Published in Volume
106, Issue 6
(January 15, 2000)J Clin Invest.
Copyright © 2000, American Society for Clinical
Functional human T-cell immunity and osteoprotegerin ligand control
alveolar bone destruction in periodontal infection
1Divisions of Periodontics and Oral Biology, and Department of
Microbiology and Immunology, Faculty of Medicine and Dentistry, the University of
Western Ontario, London, Ontario, Canada,
2Departments of Surgery and
Transplantation Immunology, Toronto General Hospital,
Ontario Cancer Institute, and Departments of Medical Biophysics and Immunology, and
4Faculty of Dentistry, University of Toronto, Toronto, Ontario,
Address correspondence to: Yen-Tung Andy Teng, Divisions of Periodontics and
Oral Biology, and Department of Microbiology and Immunology, Faculty of Medicine and
Dentistry, University of Western Ontario, London, Ontario N6A 5C1, Canada. Phone:
519-661-2111 ext. 86112; Fax: (519) 850-2316; E-mail:
Published January 15, 2000
Received for publication July 13,
2000, and accepted in revised form August 21,
Periodontitis, a prime cause of tooth loss in humans, is implicated in the increased
risk of systemic diseases such as heart failure, stroke, and bacterial pneumonia. The
mechanisms by which periodontitis and antibacterial immunity lead to alveolar bone
and tooth loss are poorly understood. To study the human immune response to specific
periodontal infections, we transplanted human peripheral blood lymphocytes (HuPBLs)
from periodontitis patients into NOD/SCID mice. Oral challenge of HuPBL-NOD/SCID mice
with Actinobacillus actinomycetemcomitans, a well-known
Gram-negative anaerobic microorganism that causes human periodontitis, activates
human CD4+ T cells in the periodontium and triggers local alveolar bone
destruction. Human CD4+ T cells, but not CD8+ T cells or B
cells, are identified as essential mediators of alveolar bone destruction.
Stimulation of CD4+ T cells by A. actinomycetemcomitans
induces production of osteoprotegerin ligand (OPG-L), a key modulator of
osteoclastogenesis and osteoclast activation. In vivo inhibition of OPG-L function
with the decoy receptor OPG diminishes alveolar bone destruction and reduces the
number of periodontal osteoclasts after microbial challenge. These data imply that
the molecular explanation for alveolar bone destruction observed in periodontal
infections is mediated by microorganism-triggered induction of OPG-L expression on
CD4+ T cells and the consequent activation of osteoclasts. Inhibition
of OPG-L may thus have therapeutic value to prevent alveolar bone and/or tooth loss
in human periodontitis.
Host inflammatory and immune responses to specific oral bacterial infections can result
in periodontal disease, i.e., periodontitis (1).
Human periodontitis is heterogeneous in etiology, but a common hallmark is alveolar bone
destruction, one of the major causes of tooth loss in human (2, 3). Interestingly, human
periodontitis has recently been implicated in the increased risks of certain systemic
disorders such as pre-term low birth weight, bacterial pneumonia, congestive heart
diseases, and stroke (4–8), possibly due to an underlying inflammatory trait
(9). About 10–12 subgingival
microorganisms have been implicated in the pathogenesis of periodontitis, including
Porphyromonas gingivalis, Prevotella intermedia,
Bacteroides forsythus, and mixed spirochetes (10). In particular, Actinobacillus
actinomycetemcomitans, a Gram-negative facultative capnophilic rod
bacterium, has been identified as the etiological agent of localized juvenile
periodontitis (LJP) and of some rapidly progressing and severe forms of periodontitis
(10–13). The prevalence of LJP is about 1–4% among teens and
young adults, and 10% among insulin-dependent diabetic patients (10). LJP is characterized by advanced alveolar bone destruction in a
molar-incisor pattern that often leads to tooth mobility and loss, resulting in
functional and aesthetic deficits.
A. actinomycetemcomitans is able to invade the gingival epithelium
(14) and releases several virulence factors
such as cytotoxins, endotoxins, and a potent leukotoxin (15–17). A.
actinomycetemcomitans infection is usually accompanied by local and systemic
antigen-specific immune responses (18–19). Earlier studies
demonstrated altered CD4+/CD8+ T-cell ratios and autologous mixed
lymphocyte reactions in LJP patients (20, 21) and the ability of T helper cells to home to
periodontal tissues in rat and mouse models of periodontitis (22–24). Further,
we have previously demonstrated that A. actinomycetemcomitans infection
in NOD/SCID mice engrafted with human peripheral blood leukocytes (HuPBLs) leads to
periodontal inflammation characterized by the infiltration of CD4+ T cells,
CD8+ T cells, CD20+ B cells, and Mac1+ macrophages
into the fibrous connective tissues adjacent to the periodontal pockets (24). These results suggested that T cells could
modulate bacterium-induced periodontal inflammation and/or alveolar bone destruction.
However, the exact role of T-cell subtypes and B cells in periodontitis, the role of
antibacterial immunity in local alveolar bone destruction, and the mechanisms by which
host immune responses contribute to alveolar bone destruction and/or tooth loss remain
To investigate the mechanism or mechanisms that regulate periodontal immunity and
alveolar bone destruction, we transplanted HuPBLs from LJP patients into NOD/SCID mice
(which lack endogenous T and B cells), generating HuPBL-NOD/SCID mice (24). Here we show that oral challenge of these
“humanized” mice with A. actinomycetemcomitans
(designated Aa-HuPBL-NOD/SCID) leads to functional activation of the
human CD4+ T cells in the periodontium and triggers local alveolar bone
destruction. In vitro stimulation of CD4+ T cells from these mice with
antigens from A. actinomycetemcomitans leads to the expression of
osteoprotegerin ligand (OPG-L, also known as TRANCE, ODF, and RANKL), a key mediator of
osteoclastogenesis and osteoclast activation (25–31). Inhibition of
OPG-L function via the decoy receptor osteoprotegerin (OPG) significantly reduces the
alveolar bone destruction detected in Aa-HuPBL-NOD/SCID mice after
bacterial inoculation, as well as the numbers of osteoclasts at the sites of local
periodontal inflammation. These results identify for the first time a critical role for
human CD4+ T cells reactive to oral microorganisms in periodontal disease.
Moreover, A. actinomycetemcomitans–triggered induction of
OPG-L expression on T cells and OPG-L–mediated osteoclast activation and
bone loss could provide one molecular explanation for the alveolar bone destruction
observed in local periodontal infection.
Construction of a model for A. actinomycetemcomitans–induced periodontal
infection in HuPBL-NOD/SCID mice.
To study the mechanism or mechanisms that regulate periodontal immunity and alveolar
bone destruction, we transplanted HuPBL into NOD/SCID mice to generate HuPBL-NOD/SCID
mice by using HuPBL from LJP patients and healthy donors, respectively (24). Four independent HuPBL-NOD/SCID chimeric mouse
strains were generated using cells from four LJP patients. In all experiments, screening
for the human CD45+ hematopoietic cell marker revealed that the level of
HuPBL engraftment in NOD/SCID mice detected over the 8-week period was
30–60% (24) (data not shown). Oral
inoculation of HuPBL-NOD/SCID mice with A. actinomycetemcomitans
resulted in periodontal inflammation characterized by the infiltration of human
CD4+ T cells, CD8+ T cells, CD20+ B cells, and
Mac1+ macrophages into the fibrous connective tissues adjacent to the
periodontal pockets at the site of infection (24). Further, we have recently demonstrated the feasibility of generating human
primary and secondary immune responses to exogenous antigens and putative pathogens
isolated from human periodontal lesions in HuPBL-SCID and HuPBL-NOD/SCID animal models
(32–33). As a result, both periodontal and splenic CD4+ T
cells from Aa-HuPBL-NOD/SCID mice were fully immunocompetent, as
assessed by their significant upregulation of the high-affinity IL-2 receptor-Q chain
expression (CD25; data not shown) and production of the T-cell growth factor IL-2
(Figure 1a) in response to in vitro restimulation
with sonicate antigens of A. actinomycetemcomitans, respectively.
Splenic CD4+ T cells from Aa-HuPBL-NOD/SCID mice failed to
produce IL-2 in response to restimulation in vitro with sonicate antigens of P.
gingivalis (Figure 1a). In addition,
the TCR-VQ and TCR-Vß repertoire of A.
actinomycetemcomitans–reactive CD4+ T cells from
Aa-HuPBL-NOD/SCID mice significantly overlapped (>83%) that
of cells isolated from periodontal surgical excisions from clinical LJP patients (data
not shown). These results indicate that bacterial antigens can activate human T cells
present in the periodontal tissues of the chimeric mice. Thus,
Aa-HuPBL-NOD/SCID chimeric mice constitute a useful and specific model
to investigate human oral pathogen-induced periodontal infection.
(a) Immunocompetence of CD4+ T cells in
Aa-HuPBL-NOD/SCID mice. IL-2 production of periodontal CD4+ T cells (Perio-CD4/T-cells) and
splenic CD4+ T cells (Aa-splenic CD4/T-cells) isolated
from A. actinomycetemcomitans–immunized
HuPBL-NOD/SCID mice is shown. Irradiated (25 Gy) HuPBL-derived autologous
monocytes/macrophages (irr-PBL) and CD4+ T cells isolated from
non–Aa-immunized HuPBL-NOD/SCID mice (no
Aa-splenic CD4/T-cells) were included as the negative
controls. Restimulation of Aa-immunized splenic CD4+ T
cells with a third-party antigen, P. gingivalis sonicate antigens
served as the specificity control (P.g.). Serial dilution of
Aa sonicate antigens did not result in significantly different
patterns of the IL-2 responses measured. Differences in IL-2 production between
the Perio-CD4/T-cell and Aa-splenic CD4/T-cell groups as compared
with the irr-PBL, no Aa-splenic CD4/T-cell, and
P.g. groups were statistically significant (P
< 0.008, paired t test). Values shown are mean IL-2
production of triplicate samples ± SD. One result representative of
three independent experiments is shown. (b) Increased alveolar bone
loss in Aa-HuPBL-NOD/SCID mice. Groups of mice as indicated were
either sham-infected or inoculated with A. actinomycetemcomitans
(Aa). Data shown are the relative amounts of alveolar bone
loss at the day of the first Aa inoculation (Day 1), by the end
of 4 weeks (4th wk), and by the end of 8 weeks (8th wk), expressed as a percentage
of the amount of bone loss in the positive control, Aa-infected
BALB/c mice (100% = 0.6 ± 0.12 mm per tooth in 8 weeks). Group I,
sham-infected NOD/SCID mice (n = 10); group II, sham-infected
chimeric NOD/SCID mice engrafted with HuPBL from four LJP patients
(n = 12); group III, NOD/SCID mice infected with
Aa (n = 16); group IV,
Aa-infected chimeric NOD/SCID mice engrafted with HuPBL from four
LJP patients (n = 32); group V, Aa-infected
chimeric NOD/SCID mice engrafted with HuPBL (N-HuPBL) from two healthy donors
(n = 12). Data shown are mean values ± SD pooled
from four independent experiments involving HuPBL engraftment from four LJP
subjects, each of which gave comparable results. Alveolar bone loss was determined
as described in Methods. AThe extent of bone loss in group IV at 8 weeks was
significantly increased as compared with all other groups (P
In humans, periodontal inflammation/infection results in destruction of alveolar bone
structure surrounding the teeth, ultimately followed by tooth loss (1–3). Thus, we analyzed whether alveolar bone loss occurs in A.
actinomycetemcomitans–infected HuPBL-NOD/SCID mice. The results
showed that periodontal and alveolar bone structures were comparable among sham-infected
NOD/SCID (Figure 1b, group I) and HuPBL-NOD/SCID
(Figure 1b, group II) mice, indicating that the
transfer of HuPBL per se did not lead to tissue loss (see Figure 3a) (24). Neither did repeated
inoculation of bacteria into nonchimeric NOD/SCID (H2d/d) mice lead to
significant levels of alveolar bone loss (Figure 1b, group III) compared with the positive control, A.
actinomycetemcomitans–infected BALB/c mice (total bone loss was
taken as “100%”). This is consistent with the findings of Baker
et al. (37) that pathogen-induced alveolar bone
loss in SCID mice is significantly less than that in immunocompetent hosts.
Histopathological lesions and bone loss in Aa-HuPBL-NOD/SCID
mice. CEJ, cemento-enamel junction, a landmark for tissue loss; ABC, alveolar bone
crest. The distance between CEJ and ABC reflects the amount of tissue or alveolar
bone loss measured as described in Methods. (a) A representative
tissue section from sham-infected HuPBL-NOD/SCID mice demonstrating the normal
periodontal and alveolar bone structures (as in Figure 1b, group II). (b, c) Typical
histopathological lesions show significant inflammatory infiltration, connective
tissue loss below CEJ (in b), and alveolar bone loss with apical
growth of sulcular epithelium below CEJ and into alveolar bone area (in
c) in Aa-HuPBL-NOD/SCID mice (as in Figure 2, group II) by the end of 8 weeks. Some
multinucleated giant cells (arrows in c) can be observed along the
surface of alveolar crestal bone. (d) Mild inflammatory infiltrates
without obvious alveolar bone loss, as evidenced by sulcular epithelium located at
CEJ, in the periodontal tissues of CD4+ T cell–depleted
Aa-HuPBL-NOD/SCID mice (as in Figure 2, group III) by the end of 8 weeks. (e,
f) In vivo depletion of CD8+ T cells (e)
(as in Figure 2, group V) or B cells
(f) (as in Figure 2, group
VI) has no apparent effects on existing periodontal inflammation accompanied by
tissue loss below CEJ (in e) or alveolar bone loss with apical growth
of sulcular epithelium below CEJ and ABC (in f). These findings are
consistent with those observed in nondepleted Aa-HuPBL-NOD/SCID
mice in b and c. (g, h) In
vivo inhibition of OPG-L via the decoy receptor OPG abrogates alveolar bone
destruction, as evidenced by sulcular epithelium located at CEJ and above ABC (in
both panels), in Aa-HuPBL-NOD/SCID mice by the end of 8 weeks (as
in Figure 4e, group III). Note that OPG
treatment does not affect periodontal inflammation observed in g and
h. Aa-infected HuPBL-NOD/SCID mice were treated
with an OPG-Fc fusion protein as described in Methods. Parts g and
h are representative of eight mice studied in this group.
Magnifications: a, b,
d–h, ×200; c,
By week 8, groups I, II, and III showed significantly less alveolar bone loss
(P < 0.01) than the positive control A.
actinomycetemcomitans–infected BALB/c mice (taken as 100%), and
about the same amount as occurred in noninfected BALB/c mice (data not shown). This
background level of spontaneous alveolar bone loss (mean = 22.6 ± 3.4% of
the positive control; see Figures 1b, 2, and 4e) has
been shown to occur even under normal circumstances without bacterial infection.
Accordingly, it has been recognized to represent a physiological alteration of the
periodontium (38). However, oral inoculation of
A. actinomycetemcomitans in NOD/SCID mice reconstituted with HuPBL
from LPJ patients (Figure 1b, group IV) led, in
time, to the development of alveolar bone destruction (mean = 82.2% of the positive
control; see Figure 3, b and c). Chimeric mice
constructed using HuPBL from each of four different LJP patients showed equivalent
levels of alveolar bone destruction induced by A. actinomycetemcomitans
(24) (data not shown). In contrast, NOD/SCID
chimeric mice reconstituted with HuPBL from healthy human donors (Figure 1b, group V) followed by inoculation with bacteria
developed only background levels of alveolar bone destruction by 8 weeks. These data
indicate that a model of A. actinomycetemcomitans–specific
periodontal infection can be produced in “humanized” NOD/SCID
mice transplanted with HuPBL from LJP, but not from normal healthy subjects.
Regulation of alveolar bone destruction by A.
actinomycetemcomitans–reactive CD4+ T cells. Groups of mice as indicated were either left untreated or inoculated with
A. actinomycetemcomitans. CD4+ T, CD8+
T, or B cells were depleted from mice using specific Ab’s and human
complement as described in Methods. Data shown are the relative amounts
(± SD) of alveolar bone loss accumulated by the end of 8 weeks,
expressed as a percentage of the bone loss in the positive control,
Aa-infected BALB/c mice (100%). Group I, sham-infected,
nondepleted NOD/SCID mice (n = 10); group II,
Aa-infected, nondepleted HuPBL-NOD/SCID mice (n
= 32); group III, Aa-infected, CD4+ T
cell–depleted NOD/SCID mice (n = 20); group IV,
sham-infected, CD4+ T cell–depleted NOD/SCID mice
(n = 9); group V, Aa-infected,
CD8+ T cell–depleted NOD/SCID mice (n =
12); group VI, Aa-infected, B cell–depleted NOD/SCID
mice (n = 16); group VII, Aa-infected NOD/SCID
mice bearing adoptively transferred Aa-reactive CD4+ T
cells (AT-CD4T) plus irradiated autologous monocytes/macrophages as APCs
(n = 10); group VIII, Aa-infected NOD/SCID
mice bearing adoptively transferred irradiated autologous monocytes/macrophages as
APCs (irr-APC; n = 6). AStatistically significant
difference in bone loss between group III and groups II, IV, V, and VI
(P < 0.005).
actinomycetemcomitans stimulates OPG-L expression on periodontal
CD4+ T cells. (a) Unstimulated HuPBL-derived CD4+ T cells stained with
OPG-FITC; negative control. (b) HuPBL-derived CD4+ T cells
restimulated with anti-TCR plus CD28 mAb’s, followed by staining with
isotypic control Ab; background control. (c) HuPBL-derived
CD4+ T cells restimulated with anti-TCR plus CD28 mAb’s,
followed by staining with OPG-FITC; positive control. (d) Periodontal
CD4+ T cells derived from Aa-HuPBL-NOD/SCID mice
stimulated with A. actinomycetemcomitans sonicate antigens,
followed by staining with OPG-FITC. Periodontal CD4+ T cells
restimulated with P. gingivalis sonicate antigens did not induce
significant membrane OPG-L expression over the background level (data not shown).
OPG-L membrane expression was determined by FACS analyses 48 hours later.
(e) Reduction of alveolar bone destruction in OPG-Fc treated
Aa-HuPBL-NOD/SCID mice. Groups of mice as indicated were
infected with A. actinomycetemcomitans followed by in vivo
treatment with soluble human OPG-Fc fusion protein. Alveolar bone loss (mean
values ± SD) was assessed at 8 weeks and normalized to the positive
control, Aa-infected BALB/c mice (100%). Group I, sham-infected
NOD/SCID mice (n = 10); group II, Aa-infected
HuPBL-NOD/SCID mice not injected with OPG-Fc (n = 16); group III,
Aa-infected HuPBL-NOD/SCID mice injected with OPG-Fc
(n = 10); group IV, Aa-infected
HuPBL-NOD/SCID mice injected with PBS (n = 8). AThe
differences in alveolar bone loss between group III and groups II and IV are
statistically significant (P < 0.002).
Human CD4+ T cells mediate alveolar bone destruction in periodontitis. We used our “humanized” mouse model to elucidate the exact
role of human T-cell subtypes and B cells in periodontitis and the role of
antibacterial immunity in local alveolar bone destruction. To assess the contribution
of different lymphocyte subsets to alveolar bone destruction, we studied the effect
of immunodepletion of CD4+ T cells, CD8+ T cells, or B cells
from Aa-HuPBL-NOD/SCID mice. Aa-HuPBL-NOD/SCID mice
depleted of CD4+ T cells showed significantly less alveolar bone
destruction by the end of 8 weeks (Figure 2,
group III: mean = 37.6% of the positive control) compared with nondepleted
Aa-HuPBL-NOD/SCID mice (Figure 2, group II). The relative reduction of alveolar bone destruction (Figure
2, group III) was significant (≈
73.6% of that of Aa-HuPBL-NOD/SCID mice) after taking into account
the background levels of spontaneous bone loss (<≈ 22.6%;
P < 0.005). The failure to achieve higher levels of
reduction of alveolar bone loss was not due to incomplete depletion of
CD4+ T cells, as the efficiency of immunodepletion was typically
95–98% by FACS analysis (24, 32, 33).
One conclusion would be that there are other cell type(s) involved in alveolar bone
loss. However, histopathologically, only a mild inflammatory infiltrate was observed
in the fibrous connective tissue adjacent to the periodontal pockets in seven of ten
depleted mice studied (compare Figure 3, b and
c, with Figure 3d). Alveolar bone loss in
sham-infected CD4+ T cell–depleted HuPBL-NOD/SCID mice (Figure
2, group IV) was not significantly different
from that in the sham-infected, nondepleted NOD/SCID mice (Figure 2, group I). By the end of 8 weeks, depletion of
CD8+ T cells (Figure 2, group V)
or B cells (Figure 2, group VI) from
Aa-HuPBL-NOD/SCID mice did not significantly reduce alveolar bone
loss (P > 0.05) compared with nondepleted
Aa-HuPBL-NOD/SCID mice (Figure 2; Figure 3, e and f). Injection of
either human complement alone or isotypic (IgG1a) control Ab into
Aa-HuPBL-NOD/SCID mice resulted in the same level of alveolar
bone loss as in nondepleted Aa-HuPBL-NOD/SCID mice (data not shown).
These data show that specific removal of CD4+ T cells prevents
inflammation and periodontal tissue damage in “humanized”
animals in response to oral microbial infection, a result consistent with data from
Baker et al., who reported that P. gingivalis–induced
alveolar bone loss in mice required CD4+ T cells (39).
To obtain direct evidence that CD4+ T cells reactive to a specific
microorganism can contribute to alveolar bone destruction, we performed adoptive
transfer experiments using purified periodontal CD4+ T cells isolated from
Aa-HuPBL-NOD/SCID mice. Purified periodontal CD4+ T
cells (1 × 106 to 2 × 106 per mouse)
were adoptively transferred to naive NOD/SCID hosts in the presence of irradiated
autologous monocytes/macrophages followed by oral inoculation with A.
actinomycetemcomitans. Transfer of these CD4+ T cells
triggered significant alveolar bone destruction in the adoptive recipients by the end
of 8 weeks (Figure 2, group VII: AT-CD4T),
comparable with that observed in nondepleted Aa-HuPBL-NOD/SCID mice
(Figure 2, group II). Adoptive transfer of
irradiated autologous monocytes/macrophages alone as APCs did not yield any
significant alveolar bone loss (Figure 2, group
VIII: irr-APC). Thus, periodontal CD4+ T cells and/or CD4+ T
cell–regulated immunity are critical for the pathogenesis of A.
actinomycetemcomitans–induced periodontal disease and
alveolar bone destruction in vivo (see also ref. 39).
A. actinomycetemcomitans stimulation triggers OPG-L expression on human
CD4+ T cells. It remains unclear how CD4+ T cells mediate alveolar bone destruction in
periodontal disease. We have previously shown that T cells stimulated via
cross-linking of their antigen receptors express a membrane-bound form of OPG-L, a
molecule of the TNF receptor superfamily (25–29). OPG-L is a key
regulator of the development and activation of osteoclasts (26–30). Loss
of OPG-L expression in gene-targeted mice results in severe osteopetrosis and a
defect in tooth eruption due to a complete loss of osteoclast development (27). Further, we have recently shown that
activated T cells can regulate systemic and local bone loss and joint destruction in
a rat model of adjuvant-induced arthritis via OPG-L expression (31). These results prompted us to investigate the role of OPG-L
in alveolar bone destruction during periodontal infections using our animal model.
To test whether CD4+ T cells activated by challenge with an oral
microorganism expressed OPG-L, CD4+ T cells from
Aa-HuPBL-NOD/SCID mice were collected and subjected to in vitro
restimulation with A. actinomycetemcomitans sonicate antigens for 2
days, followed by FACS analysis. Our data indicated that microbial stimulation did
induce OPG-L expression on the surface of CD4+ T cells (Figure 4, a–d). Interestingly, CD4+
T cells from Aa-HuPBL-NOD/SCID mice failed to produce OPG-L in
response to restimulation in vitro with a third-party species, P.
gingivalis sonicate antigens (see Figure 4 legend), indicating that OPG-L induction is antigen-specific. Moreover,
using RT-PCR we observed OPG-L mRNA expression in CD4+ T cells isolated
from the periodontal tissues of Aa-HuPBL-NOD/SCID and human LJP
patients (data not shown). To determine whether the elevated expression of OPG-L
correlated with an increase in osteoclast numbers, we determined the numbers of
osteoclasts in the alveolar crestal bone areas of molars using TRAP staining, an
enzymatic staining that specifically identifies osteoclast lineage cells (31, 36).
Whereas only a few TRAP+ osteoclasts were detected in noninfected
HuPBL-NOD/SCID mice (used as a negative control), osteoclast numbers were
significantly increased in Aa-HuPBL-NOD/SCID mice (Table 1). Thus, microbial stimulation of human
CD4+ T cells triggers the production of the key osteoclast activation
and differentiation factor OPG-L, and local microbial infections increase the numbers
of osteoclasts in the periodontal tissues. These data provide the first direct
evidence, to our knowledge, that environmental pathogens, i.e., the oral
microorganism A. actinomycetemcomitans, can activate OPG-L
production in T cells.
The numbers of TRAP+ osteoclasts in the alveolar crestal bone area of
Inhibition of OPG-L in vivo blocks alveolar bone destruction in A.
mice. To test whether OPG-L expression has biological relevance in alveolar bone
destruction during periodontal infections, that is, whether the inhibition of OPG-L
activity in vivo could reduce A.
actinomycetemcomitans–induced alveolar bone loss, we took
advantage of the existence of OPG, the natural decoy receptor of OPG-L (26–27, 31). Soluble recombinant OPG-Fc
fusion protein was injected intraperitoneally into Aa-HuPBL-NOD/SCID
mice every other day for 4 weeks. By the end of 8 weeks, the amount of alveolar bone
loss detected in OPG-Fc–treated Aa-HuPBL-NOD/SCID mice
was significantly reduced (Figure 3, g and h;
Figure 4e, group III: mean = 38.2% of the
positive control) compared with that of Aa-HuPBL-NOD/SCID mice that
were not injected with OPG-Fc (Figure 4e: group
II). This reduction of alveolar bone destruction (Figure 4e, group III) was significant (≈ 72.6% of that of
Aa-HuPBL-NOD/SCID mice) after taking account of the background
levels of spontaneous bone loss. Control injection of PBS, the vehicle for OPG-Fc,
into Aa-HuPBL-NOD/SCID mice (Figure 4e, group IV) did not significantly alter alveolar bone loss when compared
with group II. In a separate experiment, injecting ten times the amount of OPG-Fc in
vivo (n = 4) did not result in any further reduction of alveolar
bone destruction (data not shown), suggesting that (a) maximal dose inhibition was
attained, and/or (b) another cell type or types may be involved in mediating alveolar
bone loss (see below for discussion). Thus, inhibition of OPG-L via OPG significantly
decreases alveolar bone destruction and local bone resorption in
Aa-HuPBL-NOD/SCID mice. Moreover, whereas the number of osteoclasts
was significantly increased in HuPBL-NOD/SCID mice in response to microbial
infection, only a few TRAP+ osteoclasts could be detected in
Aa-HuPBL-NOD/SCID mice treated with OPG (Table 1). These results indicate that the alveolar bone
destruction observed in periodontitis is due at least in part to the action of
osteoclasts activated by OPG-L. Interestingly, inhibition of OPG-L activity via OPG
had no obvious effect on the severity of periodontal inflammation itself (Figure
3, g and h), consistent with our previous
findings in the rat arthritis model (31) and
in the collagen-induced arthritis model (40).
This observation implies that tissue inflammation and alveolar bone destruction are
relatively independent aspects of periodontal infections. In summary, our functional
data show that A. actinomycetemcomitans activates CD4+ T
cells in the periodontium and induces them to express OPG-L, and that OPG-L is a key
mediator of alveolar bone destruction in microorganism-induced periodontal infection.
Periodontitis, a prime cause of tooth loss in humans, results from the interplay between
specific bacterial infections and host immune responses. Human periodontitis is
characterized by prominent inflammatory infiltrates and is associated with irreversible
loss of alveolar bone and/or connective tissue attachment in the periodontium. We
generated HuPBL-NOD/SCID mice, using human leukocytes from LJP patients transplanted
into NOD/SCID mice (24), to elucidate the
mechanisms by which antimicrobial host immune responses contribute to alveolar bone
destruction. Oral challenge of HuPBL-NOD/SCID mice with A.
actinomycetemcomitans produced a microorganism-specific human periodontal
infection in a “humanized” mouse model.
Perturbation of the T-cell immune repertoire is often observed in chronic inflammatory
diseases such as rheumatoid arthritis (41–42). Recently, we have
shown using a rat model of arthritis that activated T cells participate in regulating
bone loss and joint destruction through their expression of OPG-L, a key mediator of
osteoclastogenesis and osteoclast activation (31). While T cells are probably not required for normal bone homeostasis (27, 29–30), we proposed that
local or systemic pathology within an osseous environment due to microbial or viral
infections, autoimmune conditions (31),
tumorigenesis or metastasis (43), or injuries
could attract T cells capable of contributing to bone remodeling through the production
Our results show that the oral microorganism A. actinomycetemcomitans
can activate OPG-L production in CD4+ T cells, and they provide, albeit
indirectly, the first evidence that environmental pathogens regulate expression of OPG-L
on pathogen-specific T cells. Recently, OPG-L–independent triggering of
osteoclast differentiation has been reported (44), a finding consistent with our observation that injection of soluble OPG
(for 4 weeks) did not completely block alveolar bone destruction in vivo (approximately
75%; Figure 4e). It must be acknowledged that there
are auxiliary factors of potential importance in the model we describe. Thus it is known
that the expression of proinflammatory cytokines (e.g., IL-1 and TNF-Q) can also
regulate the relative balance of OPG-L and OPG (osteoclastogenesis inhibitory factor) in
the bone microenvironment and/or mesenchymal tissues adjacent to bone (45–46), thus contributing to bone destruction independently of cell-mediated
immunity. Additionally, the potential contribution of local mucosal cells, dental cells,
and/or phagocytes (e.g., monocytes/macrophages, etc.; refs. 47, 48) in the early stages
of infection or inflammation has yet to be defined, either in this model or in human
Thus while our data suggest that microorganism-activated CD4+ T cells are
implicated as an important feature of alveolar bone loss in our model system, they do
not prove unequivocally that OPG-L produced by those CD4+ T cells is
responsible for the increased alveolar bone destruction seen. Selective depletion of
such cells prior to adoptive transfer would address this question but has not yet proven
to be technically feasible. Nevertheless, it is likely that CD4+ T cells of
LJP patients differ fundamentally (in phenotype and/or genotype) from those of normal
healthy subjects (49). This possibility is
supported by our recent separate study, in which CD4+ T cells isolated from
Aa-N-HuPBL-NOD/SCID mice (using HuPBL from normal healthy subjects;
Figure 1b: group V) showed significantly less OPG-L
expression by FACS analysis (data not shown). However, the underlying mechanism(s) for
any differences awaits further investigation.
In summary, our results show that CD4+ T cell–mediated immunity
is involved in the modulation of periodontal bone destruction in HuPBL-NOD/SCID mice
after oral inoculation of A. actinomycetemcomitans. In vivo and in
vitro A. actinomycetemcomitans stimulation results in the activation of
CD4+ T cells and upregulation of OPG-L. Inhibition of OPG-L function via
the decoy receptor OPG significantly reduces alveolar bone destruction in HuPBL-NOD/SCID
mice in response to oral microbial infections. Moreover, OPG treatment significantly
reduces the numbers of osteoclasts at the sites of local periodontal infection.
Experiments are currently under way to test whether the same strategy can be used to
prevent alveolar bone destruction before the full burst of periodontal inflammation and
immune activation is established. Our results suggest a critical role for human
CD4+ T cells reactive to oral microorganisms in periodontal disease.
Microbial induction of OPG-L expression on T cells and OPG-L–mediated
osteoclast activation and bone loss thus can provide a molecular explanation for the
alveolar bone destruction observed in local periodontal infections. Inhibition of the
function of OPG-L may thus have therapeutic value to prevent or to interrupt alveolar
bone and/or tooth loss in human periodontal disease.
Patients. Four LJP patients (3 males and 1 female, mean age 21 ± 4.4 years) were
recruited as blood donors for the current study. Inclusion criteria, as defined
previously (24), were: (a) positive
anti–A. actinomycetemcomitans immunofluorescence
reactions of subgingival plaque samples collected adjacent to advanced lesions, and
(b) high serum Ab titers to A. actinomycetemcomitans (Y-4 or JP2
strains; see below) as detected by ELISA. Age-matched healthy subjects without
clinical evidence of periodontal disease were recruited as controls. Informed consent
was obtained from the patients, and all protocols used were approved by the Human
Ethics Committee and the Animal Experimentation Committee of the University of
Reagents. The following reagents were commercially purchased: Ficoll-Hypaque (Pharmacia Biotech
Inc., Piscataway, New Jersey, USA); collagenase (Sigma Chemical Co., St. Louis,
Missouri, USA); mAb’s specific for human CD45+ hematopoietic
cells, human CD45RO+ T cells, and human CD19+ pan-B cells
(Serotec Ltd., Kidlington, Oxford, United Kingdom); anti-human T-cell receptor
(TCR-αß), CD25, CD28, Fc receptor, CD3, CD4, CD8, and CD14
mAb’s (PharMingen, San Diego, California, USA); rabbit and human
complements (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada); goat anti-mouse
and mouse anti-human IgG-H+L FITC-conjugated Ab’s (Bio-Rad Laboratories
Inc., Hercules, California, USA, and R&D Systems Inc., Minneapolis,
Minnesota, USA, respectively); and isotypic control Ab’s (Sigma Chemical
Co.; PharMingen). Human recombinant OPG-FITC conjugate and OPG-Fc fusion protein were
prepared as previously described (25–27, 31). All tissue culture was performed in complete
RPMI 1640 medium supplemented with 2–3% individual donor’s
serum and 10% FCS (GIBCO BRL, Burlington, Ontario, Canada).
Generation of HuPBL-NOD/SCID mice and A. actinomycetemcomitans
inoculation. At day 0, 8- to 9-week-old female NOD/SCID (H2d/d) mice were subjected to
2.5 Gy c-irradiation prior to intraperitoneal injection of 10 ×
106 to 35 × 106 mononuclear cells freshly
prepared from HuPBLs by Ficoll-Hypaque gradient centrifugation (24, 32–33). On days 1, 3, and 5, the HuPBL-NOD/SCID mice
were inoculated orally with live A. actinomycetemcomitans (100
μl of 1010 CFU/ml culture broth grown in an anaerobic chamber;
Y-4 strain [no. 43781, American Type Culture Collection, Rockville, Maryland, USA] or
JP2 strain [no. 29523, American Type Culture Collection]). Autologous HuPBL
engraftment (weekly) and bacterial challenge (3 times per week) were repeated for
3–4 consecutive weeks. The infected mice were designated
Aa-HuPBL-NOD/SCID mice. All Aa-HuPBL-NOD/SCID
mice had serum levels of 0.1–0.85mg/ml IgG Ab’s against
A. actinomycetemcomitans. All mice were housed under specific
pathogen-free conditions at the Animal Facility of the Faculty of Medicine and
Dentistry, the University of Western Ontario, following institutional guidelines.
Measurement of alveolar bone loss in mice. Changes in alveolar bone mass in Aa-HuPBL-NOD/SCID mice were
determined in maxillary molars as described (34). Briefly, horizontal bone loss, the distance between the
cementum-enamel junction (CEJ) and the alveolar bone crest (ABC) along the long axis
of either the buccal or lingual roots, was measured under a dissecting microscope on
the mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual
sites of the maxillary right and left molars (M1 and M2). The mean horizontal bone
loss in each experimental group was calculated from the pooled data sets (the
measurements of individual right and left molars, M1 and M2, per mouse). Base-line
alveolar bone height was measured at day 1 (start point), whereas values at the
fourth and eighth week were recorded as mid- and end-points, respectively. Results
for each experimental group were normalized to the mean alveolar bone loss in control
A. actinomycetemcomitans–inoculated BALB/c
(H2d/d) mice (100% ≈ 0.6 ± 0.12 mm per tooth in
an 8-week period). Statistical significance of differences in alveolar bone loss
between different groups was assessed by one-way ANOVA with a P
value of less than 0.05.
T-cell activation. Periodontal CD4+ T cells were prepared from
Aa-HuPBL-NOD/SCID mice during weeks 4–6 and restimulated
in 24-well plates with 100 μg/ml antigens from sonicated A.
actinomycetemcomitans antigens (24) in the presence of irradiated (25 Gy) autologous HuPBL-derived
monocytes/macrophages as antigen-presenting cells (APCs) (24). After 48 hours, T-cell blasts were collected by Ficoll
gradient centrifugation and sequentially immunostained with anti-human CD25 (IL-2
receptor-α chain) and FITC-conjugated goat anti-mouse IgG (H+L) Ab. The
positive controls included human Jurkat T cells and HuPBL-derived CD4+ T
cells whose TCRs were cross-linked with anti-human TCR mAb (10 μg/ml for
106 cells) and anti–Fc receptor mAb. After 24 hours, 3
× 105 to 4 × 105 cells per sample were
washed and CD25 expression was determined by flow cytometric analysis using a
Immunization with A. actinomycetemcomitans in vivo. Systemic immune responses to A. actinomycetemcomitans were tested 6
weeks after the start of the experiment. Aa-HuPBL-NOD/SCID mice were
immunized intraperitoneally with 30 μl A.
actinomycetemcomitans sonicate antigens (1 μg/μl)
mixed 1:1 with a 1:10 mixture of CFA/IFA adjuvants. After 14 days, human
CD4+ T cells were isolated from mouse spleens and purified by FACS
(purity >85–95%). Purified T cells were subjected in triplicate to
in vitro stimulation (1.5 × 104 T cells/well in U-bottom
96-well plates) using serial dilutions of A. actinomycetemcomitans
sonicate antigens (1–100 μg/ml). After 48 hours, supernatants
were collected for analysis of human IL-2 production by bioassay with
IL-2–dependent CTLL-2 cells as described (24, 35). Periodontal
CD4+ T cells from Aa-HuPBL-NOD/SCID mice served as
positive controls, while splenic CD4+ T cells from nonimmunized
HuPBL-NOD/SCID mice constituted the negative control.
Lymphocyte depletion and adoptive transfer experiments. For in vivo depletion of human lymphocytes in HuPBL-NOD/SCID mice, mAb’s
specific for human CD4+ T cells, CD8+ T cells, or B cells were
injected (150–200 μg) intraperitoneally every 5–7
days from weeks 4–8 in the presence of human complement (24, 32–33). PBLs and
splenocytes were stained with phycoerythrin-conjugated mAb’s to human
CD4, CD8, and CD20 (pan-B cells) to confirm the depletion of the corresponding cells
by FACS analysis. The efficiency of immunodepletion was typically 95–98%
without significant numbers of target cells remaining (data not shown) (24, 32–33). For adoptive
transfer, 1 × 106 to 2 × 106A. actinomycetemcomitans–reactive periodontal
CD4+ T cells were harvested from the margins of oral mucosal tissues of
Aa-HuPBL-NOD/SCID mice during week 6–8; then these
CD4+ T cells (1 × 106 to 2 ×
106) plus 3 × 106 to 5 ×
106 irradiated (25 Gy) autologous HuPBL-derived monocytes/macrophages
(as APCs) were adoptively transferred intravenously into naive NOD/SCID hosts.
Adoptive transfer was performed weekly for 3–4 consecutive weeks and in
each case immediately followed by oral inoculation with A.
actinomycetemcomitans as described above.
OPG-L expression and inhibition of OPG-L activity in vivo. To detect OPG-L expression, periodontal CD4+ T cells isolated from control
or Aa-HuPBL-NOD/SCID mice were restimulated in vitro with A.
actinomycetemcomitans sonicate antigens. OPG-L surface expression was
detected using OPG-FITC (31). To investigate
the in vivo inhibition of OPG-L by its natural decoy receptor OPG, recombinant human
OPG-Fc fusion protein was injected into mice as described previously (25–27, 31). Briefly,
Aa-HuPBL-NOD/SCID and control mice were treated intraperitoneally
every other day with PBS or OPG-Fc (1 mg/kg) between weeks 4 and 8 for a total of 14
injections per mouse. By the end of the 8th week, all mice were sacrificed, and then
alveolar bone loss was determined as described above and assessed by histology.
Osteoclasts were identified in situ using tissue sections with osteoclast-specific
tartrate-resistant acid phosphatase (TRAP) staining for determining the numbers of
positive cells as described previously (31,
We thank Mary Saunders for scientific editing of the manuscript. J.M. Penninger is
supported by Amgen Institute and the Medical Research Council (MRC) of Canada and is a
member of the Canadian Centre of Excellence for Tumor Vaccination. Y.-T.A. Teng is a
career scientist of the Ministry of Health of Ontario, Ontario, Canada. This work was
supported by grants to Y.-T.A. Teng from the London Health Sciences Centre (IRF-021-99),
the MRC of Canada (MOP-37960), Dentistry Canada Fund, and NIH (DE12969-01A2).
Conflict of interest: None.
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