OBJECTIVE: Interleukin (IL)-10 has been demonstrated to have anti-inflammatory and anti-tumour activity. Because aberrant angiogenesis is a significant pathogenic component of tumor growth and chronic inflammation, we investigated the effect of IL-10 on the production of vascular endothelial growth factor (VEGF) by the synovial fibroblasts derived from the patients with rheumatoid arthritis (RA). METHODS: Fibroblast-like synoviocytes (FLS) were cultured with transforming growth factor (TGF-beta) alone or with IL-10. The level of VEGF was measured by RT-PCR and enzyme-linked immunosorbent assay (using the 24, 48 and 72 h culture supernatants). The FLSs were cultured with TGF-b for 48 hr in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. Cell viability was assessed using MTT assay. The expressions of VEGF, ERK, AP-1 and IL-10 in the synovial tissue were quantified by immunohistochemistry. RESULTS: IL-10 exhibited the inhibitory effect on VEGF production when the FLSs were stimulated with TGF-beta. ERK and AP-1 inhibitors inhibited the TGF-beta induced VEGF production. Moreover, TGF-beta increased the phosphorylation of ERK and C-Jun, which was significantly inhibited by the IL-10. CONCLUSION: IL-10 may exert an antiangiogenic effect by inhibiting the ERK- and AP-1 mediated VEGF expression in rheumatoid synovial fibroblasts.
Anthracenes ; Arthritis, Rheumatoid ; Cell Survival ; Curcumin ; Down-Regulation ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Flavonoids ; Humans ; Immunohistochemistry ; Inflammation ; Interleukin-10 ; Interleukins ; Phosphorylation ; Transcription Factor AP-1 ; Transforming Growth Factor beta ; Transforming Growth Factors ; Vascular Endothelial Growth Factor A

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic autoimmune disease. Macrophage migration inhibitory factor (MIF) has been shown to be an important pro-inflammatory cytokine in RA. The aim of this study was to determine if the engagement of toll-like receptor 3 (TLR3) induces the production of MIF in the fibroblast-like synoviocytes (FLS) of patients with RA. METHODS: The expression of inflammatory cytokines (e.g. MIF, IL-6, IL-1beta and TNFalpha) and toll-like receptors (e.g. TLR2, TLR3 and TLR4) in the synovial tissue were quantified by immunohistochemistry. FLS were isolated from the synovial tissues of patients with RA and stimulated with TLR-3 ligand polyI:C, in the presence of a neutralizing antibody against IL-6. The concentrations of MIF and IL-6 in the culture supernatants from the FLS were measured using sandwich ELISA. RESULTS: The engagement of TLR3 with PolyI:C increased the production of MIF in FLS. The stimulatory effect of these TLR ligands showed a dose-dependent trend. The combination of TLR3 and TLR4 synergistically increased the level of MIF and IL-6 production. The addition of neutralizing antibodies against IL-6 abrogated the stimulatory effect of the ligands of TLR3 and TLR4 on the production of MIF. CONCLUSION: These results show that TLR3 engagement stimulates the production of MIF and IL-6. Therefore, the TLRs help perpetuate of RA pathogenesis through production of MIF from the FLS in patients with RA, and might provide a new therapeutic approach for the treatment of rheumatoid arthritis.
Antibodies, Neutralizing ; Arthritis, Rheumatoid ; Autoimmune Diseases ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunohistochemistry ; Interleukin-6 ; Ligands ; Macrophages ; Toll-Like Receptor 3 ; Toll-Like Receptors ; Up-Regulation

Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, IL-1 beta and tumor necrosis factor (TNF-alpha) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, IL-1 beta and TNF-alpha in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and IL-1 beta on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and IL-1 beta could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than IL-1 beta or TNF-alpha. These responses were observed in a dose- responsive manner. In addition, IL-17 or IL-1 beta neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and IL-1 beta appears to upregulate the expression of IL-23p19 in RA-SFMC.
Antibodies, Neutralizing ; Arthritis, Rheumatoid ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Humans ; Immunohistochemistry ; Interleukin-17 ; Interleukin-1beta ; Interleukin-23 ; Interleukin-23 Subunit p19 ; Joints ; Osteoarthritis ; RNA, Messenger ; RNA-Directed DNA Polymerase ; Synovial Fluid ; Synovial Membrane ; Tumor Necrosis Factor-alpha

Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, IL-1 beta and tumor necrosis factor (TNF-alpha) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, IL-1 beta and TNF-alpha in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and IL-1 beta on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and IL-1 beta could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than IL-1 beta or TNF-alpha. These responses were observed in a dose- responsive manner. In addition, IL-17 or IL-1 beta neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and IL-1 beta appears to upregulate the expression of IL-23p19 in RA-SFMC.
Antibodies, Neutralizing ; Arthritis, Rheumatoid ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Humans ; Immunohistochemistry ; Interleukin-17 ; Interleukin-1beta ; Interleukin-23 ; Interleukin-23 Subunit p19 ; Joints ; Osteoarthritis ; RNA, Messenger ; RNA-Directed DNA Polymerase ; Synovial Fluid ; Synovial Membrane ; Tumor Necrosis Factor-alpha

OBJECTIVE: Interleukin-15 (IL-15) recruits and activates synovial T cells, and IL-15 plays an important role in amplifying and perpetuating inflammation in the pathogenesis of rheumatoid arthritis (RA). Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for memory T cells in the inflamed RA synovium. This study investigated the effect of IL-15 on SDF-1 production in RA fibroblast-like synoviocytes (FLS). METHODS: The expressions of IL-15 and SDF-1 were determined from the synovium of patients with RA and osteoarthritis (OA) by performing immunohistochemistry. The expressions of SDF-1 was measured from the RA FLS that were cultured with IL-15 and IL-17 by real-time RT-PCR and ELISA. The SDF-1 expression was also measured, via ELISA, from the RA FLS stimulated by IL-15 together with the inhibitors of such intracellular signal molecules as phosphatidylinositol 3-kinase (PI 3-kinase, LY294002), STAT3 (AG490), MAP Kinase (PD98059), NF-kappaB (parthenolide) and activator protein 1 (AP-1, curcumin). RESULTS: IL-15 and SDF-1 were mainly expressed in the RA synovium compared to that of the OA synovium. IL-15 increased the SDF-1 expressions and it, and had an additive effect with IL-17 on the SDF-1 expressions in the cultured RA FLS. The IL-15 induced increase of the SDF-1 expression in the cultured RA FLS was blocked by the inhibitors of PI 3-kinase, NF-kappaB and AP-1. CONCLUSION: The SDF-1 expression was increased in the RA synovium and it was up-regulated by IL-15 in the RA FLS through the PI 3-kinase, NF-kappaB, and AP-1 pathways. These results imply that the IL-15 induced increase of the SDF-1 expressions may be involved in the immunopathogenesis of RA.
Arthritis, Rheumatoid ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Humans ; Immunohistochemistry ; Inflammation ; Interleukin-15 ; Interleukin-17 ; Memory ; NF-kappa B ; Osteoarthritis ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Phosphotransferases ; Synovial Membrane ; T-Lymphocytes ; Transcription Factor AP-1

OBJECTIVE: Interleukin-15 (IL-15) recruits and activates synovial T cells, and IL-15 plays an important role in amplifying and perpetuating inflammation in the pathogenesis of rheumatoid arthritis (RA). Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for memory T cells in the inflamed RA synovium. This study investigated the effect of IL-15 on SDF-1 production in RA fibroblast-like synoviocytes (FLS). METHODS: The expressions of IL-15 and SDF-1 were determined from the synovium of patients with RA and osteoarthritis (OA) by performing immunohistochemistry. The expressions of SDF-1 was measured from the RA FLS that were cultured with IL-15 and IL-17 by real-time RT-PCR and ELISA. The SDF-1 expression was also measured, via ELISA, from the RA FLS stimulated by IL-15 together with the inhibitors of such intracellular signal molecules as phosphatidylinositol 3-kinase (PI 3-kinase, LY294002), STAT3 (AG490), MAP Kinase (PD98059), NF-kappaB (parthenolide) and activator protein 1 (AP-1, curcumin). RESULTS: IL-15 and SDF-1 were mainly expressed in the RA synovium compared to that of the OA synovium. IL-15 increased the SDF-1 expressions and it, and had an additive effect with IL-17 on the SDF-1 expressions in the cultured RA FLS. The IL-15 induced increase of the SDF-1 expression in the cultured RA FLS was blocked by the inhibitors of PI 3-kinase, NF-kappaB and AP-1. CONCLUSION: The SDF-1 expression was increased in the RA synovium and it was up-regulated by IL-15 in the RA FLS through the PI 3-kinase, NF-kappaB, and AP-1 pathways. These results imply that the IL-15 induced increase of the SDF-1 expressions may be involved in the immunopathogenesis of RA.
Arthritis, Rheumatoid ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; Humans ; Immunohistochemistry ; Inflammation ; Interleukin-15 ; Interleukin-17 ; Memory ; NF-kappa B ; Osteoarthritis ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases ; Phosphotransferases ; Synovial Membrane ; T-Lymphocytes ; Transcription Factor AP-1

Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.
Adaptor Proteins, Vesicular Transport/genetics/metabolism ; Arthritis, Rheumatoid/*metabolism ; Blotting, Western ; Cells, Cultured ; Fibroblasts/drug effects/*metabolism ; Humans ; Immunohistochemistry ; Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/*metabolism ; Interleukin-12/pharmacology ; Interleukin-16/pharmacology ; Interleukin-17/pharmacology ; Interleukin-23/pharmacology ; Lipopolysaccharides/pharmacology ; Myeloid Differentiation Factor 88/genetics/*metabolism ; Poly I-C/pharmacology ; Polymerase Chain Reaction ; RNA, Small Interfering/genetics/physiology ; Synovial Membrane/*cytology ; Toll-Like Receptor 4/genetics/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

BACKGROUND: Stromal cell-derived factor (SDF)-1 is a potent chemoattractant for activated T cells into the inflamed Rheumatoid arthritis (RA) synovium. To determine the effect of macrophage migration inhibitory factor (MIF) on the production of SDF-1 in the inflamed RA synovium. METHODS: The expression of SDF-1 and MIF in RA and Osteoarthritis (OA) synovium was examined by immunohistochemical staining. The SDF-1 was quantified by RT-PCR and ELISA after RA fibroblast like synoviocyte (FLS) were treated with MIF in the presence and absence of inhibitors of intracellular signal molecules. The synovial fluid (SF) and serum levels of MIF and SDF-1 in RA, OA and healthy control were measured by ELISA. RESULTS: Expression of SDF-1 and MIF in synovium was higher in RA patients than in OA patients. The production of SDF-1 was enhanced in RA FLS by MIF stimulation. Such effect of MIF was blocked by the inhibitors of NF-kappaB. Concentrations of SDF-1 in the serum and SF were higher in RA patients than in OA patients and healthy control. SDF-1 and MIF was overexpressed in RA FLS, and MIF could up-regulate the production of SDF-1 in RA FLS via NF-kappaB- mediated pathways. CONCLUSION: These results suggest that an inhibition of interaction between MIF from T cells and SDF-1 of FLS may provide a new therapeutic approach in the treatment of RA.
Arthritis, Rheumatoid* ; Chemokine CXCL12* ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts* ; Humans ; Macrophages* ; NF-kappa B ; Osteoarthritis ; Synovial Fluid ; Synovial Membrane ; T-Lymphocytes

B cells play an important role in the pathogenesis of rheumatoid arthritis (RA). High levels of B cell activating factor (BAFF) are detected in autoimmune diseases. BAFF and BAFF receptor (BAFF-R) are expressed in B and T cells of RA synovium. The study was undertaken to identify the NF-kappaB signal pathway involved in the induction of BAFF-R in human B cells. Immunohistochemical staining of NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R was performed on sections of synovium from severe and mild RA and osteoarthritis (OA) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from control and RA patients and B cells were isolated from controls. BAFF-R was analyzed by flow cytometry, realtime PCR and confocal staining after treatment with NF-kappaB inhibitors. NF-kappaB p65, NF-kappaB p50, BAFF, and BAFF-R were highly expressed in severe RA synovium relative to mild RA synovium or OA synovium. BAFF-R expression was reduced by NF-kappaB inhibitors in PBMCs and B cells from normal controls. We also showed reduction in expression of BAFF-R via inhibition of the NF-kappaB pathway in PBMCs of RA patients. BAFF/BAFF-R signaling is an important mechanism of pathogenesis in RA and that BAFF-R reduction by NF-kappaB blocking therapy is another choice for controlling B cells in autoimmune diseases such as RA.
Arthritis, Rheumatoid/genetics/*metabolism/pathology/physiopathology ; B-Cell Activating Factor/genetics/metabolism ; B-Cell Activation Factor Receptor/genetics/*metabolism ; B-Lymphocytes/*drug effects/immunology/metabolism/pathology ; Cell Separation ; Cells, Cultured ; Disease Progression ; Enzyme Inhibitors/pharmacology ; Flow Cytometry ; Gene Expression Regulation/immunology ; Humans ; Immunohistochemistry ; NF-kappa B/*metabolism ; Signal Transduction/immunology ; Synovial Membrane/*pathology ; T-Lymphocytes/drug effects/immunology/metabolism/pathology ; Transcriptional Activation/drug effects

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were 11.6 +/- 12.2 cells/mm2 and 5.6 +/- 8.0 cells/mm2, respectively. The average Treg/Th17 ratio was 5.6 +/- 8.2. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury (P < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.
Acute Disease ; Creatinine/metabolism ; Forkhead Transcription Factors/metabolism ; Graft Rejection/*etiology/pathology ; Humans ; Immunoenzyme Techniques ; Interleukin-17/*metabolism ; Kidney Transplantation/*adverse effects ; Retrospective Studies ; T-Lymphocytes, Regulatory/*immunology/pathology ; Th17 Cells/*immunology/pathology ; Transplantation, Homologous