NF-{kappa}B activation and sustained IL-8 gene expression in primary cultures of cystic fibrosis airway epithelial cells stimulated with Pseudomonas aeruginosa

Theresa Joseph,1 Dwight Look,2 and Thomas Ferkol1

1Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri; and 2Department of Internal Medicine, University of Iowa, Iowa City, Iowa

Submitted 27 February 2004 ; accepted in final form 26 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The progression of lung disease in cystic fibrosis (CF) is characterized by an exuberant inflammatory response mounted by the respiratory epithelium that is further exacerbated by bacterial infection. Recent studies have demonstrated upregulation of nuclear factor-{kappa}B (NF-{kappa}B) in response to infection in genetically modified cell culture models, which is associated with expression of interleukin (IL)-8. Using human airway epithelial cells grown in primary culture, we examined in vitro activation of NF-{kappa}B in cells isolated from five CF ({Delta}F508/{Delta}F508) and three non-CF (NCF) patients in response to Pseudomonas aeruginosa. Immunofluorescence, gel-shift, and immunoblot assays demonstrated a rapid translocation of NF-{kappa}B subunits (p50 and p65) to the nucleus in both CF and NCF cell cultures. However, nuclear extracts from CF cells both before and following P. aeruginosa stimulation revealed elevated NF-{kappa}B activation compared with NCF cells. Additionally, elevated nuclear levels of the NF-{kappa}B inhibitor I{kappa}B{alpha} were detected in nuclei of CF cells after P. aeruginosa stimulation, but this increase was transient. There was no difference in IL-8 mRNA levels between CF and NCF cells early after stimulation, whereas expression was higher and sustained in CF cells at later times. Our results also demonstrated increased baseline translocation of NF-{kappa}B to nuclei of primary CF epithelial cell cultures, but intranuclear I{kappa}B{alpha} may initially block its effects following P. aeruginosa stimulation. Thus, IL-8 mRNA expression was prolonged after P. aeruginosa stimulation in CF epithelial cells, and this sustained IL-8 expression may contribute to the excessive inflammatory response in CF.

interleukin-8; I{kappa}B; nuclear factor-{kappa}B; cystic fibrosis


AIRWAY INFLAMMATION IN RESPONSE to bacterial pathogens accounts for much of the progressive, suppurative pulmonary disease that ultimately leads to the death of cystic fibrosis (CF) patients. Yet, it remains unclear whether this increased response is related to the nature of the infectious stimulus, an inherent defect of the respiratory epithelium, or both (810, 1418, 20, 33). Clinical studies have revealed elevated levels of cytokines and neutrophils in the lungs of CF patients, even those with mild lung disease or in the absence of infection (10, 15, 24, 25, 26, 32, 37). In response to similar levels of pulmonary infection (30), greater inflammation has been found in CF patients and mice (44) compared with unaffected controls. Evidence from several studies shows an association between the absence of a functional cystic fibrosis transmembrane conductance regulator (CFTR) and an altered inflammatory response to various stimuli (8, 9, 17, 41). However, a recent study using primary cultures of CF and non-CF (NCF) cells stimulated with bacterial products obtained from stationary phase growth cultures reports comparable responses in NCF vs. CF cells (6). Because exaggerated responses in CF cells could be demonstrated under specific conditions, these authors deduced that a mutant CFTR does not principally confer the exaggerated inflammatory response characteristic of CF cells.

With the use of genetically altered, immortalized epithelial cell lines, recent studies have shown that CF cells have greater activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) in response to stimulation with cytokines or live Pseudomonas aeruginosa than NCF cells (17, 45, 46). NF-{kappa}B regulates the expression of several genes encoding proinflammatory cytokines, chemokines, and adhesion molecules involved in the inflammatory response in a variety of cells, including CF cells (4, 12, 17, 22, 34, 42, 43, 46). The NF-{kappa}B family consists of five DNA binding subunits (p50, p52, p65/RelA, C-Rel/Rel, and RelB) that exist as homodimers or heterodimers, with heterodimers of p50 and p65 subunits most abundant in induced cells (4, 38, 40). In resting cells, p50 and p65 heterodimers are sequestered in the cytoplasm bound primarily to their inhibitors I{kappa}B{alpha} and I{kappa}B{beta}. After cell stimulation, I{kappa}B{alpha} is phosphorylated and degraded, resulting in unmasking of the nuclear localization signal and consequent nuclear translocation of p50 and p65 heterodimers to the nucleus. NF-{kappa}B binds to specific regulatory DNA sequences and activates transcription of interleukin (IL)-8, a potent CXC chemokine and principal chemoattractant in the CF lung in a variety of cells, including CF airway cells (17, 28, 31, 35, 42, 43, 45, 47). In addition to the cytoplasmic role of I{kappa}B{alpha} as an inhibitor of NF-{kappa}B, newly synthesized nuclear I{kappa}B{alpha} inhibits NF-{kappa}B DNA binding activity by exporting NF-{kappa}B out of the nucleus and consequently inhibiting NF-{kappa}B-dependent transcriptional activity (2, 3, 36). Thus nuclear I{kappa}B{alpha} is important in controlling the cell's response to stimuli by regulating the initial activation of NF-{kappa}B.

To date, there have been no reports of regulation of IL-8 gene expression by NF-{kappa}B activation in response to live P. aeruginosa stimulation of airway epithelial cells isolated from CF and NCF patients and grown in primary culture. Using various cell culture models of CF and NCF airway epithelial cells, we have previously reported variable IL-8 secretion in response to P. aeruginosa infection. Nevertheless, IL-8 secretion from CF airway epithelial cells was substantially greater than NCF cells when grown in submerged culture (1). In this report, we extend our previous observations by comparing nuclear translocation of NF-{kappa}B subunits, nuclear levels of I{kappa}B{alpha}, and resultant IL-8 gene expression, in primary airway epithelial cells isolated from different CF ({Delta}508/{Delta}508) and NCF human airways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Bacterial and tissue culture reagents were obtained from Sigma (St. Louis, MO), and all DNA-modifying enzymes and nucleotides were purchased from Boehringer-Mannheim (Indianapolis, IN). All research conformed to National Institutes of Health guidelines.

Cell culture and viability. Human airway epithelial cells were isolated from CF and NCF lung tissue obtained under Washington University Human Studies Committee-approved protocols. Early (3 or 4) passage cells grown in primary culture as submerged monolayers from five CF ({Delta}F508/{Delta}F508) and three NCF patients were used in this study (1). With respect to NCF patients, pulmonary CFTR genotype data were not available. None of the NCF donors had evidence of lung infection at the time of death. Cell viability of the isolated CF and NCF cell cultures before and after P. aeruginosa stimulation was assessed using the LIVE/DEAD Assay (Molecular Probes, Eugene, OR) and trypan blue exclusion.

P. aeruginosa strain PAO1 was grown in Luria-Bertani broth to exponential phase and washed twice with PBS and once with airway epithelial cell culture media (19). A dose of either 108 colony-forming units (cfu)/ml (200 bacteria/epithelial cell) or 105 cfu/ml (calculated 0.2 bacteria/epithelial cell) was used to study the effects on isolated airway epithelial cell cultures. After the cell culture monolayers were incubated with either sterile medium or P. aeruginosa strain PAO1 for 1 h at 37°C, the cells were washed twice with PBS and refed with culture medium containing 500 µg/ml of gentamicin to kill residual bacteria. Incubation of these cells was continued for 3–29 h (the total exposure time is thus indicated as 4–30 h) to study the effects of exposure to P. aeruginosa. The cell culture monolayers were again washed with cold PBS twice before the collection of whole cell extracts, nuclear extracts, and total cellular RNA that were then analyzed using techniques that included immunoblot, gel shifts, and RNA blot analyses. As positive controls, CF and NCF cells were treated with TNF-{alpha} (100 ng/ml), and nuclear extracts were analyzed using immunoblot assays and ELISA.

Immunofluorescent microscopy. Cell cultures incubated sequentially with mouse anti-pancytokeratin antibody (Sigma) followed by goat-derived, Alexa Fluor 488-conjugated anti-mouse antibody (Molecular Probes) showed specific immunofluorescent staining, indicating epithelial origin. The proportion of the secretory cells in all airway epithelial cells grown in primary culture, as determined by periodic acid-Schiff staining, was ~20%.

To observe NF-{kappa}B translocation to the nucleus, primary cultures of CF and NCF cells were grown on chamber slides (Lab-Tek; Nalgene Nunc International, Naperville, IL) and incubated with 108 cfu/ml of P. aeruginosa for 30 min. Cells were rinsed with PBS, fixed with 4% formaldehyde in PBS for 15 min, rinsed again, and permeabilized with 0.5% Triton X-100 in PBS for 15 min. The cells were then blocked with 1% BSA-PBS for 1 h, incubated with rabbit-derived, anti-p65 antibody at 1:200 dilution (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h at room temperature, and followed by a 1-h incubation at room temperature with Alexa Fluor 568-conjugated anti-rabbit antibody at 1:1,000 dilution. The cells were thoroughly washed with PBS, and cell nuclei were stained with 4',6'-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). All images were obtained using an Olympus BX60 fluorescent microscope and Axiovision software.

EMSA or gel-shift assay. Nuclear protein extracts from CF and NCF cell cultures stimulated under the conditions described above were prepared using procedures previously published (21). Protein concentrations of the nuclear extracts were determined by the Bradford-Lowry assay (Bio-Rad, Hercules, CA) (11), and extract equivalency was verified by Coomassie blue (CBB) staining of SDS-PAGE gels. Nuclear extracts (10 µg) from each sample were incubated at room temperature for 30 min with binding buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM DTT, 5 mM EDTA, 25% glycerol), poly(dI-dC) (Pharmacia, Piscataway, NJ), and the labeled [{gamma}-32P]ATP NF-{kappa}B probe (Promega, Madison, WI). Specificity of NF-{kappa}B binding was established by addition of excess unlabeled probe DNA. Supershifts of the NF-{kappa}B p50 and p65 subunits were determined by incubating the protein-DNA complexes with anti-p50 and anti-p65 antibodies (Santa Cruz Biotechnologies). DNA-protein-antibody complexes were separated using 7% PAGE. Gels were dried and subjected to autoradiography. To account for differences in protein isolation and loading, individual nuclear extracts from CF and NCF cells were similarly processed by gel-shift analysis using an SP1 probe (Promega).

Immunoblot analyses. Whole cell extracts or nuclear protein extracts prepared as described above were boiled in Laemmli buffer, and 10 µg of each sample were then resolved using 7.5% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk and incubated sequentially with NF-{kappa}B anti-p50, anti-p65, anti-I{kappa}B{alpha}, and anti-I{kappa}B{beta} antibodies (Santa Cruz Biotechnologies) followed by either anti-rabbit or anti-mouse IgG horseradish peroxidase conjugate (Sigma). The membrane was washed vigorously, and 10 ml of chemiluminescence (ECL) detection solution (Amersham, Sunnyvale, CA) were applied. Luminescence emitted from the filter was detected following exposure to photographic film. To verify equal isolation and loading of protein, nuclear extracts resolved in 7.5% SDS-PAGE gels were CBB stained and quantified by densitometry. For whole cell extracts, nitrocellulose membranes were reprobed with anti-{beta}-actin antibody to normalize for equal loading of protein and measured using densitometry.

Total I{kappa}B{alpha} ELISA. I{kappa}B{alpha} levels in nuclear extract from CF and NCF cells were estimated using total I{kappa}B{alpha} ELISA (Bio-Source International, Camarillo, CA). Values obtained were normalized to total protein levels for each sample, and mean percent values were used to compare I{kappa}B{alpha} levels in CF and NCF cell cultures following P. aeruginosa exposure.

RNA preparation and analysis. Total RNA was isolated from CF and NCF respiratory epithelial cell cultures stimulated under the conditions described above. The cell monolayers were rinsed with cold PBS and detached with a cell scraper, and total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). The total RNA was further purified using RNeasy kits (Qiagen, Valencia, CA) and subjected to Northern blot analysis. The Northern blots were hybridized with human IL-8, and GAPDH cDNA using [{alpha}-32P]-labeled probes and the resultant signals were quantified by densitometry (19, 23).

Statistical analyses. Densitometry results were expressed as means ± SD. Comparisons between groups were made using unpaired two-tailed Student's t-tests, with P values ≤ 0.05 considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased baseline p65 protein expression in whole cell extracts of CF cells. Recent studies have shown that selected CF cell lines have greater activation of NF-{kappa}B compared with matched NCF controls. To determine whether a baseline difference exists in cytoplasmic levels of NF-{kappa}B or its inhibitor I{kappa}B, we examined pretreatment expression of NF-{kappa}B and I{kappa}B in airway epithelial cells, comparing NF-{kappa}B protein subunit expression in whole cell extracts from several different CF and NCF cell cultures. Proteins from whole cell extracts were separated and sequentially immunoblotted for p65, p50, I{kappa}B{alpha}, and I{kappa}B{beta} expression. Similar levels of p50 and I{kappa}B{alpha} protein expression were found in CF and NCF cells. However, increased baseline levels of p65 protein were observed in CF cells compared with NCF cells (Fig. 1, A and B). Finally, I{kappa}B{beta} protein could not be detected, despite repeated immunoblot analysis using several different antibodies (data not shown).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1. A: relative baseline expression of NF-{kappa}B protein subunits in whole cell extracts of cystic fibrosis (CF) and non-CF (NCF) airway epithelial cells. Whole cell extracts from 4 CF and 3 NCF epithelial cell cultures not exposed to Pseudomonas aeruginosa were subjected to immunoblot analysis using p50, p65, and I{kappa}B{alpha} antibodies. The blot was then stripped and processed for anti-{beta}-actin expression to account for differences in loading. C represents control for whole cell extracts. B: quantification of NF-{kappa}B and I{kappa}B subunits in CF and NCF whole cell extracts. Protein expression was measured using densitometry and then standardized relative to {beta}-actin levels to permit comparisons. Baseline expression of NF-{kappa}B p50, p65, and I{kappa}B{alpha} protein subunits are shown, demonstrating higher cellular levels of p65 in the CF airway epithelial cells even before stimulation. NF-{kappa}B and I{kappa}B{alpha} levels in CF and NCF cells were compared using Student's t-test; *P < 0.05.

 
Nuclear translocation of NF-{kappa}B in stimulated airway epithelial cells. Using immunocytochemical techniques, we examined NF-{kappa}B nuclear translocation in primary cultures of human airway epithelial cells before and after P. aeruginosa infection. Specifically, we compared primary cultures of human CF and NCF airway epithelial cells immunostained for the NF-{kappa}B p65 protein subunit. Chamber slide cultures of CF and NCF cells were stimulated with 108 cfu/ml of P. aeruginosa for 30 min, and immunostaining for p65 subunit revealed that P. aeruginosa induced rapid translocation of NF-{kappa}B from the cytoplasm to the nucleus in both CF and NCF cells (Fig. 2).



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 2. Nuclear translocation of NF-{kappa}B in primary culture CF and NCF airway epithelial cells stimulated with P. aeruginosa. Chamber slide cultures of CF and NCF epithelial cells were stimulated with or without 108 colony-forming units (cfu)/ml of P. aeruginosa for 30 min at 37°C. Cells were then immunostained using anti NF-{kappa}B p65 rabbit antibody and visualized by fluorescent microscopy. Cell nuclei were defined using fluorescent mounting medium containing 4',6'-diamidino-2-phenylindole. The p65 subunit is stained red, and nuclear translocated NF-{kappa}B is stained magenta (a product of the merging of red and blue fluorescence). A: unstimulated CF cells; B: CF cells infected with P. aeruginosa; C: unstimulated NCF cells; D: NCF cells infected with P. aeruginosa.

 
Greater NF-{kappa}B translocation in CF epithelial cells stimulated with P. aeruginosa. To study the effect of P. aeruginosa on NF-{kappa}B activation in CF and NCF cells, we performed gel-shift assays using nuclear extracts before and after stimulation. Early passage cultures of human CF and NCF airway epithelia were treated with 108 cfu/ml of P. aeruginosa (log phase growth) for 1 h followed by washing to rid cells of free bacteria in media containing gentamicin to kill residual bacteria, and incubation proceeded for an additional 3 h. Nuclear extracts were prepared, and NF-{kappa}B protein subunits were analyzed using anti-p65 and anti-p50 antibodies (Fig. 3A). We found increased baseline activation of NF-{kappa}B in unstimulated CF cells compared with NCF cells. Furthermore, stimulation with P. aeruginosa resulted in increased activated NF-{kappa}B in nuclear extracts of CF cells compared with identically treated NCF cells (Fig. 3B). Nuclear extracts from CF and NCF cells treated with TNF-{alpha} and analyzed using an NF-{kappa}B ELISA kit (Active Motif North America, Carlsbad, CA) demonstrated a rapid and increased activation of NF-{kappa}B (data not shown), similar to the effects of P. aeruginosa stimulation of airway epithelial cells.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. A: nuclear localization and elevated NF-{kappa}B DNA binding in primary culture airway epithelial cells stimulated with P. aeruginosa. Nuclear extracts from CF and NCF epithelial cells before (NS) and after 4 h of 108 cfu/ml of P. aeruginosa (PA) stimulation were prepared and subjected to gel-shift analysis. Protein from each nuclear extract (10 µg) was incubated with binding buffer and reagents including a [{gamma}-32P]-labeled NF-{kappa}B oligonucleotide probe as described in MATERIALS AND METHODS. A representative gel-shift assay is shown. P, nuclear extract with radiolabeled oligonucleotide probe; I, nuclear extract with a mixture of radiolabeled and unlabeled NF-{kappa}B oligonucleotide. p50 And p65 nuclear extract with labeled probe coincubated with anti-p50 and anti-p65 antibody, respectively. Note the increased activity of NF-{kappa}B in the CF epithelial cells compared with the NCF cells under unstimulated and stimulated conditions, a consistent finding in all CF epithelial cells tested. B: quantitation of NF-{kappa}B activation. Relative NF-{kappa}B activation was measured using densitometry. The results showed a consistently increased intranuclear level of NF-{kappa}B binding in CF airway epithelial cells compared with similarly treated NCF cells 4 h after stimulation with P. aeruginosa. NF-{kappa}B-activated lanes were compared using t-test; *P < 0.05.

 
To confirm these observations, we further subjected the same nuclear extracts to immunoblot analysis using anti-p50 and anti-p65 antibodies (Fig. 4). Again, compared with NCF cells, we observed baseline activation of NF-{kappa}B (indicated by increased nuclear levels) in unstimulated CF airway epithelial cells and increased levels of p50 and p65 protein subunits in CF nuclei after stimulation with P. aeruginosa. Densitometric analysis of CBB-stained SDS-PAGE gels verified the differences in protein levels were not due to unequal loading of protein. Nuclear extracts from CF and NCF cell cultures that were immunoblotted for the p50 protein subunit expression also revealed trace levels of p105, a precursor of the p50 subunit, and these levels remained unchanged even with prolonged stimulation with P. aeruginosa.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Immunoblot analysis of nuclear extracts from CF and NCF airway epithelial cells. Nuclear extracts were isolated from 4 CF and 3 NCF epithelial cells that were infected with 108 cfu/ml of P. aeruginosa for 4 h and then subjected to SDS-PAGE and Western blotting using anti-p50 and anti-p65 antibodies. Uninfected cells were used as controls. Note the elevated level p50 and p65 subunits in the nuclear extracts of CF epithelial cells both at baseline and after infection. Molecular weight sizes are indicated in the right margin.

 
IL-8 mRNA expression in human CF and NCF airway epithelial cells. To correlate observed NF-{kappa}B activation with IL-8 mRNA expression, we examined total RNA isolated from CF and NCF cells for IL-8 gene expression by Northern blot analysis before and after a 4-h stimulation with 108 cfu/ml of P. aeruginosa. Blots were stripped and reprobed for GAPDH expression to account for equal loading of RNA (Fig. 5A). No difference in IL-8 gene expression was found in CF airway epithelial cells compared with NCF cells at baseline or early (4 h) after stimulation (Fig. 5B).



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 5. A: expression of IL-8 mRNA in CF and NCF airway epithelial cells by Northern blot analysis. Total RNA from 5 CF and 3 NCF airway epithelial cell cultures was isolated before and 4 h after stimulation with 108 cfu/ml of P. aeruginosa at 37°C and then subjected to Northern blot analysis. The same blot was stripped and rehybridized with a radiolabeled GAPDH probe to account for equal loading of RNA. B: quantification of IL-8 expression in CF and NCF cells using densitometry. Results are shown as ratios of IL-8 RNA/GAPDH. Note the apparent similar levels of IL-8 mRNA in CF and NCF airway epithelial cells before (NS) and 4 h after P. aeruginosa stimulation (PA). Significant difference in IL-8 expression between unstimulated and stimulated epithelial cells. *P < 0.05.

 
To determine IL-8 mRNA expression for the later time response (after 4 h of P. aeruginosa exposure) in CF and NCF airway epithelial cells, we examined total RNA at 4–24 h of 108 cfu/ml of P. aeruginosa using Northern blot analyses (Fig. 6). There was a substantial increase in IL-8 mRNA expression in stimulated CF cells at later time points of infection compared with the NCF cells, especially at 24 h, but this result was accompanied by cell loss evident as detached cells. Epithelial cell death was assessed following infection with P. aeruginosa. We found greater cell death after infection with increasing bacterial inocula (Fig. 7). Of note, there was no difference in viability between CF and NCF airway epithelial cells. Incidentally, there was very little GAPDH signal for the 24-h time point for both CF and NCF cells, further confirming cell loss.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. Comparison of IL-8 mRNA expression in primary CF and NCF respiratory epithelial cells after stimulation. IL-8 gene expression was measured before (0 h) and 4, 8, and 24 h after stimulation with 108 cfu/ml of P. aeruginosa. Total RNA isolated from CF and NCF cells isolated at different times after P. aeruginosa stimulation was subjected to Northern blot analysis. The results showed significantly elevated levels of IL-8 mRNA detection at later time points, especially 24 h after infection, for CF airway epithelial cells despite greater cell death at that bacterial inoculum. Note the loss of GAPDH signal detection in the 24-h lane with P. aeruginosa exposure for CF as well as for NCF cells.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. Comparison of cell death between CF and NCF cell cultures exposed to increasing doses of P. aeruginosa. CF and NCF cells were exposed to 105, 107, and 109 cfu/ml of P. aeruginosa for 24 h, and the cell death was estimated by fluorescent microscopy using the LIVE/DEAD assay. We found greater cell death 24 h after infection with P. aeruginosa with increasing bacterial inocula. Of note, there was no difference in viability between CF and NCF airway epithelial cells.

 
We then examined the timing of IL-8 mRNA expression while minimizing epithelial cell death by infecting CF and NCF airway epithelial cells with a lower inoculum (105 cfu/ml) of P. aeruginosa. Northern blot analysis was performed using total RNA isolated from CF and NCF cells for up to 30 h after infection (Fig. 8A). Although there was some variability between samples, increased IL-8 mRNA expression was most often observed in CF epithelial cells at later time points after infection with P. aeruginosa (8, 24, and 30 h) compared with similarly treated NCF cells (Fig. 8B). As noted before, there were no significant differences in the levels of IL-8 mRNA expression between CF and NCF cells early (4 h) after infection. Thus IL-8 mRNA expression of CF and NCF airway epithelial cells was similar soon after P. aeruginosa infection, but expression diverged after the 4-h time point and was greater in CF cells.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 8. A: CF cells exhibit a sustained IL-8 mRNA expression in response to prolonged stimulation with P. aeruginosa. Total cellular RNA was isolated from the 3 CF and NCF cells before (0 h) and after (4, 8, 24, and 30 h) stimulation with 105 cfu/ml (a lower inoculum) of P. aeruginosa to preserve cell viability. B: quantitation of IL-8 expression. Relative IL-8 expression is shown as ratios of IL-8 RNA/GAPDH RNA measured using densitometry. The results showed a consistently elevated and sustained level of IL-8 mRNA expression in CF epithelial cells compared with similarly treated NCF airway epithelial cells up to 30 h after stimulation with P. aeruginosa. *P < 0.05.

 
Increased intranuclear levels of I{kappa}B{alpha} in CF cells early after stimulation. Our results indicated a similar early response in CF and NCF airway epithelial cells to P. aeruginosa, but a later IL-8 mRNA expression profile was different in that there was a significantly increased and sustained response in CF cells. We examined the possibility that early NF-{kappa}B-DNA binding activity is being inhibited by I{kappa}B{alpha}. It has been established that NF-{kappa}B and I{kappa}B{alpha} mutually regulate the other's level and activity, resulting in differential regulation of gene expression. This regulation not only controls the transcription of NF-{kappa}B-dependent genes in resting cells but also controls the primed upregulation of NF-{kappa}B-dependent transcription of immune or proinflammatory genes in response to various external stimuli. Conversely, a network of regulatory cytokines and immune function modulators regulate NF-{kappa}B-dependent transcription of several immune response genes. An important level of regulatory control for NF-{kappa}B is through interactions with I{kappa}B{alpha} in the cytosol as well as the nucleus. Indeed, newly synthesized nuclear I{kappa}B{alpha} is purported to play a role in limiting the cell's inflammatory responses to stimuli by exporting NF-{kappa}B out of the nucleus (2, 3, 36). As shown above, NF-{kappa}B was rapidly translocated to nuclei in CF cells early after stimulation with P. aeruginosa, but we were unable to correlate this observed increased NF-{kappa}B translocation in CF airway epithelial cells to increased IL-8 mRNA expression. To determine whether the differences in IL-8 expression seen at early and late time points after infection were reflected by differences in I{kappa}B protein levels, we examined I{kappa}B{alpha} and I{kappa}B{beta} levels in nuclear extracts from different CF and NCF epithelial cells before and after 4 h of stimulation with 108 cfu/ml of P. aeruginosa using immunoblot analysis. We consistently found elevated levels of I{kappa}B{alpha} protein expression early after stimulation in CF cells compared with NCF cells (Fig. 9A). No I{kappa}B{beta} was detected in nuclear extracts of any cell (data not shown). We quantitated the I{kappa}B{alpha} in nuclear extracts at 4-, 6-, and 8-h time points of 108 cfu/ml of P. aeruginosa (Fig. 9C).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 9. A: intranuclear I{kappa}B in CF airway epithelial cells. Nuclear extracts from 4 CF and 3 NCF epithelial cell cultures were collected before and 4 h after 108 cfu/ml of P. aeruginosa stimulation and then subjected to immunoblot analysis. I{kappa}B{alpha} protein expression was consistently found to be elevated in nuclear extracts from CF respiratory epithelial cells early after stimulation with P. aeruginosa. B: time course comparison of I{kappa}B{alpha} protein expression in primary CF and NCF respiratory epithelial cells after stimulation with P. aeruginosa. Nuclear extracts from CF and NCF cell cultures were evaluated for I{kappa}B{alpha} protein expression before (0 h) and after (4, 8, and 24 h) stimulation with 108 cfu/ml of P. aeruginosa. Note the elevated level of I{kappa}B{alpha} expression at the 4-h time point. C: intranuclear I{kappa}B{alpha} in CF airway epithelial cells early after stimulation with P. aeruginosa. CF and NCF nuclear extracts (n = 3) exposed to 108 cfu/ml of P. aeruginosa for 4, 6, and 8 h were assessed for I{kappa}B{alpha} levels using ELISA. The mean percent values for each time were quantitated to compare I{kappa}B{alpha} levels in CF and NCF cells. Together, these results demonstrate increased intranuclear I{kappa}B{alpha} levels early after stimulation with 108 cfu/ml of P. aeruginosa in CF cells relative to NCF cells. *P < 0.05.

 
Finally, using the anti-I{kappa}B{alpha} antibody, we performed immunoblot analyses on nuclear extracts from CF and NCF airway epithelial cells isolated 8 and 24 h after 108 cfu/ml of P. aeruginosa stimulation. We observed similar levels of I{kappa}B{alpha} protein expression in CF and NCF cells at these later time points (Fig. 9B), although the 24-h time point was again accompanied by excessive cell death. However, nuclear extracts stimulated with 105 cfu/ml of P. aeruginosa revealed similar levels of I{kappa}B{alpha} protein expression (data not shown). Based on our observation of early, transient intranuclear expression of I{kappa}B{alpha}, we propose that intranuclear I{kappa}B{alpha} may be negatively regulating NF-{kappa}B activity, thus inhibiting early IL-8 mRNA expression.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although infection contributes to the morbidity of patients with CF, the intense host inflammatory response largely accounts for the progressive, suppurative pulmonary disease. Several lines of evidence suggest that the inflammatory response is excessive to the threat posed by infection. We tested this hypothesis using human airway epithelial cells grown in primary cultures. We describe an exaggerated inflammatory response in airway epithelial cells grown in primary culture isolated from multiple CF and NCF patients to control for variability between individuals. Our results demonstrated a constitutive and increased activation of NF-{kappa}B that was associated with later IL-8 gene expression in CF cells compared with NCF cells, which is similar to findings from other reported studies that examined genetically engineered CF and "matched" controls (17, 18, 27, 34, 45). Indeed, differences in IL-8 gene expression between CF and NCF became more apparent at times well after P. aeruginosa stimulation. The lack of an exaggerated, early IL-8 response in CF cells to stimulation was unexpected and appeared to be related to transient NF-{kappa}B inhibition by intranuclear I{kappa}B{alpha}.

Several studies comparing cytokine expression have reported on the associated upregulation of NF-{kappa}B and increased production of the cytokine IL-8 in CF phenotype cells relative to corrected or normal cells. NF-{kappa}B also induces several of the proinflammatory cytokines implicated in CF lung disease, and, therefore, it is essential to understand the NF-{kappa}B signal transduction in CF cells. Five members of the mammalian NF-{kappa}B/Rel proteins (predominantly p65/p50 dimers) have been identified based on the presence of the Rel homology domain (RHD) sequence. RHD accomplishes functions such as dimerization, interactions with different forms of I{kappa}B{alpha}, and DNA binding. NF-{kappa}B-dependent transcriptional activity is controlled by its inhibitor I{kappa}B{alpha} in the cytoplasm as well as the nucleus. For the cytoplasmic retention of NF-{kappa}B, I{kappa}B{alpha} blocks the nuclear localization signal in NF-{kappa}B, and for the nuclear block, newly synthesized free I{kappa}B{alpha} moves to the nucleus where it prevents NF-{kappa}B-dependent transcription. Additionally, nuclear NF-{kappa}B is exported back to the cytoplasm through interaction with I{kappa}B{alpha} by virtue of the nuclear export sequence present in the COOH terminus of I{kappa}B{alpha}. Multiple NF-{kappa}B binding sites that are functional have been identified in I{kappa}B{alpha} genes by promoter analyses and, reportedly, I{kappa}B{alpha} mRNA can also be upregulated through nontranscriptional means. Hence, it has been established that NF-{kappa}B and I{kappa}B{alpha} mutually regulate the other's level and activity, resulting in differential regulation of gene expression. This regulation controls not only the transcription of NF-{kappa}B-dependent genes in resting cells but also controls the primed upregulation of NF-{kappa}B-dependent transcription of immune or proinflammatory genes in response to various external stimuli. Conversely, a network of regulatory cytokines and immune function modulators regulate NF-{kappa}B-dependent transcription of several immune response genes and proinflammatory cytokines. Adding to the complexity of NF-{kappa}B and I{kappa}B{alpha} autoregulation is the reported evidence for constitutive NF-{kappa}B expression as well as constitutive phoshorylation of I{kappa}B{alpha} in several cell types. Constitutively phosphorylated I{kappa}B{alpha} reportedly regulates the stability of free I{kappa}B{alpha} or its dimerization with NF-{kappa}B subunits, and constitutive NF-{kappa}B reportedly occurs due to the instability of I{kappa}B{alpha} (4, 5, 40).

The reported dysregulation of the CF epithelium response to infection is manifested as inflammation that is disproportionately increased or prolonged in relation to the level of stimuli and may even occur in the absence of infection. High levels of proinflammatory cytokines (i.e., IL-8) and neutrophils have been detected in the respiratory epithelial lining fluid of infants with CF, and several children with CF had evidence of pulmonary inflammation in the apparent absence of infection (24). Likewise, children with CF who were infected only with Haemophilus influenzae had higher concentrations of neutrophils and IL-8 in bronchoalveolar lavage fluid compared with disease-matched controls (32). Previous investigations have also reported on the increased expression of inflammatory genes in freshly isolated cells from CF patients with identifiable bacterial infections (1, 9). In the first report, to distance their epithelial cell cultures from stimuli and bacterial exposures in the native CF lung that may have contributed to an apparent "constitutive" response, freshly isolated CF cells were grown in primary culture continuously for 2 wk after isolation. In the present study, the primary CF and NCF cell cultures were subjected to three to four passages before being tested for inflammatory gene expression. We found greater baseline expression of p65 and increased activation of NF-{kappa}B in unstimulated CF airway epithelial cells compared with NCF cells, a finding consistent with other reported studies that demonstrated higher constitutive expression of NF-{kappa}B in CF cells (17, 34, 42, 43, 46). Hence, it is possible that inflammation may occur independently of infection or that relatively minor infections induce a robust inflammatory reaction in the CF lung that does not subside. Together with previous reports, our findings lend credence to an "inflammatory phenotype" in the CF respiratory epithelium that creates an ongoing pathological inflammatory response in the CF airway to otherwise trivial stimuli.

Several investigators have used transformed CF cell culture models to examine CF epithelial responses to cytokines or bacterial stimuli and have reported an excessive inflammatory response in CF cells compared with controls (17, 27, 34, 42, 43, 45, 46). Nevertheless, others have reported contradictory results (29, 39). Very recent data by Becker et al. (6) using primary cultures of differentiated CF and NCF cells demonstrate similar levels of NF-{kappa}B activation when stimulated with bacterial products. These inconsistencies may be related to various environmental factors, including differences in cell types, culture conditions, and genetic modifications caused by immortalization and complementation. It may also be influenced by the nature (for example, live bacteria as opposed to bacterial products, exponential phase cultures to stationary phase growth cultures, or inoculum size) and timing of the infectious stimulus (the majority of the studies that have reported on differences in responses between CF and NCF cells have reportedly used doses of 108 cfu/ml or higher). In the present study, early-passage CF and NCF airway epithelial cells grown in primary culture were propagated in parallel, ensuring uniform growth conditions and similar cell composition, as determined by immunocytological staining. Our results revealed increased translocation of NF-{kappa}B in CF epithelial cells stimulated with 108 cfu/ml of P. aeruginosa and greater IL-8 mRNA expression at later time points compared with NCF cells. Studying airway epithelial cells grown in primary culture has certain limitations, including the genetic variability of isolated cells from different individuals. We attempted to minimize this factor by examining cells isolated from multiple individuals who were homozygous for the same mutant CFTR allele ({Delta}F508/{Delta}F508). Using such cells, we found a consistent, discernible inflammatory phenotype in CF cells compared with NCF controls. Another potential confounder was that our studies were performed using epithelial cell monolayers grown in submerged cultures and not at an air-liquid interface, which more closely approximates a native, respiratory epithelium (6). In future investigations, we will include such models to examine NF-{kappa}B activation in human CF epithelial cells to confirm our results.

Many studies have described an exaggerated inflammatory response in CF epithelial models, manifested by increased IL-8 secretion in response to P. aeruginosa infection. Few have compared the inflammatory response in CF and NCF cells examining IL-8 gene expression at a molecular level. We compared the timing of IL-8 mRNA expression in CF and NCF cells at different times after stimulation with P. aeruginosa. We found that P. aeruginosa induced greater NF-{kappa}B translocation in CF cultures at 4 h, yet this NF-{kappa}B activation was not accompanied by increased IL-8 mRNA expression in the same cells at the early time point of 4-h stimulation. At later times, however, the expression of IL-8 mRNA was shifted, resulting in a prolonged response of up to 30 h in CF epithelial cells, thus demonstrating a sustained IL-8 response and apparent lack of a braking mechanism for downregulation of the IL-8 gene expression. The temporal difference between CF and NCF cells in inflammatory gene expression has been reported by other investigators (27), who found increasing differences in IL-8 secretion between CF and NCF cells at later times after infection. Thus these and other observations suggest an alternative IL-8 regulatory pathway early after stimulation. But for later time points after infection, CF airway epithelial cell signaling occurred via NF-{kappa}B with associated transcription of IL-8 gene expression (12, 28, 31, 35, 43, 47). Our observations also confirmed the potential importance of blocking early events in the inflammatory cascade by targeting NF-{kappa}B activation (13).

Finally, the absence of greater IL-8 mRNA expression in CF airway epithelial cells early (4 h) after P. aeruginosa stimulation was curious, especially since we found greater activation of NF-{kappa}B in the same cells. This finding suggested that NF-{kappa}B and its effects early after infection were impeded. Several studies investigating inflammatory stimuli in leukocytes in vitro have reported on the significance of NF-{kappa}B in cytokine gene transcription and their regulation. These reports have demonstrated the central role of NF-{kappa}B in not only synchronizing its own specific promoter interactions but also associating with other transcription factors as well as repressors that may account for differential transcription of cytokine genes (7). We observed increased levels of the NF-{kappa}B inhibitor I{kappa}B{alpha} in nuclear extracts from CF airway epithelial cells at early time points of P. aeruginosa stimulation, but not at the later time points. In contrast, NCF nuclear extracts did not exhibit any difference in levels of I{kappa}B{alpha} expression at any time after infection. Together, these results indicated that early after stimulation, elevated intranuclear I{kappa}B{alpha} in CF cells blunted the initial effects of activated NF-{kappa}B. Indeed, based on these results, we suspect that translocated NF-{kappa}B also induced synthesis of its own inhibitor I{kappa}B{alpha} in the CF cell nucleus, which transiently blocked the cells' response to P. aeruginosa stimulation by associating with NF-{kappa}B bound to the IL-8 promoter (36).

In summary, we have demonstrated a rapid activation and translocation of NF-{kappa}B in primary cultures of respiratory epithelial cells from CF and NCF patients in response to P. aeruginosa. The translocation of NF-{kappa}B subunits was consistently greater in CF cells compared with NCF cells. Early after P. aeruginosa stimulation, there was no difference in IL-8 expression, which appears to be related to I{kappa}B{alpha}-mediated inhibition. At later times, though, IL-8 mRNA expression was consistently greater in CF cells and was sustained, likely contributing to the excessive inflammation in the CF lung, where relatively minor stimuli induce an intense, uncontrolled response.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The work reported in this manuscript was supported by the Cystic Fibrosis Foundation, the March of Dimes, and National Heart, Lung, and Blood Institute Grant HL-64044.


    ACKNOWLEDGMENTS
 
We thank Kathryn Akers, Sharon Favors, and Lisa Hogue for expert technical support. We also thank Dr. Joseph Zabner and Phil Karp from the University of Iowa Cystic Fibrosis Center Epithelial Cell Core for the generous supply of airway epithelial cells from non-cystic fibrosis subjects.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ferkol, Division of Pediatric Allergy and Pulmonary Medicine, Dept. of Pediatrics, 660 S. Euclid Ave., Campus Box 8208, St. Louis, MO 63110 (E-mail: ferkol_t{at}kids.wustl.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aldallal N, McNaughton EE, Manzel LJ, Richards AM, Zabner J, Ferkol TW, and Look DC. Inflammatory response in airway epithelial cells isolated from patients with cystic fibrosis. Am J Respir Crit Care Med 166: 1248–1256, 2002.[Abstract/Free Full Text]
  2. Arenzana-Seisdedos F, Thomson J, Rodriguez MS, Bachelerie F, Thomas D, and Hay RT. Inducible nuclear expression of newly synthesized I{kappa}B{alpha} negatively regulates DNA-binding and transcriptional activities of NF-{kappa}B. Mol Cell Biol 15: 2689–2696, 1995.[Abstract]
  3. Arenzana-Seisdedos F, Turpin P, Rodriguez M, Dominique T, Hay RT, Virelizer JL, and Dargemont C. Nuclear localization of I{kappa}B{alpha} promotes active transport of NF-{kappa}B from the nucleus to the cytoplasm. J Cell Sci 110: 369–378, 1997.[Abstract/Free Full Text]
  4. Baeuerle PA and Henkel T. Function and activation of NF-{kappa}B in the immune system. Annu Rev Immunol 12: 141–179, 1994.[CrossRef][ISI][Medline]
  5. Baldwin AS. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu Rev Immunol 14: 649–683, 1996.[CrossRef][ISI][Medline]
  6. Becker MN, Sauer MS, Muhlebach MS, Hirsh AJ, Wu Q, Verghese MW, and Randell SH. Cytokine secretion by cystic fibrosis airway epithelial cells. Am J Respir Crit Care Med 169: 645–653, 2004.[Abstract/Free Full Text]
  7. Blackwell TS and Christman JW. The role of NF-{kappa}B in cytokine gene regulation. Am J Respir Cell Mol Biol 17: 3–9, 1997.[Abstract/Free Full Text]
  8. Blackwell TS, Stecenko AA, and Christman JW. Dysregulated NF-{kappa}B activation in cystic fibrosis: evidence for a primary inflammatory disorder. Am J Physiol Lung Cell Mol Physiol 281: L69–L70, 2001.[Free Full Text]
  9. Bonfield TL, Konstan MW, and Berger M. Altered respiratory epithelial cell cytokine production in cystic fibrosis. J Allergy Clin Immunol 104: 72–78, 1999.[ISI][Medline]
  10. Bonfield TL, Panuska JR, Konstan MW, Hilliard KA, Hilliard JB, Ghnaim H, and Berger M. Inflammatory cytokines in cystic fibrosis lungs. Am J Respir Crit Care Med 152: 2111–2118, 1995.[Abstract]
  11. Bradford M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 72: 248–254, 1976.[CrossRef]
  12. Carter AB, Knudtson KL, Monick MM, and Hunninghake GW. The p38 mitogen-activated protein kinase is required for NF-{kappa}B-dependent gene expression. The role of TATA-binding protein (TBP). J Biol Chem 274: 30858–30863, 1999.[Abstract/Free Full Text]
  13. Christman JW, Lancaster LH, and Blackwell TS. Nuclear factor {kappa}B: a pivotal role in the systemic inflammatory response syndrome and new target for therapy. Intensive Care Med 24: 1131–1138, 1998.[CrossRef][ISI][Medline]
  14. Conese M and Assael BM. Bacterial infections and inflammation in the lungs of cystic fibrosis patients. Pediatr Infect Dis J 20: 207–213, 2001.[CrossRef][ISI][Medline]
  15. Dakin CJ, Numa AH, Wang H, Morton JR, Vertzyas CC, and Henry RL. Inflammation, infection and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 165: 904–910, 2002.[Abstract/Free Full Text]
  16. De Bentzmann S, Roger P, and Puchelle E. Pseudomonas aeruginosa adherence to remodeling respiratory epithelium. Eur Respir J 9: 2145–2150, 1996.[Abstract/Free Full Text]
  17. DiMango E, Ratner AJ, Bryan R, Tabibi, and Prince A. Activation of NF-{kappa}B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J Clin Invest 101: 2598–2606, 1998.[Abstract/Free Full Text]
  18. DiMango E, Zar HJ, Bryan R, and Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest 96: 2204–2210, 1995.[ISI][Medline]
  19. Frick AG, Joseph TD, Pang L, Rabe AM, St. Geme JW, and Look DC. Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells. J Immunol 164: 4185–4196, 2000.[Abstract/Free Full Text]
  20. Goldman MJ, Anderson GM, Stolzenberg ED, Kair UP, Zasloff M, and Wilson JM. Human {beta}-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 553–560, 1997.[CrossRef][ISI][Medline]
  21. Hart L, Lim S, Adcock I, Barnes PJ, and Chung KF. Effects of inhaled corticosteroid therapy on expression and DNA-binding activity of the nuclear factor {kappa}B in asthma. Am J Respir Crit Care Med 161: 224–231, 2000.[Abstract/Free Full Text]
  22. Jany B, Betz R, and Schreck R. Activation of the transcription factor NF-{kappa}B in human tracheobronchial epithelial cells by inflammatory stimuli. Eur Respir J 8: 387–391, 1995.[Abstract/Free Full Text]
  23. Joseph TD and Look DC. Specific inhibition of interferon signal transduction pathways by adenoviral infection. J Biol Chem 276: 47136–47142, 2001.[Abstract/Free Full Text]
  24. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, and Riches DWH. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med 151: 1075–1082, 1995.[Abstract]
  25. Konstan MW and Berger M. Current understanding of the inflammatory process in cystic fibrosis: onset and etiology. Pediatr Pulmonol 24: 137–142, 1997.[CrossRef][ISI][Medline]
  26. Konstan MW, Hilliard KA, Norvell TM, and 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 150: 448–454, 1994.[Abstract]
  27. Kube D, Sonitich U, Fletcher D, and Davis PB. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 280: L493–L502, 2001.[Abstract/Free Full Text]
  28. Li J, Johnson DX, Iazvovskaia S, Tan A, Lin A, and Hershenson MB. Signaling intermediates required for NF-{kappa}B activation and IL-8 expression in CF bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 284: L307–L315, 2003.[Abstract/Free Full Text]
  29. Massengale ARD, Quinn F Jr, Yankaskas J, Weissman D, McClellan WT, Cuff C, and Aronoff SC. Reduced interleukin-8 production by cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 20: 1073–1080, 1999.[Abstract/Free Full Text]
  30. Muhlebach MS and Noah TL. Endotoxin activity and inflammatory markers in the airways of young patients with cystic fibrosis. Am J Respir Crit Care Med 165: 911–915, 2002.[Abstract/Free Full Text]
  31. Mukaida N, Mahe Y, and 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 265: 21128–21133, 1990.[Abstract/Free Full Text]
  32. Noah TL, Black HR, Cheng PW, Wood RE, and Leigh MW. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J Infect Dis 175: 638–647, 1997.[ISI][Medline]
  33. Plotkowski MC, Chevillard M, Pierrot D, Altemayar D, Zham JM, Colliot G, and Puchelle E. Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture. J Clin Invest 87: 2018–2028, 1991.[ISI][Medline]
  34. Ramis I, Bioque G, Lorente J, Jares P, Quesada P, Rosello-Catafau J, Gelpi E, and Bulbena O. Constitutive nuclear factor-{kappa}B activity in human upper airway tissues and nasal epithelial cells. Eur Respir J 15: 582–589, 2000.[Abstract/Free Full Text]
  35. Ratner AJ, Bryan R, Weber A, Nguyen S, Barnes D, Pitt A, Gelber S, Cheung A, and Prince A. Cystic fibrosis pathogens activate Ca2+ dependent mitogen activated protein kinase signaling pathways in airway epithelial cells. J Biol Chem 276: 19267–19275, 2001.[Abstract/Free Full Text]
  36. Rodriguez MS, Thomson J, Hay RT, and Dargemont C. Nuclear retention of I{kappa}B{alpha} protects it from signal-induced degradation and inhibits nuclear factor {kappa}B transcriptional activation. J Biol Chem 274: 9108–9115, 1999.[Abstract/Free Full Text]
  37. Rosenfeld M, Gibson RL, McNamara S, Emerson J, Burns JL, Castile R, Hiatt P, McCoy K, Wilson CB, Inglis A, Smith A, Martin TR, and Ramsey BW. Early pulmonary infection, inflammation and clinical outcomes in infants with cystic fibrosis. Pediatr Pulmonol 32: 356–366, 2001.[CrossRef][ISI][Medline]
  38. Schmitz ML and Baeuerle PA. The p65 subunit is responsible for the strong transcription activating potential of NF-{kappa}B. EMBO J 10: 3805–3817, 1991.[Abstract]
  39. Schwiebert LM, Estell K, and Propst SM. Chemokine expression in CF epithelia: implications for the role of CFTR in RANTES expression. Am J Physiol Cell Physiol 276: C700–C710, 1999.[Abstract/Free Full Text]
  40. Siebenlist U, Franzoso G, and Brown K. Structure, regulation and function of NF{kappa}B. Annu Rev Cell Biol 10: 405–455, 1994.[CrossRef][ISI][Medline]
  41. Stecenko AA, King G, Torri K, Breyer RM, Dworski R, Blackwell TS, Christman JW, and Brigham KL. Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells. Inflammation 25: 145–155, 2001.[CrossRef][ISI][Medline]
  42. Tabary O, Escotte S, Couetil JP, Hubert Drusser DD, Puchelle E, and 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 155: 473–481, 1999.[Abstract/Free Full Text]
  43. Tabary O, Muelaet C, Escotte S, Antonicelli F, Hubert D, Dusser D, and Jacquot J. Interleukin-10 inhibits elevated chemokine interleukin-8 and regulated on activation normal T cell expressed and secreted production in cystic fibrosis bronchial epithelial cells by targeting the I{kappa}B kinase {alpha}/{beta} complex. Am J Pathol 162: 293–302, 2003.[Abstract/Free Full Text]
  44. Van Heeckeren A, Walenga R, Konstan MW, Bonfield T, Davis PB, and Ferkol T. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J Clin Invest 100: 2810–2815, 1997.[Abstract/Free Full Text]
  45. Venkatakrishnan A, Stecenko AA, King G, Blackwell TR, Brigham KL, Christman JW, and Blackwell TS. Exaggerated activation of NF-{kappa}B and altered I{kappa}B-{beta} processing in cystic fibrosis bronchial epithelial cells. Am J Cell Mol Biol 23: 396–403, 1999.[ISI]
  46. Weber AJ, Soong G, Bryan R, Saba S, and 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 281: L71–L78, 2001.[Abstract/Free Full Text]
  47. Yasumoto K, Okamoto S, Mukaida N, Murakami S, Mai M, and Matsushima K. Tumor necrosis factor {alpha} and interferon {gamma} synergistically induce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF{kappa}B-like binding sites on the interleukin 8 gene. J Biol Chem 267: 22506–22511, 1992.[Abstract/Free Full Text]