International Scholarly Research Network

ISRN Inflammation
Volume 2012, Article ID 578149, 6 pages
doi:10.5402/2012/578149

Research Article
Interleukin-17 Expression in the Barrett’s
Metaplasia-Dysplasia-Adenocarcinoma Sequence

J. R. Bannister,1, 2 A. L. Khan,2 D. W. Eccleston,1, 2

R. K. Deol-Poonia,2 and S. F. Hughes1
1 Department of Biological Sciences, University of Chester, Parkgate Road, Chester, CH1 4BJ, UK
2 Aintree University Hospital, NHS Foundation Trust, Liverpool L97AL, UK

Correspondence should be addressed to S. F. Hughes, stephen.hughes@chester.ac.uk

Received 18 November 2012; Accepted 14 December 2012

Academic Editors: A. Kamal and B. Kim

Copyright © 2012 J. R. Bannister et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. This pilot study evaluated the expression of the proinflammatory cytokine IL-17 along the Barrett’s metaplasia-
dysplasia-adenocarcinoma sequence by establishing the expression levels of IL-17 in columnar epithelium, intestinal metaplastic
cells, and dysplastic/glandular neoplastic cells. Immunohistochemical techniques were used to examine the accumulation of
the proinflammatory cytokine IL-17 in forty (n = 40) formalin-fixed, paraffin-embedded oesophageal archived specimens
across a range of endoscopic diagnostic categories, and a highly significant difference was found, where P ≤ 0.001, in IL-
17 expression (Kruskall Wallis and Mann-Whitney U) between all the cell types examined. There was also a strong positive
correlation (Spearman’s rank correlation) between disease progression and IL-17 expression (rs = 0.883, P < 0.001, n = 29), IL-17
expression was absent or absent/weak in columnar epithelium, weak to moderate in columnar metaplastic cells, and moderate
to strong in dysplastic/neoplastic cells, which demonstrated that the elevation of IL-17 expression occurs in the progression of
the disease. Understanding the differential expression of IL-17 between benign and malignant tissue potentially has a significant
diagnostic, prognostic, and therapeutic value. Ultimately, this selective biomarker may be employed in routine clinical practice for
the screening of oesophageal adenocarcinoma.

1. Introduction now beginning to gain greater attention [4]. Proinflam-

matory cytokines and their pathways are associated with
Oesophageal Adenocarcinoma (OAC) is the focus of intense inflammation in Barrett’s oesophagus and tumourigenesis.
research into the cause of the disease and treatment [1]. Elucidating the association between inflammation and OAC
In the context of OAC screening, a biomarker capable of may contribute to the eventual prevention of OAC [5].
reliably predicting progression to dysplasia or cancer by Further assessment of these cytokines has recently been
increased expression is highly valuable [2]. Oesophageal recommended, as these may be important targets for early
cancer develops symptoms late in the course of this disease
diagnosis and chemoprevention drugs [6, 7].
and carries a poor prognosis. In the UK, oesophageal cancer
is the eleventh most commonly diagnosed cancer and, of Inflammatory factors are commonly associated with
greater concern, the sixth most common cause of death contributing to the development of tumours; chronic inflam-
from cancer, which represented 1 : 20 of all cancer deaths in mation is often associated with malignancy [8]. Abdel-
the UK; 8,161 new cases were diagnosed in 2009 and the Latif et al. [5] concluded that chronic inflammation plays
mortality rate was 7,610 in 2010 [3]. a crucial role in the pathogenesis of the disease where
The role of inflammation in the development of OAC inflammatory cytokines are linked with advancement of
is not well understood, unlike its role in colon and breast tumourigenesis. IL-17 induces a wide variety of factors,
cancers. However, this role in the progression of OAC is which include chemokines, cytokines, acute phase proteins,
2 ISRN Inflammation

tissue remodelling proteins, antimicrobial proteins, and The rabbit polyclonal antibodies against human origin
nitric oxide. Cytokines that induce IL-17 include IL-6 IL-17 sc7927 from Santa Cruz Biotechnology (SCBT) have
and IL-8, and expression increases in both as the disease been used extensively in recent studies, including human
progresses. β-defensin, indirectly induced by IL-17, recruits immunohistochemical studies; therefore, this IL-17 antibody
dendritic cells and T lymphocytes to the inflamed site in the was selected for use by this study. The dilutions 1 : 50,
oesophagus through interaction with chemokine receptor 1 : 100, and 1 : 200 of the IL-17 antibody were prepared
(CCR) 6. Chemokine (C-C motif) ligand (CCL) 20 is up- as recommended by SCBT, and the stained tissue sections
regulated by IL-17, a chemokine that also attracts dendritic were examined via microscopy to find the initial optimum
cells and T lymphocytes to the inflamed site. β-defensins and dilution, which was 1 : 50. Further dilutions were prepared,
CCL20 serve to recruit and extend the TH 17 population at which included 1 : 30, 1 : 40, 1 : 50, and 1 : 60, and of these, the
the inflamed site [9]. IL-17 is implicated in several chronic 1 : 40 dilution was the optimum to visualise IL-17 expression
inflammatory diseases, including the autoimmune diseases, in the patient tissue samples. The same batch of this primary
rheumatoid arthritis (RA), and psoriasis [10, 11]. antibody was used throughout. Sample sections were cut at
Recent evidence suggests that the presence of infiltrating 2 μm. All positive controls were taken from a human kidney
IL-17+ cells, which include lymphocytes, mast cells, and biopsy [15].
neutrophils, in patients with OAC, may have a pathogenic Prior to staining, the antigens were unmasked at 97◦ C
role in this disease. Several studies have demonstrated a high with a target retrieval solution at pH 9.4. 30 mL of Dako
frequency of IL-17+ cells in inflammation-related cancers EnVision FLEX Target Retrieval Solution (High pH 50x) was
including OAC [12], nonsmall cell lung carcinoma, where added to 1.5 L of distilled water and treated at 97◦ C for 20
IL-17 has been implicated in metastasis of lung cancer by minutes using the Dako PT link machine (Dako).
promoting lymphangiogenesis [13] and hepatocellular car- The primary IL-17 antibody (1 : 40 dilution in Dako
cinoma where IL-17 is also believed to promote angiogenesis REAL Antibody Diluent, Santa Cruz Biotechnology, USA)
[14]. was used in this study. Staining was conducted using Dako
The aim of this pilot study was to evaluate the expression Autostainer Link 48 (Dako), which added 300 μL of undi-
of the proinflammatory cytokine IL-17 along the Barrett’s luted Dako EnVision FLEX Peroxidase-Blocking Reagent to
metaplasia-dysplasia-carcinoma sequence by establishing the each slide for 5 minutes. 300 μL of diluted IL-17 antibody
expression levels of IL-17 in columnar epithelium, intestinal was subsequently added to each slide for 30 minutes. The
metaplastic cells, and dysplastic/glandular neoplastic cells. treatment continued by adding 300 μL of undiluted Dako
EnVision FLEX+ Rabbit (Linker) to each slide for a further
15 minutes. After the Linker treatment, 300 μL of undiluted
Dako EnVision FLEX/horseradish peroxidase (HRP) was
2. Materials and Methods added to the slides for 20 minutes. This was followed by
adding a 300 μL of diluted chromogen reagent to each slide
Ethics Statement. This study, which included the use of for 5 minutes, followed by a buffer wash, and then by a
archived human tissue samples, was approved in writ- second application of 300 μL of diluted chromogen for 5
ing by the Proportionate Review Sub-Committee of the minutes. The diluted chromogen was prepared by placing 20
National Research Ethics Service Committee East Midlands- drops of Dako EnVision FLEX DAB+ Chromogen into 20 mL
Nottingham 2 Research Ethics Committee in the United of Dako EnVision FLEX Substrate Buffer. The final step
Kingdom, reference 11/EM/0384, on November 23 2011. of the automated autostainer process was to add 300 μL of
Written permission for this study was also received from the Dako EnVision FLEX Haematoxylin (Link) to each slide for
Research and Development Directorate at Aintree University 5 minutes. Between each treatment, the slides were washed
Hospital National Health Service Foundation Trust, refer- with a diluted wash buffer. The diluted wash buffer consisted
ence 451/11, on November 28 2011. of 0.5 L Dako EnVision FLEX Wash Buffer (20x) and 10 L of
Forty distal oesophageal biopsy samples embedded in distilled water. This buffer was placed into a large container
formalin-fixed paraffin blocks, originally collected between that was connected to the Autostainer.
2010 and 2011, were retrieved from the Cellular Pathology Before the cover slips were mounted, the sections were
department archive at Aintree University Hospitals National dehydrated by the Leica ST4040 system (Leica). The sections
Health Service Foundation Trust, UK; ten from each of the were dehydrated in ascending alcohols (50%, 70%, and 99%
following endoscopic examination result categories: Normal twice) for 15 seconds each using Alcohol Methylated Spirit
oesophagus, reflux oesophagitis, Barrett’s oesophagus, and 99% (Genta Medical) diluted with distilled water. The system
oesophageal adenocarcinoma. The mean age of the patients treated the slides with Xylene (Genta Medical) for 5 minutes.
involved with this study was 71 years (range: 49–88). After the section dehydration, the slides were placed into the
The IL-17 expression in columnar epithelium, columnar automated cover slipper, the Leica CV5000 (Leica).
metaplastic cells, and dysplastic/glandular neoplastic cells
was compared. A negative control was established using
an internal control on each slide (squamous mucosa), and 3. Histological Evaluation of IL-17 Staining
a positive control was established, which was taken from
normal kidney tissue. Immunohistochemistry was with IL- Two experienced pathology laboratory staff independently
17 antibody, and counter stained with haematoxylin. assessed tissue sections for the staining intensity of IL-17,
ISRN Inflammation 3

with scoring from 0 to 3 (0—no staining, 1—week intensity, 3

2—medium intensity, and 3—high intensity).

Staining intensity
2.5
The One-way nonparametric ANOVA (Kruskal-Wallis 2
test), and the Mann-Whitney U test were used to determine
1.5
if there was a difference in IL-17 expression. Pairwise
comparisons were made between the columnar epithelium, 1
intestinal metaplastic cells, and dysplastic cells/glandular 0.5
neoplastic cells. The association between disease progression 0
and the level of expression of IL-17 was evaluated using
Spearman’s rank correlation method to determine whether

(squamous mucosa)

Columnar
Positive control

metaplastic cells

glandular neoplastic cells

Neutrophils

Mast cells

Plasma cells
epithelia
Negative control

(glomeruli)

Intestinal
there was an association between disease progression and

Dysplastic/
the expression of IL-17. The original Haematoxylin and
Eosin (H&E) slides were used to compare the morphological
features of the samples with the IL-17 staining.

4. Results Cell types

Expression levels of IL-17 in columnar epithelium, intesti- Figure 1: Boxplot of the IL-17 staining intensity scores. Demonstra-
nal metaplasia, and dysplasia/glandular neoplasia were tion of the median (bold horizontal line), interquartile range (box
compared. There was significant difference between the where given) and whiskers to highest and lowest levels. Negative
expression of IL-17 between the three different cell types controls (n = 16), positive control (n = 8)—note: 1 positive control
(columnar epithelium, intestinal metaplastic cells, and dys- per batch run, columnar epithelium (n = 7), intestinal metaplastic
plastic/glandular neoplastic cells), (X 2 = 21.852, df = cells (n = 11), dysplastic/glandular neoplastic cells (n = 11),
neutrophils (n = 6), mast cells (n = 7), plasma cells (n = 1).
2, P < 0.001, n = 29, Figure 1). Squamous epithelium
included in the paraffin-embedded blocks demonstrated
absent cytoplasmic staining and acted as an internal neg-
ative control (Figure 2). Kidney-tissue positive controls
columnar epithelium to intestinal metaplasia, and onwards
demonstrated strong IL-17 expression with the collecting
towards dysplasia and glandular neoplasia. There was a
ducts (Figure 2). At the optimal concentration, columnar
strong positive correlation between disease progression and
epithelium demonstrated absent IL-17 staining in 3 of 7
IL-17 expression, and this suggests that IL-17 expression
(43%) or absent/weak staining (Figure 2) in 4 of 7 (57%).
increases as the disease progresses.
Interestingly, the staining was distributed throughout the
Immunohistochemistry staining scores were validated by
cytoplasm, and the stains were uniform for all the cell types
comparing the scores between two independent observa-
examined (n = 29). For the 11 intestinal metaplasia samples,
tions. The Cohen’s Kappa test results indicated that there was
5 of 11 (46%) demonstrated moderate staining (Figure 2), 4
“almost perfect agreement” (k = 1.00, n = 29) between
of 11 (36%) demonstrated weak staining, and 2 of 11 (18%)
the intensity scores provided by Dr. Abdul Khan and Mr.
demonstrated absent/weak staining. These staining scores
David Eccleston, both experienced histology pathology staff
were elevated when compared with columnar epithelium,
at the Aintree University Trust Hospital. Their long working
as demonstrated by the staining intensity scores, and the
relationship together within the Trust may have led to the
difference was highly significant difference (MWU, Z =
high scoring concordance.
−3.242, P = 0.001, n = 18). For the 11 dysplasia and
glandular neoplasia regions, 6 of 11 (55%) demonstrated
strong staining (Figure 2), 4 of 11 (36%) demonstrated 5. Discussion and Conclusion
moderate staining, and 1 of 11 (9%) demonstrated moder-
ate/strong staining. These staining scores were elevated when The findings, that there is a highly significant increase in IL-
compared with columnar epithelium, and also with intestinal 17 expression between columnar epithelium and intestinal
metaplasia; these were (MWU, Z = −3.598, P < 0.001, metaplastic cells, and between intestinal metaplastic cells and
n = 18) and (MWU, Z = −3.483, P < 0.001, n = 22), dysplastic/glandular neoplastic cells in the distal oesopha-
respectively. Neutrophils (n = 6) and mast cells (n = 7) gus, are novel (Figure 1). Amongst the native squamous
found in normal, Barrett’s oesophagus, and OAC cases were oesophagus and oesophageal columnar epithelium, there
all strongly positive. Surprisingly, only one case of plasma was little or no expression of IL-17. The trigger for IL-17
cells (n = 1) was found in an oesophagitis case, and it was elevated expression is not clear. There appears to be a stage
strongly positive. in the transition between columnar epithelium to intestinal
The Spearman rank correlation coefficient (non- columnar epithelium where expression of IL-17 becomes
parametric correlation analysis method) revealed that there apparent. During the investigation some IL-17 staining of
was a highly significant correlation (rs = 0.883, P < 0.001, the nuclei, but not cytoplasm, was observed in the columnar
n = 29) between IL-17 expression and the progression epithelium and squamous mucosa; this may be an indication
of the disease, where the disease progresses stepwise from of the transition phase where IL-17 becomes apparent.
4 ISRN Inflammation

(a) (b) (c)

(d) (e)

Figure 2: Expression of interleukin-17. (a) is an IL-17 positive control (human kidney). The epithelium lining the kidney collecting ducts
was strongly stained. Magnification ×100. (b) is an IL-17 negative control and demonstrates IL-17 immunohistochemistry treatment within
the oesophageal squamous mucosa. IL-17 staining is brown when present. Magnification ×200. (c) demonstrates IL-17 expression in the
columnar epithelium (without intestinal metaplasia) where IL-17 staining was absent/weak. Magnification ×200. (d) demonstrates IL-17
expression in the intestinal metaplasia where the IL-17 stain intensity was moderate. Intestinal metaplastic cells surround the goblet cells.
Magnification ×100. (e) demonstrates IL-17 expression in the glandular neoplastic cells where IL-17 stain intensity was strong. IL-17 staining
was absent within the neighbouring squamous mucosa region. Magnification ×200.

TH17 cells secrete IL-17. TH17 cells are associated with of the tumour. Examining similar serum markers against
mucosal immunity, particularly within the gastrointestinal a possible case of Barrett’s oesophagus or oesophageal
tract, and autoimmune diseases. A study examining gastric adenocarcinoma may also provide valuable diagnostic and
cancer conducted by Zhang et al. [15] found IL-17 mRNA prognostic information, which offers the prospect of an
expression in the majority of the tumour samples, and alternative cost-effective and noninvasive approach to the
expression was not detected in the patient’s normal gastric clinician.
tissue. This concurs with the finding of this study in that Recent research suggests that there is a diverse range
IL-17 expression is elevated in tissue affected by OAC at a of IL-17 producing cells amongst the immune system
level significantly greater level than that found in normal community, and innate immunity cells may secrete IL-
tissues. Radosavljevic et al. [16] studied serum IL-17 levels 17 [17]. Mast cells and neutrophils were found in nor-
in patients with colorectal carcinoma and found significantly mal, Barrett’s oesophagus, and OAC cases. Mast cells and
higher IL-17 serum levels in patients with colorectal carci- neutrophils were associated with strong IL-17 expression
noma than found in healthy subjects. Their study indicated in this study, suggesting that these innate immune cells
that IL-17, combined with other serum markers, is a valuable have an active role in maintaining high levels of IL-17
tumour marker in patients with colorectal carcinoma, which amongst the disease-affected tissues. Lin et al. [18] studied
may provide additional information about the characteristics the pathogenesis of psoriasis and demonstrated that mast
ISRN Inflammation 5

cells and neutrophils, rather than T lymphocytes, are the Acknowledgments

prominent immune cell types that express IL-17 in the
human skin. Release of IL-17 from mast cells and neutrophils Thanks to Dr Gareth Elfed Jones (University of Chester)
may be important to the pathogenesis of OAC and may with help regarding statistical advice. The authors thankfully
have a similar pattern of chronic inflammation as seen in acknowledge the University of Chester for their financial
psoriasis. support.
Clear evidence of any lymphocyte involvement in the
normal, Barrett’s oesophagus, or OAC cases was not found.
However, the stroma was affected by strong background References
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6 ISRN Inflammation

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