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Multiple NF-κB and IFN Regulatory Factor
Family Transcription Factors Regulate
CCL19 Gene Expression in Human
Monocyte-Derived Dendritic Cells
Taija E. Pietilä, Ville Veckman, Anne Lehtonen, Rongtuan
Lin, John Hiscott and Ilkka Julkunen
J Immunol 2007; 178:253-261; ;
doi: 10.4049/jimmunol.178.1.253
http://www.jimmunol.org/content/178/1/253
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Copyright © 2007 by The American Association of
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References
The Journal of Immunology
Multiple NF-␬B and IFN Regulatory Factor Family
Transcription Factors Regulate CCL19 Gene Expression
in Human Monocyte-Derived Dendritic Cells1
Taija E. Pietilä,2* Ville Veckman,* Anne Lehtonen,† Rongtuan Lin,‡ John Hiscott,‡
and Ilkka Julkunen*
D
endritic cells (DCs)3 are professional APCs that reside in
peripheral tissues where they are on alert for emerging
pathogens. DCs regulate the activation of both innate
and adaptive immune responses, and their role during microbial
infections is well recognized. Upon contact with a microbe, they
undergo a maturation process that is associated with enhanced expression of Ag presenting and costimulatory molecules and the
production of cytokines and chemokines. Eventually, DCs migrate
to a local lymph nodes where Ag presentation to naive T cells
takes place (1).
DC trafficking is tightly controlled by differential expression of
chemokine ligands and chemokine receptors. Immature DCs express CCR6 and respond to its exclusive ligand CCL20 (2). How-
*Department of Viral Diseases and Immunology, National Public Health Institute,
Helsinki, Finland; †Molecular Cancer Biology Program, Institute of Biomedicine,
Biomedicum Helsinki, Finland; and ‡Lady Davis Institute for Medical Research, Department of Microbiology and Immunology, Department of Medicine, and Department of Oncology, McGill University, Montreal, Canada
Received for publication May 24, 2006. Accepted for publication October 9, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Medical Research Council of the Academy of
Finland and the Sigrid Juselius Foundation.
2
Address correspondence and reprint requests to Dr. Taija E. Pietilä, Department of
Viral Diseases and Immunology, National Public Health Institute, Mannerheimintie
166, FI-00300 Helsinki, Finland. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; IRF, IFN regulatory factor;
ISRE, IFN-stimulated response element; ISGF3, IFN-stimulated gene factor 3; MOI,
multiplicity of infection; PDTC, pyrrolidine dithiocarbamate; DEM, diethyl maleate;
CsA, cyclosporin A; IKK␧, inducible I␬B kinase; TBK1, Tank binding kinase 1;
TRIF, Toll/IL-1R homology domain containing adaptor protein-inducing IFN-␤.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$2.00
www.jimmunol.org
ever, maturation of DCs leads to down-regulation of CCR6 and
thus loss of their responsiveness to CCL20. At the same time,
mature DCs gain responsiveness to CCL19 via induced CCR7 expression (2, 3). DCs eventually migrate to T cell zones of local
lymph nodes where T lymphocytes are also recruited by the effect
of chemokines. CCL19, and the other ligand of CCR7, CCL21, are
both expressed by stromal cells in the T cell zone of the lymph nodes
(4). Mice deficient in CCL19 and CCL21 or CCR7 display defective
DC traffic and impaired immune responses (5–7). Because DCs are
also capable of producing both CCL19 and CCL20, it is evident that
these chemokines have a unique role in DC biology.
Transcription of cytokine and chemokine genes requires the
controlled action of multiple transcription factors activated by microbes or their structural components. Activated transcription factors bind to the regulatory elements of cytokine/chemokine genes,
and may either repress or activate the transcription of the respective gene. Mammalian NF-␬B/Rel family has five members; NF␬B1 (p50), NF-␬B2 (p52), RelA (p65), RelB, and c-Rel. NF-␬B
plays an important role in the regulation of innate immunity by
activating a wide variety of immune response genes, such as proinflammatory cytokines and chemokines, and cell adhesion molecules (8 –10). NF-␬B proteins form various homo- or heterodimers
that bind to specific NF-␬B recognition sites located in the regulatory regions of many genes (10).
IFN regulatory factors (IRFs) constitute a family of nine
(IRF1–9) transcription factors that bind to a specific DNA motif
known as the IFN-stimulated response element (ISRE) (11).
Among IRF family members, IRF1, IRF3, and IRF7 have been
established as essential factors for chemokine gene expression
in response to viral or cytokine stimulation (12–16). Recently,
IRF5 has also been implicated in the regulation of cytokine and
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CCL19 chemokine has a central role in dendritic cell (DC) biology regulating DC traffic and recruitment of naive T cells to the
vicinity of activated DCs. In this study, we have analyzed the regulation of CCL19 gene expression in human monocyte-derived
DCs. DCs infected with Salmonella enterica or Sendai virus produced CCL19 at late times of infection. The CCL19 promoter was
identified as having two putative NF-␬B binding sites and one IFN-stimulated response element (ISRE). Transcription factor
binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of classical p50ⴙp65 and
alternative p52ⴙRelB NF-␬B proteins to both of the CCL19 promoter NF-␬B elements. Interestingly, Salmonella or Sendai virus
infection also increased the binding of multiple IFN regulatory factors (IRFs), STAT1, and STAT2, to the ISRE element. Enhanced
binding of IRF1, IRF3, IRF7, and IRF9 to the CCL19 promoter ISRE site was detected in Salmonella or Sendai virus-infected cell
extracts. The CCL19 promoter in a luciferase reporter construct was activated by the expression of NF-␬B p50ⴙp65 or p52ⴙRelB
dimers. IRF1, IRF3, and IRF7 proteins also activated CCL19 promoter in the presence of Sendai virus infection. CCL19 promoter
constructs mutated at NF-␬B and/or ISRE sites were only weakly activated. Ectopic expression of RIG-I (⌬RIG-I, CARDIF) or
TLR3/4 (TRIF, MyD88, IKK␧, or TBK1) signaling pathway components induced CCL19 promoter activity, suggesting that these
pathways are important in CCL19 gene expression. Our experiments reveal that expression of the CCL19 gene is regulated by a
combined action of several members of the NF-␬B, IRF, and STAT family transcription factors. The Journal of Immunology,
2007, 178: 253–261.
254
Materials and Methods
Cell culture
Leukocyte-rich buffy coats were obtained from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). PBMCs were
isolated by a density gradient centrifugation using Ficoll-Paque (Amersham Biosciences) followed by isolation of monocytes by Percoll gradient
centrifugation (Amersham Biosciences) as described previously (23, 31).
Remaining T and B cells were depleted using anti-CD3 and anti-CD19
magnetic beads (Dynal Biotech). Monocytes (2.5 ⫻ 106 cells/well) were
allowed to adhere to six-well plates (Falcon, BD Biosciences) for 1 h at
37°C in RPMI 1640 medium (Sigma-Aldrich) supplemented with antibiotics and glutamine without FCS. Nonadherent cells were removed by
washing with PBS. To obtain immature DCs, monocytes were grown in
RPMI 1640 medium (2 ml/well) supplemented with 10% FCS (Integro
BV), recombinant human-IL-4 (20 ng/ml; R&D Systems), and GM-CSF
(10 ng/ml; Biosite) for 6 –7 days. Fresh medium was added every 2 days (1
ml/well).
HEK293 cells (American Type Culture Collection (ATCC) CLR1573)
were maintained in Eagle-MEM (Sigma-Aldrich) with antibiotics and 10%
FCS. One day before transfections, 75 000 cells/well were seeded on 24well plates (Falcon) in Eagle-MEM with 2% FCS.
Infections
Salmonella enterica serovar typhimurium ATCC 14028 strain was grown
as described previously (23). A multiplicity of infection (MOI) of 5 was
used throughout the study. Sendai (Cantell strain) virus was grown as outlined by Ronni et al. (32). The hemagglutination titer of Sendai virus was
4098, and the infectivity of the stock in DCs was 6 ⫻ 109 PFU/ml (24). To
ensure 100% infection in DCs, a MOI of 50 was used.
Cytokines and inhibitors
Highly purified human leukocyte IFN-␣ and IFN-␥ were provided by the
Finnish Red Cross Blood Transfusion Service and used at 100 IU/ml. Recombinant human IL-1␤ and TNF-␣ were purchased from R&D Systems
and used at 10 ng/ml. PD98059 and SB202190 (Calbiochem) were used at
50 ␮M and 10 ␮M, respectively. Pyrrolidine dithiocarbamate (PDTC),
diethyl maleate (DEM), and cyclosporin A (CsA) were obtained from
FIGURE 1. Microbe-induced CCL19, CCL20, CXCL10, and IFN-␥
gene expression in human DCs. Monocyte-derived DCs from four individual blood donors were infected with Salmonella or Sendai virus for indicated times. A, The cells were pooled and total cellular RNA was isolated
for Northern blotting. Ethidium bromide staining of ribosomal RNA and
␤-actin probing were used to monitor equal RNA loading. B, CCL19,
CCL20, CXCL10, and IFN-␥ protein production from cell culture supernatants (blood donors were analyzed separately) was determined by
ELISA. Error bars represent SD of the means.
Alexis Biochemicals. PDTC and DEM were used at 100 ␮M unless otherwise indicated, and CsA was used at 1 ␮g/ml.
Northern blot analysis
Total cellular RNA was isolated with RNeasy Midi kit (Qiagen). Equal
amounts of RNA (10 ␮g) were size-fractionated on a 1.0% formaldehydeagarose gel, transferred to a nylon membrane (Hybond; Amersham Biosciences), and hybridized with CCL19 and CCL20 (provided by Dr. A.
Zlotnik, Neurocrine Biosciences, San Diego, CA) (33), IFN-␥ (34), and
CXCL10 (21) probes. To control equal RNA loading, ribosomal RNA
bands were stained with ethidium bromide, or membranes were hybridized
with a ␤-actin probe. The probes were labeled with [␣-32P]dATP (3000
Ci/mmol; Amersham Biosciences) by a random-primed DNA labeling kit.
Membranes were hybridized overnight in Ultrahyb buffer (Ambion) at
42°C. Membranes were washed three times with 1 ⫻ SSC/0.1% SDS at
42°C and once at 65°C, and then exposed to Kodak BioMax XAR films
(Eastman Kodak) at ⫺70°C with intensifying screens. The results were
quantitated using Kodak Digital Science 1D image analysis software.
ELISA
CCL19 and CCL20 levels from cell culture supernatants were determined
by a Duoset kit (R&D Systems). CXCL10 levels were measured using Ab
pairs and standards obtained from BD Pharmingen, and IFN-␥ determinations were conducted with an Elipair kit (Nordic Biosite).
Plasmids
DNA fragment of 1478 bp encompassing the CCL19 promoter area was
amplified from human macrophage chromosomal DNA using the following
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chemokine gene expression (17, 18). IRF9 (p48/ISGF3␥) interacts with STAT1 and STAT2 and forms a complex called IFNstimulated gene factor 3 (ISGF3) that is also able to bind to
ISRE sequences (11, 19). ISGF3 or IRF9/STAT1 complexes
have been previously associated with the regulation of chemokine gene expression (20, 21).
We have previously reported that CCL19 mRNA is induced in
human monocyte-derived DCs in response to Streptococcus pyogenes (22), Salmonella enterica serovar typhimurium (23), and
Sendai virus infections (24). Furthermore, recent findings by us
and others have shown that DCs harbor Salmonella (23, 25–27)
and that mature DCs containing Salmonella migrate toward chemokines CCL19 and CCL21 (27). At present, the mechanisms by
which CCL19 expression is regulated have been only partially revealed. Saccani and Natoli (28) have previously shown that LPSstimulated DCs up-regulate the expression of CCL19 by a methylation event occurring at lysine 9 of histone H3. Moreover, a
phenomenon called NF-␬B dimer exchange on CCL19 promoter
has been described in LPS-stimulated DCs (29). Also, the murine
CCL19 has been reported to contain a functional NF-␬B binding
site with an alternative consensus sequence that preferentially
binds RelB/p52 dimers (30).
Because CCL19 is central in DC biology, we aimed to study its
expression in human monocyte-derived DCs in detail. We have
used Salmonella or Sendai virus, which are able to induce the
expression of CCL19 in human DCs. We show that these microbes
activate different members of the NF-␬B, IRF, and STAT families
in DCs, and they bind to the CCL19 promoter NF-␬B and ISRE
elements in a coordinated fashion. These results were further confirmed by studying the transcriptional activity of the CCL19 promoter in a luciferase reporter assay.
CCL19 REGULATION IN HUMAN DENDRITIC CELLS
The Journal of Immunology
255
primers: sense, 5⬘-ACTCAGGGATCCTCACTTAATCCTAAG (BamHIsite underlined) and antisense, 5⬘-CGACTGAAGCTTAGGGGTGAAAT
GCAAGG (HindIII-site underlined) (all oligonucleotides used in the study
were obtained from DNA Technology). The resulting PCR product was
directly TA-cloned into the PCRII-vector (Invitrogen Life Technologies),
from which it was released by BamHI and HindIII digestion. The fragment
was cloned into BglII and HindIII restriction sites of the pGL3-basic (Promega) luciferase vector and named CCL19promWT thereafter. Site-specific mutations were introduced using the QuickChange II site-directed
mutagenesis kit (Stratagene), and the correct mutations were confirmed by
sequencing. The primers used for creating base mutations for relevant binding sites were as follows: NF-␬B(1), 5⬘-CACAGAATGGGACATGAAG
aaaAATTTaAGGCAGAGAAAGTGAAG; NF-␬B(2), 5⬘-GAAGCACCA
GTGAGGACAAaaaATAAAaaTAAGGAAGGGAGCAC; and ISREmut,
5⬘-CTGGGCGTTTCACATTTAaaaaCTCaaaCAAGGCC (mutated nucleotides are written in lower case).
Human p50 and p65 cDNAs have been described previously (14), and
p52 in pCDNA3 and RelB cloned in frame with a FLAG epitope in
pCDNA3 were provided by Dr. G. Natoli (Institute for Research in Biomedicine, Bellinzona, Switzerland). The human IRF1 gene was amplified
with sense, 5⬘-CTGCAGGATCCCCAACATGCCCATCACTTGGATG
and antisense, 5⬘-GCC CAGGATCCCTGCTACGCTGCACAGGGAA
TGG oligonucleotides and cloned into the BamHI site (underlined) of the
expression plasmid pBC12/CMV (35). Human IRF5 cDNA was cloned
from total cellular RNA isolated from human macrophages by PCR using
sense, 5⬘-TCGGACGGATCCACCATGAACCAGTCCATCCCAGT and
antisense, 5⬘- CCGTCTGGATCCCTATTATTGCATGCCAGCTGGGT
oligonucleotides and inserted into the BamHI site (underlined) of the
pCDNA3.1(⫹)-FLAG-tagged expression vector (36). Expression constructs for IRF3 (37), IRF7 (38), retinoic acid-inducible gene I (RIG-I) and
⌬RIG-I (39), inducible I␬B kinase (IKK␧), and Tank binding kinase 1
(TBK1) (40) have been described elsewhere. Toll/IL-1R homology domain
containing adaptor protein-inducing IFN-␤ (TRIF) and MyD88 expression
plasmids were gifts from Dr. K. Fitzgerald (University of Massachusetts
Medical School, Worcester, MA).
Transfections
HEK293 cells were seeded on 24-well plates 1 day before transfection,
after which the reporter and NF-␬B or IRF expression plasmids (100 ng/
plasmid) were introduced into cells using FuGENE6 (Roche) transfection
reagent. Renilla luciferase plasmid (10 ng/reaction; Promega) was used to
control transfection efficiency. Cells were collected 18 h after transfection.
In some experiments, the cells were infected with Sendai virus (MOI, 50)
for 24 h. Luciferase reporter assays were performed with a Dual Glo kit
(Promega) according to manufacturer’s instructions.
DNA affinity binding and Western blot analysis
Equal amounts of cells were collected, and nuclear extracts were prepared
by lysing the cells in buffer containing 10 mM HEPES-KOH, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM DTT, 1 mM Na3VO4, and a protease inhibitor
mixture (Complete; Roche). The remaining nuclei were lysed in 10 mM
HEPES, 400 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.01%
Triton X-100, 0.5 mM DTT, 1 mM Na3VO4, and a protease inhibitor mixture. Both strands of the DNA elements were synthesized with BamHI
overhangs as spacers, and the upper strand oligonucleotide was 5⬘-biotinylated (DNA Technology): CCL19 NF-␬B(1) (GGATCCGACATGAAG
GGGAATTTCAGGCAGAGAAA), NF-␬B(2) (GGATCCGAGGACAAG
GGATAAACCTAAGGAAGGG), and CCL19 ISRE (GGATCCCACAT
TTAGTTTCTCTTTCAAGGCCTTCT). Protein samples were incubated
for 2 h at 4°C with streptavidin-agarose beads (Pierce) coupled to annealed
oligonucleotides. The binding reactions were performed in binding buffer
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FIGURE 2. The effect of intracellular signaling pathway inhibitors on CCL19, CCL20, CXCL10, and IFN-␥ gene expression in DCs. Cells were
left untreated or treated with inhibitors 30 min before infection with Salmonella. A, PD98059, SB202190, PDTC/DEM, and CsA were used to block
ERK MAPK, p38 MAPK, NF-␬B, and NFAT pathways, respectively. The expression of CCL19 and CCL20 mRNAs was analyzed by Northern
blotting. Total cellular RNA was pooled from four individual blood donors. ␤-Actin probing was used to monitor equal RNA loading. A representative experiment of three is shown. B, DCs were treated with NF-␬B inhibitors PDTC or DEM with indicated concentrations and infected with
Salmonella. Inhibitors were added at the 10-h time point to ensure sufficient inhibitor concentration throughout the experiment. mRNA expression
was studied as in A at 24 h after infection using also CXCL10 and IFN-␥ probes. The Northern blotting results were quantitated by measuring the
band intensities and normalized by ␤-actin levels. Relative mRNA levels as compared with Salmonella 9 h (A) or Salmonella 24 h (B) are shown
below the corresponding probings.
256
CCL19 REGULATION IN HUMAN DENDRITIC CELLS
Results
Salmonella- and Sendai virus-induced expression of CCL19,
CCL20, CXCL10, and IFN-␥ genes in DCs
Chemokine and IFN-␥ expression was first studied at the mRNA
level by Northern blotting (Fig. 1A). DCs were left uninfected or
infected with Salmonella or Sendai virus, and total cellular RNA
was collected at different time points. Consistent with our previous
findings (23), CCL19 expression was only seen at later time points
starting from 9 h, whereas CCL20 expression peaked at 3 h and
was down-regulated thereafter. CXCL10 mRNA expression was
rapid and seen at 3-h infection after infection with Salmonella as
well as with the Sendai virus. Although CXCL10 mRNA levels
remained elevated almost 48 h after infection with Salmonella,
Sendai virus-induced CXCL10 mRNA levels returned to undetectable levels within 24 h. IFN-␥ mRNA expression was slowly increased until the 48-h time point in Salmonella-infected cells,
whereas its expression was only seen at 3 h in Sendai virus-infected cells.
Next, CCL19, CCL20, CXCL10, and IFN-␥ protein levels from
cell culture supernatants were determined by ELISA (Fig. 1B). The
results are well in line with the mRNA expression data. In monocyte-derived DCs, Salmonella appeared to be a better inducer of all
analyzed chemokine genes and IFN-␥ as compared with Sendai
virus. Typically, some constitutive CCL19 production was detected in uninfected DCs.
Cell signaling pathway inhibitors reduce CCL19 production
in DCs
To determine which intracellular signal transduction pathways are
required for CCL19 production in DCs, we treated the cells with
chemical inhibitors. PD98059 selectively blocks the activity of
ERK MAPK and has no effect on the activation of p38 or JNK (43,
44), whereas SB202190 inhibits the action of p38 MAPK (45).
PDTC and DEM are widely used as inhibitors of NF-␬B activation
(46 – 48), and CsA blocks the action of NFAT family of transcription factors (49). DCs were treated with these inhibitors 30 min
before infection with Salmonella and total cellular RNA was collected at 9 h. As shown in Fig. 2A, Salmonella-induced CCL19
mRNA levels were down-regulated to some extent in response to
all inhibitors tested. In contrast, CCL20 mRNA levels were clearly
affected by DEM and CsA. To further investigate the differential
effects of PDTC and DEM on CCL19 and CCL20 mRNA levels,
we performed a similar experiment as described above, and in-
FIGURE 3. Nucleotide sequence of the 5⬘-flanking promoter region of
the human CCL19 gene (essentially the same as the sequence derived from
accession no. AL162231). A fragment of the CCL19 promoter encompassing nucleotide residues from ⫺1437 to ⫹41 relative to the ⫹1 transcription
start site was cloned to a luciferase reporter vector and confirmed by sequencing. The sequence was compiled from the sequenced clone and the
data in accession no. AL162231. The program Vector NTI Suite was used to
analyze the sequence for putative transcription factor binding sites. Previously
identified transcription and translation initiation sites are marked with arrows,
and a putative TATA-box is underlined. Core sequences of the potential transcription factor binding sites are typed in bold face, and boxed sequences
indicate oligonucleotides used for DNA affinity binding experiments.
cluded CXCL10 and IFN-␥ in the analysis to monitor the overall
effects of the inhibitors (Fig. 2B). Consistent with the above results, CCL19 mRNA level was down-regulated dose dependently
in response to PDTC and DEM also at 24 h. However, we detected
nonspecific up-regulation of CCL20 mRNA in response to the
highest PDTC concentration. CXCL10 and IFN-␥ mRNA levels
were partially down-regulated as expected, demonstrating the importance of the NF-␬B sites in both of these genes (12, 50).
The human CCL19 promoter
To characterize transcription factors that could be involved in regulating CCL19 gene expression, we analyzed the CCL19 promoter
region using Vector NTI suite and MatInspector programs. The
detailed sequence of the promoter region is presented in Fig. 3. We
identified two putative NF-␬B binding sites located between nt
⫺62 to ⫺52 and nt ⫺363 to ⫺354 (relative to the transcription
start site) referred to as NF-␬B(1) and NF-␬B(2) hereafter. In addition, a potential consensus ISRE element was identified further
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containing 10 mM HEPES, 133 mM KCl, 10% glycerol, 2 mM EDTA, 1
mM EGTA, 0.01% Triton X-100, 0.5 mM DTT, 1 mM Na3VO4, and a
protease inhibitor mixture followed by washing the unbound proteins with
binding buffer. The oligonucleotide-bound proteins were released in SDS
sample buffer and separated on 8 or 10% SDS-PAGE gels. For direct Western blot analysis, cells were lysed in the presence of 0.5 mM DTT, 1 mM
Na3VO4, and a protease inhibitor mixture, and 20 ␮g of protein aliquots
were run on 10% SDS-PAGE gels.
Proteins were transferred onto Immobilon-P membranes (Millipore),
and specific Abs were used to visualize the protein bands on HyperMax or
Biomax film using the ECL system (Amersham Biosciences). The results
were quantitated using Kodak Digital Science 1D image analysis software.
Raising Abs in guinea pigs (anti-IRF1, anti-IRF7) and rabbits (anti-IRF3)
has been described previously (15, 24, 41). Rabbit anti-phospho-S396IRF3 was as described previously (42). Rabbit anti-IRF9 (sc-496), antiSTAT1 (sc-346), anti-STAT2 (sc-476), anti-p50 (sc-7178), anti-p65 (sc372) anti-RelB (sc-226X), and anti-actin (sc-10731) were all obtained from
Santa Cruz Biotechnology. Rabbit anti-p52 (06-413) was obtained from
Upstate Biotechnology. Anti-IRF5 was prepared in rabbits by immunizing
the animals four times at 4-wk intervals with preparative SDS-PAGE (BioRad) purified Escherichia coli-expressed GST-IRF5 fusion protein (20 ␮g/
immunization). The animals were bled 7 days after the last immunization.
HRP-conjugated anti-guinea pig (P0141) and anti-rabbit (P0448) Igs obtained from DakoCytomation were used as secondary Abs.
The Journal of Immunology
257
upstream between nt ⫺851 to ⫺842. Other putative transcription
factor binding sites displaying less significant similarity with the
transcription factor consensus sequences were found by the programs, but we focused on the three sites described above, because
it is expected that these elements would be of greatest importance
in regulating microbe-induced CCL19 gene expression in DCs.
Functionality of NF-␬B and ISRE elements in DCs
To analyze the functionality of the putative CCL19 promoter
NF-␬B sites in DCs, we performed DNA affinity binding experiments. DCs were infected with Salmonella or Sendai virus followed by isolation of cells and preparation of nuclear extracts for
promoter element binding experiments. Proteins binding to biotinylated oligonucleotides spanning the CCL19 promoter NF-␬B(1)
and NF-␬B(2) elements were pulled down and identified by Western blotting using NF-␬B-specific Abs. Both NF-␬B(1) (Fig. 4A)
and NF-␬B(2) (Fig. 4B) elements bound the classical pathway
NF-␬B components, p50 and p65, with similar efficiency (upper
two blots). The binding of p65 was clearly increased during infection with Salmonella or Sendai virus. Homodimers of p50 (and
likely p52) are constitutively transported into the nucleus, where
they may function as suppressors of gene expression (51, 52).
Basal binding of p50 was seen in uninfected cells, but its binding
was also increased simultaneously with p65 binding in response to
Salmonella or Sendai virus infection. In addition, both NF-␬B(1)
and NF-␬B(2) promoter elements were able to bind p52 and RelB
(Fig. 4; lower two blots). c-Rel binding to NF-␬B(1) and NF-␬B(2)
elements could not be detected, and no significant differences in binding of NF-␬B components were seen at later time points (48 h; data
not shown).
We then studied the functionality of the putative ISRE binding
site upstream of the NF-␬B sites. First, we characterized by direct
Western blotting whether the expression of IRF proteins was altered during Salmonella or Sendai virus infection in DCs. In Salmonella-infected cells the expression of IRF1, IRF7, IRF9, and
STAT2 proteins was induced to a different degree, whereas IRF3,
IRF5, and STAT1 protein levels remained virtually unchanged
(Fig. 5A). Sendai virus also induced the expression of IRF1, IRF7,
and IRF9, but to a lesser extent than Salmonella. In the case of IRF3,
a slower migrating form of IRF3 (representing the phosphorylated
form, see below) is detected at the 3-h time point. STAT1 protein
expression was weakly reduced in Sendai virus-infected cells.
Next, we performed DNA affinity binding experiments using
CCL19 ISRE element oligonucleotides. Interestingly, the ISRE element bound multiple IRFs, STAT1, and STAT2 (Fig. 5B). Induced IRF1 binding was detected in response to Salmonella at 9
and 24 h. Sendai virus, in contrast, increased IRF1 binding already
at 3 h. It was of interest that the m.w. of microbe-induced ISREbound IRF1 was somewhat lower as compared with that found in
uninfected cells. This difference could be due to degradation or
differential phosphorylation of IRF1. Basal IRF3 binding to the
promoter element was clearly detectable, but an activated form of
IRF3 upon Sendai stimulus was detected using a specific antiphospho-S396-IRF3. Also in case of IRF5, some basal binding of
IRF5 to the promoter element was observed in untreated cells.
However, no microbe-induced enhancement of IRF5 binding to
ISRE oligonucleotide could be detected (Fig. 5B). Strong IRF7 and
moderate IRF9 binding to the promoter element was observed in
response to Salmonella. IRF7 binding to the promoter element was
also induced in Sendai virus-infected cells, whereas no detectable
increase in IRF9 binding could be seen. Finally, both Salmonella
and Sendai virus increased the binding of STAT1 and STAT2 to
the CCL19 ISRE element, although STAT2 binding was decreased
rapidly after 3 h of Sendai virus infection.
Cytokine inducibility of CCL19 expression in DCs
As observed above, both bacterial and viral stimulation of DCs is
able to turn on CCL19 gene expression. Because CCL19 promoter
had functional ISRE and NF-␬B sites, we studied whether cytokine
stimulation is sufficient to induce CCL19 production. We stimulated DCs with STAT-activating cytokines IFN-␣ or IFN-␥, or
with NF-␬B-activating IL-1␤ or TNF-␣. For comparison, we also
infected the cells with Salmonella or Sendai virus with different
MOIs. None of the cytokine treatments alone could induce significant
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FIGURE 4. Salmonella and Sendai virus infection enhances NF-␬B
binding to CCL19 promoter DNA elements in DCs. Nuclear extracts
were prepared from DCs infected
with Salmonella or Sendai virus for
indicated times. Proteins binding to
CCL19 promoter NF-␬B(1) (A) and
NF-␬B(2) (B) DNA elements were
pulled down by DNA affinity binding
assay, separated on 10% SDSPAGE, and analyzed by Western
blotting with anti-p65, anti-p50, antiRelB, and anti-p52 Abs. The results
were quantitated by determining the
band intensities. Relative binding of
NF-␬B components, compared with
3 h control, is shown on the right panels. This experiment was performed
three times with similar results.
258
CCL19 REGULATION IN HUMAN DENDRITIC CELLS
FIGURE 5. Multiple IRF and STAT transcription factors bind to the
CCL19 promoter ISRE element in DCs. A, Expression of IRF and STAT
proteins in microbe-infected DCs. Whole cell extracts were prepared from
Salmonella or Sendai virus-infected DCs, separated on 10% SDS-PAGE,
and analyzed by direct Western blotting with anti-IRF or anti-STAT-specific
Abs. Actin staining shows equal protein loading. B, Nuclear extracts were
prepared from DCs, and proteins binding to the CCL19 ISRE element were
pulled down by DNA affinity binding assay. Bound proteins were separated on
10% SDS-PAGE (8% for detection of P-IRF3, IRF5, STAT1, and STAT2),
and analyzed by Western blotting with specific Abs as indicated in the figure.
production of CCL19 as measured by ELISA (Fig. 6). However, the
production of CCL20 could be seen after IL-1␤ stimulation but not by
TNF-␣. ⌻his is contradictory to the previous reports that indicate
CCL20 as a gene up-regulated by TNF-␣ via a NF-␬B-dependent
mechanism (53, 54). However, when IFNs were combined with
IL-1␤ or TNF-␣, elevated levels of CCL19 and CCL20 were detected
in DC supernatants. In contrast, as compared with the cytokine treatments, Salmonella-infected DCs secreted higher levels of CCL19 and
CCL20. In the case of CCL19, Salmonella MOI 100 was toxic for the
cells and the production of CCL20 was no longer dose responsive. As
compared with Salmonella, Sendai virus was a weaker inducer of
chemokine production at all tested MOIs.
Regulation of CCL19 transcription through NF-␬B and
ISRE sites
To better characterize the role of NF-␬B and ISRE sites in CCL19
gene expression, we PCR-cloned the CCL19 promoter region encompassing nucleotide residues from ⫺1437 to ⫹ 41 bp relative to
the transcription start site (Fig. 3). The promoter fragment was
cloned into pGL3basic luciferase reporter vector and was named as
CCL19promWT. We also created NF-␬B and ISRE site mutant
forms of the promoter as shown in Fig. 7A. Because primary DCs
are not suitable for conventional transfections, we transiently transfected HEK293 cells with CCL19promWT reporter and NF-␬〉 expression plasmids and examined the ability of the promoter construct
to drive the expression of the firefly luciferase gene.
CCL19promWT transfected alone did not result in reporter gene
expression in HEK293 cells. However, the expression of NF-␬B
components p50⫹p65 or p52⫹RelB led to strongly enhanced,
325- or 70-fold activation of the promoter construct, respectively
(Fig. 7B). These results demonstrate that either p50⫹p65 or
p52⫹RelB dimers can activate transcription of the CCL19 gene.
Proximal NF-␬B(1) mutant construct lost a significant proportion
of its response to cotransfected p50⫹p65 or p52⫹RelB (⬃80%).
However, mutation of NF-␬B(2) site interfered with the promoter
activity to a lesser extent; 54 or 30% of the activity was lost with
NF-␬B(2) mutant in the presence of overexpressed p50⫹p65 or
p52⫹RelB, respectively. A promoter construct containing mutations in both NF-␬B(1) and NF-␬B(2) sites showed an inhibition of
transcription comparable to that of the NF-␬B(1) mutant. These
results suggest that the proximal NF-␬B(1) site is the major NF␬B-responsive element in the human CCL19 promoter.
Next, we studied the role of IRFs in the induction of CCL19
promoter activity. Because HEK293 cells transiently transfected
with CCL19promWT and IRF1, IRF3, or IRF7 expression plasmids did not result in significant induction of promoter activity
(Fig. 7C), we used Sendai virus as an activator of IRFs. Sendai
virus has extensively been used in studies concerning the regulatory role of IRF1, IRF3, and IRF7 in cytokine gene expression (13,
14, 21). The activation of IRF5, however, has been shown to occur,
e.g., by vesicular stomatitis virus or Newcastle disease virus, but
not by Sendai virus (17). In the presence of Sendai virus infection,
all IRFs were able to activate the CCL19promWT 14- to 20-fold,
depending on the IRF protein. The greatest activation of the
CCL19 promoter was seen in response to IRF3 expression. As
expected, the expression of IRF5 during Sendai virus infection did
not induce CCL19 promoter activity (data not shown). Moreover,
IRF5 did not activate CCL19 promoter in response to vesicular
stomatitis virus infection (data not shown). The role of ISRE site
(located upstream of the two NF-␬B sites) was examined by using
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FIGURE 6. Cytokine-stimulated DCs are capable of producing CCL19
and CCL20. Monocyte-derived DCs were stimulated with purified IFN-␣
(100 IU/ml), IFN-␥ (100 IU/ml), TNF-␣ (10 ng/ml), or IL-1␤ (10 ng/ml)
alone, or in different combinations as shown in the figure. For comparison,
DCs were also infected with Salmonella or Sendai virus at indicated MOIs.
Cell culture supernatants were collected at 24 h and analyzed for CCL19
and CCL20 production by ELISA. The results are the means (⫾SD) of DCs
from four blood donors analyzed separately.
The Journal of Immunology
259
FIGURE 7. NF-␬B and IRF family members regulate CCL19 promoter
activity. A, WT and mutated sequences of human CCL19 promoter region
containing the NF-␬B(1), NF-␬B(2), and ISRE elements (mutations shown
in lower case). B, HEK293 cells were transfected with pGL3-basic backbone vector, CCL19promWT, or different CCL19 promoter NF-␬B mutant
constructs with or without NF-␬B expression plasmids as shown in the figure.
The luciferase activities indicating transcriptional activity were measured in
cell extracts collected at 18 h after transfection. C, HEK293 cells were transfected with pGL3-basic backbone vector, CCL19promWT, or an ISRE mutant
construct alone, or in combination with IRF expression plasmids. After overnight transfection, the cells were infected with Sendai virus for 24 h, after
which luciferase activities were measured. Luciferase levels were normalized
with Renilla activities and then plotted as fold induction (test plasmid/
pGL3basic) with pGL3basic activity normalized to value 1. Experiments were
performed with four replicates and repeated three times with similar results.
Representative experiments of three are shown. TF, Transcription factors; Sv,
Sendai virus.
the ISRE mutant CCL19 promoter construct (Fig. 7A). As a result,
clear inhibition of IRF-associated CCL19 promoter activity was
seen in Sendai virus-infected cells (Fig. 7C).
RIG-I and TLR pathway signaling components activate
CCL19 promoter
Having identified the transcription factors involved in the regulation of CCL19 transcription, we determined which signaling molecules upstream of IRFs would induce CCL19 promoter activation.
RIG-I has been shown to act as a cytoplasmic detector of dsRNA
synthesized by viruses. RIG-I associates with another CARD-domain containing protein, CARDIF (CARD adaptor inducing
IFN-␤, also known as IPS-1/MAVS/VISA), which is associated
with mitochondria. RIG-I and CARDIF signal downstream to
TBK1 and IKK␧ that directly phosphorylate IRF3 and IRF7. In
contrast, MyD88, a central TLR adaptor molecule, transmits a signal
in a complex pathway leading to the activation of MAPKs and NF␬B. The MyD88-independent pathway triggered by TLRs is governed
by TRIF. TLR3/4-activated TRIF is also able to activate TBK-1 and
IKK␧, leading to enhanced expression of type I IFN genes (55).
The involvement of these signaling components in the activation
of CCL19 promoter was examined using HEK293 cell transfection
approach. Plasmids encoding RIG-I, or its constitutively active
form ⌬RIG-I, CARDIF, TRIF, MyD88, IKK␧, or TBK1 were introduced in the cells together with the CCL19 promoter, and the
cells were left uninfected or infected with Sendai virus. The fulllength RIG-I-dependent activation of CCL19 promoter required
Sendai virus infection, whereas ⌬RIG-I could activate the promoter independently of virus infection (Fig. 8). Interestingly, overexpression of TRIF also activated the promoter efficiently and independently of virus infection. Ectopic expression of CARDIF,
MyD88, IKK␧, or TBK-1 directly stimulated CCL19 promoter activity, but the promoter-inducing activity was also clearly enhanced by Sendai virus infection (Fig. 8). As a whole, our experiments indicate that CCL19 promoter activity can be activated by
both the RIG-I and the TLR pathways.
Discussion
DCs play an essential role in regulating the activation of both
innate and adaptive immunity during microbial infections. DC
maturation triggered by microbes is associated with the production
of CCL19 and enhanced expression of its specific receptor, CCR7.
CCL19 thus regulates the migration of mature DCs, and it is also
involved in recruiting naive T cells to the vicinity of mature DCs
in the T cell zones of local lymph nodes (1–3). Although CCL19
has many important functions in DC biology, the molecular mechanisms regulating its gene expression have remained largely uncharacterized. In this study, we conducted a detailed analysis on
the mechanisms of Salmonella or Sendai virus-induced expression
of CCL19 gene in human monocyte-derived DCs. In addition, we
cloned the CCL19 promoter and characterized its functionality in
transfection experiments. We observed that multiple intracellular
signal transduction pathways are involved in the regulation of
CCL19 gene expression.
To obtain an insight into which signaling pathways are important for CCL19 gene expression in DCs, we used chemical inhibitors that could interfere with the activation of NF-␬B, MAPK, and
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FIGURE 8. RIG-I and TLR pathways are involved in the activation of
the CCL19 promoter transcription. HEK293 cells were transfected with
pGL3basic or CCL19promWT alone or together with RIG-I, ⌬RIG-I (constitutively active form), CARDIF, TRIF, MyD88, IKK␧, or TBK1 expression plasmids. After overnight transfection, the cells were infected with
Sendai virus for 24 h, after which luciferase activities were measured.
Luciferase levels were normalized with Renilla activities and then plotted
as fold induction (test plasmid/pGL3basic) with pGL3basic activity normalized to value 1. The data shown are the means (⫾SD) of triplicate
determinations. A representative experiment is shown.
260
efficiently interfere with IFN signaling by targeting STAT proteins
(56). Thus, the binding of Sendai virus-activated IRFs to the
CCL19 promoter ISRE site remained at a relatively low level. The
only exception was IRF3, which was rapidly and efficiently activated by Sendai virus and its binding was clearly stronger than the
one seen in Salmonella-infected DC extracts.
Unlike ectopically expressed NF-␬B dimers, which in the absence of their inhibitors (I␬Bs) are constitutively in an active form,
IRF3 and IRF7 require additional virus-induced phosphorylations
before they are transcriptionally active (57). Therefore, in transfection experiments we had to use Sendai virus infection to induce
IRF activation. Consistent with the DNA binding experiments,
we found that in transfected HEK293 cells, IRF1, IRF3, or IRF7
induced CCL19 promoter activity, and this activity was dependent on the functional ISRE site located upstream of the two
NF-␬B sites. However, we found no evidence that IRF5, which
has been described to regulate the expression of many cytokine
genes (17, 18), would be involved in CCL19 gene expression in
human monocyte-derived DCs or in promoter reporter assays.
Further experimental analyses of the pathways upstream of IRFs
revealed that the activation of both TLR and RIG-I pathways regulate CCL19 gene expression. Expression of the crucial receptor/
adapter components of the TLR3/4 pathway (MyD88, TRIF,
IKK␧, or TBK1) or the RIG-I pathway (RIG-I, ⌬RIG-I, CARDIF,
IKK␧, or TBK1) lead to efficient transcription of the CCL19 promoter reporter construct. The data indicate that the common pathways activated by Gram-negative bacteria (TLR4) or RNA viruses (TLR3 or RIG-I) led to the activation of multiple IRF
family members, which together with NF-␬B regulate CCL19
gene expression.
In this study we demonstrate that, in human monocyte-derived
DCs, both bacteria and viruses can induce CCL19 gene expression.
Efficient activation of CCL19 production correlates with the ability of
a given microbe to induce DC maturation and cytokine gene expression in general. Detailed analysis of the intracellular signal transduction pathways indicated that the CCL19 promoter is regulated by multiple transcriptional systems, at least by NF-␬B, IRFs, and STATs.
We cannot rule out the possibility that MAPK cascades and NFAT
transcription factors are also involved, because pharmacological inhibitors of these pathways also reduced CCL19 gene expression to
some extent. It was also of interest that NF-␬B- and STAT-activating
cytokines, IL-1␤ or TNF-␣ together with IFNs, were also able to
induce CCL19 production in DCs albeit in low levels. The data suggest that signals, whether they are bacteria, viruses, or cytokines,
which are able to activate NF-␬B, IRF, and/or STAT family transcription factors in DCs can also activate the CCL19 promoter in a
quantitatively, qualitatively, and timely regulated fashion.
Acknowledgments
We thank Mari Aaltonen, Hanna Valtonen, and Johanna Lahtinen for expert technical assistance.
Disclosures
The authors have no financial conflict of interest.
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