Interleukin-1β induces interleukin-6 mRNA expression and protein production in synovial cells from human temporomandibular joint

BACKGROUND: Interleukin (IL)-1 beta and IL-6 were found to be elevated in fluid from the temporomandibular joint (TMJ), although the source of these cytokines was not elucidated. There is little known about the function and response of synovial cells in the TMJ. The purpose of this study was to prepare cultured human synovial cells (HTS cells) from the TMJ and to investigate IL-6 production in HTS cells incubated with IL-1 beta. METHODS: HTS cells were isolated from temporomandibular joint synovial tissue using an outgrowth method and then cultured in Ham's F12 medium containing 10% fetal bovine serum. The HTS cells were treated with or without IL-1 beta for 3, 6, 9 and 24 h. IL-6 and soluble IL-6 receptor (sIL-6R) levels in cultured supernatant were measured by ELISA. IL-6 mRNA expression was investigated using immunocytochemistry and RT-PCR. RESULTS: HTS cells were morphologically heterogeneous. IL-1 beta increased IL-6 production in HTS cells. In those treated with IL-1 beta, several cells were strongly stained in the cytoplasm around the nucleus, while several cells were weakly stained in this area. IL-1 beta also stimulated IL-6 mRNA expression. In contrast, sIL-6R could not be detected in cells treated with or without IL-1 beta. CONCLUSIONS: IL-1 beta increased IL-6 production in synovial cells resulting from an increase in IL-6 mRNA expression. Enhanced production of IL-6, which is associated with bone resorption and inflammatory response, seems to be related to the progression of TMJ disorders.     

Regulation of HAS expression in human synovial lining cells of TMJ by IL-1beta

Hyaluronan (HA), a major glycosaminoglycan of synovial fluid, is synthesised by a class of membrane-bound HA synthase (HAS) proteins. In the present study, we investigated the regulatory roles of IL-1beta on HAS gene expression and HA production by the fibroblastic synovial lining cells. The synovial lining cells from synovial membrane in human temporomandibular joint (TMJ) were cultured and characterised using immunocytochemistry with CD14, CD44, and vimentin monoclonal antibodies. With or without treatment with IL-1beta, the production of HA was detected with radiometric assay and the expression of HAS mRNAs were analysed with a semi-quantitative reverse transcribed polymerase chain reaction (RT-PCR). HA synthesis was significantly augmented with 1ng/ml of IL-1beta for both 24 and 48h stimulation, however the production of HA declined if stimulated with 10ng/ml of IL-1beta. The expression of HAS2 and 3 mRNA were enhanced about 4.2- and 7.2-fold after 4h stimulation with 1ng/ml of IL-1beta, respectively. From these results, it is concluded that IL-1beta functions on regulating HAS expression and consequently promoting the secretion of HA in synovial lining cells from TMJ.     

Tumor necrosis factor-α increases chemokine gene expression and production in synovial fibroblasts from human temporomandibular joint

BACKGROUND: Synovitis, which is characterized by infiltration of inflammatory cells, often accompanies progression of clinical symptoms of the temporomandibular joint (TMJ). Synovial fibroblasts of the TMJ are believed to play important roles in progression of synovitis. The purpose of this study was to examine production and gene expression of chemokines by synovial fibroblasts stimulated by tumor necrosis factor-alpha (TNF-alpha). METHODS: Protein levels of chemokines were measured by enzyme-linked immunosorbent assay (ELISA). Gene expression of chemokines was analyzed by real-time polymerase chain reaction (PCR). RESULTS: Production of interleukin (IL)-8, growth-related oncogene (GRO)-alpha, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T-cell expressed and secreted (RANTES) protein by synovial fibroblasts was increased by TNF-alpha. In contrast, stromal cell-derived factor (SDF)-1alpha, macrophage inflammatory protein (MIP)-1alpha and -1beta were not detectable in conditioned media of synovial fibroblasts, with or without TNF-alpha treatment. Increases in gene expression of IL-8, GRO-alpha, MCP-1, and RANTES in response to TNF-alpha treatment were detected. CONCLUSIONS: Increased protein production and gene expression of chemokines by synovial fibroblasts in response to TNF-alpha treatment appears to play an important role in recruitment of inflammatory cells into synovium and the progression of synovitis in the TMJ.     

Microarray analysis of IL-1β-stimulated chemokine genes in synovial fibroblasts from human TMJ

BACKGROUND: Interleukin (IL)-1beta is thought to play a key role in several pathologic conditions of the temporomandibular joint (TMJ). Gene expression profile of synovial fibroblasts stimulated with IL-1beta was studied by oligonucleotide microarray analysis to elucidate candidate genes associated with intracapsular pathologic conditions of TMJ. METHODS: RNA was isolated from synovial fibroblasts from five patients after IL-1beta treatment. Gene expression profiling was performed with a GeneChip. Changes in gene expression were determined by comparing IL-1beta-treated cells with untreated cells. RESULTS: A total of 121 genes showed a greater than threefold difference in average intensity between untreated and IL-1beta-treated synovial fibroblasts in five experiments. Five chemokines were among the 10 most upregulated genes, and the most upregulated gene was CCL20. The 121 IL-1beta-responsive genes included 12 chemokines whose mRNA levels were confirmed by real-time PCR. CONCLUSION: These data should provided useful information about the pathologic conditions of TMJ, especially in support of diagnosis and therapeutic approaches to TMJ.     

Interleukin-1β increases RANTES gene expression and production in synovial fibroblasts from human temporomandibular joint

BACKGROUND: Synovial fibroblasts of temporomandibular joint (TMJ) are poorly characterized, although they have important roles in progression of temporomandibular disorders (TMD). In this study, we investigated responses of synovial fibroblasts to interleukin (IL)-1beta. METHODS: We examined gene expression profiles of synovial fibroblasts in response to IL-1beta, using Affymetrix GeneChip. Regulated upon activation normal T-cell expressed and secreted (RANTES) gene expression was confirmed by polymerase chain reaction (PCR) and real-time PCR. RANTES protein levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The RANTES was preferentially up-regulated in synovial fibroblasts by IL-1beta. The increase in RANTES gene expression in response to IL-1beta was confirmed by PCR and real-time PCR. Protein level of RANTES in synovial fibroblasts was also increased by IL-1beta. CONCLUSIONS: The RANTES, a cc-type chemokine, has chemotactic effects on lymphocytes and monocytes. Increased gene expression and protein production of RANTES in synovial fibroblasts, in response to IL-1beta, may play an important role in recruitment of inflammatory cells into synovium and progression of synovitis in TMD.     

Expression of cyclooxygenase-1 and -2 in IL-1β-induced synovitis of the temporomandibular joint

Background:  In this study, we analyzed the gene expression profile of fibroblast-like synoviocyte (FLS) cultures from the temporomandibular joint (TMJ) to identify candidate genes associated with intracapsular pathologic conditions of TMJ. Cyclooxygenase (COX)-2 was one of the genes in FLS upregulated following stimulation by interleukin (IL)-1β, a cytokine thought to play a key role in several pathological conditions. This study investigated the expression of COX-1 and COX-2 in cultured human FLS and rat TMJ synovium following stimulation with IL-1β.
Methods:  RNA was isolated from human FLS after IL-1β treatment. COX-1 and -2 expression was examined using a GeneChip and real-time polymerase chain reaction. Prostaglandin E₂ (PGE₂) levels in conditioned media from FLS were measured using enzyme-linked immunosorbent assay. Synovial tissues from TMJs of IL-1β-injected rats were examined for COX-1 and COX-2 expression by immunohistochemical staining.
Results:  Following treatment of FLS with IL-1β, expression of the COX-2 gene increased up to 8 h and peaked at 4 h, whereas COX-1 expression did not change. Stimulation with IL-1β increased the level of PGE₂ in conditioned media of cultured FLS in a time-dependent manner up to 48 h. Immunohistochemistry showed a strong positive staining for COX-2 in the lining and sub-lining synovial tissues of the TMJ of IL-1β-injected rats. In contrast, staining for COX-1 was the same in synovial tissues with and without IL-1β injection.
Conclusion:  These data suggest that COX-2 expression stimulated by IL-1β stimulates the production of PGE₂ in FLS and plays important roles in the progression of inflammation in TMJ.     

Co-expression of interleukin-1β and tumor necrosis factor α in synovial tissues and synovial fluids of temporomandibular joint with internal derangement: comparision with histological grading of synovial inflammation

BACKGROUND: It has been clarified that interleukin-1 (IL-1)beta and tumor necrosis factor (TNF)alpha play an important role in pathogenesis of various joint disease. The purpose of this study was to investigate the cellular source of IL-1beta and TNFalpha in temporomandibular joint (TMJ), and to analyze the relation between the expression of these cytokines and the intensity of TMJ synovial inflammation. METHODS: We examined 33 synovial biopsy specimens from patients with internal derangement of the TMJ by an immunohistochemical technique using specific antibodies to IL-1beta and TNFalpha. We also studied 20 synovial fluids from the patients by enzyme-linked immunosorbent assay method. These data are compared with histological grading of synovial inflammation by Gynther's system. RESULTS: Both IL-1beta and TNFalpha were predominantly localized in the synovial lining cell layer and the blood vessels of synovial biopsy specimens obtained from patients with TMJ internal derangement. A statistically significant correlation was found between the intensity of IL-1beta expression and that of TNFalpha. Additionally, the intensity of TNFalpha expression was statistically correlated with histological grading by Gynther's system. CONCLUSION: These results supported that IL-1beta and TNFalpha may be involved in the occurrence of TMJ internal derangement and that they coordinately play an role in pathogenesis of TMJ internal derangement.     

Isolation and characterization of synovial cells from the human temporomandibular joint

BACKGROUND: The synovial tissues with temporomandibular disorders (TMDs) often show chronic inflammatory changes and the synovial cells participate in the pathogenic processes of TMDs. The synovial membrane is composed of a synovial lining layer and a connective sublining layer. The synovial lining layer is made up of two kinds of cells: macrophage-like type A and fibroblastic type B cells. The aim of this study was to isolate and characterize synovial cells from the human temporomandibular joint (TMJ). METHODS: Synovial cells were isolated using an explant culture method. Then, we characterized the cultured synovial cells (SGA2 cells) using immunocytochemistry. RESULTS: SGA2 cells expressed the fibroblastic markers vimentin and prolyl 4-hydroxylase; they also expressed laminin and heat shock protein 27, all of which are markers of type B cells. However, some cells expressed the macrophage marker CD68. These CD68-positive cells simultaneously expressed laminin. CONCLUSIONS: We isolated and cultured synovial type B cells from the human TMJ, and identified the presence of intermediate type synovial lining cells, having the phenotypic properties of both type A and type B cells, among the synovial lining cells.     

Interleukin-1beta, interleukin-1 receptor antagonist, and interleukin-1 soluble receptor II in temporomandibular joint synovial fluid from patients with chronic polyarthritides.

PURPOSE: The aim of this study was to investigate whether interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra), or soluble IL-1 receptor II (sIL-1RII) in synovial fluid or plasma is associated with joint pain or signs of tissue destruction in patients with temporomandibular joint (TMJ) involvement of polyarthritides. PATIENTS AND METHODS: Forty-three patients with TMJ involvement of polyarthritides were included. TMJ resting pain, tenderness to palpation, pressure pain threshold, pain on mandibular movement, and anterior open bite were assessed. TMJ synovial fluid samples and plasma were obtained for analysis of IL-1beta, IL-1ra, and sIL-1RII. RESULTS: IL-1beta was detected in 18% of the synovial fluid samples and in 44% of the plasma samples. The concentrations of IL-1ra in plasma were lower than in the synovial fluid, whereas the opposite condition was found for sIL-1-RII. IL-1ra in synovial fluid and plasma was associated with low intensity of TMJ pain. sIL-1RII in synovial fluid was associated with low degree of anterior open bite, whereas sIL-1RII in plasma was associated with widespread musculoskeletal pain, TMJ pain and tenderness, and decreased pressure pain threshold over the TMJ. CONCLUSION: IL-1ra and sIL-1RII are present in different proportions in TMJ synovial fluid and blood plasma from patients with TMJ involvement of polyarthritis. Both of these molecules seem to influence the clinical features of these forms of TMJ inflammation.     

Immunohistochemical study of interleukin-1beta and interleukin-1 receptor antagonist in an antigen-induced arthritis of the rabbit temporomandibular joint.

BACKGROUND: In temporomandibular joint (TMJ) arthritis, there is limited knowledge of the relationship between interleukin-1beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1ra), as well as the source of these cytokines. We investigated the development of an antigen-induced arthritis in the rabbit TMJ immunohistochemically. METHODS: Unilateral TMJ arthritis was induced in 32 adult New Zealand White rabbits. From 6 h to 12 weeks after induction of arthritis, topology of IL-1beta and IL-1ra were observed. RESULT: The acute stage of induced arthritis lasted for one week after induction, thereafter it became chronic. In the early phase of the acute stage, infiltrating inflammatory cells, as well as synovial cells, produced IL-1beta and IL-1ra. In the late phase of the acute stage, the main source of these cytokines was subsynovial fibroblasts. In this phase of arthritis, IL-1beta and IL-1ra did not appear to be produced by synovial cells. From the early to intermediate phase of the chronic stage, proliferating synovial cells produced IL-1beta and IL-1ra. In this phase of the arthritis, these cytokines were also observed in a cluster formation in chondrocytes. CONCLUSION: This arthritis model shows a staging of the joint inflammation process with time. IL-1beta and IL-1ra are produced by a certain kind of cells depending on the stage of inflammation.     

Inflammatory cell and cytokine patterns in patients with chronic polyarthritis and temporomandibular joint involvement

OBJECTIVE: To investigate the occurrence of selected markers for inflammatory cells and cytokines in patients with chronic polyarthritis (CPA) and temporomandibular joint (TMJ) involvement. MATERIAL AND METHODS: Eleven patients (11 joints) with CPA and TMJ disorder were included in the study. Synovial specimens were obtained during TMJ open surgery and these were subjected to immunohistochemistry on frozen sections post-fixed with paraformaldehyde and with the cell membranes permeabilized by saponin. In all patients, the cytokines IL-1alpha, IL-1beta, IL-1ra, TNFalpha, IFNgamma, IL2, and TGFbeta were investigated using specific antibodies. The occurrence of macrophages and T-lymphocytes was investigated using immunohistochemistry with monoclonal antibodies against antigens CD68 and CD45RO, respectively. In addition, PCNA was used as a marker for cell proliferation. RESULTS: Staining of IL-1alpha, IL-1, and TGF was seen in all 11 specimens, IFN? in 1, TNFalpha in 4, and IL-2 in none. CD45RO-positive T cells were detected in 7 specimens, CD68-positive macrophages in 6, and cell proliferation seen with PCNA was noted in 8. CONCLUSIONS: The predominant cytokines of TMJ CPA were IL-1alpha, IL-1beta, and TGFbeta, and there appeared to be no differences between the subgroups (rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis) involved. Moreover, the cytokine pattern of TMJ CPA patients seemed to differ from patients with osteoarthritis, as shown in our previous study. The main difference was the absence of IFNgamma and TNFalpha in TMJ CPA patients and a stronger TGFbeta and IL-1alpha expression.     

Aspects of interleukin-8 gene expression by gingival and dermal fibroblasts stimulated with interleukin-1beta or tumour necrosis factor alpha

Fibroblast-derived interleukin (IL)-8 is thought to have an important role in the orchestration of immuno-participant cells infiltrating the skin and gingiva in response to continuously recurring bacterial infection. Therefore, the IL-8 gene expression should be under tight regulatory control and it might be temporally and spatially limited in inflammatory tissue. The purpose of this study was to examine the aspect of the IL-8 gene expression by fibroblasts stimulated with pro-inflammatory cytokines, IL-1beta and TNF-alpha. In situ hybridisation revealed that fibroblasts did not express IL-8 mRNA whereas keratinocytes and endothelial cells did in IL-1beta- or TNF-alpha-injected mice skin. However, cultured mouse dermal fibroblasts expressed not only IL-8 but also IL-1beta mRNA without stimulation by exogenous IL-1beta and TNF-alpha, and the expression was not enhanced by the exogenous cytokines. A similar result was obtained in late-passage human gingival fibroblasts. These results suggest that fibroblasts remain insensitive to IL-1beta and TNF-alpha so as to induce the IL-8 gene expression in non-inflammatory mice skin. Mouse dermal and late-passage human gingival fibroblasts in vitro are likely to be altered in phenotype into IL-8-producing cells along with the production of IL-1beta. In skin inflammation and periodontal diseases, fibroblasts may express the IL-8 gene even without an exogenous cytokine, IL-1beta or TNF-alpha, during their proliferation similar to the situation in our culture system.     

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.     

Effects of compressive loading on human synovium-derived cells

Compressive stress may be involved in temporomandibular joint (TMJ) synovitis, but its mechanism has not been fully elucidated. We hypothesized that mechanical stress to the synovial cells of the TMJ potentially causes degenerative changes in temporomandibular joint disease. We examined the effect of cyclic compressive loading on three-dimensionally engineered constructs using human TMJ synovium-derived cells in vitro. Human TMJ synovium-derived cells were cultured onto collagen scaffolds, resulting in three-dimensional constructs. Cyclic compression loading was applied to the constructs by means of a custom-designed apparatus. DNA amount, apoptotic cells, and mRNA levels for inflammatory cytokines were analyzed. The protein expression and activity of MMPs were examined. DNA amount or apoptotic cell number was unchanged by loading. MMP-2, -3, and IL-8 mRNA expression was up-regulated by the compression, and both MMP-1 and -3 protein expression and MMP-2 activity were detected. Thus, compression of human TMJ synovium-derived cells appears to modulate inflammatory cytokines.     

Comparison of cytokine level in synovial fluid between successful and unsuccessful cases in arthrocentesis of the temporomandibular joint.

PURPOSE: The aim of this study was to compare proinflammatory cytokine levels between successful cases and unsuccessful cases of arthrocentesis in patients with internal derangement of the temporomandibular joint (TMJ). PATIENTS AND METHODS: This study involved 73 joints judged as successful and 30 judged as unsuccessful of 103 joints in 100 patients diagnosed with internal derangement of the TMJ and who underwent arthrocentesis. The diluted synovial fluid before undergoing arthrocentesis was aspirated from the superior joint space. The concentrations of interleukin (IL)-1beta, tumor necrosis factor-alpha, IL-6, and IL-8 were measured using an enzyme-linked immunosorbent assay. The differences in the detection rate and concentration of each cytokine between successful cases and unsuccessful cases were analyzed statistically. RESULTS: There was a statistically significant difference between successful cases (72.2%) and unsuccessful cases (95.5%) in the detection ratio of IL-6 (P <.05). There was a statistically significant difference between successful cases (0.017 pg/100 microg protein) and unsuccessful cases (0.046 pg/100 microg protein) in the concentration of IL-1beta (P <.05). CONCLUSION: It has been shown that the presence of IL-1beta and IL-6 in synovial fluid may be indicators of possible unsuccessful treatment following arthrocentesis for internal derangement of the TMJ.     

Up-regulation of vascular endothelial growth factor in synovial fibroblasts from human temporomandibular joint by hypoxia

INTRODUCTION: Enhanced expression of vascular endothelial growth factor (VEGF) has been described in patients with internal derangement (ID). Herein, we examined the expression of VEGF in synovial fibroblasts from temporomandibular joint (TMJ) under hypoxia and investigated the regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) involved in the expression of VEGF. METHODS: Synovial fibroblasts were prepared from human TMJ. These cells were incubated under hypoxia or normoxia for the indicated time periods. VEGF levels in cultured supernatant were measured by an ELISA. VEGF mRNA isoforms and stability were assessed using RT-PCR and Northern blot analysis respectively. HIF-1alpha accumulation was evaluated by Western blotting and immunofluorescence. RESULTS: VEGF were significantly induced by hypoxia in synovial fibroblasts. In response to hypoxia, VEGF121 and VEGF165 mRNA were both remarkably increased, while there was no change in VEGF mRNA stability. The accumulation and nuclear translocation of HIF-1alpha occurred under hypoxia. CONCLUSIONS: Hypoxia may mainly induce the expression of VEGF121 and VEGF165 in synovial fibroblasts to promote inflamed angiogenesis of TMJ. HIF-1alpha, which is clearly activated in response to hypoxia, may control the expression of VEGF in synovial fibroblasts from TMJ.