Chemokine expression in CF epithelia: implications for the role of CFTR in RANTES expression

Lisa M. Schwiebert1,2, Kim Estell1, and Stacie M. Propst1

1 Department of Physiology and Biophysics and 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, Alabama 35294


    ABSTRACT
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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-alpha (TNF-alpha )- and/or interferon-gamma (IFN-gamma )-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-alpha and/or IFN-gamma (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-alpha in combination with IFN-gamma (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-kappa 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


    INTRODUCTION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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-kappa B (NF-kappa B)-mediated pathway but had no effect on IL-8 or MCP-1 expression from these cells.


    METHODS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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; Delta F508/W1282X compound heterozygote; a gift from Dr. Pam Zeitlin, Johns Hopkins University, Baltimore, MD; Ref. 37), CFBE41o- (bronchial; Delta F508 homozygote; a gift from Dr. Dieter Gruenert; Ref. 5), and Sigma CFTE29o- (tracheal; Delta F508 homozygote; a gift from Dr. Dieter Gruenert; Ref. 5) and the pancreatic adenocarcinoma cell line CFPAC-1 (Delta F508 homozygote; ATCC). Primary CF (Delta 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-beta -galactosidase reporter plasmid (each at 0.5 µg/well; Promega) or pGreen Lantern-1 [green fluorescence protein (GFP); GIBCO BRL] and pSV-beta -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 beta -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-alpha (TNF-alpha ; 100 ng/ml; R&D Systems, Minneapolis, MN) and/or interferon-gamma (IFN-gamma ; 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-alpha (100 ng/ml) and/or IFN-gamma (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 [alpha -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-kappa B binding site within this noncoding sequence, designated Delta kappa B, were confirmed via ddDNA sequencing (17). IB3-1 cells were cotransfected with pSV-beta -galactosidase, pRSV-WT-CFTR, and pGL2-basic, 1.4RANTES, 0.5RANTES, 0.2RANTES, or Delta kappa 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-beta -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-alpha in combination with IFN-gamma (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 beta -galactosidase activity.


    RESULTS
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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 Sigma CFTE29o-. IB3-1 is heterozygous for the Delta F508 CFTR mutation (Delta F508/W1282X), whereas CFBE41o- and Sigma CFTE29o- are homozygous for the Delta 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 Delta F508 CFTR mutation. For these analyses, epithelial cells were cultured in the presence and absence of the proinflammatory cytokines TNF-alpha and IFN-gamma , as these cytokines have been shown previously to upregulate chemokine expression in airway epithelial cells (33). Moreover, increased levels of TNF-alpha 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-alpha in combination with IFN-gamma 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-alpha 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-gamma either had no effect (A549) or inhibited IL-8 expression (16HBE14o-) (Fig. 1A). The CF airway epithelial cell lines IB3-1, CFBE41o-, and Sigma 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-alpha alone or TNF-alpha in combination with IFN-gamma (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-alpha together with IFN-gamma 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-alpha and/or IFN-gamma , 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-alpha and IFN-gamma at levels that are not consistent.


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Fig. 1.   Cystic fibrosis (CF) and non-CF epithelial cells express varied levels of interleukin-8 (IL-8) protein. Non-CF and CF airway epithelial cell lines (A) and primary cells (B) were stimulated with and without tumor necrosis factor-alpha (TNF-alpha ) and/or interferon-gamma (IFN-gamma ) (each at 100 ng/ml) for 18 h at 37°C. After culture, cells were harvested and counted; supernatants were analyzed for IL-8 protein content via an IL-8-specific ELISA. Results are reported as ng IL-8/106 cells (n = 6-9). ND, none detected. * P <=  0.05 compared with carrier control.

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-alpha in combination with IFN-gamma 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-alpha and/or IFN-gamma 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, Sigma CFTE29o- cells expressed ~5 ng/106 cells under basal conditions (Fig. 2A). TNF-alpha or IFN-gamma alone had no effect on MCP-1 protein expression in CFBE41o- or Sigma CFTE29o- cells yet induced expression in IB3-1 cells to ~20 ng/106 cells; TNF-alpha in combination with IFN-gamma 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-alpha together with IFN-gamma 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-alpha and/or IFN-gamma , 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-alpha and IFN-gamma , the respective cell lines do not express consistent levels of MCP-1.


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Fig. 2.   CF and non-CF epithelial cells express varied levels of monocyte chemoattractant protein-1 (MCP-1). Non-CF and CF airway epithelial cell lines (A) and primary cells (B) were stimulated with and without TNF-alpha and/or IFN-gamma (each at 100 ng/ml) for 18 h at 37°C. After culture, cells were harvested and counted; supernatants were analyzed for MCP-1 protein content via an MCP-1-specific ELISA. Results are reported as ng IL-8/106 cells (n = 6-9). ND, none detected. * P <=  0.05 compared with carrier control.

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-alpha - and/or IFN-gamma -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 Sigma 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-gamma ; TNF-alpha induced RANTES protein expression (12 ng/106 cells) in A549 cells only. However, TNF-alpha in combination with IFN-gamma 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-alpha and/or IFN-gamma had no effect on RANTES expression in these cells (Fig. 3A). In contrast, CFBE41o-, IB3-1, and Sigma CFTE29o- cells expressed no detectable RANTES protein under basal conditions or in the presence of TNF-alpha and/or IFN-gamma (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-alpha in combination with IFN-gamma 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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Fig. 3.   CF airway epithelial cells do not express RANTES protein. Non-CF and CF bronchial epithelial cell lines (A) and primary cells (B) were cultured in presence and absence of TNF-alpha and/or IFN-gamma (each at 100 ng/ml) for 18 h at 37°C. After culture, supernatants were collected and analyzed via a RANTES-specific ELISA; cells were harvested and counted to account for differences in cell number. Results are reported as ng RANTES protein/106 cells (n = 6-9). ND, none detected. * P <=  0.05 relative to carrier control.

To compare RANTES mRNA expression between non-CF and CF airway epithelia, the non-CF airway epithelial cell lines BEAS2B, A549, and 16HBE14o- and the CF airway epithelial cell lines IB3-1, CFBE41o-, and Sigma CFTE29o- as well as non-CF and CF primary airway epithelial cells were cultured in the presence and absence of TNF-alpha and/or IFN-gamma and then analyzed for mRNA expression via Northern blot analysis. As shown in Fig. 4, TNF-alpha in combination with IFN-gamma stimulated significant levels of RANTES mRNA expression (6-fold over basal levels) in BEAS2B cells; TNF-alpha or IFN-gamma alone had no effect on RANTES expression in these cells. However, TNF-alpha alone or together with IFN-gamma induced a significant increase (~2-fold and 7-fold over basal levels, respectively) in RANTES mRNA expression in the non-CF airway epithelial cell line A549 (Fig. 4). Interestingly, 16HBE14o- cells exhibited significant levels of RANTES mRNA expression in both the absence and presence of TNF-alpha and/or IFN-gamma (Fig. 4). In contrast, IB3-1, CFBE41o-, and Sigma CFTE29o- cells expressed no detectable RANTES mRNA in the presence or absence of TNF-alpha and/or IFN-gamma (Fig. 4). Importantly, non-CF and CF primary airway epithelial cells yielded patterns of RANTES mRNA expression similar to those observed in their respective cell lines. TNF-alpha together with IFN-gamma stimulated a significant increase in RANTES mRNA expression (~5-fold over basal levels) in non-CF cells yet failed to stimulate RANTES mRNA expression in CF cells (Fig. 4). Together, these findings indicate that CF airway epithelial cells express no detectable RANTES mRNA compared with their non-CF counterparts.


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Fig. 4.   CF airway epithelial cells do not express RANTES mRNA. Non-CF and CF airway epithelial cell lines and primary cells were cultured with or without TNF-alpha and/or IFN-gamma as described in Fig. 3. After stimulation, cells were harvested and total RNA was isolated and analyzed via Northern blot analysis. Northern blots were probed with a cDNA fragment specific for RANTES. Blots were then stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression to account for difference in loading. Representative autoradiograms (A) and densitometric results (B) from 3 separate experiments are shown. Lane 1, carrier control; lane 2, TNF-alpha ; lane 3, IFN-gamma ; lane 4, TNF-alpha  + IFN-gamma . Densitometric results are reported as fold induction of RANTES mRNA compared with carrier controls. * P <=  0.05.

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-alpha and IFN-gamma . The non-CF pancreatic epithelial cell line PANC-1 and the CF pancreatic cell line CFPAC-1 (Delta F508 homozygote) were utilized in these studies. As shown in Fig. 5, PANC-1 cells cultured with TNF-alpha alone or together with IFN-gamma 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-alpha alone or TNF-alpha together with IFN-gamma 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-alpha and/or IFN-gamma 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-alpha and/or IFN-gamma 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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Fig. 5.   CF and non-CF pancreatic cells express IL-8, MCP-1, and RANTES differentially. CF and non-CF pancreatic epithelial cells were cultured in presence and absence of TNF-alpha and/or IFN-gamma (each at 100 ng/ml) for 18 h at 37°C. Supernatants were collected, and cells were harvested and counted to control for differences in cell number; supernatants were analyzed via ELISA for IL-8 (A), MCP-1 (B), and RANTES (C) protein content. Results are reported as ng protein/106 cells (n = 3). ND, none detected. * P <=  0.05 relative to carrier control.

Interestingly, CF and non-CF pancreatic epithelial cells exhibited a pattern of RANTES expression similar to that observed in CF and non-CF airway epithelial cells. Specifically, PANC-1 cells expressed increased levels (between 20 and 25 ng/106 cells) of RANTES protein (Fig. 5C) and mRNA (Fig. 6) when stimulated with TNF-alpha alone or in combination with IFN-gamma ; low levels of RANTES protein (<= 1 ng/106 cells) and mRNA were observed under basal conditions or in the presence of IFN-gamma alone. CFPAC-1 cells, however, expressed little or no detectable RANTES protein (Fig. 5C) or mRNA (Fig. 6) in the presence and absence of TNF-alpha and/or IFN-gamma . Because these findings parallel the pattern of RANTES expression observed in non-CF and CF airway epithelial cells, these findings demonstrate that epithelial cells derived from multiple CF-affected organs express significantly lower amounts of RANTES than their non-CF counterparts.


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Fig. 6.   CF pancreatic cells do not express RANTES mRNA. CF and non-CF pancreatic epithelial cells were stimulated with or without TNF-alpha and/or IFN-gamma as described in Fig. 3. Cells were harvested, and total RNA was isolated and analyzed via Northern blotting as described in Fig. 4. Representative autoradiograms (A) and densitometric results (B) from 3 separate experiments are shown. Densitometric results are reported as fold induction of RANTES mRNA compared with PANC-1 carrier controls. Lane 1, carrier control; lane 2, TNF-alpha ; lane 3, IFN-gamma ; lane 4, TNF-alpha  + IFN-gamma . * P <=  0.05 relative to carrier control.

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-alpha and/or IFN-gamma . 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-alpha and/or IFN-gamma 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-alpha in combination with IFN-gamma 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-alpha and IFN-gamma (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-alpha or IFN-gamma 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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Fig. 7.   CF epithelial cells complemented with wild-type CF transmembrane conductance regulator (WT-CFTR) express RANTES protein in presence of TNF-alpha and IFN-gamma . Complemented IB3-1 (A) and CFPAC-1 (B) cells were cultured in presence and absence of TNF-alpha and/or IFN-gamma as described in Fig. 3. Supernatants were collected and analyzed via a RANTES-specific ELISA; cells were harvested and counted to account for differences in cell number. Results are reported as fold induction of RANTES protein relative to respective mock controls (n = 3-6). * P <=  0.05.


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Fig. 8.   CF epithelial cells complemented with WT-CFTR express RANTES mRNA in presence of TNF-alpha and IFN-gamma . CFPAC-1 cells complemented with WT-CFTR or a mock control were cultured in presence and absence of TNF-alpha and/or IFN-gamma as described in Fig. 3 and then harvested. Total RNA was isolated and analyzed via Northern blot analysis as described in Fig. 4. Representative autoradiograms (A) and densitometric results (B) from 3 separate experiments are shown. Densitometric results are reported as fold induction of RANTES mRNA compared with respective mock controls. Lane 1, carrier control; lane 2, TNF-alpha ; lane 3, IFN-gamma ; lane 4, TNF-alpha  + IFN-gamma . * P <=  0.05.

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-kappa 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-alpha /IFN-gamma . 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-alpha and IFN-gamma 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-alpha and IFN-gamma (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-alpha in combination with IFN-gamma 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-kappa B (Fig. 9A), confer WT-CFTR modulation of RANTES promoter activation. NF-kappa B has been implicated as an essential factor in the TNF-alpha - and/or IFN-gamma -mediated induction of RANTES gene expression (17, 21). To determine whether WT-CFTR modulates RANTES promoter activation via NF-kappa B, IB3-1 cells were transfected with the complete 1.4-kb RANTES promoter construct containing a mutant NF-kappa B site (Delta kappa B) and WT-CFTR or mock control. As shown in Fig. 9C, IB3-1 cells transfected with both WT-CFTR and the Delta kappa B RANTES promoter construct were not responsive to the effects of TNF-alpha and/or IFN-gamma , as had been observed with the intact 1.4-kb RANTES construct. Taken together, these results suggest that, in the presence of TNF-alpha and IFN-gamma , WT-CFTR modulates activation of the RANTES promoter in airway epithelial cells via an NF-kappa B-mediated pathway.


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Fig. 9.   WT-CFTR modulates RANTES promoter activation in presence of TNF-alpha and IFN-gamma . A: 1.4-kb 5' noncoding sequence of RANTES gene (modified from Ref. 17), showing mutant nuclear factor-kappa B (NF-kappa B) binding site (Delta kappa B). RANTES promoter constructs containing 1.4 kb (1.4R), 0.5 kb (0.5R), or 0.2 kb (0.2R) of 5' noncoding sequence or Delta kappa B were utilized. B: IB3-1 cells were transfected transiently with 1.4R, 0.5R, or 0.2R and WT-CFTR or a mock control. C: IB3-1 cells were transfected transiently with 1.4R or Delta kappa B construct and WT-CFTR or a mock control. Cells were then cultured in presence and absence of TNF-alpha and IFN-gamma as described in Fig. 3, harvested, counted, and processed for reporter activity. Results are reported as relative reporter activity compared with unstimulated mock controls (n = 3-5). * P <=  0.05.


    DISCUSSION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

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-alpha and IFN-gamma , 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-alpha and IFN-gamma via an NF-kappa 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-alpha and/or IFN-gamma , but not P. aeruginosa, for two reasons. First, Schuster et al. (26) detected elevated levels of TNF-alpha in the sputum of CF patients who were not infected with P. aeruginosa. Their study concluded that high concentrations of proinflammatory cytokines, including TNF-alpha , 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-alpha and IFN-gamma , 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-1beta . 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-alpha or IL-1beta ; 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-alpha alone or together with IFN-gamma , 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-alpha in combination with IFN-gamma . 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-alpha and IFN-gamma 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-kappa B. The NF-kappa B transcription factor family is composed of c-Rel, NF-kappa B1 (p50), NF-kappa 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 Ikappa B inhibitor family; members of the Ikappa B family include Ikappa Balpha , Ikappa Bbeta , and Ikappa BR (3). TNF-alpha has been reported to induce rapid phosphorylation and subsequent degradation of Ikappa B, allowing NF-kappa 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-kappa B. These authors reported that overexpression of Ikappa BR in the alveolar epithelial cell line A549 sequesters p50 homodimers, thereby facilitating the binding of a unique NF-kappa B-Rel complex to the RANTES kappa 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, Ikappa 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-kappa 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.


    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.


    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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