1 Department of Physiology and Biophysics and 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35294
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ABSTRACT |
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To delineate the
mechanisms that facilitate leukocyte migration into the cystic fibrosis
(CF) lung, expression of chemokines, including interleukin-8 (IL-8),
monocyte chemoattractant protein-1 (MCP-1), and
RANTES, was compared between CF and non-CF airway epithelia. The
findings presented herein demonstrate that, under either basal
conditions or tumor necrosis factor- (TNF-
)- and/or interferon-
(IFN-
)-stimulated conditions, a consistent pattern of
differences in the secretion of IL-8 and MCP-1 between CF and non-CF
epithelial cells was not observed. In contrast, CF epithelial cells
expressed no detectable RANTES protein or mRNA under basal conditions
or when stimulated with TNF-
and/or IFN-
(P
0.05), unlike their non-CF
counterparts. Correction of the CF transmembrane conductance regulator
(CFTR) defect in CF airway epithelial cells restored the induction of
RANTES protein and mRNA by TNF-
in combination with IFN-
(P
0.05) but had little effect on
IL-8 or MCP-1 production compared with mock controls. Transfection studies utilizing RANTES promoter constructs suggested that CFTR activates the RANTES promoter via a nuclear factor-
B-mediated pathway. Together, these results suggest that
1) RANTES expression is altered in
CF epithelia and 2) epithelial
expression of RANTES, but not IL-8 or MCP-1, is dependent on CFTR.
epithelial cells; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; inflammation
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is a lethal inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene (23). The CFTR gene encodes a 180-kDa glycosylated protein that functions as a low-conductance, cAMP-regulated chloride channel (23). Progressive pulmonary tissue damage is a hallmark of CF; however, pancreatic, liver, and intestinal dysfunction are also characteristic of CF. The CF lung disease phenotype includes thick mucus secretion and bacterial colonization of the airway with Pseudomonas aeruginosa. Recent reports suggest that the CF lung may exhibit an exaggerated immune response (19) even in the absence of bacterial infection (1, 12).
CF-associated airway inflammation is characterized by a profound influx of neutrophils into the lung; however, other types of leukocytes, including eosinophils and monocytes, have been implicated in CF airway inflammation. For example, Azzawi et al. (2) detected the presence of highly activated eosinophils in the lungs of transplanted and deceased CF patients. Moreover, Koller and co-workers (13) observed increased levels of eosinophilic cationic protein in the serum and sputum of CF patients compared with healthy nonatopic donors. These authors also demonstrated that CF eosinophils have an increased propensity to degranulate in vitro compared with eosinophils isolated from control subjects or patients with bronchial asthma (15). In addition, previous studies have reported that monocytes derived from CF patients have an increased metabolic level (34, 36) and generate increased levels of inflammatory mediators, including elastase and superoxide, compared with non-CF monocytes (9, 22, 36).
Airway epithelial cells have been described classically as barrier cells that are involved in ion and fluid homeostasis. These cells respond to a variety of environmental stimuli, resulting in the alteration of their cellular functions, such as ion transport and movement of airway secretions. In addition, airway epithelial cells may act as immune effector cells in response to endogenous or exogenous stimuli. Several studies have shown that airway epithelial cells express and secrete a variety of inflammatory mediators, including the chemokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed, and, presumably, secreted; reviewed in Ref. 28). IL-8 belongs to the C-X-C subfamily of chemokines, which displays four highly conserved cysteine amino acid residues, with the first two separated by one nonconserved residue. In contrast, MCP-1 and RANTES belong to the C-C subfamily, which exhibits two adjacent cysteine amino acid residues. The chemokine subfamilies exhibit cell selectivity with respect to chemoattraction. Members of the C-X-C subfamily primarily target neutrophils, whereas various members of the C-C subfamily target monocytes, lymphocytes, eosinophils, and basophils. Specifically, IL-8 mediates neutrophil chemotaxis; however, MCP-1 mediates monocyte and basophil chemotaxis and activation. RANTES induces chemotaxis of eosinophils, monocytes, and CD45 RO+ memory T lymphocytes. The expression of chemokines by airway epithelial cells implicates these cells in facilitating the leukocyte migration associated with airway inflammatory diseases such as CF.
Despite recent advances in understanding the cellular and molecular
basis of CF, the mechanisms that initiate and maintain CF-associated
airway inflammation remain ill defined. In an attempt to understand the
mechanisms that trigger the recruitment of leukocytes into the CF lung,
the present study examines the expression of IL-8, MCP-1, and RANTES in
CF airway epithelial cells. Results presented herein demonstrate that,
in the presence and/or absence of proinflammatory cytokines, CF airway
epithelial cells express the chemokines IL-8 and MCP-1 yet little or no
RANTES. Moreover, these results suggest that complementation of CF
epithelial cells with wild-type CFTR (WT-CFTR) to correct the CFTR
defect restored cytokine induction of RANTES expression via a nuclear
factor-B (NF-
B)-mediated pathway but had no effect on IL-8 or
MCP-1 expression from these cells.
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METHODS |
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Cell culture.
Experiments employed the following non-CF epithelial cells: the airway
epithelial cell lines BEAS2B [bronchial; American Type Culture
Collection (ATCC), Manassas, VA], 16HBE14o (bronchial; a
gift from Dr. Dieter Gruenert, University of California, San Francisco,
CA; Ref. 5), and A549 (alveolar type II; ATCC) and the
pancreatic carcinoma cell line PANC-1 (ATCC). The following CF
epithelial cells were utilized: the airway epithelial cell lines IB3-1
(bronchial;
F508/W1282X compound heterozygote; a gift from Dr. Pam
Zeitlin, Johns Hopkins University, Baltimore, MD; Ref. 37),
CFBE41o
(bronchial;
F508 homozygote; a gift from Dr. Dieter
Gruenert; Ref. 5), and
CFTE29o
(tracheal;
F508 homozygote;
a gift from Dr. Dieter Gruenert; Ref. 5) and the pancreatic
adenocarcinoma cell line CFPAC-1 (
F508 homozygote; ATCC). Primary CF
(
F508 homozygous) and non-CF airway epithelial cells (derived from
human bronchi or nasal polyps) were provided by Dr. Eric J. Sorscher
(Gregory Fleming James Cystic Fibrosis Research Center, University of
Alabama, Birmingham, AL; Ref. 6), Dr. John Engelhardt (University of
Iowa, Iowa City, IA; Ref. 38), and Dr. Nael McCarty (Emory University,
Atlanta, GA; Ref. 6). All cells were grown on Vitrogen 100 (Collagen,
Palo Alto, CA)-coated flasks; Vitrogen 100 contains a mixture of
collagen types I and IV. All airway epithelial cells were cultured in
LHC-8 medium (Biofluids, Rockville, MD) containing 5% FCS, 1%
penicillin-streptomycin, and 0.2% Fungizone. The pancreatic cells were
maintained in MEM-D-valine medium (GIBCO BRL Life Technologies, Grand Island, NY) containing 5%
FCS, 1% penicillin-streptomycin, and 0.2% Fungizone.
Correction of CFTR defect in CF epithelial cells.
CFPAC-1 cells that had been transduced previously with a virus
containing WT-CFTR or a control virus (mock) were included in these
studies (Ref. 7; Gregory Fleming James Cystic Fibrosis Research
Center). IB3-1 cells were transiently transfected as follows. Cells
were grown on Vitrogen 100-coated wells of a 12-well plate in medium
(CFPAC-1 in MEM-D-valine; IB3-1
in LHC-8) containing 5% FCS, 1% penicillin-streptomycin, and 0.2%
Fungizone. At ~50% confluence, cells were incubated with low-serum
Opti-MEM I medium (GIBCO BRL) containing Lipofectamine Plus (6 µg/well; GIBCO BRL) with pRSV-WT-CFTR (Ref. 8; a generous gift of Dr.
Erik Schwiebert, University of Alabama, Birmingham, AL) and
pSV--galactosidase reporter plasmid (each at 0.5 µg/well; Promega)
or pGreen Lantern-1 [green fluorescence protein (GFP); GIBCO
BRL] and pSV-
-galactosidase reporter plasmid (each at 0.5 µg/ml) as a mock control for 6 h at 37°C. After incubation, 2 ml
of MEM-D-valine or LHC-8 medium containing 5% FCS were added to each well, and cells were cultured for
an additional 48 h before analysis. To determine the percentage of
cells transfected transiently, GFP-positive and GFP-negative mock-transfected cells were counted using a fluorescence microscope; transfection percentages normally ranged between 40 and 50%. Mock and
WT-CFTR transfected cells were analyzed for relative transfection efficiency using the
-galactosidase reporter assay system (Promega) according to manufacturer's protocols. WT-CFTR function in
complemented IB3-1 and CFPAC-1 cells was monitored via chloride efflux
as described previously (27).
Analysis of IL-8, MCP-1, and RANTES protein expression.
To analyze IL-8, MCP-1, and RANTES protein expression, cells were
cultured in the presence and absence of tumor necrosis factor- (TNF-
; 100 ng/ml; R&D Systems, Minneapolis, MN) and/or
interferon-
(IFN-
; 100 ng/ml; R&D Systems) for 18 h at 37°C.
After culture, supernatants were harvested and prepared for ELISA of
IL-8, MCP-1, or RANTES protein content (R&D Systems); cells were
harvested and counted to account for differences in cell number. ELISAs were performed according to the manufacturer's protocol (limits of
detection <3 pg/ml).
Analysis of IL-8, MCP-1, and RANTES mRNA expression.
To analyze IL-8, MCP-1, and RANTES mRNA expression, cells were cultured
in the presence and absence of TNF- (100 ng/ml) and/or IFN-
(100 ng/ml) for 2 (IL-8, MCP-1) or 18 (RANTES) h at 37°C. After culture, total RNA was isolated from these cells with TRIzol (GIBCO BRL) and prepared for Northern blot analysis. For Northern blot
analysis, ~10 µg of total RNA were electrophoresed through a
formaldehyde-containing gel and then transferred to nitrocellulose (GeneScreen Plus, Amersham, Arlington Heights, IL). Blots were prehybridized in a buffer containing 50% formamide, 2× PIPES
buffer (0.2 M NaCl, 0.01 M KCl, 0.04 M PIPES, pH 7.5), 0.5% SDS, and denatured salmon sperm DNA (100 µg/ml; Sigma Chemical, St. Louis, MO)
for 18 h at 37°C. Blots were hybridized with a DNA fragment specific for IL-8, MCP-1, or RANTES that was randomly labeled (Random
Prime labeling kit, Promega) with
[
-32P]dATP (5 × 105
counts · min
1 · ml
1;
New England Nuclear, Boston, MA) for 18 h at 37°C. After
hybridization, blots were washed once with 2× SSC (0.3 M NaCl,
0.03 M trisodium citrate · 2H2O,
pH 7.0) for 15 min at room temperature, once with 2× SSC-1% SDS
for 15 min at 65°C, and once with 0.1× SSC for 15 min at room
temperature. Blots were then prepared for autoradiography. To control
for differences in loading, blots were stripped (0.1× SSC-1.0%
SDS, heated to 100°C, 15 min) and then hybridized with glyceraldehyde-3-phosphate dehydrogenase probe as described above. Results from these analyses were quantitated through densitometry.
Analysis of RANTES promoter activity.
To analyze RANTES promoter activity, constructs containing portions of
the RANTES promoter ligated to a luciferase reporter gene were
generated and provided by Dr. Hiro Moriuchi (National Institutes of
Health, Bethesda, MD) (17). Briefly, a 1.4-kb 5' noncoding
sequence of the RANTES gene (1.4RANTES) was cloned into pGL2-basic
(Promega). Deletion mutations, termed 0.5RANTES and 0.2RANTES, and a
site-directed mutation of an NF-B binding site within this noncoding
sequence, designated
B, were confirmed via ddDNA sequencing (17).
IB3-1 cells were cotransfected with pSV-
-galactosidase,
pRSV-WT-CFTR, and pGL2-basic, 1.4RANTES, 0.5RANTES, 0.2RANTES, or
B (each at 0.5 µg/ml) in the presence of Lipofectamine Plus as
described above (Correction of CFTR defect in CF
epithelial cells). In parallel experiments, IB3-1
cells were cotransfected with pSV-
-galactosidase, pGreen Lantern
(GFP), and the respective RANTES promoter construct as mock controls (each at 0.5 µg/well). After transfection, cells were cultured in the
presence and absence of TNF-
in combination with IFN-
(each at
100 ng/ml) for 18 h at 37°C. Cells were then harvested, and
luciferase activity was monitored via the dual-luciferase reporter
assay system (Promega) according to the manufacturer's protocol. The
percentage of cells transfected transiently, determined via GFP
expression as described above, ranged between 40 and 50%; relative
transfection efficiency was assessed via measurement of
-galactosidase activity.
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RESULTS |
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IL-8 expression in CF and non-CF airway epithelial cells.
CF-associated inflammation is characterized by a profound influx of
neutrophils into the lung. To determine whether epithelial cells
contribute to CF airway inflammation through increased expression of
the neutrophil chemoattractant IL-8, expression of IL-8 was compared
between CF and non-CF airway epithelial cells in vitro. Specifically,
IL-8 expression was analyzed in the non-CF airway epithelial cell lines
BEAS2B, 16HBE14o, and A549 and the CF airway epithelial cell
lines IB3-1, CFBE41o
, and
CFTE29o
. IB3-1 is heterozygous for the
F508 CFTR mutation (
F508/W1282X), whereas CFBE41o
and
CFTE29o
are homozygous for the
F508
CFTR mutation. IL-8 expression was also compared between primary CF and
non-CF airway epithelial cells; the CF primary cells were homozygous for the
F508 CFTR mutation. For these analyses, epithelial cells were cultured in the presence and absence of the proinflammatory cytokines TNF-
and IFN-
, as these cytokines have been shown previously to upregulate chemokine expression in airway epithelial cells (33). Moreover, increased levels of TNF-
have been detected in
bronchioalveolar lavage fluid (BALF) collected from CF patients (26).
As shown in Fig. 1, BEAS2B cells expressed
IL-8 protein under basal conditions (>20
ng/106 cells); TNF-
in
combination with IFN-
stimulated a twofold increase in IL-8 protein
expression in these cells (Fig.
1A). 16HBE14o
and A549
cells also expressed IL-8 protein under basal conditions (~0.2
ng/106 cells; Fig.
1A). TNF-
stimulation of
16HBE14o
and A549 cells induced a 10- to 100-fold increase in
IL-8 protein expression in these cells (between 2 and 20 ng/106 cells); IFN-
either had
no effect (A549) or inhibited IL-8 expression (16HBE14o
) (Fig.
1A). The CF airway epithelial cell
lines IB3-1, CFBE41o
, and
CFTE29o
each expressed low
levels of IL-8 protein (between 0 and 0.5 ng/106 cells) under basal
conditions; IL-8 expression in these cells was enhanced between 3- and
80-fold in the presence of TNF-
alone or TNF-
in combination with
IFN-
(between 1.5 and 37 ng/106
cells; Fig. 1A). In comparison, CF
and non-CF primary airway epithelial cells expressed greater amounts of
IL-8 protein (~80 ng/106 cells)
under basal conditions than their respective cell lines (Fig.
1B). Interestingly, TNF-
together
with IFN-
did not significantly affect IL-8 protein expression in
these cells (Fig. 1B). Northern blot
analysis of the CF and non-CF airway epithelial cell lines and primary
cells described above, cultured in the presence and absence of TNF-
and/or IFN-
, revealed that IL-8 mRNA expression (data not
shown) paralleled the pattern of IL-8 protein expression observed in
these cells (Fig. 1). Overall, these data suggest that CF and non-CF
airway epithelial cells express IL-8 in the presence and absence of
TNF-
and IFN-
at levels that are not consistent.
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MCP-1 expression in CF and non-CF airway epithelial cells.
Monocytes derived from CF patients have been reported to exhibit an
increased metabolic level (34, 36) and to generate increased levels of
inflammatory mediators, such as elastase and superoxide, compared with
non-CF monocytes (9, 22, 36). To determine whether CF airway epithelial
cells express the monocyte-chemoattracting and -activating agent MCP-1,
MCP-1 expression was compared between CF and non-CF airway epithelial
cell lines and primary cells in vitro. As shown in Fig.
2, BEAS2B cells expressed MCP-1 protein under basal conditions (~16
ng/106 cells); TNF- in
combination with IFN-
enhanced this expression approximately twofold
(26 ng/106 cells). In comparison,
16HBE14o
and A549 cells expressed little or no MCP-1 protein in
the absence of stimuli (Fig. 2A).
Interestingly, TNF-
and/or IFN-
induced a 60- to 140-fold
increase in MCP-1 protein expression in A549 cells yet had no effect on
MCP-1 expression in 16HBE14o
cells (Fig.
2A). The CF cell lines
CFBE41o
and IB3-1 expressed no detectable MCP-1 protein under
basal conditions; however,
CFTE29o
cells expressed ~5
ng/106 cells under basal
conditions (Fig. 2A). TNF-
or
IFN-
alone had no effect on MCP-1 protein expression in
CFBE41o
or
CFTE29o
cells yet induced expression in
IB3-1 cells to ~20 ng/106 cells;
TNF-
in combination with IFN-
enhanced MCP-1 protein expression
(~2.5 ng/106 cells) in
CFBE41o
cells only. In contrast, neither CF nor non-CF primary
airway epithelial cells expressed MCP-1 protein under basal conditions
(Fig. 2B). However, TNF-
together
with IFN-
induced a significant increase in MCP-1 expression in CF
cells (18 ng/106 cells) and non-CF
cells (42 ng/106 cells). Northern
blot analysis of the CF and non-CF airway epithelial cell lines and
primary cells described above, cultured in the presence and absence of
TNF-
and/or IFN-
, revealed that MCP-1 mRNA expression
(data not shown) paralleled the pattern of MCP-1 protein expression
observed in these cells (Fig. 2). Taken together, these findings
indicate that, although CF and non-CF primary airway epithelial cells
expressed significantly different levels of MCP-1 in the presence and
absence of TNF-
and IFN-
, the respective cell lines do not
express consistent levels of MCP-1.
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RANTES expression in CF and non-CF airway epithelial cells.
Recent studies suggest that eosinophils may play a role in the
progressive tissue damage observed in the CF lung (2). To determine
whether CF epithelial cells express the eosinophil chemoattractant RANTES, RANTES expression was compared between CF and non-CF airway epithelial cells in vitro. Specifically, the levels of RANTES were
compared, under basal and TNF-- and/or IFN-
-stimulated conditions, between the non-CF airway epithelial cell lines BEAS2B, 16HBE14o
, and A549 and the CF airway epithelial cell lines
CFBE41o
, IB3-1, and
CFTE29o
as well as between non-CF
and CF primary airway epithelial cells. As shown in Fig.
3A, BEAS2B
and A549 expressed low levels of RANTES protein (<1
ng/106 cells) under basal
conditions or when stimulated with IFN-
; TNF-
induced RANTES
protein expression (12 ng/106
cells) in A549 cells only. However, TNF-
in combination with IFN-
induced a significant increase in RANTES protein expression in both
BEAS2B cells (14 ng/106 cells)
(31, 35) and A549 cells (28 ng/106
cells) (Fig. 3A), as has
been reported previously (16). Interestingly, 16HBE14o
cells
expressed RANTES constitutively (~6
ng/106 cells); TNF-
and/or IFN-
had no effect on RANTES expression in these
cells (Fig. 3A). In contrast,
CFBE41o
, IB3-1, and
CFTE29o
cells expressed no
detectable RANTES protein under basal conditions or in the presence of
TNF-
and/or IFN-
(Fig.
3A). In comparison, non-CF and CF
primary airway epithelial cells expressed no detectable RANTES protein
in the absence of stimuli (Fig. 3B).
TNF-
in combination with IFN-
stimulated RANTES protein
expression in non-CF cells (33 ng/106 cells), yet these
proinflammatory cytokines had no significant effect on CF cells (Fig.
3B). These results suggest that CF
airway epithelial cells express no detectable RANTES protein compared with non-CF controls under basal or cytokine-stimulated conditions.
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IL-8, MCP-1, and RANTES expression in non-CF and CF pancreatic
epithelial cells.
To determine whether the pattern of IL-8, MCP-1, and RANTES expression
observed in CF airway epithelial cells was exhibited by epithelial
cells derived from CF-affected organ systems other than the lung, IL-8,
MCP-1, and RANTES expression was compared between non-CF and CF
pancreatic epithelial cell lines in the presence and absence of TNF-
and IFN-
. The non-CF pancreatic epithelial cell line PANC-1 and the
CF pancreatic cell line CFPAC-1 (
F508 homozygote) were
utilized in these studies. As shown in Fig.
5, PANC-1 cells cultured with TNF-
alone
or together with IFN-
expressed increased levels of IL-8 protein (11 and 6 ng/106 cells, respectively).
Interestingly, CFPAC-1 cells expressed approximately eightfold more
IL-8 protein (9 ng/106 cells)
under basal conditions than their non-CF counterpart; TNF-
alone or
TNF-
together with IFN-
further enhanced IL-8 protein expression
in CFPAC-1 cells (23 ng/106 cells;
Fig. 5A). A similar pattern of IL-8
mRNA expression was observed in PANC-1 and CFPAC-1 cells
(data not shown). In contrast, PANC-1 cells expressed MCP-1 protein
constitutively (between 20 and 24 ng/106 cells); TNF-
and/or IFN-
had no effect on MCP-1 protein expression in
these cells (Fig. 5B).
Interestingly, CFPAC-1 cells expressed significantly less MCP-1 protein
(between 3 and 5 ng/106 cells)
than their non-CF counterpart; TNF-
and/or IFN-
had no
effect (Fig. 5B). A similar pattern
of MCP-1 mRNA expression was observed in PANC-1 and CFPAC-1 cells (data
not shown). Together, these results demonstrate that CF and non-CF
pancreatic epithelial cells display a different pattern of IL-8 and
MCP-1 expression than that observed for the CF and non-CF airway
epithelial cells described above.
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Role of CFTR in the regulation of IL-8, MCP-1, and RANTES
expression.
To determine whether CFTR directly modulates chemokine expression in
epithelial cells, the CF epithelial cell lines CFPAC-1 and IB3-1 were
complemented with either WT-CFTR, to correct the mutant CFTR phenotype,
or a mock control and then analyzed for IL-8, MCP-1, and RANTES
expression in the presence and absence of TNF- and/or
IFN-
. Specifically, CFPAC-1 cells that had been transduced
previously with an engineered PLJ virus containing either WT-CFTR or a
mock control (7) and IB3-1 cells that were transiently transfected with
a plasmid carrying either WT-CFTR or a mock control were utilized in
these studies (8). WT-CFTR function in complemented CFPAC-1 and IB3-1
cells was monitored via chloride efflux as described previously (27).
Each cell type complemented with WT-CFTR displayed at least a twofold
increase in chloride efflux activity in the presence of cAMP nucleotide analogs compared with mock controls (data not shown); these results suggest that WT-CFTR was expressed and functional in the complemented CFPAC-1 and IB3-1 cells. Transient transfection of IB3-1 cells with
WT-CFTR did not alter the protein or mRNA expression of IL-8 or MCP-1,
either under basal or cytokine-stimulated conditions, compared with
mock controls (data not shown). Similarly, CFPAC-1 cells transduced
with a virus containing WT-CFTR expressed equivalent levels of IL-8 and
MCP-1 protein and mRNA; TNF-
and/or IFN-
did not further
enhance IL-8 or MCP-1 expression above mock control levels (data not
shown). In contrast, CFPAC-1 and IB3-1 cells that were complemented
with WT-CFTR displayed a significant increase in RANTES
protein expression (CFPAC-1, 3.3 ± 0.4 ng/106 cells or 7-fold; IB3-1, 1.0 ± 0.3 ng/106 cells or 2-fold)
when cultured in the presence of TNF-
in combination with IFN-
compared with respective mock controls (Fig.
7). CFPAC-1 containing WT-CFTR
also displayed a significant increase in RANTES mRNA when stimulated
simultaneously with TNF-
and IFN-
(9-fold greater than mock
control, Fig. 8). No significant difference in RANTES expression was observed between WT-CFTR-complemented CFPAC-1
and IB3-1 cells and their respective mock controls under basal
conditions or when stimulated with TNF-
or IFN-
separately (Figs.
7, 8). These findings, taken together with the results presented above,
demonstrate that CFTR modulates the expression of RANTES, but not IL-8
or MCP-1, at the protein and mRNA levels.
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Effect of WT-CFTR on RANTES promoter activity.
RANTES gene expression has been reported to be regulated at the level
of transcription and not mRNA stability (25). Therefore, to delineate
the role of CFTR in RANTES mRNA expression, transfection assays
utilizing RANTES promoter constructs fused to a luciferase reporter
gene were performed in IB3-1 CF airway epithelial cells complemented
with WT-CFTR and their respective mock controls; RANTES promoter
constructs were generated and provided by Dr. Hiro Moriuchi (17).
Briefly, a 1.4-kb 5' noncoding sequence of the RANTES gene was
cloned into pGL2-basic (Promega); this 1.4-kb region, 5' deletion
mutations of this region, and a site-directed mutation in an NF-B
binding site of this region (Fig.
9A) were analyzed (17). As shown in Fig. 9, IB3-1 cells transfected with the
complete 1.4-kb RANTES promoter construct in the absence of WT-CFTR
displayed low reporter activity in the presence or absence of
TNF-
/IFN-
. In contrast, IB3-1 cells transfected with the 1.4-kb
RANTES promoter construct together with WT-CFTR displayed an
approximately fourfold increase in reporter activity in the presence of
TNF-
and IFN-
compared with unstimulated controls (Fig. 9,
B and
C). To determine the regions of the
RANTES promoter that were essential for CFTR-mediated activation,
5' deletions of the RANTES promoter construct were analyzed.
Interestingly, IB3-1 cells transfected with only 0.5 kb of the 1.4-kb
RANTES promoter construct, either in the absence or presence of
WT-CFTR, exhibited increased reporter activity under basal conditions, suggesting that a negative regulatory element exists upstream of this
deletion; this activity was not responsive to the effects of TNF-
and IFN-
(Fig. 9B). However,
IB3-1 cells transfected with only 0.2 kb of the 1.4-kb RANTES promoter
construct displayed a pattern of activity similar to that observed for
the complete 1.4-kb RANTES construct. Specifically, IB3-1 cells
transfected with the 0.2-kb RANTES promoter construct together with
WT-CFTR exhibited a fourfold increase in reporter activity when
stimulated with TNF-
in combination with IFN-
compared with
unstimulated and mock controls (Fig.
9B). These findings indicate that
transcription factor binding sites located within 300 bp upstream of
the 0.2-kb construct, such as activator protein-1, signal transducer
and activator of transcription (STAT), and nuclear factor of activated T cells (NF-AT) (Fig. 9A),
mediate constitutive activation of the RANTES promoter;
however, binding sites within the 0.2-kb construct itself, including
nuclear factor IL-6 and NF-
B (Fig. 9A), confer WT-CFTR modulation of
RANTES promoter activation. NF-
B has been implicated as an essential
factor in the TNF-
- and/or IFN-
-mediated induction of
RANTES gene expression (17, 21). To determine whether WT-CFTR modulates
RANTES promoter activation via NF-
B, IB3-1 cells were transfected
with the complete 1.4-kb RANTES promoter construct containing a mutant
NF-
B site (
B) and WT-CFTR or mock control. As shown in Fig.
9C, IB3-1 cells transfected with both
WT-CFTR and the
B RANTES promoter construct were not responsive
to the effects of TNF-
and/or IFN-
, as had been observed
with the intact 1.4-kb RANTES construct. Taken together, these results
suggest that, in the presence of TNF-
and IFN-
, WT-CFTR modulates
activation of the RANTES promoter in airway epithelial cells via an
NF-
B-mediated pathway.
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DISCUSSION |
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The present study was performed to delineate the role of the airway
epithelium in the leukocyte recruitment associated with CF airway
inflammation. The data presented herein demonstrate that CF and non-CF
airway epithelial cells, in the presence or absence of the
proinflammatory cytokines TNF- and IFN-
, expressed levels of IL-8
and MCP-1 that were not consistent; therefore, these data suggest that
no differences exist in the expression of these molecules between CF
and non-CF cells. In sharp contrast, however, CF epithelial cells
expressed little or no RANTES protein or mRNA compared with non-CF
controls. Although CF and non-CF epithelial cells derived from the
pancreas, another CF-affected organ, exhibited a pattern of IL-8 and
MCP-1 expression that differed from their respective airway epithelial
counterparts, the pattern of RANTES expression was the same: CF
pancreatic epithelial cells expressed no detectable RANTES protein or
mRNA compared with non-CF controls. Importantly, insertion of WT-CFTR
into CF airway and pancreatic epithelial cells to correct the CFTR
defect restored RANTES protein and mRNA expression in the presence of
TNF-
and IFN-
via an NF-
B-mediated pathway; however,
correction of the CFTR defect had no effect on IL-8 or MCP-1 expression
in these cells. Together, these results demonstrate that
1) cytokine-induced RANTES
expression is altered in CF epithelia and
2) epithelial gene expression of
RANTES, but not IL-8 or MCP-1, is dependent on CFTR.
The studies presented herein were performed in the presence and absence
of TNF- and/or IFN-
, but not P. aeruginosa, for two reasons. First, Schuster et al.
(26) detected elevated levels of TNF-
in the sputum of CF patients
who were not infected with P. aeruginosa. Their study concluded that high
concentrations of proinflammatory cytokines, including TNF-
, may
contribute to the localized neutrophil-dominated inflammatory state
observed in the CF airway (26). Second, increasing evidence suggests that an exaggerated inflammatory response may exist in the CF lung in
the presence or absence of bacterial infection. Khan et al. (12)
demonstrated that airway inflammation may occur before the appearance
of bacterial pathogens in CF patients. These investigators examined
BALF from control and CF infants; their results demonstrated a
significant increase in the number of neutrophils and in IL-8 levels in
the BALF of CF infants who had negative cultures for common bacterial
CF-related pathogens, including P. aeruginosa and Staphylococcus
aureus, compared with normal controls. In contrast, Armstrong and co-workers (1) reported that the early onset of airway
inflammation in CF infants is due to the presence of respiratory
pathogens not previously diagnosed. Their studies demonstrated that
increased neutrophil numbers and IL-8 levels in the BALF of CF infants
correlate with bacteria and/or viral infection. However, these
authors also showed that uninfected CF infants have a greater number of
BALF neutrophils than normal controls, suggesting that an enhanced
airway inflammatory response occurs in CF patients (1). In addition,
Noah et al. (19) reported that IL-8 levels were markedly elevated in
the BALF of infected CF patients compared with levels in infected and
uninfected controls, even after standardization of IL-8 concentrations
to bacterial counts.
Although the studies presented herein indicate that CF and non-CF
airway epithelial cell lines express inconsistent differences in the
levels of IL-8 and MCP-1 in the presence and absence of TNF- and
IFN-
, the mere expression of these chemokines implicates CF
epithelia in the leukocyte migration observed in CF-associated airway
inflammation. Moreover, the observation that CF and non-CF airway
epithelial cells expressed comparable levels of IL-8, in the presence
or absence of proinflammatory cytokines, together with the observation
that complementation of CF epithelial cells did not alter IL-8
expression by these cells, suggests
1) that enhanced IL-8 expression by
CF airway epithelial cells is not the source of the increased IL-8
levels detected in uninfected CF patient BALF and
2) that cytokine-induced epithelial
IL-8 expression is regulated independently of CFTR. These findings
contradict previous studies that reported increased IL-8 expression by
CF airway epithelial cells in the presence and absence of
proinflammatory stimuli (24, 30). Ruef and co-workers (24) demonstrated that the CF airway epithelial cell line JME/CF15 secreted IL-8 in the
absence of stimuli; this expression was enhanced further in the
presence of IL-1
. From these analyses of a single CF epithelial cell
line, the authors concluded that local production of IL-8 by CF
epithelial cells may trigger significant upregulation of airway
inflammation in CF (24). In addition, Stecenko et al. (30) reported
recently that the CF airway epithelial cell lines IB3-1 and 2CF
expressed significantly more IL-8 protein than the non-CF epithelial
cell lines BEAS2B or normal human primary bronchial epithelial cells in
the presence of TNF-
or IL-1
; the WT-CFTR-complemented IB3-1 cell
line C38 expressed IL-8 levels that were similar to that expressed by
non-CF cells. However, these authors examined only two CF epithelial
cell lines, did not compare IL-8 expression between CF and non-CF
primary airway epithelial cells, and, importantly, did not demonstrate
that the complemented cell line C38 exhibited WT-CFTR activity (30).
The inability of CF epithelial cells to express RANTES protein or mRNA
suggests that these cells do not recruit eosinophils into the CF lung
via this particular C-C chemokine. Airway epithelial cells have been
shown to express C-C chemokines other than RANTES, including eotaxin
(20) and MCP-4 (32), that may facilitate eosinophil migration. To this
end, we have been unable to detect eotaxin expression in CF and non-CF
primary airway epithelial cells; however, we have observed that the
non-CF pancreatic epithelial cell line PANC-1 expresses eotaxin in
response to TNF- alone or together with IFN-
, whereas its CF
counterpart CFPAC-1 does not (L. M. Schwiebert, unpublished
observations). In a recent report by Stellato et al. (32), the authors
demonstrated that the CF airway epithelial cell line IB3-1 expressed
MCP-1 mRNA in the presence of TNF-
in combination with IFN-
.
However, the level of MCP-4 mRNA expression in IB3-1 cells was
significantly lower than that observed in the non-CF airway epithelial
cell lines BEAS2B and A549 stimulated in the same manner (32).
Interestingly, Koller et al. (14) observed that RANTES levels in the
BALF of CF patients were significantly lower than RANTES levels in the BALF of asthmatics. This report suggests that expression of RANTES by
other cell types that express this chemokine, including T lymphocytes and macrophages, may be depressed in the CF lung. At present, it is
unclear what role RANTES may play in improving CF-associated airway
inflammation and/or bacterial clearance of the CF lung.
RANTES gene expression appears to be regulated at the level of
transcription; the 3' untranslated region of RANTES mRNA does not
contain an AU-repeat element, suggesting that RANTES mRNA expression is
not regulated at the level of mRNA stability (25). Results reported
herein demonstrate that TNF- and IFN-
synergize in the induction
of RANTES expression in CF epithelial cells complemented with WT-CFTR
and that WT-CFTR modulates RANTES promoter activation via NF-
B. The
NF-
B transcription factor family is composed of c-Rel, NF-
B1
(p50), NF-
B2 (p52), RelA (p65) and RelB (reviewed in Ref.
3). Most members of this family can form homo- or heterodimers that are
retained in the cytoplasm of unactivated cells through interaction with
members of the I
B inhibitor family; members of the I
B family
include I
B
, I
B
, and I
BR (3). TNF-
has been reported
to induce rapid phosphorylation and subsequent degradation of I
B,
allowing NF-
B to translocate to the nucleus and activate transcription (3). Interestingly, a recent paper by Ray and co-workers
(21) suggests that RANTES and IL-8 are differentially regulated by
NF-
B. These authors reported that overexpression of I
BR in the
alveolar epithelial cell line A549 sequesters p50 homodimers, thereby
facilitating the binding of a unique NF-
B-Rel complex to the RANTES
B region; this binding, in turn, triggered a rapid 50- to 100-fold
induction of RANTES upon cytokine stimulation compared with control
cells (21). However, I
BR overexpression in A549 cells did not
enhance gene expression of the chemokine IL-8 (21).
It is possible that a loss of CFTR activity, i.e., loss of chloride
transport, in CF epithelial cells may alter the activity of
transcription factors such as NF-B and the subsequent induction of
RANTES gene expression. Several studies have linked the activity of ion
channels to immune responses in vivo (10, 11, 29). Cahalan and Lewis
(4) hypothesize that activating kinase-regulated chloride channels,
such as CFTR, may result in the depolarization of the membrane
potential and, thereby, may suppress mitogen-stimulated calcium influx.
To this end, Negulescu et al. (18) demonstrated that adding a permeable
cAMP analog to T lymphocytes suppressed both calcium influx and
NF-AT-mediated gene expression of IL-2 in response to T cell receptor
activation. The activity of transcription factors other than NF-AT,
including c-Fos, has also been linked to membrane depolarization and
calcium influx (29).
Defining the cellular and molecular mechanisms that trigger the migration of leukocytes into the CF lung will be critical in understanding the pathophysiology of this disease. Moreover, determining how CFTR regulates the expression of chemokines, such as RANTES, will further the understanding of airway inflammation in general.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Robert P. Schleimer for encouragement and Drs. Etty Benveniste, John Engelhardt, Nael McCarty, Hiro Moriuchi, Erik M. Schwiebert, and Theresa Strong for assistance. In addition, we thank Drs. Dale J. Benos and Eric J. Sorscher, the Department of Physiology and Biophysics, and the Gregory Fleming James Cystic Fibrosis Research Center for continued support.
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FOOTNOTES |
---|
This work was funded by grants from the Cystic Fibrosis Foundation and the American Lung Association.
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. §1734 solely to indicate this fact.
Address for reprint requests: L. M. Schwiebert, Dept. of Physiology and Biophysics, McCallum Bldg., Rm. 966, University of Alabama at Birmingham, 1918 University Blvd., Birmingham, AL 35294.
Received 29 July 1998; accepted in final form 4 December 1998.
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