Rheumatoid arthritis, a common human disease with a prevalence of about 1%, is characterized by inflammatory autoimmune responses. However, the etiopathogenesis of rheumatoid arthritis is still incompletely understood. A variety of experimental animal models has been established to investigate the pathogenesis of rheumatoid arthritis. A collageninduced arthritis model which is one of the most widely used experimental murine models is triggered by T cell responses specific to exogenous type II collagen. These T cells play a pivotal role in shaping inflammatory events in which autoantibodies, proinflammatory mediators, and innate effector cells are involved. Recently, a spontaneous arthritis model named K/BxN has been established. These mice are genetically programmed to exhibit predominance of a T cell population bearing autoantigenspecific T cell receptor molecules. Autoantigenspecific antibodies whose generation is solely dependent on the activity of autoantigen-specific T cells serve as a functional scaffold for the inflammatory events during the distal effector phase. These two models exhibit clinical and immunologic manifestations quite similar to those of rheumatoid arthritis and share a common aspect regarding that development of autoimmunity precede the inflammatory effector phase. However, these two models employ somewhat different effector pathways at the distal end-stage of arthritis. In addition to these two models, other experimental models of rheumatoid arthritis have been developed. These include spanteneous models such as TNF-alpa transgenic mice, IL-1 receptor antagonistdeficient mice and Zap-70 mutation mice, and induced models such as bacterial cell wall- and adjuvant-induced arthritis. The experimental animal models, all together, largely contribute to the improvement of Rheumatology, in terms of both the pathogenesis investigation and therapeutic approach.
Animals ; Antibodies ; Arthritis ; Arthritis, Experimental ; Arthritis, Rheumatoid* ; Autoantibodies ; Autoimmunity ; Collagen Type II ; Humans ; Interleukin-1 ; Mice ; Mice, Transgenic ; Models, Animal* ; Models, Theoretical ; Prevalence ; Receptors, Antigen, T-Cell ; Rheumatology ; Synovitis ; T-Lymphocytes

Since cancer has become the second most common cause of death, next to heart disease and approximately 20% of human population dies from cancer, it is much desired to develop therapeutic anti-tumor vaccine with safety and efficacy. Here we investigated the immunostimulatory effects of B16 freezing/thawing (F/T) anti-tumor vaccine (hereafter F/T vaccine), one of whole cell anti-tumor vaccines. To this end, we took advantage of the IL12 p40 reporter system which is designed for monitoring the induction of IL12 expression via the detection of co-expressed yellow fluorescent protein. First, we examined whether F/T vaccine can induce IL12 expression using bone marrow-derived dendritic cells (BMDCs) from IL12 p40 reporter mice. Second, we examined whether F/T vaccine can change the expression level of MHC molecules and co-stimulatory molecules during the activation of dendritic cells. Third, to dissect what component of F/T vaccines accounts for the immunostimulatory activities, we examined the effect of F/T vaccine on BMDC activation after treating it with DNase or proteinase. Lastly, we used MyD88 knockout mice to investigate whether F/T vaccine activates BMDCs in a TLRdependent manner. We found that treatment of BMDCs with F/T vaccine induced IL12 expression as well as the increase of MHC II expression and co-stimulatory molecules such as CD86. Interestingly, we also found that F/T vaccine increased CD1d expression on BMDCs, which may influence the activation of natural killer T cells known to be involved in anti-tumor immune responses. In addition, we found that treatment of F/T vaccine with proteinase but not DNase abolished its immunostimulatory effect, indicating that proteins in F/T vaccine mainly have its adjuvant activity. Furthermore, the activation of BMDCs with F/T vaccine was dependent on MyD88 adaptor molecule. Taken together, our findings in this study demonstrated that the F/T vaccine might be one of the valuable reagents to provide a new insight for underlying mechanism of whole-cell anti-tumor vaccines and an important clue for the development of better therapeutic anti-cancer vaccines.
Animals ; Cause of Death ; Dendritic Cells ; Deoxyribonucleases ; Heart Diseases ; Humans ; Indicators and Reagents ; Interleukin-12 ; Mice ; Mice, Knockout ; Natural Killer T-Cells ; Toll-Like Receptors ; Vaccines

Production of thymus-dependent antibodies by autoreactive B cells requires help from T cells. Follicular helper T (Tfh) cells are a unique lineage of CD4+ T subsets present in the follicles of peripheral lymphoid tissues which functions primarily to provide help to cognate B cells. Within germinal centers Tfh cells stimulate germinal center B cells to undergo affinity maturation, Ig class switching, and differentiation to memory B cells and plasma cells. Proposals that activity of Tfh cells is crucial for long-lived humoral autoimmunity are supported by the correlation of numbers and/or functions of Tfh cells with disease activity in many autoimmune disorders. In this review, we discuss recent findings regarding Tfh cell development and function. In addition, we discuss putative roles of Tfh cells in the pathogenesis and highlight the potential of Tfh cells as therapeutic targets in autoimmune diseases.
Antibodies ; Autoimmune Diseases ; Autoimmunity ; B-Lymphocytes ; Germinal Center ; Immunity, Humoral ; Immunoglobulin Class Switching ; Lymphoid Tissue ; Memory ; Plasma Cells ; T-Lymphocytes ; T-Lymphocytes, Helper-Inducer

Fibroblast-like synoviocytes (FLS) colocalize with leukocyte infiltrates in rheumatoid synovia. Proinflammatory leukocytes are known to amplify inflammation by signaling to FLS, but crosstalk between FLS and regulatory T cells (Tregs) remains uncharacterized. To address this possibility, we cocultured FLS lines derived from arthritic mice with Tregs. FLS that expressed the ligand for glucocorticoid-induced TNF receptor family-related gene (GITR) decreased expression of Foxp3 and GITR in Tregs in a contact-dependent manner. This effect was abolished by blocking antibody to GITR. On the other hand, the Tregs caused the FLS to increase IL-6 production. These results demonstrate that inflamed FLS license Tregs to downregulate Foxp3 expression via the GITRL/GITR interaction while the Tregs induce the FLS to increase their production of IL-6. Our findings suggest that the interaction between FLS and Tregs dampens the anti-inflammatory activity of Tregs and amplifies the proinflammatory activity of FLS, thereby exacerbating inflammatory arthritis.
Animals ; Arthritis ; Hand ; Inflammation ; Interleukin-6 ; Leukocytes ; Licensure ; Mice ; Receptors, Tumor Necrosis Factor ; Synovial Fluid ; T-Lymphocytes, Regulatory

Although rheumatoid arthritis has been known to be a common autoimmune disease characterized by chronic inflammation mainly evident in diarthrodial joints, its pathogenesis remains to be clarified. In the present study, to investigate the pathogenic signaling system taken place in the rheumatoid joints, we assessed whether synovial fluid obtained from patients with rheumatoid arthritis contains inducers for proinflammatory cytokines such as interleukin (IL)-12 and IL-23. Peritoneal macrophages isolated from IL-12/IL-23 p40-YFP reporter mice were stimulated with synovial fluid, followed by flow cytometry to screen CD11b+ and YFP-expressing cells, reflective of IL-12/IL-23 p40-producing macrophages. The expression levels of Toll-like receptor (TLR)-2 and -4, which have a potential to mediate IL-12/IL- 23 p40 induction, were determined in synovial cells obtained from a patient with rheumatoid arthritis by RT-PCR analyses. One out of 10 synovial fluid from rheumatoid arthritis patients induced IL-12/IL-23 p40 expression, while all of 10 synovial fluid from osteoarthritis patients did not. Synoviocytes constitutively expressed Toll-like receptor (TLR)-2 and -4 which are candidate receptors for IL-12/IL-23 inducers. Upon LPS stimulation, the levels of TLR-2 and -4 were downregulated and upregulated, respectively. Taken together, these results suggest that some patients with rheumatoid arthritis elicit synovitis through TLR-2- and -4-mediated induction of proinflammatory cytokines IL-12 and IL-23.
Animals ; Arthritis, Rheumatoid ; Autoimmune Diseases ; Cytokines ; Flow Cytometry ; Fluorescence* ; Humans ; Inflammation ; Interleukin-12 ; Interleukin-23 ; Interleukins ; Joints ; Macrophages ; Macrophages, Peritoneal ; Mass Screening* ; Mice* ; Osteoarthritis ; Synovial Fluid* ; Synovitis ; Toll-Like Receptors

Chronic rheumatoid arthritis (RA) is characterized by the hyperplasia of synovial tissue, which results from the combined influence of the proliferation and antiapoptosis of the synovial cells. In this study, to identify candidate factors involved in the regulation of synovial hyperplasia, the expression profile of 205 apoptosis-related genes in a rheumatoid synovium was analyzed in comparison with that in an osteoarthritis (OA) synovium using a cDNA microarray. Upregulated genes in the RA synovium include TNFR2, GRB2, RBL2, CDC25B, MAPK p38, CDK-like kinase 2, and FLICE2, whereas 5 genes including SARP1 were down-regulated relative to OA. Among them, importantly, the expression levels of GRB2 and FLICE2 genes were remarkably enhanced in RA but not OA synoviocytes in response to TNF -alpha treatment. Therefore, TNF-alpha inducibility to GRB2 and FLICE2 genes abnormally enhanced in RA synoviocytes might represent the increased transcripts of these two genes in rheumatoid sunovial tissues. Moreover, these results suggest that RA-specific signals by TNF-alpha, including GRB2 and FLICE2, are involved in the pathogenic processes of synovial hyperplasia.
Arthritis, Rheumatoid* ; Hyperplasia* ; Mass Screening* ; Oligonucleotide Array Sequence Analysis ; Osteoarthritis ; Phosphotransferases ; Receptors, Tumor Necrosis Factor, Type II ; Synovial Membrane ; Tumor Necrosis Factor-alpha

Long-lasting post-switched plasma cells (PCs) arise mainly from germinal center (GC) reactions, but little is known about the mechanism by which GC B cells differentiate into PCs. Based on our observation that the expression of the transcription factor CCAAT/ enhancer binding protein β (C/EPBβ) is associated with the emergence of post-switched PCs, we enquired whether a cell-autonomous function of C/EPBβ is involved in the program for PC development. To address this, we generated C/EPBβ-deficient mice in which the Cebpb locus was specifically deleted in B cells after transcription of the Ig γ1 constant gene segment (Cγ1).In response to in vitro stimulation, B cells from these Cebpbfl/flCγ1Cre/+ mice had defects in the induction of B lymphocyte-induced maturation protein 1 (Blimp1) and the formation of IgG1 +PCs, but not in proliferation and survival. At steady state, the Cebpbfl/flCγ1Cre/+ mice had reduced serum IgG1 titers but normal IgG2c and IgM titers. Moreover, upon immunization with T-dependent Ag, the mice produced reduced levels of Ag-specific IgG1 Ab, and were defective in the production of Ag-specific IgG1 Ab-secreting cells. These results suggest that a cellautonomous function of C/EPBβ is crucial for differentiation of post-switched GC B cells into PCs through a Blimp1-dependent pathway.

K/BxN serum can induce arthritis in normal mice because of abundant autoantibodies that trigger an innate inflammatory response in joints. To determine whether IL-17 is involved in the pathogenesis of serum-induced arthritis, we injected wild-type and IL-17(−/−) mice with K/BxN serum and evaluated them for signs of arthritis. Unlike wild-type mice, IL-17(−/−) mice did not show any signs of arthritis. IL-17 was produced predominantly by CD3⁻ CD4⁻γδTCR⁻ NK1.1⁻ Sca1(int) Thy1(hi) cells residing in the inflamed synovial tissue. When synovial cells extracted from normal joints were stimulated with IL-23 or autoantibody-containing immune complexes, a substantial fraction of Sca1(int) Thy1(hi) cells produced IL-17. Thus, we have identified a novel population of IL-17-producing innate synovial cells that play a crucial role in the development of K/BxN serum-induced arthritis.
Animals ; Antigen-Antibody Complex ; Arthritis* ; Autoantibodies ; Interleukin-17 ; Interleukin-23 ; Interleukins* ; Joints ; Mice

Adriamycin, a member of anthracycline class isolated from the culture medium of Streptomyces peucetius var. caesius, is one of the most effective and useful anti-neoplastic agents for the treatment of hematological and solid malignancy. The efficacy of adriamycin has been shown to mainly rely on free radicals generated during its inhibitory activity of DNA synthesis and its metabolism. However, the production of free radicals elicited side effects including chronic cardiotoxicityits. Such aspect occasionally limits the therapeutic use of adriamycin. This study was aimed to investigate the dose effect of adriamycin on apoptosis induction of the myocardium of left ventricle along with the effect of vitamin C on adriamycin activity. SD rats weighing 250~300 g were injected intraperitoneally with either 20 mg/kg or 5 mg/kg of adriamycin and drunk vitamin C at 1000 mg/animal/day. Rats were sacrificed at days 1, 3, 5, and 14 after injection but rats injected with 20 mg adriamycin died at day 6. The heart were paraffin -sectioned at 6 micrometer thickness, stained following TUNEL methods. Stained cells undergoing apoptotic death in myocardium of left ventricle were counted and statistically analyzed. The results were obtained as follows; 1. The muscular layer of left ventricle from nomal rats contained 4.2+/-12.3 apoptotic cells. 2. Distributions of apoptotic cells from rats treated with 20 mg/kg adriamycin were 26.7+/-43.2, 17.7+/-41.4, and 17.8+/- 31.8 at days 1, 3, and 5, respectively. 3. Distributions of apoptotic cells from rats treated with 5 mg/kg adriamycin were 10.8+/-19.5, 4.1+/-12.4, 5.6+/-10.2, 10.8+/-17.3 at days 1, 3, 5, and 14, respectively. 4. Coadministration of 20 mg/kg adriamycin and vitamin C resulted in 8.8+/-19.5, 4.1+/-12.4, and 16.2+/-33.6 apop-totic cells at days 1, 3, and 5 post administration. 5. Coadministration of 5 mg/kg adriamycin and vitamin C resulted in 17.0+/-32.3, 12.2+/-19.7, 7.1+/-14.0, and 7.2+/-16.7 apoptotic cells at days 1, 3, 5, and 14 post administration. Taken together, these results demonstrated that treatment of rats with 20 mg/kg adriamycin induced more apoptotic cells than that with 5 mg/kg adriamycin and vitamin C reduced such cytotoxic effects of adriamycin.
Animals ; Apoptosis* ; Ascorbic Acid* ; DNA ; Doxorubicin ; Free Radicals ; Heart ; Heart Ventricles ; In Situ Nick-End Labeling ; Metabolism ; Myocardium ; Paraffin ; Rats* ; Streptomyces ; Vitamins*

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. METHODS: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. RESULTS: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 (44.6+/-1.5 ng/ml) or pEBVvIL-10 (51.0+/-5.7 ng/ml) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. CONCLUSION: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.
Arthritis, Rheumatoid ; Clone Cells ; Cloning, Organism ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Gene Expression* ; Genetic Therapy ; Inflammation ; Interleukin-10 ; Joints ; Microscopy, Confocal ; Plasmids* ; Transfection