Effect of berberrubine on interleukin-8 and monocyte chemotactic protein-1 expression in human retinal pigment epithelial cell line

We examined the effects of berberrubine, a protoberberine alkaloid, on interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) expression in a human retinal pigment epithelial cell line (ARPE-19) stimulated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNF-alpha). ARPE-19 cells were cultured to confluence. Berberrubine and IL-1beta or TNF-alpha were added to the medium. IL-8 and MCP-1 protein concentrations were measured using enzyme-linked immunosorbent assay. IL-8 and MCP-1 mRNA were measured by real time polymerase chain reaction. Nuclear factor kappaB (NF-kappaB) translocation was examined by immunofluorescent staining/microscopy. Berberrubine dose-dependently inhibited IL-8 and MCP-1 protein levels in the media and mRNA expression of the cells stimulated with IL-1beta or TNF-alpha. Immunofluorescent staining/microscopy of NF-kappaB in the nucleus of unstimulated cells was faint (51+/-14 arbitrary units). Fluorescein was dense (215+/-42 or 170+/-24 arbitrary units, respectively) 30 min after stimulation with IL-1beta or TNF-alpha and was decreased to 62+/-18 or 47+/-16 arbitrary units, respectively, by berberrubine. Berberrubine dose-dependently inhibited IL-8 and MCP-1 expression and protein secretion induced by IL-1beta or TNF-alpha. Possibly, the effect on chemotactic factors may be via suppression of NF-kappaB translocation.     

Divergent effects of nitric oxide on airway epithelial cell activation

Nitric oxide (NO*) is a gaseous mediator synthesized by nitric oxide synthases. NO* is involved in the modulation of inflammation, but its role in airway inflammation remains controversial. We investigated the role of NO* in the synthesis of the chemokines interleukin-8 and monocyte chemotactic protein-1, and of intercellular adhesion molecule-1 by human airway epithelial cells. normal human bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used. interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and intercellular adhesion molecule-1 (ICAM-1) expression were measured by ELISA. mRNA was assessed by semiquantitative RTI-PCR. Interleukin-8 secretion was significantly reduced after 24h incubation with the NO* donor, sodium nitroprusside. The effect was dose-dependent. Similar results were obtained with S-nitroso-N-D,L-penicillamine and S-nitroso-L-glutathione. Inhibition of endogenous NO* with the nitric oxide synthase inhibitor N-nitro-L-arginine-methyl-ester caused an increase in IL-8 secretion by lipopolysaccharide- and cytokine-stimulated BEAS-2B cells. Sodium nitroprusside also caused a reduction in monocyte chemotactic protein-1 secretion by both cell types. In contrast, intercellular adhesion molecule-1 expression was upregulated by sodium nitroprusside. RTI-PCR results indicate that the modulation of protein levels was paralleled by modification in mRNA levels. NO* has divergent effects on the synthesis of different inflammatory mediators in human bronchial epithelial cells.     

Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.     

The expression of IL-20 and IL-24 and their shared receptors are increased in rheumatoid arthritis and spondyloarthropathy

The purpose of this study was to analyze the expression of the two proinflammatory cytokines IL-20 and IL-24 and their shared receptors in rheumatoid arthritis and spondyloarthropathy. IL-20 was increased in plasma of rheumatoid arthritis patients compared with osteoarthritis patients and IL-24 was increased in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy patients compared with osteoarthritis patients. IL-20 and IL-24 mRNA was only present at low levels in the synovium. In the synovial membrane, IL-20 protein was present in mononuclear cells and neutrophil granulocytes whereas IL-24 protein was observed in endothelial cells and mononuclear cells. IL-20 receptor type 1 and IL-22 receptor were expressed by granulocytes in the synovial fluid. In synovial fluid mononuclear cell cultures, stimulation with recombinant human IL-20 or recombinant human IL-24 induced monocyte chemoattractant protein 1 (CCL2/MCP-1) secretion, but not tumour necrosis factor alpha mRNA synthesis or IL-6 secretion. Both IL-20 and IL-24 showed correlations to CCL2/MCP-1 in plasma from rheumatoid arthritis and spondyloarthropathy patients. This study associates IL-20 and IL-24 to the synovium of rheumatoid arthritis and spondyloarthropathy and results indicate that the two cytokines contribute to disease pathogenesis through recruitment of neutrophil granulocytes and induction of CCL2/MCP-1.     

Synovial stromal cells from rheumatoid arthritis patients attract monocytes by producing MCP-1 and IL-8

Macrophages that accumulate in the synovium of rheumatoid arthritis patients play an important role in the pathogenesis of this inflammatory disease. However, the mechanism by which macrophages are attracted into the inflamed synovium and accumulate there has not been completely delineated. The results of this study show that rheumatoid arthritis synovial stromal cells produce the chemokines monocyte chemotactic protein-1 and IL-8, and these have the capacity to attract peripheral monocytes. These results suggest that one of the mechanisms by which macrophages accumulate in the inflamed synovium is by responding to the chemokines produced locally.     

Scoparone inhibits PMA-induced IL-8 and MCP-1 production through suppression of NF-kappaB activation in U937 cells

Scoparone is a major component of the shoot of Artemisia capillaris (Compositae), which has been used for the treatment of hepatitis and biliary tract infection in oriental countries. In this study, the effects of scoparone on the expression of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) and activation of nuclear factor-kappaB (NF-kappaB) were examined in U937 human monocytes activated with phorbol 12-myristate 13-acetate (PMA). Scoparone (5-100 microM) had no cytotoxic effect in unstimulated cells and concentration-dependently reversed PMA-induced toxicity in the cells stimulated with PMA. Scoparone concentration-dependently reduced the release of IL-8 and MCP-1 protein and expression of IL-8 and MCP-1 mRNA levels induced by PMA. Moreover, scoparone inhibited the levels of NF-kappaB-DNA complex and NF-kappaB activity in the cells stimulated with PMA in a concentration-dependent manner. Scoparone dose-dependently inhibited the phosphorylation of IkappaBalpha and nuclear translocation of NF-kappaB1 p50, RelA p65, and c-Rel p75. These data suggest that scoparone may inhibit the expression of chemokines (IL-8 and MCP-1) in PMA-stimulated U937 cells and a potential mechanism of scoparone may be inhibition of NF-kappaB activation, which is linked to inhibition of NF-kappaB subunits (NF-kappaB1 p50, RelA p65, and c-Rel p75) translocation via suppression of IkappaBalpha phosphorylation.     

The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor alpha (TNF-alpha)- or interleukin-1beta (IL-1beta)-induced production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor kappaB (NF-kappaB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.     

Rho-kinase mediates lysophosphatidic acid-induced IL-8 and MCP-1 production via p38 and JNK pathways in human endothelial cells

Lysophosphatidic acid (LPA), an inflammatory mediator that is elevated in multiple inflammatory diseases, is a potent activator of Rho kinase (ROCK) signaling and of chemokine production in endothelial cells. In this study, LPA activated ROCK, p38, JNK and NF-κB pathways and induced interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) mRNA and protein expression in human endothelial cells. We mapped signaling events downstream of ROCK, driving chemokine production. In summary, MCP-1 production was partly regulated by ROCK acting upstream of p38 and JNK and mediated downstream by NF-κB. IL-8 production was largely driven by ROCK through p38 and JNK activation, but with no involvement of NF-κB.     

IL-4 inhibits the expression of IL-8 from stimulated human monocytes

Peripheral blood monocytes are important mediators of inflammation via the generation of various bioactive substances, including the recently isolated and cloned chemotactic peptide IL-8. Through cytokine networking, monocyte-derived cytokines are capable of inducing IL-8 expression from non-immune cells. IL-4, a B and T lymphocyte stimulatory factor, has recently been shown to inhibit monocyte/macrophage function, including the ability to suppress monocyte-generated cytokines. We describe the in vitro inhibition of IL-8 gene expression and synthesis from LPS, TNF, and IL-1 stimulated peripheral blood monocytes by IL-4. IL-4 suppressed IL-8 production from stimulated monocytes in a dose-dependent fashion, with partial suppression observed at IL-4 concentrations as low as 10 pg/ml. The IL-4-induced suppressive effects were observed even when IL-4 was administered 2 h post-LPS-stimulation. The IL-4-induced inhibition of IL-8 mRNA expression was dependent on protein synthesis, as the suppressive effects of IL-4 were significantly negated by the addition of cycloheximide. Our findings suggest that IL-4 may be an important endogenous regulator of inflammatory cell recruitment, and adds further support to the potential role of IL-4 as a down-regulator of monocyte immune function.     

The inhibition of hyaluronan degradation reduced pro-inflammatory cytokines in mouse synovial fibroblasts subjected to collagen-induced arthritis

Hyaluronan (HA) degradation produces small oligosaccharides that are able to increase pro-inflammatory cytokines in rheumatoid arthritis synovial fibroblasts (RASF) by activating both CD44 and the toll-like receptor 4 (TLR-4). CD44 and TLR-4 stimulation in turn activate the NF-kB that induces the production of pro-inflammatory cytokines. Degradation of HA occurs via two mechanisms: one exerted by reactive oxygen species (ROS) and one controlled by different enzymes in particular hyaluronidases (HYALs). We aimed to investigate the effects of inhibiting HA degradation (which prevents the formation of small HA fragments) on synovial fibroblasts obtained from normal DBA/J1 mice (NSF) and on synovial fibroblasts (RASF) obtained from mice subjected to collagen induced arthritis (CIA), both fibroblast types stimulated with tumor necrosis factor alpha (TNF-α). TNF-α stimulation produced high mRNA expression and the related protein production of CD44 and TLR-4 in both NSF and RASF, and activation of NF-kB was also found in all fibroblasts. TNF-α also up-regulated the inflammatory cytokines, interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), and other pro-inflammatory mediators, such as matrix metalloprotease-13 (MMP-13), inducible nitric oxide synthase (iNOS), as well as HA levels and small HA fragment production. Treatment of RASF with antioxidants and specific HYAL1, HYAL2, and HYAL3 small interference RNA (siRNAs) significantly reduced TLR-4 and CD44 increase in the mRNA expression and the related protein synthesis, as well as the release of inflammatory mediators up-regulated by TNF-α. These data suggest that the inhibition of HA degradation during arthritis may contribute to reducing TLR-4 and CD44 activation and the inflammatory mediators response.     

Characterization of granulocyte chemotactic activity from human cytokine-stimulated chondrocytes as interleukin 8

Human articular chondrocytes, when stimulated with interleukin 1 beta (IL 1 beta), tumor necrosis factor-alpha (TNF-alpha), or with the double stranded RNA poly (rI).poly (rC), produce a chemotactic activity for granulocytes. The induction with IL 1 beta could be abolished by an antibody to IL 1 beta but not by an antibody to interleukin 6 (IL 6), indicating that the latter is not a mediator for the production of chemotactic activity. The inducers had no direct chemotactic effect on granulocytes. The granulocyte chemotactic factor from chondrocytes was characterized with a specific antibody against leukocyte-derived interleukin 8 (IL 8). The specificity of this antibody was demonstrated by immunochemical and biological criteria such that it could immunoprecipitate only the 6-7 kDa IL 8 protein from fibroblasts, and that it did not neutralize a structurally related monocyte chemotactic protein. This antibody against IL 8 completely neutralized the granulocyte chemotactic activity from stimulated chondrocytes. This demonstrates the identity of chondrocyte IL 8 with leukocyte- and fibroblast-derived IL 8. Our data show that leukocyte chemotaxis into the inflamed joint can be mediated by IL 8, induced in both synovial fibroblasts and chondrocytes by the inflammatory cytokines IL 1 and TNF-alpha.     

Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes

Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.