IFN-gamma/STAT1 acts as a proinflammatory signal in T cell-mediated hepatitis via induction of...

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IFN-γ/STAT1 Acts as A Pro-inflammatory Signal in T Cell-mediated

Hepatitis via Induction of Multiple Chemokines and Adhesion

Molecules: A Critical Role of IRF-1

Barbara Jaruga, Feng Hong, Won-Ho Kim, Bin Gao

Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on

Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892

Running title: Essential role of STAT1 in hepatitis

Corresponding author: Dr. Bin Gao, Section on Liver Biology, NIAAA/NIH, Park Bldg

Rm 120, 12420 Parklawn Drive, MSC 8115, Bethesda, MD 20892. Tel: 301-443-3998;

Email:bgao@mail.nih.gov

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (July 8, 2004).10.1152/ajpgi.00184.2004

Copyright © 2004 by the American Physiological Society.

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Abbreviations: Con A, Concanavalin A; JAK-STATs, Janus kinase-signal transducers

and activators of transcription; IFN-γ, interferon-γ; NKT, natural killer T; RT-PCR,

reverse transcriptase-polymerase chain reaction; MNC, mononuclear cells; PMN,

polymorphonuclear cells; EPO, eosinophil peroxidase; MPO, neutrophil

myeloperoxidase; IP-10, IFN-inducible protein 10; Mig, monokine induced by IFN; I-

TAC, IFN-inducible T cell α chemoattractant; ICAM-1, intercellular adhesion molecule-

1; VACM-1, vascular cell adhesion molecule 1; ENA-78, epithelial neutrophil-activating

peptide-78.

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ABSTRACT

We have previously shown that IFN-γ/STAT1 plays an essential role in Concanavalin A

(Con A)-induced T cell hepatitis via activation of apoptotic signaling pathways. Here we

demonstrate that IFN-γ/STAT1 also plays a crucial role in leukocyte infiltration into the

liver in T cell hepatitis. After injection of Con A, leukocytes were significantly infiltrated

into the liver, which was suppressed in IFN-γ -/- and STAT1 -/- mice. Disruption of the

IFN regulatory factor-1 (IRF-1) gene, a downstream target of IFN-γ/STAT1, abolished

Con A-induced liver injury and suppressed leukocyte infiltration into the liver.

Additionally, Con A injection induced expression of a wide variety of chemokines and

adhesion molecules in the liver. Among them, expression of ICAM-1, VCAM-1, Mig,

CCL-20, ENA-78, ITAC, and IP-10 was markedly attenuated in IFN-γ -/-, STAT1 -/- , and

IRF-1 -/- mice. In primary mouse hepatocytes, Kupffer cells, and endothelial cells, in vitro

treatment with IFN-γ activated STAT1, STAT3, and IRF-1, and induced expression of

VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 mRNA. Induction of these

chemokines and adhesion molecules was markedly diminished in STAT1-/- and IRF-1-/-

hepatic cells compared to wild-type hepatic cells. These findings suggest that in addition

to induction of apoptosis previously well documented, IFN-γ also stimulated hepatocytes,

sinusoidal endothelial cells, and Kupffer cells partly via an STAT1/IRF-1-dependent

mechanism to produce multiple chemokines and adhesive molecules responsible for

promoting infiltration of leukocytes and ultimately resulting in hepatitis.

Key words: Concanavalin A; hepatitis, IFN-γ; STAT1; IRF-1

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Alcohol consumption and hepatitis viral infection are two dominant causes of

chronic liver disease, affecting billions of people worldwide. The molecular and cellular

mechanisms underlying the pathogenesis of alcoholic liver disease and hepatitis infection

are not fully understood. Increasing evidence suggests that elevated cytokines play

important roles in the pathogenesis of liver diseases (1, 12, 19, 27, 50). For example, it

has been reported that serum levels of interferon-γ (IFN-γ) are significantly elevated in

alcoholic hepatitis (27), hepatitis B infection (14, 24), and hepatitis C infection (30, 42,

46). High levels of IFN-γ mRNA expression are also detected in the livers of patients

with chronic hepatitis C infection (30), and in hepatic infiltrating T lymphocytes and

peripheral T lymphocytes (42). It has been well documented that elevated IFN-γ is

involved in frontline defenses against viral infection through induction of antiviral

proteins and modulation of immune responses (40). However, the role of IFN-γ in liver

damage is less clear.

Interferon-γ activity is mediated through activation of the Janus kinase-signal

transducers and activators of transcription (JAK-STAT) signaling pathway. Upon IFN-γ

binding, tyrosine kinases (JAK1 and JAK2) associated with the IFN-γ receptor (IFNGR1

and IFNGR2) are activated, leading to phosphorylation and activation of STAT1.

Activated STAT1 dimerizes and translocates into the nuclei to activate transcription of a

number of genes, including IFN regulatory factor-1 (IRF-1). IRF-1 is a transcription

factor that controls transcription of many antiviral and anti-apoptotic genes (20, 39). IFN-

γ activates other STATs, such as STAT3, STAT4, STAT5, and STAT6 as well, however,

the functions of these STATs when activated by IFN-γ are less clear. IFN-γ -/- and STAT1

-/- mice have been shown to be resistant to Concanvalin A (Con A)- or LPS/D-

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galactosamine-induced liver injury (13, 18, 45, 48). Disruption of the IRF-1 gene, a

downstream target of IFN-γ/STAT1, protects against mortality associated with injection

of LPS, Con A, or P. Berghei ANKA infection (44). These results indicate that IFN-γ and

IRF-1 are both involved in experimentally induced liver injury. Furthermore, IFN-γ has

been reported to directly induce cellular apoptosis in cultured hepatocytes in an IRF-1-

dependent manner (17). Therefore, it is believed that the detrimental effects of IFN-

γ/STAT1 in liver injury is mediated partly through induction of the pro-apoptotic IRF-1

gene and consequently, induction of hepatocyte apoptosis and hepatocellular damage.

Here, we further demonstrate that the IFN-γ/STAT1/IRF-1 pathway plays an important

role in the infiltration of leukocytes in the Con A-mediated hepatitis model. As shown in

this study, IFN-γ not only targeted hepatocytes, but also sinusoidal endothelial and

Kupffer cells via an STAT1/IRF-1-dependent mechanism, resulting in the production of

chemokines (such as Mig, ENA-78, ITAC, and IP-10) and adhesion molecules (such as

ICAM-1 and VCAM-1), which may play critical roles in the infiltration of leukocytes

into the liver.

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MATERIALS AND METHODS

Materials. Anti-STAT1, anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3

(Tyr705), and anti-STAT3 antibodies were obtained from Cell Signaling Technology

(Beverly, MA). Anti-IRF-1 antibody was purchased from Santa Cruz Biotechnology, Inc

(Santa Cruz, CA). Anti-Mig antibody was obtained from R&D Systems (Minneapolis,

MN).

Mouse Models of Hepatitis Induced by Injection of Con A. Seven to eight-week

old male IFN-γ -/- mice (C57BL/6 background), IRF-1-/- mice (C57BL/6 background), and

male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME).

Seven to eight-week old male 129/SvEv background STAT1-/- mice and 129SvEv control

mice were purchased from Taconic Farms (Germantown, NY). Preliminary data showed

that C57BL/6J mice exhibited more susceptibility to Con A-induced T cell hepatitis than

129/SVEv mice. Therefore, IFN-γ-/-, IRF-1-/-, and C57BL/6J mice were injected

intravenously with Con A (12 µg/g). STAT1-/-mice and 129SvEv mice were injected

intravenously with Con A (22 µg/g). The mouse livers were then collected for detection

of neutrophil and eosinophil infiltration, H&E staining, and RT-PCR analyses at various

time points post Con A injection.

Isolation and Culture of Primary Mouse Hepatocytes, Sinusoidal Endothelial

Cells, and Kupffer Cells. Control and knockout mice weighing 20-25 g were anesthetized

with sodium pentobarbital (30 mg/kg intraperitoneally), and the portal vein was

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cannulated under aseptic conditions. The liver was subsequently perfused with EGTA

solution (5.4 mM KCl, 0.44 mM KH2PO4, 140 mM NaCl, 0.34 mM Na2HPO4, 0.5 mM

EGTA, 25 mM Tricine, pH 7.2) and DMEM (Gibco BRL, Gaithersburg, MD), and

digested with 0.075% collagenase solution. The isolated mouse hepatocytes were then

cultured in Hepato-ZYME-SFM media containing 5% fetal bovine serum (Gibco, BRL)

in rat-tail collagen coated plates for 2 h, then changed to serum-free DMEM medium for

16 h, followed by treatment with IFN-γ for various time periods.

Sinusoidal endothelial cells and Kupffer cells were isolated by collagenase

perfusion and differential centrifugation in Percoll (Sigma, St. Louis, MO) as previously

described (51). The viability of isolated sinusoidal endothelial cells was above 95% in all

isolations as determined by the Trypan blue exclusion test. The purity of sinusoidal

endothelial cells as examined by phase-contrast microscopy was 90.7% ± 5.2%. Cells

were cultured at 370C in RPMI 1640 media supplemented with fetal calf serum (20%), L-

glutamine (2mM), gentamycin (100µg/ml), and dexamethasone (1µM) in a humidified

atmosphere (100%) containing 5% CO2/95% air. Cultured cells were identified as liver

endothelial cells given immunological evidence of the von Willebrand factor. Kupffer

cells were cultured at 370C in RPMI 1640 media supplemented with 20% fetal calf serum

and gentamycin (100µg/ml) in a humidified atmosphere (100%) containing 5% CO2/95%

air. Both sinusoidal endothelial cells and Kupffer cells were cultured overnight in serum-

containing media, and then replaced with serum-free media for 4 h, followed by

stimulation with IFN-γ for various time periods.

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Western Blotting. Cells were lysed in lysis buffer (30 mM Tris, pH 7.5, 150 mM

sodium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1%

Nonidet P-40, 10% glycerol) for 20 min at 4°C, vortexed, and then centrifuged at 16,000

rpm for 10 min at 4°C. Tissues were homogenized in lysis buffer at 4°C, vortexed, and

spun at 16,000 rpm for 10 min at 4°C. The supernatants were mixed in Laemmli loading

buffer, boiled for 4 min, and then subjected to SDS-PAGE. After electrophoresis,

proteins were transferred onto nitrocellulose membranes and blotted against primary

antibodies for 16 h. Membranes were washed with TPBS (0.05% [vol/vol] Tween 20 in

phosphate-buffered saline [pH 7.4]) and incubated with a 1:4000 dilution of horseradish

peroxidase-conjugated secondary antibodies for 45 min. Protein bands were visualized by

enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ).

Determination of Liver Injury. Liver injury was determined by either

hematoxylin-eosin (H&E) staining of liver sections or by analysis of serum

aminotransferase activities. For H&E staining, livers were fixed with 10%

formalin/phosphate-buffered saline for 24 h, then sliced and stained with hematoxylin-

eosin. Serum levels of alanine aminotransaminase (ALT) and asparate aminotransferase

(AST) were quantified by measuring plasma enzyme activities of ALT and AST utilizing

a kit from Drew Scientific (Cumbria, UK).

Immunohistochemistry. Liver sections were immunostained for neutrophils as

described previously using anti-neutrophil myeloperoxidase (MPO) antibody (Lab Vision

Corporation, Fremont, CA) (15). Numbers of neutrophils in the liver sections were

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counted in 10 randomly chosen visual fields (magnification, x200), and the average from

10 selected microscopic fields was calculated.

Assay of Hepatic Eosinophil Peroxidase (EPO) Activity. Hepatic EPO activity

was measured as described previously (41). EPO enzyme activity in hepatic tissues was

calculated by subtracting the mean background optical density and expressed as change

OD490 nm/min.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RT-PCR was

performed as described previously (37). The sequences of the primers used in the study

are listed in table I. The β-actin gene was amplified as an internal control. PCR using

RNA without reverse transcription did not yield amplicons, indicating a lack of genomic

DNA contamination. The PCR bands were scanned using Storm PhosphoImager

(Molecular Dynamics, Sunnyvale, CA) and quantified using ImageQuant software

(Molecular Dynamics) and normalized to β-actin mRNA levels at each time point. Fold

induction is the relative induction compared with untreated wild-type control.

Statistical Analysis. For comparing values obtained in 3 or more groups, one-

factor analysis of variance was used, followed by Tukey’s post hoc test. Statistical

significance was taken at the P<0.05 level.

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Table I PCR primers for murine chemokines and adhesion molecules

Genes Sequences Sizes(bp)

Chemokines CCL

CCL1 (I-309) F 5’GGA TGT TGA CAG CAA GAG CA 3’R 5’TAG TTG AGG CGC AGC TTT CT 3’

176

CCL2 (MCP-1) F 5' ACT GAA GCC AGC TCT CTC TTC CTC-3'R 5' TTC CTT CTT GGG GTC AGC ACA GAC-3'

274

CCL3 (MIP-1 ) F 5' GCC CTT GCT GTT CTT CTC TGT-3'R 5’GGC AAT CAG TTC CAG GTC AGT-3'

258

CCL4 (MIP-1β) F 5’CCC ACT TCC TGC TGT TTC TC 3'R 5’GAG GAG GCC TCT CCT GAA GT 3’

237

CCL5 (RANTES) F 5'-CCC CAT ATG GCT CGG ACA CCA 3’R 5'-CTA GCT CAT CTC CAA ATA GTT GAT 3'

205

CCL6 F 5’GGC TTT GGA ATG TGT CTG GT 3’R 5’CTG GCC CCG TAG TTC TAT GA 3’

215

CCL7 (MCP-3) F 5’AAT GCA TCC ACA TGC TGC TA 3’R 5’CTT TGG AGT TGG GGT TTT CA 3’

204

CCL8 (MCP-2) F 5’ GGG TGC TGA AAA GCT ACG AG 3’R 5’ TTC CAG CTT TGG CTG TCT CT 3’

202

CCL-9 F 5’TGT TTC ACA TGG GCT TTC AA 3’R 5’TTG TAG GTC CGT GGT TGT GA 3’

226

CCL11 (Eotaxin-1) F 5’-TCC ACA GCG CTT CTA TTC CT-3’R 5’-CTA TGG CTT TCA GGG TGC AT-3’

178

CCL-12 F 5’ GGG AAG CTG TGA TCT TCA GG 3’R 5’ GGG AAC TTC AGG GGG AAA TA 3’

178

CCL-17 (TARC) F 5’ AGT GGA GTG TTC CAG GGA TG 3’R 5’ AGG GAA CAG GGA CTT CTG CT 3’

235

CCL-19 (ELC, MIP-3β)

F 5’ ATT CCA GTC ACT CCC CTG TG 3’R 5’ AGA GAA CCA GCA GGC TGA AG 3’

158

CCL-20 (LARC,MIP-3α)

F 5’ CGA CTG TTG CCT CTC GTA CA 3’R 5’ AGG AGG TTC ACA GCC CTT TT 3’

177

CCL-22 (MDC,STCP1)

F 5’ AAA TGC TCG CCA ATG ATA CC 3’R 5’ AAG GAA GCC ACC AAT GAC AC 3’

187

CCL-24 (Eotaxin-2) F 5’-ACC CCA GCT TTG AAC TCT GA- 3’R 5’-AAG GAC GTG CAG CAA GATG- 3’

180

CCL-24 (Eotaxin-2) F 5’GCC TTC TGT TCC TTG GTG TC 3’R 5’TGT ACC TCT GGA CCC ACT CC 3’

228

CCL25 (TECK) F 5’ GGG AAT CCA GAG GAC ATG AA 3’R 5’ CCT CCA GCT GGT GCT TAC TC 3’

248

CCL26 (Eotaxin-3) F 5’ GGA GGA GTT TGG GAG AAA CC 3’ R 5’ TGT GGC TGT ATT GGA AGC AG 3’

163

CCL-27 (CTACK) F 5’ ATA GAC AGC CAC TCC CAA GC 3’R 5’ ACA GTC CCT TGG AGC CTT TT 3’

187

CCL-28 (MEC) F 5’ GAG TTC ATG CAG CAT CCA GA 3’R 5’ CCT GTG TGT TCC ACG TGT TC 3’

238

Chemokines CXCLCXCL1 (KC) F 5'-TTG AAG GTG ATG CCG CCA G- 3’

R 5'-CCC AGA CTC TCA TCT CTC C- 3’205

CXCL2 (MIP-2) F 5'-GAA CAA AGG CAA GGC TAA CTG A-3'R 5'-AAC ATA ACA ACA TCT GGG CAA T-3'

204

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CXCL-4 (PF4) F 5’ GAG CCC TAG ACC CAT TTC CT 3’R 5’ GAT CTC CAT CGC TTT CTT CG 3’

200

CXCL-5 (ENA-78) F 5’ GAA AGC TAA GCG GAA TGC AC 3’R 5’ GGG ACA ATG GTT TCC CTT TT 3’

166

CXCL9 (IP-10) F-5’ CCC ACG TGT TGA GAT CAT TG-3’R 5’ AGG GGA GTG ATG GAG AGA CA 3’

205

CXCL11 (I-TAC) F 5’ AGT AAC GGC TGC GAC AAA GT 3’R 5’ GCA TGT TCC AAG ACA GCA GA 3’

225

CXCL-12 (SDF-1α) F 5’CTT CAT CCC CAT TCT CCT CA 3’R 5’GAC TCT GCT CTG GTG GAA GG 3’

171

CXCL13 (BCA-1) F 5’CAT CAT GAG GTG GTG CAA AG 3’R 5’GGG TCA CAG TGC AAA GGA AT 3’

188

CXCL14 (BRAK) F 5’CTC CAG GCC AGT TGA GAG AC 3’R 5’TGG AAG CCT TTC ACA CAC AG 3’

155

AdhesionMoleculesVCAM-1 F 5'-CAG CTA AAT AAT GGG GAA CTG-3'

R 5'-GGG CGA AAA ATA GTC CTT G-3'447

ICAM-1 F 5'-GGA GCA AGA CTG TGA ACA CG-3'R 5'-GAG AAC CAC TGC TAG TCC AC-3'

435

PECAM-1 F 5’AAC AGA AAC CCG TGG AGA TG 3’R 5’GTC TCT GTG GCT CTC GTT CC 3’

241

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Results:

IRF-1 Plays A Critical Role in Con A Injection-mediated Liver Injury. Senaldi

et al (44) reported that IRF-1-/- mice showed less mortality than control mice after

injection of a lethal dose of Con A. However, the role of IRF-1 in Con A-induced liver

injury has not been investigated. We and others have demonstrated that IRF-1 activation

correlated with liver injury in Con A-mediated hepatitis (13, 47), suggesting that IRF-1

may play an important role in T cell-mediated hepatitis. To test this hypothesis, we

compared Con A-induced liver injury in IRF-1-/- mice and control wild-type mice. As

shown in Fig. 1A, Con A-induced liver injury (elevated ALT levels) was markedly

attenuated in IRF-1-/- mice compared to wild-type mice. Liver histology revealed massive

necrosis and hepatic infiltration of leukocytes in wild-type mice but not in IRF-1-/- mice

(Fig. 1B).

IFN-γ, STAT1, and IRF-1 Play Critical Roles in Con A Injection-mediated

Infiltration of Neutrophils and Eosinophils in the Liver. To understand the mechanisms

underlying the detrimental effects of IFN-γ/STAT1/IRF-1 in T cell hepatitis, we

compared Con A-induced infiltration of neutrophils and eosinophils between wild-type

mouse livers and knockout (IFN-γ-/-, STAT1-/-, and IRF-1-/-) mouse livers. As shown in

Fig. 2A, control mouse livers contained very low numbers of neutrophils. After Con A

injection, neutrophils significantly infiltrated the liver in wild-type mice with peak effect

at 9 h. In IFN-γ-/-, STAT1-/-, and IRF-1-/- mice, hepatic infiltration induced by Con A was

markedly attenuated. The suppression of neutrophil infiltration was more prominent in

IFN-γ-/- mice compared to STAT1-/- and IRF-1-/- mice.

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Infiltration of eosinophils was determined by measuring hepatic EPO activity. As

shown in Fig. 2B, control mouse livers contained low levels of EPO activity, which was

significantly elevated 9 and 24 h post Con A injection. Elevation in EPO activity was

significantly suppressed in IFN-γ-/-, IRF-1-/-, and STAT1-/- mice compared to their

corresponding control mice.

Con A-mediated Induction of VCAM-1, IACM-1, Mig, CCL-20, ENA-78, I-

TAC, and IP-10 Expression in the Liver Is Impaired in IFN-γ--/-, STAT1-/-, and IRF-1-/-

Mice. The above data clearly showed that IFN-γ, STAT1, and IRF-1 were involved in

the infiltration of leukocytes in Con A-mediated hepatitis. To understand the mechanisms

underlying their involvement in leukocyte infiltration, expression of a variety of

chemokines and adhesion molecules was compared between wild-type mice and IFN-γ-/-

mice. As shown in Fig. 3A, Con A injection significantly induced expression of

numerous adhesion molecules and chemokines in the livers of wild-type mice, with peak

effect between 3 and 9 h after injection. Among them, induction of VCAM-1, ICAM-1,

Mig, CCL-20, ENA-78, I-TAC, and IP-10 was markedly suppressed in IFN-γ-/- mice

(Figs. 3A and 3D), whereas induction of MCP-1, Rantes, CCL-1, CCL-6, CCL-17, BCA-

1, BRAK, and PF-4 was enhanced in IFN-γ-/- mice. These findings suggest that IFN-γ

induces VCAM-1, ICAM-1, Mig, CCL-20, ENA-78, I-TAC, and IP-10 expression, but

may suppress induction of other chemokines in T cell-mediated hepatitis.

To further determine whether IFN-γ-mediated induction of adhesion molecules

and chemokines was STAT1- and IRF-1-dependent, we additionally compared

expression of these factors between wild-type and STAT1-/- or IRF-1-/- mice. As shown in

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Figs. 3B and 3C, Con A injection-mediated induction of VCAM-1, ICAM-1, Mig, CCL-

20, ENA-78, I-TAC, and IP-10 was also significantly suppressed in STAT1-/- and IRF-1-/-

mice compared to wild-type control mice. However, the magnitude of the downregulation

observed in STAT1-/- and IRF-1-/- mice relative to wild-type mice was much less

compared to the downregulation observed between IFN-γ-/- mice and wild-type mice (Fig.

3).

IFN-γ Activates STAT1, STAT3, and IRF-1 in Hepatocytes, Sinusoidal

Endothelial Cells, and Kupffer Cells. The above findings suggest that IFN-γ is important

for the induction of a variety of adhesion molecules and chemokines in Con A-mediated

hepatitis. To further understand which hepatic cell types are targeted by IFN-γ,

hepatocytes, sinusoidal endothelial cells, and Kupffer cells were isolated and stimulated

with IFN-γ in vitro. As shown in Fig. 4, IFN-γ treatment significantly induced STAT1

and STAT3 activation, and induced expression of IRF-1 protein in primary mouse

hepatocytes, which is consistent with our previous report (13). IFN-γ also activated

STAT1 and STAT3, and induced expression of IRF-1 in both sinusoidal endothelial cells

and Kupffer cells (Fig. 4).

IFN-γ Induces Expression of Chemokines and Adhesion Molecules in

Hepatocytes, Sinusoidal Endothelial Cells, and Kupffer Cells Via STAT1- and IRF-

dependent Mechanisms

The effects of IFN-γ on the induction of chemokines and adhesion molecules in

hepatocytes, sinusoidal endothelial cells, and Kupffer cells were examined. As shown in

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Fig. 5A, IFN-γ treatment induced expression of VCAM-1, ICAM-1, Mig, ENA-78, ITAC,

and IP-10, but not CCL-20 in primary mouse hepatocytes. Induction of these chemokines

and adhesion molecules by IFN-γ was markedly diminished in STAT1-/- and IRF-1-/-

mouse hepatocytes. Suppression of IFN-γ-induced I-TAC and Mig expression in IRF-1-/-

mice relative to wild-type mice was less evident in comparison to the difference between

STAT1-/- mice and wild-type mice.

In wild-type mouse Kupffer cells, IFN-γ treatment induced expression of VCAM-

1, ICAM-1, Mig, ENA-78, and ITAC, whereas in STAT1-/- and IRF-1-/- mouse Kupffer

cells, induction of these genes was diminished. IFN-γ treatment weakly induced

expression of IP-10 mRNA and failed to induce expression of CCL-20 mRNA in Kupffer

cells. Similarly, IFN-γ treatment induced significant expression of VCAM-1, ICAM-1,

Mig, ENA-78, ITAC, IP-10 mRNAs in sinusoidal endothelial cells, and induction of

these genes was suppressed in STAT1-/- and IRF-1-/- mouse sinusoidal endothelial cells.

Densitometric analyses revealed that IFN-γ induction of VCAM-1, ICAM-1, Mig,

ENA78, I-TAC was significantly reduced in STAT1-/- and IRF-1-/- Kupffer cells and

endothelial cells compared to their wild-type control cells (P<0.05 or P<0.01). IFN-γ

induction of IP-10 was also significantly diminished in STAT1-/- endothelial cells and

Kupffer cells, and IRF-1-/- endothelial cells compared to their wild-type control cells

(P<0.05 or P<0.01).

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Discussion

Previous studies suggest that IFN-γ/STAT1 contributes to Con A-induced T cell

hepatitis via induction of pro-apoptotic genes such as IRF-1 (13, 47). In the present

paper, we provide evidence demonstrating that IFN-γ/STAT1/IRF-1 also acts as a pro-

inflammatory signal in T cell mediated hepatitis. Our findings suggest that Con A

stimulates NKT cells and other cells to produce IFN-γ, which then targets hepatocytes,

sinusoidal endothelial cells, and Kupffer cells via activation of STAT1, STAT3, and IRF-

1. Consequently, activation of STAT1 and IRF-1 stimulates expression of VCAM-1,

ICAM-1, Mig, ENA-78, I-TAC, and IP-10, which along with other chemokines, attracts

neutrophils and eosinophils into the liver, resulting in hepatitis. Although we have not

examined the T cell influx in STAT1-/- and IRF-1-/- mice, it is likely that T cell infiltration

is also attenuated in these mice compared to control mice since the critical role of

neutrophils in T cell infiltration in this model has been reported (3) and neutrophil

infiltration was reduced in STAT1-/- and IRF-1-/- mice.

Both TNF-α and IFN-γ have been shown to play an essential role in the

development and progression of Con A-induced T cell hepatitis (13, 21, 22, 45). This is

probably because TNF-α and IFN-γ synergistically induce expression of several

chemokines and adhesion molecules (33, 35). Depletion of either of them results in a

marked reduction in Con A-induced liver injury and inflammation (13, 21, 22, 45). Here

we showed that expression of VCAM-1, ICAM-1, Mig, CCL-20, ENA-78, and IP-10 was

markedly attenuated in IFN-γ-/- mice compared to wild-type mice. This downregulation

was also observed in STAT1-/- and IRF-1-/- mice compared to wild-type mice, but was

less profound compared to the difference observed between IFN-γ-/- mice and wild-type

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mice (Fig. 3). Collectively, these findings suggest that IFN-γ plays a pivotal role in the

induction of VCAM-1, ICAM-1, Mig, CCL-20, ENA-78, and IP-10 in the liver during

Con A-induced T cell hepatitis partly through STAT1- and IRF-1-dependent

mechanisms. Furthermore, we provide in vitro evidence suggesting that IFN-γ induces

VCAM-1, ICAM-1, Mig, ENA-78, I-TAC, and IP-10 expression in hepatocytes,

sinusoidal endothelial cells, and Kupffer cells via STAT1- and IRF-1-dependent

mechanisms. IFN-γ activation of STATs and induction of several chemokines (such as

Mig and IP-10) in primary mouse, rat, and human hepatocytes has previously been well

documented (13, 34, 36, 37). We further showed here that IFN-γ activated STAT1 and

STAT3, and induced expression of several chemokines and adhesion molecules in

primary mouse hepatocytes as well as in sinusoidal endothelial cells and Kupffer cells.

This induction was significantly attenuated in primary STAT1-/- and IRF-1-/- cells,

suggesting that IFN-γ-mediated induction of chemokines and adhesion molecules through

STAT1- and IRF-1-dependent mechanisms (Figs. 4 and 5). IRF-1 is a transcription factor

that binds to IRF-1 response elements in the promoter regions of genes to stimulate genes

transcription (20, 39). IRF-1 response elements have been identified in the promoters of

VACM-1 (23, 33), ICAM-1 (35), Mig (35), IP-10 (32), I-TAC (10), which may provide a

molecular basis for IFN-γ induced gene expression in primary hepatocytes, endothelial

cells, and Kupffer cells via an IRF-dependent mechanism. Interestingly, IFN-γ induction

of several chemokines and adhesion molecules was not completely abolished in STAT1-/-

and IRF-1-/- cells (Fig. 5). This is probably because other STATs (such as STAT3 and

STAT5) or other IRF transcription factors activated by IFN-γ may also be involved in

IFN-γ induction of these chemokines and adhesion molecules.

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The roles of IFN-γ-controlled adhesion molecules (ICAM-1 and VCAM-1) and

chemokines (Mig, CCL-20, ENA-78, and IP-10) in hepatic inflammation and injury have

previously been investigated in several models of liver injury. For example, neutrophil

infiltration and neutrophil-mediated liver injury caused by bile duct ligation were

significantly attenuated in ICAM-1-/- mice compared to wild type mice (9). Liver

regeneration and leukocyte infiltration after partial hepatectomy were also significantly

attenuated in ICAM-1-/- mice relative to control mice (43). These findings suggest that

ICAM-1 plays an important role in leukocyte infiltration, induction of liver injury, and

promotion of liver regeneration in these models. However, the roles of VCAM-1 and

ICAM-1 in Con A-induced liver injury have been controversial. Pretreatment with anti-

VCAM-1 or anti-ICAM-1 monoclonal antibodies were shown to attenuate Con A-

induced liver injury (29, 52), but other reports failed to confirm these findings (26, 53).

These discordant findings may partly be due to the different pretreatment regimens used

in these studies (26, 29, 52, 53). Mig (CXCL9), IP-10 (CXCL10), and I-TAC (CXCL11)

belong to the CXCR3 family of chemokines that bind to CXCR3 receptors, and are

potent chemoattractants for alloantigen-primed T cells (8). Several studies reported that

expression of these chemokines are elevated in several animal models of liver injury (4,

36) and in human liver diseases (2, 11, 28, 31, 49). However, the effects of CXCR3

chemokines in liver injury remain obscure. Kakimi et al (16) reported that blocking Mig

and IP-10 protected against liver injury in viral hepatitis B transgenic mice by reducing

the recruitment of host-derived mononuclear cells into the livers, suggesting that Mig and

IP-10 are detrimental in liver injury in this model. However, other studies have suggested

that IP-10 plays a protective role against liver injury in murine models of liver injury

19

induced by acetaminophen (4). ENA-78 is a 78 amino acid, 8 kDa protein belonging to

the CXC chemokine family and has neutrophil-activating and chemoattracting properties.

Recent data suggested that ENA-78 contributes to hepatic neutrophil influx and liver

injury, but also promotes liver regeneration after partial hepatectomy via stimulation of

hepatocyte proliferation (5, 6). Taken together, VCAM-1, ICAM-1, Mig, CCL-20, ENA-

78, and IP-10 likely play important roles in hepatic neutrophil influx in Con A-induced T

cell hepatitis. The downregulation of these factors in IFN-γ-/-, STAT1-/-, and IRF-1-/- mice

after injection of Con A (Fig. 3) may contribute to decreased infiltration of neutrophils

and eosinophils in these mice relative to wild-type mice (Fig. 2). The observed decreased

infiltration of neutrophils and eosinophils may partly contribute to lesser liver injuries in

IFN-γ-/-, STAT1-/-, and IRF-1-/- mice post Con A injection compared to wild-type mice

since both neutrophils and eosinophils have been shown to play essential roles in Con A-

induced T cell hepatitis (3, 25).

In summary, in addition to its pro-apoptotic action, IFN-γ/STAT1/IRF-1 also acts

as a pro-inflammtory signal in the liver via induction of VCAM-1, ICAM-1, Mig, CCL-

20, ENA-78, and IP-10. Elevations of these chemokines and adhesion molecules have

been reported in chronic liver disease (2, 11, 28, 31, 49), which is likely mediated in part

by activation of the IFN-γ/STAT1 signal pathway as chronic liver disease is associated

with high levels of IFN-γ (14, 24, 27, 30, 42, 46) and STAT1 (7, 38). Therefore, IFN-

γ/STAT1/IRF-1 may be a potential therapeutic anti-inflammatory target to treat human

liver disease.

20

Figure legends:

Fig. 1. IRF-1 plays an essential role in Con A-induced T cell hepatitis. (A) Wild-type and

IRF-1-/- mice (5 per group) were intravenously administered Con A (12 µg/g). After 9

and 24 h, mice were sacrificed and the serum collected to measure ALT activity. Values

are shown as means ± SEM at each time point from 5 mice. **P<0.001 in comparison

with corresponding Con A-treated wild-type mice. (B) Photomicrographs of

representative mouse livers from 24-h Con A-treated wild-type and IRF-1 -/- mice with

H&E staining are shown (original magnification × 200).

Fig. 2. IFN-γ/STAT1/IRF-1 plays important roles in the infiltration of neutrophils and

eosinophils in Con A-induced T cell hepatitis. IFN-γ-/- mice, IRF-1-/- mice, and their wild-

type C57BL/6J control mice were intravenously administered Con A (12 µg/g), and

STAT1-/- mice and their wild-type 129SvEv control mice were injected intravenously

with Con A (22 µg/g). After 9 and 24 h of Con A injection, livers were collected and

immunostained with anti-MPO antibodies for detection of neutrophils (A). Eosinophils

were detected by measuring hepatic EPO activities (B). In panel A, ten fields were

randomly selected and positive cells were counted. Values in panels A and B are shown

as means ± SEM at each time point from 4 to 5 mice. *P<0.05, **P<0.001 in comparison

with corresponding Con A-treated wild-type control mice.

21

Fig. 3. IFN-γ/STAT1/IRF-1 plays important roles in the induction of chemokines and

adhesion molecules in Con A-induced T cell hepatitis. IFN-γ-/- mice, IRF-1-/- mice, and

their wild-type C57BL/6J control mice were intravenously administered Con A (12

µg/g), STAT1-/- mice and their wild-type 129SvEv control mice were injected

intravenously with Con A (22 µg/g). After various time periods, livers were collected and

used to isolate total RNA, which was subjected to RT-PCR analyses (25 cycles for I-TAC

and IP-10, 30 cycles for other genes) of various chemokines and adhesion molecules.

Liver tissues were also subjected to Western blot analysis of Mig and β-actin. Panels A-

C are representative of 3 independent experiments. Panel D, expression of VCAM-1,

ICAM-1, CCL-20, ENA-78, ITAC, IP-10 in panel A was quantified by PhosphorImager

analysis and normalized to the same number cycles (25 or 30 cycles) of RT-PCR

amplification of β-actin mRNA levels at each time point. Expression of Mig protein was

quantified by PhosphorImager and normalized to β-actin protein in Western blot analysis.

Fold induction is the relative induction compared with untreated wild-type control. The

values are shown as means ± SEM from 3 independent experiments at each time point.

*P<0.01, #P<0.05, in comparison with corresponding Con A-treated wild-type controls.

Fig. 4. IFN-γ activates STAT1 and STAT3 and induces IRF-1 protein expression in

hepatocytes, sinusoidal endothelial cells, and Kupffer cells. Primary mouse hepatocytes,

sinusoidal endothelial cells, and Kupffer cells were isolated and cultured in serum-

containing media overnight, followed by culturing in serum-free media for 4 h, and then

stimulated with IFN-γ (10 ng/ml). After various time periods, cells were harvested and

22

total protein extracts prepared for Western blot analyses using antibodies as indicated.

Data are representative of 2 independent experiments.

Fig. 5. IFN-γ induces expression of VCAM-1, ICAM-1, Mig, ITAC, and IP-10 mRNA in

hepatocytes, sinusoidal endothelial cells, and Kupffer Cells via STAT1- and IRF-1-

dependent mechanisms. Hepatocytes, sinusoidal endothelial cells, and Kupffer cells were

isolated from STAT1-/- mice and their wild-type 129SvEv control mice, and from IRF-1-/-

mice and their wild-type C57BL/6 control mice. These cells were cultured in serum-

containing media overnight, followed by culturing in serum-free media for 4 h, then

stimulated with IFN-γ (10 ng/ml). After various time periods, total RNA was isolated and

subjected to RT-PCR analyses of various chemokines and adhesion molecules as

indicated. Data are representative of 2 independent experiments.

23

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Jaruga et al. Fig. 1

AL

T (

103 U

/L)

Wild-type IRF-1 (-/-)(A)

Con A (h)

4

3

2

1

9 24

(B) Wild-type IRF-1 (-/-)

****

Jaruga et al. Fig. 2

Neu

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x200

)

100

80

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(A)Control IRF-1 (-/-) IFN-γ (-/-)

0.5

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Jaruga et al. Fig. 3

Con A (h) 0 3 6 9 24 0 3 6 9 24Wild-type IFN-γ (-/-) Wild-type STAT (-/-)

VCAM-1ICAM-1

Mig*CCL-20ENA-78I-TAC**IP-10**β-actin

Eotaxin-1Eotaxin-2MIP-1αMIP-1βMIP-2MCP-1Rantes

PECAM-1CCL-1CCL-6CCL-7CCL-8CCL-9CCL12CCL-17CCL-19CCL-22CCL-25CCL-27CCL-28BCA-1SDF-1αBRAK

KCPF-4

(A) (B)

0 3 6 9 24 0 3 6 9 24 0 3 6 9 24 0 3 6 9 24 Wild-type IRF-1 (-/-)

(C)

(D)

*Western blotting; ** PCR 25 cycles

Fol

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3

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1086420

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6543210

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IFN-γ (h) 0 0.5 1 8 0 0.5 1 8 0 0.5 1 8

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IRF-1

β-actin

Endothelial cells Kupffer cells Hepatocytes

Jaruga et al. Fig. 4

Jaruga et al. Fig. 5

IFN-γ (h) 0 3 9 24 0 3 9 24Wild-type STAT (-/-)

0 3 9 24 0 3 9 24Wild-type IRF-1 (-/-)

VCAM-1ICAM-1

MigCCL-20ENA-78I-TACIP-10

β-actin

Kupffer cells Endothelial cells

IFN-γ (h) 0 3 9 0 3 9 0 3 9 0 3 9 VCAM-1ICAM-1

MigmCCL-20ENA-78I-TACIP-10

β-actin

WT STAT(-/-) WT IRF-1(-/-)0 3 9 0 3 9 0 3 9 0 3 9

WT STAT(-/-) WT IRF-1(-/-)

(A)

(B)

Hepatocytes