IFN-γ Protects Cerulein-Induced Acute
Pancreatitis by Repressing NF- κB Activation
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J Immunol 2007; 178:7385-7394; ;
doi: 10.4049/jimmunol.178.11.7385
http://www.jimmunol.org/content/178/11/7385
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References
Takahito Hayashi, Yuko Ishida, Akihiko Kimura, Yoichiro
Iwakura, Naofumi Mukaida and Toshikazu Kondo
The Journal of Immunology
IFN-␥ Protects Cerulein-Induced Acute Pancreatitis by
Repressing NF-␬B Activation1
Takahito Hayashi,* Yuko Ishida,* Akihiko Kimura,* Yoichiro Iwakura,† Naofumi Mukaida,‡
and Toshikazu Kondo2*
A
cute pancreatitis is a common inflammatory disease of
the abdomen, with an annual incidence rate of
⬃10 –20 cases per 100,000 people in Western countries (1, 2). Acute pancreatitis is characterized by interstitial
edema, vacuolization, and parenchymal necrosis with a concomitant massive infiltration of leukocytes, mainly composed of
neutrophils (3, 4). Acute pancreatitis is commonly caused by
excessive ethanol consumption, biliary tract disease, certain
medications, and invasive procedures of the biliary and pancreatic ducts (3, 4). The treatment of acute pancreatitis consists
mainly of supportive medical therapeutic modalities such as
fluid and nutrient replenishment, and inhibition of pancreatic
proteases. However, it still has a high mortality ranging from
15–25% even at present (3, 5). Therefore, the development of a
novel therapy is required, based on the molecular clarification
of the pathogenesis.
*Department of Forensic Medicine, Wakayama Medical University, Wakayama, Japan; †Center for Experimental Medicine, Institute of Medical Science, University of
Tokyo, Tokyo, Japan; and ‡Division of Molecular Bioregulation, Cancer Research
Institute, Kanazawa University, Kanazawa, Japan
Received for publication December 8, 2006. Accepted for publication March
19, 2007.
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.
1
This work was supported by Grants-in-Aids from the Ministry of Education, Culture, Science and Technology of the Japanese Government.
2
Address correspondence and reprint requests to Dr. Toshikazu Kondo, Department
of Forensic Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama
641-8509, Japan. E-mail address: [email protected]
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
Pancreatic damage is initiated by autodigestion of the pancreas by acinar cell-derived proteases, followed by a massive
infiltration of leukocytes such as neutrophils and macrophages
(6). Infiltration of macrophages and neutrophils is an early
event of acute pancreatitis and is presumed to be involved in the
removal of necrotic cells, which are generated by autodigestion
mediated by acinar cell-derived proteases (6). Simultaneously,
the infiltrating leukocytes produce various proinflammatory cytokines, such as IL-1 and TNF-␣, and release proteinases and
reactive oxygen species (7, 8). Thus, the net effect of leukocyte
infiltration is to aggravate acute pancreatitis. This conclusion is
supported by the observations that depletion of neutrophils attenuated tissue injuries observed in acute murine pancreatitis
models (9).
IFN-␥ is mainly produced by NK cells and CD4-positive Th1
cells and has multiple effects on macrophages, NK cells, and T
lymphocytes (10, 11). Thus, IFN-␥ is presumed to have a crucial role in various immune responses, particularly those mediated by Th1 cells. Both insulin promoter-specific overexpression of IFN-␥ by the pancreas and genetic deletion of
suppressor of cytokine signaling-1, an antagonistic signal transducer of IFN-␥, leads to spontaneous development of severe
chronic pancreatitis with a predominant infiltration of lymphocytes (12, 13). These observations suggest that IFN-␥ can induce pancreas tissue injury mainly by activating T lymphocytes
in these mice.
Several lines of evidence indicate the essential involvement
of IFN-␥ in models of acute inflammatory diseases by influencing neutrophil infiltration (14 –16). Demols et al. (17) demonstrated that the administration of CD4-positive T cells into
nude mice partially exaggerated the severity of cerulein-induced acute pancreatitis, presumably due to IFN-␥ production
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We explored the pathophysiological roles of IFN-␥ in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein
injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial
edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented
intrapancreatic gene expression of TNF-␣, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-␥ in WT mice. In situ
hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-␥ mRNA.
Unexpectedly, IFN-␥ⴚ/ⴚ mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment.
Moreover, intrapancreatic gene expression of TNF-␣, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in
IFN-␥ⴚ/ⴚ mice, compared with WT mice. Cerulein activated NF-␬B, an indispensable transcription factor for gene transcription
of TNF-␣, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in
nuclear amount and DNA-binding activity of NF-␬B p65. In comparison with WT mice, IFN-␥ⴚ/ⴚ mice exhibited exaggerated and
prolonged NF-␬B activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-␥, because acetylated
Stat1 can inhibit NF-␬B activation. Indeed, IFN-␥ acetylated Stat1 and reciprocally reduced NF-␬B activation and COX-2
expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-␥ remarkably attenuated
acute pancreatitis in both WT and IFN-␥ⴚ/ⴚ mice, with reduced NF-␬B activation and COX-2 expression. Thus, IFN-␥ can have
anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-␬B activation. The Journal of Immunology, 2007, 178: 7385–7394.
THE ROLE OF IFN-␥ IN CERULEIN-INDUCED ACUTE PANCREATITIS
7386
Table I. Sequences of the primers used for RT-PCR
Sequencea
Transcript
IFN-␥
COX-2
TNF-␣
MIP-2
KC
Fas
FasL
␤-actin
a
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
(F)
(R)
Annealing Temperature (°C)
Cycle
Product Size (bp)
60
36
274
60
36
197
58
38
306
62
32
511
58
36
204
62
36
454
60
32
320
55
32
927
60
32
456
5⬘-ACTGAAGCCAGCTCTCTCTTCCTC-3⬘
5⬘-TTCCTTCTTGGGGTCAGCACAGAC-3⬘
5⬘-CTTGTACCTTTACTTCACTG-3⬘b
5⬘-AAGCACCAGGTGTCAAGTCT-3⬘c
5⬘-GACCCACTTCAAGGGAGTCTGGAAC-3⬘
5⬘-TGACTGTGGGGGGATACACCTCTCC-3⬘
5⬘-CAGCCTCTTCTCATTCCTGCTTGTG-3⬘
5⬘-CTGGAAGACTCCTCCCAGGTATAT-3⬘
5⬘-GAACAAAGGCAAGGCTAACTGA-3⬘
5⬘-AACATAACAACATCTGGGCAAT-3⬘
5⬘-GGATTCACCTCAAGAACATCCAGAG-3⬘
5⬘-CACCCTTCTACTAGCACAGTGGTTG-3⬘
5⬘-GAGAATTGCTGAAGACATGACAATCC-3⬘
5⬘-GTAGTTTTCACTCCAGACATTGTCC-3⬘
5⬘-GAGAAGGAAACCCTTTCCTG-3⬘
5⬘-ATATTCCTGGTGCCCATGAT-3⬘
5⬘-TTCTACAATGAGCTGCGTGTGGC-3⬘
5⬘-CTCATAGCTCTTCTCCAGGGAGGA-3⬘
by adoptively transferred cells, but they did not clarify the effects of the adoptive transfer on neutrophil infiltration. Moreover, accumulating evidence indicates the essential contribution
of cyclooxygenase-2 (COX-2)3 to cerulein-induced acute pancreatitis, because inhibition of the COX-2 pathway also ameliorates pancreatitis (18). Furthermore, we observed that IFN-␥,
a potential inducer of COX-2 gene expression, was detected in
neutrophils infiltrating the pancreas in the same disease model.
To address the role of IFN-␥, we compared pathological consequences of cerulein-induced pancreatitis in wild-type (WT)
and IFN-␥-deficient (IFN-␥⫺/⫺) mice. In contrast with our expectation, we discovered protective roles of IFN-␥ in ceruleininduced acute pancreatitis by repressing the activation of NF␬B, an indispensable transcription factor for COX-2 gene
expression, in neutrophils. Finally, exogenous IFN-␥ attenuated
cerulein-induced acute pancreatitis by repressing NF-␬B activation and subsequent COX-2 gene expression, even when administered after the start of cerulein injection.
were used in the following experiments (14 –16). All mice were housed individually in cages under the specific pathogen-free conditions during the experiments. All animal experiments were approved by the Committee on Animal Care and Use in Wakayama Medical University.
Materials and Methods
Measurement of pancreatic water content
Reagents and Abs
Pancreas was obtained at 12 h after the first cerulein injection to weigh a
wet tissue weight. The tissues were then freeze-dried overnight and reweighed to determine a dry tissue weight. Pancreatic water content was
calculated as the difference between the wet and dry tissue weights and
expressed as a ratio of the wet tissue weight.
Cerulein and a selective COX-2 inhibitor, NS-398, were purchased from
Sigma-Aldrich. Recombinant murine IFN-␥ and LPS were obtained from
PeproTech and Difco, respectively. A hybridoma clone, RB6-8C5, which
secretes a rat anti-mouse Ly6G mAb (19, 20), was a gift from Dr. R.
Coffman (DNAX Research Institute of Molecular and Cellular Biology,
Palo Alto, CA). The anti-Ly6G Ab was purified as described previously
and used to deplete peripheral blood granulocytes (21) The following
mAbs and polyclonal Abs (pAbs) were used in this study; rat anti-mouse
F4/80 mAb (Dainippon Pharmaceutical), rat anti-mouse IFN-␥ mAb, rat
anti-mouse Ly6G mAb, rat anti-mouse Pan NK cells mAb (BD Biosciences/BD Pharmingen), rabbit anti-myeloperoxidase (MPO) pAbs (Neomarkers), rabbit anti-mouse NF-␬B p65 pAb, goat anti-mouse IFN-␥ pAbs,
goat anti-COX-2 pAbs, goat anti-Stat1 pAbs (Santa Cruz Biotechnology),
and mouse anti-acetyl-lysine mAb (Upstate Biotechnology).
Mice
Pathogen-free 8- to 10-wk-old male BALB/c mice were obtained from Sankyo
Laboratories and designated as WT mice in this study. Age- and sex-matched
IFN-␥⫺/⫺ mice, backcrossed to BALB/c mice for at least eight generations,
Induction of acute pancreatitis
Mice were injected i.p. with cerulein (50 ␮g/kg in 0.2 ml of PBS) repeatedly every 1 h (total 10 injections) (22). In some experiments, NS-398, a
selective COX-2 inhibitor, was i.p. administered at 10 mg/kg immediately
before the first cerulein injection. In another series of experiments, recombinant murine IFN-␥ was s.c. administered WT and IFN-␥⫺/⫺ mice at a dose of
104 U/mouse at 4 h after the first cerulein injection. Preliminary experiments
demonstrated that i.p. injection of 250 ␮g of anti-mouse Ly6G (RB6-8C5)
reduced the peripheral blood neutrophil numbers below 5% of control IgGtreated animals until 2 days after the treatment (our unpublished data). Thus,
in some experiments, WT mice were injected i.p. with 250 ␮g of anti-mouse
Ly6G or isotype-matched control IgG 1 day before cerulein challenge.
Determination of serum amylase level
Whole blood samples were collected to determine serum amylase levels
with a Fuji DRI-CHEM 3500 V as instructed by the manufacturer (Fuji
Medical System).
Histopathological and immunohistochemical analyses
Pancreatic tissues were removed at the indicated time intervals after the
first cerulein injection and fixed in 4% formaldehyde buffered with PBS
(pH 7.2), embedded in paraffin, and cut to obtain 4-␮m-thick sections.
Thereafter, after the sections were stained with H&E, histopathological
changes such as vacuolization and necrosis of pancreatic acinar cells were
evaluated by an examiner without prior knowledge of the experimental
procedures, based on the following criteria: 0, none; 1, ⬍25%; 2, 25–50%;
3, 50 –75% and 4, 75–100%. A part of the pancreatic tissue was stained
with anti-MPO pAb, anti-F4/80 Ab, or anti-Pan NK cells Ab, as described
previously (15). An examiner without prior knowledge of the experimental
procedures, enumerated the numbers of infiltrating neutrophils, macrophages, or NK cells on 10 randomly chosen visual fields at ⫻400 magnification, and the average of the 10 selected microscopic fields was
calculated.
Determination of apoptotic cells
3
Abbreviations used in this paper: COX-2, cyclooxygenase-2; WT, wild type; pAb,
polyclonal Ab; MPO, myeloperoxidase; AcLys, acetyl lysine; ISH, in situ hybridization; ChIP, chromatin immunoprecipitation.
Deparaffinized sections were treated with proteinase K solution for 10 min
at 37°C, after endogenous peroxidase was inactivated with 0.3% H2O2 in
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(F), Forward primer; (R), reverse primer.
Primers were used for nested-PCR to prepare RNA probes for in situ hybridization.
b,c
The Journal of Immunology
7387
PBS. Apoptotic cells were detected by in situ Apoptosis Detection kit
(Takara Bio). The numbers of apoptotic cells were enumerated on 10 randomly chosen visual fields (⫻400) by an examiner without prior knowledge of the experimental procedures.
A double-color immunofluorescence analysis
Deparaffinized sections were incubated with PBS containing 1% normal
donkey serum and 1% BSA to reduce nonspecific reactions as previously
described (16). Thereafter, the sections were further incubated in the combination of Abs. All Abs were used at a concentration of 1 ␮g/ml. After the
incubation with fluorochrome-conjugated secondary Abs (15 ␮g/ml) at
room temperature for 30 min, the sections were observed under fluorescence microscopy.
ELISA for IFN-␥
Pancreas samples were homogenized with 0.3 ml of PBS containing Complete Protease Inhibitor Mixture (Roche) at the indicated time intervals.
The homogenates were centrifuged at 12,000 ⫻ g for 15 min. IFN-␥ levels
and total protein contents in the supernatant were measured using a specific
ELISA kit (BioSource International) and a BCA protein assay kit (Pierce),
respectively. The data were expressed as picograms of IFN-␥ per milligram
of total protein for each sample.
Semiquantitative RT-PCR analyses with Taq polymerase (Nippon Gene)
were conducted on 5 ␮g of total RNAs extracted from pancreatic tissues or
isolated neutrophils (1 ⫻ 107) using the specific sets of primers with the
optimal cycles consisting of 94°C for 1 min, optimal annealing temperature
for 1 min, and 72°C for 1 min, followed by the incubation at 72°C for 3 min
(Table I), as described previously (15, 16). The PCR products were fractionated on a 2% agarose gel and visualized by ethidium bromide staining.
The band intensities were measured using Image Analysis software (Scion
Image) and the ratios of each band to ␤-actin were calculated.
Isolation of peritoneal neutrophils
Peritoneal neutrophils were isolated as described previously (15). Briefly,
WT mice were i.p. injected with 2 ml of 3% proteose peptone (Difco) in
MEM. Eight hours later, the peritoneal cavity was washed with MEM and
the abdominal lavage fluid was collected. Thereafter, polymorphonuclear
neutrophils were isolated from the pellets of the abdominal lavage by gradient density centrifugation with Percoll (50/75%), and cultured in MEM
containing 5% FBS. The purity of the isolated neutrophils was ⬃95%, as
assessed by microscopic examination with Giemsa staining (data not
shown). Then, LPS (10 ␮g/ml) or LPS plus murine IFN-␥ (103 U/ml) was
added to culture medium. At 12 h later, the cells were collected for subsequent analyses. In some experiments, isolated neutrophils were incubated
with the pancreatic extracts, as described previously (23). Briefly, at 12 h
after the first cerulein injection, pancreas was removed from WT mice, and
homogenized on ice in PBS, followed by the centrifugation at 4,000 ⫻ g
for 5 min. After further centrifugation at 12,000 ⫻ g for 15 min, cell-free
supernatant was incubated with isolated neutrophils. Twelve hours later,
total RNA was extracted for RT-PCR analyses.
Immunoprecipitation and Western blot analysis
Pancreatic tissues or isolated neutrophils (1 ⫻ 107) were homogenized with
a lysis buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton, 1 mM
EDTA) containing Complete Protease Inhibitor Mixture (Roche), and centrifuged to obtain lysates. Transcription factors were analyzed, as described
previously (24). Briefly, the extracts immunoprecipitated with anti-Stat1
Ab, were separated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. After being immersed with the blocking buffer (10 mM
Tris-HCl (pH 7.4) containing 0.5 M NaCl, 5% skim milk, and 0.5% Tween
20), the membranes were incubated with mouse anti-acetyl lysine (AcLys)
mAb or anti-Stat1 mAb at 4°C overnight. The Ag-Ab complexes were
detected by using the ECL-Western blotting detection system (Amersham
Bioscience Japan), according to the manufacturer’s instructions. Thereafter, the membranes were reprobed with anti-Stat1 Ab. For Western blotting
for NF-␬B p65, cell lysates from neutrophils were further separated into
cytosol and nuclear fractions using the NE-PER method (Pierce) as described previously (25). Each fraction was subjected to Western blotting
using anti-NF-␬B p65.
Measurement of NF-␬B p65 DNA-binding activity
Nuclear proteins were extracted from the obtained cell pellets using the
NE-PER method (Pierce) as described previously (25). Subsequently, the
FIGURE 1. IFN-␥ expression in the pancreas of WT mice after cerulein
challenge. a and b, IFN-␥ gene expression was examined by RT-PCR as described in Materials and Methods. Representative results from eight independent experiments are shown in a. The ratios of IFN-␥ to ␤-actin were calculated and are shown in b. All values represent means ⫾ SEM (n ⫽ 8). ⴱⴱ, p ⬍
0.01, cerulein-treated WT vs untreated WT. c, Intrapancreatic IFN-␥ contents
were determined at the indicated time intervals after cerulein challenge as
described in Materials and Methods. All values represent means ⫾ SEM (n ⫽
8). ⴱⴱ, p ⬍ 0.01, cerulein-treated WT vs untreated WT mice. d–f, The in situ
detection of IFN-␥ mRNA in the pancreas of WT mice at 7 h after cerulein
challenge. ISH was conducted as described in Materials and Methods. Representative results from three independent experiments are shown here. A
squared area in d (original magnification, ⫻200) is shown in e (original magnification, ⫻400). The result using the sense probe is shown as a negative
control in f (original magnification, ⫻200). g, In situ hybridization combined
with immunofluorescence analysis was conducted to determine IFN-␥-expressing cells during cerulein-induced acute pancreatitis, as described in Materials and Methods. The images of IFN-␥ mRNA (i) and Ly6G (ii) were
digitally merged in iii. Representative results from six independent experiments are shown here (original magnification, ⫻400). h, RT-PCR analysis of
IFN-␥ mRNA expression in isolated neutrophils incubated with the pancreatic
extracts of WT mice as described in Materials and Methods. Representative
results from six independent experiments are shown here. i, The ratios of
IFN-␥ to ␤-actin were calculated and are shown here. All values represent the
mean ⫾ SEM (n ⫽ 6). ⴱⴱ, p ⬍ 0.01, vehicle treatment vs pancreatic extract
treatment.
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Extraction of total RNAs and RT-PCR
7388
THE ROLE OF IFN-␥ IN CERULEIN-INDUCED ACUTE PANCREATITIS
multiwell chemiluminescent assay was performed to detect NF-␬B p65
protein using the EZ-Detect Transcription Factor kit (Pierce), according to
the manufacturer’s instructions. The p65 contents were expressed as relative light units divided by total protein contents for each sample.
Chromatin immunoprecipitation (ChIP) assay
Isolated neutrophils (1 ⫻ 107) were applied to ChIP assay as described
previously (26). After neutrophils were incubated with LPS (10 ␮g/ml) or
LPS plus murine IFN-␥ (103 U/ml) for 12 h, the cells were cross-linked
with 1% formaldehyde for 15 min followed by resuspension in SDS-lysis
buffer. After the sonication, immunoprecipitation was conducted using antiNF-␬B p65 Abs. Then, PCR analysis was performed using specific primers
for the promoter region of the mouse COX-2 gene (forward: 5⬘-CTAATT
CCACCAGTACAGATG-3⬘ reverse: 5⬘-ACTAGGCGAGACTCAGCGA
AC-3⬘). The PCR products were analyzed on a 1% agarose gel. This primer
set covers the COX-2 promoter segment from ⫺540 to ⫺265, which contains NF-␬B-binding sites.
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FIGURE 2. a, Determination of
serum amylase levels in WT and IFN␥⫺/⫺ mice at the indicated time intervals after the first cerulein injection
(n ⫽ 8). All values represent the
mean ⫾ SEM. ⴱⴱ, p ⬍ 0.01, IFN␥⫺/⫺ vs WT. b–i, Histopathological
stained sections of the pancreas from
WT and IFN-␥⫺/⫺ (H&E staining,
original magnification, ⫻200). Representative results from eight animals
at each time point are shown here.
Histopathological alterations such as
pancreatic acinar cell vacuolization
(j) and necrosis (k) were scored at the
indicated time intervals in WT and
IFN-␥⫺/⫺ mice as described in Materials and Methods (n ⫽ 8). All values
represent the mean ⫾ SEM. ⴱⴱ, p ⬍
0.01, IFN-␥⫺/⫺ vs WT. l, Analysis of
pancreatic water content in WT and
IFN-␥⫺/⫺ mice at 12 h after the first
cerulein injection (n ⫽ 8). All values
represent the mean ⫾ SEM. ⴱⴱ, p ⬍
0.01, IFN-␥⫺/⫺ vs WT. m and n, Immunohistochemical analyses of neutrophil recruitment into the pancreas
after cerulein challenge. The samples
were obtained from WT and IFN-␥⫺/⫺
mice at the indicated time intervals, and
immunostained with anti-MPO pAbs.
Samples obtained at 12 h from WT and
IFN-␥⫺/⫺ mice are shown in m and n,
respectively. Representative results
from eight animals are shown here
(original magnification, ⫻200). The
numbers of neutrophils per a highpower microscopic field were counted
and are shown in o. All values represent
the mean ⫾ SEM (n ⫽ 8). ⴱⴱ, p ⬍
0.01, IFN-␥⫺/⫺ vs WT.
In situ hybridization (ISH)
Digoxigenin-labeled IFN-␥ sense and antisense probes were obtained
by using the DIG RNA Labeling kit (Boehringer Mannheim Biochemica) according to the manufacturer’s instructions. The sense probe
was used as a negative control. Deparaffinized sections were fixed with
4% paraformaldehyde in PBS for 15 min and incubated with 10 ␮g/ml
proteinase K in TE buffer (10 mM Tris-HCl and 1 mM EDTA) at 37°C
for 10 min. After washing with 5⫻ SSC at room temperature for 15 min,
the sections were prehybridized at 55°C for 1 h with a buffer containing
50% deionized formamide, 5⫻ SSC, and 40 ␮g/ml salmon sperm DNA.
Then, the RNA probes were added to the prehybridization buffer to 400
ng/ml and the slides were incubated under a cover at 55°C for 16 h in
a moist chamber. The section was incubated with anti-digoxigenin Abs
and positive signals were visualized with a color-substrate solution containing NBT salt and 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt for 16 h (27). After ISH, the slides were further processed
to immunofluorescence analysis using anti-MPO pAb (28).
The Journal of Immunology
7389
Results
FIGURE 3. a and b, RT-PCR analysis of gene expression for COX-2 in
the pancreas of WT and IFN-␥⫺/⫺ mice. Representative results from eight
individual animals were shown in a. The ratios of COX-2 (b) to ␤-actin
were calculated and shown here. All values represent the mean ⫾ SEM
(n ⫽ 8). ⴱⴱ; p ⬍ 0.01; ⴱ, p ⬍ 0.05; IFN-␥⫺/⫺ vs WT. c, A double-color
immunofluorescence analysis of COX-2-expressing cells in the pancreas
after cerulein treatment. The samples were immunostained with a combination of anti-Ly6G mAb and anti-COX-2 pAbs as described in Materials
and Methods. The fluorescent images were digitally merged in the bottom
column. Representative results from six independent experiments are
shown here (original magnification, ⫻400). d, Effects of NS-398, a selective COX-2 inhibitor, on cerulein-induced pancreatitis. IFN-␥⫺/⫺ mice
were treated with NS-398 or vehicle (PBS, pH7.2) as described in Materials and Methods. Serum amylase levels were determined and are shown
here. All values represent the mean ⫾ SEM (n ⫽ 5). ⴱⴱ, p ⬍ 0.01, NS-398
vs vehicle.
Statistical analysis
The means and SEMs were calculated for all parameters determined in this
study. Statistical significance was evaluated by using ANOVA or MannWhitney’s U test. A value of p ⬍ 0.05 was accepted as significant.
Intrapancreatic IFN-␥ expression in WT mice after cerulein
challenge
Cerulein injection markedly enhanced IFN-␥ mRNA expression in
pancreas, starting at 4 h and reaching a peak by 7 h after the
injection (Fig. 1, a and b). Concomitantly, intrapancreatic IFN-␥
protein levels were progressively increased from 4 h on after the
first cerulein injection (Fig. 1c). In situ hybridization analysis detected IFN-␥ mRNA in tissues containing infiltrated inflammatory
cells from 4 h on after the first cerulein injection (Fig. 1, d and e).
Controls using sense probes did not give rise to any signals, indicating the specificity of the reaction (Fig. 1f). Immunohistochemical analysis detected IFN-␥ protein in inflammatory cells recruited
into the pancreas following cerulein challenge (data not shown).
Moreover, ISH combined with immunofluorescence staining revealed that most Ly6G-positive granulocytes expressed IFN-␥
mRNA later than 7 h after the first cerulein injection (Fig. 1g). At
12 h after the first cerulein injection, IFN-␥ was detected in a small
proportion of F4/80-positive macrophages, and NK cells, in addition to Ly6G-positive granulocytes (data not shown). However, the
infiltrating neutrophils outnumbered the infiltrating macrophages
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FIGURE 4. a– d, RT-PCR analysis of gene expression for TNF-␣, KC/
CXCL1, and MIP-2/CXCL2 in the pancreas of WT and IFN-␥⫺/⫺ mice.
Representative results from eight individual animals are shown in a. The
ratios of TNF-␣ (b), KC/CXCL1 (c), and MIP-2/CXCL2 (d) to ␤-actin
were calculated and are shown here. All values represent the mean ⫾ SEM
(n ⫽ 8). ⴱⴱ, p ⬍ 0.01, IFN-␥⫺/⫺ vs WT.
7390
THE ROLE OF IFN-␥ IN CERULEIN-INDUCED ACUTE PANCREATITIS
and NK cells, 3- and 17-fold, respectively (data not shown). Moreover, when mice were rendered granulocytopenic with anti-Ly6G
Ab, intrapancreatic IFN-␥ gene expression was significantly attenuated (data not shown). Furthermore, coincubation with ceruleintreated pancreatic extracts significantly augmented IFN-␥ gene expression in isolated neutrophils (Fig. 1, h and i). Collectively, these
observations suggest that granulocytes were the major source of
IFN-␥ in this model.
Cerulein exacerbated pancreatitis and enhanced COX-2 gene
expression in IFN-␥⫺/⫺ mice
The essential contribution of COX-2 to pancreatitis has been indicated by the observation that COX-2-deficient mice exhibited
attenuated cerulein-induced pancreatitis (18, 29). Moreover, IFN-␥
can augment COX-2 gene expression in several types of cells (30 –
32). Thus, we postulated that enhanced IFN-␥ expression may promote pancreatitis by up-regulating COX-2 gene expression. To test
this hypothesis, we compared cerulein-induced pancreatitis in IFN␥⫺/⫺ with WT mice. There were no significant differences in serum amylase levels between untreated WT and IFN-␥⫺/⫺ mice
(Fig. 2a). Both WT and IFN-␥⫺/⫺ mice exhibited progressive increases in serum amylase levels from 4 h on, decreasing by 24 h
after the first cerulein injection. The elevations in serum amylase
were significantly greater in IFN-␥⫺/⫺ than WT mice (Fig. 2a).
There were no apparent morphological differences in the pancreas
of untreated WT and IFN-␥⫺/⫺ mice (Fig. 2, b and c). The interstitial tissues became moderately edematous at the earlier phase
after cerulein challenge and vacuolization and necrosis of pancreatic acinar cells became apparent along with leukocyte infiltration
after 7 h in WT mice (Fig. 2, d, f, and h). In IFN-␥⫺/⫺ mice,
FIGURE 6. The nuclear NF-␬B p65 in the pancreas of WT and IFN␥⫺/⫺ mice. a, The amounts of nuclear NF-␬B p65 in the pancreas were
measured on WT and IFN-␥⫺/⫺ mice as described in Materials and Methods. Each value represents mean ⫾ SEM (n ⫽ 6). ⴱⴱ, p ⬍ 0.01, WT vs
IFN-␥⫺/⫺ mice. b and c, Acetylated Stat1 in pancreas. Whole extracts of
pancreas were immunoprecipitated with anti-Stat1 Ab and then were subjected to Western blotting analysis using an Ab to AcLys. The same membrane was reprobed with anti-Stat1 Ab. Representative results from six
independent experiments are shown in b. The ratios of acetylated Stat1 to
total Stat1 were calculated and are shown in c. Each value represents
mean ⫾ SEM (n ⫽ 6). ⴱⴱ, p ⬍ 0.01, WT vs IFN-␥⫺/⫺ mice. d, A doublecolor immunofluorescence analysis was performed on the pancreas at 12 h
after the first cerulein injection. The analysis was performed using antiLy6G (FITC) and anti-Stat1 (Cy3), followed by the observation under a
fluorescence microscopy (original magnification, ⫻400), and signals were
merged digitally. Representative results from six individual animals are
shown here.
vacuolization and necrosis were more severe than in WT mice
(Fig. 2, e, g, i, j, and k). Moreover, pancreatic water content due to
edema was significantly higher in IFN-␥⫺/⫺ than in WT mice (Fig.
2l). The inflammatory cells infiltrated the pancreas (Fig. 2, b–i) and
neutrophil infiltration was more apparent in IFN-␥⫺/⫺ mice than in
WT mice (Fig. 2, m– o). However, macrophages were recruited to
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FIGURE 5. a and b, Detection of apoptotic acinar cells after cerulein
challenge. The samples were obtained from WT and IFN-␥⫺/⫺ mice at the
indicated time intervals, and apoptotic acinar cells were detected as described in Materials and Methods. Samples obtained at 12 h from WT and
IFN-␥⫺/⫺ mice are shown in a and b, respectively. Representative results
from eight animals are shown here (original magnification, ⫻400). c, The
numbers of apoptotic acinar cells per a high-power microscopic field were
counted and are shown here. All values represent the mean ⫾ SEM (n ⫽
8). ⴱⴱ, p ⬍ 0.01, IFN-␥⫺/⫺ vs WT. d–f, RT-PCR analysis of gene expression of Fas and FasL in the pancreas of WT and IFN-␥⫺/⫺ mice. Representative results from eight individual animals are shown in d. The ratios
of Fas (e) and FasL (f) to ␤-actin were calculated and are shown here. All
values represent the mean ⫾ SEM (n ⫽ 8). ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05,
IFN-␥⫺/⫺ vs WT.
The Journal of Immunology
7391
a similar extent in both WT and IFN-␥⫺/⫺ mice (data not shown)
and only a few NK cells infiltrated into the pancreas of WT and
IFN-␥⫺/⫺ mice (data not shown). Moreover, cerulein treatment
augmented pancreatic COX-2 gene expression in WT and IFN␥⫺/⫺ mice beginning at 7 h after injection and the enhancement
was significantly greater in IFN-␥⫺/⫺ than WT mice (Fig. 3, a and
b). COX-2 proteins were detected in Ly6G-positive neutrophils by
two-color stained immunofluorescence analysis (Fig. 3c). Furthermore, a COX-2 inhibitor, NS-398, markedly reduced cerulein-induced increases in serum amylase levels, when administered to
IFN-␥⫺/⫺ mice (Fig. 3d). Collectively, these observations suggest
that COX-2 expression is enhanced in the absence of IFN-␥ and
results in exacerbating cerulein-induced pancreatitis.
Enhanced gene expression of TNF-␣, KC/CXCL1, and MIP-2/
CXCL2 in IFN-␥⫺/⫺ mice
Neutrophil recruitment was regulated by coordinate actions of
CXC chemokines (21) and TNF-␣ (33). Hence, we examined in-
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FIGURE 7. a– d, Western blotting analyses of
NF-␬B p65 in cytosol and nuclear extracts from peritoneal neutrophils after LPS and/or IFN-␥ treatment.
Representative results of cytosol extracts and those of
nuclear extracts from six independent experiments
are shown in a and c, respectively. The ratios of
NF-␬B p65 to ␤-actin in cytosol and nuclear extracts
are shown in b and d, respectively. e, DNA-binding
activity of the nuclear NF-␬B p65 in peritoneal neutrophils after LPS and/or IFN-␥ treatment. All values
represent the mean ⫾ SEM (n ⫽ 6 experiments in
each group). ⴱⴱ, p ⬍ 0.01, vs vehicle. f, Acetylated
Stat1 was detected as described in Materials and Methods. Whole extracts of peritoneal neutrophils were immunoprecipitated with anti-Stat1 Ab and then were subjected to Western blotting analysis with an Ab
recognizing AcLys. The same membrane was reprobed
with anti-Stat1 Ab. Representative results from six independent experiments are shown here. g, The ratios of
acetylated Stat1 to total Stat1 were calculated and are
shown here. Each value represents mean ⫾ SEM (n ⫽
6). ⴱⴱ, p ⬍ 0.01, vs vehicle. h and i, RT-PCR analysis
of COX-2 gene expression in peritoneal neutrophils after LPS and/or IFN-␥ treatment. Representative results
from six independent experiments are shown in h. i, The
ratios of COX-2 to ␤-actin were calculated and shown
here. All values represent the mean ⫾ SEM (n ⫽ 6).
ⴱⴱ, p ⬍ 0.01, LPS treatment vs LPS ⫹ IFN-␥ treatment. j and k, ChIP assay was performed with antiNF-␬B p65 Ab, followed by PCR analysis of the associated DNA using specific primers of the COX-2
promoter. Representative results from six independent
experiments are shown here. The ratios of NF-␬B p65 to
input were calculated and are shown in k. All values
represent the mean ⫾ SEM (n ⫽ 6). ⴱⴱ, p ⬍ 0.01, vs
vehicle.
trapancreatic gene expression of TNF-␣, KC/CXCL1, and MIP-2/
CXCL2 in WT and IFN-␥⫺/⫺ mice. Under the experimental conditions used, the gene expression of these molecules was faintly
detected in unchallenged WT and IFN-␥⫺/⫺ mice (Fig. 4a). Cerulein challenge enhanced gene expression of TNF-␣, KC/CXCL1,
and MIP-2/CXCL2 and their increments were exaggerated in IFN␥⫺/⫺ mice, compared with WT mice (Fig. 4). Thus, enhanced
intrapancreatic neutrophil recruitment in IFN-␥⫺/⫺ mice may be
ascribed to enhanced expression of TNF-␣ and theses chemokines.
Attenuated apoptosis of pancreatic acinar cells
in IFN-␥⫺/⫺ mice
Only a few apoptotic acinar cells were observed in untreated WT
and IFN-␥⫺/⫺ mice. Cerulein increased the number of apoptotic
acinar cells in WT mice, with concomitantly enhanced gene expression of Fas and FasL. Increases in acinar cell apoptosis, and
Fas and FasL expression, were attenuated in IFN-␥⫺/⫺ mice compared with WT mice (Fig. 5). Because acinar cell apoptosis can be
7392
THE ROLE OF IFN-␥ IN CERULEIN-INDUCED ACUTE PANCREATITIS
protective for subsequent pancreatic tissue injury (34), the absence
of IFN-␥ attenuated cerulein-induced acinar cell apoptosis by reducing the expression of Fas-FasL system, and eventually aggravated pancreatic tissue injury.
Aberrant activation of NF-␬B and reciprocal attenuation of
Stat1 acetylation in IFN-␥⫺/⫺ mice
Accumulating evidence indicates that the activation of a transcription factor, NF-␬B, is indispensable for the gene transcription of
COX-2 (35) as well as TNF-␣, KC/CXCL1, and MIP-2/CXCL2
(36 –38). To address the possibility that augmented NF-␬B activation in IFN-␥⫺/⫺ mice can enhance the expression of these
genes, we determined the activation state of the NF-␬B p65 proteins in the pancreas. There was no significant difference in the
activity of NF-␬B p65 in the pancreas between unchallenged WT
and IFN-␥⫺/⫺ mice. In WT mice, the DNA-binding activity of
NF-␬B p65 started to increase at 4 h, reached a peak at 7 h, and
returned to baseline level by 24 h after the first cerulein injection
(Fig. 6a). The activity of NF-␬B p65 was significantly higher in
IFN-␥⫺/⫺ mice at 7 and 12 h than WT mice. Several lines of
evidence suggest that IFN-␥ can acetylate its major signal transducer, Stat1, and thereby repress NF-␬B activation in vitro (24).
Hence, we examined the acetylation states of Stat1. There was no
significant difference in the total amount of Stat1 in the pancreas of
WT mice and IFN-␥⫺/⫺ mice during the entire course of acute
pancreatitis (Fig. 6b). To the contrary, Stat1 acetylation was augmented in WT but not IFN-␥⫺/⫺ mice (Fig. 6, b and c). Thus, these
observations suggest that the absence of IFN-␥ reduced Stat1 acetylation and reciprocally prolonged NF-␬B activation even in vivo.
Suppression of NF-␬B activation by IFN-␥ in neutrophils
In the course of pancreatitis, COX-2 was mainly expressed by
infiltrating neutrophils (Fig. 3c) and we detected Stat1 in infiltrating neutrophils (Fig. 6d). Thus, it is probable that IFN-␥ can
act on infiltrating neutrophils and eventually modulate COX-2
expression. To address this possibility, we examined the effects
of IFN-␥ on LPS-induced NF-␬B activation in murine peritoneal neutrophils. LPS treatment markedly increased the intranuclear level of NF-␬B p65 and enhanced its DNA-binding activity in neutrophils (Fig. 7, a– e). IFN-␥ treatment reversed the
nuclear level and DNA-binding activity of NF-␬B p65 to basal
levels (Fig. 7, a and e), but reciprocally induced Stat1 acetylation
(Fig. 7, f and g), resulting in the suppression of LPS-induced
COX-2 gene expression by IFN-␥ (Fig. 7, h and i). ChIP assay
consistently demonstrated that LPS increased the amount of
NF-␬B p65 bound to the COX-2 promoter region and that IFN-␥
reversed the effects of LPS (Fig. 7, j and k). Moreover, during the
whole course of this experiment, IFN-␥ barely influence the viability of isolated neutrophils (data not shown). Thus, it is probable
that IFN-␥ can diminish NF-␬B bound to the COX-2 promoter
region by Stat1 acetylation, thereby suppressing COX-2 gene expression in neutrophils.
Alleviation of cerulein-induced pancreatic injury by IFN-␥
administration
We finally examined the effects of the administration of exogenous
IFN-␥ on cerulein-induced pancreatic injury in WT mice. Treatment with rIFN-␥ (104 U) markedly reduced the elevation in serum
amylase levels, even when administered 4 h after the initiation of
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FIGURE 8. The effects of rIFN-␥ on cerulein-induced acute pancreatitis. a, Analysis of serum amylase levels in vehicle- and IFN-␥-treated WT mice at the
indicated time intervals after the first cerulein injection (n ⫽ 8). The same experiments are repeated three times and similar results were obtained. All values
represent the mean ⫾ SEM. ⴱⴱ, p ⬍ 0.01, vehicle vs IFN-␥. b, The DNA-binding activities of nuclear NF-␬B p65 were measured on pancreas obtained from
vehicle- or IFN-␥-treated WT mice as described in Materials and Methods. Each value represents mean ⫾ SEM (n ⫽ 6). ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05, vehicle
vs IFN-␥. c and d, RT-PCR analysis of gene expression for COX-2 in the pancreas of vehicle- or IFN-␥-treated WT mice. Representative results from eight
individual animals were shown in c. The ratios of COX-2 (d) to ␤-actin were calculated and shown here. All values represent the mean ⫾ SEM (n ⫽ 8).
ⴱⴱ, p ⬍ 0.01; ⴱ, p ⬍ 0.05, vehicle vs IFN-␥. e, Analysis of serum amylase levels in vehicle- and IFN-␥-treated IFN-␥⫺/⫺ mice at the indicated time intervals
after the first cerulein injection (n ⫽ 8). All values represent the mean ⫾ SEM. ⴱⴱ, p ⬍ 0.01, vehicle vs IFN-␥.
The Journal of Immunology
cerulein injection (Fig. 8a). Concomitantly, IFN-␥ injection markedly attenuated cerulein-induced elevation in NF-␬B activation
and subsequent COX-2 expression in pancreas (Fig. 8, b– d).
Moreover, the administration of IFN-␥ (104 U) alleviated ceruleininduced acute pancreatitis also in IFN-␥⫺/⫺ mice (Fig. 8e). Collectively, these observations would indicate that IFN-␥ could attenuate cerulein-induced expression of COX-2, an essential
mediator of pancreatitis, by repressing NF-␬B activation, and
thereby alleviate pancreatic injury.
Discussion
pared with WT mice. These discrepancies can be explained by the
observations that IFN-␥ exhibited in vitro either inhibitory or stimulatory effects on the expression of these ELR-motif-positive CXC
chemokines (46 – 49), in a context-dependent manner.
Cerulein itself can markedly activate NF-␬B (50), and NF-␬B
p50-deficient mice were resistant to cerulein-induced acute pancreatitis (22). Moreover, NF-␬B activation is indispensable for
COX-2 gene expression (33). Thus, we postulated that the enhanced expression of COX-2 in IFN-␥⫺/⫺ mice might result from
augmented NF-␬B activation. Supporting this assumption, IFN␥⫺/⫺ mice exhibited prolonged NF-␬B activation in neutrophils,
which are a major cell type expressing COX-2. The activation of
NF-␬B, a master transcription factor for various proinflammatory
genes, is tightly regulated to prevent its aberrant activation, which
can lead to pathological changes. Thus, this raises a question as to
how NF-␬B activation was prolonged in the pancreas of IFN-␥⫺/⫺
mice treated with cerulein. Stat1, a major signal transducer of
IFN-␥, can regulate the transcription of target genes after phosphorylation of its tyrosine residues by JAKs (51), while in vitro,
acetylation but not tyrosine phosphorylation of Stat1 is indispensable for its interaction with NF-␬B p65, thereby impairing the
nuclear translocation of NF-␬B p65 (24, 52, 53). Here, we demonstrated that IFN-␥ deficiency reduced the amount of acetylated
Stat1 with a reciprocal enhancement of NF-␬B activity in ceruleintreated pancreas. Conversely, IFN-␥ treatment increased the
amount of acetylated Stat1 and reciprocally diminished NF-␬B
bound to the COX-2 promoter in LPS-stimulated neutrophils.
Thus, during acute pancreatitis, IFN-␥-mediated acetylation of
Stat1 can repress NF-␬B activation and subsequent COX-2 gene
expression in neutrophils in an autocrine and/or paracrine manner.
In several disease models (14, 16), the lack of IFN-␥ diminished
Fas-mediated apoptosis. Consistently, the cerulein-induced increase in apoptotic acinar cell number was attenuated in IFN-␥⫺/⫺
mice with a diminished gene expression of Fas and FasL, compared with WT mice. Fas-deficient mice exhibited exaggerated
cerulein-induced acute pancreatitis together with attenuated apoptosis of acinar cells (34). These observations would indicate that
Fas-mediated acinar cell apoptosis is protective for cerulein-induced pancreatitis. Thus, reduced acinar cell apoptosis may synergically contribute to exaggeration of cerulein-induced pancreatitis in IFN-␥⫺/⫺ mice.
At present, the treatment of acute pancreatitis is largely a supportive one consisting of rehydration, nutritional support, and/or
the use of antibiotics (3, 4). Several protease inhibitors are administered but with little effects on improvement in mortality and/or
morbidity (54, 55). Thus, the development of a novel type of treatment is urgently required. Here, we provided definitive evidence of
the protective roles of endogenously-produced IFN-␥ for ceruleininduced acute pancreatitis. Moreover, IFN-␥ markedly alleviated
cerulein-induced acute pancreatitis, even when administered 4 h
after the first cerulein injection. Although IFN-␥ is generally welltolerated by patients with various malignancies and viral hepatitis,
it exhibits dose-dependent toxicity (56 –58). Thus, our present observations suggest that IFN-␥ therapy may be feasible for a grave
disorder, acute pancreatitis, after determining its optimal dose and
schedule to avoid adverse effects.
Acknowledgments
We express our sincere gratitude to Drs. Joost J. Oppenheim and Howard A.
Young (National Cancer Institute, Frederick, MD) for their invaluable comments on the manuscript.
Disclosures
The authors have no financial conflict of interest.
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Several lines of evidence suggest the detrimental roles of proinflammatory cytokines in the pathogenesis of experimental pancreatitis (7,
8). Due to its pleiotropic biological effects on macrophages and NK
cells (10, 11), IFN-␥ is presumed to be involved in various kinds of
inflammatory diseases (14 –16) and therefore frequently classified as
a proinflammatory cytokine. Indeed, we observed that cerulein treatment augmented the intrapancreatic expression of both IFN-␥ mRNA
and protein, along with pancreatic tissue damage and massive neutrophil infiltration. IFN-␥ is presumed to be produced mainly by NK
cells, Th1 cells, and macrophages (10, 11). In contrast, IFN-␥ production by neutrophils has been documented in various inflammatory
conditions (15, 39, 40). In line with these observations, infiltrating
neutrophils appeared to be the major source of IFN-␥ during ceruleininduced acute pancreatitis as evidenced by ISH and ISH combined
with immunofluorescence staining. This notion was further supported
by our present experiments using isolated neutrophils or neutropenic
WT mice.
COX-2 and its products are crucially involved in various types
of inflammatory responses (41, 42). Evidence is accumulating
based on the anti-inflammatory effects of inhibitors that PGE2, a
product of COX-2, is indispensable for the development of acute
pancreatitis (18, 29). Supporting this notion, cerulein-induced
acute pancreatitis was markedly attenuated in COX-2-deficient
mice (18, 29). Moreover, IFN-␥ can enhance COX-2 gene expression in various types of cells including macrophages (30 –32). Furthermore, Demols et al. (17) claimed that activated CD4⫹ T cellderived IFN-␥ could be one of the injurious factors in acute
pancreatitis. These observations favor the prediction that neutrophil-derived IFN-␥ can aggravate cerulein-induced pancreatitis, by
up-regulating COX-2 gene expression. Contrary to our expectation, IFN-␥⫺/⫺ mice exhibited exaggerated cerulein-induced acute
pancreatitis as evidenced by augmented elevation in serum amylase levels and greater histopathological changes, compared with
WT mice. Moreover, COX-2 gene expression was augmented in
IFN-␥⫺/⫺ mice, compared with WT mice. These observations
would indicate that neutrophil-derived IFN-␥ has a protective
rather than pathogenic role in cerulein-induced acute pancreatitis.
The essential involvement of neutrophil infiltration in acute pancreatitis has been based on the observations that depletion of neutrophils (our unpublished data) or inhibition of neutrophil-derived
elastase attenuated acute pancreatitis in rodents (9, 43). Infiltrating
neutrophils can contribute to the pathogenesis of acute pancreatitis
by producing various proinflammatory cytokines (44, 45) and/or
releasing proteases. Indeed, COX-2, a pathogenic mediator of cerulein-induced pancreatitis, was detected predominantly in neutrophils. In particular, mouse neutrophils exhibit potent chemotactic
responses to ELR(Glu-Leu-Arg)-motif-positive CXC chemokines
such as KC/CXCL1 and MIP-2/CXCL2 and, by using a specific
receptor, CXCR2 (21, 36, 37). IFN-␥⫺/⫺ mice have been reported
to exhibit attenuated neutrophil infiltration with reduced CXC chemokine expression in several disease models (14 –16). In our
study, neutrophil infiltration and the expression of CXC chemokines were enhanced in cerulein-treated IFN-␥⫺/⫺ mice, as com-
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THE ROLE OF IFN-␥ IN CERULEIN-INDUCED ACUTE PANCREATITIS
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