Document Type : Original Article
Authors
1 Dept. Oral Medicine, Peridontology and Diagnosis. Faculty of Oral and Dental Medicine, Cairo University, Egypt
2 Dept of Oral Medicine, Periodontolgy and Diagnosis, Faculty of Dental Medicine, Cairo University, Egypt
3 Biochemistry Dept, Faculty of Medicine, Cairo University, Egypt
Abstract
Keywords
LP is a relatively common chronic
inflammatory
muco-cutaneous disease of probable immune based
etiology, and involves oral and genital mucosal
surfaces, skin, scalp and nails. OLP presents oral
mucosal lesions such as white striations, papules,
plaques, erythema, blisters or ulcers.
(1) LP is
characterized by a T cell mediated immune response
against epithelial cells, causing epithelial cell
damage and subepithelial persistent accumulation
of T lymphocytes. The mechanism involved in this
chronic inflammatory disease remains unclear.
(2-4)
Cell mediated immunity in OLP may be
regulated by various cytokines and their receptors,
(5)
so identification of the specificity of T helper
cells(Th) is one of the most important steps to reveal
pathogenesis and etiology of OLP.
* Lecturer at Department of Oral Medicine, Periodontology and Diagnosis, Faculty of Oral and Dental Medicine,
Cairo University.
** Prof. at Department of Medical Biochemistry, Faculty of Medicine, Cairo University.
Al-Azhar Journal of Dental Science
Vol. 19 - No. 1 - January 2016
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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1
Although cytotoic CD8+T lymphocytes are the
majority of intraepithelial lymphocytes and subepithelial
lymphocytes in OLP, most lymphocytes
in the lamina propria are identified as CD4+ T cells.
(5, 6)
CD4+ helper cells have been suggested to play
an important role in cytotoxic CD8+T cell activation
via T helper cell related cytokine release.
(6)
It is well accepted that two CD4+ helper T cells
subsets namely Th1 and Th2 are associated with
immunopathogenesis of OLP. Th1 and Th2 cells
are identified by their signature cytokines interferon
gamma (IFN γ) and IL-4 respectively.
(5, 7)
Recently, Th 17 cells (IL-17 producing CD4 T
cells) have been discovered as a unique subset of
T helper cells that develop along with a pathway
that distinct from the Th1 and Th2 differentiation
pathways. Th17 cells are characterized by the
production of potent pro-inflammatory cytokines
IL-17, IL-17 f, IL-21, IL-22 and IL-26, which
suggests that these cells function as pleiotropic
pro-inflammatory T helper cells.
(8, 9) IL-17 (also
referred as IL-17A) is the signature cytokine of T
helper 17 cells, which are considered a key T cell
subset involved in the etiology of autoimmune
and inflammatory disorders such as Lupus
Erythematosis, Multiple Sclerosis, Rheumaroid
Arthritis and tumor microenvironments.
(10, 11, 12)
The human IL-17A gene is a protein of 150
amino acids with a molecular weight of 15 KDa and
is secreted as disulfide linked homodimer of 30-35
KDa glyco-proteins.
(13)
There were five related cytokines identified (B,
C, D, E, F), that share 20- 50% homology to IL-
17A. IL-17A has been designated A to indicate that
it is the essential member of the IL-17 cytokines
family.
(14)
IL-17 has been shown to signal through the IL-
17R molecule and promoting production of tumor
necrosis factor alpha (TNF-α), IL-1ß IL-6, IL-8 and
granulocyte colony stimulating factor (G-CSF).
(15-17)
Thus, IL-17A functions as a pro-inflammatory
cytokine which can activate different cells such
as epithelial, endothelial, fibroblast, chondrocyte
and osteoblast producing numerous inflammatory
molecules including cytokines, chemokines,
defensins and MMPs.
(14, 18)
The receptor for IL-17A (IL-17R) is a single
pass-transmembrane protein of approximately 130
KDa, while the IL-17A cytokine expressed only
by T cells, its receptor is expressed in all tissues
examined. The activation of the receptor by IL-
17A generally results in the induction of other
proinflammatory cytokines through activation of
NK-Kß.
(19)
Although it is accepted that OLP is a localized
disease, an increasing number of studies indicate
that many significant changes in peripheral blood
are implicated in the pathogenesis.
(20, 21, 22)Several
previous investigations focused on the alteration
of T lymphocyte subsets in the peripheral blood of
OLP lesions.
(22)
Some studies found the presence of IL-17 in
the local lesion of OLP, suggesting its role in local
environments.
(23) Other studies detected the serum
and saliva levels of IL-17 in OLP patients, but
found no significant difference compared to healthy
groups.
(24)
The aim of this study was to investigate possible
correlation between serum and tissue levels ofIL-
17 as well as the expression of its tissue receptor
in order to detect their possible role in the etiopathogenesisof
OLP.
SUBJECTS AND METHODS
I. Study Population
The entire study sample comprised 30
individuals;
20 oral lichen planus (OLP) patients
were selected, in addition, 10 age and sex matched
healthy normal volunteer individuals free from any
systemic diseasewere recruited as control subjects.
A.J.D.S. Vol. 19, No. 1
EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 3
II. Inclusion and exclusion criteria
All subjects participated in this study were
selected from the Outpatient Clinic, Department of
Oral Medicine, Oral Diagnosis and Periodontology,
Faculty of Oral and Dental Medicine, Cairo
University, between February 2014 and December
2014.
A detailed medical history of each subject was
obtained according to the detailed questionnaire of
the modified Cornell Medical Index.
(20) All subjects
were free from any systemic disease and did not
receive any medication either topical or systemic
that could cause lichenoid reaction during the 3
months prior to the specimen collection. Moreover,
patients with suspected restoration-related reaction
were excluded from this study. All patients had oral
symptoms, and every case was clinically diagnosed
as erosive lichen planus (ELP) or atrophic lichen
planus(ALP). Duration of the disease ranged
from 2 to 3 months with periods of remission and
exacerbation. The lesions were bilaterally selected
and extended to involve the buccal mucosa,
labial mucosa, and tongue which varied from
one patient to another. Diagnosis was confirmed
by histopathologic examination according to the
World Health Organization’s (WHO’s) clinicopathological
diagnostic criteria for LP.
(21)
III. Ethical procedures:
All subjects were informed about the detailed
procedure and they were given written approval
consent to sign. Patients were treated after the samples
had been collected. The study was performed
between February 2014 and December 2014.
The thirty selected participants were divided into
two groups as follow:-
Group A:
It included 20 patients suffering from OLP,
3 males and 12 females. Their ages ranged from
36-48 years.
Group B:
It included 10 medically free subjects as controls,
4 males and 11 females. Their ages ranged
from 35-42 years.
VI. Collection of samples
i. Serum sample collection
Peripheral venous blood samples (5 ml) were
obtained by standardvenipuncture from subjects
using plain tubes. Samples were centrifuged. The
clarifying supernatant was filtered and stored at −20
°C until assayed.
Serum IL-17 level was determined using a
special kit (Human IL-17, ELISA Crokit ko13207p,
South Korea) with ELISA assay according to
manufacturer’s instructions.
ii. Tissue samples.
20 OLP lesion specimens were biopsied from selected
patients, and 10 normal oral mucosa (NOM)
tissues were collected from healthy volunteers receiving
ortho-gnathic surgery.
IL-17protein in tissue levels were assessed using
same ELISA kit.
Real-time RT-PCR:
Detection of tissue IL-17 receptor gene
expression by Quantitative real time polymerase
chain reaction (qRT-PCR).
Total RNA was extracted from frozen tissue
samples using the RN easy Mini Kit (Qiagen Inc)
following the manufacturer’s protocol, extracted
RNA was quantified by spectrophotometry. The
RNA integrity was assessed using agarose gel
electrophoresis and ethidium bromide staining.
2 μg of total RNA were reverse transcribed in
0.05 M Tris–HCl pH 8.3, 40 mM KCl, 7 mM MgCl2
buffer containing 0.05 μg of random hexamers,
1 mM dNTPs mix, 0.05 U/l RNase inhibitor and
200 U/l murineleukemia virus reverse transcriptase
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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1
M-MLV. Samples were incubated for 10 min at
70˚C and then 60 min at 37 C. Inactivation of the
reverse transcriptase was achieved by heating the
samples at 95C for 10 min.
Real-time RT-PCR for quantitative assessment
of mRNA expression was performed on step
one plus (Applied Biosystems, USA), reaction
contained SYBR Green Master Mix (Applied
Biosystems), gene-specific forward and reverse
primers (10 μM), cDNA and nuclease-free water.
The sequences of PCR primer pairs used for each
gene are shown in Table 1. With cycling conditions
(10 min at 95°C followed by 40 cycles of 15 s at
95°C and 60 s at 60°C). The level of expression
of each target gene was normalized relative to
the expression of GAPDH mRNA in that sample
using the ΔCt method. Relative differences in gene
expression among groups were determined using the
comparative Ct (ΔΔCt) method and fold expression
was calculated as 2
−ΔΔCt, where ΔΔCt represents
ΔCt values normalized relative to the mean ΔCt of
control samples (R).
TABLE (1)
Primer sequences used for RT-PCR(25)
Primer Sequence
IL-17
Forward primer :5′-GCTCCAGAAGGCCC TCAGACT-3′
Reverse primer : 5′-CCAGCTTTCCCTCCGCATTGA-3′
IL-17 R A Forward: 5′-AGACACTCCAGAACCAATTCC-3′,
Reverse: 5′-TCTTAGAGTTGCTCTCCACCA-3′,
GAPDH
Forward: 5`-AGA GAT ATC CCT CTG TG ATC-3`
Reverse: 5`-TAC CCC AAA GTT ATC TCA GG-3`′
V. Statistical Analysis:
Quantitative data were presented as mean,
median, standard deviation (SD), range (Minimum
– Maximum) and 95% Confidence interval (95%
CI) for the mean values. Data were explored for
normality by checking the data distribution and
using Kolmogorov-Smirnov and Shapiro-Wilk tests.
IL-17 levels data showed parametric distribution.
Student’s t-test was used to compare between the
two groups. Pearson’s correlation coefficient was
used to determine the correlation between IL-17
levels in serum, tissue and tissue receptor.
ROC (Receiver Operating Characteristic) curve
was constructed to determine the cut-off values of
IL-17 for detection of OLP. Areas under the ROC
curve (AUCs), sensitivity, specificity, predictive
values and diagnostic accuracy was calculated.
The significance level was set at P ≤ 0.05. Sta
tistical
analysis was performed with IBM
® SPSS®
Statistics Version 20 for Windows.
Comparison between the two groups
In serum, tissues as well as tissue receptor, study
group showed statistically significantly higher mean
IL-17 level than control group ( Table 2, figure 1)).
Correlation between serum and receptor IL-17
levels
There was a statistically significant positive
(direct) correlation between serum and receptor
IL-17 levels (
r = 0.940, P-value <0.001) i.e. an
increase in serum level of IL-17 is associated with
an increase in receptor level of IL-17 (Fig. 2).
Correlation between serum and tissue IL-17 levels
There was a statistically significant positive
(direct) correlation between serum and tissue IL-17
levels (
r = 0.862, P-value <0.001) i.e. an increase in
serum level of IL-17 is associated with an increase
in tissue level of IL-17 (Fig. 3).
Correlation between receptor and tissue IL-17
levels
There was a statistically significant positive
(direct) correlation between receptor and tissue
IL-17 levels (
r = 0.828, P-value <0.001) i.e. an
increase in receptor level of IL-17 is associated with
an increase in tissue level of IL-17 (Fig.4).
®
IBM Corporation, NY, USA. ®SPSS, Inc., an IBM Company.
A.J.D.S. Vol. 19, No. 1
EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 5
TABLE (2)
Descriptive statistics and results of Student’s t-test for comparison between IL-17 serum, tissue
levels as well as IL-17 tissue receptor levels in the two groups
Group Mean SD Median Minimum Maximum
95% CI
Lower
P-value
bound
Upper
bound
Serum
Study
113.3 21.5 107.0 67.4 162.8 103.2 123.4
<0.001*
Control
25.5 6.7 24.1 18.2 40.6 20.8 30.3
Receptor
Study
9.6 2.8 10.1 4.9 13.7 8.3 10.9
<0.001*
Control
1.1 0.1 1.0 0.9 1.3 1.0 1.2
Tissue
Study
142.6 26.5 142.7 97.3 201.3 130.2 155.0
<0.001*
Control
43.4 8.3 41.7 33.4 61.2 37.5 49.4
*: Significant at P ≤ 0.05
FIG (1) Bar chart representing mean and standard deviation
values of IL-17 levels in the two groups
FIG (3) Scatter diagram representing positive correlation between
serum and tissue levels of IL-17
FIG (2) Scatter diagram representing positive correlation between
serum and receptor levels of IL-17
FIG (4) Scatter diagram representing positive correlation between
receptor and tissue levels of IL-17
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Amal A. Hussine, et al. A.J.D.S. Vol. 19, No. 1
ROC curve analysis (Cut-off values)
ROC curve analysis of serum, receptor and tissue
levels of IL-17 in the present study showed cut-off
values of 40.6, 1.3 and 61.2 pg/ml, respectively.
DISCUSSION
The pathogenesis of OLP still remains poorly
understood although it is characterized by a T cell
mediated immune response against epithelial cells
causing epithelial cell damage and subepithelial
infiltration of T lymphocytes.
(26, 27)Interleukin-17
family consists of cytokines that are secreted by Th-
17 cells and have an important role in the regulation
of mucous and epithelial immune responses.
(28)
There is increasing evidence that IL-17 is
a critical pro-inflammatory cytokine in human
autoimmune diseases, and it has previously been
demonstrated to stimulate the expression of
TNF-α and IL-6 in several cell types including
keratinocytes, fibroblasts, endothelial cells and
macrophages, as well as inducing the production
of several chemokines, such as CCL-20 and
CXCL-8
(8). High levels of IL-17 and Th17 cells
can provoke an inflammatory response and are
correlated with autoimmune diseases such as
inflammatory bowel disease, rheumatoid arthritis,
multiple sclerosis and psoriasis.
(29,30)
TABLE (3)
Sensitivity, specificity, predictive values, diagnostic accuracy, Area under the ROC curve
(AUC), 95% confidence interval (95% CI) of serum and tissue IL-17 levels
IL-17 (pg/ml) Cut-off Sensitivity %
Specificity % +PV% -PV% Diagnostic
accuracy % AUC 95% CI
Serum
40.6 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000
Receptor
1.3 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000
Tissue
61.2 100.0 100.0 100.0 100.0 100.0 1.000 0.884 – 1.000
+PV: Positive Predictive Value, -PV: Negative Predictive Value
At these cut-off values, the diagnostic accuracy
of IL-17 as a marker for detecting OLP is 100.0%.
Results of ROC curve analysis are presented in
Table (3)
A previous study demonstrated that Th1 and
Th17 were scattered in lamina propria of local
lesions in OLP, in addition, there was increased
proportions of circulating Th1 and Th17 cells,
(31)
which correlates closely with our findings of
increased IL-17 in serum and tissue with increased
expression of IL-17R in the local lesions, indicating
that IL-17 mediates immune response in OLP.
Recent studies have suggested that different
lichenoid tissue reaction/ interface dermatitis
disorder, including lichen planus and psoriasis,
may share a common inflammatory signaling
pathway
(32) which supports our research studying
the IL-17 and its receptor expression in local lesions
of OLP correlating them with serum IL-17 levels.
In a study done by Ruilu et al,
(33) they observed a
large number of IL-17 + cells located in the sub
epithelial
lymphocytic infiltrate in the OLP lesions
and showed increased higher mRNA expression
of IL- 17 in OLP lesions compared to the normal
tissues. In our study we investigated these findings
by measuring IL-17 itself in local environment to
prove whether mRNA completed its transduction
A.J.D.S. Vol. 19, No. 1
EVALUATION OF THE POSSIBLE ROLE OF IL-17 AND ITS RECEPTOR 7
pathway and gave its final product IL-17. In
addition, we measured IL-17R in the local lesions
to be sure that IL-17 will perform its action. In the
present study, it was found that there is a statistically
significant relation between the increases of IL-17
in serum of patient than in normal individual.
These findings are also consistent with the
published study by Piccinni and his colleagues who
also found an elevated mRNA expression of IL-17,
together with other TH-17 type molecules in OLP
lesions compared to healthy mucosa.
(33 )Moreover,
the presence of Th17 was also identified in another
study conducted by Xie et al
(31)
Overexpression of IL-17 in OLP lesions
highlighted its potent pro-inflammatory properties
which may induce profound biological effects and
play an important role in the formation and progress
of the disease, especially that our study not only
found an increase in the local lesion, but also in
serum of the patients. This phenomenon indicated
that the overexpression of IL-17 in OLP lesions
attributes not only to the lymphocytic infiltration,
but also to other unknown regulatory mechanisms,
which is recommended for further exploration.
Finding possible techniques and materials that
control and decrease IL-17 serum levels may open
new horizons to the treatment of such medical
condition other than corticosteroids with their side
)
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