DA-9601 inhibits activation of the human mast cell line HMC-1 through inhibition of NF-κB

Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 with immune regulatory properties. The formulated ethanol extract of Artemisia asiatica Nakai (DA-9601) has been reported to have antioxidative and anti-inflammatory activities. In this report, we investigated the effect of DA-9601 on the expression of pro-inflammatory cytokines by the activated human mast cell line HMC-1 and studied its possible mechanisms of action. DA-9601 dose-dependently decreased the gene expression and production of TNF-α, IL-1β, and IL-6 on phorbol 12-myristate 13-acetate (PMA)- and calcium ionophore A23187-stimulated HMC-1 cells. In addition, DA-9601 attenuated PMA- and A23187-induced activation of NF-κB as indicated by inhibition of degradation of IκBα, nuclear translocation of NF-κB, NF-κB/DNA binding, and NF-κB-dependent gene reporter assay. Our in vitro studies provide evidence that DA-9601 might contribute to the treatment of mast cell-derived allergic inflammatory diseases.     

Production of proinflammatory cytokines in the human THP-1 monocyte cell line following induction by Tp0751, a recombinant protein of <Emphasis Type="Italic">Treponema pallidum</Emphasis>

The tissue destruction characteristic of syphilis infection may be caused by inflammation due to Treponema pallidum and the ensuing immune responses to the pathogen. T. pallidum membrane proteins are thought to be potent inducers of inflammation during the early stages of infection. However, the actual membrane proteins that induce inflammatory cytokine production are not known, nor are the molecular mechanisms responsible for triggering and sustaining the inflammatory cascades. In the present study, Tp0751 recombinant protein from T. pallidum was found to induce the production of proinflammatory cytokines, including TNF-α, IL-1βand IL-6, in a THP-1 human monocyte cell line. The signal transduction pathways involved in the production of these cytokines were then further investigated. No inhibition of TNF-a, IL-1β, or IL-6 production was observed following treatment with the SAPK/JNK specific inhibitor SP600125 or with an ERK inhibitor PD98059. By contrast, anti-TLR2 mAb, anti-CD14 mAb, and the p38 inhibitor SB203580 significantly inhibited the production of all three cytokines. In addition, pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB, profoundly inhibited the production of these cytokines. Tp0751 treatment strongly activated NF-κB, as revealed by Western blotting. However, NF-κB translocation was significantly inhibited by treatment with PDTC. These results indicated that TLR2, CD14, MAPKs/p38, and NF-κB might be implicated in the inflammatory reaction caused by T. pallidum infection.     

Nickel induces oxidative burst, NF-κB activation and interleukin-8 production in human neutrophils

Many lines of evidence have suggested that oxidative stress and inflammation play a pivotal role in the toxicity of nickel salts. Considering that neutrophils are active participants in inflammatory processes, namely by producing high amounts of reactive oxygen species, the aim of the present study was to evaluate the putative activation of human neutrophils’ oxidative burst by nickel. Subsequently, the influence of nickel in the pathways leading to NADPH oxidation in neutrophils was evaluated by measuring protein kinase C (PKC) activation. The effects of nickel on neutrophils’ nuclear factor κB (NF-κB) activation and on the production of the proinflammatory mediators interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor α were also evaluated. The results obtained showed that nickel, at concentrations that may be attained in vivo, stimulates the production of superoxide radical (O₂ ^·−), hydrogen peroxide (H₂O₂), and hypochlorous acid (HOCl) in human neutrophils in vitro, via activation of PKC. In addition, nickel was shown to activate NF-κB and to induce the production of IL-8 in these cells. These observations indicate that the sustained activation of human neutrophils by nickel may contribute for the long-term adverse effects on human health mediated by this metal.     

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-β-induced IκBα phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.     

Effects of 15-deoxy-∆<Superscript>12,14</Superscript>-prostaglandin J<Subscript>2</Subscript> on the production of IL-8 and the expression of Toll-like receptor 2 in human primary keratinocytes stimulated with lipopolysaccharide

15-deoxy-∆^12,14-prostaglandin J₂ (15d-PGJ₂) is an anti-inflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We investigated the effects of 15d-PGJ₂ on the production of IL-8 and on the expression of Toll-like receptors (TLRs) 2 in human primary keratinocytes stimulated with lipopolysaccharide (LPS). Cell proliferation was analyzed using the MTT assay, TLR2 and -4 mRNA expression was detected by RT–PCR, and IL-8 production and NF-κB p65 activities were determined by ELISA. LPS and 15d-PGJ₂ did not influence the proliferation rate at low concentrations (0.5 and 2.0 μM) in keratinocytes, and showed toxicity at high concentrations (5.0 μM). LPS, compared with control, induced the expression of TLR2 mRNA, increased IL-8 production, and enhanced NF-κB activity. 15d-PGJ₂ decreased TLR2 mRNA, increased IL-8 production, and suppressed NF-κB activity. Costimulation with LPS and 15d-PGJ₂, compared with LPS stimulation alone, decreased TLR2 mRNA (1.8-fold), increased IL-8 production (1.8-fold at 0.5 μM and 3.7-fold at 2.0 μM), and inhibited NF-κB activity (3.3-fold at 0.5 μM and 5.1-fold at 2.0 μM). TLR4 mRNA was not expressed in primary keratinocytes. These results suggest that 15d-PGJ₂ suppresses TLR2 expression and that it up-regulates the production of IL-8 by inhibiting the NF-κB signaling pathway in primary keratinocytes. Thus, 15d-PGJ₂ can have both anti- and pro-inflammatory effects, and 15d-PGJ₂-mediated IL-8 up-regulation is related to the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways.     

Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

Fluvastatin inhibits angiotensin II-induced nuclear factor kappa B activation in renal tubular epithelial cells through the p38 MAPK pathway

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been shown to reduce the progression of renal disease independent of cholesterol-lowering effect, but the mechanism of potential protective effect remains unclear. Here, we investigate the effect of fluvastatin on activation of nuclear factor-κB (NF-κB) induced by angiotensin II (AngII) in rat kidney tubule epithelial cells (NRK-52E). Electrophoretic mobility shift assays (EMSA) was used to detect NF-κB activation. Phosphorylation of cellular p38 mitogen-activated protein kinase (p38MAPK) was determined by western blot analysis. AngII stimulated the DNA-binding activity of NF-κB and phosphorylation of p38MAPK in cultured NRK-52E cells in a dose-dependent (10⁻⁹–10⁻⁶ mol/l) manner (P < 0.01). AngII (10⁻⁶ mol/l) induced a rapid (5 min) increase of the p38MAPK phosphorylation. NF-κB DNA-binding activity was increased at as early as 30 min, peaked at 2 h after AngII treatment. This stimulatory effect of AngII on NF-κB was blocked by SB203580 (a specific inhibitor of p38MAPK). Incubation of cells with fluvastatin significantly inhibited the AngII-induced NF-κB activation in a dose-dependent (10⁻⁷–10⁻⁵ mol/l) manner (P < 0.05). Exogenous mevalonate (10⁻⁴mol/l) prevented the effect of fluvastatin on NF-κB activation. These results suggest the fluvastatin reduced AngII-induced NF-κB activation via the p38MAPK pathway in NRK-52E cells. The effect is at least partly due to blocking the biosynthesis of mevalonate.     

Oxidative stress mediates chemical hypoxia-induced injury and inflammation by activating NF-κb-COX-2 pathway in HaCaT cells

Hypoxia of skin is an important physiopathological process in many diseases, such as pressure ulcer, diabetic ulcer, and varicose ulcer. Although cellular injury and inflammation have been involved in hypoxia-induced dermatic injury, the underlying mechanisms remain largely unknown. This study was conducted to investigate the effects of cobalt chloride (CoCl₂), a hypoxia-mimicking agent, on human skin keratinocytes (HaCaT cells) and to explore the possible molecular mechanisms. Exposure of HaCaT cells to CoCl₂ reduced cell viability and caused overproduction of reactive oxygen species (ROS) and oversecretion of interleukin-6 (IL-6) and interleukin-8 (IL-8). Importantly, CoCl₂ exposure elicited overexpression of cyclooxygenase-2 (COX-2) and phosphorylation of nuclear factor-kappa B (NF-κB) p65 subunit. Inhibition of COX-2 by NS-398, a selective inhibitor of COX-2, significantly repressed the cytotoxicity, as well as secretion of IL-6 and IL-8 induced by CoCl₂. Inhibition of NF-κB by PDTC (a selective inhibitor of NF-κB) or genetic silencing of p65 by RNAi (Si-p65), attenuated not only the cytotoxicity and secretion of IL-6 and IL-8, but also overexpression of COX-2 in CoCl₂-treated HaCaT cells. Neutralizing anti-IL-6 or anti-IL-8 antibody statistically alleviated CoCl₂-induced cytotoxicity in HaCaT cells. N-acetyl-L-cysteine (NAC), a well characterized ROS scavenger, obviously suppressed CoCl₂-induced cytotoxicity in HaCaT cells, as well as secretion of IL-6 and IL-8. Additionally, NAC also repressed overexpression of COX-2 and phosphorylation of NF- B κ p65 subunit induced by CoCl₂ in HaCaT cells. In conclusion, our results demonstrated that oxidative stress mediates chemical hypoxia-induced injury and inflammatory response through activation of NF-κB-COX-2 pathway in HaCaT cells.     

Cells of the synovium in rheumatoid arthritis. Dendritic cells

Dendritic cells are the major antigen-presenting and antigen-priming cells of the immune system. We review the antigen-presenting and proinflammatory roles played by dendritic cells in the initiation of rheumatoid arthritis (RA) and atherosclerosis, which complicates RA. Various signals that promote the activation of NF-κB and the secretion of TNF and IL-1 drive the maturation of dendritic cells to prime self-specific responses, and drive the perpetuation of synovial inflammation. These signals may include genetic factors, infection, cigarette smoking, immunostimulatory DNA and oxidized low-density lipoprotein, with major involvement of autoantibodies. We propose that the pathogenesis of RA and atherosclerosis is intimately linked, with the vascular disease of RA driven by similar and simultaneous triggers to NF-κB.     

Sensitization of endothelial cells to VEGI-induced apoptosis by inhibiting the NF-κB pathway

Vascular endothelial growth inhibitor (VEGI) is an endogenous inhibitor of endothelial cell growth and a promising candidate for cancer therapy. VEGI is able to inhibit tumor growth by specifically targeting the tumor neovasculature. Increasing the anti-angiogenic potential of this cytokine is of great interest for its therapeutic potential. NF-κB is known to have an integral role in TNF superfamily signaling, acting as a pro-survival factor. A role of VEGI-induced NF-κB activation in endothelial cells has yet to be described. Here we show that suppression of the NF-κB pathway can increase the apoptotic potential of VEGI. We used siRNA to deplete NF-κB or its activator IKK2 from adult bovine aortic endothelial cells. The siRNA treatments diminished VEGI-induced NF-κB activation, evidenced from a reduced extent of NF-κB nuclear translocation and diminished expression of NF-κB-target genes such as interleukins-6 and -1β. The siRNA-treated endothelial cells when exposed to VEGI exhibited a marked decrease in cell viability and a significant increase in apoptosis. These results confirm that VEGI utilizes NF-κB as a pro-survival role factor in endothelial cells. We then examined whether a combination of VEGI with NF-κB inhibitors would constitute a more potential therapeutic regiment. We found that in the presence of the NF-κB inhibitors curcumin or BMS-345541 there was a marked increase in the apoptotic potential of VEGI on endothelial cells. These findings indicate that a combination therapy using VEGI and NF-κB inhibitors could be a potent approach for cancer treatment.     

Effect of a small molecule inhibitor of nuclear factor-κB nuclear translocation in a murine model of arthritis and cultured human synovial cells

A small cell-permeable compound, dehydroxymethylepoxyquinomicin (DHMEQ), does not inhibit phosphorylation and degradation of IκB (inhibitor of nuclear factor-κB [NF-κB]) but selectively inhibits nuclear translocation of activated NF-κB. This study aimed to demonstrate the antiarthritic effect of this novel inhibitor of the NF-κB pathway in vivo in a murine arthritis model and in vitro in human synovial cells. Collagen-induced arthritis was induced in mice, and after onset of arthritis the mice were treated with DHMEQ (5 mg/kg body weight per day). Using fibroblast-like synoviocyte (FLS) cell lines established from patients with rheumatoid arthritis (RA), NF-κB activity was examined by electrophoretic mobility shift assays. The expression of molecules involved in RA pathogenesis was determined by RT-PCR, ELISA, and flow cytometry. The proliferative activity of the cells was estimated with tritiated thymidine incorporation. After 14 days of treatment with DHMEQ, mice with collagen-induced arthritis exhibited decreased severity of arthritis, based on the degree of paw swelling, the number of swollen joints, and radiographic and histopathologic scores, compared with the control mice treated with vehicle alone. In RA FLS stimulated with tumor necrosis factor-α, activities of NF-κB components p65 and p50 were inhibited by DHMEQ, leading to suppressed expression of the key inflammatory cytokine IL-6, CC chemokine ligand-2 and -5, matrix metalloproteinase-3, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The proliferative activity of the cells was also suppressed. This is the first demonstration of an inhibitor of NF-κB nuclear translocation exhibiting a therapeutic effect on established murine arthritis, and suppression of inflammatory mediators in FLS was thought to be among the mechanisms underlying such an effect.