College of Physicians and Surgeons, Columbia University, New York, New York 10032
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ABSTRACT |
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Polymorphonuclear leukocyte-dominated airway inflammation is a
major component of cystic fibrosis (CF) lung disease and may be
associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant F508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause "cell stress" and activation of nuclear factor (NF)-
B. G551D mutants also lack Cl
channel function, but CFTR is trafficked
normally. We compared the effects of CFTR mutations on the endogenous
activation of an NF-
B reporter construct. In transfected Chinese
hamster ovary cells, the mistrafficked
F508 allele caused a
sevenfold activation of NF-
B compared with wild-type CFTR or the
G551D mutant (P < 0.001). NF-
B was also activated
in 9/HTEo
/pCep-R cells and in 16HBE/pcftr
antisense cell lines, which lack CFTR Cl
channel function
but do not accumulate mutant protein in the ER. This endogenous
activation of NF-
B was associated with elevated interleukin-8
expression. Impaired CFTR Cl
channel activity as well as
cell stress due to accumulation of mistrafficked CFTR in the ER
contributes to the endogenous activation of NF-
B in cells with the
CFTR mutation.
nuclear factor-B; cystic fibrosis transmembrane conductance
regulator; chloride channel; inflammatory response; intracellular
calcium; mitogen-activated protein kinase
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INTRODUCTION |
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CYSTIC
FIBROSIS (CF) transmembrane conductance regulator (CFTR)
mutations have many effects on the physiology of respiratory epithelial
cells in addition to the expected effects on Cl
transport. One of the major clinical manifestations of CFTR mutations is excessive airway inflammation (14-17), implying
that CFTR affects the immune function of airway epithelial cells,
particularly the expression of nuclear factor (NF)-
B-dependent
proinflammatory chemokines and cytokines. Several lines of experimental
evidence suggest that CFTR dysfunction influences the exogenous
activation of epithelial immune function (8, 30). Bacteria
persist in the respiratory tract of CF patients due to decreased
antimicrobial activity in CF airway surface fluid and impair bacterial
clearance due to mucus plugging (25). Cells with CFTR
mutations have increased numbers of asialoglycolipid receptors for
common bacterial pathogens (24). Ligation of these
receptors activates NF-
B-dependent epithelial interleukin (IL)-8
production to recruit polymorphonuclear leukocytes (PMNs) from the
circulation into the airways (8). Epithelial expression of
IL-8 after bacterial stimulation is increased in cells with CFTR
dysfunction, a finding demonstrated in both matched CF and corrected
cell lines (5, 9, 27, 30) and in cftr-deficient
[cftr(
/
)] mice compared with normal control mice
(29). How CFTR dysfunction might affect the endogenous expression of NF-
B-dependent genes is less well documented.
NF-B is a transcription factor required for the expression of
many genes important in inflammation. It is complexed in the cell
cytoplasm with I
B-
and -
, which are selectively
phosphorylated, ubiquitinated, and degraded in the proteasome in
response to stimuli (2). There is both signal-dependent
and signal-independent I
B-
phosphorylation (20), a
distinction that may be relevant to the endogenous activity of NF-
B
as opposed to the activation in response to a superficial stimulus such
as bacterial attachment to surface receptors. Ligation of superficial
asialo-GM1 receptors on epithelial surfaces by bacterial
adhesins stimulates Ca2+-dependent kinase activity through
mitogen-activated protein kinase phosphorylation and NF-
B activation
(23a). NF-
B translocation may be regulated by other
intracellular factors that lead to a different pattern of I
B-
phosphorylation than that elicited by extracellular stimuli
(4). It has been proposed that the abnormal trafficking of
F508 CFTR and its accumulation in the endoplasmic reticulum (ER)
provides sufficient intracellular stress to cause NF-
B activation
(2).
The most common CFTR mutation responsible for CF in ~70% of
patients is the deletion of a phenylalanine residue (F508) in the
nucleotide binding domain. The
F508 mutant CFTR not only fails to
transport Cl
in response to cAMP but accumulates in the
ER due to abnormal maturation and folding (1, 7, 32). From
experimental data generated with adenovirus mutants targeted for
retention in the ER, Pahl and colleagues (18, 19)
proposed that homozygous
F508 mutations in CFTR would similarly
cause Ca2+ release, activation of NF-
B, and resultant
transcription of proinflammatory cytokines and chemokines. Comparison
of the endogenous activation of NF-
B in IB3 cells (W1282X/
F508),
a trafficking mutant similar to the homozygous
F508 mutation, with
that in corrected C-38 cells, which express a functional but truncated form of CFTR, was consistent with this hypothesis. The
transcriptionally active p65 component of NF-
B was found in the
nuclei of unstimulated CF cells but not of the corrected cells by
immunofluorescence labeling and was confirmed by gel shift assays
(8). Manipulations to increase CFTR trafficking to the
apical surface of the cell, such as growth at low temperature or the
addition of glycerol, decreased the amount of p65 found in the IB3 cell
nuclei. Although these observations were consistent with the hypothesis
that abnormal CFTR proteins cause endogenous activation of NF-
B,
they did not differentiate between two possible mechanisms: that
"cell stress" associated with CFTR mistrafficking leads to changes
in intracellular Ca2+ concentration
([Ca2+]i) and stimulation of NF-
B
translocation or that CFTR Cl
channel dysfunction by
itself is associated with endogenous activation of NF-
B.
Other types of CFTR mutations cause "CF-like" physiology, i.e.,
lack of Cl secretion in response to cAMP, without causing
trafficking abnormalities or resulting in the accumulation of mutant
proteins in the ER (7). Analysis of the activation of
NF-
B in such cell lines would provide the opportunity to determine
whether Cl
channel dysfunction alone is associated with
the regulation of NF-
B. The G551D CFTR mutant, in which a conserved
glycine in the ATP binding cassette is mutated, lacks Cl
channel function, but the protein is properly folded and trafficked (12). Patients with the G551D mutation have a clinical
disease that is indistinguishable from that caused by the more common
F508 mutation (10). The 9/HTEo
human
tracheal epithelial cell line transfected with a plasmid directing
constitutive expression of the CFTR regulatory (R) domain also fails to
transport Cl
in response to cAMP while expressing low
levels of wild-type CFTR message detected by RT-PCR (21).
These 9/HTEo
/pCep-R cells are readily activated by
Pseudomonas aeruginosa and express significantly more IL-8
than control cells containing the empty vector (5). The
impact of impaired CFTR Cl
channel activity on the normal
interaction of I
Bs and NF-
B is not known. Recently constructed
16HBE cell lines expressing plasmid-encoded cftr in the
sense or antisense orientation also provide the opportunity to test the
impact of CFTR Cl
channel function on the activation of
NF-
B in airway cells without the confounding issues of mistrafficked
CFTR protein (23).
In the experiments detailed in this report, several matched cell lines
with CFTR dysfunction due to lack of Cl channel function,
CFTR misfolding, mistrafficking, or both were tested for endogenous
activation of NF-
B and basal IL-8 expression. Although CFTR
mistrafficking typical of the common
F508 mutation and associated
cell stress may be responsible for the increased expression of
proinflammatory cytokines and chemokines associated with CF cells, we
sought to establish whether CFTR Cl
channel function as
well as the effects of CFTR mistrafficking contributes to endogenous
activation of NF-
B and the excessive inflammation that characterizes
CF airway pathology.
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MATERIALS AND METHODS |
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Epithelial Cell Lines
9/HTEoIL-8 Assays
RT-PCR analysis. Total RNA was isolated from confluent monolayers of IB3 and C-38 cells grown in 10-cm plates with RNeasy Mini Kit, and RT was performed with Omniscript RT (both from QIAGEN, Valencia, CA). PCR primers for human IL-8 were 5'-TACTCCAAACCTTTCCAACCC-3' and 5'-AACTTCTCCACAACCCTCTG-3'. The cells were stimulated by the addition of 0.5 ml of P. aeruginosa PAO1 (5 × 109 colony-stimulating units/ml) scraped from an agar plate and resuspended in cell culture medium.
ELISA. IL-8 in epithelial cell culture supernatants was measured with ELISA (R&D Systems, Minneapolis, MN). The cells were weaned from serum for 18 h, and the supernatants were harvested 18 h later for IL-8 ELISA. Duplicate wells were treated with trypan blue to assess epithelial viability during the assay, which was >75%. Each IL-8 data point was determined in quintuplicate, and means ± SD were calculated. Significance was evaluated with a one-way ANOVA with Dunnett's posttest (GraphPad Instat version 3.00, GraphPad Software, San Diego, CA) to test the null hypothesis that there was no difference in the amount of IL-8 produced by the epithelial cells with different CFTR function.
Activation of NF-B Detected by Luciferase Reporter
Constructs
[Ca2+]i in IB3 and C-38 Cells
Relative levels of [Ca2+]i were estimated in fluo 3-loaded IB3 and C-38 cells by fluorescence imaging. Monolayers of C-38 or IB3 cells were grown for 2 days on Lab-TekII chambered slides (Nalge Nunc International, Naperville, IL) in LHC-8 medium (Biofluids) supplemented with 10% FCS. The monolayers were washed and dye loaded with fluo 3-AM (Bio-Rad, Hercules, CA) for 0.5 h at room temperature in the dark in modified Ringer solution (145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 2 mM CaCl2, 10 mM glucose, 20 mM HEPES, and 1 mM MgSO4, pH 7.4). The cells were rinsed in Ringer solution and incubated for 30 min to allow continued cleavage of the acetoxymethyl ester. The monolayers were imaged on a Zeiss Axiovert microscope at ×40 magnification with Inovision software. Images were taken at 10-s intervals before and after the addition of stimuli, and each image is the average of eight scans. Final measurements were taken after the addition of the ionophore 4-bromo-A-23187 (Bio-Rad) to allow for correction of differences in dye loading. Images were analyzed with Scion image software (Scion, Frederick, MD). Significance was determined with an unpaired t-test to compare the mean relative fluorescence of the two populations of epithelial cells.Immunocytochemistry
Airway epithelial cells were grown to near confluence on Lab-TekII eight-chambered glass slides (Nalge Nunc International). They were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, washed three times with PBS, and blocked in 10% goat serum in Ca2+/Mg2+ PBS for 1 h at room temperature. After blocking, the cells were washed three times in PBS and incubated with a 1:100 dilution of rabbit polyclonal antibody p65 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature, washed, and then incubated with goat anti-rabbit IgG conjugated to tetramethylrhodamine B isothiocyanate (TRITC; 1:50; Zymed, South San Francisco, CA) in PBS plus 0.1% BSA plus an equal volume of bis-benzimide (50 µg/ml) for 1 h at room temperature. The cells were imaged on a Zeiss Axiovert S100 microscope with a Hamamatsu Orca II digital camera and Inovision software. 560D15 and 400DF15 band-pass filters were used for TRITC and bis-benzimide imaging, respectively. Emission data were collected with a triple-pass filter at wavelengths of 460 nm for bis-benzimide and 602 nm for TRITC. ![]() |
RESULTS |
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Activation of NF-B in Epithelial Cells With CFTR Dysfunction
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Nuclear p65 (Rel A) in Cells With CFTR Dysfunction Detected by Immunofluorescence
The presence of the p65 (Rel A) component of NF-
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Activation of NF-B in CHO Cells Expressing Wild-Type and
Mutant CFTR
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IL-8 Expression Is Increased in Cell Lines With CFTR Dysfunction
The nuclear localization of NF-
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IL-8 production could be reliably measured by ELISA in
9/HTEo and 16HBE cell culture supernatants accumulated
over 18 h after being weaned from serum. The
9/HTEo
/pCep-R cells produced more IL-8 than those
expressing the vector control (P < 0.001), and lack of
CFTR expression in the 16HBE cells expressing cftr antisense
was also associated with increased IL-8 expression (P < 0.001; Fig. 5). Thus cell lines with
defective CFTR Cl
channel activity, regardless of the
nature of the CFTR defect, had increased IL-8 production compared with
the corresponding cell line with normal CFTR activity.
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[Ca2+]i in Cells With ER Overload
Epithelial cells with mistrafficked CFTR and "ER overload" have been postulated to have increased release of Ca2+ from ER stores as a consequence, a response that could activate Ca2+-dependent kinase activity. Relative [Ca2+]i was estimated in subconfluent IB3 and C-38 cells loaded with the fluorescent indicator fluo 3-AM (Fig. 6). When the populations of the different cell types were compared, there was a consistent trend of increased [Ca2+]i associated with the IB3 cells, with relative basal fluorescence ranging from 0.6 to 0.85 compared with that in the population of C-38 cells, which had basal [Ca2+]i in the range of 0.5 to 0.75. Comparing the relative fluorescence of 10 individual cells at a single time point (30 s), the IB3 cells had a relative mean fluorescence of 0.758 ± 0.012 (SD) compared with 0.628 ± 0.08 for the C-38 cells (P < 0.001). Both cell types responded to the addition of P. aeruginosa with increased [Ca2+]i.
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DISCUSSION |
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Airway inflammation is a major factor in the pathogenesis of CF
pulmonary disease. Clinical studies have clearly established a direct
correlation between infection, IL-8 secretion, and the accumulation of
PMNs and their toxic products in the CF lung (14-17). Bacterial stimulation of cells with CFTR mutations activates
significantly greater cytokine expression than matched cells with
normal CFTR function (5, 8, 29, 30). Several reports
(8, 28) suggest that cells with CFTR dysfunction have
endogenously upregulated expression of proinflammatory cytokines and
chemokines. Young infants with CF, even without detectable evidence of
prior infection, have PMNs and increased amounts of IL-8 in their
airways (14), and proinflammatory cytokine expression is
increased in cells cultured directly from uninfected CF tissues
(28). These findings are consistent with the in vitro
studies demonstrating endogenous activation of NF-B in CF but not in
corrected cell lines. As indicated by the data in this report, lack of
CFTR Cl
channel function and the physiological
consequences of mistrafficked mutant CFTR both appear to contribute to
the endogenous activation of NF-
B in CF airway cells, which is
manifested by the increased expression of IL-8 in these cells, even in
the absence of bacterial stimulation.
There is evidence of endogenous activation of NF-B in cells with
CFTR dysfunction due to several different mechanisms. In previous
studies, IB3 cells, which express the W1282X/
F508 mutation associated with mistrafficked CFTR that accumulates in the ER, were
found to have significant amounts of the p65 component of NF-
B in
nuclei under basal conditions in which there was no nuclear NF-
B in
the corrected C-38 cells (8). Because the translocation of
NF-
B can be associated with many types of stimuli, additional consequences of accumulated mutant CFTR in the ER were also
demonstrated in the mutant cell line. The IB3 cells were found to have
higher basal [Ca2+]i, a predicted consequence
of "ER stress" (2, 18). The presence of nuclear
NF-
B in the IB3 cells could be correlated with increased IL-8
expression as estimated by RT-PCR compared with that in the control
C-38 cell line. The concentration of IL-8 in the tissue culture
supernatant was minimal in these cells after an 18-h incubation in
serum-free medium; however, the relative amount of IL-8 message was
consistent with a previous report by DiMango et al.
(8). Not all investigators have found increased
endogenous levels of IL-8 associated with CFTR mutations
(30). Cell culture conditions, effects of FCS, and
handling of the monolayers all can affect the translocation of NF-
B
and endogenous expression of IL-8. Thus it seems relevant to
demonstrate that the presence of nuclear p65 appears to be associated
with the expected biological response, namely IL-8 expression.
The activation of other NF-B-dependent pathways is not necessarily
increased in IB3 compared with C-38 cells. Stimulation of NF-
B
translocation is associated with an antiapoptotic effect in many
cell lines including respiratory epithelial cells. Rates of
apoptosis can be increased by proteasome inhibitors that
inhibit I
B degradation and block NF-
B translocation
(31). In a study published recently (23), the
rates of IB3 and C-38 cell apoptosis were equivalent both under
control conditions and after bacterial stimulation despite the observed
differences in endogenous nuclear NF-
B. Thus the association between
CFTR dysfunction, activation of NF-
B, and transcription of
NF-
B-dependent genes is somewhat selective and is not a global response.
The consequences of CFTR mistrafficking and the activation of NF-B
were further demonstrated in CHO cells that do not normally express
CFTR. The presence of the mistrafficked mutant
F508 CFTR was a
significant stimulus for NF-
B activation in CHO cells. The G551D
CFTR, which does not function appropriately as a Cl
channel but is trafficked normally to the apical surface of the respiratory epithelial cell (10), did not stimulate
NF-
B. Expression of normal CFTR from the same plasmid construct did
not cause NF-
B activation in CHO cells nor did transfection with the
empty vector or the luciferase reporter construct. Although both the
G551D and
F508 CFTR mutations are associated with clinical disease and lack of Cl
secretion in response to cAMP
(10), the major difference between these mutants is the
mistrafficking and accumulation of
F508 CFTR within the ER. Thus it
appears that the consequences of mistrafficking of
F508 are
sufficient to activate NF-
B in CHO cells.
In epithelial cells, unlike CHO cells, there appears to be a
requirement for normal CFTR function in regulating NF-B-dependent gene transcription. The homozygous
F508 or the compound
F508/W1282X mutation could activate NF-
B by two independent
mechanisms: cell stress associated with mistrafficking or effects
directly due to lack of CFTR Cl
channel activity. Cell
lines that lack CFTR Cl
function due to either the
overproduction of the CFTR R-domain or the expression of CFTR antisense
had activated NF-
B and significant amounts of endogenous IL-8
production compared with the corresponding control cell lines. The
amount of IL-8 endogenously expressed by these cell lines was greater
than that of the IB3 or C38 cells and could be quantified by a standard
ELISA assay. The presence of the p65 component of NF-
B in nuclei of
undisturbed cells grown on coverslips as well as the relative
activation of an NF-
B reporter construct compared with appropriate
controls indicates that their activated state is unlikely to be an
artifact of either cell transformation or manipulations occurring
during cell culture. Because neither the 9/HTEo
/pCep-R or
the 16HBE/cftr antisense cells have excessive CFTR accumulation in the ER, the lack of CFTR Cl
channel
function in these epithelial cells appears to be responsible for the
observed increase in NF-
B activation and IL-8 expression.
Exactly how CFTR dysfunction contributes to the activation and nuclear
localization of NF-B is unclear. There are multiple mechanisms for
NF-
B activation that can be divided into signal-dependent and
signal-independent pathways (20). In airway epithelial
cells, bacterial attachment to asialo-GM1 activates a
Ca2+-dependent signaling pathway that results in NF-
B
activation and IL-8 transcription through a cascade that includes
phosphorylation of both the p38 and extracellular signal-regulated
kinase families of mitogen-activated protein kinases
(23a). A signal-independent pathway, perhaps involving
I
B or NF-
B regulation directly, may be affected by mistrafficked
CFTR. I
B-
has been shown to be increased in IB3 cells under basal
conditions and is hypophosphorylated, enabling nuclear NF-
B with
bound I
B-
complexes to direct transcription (30).
Hypophosphorylated I
B-
may be involved in either signal-dependent or the persistent activation of NF-
B (26). Diminished
levels of cytosolic I
B-
was associated with CF (
F508/
F508)
bronchial gland cells in primary culture and correlated with the
presence of nuclear NF-
B Rel A (27). It is possible
that CFTR function, perhaps by affecting pH in specific intracellular
compartments (3), affects relevant kinase or phosphatase
activity. Abnormalities in signal transducer and activator of
transduction-1 phosphorylation in the same 9/HTEo
/pCep-R
cells have been described recently (13), indicating that
CFTR may have a role in multiple signaling pathways. These observations
reflect the complexities of CFTR involvement in the normal physiology
of the epithelial cell. Strategies such as gene therapy to correct CFTR
Cl
channel function alone, which do not ameliorate the
effects of mutant CFTR mistrafficking in the majority of airway
epithelial cells, may not be sufficient to control the inflammatory
component of CF lung disease.
These consequences of CFTR dysfunction in activating translocation of
NF-B and stimulating the expression of proinflammatory cytokines are
important in not only understanding the many roles of CFTR in normal
cell physiology but are also clinically relevant. The majority of CF
patients with the most common
F508 CFTR alleles have multiple
reasons for increased expression of inflammatory mediators in their
airways. The effects of CFTR mistrafficking and ER stress, lack of CFTR
Cl
channel function, and exogenous stimulation due to
airway infection all act to increase epithelial NF-
B translocation
and the expression of proinflammatory genes. This epithelial immune
activation does provide multiple targets for therapy. It may be
possible to devise a therapy to modulate the endogenous upregulation of
NF-
B-dependent gene transcription without compromising the
epithelial barrier function or the host inflammatory response to
exogenous airway pathogens.
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ACKNOWLEDGEMENTS |
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Expert technical assistance was provided by Robert Adamo.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-56194 (to A. Prince) and P50-HL-60293 (to P. Davis, Case Western Reserve University, Cleveland, OH). Imaging studies were performed in the Optical Microscopy Facility of the Herbert Irving Cancer Center at Columbia University (New York, NY), supported by National Center for Research Resources Grant 1-S10-RR-10506.
Original submission in response to a special call for papers on "CFTR Trafficking and Signaling in Respiratory Epithelium."
Address for reprint requests and other correspondence: A. S. Prince, Black Bldg. 416, Columbia Univ., 650 West 168th St., New York, NY 10032 (E-mail: asp7{at}columbia.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.
Received 2 October 2000; accepted in final form 29 January 2001.
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