IL-4 Suppresses the Expression and the
Replication of Hepatitis B Virus in the
Hepatocellular Carcinoma Cell Line Hep3B
This information is current as
of June 18, 2017.
Sue-Jane Lin, Pei-Yun Shu, Chungming Chang, Ah-Kau Ng
and Cheng-po Hu
J Immunol 2003; 171:4708-4716; ;
doi: 10.4049/jimmunol.171.9.4708
http://www.jimmunol.org/content/171/9/4708
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References
The Journal of Immunology
IL-4 Suppresses the Expression and the Replication of Hepatitis B
Virus in the Hepatocellular Carcinoma Cell Line Hep3B1
Sue-Jane Lin,* Pei-Yun Shu,† Chungming Chang,*‡ Ah-Kau Ng,‡¶ and Cheng-po Hu2*†
C
ytokines are known to contribute directly or indirectly to
antiviral responses (1, 2). The direct protective effects of
IFNs against viruses are mediated through the downstream
signals delivered by IFN receptors, which block one or more steps of
the life cycle of various viruses. TNF-␣ has antiviral properties that
are often synergistic with IFN-␥; however, its direct intracellular antiviral mechanisms are unclear. IL-12 and IL-18, on the other hand,
have an indirect antiviral effect through the induction of IFN-␥ after
infection with HSV or HIV (3, 4). Other cytokines, such as IL-1, IL-6,
and some chemokines, have been reported to have antiviral activities
during viral infections (5–7). IL-4 has not been considered an antiviral
cytokine; however, it has been shown to suppress PMA-induced HIV
expression at the transcriptional level in monocytic U1 cells (8). The
related cytokine, IL-13, has also been found to inhibit HIV production
in human macrophages (9).
IL-4 is produced by Th2 cells, basophils, mast cells, and NK T
(NKT)3 cells. It has a plethora of biological properties, including
*Institute of Microbiology and Immunology, National Yang-Ming University; †Department of Medical Research and Education, Taipei Veterans General Hospital; and
‡
Division of Intramural Research Affairs, National Health Research Institute, Taipei,
Taiwan, Republic of China; and ¶Department of Applied Medical Sciences, University of Southern Maine, Portland, ME 04104
Received for publication January 24, 2003. Accepted for publication August 18, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by NSC89-2320-B075-038, NSC90-2320-B075-014, and
NSC91-2320-B075-009 from the National Science Council (Taiwan, Republic of
China) and a grant from Liver Disease Prevention and Treatment Research Foundation (Taiwan, Republic of China). A.-K.N. is the recipient of a National Health Research Institute (Republic of China) Visiting Scholarship.
2
Address correspondence and reprint requests to Dr. Cheng-po Hu, Department of
Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
11217, Republic of China. E-mail address: [email protected]
3
Abbreviations used in this paper: NKT cells, NK T cells; BCP, basal core promoter;
CP, core promoter; CURS, core upstream regulatory sequence; ENII, enhancer II;
␤-gal, ␤-galactosidase; ␥c, common ␥-chain; HBsAg, hepatitis B surface Ag; HBV,
hepatitis B virus; HCC, hepatocellular carcinoma; LCL, lymphoblastoid cell line; SF,
serum-free; SPII, surface promoter II; HBeAg, hepatitis B virus e Ag.
Copyright © 2003 by The American Association of Immunologists, Inc.
the up-regulation of class II MHC expression, the activation of B
cells, and IgE class switching. IL-4 also plays a central role in
regulating the differentiation of T cells. Moreover, it inhibits proinflammatory cytokine and chemokine production in monocytes and
enhances the expression of VCAM-1 on endothelial cells. The
IL-4R complex has been detected on several cell types, including
B cells, T cells, monocytes, endothelial cells, and hepatocytes, a
finding in keeping with the broad range of biological effects of
IL-4 (10).
The hepatitis B virus (HBV) is a 3.2-kb, partially dsDNA, noncytopathic virus. Patients with a persistent infection of HBV in
hepatocytes are at a high risk of developing chronic hepatitis, cirrhosis, and/or hepatocellular carcinoma (HCC) (11, 12). It is generally believed that the elimination of HBV is mainly mediated by
CD8⫹ CTL. A balance between viral spread and the strength of the
CTL response determines whether the immune responses result in
the clearance of the virus or immunopathology. Substantial evidence obtained from studies in chimpanzees during acute HBV
infection (13), in HBV transgenic mice following adoptive transfer
of HBV-specific CTL (14), and in chronically HBV-infected patients (15) indicated that CTL may control HBV replication without the development of hepatic immunopathology (16, 17). It has
also been shown that cytokines released from CTL contribute to
noncytopathic antiviral activity against HBV (14). For instance,
injection of IFN-␣/IFN-␤ inducer poly (I-C) (18) or rIL-12 and
rIL-18 eliminated hepatocellular HBV replicative intermediates in
HBV transgenic mice (19, 20). Recombinant duck IFN-␥ and
IFN-␣ inhibited the replication of duck HBV in primary duck
hepatocytes (21, 22). In addition, NKT cells, which make up for
30 –50% of resident intrahepatic T lymphocytes, can rapidly produce high levels of IL-4 and IFN-␥ after activation (23) and inhibit
HBV replication in HBV transgenic mice (24). Thus, an antiviral
effect of the noncytotoxic cytokines seems to play an important
role in the control of HBV in the host.
In this study we found that the IL-4R complex was expressed on
all human HCC cell lines tested. To address the effect of IL-4 on
0022-1767/03/$02.00
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IL-4 has been known as a Th2 cytokine and can act on B cells, T cells, and monocytes. In this study we demonstrate that IL-4Rs
are expressed on human hepatocellular carcinoma (HCC) cells. We found that IL-4 suppresses hepatitis B surface Ag (HBsAg)
mRNA and HBsAg production in the Hep3B cell line, which contains an integrated hepatitis B virus (HBV) genome and constitutively secretes HBsAg. When Hep3B cells are further transfected with the plasmid pHBV3.6 that contains >1 U of HBV genome,
IL-4 could suppress the production of all HBV RNA and secreted HBsAg and hepatitis B virus e Ag. Furthermore, an endogenous
DNA polymerase activity assay shows a decrease in HBV DNA after IL-4 treatment. Using luciferase reporter assays we have
demonstrated that IL-4 could suppress the activity of the surface promoter II and the core promotor (CP). To delineate how IL-4
suppressed the transcription of HBV genes, we have examined the effect of IL-4 on the expression of transcription factors that are
known to bind to the core upstream regulatory sequence, which colocalizes with enhancer II of the HBV genome. Our results
demonstrate that IL-4 suppresses the expression of C/EBP␣. Furthermore, overexpression of C/EBP␣ blocked 43 and 30% of the
IL-4-mediated suppression of CP activity and IL-4-induced suppression of pregenomic RNA, respectively. Finally, we have demonstrated that mutations affecting the C/EBP␣-binding sites on core upstream regulatory sequence/enhancer II completely abolish
the IL-4-mediated suppression of CP activity. Thus, down-regulation of C/EBP␣ may be involved in the anti-HBV effect of IL-4
in Hep3B cells. The Journal of Immunology, 2003, 171: 4708 – 4716.
The Journal of Immunology
HBV, a well-differentiated HCC cell line, Hep3B, which contains
an integrated HBV genome in its chromosome and continuously
produces hepatitis B surface Ag (HBsAg) (25, 26), was chosen for
this study. Our results indicate that IL-4 suppresses the production
of HBsAg in Hep3B as well as the replication of HBV in a transiently transfected cell system. We also demonstrate that the transcription factor C/EBP␣ is involved in IL-4-mediated suppression
of HBV.
Materials and Methods
Cell culture and reagents
Binding of
125
I-labeled IL-4 to HCC cell lines
HCC cells and Raji cells (2 ⫻ 106) were resuspended in 0.2 ml of DMEM
containing 1% BSA and incubated with 125I-labeled IL-4 and various concentrations (3–500 nM) of unlabeled IL-4 at 4°C for 1 h. The separation of
cell-bound 125I-labeled IL-4 from unbound 125I-labeled IL-4 was by centrifugation of cells through a cushion in phthalate oils. The numbers of
bound IL-4 molecule per cell and the binding affinities were determined by
Scatchard plot analysis.
Measurement of HBsAg and HBeAg
Hep3B cells were seeded in 24-well plates at a concentration of 6 ⫻ 104
cells/well. After overnight incubation, the culture was refed with serumfree (SF) medium for 48 h, followed by treatment with various concentrations of IL-4 (10 –300 ng/ml) for another 48 h. Cell numbers were determined by trypan blue exclusion. HBsAg and hepatitis B virus e Ag
(HBeAg) in the culture medium were determined by ELISA (General Biological, Taiwan, Republic of China). The OD values were normalized
with cell numbers.
Plasmids and transient transfection assay
Plasmid pHBV3.6, containing ⬎1 U of HBV genome, was used for transient transfection experiments because the viral gene expression and the
replication of pHBV3.6 are similar to HBV infection in vivo (28). To
generate pSPI-Luc, pSPII-Luc, pBCP-Luc, and pCP-Luc, the XbaI-HindIII
fragments containing the surface promoter I (SPI), surface promoter II
(SPII), basal core promoter (BCP), or core promoter (CP) from
pA3SPICAT, pA3SPIICAT, pA3BCPCAT, or pA3CPCAT, respectively,
were inserted into the NheI-HindIII site of the pGL3-Basic vector (Promega, Madison, WI). The E4BP4 antisense plasmid pE4BP4-AS was generated by inserting the 378-bp BamHI-XbaI fragment, which corresponded
to the 5⬘ end region of E4BP4 from nt 117– 494, into the BamHI-XbaI site
of the pCDNA3.1 expression vector. To express C/EBP␣ in the same expression vector, the BamHI-HindIII fragment containing the C/EBP␣ open
reading frame from pCMV-C/EBP␣ was inserted into the BamHI-HindIII
site of the pCDNA3.1 expression vector. pCMV␤, a ␤-galactosidase (␤gal) expression plasmid, was used for standardization of transfection efficiency. To destroy the C/EBP␣-binding sites (underlined) on the CP (29,
30), the sequence of 5⬘-GTCTTACATAAGAGGACTCTTGGACTC
CCAGCAATGTCAACGAC-3⬘ was mutated to 5⬘-GTCTTACATCCGC
TGACTCTTGGACTCAACTAAATGTCAACGAC-3⬘. The plasmid
pmC/EBP␣-CP-Luc was then generated by inserting the mutated CP sequence into the KpnI-HindIII site of pGL3-Basic vector. Hep3B cells (4 ⫻
106) in a 10-cm plate were washed three times with SF medium and incubated for 3 h with various amounts of plasmid DNA and Lipofectamine
(Life Technologies, Grand Island, NY) as indicated for each experiment.
The culture medium was then changed to fresh DMEM containing 10%
FCS. After 18 h the transfected cells were resuspended, pooled, and reseeded at a density of 2.5 ⫻ 106 cells/plate. After overnight incubation, the
cells were cultured in SF for 2 days and then refreshed with SF medium
with or without 150 ng/ml of IL-4 for an additional 2 days.
RNA isolation and Northern blot analysis
Total cellular RNA was extracted by the RNAzol B method (Biotecx Laboratories, Houston, TX). RNA samples (30 ␮g/lane) were electrophoresed
on 1.2% formaldehyde agarose gels and transferred to nylon membranes
(Hybond-N⫹; Amersham Pharmacia Biotech, Piscataway, NJ) by a vacuum blotter (model 785; Bio-Rad, Hercules, CA). Membranes were prehybridized at 60°C for 6 h in Quick-Hyb solution (Merck, Darmstadt, Germany) and then hybridized in the same solution by adding 2 ⫻ 108 cpm/␮g
of 32P-labeled DNA probes prepared by random oligonucleotide priming of
the whole HBV genome, IL-4R␣, IL-13R␣1, ␥c, E4BP4, C/EBP␣, or a
G3PDH gene fragment. After 24 h the membranes were washed with 0.2⫻
SSC and 0.2% SDS at 60°C three times, 20 min each time, and finally
autoradiographed at ⫺70°C.
Nuclear run-on
Cells were homogenized in the buffer containing 10 mM Tris-HCl (pH
8.0), 5 mM MgCl2, 5 mM DTT, 0.3 M sucrose, and 0.1% Triton X-100 by
a Dounce homogenizer (Kontes, Vineland, NJ). Nuclear pellets were obtained by centrifugation at 36,000 ⫻ g at 1°C for 90 min. The pellets were
washed three times with the cushion buffer (50 mM Tris-HCl (pH 8.0), 0.1
mM EDTA, 5 mM MgCl2, and 0.5 mM DTT), resuspended in 40 ␮l of the
storage buffer (50 mM Tris-HCl (pH 8.3), 40% (v/v) glycerol, 5 mM
MgCl2, and 0.1 mM EDTA), and stored at ⫺70°C. For run-on experiments,
2 ⫻ 107 nuclei were reacted in 40 ␮l of transcription reaction mixture (10
mM Tris-HCl (pH 8.0), 1 mM ATP, 1 mM GTP, 1 mM CTP, 10 ␮l of 40
U/ml RNasin (Promega), 8% glycerol, and 5 ␮l of [␣-32P]UTP (3000 ␮Ci/
mmol)) for 30 min at 26°C. Unincorporated [␣-32P]UTP was removed by
a centrifugation through Ultrafree MC Centrifugal filter units (Millipore,
Bedford, MA). Nuclear RNA was isolated using the RNAzol B method and
isopropanol precipitation at 4°C for 30 min. RNA was treated with 10 U/ml
DNase (Roche, Mannheim, Germany) for 30 min at 37°C and 100 ␮g/ml
proteinase K (Roche) for 30 min at 37°C. The labeled RNA products were
partially degraded by treatment with 1 N NaOH for 10 min at 4°C. Membranes containing denatured HBV and ␤-actin control DNA were prehybridized at 42°C for 6 h in the hybridization solution (30% formamide, 0.1
M PIPES (pH 6.5), 5 M NaCl, 10% SDS, and 100 ␮g/ml Escherichia coli
tRNA) and then hybridized with equal counts per minute of nuclear RNA
per membrane at 42°C for 3 days. Membranes were washed three times
with 0.2⫻ SSC and 0.2% SDS at 60°C for 20 min and finally underwent
autoradiography at ⫺70°C.
Luciferase reporter assay
Hep3B cells were cotransfected with pSPI-Luc, pSPII-Luc, pCP-Luc,
pmC/EBP␣-CP-Luc, pBCP-Luc, and pCMV␤ expression plasmids. After
24 h cells were reseeded and cultured in SF medium for 2 days, then treated
with IL-4 (150 ng/ml) for the indicated periods. Cells were washed twice
with PBS, and the protein concentration of cell lysates was determined by
the Bradford assay (Bio-Rad). Luciferase and ␤-gal activity in 100 ␮g of
cell lysates were measured by the Luciferase Assay System and the ␤-Galactosidase Enzyme Assay System (Promega), respectively. Transfection
efficiency was normalized using ␤-gal activity.
HBV endogenous polymerase activity assay
To dissolve the envelope of HBV particles, equal protein amounts of lysates from pHBV3.6-transfected cells or equal volumes of the culture medium were treated with TNE buffer (50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, and 1 mM EDTA) containing 2% Nonidet P-40. The HBV core
particles containing the incomplete plus strand of HBV DNA were then
immunoprecipitated with rabbit anti-HBcAg antiserum. The endogenous
DNA polymerase activity in the core particles was measured by incubating
the immunoprecipitates with the polymerase buffer (50 mM Tris-HCl (pH
7.5); 40 mM NH4Cl; 10 mM MgCl2; 1% Nonidet P-40; 2% 2-ME; 12.5
␮M each of dATP, dGTP, and dTTP; and 80 ␮Ci [␣-32P]dCTP (5000
Ci/mmol)) for 3 h at 37°C. Unlabeled dCTP (12.5 ␮M) was added to fill the
remaining gaps. After washing, 200 ␮g/ml of proteinase K and 0.5% SDS
were added for 4 h at 37°C. The labeled HBV DNA was extracted with
phenol/chloroform and subjected to 1.2% agarose gel electrophoresis and
autoradiography at ⫺70°C.
Western blotting
Cells transfected with pC/EBP␣ were washed twice with ice-cold HBSS
and lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1%
Triton X-100, 2 mM PMSF, 2 ␮g/ml aprotinin, 1 ␮M leupeptin, 1 mM
Na3VO4, 80 mM pyrophosphate, and 50 mM NaF) on ice. The lysates were
subjected to SDS-PAGE. The separated proteins were then transferred onto
nitrocellulose membranes by a semidry blotter (Bio-Rad). The membrane
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
HepG2, Hep3B, HuH7, and HA22T/VGH (27) are human HCC cell lines.
An EBV-transformed lymphoblastoid cell line (LCL) and a Burkitt’s lymphoma cell line (Raji) are human B cell lines. These cell lines were cultured
in DMEM or RPMI 1640 supplemented with 10% FCS, 100 IU/ml of
penicillin, 100 ␮g/ml of streptomycin, 2 mM L-glutamine, and 1% nonessential amino acids at 37°C in 5% CO2. Recombinant human IL-4 was
purchased from R&D Systems (Minneapolis, MN). Lipofectamine was obtained from Life Technologies (Gaithersburg, MD). Actinomycin D and
cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO). 125Ilabeled IL-4 was purchased from Amersham Pharmacia Biotech (Piscataway, NJ), and anti-C/EBP␣ Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
4709
4710
was soaked in 5% skimmed milk in PBS at 4°C for 16 h and then incubated
with rabbit anti-C/EBP␣ Ab at 37°C for 1 h. After washing three times with
PBS containing 0.05% Tween 20, peroxidase-conjugated goat anti-rabbit
Ig was added, and the membranes were incubated in the buffer at room
temperature for 1 h. The signal was visualized by the ECL system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).
Results
Expression of IL-4R complex on HCC cell lines
Effect of IL-4 on the production of HBsAg and HBeAg
To study the effect of IL-4 on HBV, the production of HBsAg was
measured in a well-differentiated human HCC cell line, Hep3B,
which contains an integrated HBV genome and spontaneously secretes HBsAg. As shown in Fig. 2A, IL-4 suppresses the production of HBsAg in a dose-dependent manner. At the concentration
of 150 ng/ml of IL-4, 71% of the HBsAg is suppressed in the
culture medium. At a higher concentration (300 ng/ml) of IL-4,
there is no further suppression of HBsAg production. To investigate whether the suppression was due to an effect on the secretion
process, the amount of HBsAg in the cytosol was also measured.
Our data indicated that IL-4 inhibited the production of both secreted and cytosolic HBsAg (Fig. 2A). To rule out the possibility
that IL-4 might be acting on cellular regulatory elements upstream
of the HBV-integrated site in Hep3B, the effect of IL-4 was also
studied in Hep3B cells transiently transfected with a plasmid
pHBV3.6, which contained ⬎1-U length of the HBV genome and
retains the ability to produce mature HBV virions (28). Thus, in
pHBV3.6-transfected Hep3B cells, HBsAg can be encoded by
both the integrated and the transfected HBV DNA, whereas
HBeAg is only produced by pHBV3.6. Fig. 2B shows that the
amount of HBeAg in these cells is also curtailed in the culture
medium. These results show that IL-4 suppressed the production of
HBV proteins encoded by both the integrated and the transiently
transfected HBV genome.
Effect of IL-4 on RNA expression and replication of HBV
To investigate the mechanism by which IL-4 suppresses HBV, we
examined the effect of IL-4 on the steady state level of HBV RNA
using Northern blotting analysis. After IL-4 treatment, HBsAg
mRNA was significantly reduced at 9, 12, 18, and 24 h (Fig. 3A).
When Hep3B was further transfected with pHBV3.6, the 4.0-kb
HBsAg mRNA transcribed from the integrated HBV DNA and the
3.5-kb precore/pregenomic RNA transcribed from pHBV3.6 could
not be distinguished on the gel. However, it was evident that the
transcripts with molecular sizes of 4.0/3.5 kb (HBsAg mRNA and
precore/pregenomic RNA) and 2.4/2.1 kb (large HBsAg mRNA
and middle/major HBsAg mRNA) were significantly suppressed
by IL-4 over the various time periods studied (Fig. 3B). In addition, we measured endogenous DNA polymerase activity. The
HBV core particles in the culture supernatant or in the cytoplasm
of Hep3B transfected with pHBV3.6 were isolated and reacted
with [␣-32P]dCTP. As shown in Fig. 3C, both the nicked circular
and the linear forms of HBV DNA dramatically decreased after
treatment with IL-4 for 12, 24, and 36 h. These results clearly show
that IL-4 suppressed HBV replication.
Effect of IL-4 on the stability of HBsAg mRNA
To test whether IL-4 affected the stability of HBsAg mRNA, the
RNA samples obtained from actinomycin D-treated and untreated
Hep3B cells were subjected to Northern blotting analysis. As
shown in Fig. 4, IL-4 suppressed the expression of 4.0-kb HBsAg
mRNA of untreated cells as expected. Actinomycin D alone also
suppressed HBsAg mRNA; however, IL-4 did not affect the
amount of HBsAg mRNA in actinomycin D-treated cells. Thus,
the suppression of HBsAg mRNA production by IL-4 was not due
to an effect on the stability of mRNA.
Effect of IL-4 on the transcriptional rate of the HBsAg gene
FIGURE 1. Analysis of expression of IL-4R␣, IL-13R␣1, and ␥c in
HCC cell lines by Northern blotting. Thirty micrograms of total RNA from
LCL (lane 1), Raji (lane 2), HepG2 (lane 3), Hep3B (lane 4), HuH7 (lane
5), and HA22T/VGH (lane 6) cells was analyzed by Northern blotting
using IL-4R␣ (A), IL-13R␣1 (B), or ␥c (C) DNA fragments as probes. LCL
is a B lymphoblastoid cell line, and Raji is a Burkitt’s lymphoma cell line.
Both cell lines are the positive controls for the IL-4R␣ (4.0 kb), IL-13R␣1
(4.2 and 2.0 kb), and ␥c (3.0 and 1.6 kb) chains. The same membranes were
rehybridized with a G3PDH DNA fragment to ensure an equal amount of
RNA in each lane.
To test whether IL-4 affected the transcriptional rate of HBV
genes, the newly synthesized transcripts were detected by a nuclear
run-on experiment. There were ⬃52 and 56% decreases in the
transcriptional rate of the HBsAg gene detected in IL-4-treated
nuclei at 12 and 24 h, respectively (Fig. 5A). In addition, the transcriptional rate of HBV genes in Hep3B cells transfected with
pHBV3.6 was significantly reduced after IL-4 treatment (data not
shown). These results indicate that IL-4 may inhibit the expression
of HBV genes at the transcriptional level.
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To demonstrate the expression of IL-4R complex in HCC cell
lines, we first examined the mRNA expression of the IL-4R components, IL-4R␣-chain, common ␥-chain (␥c), and IL-13R␣1chain, by Northern blotting. A 4.0-kb transcript of IL-4R␣ (Fig.
1A) and a 4.2-kb transcript of IL-13 R␣1 genes (Fig. 1B) were
detectable in all HCC cell lines: HepG2, Hep3B, HuH7, and
HA22T/VGH cells. An additional 2.0-kb transcript of IL-13R␣1
was present in HuH7 and HA22T/VGH cells, but was only just
detectable in HepG2 and Hep3B cells. The 3.0- and 1.6-kb transcripts of ␥c (Fig. 1C) were also detected in the HCC cell lines,
Hep3B and HA22T/VGH. The LCL and Raji cell lines expressed
IL-4R␣, ␥c, and a very small amount of IL-13 R␣1 transcripts, as
expected. We further characterized the IL-4 binding sites and binding affinities of HCC cell lines using 125I-labeled IL-4 binding
assays. The numbers of IL-4 binding sites per cell and the Kd
values are listed in Table I. The Kd values were between 10⫺10 and
10⫺11 M, and the numbers of IL-4 binding sites were 2250/HepG2,
2250/Hep3B, 1246/HuH7, and 664/HA22T/VGH cell. Similar to
the results described in a pervious report (31), Raji cells, a Burkitt’s lymphoma cell line, express 2952 IL-4 receptors/cell with a
Kd of 3.1 ⫻ 10⫺10 M. For comparison, Table I also shows the
transcripts of the IL-4R components and the secretion of HBsAg in
these cell lines.
SUPPRESSION OF HBV BY IL-4
The Journal of Immunology
4711
Table I. The binding sites, binding affinities, and presence of each IL-4R transcript in human HCC cell
lines
Cell Line
HBV Genome
Receptors/Cell
Kd (M)
IL-4 R␣
IL-13 R␣1
␥c
Raji
HepG2
Hep3B
HuH7
HA22T/VGH
Unknown
⫺
⫹a
⫺
⫹b
2952
2250
2250
1246
664
3.1 ⫻ 10⫺10
7.1 ⫻ 10⫺11
1.4 ⫻ 10⫺10
1.2 ⫻ 10⫺11
3.9 ⫻ 10⫺10
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫹
a
b
Hep3B contains an integrated HBV genome in its chromosome and only expresses HBsAg.
HA22T/VGH contains an HBV genome in its chromosome and does not produce any HBV Ags.
Effect of IL-4 on the expression of E4BP4, C/EBP␣, and C/EBP␤
To further investigate the mechanism by which the transcriptional
suppression of IL-4 acts on HBV genes, we measured the effect of
IL-4 on the activity of SPI, SPII, BCP, and CP, which makes up
the BCP and the core upstream regulatory sequence (CURS). Using luciferase activity assays, we found that IL-4 significantly suppressed SPII and CP activity. As shown in Fig. 5B, IL-4 suppressed 51, 56, and 75% of the SPII activity at 12, 24, and 48 h,
respectively. Similarly, 26, 47, and 74% suppressions of CP activity were seen at 12, 24, and 48 h after IL-4 treatment, respectively (Fig. 5C). In contrast, luciferase activity driven by SPI was
not significantly affected (data not shown). We also found that IL-4
did not affect the activity of BCP (data not shown). Thus, IL-4 may
exert its suppressive effect on CP activity through the regulation
of CURS.
The CURS is colocalized with enhancer II (ENII) in the HBV
genome. Thus, the sequence of CURS/ENII not only regulates CP
activity and thus the expression of pregenomic RNA and precore
mRNA, but it also enhances the activity of the SPI, SPII, and HBx
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Effect of IL-4 on the promoter activity of HBV genes
Suppression of HBsAg by IL-4 required de novo protein
synthesis
Since the suppression of HBsAg mRNA was detected after 9 h, we
examined whether de novo protein synthesis was required for the
inhibitory effect of IL-4. Fig. 6 shows that IL-4 only suppressed
38% of HBsAg mRNA in cycloheximide-treated cells compared
with a 93% reduction in untreated Hep3B cells, suggesting that de
novo protein synthesis is required for the suppressive effect of IL-4
on HBsAg mRNA.
FIGURE 2. IL-4 suppresses the production of HBsAg and HBeAg in
Hep3B cells. A, HBsAg produced by Hep3B cells. Cells cultured in SF
medium were treated with or without IL-4 (10, 40, 60, 150, and 300 ng/ml)
for 2 days. The amount of HBsAg in the supernatants (f) or the cell lysates
(䡺) was measured by ELISA. B, HBeAg produced by Hep3B cells transfected with pHBV3.6. The plasmid HBV3.6 DNA (7.5 ␮g) was transfected
into Hep3B cells. After 18 h, the cells were pooled, reseeded, and cultured
in SF medium for 2 days. IL-4 (150 ng/ml) was added to the culture for
additional 2 days. The supernatant was collected, and the amount of HBsAg was measured by ELISA. The OD values of HBsAg and HBeAg were
normalized with cell numbers and calculated as the mean of triplicate
wells ⫾ SD. The results are expressed as a percentage of the SF control.
Independent experiments were repeated three times and showed similar
results.
FIGURE 3. IL-4 suppresses gene expression and replication of HBV. A,
Northern blotting of HBsAg mRNA. Hep3B cells cultured in SF medium
were treated with or without IL-4 (150 ng/ml) for 2 days. Total cellular
RNA was extracted at the indicated times. Thirty micrograms of RNA were
loaded in each lane and hybridized with the full-length HBV DNA. B,
Northern blotting of HBV transcripts in Hep3B cells transfected with
pHBV3.6. Hep3B cells were transfected and treated with IL-4 as described
in Fig. 2B. Total cellular RNA was extracted at the indicated times, and
Northern blotting was performed. The same membranes were rehybridized
with G3PDH DNA fragment to ensure an equal amount of RNA in each
lane. C, HBV endogenous polymerase activity assay. Hep3B cells were
transfected with pHBV3.6 and treated with IL-4 as in B. Equal protein
amounts of lysates or equal volumes of the culture medium were solubilized with Nonidet P-40. The core particles were immunoprecipitated with
a rabbit anti-HBcAg antiserum at the indicated times and assayed for HBV
endogenous polymerase activity. The nicked circular (NC) and linear (L)
forms of HBV DNA are indicated.
4712
FIGURE 4. IL-4 does not affect the stability of HBsAg mRNA. Hep3B
cells cultured in SF medium for 2 days were treated with 5 ␮g/ml of
actinomycin D for 1 h. The cells were then treated with IL-4 (150 ng/ml)
for 12 h. Total cellular RNA was extracted and analyzed by Northern
blotting. The same membranes were rehybridized with the G3PDH DNA
fragment to ensure an equal amount of RNA in each lane. The intensity of
each band was determined by densitometry. HBsAg/G3PDH ratios represent the relative intensity of HBsAg.
FIGURE 6. De novo protein synthesis is required for the suppressive
effect of IL-4. Hep3B cells cultured in SF medium were treated with 10
␮g/ml of cycloheximide (CH) for 2 h before treatment with 150 ng/ml of
IL-4. Total cellular RNA was extracted after 12 h and was analyzed by
Northern blotting using full-length HBV DNA as the probe. The same
membranes were rehybridized with G3PDH DNA fragment to ensure an
equal amount of RNA in each lane. The intensity of each band was determined by densitometry. The HBsAg/G3PDH ratios represent the relative
intensity of HBsAg.
the amount of C/EBP␤ mRNA was not altered after treatment with
IL-4, and the expression of HLF in Hep3B was below the detection
level for RT-PCR of the gene (data not shown).
E4BP4 is not involved in IL-4-mediated suppression of HBV RNA
E4BP4 has been reported to be a negative regulator for the activity
of CURS/ENII. However, its function on HBV gene expression is
controversial (29, 33). To test the involvement of E4BP4 in IL-4mediated suppression, E4BP4 antisense plasmid (pE4BP4-AS)
was introduced into Hep3B cells, and CP activity and HBV RNA
were measured. Our results showed no significant difference in
IL-4-mediated suppression of CP activity and HBV RNA between
cells transfected with pE4BP4-AS and those effects when transfected with the control plasmid pCDNA3.1 (data not shown). To
further confirm whether E4BP4 plays a role in IL-4-mediated suppression, an E4BP4-overexpressed system was used. Northern
blotting of HBV RNA in cells transfected with pCDNA3.1 or
pE4BP4 in the absence or the presence of IL-4 is shown in Fig. 7B.
After normalization of the amount of RNA using the G3PDH gene
and ␤-gal activity, the overexpressed E4BP4 did not further reduce
the amount of HBV RNA in the presence of IL-4. As shown in Fig.
7C, IL-4 suppressed 87% of 4.0/3.5-kb RNA and 77% of 2.4/
2.1-kb RNA in pCDNA3.1-tranfected cells, as expected. In
E4BP4-overexpressed cells, IL-4-mediated suppression is not
higher than that in control cells, i.e., IL-4 suppresses 79% of 4.0/
3.5-kb RNA and 54% of 2.4/2.1-kb RNA. Thus, E4BP4 does not
seem to play a role in IL-4-mediated suppression of HBV RNA.
C/EBP␣ is involved in IL-4-mediated suppression of CP activity
and HBV gene expression
FIGURE 5. IL-4 suppresses the transcriptional rate of the HBsAg gene
in Hep3B cells and the activity of SPII and CP. A, The transcriptional rate
of the HBsAg gene. Hep3B cells cultured in SF medium were treated with
or without IL-4 (150 ng/ml). The nuclei were collected after 12 and 24 h.
[␣-32P]UTP was added to the nuclei, and unincorporated [␣-32P]UTP was
removed by centrifugation. Nuclear RNA was isolated, and equal counts of
[␣-32P] UTP-incorporated, newly synthesized transcripts were used to hybridize to the HBV or ␤-actin gene. The intensity of each band was determined by densitometry. The HBsAg/␤-actin ratios represent the relative
intensity of HBsAg. B and C, Suppression of SPII (B) and CP (C) activities
by IL-4. Hep3B cells were cotransfected with pSPII-Luc or pCP-Luc and
pCMV␤. The transfected cells were cultured in SF medium (f) or were treated
with IL-4 (䡺). The same amounts of cell extracts were measured for luciferase
activity at 12, 24, and 48 h. The relative luciferase activities of pSPII-Luc (B)
and pCP-Luc (C) were normalized with the ␤-gal activity. % 2, IL-4 mediated suppression of relative luciferase activity. ⴱ, p ⬍ 0.05 (t test).
To test whether C/EBP␣ was involved in the regulation of CP
activity by IL-4, we overexpressed C/EBP␣ in IL-4-treated and
untreated Hep3B cells. While IL-4 could suppress 28.4% of the CP
activity in pCDNA3.1-transfected cells, it only suppressed 16.0%
of the CP activity in cells transfected with pC/EBP␣. Thus, overexpression of C/EBP␣ blocked 43% of the IL-4-induced suppression of CP activity (Fig. 8A). Fig. 8B shows the overexpression of
C/EBP␣ in Hep3B cells, as demonstrated by anti-C/EBP␣ Ab. Fig.
8C shows the Northern blotting of HBV RNA in cells transfected
with pCDNA3.1 or pC/EBP␣ in the absence or the presence of
IL-4. After normalization of the amount of RNA with G3PDH
gene and ␤-gal activity, the relative levels of RNA are shown in
Fig. 8D. Under SF conditions, the overexpression of C/EBP␣ was
enhanced by 1.8-fold for the 4.0/3.5-kb RNA, but had no effect on
the 2.4/2.1-kb mRNA (Fig. 8D, compare the two SF lanes), suggesting that C/EBP␣, a transcriptional activator for HBV, activates
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
promoters (32). Therefore, we focused our study on the mechanism by which IL-4 affects CP activity. It has been reported that
C/EBP␣, C/EBP␤, E4BP4, and HLF bind to box ␣ within the
CURS/ENII region and regulate the transcription of HBV genes
(29, 33). To address whether IL-4 could regulate the expression of
these transcription factors in Hep3B cells, the mRNA level of these
factors was measured by Northern blotting analysis after IL-4
treatment. We found that E4BP4 mRNA was increased 3-fold, and
C/EBP␣ mRNA was decreased 38% at 6 h after treatment with
IL-4 (Fig. 7A). However, no difference could be seen after 9 h,
indicating a transient regulation of these genes by IL-4. In contrast,
SUPPRESSION OF HBV BY IL-4
The Journal of Immunology
4713
the expression of 4.0/3.5-kb RNA, but not 2.4/2.1-kb RNA. In the
presence of IL-4, 78% of 4.0/3.5-kb RNA was suppressed in
pCDNA3.1-tranfected cells, while only 55% was suppressed in
C/EBP␣-overexpressed cells. Thus, the overexpression of C/EBP␣
blocks 30% of the IL-4-induced suppressive effect of 4.0/3.5-kb
RNA (78 vs 55%). On the contrary, the degree of suppression of
the 2.4/2.1-kb mRNA was similar in control and C/EBP␣-overexpressed cells (69 vs 72%) after treatment with IL-4. Taken together, C/EBP␣ seems to be involved in IL-4 suppression of the
production of the 4.0/3.5-kb RNA, but not of the 2.4/2.1-kb RNA.
C/EBP␣ binding sites are crucial to the effect of IL-4
Discussion
IL-4, a cytokine produced by Th2 cells, NKT cells, basophils, and
mast cells, displays a wide array of immunological functions, ranging from Th2 development, lymphocyte proliferation, and up-regulation of class II MHC expression to IgE class switch (10); it also
has a role in asthma, allergy, and resistance to intestinal nematode
infection (34, 35). Moreover, IL-4 has been shown to inhibit lipogenesis stimulated by TNF-␣, IL-1, and IL-6 in mouse hepatocytes
(36) and fibrinogen biosynthesis in the human HCC cell line
HepG2 (37). In primary human hepatocytes, IL-4 could also induce the expression of cytochrome P-450 and GST␣ (38, 39). Furthermore, it inhibits the production of haptoglobulin, albumin, and
C-reactive protein (40), but enhances the stimulatory effect of
IL-1␤ on the production of the IL-1R antagonist in human hepatocytes (41). In exploring the effect of exogenous IL-4 on Hep3B
cells and their transient transfectants, we discovered that the cytokine also had a suppressive effect on HBV gene expression.
Thus, IL-4 appears to play an important, yet complex, role in regulating the expression of viral genes and in the innate defense
mechanism in liver.
The IL-4R complex has been characterized in various cell types.
The type I receptor, which consists of IL-4R␣-chain and ␥c, is
expressed on both T and B cells. The type II receptor, which consists of IL-4R␣- and IL-13R␣1-chains, is found on endothelial
cells, and most carcinoma cells, such as ovarian carcinoma and
colon carcinoma (42, 43). In our study although all the HCC cell
lines tested expressed the IL-4R␣- and IL-13R␣1-chains, the ␥c
were also detected on Hep3B and HA22T/VGH cells. Although
FIGURE 7. E4BP4 is not involved in the IL-4-mediated suppression of
HBV gene expression. A, Northern blotting analysis of E4BP4 and
C/EBP␣ genes. Total cellular RNA of Hep3B cells was prepared as described in Fig. 3. The ratios of E4BP4/G3PDH and C/EBP␣/G3PDH represent the relative intensity of E4BP4 or C/EBP␣. B, Hep3B cells were
cotransfected with pHBV3.6, pCMV␤, and pCDNA3.1, pE4BP4. After
18 h, the pE4BP4-transfected cells were pooled, reseeded, and cultured in
SF medium for 18 h. Cells were cultured in SF medium or were treated
with IL-4 as described, and Northern blotting analysis was performed. The
␤-gal activity and the intensities of the 4.0/3.5-kb, 2.4/2.1-kb, and G3PDH
transcripts of each sample are listed at the bottom of B. Relative expression
levels of 4.0/3.5-kb and 2.4/2.1-kb HBV RNA are shown in C. The relative
level of RNA represents the ratio of the intensity of the 4.0/3.5-kb RNA
(f) or the 2.4/2.1-kb RNA (䡺) to that of the G3PDH transcript and then
normalized with the ␤-gal activity of each sample. % 2, The percent
decrease in the relative RNA level in cells cultured in the presence of IL-4.
Similar results were obtained in three independent experiments.
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To further confirm the role of C/EBP␣ in IL-4-mediated suppression, the sequences of the two C/EBP␣-binding sites on the CURS/
ENII were mutated. The nucleotide sequence from nt 1649 –1692
and the binding sites for the transcriptional factors are shown in the
upper part of Fig. 9. Mutations of C/EBP␣ binding sites 1 and 2
resulted in a 6.7-fold decrease in CP activity (compare the two SF
lanes in the lower panel). This indicates that the C/EBP␣ binding
sites are important for the activity of CURS/ENII. In addition,
while IL-4 could suppress 27% of the pCP-Luc activity, IL-4 did
not have any suppressive effect on the activity of pmC/EBP␣-CPLuc. Thus, our results demonstrated that the C/EBP␣ binding sites
of the CP are crucial for the IL-4-suppressive effect.
4714
SUPPRESSION OF HBV BY IL-4
most carcinoma cells do not express the ␥c, it has been shown that
IL-4R␣1, IL-13R␣1, and ␥c transcripts are expressed in HT-29
carcinoma cells (44). In addition, the ␥c has been shown to be a
subunit of the IL-4R complex in HT-29 cells using an 125I-labeled
IL-4 cross-linking experiment (45). It would be interesting to investigate further the expression and the functional significance of
␥c in Hep3B and HA22T/VGH cells or their clinical counterparts.
Hep3B, a well-differentiated human HCC cell line, contains an
integrated HBV genome in its chromosome and continuously produces HBsAg (25, 26). In this study we clearly demonstrated that
FIGURE 8. C/EBP␣ is involved in the IL-4-mediated suppression of
CP activity and HBV gene expression. A, Effect of pC/EBP␣ on the IL4-mediated suppression of pCP-Luc activity. Hep3B cells were cotransfected with pCP-Luc, pCMV␤, and pCDNA3.1 or pC/EBP␣. The transfected cells were cultured in SF medium or were treated with IL-4. The
same amounts of cell extracts were measured for luciferase activity at 12 h.
Relative luciferase activity of pCP-Luc was normalized with ␤-gal activity.
The percentage of IL-4-mediated suppression of pCP-Luc activity is represented by the percent decrease in pCP-Luc activity in Hep3B cells cultured in the presence of IL-4. 2, Blocking of IL-4-mediated suppression
after overexpression of pC/EBP␣. ⴱ, p ⬍ 0.05. B, Western blotting of
C/EBP␣. Cells were transfected with pC/EBP␣ as described above, and
cell lysates were collected after 2 days. Western blotting was performed
using anti-C/EBP␣ Ab and goat anti-rabbit Ig and visualized by the ECL
system. C, Northern blotting analysis of HBV genes. Hep3B cells were
cotransfected with pHBV3.6, pCMV␤, pCDNA3.1, and pC/EBP␣. The
transfected cells were cultured in SF medium or treated with IL-4 as described, and Northern blotting analysis was performed. The ␤-gal activity
and the intensities of 4.0/3.5-kb, 2.4/2.1-kb, and G3PDH transcripts of each
sample are listed at the bottom of C. Relative expression levels of 4.0/
3.5-kb and 2.4/2.1-kb HBV RNA are shown in D. The relative level of
RNA represents the ratio of the intensity of the 4.0/3.5-kb RNA (f) or the
2.4/2.1-kb RNA (䡺) to the G3PDH transcript and then normalized with the
␤-gal activity of each sample. % 2, The percent decrease in the relative
RNA level in cells cultured in the presence of IL-4. Similar results were
obtained in four independent experiments. The difference in the IL-4-mediated suppression of 4.0/3.5-kb RNA between pCDNA3.1- and pC/EBP␣transfected cells (D; f) is statistically significant (ⴱ, p ⬍ 0.05, t test.)
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FIGURE 9. C/EBP␣ binding sites are crucial for the IL-4-mediated suppression. The nucleotide sequence from nt 1649 –1692 and the binding
sites for transcriptional factors are shown at the top. Hep3B cells were
cotransfected with pCMV␤, pCDNA3.1, and pCP-Luc or pmC/EBP␣-CPLuc. The cells were cultured in SF medium or were treated with IL-4. The
same amounts of cell extracts were measured for luciferase activity at 12 h.
Relative luciferase activity of pCP-Luc was normalized with ␤-gal activity.
The effect of IL-4 on the activity of pCP-Luc or pmC/EBP␣-CP-Luc is determined as the percent decrease or increase in activity after treatment of IL-4.
The Journal of Immunology
on core particle encapsidation, the prevention of nucleocapsid assembly, or an acceleration of nucleocapsid degradation (18).
The source of IL-4 in the liver may be T cells and/or NKT cells.
A previous study indicated that most CD4⫹ and CD8⫹ intrahepatic
T cell clones isolated from chronically HBV-infected patients produce IFN-␥ and IL-4 (52). Furthermore, Schistosoma mansoniinduced Th1 and Th2 cytokines inhibited HBV replication in HBV
transgenic mice, but this parasite-induced antiviral effect was only
partially blocked in IFN-␥-deficient mice (53), implying an antiviral role played by other cytokines. In the normal liver, NKT cells
account for 30 –50% of resident intrahepatic lymphocytes, and
their role in immunity has been a focus of research. Interestingly,
when the intrahepatic NKT cells were activated by ␣-galactosylceramide-presenting hepatocytes, they predominately produced
IL-4 (54). Moreover, recent studies have shown that ␣-galactosylceramide-activated NKT cells can inhibit HBV replication in HBV
transgenic mice (24). IL-4 production by liver NKT cells, therefore, may play a role in the host defense against microbial infections of the liver, and its clinical significance remains to be further
explored.
Acknowledgments
We thank Yea-Lih Lin for providing the pSPII-Luc plasmid; Ling-Pai Ting
for providing pHBV3.6, pA3SP⌱-CAT, pA3BCP-CAT, and pA3CP-CAT;
and Sheng-Chung Lee for providing pCMV-C/EBP␣. We are grateful to
Ralph Kirby for helping revise the manuscript.
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IL-4 could bind to its receptor on Hep3B cells with high affinity,
leading to a suppression of HBV gene expression and replication.
The Hep3B cell line thus presents a suitable cell model for investigating the biochemical mechanisms underlying the effect of IL-4
on HBV expression in host cells.
Previously, IL-2, IFN-␣/␤, TNF-␣, and IFN-␥ have been shown
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mRNA and the HBsAg in Hep3B cells. It could also suppress the
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Hep3B cells transfected with pHBV3.6. The 3.5-kb precore
mRNA, which encodes the HBeAg, also appeared to be suppressed
by IL-4, since the production of HBeAg was decreased in IL-4treated Hep3B cells transfected with pHBV3.6. Presumably, IL-4
suppressed the production of the 3.5-kb pregenomic RNA, which
not only encoded the core and polymerase proteins, but also served
as a template for reverse transcription, and this resulted in the
inhibition of HBV replication. We further demonstrated that IL-4
could suppress the promoter activity of SPII and CP on HBV. In
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colocalized with the ENII that regulates the activities of all promoters. Thus, CURS/ENII is one of the most important regulatory
elements operating to control HBV gene transcription.
It has been demonstrated that IL-4 induced the expression of
E4BP4 in mouse B cells (50). In this study we showed that IL-4
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CP activity and HBV transcripts, as demonstrated by a series of
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suppressive effect of IL-4 on the 2.4/2.1-kb mRNA. E4BP4 has
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C/EBP␣ is a transcription factor found in the liver at higher
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C/EBP␣, we demonstrated that C/EBP␣ binding sites on CURS/
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The overexpression of C/EBP␣ also resulted in a 30% suppression
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Finally, the inhibitory effect of IL-4 on replication of HBV seems
to be more dramatic than its effect on RNA expression. Therefore,
other mechanisms may also contribute to the IL-4-mediated suppressive effect, for example, post-transcriptional regulation (51), an effect
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SUPPRESSION OF HBV BY IL-4