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Published ahead of print on August 22, 2002, doi:10.1164/rccm.200206-627OC
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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1248-1256, (2002)
© 2002 American Thoracic Society

Original Article

Inflammatory Response in Airway Epithelial Cells Isolated from Patients with Cystic Fibrosis

Nada Aldallal, Erin E. McNaughton, Lori J. Manzel, Autumn M. Richards, Joseph Zabner, Thomas W. Ferkol and Dwight C. Look

Departments of Pediatrics and Internal Medicine, Washington University School of Medicine, St. Louis, Missouri; and Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa

Correspondence and requests for reprints should be addressed to Dwight Look, M.D., University of Iowa College of Medicine, Department of Internal Medicine, C33A-GH, 200 Hawkins Drive, Iowa City, IA 52242. E-mail: dwight-look{at}uiowa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The concept that inflammatory gene expression is dysregulated in airway epithelial cells from patients with cystic fibrosis (CF) is controversial. To examine this possibility systematically, responses to inflammatory stimuli were compared in CF airway epithelial cell lines without versus with wild-type CF transmembrane conductance regulator (CFTR) complementation and in tracheobronchial epithelial cells from patients with versus without CF. Epithelial cell expression of the leukocyte adhesion glycoprotein intercellular adhesion molecule-1 (ICAM-1) and release of the neutrophil chemoattractant interleukin (IL)-8 were determined under basal conditions or after exposure to stimuli important in CF airway inflammatory responses. We found that uncorrected CF airway epithelial cell lines inconsistently expressed higher ICAM-1 and IL-8 levels. Human CF tracheobronchial epithelial cells in primary culture released moderately increased IL-8 only after exposure to Pseudomonas aeruginosa. In CF cells with higher IL-8 release, transient expression of wild-type CFTR using an adenoviral vector did not specifically affect cytokine levels. The results indicate that there is considerable variability in airway epithelial cell responses to inflammatory stimuli among different individuals and cell models systems. Although increased ICAM-1 and IL-8 expression are observed in some CF airway epithelial cell models, many CF cells do not exhibit significant dysregulation of these important inflammatory genes.

Key Words: adenoviral vector • Haemophilus influenzae • intercellular adhesion molecule-1 • interleukin-8 • Pseudomonas aeruginosa


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Defective expression or function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in airway epithelial cells leads to persistent and overwhelming infection and inflammation at the respiratory epithelial surface (1). Pulmonary infection and inflammation result in lung damage and cause the majority of the morbidity and mortality in patients with CF (2). Inflammation in the CF lung is remarkably compartmentalized, with infection and inflammation primarily contained in the airway lumen, whereas the alveolar space is relatively spared (3). Despite the ability of the lung to restrict the location of infection, bronchoalveolar lavage, bronchial brushings, and airway biopsies from patients with CF demonstrate an intense endobronchial inflammatory process characterized by high concentrations of proinflammatory cytokines and large numbers of neutrophils, even in patients with early, stable, or clinically mild disease (1, 4–6). Therapy aimed at modulating inflammatory responses slows the progression of CF lung disease, confirming the importance of airway inflammation to the pathophysiology of CF (7–9).

Recent evidence suggests that airway inflammation in CF is not proportionate to the level of stimulation from bacteria and other infectious agents, but rather, the molecular defect in CFTR directly modifies airway inflammatory responses. Dysregulation of airway inflammation in patients with CF may manifest as inflammation independent of infection and/or inflammation disproportionately increased or prolonged in relation to the level of stimuli. Although not a universal finding (10), some infants and children with CF have high levels of proinflammatory cytokines and neutrophils in their bronchoalveolar lavage fluid, even in the absence of detectable infection (6, 11–14). Furthermore, uninfected CF human fetal tracheal grafts have higher intraluminal concentrations of the neutrophil chemoattractant interleukin (IL)-8 and increased subepithelial leukocytes compared with non-CF grafts (15). In response to similar levels of infection, patients with CF have higher concentrations of IL-8 and neutrophils in bronchoalveolar lavage fluid compared with patients without CF (16, 17). Increased airway inflammation may occur in CF patients, even when infection is due to an organism, such as Haemophilus influenzae, that is common in both patients with and without CF (16). Likewise, after airway infection with similar quantities of Pseudomonas aeruginosa, mice homozygous for a targeted null mutation of the CFTR gene have an excessive pulmonary inflammatory response and higher mortality compared with normal animals (18). However, the possibility that airway inflammatory responses are intrinsically augmented in CF is not definitively established, and potential mechanisms that explain this effect are poorly defined.

Because epithelial cells in the airway express CFTR and participate actively in inflammatory responses (19, 20), it has been proposed that epithelial cells mediate dysregulation of inflammation in patients with CF (21, 22). Epithelial cells containing mutant CFTR genes could amplify neutrophil recruitment through constitutive and bacteria-induced release of inflammatory mediators that is disproportionate to infectious stimuli. In support of this hypothesis, certain in vitro models of CF respiratory epithelial cells demonstrate increased release of inflammatory mediators either spontaneously or after exposure to inflammatory stimuli (23–29). However, not all comparisons of CF with non-CF and/or wild-type CFTR-complemented epithelial cells reveal increased inflammatory responses in CF phenotype cells (30–34). Possible reasons for inconsistent conclusions regarding a proinflammatory phenotype in CF airway epithelial cells include derivation of cell lines from different individuals, immortalization and culture of cells under different conditions, treatment with a restricted number of stimuli, and/or variability in methods used to express wild-type CFTR. Few reports compare inflammatory responses to both bacteria and cytokine stimuli using multiple CF cell models or use CF and non-CF airway epithelial cells grown in primary culture, particularly under differentiating culture conditions.

The goal of this work was to examine systematically the hypothesis that inflammatory gene expression is altered in cells that do not express a wild-type CFTR gene using multiple in vitro CF airway epithelial cell models. Immortalized isogenic CF airway epithelial cell lines without or with CFTR complementation as well as tracheobronchial epithelial cells obtained from patients with and without CF were used for these experiments. We assessed epithelial cell surface expression of the adhesive glycoprotein for leukocytes, intercellular adhesion molecule-1 (ICAM-1), and secretion of IL-8 due to the importance of these proteins in regulation of airway inflammation in CF (4, 6, 11, 13, 35). ICAM-1 and IL-8 levels were determined before and after treatment with one of multiple cytokines or bacteria important in CF airway inflammation (4, 5). Although individual and model system variability makes it difficult to establish the presence or absence of a distinct, but subtle, CF phenotype, the results indicate that dysregulation of ICAM-1 and IL-8 gene expression is inconsistently observed in CF airway epithelial cell models.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Airway Epithelial Cells
The CF airway epithelial cell line IB3-1 (genotype {Delta}F508/W1282X) and the matched stable transfected control N6 and CFTR-complemented C38 cell lines were gifts from P. Zeitlin (Johns Hopkins University, Baltimore, MD) (36, 37). The CF airway epithelial cell line CFDEo- (genotype not determined) and the CFTR-complemented CFDE/6REP-CFTR cell line were gifts from D. Gruenert (University of California, San Francisco, CA) (38). Primary airway surface epithelial cells from human CF and non-CF tracheobronchial tissue were obtained under a protocol approved by the University of Iowa and Washington University human studies institutional review boards.

Epithelial Cell Cytokine and Bacteria Treatments
For cytokine treatment, epithelial cell monolayers were incubated with 100 U/ml of recombinant human interferon-{gamma}, tumor necrosis factor-{alpha} (TNF-{alpha}) (gifts from Genentech, San Francisco, CA), or IL-1ß (R&D Systems, Minneapolis, MN) for 24 hours as described previously (39). For bacterial treatment, aerated, log-phase cultures of H. influenzae strain 12 and P. aeruginosa strain PA01 were prepared and quantitated as described previously (40). Bacteria were incubated in 100 µg/ml gentamicin for 30 minutes, and then 108–109 CFU/ml (500–5,000 CFU/epithelial cell) of killed bacteria was incubated with epithelial cells in culture media for 24 hours.

Inflammatory Mediator Expression
ICAM-1 levels on the surface of cell monolayers were determined by enzyme-linked colorimetric immunoassay as described previously (39, 40). IL-8 released into the culture media was assayed using a quantitative sandwich enzyme-linked immunoassay kit (R&D Systems). According to the manufacturer, the sensitivity of this assay system for IL-8 is less than 10 pg/ml.

Adenoviral Vectors
Ad5cmvEGFP and Ad5cmvCFTR were generated and provided by the vector core facility of the Gene Therapy Center at the University of Iowa (http://genetherapy.genetics.uiowa.edu). A multiplicity of infection of 10–50 plaque-forming units (PFU)/cell was used to assure infection of 98% or more of cells in submerged culture conditions (41, 42). Epithelia in air-liquid interface culture conditions were infected using 8 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid applied to the apical surface to transiently disrupt tight junctions as described previously (43, 44). Adenoviruses were incubated with epithelial cells for 24 hours before cytokine or bacteria treatment, assessment of transgene expression, or electrophysiologic testing.

Adenoviral Vector Transgene Expression and Function
Green fluorescent protein expression in living epithelial cells and CFTR expression in fixed cells that underwent immunostaining with anti-human CFTR monoclonal antibody were detected by fluorescence photomicroscopy. Transepithelial electrical properties of epithelial cells cultured in air–liquid interface conditions were determined using modified Ussing chambers as described previously (45).

Statistical Analysis
Enzyme-linked immunoassays were analyzed for statistical significance using a one-way analysis of variance for a factorial experimental design. The multicomparison significance level for the one-way analysis of variance was 0.05. If significance was achieved by one-way analysis, postanalysis of variance comparison of means was performed using Scheffe F-tests (46).

Additional details on airway epithelial cell culture conditions, bacteria preparation, inflammatory mediator assay modification, and assessment of adenoviral vector transgene expression and function are provided in the online data supplement.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CF Cell Line without and with Integrated Complementation by Wild-Type CFTR
Previous reports indicate that the IB3-1 CF airway epithelial cell line releases more IL-8 under basal conditions and in response to TNF-{alpha} and P. aeruginosa compared with matched wild-type CFTR-complemented cell lines (23, 47, 48). Correction of the chloride channel defect in IB3-1 cells was originally accomplished through non–site-specific integration of wild-type truncated CFTR cDNA using a plasmid in which expression of functional CFTR is driven by an adeno-associated virus inverted terminal repeat promoter (37, 49). For our experiments, the IB3-1 parental cell line was compared with the matched transfected control N6 and CFTR-complemented C38 cell lines. Epithelial cells were exposed to one of several inflammatory stimuli, and epithelial cell surface expression of ICAM-1 and release of IL-8 were determined using specific enzyme-linked immunoassays. We found that IB3-1 cells expressed moderate constitutive levels of ICAM-1, with increased expression after incubation with interferon-{gamma}, TNF-{alpha}, IL-1ß, H. influenzae, or P. aeruginosa (Figure 1A) . However, there was no significant difference in ICAM-1 levels when IB3-1 cells were compared with N6 and C38 cells. In assays using media from the same cell monolayers, IB3-1 cells secreted low constitutive levels of IL-8 but released large amounts after treatment with TNF-{alpha}, IL-1ß, H. influenzae, or P. aeruginosa (Figure 1B). IL-8 release under basal conditions and after treatment with these inflammatory stimuli was markedly higher from IB3-1 and N6 cells compared with C38 cells. These results confirm that specific cytokines and bacteria increase ICAM-1 and IL-8 gene expression in airway epithelial cells (39, 40) and extend previous reports by demonstrating higher IL-8 release from the IB3-1 CF cell line after exposure to many different stimuli.



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  		Figure 1. CF cell line without and with integrated complementation by wild-type CFTR. IB3-1 (black bars), control sham-complemented N6 (gray bars), and wild-type CFTR-complemented C38 (white bars) cell monolayers were incubated in media without or with IFN-{gamma}, TNF-{alpha}, IL-1ß, H. influenzae, or P. aeruginosa for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Values are expressed as mean ± SD (n = 3–4), and a significant difference in IL-8 release between N6 and C38 cells is indicated by an asterisk.

 
CF Cell Line without and with Episomal Complementation by Wild-Type CFTR
To assess whether increased IL-8 secretion from IB3-1 cells could be generalized to other immortalized CF cell lines, we also tested the CFDEo- CF airway epithelial cell line. Correction of the chloride channel defect in CFDEo- cells was originally accomplished using an episomal vector system for wild-type CFTR expression to generate CFDE/6REP-CFTR cells (38). Our experiments revealed that CFDEo- cells expressed high constitutive levels of ICAM-1, and all cytokine and bacterial treatments increased cell surface expression (Figure 2A) . Release of IL-8 from CFDEo- cells increased after treatment with TNF-{alpha}, IL-1ß, H. influenzae, or P. aeruginosa (Figure 2B). In contrast to the IB3-1 cell line, the matched CFTR-complemented cells behaved identically to the uncorrected cells, with one exception. Wild-type CFTR-expressing CFDE/6REP-CFTR cells lost induction of ICAM-1 expression and IL-8 release in response to IL-1ß treatment, whereas the parental CFDEo- cell line maintained IL-1ß responsiveness. Thus, in this CF epithelial cell model, either wild-type CFTR expression or the expression system itself altered the epithelial cell response to IL-1ß, but not to other treatments.



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  		Figure 2. CF cell line without and with episomal complementation by wild-type CFTR. CFDEo- (black bars) and wild-type CFTR-complemented CFDE/6REP-CFTR (white bars) cell monolayers were incubated in media without or with the indicated cytokine or bacteria for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Values are expressed as mean ± SD (n = 3–4), and a significant difference in ICAM-1 expression or IL-8 release between CFDEo- and CFDE/6REP-CFTR cells is indicated by an asterisk.

 
Primary CF Compared with Non-CF Airway Epithelial Cells in Submerged Culture
Because of our observation that inflammatory responses were inconsistent in CF airway epithelial cell lines, we studied primary culture airway epithelial cells from patients without and with CF, thereby eliminating effects of immortalization and complementation. These experiments resulted in a comparison of cells that were not isogenic, and thus, individual differences in gene expression could be due to genotypic differences other than CFTR (50). Accordingly, cells from multiple individuals without and with CF were tested to control for individual effects on ICAM-1 and IL-8 gene expression (Table 1) . Because most cell lines do not form epithelia with characteristic transepithelial electrical properties, we initially studied primary airway epithelial cells in submerged culture conditions comparable to experiments with cell lines. We found that primary cells under these conditions expressed low basal levels of ICAM-1, with significant induction after treatment with interferon-{gamma} or bacteria, but no difference between CF and non-CF cells (Figure 3A) . All primary cell samples had low constitutive IL-8 release, with increased secretion after treatment with TNF-{alpha} or bacteria (Figure 3B). Although we observed slightly increased stimulated release of IL-8 from CF airway epithelial cells compared with cells from patients without CF, there was significant variability and overlap within treatment condition groups, with statistical significance reached only when comparing cells treated with P. aeruginosa. In dose–response experiments, increased ICAM-1 expression and IL-8 release did not occur at different inflammatory treatment concentrations when comparing cells from patients with and without CF (results not shown), indicating that mutant endogenous CFTR genes do not alter epithelial cell sensitivity to lower stimulus levels.


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  		TABLE 1. Characteristics of donors for submerged cultures of airway epithelial cells

 


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  		Figure 3. Primary CF compared with non-CF airway epithelial cells in submerged culture. Primary CF (black circles) and non-CF (white circles) airway epithelial cell monolayers in submerged culture conditions were incubated in media without or with the indicated cytokine or bacteria for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Individual patient values are plotted with the mean for the group indicated by a solid line (n = 8), and a significant difference in IL-8 release between CF and non-CF cells is indicated by an asterisk.

 
Primary CF Compared with Non-CF Airway Epithelial Cells in Air–Liquid Interface Culture
Because differentiation of epithelial cells regulates receptor polarization and CFTR expression (51, 52), we also tested primary airway epithelial cells from multiple individuals grown as epithelia under differentiating conditions at an air–liquid interface (Table 2) . Although variability among patient samples was relatively high using this cell culture system, the overall pattern of induction of ICAM-1 surface expression and IL-8 secretion in response to treatments was similar to submerged primary cell cultures. There was no significant difference in ICAM-1 expression when comparing cells from patients with and without CF (Figure 4A) . Similarly, in studies of IL-8 secretion, there were significant variability and overlap within treatment condition groups, and differences observed did not reach statistical significance under any stimulation condition (Figure 4B). These results suggest that factors other than CFTR expression exert greater control over differences in cellular responses to inflammatory stimuli in this airway epithelial cell culture system.


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  		TABLE 2. Characteristics of donors for air–liquid interface cultures of airway epithelial cells

 


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  		Figure 4. Primary CF compared with non-CF airway epithelial cells in air–liquid interface culture. Primary CF (black circles) and non-CF (white circles) airway epithelia in air–liquid interface culture conditions were incubated in media without or with the indicated cytokine or bacteria for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Individual patient values are plotted with the mean for the group indicated by a solid line (n = 8), and a significant difference in IL-8 release between CF and non-CF cells was not obtained.

 
Transient CFTR Expression and Function
To eliminate individual variability inherent in comparing genotypically different epithelial samples, we transiently expressed wild-type CFTR in CF epithelial cells and tested responses to inflammatory stimuli. A transient expression system also provided an alternate CFTR complementation strategy for CF cell lines. However, correction of potential dysregulated cellular inflammatory responses would likely require wild-type CFTR expression in the majority of CF epithelial cells due to the likelihood that each cell contributes inflammatory mediators. Accordingly, we selected an adenoviral vector for CFTR complementation based on the fact that this system is well characterized and results in transgene expression in a high percentage of cultured cells. Infection of both IB3-1 and primary CF airway epithelial cells with an adenovirus expressing green fluorescent protein resulted in transgene expression in 98% or more of cells, verifying high-efficiency infection by this expression system (Figure 5A) . Immunohistochemical staining detected faint CFTR expression in IB3-1 cells (that contain heterozygous {Delta}F508 and W1282X CFTR mutant genes) and primary CF airway epithelial cells (that contain homozygous {Delta}F508 mutant genes) that was similar to previous reports (37) (Figure 5B). Infection of both cell types with an adenoviral vector expressing wild-type CFTR augmented cellular CFTR staining in the majority of cells in the sample. Furthermore, this expression system for CFTR restored cAMP-activated transepithelial chloride current that was defective in primary CF airway epithelial cells grown as epithelia in air–liquid interface conditions (Figure 6) . The mean change (n = 8 in each group) in short circuit current after apical treatment with amiloride was 36.0 ± 22.0 µA/cm2 for CF airway epithelia infected with Ad5cmvEGFP versus 35.1 ± 15.1 µA/cm2 for CF epithelia infected with Ad5cmvCFTR. The change in current after cAMP stimulation was -0.2 ± 0.2 µA/cm2 for CF epithelia infected with Ad5cmvEGFP versus 9.4 ± 4.9 µA/cm2 for CF epithelia infected with Ad5cmvCFTR. Accordingly, these results indicate that this adenoviral vector system expresses functional wild-type CFTR in the majority of epithelial cells infected in submerged culture conditions.



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  		Figure 5. Transient green fluorescent protein or CFTR expression in CF airway epithelial cells. IB3-1 and primary CF airway epithelial cell (CF-hTBE) monolayers were infected with Ad5cmvEGFP or Ad5cmvCFTR for 24 hours under conditions that result in transgene expression in more than 98% of the cells in the monolayer. Fluorescence photomicroscopy was used to confirm green fluorescent protein expression (A) in living cells and CFTR expression (B) in cells that underwent immunostaining with anti-human CFTR monoclonal antibody (scale bar, 30 µm).

 


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  		Figure 6. Transepithelial electrophysiology after transient CFTR expression. Primary CF and non-CF airway epithelia in air–liquid interface culture conditions were not infected or infected with Ad5cmvEGFP or Ad5cmvCFTR for 24 hours and then studied in Ussing chambers. Short circuit current was determined through cell monolayers after sequential administration of amiloride (Amil), 4,4'-diisothiocyanantostilbene-2, 2'-disulfonic acid (DIDS), 3-isobutyl-1-methylxanthine + forskolin (IBMX + Fors), and bumetanide (Bumet).

 
CF Cell Line without and with Transient Complementation by Wild-Type CFTR
We initially tested effects of transient CFTR complementation on IB3-1 cells due to the observation that this cell line had much higher IL-8 secretion when compared with matched cells with integrated wild-type CFTR. There was no significant difference in constitutive or stimulated ICAM-1 expression between IB3-1 cells that were not infected with virus and cells infected with adenovirus expressing a control transgene or wild-type CFTR (Figure 7A) . In contrast to a comparison of IB3-1 cells with integrated control or CFTR complementation, there was no difference in constitutive or stimulated IL-8 release from IB3-1 cells with transient expression of a control transgene or wild-type CFTR (Figure 7B). However, a significant decrease in IL-8 secretion induced by IL-1ß treatment was observed in this cell line after infection with adenovirus expressing either transgene. The observed discrepancy using two different methods for wild-type CFTR expression in the same cell model indicates that systems for generating complementation affect whether CFTR correction alters IL-8 secretion and that non–transgene-dependent effects on epithelial cell inflammatory responses may occur.



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  		Figure 7. CF cell line without and with transient complementation by wild-type CFTR. IB3-1 cell monolayers that were not infected (black bars), infected with Ad5cmvEGFP (gray bars), or infected with Ad5cmvCFTR (white bars) were incubated in media without or with the indicated cytokine or bacteria for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Values are expressed as mean ± SD (n = 3–4), and a significant difference in IL-8 release between IB3-1 cells that were not infected with virus and cells that were infected with an adenoviral vector is indicated by an asterisk.

 
Primary CF Cells without and with Transient Complementation by Wild-Type CFTR
The effect of transient expression of wild-type CFTR on inflammatory responses in primary airway epithelial cells in submerged culture conditions was also determined. Transient CFTR expression in primary epithelial cells from selected individuals with CF allowed for comparison under isogenic conditions similar to experiments with cell lines. In experiments using cells from a patient homozygous for the CFTR {Delta}F508 mutation, we found no significant difference in constitutive or stimulated ICAM-1 expression between cells that were not infected and cells infected with adenovirus expressing a control transgene or wild-type CFTR (Figure 8A) . Stimulated release of IL-8 was slightly lower in epithelial cells infected with adenovirus expressing either transgene, and this decrease reached statistical significance when comparing cells treated with TNF-{alpha} (Figure 8B). In contrast to comparison between cells from patients with and without CF, primary CF cells expressing wild-type CFTR released similar amounts of IL-8 compared with cells expressing the control transgene. Experiments using cells from two other patients with CF (one homozygous and one heterozygous for the CFTR {Delta}F508 mutation) and a patient without CF (results not shown) gave similar results. Based on these experiments, it appears that infection with adenoviral vectors has modest effects on epithelial cell IL-8 release under some conditions. However, wild-type CFTR that is transiently expressed by this viral system does not itself affect IL-8 release from airway epithelial cells.



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  		Figure 8. Primary CF cells without and with transient complementation by wild-type CFTR. Primary CF airway epithelial cell monolayers in submerged culture that were not infected (black bars), infected with Ad5cmvEGFP (gray bars), or infected with Ad5cmvCFTR (white bars) were incubated in media without or with the indicated cytokine or bacteria for 24 hours. Levels of ICAM-1 expression on the cell surface (A) and IL-8 secretion into the culture media (B) were determined using enzyme-linked immunoassays. Values are expressed as mean ± SD (n = 3–4), and a significant difference in IL-8 release between CF cells that were not infected with virus and cells that were infected with an adenoviral vector is indicated by an asterisk.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although several potential mechanisms have been advanced, the links between CFTR gene mutation and lung damage remain unclear. For this report, a systematic study of airway epithelial cells was performed in vitro, based on the hypothesis that dysfunction of airway inflammatory responses with inappropriately high constitutive and/or infection-induced activation of airway defense genes in epithelial cells contributes to lung damage in CF. We observed much higher levels of IL-8 release in response to several inflammatory stimuli when comparing one CF airway epithelial cell line to its stable CFTR-complemented control line. In addition, a modest increase in IL-8 secretion after infection with P. aeruginosa was seen in airway epithelial cells grown in submerged primary culture from CF patients compared with patients without CF. However, other airway epithelial cell models did not have greater IL-8 release from CF phenotype cells compared with cells that express wild-type CFTR. Furthermore, differences in IL-8 secretion between primary culture epithelial cells from patients with and without CF were relatively small (with statistical significance under only one condition) compared with variability between individuals within the same group. Finally, transient expression of wild-type CFTR did not alter CF airway epithelial cell constitutive or inducible levels of ICAM-1 or IL-8. The results indicate that CF airway epithelial cell models do not have a consistent proinflammatory phenotype, although individual and model system variability makes it difficult to establish the presence or absence of a distinct, but subtle, CF phenotype.

Factors that account for inconsistencies between model systems in epithelial cell responses to inflammatory stimuli and observed alteration in CF epithelial cells compared with cells expressing wild-type CFTR are not clear. Epithelial cell lines used in many reports were derived from CF patients, with control CFTR-complemented cell lines subsequently generated using plasmid transfection or viral vector infection for transient or stable expression of wild-type human CFTR (21, 23, 31, 33, 47). Alternately, in some studies, decreased CFTR expression or function in epithelial cell lines from patients without CF was induced through expression of the CFTR regulatory domain or antisense oligonucleotides (26, 28, 53). These cellular models of CF epithelium are advantageous because they manifest characteristic electrophysiologic properties of CF cells and have isogenic control cell lines. However, variation between cell models may be caused by differences in environmental (e.g., cell source, isolation procedure, culture conditions, differentiation, viral infection) or genetic (e.g., modifying genes, immortalization, complementation) factors or modifications. For example, inclusion of serum or serum substitutes in culture media may cause significant alterations in inflammatory gene expression in many epithelial cells (47, 53; results not shown). Moreover, results from experiments with immortalized epithelial cell lines may not always be generalized to airway epithelial cell behavior in vivo because they are derived from a single individual, are not polarized, are often not diploid, and their genotype and behavior may change over time. Experimental conclusions regarding the proinflammatory phenotype of CF epithelial cells need to take these factors that affect inflammatory gene expression into account.

Primary culture airway epithelial cells were included in our studies because they have not undergone immortalization or complementation and culture conditions could be selected to allow for a more differentiated phenotype. However, the use of primary culture cells requires testing of samples from multiple subjects to control for variability in responses between individuals. The effects of genetic and environmental factors on the inflammatory response in patients that provide tissue samples for cell culture are poorly defined. Recent exposure to infection or therapy (e.g., antibiotics and antiinflammatory medication) was different between patients without and with CF that provided primary cells for our experiments. Furthermore, epithelial cells freshly isolated from the infected airway environment of CF patients have altered inflammatory gene expression, and these levels of expression may persist for days after removal from inflammatory conditions (27, 28; results not shown). Preliminary experiments indicated that ICAM-1 and IL-8 gene expression in CF airway epithelial cells decreased to a stable level of expression within 2 weeks after isolation that was similar to low levels in non-CF cells (results not shown). Therefore, cells for our studies were cultured for at least 14 days before testing to start experiments at a baseline state of inflammatory gene activation.

Mechanisms for wild-type CFTR expression or complementation appear to have different effects on inflammatory gene expression in CF airway epithelial cells. In IB3-1 and primary culture CF airway epithelial cells that demonstrated increased IL-8 release in initial comparison experiments, constitutive or stimulated ICAM-1 expression and IL-8 secretion were not affected by transient expression of functional wild-type CFTR using an adenoviral vector. The results with IB3-1 cells were particularly striking because a comparison of the parental cell line to a line derived by functional integration of wild-type CFTR cDNA revealed large differences in IL-8 release under all conditions, although its is not apparent whether the parental or complemented cell line has the more "appropriate" response. CFTR complementation strategies that rely on adenoviral vectors suffer from lack of expression of physiologic cellular levels of CFTR, lack of effect on mutant CFTR expression, and vector-dependent effects on inflammatory gene expression. For example, a proposed mechanism for excessive epithelial cell gene expression in CF involves mutation-dependent mistrafficking of CFTR protein with accumulation in the endoplasmic reticulum resulting in direct effects on cell signaling pathways (53, 54), and complementation with wild-type CFTR would presumably not alter this effect of endogenous CFTR genes with these specific mutations (e.g., {Delta}F508). Alternatively, reports suggest that loss of physiologic CFTR chloride channel function may itself affect epithelial cell inflammatory gene expression (28, 53), and complementation is likely to correct this effect if wild-type CFTR expression and chloride channel function are generated in the majority of cells in the system. Because CFTR expression from wild-type cDNA that is either stably integrated into the cellular genome or transiently functional in a nonintegrated cellular location would not be expected to alter expression of endogenous mutant CFTR genes, the reasons for differences in the effects of correction of IB3-1 and primary CF airway epithelial cells by these two strategies are not apparent.

Reports from other laboratories suggest that altered airway epithelial cell responses in CF may be mediated through dysregulation of constitutive or inducible function of the transcriptional activator, nuclear factor-{kappa}B (21, 47, 53, 55, 56). The use of ICAM-1 and IL-8 levels as markers of inflammatory gene activation in our studies is relevant for assessment of this possibility because these inflammatory mediators function to direct neutrophil recruitment at sites of infection, participate in regulation of intense airway neutrophilia characteristically seen in patients with CF, and their induction by TNF-{alpha}, IL-1ß, and bacteria is mediated by nuclear factor-{kappa}B (1, 4, 6, 11, 13, 39, 57–62). We found small differences in constitutive levels of ICAM-1 and IL-8 in all but one of the cell models in our studies, suggesting that there is minimal difference in baseline nuclear factor-{kappa}B activation in CF. In contrast, the possibility that mutant CFTR may affect nuclear factor-{kappa}B–dependent signaling in response to inflammatory stimuli is less clear because of inconsistencies between experimental models of CF airway epithelial cells. However, when significant differences in IL-8 release from CF cells compared with cells expressing wild-type CFTR were seen, differences in ICAM-1 expression were not observed. Furthermore, infection with adenoviral vectors resulted in decreased IL-8 secretion from airway epithelial cells in response to specific inflammatory stimuli regardless of the transgene expressed, without affecting ICAM-1 expression or causing cytotoxicity (42; results not shown). Therefore, the observed differences in regulation of ICAM-1 versus IL-8 expression by infectious stimuli confirm that regulatory factors other than nuclear factor-{kappa}B are also important in inflammatory gene activation in airway epithelial cells (63, 64), and these factors could account for inconsistencies in airway epithelial cell models of CF.

Defects in innate immunity result in pulmonary airways with mixtures of high levels of several inflammatory stimuli and mediators in patients with CF. Several bacteria, including H. influenzae, commonly infect the CF airway early in the course of the disease, but bacterial control of the airway is eventually surrendered to P. aeruginosa (4, 13, 16, 65). Both bacterial species are strong inducers of epithelial cell expression of inflammatory genes such as ICAM-1 and IL-8 (23, 40). The soluble factors interferon-{gamma}, TNF-{alpha}, and IL-1ß are increased in the airways of many patients with CF and also activate genes in epithelial cells that regulate airway defense responses (5, 11, 13, 19, 39, 66). Our results indicate that in vitro model systems for studying airway epithelial cell behavior may have very different responses to inflammatory stimuli and that factors other than CFTR expression exert significant control over epithelial cell responses to infection. Moreover, experimental systems that use isolated epithelial cells and single inflammatory stimuli may not recapitulate the complexity of the airway environment. Nonetheless, these studies are an initial step toward better understanding of mechanisms regulating airway inflammation that will allow for the development of better therapeutic strategies to control damaging airway inflammatory responses in patients with CF.


   Acknowledgments
 
The authors gratefully acknowledge D. Gruenert, C. Huddleston, E. Mendeloff, A. Patterson, P. Zeitlin, and the University of Iowa Center for Gene Therapy for generous gifts of cells and vectors; P. Karp and L. Ostedgaard for technical assistance; and S. Brody, G. Hunninghake, P. McCray, and M. Welsh for helpful discussion.


   FOOTNOTES
 
Supported by grants from the National Institutes of Health and the Cystic Fibrosis Foundation

This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form June 27, 2002; accepted in final form August 15, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Danel C, Erzurem SC, McElvaney NG, Crystal RG. Quantitative assessment of the epithelial and inflammatory cell populations in large airways of normals and individuals with cystic fibrosis. Am J Respir Crit Care Med 1996;153:362–368.[Abstract]
  2. FitzSimmons SC. The changing epidemiology of cystic fibrosis. J Pediatr 1993;122:1–9.[Medline]
  3. Wood RE, Boat TF, Doershuk CF. Cystic fibrosis. Am Rev Respir Dis 1976;113:833–878.[Medline]
  4. Konstan MW, Hilliard KA, Norvell TM, Berger M. Bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation. Am J Respir Crit Care Med 1994;150:448–454.[Abstract]
  5. Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, Berger M. Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 1995;152:2111–2118.[Abstract]
  6. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DWH. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med 1995;151:1075–1082.[Abstract]
  7. Auerbach HS, Williams M, Kirkpatrick JA, Colten HR. Alternate-day prednisone reduces morbidity and improves pulmonary function in cystic fibrosis. Lancet 1985;2:686–688.[CrossRef][Medline]
  8. Konstan MW, Byard PJ, Hoppel C, Davis PB. Effect of high-dose ibuprofen in patients with cystic fibrosis. N Engl J Med 1995;332:848–854.[Abstract/Free Full Text]
  9. Eigen H, Rosenstein BJ, FitzSimmons S, Schidlow DV. A multicenter study of alternate-day prednisone therapy in patients with cystic fibrosis: CF foundation prednisone trial group. J Pediatr 1995;126:515–523.[CrossRef][Medline]
  10. Armstrong DS, Grimwood K, Carlin JB, Carzino R, Gutierrez JP, Hull J, Olinsky A, Phelan EM, Robertson CF, Phelan PD. Lower airway inflammation in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 1997;156:1197–1204.[Abstract/Free Full Text]
  11. Balough K, McCubbin M, Weinberger M, Smits W, Ahrens R, Fick R. The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr Pulmonol 1995;20: 63–70.[Medline]
  12. Noah TL, Black HR, Cheng PW, Wood RE, Leigh MW. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J Infect Dis 1997;175:638–647.[Medline]
  13. Rosenfeld M, Gibson RL, McNamara S, Emerson J, Burns JL, Castile R, Hiatt P, McCoy K, Wilson CB, Inglis A, et al. Early pulmonary infection, inflammation, and clinical outcomes in infants with cystic fibrosis. Pediatr Pulmonol 2001;32:356–366.[CrossRef][Medline]
  14. Dakin CJ, Numa AH, Wang H, Morton JR, Vertzyas CC, Henry RL. Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 2002;165:904–910.[Abstract/Free Full Text]
  15. Tirouvanziam R, de Bentzmann S, Hubeau C, Hinnrasky J, Jacquot J, Péault B, Puchelle E. Inflammation and infection in naive human cystic fibrosis airway grafts. Am J Respir Cell Mol Biol 2000;23:121–127.[Abstract/Free Full Text]
  16. Muhlebach MS, Stewart PW, Leigh MW, Noah TL. Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients. Am J Respir Crit Care Med 1999;160:186–191.[Abstract/Free Full Text]
  17. Muhlebach MS, Noah TL. Endotoxin activity and inflammatory markers in the airways of young patients with cystic fibrosis. Am J Respir Crit Care Med 2002;165:911–915.[Abstract/Free Full Text]
  18. Van Heeckeren A, Walenga R, Konstan MW, Bonfield T, Davis PB, Ferkol T. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J Clin Invest 1997;100:2810–2815.[Medline]
  19. Levin SJ. Bronchial epithelial cell-cytokine interactions in airway inflammation. J Investig Med 1995;43:241–249.[Medline]
  20. Holtzman MJ, Look DC, Sampath D, Castro M, Koga T, Walter MJ. Control of epithelial immune-response genes and implications for airway immunity and inflammation. Proc Assoc Am Phys 1998;110:1–11.[Medline]
  21. DiMango E, Ratner AJ, Bryan R, Tabibi S, Prince A. Activation of NF-{kappa}B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J Clin Invest 1998;101:2598–2606.[Medline]
  22. Blackwell TS, Sencenko AA, Christman JW. Dysregulated NF-{kappa}B activation in cystic fibrosis: evidence for a primary inflammatory disorder. Am J Physiol Lung Cell Mol Physiol 2001;281:L69–L70.[Free Full Text]
  23. DiMango E, Zar HJ, Bryan R, Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest 1995;96:2204–2210.
  24. Kammouni W, Figarella C, Marchand S, Merten M. Altered cytokine production by cystic fibrosis tracheal gland serous cells. Infect Immun 1997;65:5176–5183.[Abstract]
  25. Tabary O, Zahm JM, Hinnrasky J, Couetil JP, Cornillet P, Guenounou M, Gaillard D, Puchelle E, Jacquot J. Selective up-regulation of chemokine IL-8 expression cystic fibrosis bronchial gland cells in vivo and in vitro. Am J Pathol 1998;153:921–930.[Abstract/Free Full Text]
  26. Bryan R, Kube D, Perez A, Davis P, Prince A. Overproduction of the CFTR R domain leads to increased levels of asialoGM1 and increased Pseudomonas aeruginosa binding by epithelial cells. Am J Respir Cell Mol Biol 1998;19:269–277.[Abstract/Free Full Text]
  27. Bonfield TL, Konstan MW, Berger M. Altered respiratory epithelial cell cytokine production in cystic fibrosis. J Allergy Clin Immunol 1999;104:72–78.[CrossRef][Medline]
  28. Kube D, Sontich U, Fletcher D, Davis PB. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 2001;280:L493–502.[Abstract/Free Full Text]
  29. Tabary O, Escotte S, Couetil JP, Hubert D, Dusser D, Puchelle E, Jacquot J. Relationship between I{kappa}B{alpha} deficiency, NF{kappa}B activity and interleukin-8 production in CF human airway epithelial cells. Pflügers Arch—Eur J Physiol 2001;443:S40–S44.
  30. Bedard M, McClure CD, Schiller NL, Francoeur C, Cantin A, Denis M. Release of interleukin-8, interleukin-6, and colony-stimulating factors by upper airway epithelial cells: implications for cystic fibrosis. Am J Respir Cell Mol Biol 1993;9:455–462.
  31. Black HR, Yankaskas JR, Johnson LG, Noah TL. Interleukin-8 production by cystic fibrosis nasal epithelial cells after tumor necrosis factor-{alpha} and respiratory syncycial virus stimulation. Am J Respir Cell Mol Biol 1998;19:210–215.[Abstract/Free Full Text]
  32. Schwiebert LM, Estell K, Propst SM. Chemokine expression in CF epithelia: implications for the role of CFTR in RANTES expression. Am J Physiol 1999;276:C700–C710.[Abstract/Free Full Text]
  33. Massengale ARD, Quinn F, Yankaskas J, Weissman D, McClellanWT, Cuff C, Aronoff SC. Reduced interleukin-8 production by cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 1999;20:1073–1080.[Abstract/Free Full Text]
  34. Pizurki L, Morris MA, Chanson M, Solomon M, Pavirani A, Bouchardy I, Suter S. Cystic fibrosis transmembrane conductance regulator does not affect neutrophil migration across cystic fibrosis airway epithelial monolayers. Am J Pathol 2000;156:1407–1416.[Abstract/Free Full Text]
  35. Hubeau C, Lorenzato M, Couetil JP, Hubert D, Dusser D, Puchelle E, Gaillard D. Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa. Clin Exp Immunol 2001;124:69–76.[CrossRef][Medline]
  36. Zeitlin PL, Lu L, Rhim J, Cutting G, Stetten G, Kieffer KA, Craig R, Guggino WB. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection. Am J Respir Cell Mol Biol 1991;4: 313–319.
  37. Flotte TR, Afione SA, Solow R, Drumm ML, Markakis D, Guggino WB, Zeitlin PL, Carter BJ. Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 1993;268:3781–3790.[Abstract/Free Full Text]
  38. Lei DC, Kunzelmann K, Koslowsky T, Yezzi MJ, Escobar LC, Xu Z, Ellison AR, Rommens JM, Tsui L-C, Tykocinski M, et al. Episomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells. Gene Ther 1996;3:427–436.[Medline]
  39. Look DC, Keller BT, Rapp SR, Holtzman MJ. Selective induction of intercellular adhesion molecule-1 by interferon-{gamma} in human airway epithelial cells. Am J Physiol 1992;263:L79–L87.[Abstract/Free Full Text]
  40. Frick AG, Joseph TD, Pang L, Rabe AM, St. Geme JW III, Look DC. Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells. J Immunol 2000;164:4185–4196.[Abstract/Free Full Text]
  41. Look DC, Roswit WT, Frick AG, Gris-Alevy Y, Dickhaus DM, Walter MJ, Holtzman MJ. Direct suppression of Stat1 function during adenoviral infection. Immunity 1998;9:871–880.[CrossRef][Medline]
  42. Joseph TD, Look DC. Specific inhibition of interferon signal transduction pathways by adenoviral infection. J Biol Chem 2001;276:47136–47142.[Abstract/Free Full Text]
  43. Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 1999;274: 10219–10226.[Abstract/Free Full Text]
  44. Walters RW, van't Hof W, Yi SM, Schroth MK, Zabner J, Crystal RG, Welsh MJ. Apical localization of the coxsackie-adenovirus receptor by glycosyl-phosphatidylinositol modification is sufficient for adenovirus-mediated gene transfer through the apical surface of human airway epithelia. J Virol 2001;75:7703–7711.[Abstract/Free Full Text]
  45. Zabner J, Zeiher BG, Friedman E, Welsh MJ. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J Virol 1996;70:6994–7003.[Abstract/Free Full Text]
  46. Zar JH. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall; 1984.
  47. Venkatakrishnan A, Stecenko AA, King G, Blackwell TR, Brigham KL, Christman JW, Blackwell TS. Exaggerated activation of NF-{kappa}B and altered I{kappa}B-ß processing in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol 2000;23:396–403.[Abstract/Free Full Text]
  48. Eidelman O, Srivastava M, Zhang J, Leighton X, Murtie J, Jozwik C, Jacobson K, Weinstein DL, Metcalf EL, Pollard HB. Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-{alpha}R/NF{kappa}B pathway. Mol Med 2001;7:523–534.[Medline]
  49. Kearns WG, Afione SA, Fulmer SB, Pang MC, Erikson D, Egan M, Landrum MJ, Flotte TR, Cutting GR. Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther 1996;3:748–755.[Medline]
  50. Hull J, Thomson AH. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 1998;53:1018–1021.[Abstract/Free Full Text]
  51. Imundo L, Barasch J, Prince A, Al-Awqati Q. Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface. Proc Natl Acad Sci USA 1995;92:3019–3023.[Abstract/Free Full Text]
  52. Montrose-Rafizadeh C, Guggino WB, Montrose MH. Cellular differentiation regulates expression of Cl- transport and CFTR mRNA in human intestinal cells. J Biol Chem 1991;266:4495–4499.[Abstract/Free Full Text]
  53. Weber AJ, Soong G, Bryan R, Saba S, Prince A. Activation of NF-{kappa}B in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function. Am J Physiol Lung Cell Mol Physiol 2001;281:L71–L78.[Abstract/Free Full Text]
  54. Pahl HL, Baeuerle PA. Activation of NF-{kappa}B by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers. FEBS Lett 1996;392:129–136.[CrossRef][Medline]
  55. Tabary O, Escotte S, Couetil JP, Hubert D, Dusser D, Puchelle E, Jacquot J. Genistein inhibits constitutive and inducible NF{kappa}b activation and decreases IL-8 production by human cystic fibrosis bronchial gland cells. Am J Pathol 1999;155:473–481.[Abstract/Free Full Text]
  56. Tabary O, Escotte S, Couetil JP, Hubert D, Dusser D, Puchelle E, Jacquot J. High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the I{kappa}B kinase {alpha} pathway in response to extracellular NaCl content. J Immunol 2000;164:3377–3384.[Abstract/Free Full Text]
  57. Makaida N, Mahe Y, Matsushima K. Cooperative interaction of nuclear factor-{kappa}B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem 1990;265:21128–21133.[Abstract/Free Full Text]
  58. Nakamura H, Yoshimura K, Jaffe HA, Crystal RG. Interleukin-8 gene expression in human bronchial epithelial cells. J Biol Chem 1991;266: 19611–19617.[Abstract/Free Full Text]
  59. Ledebur HC, Parks TP. Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kappa B site and p65 homodimers. J Biol Chem 1995;270:933–943.[Abstract/Free Full Text]
  60. Sharma SA, Tummuru MKR, Blaser MJ, Kerr LD. Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-{kappa}B in gastric epithelial cells. J Immunol 1998;160: 2401–2407.[Abstract/Free Full Text]
  61. Mori N, Wada A, Hirayama T, Parks TP, Stratowa C, Yamamoto N. Activation of intercellular adhesion molecule 1 expression by Helicobacter pylori is regulated by NF-kappaB in gastric epithelial cancer cells. Infect Immun 2000;68:1806–1814.[Abstract/Free Full Text]
  62. Gewirtz AT, Rao AS, Simon POJ, Merlin D, Carnes D, Madara JL, Neish AS. Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-{kappa}B pathway. J Clin Invest 2000; 105:79–92.[Medline]
  63. Carter AB, Knudtson KL, Monick MM, Hunninghake GW. The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem 1999;274:30858–30863.[Abstract/Free Full Text]
  64. Ratner AJ, Bryan R, Weber A, Nguyen S, Barnes D, Pitt A, Gelber S, Cheung A, Prince A. Cystic fibrosis pathogens activate Ca2+-dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells. J Biol Chem 2001;276:19267–19275.[Abstract/Free Full Text]
  65. Baughman RP, Keeton DA, Perez C, Wilmott RW. Use of bronchoalveolar lavage semiquantitative cultures in cystic fibrosis. Am J Respir Crit Care Med 1997;156:286–291.[Abstract/Free Full Text]
  66. Wojnarowski C, Frischer T, Hofbauer E, Grabner C, Mosgoeller W, Eichler I, Ziesche R. Cytokine expression in bronchial biopsies of cystic fibrosis patients with and without acute exacerbation. Eur Respir J 1999;14:1136–1144.[Abstract]



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