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peran dari aktivator reseptor pada ligan nuklear faktor dan proses osteoprogeterin pada kesehatan periodonsium
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Roles of receptor activator ofnuclear factor-jB ligand(RANKL) and osteoprotegerin inperiodontal health and disease
TOSHIYUKI NAGASAWA, MAKOTO KIJI , REIKO YASHIRO, DOOSADEE HORMDEE,LU HE, MELANIE KUNZE, TOMONARI SUDA, GEENA KOSHY,HIROAKI KOBAYASHI, SHIGERU ODA, HIROSHI NITTA & ISAO ISHIKAWA
Periodontitis is an inflammatory disease character-
ized by periodontal pocket formation and alveolar
bone resorption, followed by tooth loss. Porphyro-
monas gingivalis and Actinobacillus actinomycetem-
comitans are major periodontopathic bacteria
involved in various forms of periodontitis, but the
presence in or absence from periodontitis lesions of
these specific bacteria cannot simply determine the
type or severity of periodontitis. Immune responses
against periodontal pathogens may greatly affect the
course of periodontal diseases but the mechanisms
by which local immune responses against perio-
dontopathic bacteria result in alveolar bone resorp-
tion remain to be established.
A balance between bone resorption by osteoclasts
and bone formation by osteoblasts determines the
level of bone mass. Osteoclast differentiation is
regulated by osteoblasts. Receptor activator of nuc-
lear factor-jB ligand (RANKL) and its decoy receptor,osteoprotegerin, are essential molecules for osteo-
clast differentiation supported by osteoblasts. Not
only osteoblasts but also other resident cells, inclu-
ding periodontal ligament fibroblasts and gingival
fibroblasts, participate in the regulation of RANKL
and osteoprotegerin in periodontal tissue. In perio-
dontitis, infiltrated leukocytes produce inflammatory
mediators, such as interleukin-1 (IL-1) and prosta-
glandin E2, which affect RANKL and osteoprotegerin
expression by osteoblasts, periodontal ligament
fibroblasts, and gingival fibroblasts. In addition,
RANKL is also expressed in activated T cells. This
review will summarize the currently known facts
regarding RANKL and osteoprotegerin expression in
periodontal tissue, and discusses the roles and
therapeutic implications of these molecules in the
pathogenesis of periodontitis.
Immune system in the gingivaltissue
The oral cavity is the entrance to the digestive organs.
The mucosal surfaces of the digestive organs are
challenged with various antigens, including food,
drinks, commensal and/or virulent bacteria, and
viruses. Mucosal immunity is characterized by
unique T-cell subsets in the epithelium (30, 106) and
external secretion of secretory immunoglobulin A
(IgA).
Intestinal intraepithelial lymphocytes are a major
lymphocyte population, residing in close proximity to
the intestinal lumen. Almost all of the peripheral T
cells develop in the thymus, and express T-cell
receptor ab. T-cell receptor cd cells reside in theintestinal intraepithelial lymphocytes, and undergo
thymus-independent T-cell development (8, 106,
160). Intestinal intraepithelial lymphocytes increase
in response to the commensal bacteria (8, 94), and
are regarded as the first line of defense at the mucosal
surface. Lundqvist et al. (82) reported that intra-
epithelial lymphocytes in gingival tissue expressed
the cd T-cell receptor and contained cytoplasmicelectron-dense, membrane-bound granules and
multivesicular bodies, which are ultrastructural
characteristics of cytotoxic cells. These results sug-
gested that intraepithelial lymphocytes in the gingival
tissue also constitute the first line of defense against
the microflora in the oral cavity (20, 82).
65
Periodontology 2000, Vol. 43, 2007, 6584
Printed in Singapore. All rights reserved
2007 The Authors.Journal compilation 2007 Blackwell Munksgaard
PERIODONTOLOGY 2000
Mucosal surfaces of the digestive tract are bathed
with secretory IgA secreted from salivary glands and
gut. The Peyers patches and tonsils constitute the
specialized lymphoid tissue called mucosa-associ-
ated lymphoid tissue (100, 101). The Peyers patches
and tonsils develop specialized epithelial cells, and
they transport and translocate processed antigens to
the subepithelial lymphoid tissue (99). Teeth are
transmucosal organs, and are the only hard tissue in
the digestive tract; they serve as the favorable niche
for the indigenous bacteria. The junctional epithe-
lium controls the microbial challenge with its special
structural framework and through the collaboration
of its epithelial and non-epithelial cells, which pro-
vide potent antimicrobial mechanisms (18). The
junctional epithelium has been compared with
tonsillar crypt epithelium, and it may function as a
reticular epithelium, providing a favorable environ-
ment for the local immune recognition process (120).
The number of desmosomes and gap junctions that
connect junctional epithelium are small and inter-
cellular spaces are wide (114, 121), facilitating leu-
kocyte infiltration in the intercellular spaces and
diffusion of antigens from the gingival crevice into
the gingival lamina propria.
The human gingival lamina propria is highly vas-
cularized (120). The vascular network located lateral
to the junctional epithelial collar is termed the gin-
gival plexus (120). This gingival plexus extends from
the coronal to the apical termination of the junctional
epithelium (120).
Selective homing and migration of leukocytes are
determined by the expression of adhesion molecules.
For example, migration through the epidermis is
mediated by expression of intercellular adhesion
molecule-1 (ICAM-1) on keratinocytes and of leuko-
cyte-function associated antigen-1 on infiltrating
lymphocytes. In the tonsil, intraepithelial infiltration
was mediated by the expression of vascular cell
adhesion molecule-1 by epithelial cells and of very
late antigen-4 by infiltrating lymphocytes (147). The
venules of the gingival plexus constitutively express
ICAM-1 and the endothelial cell adhesion molecule-1
(ELAM-1) (90). Capillary loops expressing ICAM-1
and ELAM-1 are in close topographic association
with the inflammatory infiltrate (144). As ELAM-1
selectively binds neutrophils, expression of these
molecules might be important in the regulation of
inflammatory infiltrate in the gingiva (90).
The ICAM-1 is consistently expressed in junctional
epithelium (28), and gradient expression of ICAM-1
within the junctional epithelium is thought to be an
important mechanism for guiding neutrophils toward
the gingival sulcus (90, 144, 145). In the healthy gin-
giva, approximately 30,000 neutrophils migrate per
minute through the junctional epithelia into the
gingival crevice (118). The emigration time of neu-
trophils from vessels to the gingival sulcus is about
2030 min in rhesus monkey (122).
Neutrophils play a central role in protection against
bacteria. Systemic disorders such as cyclic neutrope-
nia, drug-induced agranulocytosis, ChediakHigashi
syndrome, and leukocyte adhesion deficiency syn-
drome are associated with a deficiency in neutrophil
cell number or function, and cause severe periodon-
titis (32, 41). Aggressive periodontitis is also related to
compromised neutrophil function (148).
Antibodies against bacteria neutralize bacterial
toxins (43) or opsonize the bacteria for effective
neutrophil phagocytosis (155). Antibody responses
in periodontal tissue include mucosal IgA responses
and systemic IgG responses (64). Overall, the IgG
response is predominant and the secretory IgA
response is minor and limited to the superficial
tissues (76). Robust antibody responses by B cells
are protective against infectious bacteria. The high
levels of serum IgG2 in localized aggressive perio-
dontitis may be helpful in localizing periodontal
destruction (81). Low antibody responses against
Tannerella forsythia, P. gingivalis, and Staphylo-
coccus aureus might relate to the early failure of
implants (72). Low antibody responses to bacterial
proteins and polysaccharides were observed in
patients with mandibular osteomyelitis, further
suggesting the relevance of antibody responses in
protection against oral bacteria (95).
If neutrophils and antibodies were not competent
enough to prevent bacterial infection, epithelial
integration would be disrupted, and influx of bacteria
and/or bacterial products would elicit more severe
inflammatory responses (Fig. 1). Early reports dem-
onstrated that B cells predominated in active lesions
compared with stable lesions in periodontitis (125),
while later studies suggested that T cells and macro-
phages are also increased in the progressive sites
compared with non-progressive sites (77, 162).
Diagnostic value of antibacterialantibodies in periodontitis
Antibody titers against periodontopathic bacteria are
higher in chronic periodontitis patients compared
with patients with generalized aggressive periodon-
titis (66, 151), and robust antibody responses to
periodontal pathogens might be protective. However,
66
Nagasawa et al.
the natural specific antibodies cannot eliminate
periodontal pathogens in the plaque biofilm. From
the diagnostic point of view, the differences in anti-
body titers between patients with aggressive and
chronic periodontitis were not large enough to
determine the type of periodontitis based on the
antibody titer. The increased IgG antibody against
periodontopathic bacteria might be regarded as the
history of active invasion.
Horibe et al. (49) reported that increased IgG
antibody to P. gingivalis in chronic periodontitis
patients decreased after periodontal treatment, sug-
gesting that the elimination of the periodontopathic
bacteria resulted in a reduction of specific antibody
against them. Mechanical debridement is effective
against P. gingivalis (27, 103). The decrease of anti-
P. gingivalis antibody was evident after periodontal
surgery (49), supporting the possibility that the
efficacy of the elimination of bacterial infection could
be monitored based on the antibody titer. In fact,
total amount of P. gingivalis in the periodontal
pockets was significantly correlated with the anti-
body titer against P. gingivalis (66). Infection with
A. actinomycetemcomitans can also be monitored
Fig. 1. Neutrophils and antibodies protect epithelial
integration from bacterial infection. Antibodies neutralize
bacterial toxins or opsonize the bacteria for effective neu-
trophil phagocytosis.When theepithelial integrationwould
be disrupted, influx of bacteria and/or bacterial pro-
ducts (lipopolysaccharide (LPS), phosphoryl choline, pro-
tease, leukotoxin, cytolethal distending toxin (CDT), etc.)
would elicit severe inflammatory responses. Inflammatory
mediators, including interleukin (IL)-1, IL-6, and prosta-
glandinE2,areproducedby lymphocytesandmacrophages,
which might affect receptor activator of nuclear factor-jBligand (RANKL) and osteoprotegerin expression in gingival
fibroblasts, periodontal ligament fibroblasts, and osteo-
blasts. PGE2, prostaglandin E2; TNF, tumor necrosis factor.
67
Roles of RANKL and osteoprotegerin
based on antibody measurement. Ishikawa et al. (54)
reported the successful treatment of PapillonLefe`vre
syndrome patients by the elimination of A. actino-
mycetemcomitans. Patients with PapillonLefe`vre
syndrome were infected with A. actinomycetemcom-
itans, and elimination of the A. actinomycetemcomi-
tans resulted in both clinical improvement and a
decrease of serum IgG antibody to A. actinomyce-
temcomitans (19). In addition, measurement of
serum IgG antibody against different serotypes of
A. actinomycetemcomitans may identify the type
of the A. actinomycetemcomitans serotype infection
(149, 151).
Recently, an association between coronary heart
disease and clinically diagnosed periodontitis has
been found in several epidemiological studies. Puss-
inen et al. (107, 108) reported that a high serum
IgA-class antibody level against P. gingivalis was
associated with an increased risk for myocardial
infarction and stroke. Beck et al. (11) also reported
that clinical signs of periodontal disease were not
associated with coronary heart disease, whereas a
systemic antibody response to the periodontopathic
bacteria was associated with coronary heart disease.
These reports suggest that measurement of antibody
titer to periodontopathic bacteria might be useful to
diagnose the association of these bacteria with cor-
onary heart disease and stroke.
Development of vaccine againstperiodontitis
The development of vaccines is an important strategy
against serious infectious diseases that do not have
effective treatments. With regard to periodontitis,
there are well-established treatment modalities
for chronic periodontitis, and the prevalences of
aggressive periodontitis and recurrent periodontitis
are low (102). However, recent findings of associa-
tions between periodontitis and other systemic dis-
eases may provide a rationale for the development of
a vaccine against periodontitis (102).
For the development of vaccines, the cell-mediated
immune system is effective against enveloped viruses
and intracellular bacteria, whereas humoral immune
responses are effective against extracellular patho-
gens. Vaccination is the most effective medical inter-
vention against viral pathogens, and vaccines against
smallpox, polio, measles, and hepatitis B have had a
great impact on world health (7). EpsteinBarr virus is
associated with the pathogenesis of tumors arising in
lymphoid or epithelial tissue (80), and a cytotoxic
T-cell-based vaccine is being developed (16). As the
association of EpsteinBarr virus and periodontitis
was suggested (130), the application of the vaccine
against EpsteinBarr virus might affect the prognosis
of periodontitis associated with the virus in the future.
Most of the periodontal pathogens are extracellular
pathogens, and host antibody responses together with
neutrophil functions are important against them.
Antibodies are produced by plasma cells differen-
tiated from B cells. B lymphocytes are classified into
two major subsets, B-1 and B-2 (conventional) B
cells. B-1 cells are unique B cells that can be distin-
guished from the other B cells (B-2 cells) by their
surface phenotype (15). Originally, B-1 cells were
identified by their expression of CD5, and were ini-
tially called CD5 B cells (15). B-1 cells are absent from
peripheral lymph nodes but constitute a substantial
fraction of B cells in the peritoneal and pleural cav-
ities (15). B-1 cells are the primary source of natural
antibody, and the antibody is polyreactive, weakly
autoreactive, and reactive with many common
pathogen-associated carbohydrate antigens such as
lipopolysaccharide and phosphoryl choline (15).
B-1 cells are activated in periodontitis tissue (5, 14,
132), and antibody to commensal bacteria is
increased in periodontitis patients (5). Phosphoryl
choline is a common bacterial antigen, and a sub-
stantial proportion of the bacteria in dental plaque
(3040%) bear phosphoryl choline antigen (118).
Antibody to phosphoryl choline is produced by B-1
cells, and is increased in periodontitis patients
(5, 118). Anti-phosphoryl choline antibodies might be
regarded as the defense system against complex
plaque bacteria but they might not be competent
enough to eliminate the bacteria. Moreover, the anti-
phosphoryl choline antibody cross-reacts with
oxidized low-density lipoprotein (oxLDL), and anti-
oxLDL antibody is detected in gingival crevicular
fluid in periodontitis patients (117). The local pro-
duction of anti-oxLDL may modify immune reactions
in cardiovascular and other systemic diseases (117).
Antibodies from B-1 cells might be important for the
immune responses against commensal bacteria, but
not effective for the elimination of the pathogenic
bacteria. As B-1 cells are potentially autoreactive,
positive selection for reactivity with self-antigens
could result in the production of detrimental auto-
reactive antibodies. B-1 cells rarely enter germinal
centers and undergo affinity maturation, indicating
that their potential for producing high-affinity anti-
bodies with harmful anti-self specificities is highly
restricted (10). B-1 cells are evolutionarily interme-
diate B cells, and produce natural antibodies to
68
Nagasawa et al.
maximize their flexibility against various kinds of
pathogens (55). B-1 cells might not be harmful for the
host unless they produce high-affinity antibody to the
host antigens. In the vaccine development, affinity
maturation of B-1 cells should be monitored and
carefully avoided to prevent autoimmune reactions.
B-2 cells reside in the lymph nodes, Peyers pat-
ches, and other peripheral lymphoid tissues, and
produce high-affinity antibodies against pathogenic
bacteria. Effective vaccination stimulates B-2 cells in
the lymph nodes to produce high avidity IgG anti-
bodies. In a non-human primate model, formalin-
killed P. gingivalis whole-cell vaccine induced serum
antibody. The induced antibody inhibited pros-
taglandin E2 production by mononuclear cells sti-
mulated with lipopolysaccharide (112). In addition,
the formalin-killed P. gingivalis whole-cell vaccine
decreased prostaglandin E2 level in gingival crevicu-
lar fluid (112). Among the P. gingivalis antigens,
fimbriae and enzymes such as arginine-specific gin-
gipains and lysine-specific gingipain have been
investigated as possible candidates for vaccine anti-
gens. Immunization with P. gingivalis fimbriae also
protected against periodontal destruction in rats (36).
Immunization of P. gingivalis fimbriae induced op-
sonic antibodies that enhance phagocytosis and
killing of P. gingivalis by neutrophils (37). Rajapakse
et al. (110) reported that immunization of arginine-
specific gingipains with adjuvant suppressed the
colonization of P. gingivalis in rats. Arginine-specific
gingipains and lysine-specific gingipain peptide vac-
cine or DNA vaccine also protected abscess forma-
tion by P. gingivalis. The protection was mediated by
Fc receptor-dependent phagocytosis (97). These re-
sults strongly suggest that vaccination against perio-
dontopathic bacteria not only suppresses the target
organism through Fc receptor-dependent phagocy-
tosis, but also neutralizes bacterial antigens to
ameliorate the harmful inflammatory reaction by the
host.
IgA antibody inhibits the adherence of the bacteria
to the host, and specific IgA antibody is protective
against mucosal infection. The characteristics of the
mucosal immune system must be taken into account
in the development of effective local vaccines to
protect specifically the mucosal infection (71).
Approximately 40% of IgA plasma cells in the gut are
derived from B-1 cells (70, 83). Commensal bacteria
in the gut are coated with B-1-derived secretory IgA
antibody, but they are stable and not eliminated,
suggesting that B-1 cells might play a role in main-
taining the normal intestinal flora (71). The rest of the
intestinal IgA plasma cells are derived from B-2 cells
initially activated in Peyers patches (15, 87). When
pathogenic bacteria penetrate the gut, B-2 cells may
be induced in Peyers patches to produce high-
affinity, narrowly tuned IgA antibodies, leading to
immune exclusion of the pathogens (71).
Porphyromonas gingivalis fimbriae are involved in
the adherence of the pathogen to the host cells,
suggesting that secretory IgA responses to the fimb-
riae might prevent the adherence and colonization of
P. gingivalis. Oral administration of fimbriae with
cholera toxin induced high-avidity salivary IgA anti-
body and serum IgG antibody in mice (92). Sharma et
al. (126) reported that biologically active domains of
P. gingivalis fimbrillin could be expressed on the
human commensal bacterium Streptococcus gordo-
nii. Oral administration with the S. gordonii induced
fimbrillin-specific serum IgG and salivary IgA anti-
bodies in rats, and suppressed the P. gingivalis-
induced alveolar bone loss. Shin et al. (128) assessed
whether edible plants can synthesize biologically
active P. gingivalis fimbrial antigen with cholera
toxin B subunit. A cDNA fragment encoding P. gin-
givalis fimbriae and cholera toxin B was cloned into a
plant expression vector, and the plasmid was trans-
ferred into potato cells. Fimbrial protein with cholera
toxin was detected in the potato cells (128). They
suggested the feasibility of the synthesized protein in
edible plants for the development of mucosal vac-
cines (128). Mucosal vaccines using edible plants or
commensal bacteria might be less painful to admin-
ister compared than conventional vaccines.
Although many reports suggest that active
immunization against periodontopathic bacteria
were effective in experimental animals, its safety for
human trial has yet to be determined. Passive
immunization is the use of specific antibody for a
particular antigen. In periodontitis, subgingival
application of monoclonal antibodies against P. gin-
givalis suppressed the colonization of P. gingivalis for
9 months in humans (17). Abiko et al. (1) generated a
human monoclonal antibody that inhibits the coag-
gregation activity of P. gingivalis. Shibata et al. (127)
constructed a functional single-chain variable frag-
ment antibody against hemagglutinin from P. gingi-
valis. The single-chain variable fragment antibody
consists of the heavy- and light-chain variable frag-
ment. The single-chain variable fragment antibody
specifically binds to the pathogens, but they lack
an Fc region, indicating that they do not activate
complements and other Fc receptor-mediated
immune responses. These antibodies might be useful
for passive immunization against periodontitis with
few side effects. However, reports on the application
69
Roles of RANKL and osteoprotegerin
of vaccines in human periodontal diseases are limited
even in passive immunization. Further study is
necessary to develop safe vaccine therapies against
periodontal diseases.
Osteoimmunology
Under physiological conditions, bone is periodically
resorbed by osteoclasts while new bone is formed by
osteoblasts (131). Osteoblasts regulate osteoclastic
bone resorption, which involves recruitment of new
osteoclasts and activation of mature osteoclasts
(131). The discovery of RANKL (4, 73, 156, 158) and its
decoy receptor, osteoprotegerin (129, 146) confirmed
the idea that differentiation and function of osteo-
clasts are regulated by osteoblasts. The recruitment
of new osteoclasts is dependent on the balance
between RANKL and osteoprotegerin in osteoblasts
(131). Mice with disrupted genes exhibited severe
osteopetrosis and a complete lack of osteoclast
activity as a result of the inability of their osteoblasts
to support osteoclastogenesis (69). Mice with a dis-
rupted RANKL gene also had abnormalities in T-cell
development and lymph node organ genesis, sug-
gesting that RANKL is also an important molecule for
immune responses (69). While T lymphocytes also
produce RANKL (56), they might not be involved in
bone resorption under physiological circumstances
because the mice lacking T lymphocytes demon-
strated normal bone metabolism (69).
In inflammatory bone resorption, however, activa-
ted T lymphocytes might mediate bone resorption
through excessive production of soluble RANKL.
RANKL is a membrane-anchored protein in osteo-
blasts, but the expression of membrane-anchored
RANKL on T cells is limited and themajority of RANKL
protein produced by T cells may be active in the sol-
uble form after shedding (58). RANKL mRNA was
detected in T lymphocytes isolated from rheumatoid
arthritis lesions, and activated T lymphocytes have
been observed to support osteoclast formation in vitro
(51). In addition, osteoprotegerin administration re-
duced bone destruction by RANKL-producing T cells
in mice with adjuvant arthritis (68). Moreover, Teng et
al. (141) isolated T lymphocytes from aggressive peri-
odontitis patients infected with A. actinomycetem-
comitans, and the T lymphocytes expressed RANKL in
response to A. actinomycetemcomitans. Severe com-
bined immunodeficient mice were reconstituted with
the T lymphocytes from the patients by adoptive
transfer, and the mice were orally infected with
A. actinomycetemcomitans (141). Adoptive transfer of
T cells from periodontitis patients caused severe bone
destruction and the bone resorption was suppressed
by osteoprotegerin, suggesting that the destruction
was mediated by soluble RANKL produced from the
transferred T cells (141). In fact, T lymphocytes isola-
ted from the periodontitis lesion express RANKL (58,
93). As T cells specific for periodontopathic bacteria
exist in the peripheral blood of periodontitis patients
(85), they might potentially express RANKL when they
encounter the specific antigens. Choi et al. (26)
reported that B lymphocytes also produce RANKL and
augment osteoclastogenesis.
Takayanagi et al. (138) reported that molecular
mechanisms of T-cell-mediated regulation of osteo-
clast formation occur through signaling cross-talk
between RANKL and interferon-c (IFN-c). T-cell pro-duction of IFN-c strongly suppresses osteoclastogen-esis by interfering with the RANKLRANK signaling
pathway. IFN-c induces rapid degradation of theRANK adapter protein, TRAF6 (tumor necrosis factor
receptor-associated factor 6), which results in strong
inhibitionof the receptor activator of nuclear factor-jBligand-induced activation of the transcription factor
NF-jB and c-Jun N-terminal kinase (138). Mice defi-cient in IFN-b signaling exhibit severe osteopeniaaccompanied by enhanced osteoclastogenesis (137).
Signal transducer and activator of transcription 1
(STAT1) is a critical mediator of gene transcription in
type I interferon (IFN-a/b) signaling that is essentialfor host defense against viruses (136). Stat1-deficient
mice showed excessive osteoclastogenesis caused by a
loss of negative regulation by IFN-b (63). However,bonemasswas increased in STAT1-deficientmice, and
the increase was caused by excessive osteoblast dif-
ferentiation (63). These unexpected phenotypes in the
STAT1-deficient mice indicate that immunomodula-
tory cytokines such as interferons also participate in
the regulation of RANKL signaling in skeletal systems
(134, 135). Arron and Choi (6) coined the term osteo-immunology to describe this interdisciplinary field ofbone biology and immunology.
In periodontitis lesions, lymphocytes, macropha-
ges, and neutrophils infiltrate the gingival connective
tissue, and interact with stromal cells, and it is
important to understand the interaction between
stromal cells (osteoblasts, periodontal ligament
fibroblasts, and gingival fibroblasts) and inflammatory
infiltrates (T and B lymphocytes and macrophages).
Macrophages and T lymphocytes produce inflamma-
tory mediators, including IL-1, IL-6, tumor necrosis
factor-a and prostaglandin E2, which can induce boneresorption indirectly by stimulating osteoblasts to
produce RANKL (140). T lymphocytes can also pro-
70
Nagasawa et al.
mote osteoclast differentiation by direct production of
RANKL. Alveolar bone resorption might be directly or
indirectly induced by the inflammatory infiltrate in
periodontal lesions (140).
Expression of RANKL andosteoprotegerin in periodontitistissue
Inflammatory cytokines, including IL-1, IL-6 and
tumor necrosis factor-a are increased in periodontitistissue, and the role of these cytokines in the patho-
genesis of periodontitis has been extensively studied
(53). Recent advances in the osteoimmunology led us
to examine the roles of RANKL and osteoprotegerin in
the pathogenesis of periodontitis. We examined the
RANKLmRNA expression in periodontitis tissue using
reverse transcription-polymerase chain reaction, and
found that RANKL-expressing sites had deeper perio-
dontal pockets compared with the RANKL-negative
sites (93). In accordance with our results, Liu et al. (79)
reported that the level of RANKL mRNA determined
using semi-quantitative reverse transcription-polym-
erase chain reaction was highest in advanced perio-
dontitis compared with the moderate periodontitis or
healthy groups. Moreover, they examined the local-
ization of RANKLmRNAexpression at the cellular level
using in situ hybridization. RANKL mRNA was ex-
pressed in inflammatory cells, mainly lymphocytes
and macrophages (79). In addition, proliferating epi-
thelium in the vicinity of inflammatory cells expressed
high levels of RANKL mRNA (79). Crotti et al. (29)
compared the RANKL and osteoprotegerin expression
in the granulomatous tissue adjacent to areas of
alveolar bone loss from periodontitis patients to the
tissue without periodontitis using immunohisto-
chemistry. Significantly higher levels ofRANKLprotein
were expressed in the periodontitis tissue, and osteo-
protegerin protein was significantly lower in the peri-
odontitis tissue. RANKL protein was associated with
lymphocytes and macrophages, and osteoprotegerin
protein was associated with endothelial cells (29).
Mogi et al. (89) examined the concentrations of
RANKL and osteoprotegerin in the gingival crevicular
fluid from periodontitis patients and healthy sub-
jects. An increased concentration of RANKL and a
decreased concentration of osteoprotegerin were
detected in gingival crevice fluid from periodontitis
patients. The ratio of the concentration of RANKL to
that of osteoprotegerin in the gingival crevice fluid
was significantly higher for periodontal disease pa-
tients than for healthy subjects (89).
These results suggested that enhanced RANKL
production might be associated with alveolar bone
resorption, and lymphocytes are one of the major
RANKL-expressing cells in periodontitis tissue. It is
difficult to evaluate the expression of RANKL directly
in osteoblasts in the periodontitis lesion, as the spe-
cific molecular markers for the osteoblasts are not
known, and they cannot be directly isolated from the
tissue. The expression of RANKL and osteoprotegerin
on fibroblastic cells, including osteoblasts, perio-
dontal ligament fibroblasts, and gingival fibroblasts,
are mainly examined using cultured cells in vitro.
Expression of RANKL andosteoprotegerin in osteoblastsstimulated with vitamin D
Vitamin D and parathyroid hormone regulate bone
metabolism in physiological conditions. Vitamin D
stimulates the osteoclast formation, and deficiency of
vitamin D results in hypocalcemia, hypophosphate-
mia, and osteomalacia. 1a,25-Dihydroxyvitamin D3(1a,25(OH)2D3), an active form of vitamin D, hasimportant roles in many biological phenomena such
as calcium homeostasis (153, 157) and bone meta-
bolism. There are genetic polymorphisms in the
human 1a,25(OH)2D3 receptor (vitamin D receptor)gene and recent reports suggested that polymor-
phisms of the vitamin D receptor gene might increase
the risk of developing early onset periodontitis
(48, 133, 159) and chronic periodontitis (31).
Yoshizawa et al. (161) generated mice that were
deficient in 1a,25(OH)2D3 receptor. These vitamin Dreceptor-deficient mice did not show any defects in
development and growth before weaning, but
showed alopecia, hypocalcemia, infertility, and
severe impairment of bone formation (161). Takeda
et al. (139) examined whether the effects of
1a,25(OH)2D3 on osteoclast formation require vita-min D receptor in osteoblasts using mice that lacked
the vitamin D receptor. When osteoblasts from nor-
mal mice and osteoclast precursor cells were
co-cultured with 1a,25(OH)2D3, osteoclasts wereformed (139). In contrast, when osteoblasts from
vitamin D receptor-deficient mice and osteoclast
precursors were co-cultured, no osteoclasts were
formed, indicating that vitamin D receptor in osteo-
blasts are essential for osteoclast formation by
1a,25(OH)2D3 (139). In addition, parathyroid hor-mone and IL-1 stimulated osteoclastogenesis by
osteoblasts from vitamin D receptor-deficient mice,
suggesting that osteoclastic bone resorption can be
71
Roles of RANKL and osteoprotegerin
maintained without 1a,25(OH)2D3 actions by otherstimulatory agents (139).
The osteoprotegerin and RANKL expression in dif-
ferentiating osteoblasts was examined to determine
the effects of 1a,25(OH)2D3 on the osteoclastogenesisby immature and mature osteoblasts (142). Osteo-
protegerin mRNA expression was increased after the
onset of mineralization compared with less mature
cultures, but the osteoprotegerin expression was not
affected by the stimulation with 1a,25(OH)2D3. Incontrast, basal RANKL mRNA expression did not
change during differentiation but was enhanced by
1a,25(OH)2D3 treatment at all times. The RANKL/os-teoprotegerin ratio was increased by 1a,25(OH)2D3treatment at all stages of osteoblastic differentiation,
but to a lesser degree in cultures after the onset of
mineralization. Accordingly, osteoclastogenesis by
immature osteoblasts stimulated with 1a,25(OH)2D3is mediated by increased RANKL expression. As os-
teoprotegerin mRNA expression in osteoblasts
increases with maturation, mature osteoblasts have
relatively decreased osteoclastogenic activity (142).
Expression of RANKL andosteoprotegerin in osteoblastsstimulated with parathyroidhormone
Parathyroid hormone functions as a major mediator
of physiological bone remodeling. Parathyroid hor-
mone-related peptide was discovered as the tumor
product that is responsible for most instances of the
syndrome of humoral hypercalcemia of malignancy
(104). It is now known that the parathyroid hormone-
related protein and parathyroid hormone genes arose
on the basis of an ancient duplication event (104).
Parathyroid hormone usually activates bone resorp-
tion but intermittent parathyroid hormone admin-
istration promotes bone formation. Intermittent
parathyroid hormone administration protected
against periodontitis-associated bone loss in a rat
ligature-induced periodontitis model (9). In ovariec-
tomized rats, intermittent parathyroid hormone
administration also reduced alveolar bone loss (86).
There are few reports about the effect of parathyroid
hormone and parathyroid hormone-related protein
on periodontitis. One paper reported the effect of
secondary hyperparathyroidism on the periodontium
of patients on hemodialysis, and secondary hyper-
parathyroidism did not have an appreciable effect on
periodontal indices, including clinical attachment
level, probing pocket depth, gingival index, and
alveolar bone loss (38).
The functions of parathyroid hormone/parathyroid
hormone-related protein receptor were investigated
using parathyroid hormone/parathyroid hormone-
related protein receptor-deficient mice (75). Most of
these deficientmice diedmid-gestation, and surviving
mice exhibited accelerated differentiation of chond-
rocytes in bone, and their bones, grown in explant
culture, were resistant to the effects of parathyroid
hormone-related protein, suggesting that parathyroid
hormone/parathyroid hormone-related protein
receptormediated the effects of parathyroidhormone-
related protein on chondrocyte differentiation (75).
To address the role of parathyroid hormone receptor
in osteoclastogenesis, Liu et al. (78) established
osteoblast cell lines that could support parathyroid
hormone-dependent osteoclastogenesis. When oste-
oclast precursors from parathyroid hormone receptor
knockout mice were co-cultured with the osteoblast
cell lines, osteoclasts were formed in response to
parathyroid hormone, indicating that receptors for the
parathyroid hormone were not present in osteoclasts
but in osteoblasts. Parathyroid hormone stimulates
osteoclast formation by binding to its receptor on
stromal/osteoblastic cells and stimulating the pro-
duction of RANKL and inhibiting the expression of
osteoprotegerin. Parathyroid hormone upregulates
RANKL mRNA in primary bone marrow stromal oste-
oblasts, with maximal sensitivity occurring late in os-
teoblast differentiation and parathyroid hormone
inhibits osteoprotegerin gene expression at all stages
of osteoblast differentiation. This suggests that the
osteoclastogenic activity of parathyroid hormone oc-
curs primarily by suppression of osteoprotegerin gene
expression in early osteoblasts and by elevation of
RANKL gene expression in mature osteoblasts (52).
The downstream signals generated by the activated
parathyroid hormone/parathyroid hormone-related
protein receptor are cyclic adenosinemonophosphate
(cAMP)/protein kinase A and phospholipase C/pro-
tein kinase C. Kondo et al. (67) evaluated the expres-
sionofRANKLandosteoprotegerin inMS1cells,which
are the conditionally transformed bone marrow stro-
mal cells. Parathyroid hormone increased RANKL and
macrophage colony-stimulating factor mRNA expres-
sion and decreased that of osteoprotegerin in MS1
cells. These effects of parathyroid hormone were
mimicked by protein kinase A stimulators 8-bromoa-
denosine-cAMP or forskolin and were blocked by the
protein kinaseA inhibitor Rp-cAMPsbut unaffected by
the protein kinase C inhibitor GF109203X (67). The
direct protein kinase C stimulator 12-o-tetradeca-
72
Nagasawa et al.
noylphorbol-13-acetate did not induce RANKL mRNA
in MS1 cells, but it did upregulate osteoprotegerin
mRNA and also antagonized osteoclast formation in-
duced by parathyroid hormone. Thus, cAMP/protein
kinase A signaling via the parathyroid hormone
receptor is the primary mechanism for controlling
RANKL-dependent osteoclastogenesis, although di-
rect protein kinase C activation may negatively regu-
late this effect of parathyroid hormone by inducing
expression of osteoprotegerin (67).
Halladay et al. (44) examined the effects of para-
thyroid hormone on the human osteoprotegerin
promoter ()5917 to +19) fused with b-galactosidasereporter gene in rat osteoblast-like UMR-106 cells.
The effect of parathyroid hormone on osteoprote-
gerin promoter expression was biphasic and con-
centration dependent. The similar biphasic response
of OPG to parathyroid hormone, parathyroid
hormone 1-31, parathyroid hormone-related protein
1-34, forskolin, 3-isobutyl-1-methylxanthine, and
dibutyryl cAMP suggested that parathyroid hormone
regulated osteoprotegerin transcription via activation
of the cAMP/protein kinase A signal transduction
pathway. They concluded that the inhibitory effects
of parathyroid hormone on osteoprotegerin are
mediated at the transcriptional level through cis ele-
ments in the proximal promoter (44).
Fu et al. (40) reported that activation of protein kin-
ase A was necessary and sufficient for the effect of
parathyroid hormone on both RANKL and osteopro-
tegerin genes. Conditional expression of a dominant-
negative form of the transcription factor cAMP-
response element-binding protein significantly re-
duced parathyroid hormone stimulation of RANKL.
Protein kinase A and cAMP-response element-binding
proteinactivity is also required for regulationofRANKL
by oncostatin M and 1a,25(OH)2D3. The dominantnegative form of cAMP-response element-binding
protein and c-fos reduced the suppression of osteo-
protegerin by parathyroid hormone. They suggested
that parathyroid hormone directly stimulates RANKL
expression via a protein kinase AcAMP-response ele-
ment-binding protein pathway. The cAMP-response
element-binding protein may be a central regulator of
RANKL expression, and the parathyroid hormone
suppression of osteoprotegerin involves cAMP-
response element-binding protein and c-fos (40).
These reports suggested that activation of protein
kinase A augments RANKL expression and protein
kinase C activation induces osteoprotegerin expres-
sion in osteoblasts (Fig. 2). Importantly, the protein
kinase AcAMP-response element-binding protein
pathway is a necessary component of transcriptional
activation of the RANKL gene regardless of the nature
of stimulus, as protein kinase A and cAMP-response
element-binding protein activity was required for the
regulation of RANKL gene expression by oncostatin M
and 1a,25(OH)2D3 (40). As oncostatin M or 1a,25(OH)2D3 do not alter protein kinase A activity, basal
protein kinase A activity is a prerequisite for the ef-
fects of both gp130 and vitamin D receptor pathways
on RANKL expression in osteoblasts (40).
n i r e g e t o r p o e t s O n i r e g e t o r p o e t s O
OsteoblastOsteoblast
L K N A R L K N A R n i r e g e t o r p o e t s O n i r e g e t o r p o e t s O
/ H T P / H T P P r H T P P r H T P
R H T P R H T P
C K P C K P A K P A K P
L K N A R L K N A R
A N R m A N R m A N R m A N R m
Fig. 2. Receptor activator of nuclear factor-jB ligand(RANKL) and osteoprotegerin expression in osteoblasts
stimulated with parathyroid hormone/parathyroid hor-
mone-related protein (PTH/PTHrP): parathyroid hor-
mone/parathyroid hormone-related protein receptor
(PTHR) is expressed on osteoblasts, and PTH/PTHrP sti-
mulates osteoblasts to activate both protein kinase A
(PKA) and protein kinase C (PKC) pathways. Activation of
PKA augments RANKL expression and inhibits osteopro-
tegerin expression in osteoblasts. Activation of PKC aug-
ments osteoprotegerin expression in osteoblasts. mRNA,
messenger ribonucleic acid.
73
Roles of RANKL and osteoprotegerin
Expression of RANKL andosteoprotegerin in humanperiodontal ligament fibroblasts
Sakata et al. (115) examined the expression of RANKL
and osteoprotegerin mRNA in human gingival kera-
tinocytes, human gingival fibroblasts, human perio-
dontal ligament fibroblasts, and human tooth pulp
cells. Osteoprotegerin mRNA was detected in gingival
fibroblasts, periodontal ligament fibroblasts, and
human tooth pulp cells, but not in human gingival
keratinocytes (115). RANKL mRNA was not detected
in these cells (115). IL-1 and tumor necrosis factor-aincreased osteoprotegerin mRNA in periodontal
ligament fibroblasts, but IL-6 and tumor necrosis
factor-b had little effect on osteoprotegerin mRNAlevels in periodontal ligament fibroblasts (115). They
suggested that osteoprotegerin synthesized by dental
mesenchymal cells locally regulates the resorption of
dental hard tissues, such as alveolar bone, cemen-
tum, and dentin (115).
As the periodontal ligament plays important roles in
alveolar bone formation and resorption in orthodon-
tic or periodontal treatment, it is an important issue
whether periodontal ligament fibroblasts, as well as
osteoblasts, can support osteoclastogenesis. Wada et
al. (150) reported that the addition of the conditioned
media fromperiodontal ligament fibroblasts tomouse
bone marrow culture inhibited osteoclast formation
in spite of the presence of 1a,25(OH)2D3. The inhibi-tory effect was most remarkable when the condi-
tionedmedia were added at the late stage of osteoclast
differentiation (150). The conditioned media from
periodontal ligament fibroblasts also suppressed pit
formation by osteoclasts on ivory slices (150). Perio-
dontal ligament fibroblasts produce osteoprotegerin,
and osteoprotegerin produced by periodontal liga-
ment fibroblasts prevented the pre-osteoclast differ-
entiation and functions (150). Hasegawa et al. (46)
reported that osteoprotegerin mRNA was downregu-
lated in periodontal ligament fibroblasts by the
application of 1a,25(OH)2D3 and dexamethasone. Incontrast, RANKL mRNA was upregulated by the same
treatment (46). When periodontal ligament fibroblasts
were co-cultured with mouse bone marrow cells
in the presence of anti-osteoprotegerin antibody
together with 1a,25(OH)2D3 and dexamethasone,mature osteoclasts were markedly induced (46). Per-
iodontal ligament fibroblasts synthesize both RANKL
and osteoprotegerin, and inactivation of osteoprote-
gerin may play a key role in osteoclast formation by
periodontal ligament fibroblasts (46).
Kanzaki et al. (60) examined the cell-to-cell inter-
actions between peripheral blood mononuclear cells
and periodontal ligament fibroblasts during osteo-
clastogenesis. Peripheral bloodmononuclear cells that
were directly co-cultured with periodontal ligament
fibroblasts formed significantly more osteoclasts than
peripheral bloodmononuclear cells thatwere cultured
alone (60). Periodontal ligament fibroblasts expressed
RANKL and osteoprotegerin mRNA, and soluble fac-
tors produced from periodontal ligament fibroblasts
inhibited the formationof osteoclasts (60). Periodontal
ligament fibroblasts might support osteoclastogenesis
through cell-to-cell contact (60).
During orthodontic tooth movement, orthodontic
force applied at the tooth crown is transmitted to the
periodontal ligament and alveolar bone, resulting in
osteoclast formation at the compression site. To
examine the effects of mechanical stress on osteo-
clastogenesis of periodontal ligament fibroblasts,
periodontal ligament fibroblasts were continuously
compressed, and co-cultured with peripheral blood
monocytes (59). The expression of RANKL mRNA in
periodontal ligament fibroblasts increased with com-
pressive force in parallel to the increase of osteoclast
formation from peripheral blood monocytes (59).
Compressive force also induces cyclooxygenase 2
mRNA expression in periodontal ligament fibroblasts,
and indomethacin inhibited the RANKL upregulation
resulting from compressive force (59). Periodontal
ligament fibroblasts induce osteoclastogenesis by
RANKL upregulation via prostaglandin E2 synthesis (59).
IL-1 increased OPG production in periodontal liga-
ment fibroblasts and, at the same time, increased
prostaglandin E2 production (116). Etodolac, a select-
ive cyclooxygenase-2 inhibitor, suppressed the in-
crease of prostaglandin E2 (116). Etodolac further
reinforced the promotion of osteoprotegerin expres-
sion by IL-1 at the mRNA and protein levels (116).
Prostaglandin E2 added to the cultures of periodontal
ligament fibroblasts decreased osteoprotegerinmRNA
levels (116). These findings suggest that the increase in
osteoprotegerin levels induced by IL-1 in periodontal
ligament fibroblasts was suppressed through prosta-
glandin E2 synthesized de novo (116). IL-1 stimulated
the expression of RANKL mRNA in periodontal liga-
ment fibroblasts, and endogenous prostaglandin E2partially mediated the IL-1-induced RANKL mRNA
expression (96). Exogenously added prostaglandin E2also augmented RANKL expression, and the expres-
sion was mediated by EP2/4 receptors (96). These
results suggested that the regulation of RANKL and
osteoprotegerin expression in periodontal ligament
fibroblasts might be similar to that in osteoblasts.
74
Nagasawa et al.
Expression of RANKL and OPG inhuman gingival fibroblasts
Fibroblasts from various tissues expressed RANKL in
the presence of 1a,25(OH)2D3 and dexamethasone inmice, suggesting that all fibroblastic cells might
potentially support osteoclastogenesis (109). How-
ever, osteoclasts are not usually observed in the gin-
gival tissue, suggesting that fibroblastic cells in the
other tissue might have different functions from
osteoblasts.
Gingival fibroblasts stimulated with lipopolysac-
charide produce osteoprotegerin, and inhibit osteo-
clastogenesis by RANKL and (93). Lipopolysaccharide
and bacterial DNA induce RANKL expression in os-
teoblasts (62, 163). Periodontal ligament fibroblasts
stimulated with A. actinomycetemcomitans lipopoly-
saccharide produce RANKL, and the RANKL expres-
sion was mediated by prostaglandin E2 (143). Bac-
terial sonicates from P. gingivalis, Treponema
denticola, and Treponema socranskii stimulated os-
teoblasts to express receptor activator of nuclear
factor-jB ligand, and the expression of RANKL wasalso mediated by prostaglandin E2 (25).
Gingival fibroblasts produce more osteoprotegerin
and less RANKL compared with periodontal ligament
fibroblasts without stimulation (50). IL-1 augmented
osteoprotegerin production in gingival fibroblasts
and periodontal ligament fibroblasts, and again,
gingival fibroblasts produced more osteoprotegerin
than periodontal ligament fibroblasts (50). Protein
kinase A inhibitor abrogated IL-1-induced osteopro-
tegerin production in gingival fibroblasts, but not in
periodontal ligament fibroblasts. Protein kinase A
activator augmented osteoprotegerin production in
gingival fibroblasts, but not periodontal ligament fi-
broblasts. Protein kinase C inhibitor suppressed IL-1-
induced osteoprotegerin production in periodontal
ligament fibroblasts, and protein kinase C activator
augmented osteoprotegerin production in periodon-
tal ligament fibroblasts. As the protein kinase C-
dependent osteoprotegerin production in periodon-
tal ligament fibroblasts was similar to the reports on
osteoblasts (67), periodontal ligament fibroblasts
exhibit some characteristics of osteoblasts (Fig. 3). In
contrast, protein kinase A-dependent osteoprotegerin
production in gingival fibroblasts was different from
these cells (Fig. 4). Gingival fibroblasts might have
the ability to suppress the osteoclastogenesis induced
by inflammatory mediators, including IL-1 and
prostaglandin E2, but extensive bone resorption
would occur if these mediators directly act on
periodontal ligament fibroblasts and osteoblasts.
Toll-like receptors play an essential role in the
recognition of microbial components (3). Similar
cytoplasmic domains allow toll-like receptors to use
the same signaling molecules as those used by the
IL-1 receptors (3). These results suggest that gingival
fibroblasts might prevent alveolar bone resorption
caused by IL-1, prostaglandin E2, and major bacterial
components such as lipopolysaccharide through the
n i r e g e t o r p o e t s O n i r e g e t o r p o e t s O A N R m A N R m
A K P A K P
n i r e g e t o r p o e t s O n i r e g e t o r p o e t s O
C K P C K P
L K N A R L K N A R A N R m A N R m
L K N A R L K N A R
L I L I - - r o t p e c e r 1 r o t p e c e r 1 g I g I - - n i a m o D e k i l n i a m o D e k i l L I L I - - 1 1
E G P E G P 2 2
Periodontal ligament fibroblastPeriodontal ligament fibroblast
Fig. 3. Receptor activator of nuclear factor-jB ligand(RANKL) and osteoprotegerin expression in periodontal
ligament fibroblasts stimulated with interleukin (IL)-1: IL-
1 receptor has immunoglobulin like domain (Ig-like do-
main) and toll-like receptor domain (TIR domain). IL-1
receptor is expressed on periodontal ligament fibroblasts,
and IL-1 stimulates human gingival fibroblasts to activate
protein kinase A (PKA) and protein kinase C (PKC) path-
ways. Activation of PKA augments RANKL expression and
inhibits osteoprotegerin expression in periodontal liga-
ment fibroblasts. Activation of PKC augments osteopro-
tegerin expression in osteoblasts. mRNA, messenger ri-
bonucleic acid.
75
Roles of RANKL and osteoprotegerin
production of osteoprotegerin, but osteoblasts and
periodontal ligament fibroblasts might augment os-
teoclastogenesis if these components penetrate into
periodontal ligament fibroblasts and osteoblasts.
Heterogeneity of fibroblasts
During the course of the study on the expression of
RANKL and osteoprotegerin in both gingival fibro-
blasts and periodontal ligament fibroblasts, we found
heterogeneity of gingival fibroblasts and periodontal
ligament fibroblasts even in the same subject (50, 93).
The difference in characteristics of these fibroblasts
might be explained by several factors, including the
differentiation, maturation, anatomical sites and
state of inflammation, and malignancies (21, 22, 34).
Mesenchymal stem cells have been identified in a
variety of adult tissues as a population of pluripotent
self-renewing cells (111). In the presence of one or
more growth factors, these cells commit to lineages
that lead to the formation of bone, cartilage, muscle,
tendon, and adipose tissue. STRO-1 and CD146/
MUC18 are known to be mesenchymal stem-cell
markers, and Seo et al. (124) isolated stem cells from
periodontal ligament tissue using monoclonal anti-
body against stem cell marker and magnetic activated
cell sorting, and implanted them into immunocom-
promised mice. The implanted cells had the capacity
to generate cementum or periodontal ligament-like
structure, suggesting that periodontal ligament con-
tains stem cells that have the potential to generate
cementum or periodontal ligament-like tissue in vivo.
Heterogeneity of periodontal ligament fibroblasts
might be partially explained by the presence of stem
cells in periodontal ligament, as stem cells in the tissue
might differentiate into a different lineage of cells.
CD40 is a surface antigen expressed on B cells, and
the CD40 ligand is expressed on activated T cells (24).
Interaction between CD40 and CD40 ligand is critical
for proliferation and isotype switching in B cells, and
mice with a disrupted CD40 gene fail to undergo
isotype switching (24). Patients with X-linked hyper-
IgM syndrome (HIGMX-1) have an abnormality in
their CD40 ligand gene, and they are unable to switch
from IgM to IgG, IgA, and IgE (24).
Brouty-Boye et al. (23) reported that culturedhuman
fibroblasts from various tissues express CD40 and
produce a distinct panel of chemokines, suggesting a
possible fundamental role of fibroblasts in immune
responses anddiseaseprocesses. CD40expressionwas
higher in gingival fibroblasts than periodontal liga-
ment fibroblasts (39, 45, 123). Dongari et al. (33)
reported that the frequency of IL-6- and IL-8-secreting
cells mirrors the frequency of cells expressing high
levels of CD40 in these cultures. In addition, they
demonstrated adirect functional relationshipbetween
CD40 expression and IL-6 or IL-8 secretion by showing
that ligation of this molecule on gingival fibroblasts,
andCD40+fibroblast subsets inparticular, upregulates
secretion of these cytokines in vitro. This report
strongly suggested that CD40+ gingival fibroblasts are
increased in inflamed gingival tissue, and signaling
through the CD40 enhances the production of
inflammatory cytokines, resulting in periodontal tis-
sue destruction. However, Wassenaar et al. (152)
investigated the CD40 ligand-induced matrix metal-
loproteinase (MMP) production by gingival fibroblasts
in the presence of cytokines that are increased in
periodontal lesions, such as IL-1b, tumor necrosisfactor-a and IFN-c. CD40 ligation on gingival fibro-blasts resulted in a decrease of MMP-1 and MMP-3
production, whileMMP-2 and tissue inhibitor ofMMP
1 (TIMP-1) production were unaffected. This down-
Gingival fibroblast
n i r e g e t o r p o e t s O
L I L I - - r o t p e c e r 1 r o t p e c e r rotpecer rotpecer 1 g I g I - - n i a m o D e k i l n i a m o D e k i l
n i r e g e t o r p o e t s O
n i r e g e t o r p o e t s O n i r e g e t o r p o e t s O A N R m A N R m
R I T R I T n i a m o D n i a m o D
A K P A K P
LILI --11Gingival fibroblast
Fig. 4. Osteoprotegerin expression
in human gingival fibroblast stimu-
lated with interleukin (IL)-1: IL-1
receptor has an immunoglobulin
like domain (Ig-like domain) and a
toll-like receptor domain (TIR do-
main). IL-1 receptor is expressed on
human gingival fibroblasts, and IL-1
stimulates human gingival fibro-
blasts to activate the protein kinase
A (PKA) pathway. Activation of PKA
augments osteoprotegerin expres-
sion in human gingival fibroblasts.
mRNA, messenger ribonucleic acid.
76
Nagasawa et al.
regulatory effect of CD40 engagement on MMP-1 and
MMP-3 production by gingival fibroblasts was also
present when MMP production was upregulated by
IL-1band tumornecrosis factor-aordownregulatedbyIFN-c (152). These results suggest that CD40 ligationon gingival fibroblasts restricts MMP-1 and MMP-3
production by gingival fibroblasts and therebymay be
an important mechanism in the retardation of further
periodontal tissue damage.
Role of fibroblasts in chronicinflammation
The resolution of immune responses is character-
ized by extensive apoptosis of activated T cells.
However, to generate and maintain immunological
memory, some antigen-specific T cells must survive
and revert to a resting G0/G1 state. Cytokines that
bind to the common c-chain of the IL-2 receptorpromote the survival of T-cell blasts, but also induce
proliferation. In contrast, soluble factors secreted by
stromal cells induce T-cell survival in a resting
G0/G1 state (105).
Pilling et al. (105) reported that IFN-a and IFN-bpromoted the reversion of blast T cells to a resting G0/
G1 configuration with all the characteristic features of
stromal cell rescue; such as high Bcl-XL expression
and low Bcl-2. Type I interferons and stromal cells
stimulated apparently identical signaling pathways,
leading to STAT1 activation. They suggested that this
mechanism might play a fundamental role in the
persistence of T cells at sites of chronic inflammation,
and that chronic inflammation was an aberrant con-
sequence of immunological memory (105).
The majority of expanded T cells generated during
an immune response are cleared by apoptosis (2).
Prevention of death in some activated T cells
enables the persistence of a memory T-cell pool (2).
Although this enables memory T cells to persist
without antigen, excessive IFN-a or IFN-c secretionmight lead to chronic inflammation (2). Gingival
fibroblasts produced comparable amounts of IFN-b(113), suggesting that gingival fibroblasts might also
be involved in the establishment of chronic gingival
inflammation by IFN-b. STAT1 activation by IFN-band IFN-c suppresses osteoclastogenesis andosteoblast proliferation (63). Accordingly, the pro-
duction of inflammatory mediators by gingival
fibroblasts might augment chronic inflammation to
combat the persistent invasion of periodontopathic
bacteria in gingival tissue with disrupted epithelial
integration.
Roles of specific periodontopathicbacteria in RANKL andosteoprotegerin expression inperiodontitis
Both P. gingivalis and A. actinomycetemcomitans are
major periodontopathic bacteria involved in various
forms of periodontitis (53). Okabayashi et al. (98)
investigated the effect of infection with viable
P. gingivalis on RANKL production in osteoblasts.
Infection with viable P. gingivalis induced RANKL
production in osteoblasts (98). Infection with P. gin-
givalis KDP136, an isogenic deficient mutant of
arginine- and lysine-specific cysteine proteinases, did
not stimulate RANKL production, suggesting that the
cysteine proteinases are involved in RANKL produc-
tion (98). In addition to the RANKL expression, os-
teoprotegerin is degraded by P. gingivalis (65).
Although gingival fibroblasts stimulated with LPS
do not produce RANKL (12, 93), A. actinomycetem-
comitans cytolethal distending toxin stimulates gin-
gival fibroblasts to produce RANKL (13). These results
suggest that both P. gingivalis and A. actinomyce-
temcomitans have unique pathways to stimulate
RANKL expression in gingival fibroblasts and osteo-
blasts.
Enamel matrix proteins andRANKL expression
Formation of the roots of the teeth is initiated by the
down-growth of Hertwigs epithelial root sheath
(42). Amelogenins are involved not only in enamel
formation, but also in the formation of the perio-
dontal attachment during tooth formation (35, 57).
The use of enamel matrix derivative has been
developed to regenerate periodontal tissues. The
enamel matrix derivative is an extract of enamel
matrix and contains amelogenins of various
molecular weights. Meta-analysis showed that
enamel matrix derivative significantly enhances
periodontal regeneration in the intra-bony perio-
dontal defects (57). The periodontal regeneration by
enamel matrix derivative is thought to mimic the
formation of the roots of the teeth (42).
After the fragmentation, the Hertwigs epithelial
root sheath gives rise to the epithelial rests of Mal-
assez. Mizuno et al. (88) reported that cultured epi-
thelial rests of Malassez are characterized by
expression of cytokeratin 8, osteoprotegerin and os-
teopontin genes, suggesting that osteoprotegerin
77
Roles of RANKL and osteoprotegerin
might play important roles in protection against root
resorption. It is interesting that amelogenin null mice
showed severe root resorption caused by the en-
hanced expression of RANKL (47). The balance be-
tween RANKL and osteoprotegerin might also be
important to maintain root homeostasis, and the
amelogenin expression might affect the RANKL
expression in the root of the teeth.
Therapeutic implications andfuture perspectives
Initial treatment reduces the number of T cells specific
for the periodontopathic bacteria (84), suggesting that
the risk for theRANKLexpressionbyactivatedTcells in
the periodontitis tissue might also be reduced by
periodontal treatment. In addition, elimination of
P. gingivalis and A. actinomycetemcomitans is pru-
dent, as they have specific mechanisms to induce
RANKL in osteoblasts and gingival fibroblasts.
As gingival fibroblasts produce osteoprotegerin in
response to lipopolysaccharide (93), inadequate
amounts of gingival tissue might be vulnerable to
lipopolysaccharide challenge. Adequate amounts of
gingival tissue contain gingival fibroblasts, which
protect bone through the production of osteoprote-
gerin against periodontal inflammation (50). Lang et
al. (74) reported that most surfaces (>80%) with
2.0 mm keratinized gingiva were clinically healthy,suggesting that minimal widths of keratinized gingiva
are compatible with periodontal health. Kennedy
et al. (61) reported a longitudinal evaluation of vary-
ing widths of attached gingiva. Thirty-two patients
with bilateral areas of inadequate attached gingiva on
the facial surface of homologous contralateral teeth
were enrolled in the study. The patients received free
gingival graft on one side, while the other side served
as an unoperated control. The patients have been
followed for 6 years. The results showed that areas of
inadequate attached gingiva did not demonstrate
additional recession or further loss of attachment. On
the experimental sides that received a free gingival
graft, the dimension of keratinized gingiva increased
and was stable for 6 years. It is of note that
examination of patients who had discontinued
participation in the study for 5 years revealed a
re-establishment of gingival inflammation on the
control sides associated with additional recession
(61). Similar changes were not observed in areas
treated by a free graft (61). It was concluded that it
was possible to maintain periodontal health and
attachment through control of gingival inflammation
despite the absence of attached gingiva (61). These
reports suggest that adequate dimensions of gingival
tissue might be needed if plaque control were not
enough to maintain epithelial integration.
It is generally accepted that a minimal amount or
absence of gingiva alone is not justification for gin-
gival augmentation, and gingival height is not a criti-
cal factor for the prevention of marginal tissue
recession (154). In conjunction with fixed or remov-
SPL SPL
LILI -- ,1,1FNT FNT --
Osteoprotegerin
Periodontal ligament fibroblast
Osteoclast
RANKL
Gingival fibroblast
Osteoblast
Fig. 5. Receptor activator of nuclear
factor-jB ligand (RANKL) and os-teoprotegerin expression in the in-
flamed periodontal tissue: T cells in
the periodontitis tissue express
RANKL. Gingival fibroblasts produce
osteoprotegerin in response to in-
terleukin (IL)-1, and suppress oste-
oclast formation. Both osteoblasts
and periodontal ligament fibroblasts
stimulated with IL-1 express RANKL.
If the inflammation extends into
periodontal ligaments and alveolar
bone, alveolar bone resorption may
be accelerated through the in-
creased expression of RANKL. LPS,
lipopolysaccharide; TNF, tumour
necrosis factor.
78
Nagasawa et al.
able prosthetic dentistry, gingival augmentation is
indicated where there are insufficient dimensions of
gingiva and the restoration margin is to be placed
intracrevicularly or where major or minor connectors
of removable partial dentures impinge on the mar-
ginal mucosa (154). To date, all the decisions made
concerning treatments are based on the clinical
morphological diagnosis. If the molecular mecha-
nisms of alveolar bone resorption are elucidated in
detail, prognosis of the periodontal lesion will be
more accurately diagnosed, and appropriate treat-
ment modalities will be proposed.
Conclusions
The tooth is a transmucosal organ and has a special-
ized immune system at the interface between the root
and the gingiva. The first line of defense in the perio-
dontal tissue comprises the neutrophils and anti-
bodies at the junctional epithelium. Neutrophils
migrate from the gingival venules to the antigenic
bacteria in the gingival sulcus through the junctional
epithelium, and protect the epithelial integration col-
laboratingwith the antibodies specific for the bacteria.
If the epithelial integration is disrupted, B cells, T cells,
and macrophages are increased in the gingival con-
nective tissue, and they secrete inflammatory media-
tors, such as IL-1 and prostaglandin E2. RANKL
expression is increased in periodontitis tissue com-
paredwith healthy gingival tissue. RANKL is expressed
on osteoblasts and activated T cells, and T cells in the
periodontitis tissue express RANKL. Gingival fibro-
blasts produce OPG in response to lipopolysaccharide
and IL-1, suggesting that they might suppress osteo-
clast formation. Gingival fibroblasts are heterogene-
ous, and some gingival fibroblasts may augment
chronic inflammation through the production of IL-6
and IFN-b. Both osteoblasts and periodontal ligamentfibroblasts stimulated with lipopolysaccharide and
IL-1 express RANKL, suggesting that once the inflam-
mation extends into the periodontal ligaments and
alveolar bone, alveolar bone resorption might be
accelerated through the increased expression of
RANKL (Fig. 5). The periodontopathic bacteria A. ac-
tinomycetemcomitans and P. gingivalis have unique
mechanisms to induce RANKL in osteoblasts and
gingival fibroblasts. RANKL and osteoprotegerin
expression might also be related to the function of
amelogenin, and regulation of odontoclast formation.
Research on osteoimmunology will shed light on
the molecular mechanisms of inflammatory bone
destruction in periodontal tissue, and novel diagnosis
and therapies in periodontics will be constructed.
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