Interleukin-6 in oral diseases: a review.

Interleukin-6 (IL-6) is a pleomorphic cytokine involved in a number of physiologic and pathologic processes including response to trauma and infection and development and progression of inflammation and malignancy. IL-6 is emerging as an important mediator and novel therapeutic target for chronic inflammatory diseases and cancer. The present study reviews the available evidence regarding the association between IL-6 and a range of oral diseases including infections (periodontal disease and endodontic infections), immunologically mediated disorders (oral lichen planus and Sjögren's syndrome) and malignancy (oral cancer and precancer). The role of common genetic variants of IL-6 in determining individual susceptibility to certain oral diseases, as well as novel therapeutic strategies based on IL-6 inhibition are also discussed.

Cytokine expression in response to root canal infection in gnotobiotic mice.

AIM: To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model. METHODOLOGY: Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor ß (TGF-ß) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05). RESULTS: The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-ß mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-ß mRNA expression during the chronic phase. CONCLUSION: Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-ß and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response.

Pattern-recognition receptors in pulp defense.

Initial sensing of infection is mediated by germline-encoded pattern-recognition receptors (PRRs), the activation of which leads to the expression of inflammatory mediators responsible for the elimination of pathogens and infected cells. PRRs act as immune sensors that provide immediate cell responses to pathogen invasion or tissue injury. Here, we review the expression of PRRs in human dental pulp cells, namely, receptors from the Toll-like (TLR) and Nod-like NLR families, by which cells recognize bacteria. Particular attention is given to odontoblasts, which are the first cells encountered by pathogens and represent, in the tooth, the first line of defense for the host. Understanding cellular and molecular mechanisms associated with the recognition of bacterial pathogens by odontoblasts is critical for the development of therapeutic strategies that aim at preventing excessive pulp inflammation and related deleterious effects.

Evaluation of chemokines and receptors in gnotobiotic root canal infection by F. nucleatum and E. faecalis.

The present study aims to evaluate the longitudinal effects of induced experimental infections in gnotoxenic animals on the expression of inflammatory chemokines and their receptors in periradicular tissues. The null hypothesis tested was that Enterococcus faecalis and Fusobacterium nucleatum had no effect on CCR5, CCL5, CXCL10, CCL2/MCP-1, CXCR2 and CCR1 expression. Two groups of five animals (n = 5) aged between 8 and 12 weeks were used in this study. The animals were anaesthetized, and coronary access was performed in the first molar on the right and left sides. Microorganisms were inoculated into the left molar, and the right molar was sealed without contamination to function as a control. Animals were sacrificed 7 and 14 days after infection, and periapical tissues were collected. The cytokine mRNA expression levels were assessed using real-time PCR. The chemokine mRNA expression levels demonstrated that the experimental infection was capable of inducing increased chemokine expression on day 7 compared to that on day 14, except for CCR5 and CCL5, which showed no changes. The gnotoxenic animal model proved to be effective and allowed evaluation of the immune response against a known infection. Additionally, this study demonstrates that gene expression of chemokines and their receptors against the experimental infection preferentially prevailed during the initial phase of induction of the periradicular alteration (i.e., on day 7 post-infection).

An overview of the dental pulp: its functions and responses to injury.

The dental pulp is a unique tissue and its importance in the long-term prognosis of the tooth is often ignored by clinicians. It is unique in that it resides in a rigid chamber which provides strong mechanical support and protection from the microbial rich oral environment. If this rigid shell loses its structural integrity, the pulp is under the threat of the adverse stimuli from the mouth, such as caries, cracks, fractures and open restoration margins, all of which provide pathways for micro-organisms and their toxins to enter the pulp. The pulp initially responds to irritation by becoming inflamed and, if left untreated, this will progress to pulp necrosis and infection. The inflammation will also spread to the surrounding alveolar bone and cause periapical pathosis. The magnitude of pulp-related problems should not be underestimated since their most serious consequence is oral sepsis, which can be life threatening, and hence correct diagnosis and management are essential. Clinicians must have a thorough understanding of the physiological and pathological features of the dental pulp as well as the biological consequences of treatment interventions.

Pulpal status of hypomineralized permanent molars.

PURPOSE: Young patients with hypomineralized teeth frequently complain of symptoms suggestive of dentin hypersensitivity. It has been proposed that these symptoms may be exacerbated by an underlying pulpal inflammation. The purpose of the study was to determine the pulpal status of hypomineralized teeth. METHODS: The experimental material comprised 25 sound and 19 hypomineralized permanent first molars obtained from children requiring dental extractions under general anesthesia. Pulp sections were processed for indirect immunofluorescence using combinations of: (1) protein gene product 9.5; (2) leukocyte common antigen; and (3) Ulex europaeus I lectin. Image analysis was then used to determine the percentage area of staining of each label. RESULTS: Innervation density was significantly greater in the pulp horn and subodontoblastic region of hypomineralized teeth than in sound teeth. Immune cells were most abundant within pulps of hypomineralized teeth exhibiting enamel loss. Vascularity was found to be similar for both hypomineralized and sound teeth, but was significantly greater in hypersensitive hypomineralized samples. CONCLUSION: This study provides biological evidence that inflammatory changes may be present within the pulpal tissue of these teeth.

A study of T and B cells in pulpal pathosis.

A study was undertaken using monoclonal antibodies to determine the types of lymphocytes present in pulpal tissues. Pulps were extirpated from teeth clinically diagnosed as normal, reversibly inflamed, or irreversibly inflamed and stained with hematoxylin and eosin and an indirect immunoperoxidase technique using monoclonal antibodies reactive to pan-B lymphocytes (B), pan-T lymphocytes (T1), and helper (T4) and suppressor (T8) T lymphocytes. T and/or B lymphocytes were observed in normal pulpal tissues with T8 lymphocytes being predominant. The pulpal tissue in the reversible group demonstrated that more than 90% of the lymphocyte population were T lymphocytes, with a T4/T8 ratio of 0.56. Higher numbers of T1, T4, T8; and B lymphocytes were observed in the pulp from teeth in the irreversible group. A ratio of 1.14 of T4/T8 lymphocytes was observed in the irreversible group. A B/T1 lymphocyte ration of 1.60 suggested this ratio might be used as an index in the immunohistological diagnosis of irreversible pulpal pathosis. There appeared to be no association between the periodontal status of the teeth and the number of immunocompetent cells observed in the pulps. An hypothesis on the regulatory functions of T4 and T8 lymphocytes as well as the interaction of T and B lymphocytes and their products in the pathogenesis of pulpal disease is presented.

Detection of immunoglobulins from explant cultures of periapical lesions.

The presence of immunoreactive cells in periapical inflammatory lesions suggests that immune responses participate in the disease process. The purpose of this study was to determine the presence and concentration of immunoglobulins in the supernatant fluids of explant cultures of periapical lesions. Ninety periapical lesions that had been contiguous with the apex of a root were removed and maintained in explant cultures for 96 h. Tissue culture medium was replenished at 24, 48, 72, and 96 h. Double diffusion in agarose assays demonstrated the presence of IgG in 100% of the 24-h supernatant fluids and IgA in 65% of the 24-h supernatant fluids. However, IgM was not detected. Radial immunodiffusion assays were used to detect and quantitate IgG, IgA, and IgM in samples of 24-h supernatant fluids from 90 explant cultures. IgG was the predominant immunoglobulin followed by IgA. A radioimmunosorbent test was used to detect and quantitate IgE in samples of 24-h supernatant fluids from 90 explant cultures. Forty of the 90 supernatant fluids contained measurable IgE. All detected immunoglobulins decreased in concentration in daily supernatant fluids with time (24, 48, 72, and 96 h) in the culture.

Presence of secretory IgA in human periapical lesions.

The concentration of secretory IgA in fluids present in the canals of 33 teeth was determined by the rocket immunoelectrophoresis technique. Except for the presence or absence of communication between the oral cavity and the root canals of the affected teeth, no other clinical finding showed significant statistical correlation with the presence of secretory IgA. The canals which were open to the oral flora had significantly higher concentrations of secretory IgA. Leaving canals open to the oral cavity may result in formation of periapical cysts.

A microbiological and immunological study of endodontic-periodontic lesions.

The microflora and humoral immune response in tissue from the periodontal pockets and root canals of five teeth with endodontic-periodontic lesions were examined. We found more microbes in the periodontal pockets than in the root canals. The flora in the periodontal pockets was dominated by rods and motile organisms, whereas that in the root canals was largely rods and cocci. We detected no spirochetes in the root canals. The cultivable microflora in the periodontal pockets comprised a high number of different species of bacteria, whereas those in the root canals included only a small number of species. There was no correlation between microbial isolates and antibody titer in the apical tissues or periodontal pockets. We conclude from these studies that the microflora of infected root canals is simple and limited, and that the local humoral immune response does not seem to affect the pathogenesis of disease directly.

Cellular immuno-competence of infected root canal contents in pathogenesis of periapical lesions.

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.

Reactivity of IgG from explant cultures of periapical lesions with implicated microorganisms.

The presence of IgG in periapical inflammatory lesions suggests that immune responses participate in the disease process. The purpose of this investigation was to study the reactivity of IgG from the supernatant fluids of explant cultures of periapical lesions with microorganisms implicated in infections of endodontic origin. Ninety periapical lesions that had been contiguous with the apex of a root were removed and maintained in explant cultures. A dot-enzyme-linked immunosorbent assay (dot-ELISA) was used to demonstrate the presence of IgG in the supernatant fluids of the explant cultures reactive with a panel of microorganisms associated with infections of endodontic origin. The percentages of reactivity by dot-ELISA follow: Bacteroides intermedius (84%), B. buccae (12%), Porphyromonas (Bacteroides) gingivalis (50%), P. endodontalis (58%), P. asaccharolyticus (17%), Peptostreptococcus micros (44%), P. anaerobius (26%), Eubacterium alactolyticum (34%), Fusobacterium nucleatum (14%), and Actinomyces israelii (6%). At least one of the three species of B. intermedius, P. gingivalis, or P. endodontalis tested gave a positive dot-ELISA with 89% of the supernatant fluids from explant cultures of periapical lesions. A lack of cross reactivity of IgG in supernatant fluids from explants of periapical lesions was demonstrated for the four strains of black-pigmented Bacteroides/Porphyromonas by dot-ELISA.

Interleukin-1 alpha and interleukin-1 beta periapical exudates of infected root canals: correlations with the clinical findings of the involved teeth.

This study quantified the concentrations of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) in the periapical exudates obtained from 69 single-rooted teeth using enzyme-linked immunosorbent assays, and examined their correlation with clinical findings of the involved teeth. Changes in the levels of these factors during root canal treatment were also investigated. The average levels of IL-1 beta (6.57 ng/ml) in periapical exudates were twice that of IL-1 alpha (3.25 ng/ml). The exudates containing pus showed significantly higher IL-1 alpha levels than those from the canals without pus (p < 0.01). The exudates from the canals with small radiolucent areas contained significantly higher IL-1 alpha levels than those from the canals with large radiolucent areas (p < 0.05). The tendency for there to be an increase in the levels of IL-1 alpha and a decrease in the levels of IL-1 beta was observed following root canal treatment. These observations suggest that IL-1 alpha and IL-1 beta are involved in the immunopathogenesis of periapical lesions and that IL-1 alpha and IL-1 beta may play different roles in the healing process of periapical lesions during root canal treatment.

Antibodies in normal and diseased pulps reactive with microorganisms isolated from deep caries.

Immunoglobulin molecules in the supernatant fluids (SF) from pulpal explant cultures have been observed to react with microorganisms implicated in infections of root canals. In this study, the reactivity of immunoglobulin molecules in the SF from normal and irreversible pulpitis pulps to six strains of predominant microorganisms isolated from the immediate layer of carious lesions above the pulps used for explant cultures was investigated using an enzyme-linked immunosorbent assay. Two ATCC strains of Eubacterium were also included in this assay. Specific antibodies to Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus, Lactobacillus acidophilus (I), (II), Streptococcus mutans, Bacteroides intermedius, Eubacterium brachy, and Eubacterium alactolyticum in the SF from the normal and irreversible pulpitis tissues were observed with a large variation of antibody levels in both groups. Immunodiffusion assays of the SF revealed that IgG was the major class of immunoglobulin in the normal as well as the irreversible groups. The presence of natural antibodies in the normal pulps suggested a possible protective role of antibodies during the invasive process of caries.

Production of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta by human polymorphonuclear neutrophils stimulated with Porphyromonas endodontalis lipopolysaccharide.

This study was undertaken to investigate the capacity of polymorphonuclear neutrophils (PMNs) to secrete Macrophage Inflammatory Protein (MIP)-1alpha and MIP-1beta after stimulation with Porphyromonas endodontalis lipopolysaccharide (LPS). Escherichia coli LPS was used as a positive control. Venous blood was collected and PMNs were isolated from healthy volunteers. Cells were cultured with various concentrations of LPS for different periods of time. Cell supernatants were assayed by enzyme-linked immunosorbent assay. The levels of chemokine secretion in PMNs stimulated with each LPS were found to be significantly higher than in the unstimulated control cells (p < 0.05), and this expression occurred in a time- and dose-dependent manner. E. coli LPS induced higher levels of cytokines than P. endodontalis LPS. These findings demonstrated that P. endodontalis LPS is capable of stimulating PMNs to produce chemotactic cytokines and suggested that PMNs stimulated with P. endodontalis LPS may play a crucial role in the inflammatory and immunopathological reactions of pulpal and periapical diseases.

[Immunological phenomena in pulpal-periapical lesions]. / Fenomeni immunitari nelle lesioni pulpo-periapicali.

Based on an extensive review of the literature, the authors examine immunological reactions in pulpal and periapical lesions. Although it has been known for some time that bacterial infection causes this pathology, attention has been recently focused on immunological factors in the ambit of the phlogistic process. The present study examines the correlation between the latter and the type of extent of antigenic response, focusing attention on their important role in the phenomena of osteoclastic activation and inhibition of bone repair. From a physiological point of view there are few inflammatory cells in dental pulp, like macrophages and T lymphocytes. When the pulp comes into contact with the antigenic substance it activates a specific and aspecific immune response: the form through the activation of B and T lymphocytes, and the latter through the action of LPS, PMN and complement. An important role in the immune response is played by the cytokines which are able to regulate the intensity and duration of the immune response against potential pathogenic agents. It was initially thought that these were only produced by lymphocytes and as a result they were known as lymphokines. Later it was observed that other cell populations were also able to produce them. Phlogosis of the periapex starts before the pulp is fully necrotic. Tissue detritus and products of bacterial derivation escape through the numerous endoparadontal communication pathways and stimulate an inflammatory response by the vascular system of the parodontal ligament. The concomitant immune reaction occurs due to the tendency to block and restrict the inflammation to the radicular channels, thus preventing its diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

[Activation of the immune system in periapical infection. 1. Lympho-monocytoid and plasma cellular elements of reactive soft tissue cells]. / Attivazione del sistema immunocompetente nella patologia del periapice infetto. Nota 1: elementi cellulari della serie linfomonocitoide, plasmacellulare e propri dei tessuti molli reattivi.

The authors have carried out a study on the immunitary mechanisms which stimulate and avoid eventual alterations of infected periapex. Above all the aim of this first study has been the microscopic and ultrastructural valuation of the cellular components that characterize the process of chronic phlogosis of periradicular tissue, lymphocytes T and B, plasmacells and macrophages, and of those even more typical of the soft reactive tissues, fibroblasts and epithelial cells. It's just the interaction among these immunocompetent cells which determines the structural change of the periapical bone whose most common image of radiotransparence make it possible to diagnose the sufference of the pulpo-periapical system.

Evaluation of chemokines and receptors in gnotobiotic root canal infection by F. nucleatum and E. faecalis

Abstract The present study aims to evaluate the longitudinal effects of induced experimental infections in gnotoxenic animals on the expression of inflammatory chemokines and their receptors in periradicular tissues. The null hypothesis tested was that Enterococcus faecalis and Fusobacterium nucleatum had no effect on CCR5, CCL5, CXCL10, CCL2/MCP-1, CXCR2 and CCR1 expression. Two groups of five animals (n = 5) aged between 8 and 12 weeks were used in this study. The animals were anaesthetized, and coronary access was performed in the first molar on the right and left sides. Microorganisms were inoculated into the left molar, and the right molar was sealed without contamination to function as a control. Animals were sacrificed 7 and 14 days after infection, and periapical tissues were collected. The cytokine mRNA expression levels were assessed using real-time PCR. The chemokine mRNA expression levels demonstrated that the experimental infection was capable of inducing increased chemokine expression on day 7 compared to that on day 14, except for CCR5 and CCL5, which showed no changes. The gnotoxenic animal model proved to be effective and allowed evaluation of the immune response against a known infection. Additionally, this study demonstrates that gene expression of chemokines and their receptors against the experimental infection preferentially prevailed during the initial phase of induction of the periradicular alteration (i.e., on day 7 post-infection).

High levels of CXC ligand 12/stromal cell-derived factor 1 in apical lesions of endodontic origin associated with mast cell infiltration.

INTRODUCTION: CXC ligand 12/stromal-derived factor-1 (CXCL12/SDF-1) is a pleiotropic chemokine that regulates the influx of a wide range of leukocytes. The aim of this study was to characterize CXCL12/SDF-1 in apical lesions (ALs) of endodontic origin, with special emphasis in associated immune cell populations. METHODS: In this case-control study, 29 individuals with chronic apical periodontitis and 21 healthy volunteers were enrolled. ALs and healthy periodontal ligament samples were obtained for tissue homogenization, immune Western blotting, and enzyme-linked immunosorbent assay to determine CXCL12/SDF-1 forms and levels. Anatomopathologic diagnosis, immunostaining for CXCL12/SDF-1, CD117-CXCL12/SDF-1, and toluidine blue were also performed to identify tissue and cell localization. Finally, a set of tissue samples were digested and analyzed by flow cytometry to identify CXCL12/SDF-1 in different immune cell populations. Data were analyzed with Stata v11 and WinDi 2.9 software, and significance was considered if P < .05. RESULTS: CXCL12/SDF-1 was predominantly identified as monomers; levels of CXCL12/SDF-1 were significantly higher in ALs compared with controls, and it was primarily localized to inflammatory infiltrates. Expression of CXCL12/SDF-1 was colocalized to mast cells in tissue sections. Furthermore, CD117(+) mast cells were the second most frequent infiltrating cells and the main CXCL12/SDF-1 expressing cells, followed by CD4(+) lymphocytes, monocytes/macrophages, neutrophils, and dendritic cells. CONCLUSIONS: ALs of endodontic origin demonstrated higher levels of CXCL12/SDF-1 compared with controls. CXCL12/SDF-1 was identified in immune cell populations, whereas mast cells represented the major CXCL12/SDF-1 expressing cells, suggesting that this chemokine might play a central role in apical tissue destruction, most probably inducing persistent recruitment of immune cells, particularly of mast cells.

MicroRNAs: new insights into the pathogenesis of endodontic periapical disease.

INTRODUCTION: Apical periodontitis is an inflammatory disease of the periradicular tissues caused by the host's immune response to infection of the root canal system. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of inflammation and the immune response; however, their role in the pathogenesis of endodontic periapical disease has not been explored. The purpose of this study was to examine the differential expression of miRNAs in diseased periapical tissues as compared with healthy controls. METHODS: We first compared miRNA profiles in diseased periapical tissues collected from patients undergoing endodontic surgery with those of healthy pulps by using microarray analyses. The target genes of the differentially expressed miRNAs were identified by using miRWalk and PubMed. Selected miRNAs linked to inflammation and the immune response were then confirmed in a separate cohort of diseased and healthy tissues by using quantitative reverse transcription-polymerase chain reaction. Healthy pulps and periodontal ligaments were used as controls. Data were normalized to the level of SNORD 44, which served as an endogenous control. RESULTS: Of the 381 miRNAs identified by using microarray, 24 miRNAs were down-regulated in diseased periapical tissues compared with controls (n = 13) (P < .003). The down-regulation of 7 miRNAs was confirmed from 9 selected miRNAs by using quantitative real-time polymerase chain reaction (n = 19) (P < .05). Target genes of these miRNAs include key mediators in the immune and inflammatory response such as interleukin-6, matrix metalloproteinase-9, and transforming growth factor-ß. CONCLUSIONS: These findings offer new insight into the pathogenesis of endodontic disease and have the potential to impact the development of new methods for prevention, diagnosis, and treatment of apical periodontitis.