Novel Role of IL-13 in Fibrosis Induced by Nonalcoholic
Steatohepatitis and Its Amelioration by IL-13R-Directed
Cytotoxin in a Rat Model
Takeshi Shimamura,* Toshio Fujisawa,* Syed R. Husain,* Mitomu Kioi,* Atsushi Nakajima,†
and Raj K. Puri*1
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-13R?2), which is colocalized with smooth muscle actin, whereas fatty liver and normal liver specimens do not express IL-13R?2.
HSCs engineered to overexpress IL-13R?2 respond to IL-13 and induce TGFB1 promoter activity and TGF-?1 production. We also
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-13R?2 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. The Journal of Immunology, 2008, 181: 4656–4665.
atohepatitis (NASH),2which can progress to cirrhosis in up to 20% of
patients (2, 4). Advanced age, obesity, insulin resistance, hyperten-
sion, and diabetes have been associated with a higher risk of devel-
oping 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
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 re-
sponses 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, 12–14).
IL-13 is a Th2 cytokine that plays a central role in various in-
flammatory diseases (15). IL-13 seems to induce tissue fibrosis by
stimulating and activating TGF-?1 (16). IL-13 binds to two known
onalcoholic fatty liver disease is one of the most common
causes of chronic liver disease in North America (1–3). It is
estimated that 3% of US population has nonalcoholic ste-
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 (21–23). 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)-ex-
pressing) 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 sub-
sequent reduction in liver collagen (24, 25). Targeting activated HSC,
not quiescent HSC, could be an appropriate strategy to eliminate es-
tablished 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 Pseudo-
monas 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 apo-
ptotic 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 glioblas-
toma, a multicenter phase 3 clinical trial (PRECISE study) is now
To determine whether IL-13 plays a role, we examined the ex-
pression of IL-13R?2 in liver biopsy samples from subjects with
*Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Thera-
pies, Center for Biologics Evaluation and Research, Food and Drug Administration,
National Institutes of Health, Bethesda, MD 20892; and†Gastroenterology Division,
Yokohama City University Graduate School of Medicine, Yokohama, Japan
Received for publication May 1, 2008. Accepted for publication July 29, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Address correspondence and reprint requests to Dr. Raj K. Puri, Tumor Vaccines
and Biotechnology Branch, Division of Cellular and Gene Therapies, Center for Bio-
logics Evaluation and Research, Food and Drug Administration, National Institutes of
Health, Building 29B, Room 2NN20 HFM-735, 29 Lincoln Drive, Bethesda, MD
20892. E-mail address: firstname.lastname@example.org
2Abbreviations used in this paper: NASH, nonalcoholic steatohepatitis; HSC, hepatic
stellate cell; ?-SMA, ?-smooth muscle actin; IL13-PE38, recombinant fusion protein
IL-13 cytotoxin; CDAA, choline-deficient L-amino acid; MTT, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide; CSAA, control (normal) choline-supple-
mented L-amino acid.
The Journal of Immunology
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
(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 (Pro-
mega). 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
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?2-
specific probe/primers set, and GAPDH or ?-actin-specific probe (5?-VIC,
3?-MGB)/primers mix (Applied Biosystems). Gene expression was normal-
ized to GAPDH to calculate the fold change in gene expression.
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-la-
beled 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
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.
TNF-?, TGF-?1, and IL-13 levels were measured in triplicate using
ELISAs (Quantikine ELISA; R&D Systems) according to the manufactur-
er’s instructions (33).
Protein synthesis inhibition assay
The in vitro cytotoxic activity of IL13-PE38 was measured by the inhibi-
tion 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 (IC50) were calculated. The inhibition of protein syn-
thesis is directly proportional to cell death.
Cell proliferation inhibition studies by 3-(4,5-dimethylthiazol-2-
yl)-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).
Male Fischer 344 rats, 6 wk of age and weighing 140–150 g, were main-
tained in a barrier facility on high-efficiency particulate air-filtered racks.
All animal studies were conducted under an approved protocol in accor-
dance 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-supple-
mented L-amino acid-defined; CSAA) diet.
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 immu-
nohistochemically 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. ?-SMA- and Sirius red-
positive 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.
Liver collagen concentration was determined by measuring hydroxyproline
protein content in liver samples using a modified method reported previ-
ously (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 citrate-
acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% sodium hydrox-
ide, 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 H2O, 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) concen-
trations from 0 to 200 ?g/ml were used to generate a standard curve.
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 ?436/?55 bp flanking the transcription start
site of the human TGFB1 promoter. HSC (0.5 ? 106cells/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 trans-
fected cells were placed in medium containing 10% TGF-?1-depleted human
serum and stimulated for 24 h. Cell lysates were analyzed for luciferase ac-
tivity (Promega) and ?-galactosidase activity (Applied Biosystems). Data are
means ? SD of triplicate determinations from three independent experiments.
?-Galactosidase activity was used for normalization of TGF-?1 promoter ac-
tivity in the luciferase assay.
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 sig-
nificance between groups was calculated by Student’s t test.
IL-13R?2 is expressed in NASH but not in normal and fatty
Clinical biopsy tissue sections from NASH liver showed moderate
to high levels of IL-13R?2 mRNA in 10 of 14 samples, whereas
4657The Journal of Immunology
fatty liver and normal liver specimens did not (Fig. 1A). Confocal
microscopy showed IL-13R?2 and ?-SMA expression in sinusoi-
dal 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 in-
crease in TNF-? levels in patients with NASH (3.37 ? 1.48 pg/ml)
compared with healthy controls (0.25 ? 0.12 pg/ml; p ? 0.001)
in NASH specimens and serum cyto-
kines 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 pos-
itive and internal controls, respec-
tively. B, All NASH and fatty liver
sections were stained for IL-13R?2
(red) and ?-SMA (green). One of the
representative specimens is shown.
?400. C, Serum TNF-?, TGF-?1, and
IL-13 levels in 17 NASH, 11 fatty
liver, and 15 healthy donors were as-
sayed 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 Stu-
dent’s t test.
4658IL-13 CYTOTOXIN AMELIORATES FIBROSIS IN NASH
and individuals with fatty liver disease (2.53 ? 0.98 pg/ml; p ?
0.001; Fig. 1C). Serum TGF-?1 levels were also significantly in-
creased in patients with NASH (62.75 ? 27.13 pg/ml) compared
with healthy controls (2.50 ? 3.20 pg/ml, p ? 0.001), but not with
fatty liver disease (103.38 ? 45.74 pg/ml) (Fig. 1C). Serum IL-13
levels almost doubled in patients with NASH (20.62 ? 8.22 pg/ml)
compared with healthy control (10.69 ? 4.14 pg/ml; p ? 0.008) or
individuals with fatty liver disease (11.00 ? 4.47 pg/ml; p ?
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 pa-
tients 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 cyto-
kines further increased the mRNA band intensity compared with ei-
ther 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-?
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 fur-
ther confirmed by real-time RT-PCR analysis (Fig. 2B). Again, the
13R?2 expression in HSCs. A, Total
RNA extracted from HSCs and hepa-
tocytes stimulated by TNF-? (50 ng/
ml), TGF-?1 (10 ng/ml), or their
combination were analyzed for IL-
13R?2 mRNA expression by RT-
PCR. Renal cell carcinoma cell line
(PMRCC), GAPDH, and IL-2 served
as positive, internal, and negative
controls, respectively. B, IL-13R?2
mRNA expression was quantified by
quantitative RT-PCR. Columns repre-
sent means ? SE of triplicate deter-
minations. C, HSCs cultured with
TNF-? (50 ng/ml), TGF-?1 (10 ng/
ml), and their combination were
stained with anti-IL-13R?2 mAb and
analyzed by flow cytometry. Repre-
sentative histograms from three sepa-
rate experiments are shown.
4659 The Journal of Immunology
combination of both TNF-? and TGF-?1 caused the highest expres-
sion of IL-13R?2 mRNA, and there was no up-regulation by IL-2.
The up-regulation of IL-13R?2 chain was confirmed by flow cy-
tometric analysis (Fig. 2C). Similar to mRNA, protein expression 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
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 ex-
pressed IL-13R?2 mRNA and when stimulated with IL-13,
TGFB1 promoter activity, TGF-?1 se-
cretion, 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-trans-
fected 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 pro-
moter activity (as
TGF-?1 luciferase assay) in IL-13R?2-
positive HSCs after 24 h of stimulation
and expressed as a ratio of stimulated
vs unstimulated cells. C, TGF-?1 se-
creted 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 ?
SD from three independent experi-
ments. 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 preincuba-
tion 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
Effect of cytokines on
4660IL-13 CYTOTOXIN AMELIORATES FIBROSIS IN NASH
TGFB1 promoter activity was significantly induced. In contrast,
TGFB1 promoter activity was not induced by IL-13 in IL-13R?2-
negative normal or mock vector-transfected HSCs (Fig. 3B). In
contrast, TGF-?1 induced TGFB1 promoter activity in both mock-
and IL-13R?2-transfected HSC. The mRNA of IL-13R?1 and IL-
4R? chains were also expressed in mock- and IL13R?2-trans-
fected HSC cells; however, no difference in expression was ob-
served between both types of cells (Fig. 3A). Thus, the IL-13R?2
chain is predominantly involved in IL-13-induced TGFB1 pro-
moter 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 stimu-
lation of these cells with TGF-?1 did not show a significant difference
in TGF-?1 production by either HSC mock (2274 ? 296.9 pg/ml)- or
IL-13R?2-transfected HSCs (2309 ? 172.5 pg/ml). Taken together,
these results suggest that IL-13 signals through IL-13R?2 in IL-
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
stimulated or stimulated with TNF-? (50 ng/ml), TGF-?1 (10 ng/ml), IL-2 (50 ng/ml), or the combination of TNF-? and TGF-?1 were incubated with
various concentrations of IL13-PE38 (0–1000 ng/ml), and inhibition of protein synthesis was measured. Bars represent means ? SD of quadruplicate
determinations, and the assay was repeated twice. B, HSCs (1 ? 104) and IL13R?2-overexpressing HSCs were incubated with various concentrations of
IL13-PE38. C, HSCs (1 ? 104) were not incubated or incubated with the combination of TNF-? (50 ng/ml) and TGF-?1 (10 ng/ml). MTT assay was
performed for determination of antiproliferative activity of IL13-PE38. Bars represent means ? SD of triplicates. D, Protein synthesis measurement of
hepatocytes incubated with IL13-PE38 (0–1000 ng/ml).
Cytotoxicity and antiproliferative effects of IL13-PE38 in IL-13R?2-positive HSCs and normal hepatocytes. A, HSCs (1 ? 104) not
in fibrotic areas but not in normal hepato-
cytes. Liver samples collected from
with IL-13R?2 (A) and mouse IgG1 (B)
Ab. Each section was stained by Mas-
son’s trichrome technique. ?40.
Expression of IL-13R?2
4661The Journal of Immunology
TGF-?1-stimulated mock HSCs and IL-13R?2-transfected HSCs. IL-13
This result showed that IL-13 increased collagen type I mRNA in IL-
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?2-
way. Our results further indicate that the TGF-?1 level induced by IL-13
ponents 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
IC50values in TNF-?- and TGF-?1-treated cells and those of and
both cytokines were 87, 104, and 12 ng/ml, respectively compared
with ?1000 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 neu-
tralized 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 (IC500.94 ng/ml) compared with mock
HSC (Fig. 4B) or normal hepatocytes (IC501000 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 cyto-
toxicity and anti-cell proliferation by IL13-PE38 are mainly reg-
ulated through binding of the IL-13 portion of IL13-PE38 to the
rates liver fibrosis in a rat model of
NASH. Liver sections after 8 wk of
CDAA diet (A and B) and after 12 wk
of diet (C and D) were stained by
Masson’s trichrome technique to ob-
serve 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 as-
say was performed in liver homoge-
nates 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 de-
creased the level of hydroxyproline.
Each bar represents the mean ? SD of
quadruplicate determinations and the
assay was repeated twice. ?, p ?
4662IL-13 CYTOTOXIN AMELIORATES FIBROSIS IN NASH
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 fol-
lowed 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 immunohistochemis-
try. 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 treat-
ments 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 con-
firmed 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 ? 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 ? 0.001). The
IL13-PE38 treatment of CDAA-fed rats significantly decreased
the levels of hydroxyproline at both time points (p ? 0.001).
These results suggest that IL13-PE38 decreased the liver fibro-
sis 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 nor-
mal 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 alkyl-
lysophospholipid level of 627 ? 99 U/L, 500 ? 68 U/L, and
492 ? 39 U/L, compared with 64 ? 11 U/L, 98 ? 22 U/L, and
165 ? 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 base-
line levels within 2 wk of treatment (data not shown).
Histological examination at necropsy did not reveal organ tox-
icity in heart, lung, kidney, and spleen of IL13-PE38-treated rats
with NASH (data not shown).
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. Inter-
estingly, IL13-PE38 was highly cytotoxic to activated HSCs ex-
pressing IL-13R?2 induced by TNF-? or TGF-?1, but not to qui-
escent 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 neutral-
ized, 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 schis-
tosomiasis and helminth parasitic diseases, IL-13 can induce
fibrosis in the absence of TGF-?1 and in this situation overex-
pressed 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 fibro-
genesis; however, in fibrosis induced by silica, M2 cells are in-
volved in the early inflammatory stage of silicosis and establish-
ment of the fibrotic process is not associated with M2 polarization
(41). In addition, as IL-13R?2 is overexpressed in M2 macro-
phages, it is possible that Kupffer cells may also be involved in
Table I. Effect of IL13-PE38 on ?-SMA-positive and Sirius red-positive areaa
Treatment (n ? no. of rats)
CDAA diet 8 wk
No treatment (n ? 4)
IL13-PE38 (n ? 4)
Normal CDAA diet 12 wk
No treatment (n ? 4)
IL13-PE38 (n ? 4)
7.21 ? 0.67 (100)
1.93 ? 0.45c(26.8)
5.87 ? 0.55 (100)
1.23 ? 0.38c(21.0)
9.84 ? 1.24 (100)
2.35 ? 0.45c(23.9)
7.14 ? 0.70 (100)
2.12 ? 0.41c(29.7)
aRats 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.
bMean ? SD. Numbers in parentheses, percent.
cp ? 0.01 vs no treatment group.
4663The Journal of Immunology
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 individ-
ual 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 fibro-
blasts 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 pa-
tients 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 sig-
naling 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 treat-
ment 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 egg-
induced pulmonary granuloma formation, chronic fungal-induced
allergic airway disease, and idiopathic interstitial pneumonia (38,
39, 45). In addition, IL13-PE38 is effective in eliminating malig-
nant 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 low-
level expression (48). Several clinical trials in patients with recur-
rent glioblastoma were initiated in which IL13-PE38 up to a con-
centration 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 tox-
icity, 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 el-
evated 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 elim-
ination of fibrosis and treatment of patients with NASH.
We thank Drs. Ramjay Vatsan and Andrew Byrnes for critical reading of
the manuscript. We are grateful to Pamela Dover for technical help, pro-
curing reagents, and general support for these studies.
The authors have no financial conflict of interest.
1. Clark, J. M., F. L. Brancati, and A. M. Diehl. 2003. The prevalence and etiology
of elevated aminotransferase levels in the United States. Am. J. Gastroenterol. 98:
2. Neuschwander-Tetri, B. A., and S. H. Caldwell. 2003. Nonalcoholic steatohepa-
titis: summary of an AASLD Single Topic Conference. Hepatology 37:
3. Kim, W. R., R. S. Brown, Jr., N. A. Terrault, and H. El Serag. 2002. Burden of
liver disease in the United States: summary of a workshop. Hepatology 36:
4. McCullough, A. J. 2004. The clinical features, diagnosis and natural history of
nonalcoholic fatty liver disease. Clin. Liver Dis. 8: 521–533.
5. Angulo, P., J. C. Keach, K. P. Batts, and K. D. Lindor. 1999. Independent pre-
dictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology
6. Dixon, J. B., P. S. Bhathal, and P. E. O’Brien. 2001. Nonalcoholic fatty liver
disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the se-
verely obese. Gastroenterology 121: 91–100.
7. Hedley, A. A., C. L. Ogden, and C. L. Johnson. 2004. Prevalence of overweight
and obesity among US children, adolescents, and adults, 1999–2002. J. Am. Med.
Assoc. 291: 2847–2850.
8. Maggioni, G. 1994. Will we have geriatric pediatric one day? Minerva Pediatr.
9. Bataller, R., and D. A. Brenner. 2005. Liver fibrosis. J. Clin. Invest. 115:
10. Day, C. P. 2004. The potential role of genes in nonalcoholic fatty liver disease.
Clin. Liver Dis. 8: 673–691.
11. Fichtner-Feigl, S., W. Strober, K. Kawakami, R. K. Puri, and A. Kitani. 2006.
IL-13 signaling through the IL-13?2 receptor is involved in induction of TGF-?1
production and fibrosis. Nat. Med. 12: 99–106.
12. Wynn, T. A. 2004. Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat. Rev.
Immunol. 4: 583–594.
13. De Lalla, C., G. Galli, L. Aldrighetti, R. Romeo, M. Mariani, A. Monno, S. Nuti,
M. Colombo, F. Callea, S. A. Porcelli, et al. 2004. Production of profibrotic
cytokines by invariant NKT cells characterizes cirrhosis progression in chronic
viral hepatitis. J. Immunol. 173: 1417–1425.
14. Shi, Z., A. E. Wakil, and D. C. Rockey. 1997. Strain specific differences in mouse
hepatic wound healing are mediated by divergent T helper cytokine responses.
Proc. Natl. Acad. Sci. USA 94: 10663–10668.
15. de Vries, J. E. 1998. The role of IL-13 and its receptor in allergy and inflam-
matory responses. J. Allergy Clin. Immunol. 102: 165–169.
16. Lee, C. G., R. J. Homer, Z. Zhu, S. Lanone, X. Wang, V. Koteliansky,
J. M. Shipley, P. Gotwals, P. Noble, Q. Chen, et al. 2001. Interleukin-13 induces
tissue fibrosis by selectively stimulating and activating transforming growth fac-
tor ?1. J. Exp. Med. 194: 809–821.
17. Murata, T., J. Taguchi, R. K. Puri, and H. Mohri. 1999. Sharing of receptor
subunits and signal transduction pathway between the IL-4 and IL-13 receptor
system. Int. J. Hematol. 69: 13–20.
TGF-?1. IL-13R?2 is activated by TGF-?1, and TGF-?1 is activated by
IL-13 through IL-13R?2 in hepatic stellate cells. Because IL13-PE38 is
cytotoxic to IL-13R?2-positive targets (e.g., fibroblasts), it breaks the vi-
cious circle and eliminates TGF-?1-induced fibrosis.
Vicious circle in HSC induced by IL-13, IL-13R?2, and
4664IL-13 CYTOTOXIN AMELIORATES FIBROSIS IN NASH
18. Kelly-Welch, A. E., E. M. Hanson, M. R. Boothby, and A. D. Keegan. 2003. Download full-text
Interleukin-4 and interleukin-13 signaling connections maps. Science 300:
19. Kawakami, K., J. Taguchi, T. Murata, and R. K. Puri. 2001. The interleukin-13
receptor ?2 chain: an essential component for binding and internalization but not
for interleukin-13-induced signal transduction through the STAT6 pathway.
Blood 97: 2673–2679.
20. Wynn, T. A., M. Hesse, N. G. Sandler, M. Kaviratne, K. F. Hoffmann,
M. G. Chiaramonte, R. Reiman, A. W. Cheever, J. P. Sypek, and
M. M. Mentink-Kane. 2004. P-selectin suppresses hepatic inflammation and fi-
brosis in mice by regulating interferon ? and the IL-13 decoy receptor. Hepa-
tology 39: 676–687.
21. Friedman, S. L. 2000. Molecular regulation of hepatic fibrosis, an integrated
cellular response to tissue injury. J. Biol. Chem. 275: 2247–2250.
22. Alcolado, R., M. J. Arthur, and J. P. Iredale. 1997. Pathogenesis of liver fibrosis.
Clin. Sci. 92: 103–112.
23. Iredale, J. P., R. C. Benyon, J. Pickering, M. McCullen, M. Northrop, S. Pawley,
and M. J. Arthur. 1998. Mechanisms of spontaneous resolution of rat liver fi-
brosis: hepatic stellate cell apoptosis and reduced hepatic expression of metallo-
proteinase inhibitors. J. Clin. Invest. 102: 538–549.
24. Friedman, S. L., and M. B. Bansal. 2006. Reversal of hepatic fibrosis: fact or
fantasy? Hepatology 43: S82–S88.
25. Wright, M. C., R. Issa, D. E. Smart, N. Trim, G. I. Murray, J. N. Primrose,
M. J. Arthur, J. P. Iredale, and D. A. Mann. 2001. Gliotoxin stimulates the
apoptosis of human and rat hepatic stellate cells and enhances the resolution of
liver fibrosis in rats. Gastroenterology 121: 685–698.
26. Debinski, W., N. I. Obiri, I. Pastan, and R. K. Puri. 1995. A novel chimeric
protein composed of IL-13 and Pseudomonas exotoxin is highly cytotoxic to
human carcinoma cells expressing receptors for IL-13 and IL-4. J. Biol. Chem.
27. Kawakami, M., K. Kawakami, and R. K. Puri. 2002. Apoptotic pathways of cell
death induced by an interleukin 13 receptor-targeted recombinant cytotoxin in
head and neck cancer cells. Cancer Immunol. Immunother. 50: 691–700.
28. Husain, S. R., and R. K. Puri. 2003. Interleukin-13 receptor-directed cytotoxin for
malignant glioma therapy: from bench to bedside. J. Neurooncol. 65: 37–48.
29. Kunwar, S., M. D. Prados, S. M. Chang, M. S. Berger, F. F. Lang,
J. M. Piepmeier, J. H. Sampson, Z. Ram, P. H. Gutin, R. D. Gibbons, et al. 2007.
Direct intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in re-
current malignant glioma: a report by the Cintredekin Besudotox Intraparenchy-
mal Study Group. J. Clin. Oncol. 25: 837–844.
30. Koppe, S., and R. M. Green. 2003. Pentoxifylline attenuates methionine choline
deficient (MCD) diet induced steatohepatitis. Gastroenterology 124(Suppl.
31. Kioi, M., M. Kawakami, T. Shimamura, S. R. Husain, and R. K. Puri. 2006.
Interleukin-13 receptor ?2 chain: a potential biomarker and molecular target for
ovarian cancer therapy. Cancer 107: 1407–1418.
32. Murata, T., N. I. Obiri, W. Debinski, and R. K. Puri. 1997. Structure of IL-13
receptor: analysis of subunit composition in cancer and immune cells. Biochem.
Biophys. Res. Commun. 238: 90–94.
33. Mawdsley, J. E., E. Joel, M. G. Macey, R. M. Feakins, L. Langmead, and
D. S. Rampton. 2006. The effect of acute psychologic stress on systemic and
rectal mucosal measures of inflammation in ulcerative colitis. Gastroenterology
34. Kawaguchi, K., I. Sakaida, M. Tsuchiya, K. Omori, T. Takami, and K. Okita.
2004. Pioglitazone prevents hepatic steatosis, fibrosis, and enzyme-altered lesions
in rat liver cirrhosis induced by a choline-deficient L-amino acid-defined diet.
Biochem. Biophys. Res. Commun. 315: 187–195.
35. Jakubzick, C., E. S. Choi, B. H. Joshi, M. P. Keane, S. L. Kunkel, R. K. Puri, and
C. M. Hogaboam. 2003. Therapeutic attenuation of pulmonary fibrosis via tar-
geting of IL-4- and IL-13-responsive cells. J. Immunol. 171: 2684–2693.
36. Yasunaga, S., N. Yuyama, K. Arima, H. Tanaka, S. Toda, M. Maeda, K. Matsui,
C. Goda, Q. Yang, Y. Sugita, et al. 2003. The negative-feedback regulation of the
IL-13 signal by the IL-13 receptor ?2 chain in bronchial epithelial cells. Cytokine
37. Farrell, G. C., and C. Z. Larter. 2006. Nonalcoholic fatty liver disease: from
steatosis to cirrhosis. Hepatology 43: S99–S112.
38. Jakubzick, C., E. S. Choi, K. J. Carpenter, S. L. Kunkel, H. Evanoff,
F. J. Martinez, K. R. Flaherty, G. B. Toews, T. V. Colby, W. D. Travis, et al.
2004. Human pulmonary fibroblasts exhibit altered interleukin-4 and interleu-
kin-13 receptor subunit expression in idiopathic interstitial pneumonia.
Am. J. Pathol. 164: 1989–2001.
39. Jakubzick, C., S. L. Kunkel, B. H. Joshi, R. K. Puri, and C. M. Hogaboam. 2002.
Interleukin-13 fusion cytotoxin arrests Schistosoma mansoni egg-induced pulmo-
nary granuloma formation in mice. Am. J. Pathol. 161: 1283–1297.
40. Kaviratne, M., M. Hesse, M. Leusink, A. W. Cheever, S. J. Davies,
J. H. McKerrow, L. M. Wakefield, J. J. Letterio, and T. A. Wynn. 2004. IL-13
activates a mechanism of tissue fibrosis that is completely TGF-? independent.
J. Immunol. 173: 4020–4029.
41. Misson, P., S. van den Bru ˆle, V. Barbarin, D. Lison, and F. Huaux. 2004. Markers
of macrophage differentiation in experimental silicosis. J. Leukocyte Biol. 76:
42. Tilg, H., and A. M. Diehl. 2000. Cytokines in alcoholic and nonalcoholic ste-
atohepatitis. N. Engl. J. Med. 343: 1467–1476.
43. Hui, J. M., A. Hodge, G. C. Farrell, J. G. Kench, A. Kriketos, and J. George.
2004. Beyond insulin resistance in NASH: TNF-? or adiponectin? Hepatology
44. Cayon, A., J. Crespo, M. Mayorga, A. Guerra, and F. Pons-Romero. 2006. In-
creased expression of Ob-Rb and its relationship with the overexpression of
TGF-?1 and the stage of fibrosis in patients with nonalcoholic steatohepatitis.
Liver Int. 26: 1065–1071.
45. Blease, K., C. Jakubzick, J. M. Schuh, B. H. Joshi, R. K. Puri, and
C. M. Hogaboam. 2001. IL-13 fusion cytotoxin ameliorates chronic fungal-in-
duced allergic airway disease in mice. J. Immunol. 167: 6583–6592.
46. Husain, S. R., and R. K. Puri. 2000. Interleukin-13 fusion cytotoxin as a potent
targeted agent for AIDS-Kaposi’s sarcoma xenograft. Blood 95: 3506–3513.
47. Kawakami, K., S. R. Husain, M. Kawakami, and R. K. Puri. 2002. Improved
antitumor activity and safety of interleukin-13 receptor targeted cytotoxin by
systemic continuous administration in head and neck cancer xenograft model.
Mol. Med. 8: 487–494.
48. Kawakami, K., B. H. Joshi, and R. K. Puri. 2000. Sensitization of cancer cells to
interleukin-13-Pseudomonas exotoxin induced cell death by gene transfer of
IL-13 receptor ? chain. Hum. Gene Ther. 11: 1829–1835.
49. Joshi, B. H., G. E. Plautz, and R. K. Puri. 2000. Interleukin-13 receptor ? chain:
a novel tumor associated antigen on malignant glioma cells. Cancer Res. 60:
4665The Journal of Immunology