Introduction

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Progressive lung disease in cystic fibrosis (CF) is largely due to persistent inflammation characterized by an abundance of activated neutrophils (1). The influx of neutrophils into CF airways has been traditionally thought to be a response to chronic infection with bacteria (2). However, recent bronchoalveolar lavage (BAL) studies have documented elevated levels of neutrophils in the lower airways of some infants with CF even in the absence of detectable infection (3, 4). These observations are consistent with the hypothesis that inflammation is abnormally regulated in CF airways. Attention has been focused on interleukin (IL)-8, a chemokine that accounts for most of the neutrophil chemoattractant activity of CF sputum (5) and is repeatedly found in elevated concentrations in the airways of patients with CF.

A potentially major source of IL-8 in the airways is the respiratory epithelium (6, 7). Since the respiratory epithelium also manifests the most important phenotypic abnormality of CF, we hypothesized that CF airway epithelial cells overproduce IL-8, and that this is linked to abnormal function of the cystic fibrosis transmembrane regulator (CFTR). To test this hypothesis, we compared IL-8 production in CF and non-CF primary nasal epithelial cell (NEC) cultures at baseline and after stimulation with tumor necrosis factor- (TNF-) or infection by respiratory syncytial virus (RSV). Nasal epithelium was used because it has the same ion transport abnormalities as lower airway epithelium in CF (8), is relatively free of inflammation early in life (4), and can be obtained from young children in a relatively noninvasive fashion. We additionally tested the effect of "correction" by transduction with adenoviral vectors carrying the wild-type CFTR complementary DNA (cDNA) of CF nasal epithelial IL-8 production. We also compared IL-8 production by an immortalized CF epithelial cell line and the same cell line permanently corrected with retroviral vectors expressing CFTR.

	Materials and Methods

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NEC Culture

Primary cultures of NECs were established using a modification of a previously described technique (9). A 3-mm bronchoscope cytology brush (BARD, Tewksbury, MA) was used to obtain brush biopsies from under the inferior nasal turbinate of pediatric patients undergoing flexible bronchoscopy; clinical characteristics of the patients are shown in Table 1. For children with CF, the diagnosis was confirmed by typical clinical manifestations and sweat chloride > 60 mEq/liter; four of the six children with CF were also known to have the F508/F508 genotype. Control children were undergoing bronchoscopy for evaluation of airway obstruction and did not have clinical features of CF. The cells were transported in room temperature RPMI 1640 medium (JRH Biosciences, Lenexa, KS) to the laboratory, where brushes and cells were placed in 3 mM dithiothreitol (DTT; Sigma Chemical Co., St. Louis, MO) and phosphate-buffered saline (PBS) for 15 min. Cells were then washed from the brush with PBS and centrifuged at 500 × g for 5 min. The cells were resuspended in serum-free bronchial epithelial cell growth medium (BEGM; Clonetics, Walkersville, MD) with added antibiotics (100 µg/ml ceftazidime, 80 µg/ml tobramycin, 100 µg/ml colistimethate sodium, and 2.5 µg/ml amphotericin B). Cells were plated on collagen-coated 25-cm² culture flasks. (Coating was created by diluting human placental collagen type IV [Sigma] to 0.05 mg/ml with sterile water and 0.02% acetic acid, adding 3 ml to flasks, drying for 18 h, and rinsing twice.) Attached cells were washed after 12 h and medium was changed every 24 h. The added antibiotics were discontinued after 72 h. Cells were grown to confluence (average 10 d), lifted with 0.1% trypsin with 1 mM ethylenediamine tetraacetic acid (EDTA) (Sigma), passaged onto collagen-coated multiwell culture plates, and again allowed to grow to 80-90% confluence. Immunohistochemical staining of cells using mouse monoclonal antibodies for human epithelial-specific antigen (NCL-ESA; Novocastra Labs, Newcastle upon Tyne, UK) or human cytokeratin 5, 6, 8, and 18 (NCL-PAN-CK; Novocastra Labs) was positive in all cells.

Stimulation of Primary NEC Cultures

Cell monolayers were incubated for 1 h with 300 µl of BEGM containing 12.5 ng/ml TNF- (Sigma) or 2 pfu/cell RSV (ATCC, Rockville, MD; purified by sucrose-gradient centrifugation as previously described [10]) or no additive as a control. After 1 h, supernatants were aspirated, wells were washed once with BEGM, and 1 ml BEGM was added to each well. Supernatants for cytokine assays were collected and stored at 80°C, and fresh medium was added at 4, 8, 24, and 48 h after the initial incubation period. After the 48-h collection, cells were detached with 0.1% trypsin with 1 mM EDTA and counted. For wells inoculated with RSV, productive infection was verified by formation of typical syncytia after placement of 48-h supernatant on HEp2 cells (ATCC) as previously described (11).

Stimulation of Primary NEC Cultures Corrected by Adenoviral Gene Transfer

In separate experiments, CF NEC cultures underwent restoration of CFTR function through a recombinant adenovirus vector driven by a cytomegalovirus (CMV) enhancer/-actin promoter (Ad5-CBCFTR) as previously described (12). Controls consisted of noninfected NEC and Ad5-CMVLacZ (CMV enhancer/CMV promoter) infected NEC from the same donors. Adenoviral vectors were obtained from the UNC Gene Therapy Core. In these experiments, CF NEC were grown to confluence in BEGM on six well plates. Cultures were incubated for 2 h with either 200 µl BEGM (noninfected control), 200 µl BEGM with 10³ pfu/cell of Ad5-CBCFTR, or 200 µl BEGM with 10³ pfu/cell of Ad5-CMVLacZ. Cells were then washed with BEGM, incubated overnight, and stimulated with 12.5 ng/ml TNF- for 1 h. Cells were again washed with BEGM and 2 ml of BEGM were added. Supernatants were collected for cytokine assay and fresh medium was added at 4, 8, and 24 h after the TNF- stimulation.

Correction of CFTR function by gene transfer was estimated with a previously described chloride-efflux assay (13). Briefly, after cytokine stimulation experiments were completed, the rate of loss of chloride in noninfected, Ad5-CBCFTR infected, was compared with that in Ad5-CMVLacZ infected cells. Wells were loaded with radioactive chloride (³⁶Cl) by incubating in 5 µCi ³⁶Cl for 1 h. Cells were washed and aliquots of isotope-free, chloride-free Ringer's solution + 10⁴ M amiloride were added and collected at 1-min intervals. To assess cyclic adenosine monophosphate (cAMP)-mediated chloride permeability, 10⁵ M forskolin (final concentration) was added after the second minute of the assay. After 10 min the cells were lysed with 0.1% sodium dodecyl sulfate (SDS) (Sigma). The amounts of ³⁶Cl in the efflux aliquots and cell layer were determined by liquid scintillation counting. Efflux curves were plotted as the percent of counts remaining in the cells versus time.

The proportion of NEC transfected with Ad5-CMVLacZ was estimated by X-gal staining at 48 h as previously described (14). Briefly, cells were washed with PBS, fixed in 0.5% glutaraldehyde for 10 min, then stained with X-gal solution (Sigma) for 3 h, washed, and stored in 70% glycerol. Noninfected cells were used as controls. The development of any blue color in the cytoplasm indicated the presence of -galactosidase activity.

Immortalized Cell Lines

To establish further whether IL-8 release was related to the presence of functioning CFTR, a third set of experiments, using three immortalized cell lines, was performed. Human papilloma virus (HPV)-immortalized tracheal epithelial cells derived from a patient with CF homozygous for F508 (CFT1 cells) have been described (15). The CFT1 cells retain a well-differentiated phenotype, including the bioelectric properties characteristic of CF (15). A cell line corrected for the apical chloride channel defect was derived from CFT1 cells by retroviral insertion of a wild-type copy of CFTR cDNA (LCFSN cells) (16). A retrovirus encoding an irrelevant transgene, LacZ, was also used to create a stable CFT1 cell line expressing LacZ (LC3 cells) (17). All cell lines were grown on plastic in Ham's F-12 base medium with seven hormones and growth supplements as described (15), and were passaged every 7 to 10 d. Cells were seeded onto collagen-coated 24-well plates, grown to confluence, and stimulated with TNF- in an identical fashion as that described for primary NEC cultures.

IL-8 Assays

IL-8 in unconcentrated cell culture supernatants was measured with an enzyme-linked immunosorbent assay (ELISA) (Quantikine IL-8; R&D Systems, Minneapolis, MN). The lower detection limit of the IL-8 assay is 10 pg/ml, and the dynamic range is 31.2 to 2,000 pg/ml. The correlation coefficient of standard curves for all assays was > 0.98. Cross-reactivity or interference with the assay by other recombinant human cytokines is below the limits of detection according to the manufacturer.

Statistical Analysis

Comparisons of cytokine production by CF and non-CF NEC were made with an unpaired t test. Noninfected, Ad5-CBCFTR infected, and Ad5-CMVLacZ infected CF cell comparisons for identical cell donors were made with repeated measures analysis of variance (ANOVA) with Bonferroni's correction for multiple comparisons after testing. Data from immortalized cell lines were compared through ANOVA with Bonferroni's correction after testing. For all culture systems, the effect of a stimulus was assessed versus unstimulated controls by paired t test at each time point. P < 0.05 was defined as statistically significant. All data were log transformed for analysis. Analyses were done with a statistical software package (InStat2; Graphpad Software Inc., San Diego, CA).

	Results

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Primary NEC Stimulation

Release of IL-8 into primary culture supernatants was assessed. Production of IL-8 was normalized to cell count for each condition and was expressed as pg/10³ cells/h. When compared with unstimulated controls, TNF- induced a significant increase in IL-8 release at 4 h after stimulation by both CF and control NEC (Figure 1). When IL-8 responses were compared in CF and non-CF control NEC, there were no differences in either baseline or TNF- stimulated IL-8 release at any time point.

View larger version (17K): [in this window] [in a new window]   Figure 1.   IL-8 release by primary culture of NEC from non-CF control children (shaded bars) and children with CF (open bars) at 4 h, 8 h, and 24 h after inoculation with medium (C) or TNF- (T). Each bar represents mean ± SEM for cultures from six separate donors. *P < 0.05 in TNF--stimulated cells versus unstimulated controls.

RSV-infected wells had a significant increase in IL-8 release at 4 h after inoculation with virus (Figure 2). For both CF and non-CF control NEC, IL-8 levels in infected wells were not statistically different from baseline levels at 8 h after inoculation with virus (data not shown) or at 24 h after inoculation with virus, but were increased at 48 h after inoculation with virus. The amount of IL-8 released by CF NEC did not differ from that released by non-CF control NEC at any time point. Plaque formation in HEp2- cell monolayers inoculated with NEC supernatants confirmed that RSV-inoculated NEC were shedding virus at 48 h after inoculation with virus (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 2.   IL-8 release by primary culture of NEC from non-CF control children (shaded bars) and children with CF (open bars) at 4 h, 24 h, and 48 h after inoculation with medium (C) or RSV (R). Each bar represents mean ± SEM for cultures from six separate donors. *P < 0.05, **P = 0.06 in RSV-infected cells versus uninfected controls.

CF NEC Correction with Adenoviral Gene Transfer

We assessed CFTR function with the chloride efflux assay. Transduction of CF cells with Ad5-CMVLacZ did not change the chloride efflux curve of these cells from that of nontransduced cells. CF cells transduced with Ad5-CBCFTR showed increased chloride efflux as compared with nontransduced and Ad5-CMVLacZ transduced CF cultures, indicating expression of functional CFTR (Figure 3). The transduction of virtually all of the Ad5-CMVLacZ cells, as indicated by X-gal staining, suggests a similar transduction efficiency in Ad5-CBCFTR transduced cells (Figure 4). Thus, transduction of CF NEC with adenoviral vectors at a multiplicity of infection (MOI) of 10³ pfu/cell restored CFTR-mediated chloride transport in most of the cells.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Chloride-efflux assay shows an increase in chloride efflux after forskolin stimulation in CF NEC transduced with Ad5-CBCFTR (squares), as compared with nontransduced cells (circles) and cells transduced with Ad5-CMVLacZ (triangles). %CPM = percent of radioactive chloride (36Cl) remaining in cells as estimated by liquid scintillation counting. The data shown are representative examples from four experiments done with cells from four separate donors.

View larger version (87K): [in this window] [in a new window]   Figure 4.   X-gal staining of (A) CF NEC infected with 103 pfu/cell Ad5-CMVLacZ and (B) CF NEC from the same donor not infected. Bar = 0.1 mm.

All three cell types (nontransduced, Ad5-CBCFTR transduced, and Ad5-CMVLacZ transduced) had increased IL-8 production 4 h after TNF- stimulation (Figure 5). However, neither baseline nor stimulated IL-8 release differed significantly among the three lines.

View larger version (16K): [in this window] [in a new window]   Figure 5.   IL-8 release by primary culture of NEC from children with CF who were not transduced (open bars), transduced with Ad5-CBCFTR (shaded bars), or transduced with Ad5-CMVLacZ (solid bars) at 4 h, 8 h, and 24 h after inoculation with medium (C) or TNF- (T). Each bar represents mean ± SEM for cultures from four separate donors. *P < 0.05 in stimulated cells versus controls.

Immortalized Cell Lines

All three immortalized cell lines showed a significant IL-8 response at 4 h after TNF- stimulation (Figure 6). When individual cell lines were compared, LCFSN (corrected CF) cells had higher baseline IL-8 release than did CFT1 or LC3 cells (P < 0.01). Among the three cell lines at 4 h after TNF- stimulation, the LCFSN (corrected CF) cells produced more IL-8 than did LC3 (sham corrected CF) cells (P < 0.05), but this difference was not significant when the data were expressed as percent of unstimulated control values. No other differences in IL-8 release after TNF- were noted among the cell lines.

View larger version (15K): [in this window] [in a new window]   Figure 6.   IL-8 release by immortalized CF tracheal epithelial cells (CFT1, open bars), CFT1 cells retrovirally corrected with CFTR (LCFSN, shaded bars), and CFT1 cells retrovirally "sham" corrected with LacZ (LC3, solid bars) at 4 h, 8 h, and 24 h after inoculation with medium (C) or TNF- (T). Each bar represents mean ± SEM for cultures from three separate experiments. *P < 0.05 in stimulated cells versus controls, and  denotes P < 0.01 in control LCFSN versus control CFT1 and control LC3.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have found that primary cultures of NEC from young children release increased amounts of the neutrophil-attracting chemokine IL-8 soon after a short exposure to TNF-, and both early and late after inoculation with RSV. These results are consistent with previously reported experiments done with human respiratory epithelial cell cultures (6, 10, 18). Because levels of both IL-8 and neutrophils are unusually high in children with CF (3, 4), we hypothesized that CF airway epithelial cells would overproduce IL-8 and that this would be related to abnormal CFTR function. However, comparison of NEC cultures from children with CF and similar cultures from children without CF showed no differences in the magnitude or duration of the IL-8 response to these stimuli.

Furthermore, restoration of CFTR function by transduction of CF cultures with Ad5-CBCFTR had no significant effect on the IL-8 response to TNF-. These results suggest that CFTR dysfunction alone does not induce excess IL-8 production under these culture conditions and in response to this stimulus. To maintain consistency of cell culture conditions throughout the study, we used a qualitative assay (chloride efflux) to assess whether cells were effectively transduced. Thus, a caveat is that we did not necessarily demonstrate full correction of CFTR function to normal levels. However, the choice of adenoviral MOI used in these experiments was based on our previously published experience, which showed that electrophysiologic correction of CF epithelial cell layers requires that a minority of cells be transduced, and that an MOI of 10³ resulted in production of -galactosidase in most cells as reflected both by X-gal staining and immunohistochemical staining (14, 19), a result matched in the current study. It is presumed that there is some variability in efficiency of adenovirus-mediated gene transfer even in cultured cells, and that this accounts for the variability in staining intensity shown in Figure 4, but it seems unlikely that an overall undercorrection of CFTR function could have accounted for our negative results.

A further finding was that immortalized CF cells did not show increased IL-8 responses compared with retrovirally corrected cells derived from the same line. This finding is consistent with that of Bédard and colleagues (20), who also compared immortalized CF and non-CF NEC. Our experiments with immortalized cell lines actually showed trends toward lower IL-8 production in the cell lines with CFTR dysfunction (CFT1 and LC3). TNF--stimulated IL-8 release expressed as a percent increase over that of unstimulated controls did not differ among the immortalized cell lines, but there appeared to be lower basal IL-8 production in the CFT1 and LC3 lines.

In order to test the general IL-8 responsiveness of CF epithelium, we stimulated our cultures with agents likely to affect both CF and non-CF airways. TNF- is a proinflammatory cytokine produced by macrophages and other cell types as part of the early inflammatory cascade in a variety of conditions (21). RSV is the most common lower respiratory pathogen in children and may be an early trigger of chronic respiratory symptoms in children with CF (24). Our results contrast with those reported by DiMango and associates who found significantly increased production of IL-8 by immortalized CF airway epithelial cells, as compared with the same cell line with corrected CFTR expression, after adherence of Pseudomonas bacteria (25). It is possible that CFTR function has significant effects on expression of factors with specific relevance to CF, such as receptors for Pseudomonas, while having little effect on more general IL-8 induction pathways, such as those stimulated in our experiments.

The use of NEC in primary culture to study respiratory epithelial functions in CF has several advantages. The bioelectric and histologic properties of epithelium on the inferior surface of the nasal turbinates in CF patients are very similar to those in the trachea and large bronchi (8, 26), suggesting similar expression of the CFTR abnormality in these portions of the airway. The cytology brush method for obtaining cells is relatively noninvasive and is not limited to patients undergoing surgical removal of airway tissue. It is therefore possible to choose cell donors based on genotype or clinical expression of disease. The use of primary cultures also avoids the potential problem of alteration in basal cytokine production by immortalization, which may have been illustrated by our data for LC3, LCFSN, and CFT1 cell lines. Furthermore, the nasal passages are not chronically inflamed early in childhood in CF (4), so that the epithelial cells obtained for culture have probably not had long-term exposure to factors that might alter their cytokine-producing characteristics. A potential limitation of this approach is that under our culture conditions the epithelium is relatively poorly differentiated. In particular, it is possible that the full effects of CFTR dysfunction on epithelial processes are not apparent unless full differentiation, including cell polarization or factors in the in vivo airway epithelial environment, are also present. Moreover, the extent to which respiratory epithelium from the upper or central lower airways functionally mimics that from small airways is unknown. The latter factor is of potential importance because current evidence indicates that obstruction of small airways is one of the earliest abnormalities in the CF lung (27).

In summary, we conclude from our data that CF NEC in primary culture do not produce excessive amounts of the neutrophil-attracting factor IL-8 in vitro, either at baseline or after stimulation with the nonspecific IL-8 inducing agents TNF- and RSV; and that CFTR dysfunction alone is not sufficient to cause significant nasal epithelial overproduction of IL-8 under the culture conditions used in our study. These findings do not support the hypothesis that epithelial IL-8 release is a primary pathologic event in CF airways. Our findings may also be consistent with those in a recent study by Armstrong and coworkers (28), in which BAL was performed in 46 newly diagnosed children with CF at a mean age of 2 mo. In the absence of infection, these children had no increase in neutrophils, neutrophil elastase, or IL-8 as compared with controls, suggesting that at a very early age, little excess IL-8 or inflammation is present in children with CF without an infectious stimulus. However, epithelial IL-8 could still play an important role in the perpetuation of a cycle of inflammation and infection, as part of a "normal" reaction to persistence of poorly cleared bacteria and activated neutrophils, or in response to a defect in resolution of inflammation, as has been suggested by studies involving the antiinflammatory cytokine IL-10 (29). Further investigation of the role of airway epithelium in the relationship among infection, inflammation, and airway secretions in CF is needed.