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Am J Physiol Lung Cell Mol Physiol 283: L1280-L1290, 2002. First published August 9, 2002; doi:10.1152/ajplung.00162.2002
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Vol. 283, Issue 6, L1280-L1290, December 2002

Regulation of RANTES promoter activation in alveolar epithelial cells after cytokine stimulation

Antonella Casola1, Allyne Henderson1, Tianshuang Liu1, Roberto P. Garofalo1,2, and Allan R. Brasier3,4

Departments of 1 Pediatrics, 2 Microbiology and Immunology, and 3 Internal Medicine and 4 Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine family of proteins implicated in a variety of diseases characterized by lung eosinophilia and inflammation, strongly produced by stimulated airway epithelial cells. Because such cytokines as tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN-gamma ) have been shown to enhance RANTES induction in airway epithelial cells and RANTES gene expression appears to be differentially regulated depending on the cell type and the stimulus applied, in this study we have elucidated mechanisms that operate to control RANTES induction on exposure to TNF-alpha and/or IFN-gamma . Our results indicate that TNF-alpha and IFN-gamma synergistically induce RANTES protein secretion and mRNA expression. RANTES transcription is activated only after stimulation with TNF-alpha , but not IFN-gamma , which affects RANTES mRNA stabilization. Promoter deletion and mutagenesis experiments indicate that the nuclear factor (NF)-kappa B site is the most important cis-regulatory element controlling TNF-induced RANTES transcription, although NF-interleukin-6 binding site, cAMP responsive element (CRE), and interferon-stimulated responsive element (ISRE) also play a significant role. TNF-alpha stimulation induces nuclear translocation of interferon regulatory factor (IRF)-3, which in viral infection binds the RANTES ISRE and is necessary for activation of RANTES transcription. However, TNF-induced IRF-3 translocation does not result in IRF-3 binding to the RANTES ISRE. Although viral infection can activate an ISRE-driven promoter, TNF cannot, indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion. Identification of molecular mechanisms involved in RANTES gene expression is fundamental for developing strategies to modulate lung inflammatory responses.

tumor necrosis factor; interferon; nuclear factor-kappa B; regulated on activation, normal T cell expressed, and presumably secreted


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

REGULATED ON ACTIVATION, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine family of proteins, strongly chemoattractant for eosinophils, basophils, monocytes, and memory T lymphocytes (1, 34). RANTES production has been implicated in a variety of diseases characterized by lung eosinophilia and inflammation, including asthma and respiratory syncytial virus (RSV) infection (2, 29, 32). Administration of RANTES in vitro produces transendothelial migration and degranulation of eosinophils (10) and in vivo eosinophilic inflammation in human upper airways (3). Concentration of RANTES is elevated in the bronchoalveolar lavage fluid of asthmatic patients (2, 38) and children with RSV bronchiolitis (11, 15, 35). Administration of a RANTES receptor antagonist or neutralizing RANTES antibodies completely blocks lung eosinophilia and inflammation in experimental models of asthma (14). Airway epithelial cells are major sites of RANTES production in these models of lung inflammation. Cytokines produced during acute inflammation can induce RANTES expression in different cell types. For example, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 increase RANTES secretion in fibroblast and epithelial cells, and interferon (IFN)-gamma has been shown to induce RANTES synergistically with TNF in epithelial and endothelial cells (24, 36).

Human RANTES gene expression appears to be differentially regulated depending on the cell type and the stimulus applied (17, 25, 26, 28, 30). Induction of transcription is an important level of control of RANTES gene expression, and different combinations of cis-regulatory elements of the promoter seem to be required for optimal levels of transcription in a variety of cell types, such as monocytes, lymphocytes, and astrocytes, after stimulation with cytokines or phorbol esters (17, 25, 26, 28, 30). In T lymphocytes, important regulatory elements of RANTES promoter activity are the nuclear factor (NF)-kappa B, NF-IL-6, NF-AT, and CD28RE binding sites (27). We and others recently showed the essential role played by the ISRE site in virus-induced RANTES transcription through activation and binding of transcription factors belonging to the interferon regulatory factor (IRF) family, specifically IRF-1, IRF-3, and IRF-7 (7, 13, 23). However, a complete analysis of the promoter cis-regulatory elements and NF involved in regulation of RANTES gene transcription after cytokine stimulation of airway epithelial cells has not been performed. The goal of this study was to define the effect of TNF-alpha and IFN-gamma stimulation on RANTES gene transcription in airway epithelial cells. Our results indicate that TNF-alpha and IFN-gamma synergistically induce RANTES protein secretion and mRNA expression. However, RANTES transcription is activated after stimulation with TNF-alpha , but not IFN-gamma , which affects RANTES mRNA stabilization. Promoter deletion and mutagenesis experiments indicate that the NF-kappa B site is the most important cis-regulatory element controlling TNF-induced RANTES transcription, although the NF-IL-6 binding site, the cAMP responsive element (CRE), and the interferon-stimulated responsive element (ISRE) also play an important role. Similar to viral infection, TNF stimulation induces nuclear translocation of IRF-3. However, TNF-induced translocation of IRF-3 does not result in IRF-3 binding to the RANTES ISRE. Furthermore, although viral infection can activate an ISRE-driven promoter, TNF cannot, indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion, similar to what we have shown for other chemokines (6). Identification of the molecular mechanisms involved in RANTES gene expression is fundamental for developing strategies to modulate lung inflammatory responses.


    METHODS
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INTRODUCTION
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Cell culture. A549 cells (American Type Culture Collection, Manassas, VA), human alveolar type II-like epithelial cells, were maintained in Ham's F-12K medium containing 10% (vol/vol) fetal bovine serum, 10 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

RANTES ELISA. Immunoreactive RANTES was quantitated by a double-antibody ELISA kit (DuoSet, R & D Systems, Minneapolis, MN) following the manufacturer's protocol.

Northern blot. Total RNA was extracted from control and infected A549 cells by the acid guanidinium thiocyanate-phenol-chloroform method (33). RNA (20 µg) was fractionated on a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to a radiolabeled RANTES cDNA, as previously described (5). After the membrane was washed, it was exposed for autoradiography using Kodak XAR film at -70°C using intensifying screens. The membrane was stripped and reprobed for beta -actin as internal control for equal loading of the samples.

mRNA half-life measurements. A549 cells were stimulated with TNF-alpha (100 ng/ml), alone or in combination with IFN-gamma (100 IU/ml) for 3 h; then 100 µM 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB), a polymerase II inhibitor (9), was added to the treated cells without a change of the medium. Total RNA was isolated at time 0 of DRB addition and at multiple times thereafter. RNA was fractionated on a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to a radiolabeled RANTES cDNA. The membrane was exposed to a PhosphorImager screen for 24 h, and images were visualized and quantitated using PhosphorImager SI analysis and ImageQuant software (Molecular Dynamics).

Plasmid construction and cell transfection. 5'-Deletion constructs of the human RANTES promoter were produced by PCR using as template the full-length human RANTES promoter from nucleotides -974 to +55 relative to the mRNA start site designated +1, cloned into the pGL2 vector (pGL2-974), as previously described (7). Upstream primers incorporating a unique KpnI restriction site were designed to produce 5'-deletions at nucleotides -220, 195, 150, and 120; the downstream oligonucleotide, containing a unique HindIII restriction site, was designed to hybridize from nucleotides +30 to +55. The PCR products were restricted with KpnI and HindIII, gel purified, cloned into the luciferase reporter gene vector pGL2 (Promega, Madison, WI), and named pGL2-220, pGL2-195, pGL2-150, and pGL2-120, as previously described (7).

Site-directed mutations of the RANTES promoter were introduced by the PCR overlap extension mutagenesis technique using pGL2-220 as template and mutagenic primers identical to the oligonucleotides used in electrophoretic mobility shift assay (EMSA; Table 1), with the exclusion of the 5'-GATC sequence, as previously described (7). The RANTES ISRE multimer was constructed by ligation of three copies of the ISRE site upstream of the nucleotide -54 of the human IL-8 luciferase promoter, as previously described (6). Before ligation, 100 pmol of the duplex oligonucleotides were phosphorylated with T4 kinase and ligated with T4 DNA ligase, and trimers were isolated by nondenaturing gel electrophoresis (PAGE).

                              
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  		Table 1.   Cis-regulatory elements of the RANTES promoter

Logarithmically growing A549 cells were transfected in triplicate in 60-mm petri dishes by DEAE-dextran, as previously described (8). Cells were incubated in 2 ml of HEPES-buffered DMEM (10 mM HEPES, pH 7.4) containing 20 µl of 60 mg/ml DEAE-dextran (Pharmacia) premixed with 6 µg of RANTES-pGL2 plasmids and 1 µg of cytomegalovirus-beta -galactosidase internal control plasmid. After 3 h, medium was removed, and 0.5 ml of 10% (vol/vol) DMSO in PBS was added to the cells for 2 min. Cells were washed with PBS and cultured overnight in 10% fetal bovine serum-DMEM. The next morning, cells were stimulated with TNF-alpha (100 ng/ml) and IFN-gamma (100 IU/ml), alone or in combination. At different time points, cells were lysed to measure independently luciferase and beta -galactosidase reporter activity, as previously described (4). Luciferase was normalized to the internal control beta -galactosidase activity. All experiments were performed in duplicate or triplicate.

EMSA. Nuclear extracts of untreated and treated A549 cells were prepared using hypotonic/nonionic detergent lysis, as previously described (8). Proteins were normalized by protein assay (Protein Reagent, Bio-Rad, Hercules, CA) and used to bind to duplex oligonucleotides corresponding to the RANTES CRE, ISRE, NF-IL-6, and NF-kappa B1 binding sites. Sequences of the oligonucleotides used for EMSA are shown in Table 1. Nuclear extracts, used for binding to the CRE site, were prepared from control and treated A549 cells that had been serum-starved before and throughout the period of stimulation for a total of 24 h.

DNA-binding reactions, using the CRE, ISRE, and NF-IL-6 probes, contained 10-15 µg of nuclear protein, 5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol (DTT), 5 mM MgCl2, 0.5 mM EDTA, 1 µg of poly(dI-dC), and 40,000 cpm of 32P-labeled double-stranded oligonucleotide in a total volume of 20 µl. DNA-binding reactions, using NF-kappa B1 probe, contained 10-15 µg of total protein, 5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM DTT, 1 µg of poly(dA-dT), and 40,000 cpm of 32P-labeled double-stranded oligonucleotide in a total volume of 20 µl. The nuclear proteins were incubated with the probe for 15 min at room temperature and then fractionated by 6% nondenaturing polyacrylamide gels (PAGE) in TBE buffer (22 mM Tris · HCl, 22 mM boric acid, and 0.25 mM EDTA, pH 8). After electrophoretic separation, gels were dried and exposed for autoradiography using Kodak XAR film at -70°C using intensifying screens. In competition assays, 2 pmol of unlabeled wild-type (WT) or mutated competitors were added at the time of probe addition. In the gel mobility supershift, commercial antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against specific transcription factors were added to the binding reactions and incubated on ice for 1 h before fractionation on 6% PAGE. In the case of CRE supershift, we used antibodies broadly reactive with different members of the activating transcription factor (ATF)/CRE binding (CREB) or activator protein-1 (AP-1) families. Anti-CREB-1 antibody also recognizes ATF-1 and CRE modulator (CREM)-1 (sc-186); anti-c-Fos recognizes c-Fos, Fos-B, Fra-1, and Fra-2 (sc-413); anti-c-Jun recognizes c-Jun, Jun-B, and Jun-D (sc-44). Preimmune serum was used as a control for any nonspecific effects of the immune antisera.

Microaffinity isolation assay. Microaffinity purification of proteins binding to the RANTES ISRE was performed using a two-step biotinylated DNA-streptavidin capture assay (8). In this assay, duplex oligonucleotides are chemically synthesized using 5'-biotin on a flexible linker (Sigma Genosys). Four hundred micrograms of 6-h-treated A549 cell nuclear extracts were incubated at 4°C for 30 min with 50 pmol of biotin-ISRE, in the absence or presence of 10-fold molar excess of nonbiotinylated WT or mutated ISRE. The binding buffer contained 8 µg of poly(dI/dC) (as nonspecific competitor) and 5% (vol/vol) glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM DTT, 5 mM MgCl2, and 0.5 mM EDTA. A 50% slurry of prewashed streptavidin-agarose beads (100 µl) was then added to the sample, which was incubated at 4°C for an additional 20 min with gentle rocking. Pellets were washed twice with 500 µl of binding buffer, and the washed pellets were resuspended in 100 µl of 1× SDS-PAGE buffer, boiled, and fractionated on a 10% SDS-polyacrylamide gel. After electrophoresis separation, proteins were transferred to polyvinylidene difluoride membranes for Western blot analysis.

Western immunoblot. Nuclear proteins were prepared as previously described, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (8). Membranes were blocked with 5% milk in Tris-buffered saline-Tween and incubated with rabbit polyclonal antibodies to IRF-1 and IRF-3 (Santa Cruz Biotechnology). For secondary detection, we used a horseradish-coupled donkey anti-rabbit antibody in the enhanced chemiluminescence assay (Amersham Life Science, Arlington Heights, IL).

Statistical analysis. Data from experiments involving multiple samples subject to each treatment were analyzed by the Student-Newman-Keuls t-test for multiple pairwise comparisons. Results were considered significantly different at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha and IFN-gamma stimulation induces RANTES gene expression in A549 cells. To determine whether cytokine stimulation of A549 cells induces RANTES protein release, cells were exposed to TNF-alpha (100 ng/ml) and IFN-gamma (100 IU/ml), alone or in combination. Cell supernatants were harvested at different times after stimulation, and RANTES protein secretion was measured by ELISA. TNF induced a time-dependent release of RANTES from A549 cells (Fig. 1). IFN-gamma treatment alone (shown only for the 24-h time point) did not upregulate RANTES secretion; however, it greatly potentiated the effect of TNF-alpha . To determine whether the increased protein release paralleled an increase in the steady-state level of RANTES mRNA, A549 cells were exposed to TNF-alpha (100 ng/ml) and IFN-gamma (100 IU/ml), alone or in combination, and total RNA was extracted from control and treated cells at different time points for Northern blot analysis. A significant increase in RANTES mRNA expression was detected 3 h after TNF exposure (Fig. 2), with no further increase in mRNA levels at later time points. IFN-gamma treatment alone did not upregulate RANTES mRNA (data not shown), but it greatly potentiated the effect of TNF-alpha . These data indicate that TNF-alpha and IFN-gamma synergistically activate RANTES gene expression, which is coupled to protein secretion, in A549 cells.


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  		Fig. 1.   Effect of tumor necrosis factor (TNF) treatment on regulated on activation, normal T cell expressed, and presumably secreted (RANTES) secretion. A549 cells were treated with TNF-alpha (100 ng/ml), alone or in combination with interferon-gamma (IFN-gamma , 100 IU/ml), and culture supernatants were assayed for RANTES production by ELISA. Values are means ± SD of 3 independent experiments performed in triplicate.



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  		Fig. 2.   Northern blot of RANTES mRNA in TNF-treated A549 cells. Cells were treated with TNF-alpha (100 ng/ml), alone or in combination with IFN-gamma (100 IU/ml). Total RNA was extracted from control and infected cells, and 20 µg of RNA were fractionated on a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to a radiolabeled RANTES cDNA probe. Membrane was stripped and reprobed with beta -actin.

TNF-alpha stimulation induces RANTES promoter activation, whereas IFN-gamma stabilizes mRNA transcripts. To determine whether TNF exposure of alveolar epithelial cells was inducing RANTES gene expression by activating its transcription, we transiently transfected A549 cells with a construct containing the first 974 nt of the human RANTES promoter linked to the luciferase reporter gene (pGL2-974). Previous studies have shown that this fragment of the promoter is sufficient to drive regulated luciferase expression in a variety of cell types (25, 26, 28). Compared with untreated cells, TNF-alpha stimulation of transfected A549 cells induced a time-dependent increase of luciferase activity (Fig. 3) that started 3 h after exposure, peaked at ~6 h, and gradually decreased by 24 h. IFN-gamma treatment did not induce RANTES promoter activation alone or in combination with TNF, suggesting that the synergistic effect in RANTES mRNA induction by the combined stimulation of TNF-alpha and IFN-gamma could be due to an effect of IFN-gamma on RANTES mRNA stabilization. Therefore, we investigated the half-life of RANTES mRNA in the presence of IFN-gamma stimulation. A549 cells were treated with TNF-alpha , alone or in combination with IFN-gamma , for 3 h, and then DRB, an inhibitor of transcription, was added to the medium. RANTES mRNA levels were analyzed in cells harvested immediately before addition of DRB (which represents time 0) and at multiple times thereafter. Inhibition of transcription caused a progressive decrease in RANTES mRNA levels in A549 cells treated with TNF-alpha (Fig. 4), with 90% reduction by 24 h after termination of transcription. In contrast, in cells treated with both TNF-alpha and IFN-gamma , the decrease in RANTES mRNA levels was much less pronounced, with a reduction of only ~33% after 24 h of DRB addition, indicating that IFN-gamma affects RANTES mRNA stability. These results indicate that the synergistic effect of these two cytokines on the upregulation of RANTES expression in alveolar epithelial cells occurs via the combination of increased RANTES gene transcription by TNF-alpha and stabilization of transcripts by IFN-gamma .


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  		Fig. 3.   RANTES promoter activation after treatment with TNF and IFN. A549 cells were transiently transfected with pGL2-974 plasmid and treated with TNF-alpha (100 ng/ml) and IFN-gamma (100 IU/ml), alone or in combination. Cells were harvested to measure luciferase activity. Uninfected plates served as controls. For each plate, luciferase was normalized to beta -galactosidase reporter activity. Values are means ± SD.



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  		Fig. 4.   RANTES mRNA half-life after cytokine stimulation. A549 cells were stimulated with TNF-alpha (100 ng/ml), alone or in combination with IFN-gamma (100 IU/ml), for 3 h, and 100 µM 5,6-dichloro-1-beta -D-ribofuranosylbenzimidizole (DRB) was added to treated cells. Total RNA was isolated, and Northern blot analysis was performed using a radiolabeled RANTES cDNA. RANTES mRNA was quantitated by exposure of membrane to a PhosphorImager screen and analysis of images by ImageQuant software. Results are representative of 1 of 2 separate assays.

We then focused on characterizing the mechanisms of RANTES promoter activation after TNF-alpha stimulation.

Effects of 5'-deletions and site mutations of the RANTES promoter sequence on TNF-inducible activity. To define the regions of the RANTES promoter involved in regulating gene expression after TNF stimulation, A549 cells were transiently transfected with plasmids containing serial 5'-to-3'-deletions of the RANTES promoter linked to the luciferase reporter gene. Cells were treated with 100 ng/ml of TNF-alpha and harvested 6 h thereafter (conditions corresponding to peak reporter gene induction) to measure luciferase activity. As shown in Fig. 4, deletions from nucleotides -974 to -220 did not affect basal or TNF-inducible luciferase activity. Further deletion to nucleotide -150 slightly reduced the basal activity of the promoter and the TNF-induced luciferase activity by ~30-40%, indicating that the sequence between nucleotides -220 and -150 is involved in RANTES promoter activation. Deletion to nucleotide -120 almost completely abolished TNF-induced luciferase activity, indicating again that the region spanning nucleotides -150 to -120 is important for promoter activation. Recent studies of the RANTES promoter have identified a CRE site at nucleotides -220 to -190 and an ISRE site in the region at nucleotides -150 to -120 of the promoter (7, 13, 25). To establish the role of the CRE and ISRE sites of the RANTES promoter in conferring responsiveness to TNF stimulation, we tested the effect of point mutations of these sites in the context of the minimal RANTES promoter fragment (-220 nt) that retains full TNF inducibility. As shown in Fig. 5, mutation of the CRE site affected basal activity and TNF inducibility of the promoter similar to deletion of the promoter region spanning nucleotides -220 to -150. Mutation of the ISRE did not affect the basal activity, but it significantly reduced TNF-induced promoter activation. Previous studies have indicated that the NF-IL-6 and two NF-kappa B binding sites located between nucleotides -110 and -30 of the promoter can play an important role in RANTES gene transcription (7, 25, 30). To determine their role in TNF-induced RANTES transcription, we introduced site-directed mutations in each of the binding sites, and we tested the mutant plasmids for TNF inducibility. The proximal NF-kappa B site is defined as NF-kappa B2 and the distal NF-kappa B site as NF-kappa B1. As shown in Fig. 5, mutation of NF-kappa B1 almost completely abolished TNF-induced promoter activation, whereas mutations of the NF-IL-6 and NF-kappa B2 sites significantly decreased luciferase activity, although to a lesser extent, indicating that NF-kappa B1 is the major regulatory site of the RANTES promoter induction after TNF stimulation.


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  		Fig. 5.   Effects of 5'-deletions and site mutations of the RANTES promoter sequence on TNF-inducible activity. A: A549 cells were transiently transfected with 5'-deletions of the human RANTES promoter and treated with TNF-alpha (100 ng/ml) for 6 h. Uninfected plates served as controls. For each plate, luciferase was normalized to beta -galactosidase reporter activity. Values are means ± SD. *P < 0.01 vs. pGL2-974. B: A549 cells were transiently transfected with site-mutated plasmids of the pGL2-220 RANTES promoter and treated with TNF-alpha (100 ng/ml) for 6 h. Uninfected plates served as controls. For each plate, luciferase was normalized to beta -galactosidase reporter activity. WT, wild type; Delta , mutation of the indicated promoter site; NF, nuclear factor; IL, interleukin; CRE, cAMP responsive element; ISRE, interferon-stimulated responsive element. Values are means ± SD. *P < 0.01 vs. pGL2-220 WT.

Role of NF-kappa B and NF-IL-6 in RANTES promoter inducibility after TNF stimulation. Because the site mutation analysis of the RANTES promoter had shown that NF-kappa B plays a major role in TNF-induced promoter activation, we performed gel shift assays to determine whether TNF addition produced changes in the abundance of DNA-binding proteins recognizing this region of the RANTES promoter.

Two DNA-protein complexes, C1 and C2, were formed from nuclear extracts of control cells on the NF-kappa B1 probe (Fig. 6A). TNF exposure markedly increased the binding of C1 and C2 as early as 1 h after stimulation, with a decrease in binding intensity at 12 h after stimulation. The sequence specificity of the different complexes was examined by competition with unlabeled oligonucleotides in EMSA (Fig. 6B). C1 and C2 were competed by the WT, but not by the mutated, oligonucleotide, indicating binding specificity. To determine the composition of the inducible complexes, we performed supershift assays, adding specific antibodies to various NF-kappa B subunits in EMSA. We did not include the antibody anti-RelB, because we and others previously showed that it is not present in epithelial cells (12, 39). The addition of anti-p50 antibody induced the appearance of a supershifted band, with a concomitant reduction of C2 intensity (Fig. 6C). Addition of anti-p65 antibody mainly supershifted C1, indicating that C1 is a p65 homodimer, whereas C2 could be a homo- or heterodimer of p50.


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  		Fig. 6.   Electrophoretic mobility shift assay (EMSA) of RANTES NF-kappa B1 binding complexes in response to TNF treatment. A: autoradiogram of time course. Nuclear extracts were prepared from control and TNF-treated cells at 1, 3, 6, and 12 h and used for EMSA. B: competition analysis. Nuclear extracts from A549 control cells or A549 cells treated with TNF for 6 h were used to bind to the NF-kappa B1 probe in the absence (--) or presence of 2 pmol of unlabeled WT or mutated (MUT) competitor in the binding reaction. C: supershift assay. Nuclear extracts of A549 cells treated with TNF for 6 h were used in the EMSA in the presence of control serum, anti-p50, anti-p52, anti-c-Rel, and anti-p65 antibodies. Arrows, supershifted bands.

Similar experiments were performed using the RANTES NF-IL-6 binding site. When nuclear extracts from A549 cells were used to bind to the NF-IL-6 probe, three complexes (C1, C2, and C3) were formed in control cells (Fig. 7A). TNF exposure markedly increased the binding of C1 and C3 as early as 1 h after stimulation, which remained similar up to 12 h after stimulation. In a competition assay, C1 and C3 were competed by the WT, but not by the mutated, oligonucleotide, indicating binding specificity (Fig. 7B). In a supershift assay, using antibodies anti-CCAAT/enhancer binding protein (C/EBP)-alpha , anti-C/EBPbeta /NF-IL-6, and anti-C/EBPdelta , the main C/EBP family members involved in promoter transactivation (31), the addition of anti-C/EBPbeta resulted in the disappearance of C1 and C3 nucleoprotein complexes and the appearance of a supershifted band (arrow), showing that C/EBPbeta is the major component of the TNF-inducible C1 and C3 (Fig. 7C). Together, the EMSA indicates that TNF stimulation induced significant changes in the composition of proteins binding to the proximal RANTES NF-IL-6 and NF-kappa B sites.


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  		Fig. 7.   EMSA of RANTES NF-IL-6 binding complexes in response to TNF treatment. A: autoradiogram of time course. Nuclear extracts were prepared from control and TNF-treated cells at 1, 3, 6, and 12 h and used for EMSA. B: competition analysis. Nuclear extracts from untreated (control) A549 cells and A549 cells treated with TNF for 6 h were used to bind to the NF-IL-6 probe in the absence (--) or presence of 2 pmol of unlabeled WT or mutated competitor. C: supershift assay. Nuclear extracts of A549 cells treated with TNF for 6 h were used in the EMSA in the presence of control serum, anti-CCAAT/enhancer binding protein (C/EBP)-alpha , anti-C/EBPbeta , and anti-C/EBPdelta . Arrow, supershifted band.

Role of CRE and ISRE binding proteins in RANTES promoter inducibility after TNF stimulation. To determine whether TNF stimulation produced changes in the abundance of DNA-binding proteins recognizing the RANTES CRE site, EMSA was performed on nuclear extracts prepared from control and TNF-treated A549 cells. The CRE sites can bind homo- and heterodimeric complexes formed by members of the CREB, ATF, Jun, and Fos transcription factor families. To exclude uncontrolled effects due to the presence of serum on c-Fos and Jun expression (20), serum-starved cells were stimulated with TNF-alpha (100 ng/ml) and harvested at different times to prepare nuclear extracts. Two binding complexes, C1 and C2, were detected in control A549 cells (Fig. 8A); TNF treatment slightly increased C1 binding, which could be detected 1 h after stimulation and persisted for the duration of the experiment. The inducible C1 complex was sequence specific, as demonstrated by its competition by an unlabeled WT, but not a mutant, oligonucleotide (Fig. 8B). To determine the composition of the TNF-inducible complex, we performed supershift assays using a panel of antibodies broadly reacting with the different members of CREB, ATF, Fos, and Jun families of transcription factors (see METHODS). Addition of anti-CREB-1 antibody caused a significant reduction of C1 complex, whereas addition of anti-ATF-2 antibody induced the appearance of a faint supershifted band (Fig. 8C, arrow), suggesting that these members of the CREB/ATF family are the major components of the CREB complexes.


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  		Fig. 8.   EMSA of RANTES CRE binding complexes in response to TNF stimulation. A: autoradiogram of time course. Nuclear extracts were prepared from control and TNF-treated cells at 1, 3, 6, and 12 h and used for EMSA. B: competition analysis. Nuclear extracts from untreated (control) A549 cells and A549 cells treated with TNF for 6 h were used to bind to the CRE probe in the absence (--) and presence of 2 pmol of WT and mutated unlabeled competitor in the binding reaction. C: supershift assay. Nuclear extracts of A549 cells treated with TNF for 6 h were used in the EMSA in the presence of control serum, anti-Jun, anti-Fos, anti-CREB-1, anti-CREB-2, and anti-activating transcription factor (ATF)-2 antibodies. Arrow, supershifted band.

Finally, we investigated changes in the abundance of DNA-binding proteins recognizing the RANTES ISRE site after TNF treatment. Three nucleoprotein complexes, C1, C2, and C3, were formed in control cells using the ISRE probe (Fig. 9A). TNF stimulation increased the binding of C2 and C3 as early as 30 min after exposure, with a peak in binding intensity 6 h after TNF stimulation (binding intensity decreased at later time points; data not shown). The sequence specificity of the ISRE complexes was examined by competition with unlabeled oligonucleotides in EMSA (Fig. 9B). C2 and C3 were competed by the WT, but not by the mutated, oligonucleotide, indicating binding specificity. RANTES ISRE binds transcription factors belonging to the ISRE family. To determine the composition of the TNF-inducible complex, preimmune serum or antibodies recognizing IRF-1, IRF-2, IRF-3, and IRF-7 were added to the binding reaction. We did not include anti-interferon consensus sequence binding protein, because this protein is expressed only in hematopoietic cell types (37). The anti-IRF-1 antibody affected C2 and C3 binding (Fig. 9C), inducing the disappearance of the complexes and the appearance of a supershifted band (arrow), indicating that IRF-1 is a major component of the TNF-inducible complexes.


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  		Fig. 9.   EMSA of RANTES ISRE binding complexes in response to TNF stimulation. A: autoradiogram of time course. Nuclear extracts were prepared from control and TNF-treated cells at 0.5, 1, 3, and 6 h and used for EMSA. B: competition analysis. Nuclear extracts from untreated (control) A549 cells and A549 cells treated with TNF for 6 h were used to bind to the ISRE probe in the absence (--) and presence of 2 pmol of unlabeled WT and mutated competitor. C: supershift assay. Nuclear extracts of A549 cells were treated with TNF for 6 h in the presence of control serum, anti-interferon regulatory factor (IRF)-1, anti-IRF-2, anti-IRF-3, and anti-IRF-7 antibodies. Arrow, supershifted band.

We and others previously showed that IRF-3 and IRF-7 play an important role in virus-induced RANTES gene transcription (7, 13). Because the absence of a supershift cannot be used to exclude the presence of IRF-3 or IRF-7 binding to the ISRE, we first investigated whether TNF stimulation was able to induce IRF-3 activation. Nuclear extracts prepared from A549 control cells and A549 cells treated with TNF for various lengths of time were used for Western blot analysis. TNF stimulation induced a time-dependent nuclear accumulation of IRF-1 and IRF-3 starting at ~3 h after exposure (Fig. 10A). We then used a two-step microaffinity isolation-Western blot to determine whether IRF-3 was binding to the RANTES ISRE after TNF stimulation. In this assay, biotinylated ISRE was used to bind nuclear extracts of control A549 cells and A549 cells treated with TNF for 6 h. ISRE binding proteins were captured by the addition of streptavidin-agarose beads and washed, and the presence of bound IRF-1 and IRF-3 was detected by Western blot. As a control, we used nuclear extracts prepared from A549 cells infected with RSV, with a multiplicity of infection of 1, for 12 h, which we previously showed is able to induce IRF-1 and IRF-3 binding to the RANTES ISRE (7). There was little binding of IRF-1 and no binding of IRF-3 in control nuclear extracts (Fig. 10B). IRF-1 binding was greatly increased after TNF stimulation and RSV infection. However, although IRF-3 from A549 cells infected with RSV was able to bind to the RANTES ISRE, IRF-3 from TNF-stimulated cells did not. IRF-1 and IRF-3 detection was abolished when 10-fold excess of ISRE oligonucleotide was included as competitor in the initial binding reaction, indicating sequence specificity. These data indicate that although TNF stimulation induces IRF-1 and IRF-3 nuclear translocation, only IRF-1 binds to the RANTES ISRE site. Finally, we investigated whether TNF stimulation was able to activate a promoter driven by the RANTES ISRE. A549 cells were transfected with a reporter gene containing multimers of the RANTES ISRE and infected with RSV or stimulated with TNF-alpha for various lengths of time. Activity of the ISRE multimer was highly inducible after RSV infection but was not significantly affected by TNF stimulation (Fig. 11), indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion.


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  		Fig. 10.   Western blot and microaffinity isolation of IRF-1 and IRF-3 protein in A549 cells treated with TNF. A: A549 cells were stimulated with TNF-alpha (100 ng/ml) and harvested at 0, 3, 6, and 12 h for preparation of nuclear extracts. Equal amounts of protein from control and treated cells were assayed for IRF-1 and IRF-3 by Western blot. B: nuclear extracts were prepared from untreated (control) A549 cells and A549 cells stimulated with TNF (100 ng/ml) for 6 h or infected with respiratory syncytial virus (RSV, multiplicity of infection = 1) for 12 h. IRF proteins were affinity purified using biotinylated ISRE in the absence (--) or presence (+) of nonbiotinylated WT competitor. After capture with streptavidin-agarose beads, complexes were eluted and assayed for IRF-1 and IRF-3 by Western blot.



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  		Fig. 11.   RANTES ISRE-driven promoter activation after viral infection and TNF stimulation. A549 cells were transiently transfected with multimers of the RANTES ISRE linked to a luciferase reporter gene. Cells were infected with RSV at multiplicity of infection of 1 or stimulated with TNF-alpha (100 ng/ml) for 3, 6, and 15 h. Cells were harvested to measure luciferase activity. Uninfected plates served as controls. For each plate, luciferase was normalized to beta -galactosidase reporter activity. Values are means ± SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The respiratory epithelium represents the principal cellular barrier between the environment and the internal milieu of the airways and is responsible for particulate clearance and surfactant secretion. After exposure to infectious agents, allergens, chemical pollutants, and particulate matter, airway epithelial cells are able to secrete a variety of proinflammatory molecules, including chemokines, which are responsible for the selective recruitment of circulating leukocytes into the lung (16, 18). RANTES is a member of the CC branch of the chemokine family, strongly chemotactic for T lymphocytes, monocytes, basophils, and eosinophils (1, 34), and has been implicated in a variety of diseases characterized by inflammation. Intercellular communication by cytokines during an inflammatory reaction is important to its orchestration and subsequent resolution. IFN-gamma and TNF-alpha are pleiotropic cytokines that often play a critical role in this process. Human RANTES gene expression appears to be differentially regulated depending on the cell type and the stimulus applied (17, 25, 26, 28, 30). Because identification of the regulatory mechanisms involved in RANTES gene transcription is important for a rational design of therapeutic agents that can block its expression in the lung, in this study we have examined the requirements for activation of RANTES gene expression after cytokine stimulation.

Our results show that IFN-gamma and TNF-alpha synergistically induce RANTES protein secretion and gene expression in alveolar epithelial cells (Figs. 1 and 2). However, the synergism does not occur at the level of RANTES gene transcription, because TNF-alpha is able to induce the RANTES promoter-driven expression of the luciferase reporter gene but not IFN-gamma (Fig. 3). Treatment of alveolar epithelial cells with IFN-gamma results in a prolonged half-life of RANTES transcripts (Fig. 4), indicating that the synergism occurs through RANTES mRNA stabilization, which has been shown to play an important role in virus-induced RANTES gene expression (19). Our results obtained in alveolar epithelial cells are different from results recently shown in fibroblasts, in which IFN-gamma and TNF-alpha stimulation synergistically activated the murine RANTES promoter (21), but are similar to results reported in astrocytes and glial cells after IL-1beta or TNF-alpha and IFN-gamma stimulation, which synergize in RANTES protein secretion and gene expression, but not transcription (22, 25). In glial cells, the half-life of RANTES mRNA in the presence of TNF-alpha and IFN-gamma was almost double that in the presence of TNF-alpha alone (22), which is very similar to our findings in alveolar epithelial cells.

Results from 5'-deletion and mutation analysis indicate that several cis-regulatory elements are required for full TNF-induced promoter activation. The shortest RANTES promoter fragment that is activated similarly to the full-length promoter after TNF-alpha stimulation comprises the first 220 bp from the start codon. Deletion of the region spanning nucleotides -220 to -195 reduces promoter inducibility ~50%, and a second deletion from nucleotides -150 to -120 almost completely abolishes it (Fig. 4). A few studies have investigated the minimal enhancer region of the RANTES promoter required to confer responsiveness to external stimuli such as cytokines or phorbol esters. In T cell and monocyte-like cell lines, the first 500 nucleotides of RANTES promoter are sufficient to confer full phorbol 12-myristate 13-acetate and ionomycin inducibility, which is lost by further deletions to nucleotide -200 (26). Differently, in phytohemagglutinin-stimulated lymphocytes, a 195-bp fragment of RANTES promoter has the same inducibility as longer fragments, whereas a 120-bp fragment is no longer inducible (28, 30). In a human astrocytoma cell line, IL-1 stimulation of RANTES promoter requires a region spanning nucleotides -220 to -120 (25), similar to our findings in alveolar epithelial cells infected with RSV (7), indicating that the minimal enhancer region required for RANTES promoter activation differs among cell types.

The site mutation analysis indicates that the CRE, ISRE, NF-IL-6, and NF-kappa B sites play an important role in TNF-induced RANTES promoter activation (Fig. 5), which is similar to our findings in alveolar epithelial cells infected with RSV (7). However, there are some important differences in the relative importance of these cis-regulatory elements in RANTES induction, as well as in the type of transcription factors binding to them, after these two different stimuli. The CRE site accounts for 40-50% of TNF-induced promoter activation, as shown by the deletion and site mutation analysis, which is similar to our findings in virus-infected cells (7). However, although TNF-alpha stimulation induces binding to the CRE site of transcription factors belonging to the CREB and ATF families (Fig. 8C), RSV infection also induces activation and binding of c-Jun (7).

Mutation of the NF-kappa B1 site almost completely abolishes TNF-induced promoter activity, indicating that the NF-kappa B site plays a fundamental role in cytokine-induced RANTES gene transcription. This result is different from our previous finding in RSV-infected cells, in which mutation of the NF-kappa B1 site accounts for a ~60% reduction of the promoter inducibility, although the composition of the DNA-nuclear complexes formed on the RANTES NF-kappa B1 site is identical in cytokine and viral stimulation, containing p65 and p50 subunits (Fig. 6C).

The situation is reversed for the ISRE site, since its mutation reduces TNF-stimulated luciferase activity ~50%, whereas it completely abolishes luciferase inducibility after RSV infection (7), indicating that the ISRE site is absolutely necessary for virus-induced RANTES promoter activation, but not for cytokine stimulation. Furthermore, the composition of the nucleoprotein complexes binding to the ISRE site after TNF-alpha addition or viral infection is quite different. Supershift assays and microaffinity isolation experiments (Figs. 9C and 10B) clearly show that TNF-alpha stimulation induces IRF-1, but not IRF-3, binding to the RANTES ISRE, even if it is able to induce IRF-3 translocation to the nucleus (Fig. 10A). This result is very different from our finding in virus-infected epithelial cells. RSV infection of A549 cells induces IRF-1, IRF-3, and IRF-7 binding to the ISRE site (8), similar to the finding of Lin et al. (23) in Sendai virus-infected cells. IRF-3 binding to the ISRE is necessary for activation of the ISRE site, as demonstrated by the inability of TNF-alpha to stimulate a RANTES ISRE-driven promoter, which is highly inducible after viral infection of alveolar epithelial cells (Fig. 11), indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion.

In conclusion, our findings indicate that cooperation among transcription factors belonging to the C/EBP, NF-kappa B, IRF, and CREB/ATF families is necessary for full transcriptional activation of the RANTES promoter after cytokine stimulation, similar to our finding in virus-infected cells, supporting again the "enhanceosome" model for RANTES gene transcription. However, the type and role of the different families of transcription factors in RANTES promoter activation in cytokine-stimulated cells are different from those in virus-infected cells, emphasizing the importance of detailed analysis of the mechanisms regulating tissue- and stimulus-specific expression of important genes, such as RANTES, to develop strategies for modulating the host immune/inflammatory responses in the lung.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants AI-01763, P01 AI-46004, and P30 ES-06676 and a Beginning Grant-in-Aid of the American Heart Association (to A. Casola).


    FOOTNOTES

Address for reprint requests and other correspondence: A. Casola, Dept. of Pediatrics, Div. of Child Health Research Center, 301 University Blvd., Galveston, TX 77555-0366 (E-mail: ancasola{at}utmb.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.

August 9, 2002;10.1152/ajplung.00162.2002

Received 23 May 2002; accepted in final form 3 August 2002.


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RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 283(6):L1280-L1290
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