Expression of Interleukin-8 in Human Obstructive

Yoshiharu Motoo, Min-Jue Xie, Hisatsugu Mouri, Norio Sawabu
Department of Internal Medicine and Medical Oncology, Cancer Research Institute,
Kanazawa University. Kanazawa, Japan
ABSTRACT
Context Obstructive pancreatitis is a specific
form of pancreatitis, which is caused by the
obstruction of the main pancreatic duct due to
tumors or some other causes. Interleukin-8 is
induced in acute pancreatitis, but its
expression in obstructive pancreatitis has not
been clarified.
Objective We attempted to provide some
insight into the significance of interleukin -8
in the pathogenesis of pancreatic fibrosis.
Patients Fifteen cases of pancreatic cancer, 7
cases of mucinous cystadenoma, 3 cases of
Vater’s papilla cancer and 9 normal
pancreases were included in this study.
Main outcome measures The obstructive
pancreatitis portions of the above pathologies
were evaluated for interleukin-8 expression
by means of immunohistochemistry and in
situ hybridization.
Results Interleukin-8 was positive in 72% of
cases of obstructive pancreatitis. The positive
rate was not significantly related to the
etiology of the obstruction (P=0.972).
Interleukin-8 was expressed in infiltrating
cells, proliferating ductular cells and acinar
cells. In contrast, normal pancreases and
tumor cells lacked interleukin-8 expression
(P<0.001 vs. obstructive pancreatitis). Both
immunohistochemistry and
in situ
hybridization demonstrated that interleukin-8
was expressed mostly in acinar cells in mild
pancreatic fibrosis, whereas it was expressed
in stromal and ductular cells in moderate and
severe pancreatic fibrosis.
Conclusions These results suggest that
interleukin-8 expression is related to the
fibrotic process in obstructive pancreatitis.
INTRODUCTION
Interleukin (IL)-8 is a member of the C-X-C
chemokine family which mediates the
recruitment
of
polymorphonuclear
neutrophils, basophils, eosinophils and
lymphocytes to inflammatory sites [1]. IL-8
acts as both a neutrophil activator and a
chemoattractant [2]. Growth-regulated gene
product/cytokine-induced
neutrophil
chemoattractant (GRO/CINC)-1 in rats
corresponds to IL-8 in humans [3].
GRO/CINC-1 consists of 72 amino acids
which are homologous to human peptides
with melanoma growth stimulatory activities,
indicating that GRO/CINC-1 has a structural
and functional homology to human IL-8 [3].
GRO/CINC-1 or IL-8 is expressed in
experimental models such as gastric ulcer [4],
ischemia-reperfused liver [5] and acute
pancreatitis [6].
In humans, serum levels of IL-8 are reported
to be an early predictor of disease severity
and complications in acute pancreatitis [7].
An anti-IL-8 neutralizing antibody inhibited
cytokine response and acute lung injury in

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139
experimental acute pancreatitis [8]. Although
IL-8 mRNA was recently reported to be
expressed in human chronic pancreatitis [9,
10], the role of IL-8 in the pathogenesis of
chronic pancreatitis is still unclear. It is often
difficult to obtain chronic pancreatitis tissue,
especially surgical specimens, in clinical
settings. Obstructive pancreatitis is a specific
form of pancreatitis, which is caused by the
obstruction of the main pancreatic duct due to
tumors or some other causes. Obstructive
pancreatitis is different from chronic
pancreatitis caused by alcohol abuse or
cholelithiasis.
However,
obstructive
pancreatitis tissue is often available at
pancreatic surgery, and analysis of IL-8
expression in obstructive pancreatitis might
provide some insight into the significance of
IL-8 in the pathogenesis of chronic
pancreatitis or pancreatic fibrosis.
In the present study, we attempted to assess
the involvement of IL-8 in human obstructive
pancreatitis.
MATERIALS AND METHODS
Patients
Fifteen cases of pancreatic cancer (14 cases of
ductal adenocarcinoma and 1 case of islet cell
carcinoma), 7 cases of mucinous
cystadenoma, and 3 cases of Vater’s papilla
cancer were studied. All cases were
diagnosed histopathologically. The mean age
of the 25 analyzed patients was 63.0±6.6
years (range: 51-74 years); 15 were males and
10 were females. Nine patients with normal
pancreases were also included in this study;
the gender (5 males, 4 females) and ages
(mean 61.0±5.1 years; range: 54-69 years) of
these patients were not significantly different
from those of pancreatic cancer patients
(P=1.000 and P=0.401, respectively).
Pancreatic Tissue
All pancreatic tissue was obtained at surgery;
25 specimens of obstructive pancreatitis
portions from the patients with the above
diseases and 9 specimens of normal
pancreatic tissue were analyzed. Normal
pancreatic tissue was obtained from patients
with illnesses other than pancreatic disease
and was confirmed to be histologically
normal.
Immediately after surgical removal, all
pancreatic tissue samples were fixed in 10%
formalin, and sectioned at a thickness of 4
µm. The sections were stained with
hematoxylin and eosin (H&E), and the
severity of obstructive pancreatitis was
graded from the viewpoint of pancreatic
fibrosis as follows: grade 1 (mild), the
percentage of fibrotic area in the total
specimen was less than 25%; grade 2
(moderate), the fibrotic involvement was
between 25 and 50%; grade 3 (severe), the
fibrotic involvement was greater than 50%.
Immunohistochemistry
We used a DAKO LSAB kit (DAKO,
Carpinteria, CA, USA) for in situ
hybridization (ISH). The sections were
incubated with a monoclonal anti-human IL-8
antibody (1:25 dilution, Genzyme,
Minneapolis, MN, USA) overnight at 4°C.
Normal mouse IgG was used as a negative
control. The immunohistochemical reactivity
of IL-8 was evaluated in each cell type as the
frequency (in percentage) of pancreatic
lobules displaying more than 20 cells which
were positive for IL-8 expression [9].
In Situ Hybridization
An ISH kit (Maxim Biotech, Inc., San
Francisco, CA, USA) was used. The
hybridization was done at 42°C overnight,
with 250 ng/mL of an oligonucleotide (30
mer) antisense human IL-8 probe (Biognostik
GmbH, Göttingen, Germany). As negative
control experiments, serial sections were
hybridized with a sense probe. These probes
were labeled with biotin using a DNA
labeling kit (Biotin-High Prime, Boehringer
Mannheim GmbH, Mannheim, Germany).

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ETHICS
Informed consent was obtained from each
patient and the study protocol conforms to the
ethical guidelines of the 1975 Declaration of
Helsinki as revised in 1983, as reflected in a
priori approval by the Kanazawa University
School of Medicine Review Committee.
STATISTICS
All data were expressed as mean, standard
deviations, range, and frequencies. The
Mann-Whitney, the Pearson chi-square, the
Fisher's exact, and the McNemar tests were
used to analyze differences in human IL-8
expression. Two-tailed P values less than 0.05
were considered statistically significant. The
SPSS version 8.0 software (SPSS Inc.,
Chicago, Illinois, USA) was used for
statistical analysis.
RESULTS
Histological Evaluation of Human
Obstructive Pancreatitis
Pathological analysis revealed the histological
characteristics of obstructive pancreatitis with
heterogeneously extended fibrosis in the
vicinity
of
damaged
parenchyma.
Mononuclear cell infiltrates and ductal
proliferation were observed. As for
histological severity, a representative figure
for each grade is shown in Figure 1: grade 1
(mild, Figure 1a), 10 cases; graded 2
Figure 1. Different grades of pancreatic fibrosis in
human chronic obstructive pancreatitis as defined in
the section "Materials and Methods: Pancreatic Tissue"
by hematoxylin and eosin staining. Mild, grade 1 (a);
moderate, grade 2 (b); severe, grade 3 (c). (Original
magnification: ×25).
Figure 2. IL-8 expression in pancreatic tissue sections
from patients with CP. IL-8 protein is localized in the
cytoplasm of acinar and stromal infiltrating cells (a).
Proliferating ductular cells were also positive for IL-8
(b). Immunohistochemistry (magnification: ×25, inset
×100).

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(moderate, Figure 1b), 6 cases; grade 3
(severe, Figure 1c), 9 cases. The 9 control
specimens showed normal pancreatic
histology (grade 0).
IL-8 Expression in Human Obstructive
Pancreatitis
IL-8 was expressed in 72.0% (18/25) of
obstructive pancreatitis specimens although
the frequency of positive cells varied
considerably among the samples. IL-8 was
localized in the cytoplasm of acinar, ductal
and inflammatory cells (Figure 2). Most IL-8-
positive-infiltrates were seen in fibrotic areas.
The relationship between the expression of
IL-8 and the histological stage of chronic
obstructive pancreatitis is shown in Figure 3.
In grade 1 (mild), IL-8 was expressed mostly
in acinar cells, whereas in grade 2 (moderate)
and 3 (severe), IL-8 expression was found to
be increased in the inflammatory infiltrates
and ductal cells.
IL-8 immunoreactivity data are shown in
Table 1. The positivity of IL-8 was not
significantly related to the etiology of
obstruction (P=0.972). In the normal human
pancreases, IL-8 immunoreactivity was
absent (P<0.001 vs. obstructive pancreatitis)
and it was not expressed in any
adenocarcinoma tissue (P<0.001 vs.
obstructive pancreatitis). In 3 (21.4%) of the
14 adenocarcinoma lesions examined, IL-8
immunoreactivity was present in infiltrates
around cancer cells (P=0.003 vs. obstructive
pancreatitis; P=0.253 vs. normal pancreas;
P=0.016 vs. cancer portion of
adenocarcinoma). These 3 cases also had
positive IL-8 immunoreactivity in the
obstructive pancreatitis portions.
IL-8 mRNA Expression in Human
Obstructive Pancreatitis
The localization of the mRNA was similar to
that of the protein. IL-8 mRNA was localized
in acinar, inflammatory and ductal cells in
serial sections of obstructive pancreatitis
(Figure 4), whereas it was not expressed in
the normal pancreas and cancer cells.
Table 1. Expression of IL-8 in human pancreatic tissues.
Disease
IL-8 positivity
Obstructive pancreatitis
18/25 (72.0%)
Etiology (P=0.972; chi-square test):
- Pancreatic cancer
11/15 (73.3%)
- Mucinous cystadenoma
5/7 (71.4%)
- Vater’s papilla cancer
2/3 (66.7%)
Adenocarcinoma (cancer portion*)
0/14 (0%)a
Adenocarcinoma (infiltrate around cancer)
3/14 (21.4%)bcd
Normal pancreas
0/9 (0%)a
* Positivity in cancer cells
P<0.001 vs. obstructive pancreatitis (Fisher's exact test)
P=0.003 vs. obstructive pancreatitis (Fisher's exact test)
P=0.253 vs. normal pancreas (Fisher's exact test)
P=0.016 vs. cancer portion (McNemar test)
Figure 3. IL-8 protein expression in pancreatic acinar,
stromal and ductular cells in different grades of
pancreatic fibrosis. IL-8 expression was semi-
quantified as defined in the section "Materials and
Methods: Immunohistochemistry".

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DISCUSSION
Major etiologies of chronic pancreatitis are
alcohol abuse and gallstones, but obstructive
pancreatitis [11] develops due to the stricture
or obstruction of the main pancreatic duct by
post-inflammatory fibrosis, stones or tumors
[12]. We attempted to assess whether IL-8
expression is involved in the pathophysiology
of obstructive pancreatitis.
In previous reports, the main source of IL-8
was inflammatory cells, including neutrophils
[13]. Saurer et al. [9] demonstrated that IL-8
mRNA is expressed in acinar, ductal and
infiltrating cells in human chronic pancreatitis
using in situ hybridization, which is
consistent with our results. However, they did
not study the expression of IL-8 protein.
Different results for IL-8 protein and mRNA
have been reported in alcoholic liver disease
[14]. In the present study, we have clearly
shown that the expression of IL-8 protein is
up-regulated in pancreatic lobules with
histopathologic alterations of obstructive
pancreatitis. The distribution of IL-8 protein
was consistent with that of IL-8 mRNA.
Therefore, IL-8 was obviously expressed in
obstructive pancreatitis, whereas it was not
found in the normal pancreases and in
pancreatic cancer. In other human chronic
diseases, including idiopathic pulmonary
fibrosis [15], and cystic fibrosis [16], an
enhanced expression of IL-8 has been
associated with fibrosis. Although no direct
link between IL-8 and pancreatic fibrosis has
been proven, Andoh et al. [17] reported that
TNF-alpha and IL-1beta regulate IL-8
production in pancreatic periacinar
myofibroblasts and that most IL-8-positive
infiltrates were located in the vicinity of
fibrotic areas.
IL-8 expression is related to the histological
activity of inflammation in inflammatory
bowel diseases [18]. In the pancreas, it is
reported that IL-8 gene expression in the
pancreatic parenchyma is frequently observed
in advanced grades of the disease. Our results
do not fully support this report. In the present
study, IL-8 was expressed mostly in acinar
cells in mild obstructive pancreatitis, whereas
its expression was increased in interstitial
infiltrating cells in moderate and severe
obstructive pancreatitis, suggesting that the
type of cells expressing IL-8 differs
depending on the histological grade of
obstructive pancreatitis. The differences
might be explained by the etiologies of
pancreatitis. Saurer’s study [9] was carried
out, for the most part, on alcoholic chronic
pancreatitis, whereas our study was on
obstructive pancreatitis.
The important early mechanism of
progressive fibrosis in chronic obstructive
pancreatitis is considered to be increased
ductular pressure and acinar damage releasing
local proinflammatory cytokines which
stimulate peri-acinar stellate cells to
accelerate pancreatic fibrosis. Animal
experiments and some clinical studies show
that obstructive pancreatitis can be reversible
if the obstruction is released. This
Figure 4. IL-8 mRNA is expressed in acinar cells (a)
and infiltrating inflammatory cells (b). In situ
hybridization (magnification: ×25; inset: ×100).

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143
reversibility could partly explain the changing
source of IL-8 during the development of
fibrosis.
The overexpression of IL-8 in pancreatic
cancer is predominant in regions exposed to
hypoxia after orthotopic implantation in the
pancreases of nude mice [19]. During the
development and progression of pancreatic
cancer, the expression of iNOS and protein
tyrosine nitration is increased, indicating the
potential involvement of oxidative stress [20].
The overexpression of iNOS has been
reported in human chronic pancreatitis [21].
A significant association between NO and IL-
8 is also reported in human chronic
obstructive pulmonary disease [22]. We
speculate that IL-8 induction is associated
with oxidative stress in obstructive
pancreatitis.
The pathophysiology of pain in chronic
pancreatitis is not yet fully understood, but it
was recently reported that IL-8 is expressed in
macrophages surrounding enlarged pancreatic
nerves [10]. Substance P is released from
sensory pancreatic nerves and directly
stimulates the release of IL-8 from
macrophages [23]. Such interaction between
inflammatory cells and nerves could be called
“neuroimmune cross talk” [23], and may be
involved in the generation of pain in chronic
pancreatitis. Our study showing the changing
source of IL-8 in different cell types in the
course of obstructive pancreatitis would
suggest a role of IL-8 in the intrinsic
maintenance of the inflammatory response,
thus sustaining the progression of obstructive
pancreatitis.
In conclusion, the present study shows that
IL-8 is expressed in the pancreas of patients
with obstructive pancreatitis with different
expression sites depending on the grade of
pancreatic fibrosis.
Received February 16
th
, 2004 - Accepted
March 9
th
, 2004
Keywords
Fibrosis; Gene Expression
Profiling; Immunohistochemistry; In Situ
Hybridization; Interleukin-8; Pancreatic
Diseases; Pancreatitis
Abbreviations GRO/CINC: growth-regulated
gene product/cytokine-induced neutrophil
chemoattractant; ISH: in situ hybridization
Correspondence
Yoshiharu Motoo
Department of Internal Medicine and Medical
Oncology
Cancer Research Institute
Kanazawa University
13-1 Takara-machi
Kanazawa 920-0934
Japan
Phone: +81-76.265.2781
Fax: +81-76.234.4523
E-mail: motoo@kenroku.kanazawa-u.ac.jp
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