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Proinflammatory and immunoregulatory mechanisms in periapical lesions

Article  in  Molecular Immunology · March 2009

DOI: 10.1016/j.molimm.2009.01.011 · Source: PubMed

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 Molecular Immunology 47 (2009) 101–113

Contents lists available at ScienceDirect

Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Review

Proinflammatory and immunoregulatory mechanisms in periapical lesions

Miodrag Čolić a,∗ , Dragan Gazivoda b , Dragana Vučević a , Saša Vasilijić a ,
Rebeka Rudolf c , Aleksandra Lukić d
a
Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11002 Belgrade, Serbia
b
Department for Oral Surgery, Clinic for Maxillofacial and Oral Surgery, Military Medical Academy, Belgrade, Serbia
c
University of Maribor, Faculty of Mechanical Engineering, Maribor, Slovenia
d
Department of Endodonotics, Faculty of Stomatology, University of Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical
Received 9 October 2008 lesions. However, little is known about how their functions are balanced and controlled at different
Received in revised form 24 December 2008 phases of lesion development. The aim of this study was to examine the relationship between the pro-
Accepted 8 January 2009
duction of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear
Available online 15 February 2009
cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of
the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high
Keywords:
production of IL-17, positive correlation between IL-17 and IFN-␥, but not between IL-17 and IL-23 pro-
Periapical lesion
Cytokine
duction. Most IL-17+ cells coexpressed IFN-␥. Asymptomatic lesions were phenotypically heterogeneous.
Cell culture The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-␥
Phenotypization which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most
Dendritic cell B-type lesions higher levels of IL-5 and TGF-␤ were observed, as well as positive correlation between the
production of TGF-␤ and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2
and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the produc-
tion of IFN-␥. IL-10 and TGF-␤ inhibited the production of both Th1 and Th17 cytokines. Dendritic cells
(DCs) from periapical lesions, composed of immature (CD83− ), and mature (CD83+ ) myeloid type DCs
and plasmacytoid (BDCA2+ ) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and
TNF-␣ than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas
the secretion of IFN-␥ was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1)
Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2
and immunoregulatory cytokines are more significant for advanced types of lesions with the predomi-
nance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating
inflammation.
© 2009 Elsevier Ltd. All rights reserved.

1. Introduction Periapical lesions are characterized histologically by fibrous and

Periapical lesions are induced by the chronic infection of dental
pulp. Microbial antigens stimulate both non-specific and specific
immune response in periapical tissue. As a consequence of these
processes and the inability of host defense mechanisms to erad-
icate infection, chronic periapical lesions are formed, with the
aim of restricting microbial invasion. In spite of numerous experi-
mental and clinical studies, specific etiologically inducing factors,
participating cells and growth mediators associated with the devel-
opment, maintenance and resolution of periapical lesions are not
fully understood (Marton and Kiss, 2000; Nair, 2004).
∗ Corresponding author. Tel.: +381 11 2662 722; fax: +381 11 2662 722.
E-mail address: vmaimi@eunet.rs (M. Čolić).
granulation tissue, proliferating epithelium or cyst infiltrated by
different inflammatory cells (Gao et al., 1988; Kopp and Schwarting,
1989; Stashenko, 1990; Marton and Kiss, 1993; Liapatas et al., 2003;
de Oliveira Rodini et al., 2004; Lukic et al., 2008). Among infiltrat-
ing leukocytes, neutrophil granulocytes are the first line of defense
which stimulate the migration of monocytes and lymphocytes.
Mononuclear cell infiltrates, composed of antigen-presenting cells
(APC), T and B lymphocytes and their effectors are characteristic of
chronic periapical processes (Marton and Kiss, 2000).
APC, especially dendritic cells (DCs) are of crucial importance
in the polarization of T helper (Th) immune responses towards
Th1, Th2, Th17 or T regulatory cells (T regs) (de Jong et al.,
2005; McGeachy and Cua, 2008). It is believed that Th1 immune
responses, mediated by interferon-␥ (IFN-␥), together with other
proinflammatory cytokines such as interleukin-1 (IL-1), IL-6 and

0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2009.01.011
102 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113
tumor necrosis factor-␣ (TNF-␣) are involved in the progression
of lesions and bone destruction. In contrast, immunosuppressive
mechanisms mediated by transforming growth factor-␤ (TGF-␤)
and Th2 cytokines (IL-4, IL-5, IL-5, IL-10) are responsible for healing
processes and the restriction of the inflammatory/immune mech-
anisms (Akamine et al., 1994; Kawashima and Stashenko, 1999;
Lukic, 2000). IL-17, by stimulating the production of IL-8, may play
a role in exacerbating inflammation in periapical lesions (Colic et
al., 2007). The role of T regs in these processes is unknown.
Experiments on IFN-␥ and IL-4 knock-out mice contradict the
proposed concept of periapical lesion development (De Rossi et al.,
2008). Therefore, new studies on cytokine network regulation at
different stages of lesion formation are necessary, using various in
vitro and in vivo animal and human models.
The aim of our study was to examine the proinflammatory and
immunoregulatory mechanisms in human periapical lesions, based
on the production of cytokines by infiltrating mononuclear cells
in vitro and to compare the cytokine production with the cellular
composition and clinical presentations of the lesions.
2. Materials and methods
2.1. Periapical lesions
Periapical lesions (n = 96) were collected from patients at the
Department for Oral Surgery, Clinic for Maxillofacial and Oral
Surgery, Military Medical Academy, Belgrade and from the Depart-
ment of Endodontics, Faculty of Stomatology, University of Belgrade
at the time of teeth extraction or apical surgery. The average age of
the patients was 34 yr (range: 18–62 yr). All patients were with-
out systemic diseases and had radiographic evidence of periapical
lesions, including periapical alveolar bone loss. The patients had
not been treated with antibiotics for 1 month before endodontic
surgery. Apical surgery involved an apicoectomy followed by curet-
tage of the lesions. When dental extraction was performed, lesions
were excised by the curettage of periodontal apical tissue. In cases
where the lesions were firmly attached to dental radices, the tissue
was removed from the extracted teeth by a scalpel. Specimens were
collected between November 2005 and April 2008. No distinctions
between specimens were made regarding the etiology or the tooth
type. For each specimen, valid informed consent was obtained from
the patient. The specimens were divided into symptomatic (n = 50)
and asymptomatic (n = 46), according to the presence or absence of
main clinical features, such as pain, moderate swelling, and other
symptoms associated with acute infection.
The tissue was immediately placed in a medium con-
sisting of RPMI-1640 (Sigma, Munich, Germany) and antibi-
otics/antimycotics, and transported to the laboratory. Some lesions,
which were processed for immunohistology, were frozen in the
tissue preserving medium and kept at −70 ◦ C.
2.2. Preparation of inflammatory cells
Inflammatory cells were isolated from the periapical lesions
using a procedure previously optimized in our laboratory (Lukic
et al., 2006). Briefly, periapical tissue was placed in a Petri dish
containing 1 ml RPMI-1640 medium and cut into 2–3 mm diame-
ter pieces using a scalpel. The tissue was then digested for 15 min
with 0.05% collagenase type IV (Sigma) and 0.02% DNAse (Sigma)
in 10 ml RPMI-1640 medium at 37 ◦ C. After that, soft tissue was
pressed through a stainless-steel mesh using a syringe plunger,
filtered through nylon gauze to remove tissue fragments, and resus-
pended in 10 ml fresh RPMI-1640 medium containing 1 mm EDTA.
The cells were washed twice by centrifugation in a RPMI medium
containing 0.5 mm EDTA at room temperature (400 g for 7 min), and
counted. Cell viability, as determined by Trypan Blue dye exclusion,
was usually between 90 and 95%. Using this method, <5% of the
non-stromal cells was retained within the rest of the tissue (unpub-
lished data). A cell suspension of total inflammatory cells (4 ml)
was layered over a 3 ml Lymphoprep gradient (Nycomed, Oslo, Nor-
way) and centrifuged at 800 × g for 20 min. Mononuclear cells were
collected from the interphase zone, washed twice in a RPMI-1640
medium containing 2% heat-inactivated fetal calf serum (FCS) (ICN,
Cost Mesa, CA, USA), and counted. Cell viability was usually >97%.
Cytospins were prepared from each sample of total inflammatory
cells and periapical lesion mononuclear cells (PL-MNC), using a
cytocentrifuge (MPW-350, Poland) on poly-l-lysine-coated glass
slides. The cytospins were stained by the May–Grünwald–Giemsa
staining method. Cytospins from the PL-MNC samples were also
processed for immunocytochemistry.
2.3. Cultures of PL-MNC
PL-MNC were cultivated in 96-wells, with round-bottomed
plates (ICN, Costa Mesa, CA) (1 × 105 cells/well, 200 ␮l) in a RPMI-
1640 medium containing 10% FCS. Phorbol myristate acetate (PMA)
(20 ng/ml) (Sigma) and Ca2+ ionophore (A 23187, 1 ␮M) (Sigma)
were used for stimulation. After 24 h, the cell supernatants were
collected, centrifuged and frozen at −70 ◦ C until the levels of
cytokines were determined. The viability of cells in the cultures
after 24 h was 80–90%.
In the experiments where the modulatory effects of cytokines
were examined, PL-MNC were stimulated with recombinant IFN-
␥ (10 ng/ml), IL-4 (5 ng/ml), IL-17 (10 ng/ml), IL-12 (5 ng/ml), IL-23
(5 ng/ml), IL-10 (1 ng/ml) or TGF-␤ (1 ng/ml) for 24 h and the super-
natants then collected and frozen. All cytokines were purchased
from R&D, Minneapolis, MN, USA.
2.4. Monoclonal antibodies
For immunostaining, anti-CD3, -CD4, -CD8, -CD14, -CD19, -
CD38 and -CD123 unconjugated monoclonal antibodies (mAbs)
were obtained from Serotec, Oxford, UK. Anti-c kit, -mast cell
tryptase, -CD1a mAbs, rabbit anti-mouse unconjugated Ig, and alka-
line phosphatase anti-alkaline phosphatase (APAAP) complex were
purchased from DAKO, Copenhagen, Denmark. BDCA2 and BDCA4
mAbs conjugated with PE were purchased from Myltenyi Biotech,
Gladbach, Germany. DC exclusion cocktail mAbs conjugated with
RPE-Cy5, anti-HLA-DR conjugated with PE or FITC, -CD83-FITC, -
CD1a-FITC and -CD3 coupled with Alexa Fluor 546 mAbs were from
Serotec.
2.5. Immunohistochemistry
Cryostat sections of the tissue (6 ␮m thickness) were fixed in
cold acetone. The cytospins were fixed with 2% pararosaniline for
2 min at room temperature. After fixation the slides were washed
with phosphate-buffered saline for 10 min and incubated with 20%
rabbit serum diluted in Tris-buffered saline (TBS), pH 7.6 and 0.05%
Tween-20 for 20 min. After washing in TBS/0.5% bovine serum albu-
min (BSA), /0.05% Tween-20, slides were incubated with the mAbs
for 60 min at room temperature in a humidified slide chamber.
The control slides were incubated with TBS. The cytospins were
then incubated with rabbit anti-mouse Ig containing 10% human
AB serum, previously inactivated at 56 ◦ C for 45 min, followed by
APAAP solution. After each incubation step, the slides were washed
with TBS/BSA/Tween-20 solution for 10 min. The AP reaction was
visualized using Fast Red as substrate. Finally, the slides were
lightly counterstained with hematoxylin, mounted in Keiser gel,
and examined by light microscopy. At least 500 cells were counted
in each cytospine.
 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113
The percentages of positive cells were determined on the basis
of total counted cells. A similar light microscopic analysis was per-
formed for the morphological identification of total inflammatory
cells. Based on the proportion of CD3+ (T cells) and CD19+ /CD38+
(B cells/plasma cells) and their ratio, the lesions were divided into
T-type (n = 54) or B-type (n = 42).
For identification of T cell/DC association, cryostat sections
were stained with anti-CD3-Alexa Fluor 546 and -CD83-FITC mAbs.
Appropriate negative controls were used, omitting one or both
mAbs. After washing, the slides were mounted and analyzed using
a confocal laser microscope (LSM 510/Axiovert 200 M, Zeiss, Jena,
Germany).
2.6. Flow cytometry
Flow cytometry was used for characterization of the DCs
and intracellular detection of cytokines. PL-MNC were resus-
pended in PBS containing 2% FCS and 0.1% sodium azide and
aliquoted into tubes (1–2 × 105 cells/tube in 100 ␮l). For identifi-
cation of DCs, the cells were stained with DC exclusion cocktail
(CD3/CD14/CD16/CD19/CD34)-RPE-Cy5 mAbs and anti-HLA-DR-
PE. For identification of DC subsets, the cells were double stained
with anti-CD86, -CD83, -CD1a or -CD123-FITC conjugated mAbs
with anti-HLA-DR-PE mAb or BDCA2- and BDCA4-PE mAbs,
together with anti-HLA-DR-FITC mAb. Negative controls were irrel-
evant isotype mAbs conjugated with RPE-Cy5, FITC or PE (Serotec).
After washing twice in PBS/sodium azide, the cells were fixed
with 1% paraformaldehyde and analyzed on an EPICS XL-MCL flow
cytometer (Coulter, Krefeld, Germany), using System IITM Software
(Coulter).
Single HLA-DR+ cells, negative for DC exclusion markers, were
considered as DCs. DC subsets were identified as double-positive
cells expressing HLA-DR and a particular DC lineage marker. Their
relative values were calculated using HLA-DR+ DCs (determined
in the DC exclusion cocktail/HLA-DR double labeling experiment)
used as 100%.
In order to identify cytokine-producing cells, PL-MNC were
stained with anti-IFN-␥-PE mAb and anti-IL-17-FITC mAb (both
from R&D) after cell permeabilization, by using the Fix & Perm
cell permeabilization kit (Caltag Laboratories, Vienna, Austria), and
following the instructions of the manufacturer. The control sam-
ples were stained with irrelevant fluorochrome conjugated mAbs
(Serotec). Single or double-positive cells were identified and their
relative values determined.
2.7. DC cultures
The PL-MNC were cultivated in 24-well plates (5 × 105 cells/
ml/well) for 4 h. The non-adherent cells were then collected. The
DCs were isolated from the non-adherent fraction by centrifugation
of the cells over different Optiprep gradients (Nycomed Pharma,
Oslo, Norway), according to the published method (Brocker et al.,
1997), and modified in our laboratory (Vasilijic et al., 2005). The
purity of the cells was higher than 75%, as judged by morphol-
ogy and strong staining with anti-HLA-DR mAb. The number of
recovered cells was between 0.5 × 103 and 1.2 × 104 /lesion. Mono-
cyte derived DCs (Mo-DCs) were generated by cultivating adherent
monocytes from the blood of patients with periapical lesions for
6 days using GM-CSF and IL-4 as previously described (Colic et al.,
2003). To induce maturation the Mo-DCs were stimulated with LPS
from E. coli (Sigma) (100 ng/ml) for 2 days. DCs (1 × 105 /ml) were
cultivated for 24 h as described for PL-MNC. Cell supernatants were
collected for analyzing the cytokines. The accessory functions of
both PL-DCs and Mo-DCs were tested in a mixed leukocyte reac-
tion (MLR) using allogeneic T cells, purified by MACS technology
(Miltenyi Biotech) as responders. DCs (0.25 × 103 ) were cultivated
103
with T cells (5 × 104 ) (ratio 1:200) in 96-well U bottom plates for 5
days. During the last 18 h of culture, the cells were pulsed with 1 ␮Ci
[3 H] thymidine (specific activity 5 Ci/mM, Amersham, Amersham
Books, UK). After cell harvesting on glass fiber filters, radioactivity
was determined by the standard liquid scintillation technique. The
results were expressed as mean count per minute (cpm) ± SD of
triplicates.
2.8. Cytokine assays
The levels of cytokines (IL-2, IL-4, IL-5, IL-10, IL-12, IFN-␥ and
TNF-␣) in PL-MNC or DC cultures were detected using a fluores-
cent bead immunoassay (Bender Med Systems, Vienna, Austria),
and flow cytometry, as described by the manufacturer. Concen-
trations of cytokines in the investigated samples were obtained
by comparing the mean fluorescence intensities of samples and
known concentrations of cytokines from standard, using the com-
mercial flow cytomix software (FF Software, BMS-FFS, Bender Med
Systems).
The concentrations of IL-12 p40, IL-23, TGF-␤ and IL-17 were
detected by using specific ELISA kits (R&D) following the instruc-
tions of the manufacturer.
2.9. Statistical analysis
Statistical analysis was performed using the Student’s t-test,
one-way analysis of variance (ANOVA) and the Spearmann correla-
tion test. p-Values of <0.05 were considered statistically significant.
3. Results and discussion
3.1. Composition of infiltrating cells in periapical lesions
To date extensive studies, mainly based on in situ immunos-
taining (Stern et al., 1982; Barkhordar and Desouza, 1988; Lukic
et al., 1990; Piattelli et al., 1991; Matsuo et al., 1992; Liapatas et al.,
2003) or flow cytometry (Sol et al., 1998; Vernal et al., 2006), have
confirmed that the cellular composition of periapical lesions varies
significantly, depending on the stage of lesion development and its
progression, histological characteristics of the lesions, the presence
or absence of clinical symptoms, and the methods used for detec-
tion and quantification of inflammatory cells. The main finding is
that lymphocytes and plasma cells are the predominant popula-
tions of infiltrating cells (Barkhordar and Desouza, 1988; Gao et al.,
1988; Lukic et al., 1990; Piattelli et al., 1991).
Our results which are based on cytological analysis of infiltrating
cells isolated from a larger sample of periapical lesions (n = 96), also
confirmed these observations and showed that the predominant
cells were lymphocytes and plasma cells, followed by granulocytes
and mononuclear phagocytes (Mo-like cells MØ, and DC-like cells).
Mast cells represented 4.2 ± 0.2% of all cells, whereas less than 2.0%
cells remained unidentified morphologically (Fig. 1A).
A higher proportion of granulocytes (more than 98% were
neutrophils), and the subsequent lower proportion of lympho-
cytes/plasma cells, observed in the clinically symptomatic lesions
are in accordance with the findings that chronic periapical lesions
are predominantly composed of mononuclear cells, whereas gran-
ulocytes are characteristics of acute periodontitis (exudative phase)
and exacerbation of chronic inflammation, as a result of new infec-
tion from the root canal (Marton and Kiss, 2000; Nair, 2004).
Neutrophils provide the first line of defense against bacte-
rial invasion from the infected root canal. Due to their effective
phagocytosis and killing function, the majority of microorgan-
isms is destroyed and eliminated, preventing them from spreading
throughout the lesion (Walton and Ardjmand, 1992; Kaufmann,
104 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113

Fig. 1. Morphological and phenotypic characterization of periapical lesions. (A) Composition of infiltrating leukocytes; (B) frequency of CD3+ (T cells) and CD19/38+ (B
cells/plasma cells); tissue distribution and staining pattern on cytospins (APAAP method); (C) phenotypic characteristics of PL-MNC. The values are given as mean ± SD; n = 87
(total lesions); n = 43 (symptomatic lesions); n = 45 (asymptomatic lesions); n = 42 (T-type lesions); n = 39 (B-type lesions); * p < 0.05; *** p < 0.005 compared to corresponding
control groups (ANOVA).

1993). Although neutrophils are also present in the granulomatous
zone of the lesion, their frequency was less abundant compared to
that in the exudative zone, and also with other infiltrating cells
(Kopp et al., 1987; Piattelli et al., 1991; Marton and Kiss, 1993).
Such histological observations correlated with the much lower fre-
quency of neutrophils observed in our asymptomatic lesions, which
are considered as chronic inflammatory processes with minimal
exudation.
Except for their protective role, neutrophils are important in the
progression of periodontitis, at both the marginal and periradicular
sites (Van Dyke and Vaikuntam, 1994). The cells cause severe dam-
age to the host tissue due to the secretion of various proteolytic
enzymes. Together with MØ, the cytokines they produce, namely
IL-1, IL-6, TNF-␣ and receptor activator of nuclear factor kB-ligand
(RANK-L), these cells are considered to be important activators of
osteoclasts, leading to the subsequent destruction of bone and den-
tal hard structures (Ataoglu et al., 2002; Radics et al., 2003; Vernal
et al., 2006). Although we did not find any statistically significant
difference in the proportion of mononuclear phagocytes between
the symptomatic and asymptomatic lesions (Fig. 1A), a statistically
significant correlation was observed between the frequencies of
granulocytes and Mo/MØ/DC (r = 0.405; p < 0.01; n = 36) in symp-
tomatic lesions. These results suggest the importance of both types
of phagocytic cells for the exacerbation of inflammation within
periapical lesions.
Different T/B cell ratios have been observed in periapical lesions
(Pulver et al., 1978; Bergenholtz et al., 1983; Torabinejad and
Kettering, 1985; Piattelli et al., 1991; Marton and Kiss, 1993; Sol
et al., 1998). Tani et al. (1992) reported that T/B cell ratio was sig-
nificantly higher in radicular cysts than in radicular granylomas,
whereas MØ were more numerous in granulomas. Other authors
suggest that the predominance of T cells is characteristic of the
initiation and development of lesions, whereas humoral immune
response mediated by B cells mainly relates to the healing pro-
cess (Akamine et al., 1994). We found that individual periapical
lesions significantly differed in the proportion of T (CD3+ ) cells and
B cells/plasma cells (CD19/38+ ). Accordingly, we divided the lesions
into T- and B-types, depending on whether CD3+ or CD19/38+ cells
predominated (Fig. 1B). Immunohistological analysis demonstrated
that both T and B lymphocyte subsets were distributed diffusely and
 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113
focally, and they formed both small and large aggregates (Fig. 1B).
The proportion of T and B cells/plasma cells did not differ between
symptomatic and asymptomatic lesions suggesting that, in both
types of lesion the cellular and humoral immune responses are
generally similar.
As expected, T-type lesions contained a higher proportion of
CD4+ cells than B-cell types, but no differences in the proportion
of CD8+ T cells were noticed (Fig. 1C). Conflicting results concern-
ing the predominant T-cell subset in human periapical lesions have
been reported (Kontiainen et al., 1986; Kopp et al., 1987; Barkhordar
and Desouza, 1988; Marton and Kiss, 1993; Sol et al., 1998). Sol et
al. (1998) identified the highest CD4/CD8 ratio in cystic lesions by
flow cytometry, which also contained a higher percentage of B cells
than granulomas. Immunohistochemical studies on experimental
periapical lesions in rats confirmed that CD4+ T cells predominate
in the early phase of lesion development, whereas in the chronic
phase CD8+ T cells slightly outnumbered CD4+ cells (Stashenko and
Yu, 1989; Kawashima et al., 1996). Some studies in humans have
shown that the development of granulomatous infiltration in peri-
apical tissue is associated with the influx of CD4+ T cells (Geratz
et al., 1995; Mielke et al., 1997). However, our study on almost
100 periapical lesions, did not suggest any relationship between
the frequency of CD4+ , CD8+ or CD19/38+ cells, and the clinical
presentation of the lesions.
Although we did not discover any significant difference in the
frequency of MØ (CD14+ T cells) between symptomatic and asymp-
tomatic or T-type versus B-type lesions (Fig. 1C), a significant
correlation was observed between the frequency of CD4+ cells and
CD14+ cells (r = 0.329; p < 0.005; n = 72) within the whole group of
lesions, suggesting a close functional association of these cells at all
stages of periapical lesion development.
Mast cells were identified, by morphological criteria and by
staining with antibodies to c-kit and mast cell tryptase. Morpholog-
ical and quantitative analyses showed that these cells represented
minor population of infiltrating leukocytes (Fig. 1C). Enzyme cyto-
chemistry and immunocytochemistry methods revealed that mast
cells were located predominantly in clusters between the central
granulation tissue and the peripheral fibrous capsule, as well as
perivascular areas in close contact with lymphocytes (Marton et
al., 1990; Piattelli et al., 1991). de Oliveira Rodini et al. (2004)
observed more numerous tryptase-positive mast cells in the regions
of active inflammation. Although, the functions of mast cells in peri-
apical lesions have never been fully elucidated, the low frequency
of IgE-producing B cells in periapical lesions suggests that these
cells are not of primary importance for hypersensitivity-type reac-
tions (Marton and Kiss, 2000). We did not find any difference in the
proportion of mast cells between symptomatic and asymptomatic
lesions. However, a higher proportion of these cells was detected
in B-type lesions, compared to the T-type lesions. In contrast, bet-
ter correlation between the frequency of CD4+ cells and mast cells
in T-type lesions (r = 0.421; p < 0.005; n = 40), than between mast
cells and CD19/38+ cells in B-type lesions (r = 0.279; p < 0.05; n = 37),
suggests the predominant involvement of mast cells in the effector
immune mechanisms mediated by T-helper cells.
3.2. Production of Th cytokines by periapical lesion mononuclear
cells
The main aim of this study focused on the production of Th
cytokines in periapical lesions. According to the current concept
of Th development, at least three different Th subsets exist: Th1,
Th2 and Th17 (McGeachy and Cua, 2008). Therefore, we mea-
sured production of IL-2 and IFN-␥ (Th1 cytokines) IL-4 and IL-5
(Th2 cytokines) and IL-17A (Th17 cytokine) by PL-MNC cultivated
in vitro in the presence of PMA + Ca2+ ionophore. The combina-
tion of PMA + Ca2+ ionophore is a commonly used procedure for
105
studying the functional potential of T cells to secrete cytokines
(Collins, 2000). In our previous paper (Colic et al., 2006) we demon-
strated that additional stimulation of PL-MNC with PMA + Ca2+
ionophore significantly enhanced the basal production of cytokines
in vitro.
IL-2, IFN-␥ and IL-17 were detected in all cultures, whereas IL-4
and IL-5 were identified in 54% and 78% of culture samples, respec-
tively (Fig. 2A). The levels of IL-2, IFN-␥ and IL-4 did not significantly
differ between symptomatic and asymptomatic lesions. However,
while the level of IL-5 was higher in asymptomatic lesions, a signif-
icantly higher level of IL-17 was observed in symptomatic lesions.
The concentrations of IFN-␥ and IL-17 were higher in T-type lesions,
whereas the concentration of IL-5 was higher in B-type lesions
(Fig. 2A). These results were not attributable to differences in the
number of T cells in T-type lesions, because similar results were
obtained when the levels of these cytokines were standardized to
the same number of T cells (data not shown).
We have demonstrated that the levels of IFN-␥ correlated
positively (r = 0.248; p < 0.05; n = 46), whereas the levels of IL-4 cor-
related negatively (r = −0.257; p < 0.05; n = 43) with the proportion
of Mo/MØ/DC. In addition, the levels of IL-4 and IL-5 correlated
positively with the number of mast cells in the whole group of
periapical lesions (r = 0.418; p < 0.01; n = 37; and r = 0.433; p < 0.005;
n = 39, respectively). MØ are the first line of local defense in response
to bacterial infection of the root canal. They are also antigen-
presenting cells for effector T cells (Kopp and Schwarting, 1989;
Ma et al., 2003). The positive correlation between IFN-␥ and MØ
is an expected finding, since IFN-␥ is the main activator of MØ,
which subsequently produce proinflammatory cytokines and other
mediators (Ma et al., 2003; Watford et al., 2003). However, IFN-␥,
as well as IFN-␥ inducing cytokines (IL-12 and IL-18), have been
shown to exert the opposite effect on bone resorption, by inhibit-
ing osteoclast formation (Takayanagi et al., 2000; Horwood et al.,
2001). Therefore, it remains better to define these dual actions of
IFN-␥ in vivo in periapical lesions, having in mind recent results on
knock-out mice which showed that endogenous IFN-␥ had no sig-
nificant effect on the pathogenesis of bone resorption in periapical
lesions (Sasaki et al., 2004). In line with these results, new data have
emerged from studies on IFN-␥ and IL-4 knock-out mice, showing
that IFN-␥ could be an endogenous suppressor of periapical lesion
development, whereas IL-4 appears to have an insignificant effect
(De Rossi et al., 2008).
The negative correlation between IL-4 and MØ could be
explained by the inhibitory effect of IL-4 on MØ functions.
Yamamoto et al. (1997) showed that gingival MØ incubated with
recombinant IL-4 rapidly died in culture by apoptosis. On the other
hand, IL-4 and GM-CSF are crucial cytokines for inducing the differ-
entiation of monocytes to DC, both in vitro and in vivo (Banchereau
et al., 2000; Cutler and Jotwani, 2004).
Th2 cells are the major source of IL-4, although mast cells and
some lymphocytes belonging to the innate immune system may
also produce this cytokine. IL-4 production by mast cells and other
cells resulting from activation of non-specific immune system may
be important in the differentiation of Th2 cells (Abbas et al., 1996;
Mekori and Metcalfe, 1999). The result of our correlation analysis
between the percentage of mast cells and the levels of IL-4 could
be explained by these findings. IL-4 stimulates humoral immune
response by inducing production of IgG4 and IgE and also inhibits
Th1 immune response. In our previous work (Colic et al., 2006) we
showed a higher proportion of IgG4+ cells in periapical lesions in
which a significant level of IL-4 was produced.
Although we detected a significant Th2 immune response in
less than 20% of periapical lesions (predominantly B-type lesions),
a clear negative correlation between the levels of IL-5 and IFN-␥
was observed only in B-type lesions (Fig. 2B). Such observations
are in line with the hypothesis that the predominance of humoral
106 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113

Fig. 2. Th cytokines in periapical lesions. (A) Production of Th1, Th2 and Th17 cytokines by PL-MNC in vitro. Values are given as mean ± SD for n = 49 (total lesions); n = 25
(symptomatic lesions); n = 23 (asymptomatic lesions); n = 24 (T-type lesions); n = 22 (B-type lesions). * p < 0.05; ** p < 0.01; *** p < 0.005 compared to corresponding control
groups (Student’s t-test). (B) A representative histogram of double immunofluorescence staining of PL-MNC with anti-IFN-␥ and anti-IL-17 mAbs (a); Correlation between
the production of IFN-␥ and IL-17 (n = 18) in symptomatic lesion (b); and between IFN-␥ and IL-5 (n = 13) in B-type lesions (c). Correlation coefficients and corresponding p
values are given (Spearmann correlation test). (C) Effect of IFN-␥, IL-17 and IL-4 on Th cytokine production by PL-MNC. Cells were stimulated with recombinant cytokines as
described in Section 2. Values are given as mean ± SD from 3 different cultures (one symptomatic, T-type; one asymptomatic T-type and one asymptomatic B-type lesion).
*
p < 0.05 compared to corresponding controls (Student’s t-test).

immune response is characteristic of an advanced stage of lesion
development and their healing (Akamine et al., 1994; Kawashima
and Stashenko, 1999).
A higher level of IL-17 in symptomatic lesions and its correla-
tion with the proportion of neutrophils (r = 0.520; p < 0.01; n = 23)
suggests that this proinflammatory cytokine is important for the
exacerbation of inflammation. It is generally accepted that IL-17
primarily acts on stromal endothelial and epithelial cells, as well
as on a subset of monocytes, to induce the secretion of proinflam-
matory mediators. These include IL-8, CXC ligand 1, TNF-␣, IL-1,
IL-6, and GM-CSF (Fossiez et al., 1996; Jovanovic et al., 1998), which
promote rapid neutrophil recruitment to the site of the infection.
Th17 cells also produce other proinflammatory cytokines such as
IL-17 A/F heterodimer, TNF-␣, IL-22 and IL-26, all of which stim-
ulate innate immunity and promote inflammation (Tesmer et al.,
2008).
 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113 107

Fig. 3. The role of IL-12 family cytokines in periapical lesions. (A) Production of IL-12 p70, IL-12 p40 and IL-23 by PL-MNC in vitro. Results are given as mean ± SD for n = 19
(total lesions); n = 10 (symptomatic lesions); n = 8 (asymptomatic lesions); n = 9 (T-type lesions); n = 9 (B-type lesions); (B) correlation between the levels of IL-12 p40/IL-12
p70 (n = 16), IL-23/IL-12 p70 (n = 16) and IL-12 p70/IFN-␥ (n = 15) in culture supernatants of total PL-MNC. Coefficient of correlations and corresponding p values are given
(Spearmann correlation test). (C) The effect of IL-12 or IL-23 on IL-17 and IFN-␥ production by PL-MNC. Values are given as mean cytokine levels ± SD (n = 3); * p < 0.05;
***
p < 0.005 compared to corresponding controls (Student’s t-test).

We previously demonstrated (Colic et al., 2007) that the pro-
duction of IL-17 was significantly higher in PL-MNC cultures than in
PB-MNC cultures. These findings are in accordance with the fact that
effector Th17+ cells are predominantly localized in inflamed tissue
(Weaver et al., 2006). In addition, the levels of IL-17 were signifi-
cantly higher in T-type PL-MNC cultures than in B-type PL-MNC, and
their concentrations in T-type lesions correlated with the propor-
tion of CD3+ and CD4+ T cells (r = 0.447; p < 0.05; n = 21 and r = 0.550;
p < 0.01; n = 21, respectively). These results are in accordance with
the knowledge that a subset of CD4+ T cells is a main producer
of IL-17. However, what remains to be tested is the role of IL-17
in asymptomatic lesions, having in mind that in a subset of asymp-
tomatic lesions with a low number of neutrophils, the production of
IL-17 was significantly higher than its median concentration (data
not shown).
The relationship between the production of IFN-␥ and IL-17, has
not been studied yet in periapical lesions, as we have shown that
the levels of IFN-␥ and IL-17 in cultures of symptomatic lesions,
but not in other types of lesions, correlated positively (r = 0.556;
p < 0.01; n = 18) (Fig. 2B), suggesting that both cytokines are impor-
tant for the exacerbation of inflammation within periapical lesions.
Further, the relationship between IFN-␥ and IL-17 at the level of
108 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113
individual cells was studied by analysing intracellular cytokine pro-
duction. Unexpectedly, we found that most Th17 cells co-expressed
IFN-␥ (Fig. 2B), but such results are generally in agreement with
already published data that in humans, as many as half of all IL-17+
cells are also IFN-␥+ (Acosta-Rodriguez et al., 2007; Wilson et al.,
2007). It is as yet unclear if these cells represent a stable phenotype
or a transitional phase, undergoing a switch from Th17 to Th1 or
vice versa. Although there are no data on the specific role of these
double-positive cells, it can be hypothesized that both cytokines, as
important mediators of inflammation, contribute to pathogenesis
of periapical lesions.
Based on our previous results we wanted to examine how the
exogenous addition of a particular Th subset cytokine modulates
the production of other Th subsets’ cytokines. As shown in Fig. 2C,
IFN-␥ inhibited the production of both IL-17 and IL-5 by PL-MNC,
IL-4 inhibited the production of IFN-␥ and IL-17, whereas IL-17 aug-
mented the production of IFN-␥, but inhibited the production of
IL-5. The mutual antagonism between Th1 and Th2 cytokines is
a well-known phenomenon (de Jong et al., 2005). In addition, it
has been shown that IL-12, IFN-␥ and IL-4 can inhibit Th17 dif-
ferentiation in both humans and mice (Harrington et al., 2005;
Acosta-Rodriguez et al., 2007; Amadi-Obi et al., 2007; Wilson et al.,
2007). IL-17 negatively regulates Th1 cell differentiation in the pres-
ence of exogenous IL-12 in vitro (Nakae et al., 2007). Our findings
on the stimulatory effect of IL-17 on IFN-␥ production contradict
already published results. One explanation for this difference might
be that the response of PL-MNC is different from that of periph-
eral blood T cells due to difference in the proportion of naive and
memory T cells. Our unpublished results show that, in contrast to
peripheral blood, CD4+ CD45RO+ (memory) T cells in periapical
lesions predominate over CD4+ CD45RA+ (naive) T cells.
3.3. Production of IL-12 family cytokines in periapical lesions
Cytokines of IL-12 family (IL-12p70 composed of p35 and p40
subunits, IL-23 composed of p19 and p40 and IL-27 composed of
Epstein-Barr virus-induced molecule 3 and p28), produced pre-
dominantly by DCs and activated MØ, are a significant link between
innate and adaptive immunity. Except for pairings with IL-12 or IL-
23, p40 can be secreted as monomer or dimer. IL-12 is a key cytokine
driving the development of Th1 cells, whereas IL-23 is important
for the final differentiation of Th17 cells (Kastelein et al., 2007).
As a result of the complex nature of Th1, Th2 and Th17 cytokine
profiles in chronic inflammatory diseases, we examined the pro-
duction of IL-12p70, IL-12p40 and IL-23 in culture supernatants of
PL-MNC. There is no report of any such study in the literature. IL-
12p70 was detected in 84% samples, IL-23 in 96% samples, whereas
IL-12p40 was detected in all cultures. As reported in Fig. 3A the
level of IL-12p40 was highest, followed by the production of IL-23
and IL-12p70. There were positive correlations between their con-
centrations (Fig. 3B). However, no significant differences in their
production were observed in asymptomatic versus symptomatic
and T-type versus B-type groups of lesions. This finding was sur-
prising, having in mind our results on the production of IFN-␥ and
IL-17. Therefore, we examined the correlation between the levels
of IFN-␥ and IL-17 with their inducers, IL-12p70 and IL-23, respec-
tively. As shown in Fig. 3B, we observed a strong positive correlation
between the production of IFN-␥ and IL-12p70 but not between
IL-23 and IL-17 (r = 0.317; p > 0.05; n = 19).
The absence of correlation between IL-23 and IL-17 is an unex-
pected phenomenon because, according to the proposed model
of Th17 differentiation, Th17+ memory cells in chronic inflamed
tissue express IL-23R and respond to IL-23 (Tesmer et al., 2008).
When explaining these results it should take into consideration
that IL-23 is not the only cytokine responsible for the develop-
ment or activation of Th17 cells. Numerous studies have shown
that the development of Th17 cells depends on IL-1, IL-6, TGF-␤
and IL-23 (McGeachy and Cua, 2008). IL-1␤, which seems to play
only a supporting role in mouse Th17 development, is the most
effective inducer of IL-17 expression in human T cells. In humans,
IL-6 and IL-23 induce a small amount of IL-17 alone and greatly
enhance Th17 differentiation in the presence of IL-1␤ (Fossiez et
al., 1996; Jovanovic et al., 1998). IL-23 upregulates IL-17 production
and promotes survival and expansion of activated or memory Th17
cells in vitro, although it is not absolutely necessary (Tesmer et al.,
2008). In addition, only IL-23R positive Th17 cells migrate to the
site of inflammation in a mouse model of multiple sclerosis. Such
results in humans are scarce and it can be supposed that, except for
IL-23, other cytokines in periapical lesions contribute to the Th17
cell development. From among them, IL-1␤, which is detected in
human periapical lesions, especially in those with active inflam-
matory processes, could be the most relevant cytokine (Marton and
Kiss, 2000).
Another explanation for the absence of IL-17/IL-23 correlation
could be that produced IL-17 acts as a down-regulator of IL-23
expression. Such hypothesis is based on our unpublished obser-
vation that depending on the stimuli used for DC maturation
induction, IL-17 may either enhance or suppress the production
of IL-23 by human Mo-DCs.
Finally, we tested how exogenous IL-12 and IL-23 modulate the
production of IFN-␥ and IL-17 by PL-MNC cultures. As shown in
Fig. 3C, IL-23, but not IL-12, stimulated the production of IL-17.
In contrast, the production of IFN-␥ was enhanced by both IL-12
and IL-23. As expected, the stimulatory effect of IL-12 was signif-
icantly higher than IL-23. What remains to be studied is whether
the stimulatory activity of IL-23 on IFN-␥ production is directed to
the IL-17+ /IFN-␥+ double-positive T cells or to the Th1 cells. These
results further demonstrate the complexity of Th1/Th17 cell inter-
actions in periapical lesions.
3.4. Dendritic cells and their cytokine production in periapical
lesions
Since the IL-12 family of cytokines are mostly produced by DCs,
another aim of this study was to phenotypically characterize these
cells in periapical lesions and to examine their contribution to the
polarization of Th1/Th17 effector immune functions.
DCs were characterized in PL-MNC by double staining with
DC exclusion cocktail of mAbs and anti-HLA-DR mAb. HLA-DR+
cells, negative for CD3/CD14/D16/CD19/CD34, were considered
as DCs (Fig. 4A). Their frequency varied between 2.8 and 8.8%
(mean ± SD = 4.8 ± 3.4; n = 11) and was much higher than in periph-
eral blood (data not shown). Similar results were obtained in our
previous study (Lukic et al., 2006), when DCs were characterized
using flow cytometry, as HLA-DR+ CD19− CD3− CD14− cells. Most
DCs were CD86+ but only half of them expressed CD83, a marker of
mature DCs, suggesting that PL-DCs are both phenotypically mature
and immature (Fig. 4A).
According to the concept of DC differentiation and migration,
CD83− DCs are most probably immature cells, capable of capturing
and processing microbial antigens within inflamed tissue, such as
periapical lesion. Upon migration to the draining lymph node, and
their subsequent maturation, DCs activate naive T cells and trig-
ger specific T-cell immune responses. This causes clonal expansion
of naive T cells and their differentiation into effector and memory
T cells with the capability of migrating to the site of inflamma-
tion, where they perform different functions (Banchereau et al.,
2000; Palucka et al., 2002). In chronic inflammation, a number of
DCs are retained at the site and undergo local maturation mani-
fested by the upregulation of costimulatory molecules (DC80, CD86,
CD40), and expression of CD83. In addition, new blood DC precur-
sors migrate to the tissue and transform into inflammatory DCs
 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113 109

Fig. 4. Dendritic cells in periapical lesions. (A) Phenotypic characteristics of DCs in PL-MNC (a and b) or tissue (c). PL-MNC were stained with DC exclusion cocktail of mAbs
conjugated with RPE-Cy5 and HLA-DR-PE. A representative histogram is presented (a). Relative values (n = 6) of DC subsets (compared to total HLA-DR+ DCs) are presented
(b). Confocal image of a periapcial lesion showing close association of CD83+ DCs (green) with CD3+ T cells (red) (c); (B) DCs isolated from PL-MNC stained with an APAP
method with isotype control mAb (a) or HLA-DR mAb (b). Accessory function of PL-DCs and control Mo-DCs in MLR. DCs were cultivated with purified allogeneic T cells as
described in Section 2. Values are given as mean cpm of triplicates from one experiment (c); (C) production of IL-10, IL-12 p70, IL-23 and TNF-␣ by PL-DCs and Mo-DCs in
culture. Values are given as mean ± SD of 4 different cultures; * p < 0.05; ** p < 0.01; *** p < 0.005 compared to PL-DCs (Student’s t-test). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)

(Lopez-Bravo and Ardavin, 2008). Many proinflammatory media-
tors, such as IL-1␤, TNF-␣, IL-6 and prostaglandin E2, which are
found in periapical lesions (Nair, 2004), are well-known DC mat-
uration stimuli (Banchereau et al., 2000). The close association
between CD83+ and T cells in vivo observed with the confocal
microscope (Fig. 4A) support the hypothesis that DCs are potent
stimulators of local immune response in periapical lesions. Similar
observations have been reported in marginal periodontitis lesions
(Jotwani et al., 2001), and radicular granuloma (Kaneko et al., 2008).
We detected a relatively low percentage of CD1a+ , Langerhans-
type DCs (Fig. 4A). As CD1a+ DCs are predominantly localized within
epithelium, especially in radicular cysts (Suzuki et al., 2001), the
results of this study suggest that the epithelium was not a domi-
nant component in our samples of periapical lesions. CD1a− DCs
probably comprise both resident and inflammatory DCs localized
outside the epithelium (Banchereau et al., 2000).
A novel finding of this study was the identification of plasma-
cytoid DCs in periapical lesions using the three markers CD123,
BDCA2 and BDCA4. BDCA2 is more specific for plasmacytoid DCs
(Villandagos and Young, 2008) and this is probably the reason why
the percentage of BDCA2+ cells was the lowest. In our previous study
we characterized plasmacytoid DCs according to the coexpression
of HLA-DR and CD123 (Lukic et al., 2006). A slightly higher per-
centage of HL-DR+ CD123+ cells than BDCA2+ cells suggest that
110 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113

Fig. 5. Immunoregulatory cytokines in periapical lesions. (A) Production of IL-10 and TGF-␤ (a) in PL-MNC. Values are given as mean ± SD for n = 31 (total lesions); n = 13
(symptomatic lesions); n = 16 (asymptomatic lesions); n = 15 (T-type lesions); n = 12 (B-type lesions); correlation between TGF-␤ and IL-10 in asymptomatic lesions (n = 16)
(b) and B-type lesions (n = 11) (c). Coefficient of correlations and corresponding p values are given (Spearmann correlation test). (B) Effect of TGF-␤ or IL-10 on Th1/Th2/Th17
cytokine production by PL-MNC. Values are given as mean ± SD for 3 PL-MNC cultures (two asymptomatic B-type and one symptomatic T-type lesions). * p < 0.05; ** p < 0.01
compared to corresponding controls (Student’s t-test).

CD123 might be expressed in other cells. A low level of CD123
was also detected in indoleamine 2.3-dioxygenase-positive DCs of
myeloid origin. These cells are involved in the induction of toler-
ance, and suppression of the immune response (Mellor and Munn,
2004). Therefore, the exact nature of CD123+ DCs and their func-
tion in periapical lesions remains to be elucidated. The functional
significance of plasmacytoid DCs in chronic inflammatory lesions
is insufficiently known but they could be significant for the patho-
genesis of virally induced periapical lesions, and the production of
type 1 IFNs. A number of periapical lesions were found to be caused
by viruses (Sabeti et al., 2003; Slots et al., 2003). Alternatively, this
DC subset may regulate inflammatory/immune responses (Jahnsen
et al., 2002; Villandagos and Young, 2008).
In order to examine the functional characteristics of PL-DCs, we
succeeded in isolating them from several periapical lesions with a
higher number of infiltrating leukocytes. We showed that PL-MNC
(purity higher than 75%) had similar potential for stimulating allo-
geneic T-cell response as control Mo-DCs (Fig. 4B). PL-DCs produced
a higher level of IL-23, moderate level of IL-12 p70, and low level
of IL-10 and TNF-␣. Such results clearly indicate than PL-DCs are
capable of stimulating both Th1 and Th17 cells. In contrast, Mo-DCs
produced lower levels of both IL-12 p70 and IL-23 and higher levels
of IL-10 and TNF-␣, than PL-DCs (Fig. 4B). These differences could
be explained by the fact that DCs from periapical lesions are more
heterogeneous and differently stimulated in vivo, compared to Mo-
DCs. As described in Section 2, Mo-DCs were treated with LPS (a
TLR4 agonist) to promote maturity.
3.5. Immunoregulatory cytokines in periapical lesions
Under normal conditions, proinflammatory mechanisms must
be tightly controlled in order to prevent excessive tissue destruction
and promote autoimmune processes. TGF-␤ and IL-10 are two very
important immunoregulatory cytokines (Li et al., 2006; Couper et
al., 2008), and in order to study their contribution to the pathogen-
esis of periapical lesions, we measured their production in PL-MNC
cultures. The levels of both cytokines were detectable in all sam-
ples. The results presented in Fig. 5A show that the concentrations
 M. Čolić et al. / Molecular Immunology 47 (2009) 101–113
of TGF-␤ and IL-10 did not significantly differ between clinically
different groups of lesions, suggesting that anti-inflammatory pro-
cesses are equally controlled, regardless of whether the lesions are
symptomatic or asymptomatic. In contrast to IL-10, B-type lesions
produced significantly higher levels of TGF-␤, than T-type lesions
(Fig. 5A). In spite of these differences, there were positive correla-
tions between the levels of IL-10 and TGF-␤ in asymptomatic lesions
and B-type lesions (Fig. 5A). Such findings suggest that immunoreg-
ulatory mechanisms are more operative in chronic asymptomatic
lesions with the predominance of humoral immune response and
are supportive of the hypothesis that such processes are characteris-
tic of an advance stage in the development and healing of periapical
lesions.
Different findings support this hypothesis. TGF-␤ is a well-
known healing-promoting cytokine (Amento and Beck, 1991). It is
also involved in the stimulation of IgA production (van Vlasselaer
et al., 1992). A large number of B cells/plasma cells in periapical
lesions are IgA+ (Torres et al., 1994). In addition, in our previous
work we found that B-type lesions contained a significantly higher
proportion of T regs (CD4+ CD25hi Foxp3+ ) than T-type lesions
(Colic et al., manuscript submitted). It is generally known that
the development of T regs depends on TGF-␤ and that these cells
exert the immunoregulatory activity by secreting IL-10 and TGF-
␤ (Chen et al., 2003). IL-10 is also produced by Th2 cells, which
are more dominant in B-type lesions, and by other cells of the
innate immunity. Low production of IL-10 by immunostimulatory
PL-DCs, as confirmed in this study, is also in agreement with the
fact that immunostimulatory mechanisms are counterbalanced by
immunoregulatory cytokines.
In order to examine how such mechanisms are operative at
the levels of PL-MNC, we added exogenous IL-10 and TGF-␤ to
cell cultures and measured the production of Th1, Th2 and Th17
cytokines. As shown in Fig. 5B, both TGF-␤ and IL-10 inhibited to
the same extent IFN-␥, IL-2, TNF-␣ and IL-17 produced by PL-MNC,
whereas no significant effect was observed on the production of
IL-5. IL-10 did not significantly modulate the production of TGF-
␤ and vice versa. These results, although performed on a small
number of PL-MNC cultures (two asymptomatic B-type and one
symptomatic T-type lesions), are in agreement with previous pub-
lications on peripheral blood T cells (Rowan et al., 2008) and confirm
that both TGF-␤ and IL-10 can inhibit Th1 and Th17 immune
responses.
A number of recent publications confirmed an interplay
between Th17 cells and T regs (Bettelli et al., 2007; Oukka, 2007).
Although TGF-␤ is important for both the development of T regs
and Th17 cells, it seems that IL-6, together with TGF-␤, promotes
the development of IL-17 and inhibits the development of T regs
(Bettelli et al., 2007). However, some murine models of autoim-
mune disease have shown that TGF-␤ and IL-6 upregulate IL-17
production but fail to upregulate the proinflammatory cytokines
crucial for inflammation (McGeachy et al., 2007). The explana-
tion for such an unexpected finding is that under such conditions
T cells also produce IL-10 with potent anti-inflammatory activ-
ity. The double-positive cell population, IL-17+ /IL-10+ , which is
also described by other authors (Stumhofer et al., 2007), seems
to have an important protective function by limiting inflamma-
tion and tissue damage normally caused by IL-17 (Tesmer et al.,
2008).
Based on all these new findings, it would be useful to exam-
ine the mutual relationship between the frequency of T regs and
IL-17+ /IL-10+ cells, as well as the production of IL-17, TGF-␤, IL-10
and IL-6 in periapical lesions. Such experiments are presently in
progress in our laboratory.
In conclusion, our results suggest that the development of
human periapical lesions is a dynamic process in which differ-
ent inflammatory cells and their secretory products are involved.
111
Based on the production of cytokines by PL-MNC in vitro it can
be concluded that the Th cytokine network is complex and that
Th1, Th2 and Th17 immune responses are generally antagonistic.
Th1 and Th17 immune responses, which dominate over Th2, are
more important for T-cell-mediated pathological processes. Th2
and immunoregulatory cytokines are more significant for advanced
types of lesions with the predominance of B cells/plasma cells,
whereas the Th17 immune response seems to play a dominant role
in the exacerbation of inflammation.
Acknowledgments
This study was supported by a grant (VMA/06-08/B.3) of Military
Medical Academy, Belgrade, Serbia. The authors thank I. Majs-
torovic, P. Milosavljevic and J. Stamenkovic for technical assistance.
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