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Receptor activator of NF-κB ligand and osteoprotegerin expression in chronic
apical periodontitis: possible association with inflammatory cells
FAN Rong, SUN Bin, ZHANG Cheng-fei, LÜ Ya-lin, XUAN Wei, WANG Qian-qian and YIN
Xing-zhe
Department of Stomatology, Beijing Anzhen Hospital, Capital Medical University, Beijing
100029, China (Fan R, Lü YL and Xuan W)
Department of Special Dentistry, Peking University School and Hospital of Stomatology, Beijing
100081, China (Fan R, Zhang CF, Wang QQ and Yin XZ)
Department of Oral and Maxillofacial Surgery, School of Stomatology, Capital Medical
University, Beijing 100050, China (Sun B)
Comprehensive Dental Care, Endodontics, Faculty of Dentistry, the University of Hong Kong,
Hong Kong SAR, China (Zhang CF)
Correspondence to: Dr. ZhANG Cheng-fei, Area of Endodontics, Comprehensive Dental Care,
Faculty of Dentistry, the University of Hong Kong HKSAR, China
(Email: [email protected])
Key Words: apical periodontitis; receptor activator of nuclear factor kappa B ligand;
osteoprotegerin; inflammation; bone resorption; immunohistochemistry
Background Receptor activator of NF-κB ligand (RANKL) and osteoprotegerin
(OPG) have been recently shown to play important roles in bone resorption. The aim
of this study was to investigate the possible association between the expression of
bone resorption regulators (RANKL and OPG) and inflammatory cell infiltration in
chronic periapical periodontitis.
Methods
The samples of chronic periapical lesions (n = 40) and healthy periapical
tissues (n = 10) were examined for immunohistochemical analysis of RANKL and
OPG. Lesion samples were further analysed the inflammatory infiltration condition.
The inflammatory cell infiltration was scored in relation to immunohistochemical
reactivity for CD3, CD20 and CD68.
Results
The number of RANKL-positive cells and the ratio of RANKL/OPG in
chronic apical periodontitis were significantly higher than those in healthy periapical
tissues (P<0.001). The number of RANKL-positive cells was higher in lesions with
1
severe inflammatory infiltration than in those with light inflammatory infiltration
(P<0.05). Significantly increased RANKL expression was found with T lymphocytes
(CD3+), macrophages (CD68+) and B lymphocytes (CD20+) infiltration (P<0.05). No
association was found between the ratio of RANKL/OPG and inflammatory cell
infiltration.
Conclusions
RANKL expression was increased with T, B lymphocytes and
macrophages infiltration, respectively in chronic periapical lesions. RANKL appears
to be closely related to periapical inflammatory infiltrates. The relative ratio of
RANKL/OPG may be a key determinant of RANKL-mediated bone resorption.
Chronic apical periodontitis occurs as a result of local inflammatory response, which
is mediated by infiltrated inflammatory cells and their products.1 The inflammatory
process ultimately results in periapical bone resorption, which is the unique function
of osteoclasts.2 Receptor activator of NF-κB ligand (RANKL), its receptor, RANK,
and osteoprotegerin (OPG) play crucial roles in regulating the differentiation,
activation and survival of osteoclasts in both health and disease.3,4 RANKL induces
bone destruction, and its natural decoy receptor, OPG protects against bone
destruction by preventing the binding of RANKL with its receptor RANK.5,6
During normal bone remodeling, osteoblasts are thought to provide RANKL that
stimulates bone resorption. Abnormal activation of the immune system leads to bone
destruction
in
diseases
such
as
rheumatoid
arthritis
and
periodontitis.3,4
Osteoimmunology is an emerging field of research dedicated to investigating the
interactions between the immune and skeletal systems.7 Chronic periodontitis share
some of the features of periapical periodontitis.1,8 It is evident that the major sources
of RANKL mediating periodontal bone destruction in vivo are lymphocytes and
macrophages. 4,9
RANKL and OPG have been recently shown to play critical roles in periradicular
bone destruction.10,11 However, the precise mechanisms of periapical inflammatory
bone resorption remains unclear. Periradicular infiltration is mainly characterized by
2
macrophages, and T and B lymphocytes.12 These cells may be important stimulators
of osteroclastogenesis. It is generally accepted that T lymphocytes have multiple
biological activities and have received considerable attention because of its central
role in the concept of osteoimmunology.7,13,14 The role of macrophages in
RANKL-induced bone resorption is controversial. High RANKL levels were related
to the monocyte-macrophage activity in six periapical granulomas.15 Conversely,
other reports revealed that activated macrophages do not express RANKL in vivo.16
Data are accumulating in support of B cells involvement in bone resorption.4,17
However, there are still few reports discussing the effect of B lymphocytes on
periapical bone loss.
It is general believed that the balance between RANKL and OPG is important for
regulating osteoclastogenesis.5 The relative ratio of RANKL/OPG may also correlate
with the clinical severity of periodontitis.18 To date, little information is available
about whether the RANKL/OPG ratio is related to the severity of periapical
inflammation.
Accordingly, the present study aimed to investigate the expression of RANKL, OPG
and T, B lymphocytes and macrophages immunohistochemically in periapical lesions,
to evaluate whether the level of RANKL or RANKL/OPG ratio is related to
inflammatory cells infiltration.
MATERIAL AND METHODS
Tissue Preparation
This study obtained institutional review board approval by the Ethical Committee of
Peking University Health Science Center. A total of 50 subjects were included in this
study. Written and informed consents from all patients were obtained before sample
collection.
The diseased tissue samples comprised forty chronic periapical lesions. The samples
were obtained from 40 patients, aged 16-60 (average 37) years, subjected to periapical
surgery and curettage of the tissue as part of the clinical treatment. All patients had
radiographic evidence of periapical lesions and disappearance of the periodontal
3
ligament space. The control group comprised ten samples of clinically healthy apical
periodontal ligament obtained from permanent premolars extracted for orthodontic
purposes from 10 subjects, (age, 18-23 years) presenting good oral health. All these
premolars were with the completion of root development and without periapical
pathosis. The periodontal ligament was scraped off the roots with a sterile blade. All
patients were negative for systemic diseases and were not under antibiotics for the last
2 months. All samples were immediately fixed in 10% neutral buffered formalin
overnight and embedded in paraffin.
Immunohistochemistry
EnVision 2-step immunoperoxidase method was applied to detect the expression of
RANKL, OPG, CD3 (T lymphocyte marker), CD20 (B lymphocyte marker) and
CD68 (macrophage marker). Consecutive sections were cut and stained using
Envision peroxidase procedure (Dako, Glostrup, Denmark), according to the
manufacturer's protocol. Briefly, 4-μm thick sections were deparaffinized and
dehydrated followed by antigen retrieval, which was performed by autoclave in 10
mM citrate buffer (pH=6.0) for 10min (120℃, 2 atm). Endogenous peroxidase
activity was blocked with 3% hydrogen peroxide for 15 min.
After rinsing 3 times with PBS, sections were incubated for 30 min at room
temperature (CD3, CD20 and CD68) or at 4°C overnight (RANKL and OPG) with
the primary antibodies. Primary antibodies used in this study were as follows:
anti-OPG antibodies (mAb8051; R&D Systems, Minneapolis, MN, USA; dilution
1:300), anti-RANKL antibodies (mAb626; R&D Systems; dilution 1:100), rabbit
polyclonal anti-CD3 (Neomarkers, Fremont, CA; dilution 1:200) to detect T
lymphocytes, rabbit polyclonal anti-CD20 (Neomarkers; dilution 1:400) to detect B
lymphocytes, and mouse monoclonal anti-CD68 (Neomarkers; dilution 1:50) to detect
macrophages. The sections were subsequently incubated with a polymer-peroxidase
complex and 3,3′-diaminobenzidine (DAKO), and counterstained with haematoxylin.
Negative controls with the omission of the primary antibody were performed for each
case.
4
Cell Counting
One section from each periapical lesion specimen was stained with haematoxylin and
eosin
for
histologic
analysis
and
grading
of
inflammation
prior
to
immunohistochemistry. According to Tsai et al19, each specimen was graded at ×200
magnification as: grade - light, inflammatory cells less than 1/3 per field; grade moderate, inflammatory cells between 1/3 and 2/3 per field; and grade - severe,
inflammatory cells more than 2/3 per field.
The number of positively stained cells for RANKL or OPG was counted in 10
consecutive microscopic high-power fields (×400). According to Liapatas et al20,
immunohistochemical reactivity for CD3, CD20 and CD68 was classified into five
categories according to the percentage of positively staining cells as follows: 0= no
staining; 1= light, less than 10%; 2= moderate, 10-25%; 3= intense, 25-50%; and 4=
very intense, more than 50%.
Statistical Analysis
Data were expressed as mean ± standard deviation (SD). Differences among groups
were assessed using Student’s t test or one-way analysis of variance (ANOVA).
Statistical significance was set at P < 0.05. All data were analyzed by SPSS (Ver. 13.0,
SPSS Inc., USA).
RESULTS
All periapical lesions showed positive immunoreactivity to RANKL and OPG (Figure
1A, C) and showed various degrees of inflammatory cell infiltration. RANKL protein
was mainly in the nucleus of the lymphocytes, plasma cells, macrophage-like cells,
epithelial cells and fibroblast-like cells. OPG protein was found mostly in the
cytoplasm of the oral epithelium and inflammatory cells. RANKL and OPG were also
expressed in the samples of normal controls (Figure 1B, D). The presence of CD3,
CD20, CD68 antigens varied from light to intense in all periapical lesion specimens
(Figure 2). The normal control subjects showed no or minimal inflammatory
5
infiltration. Negative controls were obtained by replacing the primary antibodies by
PBS showing no stained cells.
As shown in Table 1, Periapical lesion tissues had a significantly higher number of
RANKL-positive cells (P < 0.001). There were no differences in OPG expression
between periapical lesions and controls ( P > 0.05). Periapical lesion tissues showed a
significantly higher ratio of RANKL/OPG ( P < 0.001).
The number of RANKL-positive cells was significantly higher in periapical lesions
with servere inflammation than those with light inflammation ( P < 0.05) (Table 2).
There was a significant association between the number of RANKL-positive cells and
the degree of CD3, CD20 and CD68 infiltration, respectively ( P < 0.05) (Table 3).
Moreover, the ratio of RANKL/OPG did not show association with the degree of
periapical inflammation, as well as CD3+, CD20+ and CD68+ infiltration ( P > 0.05)
(Table 4, 5).
DISCUSSION
In the present study, the number of RANKL-positive cells, as well as the
RANKL/OPG ratio in periapical lesions were significantly higher than those in
healthy periapical tissues. The results are consistent with previous studies.11,14
RANKL expression was also detected in some healthy periapical tissues. Such
findings, in accordance with Menezes et al11, may account for a regulatory role on
osteoclasts to maintain a normal bone homeostasis. Furthermore, it could imply that
only the existence and abundance of RANKL might not always lead to bone
resorption. It is suggested whether bone resportion or bone formation occurs depends
critically on the RANKL/OPG ratio, rather than a single value of RANKL or OPG.5
The number of RANKL-positive cells was found to increase with the grade of
periapical inflammation, suggesting a clear positive relation between high RANKL
expression and leukocytes activities during periapical bone loss. Furthermore, the
number of RANKL-positive cells was found to be closely related to the degree of the
main inflammatory cells, including T, B lymphocytes and macrophages. Lesions with
intense infiltrated T cells showed a significantly higher RANKL expression than those
6
with light or moderate infiltrated T cells. T cells may play an important role not only
as an initial trigger but also as a constant stimulator of bone resorption.21 In
pathological conditions, the effects of T cells on osteoclastogenesis depend on a
dynamic balance between positive and negative factors that they express.21 T help
type 1 (Th1) immune response is involved in the progession of periapical lesion and
bone destruction, whereas Th2 cytokines are more significant for the restriction of
immune mechamisms.13,14 The balance between Th1 and Th2 cytokines may
determine the overall biological events in the lesions.14 Previous studies in
periodontitis have elucidated that activated CD4+ T cells express RANKL in vivo and
activated CD8+ T cells suppress osteoclastogenesis in vitro.22,23 Further studies should
be done to evaluate the roles of T-lymphocyte subsets in periapical bone loss,
importantly, to identify the osteoclastogenic T-cell subset. In a recently work, most of
RANKL+ cells were fibroblastic, but few of them were T cells in vitro.16 Other studies
have also shown that gingival fibroblasts and periodontal ligament cells can be
stimulated to produce RANKL via microbial challenge or contact with bacterial
products.9
A significant association was found between the number of RANKL-positive cells
and the degree of infiltrated macrophages. Our findings are in agreement with several
studies.11,24,25 Macrophages may also participate in osteoclast formation, either by
producing proinflammatory cytokines or by serving themselves, as osteoclast
precursors.9,26 However, it is not sure wheather these cells were indeed the source of
this RANKL or merely binding RANKL derived from other cells, because
macrophages may also express RANK. Yoshinaga et al27 proposed no report has
clearly shown that macrophages produce RANKL in vitro. Despite some controversies,
the current consensus is that activated macrophages would participate in the formation
and perpetuation of the periapical lesion and bone destruction. Further studies are
needed to clarify this issue.
Accumluating evidence indicate that B cells not only produce bacteria-reactive IgG,
but also contribute to pathogenic bone resportion.9,28 In periodontal diseases, B and T
cells are proved to be the mainly celluar source of RANKL in vivo.4 B cells may also
7
participate in osteoclast formation, either by expressing RANKL or by serving
themselves, as osteoclast progenitor cells.29 Morever, osteoblasts support commitment
and differentiation of all stages of B cells development.28 However, what is often
overlooked is the close relationship between B cells and bone cells. Only one report
was found discussing the potential role of B cells in RANKL-mediated periapical
bone resorption, showing that lymphocytes were the predominant leukocyte cells, but
monocytes and dendritic cells were mainly responsible for synthesizing RANKL in
six periapical granulomas.15
Taken together, close relationships were found between the number of
RANKL-positive cells and the degree of macrophages and T, B lymphocytes
infiltration respectively, suggesting that these leukocytes may be the three most likely
sources of RANKL in the periapical bone resorption. However, as a limitation of the
immunohistochemistry technique, we were unable to definitively identify the cellular
sources of RANKL. And not all inflammatory cells in periapical lesions are in a state
of activation.26 Further studies should be carried out to precisely identify the
distribution of RANKL-expressing cells.
In the present study, the ratio of RANKL/OPG is not directly related to periapical
inflammatory infiltration. The results are not unexpected, and similar observations
were described by Geusens et al.30 RANKL-mediated osteoclastogenesis is not
responsible for all pathological bone destruction. Other cytokines, such as
interleukin-1(IL-1) and tumor necrosis factor α (TNF-α) can promote osteoclast
formation by a fully or partilly independent-RANKL pathway.31 Our results can be
further explained by a recent study that the RANKL/OPG ratio was associated with
the progressive or stable nature of periapical granulomas.32 The progression and
expansion of the disease and the occurance of bone destruction are initially trigged by
the abnormal host immune response determined by inflammatory cells. Inflammatory
infiltration in the periapex spaces seems to be a critical step in both immune responses
and bone destruction. Although inflammation and osteoclastogenesis are different
processes in the periapical region, proinflammatory cytokines act as major co-factors
in osteoclast formation and activation. The outcomes of these pathological events are
8
determined by the balance between pro- and anti-inflammatory cytokines.8 Clinically,
it is unlikely that periradicular bone destruction would occur in the absence of
inflammation, but inflammation may occur in the absence of bone destruction.
In conclusion, the expression of RANKL, but not the RANKL /OPG ratio, was
significantlly associated with the grade of periapical inflammation. Morever, the
expression of RANKL, but not the RANKL/OPG ratio, was also increased with the
severity of macrophages or T-, B- lymphocytes. Our findings implied that RANKL
appears to be closely related to periapical inflammatory infiltrates, and the relative
ratio of RANKL/OPG may be a key determinant of RANKL-mediated bone
resorption.
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11
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Figure 1. Immunohistochemical analysis of RANKL and OPG expression in chronic periapical lesions and healthy
periapical tissues (original magnification × 400). 1A: RANKL protein in periapical lesions. 1B: RANKL protein in
healthy periapical tissues. 1C: OPG protein in periapical lesions. 1D: OPG protein in healthy periapcial tissues
(arrows)
Figure 2. Immunohistochemical staining for major inflammatory cells in chronic periapical lesions (original
magnification × 400). 2A: CD3 marker for T lymphocytes. 2B: CD20 marker for B lymphocytes. 2C: CD68
marker for macrophages (arrows).
Table 1. the semiquantitative analysis of lesion and control groups immunostained for RANKL and OPG
Groups
RANKL
OPG
Lesions ( n = 40 )
42.03 ± 8.07 *
27.08 ± 4.56
1.57 ± 0.25*
Controls ( n = 10 )
13.61 ± 3.54
24.17± 4.58
0.48 ± 0.24
* P < 0.001, compared with the control group
12
RANKL/OPG
ratio
Table 2.
The number of RANKL-positive cells and the ratio of RANKL/OPG according to
the grade of inflammation
The grade of
The number of
The ratio of
inflammation
RANKL-positive cells
RANKL/OPG
light
(n=8)
35.74 ± 9.02 *
1.60 ± 0.26
moderate ( n = 17 )
42.08 ± 8.07
1.52 ± 0.21
severe
45.34 ± 5.64
1.61 ± 0.15
( n = 15 )
* P < 0.05 compared with severe inflammation.
Table 3.
The number of RANKL-positive cells and the ratio of RANKL/OPG according to
the degree of inflammatory cells
The degree of
inflammatory cells
T lymphocytes
Light
The number of
RANKL-positive cells
The ratio of
RANKL/OPG
(CD3）
(n=9)
39.49 ± 8.84 *
1.56 ± 0.17
Moderate ( n = 20 )
40.25 ± 7.82 †
1.53 ± 0.23
Intense
47.35 ± 5.62
1.64 ± 0.17
( n = 11 )
B lymphocytes（CD20）
Light
(n=9)
36.16 ± 8.14 *
1.55 ± 0.27
Moderate
( n = 19 )
41.96 ± 8.13
1.57± 0.21
Intense
( n = 12 )
46.55 ±4.88
1.58 ± 0.14
Macrophages （CD68）
13
Light
( n = 12 )
37.59 ± 9.33 *
1.55 ± 0.28
Moderate
( n = 18 )
42.93 ± 7.05
1.53 ± 0.18
Intense
( n = 10 )
45.74 ± 6.19
1.66 ± 0.10
*
P < 0.05, significant difference between light and intense staining
†
P < 0.05, significant difference between moderate and intense staining