The line of HCV transgenic (Tg+) mice carrying a transgene that encodes the core, E1 and E2 proteins of type 1b HCV has been described previously (Kawamura et al., 1997
). The expression of the transgene is under the control of a liver-specific murine albumin promoter. This line of HCV transgenic mice was originally created in FVB background and bred to BALB/c mice to generate (FVB × BALB/c) F1 mice (H-2q/d
). Transgenic mice were typed by PCR as previously described (Kawamura et al., 1997
). Age- and sex-matched littermates negative for expression of HCV (Tg−) were used as a control.
Isolation and culture of murine hepatocytes
Murine hepatocytes were isolated by in situ perfusion via the portal vein with collagenase IV solution (Gibco BRL), according to standard techniques as described (Klaunig et al., 1981
). Cells were washed three times in RPMI supplemented with 10% FCS, 1% penicillin and streptomycin, and 2 mM glutamine before being plated into Primaria 24- or 6-well plates in William’s medium supplemented with insulin (10 μg/ml), EGF (50 ng/ml) and glucagon (10 μg/ml), as described (Enat et al., 1984
). Non-adherent cells, including contaminating lymphocytes, were removed by washing 4 h after plating, and the hepatocytes were then cultured for 24 to 48 h.
Activation of T cells
Splenic T cells were isolated from non-transgenic controls, and cultured in vitro for 24 h in the presence of 5 μg/ml Concanavalin A (Con A) in RPMI medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 μg/ml), HEPES buffer, and L-glutamine (2 mM). Gradient centrifugation was performed over Ficoll-Hypaque immediately before use to maximize the viability of activated T cells used in co-culture. Freshly isolated ex-vivo splenic T cells were used as resting T cell controls. In addition, cells were also activated by co-culture of splenic T cells from C57/B6 mice (H-2b) with non-transgenic splenocytes (H-2q/d) for 48–72 h.
Co-culture of activated T cells and primary cultured hepatocytes
Four million activated, unfractionated T cells were co-cultured with adherent HCV transgenic or non-transgenic hepatocytes. After incubation for indicated periods of time, lymphocytes were harvested by pipetting. Hepatocytes remain adherent after pipetting. After one wash in PBS, viable lymphocytes were counted using a hemacytometer. Trypan blue was used to exclude non-viable cells. In some experiments, co-cultures were performed by adding antibody, 10 μg/ml anti-FasL (Ab-1, Oncogene Research Products, Boston, MA), or the isotype control antibody, and then apoptosis was measured in CD4+ and CD8+ T cells.
Staining of apoptotic cells by annexin V
0.1–0.5 × 106 recovered lymphocytes were stained for 30 min in U-bottomed 96 plates with FITC-conjugated anti-CD4 (BD Pharmingen, San Diego, CA) or anti-CD8 monoclonal antibody (BD Pharmingen, San Diego, CA), dissolved in PBS containing 1% FCS and 0.1% sodium azide. Thirty minutes before FACS analysis, cells were then stained with PE-conjugated annexin V (BD Pharmingen, San Diego, CA) and propidium iodide (PI; BD Pharmingen, San Diego, CA), as suggested by the manufacturer. During FACS analysis, a live acquisition gate was placed on lymphocytes based on FSC/SSC profile. Side and forward angle light scattering was used to electronically gate the cells of choice and to exclude debris. Ten thousand events within the gate region were collected for each sample. Rare contaminating hepatocytes were excluded on the basis of FSC/SSC. The percentages of CD4+ and CD8+ T cells and the percentages of CD4+ and CD8+ T cells that were annexin V and/or PI positive were measured by FACScan using CellQuest software (BD, San Jose, CA). The absolute numbers of viable CD4+ and CD8+ T cells were calculated by multiplying the total number of viable lymphocytes recovered from individual co-cultures with the percentages of CD4+ and CD8+ T cells, respectively.
Adoptive transfer and in vivo activation
Splenocytes from non-Tg littermates were activated with conA as above. Forty-eight hours later, five to ten million CFSE-labeled cells were injected via the portal circulation into HCV Tg mice as described (Huang et al., 1994
). Non-Tg littermates were used as controls. Sixteen hours after injection, the animals were sacrificed, and liver and spleen lymphocytes were harvested. The degree of apoptosis of CFSE labeled cells was analyzed as described above. In some experiments, HCV Tg+ or non-Tg mice were treated with 20 μg/mouse of anti-FasL Ab (Ab-1, Oncogene Research) or the appropriate isotype control prior to adoptive transfer of cells.
Western blotting analysis
200 μg of cell lysates of freshly isolated transgenic and non-transgenic hepatocytes were electrophoresed in 10% SDS-PAGE and transferred to nitrocellulose membrane. Immunodetection was performed using the enhanced chemiluminescence technique (NEN Life Science Products, Boston, MA). The following specific antibodies were used in the study: polyclonal rabbit anti-mouse Fas ligand antibodies (Ab-1, Oncogene Research Products, Boston, MA), polyclonal anti-mouse β-Actin, polyclonal rabbit anti-serum to galectin-1 (gift of Dr. Linda Baum; University of California, Los Angeles, CA), and a monoclonal antibody against TRAIL (clone N2B2; gift of Dr. Hideo Yagita, Juntendo University, Japan). Mouse beta actin (clone C-11, Santa Cruz) was used as a control, and expression of FasL was normalized to beta actin. Densitometry was performed using the NIH Image software downloaded from the NIH web site.
RNA isolation and RT-PCR
Liver tissue from Tg+ or non-Tg littermates was harvested, and total RNA was isolated using the TriZol reagent (Invitrogen, Inc. Carlsbad, California) according to the manufacturer’s instructions. For RT-PCR, 2 μg of total RNA was treated for 15 min at room temperature with 2 U of amplification grade DNase I (Invitrogen, Inc. Carlsbad, California) to remove the genomic DNA. The cDNA was generated from 2 μg of total RNA using Ready-To-Go First-Strand Beads (Amersham Biosciences, Piscataway, NJ). RT-PCR amplifications of cDNA were carried out using the PTC-100 Thermal Controller (MJ Research, Inc. Waltham, MA) as follows: 5 min at 94 °C, 35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C extension, followed by 10 min at 72 °C. The PCR products were analyzed on 1.5% agarose gel, stained with ethidium bromide, and visualized with UV light. The sense primer for FasL was 5′-TCATCTTGGGCTCCTCCAGGGTCAG-3′, and the antisense primer was 5′-GGCTTTGGTTGGTGAACT-CACGGAG-3′ (nucleotides 185–483), which gave a 298-bp PCR product.
Flow cytometric analysis of FasL expression
For analysis of surface expression of FasL on hepatocytes, a total of 1 × 106 freshly isolated hepatocytes were stained with an optimal dilution of PE-anti-mouse Fas Ligand antibody (MFL3 clone, Pharmingen, San Diego, CA). After several washes with PBS, hepatocytes were incubated in blocking solution (PBS containing 5% normal mouse serum) at 4 °C for 30 min and then with PE-conjugated anti-FasL at 4 °C for 30 min. Fluorochrome-conjugated isotype-matched Abs were used as controls for non-specific binding. In all experiments, cells were taken from either Tg+ or non-Tg littermates as controls. Data were acquired on a FACScan flow cytometer. Data were analyzed using CellQuest software (BD Biosciences, San Jose, CA). Similar experiments were performed to analyze the expression of FasL and Fas on lymphocytes. In these experiments, lymphocytes were activated as above, then co-cultured with either Tg+ or non-Tg hepatocytes. FasL and Fas expressions were measured as above.
Following co-culture with either Tg+ or non-Tg hepatocytes, lymphocytes were collected, purified using Ficoll-Hypaque centrifugation, and the pellet was lysed by addition of lysis buffer. The lysates of 2 × 106 cells were incubated on ice for 10 min and centrifuged at 10,000 × g for 1 min. The supernatant was collected and used as cell lysate. The activity of caspase-3 was measured using caspase-3 colorimetric Assay kit (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer. Briefly, lysates were incubated with caspase-3 colorimetric substrate (DEVD-pNA) for 2 h at 37 °C in a 96-well flat bottom microplate. Upon cleavage of the substrate, the liberated chromophore pNA is spectrophotometrically detected. The pNA light emission was quantified using a microtiter plate reader at 405 nm. The level of caspase-3 enzymatic activity in the cell lysate is directly proportional to the color reaction.
The concentration of tumor necrosis factor α (TNF α) was measured using a TNF α ELISA kit (Endogen, Woburn, MA) according to manufacturer’s specifications.
Statistical analysis was performed using the StatView software (SAS Institute Inc., Cary, NC). Analysis was performed using student t test for parametric data and Mann–Whitney test for non-parametric, as appropriate, and Kruskal–Wallis for comparison of multiple groups.