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 expression of macrophage migration inhibitory factor is correlated with receptor activator of nuclear factor kappa B ligand in induced rat periapical lesions.

INTRODUCTION: Macrophage migration inhibitory factor (MIF) has been defined as a key cytokine in regulation of innate and adaptive immunity. The purpose of this study was to investigate the immunohistochemical localization of MIF and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) protein during the development of periapical lesions in rats. METHODS: Apical periodontitis was induced in Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were randomly killed at 0, 7, 14, 21, 28, and 35 days after pulp exposure. The jaws that contained the first molar were obtained and were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and double immunofluorescence staining. RESULTS: From day 0 to day 35, the areas of periapical bone loss increased and seemed to be stabilized on day 35. A few MIF-positive and RANKL-positive cells and osteoclasts could be observed on day 7, and all climaxed on day 14. From day 21 to day 35, the expression of MIF and RANKL protein decreased, and fewer osteoclasts could be observed. CONCLUSIONS: These findings showed that MIF might be associated with the differentiation of osteoclasts in the periapical lesions. MIF contributes to the pathogenesis of the periapical lesions through the induction of RANKL protein.

Evidence supporting a protective role for th9 and th22 cytokines in human and experimental periapical lesions.

INTRODUCTION: The development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice. METHODS: Periapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via real-time polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated. RESULTS: IL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals. CONCLUSIONS: Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability.

Increased gene expression of Toll-like receptors and antigen-presenting cell-related molecules in the onset of experimentally induced furcation lesions of endodontic origin in rat molars.

INTRODUCTION: Early immunopathogenic mechanisms behind pulp infection-induced furcal inflammation have not been well understood. To address the immunopathology of the pulp infection-induced furcal region of the periodontal ligament (PDL), we performed immunohistochemical and quantitative gene expression analyses for toll-like receptors (TLRs) in the furcal PDL of rat molars subjected to unsealed or sealed pulpotomy. METHODS: Furcal inflammation in rat molars was generated by making unsealed pulpotomies that were exposed to the oral environment for 24 hours. Pulpotomized teeth sealed with a temporary filling material and untreated normal teeth served as controls. Gene expression was analyzed with laser capture real-time polymerase chain reaction for TLR-2, TLR-4, and antigen presenting cell (APC)-related molecules (class II MHC, CD83, and CD86). Immunohistochemistry for TLR-2 and TLR-4 was also performed. RESULTS: Messenger RNA expression levels of TLRs and the APC-related molecules in the furcal periodontal ligament were significantly up-regulated in teeth with unsealed pulpotomy. Immunohistochemistry for unsealed pulpotomized teeth revealed that TLRs-expressing cells were predominantly distributed within the PDL beneath the furcal dentin. CONCLUSIONS: These results suggested the involvement of innate immune mechanisms involving TLRs and resulting activation of APCs in the early pathogenesis of pulp infection-induced furcal inflammation.

Artificial dental pulp exposure injury up-regulates antigen-presenting cell-related molecules in rat central nervous system.

INTRODUCTION: Bacterial infection and resulting inflammation of the dental pulp might not only trigger neuroimmune interactions in this tissue but also sensitize the central nervous system (CNS) such as the thalamus via nociceptive neurons. Thus, immunopathologic changes in the rat thalamus that take place after pulp inflammation were investigated. METHODS: Pulp exposure was made in mandibular right first molars of 5-week-old Wistar rats. After 24 hours, the thalamus was retrieved and subjected to either immunohistochemistry for class II major histocompatibility complex (MHC) molecules and glial fibrillary acidic protein (GFAP) or mRNA expression analysis of antigen-presenting cell-related molecules and N-methyl-D-aspartate receptor 2D subunit (NR2D) by means of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: At 24 hours after pulp exposure, the density of class II MHC molecule-expressing and GFAP-expressing cells was increased in the contralateral thalamus. Gene expression analysis revealed the up-regulation of class II MHC molecules, CD80, CD83, CD86, and NR2D in the contralateral thalamus, as compared with the ipsilateral thalamus. CONCLUSIONS: These results suggest the signal of pulp inflammation induces neuronal activation in the CNS.

Influence of selective immunosuppressive drugs on the healing of exposed dogs' dental pulp capped with mineral trioxide aggregate.

INTRODUCTION: Immunosuppressive drugs are used in clinical medicine for a variety of disorders, but their effects on the reparative capacity of the dental pulp are unknown. This study evaluated the influence of selected immunosuppressive drugs on pulpal tissue healing after direct pulp capping of mechanically exposed dog's teeth with mineral trioxide aggregate (MTA). METHODS: Ten healthy male dogs were assigned into 5 experimental groups: a control group in which no drug was received and 4 experimental groups in which the immunosuppressive drugs prednisone, mycophenolate mofetil, sirolimus, and cyclosporine A were administered 45 days before the operative procedures and until the dogs were killed. Class V cavities were prepared on the buccal surfaces of 12 teeth in each dog. In each cavity, the pulp was exposed and capped with MTA. The pulpal tissue responses to capping material were assessed 65 days postoperatively. RESULTS: Compared with the control group, variable responses was recorded in the groups treated with mycophenolate mofetil, sirolimus, and cyclosporine A, which were characterized by moderate to severe inflammatory reactions, tissue necrosis, and total absence of hard tissue bridging. Pulpal tissue responses in the group treated with prednisone were characterized by inflammatory cell infiltration, limited tissue necrosis, as well as partial to complete hard tissue bridging. CONCLUSIONS: From these findings, it seemed evident that acceptable repair of the dentin-pulp complex, eg, wound healing with hard tissue formation after capping with MTA, is unlikely with mycophenolate mofetil, sirolimus, or cyclosporine A immunosuppressive drug therapy.

Regulatory T cells in mouse periapical lesions.

INTRODUCTION: T-regulatory (Treg, CD4+ FOXP3+) cells constitute a unique subpopulation of CD4+ T cells that inhibit T-cell responses and prevent disease development/exacerbation in models of autoimmunity. In the present study, we tested the hypothesis that Treg cells are induced in periapical lesions by dental pulp infection. METHODS: In situ hybridization (ISH) was used to localize FOXP3+ cells on day 21 after pulp exposure of the first molar teeth and infection with bacteria from the oral environment. FOXP3/GFP knock-in transgenic mice were used to quantify FOXP3+ Treg cells that infiltrate into periapical lesions by flow cytometry on days 7, 14, and 21 after infection. Periodontal ligament from uninfected teeth served as a negative control. RESULTS: ISH showed strong signals that showed the presence of FOXP3+ cells mainly at the periphery of periapical lesions. In contrast, no positive cells were present in the periodontal ligament of uninfected controls. Flow cytometry showed an increase in the number of FOXP3+ Treg beginning between day 7 and day 14 (0.69% of the infiltrate) after infection and increased to day 21 (0.94%) (p < 0.05 and p < 0.001, respectively, vs uninfected controls). Treg were also increased in number in draining cervical lymph nodes after pulpal infection. CONCLUSIONS: These results show that Treg cells are induced to infiltrate into periapical lesions by pulpal infection and suggest that they increase in a time-dependent manner.

IL-1 alpha and TNF-alpha expression in early periapical lesions of normal and immunodeficient mice.

T-helper and B-lymphocytes may contribute to mechanisms that result in bone-resorptive cytokine production in periapical lesion. Mice with severe combined immunodeficiency (scid) lack functional B- and T-cell immunity. The purpose of this study was to investigate the progression of pulp necrosis and the histomorphometric features of periapical lesions in scid vs. normal mice. The expression of the bone-resorptive cytokines IL-1 alpha and TNF-alpha was also investigated. Sixteen five-week-old homozygous scid mice and 14 normal BALB/cJ mice were used. The pulps of mandibular first molars were exposed for 1, 2, 3, or 4 weeks. Blocks of tissue containing the mandibular teeth and supporting structures were processed for both light microscopic examination and immunohistochemical staining for IL-1 alpha dna TNF-alpha. Central sections were randomized, their images were blindly digitized into a computer, and the areas of the lesions surrounding the distal root apices were measured. The cells that stained positively for the cytokines in the same area of adjacent sections were counted. Pulp necrosis progressed at similar rates in teeth from both strains. A progressive and significant increase in the periapical lesion size in both strains was observed. The scid mice lesions were significantly smaller than the controls at only the three-week period. There was heavy cytokine staining in periapical lesions from both strains, especially in areas that contained a mixed inflammatory infiltrate or fibroblasts. The number of positively staining cells was proportional to the lesion size. Therefore, pulpal and periapical pathosis were independent of the presence of functional T- and B-cells in this model.

Immunolocalization of bone-resorptive cytokines in rat pulp and periapical lesions following surgical pulp exposure.

The bone-resorptive cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) have been implicated in the pathogenesis of many chronic inflammatory diseases, including pulpitis and apical periodontitis.To further elucidate their role in these disorders, we have identified cells that express IL-1 alpha and TNF alpha in infected pulps and in developing rat periapical lesions after surgical pulp exposure. As detected by immunohistochemistry, IL-1 alpha- and TNF alpha-positive cells were present as early as 2 days after pulp exposure in both the pulp and periapical region. The numbers of cytokine-expressing cells increased up to day 4 in the pulp and up to day 30 in the periapex. In contrast, cells expressing IL-1 beta and TNF beta, the homologous forms of these mediators, were not found in pulp or periapical lesions during this period. Cells expressing IL-1 alpha and TNF alpha were identified primarily as macrophages and fibroblasts, with occasional staining of polymorphonuclear leukocytes. Osteoblasts and osteoclasts were also positive, whereas lymphocytes were negative. In general, cytokine-expressing cells were located proximal to abscesses and the root apex. These findings demonstrate that cells that express bone-resorptive cytokines IL-1 alpha and TNF alpha are present immediately after pulp exposure in this model, which supports the hypothesis that these mediators play a key role in pulpal and periapical pathogenesis, including the concomitant bone destruction. They also indicate that both resident connective tissue cells as well as infiltrating cells express bone-resorptive cytokines in response to infection in these lesions.

Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth.

The condition of the pulp tissue was classified into seven groups according to the depth of carious lesions from stage (S) 0 (non-carious teeth) to S6 (exposed pulp). A substantial change in the infiltration of immunocompetent cells occurred between S3 and S4; all types were markedly increased in S4 as compared to S3, with a remarkable increase in the number of helper T lymphocytes, B-lineage cells, neutrophils and macrophages. Therefore, the pulpal immune reaction to carious stimuli could be classified into early (S1-S3) and advanced phases (S4-S6). In the early phase a cellular immunoresponse would be induced by T-lineage cells, and in the advanced phase the humoral immunoresponse is furthered by B-lineage cells concomitant with the destruction of pulp tissue by proteolytic enzymes released from infiltrating neutrophils and macrophages. Human dental pulp is thus equipped with a functional immune response that is sufficient as a biodefensive mechanism. Dental caries should be treated before S4.

Quantitative analysis of immunoglobulins and inflammatory factors in human pulpal blood from exposed pulps.

The levels of immunoglobulins (IgG, IgA, and IgM) and inflammatory factors (elastase, prostaglandin E2, interleukin-1 alpha, interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha) in the blood of human dental pulp were quantified, using enzyme-linked immunosorbent assays. The pulpal blood samples were obtained with nylon fiber pellets from normal and inflamed dental pulp at pulp sites exposed on pulpectomy. Statistically significant differences between normal and inflamed pulp were found in the levels of IgG (p < 0.01), IgA (p < 0.05), IgM (p < 0.01), elastase (p < 0.05), and prostaglandin E2 (p < 0.01). These findings indicate that these factors play an important role in the pathogenesis of pulpal disease, and the sampling methods used in this study are useful for examination of pulpal inflammation.

Effect of injury on pulpal levels of immunoreactive substance P and immunoreactive calcitonin gene-related peptide.

Neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P are present in dental pulp in relatively high concentrations. Previous studies have demonstrated that the staining density of immunoreactive CGRP (iCGRP) changes in dental pulp after tissue injury. This study evaluated injury-related changes in levels of both immunoreactive CGRP (iCGRP) and immunoreactive substance P (iSP) in dental pulp using radioimmunoassays. After pulpal exposure, iSP levels decreased to about 10% of baseline values, while iCGRP levels decreased to about 45% of baseline measures. After dentin exposure with acid etch, iSP levels decreased to about 10 to 20% of baseline measures, while iCGRP levels decreased to 60% of baseline values. For both forms of injury, iSP decreased to a greater extent than did iCGRP levels. Collectively, these findings indicate that pulpal neuropeptides undergo dynamic, injury-specific, and peptide-specific responses following trauma to dental pulp.