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* Laboratoire de Biologie Cellulaire, UPRES 1833, Université Paris5;
Service de Chirurgie, Hôpital Cochin, APHP;
Laboratoire de Biochimie, Hôpital Cochin, APHP;
Département d’Hématologie, Institut Cochin, INSERM U567, Paris, France; and
|| Laboratoire de Biologie Cellulaire, INSERM U327, Faculté Xavier Bichat, Paris, France
1Correspondence: Service de Chirurgie, Hôpital Cochin, 75674 Paris Cedex 14, France. E-mail: filomena.conti{at}cch.aphp.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: liver transplantation • IL-4
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Liver cirrhosis due to hepatitis C virus (HCV) infection is currently the leading indication for adult LT (2
, 3)
. Persistence of HCV and recurrence of hepatitis C are the rule after LT (4)
, resulting in accelerated progression toward fibrosis (5
, 6)
.
The mechanisms underlying allograft rejection and accelerated progression of hepatitis C in this setting are poorly understood. Early events in both complications include inflammation and activation of T lymphocytes, which are essential to initiate and maintain the cell-mediated immune response (7
, 8)
. Interleukin-2 (IL-2) is involved in acute rejection (9)
and in the progression of liver damage during chronic hepatitis C (10)
. Current immunosuppressive protocols markedly reduce IL-2 production, whereas the production of other cytokines, such as IL-4, is less sensitive to their inhibitory effects (11
, 12)
. We have previously shown that IL-4 is overexpressed in liver grafts during acute and chronic rejection (13)
. We also recently reported that, in patients with severe recurrent hepatitis C, IL-4 is overexpressed in the graft and IL-4 production by peripheral mononuclear cells is enhanced, relative to patients with minimal recurrence and to HCV-negative recipients (14)
. IL-4 overexpression might thus be involved in the onset and progression of hepatic lesions in recipients receiving potent anti-IL-2 immunosuppressive therapy.
IL-4 is a cytokine that controls cell growth and regulates the immune system (15
, 16)
. IL-4 exerts its effects by binding to and stimulating its transmembrane receptor (IL-4R). In epithelial cells, IL-4R consists of an 
chain that binds IL-4 with high affinity and of a 
-chain shared by IL-13R (17
, 18)
. IL-4 induces tyrosine phosphorylation of IL-4R by receptor-associated Janus kinases [janus-activated kinase (JAK)]-1 and JAK-3 (19)
. This receptor-kinase complex then recruits and activates signal transducer and activator of transcription (STAT)-6 (20
, 21)
. Subsequently, tyrosine-phosphorylated STAT6 homodimerizes and translocates to the nucleus, where it binds distinct promoter regions on DNA and regulates specific gene transcription (22)
. IL-4 exerts anti-inflammatory effects by down-regulating T-helper 1 (Th1) cell activity (23)
and promotes T cell differentiation toward a Th2 phenotype (24)
. However, IL-4 can also induce apoptosis of certain cell types, including human endothelial cells (25)
and stimulated monocytes (26)
. In rats, IL-4 induces lethal hepatitis due to hepatocyte apoptosis (27)
, a phenomenon largely independent of immune cells present in the liver (28)
. Hepatocyte apoptosis is an important pathogenic mechanism in liver allograft rejection (29
, 30)
and also in chronic hepatitis C (31
, 32)
.
We therefore postulated that IL-4 could contribute to liver graft damage during allograft rejection, and during recurrent severe chronic hepatitis C after LT, by inducing hepatocyte apoptosis. Here we examined the proapoptotic effects of IL-4 on hepatocellular cell lines and on primary human hepatocytes and investigated the underlying mechanisms.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell lines
HepG2 cells (derived from a human hepatoblastoma) and Huh7 cells (derived from a hepatocellular carcinoma) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO) containing 10% FCS, 1% pyruvate (GIBCO), and 1% penicillin-streptomycin (GIBCO). Cells were plated in 6-well dishes (106 cells/well) and then, at 70% confluence, were treated with recombinant human IL-4 (R&D Systems Europe, Lille, France) at final concentrations of 10, 50, 100, and 500 ng/ml or with an agonistic anti-Fas antibody (0.5 µg/ml; CH11, Immunotech, Marseilles, France), or with recombinant IL-4 plus an anti-human IL-4 monoclonal antibody (mAb) (100 ng/ml; R&D Systems) for 24 or 48 h.
Isolation and primary culture of human hepatocytes
Normal liver tissue was obtained from patients undergoing partial hepatectomy for metastases or benign tumors. Dissociation was based on a two-step collagenase perfusion method (33)
. Visible vessels were perfused first with HEPES-EDTA buffer and then with HEPES containing 0.05% collagenase (GIBCO) and 5 mM CaCl2 at a flow rate of 10 ml/catheter/min (Masterflex peristaltic pump, Bioblock, France) for 30 min. Liver fragments were gently shaken to free loose liver cells that were then filtered and centrifuged. Hepatocytes were purified by Percoll density gradient centrifugation, and viability was determined by trypan blue dye exclusion. Hepatocytes were resuspended in Leibovitz L15 medium (GIBCO) with 100 IU/l insulin, 26 mM NaHCO3, 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. At the end of the attachment period (12–24 h), the medium was replaced with medium containing hydrocortisone (10–6 M). The medium was renewed every day. Hepatocytes were treated 4 days after isolation for 2, 4, 6, 12, or 24 h with anti-Fas antibody (CH11), recombinant human IL-4 (10, 50, 100, 500, and 1000 ng/ml),or human IL-4 plus an anti-human IL-4 mAb (100 ng/ml).
Reverse transcriptase-polymerase chain reaction for IL-4 mRNA detection
Total RNA was prepared with the RNeasy minikit (Qiagen, Courtaboeuf, France) according to the manufacturer’s recommendations. cDNA synthesis was carried out for 90 min at 42°C in a reaction mixture containing 2 U of RNase inhibitor (Promega, Charbonnieres, France), 3 U of avian myeloblastosis virus (AMV) reverse transcriptase (AMV-RT; Promega), 100 ng of specific reverse primers (Sigma-Genosys Ltd, Saint-Quentin Fallavier, France), and 2.5 mM DNTP (Promega) for 1 µg of total RNA. The cDNAs were stored at –20°C.
Amplification was achieved by adding 2 µl of cDNA to a polymerase chain reaction (PCR) mixture containing 2.5 U of TaqDNA polymerase (Promega), 2 mM MgCl2, 1 mM DNTP, and 100 ng of each primer and PCR buffer (Promega) in a final volume of 50 µl. PCR was run in a thermal cycler (Perkin-Elmer Cetus 480) for 35 cycles as follows: a denaturation step (94°C for 1 min), an annealing step (56°C for 45 s), and an elongation step (72°C for 1 min).
The following primers (Sigma-Genosys Ltd.), published elsewhere (34)
, were used: IL-4R sense: 5'-CTGGAGCACAACATGAAAAGG-3'; IL-4R antisense: 5'-AGTCAGGTTGTCTGGACTCT-3', yielding a PCR product of 510 bp. Each reverse-transcribed mRNA was internally controlled with the 28S ribosomal housekeeping gene (35)
for which the primers were designed with Oligo6 software (sense: 5'-TTGAAAATCCGGGGGAGAG3-'; antisense: 5'-ACATTGTTCCAACATGCCAG-'3) yielding a PCR product of 100 bp. PCR products were analyzed on 2% agarose gel containing ethidium bromide.
Immunohistochemical methods
Cells were resuspended in PBS (105 cells/ml) and then cytocentrifuged on Super Frost Plus slides (CML, Nemours, France); 5 µm sections of frozen normal liver were placed on the same type of slides, air-dried overnight at room temperature, fixed for 10 min in acetone, and stored at –20°C until immunohistochemical analysis.
IL-4R expression and STAT6 activation were evaluated with an indirect immunoenzymatic method using alkaline phosphatase/antialkaline phosphatase complexes as described previously (36)
. For intracellular staining, cells were first permeabilized with 0.1% Triton X-100. Anti-human-IL-4R mAb (R&D Systems, dilution 1/10), anti-P-STAT6 polyclonal antibody (pAb; dilution 1/50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-rabbit immunoglobulin (only for polyclonal primary antibody; DAKO, Golstrup, Denmark), and then rabbit anti-mouse immunoglobulin (DAKO) and alkaline phosphatase/antialkaline phosphatase complexes (DAKO) were used. Alkaline phosphatase activity was revealed for 20 min in fast-red TR (1 mg/ml) and naphthol-phosphate (0.2 mg/ml) solutions (Sigma) containing levamisole (0.24 g/ml; Sigma). The slides were counterstained with Harris hematoxylin. In negative controls, the primary antibody was omitted or replaced by an irrelevant antibody at the same dilution. PBMC cytospin slides were used as positive controls.
Electrophoretic mobility shift assay (EMSA)
EMSA, which requires a large number of cells, was only performed on HepG2 cells. After incubation with human IL-4 for 30 min, HepG2 cells were resuspended and incubated in buffer A: 20 mM HEPES pH 7.9, 10 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF, containing a protease inhibitor cocktail plus 0.2% Nonidet P-40 (Roche Diagnostics, Meylan, France) for 5 min on ice and were then centrifuged, as described previously (37)
. The pellet (nuclear extract) was incubated for 30 min on ice in buffer B (20 mM HEPES pH 7.9, 420 mM NaCl, 1 mM EDTA, 10 mM KCl, 1 mM DTT, 1 mM PMSF, and 20% glycerol, containing a protease inhibitor cocktail) and was then clarified. The protein concentration of nuclear extracts was measured with BCA kits (Pierce Biotechnology, Brebieres, France). Then, 10 µg of protein were incubated with 16 pmol of 32P-labeled beta-casein promoter-derived probe (5'-AGATTTCTAGGATTCAAATC-3') and separated on 6% nondenaturing polyacrylamide gel. Supershift assays were performed by incubating the nuclear extracts with an anti-human STAT6 pAb (S-20, Santa Cruz Biotechnology) for 30 min with the probe (38)
.
Inhibition of STAT6 by STAT6 small interfering RNA transfection of cultured cells
STAT6 was silenced by using the STAT6 SMARTpool small interfering RNA (siRNA) reagent (Euromedex, Mudolsheim, France). This experiment used four siRNA sequences targeting human STAT6. HepG2 cells or human hepatocytes (5x105 per well) in 6-well plates were incubated overnight in normal DMEM medium supplemented with 10% FCS to reach 50–70% confluency. Cells were transfected with 100 nM siRNA in the presence of 10 µl of Lipofectamine 2000 (Invitrogen SARL, Cergy Pontoise, France) and 1 ml of FCS-free growth medium; 6 h after transfection, 1 ml of DMEM medium with 10% of FCS was added to each well (39)
. Then, 48 h after transfection, cells were incubated in fresh medium and stimulated with human IL-4 (50 ng/ml) for 30 min before studying STAT6 activation or for 48 h (HepG2 cells) and 24 h (human hepatocytes) before apoptosis was studied.
Measurement of STAT6 activation by flow cytometry
Cells were stimulated with human recombinant IL-4 for 30 min. After being washed, cells were first permeabilized with FACS permeabilizing solution. STAT6 phosphorylation at tyrosine 641 was assessed by staining with a mouse anti-human phosphorylated STAT6 antibody (dilution 1/50; BD Biosciences, Pharmingen, Le Pont De Claix, France) followed by FITC-labeled goat anti-mouse immunoglobulin (dilution 1/20; Caltag, Burlingame, CA, USA). Fluorochrome-conjugated irrelevant isotype (mouse IgG) was used as a negative control. Cell staining was then analyzed with a FACSCalibur flow cytometer (BD Biosciences) and Cellquest software.
Annexin V and propidium iodide staining
Annexin V-FITC was used to detect and quantify early-stage apoptosis, and propidium iodide (PI) was to detect necrosis with the Annexin V-FITC kit (Immunotech, Marseille, France), following the manufacturer’s recommendations. Cells were suspended in binding buffer containing Ca+2 and incubated with 1 µg/ml Annexin V-FTIC and 1 µg/ml PI. Apoptosis and necrosis were measured with a FACSCalibur and Cellquest software. A total of 5000 cells was acquired and analyzed in each sample. FITC-positive and PI-negative cells were considered apoptotic, whereas PI-positive cells were considered necrotic and unstained cells were considered normal viable cells.
TUNEL assay
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) technique was used to detect DNA fragmentation on cytospun slides from the same samples as those used for immunohistochemical experiments. The in situ cell death detection-activating protein (AP) kit was used according to the manufacturer’s instructions (Boehringer, Mannheim, Germany). Slides were incubated with an end-labeling mixture containing TdT and fluorescein-labeled nucleotides for 60 min and then with an antifluorescein antibody conjugated with alkaline phosphatase for 30 min. Alkaline phosphatase activity was revealed in fast-red TR and naphthol-phosphate solution containing levamisole. The slides were counterstained with hematoxylin. A negative control without TdT was included in each procedure.
Changes in mitochondrial transmembrane potential DiOC6
Mitochondrial transmembrane potential was evaluated with the lipophilic cation DiOC6 (3
; 3,3'-dihexyloxacarbocyanine iodide). Cells were incubated for 15 min with 40 nM DiOC6 (3
; Molecular Probes, Leiden, Netherlands) and 1 µg/ml PI (Immunotech) at room temperature (40)
. Acquisition and analysis of 5000 cells were performed on a FACSCalibur with Cellquest software. The population highly fluorescent for DiOC6 (3)
was considered to represent viable cells with unaltered mitochondrial transmembrane potential, and the dull population was considered to represent apoptotic cells with decreased mitochondrial transmembrane potential.
Caspase-3, -8, and -9 activity assay
Caspase-3, -8, and -9 activities were evaluated with commercial kits (VWR International, France) containing specific substrates, following the manufacturer’s recommendations. Cells were resuspended in 1 µM Phiphilux G1D2 substrate solution (containing the sequence of the caspase 3 cleavage site: GDEVDGI) or 1 µl of FITC-IETD-FMK (caspase 8 substrate) or 1 µl of FITC-LEHD-FMK (caspase 9 substrate) for 1 h. Then, cells were washed and fluorescence of 5000 cells was measured on a FacsCalibur.
Preparation of cytosolic and mitochondrial extracts and Western blot analysis
Protein was isolated from cytosolic and mitochondrial extracts of 1 x 107 HepG2 cells as described previously (41)
for Bid and Bax detection. This experiment was not done on human hepatocytes, because of the large number of cells required. After incubation with IL-4, adherent and nonadherent cells were removed and centrifuged, and the pellets were kept at –80°C until use. Briefly, cell pellets were resuspended and homogenized in buffer A (70 mM sucrose, 210 mM mannitol, 2 mM HEPES, and 2 mM EDTA, pH 7.4) with a Wheaten Dounce glass homogenizer and were centrifuged for 10 min at 500 g. The supernatants were further centrifuged at 10,000 g for 30 min, and the supernatants (cytosol fraction) were collected. The pellet (mitochondria) was washed again in buffer A by centrifugation at 600 g for 5 min to remove any contaminating particles and was then recovered by centrifugation at 12 000 g for 15 min. The isolated mitochondria were resuspended in buffer A. The protein concentrations of the cytosolic and mitochondrial fractions were assayed with a BCA kit (Pierce Biotechnology).
The mitochondrial and cytosolic fractions were screened for Bid and Bax. Equal amounts of the fractions were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Free sites were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20. Bid and Bax were detected by using either a rabbit anti-human Bid pAb (Santa Cruz Biotechnology) or a rabbit anti-human Bax pAb (BD Biosciences) both at 1:1000 dilution. For signal detection, blots were incubated with peroxidase-labeled goat anti-rabbit immunoglobulin. Enhanced chemiluminescence reagents were used according to the manufacturer’s instructions (Pierce Perbio Science, Brebieres, France) and blots were exposed to Kodak X-oMAt UV film. An anti-ß-actin antibody (Sigma) and an antiporine antibody (Santa Cruz Biotechnology) were used to control the purity of the cytosolic and mitochondrial fractions, respectively.
Expression of Fas (CD95) and Fas ligand (CD95L)
CD95 and CD95L mRNAs were detected by reverse transcriptase (RT)-PCR as described previously (42)
. For CD95 DNA (246 bp), the sense primer was 5'-CACTATTGCTGGAGTTCAT-3' and the antisense primer 5' CTGAGTCACTAGTAATGTCC-3'. PCR was performed for a total of 35 cycles at 95°C for 30 s, 55°C for 45 s, and 72°C for 1 min. The primers used for CD95L (520 bp) were as follows: sense 5'- ATAGGATCCATGTTTCTGCTCTTCCACCTACAGAAGGA-3' and antisense 5'-ATAGAATTCTGACCAAGAGAGGAGCTCAGATACGTTGAC-3', using 35 cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min (43)
. PCR products were analyzed on 2% agarose gels after ethidium bromide staining; the expected molecular masses were 246 bp for CD95 and 520 bp for CD95L.
The protein expression of Fas and Fas ligand on HepG2 cells and human hepatocytes, in the presence and absence of IL-4 alone or combined with a Fas antagonist (ZB4) (Immunothech, Marseille, France), was assessed by flow cytometry, with or without cell permeabilization with FACS permeabilizing solution, after being stained with a mouse anti-human Fas-L antibody or a mouse anti-human Fas antibody (BD Biosciences), followed by incubation with FITC-labeled goat anti-mouse immunoglobulin (Caltag, Burlingame, CA, USA). A total of 5000 cells was acquired with a FACSCalibur in each sample.
Statistical analysis
All data are mean ± SD. The significance of differences between the different groups was determined by one-way ANOVA and by the Mann-Witney test; P values of <0.05 were considered statistically significant. Statvies IV software was used (Abacus Concepts, Berkeley, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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Immunohistochemical results
Some PBMC (positive control) showed intense red staining characteristic of IL-4R (data not shown), while negative controls (omitting the primary antibody or using an irrelevant antibody) were consistently negative (Fig. 2
A). All the study cells (HuH7 cells, HepG2 cells, and primary human hepatocytes) expressed IL-4R (Fig. 2A
). In normal liver sections, many cell types expressed IL-4R, including hepatocytes, sinusoidal cells, and infiltrating cells (Fig. 2A
).
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IL-4 activation of STAT6 in vitro
Results of immunohistochemistry and flow cytometry
The capacity of IL-4 to induce STAT6 activation was analyzed by immunohistochemical and flow cytometric detection of P-STAT6 with two anti-P-STAT6-specific antibodies. By immunohistochemical staining, HepG2 cells and human hepatocytes treated with IL-4 (50 ng/ml) for 30 min expressed P-STAT6 (Fig. 2B
), whereas untreated cells were consistently negative (Fig. 2B
). P-STAT6 was detected in both the cytoplasm and the nucleus. STAT6 activation by IL-4 was confirmed by the detection of P-STAT6 by flow cytometry (10 experiments were done with each cell type): higher percentages of cells expressed P-STAT6 among IL-4-treated cells (31.9±4.4% of HepG2 cells and 34.8±3.3% of human hepatocytes) than among untreated cells (2.2±0.6% and 1.9±0.7%, respectively, P<0.0001 and P<0.0001, Fig. 3
A, B).
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EMSA results
To determine whether IL-4-induced activation was related to nuclear translocation of STAT6, we tested the ability of nuclear proteins to bind the STAT6 consensus DNA binding sequence in an EMSA with a STAT6-responsive element from the beta-casein promoter as probe. P-STAT6 was not detected in nuclear extracts from HepG2 cells cultured without IL-4. In contrast, after incubation with IL-4 (10, 50, 100, or 500 ng/ml for 30 min), P-STAT6 was detected in nuclear extracts from HepG2 cells and formed a complex with the probe (Fig. 3c
). This complex was entirely supershifted by incubation with the anti-STAT6 antibody, indicating the presence of active and functional STAT6 in the DNA-bound complex. Maximal expression of nuclear P-STAT6 was obtained at a final IL-4 concentration of 50 ng/ml and with an incubation period of 30 min.
Inhibition of STAT6 activation by anti-IL4 and STAT6 siRNA
First, STAT6 activation was investigated by flow cytometric detection of P-STAT6 on cells treated with human IL-4 (50 ng/ml) with or without anti-human IL-4 (100 ng/ml). IL-4 stimulation significantly increased the percentage of cells showing STAT6 activation and anti-human IL-4 inhibited STAT6 activation (5.1±0.91 and 5.7±1.8% in HepG2 cells and human hepatocytes, P<0.0001 vs. IL-4-stimulated cells, NS vs. unstimulated cells; 10 experiments were done with each cell type; Fig. 3A
, B).
Second, HepG2 cells and human hepatocytes were stimulated with human IL-4 after STAT6 siRNA transfection before P-STAT6 expression was examined by flow cytometry. STAT6 activation by IL-4 was significantly lower in transfected HepG2 cells (7.2±1.8%) and in transfected human hepatocytes (5.8±0.3%; Fig. 4
A, B) than in nontransfected cells (P<0.0001 and P<0.0001, respectively).
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IL-4-induced apoptosis of human hepatic cells
Study cells (Huh7, HepG2, and primary human hepatocytes) were treated with the anti-Fas antibody CH11 (0.5 µg/ml), which has previously been shown to induce apoptosis of HepG2 and Hep3B cells (44
, 45)
. Cells were cultured without stimulation, with anti-Fas (0.5 µg/ml), or with IL-4 at a final concentration of 10–500 ng/ml for 4–48 h, and then apoptosis was evaluated by flow cytometry using annexin V-PI staining. Fifteen annexin V-PI binding experiments were done with each cell type.
HuH7 cell viability was unaffected by the different treatments and incubation times (data not shown), confirming the resistance of this cell line to apoptosis (45)
.
Apoptosis in untreated HepG2 cells reached 3.1 ± 0.7% (% of annexin V+/PI- cells) after 48 h. Anti-Fas strongly enhanced apoptosis (47.9±7.9% after 48 h, P<0.001 vs. untreated cells; Fig. 4A
). IL-4 also increased apoptosis (Fig. 4A
), which reached 17.6 ± 4.1, 15.4 ± 1.1, and 18.2 ± 2.5%, at final IL-4 concentrations of 50, 100, and 500 ng/ml, respectively (P=0.009, 0.01, and 0.008 vs. untreated cells), after 48 h of incubation.
Untreated human hepatocytes showed minimal apoptosis (4.4±0.7%) after 24 h of culture. Anti-Fas significantly enhanced apoptosis (29.0±3.4%, P<0.001 vs. untreated hepatocytes) after 24 h of incubation (Fig. 4A
). IL-4 induced apoptosis in a time- and concentration-dependent manner (Fig. 4B
, C). After 24 h, apoptosis reached 24.4 ± 4.9, 27.7 ± 3.6, and 29.9 ± 5.4%, respectively, at IL-4 concentrations of 50, 100, and 500 ng/ml (P=0.0002, P<0.0001, and P<0.0001, respectively, vs. untreated cells; Fig. 4B
).
To confirm these results, the TUNEL assay was applied to cytospun cells from the same experiments. The results were similar to those obtained with annexin V staining. The number of TUNEL-positive cells was minimal in untreated HepG2 cells and hepatocytes (Fig. 2C
) and increased after anti-Fas and human IL-4 treatment (Fig. 2C
). Staining was nuclear, and chromatin condensation was observed in some cells.
Inhibition of IL-4-induced apoptosis by anti-human IL-4 and STAT6 siRNA
The effect of anti-human IL-4 and STAT6 siRNA on IL-4-induced apoptosis was evaluated in terms of annexin V–PI staining. In HepG2 cells, anti-human IL-4 (100 ng/ml for 48 h) inhibited apoptosis induced by 50 ng/ml IL-4 (3.2±0.7 vs. 17.6±4.1% in HepG2 cells treated with IL-4 alone, P=0.004; Fig. 4A
). In primary human hepatocytes, anti-human IL-4 (100 ng/ml for 24 h) also inhibited apoptosis induced by IL-4 (3.4±1.4 vs. 24.4±4.9% in hepatocytes treated with IL-4 alone, P=0.0005; Fig. 4A
). To investigate the role of STAT6 activation in IL-4-induced apoptosis, HepG2 cells and human hepatocytes were transfected with STAT6 siRNA before treatment with human IL-4 for 48 and 24 h, respectively. Transfection inhibited IL-4-induced apoptosis of HepG2 cells (5.4±1.8%) and human hepatocytes (4.0±1.3%; P=0.004 and P=0.0007, respectively, vs. nontransfected cells).
Mechanisms of IL-4-induced apoptosis
IL-4-induced apoptosis involves the mitochondrial pathway
Induction of apoptosis in many systems has been related to initiation of mitochondrial damage, including a reduction or loss of the mitochondrial membrane potential, which may be an early signaling event (46)
. To determine if the mitochondrial transmembrane potential was altered early during IL-4-induced apoptosis, leakage of DiOC6 fluorescent probe, reflecting the loss of mitochondrial transmembrane potential (47)
, was measured in HepG2 cells and human hepatocytes during apoptosis induced by anti-Fas antibody (positive control) and by IL-4 (10 experiments were done with each cell type).
The number of DiOC6-negative HepG2 cells was minimal (4.5±0.9%) in untreated cells but increased (25.9±4.5%) after incubation with anti-Fas for 48 h (P<0.0001 vs. untreated cells; Fig. 5
A). The percentage of depolarized cells reached 13.1 ± 1.5, 18.8 ± 3.8, and 22.1 ± 4.3% after 48 h of incubation with 50, 100, and 500 ng/ml IL-4, respectively (P=0.04, P=0.001, and P=0.005, vs. untreated cells).
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In human hepatocytes, DiOC6-negative cells represented 3.0 ± 0.7% of untreated cells and reached 29.8 ± 5.0% after 24 h of anti-Fas treatment (P<0.001 vs. untreated cells; Fig. 5A
) and 17.0 ± 3.8, 24.8 ± 4.2, and 26.1 ± 6.7%, respectively, after 24 h of treatment with 50, 100, and 500 ng/ml IL-4 (P=0.001, P<0.001, and P<0.001, vs. untreated cells, respectively; Fig. 5A
).
IL-4-induced apoptosis results from caspase-3, -8, and -9 activation
We then examined whether IL-4-induced apoptosis involved the activation of upstream caspase-3, caspase-8, and/or effector caspase-9. The ability of HepG2 cells treated with IL-4 (50 ng/ml for 48 h) to cleave fluorigenic caspase-3, -8, or -9 substrates was evaluated (10 experiments were done each cell type). The percentages of cells showing caspase activation were 14.9 ± 3.5, 18.7 ± 3.9, and 32.7 ± 3.3% (vs. 2.8±0.4, 4.6±0.3, and 4.5±1.2%, respectively, in untreated HepG2 cells, P=0.005, P=0.04, and P=0.02; Fig. 5B, C, D
). The mean percentages of HepG2 cells able to cleave fluoregenic caspase-3, -8, and -9 after treatment with anti-Fas were 19.4 ± 0.4, 30.6 ± 10.0, and 48.1 ± 7.1%, respectively (P=0.001, P<0.001, and P<0.001 vs. untreated cells; Fig. 5B, C, D
).
In human hepatocytes, caspase-3, -8, and -9 activities were enhanced after 24 h of incubation with IL-4: 16.9 ± 3.7% for caspase-3 (P<0.001 vs. untreated cells), 12.7 ± 1.1% for caspase-8 (P=0.002 vs. untreated cells), and 15.9 ± 1.3% for caspase-9 (P=0.008 vs. untreated cells, Fig. 5B, C, D
). Caspase-3, -8, and -9 activities in cells treated with anti-Fas 0.5 µg/ml were 18.1 ± 1.9, 12.8 ± 1.9, and 19.7 ± 4.9%, respectively, compared with 3.4 ± 1.2, 3.1 ± 0.3, and 2.3 ± 0.7% in untreated hepatocytes (P<0.001, P=0.001, and P=0.008; Fig. 5B, C, D
).
We also investigated the role of the STAT6 pathway in caspase 3 activation by means of flow cytometry. Incubation of HepG2 cells and human hepatocytes with IL-4 plus STAT6 siRNA drastically inhibited the capase 3 activation induced by IL-4 (3.2±1.3%).
IL-4-induced apoptosis involves Bid cleavage and Bax mitochondrial translocation
The roles of Bid and Bax activation and Bax mitochondrial translocation in IL-4-induced apoptosis were investigated by Western blot analysis of HepG2 cells. In untreated cells, Bid was detected as a 22 kDa protein (Fig. 6
). After IL-4 treatment, a smaller cleaved fragment of 15 kDa appeared (Fig. 6)
. When cells were treated with IL-4 plus anti-IL-4 or with IL-4 plus STAT6 siRNA, Bid was not cleaved but was detected, as in untreated cells, as a 22 kDa protein (Fig. 6)
. IL-4 treatment enhanced Bax expression in mitochondria (Fig. 6)
, an effect reversed by the addition of anti-IL-4 or STAT6 siRNA (Fig. 6)
. These results suggest that IL-4 induces Bid activation and cleavage and promotes Bax translocation to mitochondria, effects inhibited by anti-IL-4 antibody and by STAT6 si RNA.
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IL-4 neither up-regulates Fas expression nor induces Fas ligand expression
As caspase 8 activity was increased during IL-4-induced apoptosis, we investigated the possible involvement of the Fas signaling pathway. Fas mRNA expression was not enhanced in HepG2 cells or human hepatocytes after incubation with IL-4 at various concentrations (Fig. 7
). Fas ligand was detected in stimulated PBMC (positive control) but not in hepatocellular cells (Fig. 7)
.
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Fas and FasL protein expression was analyzed by means of flow cytometry, with or without cell membrane permeabilization, to evaluate membrane and intracellular expression of these molecules. The results were similar with and without permeabilization and were compatible with those obtained by RT-PCR. They showed that Fas was expressed by HepG2 cells (40.4±3.5% of cells) and by human hepatocytes (47.8±8.3% of cells), whereas FasL was not substantially expressed by these hepatocellular cells. The expression of Fas and FasL was not influenced by IL-4 incubation.
To further investigate the possible role of Fas in IL-4-induced apoptosis, we examined the effect of Fas antagonist ZB4. No reduction in IL-4-induced apoptosis was observed (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, mast cells (48)
, and stimulated monocytes (26)
, whereas it is antiapoptotic for B cells (49)
. In rat models, IL-4 can induce lethal hepatitis mediated by hepatocyte apoptosis (27
, 28)
. We have previously shown that increased IL-4 production by peripheral blood mononuclear cells correlates with increased IL-4 expression in the graft during allograft rejection and severe recurrent hepatitis C in liver transplant recipients (13)
. This IL-4 overexpression in patients receiving IL-2-inhibiting immnosuppressive therapy could play a role in liver injury by inducing hepatocyte apoptosis. Indeed, hepatocyte apoptosis appears to be a key contributor to liver injury in a wide range of acute and chronic liver diseases (50)
.
Our results show for the first time that human recombinant IL-4 induces apoptosis of HepG2 cells and primary human hepatocytes; in contrast, Huh7 cells, which are resistant to apoptosis (45)
, were also resistant to apoptosis induced by IL-4. We also show that HepG2 cells and human hepatocytes express IL-4R. In our hands, IL-4 did not induce apoptosis of biliary cells, which do not express IL-4R (data not shown). The IL-4 concentrations used here were similar to those used in previous in vitro studies (50 ng/ml; refs. 27
, 51
), although no information is available on IL-4 concentrations in liver or serum after liver transplantation. In a model of rat hepatitis induced by IL-4, the IL-4 serum concentration reached 1800 pg/ml (52)
. In HIV-Trypanosoma cruzi coinfection, IL-4 serum levels reached 6200 pg/ml (53)
. Intrahepatic concentrations are unknown.
IL-4 acts by binding and stimulating its specific receptor IL-4R (54)
. Binding of IL-4 to IL-4R triggers phosphorylation of JAK-1 and JAK-3 (55)
, leading to the activation of the major IL-4 signaling pathway known as STAT-6 (18)
. Tyrosine-phosphorylated STAT-6 forms homodimers and translocates to the nucleus, where it binds IL-4-responsive elements and induces gene transcription (21)
. Our results show that IL-4 interaction with its receptor activates the STAT6 signaling pathway in HepG2 cells and in human hepatocytes. STAT6 phosphorylation was detected after 30 min of incubation with IL-4, and STAT6 nuclear translocation was detected with a supershift assay. One major finding of this study is that the STAT-6 signaling pathway is required for IL-4-mediated apoptosis, as blockade of STAT6 activation by STAT6 siRNA and by anti-IL-4 inhibited IL-4-mediated apoptosis.
We then investigated the pathways connecting STAT6 activation with the initiation of hepatocyte apoptosis. Maintenance of mitochondrial membrane potential is critical for hepatocyte survival, and its reduction is an early event in apoptosis (40)
. In our hands, an agonistic anti-Fas antibody and IL-4 generated a similar decrease in mitochondrial membrane potential. Caspases play a major role in the initiation and execution of apoptosis induced by a variety of stimuli (56
, 57)
, and we found that caspase-3, -8, and -9 were activated during both IL-4-induced and anti-Fas-induced apoptosis of cultured hepatocytes.
As mitochondria and caspase-3, -8, and –9 were similarly involved in apoptosis induced by IL-4 and anti-Fas, we investigated whether the Fas/FasL pathway was required for IL-4-induced hepatocyte apoptosis. No change in Fas or Fas ligand expression was observed in HepG2 cells or hepatocytes cultured with IL-4, indicating that IL-4-induced apoptosis occurs independently of this pathway. Moreover, blockade of the Fas/FasL pathway by an antagonistic anti-Fas antibody failed to inhibit IL-4-induced apoptosis. Of note, in the rat model of IL-4-induced lethal hepatitis, apoptosis is Fas-FasL-independent, suggesting a similar mechanism (28)
.
IL-4R intracellular segments contain distinct domains responsive for IL-4 signal transduction, but they do not contain death domains, such as Fas, present in some receptors, that directly transduce apoptotic signals (28)
. Apoptosis induced by IL-4 may thus be mediated by activation of STAT6, which is a key regulator of IL-4-promoted gene transcription. In this study, activation of the STAT6 pathway, directly or indirectly, induced the activation of caspase-8 and/or of Bid and Bax, resulting in mitochondrial changes. Bid and Bax are cytosolic proteins that can themselves mediate mitochondrial changes. Their activation (especially that of Bid) can be triggered by many stimuli, including activated caspase-8 (58)
. Cleaved Bid interacts with Bax to trigger a change in the Bax conformation, leading to dimerization (or oligomerization) and integration into the outer mitochondrial membrane; this forms a channel allowing cytochrome c to escape into the cytosol, in turn activating caspases by binding to Apaf-1 (59
, 60)
. Thus, activated STAT6 might promote the gene transcription of proapoptotic proteins or another way that can activate proteins such as Bax and Bid, which are critical for apoptosis.
In conclusion, this study is the first to show that IL-4 has a potent proapoptotic action on human hepatocytes. This effect requires IL4-R binding and STAT6 activation, which are followed by a decrease in mitochondrial membrane potential and by caspase activation. These findings may have therapeutic implications: drugs able to inhibit STAT6, or immunosuppressive drugs with anti-IL4 activity, might be beneficial in liver transplant recipients undergoing rejection or recurrent HCV infection. High IL-4 producers might represent a subset of liver transplant recipients warranting more intensive management.
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ACKNOWLEDGMENTS |
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Received for publication May 17, 2006. Accepted for publication December 6, 2006.
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REFERENCES |
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stimulated human epithelial cells. FEBS Lett. 579,173-178[CrossRef][Medline]
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