Gene Expression Profiling of IL-17A-Treated Synovial Fibroblasts from the Human Temporomandibular Joint.

Synovial fibroblasts contribute to the inflammatory temporomandibular joint under pathogenic stimuli. Synovial fibroblasts and T cells participate in the perpetuation of joint inflammation in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. IL-17 is an inflammatory cytokine produced primarily by Th17 cells which plays critical role in the pathogenesis of numerous autoimmune and inflammatory diseases. Here, we investigated the roles of IL-17A in temporomandibular joint disorders (TMD) using genome-wide analysis of synovial fibroblasts isolated from patients with TMD. IL-17 receptors were expressed in synovial fibroblasts as assessed using real-time PCR. Microarray analysis indicated that IL-17A treatment of synovial fibroblasts upregulated the expression of IL-6 and chemokines. Real-time PCR analysis showed that the gene expression of IL-6, CXCL1, IL-8, and CCL20 was significantly higher in IL-17A-treated synovial fibroblasts compared to nontreated controls. IL-6 protein production was increased by IL-17A in a time- and a dose-dependent manner. Additionally, IL-17A simulated IL-6 protein production in synovial fibroblasts samples isolated from three patients. Furthermore, signal inhibitor experiments indicated that IL-17-mediated induction of IL-6 was transduced via activation of NFκB and phosphatidylinositol 3-kinase/Akt. These results suggest that IL-17A is associated with the inflammatory progression of TMD.

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

BACKGROUND: Interleukin-1ß (IL-1ß) 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-1ß or TNF-α to determine which genes were altered. METHODS: Ribonucleic acid was isolated from SFCs after IL-1ß 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-1ß or TNF-α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL-1ß 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-1ß- or TNF-α stimulation. The mRNA and protein levels of MIP-3α increased in response to IL-1ß 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-1ß- and TNF-α-stimulated MIP-3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors. CONCLUSION: Interleukin-1ß 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-1ß or TNF-α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.

The anti-inflammatory effect of cyclooxygenase inhibitors in fibroblast-like synoviocytes from the human temporomandibular joint results from the suppression of PGE2 production.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely used for the management of pain and inflammation. However, little remains known about the effects of NSAIDs on synovitis of the human temporomandibular joint (TMJ). The aims of this study were to investigate the potential anti-inflammatory effects of NSAIDs on synovitis of the TMJ and the inflammatory effects of PGE2 on fibroblast-like synoviocytes (FLS) derived from the TMJ. METHODS: Human synovial tissue was obtained from patients with internal derangement who underwent arthroscopy of the TMJ. FLSs were prepared from the tissues using the outgrowth method. A COX inhibitor (indomethacin or celecoxib) was added to the IL-1ß-stimulated cells in culture. The cells were also stimulated with PGE2 or an EP agonist. The PGE2 production and COX-2 and IL-6 expression levels were examined using enzyme-linked immunosorbent assays, real-time PCR, and a microarray analysis. RESULTS: COX inhibitors decreased not only PGE2 production, but also the expression of COX-2 and IL-6 in FLS stimulated with IL-1ß. EP2 and EP4 were both expressed in the FLS, and the treatment with EP2 and EP4 agonists induced IL-6 production in these cells. CONCLUSION: The COX inhibitors indomethacin and celecoxib reduce the expression of inflammatory factors, such as COX-2 and IL-6, in FLS from the TMJ via suppression of PGE2 production. EP2 and EP4 were the main receptors for PGE2 present in the FLS. The approach used in this study may be useful for revealing how drugs such as NSAIDs affect the cellular functions of FLS from the TMJ.

MicroRNA-204 regulates osteogenic induction in dental follicle cells.

The dental follicle is an ectomesenchymal tissue surrounding developing tooth germ that contains osteoblastic-lineage-committed stem/progenitor cells. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. The aim of this study was to investigate the key regulators of miRNA during osteogenic differentiation in human dental follicle cells (hDFC). We analyzed miRNA expression profiles in hDFC during osteoblastic differentiation. Expression of miR-204 was decreased in hDFC during osteogenic induction on microarray analysis. Real-time and RT-PCR analysis also showed that the expression of miR-204 was decreased in all three hDFC during osteogenic differentiation. To investigate whether miR-204 has an effect on osteogenic differentiation, miR-204 was predicted to target alkaline phosphatase (ALP), secreted protein acidic and rich in cysteine (SPARC), and Runx2 in the in the 3'-UTRs by in silico analysis. When miR-204 was transfected into hDFC, the activity of ALP and protein levels of SPARC and Runx2 were decreased. mRNA levels of ALP, SPARC and Runx2 were also decreased by miR-204 transfection. Our data suggest that miR-204 negatively regulates the osteogenic differentiation of hDFC by targeting the bone-specific transcription factor Runx2, the mineralization maker ALP and the bone extracellular matrix protein SPARC.

Capacity of Human Dental Follicle Cells to Differentiate into Neural Cells In Vitro.

The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells (hDFCs) have the capacity to commit to differentiation into multiple cell types. Here we investigated the capacity of hDFCs to differentiate into neural cells and the efficiency of a two-step strategy involving floating neurosphere-like bodies for neural differentiation. Undifferentiated hDFCs showed a spindle-like morphology and were positive for neural markers such as nestin, ß-III-tubulin, and S100ß. The cellular morphology of several cells was neuronal-like including branched dendrite-like processes and neurites. Next, hDFCs were used for neurosphere formation in serum-free medium containing basic fibroblast growth factor, epidermal growth factor, and B27 supplement. The number of cells with neuronal-like morphology and that were strongly positive for neural markers increased with sphere formation. Gene expression of neural markers also increased in hDFCs with sphere formation. Next, gene expression of neural markers was examined in hDFCs during neuronal differentiation after sphere formation. Expression of Musashi-1 and Musashi-2, MAP2, GFAP, MBP, and SOX10 was upregulated in hDFCs undergoing neuronal differentiation via neurospheres, whereas expression of nestin and ß-III-tubulin was downregulated. In conclusion, hDFCs may be another optimal source of neural/glial cells for cell-based therapies to treat neurological diseases.

Plasma rich in growth factors stimulates proliferation, migration, and gene expression associated with bone formation in human dental follicle cells.

BACKGROUND/PURPOSE: Plasma rich in growth factors (PRGFs), which is prepared from autologous blood from patients, has been reported with regards to bone regeneration for dental implants. Human dental follicle cells (hDFCs) have the capacity to commit to multiple cell types such as the osteoblastic lineage. The aim of this study is to evaluate the effects of PRGFs for mineralization in hDFCs. MATERIALS AND METHODS: PRGFs was prepared from whole blood centrifuged at 460g for 8 minutes. hDFCs isolated from the dental follicle with collagenase/dispase were cultured with growth medium or osteogenic induction medium (OIM) containing PRGFs or fetal bovine serum. Concentrations of the growth factors were examined using an enzyme-linked immunosorbent assay kit. A cell migration assay was used for two-dimensional movement. Gene expressions were examined with real-time polymerase chain reaction using a DyNAmo SYBR Green quantitative polymerase chain reaction kit. RESULTS: The platelet concentration in PRGF Fraction 2 was 2.14-fold higher than in whole blood. White blood cells were not detected in PRGFs. Transforming growth factor-ß levels were higher than insulin-like growth factor-1, platelet-derived growth factor-AB and -BB, and vascular endothelial growth factors in PRGF Fraction 2. Proliferation and migration by hDFCs increased in OIM supplemented with PRGFs in a dose-dependent manner and were higher in hDFCs cultured in OIM plus 10% PRGFs compared with OIM plus 10% fetal bovine serum. PRGFs upregulated the gene expression of type I collagen, osteomodulin, alkaline phosphatase, bone morphogenic protein-4, and transforming growth factor-ß in hDFCs. CONCLUSION: PRGFs may promote bone regeneration due to it including high levels of growth factors.

Differentiation of the human mesenchymal stem cells derived from bone marrow and enhancement of cell attachment by fibronectin.

The ability of human mesenchymal stem cells (hMSC) to differentiate into osteoblasts was examined through the use of osteogenic induction medium (MSCOIM) cultures. hMSC first attached to the dish surface and exhibited fibroblast-like spindle shapes, and after proliferation, formed cuboidal shapes. Calcium assays and the use of von Kossa and alizarin red S staining showed that hMSC were capable of mineralization when cultured in MSCOIM. Gene expressions of Cbfa-1 and BMP-4, which are markers for osteogenic differentiation, were also increased during the hMSC differentiation into osteoblasts. When compared to albumin (Alb)-coated dishes, microscopic observation documented enhanced cell attachment and spreading when hMSC were cultured on fibronectin (FN)-coated dishes. Adherent cell numbers also exhibited a greater increase on the FN-coated dishes during earlier culture stages than that seen for the Alb-coated dishes. These findings suggest that hMSC have the capability to differentiate into osteoblasts and that FN can stimulate the attachment and spreading of the hMSC.

Bone morphogenetic protein 6 stimulates mineralization in human dental follicle cells without dexamethasone.

OBJECTIVE: The aim of this study is to investigate the osteogenic differentiation human dental follicle cells (hDFCs) cultured with in osteogenic induction medium (OIM) without dexamethasone (DEX), and to analyze the gene expression profile during osteogenic differentiation. METHODS: hDFCs, which isolated from dental follicle tissue from impacted third molar teeth, were cultured with OIM with or without DEX. Osteogenic differentiation of hDFCs was examined using Alkaline phosphatase activity and Arizarin red staining. Gene expression analysis was performed by Microarray and real time-PCR. RESULTS: We showed that hDFCs have the capacity to differentiate into osteogenic lineages in osteogenic induction medium lacking DEX. We also analyzed gene expression profiling of hDFCs during osteogenic differentiation. BMP6 is up-regulated in both the presence and absence of DEX. In addition, BMP6 enhances gene expression levels of DLX-5, Runx2, and Osterix, which are transcription factors associated with osteogenic differentiation. BMP6 also stimulates phosphorylation of Smad1/5/8 which are transcription factors associated with BMP signalling at protein levels. Additionally BMP6 stimulates mineralization of hDFCs monolayers examined by Arizarin red S staining. CONCLUSION: These findings suggest that hDFCs can differentiate to osteogenic lineage cells osteogenic induction medium without DEX, and BMP6 is a key gene in the osteogenic differentiation of hDFCs, and has therapeutic utility for bone regeneration and bone research.

Microarray analysis of IL-1beta-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.

Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus.

BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. METHODS: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.

Tumor necrosis factor-alpha 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.

Anti-inflammatory effect of linear polarized infrared irradiation on interleukin-1beta-induced chemokine production in MH7A rheumatoid synovial cells.

We examined the anti-inflammatory effect of infrared linear polarized light irradiation on the MH7A rheumatoid fibroblast-like synoviocytes (FLS) stimulated with the proinflammatory cytokine interleukin (IL)-1beta. Expression of messenger ribonucleic acids (mRNAs) encoding IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted), growth-related gene alpha (GROalpha), and macrophage inflammatory protein-1alpha (MIP1alpha) was measured using real-time reverse transcription polymerase chain reaction, and the secreted proteins were measured in the conditioned media using enzyme-linked immunosorbent assays. We found that irradiation with linear polarized infrared light suppressed IL-1beta-induced expression of IL-8 mRNA and, correspondingly, the synthesis and release of IL-8 protein in MH7A cells. This anti-inflammatory effect was equivalent to that obtained with the glucocorticoid dexamethasone. Likewise, irradiation suppressed the IL-1beta-induced expression of RANTES and GROalpha mRNA. These results suggest that the irradiation of the areas around the articular surfaces of joints affected by rheumatoid arthritis (RA) using linear polarized light may represent a useful new approach to treatment.

Interleukin-1beta stimulates interleukin-8 production and gene expression in synovial cells from human temporomandibular joint.

PURPOSE: The aims of the present study were to isolate and characterize cultured synovial cells from human temporomandibular joint (TMJ) specimens and to investigate the effect of interleukin (IL)-1beta on IL-8 production and gene expression in those cells. MATERIALS AND METHODS: Synovial cells (HTS cells) were isolated from TMJ synovial tissues using an outgrowth method and then primary cultured. The cells were examined for cell-specific markers of fibroblast, macrophage, and dendritic cells using immunocytochemistry. HTS cells were then treated with IL-1beta, and amounts of IL-8 were measured by enzyme-linked immunosorbent assay. IL-8 production and expression were also investigated using immunocytochemistry and a reverse transcription-polymerase chain reaction method. RESULTS: HTS cells were positive for the fibroblast-specific markers, such as vimentin and propyl 4-hydroxylase. The macrophage or dendritic cell markers and HLA class II antigen were negative. Furthermore, IL-1beta enhanced IL-8 production in HTS cells in a time- and dose-dependent manner and stimulated IL-8 gene expression. CONCLUSIONS: HTS cells may provide important advantages for studies of the cellular and molecular mechanisms in the TMJ. In addition, we found that IL-1beta stimulated IL-8 production through an increase in IL-8 gene expression in HTS cells, which may be associated with the increase of infiltrating inflammatory cells seen in the synovial membrane of TMJ disorders.

Interleukin-1 beta 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.