Bone Marrow T Cells are Superior to Splenic T Cells to Induce Chimeric Conversion After Non-Myeloablative Bone Marrow Transplantation

BACKGROUND/AIMS: The bone marrow functions not only as the primary B-lymphocyte-producing organ but also as a secondary lymphoid organ for CD4 and CD8 cell responses and a site of preferential homing and persistence for memory T cells. Bone marrow T (BM-T) cells are distinguished from peripheral blood T cells by surface phenotype, cytokine secretion profile, and immune functions. In this study, we evaluated the alloreactive potential of donor lymphocyte infusion (DLI) using BM-T cells in mixed chimerism compared to that using spleen T (SP-T) cells. METHODS: Cells were prepared using established procedures. BM-T cells were obtained as a by-product of T-cell depletion in BM grafting and then cryopreserved for subsequent DLI. We performed DLI using BM-T cells in allogeneic mixed chimera mice on post-BMT day 21. RESULTS: When the same dose of T cells, 5-10x10(5) (Thy1.2+), fractionated from BM and spleen were administered into mixed chimeras, the BM-T group showed complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the SP-T group showed persistent mixed chimerism, with pathological signs of GVHD in the liver and intestine. CONCLUSIONS: Our results suggest that DLI using BM-T cells, even in small numbers, is more potent at inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice without concerns about GVHD.

Increased Indoleamine 2,3-Dioxygenase Expressing CD11c+ CD11b+ Dendritic cells in Oral Tolerance to Type II Collagen / 대한류마티스학회지

OBJECTIVE: Indoleamine 2, 3-dioxygenase (IDO), an immuno suppression enzyme, is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. IDO inhibits T cell proliferation, induces T cell apoptosis, and plays a fundamental role in autoimmunity and allergy. We investigated which subtype of dendritic cells (DCs) is involved in IDO expression and the generation of regulatory T cells during the induction of oral tolerance in type II collagen-induced arthritis (CIA). METHODS: Type II Collagen was fed to DBA/1J mice before immunization. Changes in DC subtypes and induction of regulatory T cell in orally tolerized CIA mice were analyzed. Whether the effect of DC subtype was modulated by the IDO expression, was determined by flow cytometry (FACs) and confocal microscopy. RESULTS: IDO expression of CD11c+ DCs was higher in orally tolerized CIA mice than in non-tolerized CIA mice. CD11b+ DCs of the CD11c +DCs, subtype was higher in the induction of in IDO expression. Our data suggest that these IDO expressing DCs of oral tolerized mice suppressed type II collagen-specific T cell proliferation and favored the differentiation of naive CD4+ T cells into regulatory T cells. Especially, CD11c+CD11b+ DCs expressed IDO, which is known to be associated with regulatory T cell induction. CONCLUSION: We observed that oral tolerance induced the increase in IDO-expressing CD11c+CD11b+ DCs, which appeared to induce regulatory T cells. IDO-expressing CD11c+CD11b+ DCs are involved in oral tolerance, which may provide a new therapeutic approach for the treatment of rheumatoid arthritis.

Practical Evaluation of Engraftment and Mixed Chimerism Using PCR Amplification of a Microsatellite in the Class II Eb Gene in Murine MHC-mismatched, Nonmyeloablative Bone Marrow Transplantation / 대한혈액학회지

BACKGROUND: Although engraftment following murine allogeneic bone marrow transplantation (BMT) is most commonly confirmed by H2 typing using flow cytometry, recipient mice can be seriously injured during peripheral blood (PB) sampling. Therefore, we developed an alternative DNA-based assay that does not require the large volume of PB necessary for flow cytometry. METHODS: A minute volume of PB from the tail vein was used to evaluate the engraftment by PCR amplification of a microsatellite in the class II Eb gene. Dilution experiments were performed to evaluate the sensitivity of this assay for detecting donor cells in mixed cell populations compared with flow cytometry analysis. RESULTS: Early engraftment and mixed chimerism were confirmed, based on the length variation of the microsatellite in the class II Eb gene. The degree of donor chimerism in the donor-recipient cell mixture could be estimated semiquantitatively in a dilution experiment. The sensitivity of this assay by the naked eye approached 10% of the degree of donor chimerism. CONCLUSION: PCR amplification of a microsatellite in the class II Eb gene can be a useful alternative to flow cytometry for evaluating early engraftment and mixed chimerism following murine nonmyeloablative BMT.

Down-regulation of Membrane Bound CD27 Expression Mediated by Protein Kinase C-beta and Protein Kinase C-delta in B Cells / 대한류마티스학회지

OBJECTIVE: CD27 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed on T, B, and NK cells. The signaling via CD27 plays pivotal roles in T-T and T-B interaction. CD27 is a useful marker in assessing the number of circulation B cells and B cell subsets because it permits one step identification of the major B cell compartments, CD27- naive and CD27+ memory B cells as well as CD27high plasma cells. We have analyzed the mechanisms underlying the regulation of CD27 expression. METHODS: Isolation B cells and Raji cells were cultured with PMA. The levels of cell surface CD27 and CD 27 mRNA were analyzed by FACs staining and RT-PCR. Raji cells were cultured with phorbol 12-myristate 13-acetate (PMA), with or without pretreated shedding inhibitor BB3103 and TAPI-1. sCD27 was measured in culture supernatant by ELISA. Cell lysates were analyzed for PKC isotype activation by Western blot. We used PKC inhibitor Ly379196 and rottlen. RESULTS: Membrane expression of CD27 was down-regulated after PMA stimulation without cytotoxic effect in B cells. Furthermore, PMA treatment could directly reduce CD27 mRNA without intermediate protein synthesis in B cells. In contrast, PMA treatment induced soluble form of CD27 (sCD27), which was shed from the cell surface and was found in PMA treatment B cell culture supernatant. PMA-induced sCD27 proteins were decreased with shedding inhibitor BB3103 and TAPI-1. PMA-induced down regulation of CD27 expressions were quenched with protein kinase C (PKC) inhibitor Staurosporin, PKC-beta inhibitor rottlerin and PKC-delta inhibitor Ly379196. CONCLUSION: These data suggest that PMA-induced activation of PKC plays a crucial role in down-regulation of the expression of the CD27 and up-regulation of the shedding of the CD27 in human B cells.

Selective Expansion of TCR V beta 3+CD4+ T Cells in Collagen-induced Arthritis in DBA/1 Mice

BACKGROUND: Collagen-induced arthritis (CIA) in mice is animal model of autoimmune disease known as rheumatic arthritis in human. We investigated CII-specific CD4+ T cell receptor usage in CIA mice. METHODS: In CIA model, draining lymph node (dLN) CD4+ T cells and splenocytes at 3rd, 5th, 8th week, we investigated CII-specific T cell proliferation, production of IL-17, IFN-gamma, TNF-alpha, IL-4 and IL-10. And we also performed anti-CII IgG Ab measurements in serum level, TCRVbeta usage and T cell clonality with RT-PCR-SSCP analysis. Also, we performed proliferative response against CII when CII-specific T cell subset is deleted. RESULTS: CIA mice showed more increase in the serum level of anti-CII IgG than normal mice after induction of arthritis. And the level of anti-CII IgG2a in CIA mice was increased after 3rd week after primary immunization, while anti-CII IgG1 was decreased. Draining LN CD4+T cells have proliferated against CII stimulation at 3rd week after 1st immunization. CD4+T cells derived from dLN of CIA mice produced proinflammatory cytokine IFN-gamma, IL-17 etc. Draining LN CD4 T cells of CIA presented higher proportion of CD4+Vbeta +subsets compared to those of normal mice at 3rd week after 1st immunization, and they were increased in proportion by CII stimulation. Draining LN CD4+ T cells without TCRVbeta +/Vbeta 8.1/8.2+/Vbeta 10b+cells were not responsive against CII stimulation. But, CII-reactive response of TCRVbeta 3-/Vbeta 8.1/8.2-/Vbeta 10b- T cells was recovered when Vbeta 3+ T cells were added in culture. CONCLUSION: Our results indicate that CD4+Vbeta 3+ T cells cells are selectively expanded in dLN of CIA mice, and their recovery upon CII re-stimulation in vitro, as well as the production Th1-type cytokines, may play pivotal role in CIA pathogenesis.

IL-4 Induces CD4+CD25+ Regulatory T Cells from CD4+CD25- T Cells in Peripheral Blood / 대한류마티스학회지

OBJECTIVE: The CD4+CD25+ regulatory T cells (Treg) can be induced by TGFbeta and IL-10 in the periphery, and understanding the biological function of cytokine-induced Treg is critically important for the control of autoimmune diseases. We investigated the IL-4-induced CD4+CD25+ regulatory T cells in human PBMCs, which were derived from the CD4+CD25- T cells. METHODS: The CD4+CD25- T cells from human PBMC were isolated by MACS and cultured in the presence of IL-4 or absence of IL-4. The presence and phenotype of induced CD4+CD25+ T cells were determined by flow cytometry. Supressive activity of induced CD4+CD25+ T cells were assessed by culturing CD4+CD25- and CD4+CD25+ T cells with anti-CD3 monoclonal antibodies and antigen-presenting cells, followed by proliferation RESULTS: After 5 days, significant amount of CD4+CD25+ T cells were generated from the CD4+CD25- T cells cultured with anti-CD3 antibody in the presence of IL-4. These IL-4 induced CD4+CD25+ T cells presented with similar phenotype to natural occurred Treg cells, including CD45RO(hi), CD45RA(lo), CTLA-4(hi), OX40(hi), CD62L(hi) and HLA-DR(hi), and also exhibited high expression of Foxp3 molecule. In addition, the IL-4 induced CD4+CD25+ T cells can suppress the proliferative responses against anti-CD3. The regulatory property of IL-4 induced CD4+CD25+ T cell was partially abrogated after treatment with anti-IL-10 and anti-TGFbeta antibodies. CONCLUSION: These data indicate that IL-4 induced CD4+CD25+ Treg cells can be generated from the CD4+CD25- T cells in the peripheral blood, and may contribute to the important immunoregulatory function in human.

Immune Regulatory Function of Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase in Orally Tolerance to Type II Collagen-induced Animal Model

BACKGROUND: Immune regulatory dendritic cells (DCs) play an important role in maintaining self-tolerance. Recent evidences demonstrate that DCs expressing indoleamine 2,3-dioxygenase (IDO), which is involved in tryptophan catabolism, play an important role in immunoregulation and tolerance and induce T cell apoptosis. This study was devised to examine the role of IDO in the oral tolerance induction in collagen-induced arthritis (CIA) mouse model. METHODS: Beginning 2 weeks before immunization, CII was fed six times to DBA/1 mice and the effect on arthritis was assessed. In tolerized mice, CD11c+ DCs were isolated and stimulated with CII, IFN-gamma, and LPS with or without IDO inhibitor, 1-methyl-DL-tryptophan (1-MT) and IDO expression by CD11c+ DCs was analyzed using FACS and RT-PCR. The expression of IDO, MHC II, CD80, and CD86 by CD11c+ DCs were examined using confocal microscopy. Regulatory effect of CD11c+ DCs on Ag-specific T cell proliferative response to CII was examined by mixed lymphocyte reaction (MLR) with or without 1-MT. RESULTS: The proportion of IDO-expressing CD11c+ DCs was slightly higher in tolerized mice than in CIA mice and significantly increased after stimulation with CII, IFN-gamma, and LPS in an IDO- dependent manner. On confocal microscopic examination, the expression of IDO was higher and those of MHC II and CD86 were lower in CD11c+ DCs from tolerized mice compared to those from CIA mice. On MLR, CD11c+ DCs from tolerized mice inhibited T cell proliferative response to CII in an IDO-dependent manner. CONCLUSION: Enhanced IDO expression by CD11c+ DCs from tolerized mice may contribute to the regulation of proliferative response of CII-reactive T cells and could be involved in the induction of oral tolerance to CII.

Regulation of Interleukin-17 Production in Patients with Rheumatoid Arthritis by Phosphoinositide 3-kinase (PI3K)/ Akt and Nuclear Factor KappaB (NF-kappaB) Dependent Signal Transduction Pathway

Inflammatory mediators has been recognized as an important role in the pathogenesis of rheumatoid arthritis (RA). IL-17 is increasingly recognized as an important regulator of immune and inflammatory responses, including induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence of the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. However, the signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in the regulation of IL-17 production in RA. PBMC were separated from RA (n=24) patients, and stimulated with various agents (anti CD3, anti CD28, PHA, ConA, IL-15). IL-17 levels were determined by sandwich ELISA and RT-PCR. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody, PHA, IL-15 or MCP-1 (P<0.05). ConA also strongly induced IL-17 production (P<0.001), whereas TNF-alpha, IL-1beta, IL-18 or TGF-beta did not. IL-17 was detected in the PBMC of patients with osteoarthritis (OA) but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K-Akt pathway and activation of the PI3K-Akt pathway resulted in a pronounced augmentation of nuclear factor kappaB (NF-kappaB). IL-17 production by activated PBMC in RA is completely or partially blocked in the presence of NF-kappaB inhibitor PDTC and PI3K- Akt inhibitor, wortmannin and LY294002, respectively. Whereas the inhibition of AP-1 and extracellular signal-regulated kinase (ERK)1/2 did not affect IL-17 production. These results provide new insight into that PI3K/Akt and NF-kappaB dependent signal transduction pathway could be involved in the overproduction of key inflammatory cytokine, IL-17 in rheumatoid arthritis.

Feedback Control of Cyclooxygenase-2 Expression by Prostaglandin E2 in Rheumatoid Synoviocytes

Objective: The role of prostaglandin E2 (PGE2) in the etiopathogenesis of immune and inflammatory diseases has become the subject of recent debate. To determine the role of PGE2 in rheumatoid arthritis (RA), we tested the effect of exogenous PGE2 on the production of cyclooxygenase-2 (COX-2) by rheumatoid synoviocytes. METHODS: Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and cultured in the presence of PGE2. The COX-2 mRNA and protein expression levels were determined by RT-PCR and Western blot analysis, respectively. The PGE2 receptor subtypes in the FLS were analyzed by RT-PCR. Electrophoretic mobility shift assay (EMSA) was used to measure the NF-kappaB binding activity for COX-2 transcription. The in vivoeffect of PGE2 on the development of arthritis was also tested in collagen induced arthritis (CIA) animals. RESULTS: PGE2 (10(-11) to 10(-5) M) dose-dependently inhibited the expression of COX-2 mRNA and the COX-2 protein stimulated with IL-1beta, but not COX-1 mRNA. NS-398, a selective COX-2 inhibitor, displayed an additive effect on PGE2-induced COX-2 downregulation. The FLS predominantly expressed the PGE2 receptor (EP) 2 and EP4, which mediated the COX-2 suppression by PGE2. Treatment with anti-IL-10 monoclonal antibodies partially reversed the PGE2-induced suppression of COX-2 mRNA, suggesting that IL-10 may be involved in modulating COX-2 by PGE2. Experiments using an inducer and an inhibitor of cyclic AMP (cAMP) suggest that cAMP is the major intracellular signal that mediates the regulatory effect of PGE2 on COX-2 expression. EMSA revealed that PGE2 inhibited the binding of NF-kappaB in the COX-2 promoter via a cAMP dependent pathway. In addition, a subcutaneous injection of PGE2 twice daily for 2 weeks significantly reduced the incidence and severity of CIA as well as the production of IgG antibodies to type II collagen. CONCLUSION: Our data suggest that overproduced PGE2 in the RA joints may function as an autocrine regulator of its own synthesis by inhibiting COX-2 production and may, in part, play an anti-inflammatory role in the arthritic joints.

Expression of Co-stimulatory Molecules and STAT/SOCS Signaling Factors in the Splenocytes of Mice Tolerized against Arthritis by Oral Administration of Type II Collagen

Oral administration of antigen has long been used in the induction of immune tolerance in various animal models of autoimmune diseases including rheumatoid arthritis (RA). Alleveation of arthritogenic symptoms has been reported from RA patients who received oral administration of type II collagen (CII) without side effects, however its rather inconsistent therapeutic efficacy and variation among patients calls for more detailed investigation on the mechanism of oral tolerance to be settled as regular treatment for RA. In an attempt to understand the immunogenic processes underpinning tolerance induction by orally administered CII, we analyzed changes in the expression of costimulatory molecules and STAT/SOCS signaling messengers in the mouse model of collagen induced arthritis (CIA). We found thatin the spleen of CIA mice, that has been undergone repeated oral feeding of CII prior to the induction of arthritis, showed increased promortion of CTLA4 expressing lymphocytes than in the spleen of PBS fed control. On the other hand, cells expressing CD28 or ICOS were decreased in the spleen of tolerized mice. Tolerance induction by oral CII administration also enhanced the expression of STAT6 in both RNA and protein level, while not affecting the expression of STAT3. The expression of SOCS3, which hasbeen known to transmit STAT-mediated signals from Th2 type cytokines, remained unchanged in the spleen of tolerized mice. Interestingly transcript of SOCS1, which has been associated with Th1 related pathways, was only visible in the spleen of tolerized but not of control mice, suggesting that as in the case of IL-6 signaling, it may exert a feed back inhibition toward the Th1 type stimulation.

Studies on the Cellular Immune Response in Animal Model of Arthritis after the Induction of Oral Tolerance

Oral administration of antigen has long been considered as a promising alternative for the treatment of chronic autoimmune diseases including rheumatoid arthritis (RA), and oral application of type II collagen (CII) has been proven to improve pathogenic symptoms in RA patients without problematic side effects. To further current understandings about the immune suppression mechanisms mediated by orally administered antigens, we examined the changes in IgG subtypes, T-cell proliferative response, and proportion of interleukin (IL)-10 producing Th subsets in a time course study of collagen induced arthritis (CIA) animal models. We found that joint inflammation in CIA mouse peaked at 5 weeks after first immunization with CII, which was significantly subdued in mice pre-treated by repeated oral administration of CII. Orally tolerized mice also showed increase in their serum level of IgG1, while the level of IgG2a was decreased. T-cell proliferation upon CII stimulation was also suppressed in lymph nodes of mice given oral administration of CII compared to non-tolerized controls. When cultured in vitro in the presence of CII, T-cells isolated from orally tolerized mice presented higher proportion of CD4+ IL-10+ subsets compared to non-tolerized controls. Interestingly, such increase in IL-10 producing cells were obvious first in Peyer's patch, then by 5 weeks after immunization, in mesenteric lymph node and spleen instead. This result indicates that a particular subset of T-cells with immune suppressive functions might have migrated from the original contact site with CII to inflamed joints via peripheral blood after 5 weeks post immunization.

Urinary Excretion of Rantes is Elevated in Lupus Nephritis / 대한류마티스학회지

OBJECTIVE: Infiltrating T cells and monocytes have been implicated in the pathogenesis of lupus nephritis (LN). Chemokines may play a key role in the recruitment of these cells. We investigated whether RANTES (regulated on activation normal T cell expressed and secreted), one of the CC chemokine family, may be involved in the pathogenesis of LN. METHODS: We measured the levels of RANTES in sera and urine from 87 systemic lupus erythematosus (SLE) patients and 78 healthy controls using ELISA. Clinical and laboratory assessment including SLE disease activity index (SLEDAI) were performed at the time of sampling. RESULTS: Serum RANTES levels were significantly higher in the patients with SLE than in healthy controls (115.0+/-5.6 vs. 91.5+/-4.0 pg/ml, p=0.001, mean+/-SEM). Serum RANTES levels correlated well with anti-dsDNA antibody titer (r=0.29, p<0.05) and inversely with serum complement C4 level (r=-0.28, p<0.05). Urinary RANTES/creatinine ratios were significantly higher in patients with nephritis than those without (3.4+/-0.4 vs. 2.2+/-0.3, p=0.004), while serum RANTES level was not different between patients with nephritis and those without. Moreover, urinary RANTES/creatinine ratio positively correlated with urine protein/creatinine ratio (r=0.41, p<0.001). CONCLUSIONS: Our results demonstrate that serum RANTES was elevated in patients with SLE and urinary excretion of RANTES was strongly associated with presence of nephritis. These data suggest that RANTES may be expressed in renal inflammatory sites and may participate in the pathogenesis of LN possibly by augmenting the recruitment of T cells and monocytes.

Elevated serum interleukin 15 (IL-15) in Behcet's disease / 대한류마티스학회지

OBJECTIVE: To evaluate clinical significance of interleukin 15 (IL-15) in patients with Behcet's disease (BD). METHODS: Serum samples were obtained from 31 patients with BD and 29 healthy controls. BD patients were divided into active and inactive group according to the presence of clinical manifestations on the day of sampling. Serum levels of IL-15 and IL-8 were measured by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum levels of IL-15 and IL-8 were significantly higher in BD patients than in healthy controls (117.2+/-26.2 pg/ml versus 51.8+/-15.4 pg/ml, p<0.01, 287.7+/-100.9 pg/ml versus 138.5+/-17.2 pg/ml, p<0.01, respectively). There was a significant correlation between serum levels of IL-15 and IL-8 IL-15 levels compared with those without it (161.1+/-68.8 pg/ml versus 96.4+/-3 4 . 8pg/ml, p<0.05). Serum levels of IL-15 and IL-8 tended to be higher in active group than in inactive group, but didn't reach statistical significance. CONCLUSION: Serum level of IL-15 was elevated in patients with BD, especially those with uveitis, but it did not seem to be useful as a marker of disease activity in BD.

Inhibition of Vascular Endothelial Growth Factor Expression in Cultured Rheumatoid Synoviocytes by Nonsteroidal Anti-inflammatory Drugs / 대한류마티스학회지

OBJECTIVE: Vascular endothelial growth factor (VEGF),a potent angiogenic, permeability-enhancing cytokine plays an important role in chronic inflammatory process of rheumatoid arthritis (RA).Nonsteroidal anti-inflammatory drugs (NSAIDs)are the most widely used drugs for the treatment of RA.However, the effect of NSAIDs on angiogenesis in rheumatoid synovium is unclear.In this study,we investigated the effects of NSAIDs such as indomethacin (IDC) on TGF-beta-induced VEGF production in rheumatoid synoviocytes. METHODS: Fibroblast-like synoviocytes (FLS)from RA were stimulated with T G F -beta(10 ng/ml)for 24hr in the presence of the various concentrations of IDC. The levels of VEGF were measured in culture supernatant by ELISA.In addition, COX-2 and VEGF mRNA expression of cultured FLS were evaluated by RT-PCR. RESULTS:VEGF production from FLS was significantly increased in the presence of TGF-beta.IDC exerted a dose-dependent inhibitory effect on the production of VEGF induced by TGF-beta.RT-PCR analysis showed that IDC also inhibited TGF-beta-induced COX-2 and VEGF mRNA expression in cultured FLS by a dose-dependent manner. CONCLUSION:Our results demonstrate that NSAIDs inhibit VEGF production and the expression of its mRNA and COX-2 mRNA in synovial cells of RA patients.These findings suggest that NSAIDs may suppress progression and perpetuation of rheumatoid synovitis by anti-angiogenic activity.

Effects of Hydroxychloroquine on the Metabolism of Fas ligand of T cells / 대한류마티스학회지

OBJECTIVE: Hydroxychloroquine (HCQ) is a drug that has been used to treat autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus. However, the specific mechanism for its pharmacologic action has been largely unknown. It has been reported that dysregulation of lymphocytic apoptosis mediated by Fas ligand (FasL) and Fas is associated with the development of autoimmune diseases and HCQ induces apoptosis in peripheral blood lymphocytes. These reports suggest that HCQ may exert its pharmacologic effects through the modulation of FasL and Fas. Therefore, we are intended to investigate the effects of HCQ on the regulation of FasL and Fas. Jurkat cells or peripheral blood mononuclear cells (PBMNC) were treated with varying concentrations of HCQ. Semiquantative reverse transcription- polymerase chain reaction, Western blotting, flow cytometry, and ELISA were used for this study. HCQ at nontoxic concentrations( 50~150 micrometer) caused a dose dependent increase of FasL mRNA expression and FasL in cell lysates. HCQ inhibited the release of intracellular 40 kDa FasL by Jurkat cells which were pulse-stimulated with PHA (50 microgram/ml). Jurkat cells activated with PHA increased membrane bound FasL (mFasL) expression (24.5+/-4.3%), however Jurkat cells pretreated with HCQ(150 micrometer) followed by PHA administration did not further increase mFasL expression (26.8+/-1.6%). Addition of different concentrations of HCQ to the cultured PBMNC stimulated with PHA for 24 hours showed increase of soluble FasL (sFasL). The levels of sFasL treated with HCQ zero, 50, 150 and 300 micrometer for 24 hours were 38.6+/-3.0, 43.4+/-5.1, 77.0+/-3.6(P<0.05) and 72.3+/-8.1pg/ml(P<0.05) respectively. However, fas metabolism was not affected by HCQ. CONCLUSION: These results suggest that HCQ may exhibit its pharmacological effects by upregulation of FasL gene expression and increased production sFasL without any influence on the Fas metabolism of T cells.

Measurement of Serum Fas Ligand (FasL), FasL-Fas Complex and FasL-IgG Complex in Patients with Rheumatic Diseases / 대한류마티스학회지

OBJECTIVE: To quantify the soluble Fas ligand (sFasL) and to measure FasL-Fas complex and FasL-IgG complex in the sera of patients with various rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), and adult onset Still? disease (AOSD). METHODS: Serum samples were obtained from 37 patients with SLE, 40 with RA, 30 with SSc, 20 with AOSD, and 40 healthy controls. The serum sFasL, FasL-Fas complex, and FasL-IgG complex were measured using a sandwich enzyme-linked immunoabsorbent assay. Hospital medical records were retrospectively reviewed for clinical and laboratory characteristics in patients with SLE. Disease activity in SLE patients was assessed by the SLE Disease Activity Index (SLEDAI) score. RESULTS: In patients with SLE, serum sFasL levels (383.1+/-208.9pg/ml) were significantly higher (p<0.001) than those of healthy controls (192.0+/-84.7pg/ml). sFasL levels in patients with RA (150.8+/-30.7pg/ml, p=0.014), SSc (115.4+/-13.5pg/ml, p<0.001), and AOSD (137.5+/-12.9pg/ml, p=0.001) were significantly lower compared with healthy controls. The frequencies of positive FasL-Fas complex and FasL-IgG complex were higher in patients with SLE (56.8%, 56.8% respectively) than in healthy controls (2.5%, 0% respectively) (p<0.001). All patients with RA or AOSD were negative for FasL-Fas complex and FasL-IgG complex. No patients with SSc were positive for FasL-Fas complex. On the other hand, the positive frequency of FasL-IgG complex was greater in patients with SSc (16.7%) than in healthy controls (0%)(p=0.012). Serum levels of FasL-IgG complexes in active SLE patients (OD 0.467+/-0.050) were tended to be lower than those in inactive SLE patients (OD 0.509+/-0.055)(p=0.060). SLEDAI score was tended to be negatively correlated with the serum levels of FasL-IgG complex in patients with SLE (r=-0.308, p=0.068). CONCLUSION: These results suggest that FasL may possibly play a role in the pathogenesis of SLE.

Measurement of Serum Fas Ligand (FasL), FasL-Fas Complex and FasL-IgG Complex in Patients with Rheumatic Diseases / 대한류마티스학회지

OBJECTIVE: To quantify the soluble Fas ligand (sFasL) and to measure FasL-Fas complex and FasL-IgG complex in the sera of patients with various rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc), and adult onset Still? disease (AOSD). METHODS: Serum samples were obtained from 37 patients with SLE, 40 with RA, 30 with SSc, 20 with AOSD, and 40 healthy controls. The serum sFasL, FasL-Fas complex, and FasL-IgG complex were measured using a sandwich enzyme-linked immunoabsorbent assay. Hospital medical records were retrospectively reviewed for clinical and laboratory characteristics in patients with SLE. Disease activity in SLE patients was assessed by the SLE Disease Activity Index (SLEDAI) score. RESULTS: In patients with SLE, serum sFasL levels (383.1+/-208.9pg/ml) were significantly higher (p<0.001) than those of healthy controls (192.0+/-84.7pg/ml). sFasL levels in patients with RA (150.8+/-30.7pg/ml, p=0.014), SSc (115.4+/-13.5pg/ml, p<0.001), and AOSD (137.5+/-12.9pg/ml, p=0.001) were significantly lower compared with healthy controls. The frequencies of positive FasL-Fas complex and FasL-IgG complex were higher in patients with SLE (56.8%, 56.8% respectively) than in healthy controls (2.5%, 0% respectively) (p<0.001). All patients with RA or AOSD were negative for FasL-Fas complex and FasL-IgG complex. No patients with SSc were positive for FasL-Fas complex. On the other hand, the positive frequency of FasL-IgG complex was greater in patients with SSc (16.7%) than in healthy controls (0%)(p=0.012). Serum levels of FasL-IgG complexes in active SLE patients (OD 0.467+/-0.050) were tended to be lower than those in inactive SLE patients (OD 0.509+/-0.055)(p=0.060). SLEDAI score was tended to be negatively correlated with the serum levels of FasL-IgG complex in patients with SLE (r=-0.308, p=0.068). CONCLUSION: These results suggest that FasL may possibly play a role in the pathogenesis of SLE.