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.

     

Tumor necrosis factor α–mediated cleavage and inactivation of SirT1 in human osteoarthritic chondrocytes

Objective

The protein deacetylase SirT1 positively regulates cartilage‐specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression.

Methods

Cartilage‐specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope‐tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope‐specific antibodies. The 75‐kd SirT1‐reactive protein present in TNFα‐treated extracts was identified by mass spectroscopy, and its amino‐terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα.

Results

TNFα‐treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full‐length 110‐kd protein and a smaller 75‐kd fragment. The 75‐kd SirT1 fragment was found to lack the carboxy‐terminus. Cathepsin B was identified as the TNFα‐responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75‐kd SirT1 fragment.

Conclusion

These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B–mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage‐specific gene expression evident in these TNFα‐treated cells.

     

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.

     

Tumor necrosis factor α and interleukin‐1β modulate calcium and nitric oxide signaling in mechanically stimulated osteocytes

Objective

Inflammatory diseases often coincide with reduced bone mass. Mechanoresponsive osteocytes regulate bone mass by maintaining the balance between bone formation and resorption. Despite its biologic significance, the effect of inflammation on osteocyte mechanoresponsiveness is not understood. To fill this gap, we investigated whether the inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin‐1β (IL‐1β) modulate the osteocyte response to mechanical loading.

Methods

MLO‐Y4 osteocytes were incubated with TNFα (0.5–30 ng/ml) or IL‐1β (0.1–10 ng/ml) for 30 minutes or 24 hours, or with calcium inhibitors for 30 minutes. Cells were subjected to mechanical loading by pulsatile fluid flow (mean ± amplitude 0.7 ± 0.3 Pa, 5 Hz), and the response was quantified by measuring nitric oxide (NO) production using Griess reagent and by measuring intracellular calcium concentration ([Ca²⁺]_i) using Fluo‐4/AM. Focal adhesions and filamentous actin (F‐actin) were visualized by immunostaining, and apoptosis was quantified by measuring caspase 3/7 activity. Cell‐generated tractions were quantified using traction force microscopy, and cytoskeletal stiffness was quantified using optical magnetic twisting cytometry.

Results

Pulsatile fluid flow increased [Ca²⁺]_i within seconds (in 13% of cells) and NO production within 5 minutes (4.7‐fold). TNFα and IL‐1β inhibited these responses. Calcium inhibitors decreased pulsatile fluid flow–induced NO production. TNFα and IL‐1β affected cytoskeletal stiffness, likely because 24 hours of incubation with TNFα and IL‐1β decreased the amount of F‐actin. Incubation with IL‐1β for 24 hours stimulated osteocyte apoptosis.

Conclusion

Our results suggest that TNFα and IL‐1β inhibit mechanical loading–induced NO production by osteocytes via abrogation of pulsatile fluid flow–stimulated [Ca²⁺]_i, and that IL‐1β stimulates osteocyte apoptosis. Since both NO and osteocyte apoptosis affect osteoclasts, these findings provide a mechanism by which inflammatory cytokines might contribute to bone loss and consequently affect bone mass in rheumatoid arthritis.

     

Tumor necrosis factor‐α and interferon‐γ directly impair epithelial barrier function in cultured mouse cholangiocytes

Abstract: Background/Aims: In primary biliary cirrhosis (PBC), cytokines from CD4 ⁺ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8 ⁺ T lymphocytes. Recently, we reported that immunolocalization of 7H6 – a tight junction (TJ)‐associated protein – was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines – tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐2 and 4 (IL‐2 and 4) – on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine‐induced changes in paracellular permeability. Methods: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and ³H‐inulin flux. We also performed immunostaining and immunoblotting for TJ‐associated proteins – claudin‐1 and ‐3, occludin, zonula occluden‐1 (ZO‐1) and 7H6. Results: TNF‐α and IFN‐γ, but neither IL‐2 nor IL‐4, significantly decreased TER (P < 0.005). ³H‐inulin flux studies confirmed IFN‐α‐induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ‐associated proteins were well preserved in TNF‐α‐ or IFN‐γ‐treated cells. Ursodeoxycholic acidhas been found to have no inhibitory effect on increased paracellular permeability induced by TNF‐α or IFN‐γ. Conclusion: These findings show that TNF‐α and IFN‐γ disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.     

Extended indications for anti‐tumor necrosis factor‐α therapy

Tumour necrosis factor‐α is a pleiotropic cytokine which has a broad range of actions in inflammation, infection and immunity. TNF‐α is supposed to play a crucial role in the pathogenesis of various autoimmune diseases. TNF‐α blocking agents have been demonstrated to be highly effective in the treatment of rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile rheumatoid arthritis. TNF‐α inhibitors also have been tried with other rheumatic diseases and have emerged as promising treatments. We here review the current evidences of effectiveness of the anti‐TNF‐α therapy in various autoimmune diseases.