CCAAT/enhancer binding protein β Induces Post-Switched B Cells to Produce Blimp1 and Differentiate into Plasma Cells

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.

CCAAT/enhancer binding protein β Induces Post-Switched B Cells to Produce Blimp1 and Differentiate into Plasma Cells

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.

Aged Sanroque Mice Spontaneously Develop Sjögren's Syndrome-like Disease

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disorder that affects mainly salivary and lacrimal glands, but its cause remains largely unknown. Clinical data indicating that SS occurs in a substantial proportion of patients with lupus points to common pathogenic mechanisms underlying the two diseases. To address this idea, we asked whether SS develops in the lupus-prone mouse strain sanroque (SAN). Owing to hyper-activation of follicular helper T (Tfh) cells, female SAN mice developed lupus-like symptoms at approximately 20 wk of age but there were no signs of SS at that time. However, symptoms typical of SS were evident at approximately 40 wk of age, as judged by reduced saliva flow rate, sialadenitis, and IgG deposits in the salivary glands. Increases in serum titers of SS-related autoantibodies and numbers of autoantibody-secreting cells in cervical lymph nodes (LNs) preceded the pathologic manifestations of SS and were accompanied by expansion of Tfh cells and their downstream effector cells. Thus, our results suggest that chronic dysregulation of Tfh cells in salivary gland-draining LNs is sufficient to drive the development of SS in lupus-prone mice.

Autoantibody-Mediated Dysfunction of Salivary Glands Leads to Xerostomia in SKG Mice

Sjögren's syndrome (SS) is a chronic heterogeneous disease that mainly affects exocrine glands, leading to sicca syndromes such as xerostomia. Despite the second highest prevalence rate among systemic autoimmune diseases, its pathophysiology remains largely unknown. Here we report that SKG mice, a cardinal model of Th17 cell-mediated arthritis, also develop a secondary form of SS-like disorder upon systemic exposure to purified curdlan, a type of β-glucan. The reduced production of saliva was not caused by focal immune cell infiltrates but was associated with IgG deposits in salivary glands. Sera from curdlan-injected SKG mice contained elevated titers of IgG (predominantly IgG1), autoantibody to the muscarinic type 3 receptor (M3R) and inhibited carbachol-induced Ca2+ signaling in salivary acinar cells. These results suggest that the Th17 cells that are elicited in SKG mice promote the production of salivary gland-specific autoantibodies including anti-M3R IgG; the antibodies are then deposited on acinar cells and inhibit M3R-mediated signaling required for salivation, finally leading to hypofunction of the salivary glands. This type II hypersensitivity reaction may explain the origin of secondary SS occurring without focal leukocyte infiltrates.

STX0119 Ameliorates Arthritis in SKG Mice via Inhibiting T Helper 17

Rheumatoid arthritis (RA) is an autoimmune disease with chronic and excessive inflammation. Upregulation of interleukin (IL)-17 is involved in the pathogenesis of RA. STX0119 is a specific inhibitor of signal transducer and activator of transcription 3 (STAT3) as a potential target for the treatment of RA. STAT3 is a member of DNA-binding molecules that regulates the expression of proinflammatory cytokines involved in the pathogenesis of RA. The objective of this study was to determine whether STX0119 could inhibit STAT3 and IL-17. We demonstrated that STX0119 decreased T helper (Th) 17 differentiation and IL-17 expression in vitro. STX0119 also improved the severity of zymosan induced arthritis and reduced joint inflammation. STX0119 reduced the proliferation of Th17 and phosphorylated STAT3 expression while increasing Treg differentiation and phosphorylated STAT5 expression. Moreover, STX0119 decreased the expression of IL-6 and -17 but not IL-10. These findings suggest that STX0119 can be used to treat autoimmune RA through inhibiting the activation of STAT3.

Interleukin 17-expressing Innate Synovial Cells Drive K/Bxn Serum-induced Arthritis

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.

Early Growth Response-1 Plays a Non-redundant Role in the Differentiation of B Cells into Plasma Cells

Early growth response (Egr)-1 is a Cys2-His2-type zincfinger transcription factor. It has been shown to induce survival and proliferation of immature and mature B cells, respectively, but its role in the differentiation of B cells into plasma cells remains unclear. To examine the effects of Egr-1 deficiency on the activation of B cells, naive B cells from Egr1-/- mice and their wild-type (WT) littermates were activated to proliferate and differentiate, and then assayed by FACS. Proportions of cells undergoing proliferation and apoptosis did not differ between Egr1-/- and WT mice. However, Egr1-/- B cells gave rise to fewer plasma cells than WT B cells. Consistently, Egr1-/- mice produced significantly lower titer of antigen-specific IgG than their WT littermates upon immunization. Our results demonstrate that Egr-1 participates in the differentiation program of B cells into plasma cells, while it is dispensable for the proliferation and survival of mature B cells.

Insights into the Role of Follicular Helper T Cells in Autoimmunity

Follicular helper T (TFH) cells are recently highlighted as their crucial role for humoral immunity to infection as well as their abnormal control to induce autoimmune disease. During an infection, naive T cells are differentiating into TFH cells which mediate memory B cells and long-lived plasma cells in germinal center (GC). TFH cells are characterized by their expression of master regulator, Bcl-6, and chemokine receptor, CXCR5, which are essential for the migration of T cells into the B cell follicle. Within the follicle, crosstalk occurs between B cells and TFH cells, leading to class switch recombination and affinity maturation. Various signaling molecules, including cytokines, surface molecules, and transcription factors are involved in TFH cell differentiation. IL-6 and IL-21 cytokine-mediated STAT signaling pathways, including STAT1 and STAT3, are crucial for inducing Bcl-6 expression and TFH cell differentiation. TFH cells express important surface molecules such as ICOS, PD-1, IL-21, BTLA, SAP and CD40L for mediating the interaction between T and B cells. Recently, two types of microRNA (miRNA) were found to be involved in the regulation of TFH cells. The miR-17-92 cluster induces Bcl-6 and TFH cell differentiation, whereas miR-10a negatively regulates Bcl-6 expression in T cells. In addition, follicular regulatory T (TFR) cells are studied as thymus-derived CXCR5+PD-1+Foxp3+ Treg cells that play a significant role in limiting the GC response. Regulation of TFH cell differentiation and the GC reaction via miRNA and TFR cells could be important regulatory mechanisms for maintaining immune tolerance and preventing autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Here, we review recent studies on the various factors that affect TFH cell differentiation, and the role of TFH cells in autoimmune diseases.

Gut-residing Microbes Alter the Host Susceptibility to Autoantibody-mediated Arthritis

K/BxN serum can transfer arthritis to normal mice owing to the abundant autoantibodies it contains, which trigger innate inflammatory cascades in joints. Little is known about whether gut-residing microbes affect host susceptibility to autoantibody-mediated arthritis. To address this, we fed C57BL/6 mice with water containing a mixture of antibiotics (ampicillin, vancomycin, neomycin, and metronidazol) for 2 weeks and then injected them with K/BxN serum. Antibiotic treatment significantly reduced the amount of bacterial genomic DNA isolated from fecal samples, in particular a gene encoding 16S ribosomal RNA derived from segmented filamentous bacteria. Arthritic signs, as indicated by the arthritic index and ankle thickness, were significantly attenuated in antibiotic-treated mice compared with untreated controls. Peyer's patches and mesenteric lymph nodes from antibiotic-treated mice contained fewer IL-17-expressing cells than those from untreated mice. Antibiotic treatment reduced serum C3 deposition in vitro via the alternative complement pathway. IL-17-/- congenic C57BL/6 mice were less susceptible to K/BxN serum-transferred arthritis than their wild-type littermates, but were still responsive to treatment with antibiotics. These results suggest that gut-residing microbes, including segmented filamentous bacteria, induce IL-17 production in GALT and complement activation via the alternative complement pathway, which cause the host to be more susceptible to autoantibody-mediated arthritis.

The Niche of Follicular Helper T Cells in Systemic Autoimmune Diseases / 한양의대학술지

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.

Rheumatoid Fibroblast-like Synoviocytes Downregulate Foxp3 Expression by Regulatory T Cells Via GITRL/GITR Interaction

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.

Deficiency of Foxp3+ Regulatory T Cells Exacerbates Autoimmune Arthritis by Altering the Synovial Proportions of CD4+ T Cells and Dendritic Cells

BACKGROUND: CD4+Fop3+ regulatory T cells (Tregs) are needed to maintain peripheral tolerance, but their role in the development of autoimmune arthritis is still debated. The present study was undertaken to investigate the mechanism by which Tregs influence autoimmune arthritis, using a mouse model entitled K/BxN. METHODS: We generated Treg-deficient K/BxNsf mice by congenically crossing K/BxN mice with Foxp3 mutant scurfy mice. The arthritic symptoms of the mice were clinically and histopathologically examined. The proportions and activation of CD4+ T cells and/or dendritic cells were assessed in the spleens, draining lymph nodes and synovial tissue of these mice. RESULTS: K/BxNsf mice exhibited earlier onset and more aggressive progression of arthritis than their K/BxN littermates. In particular, bone destruction associated with the influx of numerous RANKL+ cells into synovia was very prominent. They also contained more memory phenotype CD4+ T cells, more Th1 and Th2 cells, and fewer Th17 cells than their control counterparts. Plasmacytoid dendritic cells expressing high levels of CD86 and CD40 were elevated in the K/BxNsf synovia. CONCLUSION: We conclude that Tregs oppose the progression of arthritis by inhibiting the development of RANKL+ cells, homeostatically proliferating CD4+ T cells, Th1, Th2 and mature plasmacytoid dendritic cells, and by inhibiting their influx into joints.

Deficiency of Foxp3+ Regulatory T Cells Exacerbates Autoimmune Arthritis by Altering the Synovial Proportions of CD4+ T Cells and Dendritic Cells

BACKGROUND: CD4+Fop3+ regulatory T cells (Tregs) are needed to maintain peripheral tolerance, but their role in the development of autoimmune arthritis is still debated. The present study was undertaken to investigate the mechanism by which Tregs influence autoimmune arthritis, using a mouse model entitled K/BxN. METHODS: We generated Treg-deficient K/BxNsf mice by congenically crossing K/BxN mice with Foxp3 mutant scurfy mice. The arthritic symptoms of the mice were clinically and histopathologically examined. The proportions and activation of CD4+ T cells and/or dendritic cells were assessed in the spleens, draining lymph nodes and synovial tissue of these mice. RESULTS: K/BxNsf mice exhibited earlier onset and more aggressive progression of arthritis than their K/BxN littermates. In particular, bone destruction associated with the influx of numerous RANKL+ cells into synovia was very prominent. They also contained more memory phenotype CD4+ T cells, more Th1 and Th2 cells, and fewer Th17 cells than their control counterparts. Plasmacytoid dendritic cells expressing high levels of CD86 and CD40 were elevated in the K/BxNsf synovia. CONCLUSION: We conclude that Tregs oppose the progression of arthritis by inhibiting the development of RANKL+ cells, homeostatically proliferating CD4+ T cells, Th1, Th2 and mature plasmacytoid dendritic cells, and by inhibiting their influx into joints.

IL-12 p40-Expressing Immune Cells Revealed by Cytokine Reporter Mouse System / 체질인류학회지

Interleukin-12 (IL-12), consisting of p35 and p40, plays important roles in linking innate and adaptive immunity. While p35 is constitutively expressed, IL-12 p40 gene expression is induced upon activation by Toll-like receptor ligands. Recently, with gene targeting technology, the cytokine IL-12 p40 reporter mouse has been developed to express the p40 gene linked via a viral IRES element with yellow fluorescence protein (YFP) fluorescent reporter. We investigated whether this novel system would be useful to reveal IL-12 p40-producing immune cells. We first investigated whether macrophages and dendritic cells from these mice faithfully reported p40 induction. Next, we tested if microglial cells, macrophages in the brain, could induce IL-12 p40. Finally we tested whether B cells could produce IL-12 p40 because there were very few reports for IL-12 production by B cells. Our results confirmed that macrophages and dendritic cells are main producer of IL-12 p40. Then, we found that microglial cells could produce IL-12 p40 upon stimulation with various TLR ligands. Finally we found that a subset of B cells could produce IL-12 p40 in TLR9-dependent manner. Taken all together, our system will be a valuable tool to identify the type of immune cells that produce IL-12 p40.

The Immunostimulatory Effect of B16 Freezing/thawing Anti-tumor Vaccine / 체질인류학회지

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.

Screening of Interleukin-12/interleukin-23 p40 Inducers in Rheumatoid Synovial Fluids by Fluorescence Reporter Mouse System / 체질인류학회지

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.

Lessons for the pathogenesis of rheumatoid arthritis acquired from experimental animal models / 한양의대학술지

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.

Highly Efficient Gene Expression in Rabbit Synoviocytes Using EBV-Based Plasmid

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.

Effect of Vitamin C on the Expression of Apoptosis in the Adriamycin-Treated Rats / 체질인류학회지

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.

Screening of Genes Regulating TNF-alpha-mediated Synovial Hyperplasia in Rheumatoid Arthritis / 체질인류학회지

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.