Immunological profile of teeth with inflammatory periapical disease from chronic liver disease patients.

AIM: To evaluate the mRNA expression levels of the cytokines interferon-γ, tumour necrosis factor-α, interleukin (IL)-1ß, IL-10, IL-6, VEGF, and AGT and the chemokine CCL2/MCP-1 in periapical interstitial fluid associated with root canal infections before and after the reduction of the bacterial load using a cleaning procedure. METHODOLOGY: The case group included 11 patients with chronic liver disease, and the control group included 11 healthy patients. Clinical samples were taken from teeth with pulp necrosis. After cleaning and drying the canal, three paper points were introduced into the root canal and passed through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later to characterize those gene expression levels using real-time PCR. The data were subjected to the Shapiro-Wilk and the Wilcoxon tests. RESULTS: In the control group, significantly increased expression of the pro-inflammatory cytokines IFN-γ and TNF-α was observed in teeth with restrained bacterial loads (day 7) (P < 0.05). Similarly, increased TNF-α expression was found on day 7 in the liver group (P < 0.05). No differences were observed in the expression levels of the IL-1ß, IL-10 and, IL-6, MCP-1/CCL-2 and VEGF between the first collection (day 0) and second collection (day 7), over time in either group. CONCLUSION: Chronic liver disease patients exhibited sufficient immunologic ability showing relatively similar expression levels of cytokines, chemokines and angiogenic factors in periapical samples compared with the responses from no-chronic liver disease patients. The outcomes of this study suggest that liver impairment did not compromise the periapical immune response.

Periapical fluid RANKL and IL-8 are differentially regulated in pulpitis and apical periodontitis.

The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P < 0.05), whereas OPG was under the detection limit in most samples. In contrast, IL-8 levels were significantly lower in SIP compared to AAP (P < 0.05). Spearman's correlation analysis between RANKL and IL-8 revealed a significantly (P < 0.05) negative correlation between the two measures (rho = -.44). The results of this study suggest that, in the development of apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8.

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 TCRαß(+) 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-1ß 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.

The impact of chlorhexidine-based endodontic treatment on periapical cytokine expression in teeth.

INTRODUCTION: Root canal treatment typically involves cleaning and shaping procedures followed by treatment with antibacterial endodontic dressing between appointments and, ultimately, 3-dimensional,hermetic filling. Chlorhexidine (CHX) is effective as an irrigation solution and is used as an endodontic dressing. The aim of this study was to examine the influence of CHX on periapical cytokine expression. METHODS: Expression levels of the cytokines interferon γ, tumor necrosis factor α, interleukin (IL)-1ß, IL-17A, IL-10, and the chemokine monocyte chemoattractant protein-1 (CCL2/MCP-1) were assayed by real-time polymerase chain reaction immediately after root canal cleaning and 15 days later. RESULTS: Messenger RNA expression of IL-1ß, interferon γ, IL-10, and CCL2/MCP-1 was increased on day 15 in teeth without endodontic dressing. No statistical change was observed in the messenger RNA expression of cytokines when comparing sampling times for teeth that received endodontic dressing. CONCLUSIONS: The results show that CHX application between appointments prevented the increase of both proinflammatory and immunoregulatory cytokines 15 days after the dental procedure.

Mesenchymal stem cells from periapical lesions modulate differentiation and functional properties of monocyte-derived dendritic cells.

Immunoregulatory mechanisms within periapical lesions (PLs) are as of yet unexplored. Considering the crucial role of DCs in controlling the immune response within PLs, the immunomodulatory properties of mesenchymal stem cells (MSCs), and the colocalization of MSCs and DCs in situ, we wondered whether MSCs from PLs modulate the development and functions of DCs. Using a model of monocyte-derived DCs, we showed that PL-MSCs inhibited differentiation of DCs via soluble factors, of which IL-6 had a minor effect, but did not impair their subsequent maturation induced by pro-inflammatory cytokines. However, upon maturation such DCs favored the production of Th2/Th17 cytokines by allogenic CD4(+) lymphocytes in coculture, compared with mature DCs differentiated without PL-MSCs. PL-MSC-differentiated DCs, cultivated with pro-inflammatory cytokines and PL-MSCs, although phenotypically mature, exhibited poor allostimulatory activity, induced anergy, Th2 polarization, differentiation of suppressive CD4(+) CD25(high) CD39(+) Treg-cell subsets via IDO-1-, ILT-3-, and ILT-4-dependent mechanisms, and increased production of TGF-ß in the coculture. In contrast, DCs cultivated with PL-MSCs only during maturation stimulated proliferation and Th1 polarization of CD4(+) T cells in an IL-12-independent manner. In conclusion, PL-MSCs significantly modulate the development and functions of DCs, depending on the phase of DCs development during which the interaction occurs.

Interactions between immune system and mesenchymal stem cells in dental pulp and periapical tissues.

The recent isolation and characterization of mesenchymal stem cells (MSCs) in dental tissues constitutes a major step forward in the development of new treatment strategies. MSCs are essential for dental pulp repair and the success of regenerative endodontic procedures. It is important to understand that immune cells and cytokines can affect stem cell function, which can impact their healing potential. On the other hand, stem cells are immunoprivileged and have the ability to modulate immune and inflammatory responses, which can be utilized to improve treatments outcome. This review addresses both aspects of this interaction and suggests that any change on both sides can tip the balance in favour of either persistence of inflammation or healing. Finally, the therapeutic relevance of the interaction between MSCs and immune system relative to current treatments is discussed, and future research and treatment perspectives are suggested.

Monocyte chemotactic protein-3: possible involvement in apical periodontitis chemotaxis.

AIM: To study the expression of monocyte chemotactic protein-3 (MCP-3, also known as chemokine CCL-7) in tissue from apical lesions (AL) and to associate MCP-3 expression with symptomatic or asymptomatic apical periodontitis. METHODOLOGY: To determine the expression of MCP-3 in AL, biopsies obtained during tooth extraction procedures were fixed, subjected to routine processing and diagnosed as apical granuloma (AG) (n = 7) or radicular cyst (RC) (n = 5). As controls, apical periodontal ligament (PDL) specimens from healthy premolars extracted for orthodontics reasons were included (n = 7). All specimens were immunostained for MCP-3 and examined under a light microscope. In addition, homogenates from AL (n = 14) and healthy PDL samples (n = 7) were studied through immunowestern blot. Finally, periapical exudates samples were collected from root canals of teeth having diagnosis of symptomatic (n = 14) and asymptomatic apical periodontitis (n = 14) during routine endodontic treatments and analysed by immunowestern blot and densitometry. RESULTS: MCP-3 was detected in AG and RC and localized mainly to inflammatory leucocytes, whereas no expression was observed in healthy PDLs. MCP-3 was also detected in periapical exudate, and its levels were significantly higher in symptomatic than in asymptomatic apical periodontitis. CONCLUSIONS: MCP-3 was expressed in AL and its levels associated with clinical symptoms. MCP-3 might play a role in disease pathogenesis, possibly by stimulating mononuclear chemotaxis.

A controversial role for IL-12 in immune response and bone resorption at apical periodontal sites.

Periapical lesions are inflammatory conditions of tooth periapical tissues, triggered by dental pulp infection and characterized by exudation of immune cells to the affected tissues and production of inflammatory mediators such as cytokines. The inflammatory periapical reaction is mainly driven by Th1, Th2, and Th17 responses, and such polarization may modulate progression of the disease and expression of bone proresorptive cytokines. IL-12 is a potent inducer of IFN-γ production, which stimulates Th1 effector cells. Many evidences have shown a positive correlation between the bone resorptive cytokine IL-1ß and the production of IL-12 and IFN-γ. Furthermore, IL-12 may have a potential role in the release of bone resorptive mediators and blockade of Th2 cytokines, affecting the progression of periapical bone loss. Nevertheless, IL-12 and IFN-γ have also been described as suppressors of osteoclast differentiation and activation, favoring bone maintenance. This paper focuses on the controversial roles of IL-12 in periapical lesions.

[Formation of plasma cell infiltrates in gingiva of patients with chronic apical periodontitis].

The structure of leukocyte infiltrates in gingiva of 80 patients of various ages and gender with chronic apical periodontitis was studied using light microscopy with the application of monoclonal antibodies detecting CD38 antigen. Gingival tissues of practically of all the patients with periodontitis contained 2 types of leukocyte infiltrates: infiltrates with the low plasma cell content and high numbers of neutrophils and structures with high number of plasmocytes and low concentration of neutrophilic granulocytes. Within the gingival epithelium in patients with chronic apical periodontitis CD38 + cells were absent. The histogenesis of gingival leukocyte infiltrates in chronic apical periodontitis is discussed with the special emphasis on the role of plasma cells in the development of the pathological process.

The potential role of suppressors of cytokine signaling in the attenuation of inflammatory reaction and alveolar bone loss associated with apical periodontitis.

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which downregulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development.

Kinetic study of immunohistochemical colocalization of antigen-presenting cells and nerve fibers in rat periapical lesions.

Immune and nervous systems play key roles in periapical pathosis; however, their spatial associations, which may be a prerequisite for paracrine interactions in the progression of periapical lesions, remain to be examined. In this study we examined the distribution and frequency of spatial associations between major histocompatibility complex class II molecule-expressing antigen-presenting cells (APCs) and protein gene product 9.5-immunoreactive nerve fibers in experimentally induced rat periapical lesions using double-immunofluorescence staining and confocal laser scanning microscopy. When active lesion expansion started, macrophage-like APCs frequently associated with nerve fibers around the apex. When the lesions were starting to stabilize, however, close associations between APCs with dendritic morphology and nerve fibers were found mostly in the periphery of lesions. CD86+ mature dendritic cells were also observed in this area. These findings suggest that functional interactions between APCs and nerve fibers may play some roles in the development of self-defense reactions in periapical lesions.

An immunoelectron-microscopic study of class II major histocompatibility complex molecule-expressing macrophages and dendritic cells in experimental rat periapical lesions.

Previous studies have demonstrated that heterogeneous populations of class II major histocompatibility complex (MHC) molecule-expressing non-lymphoid cells, ultrastructurally classified as macrophages and dendritic cell (DC) cell-like cells, comprise the major immune cell population in experimental periapical lesions in rat molars. In this study, the temporal changes in relative proportions of the two types of cells were examined, on the hypothesis that they are involved in different aspects of the pathogenesis of the lesions. The lesions were induced by making surgical pulp exposures in mandibular first molars of 5-week-old Wistar rats. Observation periods were set at 0 (normal), 3, 14, 28, and 56 days. Non-lymphoid cells immunoreactive to OX6 (reactive to class II MHC molecules) were classified as macrophages and DC cell-like cells according to their ultrastructure, and the frequencies of the two types of cells were assessed at each time-point. ED1 (reactive to nearly all macrophages and DCs) was also used to identify macrophages and DC cell-like cells. At 3 days, most OX6+ cells and ED1+ cells in the periapical tissue had the ultrastructural appearance of newly recruited macrophages. At 14 days, when the lesion was actively expanding, there were significantly more OX6+ macrophages than OX6+ DC cell-like cells (P<0.01). However, at 28 days, when lesion expansion had ceased, DC cell-like cells significantly outnumbered OX6+ macrophages (P<0.01); this remained constant at 56 days. Cell-to-cell contact between OX6+ non-lymphoid cells and OX6- lymphocytes, suggesting a functional interaction, was most frequently seen at 28 days. These results support the notion that class II MHC molecule-expressing macrophages play some part in the initial lesion expansion, and suggest that DC cell-like cells may primarily be involved in immune defence against perpetuated antigenic challenges following lesion stabilization.

Endothelial cell and stromal antigens in human periapical granulation tissue.

Periapical granulation tissue consists of vasculature of varying sizes and types, infiltrating cells, and other stromal elements. We examined the differential expression of endothelial and stroma antigens in this tissue to determine their tissue distribution in order to obtain hints on their functions. Some of the antigens examined were present only in the endothelial lining of vasculature, including high endothelial venules (e.g. CD31 and CD105), whereas others were more widely expressed by both vascular and stromal elements (e.g. CD29, CD63, CD44, and CD151). Immunohistochemical analysis using monoclonal antibodies specific to certain tissue compartments revealed the tissue architecture more precisely and the expression of certain antigens in the tissue suggested special roles for these antigens. Tissue distribution of CD63, CD143, CD147, and CD151 in periapical granulation tissue is first reported in the present study.

CD45/isotypes expression in the immune cells of human periapical lesions.

Expression of some leukocyte antigens (including CD45) and its isoforms (CD2, CD4, CD5, CD6, CD7, and CD8) was examined in the human periapical granulation tissue samples in the present study. The majority of the infiltrating cells expressed heavy molecular-weight isoforms of the CD45 antigen. Expression of CD2, CD5, CD6, and CD7 antigens was also detected, implying significant roles for these antigens in the immune reaction taking place in periapical lesions. This suggests that the immune response taking place at the periapical region is predominantly cellular and the humoral responses to antigenic challenge are conducted mainly by regional lymph nodes.

Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1.

Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 microm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation.

Effects of formocresol alone vs. formocresol with eugenol on macrophage adhesion to plastic surfaces.

PURPOSE: The purpose of this study was to compare the in-vitro effects of a European-based formocresol formulation that incorporates eugenol with formocresol alone on the adhesion of macrophages to plastic surfaces. METHODS: Macrophages were obtained from Wistar rats. The adherence capacity of macrophages to a plastic surface was determined. Assays were carried out in Eppendorf tubes incubated for 15 min at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. RESULTS: Results showed that both formocresol/eugenol and formocresol alone significantly decreased the adherence index of macrophages. The formocresol formulation that incorporated eugenol was more potent in inhibiting macrophage adhesion than formocresol alone. CONCLUSIONS: Taking into account that adherence to a substrate is the first step in the phagocytic process of macrophages and in antigen presentation, both formocresol formulations could inhibit macrophage function and modulate immune and inflammatory responses in dental pulp and periapical tissues.

Detection of IgA subclasses and J chain mRNA bearing plasma cells in human dental periapical lesions by in situ hybridization.

Humoral immune responses are implicated in the pathogenesis of human dental periapical lesions. To elucidate whether IgA-associated immune systems play a role in the lesions, the in situ hybridization technique was used to detect J chain mRNA expression, which is correlated with the secretion of dimeric IgA. In addition, IgA subclass mRNA-expressing cells were also investigated by double target in situ hybridization (ISH) methodology using digoxigenin- and biotin-labeled IgA subclass specific oligonucleotide probes. This double target ISH technique involved immunochemical detection with an alkaline phosphatase-conjugated antibody and a peroxidase conjugated avidin-biotin complex system to detect IgA subclass mRNA in the formalin-fixed, paraffin wax embedded periapical tissue sections. Twenty-four biopsy samples (16 periapical granulomas and 8 radicular cysts) were examined. IgA subclass mRNA positive plasma cells were detected in all samples. IgA1 mRNA-expressing cells were predominant both in granulomas and cysts (mean = 75.3 +/- 11.2%, 64.8 +/- 21.3%, respectively), and the IgA1 proportion was higher in granulomas than in cysts, although no significant difference was seen between the two lesions (p = 0.132). J chain mRNA positive cells were very sparsely detected in 21/24 cases. The median percentages of J chain mRNA positive cells/IgA mRNA positive plasma cells (4.7%, range 0.3-13.6%) in cysts were significantly higher than in granulomas (1.3%, range 0-7.7%; p = 0.03). This result supports the hypothesis that dimeric IgA may be more actively produced in radicular cysts than in granulomas. These features are thought to reflect the local activation of the periapical immune system in response to environmental factors and indicate that secretory IgA mediated immune defense systems appear to play little role in these lesions. Our results indicate that the IgA-associated immune response in periapical lesions is more similar to serum or systemic IgA responses than to mucosa-associated immune responses where dimeric IgA predominates.

Immunohistochemical examination of the distribution of macrophages and CGRP-immunoreactive nerve fibers in induced rat periapical lesions.

To clarify the role of calcitonin gene-related peptide (CGRP) in the development of periapical lesions, we examined the distribution of CGRP-immunoreactive (IR) nerve fibers and macrophages, and the behavior of bone tissues in experimentally induced rat periapical lesions by immunohistochemical and quantitative methods. Although no extensive changes were observed at 7 days after pulp exposure, CGRP-IR nerve fibers increased in number until 28 days with a decrease thereafter. These neural changes were closely correlated with the alteration in number of macrophages except on day 7 when macrophages were significantly increased in number as compared with control rats. Tissue repair began to take place and a decrease in number of osteoclasts was observed when the density of CGRP-IR nerve fibers reached a peak. These results suggested that there might be a close relationship between macrophages and CGRP-IR nerve fibers and that CGRP-IR nerve fibers might participate in tissue repair in experimentally induced rat periapical lesions.

[Periapical lesions of mixed etiology: bacterial and foreign body]. / Lesioni periapicali ad eziologia mista: batterica e da corpo estraneo.

After a review of the literature on periapical lesion pathogenesis, we studied histological, immunological, and bacteriological examinations of 10 overfilled teeth with periapical lesions. We found, in our research, a bacteriological etiology with foreign body reactions.

Expression of inflammatory cytokine genes in vivo by human alveolar bone-derived polymorphonuclear leukocytes isolated from chronically inflamed sites of bone resorption.

Alveolar bone-derived polymorphonuclear leukocytes (PMNs) were characterized for their ability to produce inflammatory cytokines such as interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF alpha), and IL-6 in vivo. Periapical exudates (PE) were collected from periapical lesions with chronic periapical periodontitis through root canals. Cells and noncellular supernatants were then isolated by centrifugation. The concentration of cytokines present in the noncellular supernatants were determined by ELISA. High concentrations of IL-1 alpha, IL-1 beta, and IL-6 were detected in PE, however, TNF alpha was not. PE contains predominantly PMNs ( > 95% of residing cells) with a few percent of lymphocytes and/or macrophages. These alveolar bone-derived PMNs were purified by the Ficoll-Hypaque gradient method and were analyzed for cytokine mRNA expression using the cytokine-specific reverse-transcription polymerase chain reaction. Highly purified PMNs ( > 99.5%) isolated from PE expressed significant levels of mRNA for IL-alpha, IL-1 beta, and TNF alpha. IL-6 mRNA was not detected, although a high concentration of IL-6 was detected in supernatants of PE by ELISA. The IL-6 secretion in PE could be derived from macrophages, T lymphocytes, osteoblasts, or fibroblasts around periapical lesions. These data strongly suggest that human PMNs derived from alveolar bone can spontaneously produce IL-1 alpha, IL-1 beta, and TNF alpha at sites of inflammation, and probably initiate inflammation and regulate augmentation of bone resorption in vivo.