T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts

archives of oral biology 54 (2009) 156–161

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T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts N. Ihan Hren a,*, A. Ihan b a b

Clinical Department of Maxillofacial and Oral Surgery, University Medical Centre Ljubljana, Zalosˇka 2, 1000 Ljubljana, Slovenia Institute for Microbiology and Immunology, Medical Faculty Ljubljana, Zalosˇka 4, 1000 Ljubljana, Slovenia

article info

abstract

Article history:

Introduction: Radicular cysts (RCs) are periapical lesions resulting in jaw bone destruction.

Accepted 21 September 2008

The inflammatory dental periapical granuloma (PG) is considered to be the origin of RC formation; however the mechanism of RC development remains unclear.

Keywords:

Methods: Cell suspension from the surgically extirpated tissue of 27 RCs and 25 PGs was

Periapical granuloma

obtained. Bacteriological analysis of the PG tissue samples was performed in order to define

Radicular cyst

two major groups of PG according to the prevailing causative bacterial infection: the

Cytokines

streptococcal PG (PG-S, n = 10) and the anaerobe PG (PG-A, n = 9) group. The inflammatory

Th1 cells

response of tissue infiltrating lymphocytes was assessed by following T lymphocyte activa-

Th2 cells

tion (HLA-DR expression) as well as interferon g (IFN-g) and interleukin 4 (IL-4) production which were evaluated by the flow cytometry. Results: In comparison to RC both types of PG contained a higher proportion of activated T cells (HLA-DR) and lower proportion of IL-4 producing cells. PG-A tissue contained increased percentage of CD3 cells and increased percentage of T helper 1 (Th1) cells in comparison with PG-S. In RC the IFN-g production is higher than in streptococcal PG-S but similar as in PG-A. Discussion: Tissue infiltration by Th2 cells and IL-4 production is likely to play an etiopathogenic role in RC formation. # 2008 Elsevier Ltd. All rights reserved.

A pathological immune response to stimuli from infected dental root canals results in the development of two types of periapical inflammatory lesions in the jaw bones, periapical granulomas (PGs) and radicular cysts (RCs).1 PG and RC represent two different developmental stages of the same inflammatory process2 caused by a non-specific multibacterial infection.3 PG is histologically defined as a chronical granulation tissue with epithelial rest of Malassez. In some PGs, proliferation of epithelial rest of Malassez may lead to the development of the inflammatory RC.4 The epithelial rests of Malassez in PG may be stimulated to proliferate by inflammatory stimuli. The early developmental forms of cysts – bay or pocket cysts5 – communicate with the

root canal. RCs however, do not communicate with the root canals or any other openings. RCs contain no bacteria,6,7 although the invasion of living bacteria is necessary for the development of PG.8 Both lesions grow by local bone resorption carried out by osteoclasts,9 but the dynamic equilibrium between defensive and destructive inflammatory mechanisms may provide a pathobiological basis for better understanding of PG10 and RC pathogenesis. Both cellular and humoral specific immune responses are activated within periapical lesions, among them T helper cells play an important role.11 The T cells are the most numerous in periapical lesions, and play an important role in their formation.12 Their activation and function is not known yet,

* Corresponding author. Tel.: +386 1 522 4751; fax: +386 1 522 24 95. E-mail address: [email protected] (N. Ihan Hren). 0003–9969/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2008.09.014

archives of oral biology 54 (2009) 156–161

since many regulatory circuits are activated not only within the immune system but also in interaction with other cells, e.g. oral epithelium and oral bacteria.13 Also the herpesviruses together with endodontopathic bacteria, may play major roles in the etiopathogenesis of periapical pathosis.14 The presence of bacteria in PG was confirmed,8 but only in their apical area and not they are not present in tissue of periapical lesion.15 Inflammatory cells in periapical lesions were immunohistochemically identified,16,17 but the exact mechanisms why a sequel of some PG formation is the proliferation of the epithelial cell rests of Malassez and an inflammatory RC formation, also the enlargement of RC are still not clear. As there is strong evidence that host-derived factors such as cytokines are involved in the pathogenesis of PG and RC.1 The RC enlargement could be abundant due to the bone resorption; IL-1 has been implicated as a central mediator of bone destruction.18 Both proinflammatory Th1 and antiinflammatory Th2 cytokines might modulate IL-1 expression, implicated as central mediators of periapical bone resorption and activity of macrophages.19 We have, in our previous studies, investigated the inflammatory properties of distinct oral bacteria species in PG20,21 in in vitro lymphocyte cultures22 and in a mouse model of bacterial infection.23 Using both experimental models we demonstrated significant differences in the inflammatory process induced by oral streptococci and oral anaerobic bacteria (Bacteroides spp.), the latter showing increased levels of cell infiltration, cytotoxic cell activation and IFN-g secretion. We also demonstrated that dendritic cells primed in vitro with antigenes of distinct commensal oral bacteria species (Bacteroides, Streptococcus, Propionibacterium) induced different inflammatory responses; this may have important consequences in understanding various immunopathologic manifestations in oral cavity.24 The purpose of the present study was to characterize the inflammatory response of the RC and PG tissue infiltrating lymphocytes by following T lymphocyte activation (HLA-DR expression) as well as interferon g (IFN-g) and interleukin 4 (IL4) productions, evaluated by the flow cytometry. In addition, bacteriological analysis of PG tissue was performed in order to define two major groups of PG according to the prevailing causative bacterial infection: the streptococcal PG (PG-S) and the anaerobe PG (PG-A) group.

1.

Materials and methods

Tissue samples of PGs and RCs were collected during the standard sterile operation procedures. The study was approved by the Slovenian Ethics Committee, and informed consent was obtained from consecutively enrolled patients. The tissue samples were obtained from 52 patients; 27 of them had surgical extirpation of large RC (more than 15 mm in diameter) to avoid small cysts where misdiagnosed RC are possible7 and 25 of them had surgical extirpation of PG during the apiectomy. To prevent bacterial contamination, subjects did not have previous root exposures and the PG tissue was collected during apiectomy only in upper jaws, with marginal gingiovectomy approach after the hibisept mouth washing. Then tissue were separated and part for microbiological culturing crumbled in sterile way.

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The material obtained at surgical procedures was sent for standard histopathological examination, based on hematoxylin–eosin staining. The diagnosis of PG was made if there was fibrous and granulation tissue, which often contained foamy macrophages, cholesterol granuloma and islands of epithelium (rests of Malassez). The diagnosis of RC was made if fibrous and granulation tissue from the cyst wall was lined by squamous epithelium. Epithelial lining and connective tissue may exhibit Rushton bodies, cholesterol granuloma and varying degree of inflammation. To avoid contamination with oral bacteria all the patients with oral communication of the periapical lesion directly or through the tooth were excluded at the time of the surgical procedure. Both (PG and RC) lesions were exposed during the standard sterile surgical procedures with gingival marginal incision and vertical relieving incisions. The PG and RC tissue was removed from the jaw bone and a part of it was transferred to a bottle containing 1.0 ml of sterile isotonic phosphate buffered saline (pH 7.4—for flow cytometry). For bacteriological analysis the remaining part of PG tissue samples was transferred into bottles containing 1.0 ml of sterile tioglicolate anaerobe broth (pH 7.2). The tissue samples were disaggregated by sterile pincers and then gently agitated in a tube containing 1 ml of sterile tioglicolate broth, before being homogenised and cultured. The bacterial strains from the tissue samples were initially categorised by their atmospheric requirement, Gram staining and the catalase reaction. Facultative streptococci were identified with the API 20 Strep. System (API, Biomerueux, France). Strict anaerobes were identified with the API 20A system. For flow cytometric analysis the tissue samples were rinsed with RPMI and mechanically dispersed with Medimachine (Becton Dickinson, Mountain View, California, USA). The suspensions were then transferred to a tube containing RPMI supplemented with collagenase (625 collagen digestion units—CDU/ml; Sigma, Missouri, USA). The capped tubes were incubated at 37 8C for 20 min and titrated gently every 5 min through a wide-bore pipette to assist disintegration. The cells were washed twice with PBS and prepared for the flow cytometry. Flow cytometry analysis was performed using a fluorescence cell counter (FACS Calibur Becton Dickinson, Mountain View, California, USA). Fluorochrome conjugated monoclonal antibodies were purchased from Becton Dickinson (Mountain View, California, USA). A two-parameter analysis was performed to determine percentage of T cells (CD3—FITC; clone UCHT1), CD4 T cells (CD4—PerCP clone RPA-T4), CD8 T cells (CD8—PerCP; clone RPA-T8), as well as the expression of HLA-DR (anti HLADR—PE; clone G46-6). The cells were labelled with monoclonal antibodies and analyzed with Becton Dickinson Cell Quest software (Becton Dickinson, Mountain View, California, USA). The lymphocytes were initially located by CD45 (clone HI30) expression and then gated according cell size (FCS parameter) and cell granularity (SSC parameter) on FCS/SSC dot-plot. A control of viable cells (LIVE/DEAD kit, Molecular Probes, Oregon, USA) was included in the analysis. For the detection of intracellular IFN-g (anti hu-INF-g FITC; clone XMG1.2) and IL-4 (anti hu-IL-4—PE; clone 11B11) the cells obtained from RCs were isolated by gradient centrifugation on Ficoll and cultured in

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3  105 ml 1 in RPMI, supplemented with FCS (10%) and stimulated for 6 h with ionomycin/PMA and breferdinA (SIGMA), the inhibitor of exocytose. At least 1000 gated cells were analyzed for each test and signals from the two light scatters and the four fluorescence parameters were analyzed. We used standard methods of statistical analysis. The Student–Newman–Keuls test of one way ANOVA was used for comparison of the chosen immune parameters; P < 0.05 was accepted as significant.

2.

Results

Flow cytometric analysis of tissue infiltrating lymphocytes from RC and PG tissues was employed to determine T lymphocyte activation (Fig. 1) and cytokine production— IFN-g and interleukin 4 (IL-4) induced by ionomycin/PMA stimulation. Table 1 represents the results of the flow cytometric analysis. We demonstrated that both types of PG contained a significantly higher proportion of activated (HLADR) T cells compared to the RC tissue. In contrast, the production of IL-4 was higher in RC tissue compared to PG. 25 samples of PGs were microbiologically analyzed, 22 yielded a positive bacterial growth when homogenised and cultured. In 10 samples Streptococcus sp. was predominant (more than 90% of colonies) isolate (10 PG-S), in 7 samples predominantly P. acnes and in 2 samples Bacteroides sp. were predominant (9 PG-A). In other samples Lactococcus sp., Bacillus sp. and Escherichia coli were isolated. RC samples were clinically asymptomatic and were assumed to be sterile (3, 18). The PGAs showed a significantly increased (P < 0.05) percentage of CD3 cells and significantly increased (P < 0.01) percentage of Th1 cells in comparison with the PG-Ss.

3.

Discussion

In the presented study lymphocyte characteristics of RC and PG were compared. We studied the T cells populations and the

Table 1 – Mean percentages (with standard deviations) of distinct T lymphocyte phenotypes obtained from RC, PGS (infected by streptococci) and PG-A (infected by anaerobes). The significant differences between RC and PG-S and between RC and PG-A as analyzed by the T-test are marked by parentheses. RC (S.D.), N = 27 % CD3 % CD4 % CD8 % HLA DR/CD8 % cIFNg/CD3 % cIL-4/CD3 Th1/Th2

34.29 18.12 16.17 9.65 10.30 8.09 1.61

(19.51) (11.50) (9.85) (5.86) (4.51) (5.86) (1.04)

PG-S (S.D.), N = 10 45.5 20.2 25.3 71.3 5.5 2.4 2.9

(19.38) (11.15) (9.81) (10.23)*** (4.03)** (1.43)** (2.39)

PG-A (S.D.), N=9 54.8 28.1 26.7 71.2 12.7 1.3 10.0

(13.50)* (11.17) (5.15)* (14.41)*** (5.95) (0.52)*** (5.59)***

RC, radicular cysts; PG-S, periapical granuloma infected by streptococci; PG-A, periapical granuloma infected by anaerobes; % CD3, percentage of CD3 cells; % CD4, percentage of CD4 cells; % CD8, percentage of CD8 cells; % HLA DR/CD8, percentage of HLA DR+CD8+ cells; % cIFNg/CD3, percentage of CD3 cells producing interferon g; % cIL-4/CD3, percentage of CD3 cells producing interleukin 4. * P < 0.05. ** P < 0.01. *** P < 0.001.

production of IFN-g and IL-4, the major Th1 and Th2 cytokines in human PG and RC by flow cytometry. We found out that the type of the immune response in the PG tissue is significantly determined by their apically resident bacteria. So the comparison of RC characteristics was done separately for both PG groups: streptococcal and anaerobic. The PGs with anaerobic bacteria were infiltrated with increased levels of T cells and Th1 cells compared to streptococcal PG. We also found differences in T cell populations between PG and RC. In RC the percentage of lymphocytes was smaller than in both PGs, significantly less in comparison with anaerobic PG. The portion of T helper (CD4+) cells and portion of cytotoxic T (CD8+) cells in RCs is similar as

Fig. 1 – Flow cytometric analysis of activated CD8 cells. Cells were labelled with monoclonal antibodies CD8 and anti-HLA DR. Lymphocytes were initially located by CD45 expression and then gated on FCS/SSC dot-plot and the percentage of activated cytotoxic cells (HLA DR positive and CD8 positive—found in upper/right quadrant) was obtained on CD8/HLA DR dot-plot.

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in both PG groups. The differences of the proportions of these immunocompetent cells between PG and RC have been studied before and the studies also didn’t show significant differences in the proportion of CD4 and CD8 T-cell subsets.11 In comparison to RC, both types of infected PG tissue contained higher proportions of activated (HLA-DR) T cells in our study. That may be explained by the fact, that PG is a result of a chronic non-specific inflammation caused by bacterial invasion from an infected root canal. On the other hand, the induction of RC growth is most probably the consequence of a specific T cell mediated inflammatory process that persists without living bacteria in RC walls.25 From our data it may therefore be concluded that a high proportion of activated (HLA-DR) T cells is a characteristic of PG, what is consistent with the published results.26,27 We also found a high production of IL-4 in RC compared to both PG groups. The high IL-4 activity in RC represents an antigen specific, Th2 differentiated immune response. This is consistent with the study of Takeichi et al.28 reporting weak iNOS synthesis in RC which is associated with predominant Th2 response. Walker et al.29 reported greater number of Th2 cells in both PG and RC with IL-4 being more frequent in RC; while IL-4 positive cells in PG were found by de Sa et al.30 In contrary to our results some studies reported that IL-4 was completely missed in cells of PGs and they were IFN-g positive; but the opposite was a characteristic of periapical regenerative tissues.31,32 Therefore, the non-specific inflammatory response to infection in PG, followed by a T cell mediated chronic inflammation of Th2 type, may predispose RC induction and growth. The findings about IFN-g productions in RC in our study are determined by the big difference in IFN-g in anaerobic and streptococcal PGs. In the anaerobic PG the Th1 response by higher IFN-g is characteristical. In RC the IFN-g production is higher than in streptococcal PG but similar as in anaerobic one. So the Th1/Th2 relation is similar in RC and streptococcal PG, but is more cytotoxic and Th1 predominant in anaerobic PG. It is believed that Th1 immune response is involved in the progression of periapical lesion and bone destruction,18,33,34 whereas immunosuppressive mechanisms mediated by Th2 cytokines are of importance in healing processes occurring in the late stages of lesion development and down-regulation of the inflammatory immune mechanisms.35 But the results obtained in human and animal models varied significantly and the mechanisms involved in the formation of RC are controversial. The important role of IFN-g and IL-10 in the pathogenesis of experimentally induced periapical lesions was suggested by a study with knockout mices, whereas IL-4 appears to present a nonsignificant effect on periapical modulation of PG, RCs were not included in that study.15 The opposite has been shown in another study with knock-out mice that IFN-g do not have a significant effect on bone resorption in periapical lesions, suggesting possible functional redundancy in proinflammatory pathways.36 In contrast the same group showed that IL-10, but not IL-4, suppressed infection-stimulated bone resorption in vivo.37 Multiple mechanisms are involved in the pathologic changes associated with periapical lesions. Nowadays the concept of proinflammatory Th1 and anti-inflammatory Th2

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subsets of CD4+ T helper cells as immunomodulators of immune responses and inflammatory diseases needs to be reconsidered, because recent data indicate that IL-17 (produced by T helper cells) is distinct from the traditional Th1 and Th2 subsets.38 IL-23 induces the differentiation of natı¨ve CD4+ T cells into highly pathogenic T helper cells Th17, that produce IL-17, IL-17F, IL-6 and TNF-alpha but not IFN-g and IL-4.39 The IL-17 is proinflammatory cytokine which contributes to bone destruction in reumathoid arthritis by stimulation of multiple inflammation mediators as IL-1, IL-6, TNF-alpha, NOS-2, chemokines, etc.; but it is essential in host defense against pathogens susceptible to neutrophils.40 The role of IL-17 in periodontal disease was not obvious (is controversial). While IL-17 has been implicated in a few of studies of severe PD41 its role in this disease remains largely undefined. Whereas IL-17 is clearly bone destructive in the context of arthritis, its potent effects on neutrophils suggest that IL-17 could play a defensive role in PD, ultimately exerting a net bone-protective effect. The overall influence of IL-17 is bone protective, largely through control of chemokine expression and neutrophil recruitment. Thus, the positive influence of IL-17 on neutrophil regulation outweights its potential bone-destructive signals.42 So the role of Th17 cells in periapical lesions must be clarified in the future, also gender differences should be taken into account because of gender-dependent effect a IL-17 signaling.43 The mechanism of cyst development and enlargement involves bone resorption.44 IL-1 has been implicated as a central mediator of bone destruction18 via stimulation of receptor activator of nuclear factor kB ligand (RANKL) expressed by osteoblasts and lymphocytes.45 Other mediators, particularly those derived from T cells, may also play critical roles by modulating inflammation on RANKL expression.18 Distinct positive immunoreactivity to RANKL was demonstrated in RC as in other osteolytic lesions of the facial skeleton,46 there were also no differences in the presence of RANKL in PGs and RCs.47 Both proinflammatory Th1 and antiinflammatory Th2 cytokines might modulate IL-1 expression, implicated as central mediator of periapical bone resorption and activity of macrophages.19 There is a report that stimulation of IL-1 beta release from dental pulp cells due to anaerobe Porphyromonas endodontalis correlates with the progression of periapical inflammation.44 IL-6 stimulates bone resorption,48 but IL-4 and IFN-g exert an inhibitory effect on bone resorption through negative effects on osteoclasts.49 IL-4 suppresses the synthesis of proinflammatory cytokines, including IL-1 and TNF-a,50 which promote bone resorption. IL-4 also stimulates the expression of alkaline phosphatase and other substances of mineralisation. So the mechanisms of RC enlargement are abundant and still unclear. Cytokines that promote epithelial cell proliferation play an important role in formation of cysts.23 Bone-remodelling cytokines such as IL-1 and IL-6 were shown to be present in the RC epithelium51; high IL-6 synthesis is characteristic in cultured fibroblasts isolated from RC.52 The epidermal and basic fibroblast growth factor receptors are expressed in inflammatory periapical lesions.4,53 IL-6 may contribute to the high proportion of IL-4 secreting T cells because of its ability to stimulate Th2 cell proliferation.54 After cyst formation and in the absence of antigen stimulation, the high cytokine content of the RC may inhibit T cell activation. Therefore, a low

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number of activated T cells in our study is not unexpected, as it is also described pathohistologically—the epithelial lining in larger cysts becomes flattened and the number of inflammatory cells decreases progressively.55 In our previous work23 we provided an evidence that Th1type cytokine production is increased in periapical lesions predominantly colonised by anaerobic bacteria compared to periapical lesions predominantly colonised by streptococci. Our results indicated more extended inflammation and tissue damage in periapical lesions colonised by anaerobic bacteria. We also established a mouse model of chronic bacterial infection (cotton trap) and provided an evidence that anaerobic bacteria (Bacteroides sp.) are more effective in inducing cytotoxic immunity and Th1 response compared to oral streptococci.21 Our present results are consistent with our previous work as we confirmed a correlation between the resident bacteria and the type of the immune response in PG. In PGs colonised by anaerobic bacteria, we demonstrated significantly increased levels of cell infiltration, cytotoxic cell activation and IFN-g secretion. On the other hand, the secretion of IL-4 in PG-A was low—hence the difference in Th1/Th2 ratio between RC and PG-A. Our results correspond to the study of Stashenko et al.56 which describes a Th1 immune response promoted by Porphyromonas gingivalis. In PG-S both types of cytokine secretion (IFN-g and IL-4) were significantly lower in comparison to those in RC. However, by comparing the cytokine profile of the RC and PG it is not possible to simply conclude which type of bacterial infection is able to induce RC formation from PG tissue. Our work suggests that anaerobes are strong inducers of Th1 response, that include TNF alfa, IL-1beta and IL-6 secretion—all cytokines are strong osteoclasts inducers and promote bone resorption which is one of the characteristics of PGs and RCs growth. Therefore, an infection of tissue with bacterial inducers of Th1 response, may be an important factor in bone resorption. In that aspect our data are consistent with published reports demonstrating that Th1 cells play an important role in the bone resorption process during local inflammation. Superinfection with Th2 inducers may additionally contribute in epithelium growth, which can determine PGs transformation in RC or RC growth. Our results established that high interleukin 4 (IL-4) production is a characteristic of established RC, while a high proportion of activated (HLA DR) T cells is a characteristic of PG without differences of their prevalent apically resident bacteria. The anaerobe PG tissue contained increased percentage of CD3 cells and increased percentage of Th1 cells in comparison with streptococcal PG. In RC the IFN-g production is higher than in streptococcal PG but similar as in anaerobe PG. Regarding these results the speculation about the type of bacteria which predispose the periapical lesion’s development is impossible, but these influence of causative bacteria should be reconsidered in pathogenesis of PG and RC. A better understanding of PG pathogenesis could open some new possibilities for prevention of RC formation and the resulting tooth and bone loss.

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