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The Journal of Immunology, 2002, 168: 5621-5628.
Copyright © 2002 by The American Association of Immunologists

Induction of Macrophage-Inflammatory Protein-3{alpha} Gene Expression by TNF-Dependent NF-{kappa}B Activation1

Shinichi Sugita*,{dagger}, Tomoko Kohno{dagger}, Kazuo Yamamoto{dagger}, Yoshitaka Imaizumi{dagger}, Hisayoshi Nakajima*, Tadayuki Ishimaru* and Toshifumi Matsuyama2,{dagger}

* Department of Obstetrics and Gynecology, Nagasaki University School of Medicine, and Nagasaki University Medical Skill Junior College, Nagasaki, Japan; and {dagger} Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medical Sciences, Nagasaki, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophage-inflammatory protein-3{alpha} (MIP-3{alpha}), also designated as liver and activation-regulated chemokine (LARC), Exodus, or CCL20, is a recently identified CC chemokine that is expected to play a crucial role in the initiation of immune responses. In this study, we describe that MIP-3{alpha} expression is under the direct control of NF-{kappa}B, a key transcription factor of immune and inflammatory responses. Overexpression of the p65/RelA subunit of NF-{kappa}B significantly increased the MIP-3{alpha} mRNA level. MIP-3{alpha} transcription was stimulated by TNF, and this stimulation was inhibited by an NF-{kappa}B inhibitor, I-{kappa}B{alpha} superrepressor. Analysis of the human MIP-3{alpha} promoter demonstrated a functional NF-{kappa}B site responsible for its expression. We also show that MIP-3{alpha} expression is induced in LPS-treated mouse livers that were primed with Propionibacterium acnes, which developed massive liver injury with infiltration of inflammatory cells. This induction was fully dependent on the TNF signaling cascade, because it was not observed in the livers of TNFR1-deficient mice. Furthermore, pretreatment with gliotoxin, an inhibitor of NF-{kappa}B activity, abrogated the P. acnes/LPS-induced MIP-3{alpha} expression of wild-type mice. These results clearly demonstrate that MIP-3{alpha} gene expression is dependent on NF-{kappa}B activity in vitro, and indicate that the TNFR1-mediated TNF signaling cascade that leads to NF-{kappa}B activation plays an essential role in MIP-3{alpha} expression in the murine liver injury model.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor is a pleiotropic cytokine that triggers a variety of biological effects, such as immunity, inflammation, proliferation or apoptosis, insulin resistance, and differentiation, on various types of cells (1). Two different types of TNFR, TNFR1 and TNFR2, mediate these effects; however, the majority of the biological effects of TNF are mediated by TNFR1. Activated TNFR1 forms a signaling complex with a number of proteins, such as TNFR-associated death domain protein, Fas-associated death domain protein, TNFR-associated factor 2, and receptor-interacting protein, which initiate intracellular signaling pathways. Cellular transcription factors then become activated and induce the expression of a large number of bioactive genes, many of which are controlled by NF-{kappa}B (1).

NF-{kappa}B is a ubiquitously expressed transcription factor, usually found as a heterodimer of p50 and p65 (Rel A). Without stimulation, the heterodimer is associated with a member of the inhibitory I-{kappa}B family of proteins and is sequestered in the cytoplasm. Among the I-{kappa}B family proteins, the best-characterized molecule is I-{kappa}B{alpha}. Treatment of cells with various stimuli, including mitogens, cytokines, viruses, bacterial LPS, radiation, and some anticancer drugs, leads to the phosphorylation of I-{kappa}B{alpha} on serines 32 and 36. The phosphorylated I-{kappa}B{alpha} is then rapidly ubiquitinated and subsequently degraded by the 26S proteasome. The released NF-{kappa}B then translocates into the nucleus, where it binds to specific NF-{kappa}B elements and activates many important genes (1).

Recently, a wide variety of transcriptional targets of NF-{kappa}B have been reported. The group of NF-{kappa}B-regulated genes that encode proteins playing important roles in inflammation and immune responses includes proinflammatory cytokines, such as IFN-{beta}, IL-2, IL-6, and TNF (1, 2, 3); chemokines, such as monocyte chemoattractant protein-1, IL-8, RANTES, and macrophage-inflammatory protein (MIP)3-1{alpha} (4, 5, 6, 7); and genes related to Ag presentation, such as MHC molecules, the peptide transporter TAP1, and the protease low molecular weight protein 2 (8, 9, 10). Many cell adhesion molecules, such as VCAM-1, ICAM-1, E-selectin, and the matrix-degrading proteases matrix metalloproteinase-9 and urokinase-type plasminogen activator, are also regulated by NF-{kappa}B (1, 11, 12, 13). NF-{kappa}B has also been implicated in the regulation of cell proliferation. Recently, NF-{kappa}B was found to stimulate the transcription of cyclin D1, a key regulator of G1 checkpoint control, providing evidence for a direct link between NF-{kappa}B activity and cell cycle regulation (14). A requirement of NF-{kappa}B for preventing apoptosis induced by TNF, ionizing radiation, and anticancer drugs has also been shown (15, 16, 17). The candidate genes are c-inhibitor of apoptosis, X-linked inhibitor of apoptosis, members of the Bcl-2 family A1 and Bcl-xL, immediate early response gene X-1L, A20, and manganese superoxide dismutase (18, 19).

Although many genes have been found, as described above, it is evident that not all of the NF-{kappa}B response genes have been identified at present. Therefore, we performed a subtractive hybridization approach to identify NF-{kappa}B response genes. One of the clones obtained was the CC chemokine MIP-3{alpha}, also called liver and activation-regulated chemokine, Exodus, or CCL20, which was recently identified as a chemoattractant factor for certain dendritic cells and T cells (20, 21, 22, 23, 24). Because little is known about the molecular mechanism of MIP-3{alpha} transcription, we next examined the role of NF-{kappa}B in its expression in vitro and in vivo. In this study, we provide lines of evidence that MIP-3{alpha} gene expression is under the control of NF-{kappa}B activity in vitro. We also found that MIP-3{alpha} expression is induced in Propionibacterium acnes/LPS-treated mouse livers, which develop massive liver injury that pathologically mimics fulminant hepatitis in humans (25). Finally, functional analysis of the human MIP-3{alpha} promoter revealed an NF-{kappa}B binding site responsible for its induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and experimental reagents

HeLa cells and HT1080 cells were grown in DMEM supplemented with 10% FCS and 100 U/ml penicillin-streptomycin. The HISR, a clone of HeLa cells expressing the I-{kappa}B{alpha} superrepressor (I-{kappa}B{alpha}SR), was established by transfection of pcDNA3-I-{kappa}B{alpha}SR. A control cell clone with the stably integrated pcDNA3 vector alone was also established and maintained, as described. The medium, FCS, and G418 were purchased from Life Technologies (Rockville, MD). Human TNF and LPS (from Escherichia coli O127:B8) were purchased from Sigma (St. Louis, MO). P. acnes (ATCC 11827) was grown and heat killed, as previously described (26).

DNA constructs and transfection

cDNA cloning of I-{kappa}B{alpha} was done by RT-PCR and was confirmed by DNA sequencing. Site-directed mutagenesis was performed, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA), to generate pcDNA3-I-{kappa}B{alpha}SR bearing a serine-to-glycine substitution at residue 32 and a serine-to-alanine substitution at residue 36 of human I-{kappa}B{alpha}. These mutations were confirmed by DNA sequencing. The pNF-{kappa}B luciferase reporter plasmid was purchased from Stratagene. Mouse MIP-3{alpha} cDNA was generated by RT-PCR, cloned into the pCR2.1 vector (Invitrogen, San Diego, CA), and confirmed by DNA sequencing. To isolate the MIP-3{alpha} regulatory sequences lying upstream of the coding region, PCR amplification of human genomic DNA was performed on the basis of the information about the genomic sequences of MIP-3{alpha} on chromosome 2 (GenBank accession AC027560 and AC073065). The amplified products were directionally cloned into pGL2-basic (Promega, Madison, WI), and DNA sequencing was performed. Site-directed mutagenesis was performed, as described above, to inactivate the putative NF-{kappa}B site on the promoter. All transfections were conducted with the FuGENE 6 reagent (Roche, Indianapolis, IN).

Northern blot analysis

Total RNA was isolated with the ISOGEN (NipponGene, Tokyo, Japan) reagent, according to the manufacturer’s protocol. Aliquots of RNA (10 µg) were fractionated in 1% agarose-formaldehyde gels, transferred onto Gene Screen Plus hybridization transfer membranes (NEN Life Science Products, Boston, MA), and immobilized with a UV cross-linker. cDNA fragments were labeled with the Redi-Prime II labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden) in the presence of [{alpha}-32P]dCTP. The hybridization signals were visualized by the image analyzer BAS 5000 system (Fuji Film, Tokyo, Japan).

Western blot analysis

Cells were washed with TBS and resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF). After an incubation on ice for 15 min, Nonidet P-40 was added to a final concentration of 0.5%, and then the cells were centrifuged at 12,500 rpm for 30 s at 4°C. The supernatant containing the cytoplasmic fraction was recovered. The protein from the cytoplasmic extracts was fractionated on 10% SDS-polyacrylamide gels, transferred onto Immobilon-P membranes (Millipore, Bedford, MA), and incubated with the anti-I-{kappa}B{alpha} Ab (Santa Cruz Biotechnology, Santa Cruz, CA), followed by an incubation with HRP-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech). The blots were visualized with the ECL detection system (Amersham Pharmacia Biotech).

Reporter assays

To analyze the NF-{kappa}B activity, cells seeded into six-well plates were transfected with 1 µg pNF-{kappa}B luciferase reporter plasmid and 1 ng pRL{beta}-actin luciferase (27) as an internal control. The total amount of transfected DNA was kept at 2 µg by supplementation with pcDNA3. To analyze the MIP-3{alpha} promoter activity, HeLa cells were cotransfected with 1 µg pGL2 reporter plasmid containing either the wild-type or mutant MIP-3{alpha} promoter and 1 ng pRL{beta}-actin luciferase, together with 1 µg pcDNA3 or pcDNA3-I-{kappa}B{alpha}SR. Twenty-four hours after transfection, cells were treated with or without TNF for 3 h, and then extracts were prepared and the luciferase activity was determined with the dual-luciferase reporter assay system (Promega), according to the manufacturer’s protocol. The luciferase activity was normalized with the Renilla luciferase activity from the internal control.

Subtractive hybridization

Total RNA was prepared from both HeLa cells transiently expressing p65 and HISR. Poly(A)+ RNA was purified from total RNA using an mRNA purification kit (Amersham Pharmacia Biotech). Purified mRNA (2 µg) was reverse transcribed and subjected to subtractive hybridization with a PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA). After hybridization, differential transcripts were selectively amplified, and were ligated with the pCR2.1 vector using the Topo-TA cloning kit (Invitrogen). Partial cDNA sequences were determined and compared with the entries in the GenBank database using the BLAST SEARCH program. For subsequent Northern blot analysis, radiolabeled probes were generated from the subtracted cDNAs. Total RNA from HeLa cells transiently expressing p65 and from HISR was fractionated and transferred to the membranes, and Northern blot analyses were performed using these probes.

In vivo MIP-3{alpha} expression

Healthy 6- to 8-wk-old TNFR1-/- mice (28) and age-matched C57BL/6 control mice received an i.p. injection of 10 mg P. acnes suspended in 300 µl PBS. Seven days later, the mice received an i.p. injection of 20 µg LPS in 300 µl PBS. In some cases, the mice were pretreated with 20 µg gliotoxin (29, 30) (Sigma) by i.p. injection 3 h before the LPS injection. Control mice were treated with 300 µl PBS only. Ninety minutes or three hours after the LPS injection, the mice were sacrificed and total RNA was prepared from the liver for Northern blot analysis. At least three mice were examined for each set of experiments. The animal protocols described above were approved by the Nagasaki University Animal Research Center Committee.

Nuclear extract preparation

Nuclear extracts were prepared according to the method of Zhou et al. (31). Briefly, tissues were homogenized in 1 ml ice-cold lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, supplemented with a protease inhibitor mixture tablet (one tablet per 10 ml; Roche)). The homogenates were kept on ice for 15 min, and then 25 µl 10% Nonidet P-40 was added. After a brief vortexing, they were incubated on ice for 20 min and centrifuged at 12,500 rpm for 30 s at 4°C. The pellets of nuclei were resuspended in 200 µl extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF, supplemented with a protease inhibitor mixture tablet (one tablet per 10 ml)) and were kept on ice for 30 min. The nuclear suspension was centrifuged at 12,500 rpm for 15 min at 4°C to collect the supernatants containing nuclear proteins.

To prepare the nuclear extracts from HeLa cells, 2 x 106 cells were washed and resuspended in buffer A, and then cytoplasmic fractions were removed, as described above, in the Western blot analysis. The pellets of nuclei were resuspended in 50 µl buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) at 4°C for 15 min. The nuclear suspension was centrifuged at 15,000 rpm for 5 min at 4°C to collect the supernatants containing nuclear proteins.

Gel shift analysis

To examine the NF-{kappa}B activity in the mouse livers, an aliquot of the nuclear extracts (5 µg) was incubated in a reaction buffer (10 mM HEPES, pH 7.9, 100 mM NaCl, 0.4 mM MgCl2, 0.3 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10% glycerol, and 100 µg/ml poly(dI-dC)). In some cases, a 20-fold molar excess of unlabeled double-stranded oligonucleotide was added as a competitor. After a 20-min incubation on ice, ~10 kcpm 32P end-labeled double-stranded oligonucleotide containing a consensus NF-{kappa}B-binding sequence (5'-GGGCTGGGGAATCCCGCTAA-3') was added to the reaction, which was then incubated for an additional 30 min at room temperature. The same oligonucleotide in the unlabeled form was used as the wild-type competitor. The mutant competitor sequence was 5'-GGGCTGTGGAATCACGCTAA-3'. In some cases, the reactions were further incubated with anti-p65 or anti-p50 Abs (Santa Cruz Biotechnology) at room temperature for 30 min. To examine the binding of NF-{kappa}B to the putative NF-{kappa}B site of the MIP-3{alpha} promoter, a gel shift analysis was performed using nuclear extracts from TNF-treated or untreated HeLa cells with the same protocol as described above. The labeled double-stranded oligonucleotide was derived from the sequence of the MIP-3{alpha} promoter containing the putative NF-{kappa}B site (5'-CACATGGGGTTTTCCCCATTGA-3'). The same oligonucleotide in the unlabeled form was used as the wild-type competitor. The mutant competitor sequence was 5'-CACATGTTTTTTTCCCCATTGA-3'. In some cases, the reactions were further incubated with anti-p65 or anti-p50 Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment of a cell line with impaired NF-{kappa}B activity

To establish a cell line in which the NF-{kappa}B activity is impaired, we isolated drug-resistant clones and examined them for I-{kappa}B{alpha}SR expression. One of the clones, designated as HISR, showed strong and constitutive I-{kappa}B{alpha} expression, as revealed by Northern blot analysis (Fig. 1GoA). I-{kappa}B{alpha} protein in control cell line was almost completely degraded after a 10-min incubation with TNF and returned to the untreated level by 30 min (Fig. 1GoB). In contrast, in HISR, the mutant I-{kappa}B{alpha} protein was continuously present after a 60-min incubation with TNF (Fig. 1GoB, HISR, I-{kappa}B{alpha} SR), along with the continuous presence of endogenous I-{kappa}B{alpha} (Fig. 1GoB, HISR, I-{kappa}B{alpha}). Similar effect of exogenous I-{kappa}B{alpha} on the stability of endogenous I-{kappa}B{alpha} was reported previously (32). Then, we examined NF-{kappa}B activation in response to TNF stimulation with an NF-{kappa}B-dependent luciferase reporter plasmid, pNF-{kappa}B luciferase (Fig. 1GoC), which contains five tandem repeats of the NF-{kappa}B site upstream of a basic promoter element (TATA box) and the luciferase gene. In HISR, the TNF-induced activation of NF-{kappa}B was blocked, although not completely, as compared with the control cells. As the inhibition of NF-{kappa}B activation renders cells sensitive to TNF-induced cell death (15, 16, 17, 18, 19, 33), we then investigated the sensitivity of HISR to TNF. The HISR became sensitive to TNF, and remarkable cell death was observed by 12 h, but not control cells (Fig. 1GoD). These data demonstrate that the biological activity of NF-{kappa}B is specifically impaired in HISR.



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  		FIGURE 1. Establishment of HISR with blocked NF-{kappa}B activity. A, Strong expression of I-{kappa}B{alpha}SR mRNA in HISR, a clone of HeLa cells stably transfected with pcDNA-I-{kappa}B{alpha}SR. For Northern blot analysis, the blot was hybridized with a 32P-labeled human I-{kappa}B{alpha} cDNA probe, and then rehybridized with a 32P-labeled {beta}-actin cDNA probe. B, Western blot analysis of I-{kappa}B{alpha}SR in HISR. HISR and control vector transfectant were treated with TNF (50 ng/ml) for the indicated time. Cytoplasmic proteins were analyzed by immunoblotting with anti-I-{kappa}B{alpha} Ab. C, Reduced NF-{kappa}B activity in response to TNF in HISR. HISR and control vector transfectants were transfected with the pNF-{kappa}B luciferase reporter plasmid with an internal control luciferase vector. After 24 h, cells were incubated with or without TNF for 3 h, and then relative luciferase activity was determined. The results shown are averages of three independent experiments with SE bars. D, Increased sensitivity to TNF-induced cell death in HISR. HISR and control vector transfectants were incubated with or without TNF (20 ng/ml) for 12 h, and cell viability was determined by trypan blue staining. The results shown are averages of three independent experiments with SD bars.

 
cDNA subtractive hybridization to identify novel genes controlled by NF-{kappa}B

Next, we performed cDNA subtractive hybridization between HeLa cells transfected with the p65/RelA expression plasmid and HISR, to identify novel NF-{kappa}B response genes. Using the prepared mRNA from both cell lines, we performed a Northern blot analysis to verify the quality of the mRNA samples by confirming that A1 (18), a direct transcriptional target of NF-{kappa}B, was specifically detected in the mRNA sample from HeLa cells expressing p65 (data not shown). The transcripts were converted to cDNAs by reverse transcription and were subjected to subtractive hybridization, as previously reported (34). Differential transcripts were selectively amplified by suppression PCR and were inserted into cloning vectors. Radiolabeled probes were generated from the subtracted cDNAs, and Northern blot analysis was performed to screen for the cDNA clones whose expression was specifically up-regulated in the p65 transfectant. Among the 131 nonredundant cDNA clones, 17 clones showed clear p65-dependent expression. As shown in Table IGo, 7 clones had coding sequences that were previously reported as transcriptional targets of NF-{kappa}B. Another 7 clones were identical to expressed sequence tag clones. One clone had no significant homology with entries in the expressed sequence tag database, but corresponded to a human BAC clone. The remaining 2 clones were identical to genes whose coding amino acids and functions had been characterized, but not reported as NF-{kappa}B-regulated genes, and one of them is the recently identified CC chemokine, MIP-3{alpha}.


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  		Table I. Summary of the subtracted cDNA clones that were specifically up-regulated in a p65-dependent fashion

 
TNF induces MIP-3{alpha} gene expression in an NF-{kappa}B-dependent manner

It has been reported that MIP-3{alpha} expression could be induced by some proinflammatory cytokines (20, 21). However, the precise transcriptional regulation of the MIP-3{alpha} gene has not been revealed, and most importantly, it is not known whether NF-{kappa}B is involved in the expression. As clearly demonstrated in Fig. 2Go, ectopic expression of p65 can induce MIP-3{alpha} expression in HeLa cells. We also confirmed that TNF stimulation induced MIP-3{alpha} expression in HeLa cells and HT1080 cells, but not in Jurkat cells (data not shown).



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  		FIGURE 2. Up-regulation of the MIP-3{alpha} gene expression in p65-transfected HeLa cells. Total RNA from p65-transfected HeLa cells and HISR was analyzed by Northern blotting, as described in Fig. 1GoA.

 
MIP-3{alpha} expression is induced in wild-type, but not in TNFR1-/-, mouse livers treated with LPS after P. acnes priming

To investigate MIP-3{alpha} expression and its NF-{kappa}B dependence in vivo, we used a mouse hepatitis model, in which priming with P. acnes and subsequent LPS injection generates intrahepatic TNF. The produced cytokines may subsequently activate NF-{kappa}B in the liver. MIP-3{alpha} expression was not detected in the livers of control mice (Fig. 3Go, lane 1), or those treated with P. acnes alone (Fig. 3Go, lane 2) or LPS alone (Fig. 3Go, lanes 5 and 6). However, the LPS challenge after P. acnes priming clearly induced MIP-3{alpha} expression in the wild-type mouse livers in a time-dependent fashion (Fig. 3Go, lanes 3 and 4). It was reported that cytokine-induced NF-{kappa}B activation was impaired in TNFR1-/- mouse livers (35), and LPS challenge after P. acnes priming caused few pathological changes (25). As shown in Fig. 3Go, there was no induction of MIP-3{alpha} in the TNFR1-/- mouse livers, either in the presence or absence of P. acnes/LPS treatment (Fig. 3Go, lanes 5–8). These results indicate that TNF/TNFR1-mediated signaling is required for the induction of MIP-3{alpha} in P. acnes/LPS-treated mouse livers, and suggest that NF-{kappa}B activation plays a critical role in the MIP-3{alpha} expression.



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  		FIGURE 3. LPS induced MIP-3{alpha} expression in P. acnes-primed mouse livers. Wild-type or TNFR1-/- mice were primed with (lanes 2–4 and 8–10) or without (lanes 1, 5, 6, and 7) P. acnes. Seven days later, mice were i.p. injected with (lanes 3–6, 9, and 10) or without (lanes 1, 2, 7, and 8) 20 µg LPS. After the LPS injection, total RNA was prepared from the livers at the indicated times. Northern blot analysis was performed, as described in Fig. 1GoA.

 
Gliotoxin, an inhibitor of NF-{kappa}B activation, abolishes the induction of MIP-3{alpha} expression in P. acnes/LPS-treated mice livers

To confirm the dependence of MIP-3{alpha} expression on NF-{kappa}B activity in vivo, we examined the effects of gliotoxin on the MIP-3{alpha} induction. The effect of gliotoxin was reported to be NF-{kappa}B specific because the toxin did not affect activation of the transcription factor NF-AT or of STAT, or the activity of the constitutively DNA-binding transcription factors Oct-1 and CREB (29). Although the mechanism by which gliotoxin inhibits NF-{kappa}B activation remains unknown, it was demonstrated to inhibit the degradation of I-{kappa}B (30). P. acnes-primed wild-type mice were pretreated with gliotoxin, and then received an LPS injection. To examine the NF-{kappa}B activity, a gel shift analysis was performed with the liver nuclear extracts, using a radiolabeled NF-{kappa}B consensus probe. The LPS challenge after P. acnes priming induced the DNA-protein complex in the nuclear extracts from wild-type mouse livers (Fig. 4Go, A and B, lanes 1 and 2). The complex formation was disturbed by the wild-type competitor of the NF-{kappa}B oligonucleotide, but not by the competitor with mutations in the consensus NF-{kappa}B sequence (Fig. 4GoB, lanes 3 and 4). The complex was either eliminated or supershifted in the presence of p65 or p50 Abs (Fig. 4GoB, lanes 5 and 6). It is important to note that NF-{kappa}B activation was not induced by P. acnes/LPS treatment in TNFR1-/- mouse livers (Fig. 4GoA, lane 5). In addition, pretreatment with gliotoxin, before the LPS injection, abrogated the induction of NF-{kappa}B activation (Fig. 4GoA, lane 3). Northern blot analysis revealed that gliotoxin pretreatment dramatically inhibited the MIP-3{alpha} expression following P. acnes/LPS treatment (Fig. 5Go). Therefore, we concluded that MIP-3{alpha} expression is dependent on NF-{kappa}B activation in vivo, and that the TNFR1-mediated TNF signaling cascade that leads to NF-{kappa}B activation is essential for MIP-3{alpha} expression in the liver injury model.



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  		FIGURE 4. Gliotoxin inhibits NF-{kappa}B activation in the liver injury model. A, Liver nuclear extracts were prepared from wild-type mice untreated (lane 1) or treated with P. acnes/LPS (lane 2) or with P. acnes/gliotoxin/LPS (lane 3), or from TNFR1-/- mice untreated (lane 4) or treated with P. acnes/LPS (lane 5). The nuclear extracts were incubated with a 32P end-labeled NF-{kappa}B consensus oligonucleotide, followed by gel electrophoresis. The black arrow indicates the NF-{kappa}B-containing complex. B, Nuclear extracts from wild-type mouse livers untreated (lane 1) or treated with P. acnes/LPS (lanes 2–6) were incubated with the labeled NF-{kappa}B probe, in the presence of competitors or Abs. The unlabeled consensus NF-{kappa}B oligonucleotide (lane 3) or the mutant NF-{kappa}B oligonucleotide (lane 4) was added as a competitor in a 20-fold molar excess to the binding reaction. Abs against p65 (lane 5) or p50 (lane 6) were added to the reaction for a supershift assay. The black arrow indicates the NF-{kappa}B-containing complex. The asterisk indicates the supershifted band.

 


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  		FIGURE 5. Abrogated MIP-3{alpha} induction by an NF-{kappa}B inhibitor, gliotoxin, in the mouse liver injury model. Wild-type mice were primed with or without P. acnes. Seven days later, the primed mice were pretreated with or without 20 µg gliotoxin and received an i.p. injection of 20 µg LPS. After 3 h, total RNA was prepared and Northern blot analysis was performed, as described in Fig. 1GoA.

 
The human MIP-3{alpha} promoter contains an NF-{kappa}B site responsible for its TNF-inducible expression

To verify a direct role of NF-{kappa}B in the regulation of MIP-3{alpha} expression, we isolated and characterized the regulatory sequences of the human MIP-3{alpha} gene. The cloned 5'-upstream sequence of MIP-3{alpha} gene is identical to the reported sequence, except for one base substitution (A to T) at position -142 relative to the first nucleotide in the human MIP-3{alpha} cDNA (GenBank/EMBL/DDBJ accession D86955). Because repeated amplifications of five independent clones revealed the same substitution, we regarded it as a personal polymorphism. Using the reporter plasmid (Fig. 6GoA), the MIP-3{alpha} promoter activity was assayed with or without TNF stimulation. As shown in Fig. 6GoB, the -874/+58 reporter construct showed minimal basal activity on its own. TNF stimulation enhanced the construct’s expression by 20-fold, and this enhancement was completely inhibited by cotransfection of the I-{kappa}B{alpha}SR expression plasmid (Fig. 6GoB). This result further supports the NF-{kappa}B-dependent expression of the MIP-3{alpha} gene with TNF stimulation. Alignment of the MIP-3{alpha} promoter sequences revealed a putative NF-{kappa}B binding site on the lower strand, at a position between -82 and -91 relative to the first nucleotide in the human MIP-3{alpha} cDNA sequence (GGGGTTTTCC). Site-directed mutagenesis was performed to introduce the mutation into the consensus NF-{kappa}B site between -82 and -91 (GTTTTTTTCC; -874/+58 m{kappa}B). With the luciferase assay, this mutation was found to decrease the basal activity of the MIP-3{alpha} promoter. Furthermore, this mutation greatly impaired the TNF inducibility of the MIP-3{alpha} promoter activity (Fig. 6GoB). In a gel shift assay using a probe containing the NF-{kappa}B sequence found in the MIP-3{alpha} promoter, TNF-induced NF-{kappa}B activity was readily detected (Fig. 6GoC, lane 2), which was not competed by the mutant competitor, including the same {kappa}B mutation introduced in the promoter assay (Fig. 6GoC, lane 4). These findings demonstrate that the human MIP-3{alpha} promoter contains a functional NF-{kappa}B site between -82 and -91 that is responsible for its expression, and that MIP-3{alpha} is a chemokine under the direct control of NF-{kappa}B.



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  		FIGURE 6. The human MIP-3{alpha} promoter contains a consensus NF-{kappa}B site responsible for its TNF-stimulated induction. A, Schematic representation of luciferase reporter constructs containing the human MIP-3{alpha} promoter (-874/+58) or the NF-{kappa}B site mutant promoter (-874/+58 m{kappa}B). The putative NF-{kappa}B site is indicated. B, NF-{kappa}B site-dependent activation of the MIP-3{alpha} promoter. HeLa cells were cotransfected with the -874/+58 MIP-3{alpha} promoter-luciferase reporter plasmid or the putative NF-{kappa}B site mutant -874/+58 m{kappa}B-luciferase reporter plasmid with an internal control luciferase vector, together with pcDNA3-I-{kappa}B{alpha}SR or an empty pcDNA3 vector. After 24 h, cells were treated with or without TNF for 3 h. The results shown are the averages of three independent experiments with SE bars. C, Binding of NF-{kappa}B to the NF-{kappa}B site of the MIP-3{alpha} promoter. Nuclear extracts from HeLa cells untreated (lane 1) or treated with TNF (lanes 2–6) were incubated with the labeled probe containing the putative NF-{kappa}B site of the MIP-3{alpha} promoter. The same unlabeled NF-{kappa}B oligonucleotide (lane 3) or the mutant NF-{kappa}B oligonucleotide (lane 4) was added as competitor in a 20-fold molar excess to the binding reaction. Abs against p65 (lane 5) or p50 (lane 6) were added to the reaction for a supershift assay. The black arrow indicates the NF-{kappa}B-containing complex. The asterisk indicates the supershifted band.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated a direct link between the immunomodulator NF-{kappa}B and the inflammatory mediator MIP-3{alpha}, which both play important roles in the development of inflammation. After identifying MIP-3{alpha} as a new transcriptional target of NF-{kappa}B/p65, we further performed experiments both in vitro and in vivo to clarify the regulatory mechanism. Functional analysis of the MIP-3{alpha} promoter revealed an NF-{kappa}B site, at a position between -82 and -91, which is responsible for the TNF-dependent induction (Fig. 6Go). Although inactivation of the consensus NF-{kappa}B site greatly reduced the gene expression, a marginal response to TNF stimulation still remained. Because cotransfection of the I-{kappa}B{alpha}SR expression plasmid with the NF-{kappa}B site mutant reporter (-874/+58 m{kappa}B) completely abolished the response to TNF stimulation (data not shown), an unidentified NF-{kappa}B element(s) together with the element we identified may regulate MIP-3{alpha} expression cooperatively or synergistically. Our preliminary data from the deletion analysis of the MIP-3{alpha} promoter indicate that, in addition to the -82/-91 NF-{kappa}B site, the region from -258 to -163 is required for the maximal induction of the reporter gene by TNF stimulation. In this region, an NF-{kappa}B-like sequence was found on the lower strand, at a position between -210 and -219 (GGGAGAATCC). Deletion of this region moderately reduced the TNF response of the promoter (data not shown). This suggests that the NF-{kappa}B-like element between -210 and -219 possesses an additional, but not essential, role in the regulation of MIP-3{alpha} transcription, even though the sequence deviates from the consensus NF-{kappa}B sequence. Thus, the TNF responsiveness of MIP-3{alpha} expression may be dignified by collaborative action of the strong and the weak NF-{kappa}B response elements.

A number of in vivo studies have highlighted the importance of cytokine/chemokine biology in both acute and chronic liver disease (36, 37). In addition, because MIP-3{alpha} was first identified as a novel inducible CC chemokine expressed mainly in the liver (20), we reasoned that the liver would be the best organ to examine MIP-3{alpha} induction in vivo. It is known that P. acnes priming and LPS challenge in mice cause massive liver injury, which consists of priming and eliciting phases (25). P. acnes priming induces mononuclear cell infiltration into the liver lobules and granuloma formation (25). The subsequent LPS injection elicits acute and massive hepatic injury, with a concomitant release of various cytokines, such as IL-1{beta}, IL-6, IL-8, IL-12, IL-18, TNF, and IFN-{gamma} (38, 39, 40, 41, 42). Among these, TNF is a crucial factor to mediate hepatocyte apoptosis and hepatic necrosis, because TNFR1-/- mouse livers showed few pathological changes after the LPS challenge (data not shown) (25). In accordance with these pathologies, MIP-3{alpha} expression was induced by the LPS challenge after P. acnes priming in the wild-type mouse livers, but not in the TNFR1-/- mouse livers (Fig. 3Go).

We propose in this study that the difference between the wild-type and TNFR1-/- mice in MIP-3{alpha} induction may originate from the two pathogenesis steps. The first one is the NF-{kappa}B-independent intrahepatic granuloma formation in the priming phase. TNFR1-/- mice showed no apparent granuloma formation after priming with P. acnes (data not shown) (25). A significant reduction in the number of granulomas was also seen in IFN-{gamma}-/- or M-CSF-/- mice (42, 43). As neither IFN-{gamma} nor M-CSF is an activator of NF-{kappa}B, the granuloma formation is probably independent of the NF-{kappa}B activity. In addition, without P. acnes priming, the LPS challenge could not induce MIP-3{alpha} even in wild-type mice (Fig. 3Go). From these results, together with a previous report that the number and the size of the granulomas paralleled the levels of TNF, IL-12, and IL-18 (41), it seems that the intrahepatic granuloma formation induced by P. acnes is essential for MIP-3{alpha} induction. The second step in the pathogenesis is NF-{kappa}B-dependent activation of responsible genes, including MIP-3{alpha}, in the eliciting phase. Although it has been reported that the P. acnes/LPS treatment produces various cytokines, as described above, our results with the TNFR1-/- livers clearly indicate that NF-{kappa}B is induced exclusively by TNF in this model. It was reported that TNFR2 can mediate TNF signaling and activate NF-{kappa}B (44); this result showed that TNFR2 did not compensate for the TNFR1 defect in the activation of NF-{kappa}B in MIP-3{alpha} induction. This may be due to the cell type specificity of TNFR2-mediated NF-{kappa}B activation. A previous report described that TNFR2 has only a minor role in the liver injury by the injection of mice with bacterial LPS (45).

In conclusion, the current study demonstrates that MIP-3{alpha} gene expression is induced by the TNF-activated transcription factor NF-{kappa}B. This implies the possibility that some of the various biological effects of TNF are mediated by MIP-3{alpha} via NF-{kappa}B activation.


   Acknowledgments
 
We thank Dr. N. Mori for providing materials, Dr. R. Moriuchi for technical advice, and Drs. A. Koda, H. Ichinose, and M. Miyazaki for encouragement.


   Footnotes
 
1 This work is supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, and by a grant from NDR Corporation (Gifu, Japan). Back

2 Address correspondence and reprint requests to Dr. Toshifumi Matsuyama, Division of Cytokine Signaling, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Medical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail address: tosim{at}net.nagasaki-u.ac.jp Back

3 Abbrevation used in this paper: MIP, macrophage-inflammatory protein. Back

Received for publication September 26, 2001. Accepted for publication March 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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