Enterococcus faecalis’in PMNL fonksiyonları üzerine etkisi: bir in vitro çalışma

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Introduction
Bacteria are the main causative factor in the development of periapical inflammation (1,2). The main
objective of endodontic therapy is, therefore, to eliminate bacteria from the infected root canal and to
prevent reinfection. Complete chemomechanical
preparation and careful obturation are essential to
avoid reinfection of the root canal space. Even after
cleaning, shaping and irrigation with disinfectants,
total elimination of bacteria is difficult to achive
complete debridement in all cases (3). However, these procedures undoubtly reduce the number of viable
microorganisms in the root canal system. Therefore,
reinfection or chronic periapical inflammation of
root-filled teeth may be due to residual bacteria within the root canal system and dentinal tubules (4,5).
Various bacteria can be isolated within an infected root canal system where anaerobes predominate (6). These microorganisms are generally present
in all parts of the root canal system, and found at
varying depths within the dentinal tubules from the
pulpal end (7,8). Berutti et al. have shown that bacteria is found within dentinal tubules in deep regions of dentine even after the root canal irrigation (8).
Accordingly, in an in vitro study by Weigner et al. penetration time of bacteria into human root dentine
up to a depth of at least 150 μm was found to be 4 weeks (9). There are regional variations in the extent of
bacterial invasion of dentinal tubules in the infected
root canals (10). In turn, cervical tubules are invaded
to a greater extent than the midroot tubules, which
are invaded more than those of the apical region.
E. faecalis is gram-positive, facultatively anaerobic,
coccal bacterium that causes a wide variety of infections in humans (11). These enteric bacteria are also
part of the normal flora of the oral cavity and can be
present in the infected root canal during endodontic
treatment (12). E. faecalis is the most common and
occasionally the single isolated bacteria from root ca-
* Dental Services, Turkish Land Forces
** Department of Immunology, Gülhane Military Medical Faculty
*** Sarıkamış Military Hospital
**** Department of Endodontics, Gülhane Military Medical Academy
***** Department of Microbiology and Clinical Microbiology, Gülhane Military
Medical Faculty
This work was supported by grants from Gulhane Military Medical Academy,
and not presented in any scientific meeting before.
Reprint request: Dr. Uğur Muşabak, Department of Immunology, Gülhane
Military Medical Faculty, Etlik-06018, Ankara, Turkey
E-mail: umusabak@hotmail.com
Date submitted: February 01, 2010 • Date accepted: May 07, 2010122 • June 2010 • Gulhane Med J Oktay et al.
nals of teeth with persistent periapical periodontitis
(13,14). Especially, these single species of organisms
were found to be one of the predominant bacteria in
the teeth in which root canal treatment failed (15).
Pinheiro et al. have found E. faecalis in 52.94% of canals with bacterial growth (16). This microorganism
has demonstrated the capacity to survive in an environment in which there are scant available nutrients
and in which commensality with other bacteria is minimal (17). There have been many studies to identify
the possible mechanism that would explain how E.
faecalis could survive and grow within root canal system and reinfect an obturated root canal (18). It was
shown that E. faecalis persists in harsh environmental
conditions which exist in the endodontically treated
tooth due to inherent antimicrobial resistance and
adaptation to changed environment conditions (19).
Many kinds of immune cells such as polymorphonuclear neutrophils (PMNs), osteoclasts, lymphocytes, plasma cells, monocytes, macrophages, epithelial
cells play a role in the state of early periradicular periodontitis via cell-to-cell contact or their secretory
products (20). This pathological process is modulated by interactions between the cellular and soluble
components of the immune systems. PMNs are the
first cells that migrate into the tissues in response to
invading pathogens. It is well known that a variety of
neutrophil chemoattractants produced by different
cells sources such as host endothelial, epithelial, and
stromal cells are involved in PMNL migration to the
site of infection. In this context, IL-8 is a soluble chemoattractant produced by endothelium in response
to infectious agents and activates neutrophils (21).
However, not only immune cells and their secretory
products but also certain bacterial substances play a
crucial role in the inflammation process. It has previously been reported that most bacteria produce a
heterogeneous mixture of neutrophil chemotactic
agents and contribute to neutrophil recruitment to
the site of infection during inflammatory response
(22). Sannomiya et al. have demonstrated that E. faecalis derived nonformylated peptides, in particular
cAM373 and cPD1, are potent chemotactic factors
and inducers of lysosomal granule enzyme secretion
for rat peritoneal neutrophils (22).
Although there have been many investigation related to immunity against E. faecalis, immunopathogenic mechanisms of persistence of these microorganisms in root canals are still unknown. Therefore,
we investigated in vitro effects of E. faecalis on PMNL
functions in the present study. In addition, we studied whether phytohemagglutinin (PHA)-stimulated
mononuclear cell (MNC) culture supernatants could
affect neutrophil functions against E. faecalis.
Material and Methods
Donors: This study was carried out on 3 male and 3
female healthy donors, aged between 27 and 39 years. They had no immunological or metabolic disorders and took no medication that could alter the immunological parameters. All subjects were informed
about the aim and procedures of the study and gave
their consent.
Preparation of E. faecalis culture supernatants: E. faecalis strain ATCC29212 was cultured in brain heart
infusion broth. Bacteria were grown at 37˚C in aerobic condition (85% N2
and 5% CO2
) for 3 days.
Bacterial cell suspensions were harvested and washed
in 20 ml of phosphate-buffered saline (PBS) containing the protease inhibitor phenylmethylsulonylfluoride (PMSF, Sigma Chemical Co., St. Louis, MO,
USA). Washed E. faecalis were disrupted by sonication
(Sonic Dismembrator, Model 550, Fisher Scientific,
Pittsburgh, PA, USA) on ice for 5 minutes with 30-sec
pulses. The homogenate was centrifuged at 12.000
xg in a Sorvall RC5C (Sorvall İnstruments, DuPont,
Wilmington, DE, USA) for 20 minutes at 4˚C. Then,
the supernatant was collected, and stocked in deep
freezer at -20˚C. Frozen samples were allowed to thaw
at room temperature before use (23).
Preparation of human PMNL cultures: Peripheral blood samples from six healthy donors were drawn in
sterile heparin-treated tubes. Immediately after phlebotomy, PMNLs and MNCs isolated from the whole
blood samples through density gradient centrifugation. Double gradient of Ficoll-Hypaque Separating
Solutions (histopaque 1077 and 1119, Sigma Chemical
Co., St. Louis, MO, USA) were used for this purpose.
Initially, histopaque 1077 (3 ml) was carefully layered
onto the histopaque 1119 (3 ml). Then, 6 ml of whole
blood was placed onto the upper gradient of tube and
centrifuged at 700 g for 30 minutes at room temperature. PMNL and MNC layers were harvested from
the interfaces by the aid of sterile Pasteur pipette
and washed twice with RPMI-1640 medium (Gibco,
Invitrogen Corporation, Scotland, UK), separately.
They were then resuspended in 2 ml of same medium. The viability of both cell populations was more
than 97% by staining with acridine-orange and ethidium bromide. These cell populations were analyzed
by flow cytometry (FACSCalibur, Becton Dickinson,
San Jose, CA, USA). In this way, the percentages of
PMNLs and MNCs in cell suspentions were found to
be 99.6% and 98.8%, respectively. PMNL and MNC
suspensions were adjusted to 1x10
6
cells/ml by RPMI, Volume 52 • Issue 2 Enterococcus faecalis and PMNL functions • 123
and supplemented with 10% heat-inactivated newborn calf serum (NCS) (Gibco), 100 IU/ml penicillin,
100 mg/ml streptomycin (Sigma Chemical), and 2
mM/ml L-glutamin (Gibco) (19,23,24).
Firstly, stimulated and unstimulated MNC culture
supernatants were prepared for using PMNL cultures.
For this purpose, MNCs cultured in 24-well plates
(Nunc Brand Products) in the absence or presence
of phytohaemagglutinin-M (PHA-M, Gibco) at 37°C
in a humudified 5% CO2
atmosphere for 4 hours.
PHA-M was used at a final concentration of 15 μg/
ml in the cultures. At the end of 4 hours, the MNC
cultures were centrifuged at 400 g for 10 minutes at
room temperature. Then, culture supernatants from
stimulated and unstimulated samples were separately
pooled and aliquoted for PMNL cultures. The levels of
interferon (IFN)-γ in culture supernatants were measured by enzyme immunoassay (DIAsource, Nivelles,
Belgium).
In first two sets of experiments, PMNL cultures
were incubated with RPMI or E. faecalis culture supernatants at 37ºC in a 5% CO2
humidified atmosphere
for 4 hours. The volumes of RPMI or E. faecalis culture supernatants were equivalent to those of PMNL
cultures in these experiments. In third set of experiments, PHA-stimulated MNC culture supernatants
of donors were added PMNL cultures with E. faecalis
culture supernatants and the incubation was maintained for 4 hours at 37ºC in a 5% CO2
humidified
atmosphere. So, the effects of soluble factors derived
from active MNCs on the PMNL functions against E.
faecalis were tested. Culture supernatants and PMNL
cultures were used in equal volume in third set of
experiments. Optimal volumes determined in our
previous experiences were used in these experiments
(data not shown).
Functional analysis of neutrophils: The Transwell
(TW) system consisting of inserts (containing 3 mm
polycarbonate membrane) and 24-well plates were
used for migration experiments. After the inserts
were washed with RPMI into the wells of plates, they
were transferred to other wells. Interleukin-8 (IL-8)
(RD Systems, Minneapolis, MN, USA) was used as a
chemoattractant and put in the wells in 500 μl of
RPMI medium. The concentrations of IL-8 were adjusted according to the manufacturer’s instructions.
This chemoattractant was not put in control wells.
First, 500 μL of cell suspensions (1x10
6
PMNLs/ml)
obtained at sets of experiments were added into each
TW insert and placed into the 24-well plate, which
had already been filled with 500 μL of RPMI medium
or chemokine. Then, TW apparatus were incubated
in a 37°C humidified CO2
incubator for 2 hours. After
2 hours of incubation, the TW inserts were lifted, and
bases of the inserts were washed with RPMI medium.
Migrated cells in the wells were counted with a hemocytometer. At the end of this process, the viability
of the cells was more than 95% (21-24).
The evaluation of oxidative burst activity of PMNLs
was performed by flow cytometry Bursttest kit
(Orpegen Pharma, Heidelberg, Germany) used for
quantitative determination of neutrophil oxidative
burst. It contains unlabelled opsonized E. coli bacteria as particulate stimulus, phorbol 12-myristate
13-acetate (PMA) as high stimulus, and N-formylMetLeuPhe (fMLP) as low physiological stimulus, and
dihydrorhodamine (DHR) as a fluorogenic substrat.
PMNLs obtained at the end of the second set of experiments were incubated with the various stimuli at
37ºC, a sample without stimulus was used as negative
control. Upon stimulation, the PMNLs that produced
ROS were then analyzed as well as their mean fluorescence intensity (MFI) (21-24).
Statistical analysis: All statistical analyses were
performed by using SPSS (SPSS 10.0) package. The
Friedman test was used for multiple statistical comparisons. However, the Wilcoxon signed rank test
was used to compare two paired samples. P values less
than or equal to 0.05 were evaluated as statistically
significant.
Results
Detectable IFN levels were only observed in two
unstimulated MNC cultures (53 pg/ml and 104 pg/
ml, respectively), whereas all stimulated MNC cultures had detectable IFNγ levels (mean±SD: 621±329
pg/ml; minimum-maximum: 123-989 pg/ml).
Chemotactic activity was found significantly higher in PMNL cultures with E. faecalis culture supernatants than those of PMNL cultures without E. faecalis
and MNC culture supernatants (33±8% vs 26±9%,
p<0.05) (Figure 1a). PMNL cultures with E. faecalis
and stimulated MNC culture supernatants had also
higher chemotactic activity compared to PMNL cultures without E. faecalis and stimulated MNC culture
supernatants (40±5% vs 26±9%, p<0.05). There was
no statistically significant difference between PMNL
cultures with E. faecalis culture supernatants and
with E. faecalis and stimulated MNC culture supernatants with respect to chemotactic activity (33±8% vs
40±5%, p>0.05).
Oxidative burst activity was found significantly
higher in PMNL cultures with E. faecalis culture supernatants than those of PMNL cultures without E.
faecalis and stimulated MNC culture supernatants
(461±159 MFI vs 342±123 MFI, p<0.05) (Figure 1b). 124 • June 2010 • Gulhane Med J Oktay et al.
PMNL cultures with E. faecalis and stimulated MNC
culture supernatants had also higher oxidative burst
activity compared to PMNL cultures with E. faecalis culture supernatants and without E. faecalis and
stimulated MNC culture supernatants (665±109 MFI
vs 461±159 MFI and 665±109 MFI vs 342±123 MFI,
respectively, p<0.05).
Discussion
In the present study, we evaluated the direct effect
of E. faecalis on the functions of PMNLs, using PMNL
cultures. To assess the effect of E. faecalis on the priming of neutrophils, we used PMNLs incubated in an
environment that was rich in cytokines secreted by
stimulated MNCs. To attain such an environment,
MLCs were stimulated with PHA, which has been
known to induce the secretion of several cytokines,
including IFNγ and TNFα.
Our results showed that E. faecalis derived substances increased chemotactic and oxidative burst activities of PMNLs in the cultures with or without MNC
secretory products. These findings support the studies by Sannomiya et al. and Ember et al. who revealed
that some of the E. faecalis derived substances, sex
pheromones and their inhibitory peptides, were found to be chemotactic for human and rat neutrophils
and also to induce superoxide production and lysosomal enzyme secretion (22,25). These peptides are
one of the most cited virulence factors of E. faecalis
related to endodontic infection and the periradicular
inflammatory response (26). Aggregation substance,
surface adhesins, lipoteichoic acid, extracellular superoxide production, the lytic enzymes gelatinase
and hyaluronidase, and the toxin cytolysin are the
other important virulance factors of E. faecalis (26).
Superoxide anion derived from E. faecalis is also a
highly reactive oxygen radical involved in cell, and
tissue damage in inflammatory disease may react
with a precursor in plasma to generate a factor that is
chemotactic for neutrophils (27).
On the other hand, there are some reports in literature that seem contrary to our results. In the study of
Shon et al., they have found that sonicated extracts of
E. faecalis suppress PMNs recruiting activity by downregulating α4 integrin expression, and proposed this
microorganism may play a crucial role in persistent
apical periodontitis (23,27). In addition, Vanek et al.
have reported that E. faecalis aggregation substance
promotes opsonin independent binding to human
PMNLs via a complement receptor type 3 mediated
mechanism (28). The resistance of this microorganism that gives damage human PMNLs has been explained with this non-opsonic binding of E. faecalis to
PMNs (29). Furthermore, both extracellular superoxide production and phagosomal oxidant production
of PMNLs against E. faecalis were found to be higher
than those against the control strains lacking aggregation substance (29). Thus, oxidative burst activity
of PMNLs may be a possible contribution to tissue
damage in the endodontic infections with E. faecalis.
Figure 1. Chemotactic (a) and oxidative burst activity (b) of PMNLs. Bars in Mean±SD notation represent the data from unmixed PMNL cultures
(1) and PMNL cultures mixed with E. faecalis culture supernatants alone (2) or combined with stimulated MNC culture supernatants (3). p values
were indicated above the bars when there was a level of significance less than or equal to 0.05.
1 2 3
10
20
30
40
50
Chemotaxis(%)
p=0.027
p=0.027
(a)
250
500
750
Oxidatve burst (MFI)
p=0.028
p=0.028
p=0.046
(b)
1 2 3Volume 52 • Issue 2 Enterococcus faecalis and PMNL functions • 125
We also demonstrated that MNC secretory products increase the oxidative burst activity of PMNLs
induced by E. faecalis. In our previous study, we observed that migration ability and reactive oxygen species production of PMNLs were increased by MNCs in
mixed cultures (30). A plenty of cytokines have been
shown to act as priming agents for PMNs, including
IL-8 and TNFα (31). IFNγ also has been demonstrated
to enhance, or prime, increased reactive oxygen species production in combination with a secondary stimulus (32). In this context, Berton et al. were the first
to observe the priming effect of IFNγ on PMN oxidative burst (33). In their study, increased O2
consumption and O2
-
production of PMNLs against either fMLP,
concanavalin A, or lipopolysaccharide were observed
in PMNLs pretreated with IFNγ (33). However, it was
shown that the priming effect of IFNγ on PMNLs may
be specific to stimuli that act via membrane associated receptors. The other modulatory effect of IFNγ on
PMNLs is to stimulate the production of the cytotoxic agent nitric oxide by a variety of cells, including
macrophages and neutrophils, and to cause undesirable cell and tissue damage (34).
This in vitro study supports the previous studies on
the effects of E. faecalis on neutrophil functions. In
view of our findings, we suggest that a direct effect of
E. faecalis on neutrophil functions seems improbable.
MNC products such as IFNγ play an important role in
innate immune response by priming neutrophils that
lead to an enhanced antimicrobial activity. As a consequence of these results, increased PMNL functions
by E. faecalis and MNC products may have possible
contributions to tissue damage in the endodontic infections with E. faecalis. However, in the future, other
comparative in vivo and in vitro studies including patients and healthy individuals should be carried out
to explain different clinical courses of the endodontic
infections by E. faecalis.