Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells

Abstract

Rationale: Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease. Objectives: To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia. Methods: We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture. Measurements and Main Results: cAMP-stimulated non-CF ionocytes do not secrete Na⁺ or Cl^- into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl^-/bicarbonate exchange. Non-CF club cells secrete Na⁺ and Cl^- to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties. Conclusions: We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance.

Keywords: club cells; cystic fibrosis; elexacaftor; ionocytes; ivacaftor; tezacaftor.

Figure 1.

Self-referencing ion-selective microelectrode (SRISM) system setup. (A) Schematic representation of the SRISM system for ion flux measurements using double electrodes in primary human bronchial epithelial (pHBE) cell culture. A video camera connected to a microscope facilitates placement of the microelectrodes. Movement of the microelectrodes is computer controlled. The electrodes are connected to an amplifier and data acquisition system for offline analysis. (B) We tested the spatial resolution of our SRISM experimental protocol by measuring the ability to resolve two NaCl point sources consisting of borosilicate micropipettes with a ∼5-μm tip diameter, filled with 1 M NaCl in 1% agarose at pH 6.5 and separated by 10 μm. Our SRISM system resolved the Na⁺, H⁺, and Cl⁻ fluxes emanating from the two artificial sources. MitoTracker Deep Red FM (100 nM) was used to locate mitochondria-rich (C) ionocytes and (D) club cells in pHBE live cell culture. We marked the position of the cell of interest by scraping the preparation surface with two perpendicular linear markings (located 100 μm away from the cell), the projections of which intersected at the position of the cell. The markings were produced with a ∼5-μm tipped tool dragged along the cell culture using a computer-controlled micromanipulator. The cell identity was verified by staining with ionocyte-specific marker FOXI1 and club cell–specific marker SCGB1A1. (E) FOXI1-, BSND-, and MitoTracker-labeled ionocyte. Scale bars in B–E, 10 μm.

Figure 1.

Self-referencing ion-selective microelectrode (SRISM) system setup. (A) Schematic representation of the SRISM system for ion flux measurements using double electrodes in primary human bronchial epithelial (pHBE) cell culture. A video camera connected to a microscope facilitates placement of the microelectrodes. Movement of the microelectrodes is computer controlled. The electrodes are connected to an amplifier and data acquisition system for offline analysis. (B) We tested the spatial resolution of our SRISM experimental protocol by measuring the ability to resolve two NaCl point sources consisting of borosilicate micropipettes with a ∼5-μm tip diameter, filled with 1 M NaCl in 1% agarose at pH 6.5 and separated by 10 μm. Our SRISM system resolved the Na⁺, H⁺, and Cl⁻ fluxes emanating from the two artificial sources. MitoTracker Deep Red FM (100 nM) was used to locate mitochondria-rich (C) ionocytes and (D) club cells in pHBE live cell culture. We marked the position of the cell of interest by scraping the preparation surface with two perpendicular linear markings (located 100 μm away from the cell), the projections of which intersected at the position of the cell. The markings were produced with a ∼5-μm tipped tool dragged along the cell culture using a computer-controlled micromanipulator. The cell identity was verified by staining with ionocyte-specific marker FOXI1 and club cell–specific marker SCGB1A1. (E) FOXI1-, BSND-, and MitoTracker-labeled ionocyte. Scale bars in B–E, 10 μm.

Figure 2.

Ionocytes regulate airway surface liquid pH. Effect of forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM) stimulation on non–cystic fibrosis (non-CF) pulmonary ionocyte transport of (A) Na⁺ (n = 9 from four donors), (B) H⁺ (n = 16 from five donors), and (C) Cl⁻ (n = 16 from five donors). Effect of forskolin plus IBMX stimulation on CF ionocyte transport of (D) Na⁺ (n = 10 from four donors), (E) H⁺ (n = 14 from six donors), and (F) Cl⁻ (n = 4 from three donors). Effect of 24-hour incubation of CF ionocytes with elexacaftor (3 μM), tezacaftor (3 μM), and ivacaftor (1 μM) (ETI) on forskolin plus IBMX–stimulated transport of (G) Na⁺ (n = 11 from four donors), (H) H⁺ (n = 13 from six donors), and (I) Cl⁻ (n = 10 from four donors). Total transport of (J) Na⁺, (K) H⁺, and (L) Cl⁻ and maximum transport rate of (M) Na⁺, (N) H⁺, and (O) Cl⁻ by ionocytes from non-CF, CF, and CF treated with ETI for 24 hours (CF + ETI) preparations. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

Figure 3.

Bicarbonate transport by ionocytes in non–cystic fibrosis (non-CF) primary human bronchial epithelial cell culture. Effect of 15-minute incubation of non-CF ionocytes with bafilomycin A1 (100 μM) on forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM)-stimulated (A) transport of H⁺ (black trace, n = 8 from five donors). (B) Total H⁺ transport and (C) maximum transport rate of H⁺ by ionocytes from non-CF, non-CF treated with bafilomycin A1 (non-CF + BafA1), and CF preparations. (D) Confocal immunofluorescence of BSND (ionocyte marker), FOXI1 (ionocyte marker), ATP6V0C (V-type ATPase marker), and merged image of non-CF ionocytes. Scale bars, 5 μm. Effect of 15-minute incubation with acetazolamide (ACTZ; 100 μM) on forskolin plus IBMX–stimulated non-CF ionocytes on (E) H⁺ flux (black trace, n = 14 from six donors). (F) Total transport of H⁺ and (G) maximum transport rate of H⁺ by ionocytes from non-CF, non-CF treated with ACTZ (non-CF + ACTZ), and CF preparations. (H) Effect of HCO₃⁻-free bathing saline solution on forskolin plus IBMX–stimulated non-CF ionocytes on H⁺ (black trace, n = 7 from four donors). (I) Total transport and (J) maximum transport rate of H⁺ by ionocytes from non-CF, non-CF incubated in HCO₃⁻-free bathing saline (non-CF + HCO₃⁻-free), and CF preparations. (K) Effect of combined treatment with ACTZ and HCO₃⁻-free bathing saline on forskolin plus IBMX–stimulated non-CF pulmonary ionocytes on H⁺ (black trace, n = 7 from four donors). (L) Total transport and (M) maximum transport rate of H⁺ by non-CF ionocytes. Red trace represents the H⁺ measured under control conditions and shown in Figure 2B. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

Figure 4.

Bicarbonate secretion involves the coordinated function of apical cystic fibrosis transmembrane conductance regulator (CFTR) and AE2 (anion exchanger 2) with basolateral NHE2 (Na⁺/H⁺ exchanger 2). Effect of 15-minute incubation with CFTR_inh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) ionocytes on (A) H⁺ flux (black trace), (B) total transport of H⁺, and (C) maximal H⁺ transport rate (n = 9 from four donors). Effect of 15-minute incubation with DIDS (500 μM) on forskolin plus IBMX–stimulated non-CF ionocytes on (E) H⁺ flux (black trace), (F) total transport of H⁺, and (G) maximal H⁺ transport rate (n = 8 from two donors). Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF primary human bronchial epithelial (pHBE) ionocytes on (I) H⁺ flux (black trace, n = 6 from three donors), (J) total transport of H⁺, and (K) maximal transport rate of H⁺. Effect of 15-minute incubation with basolateral amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (L) H⁺ flux (black trace, n = 7 from three donors), (M) total transport of H⁺, and (N) maximal transport rate of H⁺. Effect of 15-minute incubation with EIPA (5 μM basolateral and apical) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (O) H⁺ flux (black trace, n = 8 from three donors), (P) total transport of H⁺, and (Q) maximal transport rate of H⁺. Red trace represents the H⁺ measured under control conditions and shown in Figure 2B. Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (S) Na⁺ flux (black trace, n = 5 from three donors), (T) total transport of Na⁺, and (U) maximal transport rate of Na⁺. Red trace represents control Na⁺ flux (Figure 2A). Positive flux values indicate ion movement from the basolateral side into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control Na⁺ flux (Figure 2A). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Tukey post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Confocal immunofluorescence of non-CF ionocytes of (D) BSND (ionocyte marker), FOXI1 (ionocyte marker), CFTR, and merged image; (H) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC4A2 (AE2 channels), and merged image; (R) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC9A2 (NHE2 marker), and merged image; and (V) BSND (ionocyte marker), FOXI1 (ionocyte marker), SCNN1B (epithelial Na⁺ channel marker), and merged image. Scale bars in D, H, R, and V, 10 μm. DIDS = 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; EIPA = 5-(N-Ethyl-N-isopropyl)-amiloride; ns = no significant difference.

Figure 4.

Bicarbonate secretion involves the coordinated function of apical cystic fibrosis transmembrane conductance regulator (CFTR) and AE2 (anion exchanger 2) with basolateral NHE2 (Na⁺/H⁺ exchanger 2). Effect of 15-minute incubation with CFTR_inh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) ionocytes on (A) H⁺ flux (black trace), (B) total transport of H⁺, and (C) maximal H⁺ transport rate (n = 9 from four donors). Effect of 15-minute incubation with DIDS (500 μM) on forskolin plus IBMX–stimulated non-CF ionocytes on (E) H⁺ flux (black trace), (F) total transport of H⁺, and (G) maximal H⁺ transport rate (n = 8 from two donors). Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF primary human bronchial epithelial (pHBE) ionocytes on (I) H⁺ flux (black trace, n = 6 from three donors), (J) total transport of H⁺, and (K) maximal transport rate of H⁺. Effect of 15-minute incubation with basolateral amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (L) H⁺ flux (black trace, n = 7 from three donors), (M) total transport of H⁺, and (N) maximal transport rate of H⁺. Effect of 15-minute incubation with EIPA (5 μM basolateral and apical) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (O) H⁺ flux (black trace, n = 8 from three donors), (P) total transport of H⁺, and (Q) maximal transport rate of H⁺. Red trace represents the H⁺ measured under control conditions and shown in Figure 2B. Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (S) Na⁺ flux (black trace, n = 5 from three donors), (T) total transport of Na⁺, and (U) maximal transport rate of Na⁺. Red trace represents control Na⁺ flux (Figure 2A). Positive flux values indicate ion movement from the basolateral side into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control Na⁺ flux (Figure 2A). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Tukey post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Confocal immunofluorescence of non-CF ionocytes of (D) BSND (ionocyte marker), FOXI1 (ionocyte marker), CFTR, and merged image; (H) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC4A2 (AE2 channels), and merged image; (R) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC9A2 (NHE2 marker), and merged image; and (V) BSND (ionocyte marker), FOXI1 (ionocyte marker), SCNN1B (epithelial Na⁺ channel marker), and merged image. Scale bars in D, H, R, and V, 10 μm. DIDS = 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; EIPA = 5-(N-Ethyl-N-isopropyl)-amiloride; ns = no significant difference.

Figure 5.

Club cells secrete Na⁺ and Cl⁻. Effect of forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM) stimulation on non–cystic fibrosis (non-CF) club cell transport of (A) Na⁺ (n = 15 from five donors), (B) H⁺ (n = 36 from seven donors), and (C) Cl⁻ (n = 21 from six donors). Effect of forskolin plus IBMX stimulation on CF club cell transport of (D) Na⁺ (n = 11 from four donors), (E) H⁺ (n = 17 from five donors), and (F) Cl⁻ (n = 5 from three donors). Effect of 24-hour incubation of CF club cells with elexacaftor (3 μM), tezacaftor (3 μM), and ivacaftor (1 μM) (ETI) on forskolin plus IBMX–stimulated transport of (G) Na⁺ (n = 7 from three donors), (H) H⁺ (n = 14 from four donors), and (I) Cl⁻ (n = 7 from four donors). Total transport of (J) Na⁺, (K) H⁺, and (L) Cl⁻ and maximum transport rate of (M) Na⁺, (N) H⁺, and (O) Cl⁻ by club cells from non-CF, CF, and CF treated with ETI for 24-hour (CF + ETI) preparations. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

Figure 6.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transport by club cells. Effect of 15-minute incubation with CFTR_inh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) club cells on (A) Na⁺ flux (black trace, n = 10 from four donors), (B) total transport of Na⁺, (C) maximal transport rate of Na⁺, (E) H⁺ flux (black trace, n = 17 from six donors), (F) total transport of H⁺, (G) maximal transport rate of H⁺, (H) Cl⁻ flux (black trace, n = 7 from three donors), (I) total transport of Cl⁻, and (J) maximal transport rate of Cl⁻. Effect of 15-minute incubation with bumetanide (100 μM basolateral) on forskolin plus IBMX–stimulated non-CF club cells on (K) H⁺ flux (black trace, n = 6 from three donors), (L) total transport of H⁺, (M) maximal transport rate of H⁺, (N) Cl⁻ flux (black trace, n = 6 from three donors), (O) total transport of Cl⁻, and (P) maximal transport rate of Cl⁻. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control ion flux (Figures 2A and 2B). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (D) Confocal immunofluorescence of SCGB1A1 (club cell marker), CFTR, and merged image of a club cell. CFTR was localized at the apical membrane of ionocytes. Scale bars represents 10 μm. ns = no significant difference.

Figure 6.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transport by club cells. Effect of 15-minute incubation with CFTR_inh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) club cells on (A) Na⁺ flux (black trace, n = 10 from four donors), (B) total transport of Na⁺, (C) maximal transport rate of Na⁺, (E) H⁺ flux (black trace, n = 17 from six donors), (F) total transport of H⁺, (G) maximal transport rate of H⁺, (H) Cl⁻ flux (black trace, n = 7 from three donors), (I) total transport of Cl⁻, and (J) maximal transport rate of Cl⁻. Effect of 15-minute incubation with bumetanide (100 μM basolateral) on forskolin plus IBMX–stimulated non-CF club cells on (K) H⁺ flux (black trace, n = 6 from three donors), (L) total transport of H⁺, (M) maximal transport rate of H⁺, (N) Cl⁻ flux (black trace, n = 6 from three donors), (O) total transport of Cl⁻, and (P) maximal transport rate of Cl⁻. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control ion flux (Figures 2A and 2B). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (D) Confocal immunofluorescence of SCGB1A1 (club cell marker), CFTR, and merged image of a club cell. CFTR was localized at the apical membrane of ionocytes. Scale bars represents 10 μm. ns = no significant difference.

Figure 7.

Models of ion transport. Schematic model of working hypothesis of how (A) pulmonary ionocytes and (B) club cells function in primary human bronchial epithelial cell culture stimulated with forskolin plus 3-isobutyl-1-methylxanthine. AE2 = anion exchanger 2; CFTR = cystic fibrosis transmembrane conductance regulator; NHE2 = Na⁺/H⁺ exchanger 2; NKCC = Na⁺:K⁺:2Cl⁻ cotransporter.