1
Supporting Materials and Methods
Suppression of innate immunity (natural killer cell/interferon-) in the
advanced stages of liver fibrosis in mice
Won-Il Jeong,1,4 Ogyi Park,4 Yang-Gun Suh,1 Jin-Seok Byun,1 So-Young Park,1 Earl
Choi,1 Ja-Kyung Kim,2 Hyojin Ko,3 Hua Wang,4 Andrew M. Miller,4 and Bin Gao4
Animals. C57BL/6J and IFN-γ-/- mice on a C57BL/6 background were purchased from
the Jackson Laboratory (Bar Harbor, ME). IFN-γ-/-SOCS1-/- mice were kindly provided
by Dr. James Ihle, St. Jude Children’s Research Hospital (Memphis, TN). All mice used
in the present study were housed in a specific pathogen–free facility and were cared for in
accordance with National Institutes of Health (NIH) guidelines.
Materials. Polyinosinic-polycytidylic acid (Poly I:C), Gey’s balanced salt solution,
OptiPrep, Carbon tetrachloride (CCl4), Collagenase type I , 4-methylpyrazole (4-MP),
CD437, retinol, all trans and 9-cis retinaldehyde (Rald) or retinoic acid (RA) were
purchased from Sigma (St. Louis, MO). Methoprene acid (MA) was purchased from
BIOMOL Research Laboratories Inc (Plymouth Meeting, PA). Recombinant murine IFNγ was obtained from R&D Systems Inc (Minneapolis, MN) (the activity of IFN-γ is 8.4
IU/ng)
Serum
cytokines,
histology,
immunohistochemistry,
immunocytochemistry,
determination of hepatic hydroxyproline content. Serum levels of IFN-γ were measured
with Cytometric Bead Array (CBA) Method (BD Biosciences, Mountain View, CA).
After routine processing of fixed liver tissue or fixing cultured HSCs with 4%
paraformaldehyde, immunostaining was performed using antibodies of alpha-smooth
muscle actin (α-SMA) (Dako, Carpinteria, CA) and
phospho-STAT1 (pSTAT1) (Cell
signaling, Danvers, MA), and ABC kit (Vector laboratories, Burlingame, CA). DAB
2
(Zymed, San Francisco, CA) was used as the chromagen/substrate. Hepatic
hydroxyproline was also measured as described previously.1, 2
Flow cytometric analysis of NK cells and NKG2D expression. NKG2D expression on
NK cells were determined by using anti-NK1.1, anti-CD3 (BD PharMingen, San Diego,
CA), and anti-NKG2D antibodies (eBioscience, San Diego, CA) by FACSCalibur (BD
Biosciences, Mountain View, CA).
Reverse transcriptase-polymerase chain reaction (RT-PCR). The RT-PCR was carried
out and the primers were used as described previously.3, 4
Isolation and culture of hepatic stellate cells, cytotoxicity assay and cell proliferation.
Mouse liver HSCs were isolated via in situ collagenase perfusion and differential
centrifugation on Optiprep (Sigma) density gradients, as previously described.1, 2 The
isolated HSCs were resuspended in RPMI 1640 medium containing 0.5 % or 20% fetal
bovine serum with or without 5mM 4-MP, 1 μM all trans and 9-cis of Rald or RA, 1 μM
of CD437 and MA and then plated onto 24-well plates at a density of 1 × 104 cells per
well or onto 6-well plates at a density of 1 × 105 cells per well. On several time points,
some HSCs were collected for RT-PCR. On day 3 or 7, HSCs were cultured in serumfree medium for an additional 24 h. These cells were designated as D4 or D8 HSCs.
These HSCs were then treated with IFN-γ for 30 min for immunohistochemistry
experiments and Western blot or 24 h for cell proliferation assays. Cell proliferation and
cytotoxicity assay were performed as described previously.1, 2
Isolation of NK cells, Cytotoxicity assay, Co-culturing and antibody treatment of
NKG2D and TGF-β. NK cells were isolated from mouse livers as described1, 2 and used
as effector cells. HSCs isolated from mouse liver and Yac-1 cells, a murine T-lymphoma
cell line sensitive to NK-cells, were used as target cells.5 Cytotoxicity assay of NK cells
against HSCs and Yac-1 with or without treatment of TGF-β antibody was described
previously.2 Cultured D0-8 HSCs and Yac-1 cells were used as target cells and
cytotoxicity was measured using Calcein-AM release (target cells/effector cells = 1/5 or
3
1/10). For the measurement of IFN-γ in supernatant, NK cells were co-cultured with D08 HSCs with or without treatment of NKG2D antibody for 24 hrs in serum free medium.3
Western Blot. Liver tissue or isolated HSCs were homogenized in lysis buffer (30
mmol/L Tris, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L phenylmethylsulfonyl
fluoride, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, phosphotase
and protease inhibitors). Western blot analyses were performed with 40-80μg protein
from liver or isolated HSCs homogenates using anti-α-smooth muscle actin (SMA)
(1:1000 dilution, Sigma), anti-TGF-ß1, anti-phospho-STAT1, anti-STAT1 (1:1000
dilution, Cell signaling technology) and SOCS1 (1:200 dilution, US Biological).
Measurement of retinoids with HPLC. Cells were quantified and extracted as described.6
HPLC measurements were performed using a Hewlett–Packard 1100 HPLC equipped
with a Zorbax Eclipse 5 μm XDB-C18 analytical column (250 X 4.6 mm; Agilent
Technologies Inc, Palo Alto, CA). A linear gradient solvent system: 5% acetic acid
aqueous solution/MeOH from 55:45 to 35:65 in 40 min; the flow rate was 1 mL/min.
Peaks were detected by UV absorption (330 nm for retinol and 360 nm for the
others) with a diode array detector. Acitretin was used as internal standard. All standard
reagents and solvents were purchased from Sigma. LC-MS measurements were
performed for double checking of compounds on micromass/Waters LCT Premier
Electrospray Time of Flight mass spectrometer coupled with a Waters HPLC system.
4
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