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The Journal of Immunology, 2003, 171: 3233-3244.
Copyright © 2003 by The American Association of Immunologists

Crucial Role of IL-4/STAT6 in T Cell-Mediated Hepatitis: Up-Regulating Eotaxins and IL-5 and Recruiting Leukocytes

Barbara Jaruga, Feng Hong, Rui Sun, Svetlana Radaeva and Bin Gao1

Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-{gamma}/STAT1, IL-6/STAT3, or TNF-{alpha}/NF-{kappa}B signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 -/- and STAT6-/- mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6-/- mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4-/- and STAT6-/- mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell-mediated immune responses play important roles in the pathogenesis of a variety of human liver disorders (1, 2, 3), including autoimmune liver disease (4), viral hepatitis (5, 6), and alcoholic liver disease (7). Injection of Con A is a well-established model to study T cell-mediated hepatitis that closely resembles the pathology of human autoimmune hepatitis (8). The action of T cells in the liver is in part mediated through release of a variety of cytokines, which target liver cells and immune cells via activation of multiple signaling cascades, including the signal transducers and activators of transcription factor (STAT1, -2, -3, -4, -5a, -5b, and -6) family members (9, 10, 11). For example, activation of STAT1 by IFN-{gamma} is essential for the development of liver injury in Con A-induced hepatitis, (12), whereas activation of STAT3 by IL-6 protects against liver injury (13, 14). Administration of Con A also activates STAT4, STAT5, and STAT6 in the liver (12), but their precise roles remain obscure.

The T cell-derived cytokines IL-4 and IL-13 specifically activate STAT6, which plays important roles in Th2 differentiation, tissue adhesion, inflammation, and a variety of effects on hemopoietic tissues (15, 16, 17). On binding of IL-4 (or IL-13) to its receptor (IL-4R{alpha}), IL-4R{alpha} dimerizes either with IL-2R{gamma} to form the type I IL-4R or with the IL-13R{alpha}1 to form the type II IL-4R. Dimerization of the receptor triggers activation of adjacent Janus kinase (JAK)2 1 and JAK3 tyrosine kinases, which phosphorylate tyrosine residues in the cytoplasmic domain of IL-4R{alpha}. This receptor-kinase complex then recruits and activates several proteins containing phosphotyrosine binding or Src homology 2 domains such as the STAT6 family member. Subsequently, activated STAT6 homodimerizes and translocates into the nucleus, where it regulates gene transcription (15, 16, 17). In various liver injury models, IL-4 and IL-13 have been shown to be both protective and deleterious. Injection of either IL-4 or IL-13 reduces hepatic ischemia/reperfusion injury (18, 19), and it was observed that STAT6-deficient mice are highly susceptible to endotoxin-induced liver injury (20). Conversely, IL-4 accelerates severe hepatitis in mice deficient in suppressor of cytokine signaling (SOCS)-1 proteins through activation of NK T cells (NKT cells) (21), and it is believed that IL-4 plays a key role in Con A-induced hepatitis via augmentation of V{alpha}14 NKT cell-mediated cytotoxicity (22). However, it was reported that adenoviral gene transfer of IL-4 caused severe hepatitis largely independent of any immune cells in the liver (23), and administration of IL-4 completely restored Con A-induced liver injury in {beta}2-microglubin-deficient mice lacking NKT cells (24), suggesting that IL-4 mediates liver injury through other mechanisms outside of NKT cells. Here, we demonstrate that IL-4 activation of STAT6 up-regulates eotaxin expression in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, resulting in eosinophil and neutrophil recruitment into the liver and leading to hepatitis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Anti-STAT1, anti-phospho-STAT1 (Tyr701), anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-phospho-STAT6 (Tyr641), anti-STAT6, and anti-phospho-I{kappa}B Abs were obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-STAT4 (Tyr693) was obtained from Zymed Laboratories (South San Francisco, CA). Anti-Bcl-xL Ab was obtained from BD PharMingen (San Diego, CA). Anti-IFN regulatory factor-1 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-murine monocyte IFN-{gamma}-inducible protein (Mig) and anti-murine eotaxin Abs were obtained from R&D Systems (Minneapolis, MN). The anti-murine eotaxin Ab is able to neutralize all mouse eotaxins. Murine IL-4 and human IL-4 were purchased from Biosource International (Camarillo, CA). The sources of other Abs are indicated in the relevant methods sections below.

Mouse models of hepatitis induced by injection of Con A.

Male STAT6-/- mice (7–8 wk old; BALB/c background), control male BALB/cJ mice, male IL-4-/- mice (7–8 wk old; C57BL/6 background), and male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). To induce hepatitis, STAT6-/- mice and BALB/cJ mice were injected i.v. with Con A (16–20 µg/g). IL-4-/- mice and C57BL/6J mice were injected i.v. with Con A (10–12 µg/g). All mice were sacrificed within 24 h postinjection, and serum and livers were collected for in vitro experiments.

Isolation and culture of primary mouse hepatocytes and sinusoidal endothelial cells

Mice weighing 20–25 g were anesthetized with sodium pentobarbital (30 mg/kg i.p.), and the portal vein was 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 (Life Technologies, Gaithersburg, MD) and digested with 0.075% collagenase solution. The isolated mouse hepatocytes were then cultured in Hepato-ZYME-SFM medium (Life Technologies) in rat tail collagen-coated plates for 24 h and then cultured in serum-free DMEM and treated with IL-4 for various time periods.

Sinusoidal endothelial cells were isolated by collagenase perfusion and differential centrifugation in Percoll (Sigma-Aldrich, St. Louis, MO) as previously described (25). The viability of isolated sinusoidal endothelial cells was >95% in all isolations as determined by trypan blue exclusion test. The purity of sinusoidal endothelial cells as examined by phase contrast microscopy was 90.7 ± 5.2%. Cells were cultured in RPMI 1640 supplemented with FCS (20%), L-glutamine (2 mM), gentamicin (100 µg/ml), and dexamethasone (1 µM) at 37°C in a 100% humidified atmosphere containing 5% CO2–95% air. Cultured cells were identified as liver endothelial cells through immunological evidence of von Willebrand factor.

Western blotting

Tissues were homogenized in lysis buffer (30 mM Tris (pH 7.5), 150 mM sodium chloride, 1 mM PMSF, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10% glycerol) at 4°C, vortexed, and centrifuged at 16,000 rpm at 4°C for 10 min. 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 Abs for 16 h. Membranes were washed with 0.05% (vol/vol) Tween 20 in PBS (pH 7.4) and incubated with a 1/4000 dilution of HRP-conjugated secondary Abs for 45 min. Protein bands were visualized by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ).

H&E staining of liver sections

After fixation of the livers with 4% formalin/PBS, livers were sliced and stained with H&E.

Analysis of alanine aminotransaminase (ALT) and aspartate aminotransferase (AST) activities

Liver injury was quantified by measuring plasma enzyme activities of ALT and AST using a kit purchased from Sigma-Aldrich.

Immunohistochemistry

Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated with PBS, followed by proteinase K digestion (30 µg/ml in 100 mmol/L Tris-HCl buffer, 50 mmol/L EDTA (pH 8.0); 30 min at 37°C). Next, sections were incubated in 0.3% H2O2 in methanol to block endogenous peroxidase activity. Nonspecific binding sites were blocked by a 30-min incubation period in 50% FCS, 0.1% BSA, followed by another 30 min in 1% BSA, 0.1% fish gelatin. Sections were incubated with 1/50 diluted primary Abs overnight at 4°C. Biotinylated secondary Abs and ABC Reagent were applied according to the manufacturer’s instructions (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). Color development was induced using 3,3'-diaminobenzidine substrate during a 5- to 10-min incubation period. Using this substrate, specific staining was visualized by light microscopy.

Liver sections were immunostained using anti-neutrophil myeloperoxidase (MPO) Ab (Lab Vision, Fremont, CA) for neutrophils and anti-F4/80 Ab (Novus Biologicals, Cambridge, U.K.) for macrophages. Eosinophils were detected by measuring cyanide-resistant eosinophil peroxidase (EPO) activity in situ. The numbers of neutrophils, eosinophils, and macrophages in the liver were counted in 10 randomly chosen visual fields (magnification, x200) of the sections, and the average of 10 selected microscopic fields was calculated.

Assay of hepatic EPO activity

Hepatic EPO activity was measured as described previously (26). The EPO enzyme activities of the hepatic tissues were calculated by substracting the mean background OD and are expressed as change of OD490 per minute.

RT-PCR

RT-PCR was performed as described previously (27). The sequences of the primers used in the study are listed in Table I. The {beta}-actin gene was also 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).


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  		Table I. PCR primers used in this paper

 
Cytotoxicity assay

Mouse hepatocytes isolated from wild-type or knockout mice were seeded on rat tail collagen-coated 48-well microtiter plates at a density of 2 x 104 viable cells/well. After the cells were treated with IL-4 or saline for various time points, cytotoxicity was assessed by measuring AST activity, a specific enzyme for hepatocytes. Cytolytic activity of hepatic mononuclear cells (MNCs) was also tested against mouse hepatocytes using a 4-h AST release assay. Hepatic MNCs were added onto cultured mouse hepatocytes at the indicated effector to target ratios. After a 4-h incubation, the supernatant was harvested, and AST activity was measured. The specific cytotoxicity was calculated as [(ASTexperimental - ASTspontaneous)/(ASTmaximum - ASTspontaneous)] x 100%.

EMSA

EMSAs were performed in 20-µl volumes with 20 mM Tris-HCl (pH 7.9), 1.5% glycerol, 50 µg/ml BSA, 1 mM DTT, 0.5 mM PMSF, 2 µg of poly(deoxyinosinic-deoxycytidylic) acid, 1 ng of 32P-labeled probe, and 10 µg of nuclear extract. Reactions were incubated at 25°C for 20 min and subsequently analyzed by electrophoresis through nondenaturing stock polyacrylamide gels (6% or 10%) in 0.5 x TBE buffer containing 44.5 mM Tris-HCl (pH 8.2), 44.5 mM boric acid, and 1 mM EDTA. After prerunning the gel at 100 V for 2 h, electrophoresis was performed at 270 V for 2 h at 4°C. The gels were then analyzed by the PhosphorImager ImageQuant program (Molecular Dynamics). The NF-{kappa}B-binding site in the double-stranded oligonucleotide 5'-AGT TGA GGG GAC TTT CCC AGG-3' was used as a probe to determine NF-{kappa}B binding.

ELISA

Plasma levels of cytokines were measured using the standard ELISA sandwich kits as specified by the manufacturer (Biosource International).

Isolation of liver MNCs and adoptive transfer

Wild-type and knockout murine livers were removed and pressed through a 200-gauge stainless steel mesh. The liver cell suspension was collected and suspended in RPMI 1640 (Life Technologies). Parenchymal cells (pellet) were separated from MNC (supernatant) by centrifugation at 50 x g for 5 min. Supernatants containing MNC were collected, washed in PBS, and resuspended in 40% Percoll in RPMI 1640. The cell suspension was gently overlaid onto 70% Percoll and centrifuged for 20 min at 750 x g. MNC were collected from the interphase, washed twice in PBS, and resuspended in RPMI 1640.

Adoptive hepatic MNC transfer was performed as described previously (28). Briefly, 50 µl of hepatic MNCs (5 x 106 cells) in PBS were injected into the lateral left lobe of the liver at a rate of 10 µl/s using a 29-gauge needle attached to a 1-ml syringe, followed by i.v. injection of Con A (15 µg/g for B6 mice and 20 µg/g for BABL/c mice). Liver injury was assessed 9 h later by serum ALT levels and histological analysis.

Flow cytometry analysis of CD4+ T cell and NKT cell activation in the liver after administration of Con A

Activation of CD4+ T cells was determined by anti-CD4 plus anti-CD69 (early activation marker) (BD PharMingen) through a FACS (FACSCalibur; BD Biosciences, San Jose, CA). NKT cell numbers in C57BL/6J wild-type mice and C57BL/6J background IL-4-/- mice were determined using anti-NK1.1 plus anti-CD3 Abs or anti-DX5 plus anti-CD3 Abs (BD PharMingen), whereas anti-DX5 and anti-CD3 Abs were used to determine NKT cell numbers in BABL/cJ wild-type mice and BABL/cJ background STAT6-/- mice, because BALB/c mice lack expression of the NK1.1 Ag.

Statistical analysis

For comparing values obtained in three or more groups, one-factor ANOVA was used, followed by Tukey’s post hoc test. Statistical significance was taken at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of STAT6 in the liver and spleen of Con A-induced hepatitis: involvement of IL-4

As shown in Fig. 1A, injection of Con A rapidly induced STAT6 tyrosine phosphorylation with the peak effect occurring between 1 and 3 h and returning to basal levels at 6 h. The levels of STAT6 proteins were unchanged. To localize activated STAT6 in the liver after Con A administration, isolated livers were immunostained with anti-phospho-STAT6 Abs and examined by light microscopy. Levels of phosphorylated STAT6 were undetectable in control livers injected with PBS, whereas strong tyrosine-phosphorylated STAT6 staining was detected in the nuclei of hepatocytes as well as in some nonparenchymal cells of Con A-treated livers.



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  		FIGURE 1. IL-4-mediated activation of STAT6 in the liver and spleen after injection of Con A. A, Wild-type and IL-4-/- mice were injected with Con A (12 µg/kg); at various time points, total liver and spleen protein extracts were immunoblotted with anti-phospho (p)-STAT6 and anti-STAT6 Abs. B, A representative immunostaining of phosphorylated STAT6 in the liver after administration of Con A for 3 h (original magnification, x400). Phosphorylated STAT6 was stained as relatively large brown granules in the nuclei of the hepatocytes (representatives are indicated by arrows) of Con A-treated livers. Positive staining was also detected in some nonparenchymal cells (representatives are indicated by arrowheads) of Con A-treated livers. C, Primary mouse hepatocytes (MH), Hep3B cells, HepG2 cells, and hepatic sinusoidal endothelial (ME) cells were treated with murine or human IL-4 (50 ng/ml) for various time points. Total protein extracts were immunoblotted with various Abs as indicated. D, Total RNA from primary mouse hepatocytes, HepG2, Hep3B cells, hepatic sinusoidal endothelial cells, and mouse spleen were subjected to RT-PCR using various primers as indicated. Data are representative of three independent experiments with similar results in all panels.

 
STAT6 is specifically activated by IL-4 and IL-13 (16, 17, 29), and the levels of IL-4 are markedly elevated in Con A-induced hepatitis (30). Therefore, we hypothesized that IL-4 may be the major factor responsible for activation of STAT6 in the liver and spleen after administration of Con A. To test this hypothesis, IL-4-deficient mice were used. As shown in Fig. 1A, Con A injection-induced STAT6 activation was significantly suppressed in IL-4-/- mice compared with wild-type mice. The levels of STAT6 proteins were unchanged, indicating that IL-4 is the major cytokine responsible for STAT6 activation in Con A-mediated hepatitis.

To further confirm IL-4 activation of STAT6 in hepatocytes and sinusoidal endothelial cells, we examined STAT6 activation and IL-4 receptor expression in primary mouse hepatocytes, human hepatoma cells, and primary sinusoidal endothelial cells. As shown in Fig. 1C, treatment with IL-4 induced strong STAT6 activation in primary mouse hepatocytes, HepG2 cells, Hep3B cells, and sinusoidal endothelial cells. Weak STAT3 activation was also detected in these cells; whereas no significant STAT1 activation was detected (Fig. 1C). RT-PCR in Fig. 1D showed that primary mouse hepatocytes, human HepG2 cells, and Hep3B cells express high levels of IL-4R{alpha} and IL-13R{alpha}1, whereas expression of IL-13R{alpha}2 and IL-2R{gamma} was undetectable. Interestingly, mouse sinusoidal endothelial cells and splenocytes express high levels of IL-4R{alpha}, IL-13R{alpha}1, IL-13R{alpha}2, and IL-2R{gamma}. Taken together, these findings indicate that IL-4 directly targets hepatocytes via type II IL-4 R (IL-4R{alpha}/IL-13R{alpha}1), and IL-4 targets hepatic sinusoidal cells and splenocytes via both type I IL-4 R (IL-4R{alpha}/IL-2R{gamma}) and type II IL-4 R (IL-4R{alpha}/IL-13R{alpha}1).

Activation of IL-4/STAT6 is essential for development of liver injury in Con A-induced hepatitis

To define the role of STAT6 activation, we compared Con A-induced hepatitis in STAT6-/- and STAT6+/+ mice. As shown in Fig. 2A, injection of Con A significantly elevated serum ALT activity in wild-type mice, but not in STAT6-/- mice. Examination of liver pathology showed massive necrosis in control wild-type mice, but not in STAT6-/- mice. A representative liver histology sample from Con A-injected STAT6+/+ and STAT6-/- mice after 9 h is shown in Fig. 2B.



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  		FIGURE 2. IL-4/STAT6 plays an essential role in Con A-induced murine hepatitis. A, Wild-type and STAT6-/- mice (5 per group) were injected with Con A (16 µg/kg); serum ALT activity was determined 9 and 24 h later. B, Photomicrographs of representative H&E-stained mouse livers treated with Con A for 24 h (original magnification: left, x100, right, x400). Yellow arrows indicate massive necrosis observed in the liver. C, Wild-type and STAT6-/- mice (5 per group) were injected with Con A (15 µg/kg), serum IL-4 levels were determined at various time points. D, Wild-type and IL-4-/- mice (5 per group) were injected with Con A (12 µg/kg); serum ALT activity was determined 9 h later. Data are representative of three independent experiments with similar results in all panels.

 
Because serum IL-4 levels are markedly elevated in Con A-induced hepatitis (31), we compared serum IL-4 levels in both wild-type and STAT-6-/- mice after injection of Con A. As shown in Fig. 2C, Con A injection-induced elevation of IL-4 was significantly attenuated in STAT-6-/- mice compared with wild-type mice. The role of IL-4 in Con A-induced liver damage was also examined in IL-4-deficient mice. As shown in Fig. 2D, serum ALT levels were significantly elevated after administration of Con A in wild-type mice but not in IL-4-/- mice. Examination of liver pathology showed massive necrosis in control wild-type mice, but not in IL-4-/- mice (data not shown). Taken together, these findings suggest that IL-4-induced STAT6 activation is vital for development of Con A-induced hepatitis.

Absence of liver injury in IL-4-/- and STAT6-/- mice after administration of Con A is not due to dysregulation of IFN-{gamma}/STAT1, IL-6/STAT3, and TNF-{alpha}/NF-{kappa}B signaling

We have previously shown that Con A-induced hepatitis is controlled by the mutual antagonism of STAT1 and STAT3 (12). Therefore, we examined whether the equilibrium of activity between STAT1 and STAT3 was dysregulated in STAT6-/- mice as a potential mechanism for the deleterious role of STAT6 in liver injury. As shown in Fig. 3A, Con A-mediated activation of STAT1 and STAT3 was slightly increased in STAT6-/- mice and IL-4-/- mice compared with their corresponding wild-type mice. Consistent with the slight increase in STAT1 and STAT3 activation, induction of the STAT1-controlled proapoptotic IFN regulatory factor-1 protein, and the STAT3-controlled anti-apoptotic Bcl-xL protein were also slightly induced in knockout mice. The difference in STAT1 and STAT3 activation between wild-type and IL-4-/- mice were minor (Fig. 3A), evidence that STAT1 and STAT3 are activated independently of IL-4.



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  		FIGURE 3. Absence of liver injury in STAT6-/- mice after administration of Con A is not due to dysregulation of IFN-{gamma}/STAT1, IL-6/STAT3, and TNF-{alpha}/NF-{kappa}B signaling. A, STAT6-/- and wild-type controls were injected with Con A (16 µg/kg); IL-4-/- mice, and wild-type controls were injected with Con A (12 µg/kg); at various time points, total liver protein extracts were immunoblotted using various Abs as indicated. Nuclear extracts were also analyzed by gel mobility shift analysis using a double-stranded oligonucleotide containing NF-{kappa}B binding sites. Data are representative of three independent experiments with similar results. B, STAT6-/- mice and wild-type controls were injected with Con A (16 µg/kg); IL-4-/- mice and wild-type controls were injected with Con A (12 µg/kg); at various time points, serum TNF-{alpha} levels were measured using ELISA. Values are shown as means ± SEM. from four mice at each time point. *, p < 0.01 in comparison with corresponding Con A-treated wild-type groups. p, phospho-.

 
Next we examined whether TNF-{alpha}/NF-{kappa}B signaling, which has been implicated in Con A-induced liver cell damage (32), was dysregulated in STAT6-/- and IL-4-/- mice. As shown in Fig. 3B, elevation of serum TNF-{alpha} levels after administration of Con A increased 2- to 3-fold in STAT6-/- and IL-4-/- mice compared with wild-type mice. Administration of Con A rapidly induced activation of NF-{kappa}B in wild-type mice as demonstrated by detection of phosphorylated I{kappa}B and NF-{kappa}B binding, which was not significantly down-regulated in either STAT6-/- mice or IL-4-/-. These findings indicate that TNF-{alpha}/NF-{kappa}B signaling was not significantly dysregulated in STAT6-/- and IL-4-/- mice.

Con A-mediated activation of T cells and NKT cells is not impaired in IL-4-/- and STAT6-/- mice

Activation of CD4+ and NKT cells, which plays an important roles in Con A-induced hepatitis (8, 28), was examined in IL-4-/- and STAT-6-/- mice. Activation of CD4+ T cells was evaluated by FACS of the activation marker CD69+. As shown in Fig. 4, administration of Con A caused a significant increase in the activation of CD4+ T cells (CD4+CD69+ double-positive cells in the upper right quadrant) in wild-type mice. Similar activation was also observed in STAT6-/- mice and IL-4-/- mice. NKT cells are rapidly activated and then depleted after administration of Con A (8, 28), demonstrating that NKT cell activation results in depletion, which could be an index of NKT cell activation. As shown in Fig. 4A, injection of Con A led to significant depletion of NKT cells (CD3+NK1.1+ double-positive cells in the upper right quadrant) in both wild-type B6 mice and IL-4-/- mice, whereas NK cells (NK1.1+CD3-) were not down-regulated. To further confirm NKT cell depletion, hepatic lymphocytes were also stained with DX5 and CD3 to detect NKT-like cells. Although the cluster of DX5+CD3+ cells is not clear as NK1.1+CD3+ cells, Fig. 4A shows that hepatic DX5+CD3+ cells markedly declined after administration of Con A. Similarly, administration of Con A also caused significant depletion of CD3+DX5+ NKT cells in wild-type BALB/c mice as well as BALB/c background STAT6-/- mice.



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  		FIGURE 4. Activation of CD4+ T and NKT cells after administration of Con A is not impaired in IL-4-/- and STAT6-/- mice. Wild-type and IL-4-/- mice (A), and wild-type and STAT6-/- mice (B) were injected with Con A; after 3, 6, or 9 h, hepatic lymphocytes were isolated. The surface expression of CD3+CD69+, NK1.1+CD3+, and DX5+CD3+ (for IL-4-/- and wild-type B6 mice), or DX5+CD3+ (for STAT6-/- and wild-type BALB/c mice) was analyzed by flow cytometry. A representative example of three independent experiments is shown. The upper right quadrant in each panel shows CD3+CD69+, NK1.1+CD3+, or DX5+CD3+ double-positive cells (percentage of the total hepatic MNCs).

 
Adoptive transfer of wild-type mouse hepatic MNCs completely restores Con A-induced liver injury in IL-4-/- mice but only weakly restores it in TAT6-/- mice

To further determine whether the absence of Con A-induced liver injury in IL-4-/- and STAT6-/- mice is due to defects in hepatic MNCs, adoptive transfer of wild-type hepatic MNCs was performed. As shown in Fig. 4A, adoptive transfer of wild-type murine hepatic MNCs, but not IL-4-/- mouse hepatic MNCs, completely restored Con A-induced liver injury in IL-4-/- mice, whereas the same adoptive transfer only weakly restored Con A-induced liver injury in STAT6-/- mouse. Liver histology showed that Con A injection did not cause significant necrosis in the livers of IL-4-/- mice but caused massive necrosis in these mice after adoptive transfer of wild-type hepatic MNCs (Fig. 5B). The same transfer did not restore Con A injection-induced massive necrosis in STAT6-/- mice (Fig. 5B). Next, we examined whether weak restoration of hepatocyte damage in STAT6-/- mouse after adoptive transfer of wild-type mouse hepatic MNCs was due to STAT6-/- hepatocyte resistance to wild-type mouse hepatic MNC killing. As shown in Fig. 5C, Con A-activated hepatic MNCs induced similar killing in both wild-type and STAT6-/- mouse hepatocytes. Moreover, we also examined the effects of IL-4 on hepatocyte death because IL-4 has been shown to induce apoptosis of several cell types (33, 34, 35). As shown in Fig. 4D, treatment with IL-4 did not significantly induce hepatocyte death.



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  		FIGURE 5. Adoptive transfer of wild-type hepatic MNCs completely restores Con A-induced liver injury in IL-4-/- mice but only weakly restores it in STAT-6-/- mice. A and B, Total hepatic MNCs (5 x 106 cells) isolated from wild-type C57BL/6 mice (WT) or IL-4-/- mice were injected into the livers of IL-4-/- mice, followed by injection i.v. of Con A (15 µg/g); or total hepatic MNCs (5 x 106 cells) isolated from wild-type BALB/c mice (WT) or STAT-6-/- mice were injected into the livers of STAT-6-/- mice, followed by injection i.v. of Con A (20 µg/g). Serum ALT levels were measured 9 h later (A) and liver histology was determined by H&E (B). Yellow arrows indicate massive necrosis observed in the liver. C, Hepatic MNCs were isolated from wild-type mice that were injected with Con A (20 µg/g) for 2 h, and their cytotoxicity was tested against wild-type mouse hepatocytes and STAT6-/- mouse hepatocytes at the indicated E:T ratios as described in Materials and Methods. D, Wild-type mouse hepatocytes were incubated with IL-4 or saline for various time points; cell death was determined by measuring the ALT levels as described in Materials and Methods.

 
STAT6 plays a critical role in Con A injection-mediated infiltration of neutrophils and eosinophils in the liver

Next, we compared Con A-induced infiltration of neutrophils, eosinophils, and macrophages in STAT6-/- and wild-type mouse livers. As shown in Fig. 6A, significant numbers of macrophages (Kupffer cells) were detected in normal mouse livers, but neutrophils and eosinophils were not detected. Con A injection caused significant macrophage death, declining macrophage numbers 12 and 24 h postinjection in wild-type mice. Con A injection-induced macrophage death was also observed in STAT6-/- mice but was less evident. In contrast to a decrease in macrophages, neutrophils and eosinophils significantly infiltrated the liver after injection of Con A. Infiltration in STAT 6-/- mice (Fig. 6, A and B) was markedly attenuated. Con A-induced infiltration of neutrophils and eosinophils in the liver was also significantly diminished in IL-4-/- mice compared with wild-type mice (data not shown). To further confirm eosinophil infiltration, we measured the EPO activity. As shown in Fig. 6C, adminstration of Con A significantly elevated EPO activity in the livers of wild-type mice but not in STAT6-/- mice.



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  		FIGURE 6. STAT6 plays a critical role in Con A injection-mediated infiltration of neutrophils and eosinophils in the liver. A, Wild-type BALB/c mice and STAT6-/- mice were injected with Con A (16 µg/g) for 12 and 24 h. Liver sections were prepared and subjected to H&E and immunohistochemistry using anti-MPO and anti-F4/80 Abs to detect neutrophils and macrophages, respectively. Eosinophils were detected by measuring cyanide-resistant EPO activity in situ and slides were counterstained with hematoxylin. B, Ten fields were randomly selected and positive cells were counted. Values are shown as means ± SEM from four mice at each time point. *, p < 0.05, **, p < 0.01 in comparison with corresponding Con A-treated wild-type group at the same time points. C, Wild-type BALB/c mice and STAT6-/- mice were injected with Con A (16 µg/g) for 12 and 24 h. Hepatic EPO activities were measured. Values are shown as means ± SEM from three mice at each time point. **, p < 0.01 in comparison with corresponding Con A-treated wild-type group at the same time points.

 
IL-4/STAT6 plays a critical role in Con A injection-induced elevation of eotaxins and IL-5

To define the molecular mechanisms underlying attenuation of Con A-induced infiltration of neutrophils and eosinophils in STAT6-/- and IL-4-/- mice compared with wild-type mice, expression of a variety of chemokines and adhesion molecules in the liver was examined. As shown in Fig. 7A, Con A injection significantly induced expression of a number of chemokines and adhesion molecules in the livers of wild-type mice. Induction of the majority remained unchanged or slightly enhanced in STAT6-/- mice compared with wild-type mice except for eotaxin-1 and eotaxin-2. Con A injection-mediated induction of eotaxin-1 and eotaxin-2 expression was significantly attenuated in STAT6-/- mice. Next, we compared serum levels of eotaxins in wild-type mice and knockout mice. As shown in Fig. 7B, Con A injection significantly elevated serum levels of eotaxins in wild-type mice, with peak effect occurring at 3 and 6 h after injection of Con A. This elevation was markedly attenuated in STAT 6-/- mice and IL-4-/- mice.



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  		FIGURE 7. STAT6 plays a critical role in Con A injection-induced expression of eotaxins in the liver. A, Wild-type BALL/c mice and STAT6-/- mice were injected with Con A (16 µg/g) for various time points. Total RNA samples were prepared and subjected to RT-PCR using various primers as indicated, or total liver protein extracts were prepared and immunoblotted with an anti-Mig Ab (*). B, Wild-type BALB/c mice and STAT 6-/- mice were injected with Con A (16 µg/g); wild-type B6 mice and IL-4-/- mice were injected with Con A (12 µg/g). At various time points, serum levels of eotaxins were measured using an ELISA kit. (three to four mice per time points). C, Primary hepatocytes and liver sinusoidal endothelial cells were isolated from wild-type and STAT6-/- mice and stimulated with murine IL-4 (50 ng/ml) for various time periods. Total RNA samples were prepared and subjected to RT-PCR using primers as indicated. D, Human hepatoma HepG2 cells were stimulated with human IL-4 (50 ng/ml) for various time periods. Total RNA samples were prepared and subjected to RT-PCR using primers as indicated. MCP, monocyte chemoattractant protein, MIP, macrophage-inflammatory protein; IP-10, IFN-{gamma}-inducible protein-10; PECAM-1, platelet endothelial cell adhesion molecule-1.

 
The above data showed that Con A injection induced expression of eotaxins in the liver; however, the liver cell type responsible for eotaxin production was not known. To answer this question, we examined the effects of IL-4 on the expression of eotaxins in mouse hepatocytes and hepatic sinusoidal endothelial cells. As shown in Fig. 7C, IL-4 treatment markedly induced expression of eotaxin-1 and eotaxin-2 in primary mouse hepatocytes and sinusoidal endothelial cells isolated from wild-type mice. This induction was not observed in cells isolated from STAT6-/- mice. Moreover, IL-4 induction of eotaxin-1, eotaxin-2, and eotaxin-3 mRNA expression was also observed in human hepatocellular carcinoma HepG2 cells (Fig. 7D).

Finally, we examined the serum levels of IL-5, which has been shown to play an important role in infiltration of eosinophils and liver injury in Con A-induced hepatitis (30). As shown in Fig. 8, administration of Con A markedly elevated serum levels of IL-5, which peaked at 6 and 12 h. In both STAT6-/- and IL-4-/- mice, the elevation was significantly suppressed compared with corresponding controls.



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  		FIGURE 8. IL-4/STAT6 plays a critical role in Con A injection-induced elevation of serum IL-5 levels. Wild-type BABL/c mice and STAT 6-/- mice were injected with Con A (16 µg/g); wild-type B6 mice and IL-4-/- mice were injected with Con A (12 µg/g). At various time points, serum levels of IL-5 were measured using an ELISA kit (three to four mice per time points).

 
Eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration

The role of IL-5 in Con A-induced liver injury and eosinophil infiltration has been previously demonstrated (30); however, the role of eotaxins in this model is not clear. To test the biological significance of elevated eotaxins in Con A-mediated hepatitis, the anti-eotaxin neutralizing Ab was used. As shown in Fig. 9, administration of an anti-eotaxin neutralizing Ab markedly attenuated Con A-induced liver injury (Fig. 9A), infiltration of neutrophils and eosinophils (Fig. 9, B and C). Furthermore, hepatic EPO activity in anti-eotaxin-treated group after Con A injection was significantly lower than that in IgG-treated group (Fig. 9D), which further confirmed that infiltration of eosinophils in the liver was attenuated after anti-eotaxin treatment.



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  		FIGURE 9. Blocking eotaxins attenuates Con A-induced liver injury and leukocyte infiltration in the liver. Wild-type BABL/c mice were injected with Con A (16 µg/g). After 30 min, anti-eotaxin antibody (0.5 mg/mice i.v.) or anti-IgG control Ab were injected. A, Serum ALT levels were measured 12 h after Con A injection. Values are shown as means ± SEM from three mice. *, p < 0.05 in comparison with the IgG-treated group. B, Liver sections were prepared and stained with an anti-MPO Ab to detect neutrophils. Eosinophils were detected by measuring cyanide-resistant EPO activity in situ and slides were counterstained with hematoxylin. (C) Ten fields were randomly selected and positive cells were counted. Values are shown as means ± SEM from three mice. *, p < 0.05 and **, p < 0.01 in comparison with corresponding IgG-treated group. D, Hepatic EPO activities were measured 12 h after Con A injection. Values are shown as means ± SEM from three mice. *, p < 0.05 in comparison with the IgG-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Administration of Con A rapidly induces STAT6 activation in the liver and the spleen. Such activation is almost completely abolished in IL-4-/- mice (Fig. 1), suggesting that IL-4 is the major cytokine responsible for STAT6 activation in this model. IL-4/STAT6 signaling is essential for development of Con A-mediated liver damage because IL-4-/- and STAT6-/- mice are resistant to Con A-induced hepatitis (Fig. 2). Activation of IFN-{gamma}/STAT1, IL-6/STAT3, and TNF-{alpha}/NF-{kappa}B signaling pathways, which has been reported to tightly control Con A-mediated hepatitis (13, 32), was slightly enhanced or remained unchanged in IL-4-/- and STAT6-/- mice (Fig. 3), suggesting that absence of liver injury in IL-4-/- and STAT6-/- mice after administration of Con A is not due to dysregulation of these cytokines and their signal cascades. Currently, it is generally believed that the role of IL-4 in Con A-mediated hepatitis is mediated via enhancing the cytotoxicity of NKT cells against hepatocytes (22). The important role of IL-4/STAT6 in NKT-mediated cytotoxicity against hepatocytes is also evidenced in a hepatitis model of SOCS-1-deficient mice (21). Although we (Fig. 5C) and others (21, 22) demonstrate that Con A-activated liver NKT cells are cytotoxic against hepatocytes in vitro, whether NKT cells also actively participate in killing hepatocytes in vivo is not known. Several lines of evidence suggest that although NKT cells play a key role in releasing several cytokines (such as IL-4, IL-5), they may only play a minor role in killing hepatocytes in vivo. 1) Liver NKT cells were rapidly depleted 3 h after injection of Con A (Fig. 4 and Reference28); however, massive liver injury was observed 9 h later. 2) Activated liver MNCs have similar ability in killing hepatocytes from wild-type mice and STAT 6-/- mice, but adoptive transfer wild-type liver MNCs only weakly restored Con A-induced liver injury, suggesting that activated liver MNCs are unable to cause massive hepatocyte damage in STAT6-/- mice. 3) Administration of IL-4 has been shown to completely restore Con A-induced liver injury in {beta}2-microglobulin-deficient mice lacking NKT cells (24). 4) Thre is no evidence suggesting that NKT cells directly contact hepatocytes in vivo. Finally, 5), the ratio of NKT cells/hepatocytes in the liver is less than 1:100, which is much lower than the ratio of NKT cells to hepatocytes (50:1, 40:1), used in vitro cytotoxicity assay. In contrast, neutrophils and eosinophils were accumulated in the liver after injection of Con A, and accumulation of these cells was located in necrotic areas. Such accumulation is not just a paraphenomenon, because depletion of these cells markedly suppressed Con A-induced liver injury (30, 36). Taken together, these findings suggest that enhancement liver NKT cytotoxicity is probably not the major mechanism contributing to the critical role of IL-4 in vivo in Con A-mediated hepatitis. Here, we demonstrate that the pivotal role of IL-4 in Con A-mediated hepatitis is mediated via a STAT6-dependent pathway by inducing expression of eotaxins in hepatocytes and sinusoidal endothelial cells and by inducing IL-5 expression in NKT cells. Subsequently, neutrophils and eosinophils were attracted into the liver, resulting in liver injury. We have integrated these findings in a new model (summarized in Fig. 10) that depicts the cellular and molecular mechanisms underlying the pivotal involvement of IL-4/STAT6 in T cell-mediated hepatitis.



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  		FIGURE 10. A model illustrating the pivotal role of IL-4/STAT6 in Con A-induced hepatitis. Administration of Con A activates NKT cells to produce IL-4. IL-4 then stimulates hepatocytes and sinusoidal endothelial cells to secret eotaxins and enhances IL-5 production via a STAT6-dependent pathway, followed by attracting neutrophils and eosinophils into the liver and leading to hepatitis. IL-4-mediated enhancement of the cytotoxicity of NKT cells may also play a minor role in killing hepatocytes.

 
In our model, IL-4 produced by Con A-activated NKT cells directly stimulates hepatocytes and sinusoidal endothelial cells to produce eotaxins and NKT cells to produce IL-5 in the liver in a STAT6-dependent mechanism. Con A injection-induced elevation of eotaxins and IL-5 are markedly suppressed in IL-4-/- and STAT6-/- mice (Fig. 7, A and B, and Fig. 8), suggesting that IL-4/STAT6 is responsible for eotaxin and IL-5 induction in vivo in Con A-mediated hepatitis. Data from Louis et al. (30) suggest that NKT cells are the primary source of IL-5 production in Con A-mediated hepatitis. Because elevation of IL-5 is significantly attenuated in IL-4-/- and STAT6-/- mice (Fig. 8), it is likely that NKT cells produce IL-5 via IL-4- and STAT6-dependent mechanisms. In addition to NKT cells, T cells, mast cells, and eosinophils also produce IL-5 (37). Whether these cells also contribute to elevation of IL-5 in Con A-mediated hepatitis requires further studies. Treatment with IL-4 significantly induces expression of eotaxins in both primary mouse hepatocytes and sinusoidal endothelial cells (Fig. 7C), which is mediated in a STAT6-dependent pathway because IL-4 induction of eotaxins is not observed in STAT6-deficient mouse hepatocytes and sinusoidal endothelial cells (Fig. 7C). In addition, IL-4 induces expression of eotaxins in human hepatoma HepG2 cells (Fig. 7D). Finally, we show that hepatocytes express type II IL-4R (IL-4R{alpha}/IL-13R{alpha}1) and sinusoidal endothelial cells express both type I IL-4R (IL-4R{alpha}/IL-2R{gamma}) and type II IL-4R (IL-4R{alpha}/IL-13R{alpha}1) (Fig. 1D), which provide a molecular basis for direct targeting of these cells by IL-4.

In the next step of our model, IL-4/STAT6-induced eotaxins and IL-5 synergistically induce accumulations of neutrophils and eosinophils in the liver, leading to hepatitis. The critical effect of IL-5 in liver injury and eosinophil infiltration in Con A-mediated hepatitis has been previously demonstrated in IL-5-deficient mice and by depletion of IL-5 with neutralizing IL-5 Abs (30). It is believed that IL-5 is responsible for eosinophil precursor maturation, proliferation, and trafficking within tissues through CC chemokine receptors (CCR3) (37, 38, 39). Although the physiological significance of eotaxins has been extensively investigated (40, 41, 42, 43, 44), its role in liver disease remains poorly characterized. Here we demonstrate that eotaxins play a vital role in liver injury in Con A-mediated hepatitis. As shown in Fig. 9, depletion of eotaxins markedly attenuated Con A-induced liver injury and infiltration of leukocytes. Eotaxin is a CC chemokine that was originally identified as a potent and selective stimulus for the migration of eosinophils from the small blood vessels into the tissues by acting on CCR3 (40, 41, 42, 43, 44). Subsequently, eotaxin has also been found to stimulate migration of other leukocytes and lymphocytes via other chemokine receptors (45, 46, 47). Here we showed that blocking eotaxin with a neutralizing Ab not only suppressed infiltration of eosinophils but also attenuated infiltration of neutrophils, suggesting that elevation of eotaxins is important in attracting both eosinophils and neutrophils into the liver in Con A-mediated hepatitis. Increasing evidence suggests that IL-5 and eotaxin are the two most important factors responsible for regulating eosinophil locomotion and are known to cooperatively recruit eosinophils to inflamed tissues via targeting different steps (48, 49, 50). Thus, it is plausible that IL-5 and eotaxins synergistically induce the infiltration of eosinophils into the liver in Con A-mediated hepatitis.

In contrast to eotaxins and IL-5, induction of various chemokines, adhesion molecules (Fig. 7A), and cytokines (Fig. 3) in the liver after administration of Con A was not attenuated in IL-4-/- mice (data not shown) and STAT 6-/- mice (Fig. 7A). These chemokines and cytokines have been shown to be involved in inflammation (51, 52, 53). They may also play an important role in infiltration of neutrophils in the livers of wild-type mice as well as STAT6-/- mice after injection of Con A because significant infiltration of neutrophils was still observed in STAT6-/- mice (Fig. 6). Although significant infiltration of neutrophils and induction of many chemokines were detected in the livers of Con A-treated IL-4-/- and STAT6-/- mice, liver injury was markedly attenuated in these mice, suggesting that infiltration of neutrophils alone is not enough for inducing massive liver injury. In contrast, the absence of eosinophils correlates with a lack of liver injury in IL-4-/- and STAT6-/- mice, indicating that eosinophils are essential for Con A-indued liver injury. Indeed, it has been shown that depletion of eosinophils completely prevents Con A-induced liver damage (30), whereas depletion of neutrophils only partially suppresses liver injury in this model (36). Eosinophils cause tissue damage through the release of highly cytotoxic granular proteins, cytokines, chemokines, eosinophil cationic protein, EPO, eosinophil protein, eosinophil-derived neurotoxin, major basic protein, lipid mediators (such as LTC4 and platelet-activating factor), and toxic oxygen metabolites (54, 55, 56), which could all contribute to liver tissue injury in Con A-mediated hepatitis.

Elevated IL-4 and eosinophil infiltration have been reported in a wide variety of human liver diseases, including chronic hepatitis C (57, 58, 59), drug-induced liver disease (60, 61), primary biliary cirrhosis (62, 63), and liver transplantation (64). It is very likely that activation of IL-4/STAT6 and the consequent infiltration of eosinophils are also involved in a variety of human liver diseases. Therefore, modulation of the IL-4/STAT6 signaling pathway in vivo could offer a novel approach in the treatment of these human liver disorders.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Bin Gao, Section on Liver Biology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Park Building, Room 120, 12420 Parklawn Drive, MSC 8115, Bethesda, MD 20892. Email address: bgao{at}mail.nih.gov Back

2 Abbreviations used in this paper: JAK, Janus kinase; SOCS, suppressor of cytokine signaling; NKT, natural killer T; MNC, mononuclear cell; EPO, eosinophil peroxidase; MPO, neutrophil myeloperoxidase; Mig, monocyte IFN-{gamma}-inducible protein; ALT, alanine aminotransaminase; AST, aspartate aminotransferase. Back

Received for publication September 26, 2002. Accepted for publication July 16, 2003.


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 Results
 Discussion
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