Eun-Kyung Bae

Expression of human TIM-3 and its correlation with disease activity in rheumatoid arthritis

Objective: T-cell immunoglobulin- and mucin-domain-containing molecule 3 (TIM-3) is a novel transmembrane protein that is involved in the regulation of T-helper 1 (Th1)-cell-mediated immunity. This study was undertaken to investigate the expressions of TIM-3 and its ligand galectin 9 (Gal-9) with respect to disease activity in rheumatoid arthritis (RA). Methods: Blood was collected from 39 RA patients and 31 healthy controls. Blood leucocyte TIM-3 and Gal-9 mRNA levels and RA disease activity were determined. Synovial tissue (ST) from five RA patients and five osteoarthritis (OA) patients were examined for TIM-3 mRNA expression and were also analysed for TIM-3 by immunohistology. Results: TIM-3 mRNA expression was significantly higher in the ST of RA patients than in the ST of OA patients. TIM-3 was expressed in the synovial sublining area in ST of RA patients but not in OA ST. TIM-3 mRNA expression from peripheral blood mononuclear cells (PBMCs) of RA patients was negatively correlated with the 28-joint Disease Activity Score (DAS28). Gal-9 mRNA level in PBMCs of RA patients was higher than in healthy controls, and was significantly higher in patients with low disease activity compared to those with moderate to high disease activity. Gal-9 mRNA expression in PBMCs of RA patients was positively correlated with forkhead box P3 (FoxP3) mRNA expression. Conclusion: TIM-3 and its interaction with Gal-9 are closely associated with RA disease activity and may play an important role in the pathogenesis of RA. In addition to the negative regulatory effect of Gal-9 mediated through the TIM-3–Gal-9 pathway, Gal-9 may exert its suppressive effect on RA disease activity by modulation of regulatory T (Treg) cells.

Slug suppression induces apoptosis via Puma transactivation in rheumatoid arthritis fibroblast-like synoviocytes treated with hydrogen peroxide

Inadequate apoptosis contributes to synovial hyperplasia in rheumatoid arthritis (RA). Recent study shows that low expression of Puma might be partially responsible for the decreased apoptosis of fibroblast-like synoviocytes (FLS). Slug, a highly conserved zinc finger transcriptional repressor, is known to antagonize apoptosis of hematopoietic progenitor cells by repressing Puma transactivation. In this study, we examined the expression and function of Slug in RA FLS. Slug mRNA expression was measured in the synovial tissue (ST) and FLS obtained from RA and osteoarthritis patients. Slug and Puma mRNA expression in FLS by apoptotic stimuli were measured by real-time PCR analysis. FLS were transfected with control siRNA or Slug siRNA. Apoptosis was quantified by trypan blue exclusion, DNA fragmentation and caspase-3 assay. RA ST expressed higher level of Slug mRNA compared with osteoarthritis ST. Slug was significantly induced by hydrogen peroxide (H2O2) but not by exogenous p53 in RA FLS. Puma induction by H2O2 stimulation was significantly higher in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. After H2O2 stimulation, viable cell number was significantly lower in Slug siRNA-transfected FLS compared with control siRNA-transfected FLS. Apoptosis enhancing effect of Slug siRNA was further confirmed by ELISA that detects cytoplasmic histone-associated DNA fragments and caspase-3 assay. These data demonstrate that Slug is overexpressed in RA ST and that suppression of Slug gene facilitates apoptosis of FLS by increasing Puma transactivation. Slug may therefore represent a potential therapeutic target in RA.

The Role of Raf Kinase Inhibitor Protein in Rheumatoid Fibroblast-like Synoviocytes Invasiveness and Cytokine and Matrix Metalloproteinase Expression

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-κB pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-α-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-α-induced ERK and NF-κB activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-α-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

Phenotypic Characterization and Invasive Properties of Synovial Fluid-Derived Adherent Cells in Rheumatoid Arthritis

The present study aimed at characterizing the phenotype and functions of adherent synovial fluid (SF) cells derived from rheumatoid arthritis (RA), comparing with fibroblast-like synoviocytes (FLS) derived from RA synovial tissue (ST). Adherent SF-derived cells were spindle-shaped from passages 1–6 under light microscopy. The cell surface marker profile in SF-derived cells from passage 1–6 was similar to that of ST-derived FLS. Levels of MMP-1 and MMP-3 were not significantly different between SF-derived cells and ST-derived FLS (p = 0.20 and p = 0.40, respectively). There was no significant difference in the optical density value between two cell types in the cell invasion assay (p = 0.10). SF-derived adherent cells have a fibroblast-like phenotype from very early culture passages and have the potential to produce MMPs with the invasive capacity to degrade cartilage, identical to ST-derived FLS. Therefore, these cells could substitute for ST-derived FLS in studying the pathogenesis of RA.

Microenvironment Effects on Promoting Upregulation of Matrix Metalloproteinases in Bcl-2-Overexpressing Renal Cell Carcinoma as a Response to Doxorubicin Treatment Inducing the Production of Metastasis

Dysfunction in apoptosis has been suggested to play an important role in the development of a distant metastasis. The Bcl-2 gene plays a key role in the response to chemotherapeutic agents, and its upregulation protects the cells from apoptosis by inactivating the Bax proteins through heterodimerization of Bcl-2/Bax. However, there is no direct evidence showing that the upregulation of Bcl-2 increases the antiapoptotic effects against chemotherapeutic agents and is associated with the production of a distant metastasis. In this study, the role of Bcl-2 in the production of distant metastasis was investigated by examining the activity of caspase-3 and the expression of matrix metalloproteinases (MMPs) after transfecting the Bcl-2 gene into human renal cell carcinoma cells (SN12C). In addition, the production of a distant metastasis was examined in an orthotopic animal model. In vitro, the SN12C/smb2 cells were more resistant to doxorubicin (DXR) than the untreated parental cells. The IC50 of the SN12C/smb2 was 50% higher than that of the parental cells. In addition, the caspase-3 activity of the SN12C/smb2 cells was lower than that of the parental cells after the DXR treatment. On the other hand, there was no difference in the expression of MMP-2 and MMP-9 between the SN12C and SN12C/smb2 cell lines. However, the SN12C/smb2 cells had a higher metastatic potential than the parental cells in the orthotopic animal model. Unlike the results from the in vitro analysis, the expression of MMP-2 and MMP-9 in the kidney tumors produced by the SN12C/smb2 cells was higher than in the kidney tumors produced by the SN12C/vector. These results show that the upregulation of Bcl-2 in human renal cell carcinoma cells induces drug resistance to DXR. Moreover, Bcl-2 induced the expression of MMP in tumors grown in the orthotopic sites even though no appreciable effects were observed in the in vitro condition. When the in vitro and in vivo data were combined, it appears that the Bcl-2 gene initially affects the response to DXR. The cells that survive the DXR treatment then have a chance to become metastatic by increasing the levels of MMP in an orthotopic environment.

Increased α-defensin-1 expression in Korean patients with Behcet's disease

OBJECTIVES The aim of this study is to measure α-defensin-1 expression in the peripheral blood of patients with Behçets disease (BD) and healthy control (HC) and to assess the association between α-defensin-1 expression and clinical features of BD. METHODS Our patients fulfilled the diagnostic criteria of the international BD study group. ELISA and real-time PCR were performed to measure α-defensin-1 protein level in the sera and α-defensin-1 mRNA level in peripheral blood mononuclear cells (PBMC), respectively. RESULTS The α-defensin-1 mRNA expression was significantly higher in BD patients (n=59) than HC (n=34) (0.49±0.10 vs. 0.19±0.45, P=0.03). The level of α-defensin-1 mRNA and protein was significantly higher in active patients than inactive patients (n=15, 0.91±0.28 vs. n=44, 0.35±0.09, P<0.001 and n=21, 7.50±2.14ng/ml vs. n=50, 3.32±0.96ng/ml, P=0.001, respectively). The level of α-defensin-1 mRNA was significant higher in patients with arthritis (n=20) than those without arthritis (n=39). CONCLUSION α-defensin-1 mRNA and protein levels are significantly increased in BD patients, especially in active BD patients. Furthermore, α-defensin-1 mRNA is over-expressed in the PBMC of BD patients with arthritis. The present study suggests that α-defensin-1 may be involved in the pathogenesis of BD and can be used as valuable biologic marker for estimation of disease activity in BD.