Production of IL‐10 and IL‐12 by antigen‐presenting cells in periapical lesions

J Oral Pathol Med (2010) 39 : 690–696 Background: Interferon‐γ (IFN‐γ) plays an important role in the pathogenesis of periapical lesions. Its expression is up‐regulated by interleukin (IL)‐12) and down‐regulated by IL‐10. The aim of this work was to study the cellular source of these cytokines and their mutual interactions in human periapical lesions. Methods: Mononuclear cells, macrophages and dendritic cells were isolated from periapical lesions using plastic adherence and osmotic gradients. Cytokines were measured in culture supernatants by a microbeads fluorescence assay. Phenotypic characteristics of cells were studied by immunocytochemistry, whereas allostimulatory activity of antigen‐presenting cells was tested using a mixed leukocyte reaction. Results: We observed the positive correlations between the levels of IL‐12 and IFN‐γ as well as IL‐12 and IL‐10 in cultures of mononuclear cells. As IL‐10 and IL‐12 are produced by dendritic cells and activated macrophages, we examined their contribution to the production of these cytokines. Macrophages, CD14⁺ adherent cells, produced high levels of IL‐10 and very low levels of IL‐12. In contrast, non‐adherent, strongly HLA‐DR⁺ dendritic cells, potent stimulators of the alloreactive T‐cell response, produced low levels of IL‐10 and moderate levels of IL‐12. Dendritic cells stimulated the production of IFN‐γ by allogeneic CD4⁺ T cells. In contrast, the level of IFN‐γ was significantly decreased and the production of IL‐10 was enhanced by addition of macrophages to the culture system. Conclusion: Our results suggest that a fine balance between the production of IL‐10 and IL‐12 by different antigen‐presenting cells, through IFN‐γ, may control the course of chronic inflammation in periapical lesions.     

Interleukin-17 plays a role in exacerbation of inflammation within chronic periapical lesions

Interleukin (IL)-17 plays an important role in inflammation and certain autoimmune diseases. However, its role in the pathogenesis of chronic dental periapical lesions has not been studied. Periapical lesion mononuclear cells (PL-MNC) were isolated from inflammatory cells and phenotypically analyzed by immunocytochemistry. The cells were cultured in vitro and IL-17 and IL-8 were measured in the culture supernatants. Controls were peripheral blood (PB) MNC. The level of IL-17 and the proportion of neutrophils were significantly higher in symptomatic lesions. In addition, the production of IL-17 was higher in culture supernatants of PL-MNC isolated from lesions with a predominance of T cells, and the IL-17 concentration correlated with the proportion of CD3+ and CD4+ cells. There was a positive correlation between the levels of IL-17 and IL-8 in the group of symptomatic lesions. The relationship between these cytokines was additionally confirmed on the basis of augmented production of IL-8 by both PL-MNC and PB-MNC treated with IL-17. Our results suggest that IL-17, by stimulating the production of IL-8, may play a role in exacerbating inflammation within chronic periapical lesions.     

Aggressive and Chronic Periodontitis Correlate With Distinct Cellular Sources of Key Immunoregulatory Cytokines

Background: Chronic periodontitis (CP) and aggressive periodontitis (AP) are inflammatory diseases and the main cause of dental loss in adults. We aimed to investigate the expression of adhesion molecules and the source of proinflammatory and anti‐inflammatory cytokines in circulating mononuclear cells from patients with CP and AP. Methods: Peripheral blood mononuclear cells from healthy controls and CP or AP patients were collected. The expression of the cell adhesion molecules CD11a and CD11b, and the cellular sources of interleukin (IL)‐4, IL‐10, IL‐12, interferon‐γ, and tumor necrosis factor‐α by distinct subpopulations of circulating leukocytes were determined using flow cytometry. Results: The expression of CD11a, but not CD11b, was significantly higher within the CD4⁺ and CD8⁺ T cells in CP and AP than in healthy controls. The frequencies of tumor necrosis factor‐α–expressing CD4⁺ T cells and CD14⁺ cells were higher in AP and CP, compared to healthy controls, respectively. Moreover, the frequency of IL‐10 expressing CD14⁺ cells was higher in CP, but not AP, compared to healthy controls CD4⁺ T cells committed to IL‐4 production was higher in CP than in healthy controls. Conclusion: These results suggest the participation of CD11a in the pathogenesis of periodontal lesions and show distinct cellular sources of immunoregulatory cytokines in AP versus CP.     

Kinetics of Th17‐related cytokine expression in experimentally induced rat periapical lesions

Th17‐related cytokines are essential factors in various pathological states, including inflammatory bone destruction. This study investigated the contribution of Th17‐related cytokines to the progress of experimentally induced rat periapical lesions. Periapical pathoses were induced by unsealed exposure of the pulp chamber of the lower first molars. A variety of immunocompetent cells, including CD68⁺ macrophages, Ia antigen⁺ cells and TCRαβ⁺ T cells, were observed in the lesions. The expression levels of Th17‐related cytokines, IL‐17 and IL‐23, and of pro‐inflammatory cytokines, IL‐1β and IL‐6, were significantly increased at 14 days (expansion stage) compared with normal periapical tissues. The expression levels of Foxp3, a regulatory T cell (Treg)‐related gene, and of IL‐10, an anti‐inflammatory cytokine, were higher at 28 days (chronic stage) than at 14 days. These findings suggest that Th17‐related cytokines may be primary contributors to the initiation of periapical bone destruction, and that lesion expansion may be regulated by anti‐inflammatory mediators.     

Distribution of interleukin-2 receptor α-chain and cells expressing major histocompatibility complex class II antigen in chronic human periapical lesions

In situ distribution of CD2⁺ T-lymphocytes, CD4⁺ and CD8⁺ T-cell subsets, CD14⁺ macrophages, interleukin-2 receptor α-chain (IL-2Rα) and class II major histocompatibility complex antigen (major histocompatibility complex class II, HLA-DR) expressing cells were determined in 14 chronic human periapical granulomas by imrnunohistochemical method using monoclonal antibodies. CD2⁺ lymphocytes were rather evenly distributed within the classical granulation tissue and comprised 55% of the mononuclear cells. Macrophages were distributed all over the periapical area, but their proportion was much less than that of T lymphocytes. Both small, lymphocyte-like mononuclear cells and larger mononuclear cells resembling macrophages displayed mild to strong circumferential staining with the anti-HLA-DR antibody. The majority of lymphocytes expressed IL-2Rα indicating the activated state of T cells within the lesion.     

Imbalance of Interleukin-17+ T-cell and Foxp3+ Regulatory T-cell Dynamics in Rat Periapical Lesions

Introduction

Interleukin (IL)-17⁺ T-helper (Th17) cells and Foxp3⁺ regulatory T (Treg) cells are CD4⁺ T-helper cells with reciprocal functions in immunology and bone metabolism. The present study aimed to investigate the expression dynamics of Th17 and Treg cells in rat periapical lesions as well as their correlation with bone resorption.

Methods

Experimental pulp exposures were made in the lower first molars of 28 Wistar rats to induce periapical lesions. Rats were killed on days 0, 7, 21, and 35. Mandibles were prepared for micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis.

Results

Through 3-dimensional and 2-dimensional measurements, the volume and area of periapical lesions visibly increased from day 7 to day 21 and then expanded slowly between days 21 and 35. IL-17–positive cells markedly increased from day 7 to day 35. However, Foxp3-positive cells remained at low levels until day 21 and then dramatically increased by day 35. The IL-17⁺/Foxp3⁺ ratio and number of osteoclasts simultaneously increased from day 7 to day 21 and then decreased on day 35. Finally, the distinct distribution of CD4⁺/IL-17⁺ Th17 and CD4⁺/Foxp3⁺ Treg cells was observed on days 7 and 35.

Conclusions

Our findings imply the imbalance of IL-17⁺ T cell and Foxp3⁺ Treg cell dynamics in induced periapical lesions, which may play an important role in periapical lesion progression.     

Production of interleukin-8 in vitro by mononuclear cells isolated from human periapical lesions

INTRODUCTION: Interleukin-8 (IL-8) is an important mediator of inflammation. However, little is known about its production in chronic dental periapical lesions and this was the main aim of this work. METHODS: Inflammatory cells were isolated from clinically different periapical lesions and analyzed by morphological criteria. The mononuclear cells were isolated, phenotypically analyzed by immunocytochemistry and cultivated in vitro. IL-8 was measured in culture supernatants of these periapical lesion mononuclear cells (PL-MNC) using a microbeads fluorescence assay. RESULTS: We found a relatively high production of IL-8 in 19 out of 21 periapical lesions included in the study. The level of IL-8 and the proportion of neutrophil granulocytes were significantly higher in the group of symptomatic lesions, compared to the asymptomatic lesions, but there was no statistically significant correlation between these parameters. According to the predominance of CD3(+) T cells and Ig(+)/CD19(+) B cells and plasma cells, lesions were divided into T-type and B-type lesions, respectively. The levels of IL-8 were significantly higher in the culture supernatants of PL-MNC in the T-type lesions and were positively correlated with the proportion of macrophages/dendritic cells (CD11c(+) cells) and CD4(+) T cells. Such a correlation was not shown in B-type lesions. CONCLUSION: These results suggest that PL-MNC are a significant source of IL-8, which is probably an important chemokine for the migration and function of different cell types at the site of chronic inflammation.     

Immunohistological analysis of Tannerella forsythia‐induced lesions in a murine model

Tannerella forsythia has been implicated as a defined periodontal pathogen. In the present study a mouse model was used to determine the phenotype of leukocytes in the lesions induced by subcutaneous injections of either live (group A) or nonviable (group B) T. forsythia. Control mice (group C) received the vehicle only. Lesions were excised at days 1, 2, 4, and 7. An avidin‐biotin immunoperoxidase method was used to stain infiltrating CD4⁺ and CD8⁺ T cells, CD14⁺ macrophages, CD19⁺ B cells, and neutrophils. Hematoxylin and eosin sections demonstrated lesions with central necrotic cores surrounded by neutrophils, macrophages and lymphocytes in both group A and group B mice. Lesions from control mice exhibited no or only occasional solitary leukocytes. In both groups A and B, neutrophils were the dominant leukocyte in the lesion 1 day after injection, the numbers decreasing over the 7‐day experimental period. There was a relatively low mean percent of CD4⁺ and CD8⁺ T cells in the lesions and, whereas the percent of CD8⁺ T cells remained constant, there was a significant increase in the percent of CD4⁺ T cells at day 7. This increase was more evident in group A mice. The mean percent of CD14⁺ macrophages and CD19⁺ B cells remained low over the experimental period, although there was a significantly higher mean percent of CD19⁺ B cells at day 1. In conclusion, the results showed that immunization of mice with live T. forsythia induced a stronger immune response than nonviable organisms. The inflammatory response presented as a nonspecific immune response with evidence of an adaptive (T‐cell) response by day 7. Unlike Porphyromonas gingivalis, there was no inhibition of neutrophil migration.     

Interleukin‐17/T‐helper 17 cells in an atopic dermatitis mouse model aggravate orthodontic root resorption in dental pulp

Interleukin (IL)‐17 is an important mediator of orthodontically induced inflammatory root resorption (OIIRR). However, its role in the dental pulp (DP) has not been studied. The aim of this study was to investigate, using an atopic dermatitis (AD) model, how IL‐17 contributes to OIIRR in DP. Atopic dermatitis is the most common IL‐17‐associated allergic disease. Atopic dermatitis model mice (AD group) and wild‐type mice (control group) were subjected to an excessive orthodontic force. The localization of T‐helper (Th)17 cells, IL‐17, IL‐6, and keratinocyte chemoattractant (KC; an IL‐8‐related protein in rodents) were determined in DP. In addition, CD4⁺ T cells, including IL‐17 production cells, were obtained from patients with AD and from healthy donors, and the effects of IL‐17 on the production of IL‐6 and IL‐8 were investigated using a co‐culture of CD4⁺ T cells with human dental pulp (hDP) cells stimulated with substance P (SP). Immunoreactivity for Th17 cells, IL‐17, IL‐6, and KC was increased in DP tissue subjected to orthodontic force in the AD group compared with DP tissue subjected to orthodontic force in the control group. The cells obtained from the AD patients displayed increased IL‐6 and IL‐8 production. These results suggest that IL‐17 may aggravate OIIRR in DP.     

Memory T cell subsets in healthy gingiva and periodontitis tissues

1 Background

In the gingival sulcus, effective and balanced innate and adaptive immune responses against subgingival plaque microbiome are crucial to maintain immune homeostasis. In this study, we investigated the memory T cell subsets in healthy gingiva and periodontitis tissues.

2 Methods

Anatomical localization of T cells (CD3⁺, CD4⁺, and CD8⁺) in healthy gingiva and periodontitis tissues were examined immunohistochemically. Subsets of memory T cells from isolated gingival cells were analyzed by flow cytometry using a cocktail of monoclonal antibodies (anti‐CD69, anti‐CD103, anti‐CD45RA, anti‐CCR7, anti‐CD28, and anti‐CD95). Intracellular cytokine staining of interleukin (IL)‐17 and interferon (IFN)‐γ expression on memory T cells in periodontitis tissues was also investigated.

3 Results

We found that healthy gingiva contains two memory T cell populations; a CD69^? recirculating population and a CD69⁺ gingiva‐resident memory T cell population. CD4⁺ T cells with transitional memory (T_TM) phenotype (CD45RA^?CCR7^?CD28⁺CD95⁺) constitute the major subset within these two populations. A significant increase in the proportion of CD4⁺CD69⁺CD103^? memory T cells was observed in periodontitis tissues compared with healthy gingiva. CD4⁺ memory T cells from periodontitis tissues produced either IL‐17 or IFN‐γ whereas CD8⁺ memory T cells produced only IFN‐γ.

4 Conclusions

Our findings suggest that recirculating and gingiva‐resident memory T cells could represent an important part of the immune surveillance network in the connective tissue, maintaining periodontal homeostasis. Imbalance of subgingival bacterial communities could damage gingival barrier allowing bacterial antigens to get access to the deeper connective tissue where they activate memory T cells leading to deleterious inflammation; a hallmark of periodontitis.     

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry

AIM: To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. METHODOLOGY: Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. RESULTS: Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). CONCLUSIONS: Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.     

Identification of T‐cell epitopes of Porphyromonas gingivalis heat‐shock‐protein 60 in periodontitis

The heat shock proteins (hsp) of bacterial species are considered to be involved in regulating the autoimmune mechanism in human diseases due to the considerable homology of their sequences with human hsp. To elucidate how stress proteins contribute to the immunopathogenesis of periodontitis, mononuclear cells from gingival connective tissue of 10 periodontitis patients were simulated with Porphyromonas gingivalis hsp60. T‐cell lines reactive to P. gingivalis hsp60 were established from each patient to define T‐cell epitope specificities. Anti‐P. gingivalis IgG antibody titres were elevated in all patients. We could establish P. gingivalis hsp‐reactive T‐cell lines from gingival mononuclear cells that were mixtures of CD4⁺ and CD8⁺ cells. Of 108 overlapping synthetic peptides spanning the whole P. gingivalis hsp60 molecule, 10 peptides with epitope specificities for T‐cells were identified, and were identical to those reported be B‐cell epitopes in periodontitis.     

Interleukin‐2, interleukin‐6 and T regulatory cells in peripheral blood of patients with Behçet’s disease and recurrent aphthous ulcerations

J Oral Pathol Med (2012) 41 : 73–79 Background: One of the factors involved in the pathogenesis of Behçet disease (BD) and recurrent aphthous ulcerations (RAU) is a cell‐mediated immune response in which several cytokines (interleukin‐2, interleukin‐6) and T regulatory cell (T reg cell) population seem to play a major role. The aim of this study was to measured the interleukin‐2 (IL‐2), interleukin‐6 (IL‐6) levels and analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells in peripheral blood from patients with BD and RAU. In addition; we also analysed peripheral blood from healthy subjects for comparison. Methods: Thirty patients (15 men and 15 women) with BD, 30 patients (12 men and 18 women) with RAU and 15 healthy control subjects (nine men and six women) participated in the study. Analysis of CD4⁺ CD25⁺ Foxp‐3⁺ Treg cells, IL‐2 and IL‐6 levels have been measured in flow cytometry. Results: No statistical differences were observed in the serum levels of IL‐2 and IL‐6 between BD and RAU patients, and healthy subjects. Although there were no statistical differences in the number of CD4⁺ CD25⁺ Foxp‐3⁺ cells between groups, there were statistically significant differences in the number of CD4⁺ CD25^bright Treg cells. CD4⁺ CD25^bright Treg cells were significantly increased in BD and RAU patients compared to healthy subjects. Statistical analysis revealed no difference according to the number of CD4⁺ CD25^bright cells between BD and RAU patients. Conclusions: These results indicate that CD4⁺ CD25^bright T regulatory cells may be contributing factor in the pathogenesis of BD and RAU.     

Toll‐like receptor 2‐mediated modulation of growth and functions of regulatory T cells by oral streptococci

This study was designed to determine whether oral streptococci modulate the growth and functions of regulatory T cells. Heat‐killed cells of wild‐type strains of Streptococcus gordonii and Streptococcus mutans induced the Toll‐like receptor 2 (TLR2) ‐mediated nuclear factor‐κB (NF‐κB) activation, but their lipoprotein‐deficient strains did not. Stimulation with these streptococci resulted in a significant increase in the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in splenocytes derived from both TLR2^+/+ and TLR2^?/? mice, but the level of increase in TLR2^+/+ splenocytes was stronger than that in TLR2^?/? splenocytes. Both strains of S. gordonii enhanced the proliferation of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells isolated from TLR2^+/+ mice at the same level as those from TLR2^?/? mice in an interleukin‐2‐independent manner. However, wild‐type and lipoprotein‐deficient strains of both streptococci did not enhance the suppressive activity of the isolated regulatory T cells in vitro, but rather inhibited it. TLR ligands also inhibited the suppressive activity of the regulatory T cells. Inhibition of the suppressive activity was recovered by the addition of anti‐IL‐6 antibody. Pretreatment of antigen‐presenting cells with the NF‐κB inhibitor BAY11‐7082 enhanced the suppressive activity of the regulatory T cells. These results suggested that interleukin‐6 produced by antigen‐presenting cells inhibits the suppressive activity of the regulatory T cells. Wild‐type strain, but not lipoprotein‐deficient strain, of S. gordonii reduced the frequency of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in the acute infection model, whereas both strains of S. gordonii increased it in the chronic infection model mice. Hence, this study suggests that oral streptococci are capable of modulating the growth and functions of regulatory T cells in vitro and in vivo.     

Expression of CD163, interleukin‐10, and interferon‐gamma in oral squamous cell carcinoma: mutual relationships and prognostic implications

Tumor‐associated macrophages (TAMs) and their associated inflammatory cytokines represent the major inflammatory component of the stroma of many tumors and can affect prognosis in the case of neoplasms. The objective of this study was to determine the prognostic significance of CD163⁺ cells, interleukin‐10 (IL‐10), and interferon‐gamma (IFN‐γ) in oral lesions associated with oral squamous cell carcinoma (OSCC). The levels of CD163, IFN‐γ, and IL‐10 in the tissue samples of 240 patients with OSCC and 58 patients with other oral lesions were assessed by immunohistochemistry. Individuals with low IFN‐γ levels, high IL‐10 levels, and low CD163 levels were of special concern with respect to OSCC progression. We found that high levels of CD163, or a combination of low IFN‐γ levels, high IL‐10 levels, and low CD163 levels, were associated with poorer overall survival (OS). CD163⁺ cells provide better predictive power for OS in comparison with traditional markers, such as clinical stage and lymph node metastasis. Therefore, CD163⁺ cells may be effective prognostic predictors of OSCC. IL‐10 may also indicate poor outcomes when IFN‐γ secretion is low and the cells are CD163^?.     

Role of Smoking and Type 2 Diabetes in the Immunobalance of Advanced Chronic Periodontitis

Background : This study evaluates the tissue levels of interleukin (IL)‐17⁺, IL‐15⁺, Foxp3⁺ cells, fibrosis, and plasma B‐cell infiltration in sites with chronic periodontitis in smokers and subjects with type 2 diabetes. Methods: Gingival biopsies were harvested from the following groups: systemically and periodontally healthy subjects (healthy group, n = 10); non‐smokers and subjects with advanced periodontitis and without diabetes (non‐risk factor/periodontitis group, n = 10); heavy smokers with advanced periodontitis and without diabetes (smoking/periodontitis group, ≥20 cigarettes per day for at least the past 5 years, n = 10); and non‐smoking poorly controlled subjects with diabetes (glycated hemoglobin levels ≥9%) with advanced periodontitis (diabetes mellitus/periodontitis group [DMP], n = 10). The number of IL‐17⁺, IL‐15⁺, and Foxp3⁺ cells was analyzed by immunohistochemistry, whereas the amount of fibrosis and plasma B‐cell infiltration in gingival tissue was analyzed by histomorphometry. Results: The number of Foxp3⁺ cells was significantly higher in the periodontitis groups compared to the healthy group (P <0.05). The DMP group presented higher levels of Foxp3⁺ cells than other periodontitis groups (P <0.05). The levels of IL‐15⁺ and IL‐17⁺ cells and the amount of fibrosis were higher in the DMP group than in the other groups (P <0.05). There was a trend for a decreased B‐cell infiltration in the DMP group (P >0.05). There was a slightly significant negative correlation between B‐cell infiltration and the amount of fibrosis (P <0.05). Conclusion: Upregulation of IL‐17⁺, IL‐15⁺, and Foxp3⁺ cells and increased amounts of fibrosis were observed in chronic periodontitis sites in subjects with type 2 diabetes, suggesting that periodontitis development in these subjects may be influenced by the T helper 17/T regulatory axis.