Proinflammatory and immunoregulatory mechanisms in periapical lesions

Molecular Immunology 47 (2009) 101–113

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Review

Proinflammatory and immunoregulatory mechanisms in periapical lesions ˇ c´ a,∗ , Dragan Gazivoda b , Dragana Vuˇcevic´ a , Saˇsa Vasilijic´ a , Miodrag Coli Rebeka Rudolf c , Aleksandra Lukic´ d a

Institute for Medical Research, Military Medical Academy, Crnotravska 17, 11002 Belgrade, Serbia Department for Oral Surgery, Clinic for Maxillofacial and Oral Surgery, Military Medical Academy, Belgrade, Serbia University of Maribor, Faculty of Mechanical Engineering, Maribor, Slovenia d Department of Endodonotics, Faculty of Stomatology, University of Belgrade, Serbia b c

a r t i c l e

i n f o

Article history: Received 9 October 2008 Received in revised form 24 December 2008 Accepted 8 January 2009 Available online 15 February 2009 Keywords: Periapical lesion Cytokine Cell culture Phenotypization Dendritic cell

a b s t r a c t Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear 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 production of IL-17, positive correlation between IL-17 and IFN-␥, but not between IL-17 and IL-23 production. Most IL-17+ cells coexpressed IFN-␥. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-␥ which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most 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 production 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 predominance 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 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 eradicate infection, chronic periapical lesions are formed, with the aim of restricting microbial invasion. In spite of numerous experimental and clinical studies, specific etiologically inducing factors, participating cells and growth mediators associated with the development, 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. ˇ c). ´ E-mail address: [email protected] (M. Coli 0161-5890/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2009.01.011

Periapical lesions are characterized histologically by fibrous and 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 infiltrating 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

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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 mechanisms (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 Department 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 without 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 curettage 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 consisting of RPMI-1640 (Sigma, Munich, Germany) and antibiotics/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 diameter 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 resuspended 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 (unpublished data). A cell suspension of total inflammatory cells (4 ml) was layered over a 3 ml Lymphoprep gradient (Nycomed, Oslo, Norway) 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 RPMI1640 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 supernatants 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 alkaline 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 albumin (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.

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The percentages of positive cells were determined on the basis of total counted cells. A similar light microscopic analysis was performed 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 resuspended in PBS containing 2% FCS and 0.1% sodium azide and aliquoted into tubes (1–2 × 105 cells/tube in 100 ␮l). For identification of DCs, the cells were stained with DC exclusion cocktail (CD3/CD14/CD16/CD19/CD34)-RPE-Cy5 mAbs and anti-HLA-DRPE. 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 irrelevant 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 samples 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 morphology and strong staining with anti-HLA-DR mAb. The number of recovered cells was between 0.5 × 103 and 1.2 × 104 /lesion. Monocyte 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 reaction (MLR) using allogeneic T cells, purified by MACS technology (Miltenyi Biotech) as responders. DCs (0.25 × 103 ) were cultivated

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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 fluorescent bead immunoassay (Bender Med Systems, Vienna, Austria), and flow cytometry, as described by the manufacturer. Concentrations 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 commercial 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 instructions 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 correlation 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 immunostaining (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 detection and quantification of inflammatory cells. The main finding is that lymphocytes and plasma cells are the predominant populations 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 lymphocytes/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 granulocytes are characteristics of acute periodontitis (exudative phase) and exacerbation of chronic inflammation, as a result of new infection from the root canal (Marton and Kiss, 2000; Nair, 2004). Neutrophils provide the first line of defense against bacterial invasion from the infected root canal. Due to their effective phagocytosis and killing function, the majority of microorganisms is destroyed and eliminated, preventing them from spreading throughout the lesion (Walton and Ardjmand, 1992; Kaufmann,

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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 frequency 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 damage 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 dental 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 symptomatic 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 significantly 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 process (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

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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 concerning 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 periapical 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 asymptomatic 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. Morphological and quantitative analyses showed that these cells represented minor population of infiltrating leukocytes (Fig. 1C). Enzyme cytochemistry 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 periapical 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 reactions (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, better 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 measured 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 combination of PMA + Ca2+ ionophore is a commonly used procedure for

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studying the functional potential of T cells to secrete cytokines (Collins, 2000). In our previous paper (Colic et al., 2006) we demonstrated 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, respectively (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 significantly 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 correlated 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 antigenpresenting 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 inhibiting 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 significant 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 differentiation 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

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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 correlation 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 proinflammatory 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 stimulate innate immunity and promote inflammation (Tesmer et al., 2008).

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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 production 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 significantly higher in T-type PL-MNC cultures than in B-type PL-MNC, and their concentrations in T-type lesions correlated with the proportion 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 asymptomatic 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 important for the exacerbation of inflammation within periapical lesions. Further, the relationship between IFN-␥ and IL-17 at the level of

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individual cells was studied by analysing intracellular cytokine production. 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 augmented 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 differentiation 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 presence 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 peripheral 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 predominantly by DCs and activated MØ, are a significant link between innate and adaptive immunity. Except for pairings with IL-12 or IL23, 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 production 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. IL12p70 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 concentrations (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 surprising, 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, respectively. 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 unexpected 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 development 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 inflammatory 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 observation 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 significantly 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 interactions 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 peripheral 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 trigger 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 inflammation, 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 manifested by the upregulation of costimulatory molecules (DC80, CD86, CD40), and expression of CD83. In addition, new blood DC precursors migrate to the tissue and transform into inflammatory DCs

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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 mediators, such as IL-1␤, TNF-␣, IL-6 and prostaglandin E2, which are found in periapical lesions (Nair, 2004), are well-known DC maturation 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+ , Langerhanstype 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 dominant 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 plasmacytoid 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 percentage of HL-DR+ CD123+ cells than BDCA2+ cells suggest that

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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 tolerance, and suppression of the immune response (Mellor and Munn, 2004). Therefore, the exact nature of CD123+ DCs and their function 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 pathogenesis 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 allogeneic 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 MoDCs. 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 pathogenesis of periapical lesions, we measured their production in PL-MNC cultures. The levels of both cytokines were detectable in all samples. The results presented in Fig. 5A show that the concentrations

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of TGF-␤ and IL-10 did not significantly differ between clinically different groups of lesions, suggesting that anti-inflammatory processes 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 correlations between the levels of IL-10 and TGF-␤ in asymptomatic lesions and B-type lesions (Fig. 5A). Such findings suggest that immunoregulatory 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 characteristic of an advance stage in the development and healing of periapical lesions. Different findings support this hypothesis. TGF-␤ is a wellknown 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 publications 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 autoimmune 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 explanation for such an unexpected finding is that under such conditions T cells also produce IL-10 with potent anti-inflammatory activity. 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 inflammation and tissue damage normally caused by IL-17 (Tesmer et al., 2008). Based on all these new findings, it would be useful to examine 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 different inflammatory cells and their secretory products are involved.

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