Novel Role of IL-13 in Fibrosis Induced by Nonalcoholic Steatohepatitis and Its Amelioration by IL-13R-Directed Cytotoxin in a Rat Model

Nonalcoholic steatohepatitis (NASH), the most common cause of chronic liver fibrosis, progresses to cirrhosis in up to 20% of patients. We report that hepatic stellate cells (HSC) in sinusoidal lesions of liver of patients with NASH express high levels of high-affinity IL-13R (IL-13Rα2), which is colocalized with smooth muscle actin, whereas fatty liver and normal liver specimens do not express IL-13Rα2. HSCs engineered to overexpress IL-13Rα2 respond to IL-13 and induce TGFB1 promoter activity and TGF-β1 production. We also developed NASH in rats by feeding a choline-deficient l-amino acid diet. These rats developed liver fibrosis as assessed by H&E staining, Masson’s trichrome and Sirius red staining, and hydroxyproline assays. Treatment of these rats with IL-13R-directed cytotoxin caused a substantial decline in fibrosis and liver enzymes without organ toxicity. These studies demonstrate that functional IL-13Rα2 are overexpressed in activated HSCs involved in NASH and that IL-13 cytotoxin ameliorates pathological features of NASH in rat liver, indicating a novel role of this cytotoxin in potential therapy.

N onalcoholic fatty liver disease is one of the most common causes of chronic liver disease in North America (1)(2)(3). It is estimated that 3% of US population has nonalcoholic steatohepatitis (NASH), 2 which can progress to cirrhosis in up to 20% of patients (2,4). Advanced age, obesity, insulin resistance, hypertension, and diabetes have been associated with a higher risk of developing cirrhosis in NASH cases (5,6). Given the current epidemic of obesity, particularly in children, it has been reported that the national health care burden related to cirrhosis due to NASH will continue to increase (7,8). Genetic factors including hereditary hemochromatosis, angiotensinogen, and TGF-␤1 genes have been also associated with progression of fibrosis to NASH (9,10).
The end results of many inflammatory and tissue repair responses are the development of fibrosis (11,12). Fibrosis is of particular concern in numerous persistent inflammatory diseases driven by Th2 responses leading to active fibrogenesis, including those resulting from pathogens such as Leishmania donovani and Schistosoma mansoni parasites and hepatitis viruses (9,(12)(13)(14).
IL-13 is a Th2 cytokine that plays a central role in various inflammatory diseases (15). IL-13 seems to induce tissue fibrosis by stimulating and activating TGF-␤1 (16). IL-13 binds to two known chains, IL13R␣1 and IL13R␣2. IL13R␣1 chain is a low-affinity receptor that forms a heterodimer with IL-4R␣ chain to form a high-affinity IL-13R and mediate signal transduction through the JAK-STAT-6 pathway (17,18). IL13R␣2, on the other hand, binds IL-13 with high affinity and was not found to mediate signaling even though it was internalized after binding to IL-13 (19). IL-13R␣2 was shown to act as a decoy receptor in murine system (9,20). We recently reported that IL-13 can signal through IL13R␣2, in a STAT-6-independent manner, in murine macrophage cell line and that IL-13 is involved in fibrosis through the TGF-␤1 pathway (11). However, it is not known whether IL-13 and its receptor are involved in NASH.
Hepatic stellate cells (HSC) play a central role in the development and resolution of liver fibrosis (21)(22)(23). Several types of cytokines, e.g., IL-6, IFN-␥, TGF-␤1, TNF-␣, endothelin-1, and platelet-derived growth factor, which regulate the inflammatory response to injury, cause HSC trans differentiation from the quiescent phenotype to the activated myofibroblast-like (␣-smooth muscle actin (␣-SMA)-expressing) phenotype (9). HSCs are responsible for the majority of extracellular protein deposition in liver fibrosis, and recovery from established fibrosis can occur through the apoptosis of HSC and subsequent reduction in liver collagen (24,25). Targeting activated HSC, not quiescent HSC, could be an appropriate strategy to eliminate established liver fibrosis.
Because IL-13R are overexpressed on cancer cells, we have developed a recombinant fusion protein, IL-13 cytotoxin (IL13-PE38), composed of human IL-13 and a mutated form of Pseudomonas exotoxin to target these receptors (26). After binding to IL-13R on the cell surface, IL13-PE38 prevents the initiation of protein synthesis, leading to cell death through necrotic and apoptotic pathways (27). IL13-PE38 mediates antitumor effects in IL-13R␣2-positive cancer cells in vitro and animal models of human cancer (28). After the successful completion of several phase 1 and 2 clinical trials with IL13-PE38 in patients with recurrent glioblastoma, a multicenter phase 3 clinical trial (PRECISE study) is now completed (29).
To determine whether IL-13 plays a role, we examined the expression of IL-13R␣2 in liver biopsy samples from subjects with NASH and fatty liver disease. We also examined whether HSCs express IL13R␣2, respond to IL-13 and IL13-PE38 mediates IL-13R-specific cytotoxicity to HSC. We developed an animal model of NASH induced by choline-deficient L-amino acid-defined (CDAA) diet (30) and examined the role of IL-13R in vivo on liver fibrosis after treating rats with IL13-PE38. Finally, we studied the mechanism of IL-13 induced fibrosis through the TGF-␤1 pathway in activated HSCs.

Materials and Methods
Cell culture, reagents, tissue specimens, and serum samples Hepatocytes and HSCs (Sciencell) were cultured in hepatocyte or HSC medium containing growth factors. The LI-90 cell line exhibiting human HSC characteristics was kindly provided by Human Science Cell Bank (Saitama, Japan). Clinical liver biopsy samples and sera from NASH, fatty liver, and normal pathologically diagnosed subjects were obtained from Yokohama City University Hospital (Yokohama, Japan). rIL13-PE38 was generated as previously described (26).

RT-PCR and real-time TaqMan RT-PCR
Total RNA was extracted from clinical samples and cell lines with TRIZOL reagent (Invitrogen) followed by treatment with DNase I (Promega). The total RNA was reverse transcribed as described earlier (31,32). For neutralization of TGF-␤ activity, HSCs were preincubated with anti-TGF-␤ Abs (clone 1D11; R&D Systems) for 1 h before the addition of IL-13.
Quantitative PCR reactions were performed using the ABI PRISM 7700 sequence detection system (Applied Biosystems). cDNA (50 ng) was added to a reaction volume (30 l) containing 1x TaqMan PCR master mix, IL-13R␣2specific probe/primers set, and GAPDH or ␤-actin-specific probe (5Ј-VIC, 3Ј-MGB)/primers mix (Applied Biosystems). Gene expression was normalized to GAPDH to calculate the fold change in gene expression.

Confocal microscopy
Tissue sections were deparaffinized by xylene and washed with various concentrations of ethanol and PBS. Sections were incubated with anti-␣-SMA mAb (Calbiochem) or isotype control (IgG) followed by FITC-labeled secondary Ab and incubated with Cy3-conjugated anti-IL-13R␣2 mAb (Cell Sciences and Amersham Biosciences). Sections were washed in PBS and mounted in Vectashield (Vector Laboratories) for confocal microscopy.

Flow cytometry
For the detection of IL-13R␣2 expression on HSCs, the adherent HSCs were detached and stained with anti-IL-13R␣2 Ab (R&D Systems) or IgG1 (Sigma-Aldrich) isotype control followed by FITC-labeled secondary Ab and analyzed by FACScan (BD Biosciences). HSCs were stimulated in the presence or absence of human TNF-␣, TGF-␤1, IL-4, or IL-13 alone or in their combinations for 48 h.

Protein synthesis inhibition assay
The in vitro cytotoxic activity of IL13-PE38 was measured by the inhibition of protein synthesis as described earlier (31). All assays were done in quadruplicate, and the concentrations of IL13-PE38 causing 50% inhibition of protein synthesis (IC 50 ) were calculated. The inhibition of protein synthesis is directly proportional to cell death.

Cell proliferation inhibition studies by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay
HSCs were seeded in 96-well plates with cytokines for 48 h, washed with PBS three times, and treated with various concentrations of IL13-PE38 for 72 h. After treatment, the cells were incubated with MTT (0.5 mg/ml; Sigma-Aldrich) in medium at 37°C for 2 h and then with isopropanol at room temperature for 1 h. The optical density was determined at 595 nm using Spectra Max 5 (Molecular Devices).

Animals
Male Fischer 344 rats, 6 wk of age and weighing 140 -150 g, were maintained in a barrier facility on high-efficiency particulate air-filtered racks. All animal studies were conducted under an approved protocol in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. CDAA diet was obtained in pellets (Dyets), and its composition is described in Ref. 34. Normal diet group animals were fed a control (choline-supplemented L-amino acid-defined; CSAA) diet.

Treatment protocol
Twenty rats fed with CDAA diet were divided into control and IL13-PE38 treatment groups. IL13-PE38 (50 g/kg) was administered i.p. three times on alternate days, and organs were collected 7 days posttreatment. Liver samples were harvested on wks 8 and 12 after starting the diet.

Histology and immunohistochemical examination
The right lobes of all rat livers in 5-m-thick sections were fixed in 10% formalin for 24 h, embedded in paraffin, and then processed for Masson trichrome and Sirius red staining. The activated stellate cells were immunohistochemically assessed using IL-13R␣2 and ␣-SMA mAbs. To avoid nonspecific reaction by endogenous biotin and peroxidase in hepatocytes, we used Dako Envision Plus system (Dako) with diaminobenzidine as chromogen and counterstained with hematoxylin. ␣-SMAand Sirius redpositive areas in the liver were quantified using a Provis microscope (Olympus) equipped with a charge-coupled device camera, and subjected to computer-assisted image analysis with image J (National Institutes of Health, Bethesda, MD) software. Ten randomly selected different areas per specimen were analyzed. The area of Sirius red-and ␣SMA-positive cells was calculated as the percentage of the total area of the specimen.

Hydroxyproline assay
Liver collagen concentration was determined by measuring hydroxyproline protein content in liver samples using a modified method reported previously (35). Briefly, liver samples were homogenized in PBS, pH 7.4, and then digested in 6 N HCl (final concentration, 6 N) for 18 h at 110°C. After filtration of the hydrolysate through a 0.45-m pore size Millipore filter, samples were evaporated in a rotary evaporator. Five microliters of citrateacetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydroxide, and 1.2% glacial acetic acid, pH 6.0) and 100 l of chloramine-T solution (282 mg of chloramine-T, 2 ml of n-propanol, 2 ml of H 2 O, and 16 ml of citrate-acetate buffer) were added to samples and left at room temperature for 20 min. Next, 100 l of Ehrlich's solution (Sigma-Aldrich) were added to each sample, and the samples were incubated for 15 min at 65°C. Samples were cooled for 10 min and read at 550 nm using Spec-traMax M5 (Molecular Devices). Hydroxyproline (Sigma-Aldrich) concentrations from 0 to 200 g/ml were used to generate a standard curve.

Luciferase assay
A plasmid containing the human TGFB1 promoter linked to luciferase reporter gene was provided by J. Nam (National Cancer Institute, National Institutes of Health). This plasmid contains Ϫ436/ϩ55 bp flanking the transcription start site of the human TGFB1 promoter. HSC (0.5 ϫ 10 6 cells/ml) in a 96-well plate were transiently transfected with the luciferase reporter plasmid pTGF-␤ 1 -luciferase (250 ng) and the pSV-␤-galactosidase vector (25 ng; Promega) by overnight incubation with HVJ-E loaded with plasmid vector. The transfected cells were placed in medium containing 10% TGF-␤1-depleted human serum and stimulated for 24 h. Cell lysates were analyzed for luciferase activity (Promega) and ␤-galactosidase activity (Applied Biosystems). Data are means Ϯ SD of triplicate determinations from three independent experiments. ␤-Galactosidase activity was used for normalization of TGF-␤1 promoter activity in the luciferase assay.

Statistical analysis
Cytokine serum levels in patients with NASH, fatty liver, and healthy donors were analyzed by ANOVA. The fibrotic areas in tissue sections from control and IL13-PE38-treated groups were also analyzed by ANOVA. Statistical significance between groups was calculated by Student's t test.

IL-13R␣2 is expressed in NASH but not in normal and fatty liver tissues
Clinical biopsy tissue sections from NASH liver showed moderate to high levels of IL-13R␣2 mRNA in 10 of 14 samples, whereas fatty liver and normal liver specimens did not (Fig. 1A). Confocal microscopy showed IL-13R␣2 and ␣-SMA expression in sinusoidal lesions in the NASH liver samples and when both images were merged, IL-13R␣2 and ␣-SMA coexpressed in HSC (Fig. 1B). All 14 tissue samples showed histological evidence of fibrosis. In sharp contrast to and consistent with RT-PCR results, IL-13R␣2 expression was not detected in sinusoidal lesions of normal and fatty liver specimens (Fig. 1B). Because HSCs show strong ␣-SMA expression when trans-differentiated from the quiescent phenotype to the activated myofibroblast-like phenotype, these re-sults indicate that IL-13R␣2 expression was also detected on the activated HSC, but not in quiescent HSC or hepatocytes.

TNF-␣, TGF-␤1, and IL-13 cytokines are increased in sera of patients with NASH but not in healthy subjects
As TNF-␣, TGF-␤1, and IL-13 are implicated in inflammation and fibrosis, we analyzed sera from 17 patients with NASH, 11 with fatty liver, and 15 healthy subjects that showed a significant increase in TNF-␣ levels in patients with NASH (3.37 Ϯ 1.48 pg/ml) compared with healthy controls (0.25 Ϯ 0.12 pg/ml; p Ͻ 0.001) FIGURE 1. IL-13R␣2 expression in NASH specimens and serum cytokines levels in subjects with NASH. A, Total RNA extracted from 14 patients with NASH, 4 with fatty liver, and 2 normal livers were analyzed for IL-13R␣2 mRNA expression by RT-PCR. Renal cell carcinoma cell line (PMRCC) and GAPDH served as positive and internal controls, respectively. B, All NASH and fatty liver sections were stained for IL-13R␣2 (red) and ␣-SMA (green). One of the representative specimens is shown. ϫ400. C, Serum TNF-␣, TGF-␤1, and IL-13 levels in 17 NASH, 11 fatty liver, and 15 healthy donors were assayed in triplicate by ELISA. Each circle indicates the average of three different determinations. p values for significant difference between groups were determined by ANOVA and Student's t test. and individuals with fatty liver disease (2.53 Ϯ 0.98 pg/ml; p Ͻ 0.001; Fig. 1C). Serum TGF-␤1 levels were also significantly increased in patients with NASH (62.75 Ϯ 27.13 pg/ml) compared with healthy controls (2.50 Ϯ 3.20 pg/ml, p Ͻ 0.001), but not with fatty liver disease (103.38 Ϯ 45.74 pg/ml) (Fig. 1C). Serum IL-13 levels almost doubled in patients with NASH (20.62 Ϯ 8.22 pg/ml) compared with healthy control (10.69 Ϯ 4.14 pg/ml; p ϭ 0.008) or individuals with fatty liver disease (11.00 Ϯ 4.47 pg/ml; p ϭ 0.019; Fig. 1C).

TNF-␣ and TGF-␤1 up-regulate IL-13R␣2 expression in HSC
Because TNF-␣ and TGF-␤1 levels were elevated in serum of patients with NASH, we examined whether these cytokines enhanced the expression of IL-13R␣2 in HSC and normal hepatocytes cultured with TNF-␣ and/or TGF-␤1. IL-13R␣2 mRNA expression was strongly induced by each cytokine. The combination of both cytokines further increased the mRNA band intensity compared with either cytokine alone. In contrast to HSC, IL-13R␣2 mRNA expression was not induced by these cytokines in normal hepatocytes ( Fig. 2A). Similarly, type I collagen mRNA was also up-regulated by TNF-␣ and TGF-␤1, and their combination showed the highest up-regulation. As expected, a renal cell carcinoma cell line (PMRCC), a positive control, did not express type I collagen. As a control, IL-2 did not up-regulate either IL-13R␣2 or collagen type I mRNA expression. The induction of IL-13R␣2 mRNA expression by cytokines was further confirmed by real-time RT-PCR analysis (Fig. 2B). Again, the The up-regulation of IL-13R␣2 chain was confirmed by flow cytometric analysis (Fig. 2C). Similar to mRNA, protein expression was up-regulated by TNF-␣ or TGF-␤1, and the highest up-regulation was observed with both cytokines. IL-2 did not enhance IL-13R␣2 protein expression (data not shown). Consistent with a previous report, both IL-4 and IL-13 increased IL-13R␣2 mRNA expression and protein levels, but increase of IL-13R␣2 protein on the cell membrane was not observed (36). The combination of IL-4 and TNF-␣ or IL-13 and TNF-␣ did not further increase IL-13R␣2 protein expression com-pared with TNF-␣ alone (data not shown). Comparable results were obtained in the experiments using the HSC LI-90 cell line (data not shown).

Role of IL-13R␣2 and other receptor chains in IL-13 signaling in HSC
As increased IL-13 level in the serum and IL-13R␣2 expression in the liver specimens are detected in NASH patients, we examined whether IL-13 signal is activated in HSC expressing IL-13R␣2. In addition, we examined the expression and roles of IL-13R␣1 and IL-4R␣ chains in HSCs. HSCs transfected with IL-13R␣2 expressed IL-13R␣2 mRNA and when stimulated with IL-13, . Effect of cytokines on TGFB1 promoter activity, TGF-␤1 secretion, and signaling through IL-13R␣2 in HSCs. A, RT-PCR shows increased expression of IL-13R␣2 mRNA in IL-13R␣2-transfected HSCs compared with parent and mock-transfected HSCs. However, expression of IL-13R␣1 and IL-4R␣ mRNA was not changed. B, IL-13 (10 ng/ml) and TGF-␤1 (2 ng/ml) induce TGFB1 promoter activity (as determined by TGF-␤1 luciferase assay) in IL-13R␣2positive HSCs after 24 h of stimulation and expressed as a ratio of stimulated vs unstimulated cells. C, TGF-␤1 secreted by IL-13R␣2-positive HSCs stimulated with IL-13 (10 ng/ml) and TGF-␤1 (2 ng/ml) was measured by ELISA. Data are expressed as means Ϯ SD from three independent experiments. D, Type I collagen mRNA was induced by IL-13 (10 ng/ml) treatment in IL-13R␣2-positive HSCs, and this induction was inhibited by preincubation with anti-TGF-␤ Ab (10 g/ml). IL-13 did not induce type I collagen mRNA in mock HSCs. Cells were also incubated with TGF-␤1 (10 ng/ml) as a positive control. TGFB1 promoter activity was significantly induced. In contrast, TGFB1 promoter activity was not induced by IL-13 in IL-13R␣2negative normal or mock vector-transfected HSCs (Fig. 3B). In contrast, TGF-␤1 induced TGFB1 promoter activity in both mockand IL-13R␣2-transfected HSC. The mRNA of IL-13R␣1 and IL-4R␣ chains were also expressed in mock-and IL13R␣2-transfected HSC cells; however, no difference in expression was observed between both types of cells (Fig. 3A). Thus, the IL-13R␣2 chain is predominantly involved in IL-13-induced TGFB1 promoter activation. ELISA confirmed the increased level of TGF-␤1 production by the stimulation of IL-13 in IL-13R␣2-positive HSCs, whereas it was not detected in normal and mock vector-transfected HSC (Fig. 3C). IL-4 stimulation did not increase TGF-␤1 production in both types of HSC, indicating specificity for IL-13 and IL-13R␣2. For a positive control, HSC mock-and IL-13R␣2-transfected HSCs were stimulated with TGF-␤1. The stimulation of these cells with TGF-␤1 did not show a significant difference in TGF-␤1 production by either HSC mock (2274 Ϯ 296.9 pg/ml)-or IL-13R␣2-transfected HSCs (2309 Ϯ 172.5 pg/ml). Taken together, these results suggest that IL-13 signals through IL-13R␣2 in IL-13R␣2-positive HSC but not in IL-13R␣2-negative HSCs and that the type II IL-4R complex does not participate in TGF-␤1 production.
To confirm that IL-13/IL-13R␣2 signaling induces fibrosis through TGF-␤1 production, we measured collagen type I mRNA in IL-13-and  TGF-␤1-stimulated mock HSCs and IL-13R␣2-transfected HSCs. IL-13 stimulation was blocked by preincubation with anti-TGF-␤ Ab (Fig. 3D). This result showed that IL-13 increased collagen type I mRNA in IL-13R␣2-transfected HSCs and that anti-TGF-␤ Ab prevented the collagen type I induced by IL-13. In contrast, IL-13 and anti-TGF-␤ Ab did not affect collagen type I induction in mock HSCs. Therefore, in IL-13R␣2positive HSC, IL-13 can induce collagen type I through the TGF-␤ pathway. Our results further indicate that the TGF-␤1 level induced by IL-13 treatment was enough to produce collagen type I, one of the critical components of liver fibrosis.

IL13-PE38 is cytotoxic to HSCs expressing IL-13R␣2 but not to HSC or hepatocytes devoid of IL-13R␣2
Because TNF-␣ and TGF-␤1 induced IL-13R␣2 expression in HSCs at both the mRNA and protein levels, we examined whether IL-13R␣2 are functional and whether these cells become targets for IL13-PE38. HSCs were least sensitive to the cytotoxic effect of IL13-PE38. However, HSCs stimulated with TNF-␣, TGF-␤1, or both cytokines become susceptible to IL13-PE38 (Fig. 4A). The IC 50 values in TNF-␣-and TGF-␤1-treated cells and those of and both cytokines were 87, 104, and 12 ng/ml, respectively compared with Ͼ1000 ng/ml in control HSCs. Consistent with the lack of effect of IL-2 on IL-13R␣2 expression, IL-2-treated cells did not show cytotoxicity to IL13-PE38. The IL13-PE38 activity was neutralized by an excess of IL-13, indicating a receptor-specific effect (not shown). HSCs transfected with IL-13R␣2 showed the highest sensitivity to IL13-PE38 (IC 50 0.94 ng/ml) compared with mock HSC (Fig. 4B) or normal hepatocytes (IC 50 1000 ng/ml) (Fig. 4D). Protein synthesis inhibition results were confirmed by MTT assay. IL13-PE38 inhibited cell proliferation in a dose-dependent manner of HSCs stimulated by TNF-␣ plus TGF-␤1 but not of normal untreated HSC (Fig. 4C). These data indicate that specific cytotoxicity and anti-cell proliferation by IL13-PE38 are mainly regulated through binding of the IL-13 portion of IL13-PE38 to the IL-13R␣2 chain.  A and B) and after 12 wk of diet (C and D) were stained by Masson's trichrome technique to observe liver fibrosis. A, No treatment group 8 wk after CDAA diet. B, IL13-PE38-treated (50 g/kg on alternate days for three treatments) group after 8 wk of CDAA diet. C, No treatment group after 12 wk CDAA diet. D, IL13-PE38-treated group after 12 wk of CDAA diet. E, Hydroxyproline assay was performed in liver homogenates to quantify liver fibrosis. The level of hydroxyproline in the liver was increased by CDAA diet for 8 and 12 wk compared with basal diet. IL13-PE38 treatment significantly decreased the level of hydroxyproline. Each bar represents the mean Ϯ SD of quadruplicate determinations and the assay was repeated twice. ‫,ء‬ p Ͻ 0.001.

IL13-PE38 eliminates IL-13R␣2-positive fibrotic cells and hepatic fibrosis in rat model of NASH
During the pathogenesis of human NASH, hepatic steatosis is followed by inflammation, oxidative damage, and fibrosis in liver (37). To simulate these clinical features of NASH, we fed rats a CDAA diet to induce liver fibrosis as reported previously (30,34). Rats were fed CDAA diet for 8 and 12 wk, and then liver samples were analyzed for IL-13R␣2 expression by immunohistochemistry. Fibrotic livers stained with Masson's trichrome showed strong to moderate staining for IL-13R␣2 in areas of fibrosis, whereas no staining was seen in hepatocytes, portal vein, and bile duct (Fig.  5A). Staining of similar sections with isotype control Ab did not show any specific staining (Fig. 5B).
Masson's trichrome staining showed reduction of fibrotic areas (Fig. 6, B and D) in CDAA-fed rats for 8 and 12 wk and when treated with IL13-PE38 (50 g/kg on alternate days for three treatments on wks 9 and 13), whereas untreated control rat livers showed distinct fibrosis (Fig. 6, A and C). Livers from rats fed with CDAA diet showed extensive accumulation of fibrosis as confirmed by Sirius red staining (not shown). Quantitative analysis showed that the average Sirius red-positive area in liver sections of CDAA-fed rats for 8 and 12 wk was reduced by 73% (from 7.21% to 1.93%) and 76% (from 9.84% to 2.35%), respectively, by IL13-PE38 (Table I). The average of ␣-SMA-stained areas was also reduced by 79% and 70% in the 8-and 12-wk models, respectively (Table I). Thus, IL13-PE38 significantly ( p Ͻ 0.01) reduced the fibrosis area in the livers of CDAA-fed rats. To further confirm these results, we performed quantitative hydroxyproline assays in rat livers fed with CDAA and control CSAA diets. As shown in Fig. 6E, the level of hydroxyproline in livers of CDAA diet fed rats was significantly increased at both 8 wk and 12 wk of feeding compared with control diet (CSAA)-fed rats ( p Ͻ 0.001). The IL13-PE38 treatment of CDAA-fed rats significantly decreased the levels of hydroxyproline at both time points ( p Ͻ 0.001). These results suggest that IL13-PE38 decreased the liver fibrosis and support our histological analysis of fibrosis in NASH liver (Fig. 6, A-D).

Serum chemical changes and organ histology in control and CDAA-fed rats and after IL13-PE38 treatment
There was no significant difference between the CDAA-and normal diet (CSAA)-fed rats in terms of the total amount of calories consumed. Body weights of rats in both groups were also similar (data not shown). Consistent with NASH-induced pathology, rats fed with the CDAA diet for 8 wk showed an increased serum alanine aminotransferase, aspartase aminotransferase, and alkyllysophospholipid level of 627 Ϯ 99 U/L, 500 Ϯ 68 U/L, and 492 Ϯ 39 U/L, compared with 64 Ϯ 11 U/L, 98 Ϯ 22 U/L, and 165 Ϯ 32 U/L, in rats fed with CSAA diet, respectively. However, rats fed with the CDAA diet for 8 wk and then treated with IL13-PE38, showed reversal of enzyme elevation normalizing to baseline levels within 2 wk of treatment (data not shown).
Histological examination at necropsy did not reveal organ toxicity in heart, lung, kidney, and spleen of IL13-PE38-treated rats with NASH (data not shown).

Discussion
We demonstrate that IL-13R␣2 is expressed in sinusoidal lesions of liver from patients with NASH. These receptors coexpressed with ␣-SMA in HSC. In vitro data show that IL-13 is functional in HSCs and it signals through IL-13R␣2 leading to activation of TGFB1 promoter activity, TGF-␤1 production and fibrosis. Interestingly, IL13-PE38 was highly cytotoxic to activated HSCs expressing IL-13R␣2 induced by TNF-␣ or TGF-␤1, but not to quiescent IL-13R␣2-negative HSCs or hepatocytes. CDAA-fed rats developed NASH with prominent areas of fibrosis and fibrotic cells expressing IL-13R␣2. Treatment of these animals with IL13-PE38 significantly decreased fibrosis.
IL-13R␣2 expression has been reported on fibroblasts in human fibrotic diseases including idiopathic interstitial pneumonia and schistosomiasis (38,39). We demonstrate for the first time that the IL-13R␣2 is expressed in liver specimens obtained from patients with NASH. Resting HSC fibroblasts did not express IL-13R␣2; however, TGF-␤1 and TNF-␣ induced high levels of IL-13R␣2. This is consistent with a high level of expression of IL-13R␣2 in activated fibroblasts in livers with NASH. IL-13 mediates tissue fibrosis by regulating the production and activation of TGF-␤1, a known mediator of fibrosis (16). Using an IL-13-transgenic mouse that overexpresses IL-13 in the lung, Elias and colleagues showed that IL-13 is a potent inducer of matrix metalloproteinase-9 and TGF-␤1. They also showed that when TGF-␤1 activity is neutralized, collagen deposition in IL-13-transgenic mice is substantially decreased indicating a direct functional link between IL-13 and TGF-␤1. Fichtner-Feigl et al. (11) showed that IL-13 induces TGF-␤1 promoter and secretion in hemopoietic cells through IL-13R␣2 and leading to fibrosis. However, in the context of schistosomiasis and helminth parasitic diseases, IL-13 can induce fibrosis in the absence of TGF-␤1 and in this situation overexpressed IL-13R␣2 acts as a soluble decoy receptor that decreases fibrosis (40). In this case, alternatively activated macrophages (M2) induced by IL-13 in vivo may play a critical role in fibrogenesis; however, in fibrosis induced by silica, M2 cells are involved in the early inflammatory stage of silicosis and establishment of the fibrotic process is not associated with M2 polarization (41). In addition, as IL-13R␣2 is overexpressed in M2 macrophages, it is possible that Kupffer cells may also be involved in a Rats were fed the CDAA diet for 8 or 12 wk. After treatment, rats were sacrificed, and livers were stained with ␣-SMA and Sirius red stain. ␣-SMA-positive and Sirius red-positive areas were measured as described in Materials and Methods. b Mean Ϯ SD. Numbers in parentheses, percent. c p Ͻ 0.01 vs no treatment group.
fibrosis, although it is not known at what stage of pathogenesis these cells may be involved. Furthermore, the production of TNF-␣ is one of the earliest events in many types of liver fibrotic diseases, triggering the production of other cytokines that together recruit inflammatory cells, kill hepatocytes, and initiate a healing process that includes fibrogenesis (42). Although the molecular mechanism of development of fibrosis may be different in individual disease situations, high-level expression of surface IL-13R␣2 is detected in fibrotic cells in many fibrotic diseases. Because IL-13R␣2 is expressed in activated HSCs but not on quiescent HSCs and hepatocytes in NASH liver specimens, IL13-PE38 targeting IL-13R␣2 could be a potential antifibrotic agent. The mechanism of up-regulation of IL-13R␣2 in NASH fibroblasts is not clear. It is hypothesized that a multitude of events are involved in this up-regulation and fibrosis. We observed increased serum levels of TNF-␣, IL-13, and TGF-␤1 in patients with NASH. Increased serum levels of TNF-␣ and TGF-␤1 in patients with NASH and fatty liver disease have also been reported along with increased gene expression of TGF-␤1 in liver tissues of patients with NASH (43,44). Because our in vitro data demonstrated that IL-13R␣2 expression in HSCs is strongly induced by TNF-␣ and TGF-␤1, it is hypothesized that IL-13R␣2 expression in HSCs might be induced by these cytokines in vivo. As IL-13R␣2 signaling leads to the activation of TGF-␤1 and TGF-␤1 activates expression of IL-13R␣2. These results indicate that a vicious cycle of mutual activation is possible in HSCs as shown in Fig. 7. Thus, IL-13R␣2 could be one of the key molecules that participate in the development of liver fibrosis in patients with NASH. Therefore, IL-13R␣2 could be an important therapeutic target for the treatment of TGF-␤1-mediated fibrosis such as NASH. Because IL13-PE38 is cytotoxic to IL-13R␣2-positive target (fibroblasts) cells, it breaks the vicious circle and eliminates TGF-␤1-induced fibrosis.
IL13-PE38 is extremely effective in attenuating S. mansoni egginduced pulmonary granuloma formation, chronic fungal-induced allergic airway disease, and idiopathic interstitial pneumonia (38,39,45). In addition, IL13-PE38 is effective in eliminating malignant tumors derived from glioblastoma, AIDS-associated Kaposi sarcoma, ovarian carcinoma, and head and neck cancer in vitro and in vivo (28,31,46,47). IL-13R␣2 chain binds to IL13-PE38 to exert its cytotoxicity, but not to cells with any expression or lowlevel expression (48). Several clinical trials in patients with recurrent glioblastoma were initiated in which IL13-PE38 up to a concentration of 0.5 g/ml (total, 45.4 g) was extremely well tolerated by normal brain devoid of IL-13R␣2 (29,49).
Although IL13-PE38 mediated remarkable antifibrotic effects, no visible toxicity or features such as weight loss and inactivity or lethargy were observed in rats receiving treatment. Previous IL13-PE38 toxicity studies have reported that none of the mice and rats showed any change in hepatic transaminases, hematological toxicity, or vascular leak syndrome at maximum tolerated doses (28). Similar study in cynomolgus monkeys after i.v. injection of IL13-PE38 (50 g/kg for 5 days) caused reversible elevation of hepatic transaminases and creatinine kinase with subsequent decline to normal levels. In the current study, IL13-PE38 normalized the elevated liver enzymes caused by fibrosis and caused no histological changes in vital organs.
In conclusion, HSCs in NASH liver specimens express high levels of functional IL-13R␣2, respond to IL-13, induce TGFB1 promoter activity, and cause TGF-␤1 production. Because IL-13R␣2-positive cells were diminished by IL13-PE38 treatment, it may be an important therapeutic target for the treatment of TGF-␤1-mediated fibrosis such as NASH. Therefore, further studies should be performed to explore the potential of IL13-PE38 in elimination of fibrosis and treatment of patients with NASH.