis (day 29). The proportion of ciliated cells remained lower than in control conditions after IL-13 treatment (single staining, GT335: compare –IL-13 and +IL-13). Numerous MUC5AC-positive cells (Anti-M1) were observed in the IL-13–treated conditions compared with control conditions. Accordingly, the proportion of epithelial cells with ezrin located within the apical membrane domain was lower in the presence of the cytokine. Note that ezrin staining is absent in MUC5AC-positive cells (+IL-13, Anti-M1/anti-ezrin). Bar, 10 μm. (b) The effect of IL-13 on CBF was determined on partially differentiated spheroids (21 days in suspension after confluence). The CBF was measured after 1, 3, and 6 days. CBF does not change significantly after 1 and 3 days of continuous treatment with IL-13. A significant concentration-dependent downregulation of the CBF values is observed after 6 days of continuous stimulation (0, 1, 10, 100 ng/ml of IL-13). The values are expressed as mean ± SD. The Student t test was used to compare IL-13–stimulated cells and controls at the same incubation time. A value of P < 0.05 was accepted as statistically significant (* P < 0.02; **P < 0.01; ***P < 0.0001).