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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