Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0104108 on March 23, 2004

Published online before print March 23, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0104108v1
75/6/1056    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsumura, T.
Right arrow Articles by Onozaki, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsumura, T.
Right arrow Articles by Onozaki, K.
(Journal of Leukocyte Biology. 2004;75:1056-1061.)
© 2004 by Society for Leukocyte Biology

TGF-ß down-regulates IL-1{alpha}-induced TLR2 expression in murine hepatocytes

Takayuki Matsumura*,{dagger}, Hidetoshi Hayashi*, Takemasa Takii*, Caroline F. Thorn{ddagger}, Alexander S. Whitehead{ddagger}, Jun-ichiro Inoue{dagger} and Kikuo Onozaki*,1

* Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan;
{ddagger} Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, Philadelphia; and
{dagger} Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Japan

1 Correspondence: Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan. E-mail: konozaki{at}phar.nagoya-cu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that the proinflammatory cytokine interleukin (IL)-1{alpha} can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-ß (TGF-ß) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-ß up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1{alpha} at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1{alpha} but not TGF-ß or the combination of TGF-ß and IL-1{alpha}. TLR2 promoter activity and nuclear factor (NF)-{kappa}B activation by IL-1{alpha} were inhibited by TGF-ß treatment. Pretreatment with TGF-ß strongly suppressed IL-1{alpha}-induced TLR2 promoter activity and NF-{kappa}B activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1{alpha} up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-ß. These results suggest that TGF-ß suppresses the induction of TLR2 expression by IL-1{alpha} through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1{alpha} and TGF-ß in regulating TLR2-mediated SAA production in hepatocytes.

Key Words: bacterial lipopeptide • serum amyloid A • type I IL-1R


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toll is the type I transmembrane protein that controls the dorsoventral pattern formation during embryogenesis of Drosophila melanogaster and the adult antifungal immune response [1 ]. Recently, mammalian homologues of the Toll-like receptor (TLR) have been identified, and 10 members have been cloned to date. TLRs, interleukin-1 receptor (IL-1R), and IL-18R share a common signal-transduction pathway through their Toll/IL-1R homologous region domains. In mammals, stimulation of TLR or IL-1R leads to the sequential activation of the adaptor protein myeloid differentiation factor 88, the IL-1R-associated kinases, tumor necrosis factor (TNF) receptor-associated factor 6, and eventually, the inhibitor of {kappa}B (I{kappa}B) kinase complex (IKK{alpha}, ß, {gamma}). The nuclear factor (NF)-{kappa}B/Rel family of transcription factors is maintained in the cytoplasm as inactive complexes with inhibitory proteins called I{kappa}Bs. The IKK complex phosphorylates the I{kappa}Bs, targeting them for ubiquitination and degradation by the proteasome. Degradation of I{kappa}B liberates NF-{kappa}B/Rel dimers, which translocate to the nucleus and augment the expression of NF-{kappa}B-responsive genes such as defense-related and antiapoptotic genes [1 2 3 ].

TLR2 has been shown to function as a pattern recognition receptor for diverse bacteria and their products, including the mycobacterial arabinose-capped lipoarabinomannan, mannosylated phosphatidylinositol, peptidogycan, lipopolysaccahride (LPS) from Leptospira interrogans and Porphyromonas gingivalis, and lipoproteins (LPs) from various sources [1 ]. Bacterial LPs (BLPs), which are expressed by all bacteria, are potent activators for TLR2 [4 5 6 ].

We have studied the regulation of TLR2 and TLR4 gene expression in murine tissues, especially liver, and found that TLR2, but not TLR4, mRNA was up-regulated by proinflammatory cytokines (IL-1{alpha} or TNF-{alpha}) or TLR4 agonist (LPS) but not by IL-6 [7 ]. Recently, we found that IL-1{alpha} was the most potent in up-regulating functional TLR2 expression in primary-cultured murine hepatocytes [8 ]. Transforming growth factor-ß (TGF-ß) is a multifunctional immunomodulator with immunosuppressive effects. TGF-ß is produced at a later phase in inflammation and works as a negative regulator of inflammation. However, the regulatory mechanism of TGF-ß for inflammation is largely unknown. In the present study, we investigated the effect of TGF-ß on TLR2 expression in primary-cultured murine hepatocytes. In this study, we show that TGF-ß inhibits functional TLR2 expression by IL-1{alpha} in primary mouse hepatocytes and that this inhibition is regulated at the transcriptional level. We also show that TGF-ß suppresses TLR2 promoter activity and NF-{kappa}B activation by IL-1{alpha} through down-regulation of type I IL-1R (IL-1RI) expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Human recombinant (rHu)-TGF-ß was purchased from R&D Systems (Minneapolis, MN). rHu-IL-1{alpha} (2.0x107 U/mg) was kindly provided by Dainippon Pharmaceutical Co. (Osaka, Japan). Synthetic bacterial lipopeptide Pam3-Cys-Ala-Gly-OH (BLP), corresponding to the N-terminal region of a BLP, was purchased from Bachem (Torrance, CA). Actinomycin D was purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mouse TLR2 antibody and anti-ß-actin mouse monoclonal antibody (mAb) were obtained from eBioscience (San Diego, CA) and Sigma Chemical Co., respectively. Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT).

Mice
Female ICR mice (7 weeks of age) were purchased from Charles River (Yokohama, Japan) and were maintained under specific, pathogen-free conditions with food and water available ad libitum. Animals were used in experiments after 1 week acclimation. The mice were treated according to the guideline of the ethical committee of the Graduate School of Pharmaceutical Sciences, Nagoya City University (Japan).

Mouse hepatocyte cultures
Mouse hepatocytes were isolated using a modification of the collagenase method and cultured as described previously [8 ]. Briefly, the liver was perfused in situ with 0.0125% collagenase (Sigma Chemical Co.) through the portal vein. The total liver cells isolated were centrifuged five times at 50 g for 1 min at 4°C to remove nonparenchymal cells. The cells were finally suspended in Williams’ medium E containing 10% heat-inactivated FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, 10–8 M dexamethasone, and 10–7 M insulin at a density of 5 x 105 cells/ml. The cell suspension (10, 4, and 0.5 ml) was cultured in collagen-coated, 100 60-mm dishes and 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ), respectively. The cell suspension contained >95% hepatocytes, according to the presence of dinuclear phenotype detected by staining with diamidinophenyl indole, and the viability of the cells was >80% by trypan blue exclusion. If the viability were <80%, the cells were not used. After incubation for 4 h at 37°C in 5% CO2 in air, nonadherent cells were removed, and fresh medium was added. After another 20 h incubation, the hepatocytes were washed twice and then stimulated with various reagents in fresh medium.

Hepatoma cells
Murine hepatoma cell line Hepa 1-6, a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [9 ], was a gift of Dr. De Yang (National Cancer Institute, Frederick, MD). The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 2 mM glutamine and 10% FBS.

RNA extraction and Northern blot analysis
Total RNA was extracted from hepatocytes seeded in 100-mm plates, and Northern blot analysis was performed as described previously [8 ]. Total RNA (30 µg) was separated in a 1% agarose gel containing 2% formaldehyde and transferred onto a filter, Hybond-N+ (Amersham Pharmacia Biotech, Buckinghamshire, UK), with 20x saline sodium citrate. A 548-bp murine TLR2 fragment and a 392-bp murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) fragment as DNA probes were obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using mRNA derived from RAW264.7 and EL-4 6.1 C10 cells, respectively. Primers used for TLR2 (548 bp) were 5'-GGCCAGGTTCCAGTTTTCAC-3' and 5'-GGAACAACGAAGCATCTGGG-3', and those for GAPDH (392-bp) were 5'-TGGTCTACAGGATCCAGTATGACTCC-3' and 5'-TGATGGCATGGATCCTGGTCATGAGC-3' [8 ]. The TLR2 fragment was cloned into the HindIII- and BamHI-digested pcDNA3.1 vector. The GAPDH fragment was cloned into the BamHI site of the pGEM-3Z vector.

Preparation of cell extracts and Western blot analysis
Preparation of cell extracts from hepatocytes seeded in 60-mm dishes and Western blot analysis were performed as described previously [8 ]. After the cells were treated for indicated periods of time, the medium was removed. The cells were washed three times with ice-cold phosphate-buffered saline, and the plates were chilled on ice immediately. An amount equal to 0.4 ml ice-cold lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 120 mM NaCl, 0.5% Triton-X, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml antipain, 100 µg/ml benzamidine hydrochloride, 50 µg/ml aprotinin, 100 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride) was added into the plates and stirred for 15 min. The cells lysates were collected by a rubber polishman and sonicated. The cell debris was pelleted by 5 min centrifugation in a microcentrifuge, and the supernatants were collected. For Western blots, equal amounts of proteins were suspended in sodium dodecyl sulfate (SDS) sample buffer. After boiling for 5 min, proteins were stored at –80°C until they were used. The proteins were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) microporous membrane, ImmobilonTM PVDF (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20, membranes were incubated with rabbit serum anti-mouse TLR2 (eBioscience) or anti-ß-actin mouse mAb (Sigma Chemical Co.) as a loading control at 1:1000 dilution and then with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG; 1:10,000, Jackson ImmunoReseach, West Grove, PA) or anti-mouse Ig HRP-linked whole antibody (1:5000, Amersham Pharmacia Biotech), respectively. The reactive proteins were detected with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) and were analyzed by a chemiluminescence image analyzer, LAS-1000 (Fuji Photo Film, London, UK).

Measurement of murine TLR2 promoter activity, NF-{kappa}B activation, and serum amyloid A (SAA) promoter activity
Primary-cultured mouse hepatocytes were transiently transfected with pGL3-TLR2 promoter construct [8 ] (for TLR2 promoter reporter gene assay), pGL3-4{kappa}B wild-type (wt) construct (for NF-{kappa}B reporter gene assay), kindly provided by Dr. Takashi Okamoto (Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences), and pGL3-BALB/c-Saa1 promoter construct or pGL3-BALB/c-Saa2 promoter construct [10 ] (for Saa promoter reporter gene assay) in the presence of pCMV–ß-galactosidase (ß-gal) and pCMV in 24-well plates by lipofection using effectene (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Luciferase assay was performed with the Luciferase reporter gene assay kit (Roche, Germany) as described previously [8 ]. Statistical significances were calculated using Student’s t-test (*, P<0.05; **, P<0.01).

RT reaction and PCR analysis
The RT reaction was performed as described previously [7 ]. Reaction mixtures were diluted fivefold with ddH2O to give cDNA stocks that were stored at 4°C until PCR analysis. Primers used for IL-1RI were 5'-TCTTTGGTTTGTACCTGCCA-3' and 5'-TATTACTCGTGTGACCGGAT-3' [11 ]; for IL-1RII, 5'-GAGCAAATGTCTGTGGAACT-3' and 5'-ATGATGCTGGTATTGTCTCC-3' [11 ]; for GAPDH, 5'-TCGGTGTGAACGGATTTGGC-3' and 5'-CTCTTGCTCAGTGTCCTTGC-3' [7 ]. PCR reactions contained 1x PCR buffer, 0.4 mM deoxy-unspecified nucleoside 5'-triphosphates, 2.5 ng forward and reverse primers, 0.005 U Ampli Taq GoldTM DNA polymerase (Perkin Elmer, Wellesley, MA), and 5 µl cDNA solution in a 10-µl vol. IL-1RI, IL-1RII, and GAPDH cDNAs were amplified for 25 cycles of 94°C (denaturation) for 1 min, 58°C (annealing) for 1 min, and 72°C (primer extension) for 1 min. PCR products (5 µl) were analyzed on 1.5% agarose gels in the presence of ethidium bromide. Fragments of 400 bp (IL-1RI, 473–872), 400 bp (IL-1RII, 477–876), and 1035 bp (GAPDH, 57–1091) were generated; the restriction endonuclease patterns of each were consistent with those of the respective genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TGF-ß inhibits TLR2 mRNA and protein expression by IL-1{alpha} in murine hepatocytes
We determined the level of TLR2 mRNA in primary-cultured murine hepatocytes treated with TGF-ß, IL-1{alpha}, or the combination of TGF-ß and IL-1{alpha} for 6, 12, and 24 h by Northern blot analysis using a specific probe for murine TLR2 (Fig. 1A ). The expression level of TLR2 mRNA was significantly augmented by IL-1{alpha} treatment, and the level sustained up to 24 h, which is consistent with our previous observation [8 ]. It is interesting that TGF-ß could weakly up-regulate TLR2 mRNA expression at 12 and 24 h and partially inhibit IL-1{alpha}-induced TLR2 mRNA expression at all time points. To examine whether TGF-ß also suppresses TLR2 protein expression induced by IL-1{alpha}, we determined the TLR2 protein levels in primary-cultured hepatocytes treated with these cytokines for 24 h by Western blot analysis (Fig. 1B) . The changes in TLR2 protein were similar to those observed for TLR2 mRNA. Similar effects of TGF-ß and IL-1{alpha} on TLR2 protein expression were observed in murine hepatoma cell line Hepa 1-6 (Fig. 1C) , indicating that these cytokines affect hepatocytes directly.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 1. TGF-ß inhibits IL-1{alpha}-induced TLR2 mRNA and protein expression in primary-cultured murine hepatocytes. (A) Mouse hepatocytes were treated with or without rHu-IL-1{alpha} (10 U/ml), rHu-TGF-ß (100 PM), or the combination of rHu-IL-1{alpha} (10 U/ml) and rHu-TGF-ß (100 PM), and total RNA was extracted at the indicated periods of time. The levels of TLR2 and GAPDH mRNA were determined by Northern blot analysis. Relative intensity of TLR2 mRNA was shown by normalization with GAPDH mRNA. (B and C) Hepatocytes (B) or Hepa 1-6 cells (C) were treated with or without rHu-IL-1{alpha} (10 U/ml), rHu-TGF-ß (100 PM), or the combination of them. The cells were lysed at 24 h, and equal amounts of protein were subjected to SDS-PAGE. Western blotting was performed with anti-mouse TLR2 or anti-ß-actin. Similar results were obtained in three independent experiments.

 
TGF-ß suppresses IL-1{alpha}-induced TLR2 expression in murine hepatocytes
The SAA gene family encodes constitutive (C-SAA) and acute-phase (A-SAA) proteins [12 ]. In the mouse, the A-Saa genes have been categorized further according to the principal sites of their expression, as hepatic (Saa1 and Saa2) and extra-hepatic (Saa3) [13 ]. We previously reported that the TLR2 ligand BLP is capable of signaling through TLR2 to induce hepatocytes to produce A-SAA [8 ]. To determine whether TGF-ß suppresses IL-1{alpha}-induced TLR2 expression in primary-cultured murine hepatocytes, we performed luciferase assays following transfection with mouse Saa1 and Saa2 promoter reporter constructs [10 ] (Fig. 2 ). Pretreatment with IL-1{alpha} up-regulated the basal level of the promoter activity. BLP-mediated activation of the two Saa promoters was augmented by pretreatment with IL-1{alpha} but not with TGF-ß or the combination of TGF-ß and IL-1{alpha}. These data suggest that TGF-ß cannot up-regulate TLR2 expession and that TGF-ß actively suppresses IL-1{alpha}-induced TLR2 expression.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effects of pretreatments with IL-1{alpha} and TGF-ß on BLP-induced Saa promoter activity in primary-cultured murine hepatocytes. Cells cotransfected with pGL3-BALB/c-Saa1 promoter or pGL3-BALB/c-Saa2 promoter constructs in the presence of pCMV-ß-gal were treated with rHu-IL-1{alpha} (10 U/ml), rHu-TGF-ß (100 PM), or the combination of rHu-IL-1{alpha} (10 U/ml) and rHu-TGF-ß (100 PM) for 24 h before BLP (1 µg/ml) treatment for 6 h. The luciferase activity was normalized with ß-gal activity in the same well. The average (mean±SD) of triplicate wells is shown. *, **, Significant differences compared with medium alone. Experiments were conducted three times, and similar results were obtained.

 
TLR2 promoter activity and NF-{kappa}B activation by IL-1{alpha} are inhibited by TGF-ß in murine hepatocytes
We previously showed that NF-{kappa}B activation contributes to IL-1{alpha}-induced TLR2 promoter activity in primary-cultured murine hepatocytes [8 ]. To determine whether TGF-ß affects the level of TLR2 transcription, luciferase reporter assays using mouse TLR2 promoter reporter and NF-{kappa}B reporter constructs were performed (Fig. 3 ). TLR2 promoter activity and NF-{kappa}B activation were significantly augmented by treatment with IL-1{alpha} but not with TGF-ß. Both of the activities induced by IL-1{alpha} could be substantially inhibited by cotreatment with TGF-ß for 24 h. Thus, the inhibitory effect of TGF-ß acts at the transcriptional level. In contrast to the result of 24 h cotreatment, there was no effect of TGF-ß on IL-1{alpha}-induced TLR2 promoter activity or NF-{kappa}B activation after only 6 h of treatment. These data suggest that TGF-ß does not affect IL-1{alpha} signaling directly.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Effects of IL-1{alpha} and TGF-ß on TLR2 promoter activity and NF-{kappa}B activation in primary-cultured murine hepatocytes. Hepatocytes were cotransfected with pGL3-TLR2 promoter or pGL3-4{kappa}B construct and pCMV-ß-gal. Following transfection, the cells were treated with rHu-IL-1{alpha} (10 U/ml), rHu-TGF-ß (100 PM), or the combination of rHu-IL-1{alpha} (10 U/ml) and rHu-TGF-ß (100 PM) for 6 h and 24 h. The luciferase activity was normalized with ß-gal activity in the same well. The average (mean±SD) of triplicate wells is shown. *, **, Significant differences compared with IL-1{alpha} alone. Experiments were conducted three times, and similar results were obtained.

 
TGF-ß down-regulates IL-1RI expression in murine hepatocytes
TGF-ß modulates IL-1R expression in positive and negative manners [14 15 16 ]. We determined whether TGF-ß affects IL-1RI mRNA expression in primary-cultured murine hepatocytes. RT-PCR was used, as IL-1RI mRNA levels in control hepatocytes were below the limit of detection by Northern blot analysis (data not shown). As shown in Figure 4 , IL-1RI mRNA was significantly down-regulated by TGF-ß treatment. In contrast, IL-1{alpha} treatment up-regulated IL-1RI mRNA in accordance with our previous data [17 ]. However, the inductive effect of IL-1{alpha} was abolished by cotreatment with TGF-ß. We were unable to detect expression of mRNA specifying the decoy receptor IL-1RII [18 ] under any of the experimental conditions used (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Regulation of IL-1RI mRNA expression in primary-cultured hepatocytes. Mouse hepatocytes were treated with or without rHu-IL-1{alpha} (10 U/ml), rHu-TGF-ß (100 PM), or the combination of rHu-IL-1{alpha} (10 U/ml) and rHu-TGF-ß (100 PM), and total RNA was extracted at the time-points indicated. IL-1RI and GAPDH mRNA levels were determined by RT-PCR. The representative data of three independent experiments are shown.

 
Next, we attempted to determine whether TGF-ß decreases cell-surface IL-1RI in primary-cultured murine hepatocytes by binding assay using [125I] IL-1. However, specific [125I] IL-1-binding activity was too low in control hepatocytes to permit accurate analysis (data not shown). We then examined whether a 12-h pretreatment with TGF-ß affects NF-{kappa}B activation and TLR2 promoter activity (Fig. 5 ) by IL-1{alpha}. Such exposure to TGF-ß strongly suppressed IL-1{alpha}-induced NF-{kappa}B activation and TLR2 promoter activity that could otherwise be induced by a subsequent treatment with IL-1{alpha}. This finding was consistent with the down-regulation of IL-1RI mRNA expression described above. These results suggest that TGF-ß suppresses IL-1{alpha}-mediated NF-{kappa}B activation and TLR2 promoter activity by down-regulating IL-1RI expression.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Effects of IL-1{alpha} and TGF-ß on TLR2 promoter activity and NF-{kappa}B activation in primary-cultured murine hepatocytes. Hepatocytes were cotransfected with pGL3-TLR2 promoter or pGL3-4{kappa}B construct and pCMV-ß-gal. Following transfection, the cells were treated with or without rHu-TGF-ß (100 PM) for 12 h before rHu-IL-1{alpha} (10 U/ml) treatment for 12 h. The luciferase activity was normalized with ß-gal activity in the same well. The average (mean±SD) of triplicate wells is shown. **, Significant differences compared with no TGF-ß pretreatment. Experiments were conducted three times, and similar results were obtained.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many cell types, including macrophages, monocytes, and neutrophils, produce IL-1 [19 ]. It exhibits many effects on hepatocytes, such as induction of acute-phase proteins and nitric oxide synthase. Recently, we have reported that IL-1{alpha} augments the BLP-mediated response of murine hepatocytes by up-regulating TLR2 mRNA and protein expression [8 ]. In the liver, cells other than hepatocytes, such as Kupffer cells and hepatic stellate cells, mainly produce TGF-ß [20 ], which is produced at a later phase in inflammation and acts as a negative-inflammatory regulator: It has been reported to inhibit the production of H2O2, IL-1, IL-6, and TNF-{alpha} by macrophages/monocytes [21 , 22 ]. However, the molecular mechanism of TGF-ß action is largely unknown. In the present study, we show that TGF-ß inhibits IL-1{alpha}-induced TLR2 expression in murine hepatocytes.

Treatment of murine hepatocytes with TGF-ß alone augmented the expression of TLR2 at the mRNA and protein levels. However, TGF-ß inhibited the induction of IL-1RI mRNA and protein expression mediated by IL-1{alpha}. Although it has been reported that TGF-ß regulates gene expression at transcription level and mRNA stability [23 24 25 ], in our study, TGF-ß inhibited the TLR2 promoter activity and NF-{kappa}B activation that could be induced by IL-1{alpha} (Fig. 3) . The stability of TLR2 mRNA was not affected by TGF-ß (data not shown). Thus, the inhibitory effect of TGF-ß appeared to act at the transcriptional level. The reporter gene assay for TLR2 promoter activity and NF-{kappa}B activation suggested that TGF-ß does not directly affect IL-1 signaling, as 6 h of TGF-ß treatment (but not 12 h) was insufficient to inhibit the augmenting effect of IL-1. Therefore, we consider it likely that TGF-ß acts in murine hepatocytes by modulating IL-1R expression.

TGF-ß is known to regulate IL-1R expression in positive and negative manners [14 15 16 ]. As expected in primary-cultured murine hepatocytes, TGF-ß decreased IL-1RI mRNA levels in control cells and also inhibited the IL-1{alpha}-induced up-regulation of IL-1RI mRNA (Fig. 4) . In addition, positive indices of IL-1 signaling such as NF-{kappa}B activation and TLR2 promoter activity were strongly inhibited by TGF-ß pretreatment (Fig. 5) . These results suggest that TGF-ß down-regulates cell-surface IL-1RI, although we were unable to determine the level of cell-surface IL-1RI, as it was at the limit of detection. Our data may also suggest that although IL-1{alpha} up-regulates IL-1RI to sustain and enhance its capacity to signal and induce gene targets such as TLR2, TGF-ß can inhibit such up-regulation of IL-1RI expression and thereby suppress the induction of functional TLR2 expression by IL-1{alpha}. This hypothesis was supported by evidence that TGF-ß inhibited TLR2 mRNA expression by IL-1{alpha} at late phase such as 12 and 24 h but not at early phase (Fig. 1) . The inhibitory effect of TGF-ß was specific to IL-1, as TGF-ß did not down-regulate TLR2 expression induced by TNF-{alpha}, LPS, or BLP (data not shown).

Recently, we found that BLP is an inducer of A-SAA in murine hepatocytes [8 ]. A-SAA acts as a chemoattractant for immune cells such as monocytes, polymorphonuclear cells (PMN), mast cells, and T lymphocytes [26 ]. A-SAA is also an activator of PMN antimicrobial functions, such as induction of degranulation, phagocytosis, and enhancement of anti-Candida activity [27 ]. Our findings, therefore, suggest that positive and negative regulation of hepatic TLR2 expression by IL-1{alpha} and TGF-ß contributes to the control of host-defense mechanisms against pathogens by changing the hepatocyte response to bacteria or bacterial products.

In conclusion, we have demonstrated that the anti-inflammatory cytokine TGF-ß inhibits functional TLR2 expression by the proinflammatory cytokine IL-1{alpha} in primary-cultured murine hepatocytes and that this inhibition is regulated at the transcriptional level. Our data suggest that TGF-ß suppresses IL-1{alpha}-mediated NF-{kappa}B activation, TLR2 promoter activity, TLR2 mRNA expression, and TLR2 expression by down-regulating IL-1RI expression. The elucidation of the reciprocal biological and physiological effects of IL-1{alpha} and TGF-ß will contribute to our understanding of the regulatory mechanism of TLR2 expression during systemic inflammation and infection.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a Grant-in Aid for Scientific Research (B) from the Japan Society for the Promotion of Science. We thank Dr. Takashi Okamoto (Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences) for providing pGL3-4{kappa}B wt construct and B. S. Y. Taniguchi (Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University) for technical help.

Received January 12, 2004; revised February 5, 2004; accepted February 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Heldwein, K. A., Golenbock, D. T., Fenton, M. J. (2001) Recent advances in the biology of Toll-like receptors Modern Aspects of Immunobiology 1,249-252[Medline]
  2. Baeuerle, P. A., Baichwal, V. R. (1997) NF-{kappa}B as a frequent target for immunosuppressive and anti-inflammatory molecules Adv. Immunol. 65,111-137[Medline]
  3. Baichwal, V. R., Baeuerle, P. A. (1997) Activate NF-{kappa}B or die? Curr. Biol. 7,R94-R96[CrossRef][Medline]
  4. Hoffmann, P., Heinle, S., Schade, U. F., Loppnow, H., Ulmer, A. J., Flad, H. D., Jung, G., Bessler, W. G. (1988) Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues Immunobiology 177,158-170[Medline]
  5. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T., Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T., Brennan, P. J., Bloom, B. R., Godowski, P. J., Modlin, R. L. (1999) Host defense mechanisms triggered by microbial lipoproteins through Toll-like receptors Science 285,732-736[Abstract/Free Full Text]
  6. Aliprantis, A. O., Yang, R. B., Mark, M. R., Suggett, S., Devaux, B., Radolf, J. D., Klimpel, G. R., Godowski, P., Zychlinsky, A. (1999) Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2 Science 285,736-739[Abstract/Free Full Text]
  7. Matsumura, T., Ito, A., Takii, T., Hayashi, H., Onozaki, K. (2000) Endotoxin and cytokine regulation of Toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes J. Interferon Cytokine Res. 20,915-921[CrossRef][Medline]
  8. Matsumura, T., Degawa, T., Takii, T., Hayashi, H., Okamoto, T., Inoue, J., Onozaki, K. (2003) TRAF6-NF-{kappa}B pathway is essential for IL-1-induced TLR2 expression and its functional response to TLR2 ligand in murine hepatocytes Immunology 109,127-136[CrossRef][Medline]
  9. Darlington, G. J., Bernhard, H. P., Miller, R. A., Ruddle, F. H. (1980) Expression of liver phenotypes in cultured mouse hepatoma cells J. Natl. Cancer Inst. 64,809-819
  10. Thorn, C. F., Whitehead, A. S. (2002) Differential transcription of the mouse acute phase serum amyloid A genes in response to pro-inflammatory cytokines Amyloid 9,229-236[Medline]
  11. Yoshimoto, T., Takeda, K., Tanaka, T., Ohkusu, K., Kashiwamura, S., Okamura, H., Akira, S., Nakanishi, K. (1998) IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN-{gamma} production J. Immunol. 161,3400-3407[Abstract/Free Full Text]
  12. Uhlar, C. M., Burgess, C. J., Sharp, P. M., Whitehead, A. S. (1994) Evolution of the serum amyloid A (SAA) protein superfamily Genomics 19,228-235[CrossRef][Medline]
  13. Ramadori, G., Sipe, J. D., Colten, H. R. (1985) Expression and regulation of the murine serum amyloid A (SAA) gene in extrahepatic sites J. Immunol. 135,3645-3647[Medline]
  14. Dubois, C. M., Ruscetti, F. W., Palaszynski, E. W., Falk, L. A., Oppenheim, J. J., Keller, J. R. (1990) Transforming growth factor ß is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action J. Exp. Med. 172,737-744[Abstract/Free Full Text]
  15. McGee, D. W., Beagley, K. W., Aicher, W. K., McGhee, J. R. (1993) Transforming growth factor-ß and IL-1ß act in synergy to enhance IL-6 secretion by the intestinal epithelial cell line, IEC-6 J. Immunol. 151,970-978[Abstract]
  16. Silvera, M. R., Sempowski, G. D., Phipps, R. P. (1994) Expression of TGF-ß isoforms by Thy-1+ and Thy-1– pulmonary fibroblast subsets: evidence for TGF-ß as a regulator of IL-1-dependent stimulation of IL-6 Lymphokine Cytokine Res. 13,277-285[Medline]
  17. Ito, A., Takii, T., Soji, T., Onozaki, K. (1997) Endotoxin-induced upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of mice: role of cytokines J. Interferon Cytokine Res. 17,55-61[Medline]
  18. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Giri, J. G., Dower, S. K., Sims, J. E., Mantovani, A. (1993) Interleukin-1 type II receptor: a decoy target for IL-1 that is regulated by IL-4 Science 261,472-475[Abstract/Free Full Text]
  19. Dinarello, C. A. (1996) Biologic basis for interleukin-1 in disease Blood 87,2095-2147[Abstract/Free Full Text]
  20. Kmiec, Z. (2001) Cooperation of liver cells in health and disease Adv. Anat. Embryol. Cell Biol. 161,III-XIII, 1–151[Medline]
  21. Bogdan, C., Nathan, C. (1993) Modulation of macrophage function by transforming growth factor ß, interleukin-4, and interleukin-10 Ann. N. Y. Acad. Sci. 685,713-739[Medline]
  22. Pilette, C., Ouadrhiri, Y., Van Snick, J., Renauld, J. C., Staquet, P., Vaerman, J. P., Sibille, Y. (2002) IL-9 inhibits oxidative burst and TNF-{alpha} release in lipopolysaccharide-stimulated human monocytes through TGF-ß J. Immunol. 168,4103-4111[Abstract/Free Full Text]
  23. Mehra, A., Wrana, J. L. (2002) TGF-ß and the Smad signal transduction pathway Biochem. Cell Biol. 80,605-622[CrossRef][Medline]
  24. Burton, T. R., Kashour, T., Wright, J. A., Amara, F. M. (2003) Cellular signaling pathways affect the function of ribonucleotide reductase mRNA binding proteins: mRNA stabilization, drug resistance, and malignancy Int. J. Oncol. 22,21-31[Medline]
  25. Hayashi, H., Inoue, Y., Tsutsui, H., Okamura, H., Nakanishi, K., Onozaki, K. (2003) TGFß down-regulates IFN-{gamma} production in IL-18 treated NK cell line LNK5E6 Biochem. Biophys. Res. Commun. 300,980-985[CrossRef][Medline]
  26. Uhlar, C. M., Whitehead, A. S. (1999) Serum amyloid A, the major vertebrate acute-phase reactant Eur. J. Biochem. 265,501-523[Medline]
  27. Badolato, R., Wang, J. M., Stornello, S. L., Ponzi, A. N., Duse, M., Musso, T. (2000) Serum amyloid A is an activator of PMN antimicrobial functions: induction of degranulation, phagocytosis, and enhancement of anti-Candida activity J. Leukoc. Biol. 67,381-386[Abstract]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
T. Lu, L. Tian, Y. Han, M. Vogelbaum, and G. R. Stark
Dose-dependent cross-talk between the transforming growth factor-beta and interleukin-1 signaling pathways
PNAS, March 13, 2007; 104(11): 4365 - 4370.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
J. L. Reyes, L. I. Terrazas, B. Espinoza, D. Cruz-Robles, V. Soto, I. Rivera-Montoya, L. Gomez-Garcia, H. Snider, A. R. Satoskar, and M. Rodriguez-Sosa
Macrophage Migration Inhibitory Factor Contributes to Host Defense against Acute Trypanosoma cruzi Infection.
Infect. Immun., June 1, 2006; 74(6): 3170 - 3179.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. M. Shayakhmetov, Z.-Y. Li, S. Ni, and A. Lieber
Interference with the IL-1-Signaling Pathway Improves the Toxicity Profile of Systemically Applied Adenovirus Vectors
J. Immunol., June 1, 2005; 174(11): 7310 - 7319.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. A. Ruiz, A. Shkoda, S. C. Kim, R. B. Sartor, and D. Haller
IL-10 Gene-Deficient Mice Lack TGF-{beta}/Smad Signaling and Fail to Inhibit Proinflammatory Gene Expression in Intestinal Epithelial Cells after the Colonization with Colitogenic Enterococcus faecalis
J. Immunol., March 1, 2005; 174(5): 2990 - 2999.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0104108v1
75/6/1056    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Matsumura, T.
Right arrow Articles by Onozaki, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsumura, T.
Right arrow Articles by Onozaki, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS