Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

高迁移率族蛋白B1对牙周膜成纤维细胞表达细胞因子影响的研究

目的观察高迁移率族蛋白B1（HMGB1）对人牙周膜成纤维细胞表达白细胞介素-6（IL-6）、破骨细胞核因子κB受体活化因子（RANKL）、骨保护因子（OPG）的影响,初步探讨HMGB1在牙周疾病的作用。方法采用原代组织块培养法,培养人牙周膜成纤维细胞,用第4~6代的细胞进行实验。分别用10、30、100 ng·mL-1质量浓度的HMGB1孵育牙周膜成纤维细胞24 h后,RT-PCR检测IL-6、RANKL、OPG的mRNA表达;Western blot法检测RANKL、OPG的蛋白表达。均以0 ng·mL-1质量浓度组为对照,所得数据用单因素方差分析处理。结果HMGB1在10、30、100 ng·mL-1质量浓度时,细胞中的RANKL/OPG mRNA的比值增高（P<0.05）,100 ng·mL-1质量浓度时细胞中的IL-6 mRNA的表达量增高（P<0.05）。Western blot检测结果显示10 ng·mL-1质量浓度组的RANKL/OPG的比值有明显增高。结论一定浓度的HMGB1可使牙周膜细胞中的RANKL/OPG比值增高,还会诱导炎症因子IL-6 mRNA表达上调。提示HMGB1可能会在牙周炎的发病以及炎症进展中发挥作用。     

高迁移率族蛋白1在慢性牙周炎病变牙龈组织中的表达

目的 研究慢性牙周炎病变牙龈组织中高迁移率族蛋白1（HMGB1）的表达。方法 提取健康志愿者外周血单核细胞（PBMC）,以1 pg·mL-1的细菌脂多糖（LPS）刺激细胞,24 h后用免疫荧光染色法检测HMGB1的表达,48 h 后用酶联免疫吸附试验检测细胞上清液中HMGB1的表达;分别以50 ng·mL-1肿瘤坏死因子-α（TNF-α）和100 ng? mL-1 HMGB1刺激PBMC,48 h后检测细胞上清液中HMGB1和TNF-α的表达。另外收集健康者和慢性牙周炎患者的牙龈组织和龈沟液,分别检测牙龈组织和龈沟液内HMGB1的表达。结果 LPS刺激PBMC 24 h后,HMGB1自细胞核移出至细胞质中;刺激48 h后,细胞上清液中HMGB1的表达量明显高于对照组（P<0.01）。TNF-α和HMGB1分别刺激 PBMC 48 h后,上清液中HMGB1和TNF-α的表达水平较对照组亦有明显增强（P<0.01）。在慢性牙周炎牙龈组织上皮钉突下方浸润的细胞中,HMGB1自细胞核转移至细胞质和细胞外;其龈沟液内HMGB1的表达量也明显高于健康对照组（P<0.01）。结论 HMGB1可能在牙周炎病理进程中有重要作用。     

高速泳动族蛋白盒1与正畸牙移动的相关性研究

正畸牙受矫治力作用后,其牙周组织将发生一系列的生物化学反应,多种细胞因子和激素参与了反应的整个过程。高速泳动族蛋白盒1(HMGB1)是一种重要的晚期炎症因子,参与骨组织改建并与成纤维细胞相互作用,据此推测其可能参与正畸牙移动过程中的牙周组织改建。本文就HMGB1与炎症反应、骨组织改建、成纤维细胞、牙周炎,正畸牙移动的生物学基础等研究现状作一综述。     

The expressions of IGF‐1, BMP‐2 and TGF‐β1 in cartilage of condylar hyperplasia

Summary Condylar hyperplasia is a complex post‐natal growth abnormality of the mandible and condyle, which leads to facial asymmetry. We investigated the distributions of insulin‐like growth factors (IGF‐1), bone morphogenetic protein‐2 (BMP‐2) and transforming growth factor‐β1 (TGF‐β1) in cartilage of condylar hyperplasia and revealed relationships between age and the cartilaginous thickness. Twenty patients with condylar hyperplasia were divided into four histopathological types. The cartilaginous thickness and age in different histological types were analysed, and the localizations of IGF‐1, BMP‐2 and TGF‐β1 were detected by immunohistochemistry analysis. The cartilaginous thickness of condylar hyperplasia significantly increased. The cartilaginous thickness of type III was significantly thicker than type I and type II, Bivariate correlation revealed a significant correlations between age and the cartilaginous thickness (r = 0·68, P = 0·01). However, the expressions of IGF‐1, BMP‐2 and TGF‐β1 were the strongest in type I. In almost all types of condylar hyperplasia, the presence of IGF‐1 and BMP‐2 was found mainly in the proliferative chondrocyte layer and the hypertrophic chondrocyte layer, and only a few in the calcified chondrocyte layer. The presence of TGF‐β1 widely distributed from the fibrous articular surface to the calcified cartilage. These findings suggest that the proliferative activity of cartilage in condylar hyperplasia is strongly associated with age and cartilaginous thickness. Therefore, the four pathological types of condylar hyperplasia seem more likely to be four discontinuous stages.     

Human gingival fibroblasts release high‐mobility group box‐1 protein through active and passive pathways

Introduction:  The nuclear protein high‐mobility group box‐1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS‐stimulated HGF. Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time‐dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

Association between condylar morphology and changes in bony microstructure and sub‐synovial inflammation in experimental temporomandibular joint arthritis

J Oral Pathol Med (2011) 40 : 111–120 Background: In juvenile idiopathic arthritis involvement of the temporomandibular joints (TMJs) is often associated with mandibular growth deviations. The relation between the growth deviations and severity of the inflammation, condylar shape, the micro‐architecture, and the quality of the bone has not previously been investigated. This paper studies the effect on the bony structures in mandibular condylar development in rabbits with antigen‐induced arthritis. Methods: Included were 42 juvenile rabbits with ovalbumin‐induced arthritis of the TMJs treated with intraarticular saline, intraarticular etanercept or subcutaneous etanercept. A TMJ from each animal was scanned using micro‐computed tomography and structural parameters were calculated. Three‐dimensional reconstructions of the mandibular condyle were scored blindly as normal or abnormal. TMJs were stratified for condylar morphology and were evaluated against data on trabecular structural parameters, inflammation, degree of mineralization, overall mandibular growth, and mineral apposition rate. Results: Abnormal morphology were seen in 15/32 animals available for data analysis. Erosions were an uncommon finding. Abnormal morphology was strongly related to the degree of inflammation. The trabecular separation was larger in group with abnormal morphology than in the group with normal morphology. Abnormal condylar morphology was not associated with overall mandibular growth. No differences were observed in mineral apposition rate. No differences in structural parameters were seen according to treatment modality. Conclusion: We showed that severe inflammation in the TMJs during mandibular development was associated with morphological changes in the mandibular condyle. These changes were predominantly seen at the macro‐morphological level and only very few differences were structural.     

Transcriptional regulation of proteoglycan 4 by 17β‐estradiol in immortalized baboon temporomandibular joint disc cells

Temporomandibular joint disorders (TMDs) affect a significant portion of the population of the USA, with the majority of those seeking treatment being women of childbearing age. Owing to this striking sexual dimorphism it has been postulated that sex hormones play a role in the maintenance of normal temporomandibular joint (TMJ) function. Proteoglycan 4 (PRG4) is a secreted lubricating molecule required for maintaining low frictional levels within articular joints; however, its role in the TMJ is not well characterized. In this study we describe the development of immortalized baboon cells isolated from specific regions of the TMJ disc and their use in the investigation of PRG4 expression and localization patterns in the TMJ. We identified conserved estrogen response elements within the 5′ flanking region of the PRG4 gene of several species, and found that treatment of baboon TMJ disc cells with estrogen led to reduced PRG4 promoter activity and reduced expression of PRG4 mRNA in vitro. The observed negative regulation of PRG4 by estrogen could lead to increased friction and degradation of joint components over time. This study, for the first time, provides evidence of the regulatory potential of estrogen on PRG4 gene expression and suggests a novel etiology for the gender disparity observed among TMD patients.