Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers

Abstract

Recent studies have shown that airway inflammation dominated by neutrophils, ie, polymorphonuclear cells (PMN) was observed in infants and children with cystic fibrosis (CF) even in the absence of detectable infection. To assess whether there is a CF-related anomaly of PMN migration across airway epithelial cells, we developed an in vitro model of chemotactic migration across tight and polarized CF(15) cells, a CF human nasal epithelial cell line, seeded on porous filters. To compare PMN migration across a pair of CF and control monolayers in the physiological direction, inverted CF(15) cells were infected with increasing concentrations of recombinant adenoviruses containing either the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, the DeltaF508 CFTR cDNA, or the beta-galactosidase gene. The number of PMN migrating in response to N-formyl-Met-Leu-Phe across inverted CF(15) monolayers expressing beta-galactosidase was similar to that seen across CF(15) monolayers rescued with CFTR, whatever the proportion of cells expressing the transgene. Moreover, PMN migration across monolayers expressing various amounts of mutated CFTR was not different from that observed across matched counterparts expressing normal CFTR. Finally, PMN migration in response to adherent or Pseudomonas aeruginosa was equivalent across CF and corrected monolayers. The possibility that mutated CFTR may exert indirect effects on PMN recruitment, via an abnormal production of the chemotactic cytokine interleukin-8, was also explored. Apical and basolateral production of interleukin-8 by polarized CF cells expressing mutated CFTR was not different from that observed with rescued cells, either in baseline or stimulated conditions. CF(15) cells displayed a CF phenotype that could be corrected by CFTR-containing adenoviruses, because two known CF defects, Cl(-) secretion and increased P. aeruginosa adherence, were normalized after infection with those viruses. Thus, we conclude that the presence of a mutated CFTR does not per se lead to an exaggerated inflammatory response of CF surface epithelial cells in the absence or presence of a bacterial infection.

Figure 1.

Comparison of PMN migration across right-side-up or inverted CF₁₅ monolayers. PMN were either added to the basolateral or the apical side of confluent monolayers and driven to transmigrate for 1 hour 30 minutes in response to various concentrations of fMLP. PMN migration across right-side-up monolayers is expressed as a percentage of that obtained across inverted monolayers. Values are means ± SEM of three to four monolayers of four different experiments. Absolute values (in millions) of PMN recovered across the inverted epithelia were, from left to right: 1.17 ± 0.06, 3.7 ± 0.25, 2.79 ± 0.07, 1.18 ± 0.11. The TER, measured at the beginning of the experiment (ie, without fMLP or PMN) and shown in parentheses (means ± SEM, n = 3 to 4), are not significantly different between both groups. **P < 0.05, ***P < 0.001 versus corresponding controls.

Figure 2.

Transepithelial Cl⁻ current of inverted or right-side-up CF₁₅ monolayers infected with Ad RSV βgal or Ad RSV CFTR. Inverted CF₁₅ monolayers were infected for 16 hours at a MOI of 500 of Ad CFTR (A) or Ad βgal (B), and short-circuit current (I_sc) was measured 1 day after. Baseline I_sc values were 7.0 and 1.7 μA/cm², respectively. The following drugs were added: 100 μmol/L amiloride (A), followed by a low Cl⁻ solution containing amiloride (low Cl⁻), and 50 μmol/L forskolin (F). The monolayers had a TER higher than 400 Ω.cm and, in the Ad βgal group, ∼60% of blue cells were revealed after X-Gal staining. Right-side-up monolayers were infected for 8 hours at a MOI of 500 of Ad RSV CFTR (C) and were processed as described above, except that forskolin was 100 μmol/L, followed by the addition of 200 μmol/L 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). Matched monolayers infected with Ad βgal displayed ∼35% of blue cells.

Figure 3.

A: Examples of membrane currents recorded from a cell infected with Ad RSV CFTR (solid line) or Ad RSV βgal (dotted line) in the presence of 100 μM amiloride. Whereas exposure of the CFTR-expressing cells to 50 μM forskolin induced reversible inward currents, the drug was without effect on membrane currents of the cells infected with Ad βgal. Bar indicates the duration of forskolin superfusion. B: Distribution of basal and forskolin-stimulated membrane currents (pA/pF) as recorded in cells infected with Ad CFTR (n = 8) or Ad βgal (n = 8). Basal and stimulated currents were markedly enhanced in cells expressing normal CFTR. More than 90% of the cells infected with Ad βgal were blue.

Figure 4.

A and C: PMN migration across inverted CF and corrected monolayers. The monolayers were infected with various MOI of either Ad RSV CFTR or Ad RSV βgal (A), or Ad CMV CFTR or Ad CMV eGFP (C) before assessing PMN migration. PMN were added to the basolateral side of the monolayers and driven to transmigrate for 1 hour (A) or 1 hour 30 minutes (C) in response to 50 nmol/L fMLP present in the opposite compartment. Values are means ± SEM of three monolayers. B and D: effect of increasing viral MOI on transepithelial Cl⁻ currents. Part of the monolayers were used in parallel to perform electrical measures as described for Figure 2, A and B ▶ . The combined effect of low Cl⁻ and forskolin is presented for both groups of monolayers infected with viruses containing either the RSV (B) or the CMV (D) promoter. In the Ad βgal-infected group, the number of blue cells after X-Gal staining increased from ∼20% (MOI 50) to ∼90% (MOI 500).

Figure 5.

PMN migration across inverted CF₁₅ monolayers expressing normal or mutated CFTR. The monolayers were infected or not with various MOI of either Ad CB CFTR or Ad CB ΔF508 before measuring PMN migration. PMN were added to the basolateral side of the monolayers and allowed to transmigrate for 1 hour 30 minutes in response to 50 nmol/L fMLP. A: Values of a typical experiment done in triplicate. B: The pooled data of three experiments. Each group of monolayers infected with a given MOI of Ad CB ΔF508 is expressed as a percentage of its corresponding group (ie, infected with the same MOI of Ad CB CFTR). For each condition, data are means ± SEM of the nine samples pooled. Electrical measures done in parallel showed that monolayers infected with a MOI of 250 of Ad CB CFTR or Ad CB ΔF508 displayed a ΔI_sc of 49 ± 6 μA/cm (n = 8) and 1 ± 0.3 μA/cm (n = 5), respectively, in response to low Cl⁻ and forskolin.

Figure 6.

PMN migration across inverted monolayers rescued or not with CFTR, in response to adherent PAO1. Confluent CF₁₅ cells were infected or not with Ad RSV CFTR or Ad RSV βgal (MOI 500), before being incubated with PAO1 on their apical surface. The absolute values of three experiments done in quadruplicate were pooled. Data are means ± SEM of the 12 samples pooled. In parallel, monolayers that were neither infected with viruses nor challenged with PAO1 were assessed for migration in response to 50 nmol/L fMLP as a means of comparison. In the Ad βgal-infected groups, X-Gal staining revealed between ∼40% and ∼80% of blue cells and, in the Ad CFTR-infected groups, the response to low Cl⁻ and forskolin varied between 28 and 57 μA/cm².

Figure 7.

IL-8 production by polarized CF₁₅ cells expressing normal or mutated CFTR. CF₁₅ monolayers on inserts were infected or not with Ad RSV CFTR or Ad RSV βgal (MOI 500) (A, B) or Ad CB CFTR or Ad CB ΔF508 (MOI 250) (C, D) before incubation with various concentrations of TNF-α. The apical (A, C) and basolateral (B, D) supernatants were collected after 4 hours and assayed for IL-8 production. A and B: Data are means ± SEM (n = 3) of a typical experiment. C and D: The data of three experiments were pooled and presented as a fold increase over controls (monolayers corrected with CFTR and not submitted to TNF-α) and are means ± SEM of the eight to nine samples.

Figure 8.

PAO1 adherence to CF₁₅ cells rescued or not with CFTR. Confluent CF₁₅ cells were infected or not with Ad RSV CFTR or Ad RSV βgal (MOI 500), before being incubated with PAO1. The data of three experiments were pooled and presented as a percentage of bacteria adhering to uninfected CF₁₅ cells (means ± SEM of the 12 samples pooled). The number of bacteria adhering to uninfected cells, determined in the third experiment, was of 13 ± 1 PAO1/CF₁₅ cell (mean ± SEM, n = 4 wells). ***P < 0.001 versus uninfected CF₁₅ cells, ^§§P < 0.05 versus cells infected with Ad βgal.