Evaluation of changes in oral health-related quality of life over time in patients with Sjögren's syndrome.

OBJECTIVES: To assess oral health-related quality of life (OHRQoL) and changes in OHRQoL in 3 years of patients with Sjögren's symdrome (SS). METHODS: Thirty-five SS patients and 23 non-SS individuals were enrolled. OHRQoL were quantitatively evaluated using the shortened Oral Health Impact Profile (OHIP-14). After 3 years, 22 patients and 14 controls tool the OHIP-14 survey again. RESULTS: The SS group had a significantly higher OHIP-14 score, which indicated a lower OHRQoL, than the non-SS group. Among individual questions in the OHIP-14, scores for 'trouble pronouncing words', 'uncomfortable to eat foods', 'self-conscious', and 'diet unsatisfactory' were markedly higher in the SS group than in the non-SS group. The OHIP-14 score significantly increased in 3 years in the SS group. Furthermore, there was an inverse correlation between the change rate of salivary flow rate and change of OHIP-14 scores in 3 years in patients with SS whose OHIP-14 score increased. Scores for 'irritable with other people', 'difficulty doing usual jobs', 'felt life less satisfying', and 'unable to function' significantly increased in 3 years. CONCLUSION: In SS, OHRQoL decreased in 3 years, which was associated with a decrease in saliva secretion. Moreover, troubles related to psychosocial aspects in SS patients were found to intensify over time.

IL-2-Anti-IL-2 Monoclonal Antibody Immune Complexes Inhibit Collagen-Induced Arthritis by Augmenting Regulatory T Cell Functions.

IL-2 induces regulatory T cells (Tregs) and reduces disease severity, such as in graft-versus-host disease and systemic lupus erythematosus. To investigate the regulatory network of IL-2 in rheumatoid arthritis, we examined the effects of IL-2-anti-IL-2 mAb immune complexes (IL-2ICs) in a rheumatoid arthritis model of collagen-induced arthritis (CIA). CIA was induced in male DBA/1 mice by two immunizations with type II collagen at 3-wk intervals. IL-2ICs were prepared by mixing 5 µg of an anti-IL-2 mAb (clone JES6-1D) with 1 µg of mouse IL-2 and were injected i.p. every day for 3 d. Mouse paws were scored for arthritis using a macroscopic scoring system. Th1, Th2, Th17, and Tregs were analyzed by flow cytometry. Joint histopathology was examined by H&E and immunohistochemical staining. Treg functions were examined by studying in vitro suppression using flow cytometry. IL-2IC administration effectively elicited a 1.6-fold expansion of CD4+Foxp3+ Tregs in peripheral blood cells relative to that found in control mice. IL-2IC treatment significantly inhibited arthritis in CIA mice. Histopathological examination of joints revealed inhibited synovial cell proliferation and IL-17, IL-6, and TNF-α levels but increased Foxp3+ Tregs after IL-2IC treatment. Flow cytometric examination of spleen cells revealed reduced IFN-γ- and IL-17-producing cells and increased IL-10-producing Tregs after IL-2IC treatment. The suppressive activities of CD4+CD25+ Tregs induced by IL-2ICs were stronger than those in untreated mice. IL-2ICs inhibited arthritis by augmenting not only Treg numbers but also Treg functions, which play regulatory roles in autoimmune arthritis.

A comparative proteomics study of a synovial cell line stimulated with TNF-α.

To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor-α (TNF-α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF-α. Next, we selected 269 differentially produced proteins that were detected only under TNF-α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF-α-stimulated MH7A cells, we observed substantial production of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins such as BH3-interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase-3. These results indicate that the upregulation of plasminogen-activator inhibitor 2 and apoptosis-regulating proteins in synovial cells in response to TNF-α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.

Salivary epidermal growth factor (EGF) in Sjögren's syndrome: association between salivary EGF levels and the severity of intraoral manifestations.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease characterized by lymphocytic infiltration of the exocrine glands, especially the salivary and lacrimal glands. As a result of salivary gland dysfunction, most patients with SS have xerostomia, related to a reduced salivary flow rate. In addition to the discomfort due to xerostomia, dry mouth can cause various intraoral manifestations such as refractory stomatitis, ulcer and atrophic changes in the oral mucosa and tongue, and patients' quality of life (QOL) is impaired severely. These manifestations are believed to be caused mainly by a decrease in the clearance in the oral cavity owing to hyposalivation. However, since saliva has several beneficial physiological effects on the intraoral environment, qualitative changes in sialochemistry should also be considered a cause of the refractory intraoral manifestations in SS. Salivary epidermal growth factor (EGF) is considered an important cytoprotective factor against injuries, and it contributes to wound healing in the oral cavity. We evaluated changes in salivary EGF levels and assessed the association between salivary EGF levels and the severity of intraoral manifestations in SS patients. The results showed that the salivary EGF levels decreased with the progression of SS, and this deterioration in saliva quality as well as hyposalivation could play a role in the pathogenesis of refractory intraoral manifestations in SS patients. Our findings provide new target for therapeutic intervention in SS.

Blocking c-Met signaling enhances bone morphogenetic protein-2-induced osteoblast differentiation.

We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c-Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c-Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3-E1 murine pre-osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)-2 or osteoblast-inducer reagent in the presence or absence of either HGF antagonist (NK4) or c-Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme-linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription-PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP-2-stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast-inducer reagent-stimulated MC3T3-E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c-Met signaling pathway. However, both mitogen-activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP-2-stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP-2-stimulated C2C12 cells. These results indicate that although the c-Met-MEK-ERK-Smad and c-Met-PI3K-AKT-Smad signaling pathways positively regulate osteoblast differentiation, c-Met signaling negatively regulates osteoblast differentiation, independent of the MEK-ERK-Smad and PI3K-AKT-Smad pathways. Therefore, blocking c-Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA.

Rapid decrease in salivary epidermal growth factor levels in patients with Sjögren's syndrome: A 3-year follow-up study.

OBJECTIVES: To assess changes in salivary epidermal growth factor (EGF) levels within three years and investigate the correlation between these changes and the severity of intraoral manifestations in patients with Sjögren's syndrome (SS). METHODS: Twenty-three SS patients (14 primary SS and 9 secondary SS) and 14 controls were followed up for three years. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). Changes in salivary flow rate, EGF level, and severity of intraoral manifestations were analyzed, along with associations among them. RESULTS: The OHIP-14 score significantly increased and the total salivary EGF output significantly decreased after three years in the SS group (10.2 ± 8.8 vs. 12.6 ± 9.2, p = 0.040; 10158.4 ± 9820.9 vs. 8352.8 ± 7813.3 pg/10 min, p = 0.032), though the salivary flow rate did not change. The decrease in total EGF output was especially high in patients with long disease duration and poor oral health-related quality of life (OHRQoL). In patients with poor OHRQoL, the change in total EGF output significantly correlated with the OHIP-14 score (r = - 0.847, p = 0.008). However, there was no correlation between the change in salivary flow rate and the OHIP-14 score. CONCLUSIONS: The rapid decrease in salivary EGF level contributes to the progression of intraoral manifestations of SS.

Differential regulation of c-Met signaling pathways for synovial cell function.

We previously demonstrated that blocking the hepatocyte growth factor (HGF) receptor, c-Met, using a HGF antagonist, NK4, inhibited arthritis in a rheumatoid arthritis (RA) model mice. In the present study, we investigated the role of c-Met signaling in synovial cell function. We demonstrated that synovial tissues from RA patients and MH7A cells, a human RA synovial cell line, expressed HGF and c-Met. HGF and c-Met expression in RA synovium was increased compared to osteoarthritis synovium suggesting increased c-Met signaling in RA synovial cells. The c-Met inhibitor, SU11274, inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated MH7A cells. MEK and PI3K inhibitors suppressed production of matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) by MH7A cells, suggesting that c-Met-MEK-ERK and c-Met-PI3K-AKT pathways are involved positively regulating MH7A cell function. Although SU11274 suppressed MMP-3 and VEGF production it enhanced PGE2 production by MH7A cells suggesting that negative regulation by c-Met signaling, independent of the MEK-ERK and PI3K-AKT pathways, is involved in PGE2 production. Blocking c-Met signaling may be therapeutically useful to inhibit angiogenesis and cartilage and bone destruction by inhibiting VEGF and MMP-3 production, while enhancing PGE2 production in synovial cells in RA.

Correlation between salivary epidermal growth factor levels and refractory intraoral manifestations in patients with Sjögren's syndrome.

OBJECTIVE: To assess changes in salivary epidermal growth factor (EGF) levels and the correlation between these levels and the severity of intraoral manifestations in Sjögren's syndrome (SS). METHODS: Forty SS patients and 23 controls were enrolled. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). The associations among salivary flow rate, EGF levels and the severity of intraoral manifestations were analyzed. RESULTS: The total salivary EGF output was significantly decreased in the SS patients compared with the controls (9237.6 ± 8447.0 vs. 13296.9 ± 7907.1 pg/10 min, respectively, p = 0.033). In the SS patients, total EGF output and salivary flow rate showed a strong positive correlation (rs = 0.824, p = 0.0005), while total EGF output and disease duration showed a negative correlation (rs = -0.484, p = 0.008). Further, total EGF output was significantly correlated with the OHIP-14 score (rs = -0.721, p = 0.012). CONCLUSIONS: The salivary flow rate and EGF levels are decreased in SS, and this deterioration in saliva quality causes refractory intraoral manifestations. Our findings have provided new therapeutic targets for SS.

Molecular targeting of hepatocyte growth factor by an antagonist, NK4, in the treatment of rheumatoid arthritis.

INTRODUCTION: Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice. METHODS: Arthritis was induced in SKG mice by a single intraperitoneal injection of ß-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after ß-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells. RESULTS: The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of ß-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells. CONCLUSIONS: These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4⁺ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.

Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation.

We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

Effect of transient expression of fluorescent protein probes in synovial and myoblast cell lines.

Here, we investigate the appropriate fluorescent proteins for use in the culture of synovial MH7A and myoblast C2C12 cells. Fluorescent signal intensities of 3 different fluorescent proteins were examined in these cell lines. The fluorescent intensity of transiently expressed AcGFP, DsRed, and mStrawberry were examined in these cell lines, and the influence of the amount of plasmid used on transfection efficiency and cell viability were investigated.

The protective role of host Toll-like receptor-4 in acute graft-versus-host disease.

BACKGROUND: Mutations in Toll-like receptor (TLR)-4 have been associated with the hyporesponsiveness of macrophages to lipopolysaccharide, possibly reducing the risk of acute graft-versus-host disease (GVHD). However, TLR-4 mutations may also increase the risk of intestinal damage and microbial infection, thereby accelerating acute GVHD. METHODS: In this study, we investigated the role of TLR-4 in triggering acute GVHD using C3H/HeJ mice with disrupted TLR-4 and C3H/HeN mice with intact TLR-4 as recipients in an acute GVHD model. RESULTS: TLR-4 expression was significantly increased in the intestines and livers from acute GVHD mice. TLR-4-mutant C3H/HeJ hosts that received C57BL/6 (B6) donor cells developed significantly more severe GVHD than TLR-4-intact C3H/HeN hosts receiving B6 donor cells. Antibiotic treatment prolonged the survival of C3H/HeN-host GVHD mice but reduced the survival of C3H/HeJ-host GVHD mice. C3H/HeJ-host GVHD mice showed increased lipopolysaccharide levels in the blood, donor cell and CD68+ cell infiltration, tumor necrosis factor-α mRNA expression, and more apoptotic cells in the intestine compared with C3H/HeN host GVHD mice. In contrast, intestinal cyclooxygenase-2, prostaglandin E2, and hepatocyte growth factor expression in C3H/HeJ-host GVHD mice were significantly decreased compared with C3H/HeN-host GVHD mice. CONCLUSIONS: Our results indicated that host TLR-4 is crucial for the induction of tissue protective factors and for protection against intestinal cell apoptosis during acute GVHD.

Effects of the novel immunosuppressant FTY720 in a murine rheumatoid arthritis model.

We investigated the effects and mechanisms by which FTY720 (FTY) inhibits arthritis development in the SKG mouse rheumatoid arthritis (RA) model. FTY (1mg/kg/day) administration suppressed the progression of laminarin-induced arthritis in SKG mice. FTY treatment decreased IL-6 and TNF-alpha expression in synovial fibroblast cells and the number of inflammatory cells overall. Bone destruction was also suppressed by treatment with FTY. The numbers of CD4(+) and CD8(+) T cells were significantly increased in the thymus and decreased in the spleen in FTY-treated SKG mice. FTY enhanced IL-4 production by CD4(+) T cells stimulated by allogeneic spleen cells and inhibited prostaglandin E(2) (PGE(2)) production by a TNF-alpha-stimulated synovial fibroblast cell line. These results indicate that FTY can inhibit arthritis in SKG mice via sequestration of autoimmune CD4(+) T cells in the thymus, enhancement of Th2 immune responses, and inhibition of PGE(2) production by synovial cells.

Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma.

The tight-skin (TSK/+) mouse, a genetic model of systemic sclerosis (SSc), develops cutaneous fibrosis and defects in pulmonary architecture. Because hepatocyte growth factor (HGF) is an important mitogen and morphogen that contributes to the repair process after tissue injury, we investigated the role of HGF in cutaneous fibrosis and pulmonary architecture defects in SSc using TSK/+ mice. TSK/+ mice were injected in the gluteal muscle with either hemagglutinating virus of Japan (HVJ) liposomes containing 8 mug of a human HGF expression vector (HGF-HVJ liposomes) or a mock vector (untreated control). Gene transfer was repeated once weekly for 8 weeks. The effects of HGF gene transfection on the histopathology and expression of tumor growth factor (TGF)-beta and IL-4 mRNA in TSK/+ mice were examined. The effect of recombinant HGF on IL-4 production by TSK/+ CD4+ T cells stimulated by allogeneic dendritic cells (DCs) in vitro was also examined. Histologic analysis revealed that HGF gene transfection in TSK/+ mice resulted in a marked reduction of hypodermal thickness, including the subcutaneous connective tissue layer. The hypodermal thickness of HGF-treated TSK/+ mice was decreased two-fold to three-fold compared with untreated TSK/+ mice. However, TSK/+ associated defects in pulmonary architecture were unaffected by HGF gene transfection. HGF gene transfection significantly inhibited the expression of IL-4 and TGF-beta1 mRNA in the spleen and skin but not in the lung. We also performed a mixed lymphocyte culture and examined the effect of recombinant HGF on the generation of IL-4. Recombinant HGF significantly inhibited IL-4 production in TSK/+ CD4+ T cells stimulated by allogeneic DCs. HGF gene transfection inhibited IL-4 and TGF-beta mRNA expression, which has been postulated to have a major role in fibrinogenesis and reduced hypodermal thickness, including the subcutaneous connective tissue layer of TSK/+ mice. HGF might represent a novel strategy for the treatment of SSc.