Proinflammatory cytokines induce liver and activation-regulated chemokine/macrophage inflammatory protein-3{alpha}/CCL20 in mucosal epithelial cells through NF-{kappa}B

Satoru Fujiie1,2, Kunio Hieshima2, Dai Izawa2, Takashi Nakayama2, Ryuichi Fujisawa2, Harumasa Ohyanagi1 and Osamu Yoshie2

1 Departments of Surgery II and
2 Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

Correspondence to: Correspondence to: K. Hieshima


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Liver and activation-regulated chemokine (LARC)/CCL20 is expressed by surface-lining epithelial and epidermal cells, and is likely to link innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. Here we examined the mechanism of LARC expression in epithelial-type cells. Either IL-1ß or tumor necrosis factor (TNF)-{alpha} strongly induced LARC mRNA in intestinal cell lines Caco-2 and T84, while both were effective on HEK 293T cells. Induction of LARC was also demonstrated in the intestinal epithelium of BALB/c mice upon treatment with IL-1{alpha} or TNF-{alpha}. Transient transfection assays using murine LARC promoter–reporter constructs identified a region essential for IL-1ß- or TNF-{alpha}-induced promoter activation in Caco-2 and 293T cells. Using site-directed mutagenesis, we demonstrated that an NF-{kappa}B site located between –96 and –87 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1ß- or TNF-{alpha}-induced promoter activation in Caco-2 and 293T cells. Electrophoretic mobility shift assays demonstrated that p50/p65 heterodimer and p65 homodimer of NF-{kappa}B bound to this site in 293T cells upon treatment with IL-1ß and TNF-{alpha}, and p50/p65 heterodimer bound to this site in Caco-2 cells upon treatment with IL-1ß. Co-expression of constitutively active p65 strongly activated the promoter construct carrying the intact NF-{kappa}B site in 293T and Caco-2 cells. Collectively, LARC expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-{alpha} primarily through activation of NF-{kappa}B.

Keywords: chemokine, epithelial cell, IL-1, NF-{kappa}B, tumor necrosis factor, small intestine


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Chemokines are a group of small (6–14 kDa), mostly basic, heparin-binding cytokines that play pivotal roles in inflammatory and immunological responses by inducing directed migration of specific types of leukocytes (1). In humans, >40 members have been identified (2). Based on the arrangement of the N-terminal conserved cysteine residues, chemokines are now divided into four subfamilies: CXC, CC, C and CX3C (2). Their biological effects are mediated through a family of seven transmembrane, G protein-coupled receptors (3). At present, 18 functional chemokine receptors have been identified (2). From the functional point of view, chemokines can be roughly divided into two groups, i.e. inflammatory and immune (system) chemokines (4). Inflammatory chemokines are those that are strongly up-regulated by proinflammatory signals, attract mainly neutrophils, monocytes and/or eosinophils, and play major roles in acute and chronic inflammatory responses. Immune (system) chemokines, on the other hand, are mostly expressed constitutively in some lymphoid and other tissues, selectively attract lymphocytes and dendritic cells, and are involved in the development, maintenance and function of the immune systems (4). It is, however, likely that these two functional groups are not mutually exclusive, but are in fact substantially overlapped in the host defense mechanisms.

Liver and activation-regulated chemokine (LARC) (5), also designated macrophage inflammatory protein (MIP)-3{alpha}/exodus-1/CCL20 (2,6,7), is a CC chemokine originally reported to be expressed selectively in the lung and liver, and chemotactic for lymphocytes but not for monocytes or neutrophils (5). Subsequent studies, however, have demonstrated that LARC is primarily expressed in small intestine and colon where it is constitutively expressed by follicle-associated epithelium (8,9). Later, epidermal keratinocytes, especially in inflamed skin lesions such as psoriasis and atopic dermatitis, have also been shown to express LARC (1013). Its receptor was identified and termed CCR6 (1417). CCR6 has been shown to be expressed on the surface of immature dendritic cells, most B cells, and most {alpha}4ß7+ intestine-homing memory T cells and a fraction of cutaneous lymphocyte antigen (CLA)+ skin-homing memory T cells (17). Furthermore, mice with targeted disruption of the CCR6 gene demonstrated selective lack of myeloid-lineage dendritic cells in the subepithelial dome region of intestinal lymphoid follicles and severely impaired immune responses to orally administered antigen and enteropathic rotavirus (18). Thus, LARC and its receptor CCR6 are likely to be the key elements in the mucosal immunity (8,18,19).

Previously, we have shown that normal epidermal keratinocytes express LARC upon stimulation with proinflammatory cytokines such as IL-1 and tumor necrosis factor (TNF)-{alpha} (13). Given the importance of LARC in the mucosal immunity, it is of great interest to elucidate the regulatory mechanism of LARC expression in intestinal epithelial cells. Here, we demonstrate that LARC expression in intestinal epithelial cells is also induced upon stimulation with IL-1 and TNF-{alpha} and NF-{kappa}B play the major role in cytokine-induced LARC expression.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines and reagents
Caco-2 and T84 (human colon carcinoma cell lines) and HEK 293T (a human embryonic kidney epithelial cell line) were maintained in Dulbecco's MEM containing 50 µg/ml of kanamycin and supplemented with 10% FCS, non-essential amino acids and 2 mM L-glutamine. Recombinant IL-1{alpha}, IL-1ß, TNF-{alpha}, IFN-{gamma} and MIP-3{alpha}/CCL20 were purchased from PeproTech (Rocky Hill, NJ). Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St Louis, MO). Anti-p50 antibody (sc-114 x), anti-p65 antibody (sc-372 x), anti-RelB antibody (sc-226 x), anti-c-Rel antibody (sc-70 x) and anti-p52 antibody (sc-298 x) were purchased from Santa Cruz (Santa Cruz, CA).

RT-PCR analysis
Total RNA was prepared by using Trizol reagent (Gibco/BRL, Gaithersburg, MD) and RNeasy (Qiagen, Hilden, Germany). Reverse transcription of total RNA (1 µg) was carried out using oligo(dT)18 primer and SuperScript II reverse transcriptase (Gibco/BRL). First-strand DNA (20 ng total RNA equivalent) and original total RNA (20 ng) were amplified in a final volume of 20 µl containing 10 pmol of each primer and 1 unit of Ex-Taq polymerase (Takara, Kyoto, Japan). The primers used were: +5'-TACTCCACCTCTGCGGCGAATCAGAA-3' and –5'-GTGAAACCTCCAACCCCAGCAAGGTT-3' for LARC, and +5'-GCCAAGGTCATCCATGACAACTTTGG-3' and –5'-GCCTGCTTCACCACCTTCTTGATGTC-3' for G3PDH. Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s, and extension at 72°C for 30 s (5 min for the last cycle) for 31–35 cycles for LARC and 27 cycles for G3PDH. Amplification products (10 µl each) were subjected to electrophoresis on 2% agarose and stained with ethidium bromide.

ELISA
A sandwich-type ELISA for LARC/CCL20 was developed using mouse anti-human MIP-3{alpha}/CCL20 mAb (67310.111) (Genzyme/Techne, Cambridge, MA) for capture and biotinylated goat anti-human MIP-3{alpha}/CCL20 polyclonal antibody (R & D Systems) for detection. For standardization of assay, serially diluted recombinant MIP-3{alpha}/CCL20 was included on each ELISA plate. The detection range was typically between 20 pg/ml and 1 ng/ml.

Animals
Male BALB/c mice at 8 weeks of age were purchased from CLEA (Tokyo, Japan). All animal experiments described in this report were approved by the institutions and performed under the guidelines of our animal facilities. Mice were injected i.p. with 1 ml of PBS without or with IL-1{alpha} (1 µg/ml) or TNF-{alpha} (4 µg/ml). After 5 h, mice were sacrificed and the jejunum was removed. Tissue specimens with or without Peyer's patches were prepared for immunohistochemistry and RT-PCR analysis.

Immunohistochemistry
Tissue specimens were fixed in periodate–lysine–paraformaldehyde for 1 h, rinsed in 10% and 20% sucrose in PBS, each for 1 h, and embedded in the OCT compound. Tissue sections (4 µm) were made by a cryostat and air-dried. Unless otherwise stated all subsequent washes were done with ice-cold PBS for 5 min 3 times. To prevent non-specific staining, tissue sections were treated with PBS containing 2.5% normal rabbit serum and 0.2% mAb to mouse CD32/16 (IM2807) (Beckman Coulter, Fullerton, CA) for 30 min at 44°C. The sections were incubated overnight at 4°C with either goat polyclonal anti-rat/mouse MIP-3{alpha}/CCL20 (AF540) (R & D Systems) or normal goat IgG (41108C) (Genzyme/Techne, Minneapolis, MN), both at a working concentration of 2 µg/ml. The sections were then incubated with biotinylated rabbit anti-goat IgG (BA-5000) (Vector, Burlingame, CA; 1:100 dilution) for 1 h. To block endogenous biotin and peroxidase, sections were incubated with Biotin Block System (X-0590) (Dako, Capinteria, CA) and with 0.3% H2O2 in methanol respectively for 30 min. After three washes with PBS, sections were incubated with avidin–biotin–peroxidase complex (Vectastatin ABC kit, PK-4000; Vector) for 1 h. Sections were counterstained with hematoxylin

Transfection and luciferase assay
The promoter region of mouse LARC gene was cloned from the genomic DNA of C57BL/6 mouse by PCR using primers based on the reported sequence (9). To generate the promoter–reporter construct (pGL3-mLARC), the promoter region was inserted into XhoI–HindIII sites in pGL3-luciferase (Promega, Madison, WI). HEK 293T cells were preincubated with 25 µM chloroquine for 1 h and transfected with 3.5 µg of each plasmid together with 1 µg of pSV-ß-galactosidase by calcium phosphate precipitation as described previously (20). After 7–12 h post-transfection, medium was changed for fresh medium. Cells were further cultured for 36–42 h without or with various stimulants. Caco-2 cells were transfected by electroporation. After 12 h post-transfection, medium was changed for fresh medium. Cells were further cultured without or with various stimulants for 24–36 h. Cells were harvested and luciferase activity was determined by using the Luciferase Assay Kit (Promega). The luciferase activity was normalized by the ß-galactosidase activity used as an internal control for transfection efficiency. All assays were done in duplicate or triplicate and were repeated at least 3 times. The expression vector pcDNA3.1-(His)6-p65 encoding the NF-{kappa}B subunit p65 with additional hexa-histidines [(His)6] and anti-Xpress epitopes (Invitrogen, Carlsbad, CA) was kindly provided by Dr T. Okamoto (Nagoya-City University, Japan).

Electrophoretic mobility shift assay (EMSA)
EMSA was performed essentially as described previously (21,22). The probe was a 32P-end-labeled DNA fragment containing an NF-{kappa}B-like motif located in the proximal region of mouse LARC promoter ({kappa}B/mLARC, 5'-ATCAATGGGGGAAAACCCCGGGTGAG-3'). Nuclear extracts were prepared as described previously (22). Approximately 6 µg of nuclear extract was used. The binding reaction was carried out in a 20-µl reaction mixture containing 20 mM HEPES–KOH (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 5000 c.p.m. (equivalent to ~0.2 ng) of probe and 0.2 µg of poly(dI–dC) (Rosh Diagnostics, Laval, Quebec, Canada). Before adding the probe, the mixture was kept on ice for 20 min. In supershift or competition experiments, 2 µg of specific antibodies or 100-fold molar excess, cold annealed oligonucleotides ({kappa}B/mLARC, see above, and {kappa}Bmut/mLARC, 5'-ATCAATGGGGCAAAAGTCCGGGTGAG-3') were included together with the non-specific competitor poly(dI–dC). After 30 min incubation at 4°C, samples were fractionated on a 5% polyacrylamide gel (acrylamide/bisacrylamide, 40:1) containing 45 mM Tris–borate and 1 mM EDTA (pH 8.0) at 4°C. Electrophoresis proceeded until the bromophenol blue tracking dye migrated for 12 cm in the gel. The gel was dried, exposed to a PhosphoImager screen (Molecular Dynamics, Sunnyvale, CA) and analyzed using ImageQuant software (Molecular Dynamics).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of LARC mRNA by IL-1ß and TNF-{alpha}
Previously, we demonstrated highly selective expression of LARC in small intestine and colon of normal mice (9). In situ hybridization denoted a constitutive expression at follicle-associated epithelium and strong induction in most parts of mucosal epithelium upon systemic treatment with LPS (9). Therefore, we wished to examine the mechanism of LARC expression in the intestinal epithelial cells. We mainly used colon carcinoma cell lines, Caco-2 and T84, because they have been widely used as models for intestinal mucosal cells. We also used human embryonic kidney epithelial-type HEK 293T cells because this cell line was highly efficient in transfection assays. Cells were cultured without or with various stimulants for 24 h and examined for expression of LARC mRNA by RT-PCR. As shown in Fig. 1Go, LARC was expressed in untreated Caco-2 cells at low levels but strongly induced upon treatment with IL-1ß but not with TNF-{alpha}. In contrast, LARC expression was strongly induced in T84 cells by TNF-{alpha} but not by IL-1ß. In the case of HEK 293T cells, both IL-1ß and TNF-{alpha} were similarly effective and also highly synergistic. On the other hand, IFN-{gamma} or LPS had little inducing effect. In fact, IFN-{gamma} consistently suppressed constitutive expression of LARC in Caco-2 and T84 cells. Collectively, either IL-1ß or TNF-{alpha} was effective on induction of LARC, depending on the cell background.



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Fig. 1. Induction of LARC mRNA by IL-1ß and TNF-{alpha}. Total RNA was prepared from Caco-2, T84 cells and HEK 293T cells treated without or with IL-1ß (1 ng/ml), TNF-{alpha} (50 ng/ml), IL-1ß (1 ng/ml) + TNF-{alpha} (50 ng/ml), IFN-{gamma} (100 ng/ml) and LPS (30 ng/ml) as indicated. After 24 h, RT-PCR analysis was carried out for LARC and G3PDH. Representative results from three independent experiments are shown.

 
Secretion of LARC protein
We next examined production of LARC protein by using a sandwich-type ELISA for LARC. As shown in Fig. 2Go, Caco-2 cells secreted large amounts of LARC upon treatment with IL-1ß. No such secretion was seen in untreated Caco-2 cells or those treated with TNF-{alpha}, IFN-{gamma} or LPS. TNF-{alpha}, however, enhanced IL-1ß-induced LARC secretion in a dose-dependent fashion. This might be due to weak enhancing effects of TNF-{alpha} on IL-1ß-induced LARC mRNA expression (Fig. 1Go). In the case of HEK 293T cells, on the other hand, both IL-1ß and TNF-{alpha} induced secretion of LARC protein, and their effects were highly synergistic as their effects on induction of LARC mRNA (Fig. 1Go).



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Fig. 2. Secretion of LARC protein by IL-1ß and TNF-{alpha}. Caco-2 and HEK 293T cells were cultured in a six-well plate dish (~5x105 cells/well) and were treated without or with IL-1ß (1 ng/ml), TNF-{alpha} (50 ng/ml), IL-1ß (1 ng/ml) + TNF-{alpha} (0.5 ng/ml), IL-1ß (1 ng/ml) + TNF-{alpha} (5 ng/ml), IL-1ß (1 ng/ml) + TNF-{alpha} (50 ng/ml), IFN-{gamma} (100 ng/ml) and LPS (30 ng/ml) as indicated. After 24 h, LARC secreted in culture supernatant was measured by ELISA. All assays were done in triplicate to obtain means ± SD. Representative results from three independent experiments are shown.

 
Induction of LARC in mice
We next wished to confirm induction of LARC by IL-1 and TNF-{alpha} in normal intestinal epithelium. For this purpose, we treated BALB/c mice with IL-1{alpha} or TNF-{alpha} and prepared intestinal tissue specimens after 5 h. As shown in Fig. 3Go, LARC mRNA was clearly induced in the jejunum without Peyer's patches by the treatment with IL-1{alpha} or TNF-{alpha}. In contrast, LARC mRNA was constitutively expressed at high levels in the Peyer's patches and hardly enhanced any further by IL-1{alpha} or TNF-{alpha}. As shown in Fig. 4Go, constitutive production of LARC protein was clearly detected at the follicle-associated epithelium but not in the normal epithelium. However, signals of LARC protein were clearly enhanced in the latter after the treatment with IL-1{alpha} (Fig. 4CGo) or TNF-{alpha} (Fig. 4DGo). We also detected some cells within the follicles strongly positive for LARC after treatment with IL-1{alpha} or TNF-{alpha}. The identity of these cells remains unknown at present.



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Fig. 3. Induction of LARC in small intestine of BALB/c mice. Tissue specimens were obtained from mice sacrificed at 5 h after injection with 1 ml of PBS without or with IL-1{alpha} (1 µg/ml) or TNF-{alpha} (4 µg/ml). Total RNA was prepared from Peyer's patches and jejunum without Peyer's patches. RT-PCR analysis was carried out for LARC and G3PDH. Representative results from three independent experiments are shown.

 


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Fig. 4. Staining of LARC-producing cells in small intestine. Tissue specimens were obtained from BALB/c mice sacrificed at 5 h after injection with 1 ml of PBS without or with IL-1{alpha} (1 µg/ml) or TNF-{alpha} (4 µg/ml). Tissue sections were stained with either normal goat IgG (A) or goat anti-mLARC (B, C and D) using the ABC method. Sections were counterstained with hematoxylin. Magnification x200.

 
Promoter assays using luciferase-reporter constructs
Induction of LARC by IL-1{alpha} and TNF-{alpha} promoted us to analyze the transcriptional elements in the mouse LARC promoter region (9). As shown in Fig. 5Go(A), the TFSEARCH program (24) revealed a number of potential transcriptional element sites such as MyoD, Ets, Sp-1 and NF-{kappa}B within the mouse LARC promoter region of 308 bp from the transcriptional start. Due to a long stretch of sequence consisting of GA and GGA repeats starting from –308, we were unable to isolate clones containing sequences upstream of the repeat sequence (9). We therefore generated a reporter construct pGL3-mLARC by fusing the promoter region from –299 to +6 to the luciferase reporter gene. We transfected this construct into Caco-2 and HEK 293T cells, and measured luciferase activity after incubation of the cells without or with various stimulants for 36–42 h. The results are shown in Fig. 5Go(B). Even though transfection efficiency of Caco-2 cells was extremely low, IL-1ß but not TNF-{alpha}, IFN-{gamma} or LPS significantly induced luciferase expression. On the other hand, both IL-1ß and TNF-{alpha} strongly induced luciferase expression in HEK 293T cells. These results were highly consistent with the effects of IL-1ß and TNF-{alpha} on expression of LARC mRNA in these cells (Fig. 1Go), supporting that the promoter region from –299 to +6 contained the regulatory elements necessary and sufficient for induction of LARC by IL-1ß or TNF-{alpha}.



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Fig. 5. Analysis of the mouse LARC promoter. (A) The nucleotide sequence of the mouse LARC promoter region. Potential regulatory elements revealed by the TFSEARCH program (24) are indicated by arrows. The TATA box is boxed and the transcriptional initiation site is indicated by +1. (B) Transcriptional activation of the mouse LARC promoter–luciferase construct. Caco-2 and HEK 293T cells were transfected with the LARC promoter–luciferase construct (pGL3-mLARC) and treated without or with IL-1ß (1 ng/ml), TNF-{alpha} (50 ng/ml), IFN-{gamma} (100 ng/ml) and LPS (30 ng/ml). After 36–42 h, relative luciferase activities were determined using a luminometer. All assays were done in triplicate to obtain means ± SD. Representative results from three independent experiments are shown.

 
To narrow down the region of the LARC promoter involved in induction by IL-1ß or TNF-{alpha}, we generated LARC promoter–reporter constructs with serially deleted 5' ends and transfected them into Caco-2 and HEK 293T cells. As shown in Fig. 6Go(A), the region from –105 and –86 appeared to be necessary and mostly sufficient for induction of luciferase activity in Caco-2 cells by IL-1ß, and HEK 293T cells by IL-1ß and TNF-{alpha}. As shown in Fig. 5Go(A), the region from –105 and –86 contains an NF-{kappa}B-like site. To address the importance of this element in response to IL-1ß and TNF-{alpha}, we transfected Caco-2 and HEK 293T cells with full-length constructs without (wt) or with mutations at this site (mut). As shown in Fig. 6Go(B), the mutation in the NF-{kappa}B -like site completely abrogated the response of the promoter to IL-1ß in Caco-2 cells or to IL-1ß and TNF-{alpha} in HEK 293T cells.



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Fig. 6. Deletion and mutation analyses of the mouse LARC promoter. (A) Deletion analysis. Caco-2 and HEK 293T cells were transfected with a series of LARC promoter–luciferase constructs indicated by the 5'-ends and incubated without (open bars) or with 1 ng/ml of IL-1ß (black bars) or 50 ng/ml of TNF-{alpha} (hatched bars). After 36–42 h, relative luciferase activities were determined using a luminometer. All assays were done in triplicate to obtain means ± SD. Representative results from three independent experiments are shown. (B) Mutational analysis. Caco-2 and HEK 293T cells were transfected with full-length LARC promoter–luciferase constructs with the intact (wt) or mutated (mut) NF-{kappa}B site, and incubated without (open bars) or with 1 ng/ml of IL-1ß (black bars) or 50 ng/ml of TNF-{alpha} (hatched bars). After 36–42 h, relative luciferase activities were determined using a luminometer. All assays were done in triplicate to obtain means ± SD. Representative results from three independent experiments are shown.

 
Specific binding of p50/p65 and p65/p65 to the NF-{kappa}B site
To demonstrate specific binding of nuclear proteins to the putative NF-{kappa}B site located at –96 to –87, we next carried out EMSA. As shown in Fig. 7Go(A), both IL-1ß and TNF-{alpha} induced specific binding of nuclear proteins to the NF-{kappa}B site in HEK 293T cells. IL-1ß showed the same effect on Caco-2 cells. The induced band disappeared in the presence of 100-fold excess of the wild-type probe but not of the mutant probe. Both IL-1ß and TNF-{alpha} induced formation of nuclear protein–DNA complexes termed I and II in HEK 293T cells. On the other hand, IL-1ß induced only nuclear protein–DNA complex corresponding to complex I in Caco-2 cells.



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Fig. 7. Specific binding of nuclear proteins to the NF-{kappa}B site. (A) Induction of specific binding of nuclear proteins to the NF-{kappa}B site by IL-1ß and TNF-{alpha}. EMSA was performed using nuclear extracts from HEK 293T cells treated without or with IL-1ß (1 ng/ml) and/or TNF-{alpha} (50 ng/ml), singly and in combination, and from Caco-2 cells treated without or with IL-1ß (1 ng/ml) for 24 h. For cold competition, a 100-fold excess of the wild-type NF-{kappa}B site oligonucleotide (wt) or the mutant NF-{kappa}B site oligonucleotide (mut) was included. Specific bands are indicated as complex I and complex II. (B) Supershift assays. HEK 293T cells were treated with IL-1ß (1 ng/ml) + TNF-{alpha} (50 ng/ml) for 24 h. Caco-2 cells were treated with IL-1ß (1 ng/ml) for 24 h. EMSA was carried out in the absence or presence of anti-p50, anti-p65, anti-RelB, anti-c-Rel or anti-p52 as indicated. (C) Binding of overexpressed p65 to the NF-{kappa}B site was examined using nuclear extracts from HEK 293T cells transfected with an expression vector for p65 [(His)6-p65] or control vector (vehicle only). Representative results from at least two independent experiments are shown.

 
To identify nuclear proteins in these complexes, we next carried out supershift assays using antibodies to p50, p65, RelB, c-Rel and p52. As shown in Fig. 7Go(B), anti-p65 almost completely supershifted both complex I and complex II formed by HEK 293T cells, while anti-p50 mainly supershifted complex I. On the other hand, anti-RelB, anti-c-Rel or anti-p52 had no such effects separately or even in combination with anti-p50. These results suggested that complex I and complex II were mainly p50/p65 heterodimer and p65 homodimer respectively. Similar analyses were next carried out using the nuclear extracts from Caco-2 cells. Complex I induced by IL-1ß was clearly supershifted by anti-p50 or anti-p65 (Fig. 7BGo). No such effects were seen with anti-RelB, anti-c-Rel or anti-p52, alone or even in combination with anti-p50. We also examined nuclear extracts from HEK 293T cells transfected with an expression vector for p65 [pcDNA3.1-(His)6-p65], which encodes p65 with additional hexa-histidines [(His)6] and anti-Xpress epitopes. Taking its slightly larger size than the endogenous p65 into consideration, the DNA–protein complex formed by overexpressed (His)6-p65 migrated identically to endogenous complex II and was completely supershifted by anti-p65 (Fig. 7CGo). Collectively, these results strongly suggested that the NF-{kappa}B-like site located at –96 to –87 in the LARC promoter was indeed the binding site of NF-{kappa}B proteins, and complex I and complex II were mostly p50/p65 heterodimer and p65 homodimer respectively.

Overexpression of p65 activates LARC promoter
To further address the role of p65 of NF-{kappa}B in transcriptional activation of the LARC promoter, we transfected Caco-2 and HEK 293T cells with the full-length promoter–luciferase constructs with the wild-type (wt) or mutated (mut) NF-{kappa}B site together with the expression vector for (His)6-p65 [pDNA3-(His)6-p65]. The luciferase activity was measured after 24 h. As shown in Fig. 8Go, overexpression of p65 indeed resulted in strong induction of luciferase activity through the wild-type (wt) but not mutated (mut) NF-{kappa}B site in both Caco-2 and HEK 293T cells.



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Fig. 8. Transactivation of the LARC promoter by overexpression of p65. Caco-2 and HEK 293T cells were co-transfected with the full-length LARC promoter–luciferase constructs with the intact (wt) or mutated (mut) NF-{kappa}B site and an expression vector for (His)6-p65 (closed bar) or a control vector (open bar). After 24 h, relative luciferase activities were determined using a luminometer. All assays were done in triplicate to obtain means ± SD. Representative results from three independent experiments are shown.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
LARC is a CC chemokine selectively expressed by the surface-lining cells such as mucosal epithelial cells and epidermal keratinocytes (8,9,13). Its receptor CCR6 is expressed on immature dendritic cells, B cells and memory T cells (17). Thus, it is likely that LARC is an important mediator for both initiation phase and effector phase of immune responses. In this study, we examined the mechanism of LARC expression in mucosal epithelial cells. We have shown that LARC is highly inducible by proinflammatory cytokines such as IL-1 and TNF-{alpha} (Figs 1–4GoGoGoGo). Previously, we have also shown that LARC is highly inducible in epidermal keratinocytes by IL-1 and TNF-{alpha} (13). Thus, these proinflammatory cytokines appear to be the universal inducers of LARC in surface-lining cells. However, TNF-{alpha} was much less effective than IL-1ß for induction of LARC in Caco-2 cells, while the opposite was true in the case of T84 cells (Fig. 1Go). Schuerer et al. also reported that, while IL-8/CXCL8 expression in intestinal cell lines such as HT29 and SW620 was inducible by both IL-1ß and TNF-{alpha}, IL-8 expression in Caco-2 was inducible only by IL-1ß (23). Thus, the effects of IL-1ß and TNF-{alpha} on induction of LARC may be highly dependent on the cellular background. It was also unexpected that LPS was totally unable to induce LARC expression in mucosal epithelial cells even though mice treated with LPS strongly up-regulated LARC expression in intestinal epithelium (9). Thus, induction of LARC expression by intestinal mucosal cells upon systemic treatment with LPS might be mediated through induction of IL-1 and TNF-{alpha} from LPS-activated macrophages or other cells.

We have further demonstrated that induction of LARC by IL-1ß and TNF-{alpha} is critically mediated by NF-{kappa}B (Figs 5–7GoGoGo). NF-{kappa}B is an important transcriptional factor for inflammatory and immunological responses. NF-{kappa}B is known to be involved in expression of various cytokines, acute-phase proteins and adhesion molecules (25). NF-{kappa}B has also been shown to play essential roles in induction of a number of inflammatory chemokines such as IL-8, monocyte chemoattractant protein-1 (MCP-1)/CCL2, regulated upon activation, normal T-cell expressed and secreted (RANTES)/CCL5 and eotaxin/CCL11 (2630). The present results extend its list of target to LARC.

A synergy between NF-{kappa}B and AP-1 has been shown for transcriptional activation of IL-8, MCP-1 and RANTES (26,27,3134). In contrast to their promoters, however, the mouse LARC promoter contains no consensus AP-1 binding site within the 308 nucleotide sequence upstream from the transcriptional initiation site (Fig. 5AGo). When we were about to complete the present work, we obtained a working draft sequence of the human LARC gene from GenBank (AC027560/AC073065). The human promoter shows ~75% identity to the mouse promoter. Concerning the potential regulatory elements, the TATA box, the NF-{kappa}B site and only one upstream Ets-1 site are conserved between the mouse and human promoters. This further supports the importance of the NF-{kappa}B site for LARC gene expression. Ets-1 is a transcription factor involved in constitutive and/or inducible expression of cytokines such as TNF-{alpha}, granulocyte macrophage colony stimulating factor (GM-CSF) and platelet factor 4 (PF4)/CXCL4 (3539). The role of c-Ets-1 in LARC expression thus remains to be seen.

Both IL-1ß and TNF-{alpha} induced specific binding of p50/p65 heterodimer and p65/p65 homodimer to the NF-{kappa}B site in HEK 293T cells. On the other hand, IL-1ß induced specific binding of only p50/p65 heterodimer to the NF-{kappa}B site in Caco-2 cells. Since the p65 subunit but not p50 subunit contains the transcriptional activation domain, this may explain why HEK 293T cells showed stronger induction of luciferase activity than Caco-2 cells.

Considering its attraction of dendritic cells and lymphocytes, LARC appears to be a typical immune chemokine (4). However, its highly inducible nature by typical proinflammatory cytokines such as IL-1ß and TNF-{alpha} may also place it in a rank of inflammatory chemokines. Thus, LARC is likely to be a unique chemokine playing a role in both innate and acquired immunity. Since LARC is constitutively expressed by intestinal follicle-associated epithelium (Fig. 4Go), it would be of great interest to see whether this constitutive expression is mediated by IL-1ß, TNF-{alpha} or even some other factors delivered from the underneath lymphoid tissues.

After the completion of this work, Izadpanah et al. also reported up-regulation of LARC expression in intestinal epithelial cells by IL-1 and TNF-{alpha} (40). They employed a mutant I{kappa}B{alpha} to demonstrate the role of NF-{kappa}B in the induction of LARC by these proinflammatory cytokines.


    Acknowledgments
 
We are grateful to Dr T. Okamoto and Dr T. Tetsuka for pcDNA3.1-(His)6-p65. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


    Abbreviations
 
ABC avidin–biotin–peroxidase
EMSA electrophoretic mobility shift assay
LPS lipopolysaccharide
LARC liver and activation-regulated chemokine
MCP monocyte chemoattractant protein
MIP macrophage inflammatory protein
RANTES regulated upon activation, normal T-cell expressed and secreted.
TNF tumor necrosis factor

    Notes
 
Transmitting editor: K. Sugamura

Received 14 May 2001, accepted 28 June 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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