The anti-malaria agent artesunate inhibits expression of vascular endothelial growth factor and hypoxia-inducible factor-1�� in human rheumatoid arthritis fibroblast-like synoviocyte

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.     

Adiponectin inhibits endothelial synthesis of interleukin-8

Adiponectin is an antiatherogenic adipokine that inhibits inflammation by mechanisms that are not completely understood. We explored the effect of adiponectin on endothelial synthesis of interleukin-8 (IL-8), a pro-inflammatory chemokine that plays a role in atherogenesis. Adiponectin decreased the secretion of IL-8 from human aortic endothelial cells (HAEC) stimulated with tumor necrosis factor-alpha (TNF-alpha). Adiponectin also inhibited IL-8 mRNA expression induced by TNF-alpha. Phosphorylation of IkappaB-alpha was decreased by adiponectin, but phosphorylation of ERK, SAPK/JNK, and p38MAPK were unaffected. Adiponectin increased intra-cellular cAMP levels in HAEC in a dose-dependent manner; PKA activity was also increased. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was inhibited by pretreatment with Rp-cAMP, a PKA inhibitor. These observations suggest that adiponectin inhibits IL-8 synthesis through inhibition of a PKA dependent NF-kappaB signaling pathway. We also showed that adiponectin enhances Akt phosphorylation. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was abrogated in part by pretreatment with the PI3 kinase inhibitor LY294002 or by Akt siRNA transfection, suggesting that Akt activation might inhibit IL-8 synthesis induced by TNF-alpha. We conclude that inhibition of NF-kappaB and activation of Akt phosphorylation may mediate adiponectin inhibition of atherosclerosis.     

Thalidomide inhibits tumor necrosis factor-alpha-induced interleukin-8 expression in endometriotic stromal cells, possibly through suppression of nuclear factor-kappaB activation

OBJECTIVE: Endometriosis, a common disease among women of reproductive age, is characterized by the presence of endometrial-like tissue outside the uterus. TNF-alpha induces IL-8 production in endometriotic cells through nuclear factor-kappaB (NF-kappaB) activation. Thalidomide (Thal) inhibits inflammation by down-regulating the expression of proinflammatory cytokines in tumor cells and inflammatory cells. However, the mechanism of Thal action in human endometriotic stromal cells has not yet been elucidated. MAIN OUTCOME MEASURES: We examined whether Thal abrogates TNF-alpha-induced up-regulation of IL-8 expression in endometriotic stromal cells. RESULTS: Here, we show 1) that treatment of endometriotic stromal cells with TNF-alpha increased the expression of phosphorylated IkappaBalpha and degradation of total IkappaBalpha, which in turn activates NF-kappaB; 2) Thal significantly inhibits the TNF-alpha-induced expression of phosphorylated IkappaBalpha and degradation of IkappaBalpha; 3) TNF-alpha activation induced increased nuclear translocation of NF-kappaB, which was inhibited by pretreatment with either Thal or N-tosyl-L-phenylalanine chloromethyl ketone, an NF-kappaB inhibitor. Thal did not enhance the N-tosyl-L-phenylalanine chloromethyl ketone's action; and 4) Pretreatment with Thal reduced TNF-alpha-induced IL-8 protein production as well as mRNA expression. CONCLUSION: The current study showed for the first time that Thal treatment attenuated the expression of IL-8 by reducing TNF-alpha-induced NF-kappaB activation.     

Mitogen-activated protein kinase kinase 3 is a pivotal pathway regulating p38 activation in inflammatory arthritis

p38 mitogen-activated protein kinase (MAPK) regulates cytokines in arthritis and is, in turn, regulated by MAPK kinase (MKK) 3 and MKK6. To modulate p38 function but potentially minimize toxicity, we evaluated the utility of targeting MKK3 by using MKK3(-/-) mice. These studies showed that TNF-alpha increased phosphorylation of p38 in WT cultured synoviocytes but that p38 activation, IL-1beta, and IL-6 expression were markedly lower in MKK3(-/-) synoviocytes. In contrast, IL-1beta or LPS-stimulated p38 phosphorylation and IL-6 production by MKK3(-/-) synoviocytes were normal. Detailed signaling studies showed that NF-kappaB also contributes to IL-6 production and that TNF-alpha-induced NF-kappaB activation is MKK3-dependent. In contrast, LPS-mediated activation of NF-kappaB does not require MKK3. To determine whether this dichotomy occurs in vivo, two inflammation models were studied. In K/BxN passive arthritis, the severity of arthritis was dramatically lower in MKK3(-/-) mice. Phospho-p38, phospho-MAPK activator protein kinase 2, IL-1beta, CXC ligand 1, IL-6, and matrix metalloproteinase (MMP) 3 levels in the joints of MKK3(-/-) mice were significantly lower than in controls. Exogenous IL-1beta administered during the first 4 days of the passive model restored arthritis to the same severity as in WT mice. In the second model, IL-6 production after systemic LPS administration was similar in WT and MKK3(-/-) mice. Therefore, selective MKK3 deficiency can suppress inflammatory arthritis and cytokine production while Toll-like receptor 4-mediated host defense remains intact.     

Tumor necrosis factor-alpha induces vascular endothelial growth factor-C expression in rheumatoid synoviocytes

OBJECTIVE: To determine the expression of vascular endothelial growth factor-C (VEGF-C) in the synovial fluid of patients with rheumatoid arthritis (RA) and to investigate the regulation of VEGF-C production by major proinflammatory cytokines in fibroblast-like synoviocytes (FLS). METHODS: The concentrations of VEGF-C, tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) were measured using an ELISA method in synovial fluids obtained from 20 patients with RA and 20 with osteoarthritis (OA). Primary cultured RA FLS were stimulated with TNF-alpha or IL-1beta, and the expression levels of VEGF-C mRNA and protein were assessed by quantitative real-time polymerase chain reaction and ELISA. RESULTS: Significantly higher levels of VEGF-C were found in RA synovial fluids compared to OA synovial fluids. VEGF-C levels showed a highly significant correlation with the levels of both TNF-alpha and IL-1beta in the synovial fluid of patients with RA. TNF-alpha stimulation significantly increased VEGF-C mRNA and protein expression in RA FLS in a dose-dependent manner. A tendency to increased expression of VEGF-C was also observed after IL-1beta stimulation in FLS. CONCLUSION: Overexpression of VEGF-C in FLS by stimulation with TNF-alpha may play an important role in the progression of synovial inflammation and hyperplasia in RA by contributing to local lymphangiogenesis and angiogenesis.     

Control of fibroblast-like synoviocyte proliferation by macrophage migration inhibitory factor

OBJECTIVE: The hyperplasia of fibroblast-like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation. METHODS: Human RA FLS were treated with recombinant MIF, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and/or anti-MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor kappa B (NF-kappa B) and mitogen-activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively. RESULTS: FLS proliferation was significantly increased by MIF. IL-1 beta and TNFalpha also induced proliferation, but these effects were prevented by neutralization with anti-MIF mAb. Activation of NF-kappa B was induced by IL-1 beta, but not by MIF. Anti-MIF mAb had no effect on IL-1 beta-induced NF-kappa B nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal-regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF-kappa B antagonism, prevented the effect of MIF on FLS proliferation. CONCLUSION: These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL-1 beta and TNFalpha. In addition, the effects of MIF on FLS activation are independent of NF-kappa B, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.     

Simvastatin inhibits production of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha in fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy. METHODS: RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay. RESULTS: Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate. CONCLUSION: Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.     

Activation of synovial cell p38 MAP kinase by macrophage migration inhibitory factor

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine important in animal models of rheumatoid arthritis (RA). We investigated the utilization by MIF of mitogen activated protein (MAP) kinase signalling pathways in the stimulation of fibroblast-like synoviocytes (FLS), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin 6 (IL-6) and IL-8 expression. METHODS: Cultured human RA FLS were treated with recombinant MIF. Activation of MAPK was measured by Western blotting and blocked using specific inhibitors. The expression of COX-2, PGE(2), IL-6, and IL-8 were measured using flow cytometry, ELISA, and real-time polymerase chain reaction. RESULTS: MIF induced the phosphorylation of FLS p38 and extracellular-signal regulated kinase (ERK) MAP kinase. MIF significantly induced COX-2 and IL-6 protein and mRNA expression as well as PGE(2) and IL-8 production. Antagonism of p38 MAP kinase inhibited MIF induction of COX-2, PGE(2), and IL-6. In contrast, antagonism of ERK had no effect on COX-2, PGE(2), or IL-6. Neither antagonist inhibited MIF-induced IL-8. CONCLUSION: MIF activates RA FLS COX-2 and IL-6 expression via p38 MAP kinase activation and induces IL-8 via p38 and ERK MAP kinase-independent pathways.     

Interleukin-1beta and tumor necrosis factor-alpha induce chemokine and matrix metalloproteinase gene expression in human colonic subepithelial myofibroblasts

BACKGROUND: Colonic subepithelial myofibroblasts may play a role in the inflammatory responses and in extracellular matrix (ECM) metabolism. In this study, we investigated the effects of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and ECM turnover (proliferation of subepithelial myofibroblasts, and secretion of ECM and matrix metalloproteinases (MMPs)) in colonic subepithelial myofibroblasts. METHODS: Human colonic subepithelial myofibroblasts were isolated using the method described by Mahida et al. Chemokine and MMP expressions were determined by ELISA and Northern blotting. Nuclear factor (NF)-kappaB and NF-IL6 DNA binding activities were evaluated by electrophoretic gel mobility shift assays (EMSA). RESULTS: IL-1beta and TNF-alpha did not affect the proliferation of subepithelial myofibroblasts, but stimulated the secretion of types I and IV collagens weakly. Unstimulated subepithelial myofibroblasts secreted a large amount of MMP-2, but a small amount of IL-8, MCP-1 and MMP-1. IL-1beta and TNF-alpha both induced a dose- and time-dependent increase in IL-8, MCP-1 and MMP-1 secretion, and weakly stimulated MMP-2 secretion. IL-1beta and TNF-alpha both rapidly evoked NF-kappaB DNA-binding activity. The inhibition of NF-kappaB activation markedly blocked both IL-1beta- and TNF-alpha-induced IL-8 and MCP-1 mRNA expression, but did not affect MMP-1 mRNA expression. CONCLUSIONS: These observations indicate that chemokine secretion and ECM metabolism are collectively regulated by the proinflammatory cytokines, IL-1beta and TNF-alpha, in colonic subepithelial myofibroblasts. Thus, colonic subepithelial myofibroblasts may play an important role in the pathophysiology of inflammation in the colon.     

Heterogeneous requirement of IkappaB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: implications for therapy

OBJECTIVE: To investigate the potential role of IkappaB kinase 1 (IKK-1) and IKK-2 in the regulation of nuclear factor kappaB (NF-kappaB) activation and the expression of tumor necrosis factor alpha (TNFalpha), as well as interleukin-1beta (IL-1beta), IL-6, IL-8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA). METHODS: Recombinant adenoviruses expressing beta-galactosidase, dominant-negative IKK-1 and IKK-2, or IkappaBalpha were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte-derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus-induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined. RESULTS: IKK-2 was not required for lipopolysaccharide (LPS)-induced NF-kappaB activation or TNFalpha, IL-6, or IL-8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFalpha, and IL-1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK-2 was also required for LPS-induced NF-kappaB activation and IL-6 or IL-8 production. In RA synovial membrane cells, IKK-2 inhibition had no effect on spontaneous TNFalpha production but significantly reduced IL-1beta, IL-6, IL-8, VEGF, and MMPs 1, 2, 3, and 13. CONCLUSION: Our study demonstrates that IKK-2 is not essential for TNFalpha production in RA. However, because IKK-2 regulates the expression of other inflammatory cytokines (IL-1beta, IL-6, and IL-8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.     

Taurine chloramine inhibition of cell proliferation and cytokine production by rheumatoid arthritis fibroblast-like synoviocytes

OBJECTIVE: To examine whether taurine (Tau) or its physiologic chlorinated derivative, taurine chloramine (Tau-CI), affects proliferation of, and proinflammatory cytokine (interleukin-6 [IL-6] and IL-8) production by, fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. METHODS: FLS, isolated from the synovial tissue of 19 RA patients and cultured in vitro for 3-6 passages, were stimulated with the recombinant human cytokines IL-1beta (1 ng/ml), tumor necrosis factor alpha (TNFalpha; 10 ng/ml), or IL-17 (10 ng/ml) in the presence of either Tau or Tau-Cl, which were added at concentrations of 50-500 microM. Tau and Tau-Cl were added simultaneously with, 2 hours before, or 24 hours after the stimuli. The concentrations of IL-6 and IL-8 were determined in culture supernatants using specific enzyme-linked immunosorbent assays. Proliferation of FLS was estimated on the basis of 3H-thymidine incorporation into the cells, which were cultured for 72 hours in the presence of recombinant human basic fibroblast growth factor (bFGF) (1 ng/ml) and Tau or Tau-Cl, which were added simultaneously at the beginning of the culture. RESULTS: Cultured in vitro, RA FLS spontaneously secreted low levels of IL-6 and IL-8, but when RA FLS were stimulated with IL-1beta, TNFalpha, or IL-17, significantly higher amounts of IL-6 and IL-8 were produced. Tau-Cl, but not Tau, inhibited cytokine-triggered synthesis of IL-6 (50% inhibitory concentration [IC50] approximately 225 microM) and IL-8 (IC50 approximately 450 microM) when added simultaneously with the stimuli. However, IL-17-induced production of IL-8 was not affected by Tau-Cl. In the cells prestimulated with IL-1beta for 24 hours, Tau-Cl still inhibited synthesis of IL-6, but did not affect IL-8 production. Moreover, Tau-Cl inhibited spontaneous and bFGF-triggered proliferation of FLS in a dose-dependent manner. Neither Tau nor Tau-Cl affected cell viability. CONCLUSION: The results of these studies demonstrate that Tau-Cl inhibits production of proinflammatory cytokines by RA FLS, as well as proliferation of these cells. Thus, Tau-Cl may act as a physiologic modulator of FLS functions related to their pathogenic role in RA.