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Infection and Immunity, May 1999, p. 2125-2130, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Bacterium-Dependent Induction of Cytokines in
Mononuclear Cells and Their Pathologic Consequences In Vivo
Yanling
Jiang,*
Luciano
Magli, and
Michael
Russo
Department of Endodontics, Boston University
School of Dental Medicine, Boston, Massachusetts 02118
Received 3 December 1998/Accepted 9 February 1999
 |
ABSTRACT |
Viridans streptococci are a heterogeneous group of gram-positive
bacteria that are normal inhabitants of the mouth. These organisms are
thought to contribute significantly to the etiology of infective
endocarditis, although recently they have been implicated in serious
infections in other settings. Another group of oral bacteria,
gram-negative anaerobes, is associated with chronic dental infections,
such as periodontal diseases or endodontic lesion formation. We
evaluated the ability of the oral pathogens Streptococcus
mutans and Porphyromonas endodontalis to induce a
pathogenic response in vivo, with the goal of quantifying the inflammatory response in soft tissue by measuring leukocyte recruitment and hard tissues by measuring osteoclastogenesis. S. mutans
induced a strong inflammatory response and was a potent inducer of
osteoclast formation, while P. endodontalis was not. To
further study the mechanisms by which P. endodontalis and
S. mutans elicit significantly different levels of
inflammatory responses in vivo, we tested the capacity of each to
induce production of cytokines by mononuclear cells in vitro. S. mutans stimulated high levels of interleukin-12 (IL-12), gamma
interferon (IFN-
), and tumor necrosis factor alpha (TNF-
), all of
which are associated with inflammation, enhanced monocyte function, and
generation of a Th1 response. In contrast, P. endodontalis
stimulated production of IL-10 but not of TNF-, IL-12, or IFN-.
These results demonstrate that oral pathogens differ dramatically in
their abilities to induce inflammatory and immunoregulatory cytokines.
Moreover, there is a high degree of correlation between the cytokine
profile induced by these bacteria in vitro and their pathogenic
capacity in vivo.
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INTRODUCTION |
Several different bacteria have been
associated with oral infections (1, 27, 34). However, the
mechanisms by which specific bacteria cause pathogenic lesions are not
completely understood. Bacterial contamination of the dental pulp in
decayed teeth inevitably leads to pulpitis and necrosis. The pathogens that cause pulpitis are derived from dental plaque, and are, for the
most part, gram-positive bacteria such as viridans streptococci. Oral
streptococci are also important agents in infective endocarditis and
are suspected to contribute to the etiology in over 50% of cases
(16, 31). Approximately 20% of viridans streptococcus endocarditis are caused by Streptococcus mutans
(5). In addition, viridans group streptococci have been
described as responsible for the alpha-streptococcal shock syndrome in
neutropenic patients (19). The mechanisms by which viridans
streptococci cause bacteremia associated with severe clinical
manifestations have not been elucidated. In contrast to oral
streptococci, which appear to cause acute or subacute infections,
gram-negative anaerobes are primarily associated with chronic oral
infections such as periodontal disease or endodontic lesions. For
example, Porphyromonas endodontalis has been found in half
of chronic endodontic lesions and is almost exclusively found in
infections of endodontic origin, suggesting a specific association of
P. endodontalis and the dental pulp (4, 32).
The innate immune response associated with infections is linked to the
adaptive immune response by cytokine production. For example, bacteria
can initiate cell-mediated immunity by stimulating macrophages to
produce interleukin-12 (IL-12), which can then promote gamma interferon
(IFN-) production by natural killer cells. IFN--activated
macrophages then secrete more IL-12 as a result of positive feedback,
leading to cell-mediated Th1 responses. IFN- also activates
macrophages to produce tumor necrosis factor alpha (TNF-), which in
turn maintains the activated state of the macrophage (16).
TNF- is a potent proinflammatory cytokine that can stimulate
chemokine production by endothelial cells and fibroblasts in vitro
(25). In addition to its protective role in the host defense
against infectious agents, TNF- contributes to bone resorptive
activity and has been implicated in pathologic bone resorption
(11, 21, 28). An effective host defense against bacterial
invasion is characterized by the vigorous recruitment and activation of
inflammatory cells, which are modulated by the coordinated expression
of both pro- and anti-inflammatory cytokines. IL-10, which is produced
by monocytes and Th2 lymphocyte subsets, inhibits IFN- synthesis by
Th1 cells and inhibits the production of proinflammatory cytokines such
as IL-12 and TNF-. The inhibiting effects of IL-10 on cytokine
production correlate with their antiinflammatory effects in vivo
(9). Therefore, IL-10, IFN-, IL-12, and TNF- have
important and cross-regulatory roles in infection (29).
In data presented here, we examined the capacities of S. mutans and P. endodontalis to stimulate leukocyte
recruitment and osteoclastogenesis in vivo and cytokine production by
mononuclear cells in vitro. This was accomplished in an animal model
that has recently been shown to be an effective in describing the host response to oral pathogens (35). The results demonstrate
that S. mutans stimulates considerable inflammatory cell
recruitment and osteoclast formation in vivo and is a strong inducer of
proinflammatory cytokine induction in vitro whereas P. endodontalis is not. These results should provide insight into the
inflammatory response of S. mutans and its capacity to
stimulate the host in oral infections and in bacterial endocarditis.
| |
MATERIALS AND METHODS |
Bacteria.
P. endodontalis ATCC 35406 (generously
provided by Ann Tanner [Forsyth Dental Center, Boston, Mass.]) was
cultured under strict anaerobic conditions (85% N2, 10%
H2, and 5% CO2) on Trypticase soy-brain heart
infusion-yeast extract-blood agar containing hemin and vitamin
K1 (Northwest Laboratory, Waterville, Maine). S. mutans LM7 and OMZ175, grown in Todd-Hewitt broth (Becton
Dickinson, Cockeysville, Md.), were generously donated by F. Oppenheim
(Boston University Goldman School of Dental Medicine, Boston, Mass.). The bacteria, used at early- to mid-log-phase growth, were heat killed
by boiling for 10 min and then washed three times with phosphate-buffered saline (PBS) as described previously (7). The amount of each bacterium was quantitated spectrophotometrically according to a standard curve established by colony formation on
bacterial plates.
Mononuclear cell stimulation.
Human peripheral blood was
obtained from healthy volunteers by venipuncture followed by the
addition of heparin (100 U per 50 ml of blood). Peripheral blood
mononuclear cells (PBMC) were isolated from the leukocyte-rich buffy
coat following centrifugation over a gradient of Ficoll-Hypaque.
Autologous serum was obtained by centrifugation of nonheparinized
venous blood. PBMC (3 × 105 per well) were incubated
in polypropylene cell culture plates containing the indicated stimulus
in a final volume of 250 µl of RPMI tissue culture medium
supplemented with 5% autologous serum. All solutions and reagents had
nondetectable endotoxin levels (<30 pg/ml) as determined by the
Limulus amebocyte lysate assay (Sigma, St. Louis, Mo.).
After 24 h, 96 h, or 7 days of stimulation, supernatants were
harvested for evaluation of induced cytokine levels by enzyme-linked
immunosorbent assay (ELISA).
ELISAs for cytokines.
Antibody pairs for human IL-12 p70
(R&D Systems, Minneapolis, Minn.), IFN-, TNF-, and IL-10
(PharMingen, San Diego, Calif.) were used in the sandwich ELISA. These
assays were sensitive to 10 pg/ml of cytokine per ml. Other ELISA
reagents were purchased from Kiekegaard & Perry Laboratories,
(Gaithersburg, Md.). ELISAs were performed as we described previously
(6). Briefly, 96-well plates were incubated with capture
monoclonal antibody at 4°C overnight. Nonspecific bind sites were
blocked by 1% bovine serum albumin for 1 h at room temperature.
After being rinsed with PBS-Tween 20 buffer, the samples were added and
incubated at room temperature for 1 h. Biotinylated detecting
antibody to the cytokine was added to the samples and incubated for
1 h after the plate was washed with PBS-Tween buffer. Horseradish
peroxidase-strepavidin and color metric system
(tetramethylenebenzidine) were used as described by the manufacturer
(KPL, Kiekegaard & Perry Laboratories). The plates were read at 450 nm
within 30 min with an ELISA plate reader (Molecular Devices).
Bacteria inoculation and immunohistology studies.
The host
response to bacteria was investigated in both soft and hard tissue,
using the calvarial model that was recently shown to be effective in
characterizing the response to oral pathogens (35). CD-1
outbred mice, 12 to 14 weeks of age, were obtained from Charles River
Laboratories (Boston, Mass.). Mice were inoculated with 108
live P. endodontalis or S. mutans in 50 µl of
PBS. Bacteria were applied by supraperiosteal injection in the
mid-calvarial region after complete removal of hair with a microshaver
(Conair). Vehicle (PBS) alone was injected as a negative control. Mice
were sacrificed 2, 5, and 10 days after injection of bacteria. The
calvarial samples with attached scalp were harvested, fixed in 4%
paraformaldehyde for 24 h at 4°C, and decalcified in
EDTA-glycerin-PBS (pH 7.0). After decalcification, specimens were
immersed in 30% sucrose-PBS overnight at 4°C, snap frozen, and cut
into 5-µm sections with a cryostat according to published procedures
(12). The tissue sections were stained with hematoxylin and
eosin to count inflammatory cells or tartrate-resistant acid
phosphatase (TRAP) to aid in identifying osteoclasts.
Statistical analysis.
Statistical analysis was performed
with one-way analysis of variance with Duncan's post hoc test, to
determine the significance of cytokine production between control and
bacterium-stimulated samples. Significance was determined at
P < 0.05.
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RESULTS |
The response to P. endodontalis and S. mutans in vivo.
To determine whether different oral bacteria
stimulate disparate host responses, P. endodontalis and
S. mutans were injected into the mouse calvaria (scalp),
adjacent to the periosteum. The number of inflammatory cells recruited
and number of osteoclasts formed were quantified. The results
demonstrate that S. mutans elicits a pronounced inflammatory
infiltrate in mouse calvarial soft tissue, while P. endodontalis elicits a relatively mild inflammatory cell
infiltrate (Fig. 1). While both S. mutans and P. endodontalis stimulated a peak of
inflammatory cell recruitment at 2 days, S. mutans produced
a much more sustained inflammatory infiltrate. At 5 and 10 days,
S. mutans enhanced inflammatory cell recruitment 30- and
20-fold, respectively, while P. endodontalis stimulated increases of only 5- and 3-fold, respectively, compared to vehicle alone.
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FIG. 1.
Quantitative assessment of leukocyte recruitment
following injection of heat-killed S. mutans and P. endodontalis. Heat-killed S. mutans (S.m.) and P. endodontalis (P.e.) (108 bacteria/scalp) were injected
into CD-1 wild-type mice. Vehicle alone was injected as a negative
control (C). Mice were sacrificed at 0, 2, 5, and 10 days after
injection. Sections were stained with hematoxylin and eosin. Image
analysis was used to quantify the total number of leukocytes per field
at a magnification of ×1,000. Eighteen fields were examined per
section. Five specimens were examined per data point.
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|
To assess the impact of
S. mutans or
P. endodontalis infection on hard tissue, we measured
osteoclastogenesis and the percent
bone surface covered by osteoclasts.
S. mutans induced formation
of a large number of
osteoclasts, which were identified as large
multinucleated
TRAP-positive cells in lining the bone surface.
S. mutans
stimulated a sevenfold-greater increase in both the
number of
osteoclasts and the percent bone surface covered by
osteoclasts
compared to
P. endodontalis at the 5-day time point
(Fig.
2 and
3).
Thus, the magnitude of the induced osteoclastogenesis
agrees well with
the results obtained by measuring the recruitment
of inflammatory
cells, indicating that
S. mutans stimulates a
more vigorous
host response than
P. endodontalis. With the negative
control (vehicle alone), there was virtually no stimulation of
osteoclast formation.
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FIG. 2.
Quantitative assessment of osteoclast formation
following injection of heat-killed S. mutans and P. endodontalis. Heat-killed S. mutans (S.m.) and P. endodontalis (P.e.) (108 bacteria/scalp) were injected
into CD-1 wild-type mice. Vehicle alone was injected as a negative
control (C). Mice were sacrificed at 0, 2, 5, and 10 days after
injection. Sections were stained with TRAP. Image analysis was used to
quantify the total number of osteoclasts per millimeter at a
magnification of ×1,000. Five specimens were examined per data
point.
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FIG. 3.
Quantitative assessment of osteoclast activity following
injection of heat-killed S. mutans (S.m.) and P. endodontalis (P.e.). The results demonstrated in Fig. 2 were
analyzed to quantify the percent bone surface covered by osteoclasts at
a magnification of ×1,000. Five specimens were examined per data
point.
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|
In the experiments described above, we used heat-killed bacteria to
simplify interpretation of the experimental results since
the
confounding influence of bacterial survival in vivo is eliminated.
Since it is possible that heat inactivation of the bacteria may
had
altered their true capacity to induce a host response, we
repeated the
experiments with live bacteria in vivo. Similar results
were obtained,
with
S. mutans inducing a greater degree of inflammatory
response (tissue necrosis and inflammatory cell infiltration)
and
osteoclast activity (data not shown). Thus, the host response
in vivo
induced by inoculation with either heat-killed or live
S. mutans is dramatically more pronounced than that stimulated
by
P. endodontalis. Other investigators have found that
injection
of either heat-killed or live oral bacteria in this model
produces
similar inflammatory responses (
35).
Bacterium-stimulated cytokine production.
To explore the
mechanisms to account for differences in the inflammatory response and
osteoclast activation induced by S. mutans or P. endodontalis infection, we examined the capacities of these
bacteria to stimulate production of a proinflammatory cytokine profile
by mononuclear cells in vitro. Since IL-12 is produced by
bacterium-stimulated macrophages and bridges the innate immune response
and cell-mediated immune response, we examined IL-12 production
stimulated by these two bacteria. The results demonstrate that S. mutans significantly increased IL-12 production compared to
unstimulated controls at ratios of 1, 10, and 100 bacteria per PBMC
(Fig. 4). The highest levels of
production of IL-12 by PBMC were 92-fold above unstimulated control
levels at a ratio of 10 bacteria per PBMC. In contrast, P. endodontalis stimulated only a minimal increase in IL-12
production compared to unstimulated controls. Since IFN- is a
powerful activator for macrophages and IL-12 increases inflammatory
responses by enhancing the production of IFN- (8, 17), we
examined bacterium-stimulated IFN- production. Figure
5 shows that S. mutans
stimulated high levels of IFN- by PBMC. S. mutans
stimulated IFN- production approximately 22-fold over unstimulated
controls at a ratio of 10 bacteria per PBMC, while P. endodontalis induced none.
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FIG. 4.
S. mutans stimulates high levels of
production of IL-12 by PBMC. PBMC were stimulated by the indicated
ratios of bacteria to PBMC. Supernatants were collected after 24 h
and analyzed by ELISA for IL-12. The results are representative of
three experiments. Each data point is the mean of three triplicate
samples. By one-way analysis of variance with Duncan's post hoc test,
S. mutans (S.m.) stimulation significantly increased IL-12
production compared to unstimulated controls (C) (<10 pg/ml) at ratios
of 1, 10, and 100 (P < 0.05). P.e., P. endodontalis.
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FIG. 5.
S. mutans stimulates high levels of
production of IFN- by PBMC. PBMC were stimulated by the indicated
ratios of bacteria to PBMC. Supernatants were collected after 24 h
and analyzed by ELISA for IFN-. The results are representative of
three experiments. Each data point is the mean of triplicate samples.
By one-way analysis of variance with Duncan's post hoc test, S. mutans (S.m.) stimulation significantly increased IFN-
production compared to unstimulated controls (C) (0.4 ng/ml) at ratios
of 1, 10, and 100 (P < 0.05). P.e., P. endodontalis.
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TNF-
is an important proinflammatory cytokine. It can promote
inflammatory cell infiltration leading to a vigorous host response
and
can directly stimulate osteoclast formation and activity
(
24).
Therefore, we examined TNF-
induction by putative
endodontic
pathogens and found that
S. mutans stimulated
high levels of TNF-
production by PBMC whereas
P. endodontalis was a relatively weak
inducer (Fig.
6).
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FIG. 6.
S. mutans (S.m.) stimulates high and P. endodontalis (P.e.) stimulates low levels of production of
TNF-. PBMC were stimulated by the indicated ratios of bacteria to
PBMC. Supernatants were collected after 24 h and analyzed by ELISA
for TNF-. The results are representative of three experiments. Each
data point is the mean of triplicate samples. By one-way analysis of
variance with Duncan's post hoc test, bacterial stimulation
significantly increased TNF- production compared to unstimulated
controls (C) at ratios of 1, 10, and 100 (P < 0.05).
Unstimulated PBMC secreted undetectable levels (<10 pg/ml) of
TNF-.
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IL-10 inhibits IL-12, IFN-
, and TNF-
production and is considered
an antiinflammatory cytokine. In contrast to the above
findings,
P. endodontalis stimulated high levels of IL-10 production
(2,600 pg/ml) whereas
S. mutans stimulated low levels (178 pg/ml)
(Fig.
7). Compared to
S. mutans,
P. endodontalis stimulated 15-fold
more IL-10
production.
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FIG. 7.
P. endodontalis (P.e.) stimulates high levels
of production of IL-10 by PBMC. PBMC were stimulated by the indicated
ratios of bacteria to PBMC. Supernatants were collected after 24 h
and analyzed by ELISA. The results are representative of three
experiments. Each data point is the mean of triplicate samples. By
one-way analysis of variance with Duncan's post hoc test, bacterial
stimulation significantly increased IL-10 production compared to
unstimulated controls at ratios of 1, 10, and 100 (P < 0.05). Unstimulated controls secreted undetectable levels of (<10
pg/ml) of IL-10. S.m. S. mutans.
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To further investigate the capacity of each of the two bacteria to
stimulate production of an inflammatory cytokine profile,
time-dependent cytokine induction was assessed over periods of
1, 4, and 7 days. This would take into account potential cell-to-cell
interactions that occur from different leukocyte subsets found
in
mononuclear cell populations. Figures
8
to
11 demonstrate that
the patterns of cytokine induction by both
bacteria are consistent
with time.
S. mutans stimulated high
levels of IL-12 (Fig.
8)
and IFN-
(Fig.
9) at 1-, 4-, and 7-day time points,
while
P. endodontalis did not. The levels of TNF-
production decreased
with time for both bacteria but kept the same
order of potency.
Thus,
S. mutans was a more efficacious
stimulator than
P. endodontalis (Fig.
10). For IL-10 induction, the order was
reversed, as
P. endodontalis stimulated high levels and
S. mutans stimulated low levels of
IL-10 (Fig.
11). These findings are not restricted
to the strain
of
S. mutans used here (OMZ175), since similar
results were obtained
with another strain, LM7 (data not shown).
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FIG. 8.
Bacterium-stimulated production of IL-12 by PBMC at 1, 4, and 7 days. PBMC were stimulated by 10 bacteria per PBMC.
Supernatants were collected at the indicated times and analyzed by
ELISA for IL-12. The results are representative of two experiments.
Each data point is the mean of three triplicate samples. By one-way
analysis of variance with Duncan's post hoc test, S. mutans
(S.m.) stimulation significantly increased IL-12 production compared to
P. endodontalis (P.e.) at all time points (P < 0.05).
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FIG. 9.
Bacterium-stimulated production of IFN- by PBMC at 1, 4, and 7 days. PBMC were stimulated by 10 bacteria per PBMC.
Supernatants were collected at the indicated times and analyzed by
ELISA for IFN-. The results are representative of two experiments.
Each data point is the mean of triplicate samples. By one-way analysis
of variance with Duncan's post hoc test, S. mutans (S.m.)
stimulation significantly increased IFN- production compared to
P. endodontalis (P.e.) at all time points (P < 0.05).
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FIG. 10.
Bacterium-stimulated production of TNF- by PBMC at
1, 4, and 7 days. PBMC were stimulated by 10 bacteria per PBMC.
Supernatants were collected at the indicated times and analyzed by
ELISA for TNF-. The results are representative of two experiments.
Each data point is the mean of triplicate samples. By one-way analysis
of variance with Duncan's post hoc test, S. mutans (S.m.)
stimulation significantly increased TNF- production compared to
P. endodontalis (P.e.) at all time points (P < 0.05).
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FIG. 11.
Bacterium-stimulated production of IL-10 by PBMC at 1, 4, and 7 days. PBMC were stimulated by 100 bacteria per PBMC.
Supernatants were collected at the indicated times and analyzed by
ELISA. The results are representative of two experiments. Each data
point is the mean of triplicate samples. By one way analysis of
variance with Duncan's post hoc test, P. endodontalis
(P.e.) stimulation significantly increased IL-10 production compared to
S. mutans (S.m.) at all time points (P < 0.05).
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| |
DISCUSSION |
We demonstrate here that two putative oral pathogens are
considerably different in their capacities to stimulate human PBMC to
produce inflammatory and immunoregulatory cytokines. Interestingly, S. mutans, which stimulates high levels of Th1 cytokines
such as IL-12 and IFN-, also stimulates high levels of the
proinflammatory cytokine TNF-. In contrast, P. endodontalis, which induces little or none of the above cytokines,
appears to be potent in inducing IL-10, an anti-inflammatory cytokine.
It is possible that IL-10 induced in response to P. endodontalis inhibits expression of other cytokines by the
mononuclear cells. Regardless of the mechanism, it is clear that the
inherent properties of an invading bacterium influence the cytokine
profile that is ultimately produced.
The proinflammatory cytokine profile that was induced in human PBMC
corresponded well with both quantitative and qualitative measures of
the inflammatory response observed in vivo in mice. That human and
murine leukocytes would behave similarly is not surprising given the
large body of evidence indicating similarities in various immune
responses (15, 23). When injected in vivo, S. mutans induced formation of a dense inflammatory cell infiltrate and bone resorptive activity compared to P. endodontalis.
This finding is consistent with the production of TNF- and IFN-
stimulated by S. mutans and the relative lack of their
expression induced by P. endodontalis.
S. mutans gained notoriety in the 1960s as a cariogenic
pathogen; its virulence factors related to caries formation have been extensively studied (2). Bacteriemias of S. mutans are capable of causing significant morbidity and mortality
through bacterial endocarditis or other complications such as
respiratory distress syndrome (14, 22, 30). The mechanisms
by which S. mutans produce an inflammatory response and
causes tissue damage have not been elucidated. Our results demonstrated
that S. mutans is able to elicit cell-mediated Th1 responses
by stimulating expression of IL-12, IFN-, and TNF- in mononuclear
cells. We postulate that the exuberant production of these cytokines
stimulated by S. mutans may contribute to pathological
process of the infection since Th1 cytokines can exacerbate
inflammatory pathology by enhancing chemokine expression and
recruitment of inflammatory cells (3, 10, 18, 20, 26).
In contrast to S. mutans, P. endodontalis (which
is associated with chronic infections of the dental pulp) has been
found to be of low virulence in experimental monoinfections but seems to play an essential role in anaerobic mixed infection (33). The reasons for this interesting phenomenon have not been explained. In
the present study, we demonstrate that P. endodontalis
induces production of high levels of IL-10 but low levels of IL-12,
IFN-, and TNF-. Consistent with this cytokine profile, in vivo,
P. endodontalis elicits mild inflammatory infiltration and
bone resorption activity. It is possible that the cytokine profile
induced by P. endodontalis (i.e., IL-10) contributes to its
low virulence in monoinfections. However, in mixed infections, the
"stealth" property of this microorganism may help itself and other
bacteria to invade the host without causing strong host responses, so
that the pathogens can reside in infected sites for long periods and grow to large numbers. The stealth properties of P. endodontalis are consistent with finding that a bacterium of the
same genus, Porphyromonas gingivalis, has similar
properties. P. gingivalis is poorly recognized by cells of
the innate host defense system (13), and over 1,000 times
more bacteria are required to stimulate high levels of chemokine
production compared to S. mutans (6). Thus, both
P. endodontalis and P. gingivalis seem to possess
stealth properties, which may provide a selective advantage to the
bacteria by enabling them to avoid host defense mechanisms. It is
possible that this represents an important aspect of their pathologic potential.
From our results, we conclude that specific bacteria can induce the
synthesis of a profile that is consistent with up-regulation of the
host response or one that is not. Thus, the type of host response
generated is considerably different for specific bacteria and is
reflected by the cytokine profile induced in vitro. This conclusion is
supported by results demonstrating that the nature of the cytokine
profile induced in vitro is consistent with the capacity of the
bacteria to produce an inflammatory infiltrate and stimulate pathologic
osteoclast activity in vivo.
| |
ACKNOWLEDGMENTS |
We thank John Richardson for help in the histologic analysis of
inflammatory cell infiltrates.
This study was partially supported by American Association of Endodontists.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 700 Albany St.,
Rm. W-201, Boston University Medical Center, Boston, MA 02118. Phone: (617) 638-4987. Fax: (617) 638-4924. E-mail:
yljiang{at}acs.bu.edu.
Editor:
J. R. McGhee
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Infection and Immunity, May 1999, p. 2125-2130, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
