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Expression of interleukin-18, interferon-g and interleukin-10 inhepatocellular carcinoma
Chok Seng Chia a, Kechen Ban a, Hairuszah Ithnin a, Harjit Singh b, R. Krishnan b,Suryati Mokhtar b, Nik Malihan c, Heng Fong Seow a,d,*
a Department of Clinical Laboratory Science, Faculty of Medicine and Health Science, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysiab Department of Hepato-Pancreato-Biliary Surgery, Selayang Hospital, Selangor, Malaysia
c Department of Pathology, Selayang Hospital, Selangor, Malaysiad Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia
Received 7 May 2002; accepted 12 July 2002
Abstract
This is the first report on the detection of IL-18, IFN-g and IL-10 proteins in hepatocelllular carcinoma. In the apparently normal
surrounding tissue, 13 out of 17 paired specimens showed positive immunoreactivity to IL-18 (76.5%) compared with six out of 17 in
the tumour portion (35.3% of specimens). Thus, a significantly higher number of IL-18 positive specimens was found in the
hepatocytes of apparently normal surrounding tissue compared with the tumour (P�/0.018). In contrast, the number of specimens
with positive immunoreactivity to the antibody against the Th1 cytokine, IFN-g expression in the hepatocytes was lower. Only one
specimen from the apparently normal surrounding tissue (one out of 17; 5.9%) and three other specimens from the tumour portion
(three out of 17; 17.6%) had positive immunoreactivity. Similarly, the expression of the Th2 cytokine, IL-10 in normal (four out of
17; 23.5%) and tumour portions (five out of 17; 29.4%) was also low. Thus, there did not appear to be predominant Th2 immune
response as denoted by IL-10 expression. Using the Spearman correlation rank test, a significant correlation between IL-18
expression in the apparently normal surrounding tissue and high a-foetoprotein (AFP) levels of �/350 IU/l. No correlation between
IL-18 expression in the tumour portion and clinicopathological factors was found. There was also no correlation found between IL-
18 and the other cytokines, namely, IFN-g and IL-10 expression These new findings provide additional information on the type of
cytokines expressed in the tumour microenvironment and give a further insight into the role of cytokines in the pathogenesis of
cancer which is critical for the development of effective immunotherapeutic approaches for cancer therapy in the future.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Cytokines; Hepatocelllular carcinoma; Immunohistochemistry
1. Introduction
In the last decade, there has been a massive explosion
in our knowledge on the identity and functions of
cytokines. These discoveries have been rapidly applied
to the prevention and treatment of a diverse array of
diseases ranging from malignancies, infectious diseases,
autoimmune disorders and allergies [1]. The cell-
mediated response depends on cytotoxic and helper T
cell activities and function through the actions of
cytokines to regulate cells such as macrophages and
natural killer (NK) cells produced in the cells including
those in the liver are essential in defending the host
against infectious agents such as hepatitis B virus
(HBV). Various cytokines have been documented in
the liver host defence [2], liver injury [3] and liver
regeneration [4]. High levels of macrophage inhibitory
factor (MIF) have been detected in the sera of patients
with hepatocellular carcinoma (HCC) and liver cirrhosis
suggesting an association between the overexpression of
MIF and hepatocarcinogenesis [5]. Other cytokines such
as human monokine induced by interferon (IFN)-g(hMIG), interleukin (IL)-8, macrophage inflammatory
protein-1 (MIP-1)-a and b have also, been found to be
expressed in HCC. These chemokines have been sug-
* Corresponding author. Tel.: �/60-389-48-6101x8511; fax: �/60-
389-42-6957
E-mail address: shf@medic.upm.edu.my (H.F. Seow).
Immunology Letters 84 (2002) 163�/172
www.elsevier.com/locate/
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 1 7 6 - 1
gested to play a role in the recruitment of lymphocytes
to these tumours in vivo [6]. Thus, cytokines may be
beneficial in the host immune response against tumours.
Several evidence support the hypothesis that a bene-ficial immunological control of tumour growth is
associated with the development of cell-mediated cyto-
toxicity either through specific T cells [7] or non-antigen
specific NK cells [8]. The generation of such cells are
under the control of cytokines such as, IL-2 and IFN-g,
produced by T helper 1 (Th1) cells [9]. IL-18, also
known as IFN-g inducing factor, has been shown to be a
strong co-factor for Th1 development [10]. IL-10, on theother hand, is associated with development of the Th2
response has been shown to inhibit the development of
cellular immune responses via a number of mechanisms
[11]. Thus, tumour cells can also evade the immune
surveillance system by various mechanisms such as the
disruption of Th1/Th2 balance, destruction of immune
effector cells by Fas/Fas ligand and production of
immunosuppressive cytokines such as transforminggrowth factor-b (TGF-b) and IL-10 [12]. In addition,
there is increasing evidence that the tumour microenvir-
onment may also contribute to cancer progression [13].
Since the cytokines expressed at the tumour site may
play important roles in determining a successful immune
response against tumours as well as cancer pathogenesis
and progression, this study has been undertaken to
determine the expression of IL-18, IFN-g and IL-10 inHCC which is a common cancer in Malaysia and other
parts of Asia such as China, Taiwan and Hong Kong. In
Malaysia, a large proportion of HCC occurs in patients
who are chronic carriers of the human HBV [14].
2. Materials and methods
2.1. Production of polyclonal antibodies to IL-18
Recombinant human IL-18 (rhIL-18) produced in our
laboratory was purified as previously described [15].
One New Zealand white rabbit was immunised with 100
mg of rhIL-18 emulsified in an equal volume of complete
Freund’s adjuvant (Sigma, USA). Two booster injec-
tions were administered at fortnightly intervals with 50
mg of rhIL-18 emulsified in incomplete Freund’s adju-vant. Blood (4 ml) was collected from the ear vein prior
to immunisation. Subsequent bleeds were collected at 2,
4 and 8 weeks after the first immunisation. The
immunoglobulin fraction IgG was purified from whole
serum using Thiophilic adsorption chromatography
(Clontech, USA). Briefly, 1 ml of serum was diluted in
9 ml of sample buffer (50 mM sodium phosphate; 0.55
M sodium sulphate pH 7.0). Thiophilic resin (1 ml) waswashed with ten column volumes of equilibration buffer
(50 mM sodium phosphate; 0.5 M sodium sulphate
pH7.0). The diluted serum was then adsorbed to the
resin and after extensive washings, the IgG was eluted in
5 ml of 50 mM sodium phosphate.
2.2. Tissues
Seventeen pairs of formalin-fixed, paraffin-embedded
liver tumour and the adjacent tissue from confirmed
HCC cases were collected from Selayang Hospital,
Selangor dated from September 2000 till August 2001.
This hospital is the National Reference Centre for Liver
Diseases in Malaysia.
2.3. Immunohistochemistry
Sections of 5 mm thickness were cut from the paraffin
blocks, dried at 37 8C, deparaffinized in xylene and
rehydrated in graded ethanols before the antigen
retrieval step by microwave treatment for 10 min at
560 W.
The tissue sections were quenched with peroxidase
block for 10 min, washed three times with phosphate-buffered saline (PBS) and then incubated for 30 min
with the 100-fold diluted polyclonal antibody to IL-18
produced as described in Section 2.1. After three washes
with PBS, the tissue sections were incubated with
streptavidin and biotin secondary antibody (LSAB kit,
DAKO Ltd., USA) for 15 min, washed in PBS and then
incubated with DAB plus substrate chromogen (DAKO
Ltd., USA) for 5 min. The sections were then rinsed indistilled water followed by counterstaining with haema-
toxylin. With the negative control sections, the primary
antibody was substituted with PBS. For the detection of
IFN-g and IL-10, the monoclonal antibodies were
purchased from Santa Cruz Technology, USA. The
tissue sections were incubated with diluted normal
blocking serum for 1 h followed by these antibodies
(1:50 dilution) for 30 min in a wet chamber. They werethen incubated with goat-anti-mouse secondary anti-
body in the DAKO EnvisionTM kit for 30 min at room
temperature followed by incubation with 3,3-diamino-
benzidine (DAB) plus substrate chromogen for 5 min.
The sections were then rinsed in distilled water followed
by counterstaining with haematoxylin.
2.4. Scoring of immunoreactivity
The immunoreactive score was determined by the sum
of extension and intensity as previously reported [16,17].
The intensity of staining was scored as 0 for negative, 1
for weakly postive, 2 for moderately positive and 3 for
strongly positive. The extent of positive immunoreactiv-
ity was estimated as 0 for negative, 1 for positive
immunoreactivity for 1�/25% of the cells, 2 for 26�/
50%, 3 for 51�/75% and 4 for 76�/100%. The combined
staining score (score from intensity plus score from the
extent of positive immunoreactivity) ]/3 was considered
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172164
as positive immunoreactivity. The slides were viewed
using the digitalised Olympus BX50 microscope and
images were captured at selected magnification using the
Olympus pm10-sp35 camera attached.
2.5. Statistical analysis
Data analysis was performed with the Statistical
Package of Social Science (SPSS) v10.0.1. The signifi-
cance differences between categorical variables werecompared using the Fischer’s exact probability test.
Values of P B/0.05 were considered to be statistically
significant. Correlation between factors was evaluated
using the Spearman rank correlation coefficient test.
3. Results
3.1. Higher IL-18 expression in apparently normal
adjacent tissues
IL-18 was detected in the hepatocytes of the appar-ently normal tissue adjacent to the tumour (13 of the 17
specimens; Table 1) whereas in the tumour portion, only
six of the 17 specimens showed positive immunoreactiv-
ity (Table 1). Fig. 1 shows the representative slides for
positive immunoreactivity to the antibody against IL-
18. The specimens considered to have intense (three
specimens), moderate (six specimens), weak (four speci-
mens) and negative immunoreactivity in the apparentlynormal tissue surrounding the tumour are as shown in
Fig. 1a, c, d and f, respectively. Representative slides
showing IL-18 immunoreactivity in the tumour portion
for moderate (one specimen) and weak (five specimens)
immunostaining are as shown in Fig. 1b and e,
respectively.
There was a 2-fold higher number of cases with IL-18
positive immunoreactivity in the apparently normal
tissue compared with the tumour portion. Statistical
analysis showed that this was significant (P�/0.018).Analysis of the case distribution of IL-18 immunoreac-
tivity in the adjacent tissue with the extent of the area
versus intense and moderate immunostaining showed
that not only was there a higher rate of immunoposi-
tivity for IL-18 (nine cases), the extent of the intense
immunostaining was large (76�/100%) compared with
the tumour portion (one case with moderate staining
over 51�/75% of area; five with weak staining) (Fig. 2).The correlation between IL-18 expression with sex,
age and race (Table 1) as well as with clinicopathological
factors such as tumour size, infection with HBV,
cirrhosis and AFP levels was made (Table 2). Using
the Spearman correlation rank test, it was found that
there was a significant correlation between IL-18
immunoreactivity in the apparently normal tissue and
high AFP levels�/350 IU/l (P�/0.015) (Table 2). How-ever, no correlation between IL-18 expression in the
tumour and the clinicopathological factors was found.
There was also no correlation between IL-18 and IFN-gor IL-10 expression (Table 3).
3.2. Low incidence of expression of IFN-g in apparently
normal and tumour tissue
In three out of the 17 paired specimens, positive
immunoreactivity to IFN-g monoclonal antibody was
detected in the hepatocytes of the adjacent normal tissue(Fig. 3a) and only in one out of the 17 tumour tissue
(Fig. 3b). The negative controls for the normal and
tumour tissues are shown in Fig. 3c and d, respectively.
The expression of IFN-g in the two specimens from
the adjacent normal tissue was intense, in contrast to the
weak and granular-like staining for one specimen from
Table 1
Relationship between IL-18, IFN-g and IL-10 expression with sex, race and age
Variables IL-18 IFN-g IL-10
Surrounding tissue Tumour Surrounding tissue Tumour Surrounding tissue Tumour
� � P � � P � � P � � P � � P � � P
Sex
Male 11 3 0.579 6 8 0.243 3 11 0.824 1 13 0.824 4 10 0.421 4 10 0.676
Female 2 1 0 3 0 3 0 3 0 3 1 2
Race
Malay 3 3 0.261 1 5 0.099 2 4 0.272 0 6 0.647 0 6 0.139 2 4 0.605
Chinese 10 1 5 6 1 10 4 7 3 8
Age
B60 7 2 0.665 3 6 0.627 2 7 0.547 0 9 0.471 1 8 0.247 3 6 0.563
]60 6 2 3 5 1 7 1 7 3 5 2 6
Total 13 4 6 11 3 14 1 16 4 13 5 12
�, Positive immunoreactivity; �, negative immunoreactivity.
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172 165
Fig. 1. Immunostaining with anti-IL-18. Intense immunostaining was detected in the hepatocytes of the apparently normal tissue surrounding the
tumour in three specimens (a). The representative slides show moderate staining in a tumour (b) and apparently normal tissue surrounding the
tumour (c) and weak immunostaining in the normal vs. tumour in (d) and (e), respectively. Negative immunoreactivity is shown in (f) in the normal
tissue (200�/ magnification).
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172166
normal and the other from the tumour tissue. In
addition, all the staining covered only 26�/50% of the
area for both the tumour and surrounding tissue (Fig.
2).Statistical analysis showed no significant difference in
the incidence of IFN-g expression in the tumour versus
the normal tissue. No correlation was found between the
IFN-g expression, sex, race and age (Table 1) as well as
clinicopathological factors (Table 2). There was also no
correlation in the expression of IFN-g and the other
cytokines (Table 3).
3.3. Expression of IL-10 in apparently normal and
tumour tissues
IL-10 immunoreactivity was detected in the hepato-cytes of apparently normal tissue adjacent to the tumour
in four out of the 17 (23.5%) compared with five out of
the 17 tumour (29.4%) specimens (Table 1). The
representative slides for intense and weak immunoreac-
tivity for the apparently normal tissues are as shown in
Fig. 4a and c, respectively. The corresponding immu-
nostaining for the tumour portion are is shown in Fig.
4b and d, respectively.Fig. 2 shows the case distribution of IL-10 expression
with the intensity and extent of the area. The immunos-
taining was either intense or weak and the extent of
moderate staining is at 51�/75% of the area (one in
normal vs. none in tumour) whereas for weak staining, it
could either be 26�/50% (two cases for both apparently
normal and tumour tissue) or 76�/100% (only one for
both normal and tumour portion) of the area. Overall, it
appeared that for both apparently normal and tumour
tissue, the number of positive IL-10 cases were low, the
staining was only moderate or weak and extent of the
area with staining is similar.
There was no correlation between tumour IL-10
expression and clinicopathological factors (Table 2) as
Fig. 2. Case distribution of IL-18, IFN-g and IL-10 expression in the surrounding and tumour tissue as shown by the extent of the area and intensity
of immunostaining.
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172 167
well as with other cytokines (Table 3) but it should be
kept in mind that this could be due to the small number
of samples.
4. Discussion
IL-18 has been initially known as a factor that is
primarily involved in the inflammatory immune re-
sponse and is a potent IFN-g inducing factor [18].
However, more recently, it has been reported that IL-18
has the capacity to stimulate innate immunity and both
Th1 and Th2 mediated responses [19]. IL-18 exerts anti-
tumour action via a number of mechanisms such as
enhancement of NK cell activity [20], induction of
apoptosis via Fas/Fas ligand interaction and inhibition
of angiogenesis [21]. In the present study, the number of
cases with positive IL-18 immunoreactivity is signifi-
cantly higher (2-fold) in the apparently normal tissue
compared with the tumour portion of the same indivi-
dual. This appears to be consistent with a previous study
showing that the presence of IL-18 transcript is much
Table 2
Relationship between IL-18, IFN-g and IL-10 expression and clinicopathological data
Variables IL-18 IFN-g IL-10
Surrounding tissue Tumour Surrounding tissue Tumour Surrounding tissue Tumour
� � P � � P � � P � � P � � P � � P
Tumour size (cm)
B5 6 1 0.441 4 3 0.145 2 5 0.360 1 6 0.421 3 4 0.162 3 4 0.314
�5 7 3 2 8 1 9 0 10 1 9 2 8
HbsAg
Positive 9 1 0.162 3 7 0.484 2 8 0.641 0 10 0.412 4 6 0.088 3 7 0.686
Negative 4 3 3 4 1 6 1 6 0 7 2 5
HCV
Positive 1 1 0.426 1 1 0.426 1 1 0.331 0 2 0.882 0 2 0.574 2 0 0.074
Negative 12 3 12 3 2 13 1 14 4 11 3 12
Cirrhosis
Present 11 4 0.750 6 9 0.750 3 11 0.800 1 14 0.938 3 12 0.250 4 11 0.750
Absent 1 0 0 1 0 1 0 1 1 0 0 1
Unknown 1 0 0 1 0 1 0 0 0 1 1 0
AFP(IU/l)
�350 10 0 0.015 4 6 0.516 3 7 0.17 0 10 0.412 3 7 0.441 4 6 0.278
B350 3 4 2 5 0 7 1 6 1 6 1 6
6
Total: 13 4 6 11 3 14 1 16 4 13 5 12
�, Positive immunoreactivity; �, negative immunoreactivity.
Table 3
Relationship between IL-18, IFN-g and IL-10 expression
Variables IL-18 IFN-g IL-10
Surrounding tissue Tumour Surrounding tissue Tumour Surrounding tissue Tumour
� � P � � P � � P � � P � � P � � P
IL-18
Positive 3 10 0.421 1 5 0.353 4 9 0.300 2 4 0.605
Negative 0 4 0 11 0 4 3 8
IFN-gPositive 3 0 0.421 1 0 0.353 1 2 0.465 0 1 0.765
Negative 10 4 5 11 2 12 5 11
IL-10
Positive 4 0 0.300 2 3 0.605 1 2 0.465 0 5 0.765
Negative 9 4 4 8 2 12 1 11
Total 13 4 6 11 3 14 1 16 4 13 5 12
�, Positive immunoreactivity; �, negative immunoreactivity.
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172168
higher in the normal compared with the tumour colon
cells of the same individual [22]. The latter study also
indicated that the IL-18 expressed in the tumour will
most likely not produce bioactive protein because of the
lack of the interleukin-1b converting enzyme (ICE)
transcript. ICE activity is required to cleave the pre-
cursor form of IL-18 in order to produce a biologically
active IL-18. In contrast, IL-18 and ICE transcripts
have been detected in the corresponding normal colon
cells suggesting the bioactive IL-18 is most likely
produced by the adjacent normal cells [22]. So far, the
role of IL-18 produced in the surrounding normal tissue
in the anti-tumour immune response or cancer progres-
sion is still unknown. IL-18 alone or in combination
with IL-12 can be a potent adjuvant for reducing
tumour growth and metastasis in animal tumour models
[21]. Since our initial study has indicated the presence of
IL-18 in HCC, it is worthwhile performing further
studies to investigate whether the IL-18 detected is
biologically active and to elucidate the role of IL-18 in
liver and other cancers.
A previous study showed that IFN-g transcript was
expressed in HCC but not the protein [23]. Since IFN-gpromotes a Th1 response, which will be favourable for
the induction of the anti-tumour response, the absence
of IFN-g protein in the majority of the specimens in this
study suggests a favourable environment for evasion of
the immune surveillance system. The very low number
of cases with IFN-g immunoreactivity is consistent with
the hypothesis that tumour cells are allowed to survive
due the lack of a predominant Th1 response required for
the anti-tumour response. Further studies on factors
Fig. 3. Representative slides showing the immunohistochemical staining with anti-IFN-g. Moderate expression of IFN-g was detected in the
hepatocytes of the apparently normal tissue surrounding the tumour (a) and weak expression in the tumour (b). The negative control for the normal
and tumour tissue are as shown in (c) and (d) (200�/ magnification).
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172 169
that can contribute to enhance tumour cell survival
rather than death via apoptosis need to be investigated.
The presence of IFN-g in the tumour has been
associated with the rejection of the tumour. Non-
immunogenic tumour cells of high metastasing potential
and with the IFN-g gene insertion when injected into
syngeneic mice have a lower ability than the parental
cells to develop into tumours [24]. However, other
studies have shown that the presence of IFN-g may
not be beneficial, as the administration of IFN-g did not
inhibit the growth of the tumour in animal experimental
models. Thus, the role of IFN-g in the anti-tumour
response requires further investigation.
It is generally thought that IL-10 has the capacity to
suppress cellular responses via a number of ways such as
the inhibition of cytotoxicity and cytokine production
by tumour-specific T cells [25], suppression of the
tumoricidal activity of macrophages [26] and inhibition
of dendritic cell functions against tumour cells [27,28].
Perhaps, this will allow for progressive tumour growth
Fig. 4. Immunostaining with anti-IL-10. Intense staining was detected in the apparently normal tissue surrounding the tumour (a) and moderate
staining in the tumour (b). A representative slide showing weak staining in the normal surrounding tissue and in the tumour is as shown in (c) and (d),
respectively (200�/ magnification).
C.S. Chia et al. / Immunology Letters 84 (2002) 163�/172170
by prevention of the cellular immune responses. This is
consistent with the elevation of IL-10 expression in a
variety of human cancers including melanoma, non-
small cell lung carcinoma, gastric and colon adenocar-
cinoma [29,30]. Thus, according to this hypothesis, a
higher percentage of the tumour is expected to have
positive immunoreactivity for IL-10 as the presence of
IL-10 in the tumour microenvironment will allow for
suppression of the productive cellular immune response
that contributes to the anti-tumour effect. Our results
have shown that, a higher percentage of cells in the
tumour portion (five out of 17; 29.4%) produce IL-10,
and only one out of 17 (5.9%) tumour was positive for
IFN-g but it should be kept in mind that, the number of
samples available for this study was small. In the
apparently normal tissue adjacent to the tumour, only
one specimen was found to be have positive immunor-
eactivity for both IL-10 and IFN-g and two specimens
expressed IFN-g only. However, the immunoreactivity
for IFN-g in these cases is very weak. Due to the small
number of samples, it is difficult to be definitive that the
hypothesis that a predominant Th2 response exists in
the tumour microenvironment is upheld but the lack of
IL-10 in a large proportion of the tumours suggests a
lack of a predominant Th2 response.
The correlation between IL-18, IL-10 and IFN-gexpression was investigated due to a number of reasons.
Amongst the many biological activities of IL-18 and IL-
10 such as in anti-angiogenesis and immunosuppression,
respectively, IL-18 has been shown to inhibit production
of IL-10 in vitro [31], the correlation between IL-18 and
IL-10 production will give further insight into the
possible roles of these two cytokines in the anti-tumour
response. In this study, we found no relationship
between the expression of IL-10, IL-18 and IFN-g in
both tumour and the surrounding tissues. As IL-18 can
stimulate Th1 or Th2 responses depending on its
cytokine milieu [19], it will be interesting to further
investigate the profile of other cytokines.
In addition, this study needs to be further extended to
find out the phenotype and activation status of other
cells such as the T-lymphocytes in the tumour micro-
environment. One of the limitations of immunohisto-
chemical staining is that the amount of protein cannot
be quantitated and the intensity of staining can some-
times be fairly subjective. With the advances in laser
capture microdissection techniques and real-time poly-
merase chain reaction, further studies can be done to
analyse the level of the transcript for various cytokines.
The latter approach combined with immunohistochem-
ical staining of the specific cell types will provide
additional information on the source of the cytokines
produced at the tumour microenvironment. Thus, these
studies will contribute to the development of rationale
new therapeutic approaches for the treatment of cancer.
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