NF‐κB–regulated expression of cellular FLIP protects rheumatoid arthritis synovial fibroblasts from tumor necrosis factor α–mediated apoptosis

Objective

Little apoptosis has been observed in rheumatoid arthritis (RA) synovial tissues. Tumor necrosis factor α (TNFα) is expressed in the joints of patients with RA, yet RA synovial fibroblasts are relatively resistant to apoptosis induced by TNFα. Recently, we demonstrated that FLIP is highly expressed in the RA joint. These studies were performed to determine if TNFα‐induced NF‐κB controls the expression of FLIP long (FLIP_L) and FLIP short (FLIP_S) in RA synovial fibroblasts and to determine the role of FLIP in the control of TNFα‐induced apoptosis.

Methods

RA synovial fibroblasts were isolated from RA synovial tissues and used between passages 3 and 9. RA synovial or control fibroblasts were sham infected or infected with a control adenovirus vector or one expressing the super‐repressor IκBα (srIκBα). The cells were stimulated with TNFα or a control vehicle, and expression of FLIP_L and FLIP_S was determined by isoform‐specific real‐time polymerase chain reaction and Western blot analysis. Cell viability was determined by XTT cleavage, and apoptosis was determined by annexin V staining, DNA fragmentation, and activation of caspases 8 and 3.

Results

TNFα induced the expression of both isoforms of FLIP messenger RNA (mRNA) in RA synovial fibroblasts; however, FLIP_L was the dominant isoform detected by Western blot analysis. In control fibroblasts, TNFα induced the expression of FLIP_L and FLIP_S mRNA and protein. The TNFα‐induced, but not the basal, expression of FLIP was regulated by NF‐κB. When NF‐κB activation was suppressed by the expression of srIκBα, TNFα‐mediated apoptosis was induced. TNFα‐induced apoptotic cell death was mediated by caspase 8 activation and was prevented by the ectopic expression of FLIP_L or the caspase 8 inhibitor CrmA.

Conclusion

The TNFα‐induced, but not the basal, expression of FLIP is regulated by NF‐κB in RA synovial fibroblasts. The resistance of RA synovial fibroblasts to TNFα‐induced apoptosis is mediated by the NF‐κB–regulated expression of FLIP. These observations support the role of NF‐κB and FLIP as attractive therapeutic targets in RA.

     

Chondrocyte AMP‐activated protein kinase activity suppresses matrix degradation responses to proinflammatory cytokines interleukin‐1β and tumor necrosis factor α

Objective

Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.

Methods

Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr¹⁷² in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.

Results

Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.

Conclusion

Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.

     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

RhoA‐mediated,tumor necrosis factor α–induced activation of NF‐κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective

Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF‐κB in tumor necrosis factor α (TNFα)–stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα‐induced activation of RhoA and NF‐κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

Methods

Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull‐down assay. NF‐κB DNA binding activity and nuclear translocation of NF‐κB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

Results

TNFα stimulation elicited a robust increase in RhoA activity in a dose‐dependent manner, and SMV mitigated this increase. TNFα also hastened NF‐κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin‐1β (IL‐1β) and IL‐6. SMV prevented the increase in NF‐κB activation and rise in IL‐1β and IL‐6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF‐κB and RhoA. Furthermore, cotransfection with a dominant‐negative mutant of RhoA demonstrated that the TNFα‐induced signaling pathway involved sequential activation of RhoA, leading to NF‐κB activation and, ultimately, to secretion of cytokines.

Conclusion

This study identifies RhoA as the key regulator of TNFα‐induced NF‐κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.

     

Tumor necrosis factor α‐mediated joint destruction is inhibited by targeting osteoclasts with osteoprotegerin

Objective

To study the effects of osteoclast‐targeted therapies, such as osteoprotegerin (OPG) and pamidronate, on joint inflammation and bone destruction using a tumor necrosis factor α (TNFα)‐transgenic mouse model.

Methods

Mice were placed into 5 groups that received either OPG, pamidronate, a combination of both agents, infliximab as a positive control, or phosphate buffered saline as a negative control. Treatment was initiated at the onset of arthritis, continued over 6 weeks, and thereafter, the clinical, radiologic, and histologic outcomes were assessed.

Results

A significant improvement in clinical symptoms, as assessed by the reduction of paw swelling, was only found in the infliximab group, whereas all other treatment groups failed to show significant improvement. However, when assessing structural damage with radiographic analysis, a significant retardation of joint damage was evident in animals treated with OPG (55% reduction of erosions), pamidronate (50% reduction of erosions) the combination therapy of OPG and pamidronate (64% reduction of erosions), and with infliximab (66% reduction of erosions). Confirming these data, quantitative histologic analysis revealed a significant reduction in the size of bone erosions in all treatment groups (OPG 56%, pamidronate 53%, OPG and pamidronate 81%, and infliximab 46%) compared with the control group. Furthermore, a significant reduction of osteoclast numbers was seen in animals treated with OPG alone or in combination with pamidronate as well as in animals treated with infliximab.

Conclusion

These data suggest that OPG alone or in combination with bisphosphonates is an effective therapeutic tool for the prevention of TNFα‐mediated destruction of bone by reducing the number of bone‐resorbing cells in the inflammatory tissue.

     

Interleukin‐6 receptor shedding is enhanced by interleukin‐1β and tumor necrosis factor α and is partially mediated by tumor necrosis factor α–converting enzyme in osteoblast‐like cells

Objective

Interleukin‐6 (IL‐6) and soluble IL‐6 receptor (sIL‐6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL‐6R levels in response to the inflammatory cytokines IL‐1β and tumor necrosis factor α (TNFα) and to determine the role of TNFα‐converting enzyme (TACE) in this process.

Methods

Levels of sIL‐6R in the culture media of MG63 and SAOS‐2 osteoblast‐like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL‐6R and TACE to determine the potential involvement of TACE in IL‐6R shedding.

Results

IL‐1β and TNFα increased the levels of sIL‐6R in the culture media of MG63 osteoblast‐like cells. This effect was not influenced by cycloheximide or 5,6‐dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL‐1β and TNFα had no influence on the alternatively spliced form of IL‐6R RNA. Levels of sIL‐6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS‐2 cells with expression plasmids for IL‐6R and TACE produced a dose‐dependent increase in sIL‐6R levels.

Conclusion

IL‐1β‐ and TNFα‐mediated induction of IL‐6R shedding in osteoblast‐like cells is at least partly dependent on TACE activation.

     

Expression of Toll‐like receptors 2 and 4 in rheumatoid synovial tissue and regulation by proinflammatory cytokines interleukin‐12 and interleukin‐18 via interferon‐γ

Objective

To study the expression of Toll‐like receptor 2 (TLR‐2) and TLR‐4 and its association with proinflammatory cytokines in synovial tissue from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals.

Methods

Synovial tissue specimens from 29 RA patients were stained for TLR‐2, TLR‐4, and proinflammatory cytokines (interleukin‐1β [IL‐1β], IL‐12, IL‐17, IL‐18, and tumor necrosis factor α [TNFα]). The expression of TLR‐2, TLR‐4, and cytokines as well as the degree of inflammation in synovial tissue were compared between patients with RA, patients with OA (n = 5), and healthy individuals (n = 3). Peripheral blood mononuclear cells (PBMCs) were incubated with IL‐12 and IL‐18, and TLR expression was assessed using fluorescence‐activated cell sorter analysis. Production of TNFα and IL‐6 was measured using Luminex bead array technology.

Results

In RA synovial tissue, the expression of TLR‐2 was slightly higher than that of TLR‐4. Interestingly, both TLR‐2 and TLR‐4 were expressed at higher levels in moderately inflamed synovium, as compared with synovial tissue with no or severe inflammation. TLR expression in both the lining and the sublining was associated with the presence of IL‐12 and IL‐18, but no other cytokines, in the lining. The expression of both TLRs was low in synovial tissue from OA patients and healthy donors. Stimulation of PBMCs with IL‐12 and IL‐18 resulted in increased expression of both TLR‐2 and TLR‐4; this could be blocked with anti–interferon‐γ (anti‐IFNγ) antibodies, suggesting a role for IFNγ. Lipopolysaccharide‐ or lipoteichoic acid–mediated triggering of PBMCs incubated with IL‐12/IL‐18 or IFNγ led to an increased production of both TNFα and IL‐6, indicating the functionality of TLR‐2 and TLR‐4.

Conclusion

TLR‐2 and TLR‐4 are expressed in synovial tissue of patients with clinically active disease and are associated with the levels of both IL‐12 and IL‐18. The synergistic effect of IL‐12 and IL‐18 on T cell IFNγ production seems to regulate expression of TLR‐2 and TLR‐4 in the synovial tissue of RA patients.

     

Association of interleukin‐18 expression with enhanced levels of both interleukin‐1β and tumor necrosis factor α in knee synovial tissue of patients with rheumatoid arthritis

Objective

To examine the expression patterns of interkeukin‐18 (IL‐18) in synovial biopsy tissue of patients with rheumatoid arthritis (RA), and to determine whether expression of this primary cytokine is related to the expression of other cytokines and adhesion molecules and related to the degree of joint inflammation.

Methods

Biopsy specimens of knee synovial tissue either without synovitis (n = 6) or with moderate or severe synovitis (n = 11 and n = 12, respectively) were obtained from 29 patients with active RA. Paraffin‐embedded, snap‐frozen sections were used for immunohistochemical detection of IL‐18, tumor necrosis factor α (TNFα), IL‐1β, IL‐12, and IL‐17. Furthermore, adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E‐selectin, and cell markers CD3, CD14, and CD68 were stained.

Results

IL‐18 staining was detectable in 80% of the RA patients, in both the lining and sublining of the knee synovial tissue. IL‐18 expression in the synovial tissue was strongly correlated with the expression of IL‐1β (in the sublining r = 0.72, in the lining r = 0.71; both P < 0.0001) and TNFα (in the sublining r = 0.59, P < 0.0007, and in the lining r = 0.68, P < 0.0001). In addition, IL‐18 expression in the sublining correlated with macrophage infiltration (r = 0.64, P < 0.0007) and microscopic inflammation scores (r = 0.78, P < 0.0001), and with the acute‐phase reaction as measured by the erythrocyte sedimentation rate (r = 0.61, P < 0.0004). Interestingly, RA synovial tissue that coexpressed IL‐18 and IL‐12 demonstrated enhanced levels of the Th1‐associated cytokine IL‐17.

Conclusion

Our results show that expression of IL‐18 is associated with that of IL‐1β and TNFα and with local inflammation in the synovial tissue of patients with RA. In addition, synovial IL‐18 expression correlates with the acute‐phase response. These data indicate that IL‐18 is a primary proinflammatory cytokine in RA that drives the local production of IL‐1β and TNFα.

     

Induction of lipocalin‐2 expression in acute and chronic experimental liver injury moderated by pro‐inflammatory cytokines interleukin‐1β through nuclear factor‐κB activation

Background: Lipocalin‐2 (LCN2) belongs to the lipocalin superfamily, sharing a barrel‐shaped tertiary structure with a hydrophobic pocket and an ability to bind lipophilic molecules. LCN2 has recently emerged as an important modulator of cellular homeostasis in several organs, i.e. heart, lung and kidney, but little is known about the expression of LCN2 in acute and chronic liver injury. Aims: In this study, we wanted to analyse the expression and regulation of LCN2 in models of acute and chronic experimental liver injury. Materials and methods: We analysed LCN2 expression in livers of rats subjected to bile duct ligation or repeated doses of carbon tetrachloride and tested the impact of various pro‐inflammatory cytokines in cultured primary liver cells. Results: By using primary cultures of hepatic stellate cells and hepatocytes isolated from normal and injured rat livers, we found a significant LCN2 expression in early hepatic stellate cell cultures, a lower expression in fully transdifferentiated myofibroblasts and no expression in freshly isolated hepatocytes. However, LCN2 expression and secretion in hepatocytes increased dramatically during culturing. In addition, chronic in vivo liver injury resulting from both bile duct ligation and repeated application of carbon tetrachloride resulted in rapid and well‐sustained induction of LCN2 expression. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2 production. LCN2 is strongly induced in both primary hepatocytes and immortalized hepatocellular carcinoma cell line HepG2 by the pro‐inflammatory cytokine interleukin‐1β via nuclear factor‐κB activation, but not by the profibrotic cytokines platelet‐derived growth factor and transforming growth factor‐β. Conclusion: LCN2 expression shows clear correlation to liver damage and resulting inflammatory responses, rather than to the degree of liver fibrosis, which in fact may imply a distinct diagnostic value as an early biomarker of liver inflammation.