Enhanced integrative repair of the porcine meniscus in vitro by inhibition of interleukin-1 or tumor necrosis factor alpha

OBJECTIVE: To examine the hypotheses that increasing concentrations of interleukin-1 (IL-1) or tumor necrosis factor alpha (TNFalpha) inhibit the integrative repair of the knee meniscus in an in vitro model system, and that inhibitors of these cytokines will enhance repair. METHODS: Explants (8 mm in diameter) were harvested from porcine medial menisci. To simulate a full-thickness defect, a 4-mm-diameter core was removed and reinserted. Explants were cultured for 14, 28, or 42 days in the presence of 0-1,000 pg/ml of IL-1 or TNFalpha. Explants were also cultured in the presence of IL-1 or TNFalpha with IL-1 receptor antagonist (IL-1Ra) or TNF monoclonal antibody (mAb). At the end of the culture period, biomechanical testing, cell viability, and histologic analyses were performed to quantify the extent of repair. RESULTS: Mechanical testing revealed increased repair strength, cell accumulation, and tissue formation at the interface over time under control conditions. Pathophysiologic concentrations of both IL-1 and TNFalpha significantly decreased repair strength, cell migration, and tissue formation at the interface. The addition of IL-1Ra or TNF mAb to explants prevented the effects of IL-1 or TNFalpha, respectively. CONCLUSION: Our findings document that physiologically relevant concentrations of IL-1 and TNFalpha inhibit meniscal repair in vitro and therefore may also inhibit meniscal repair during arthritis or following joint injury. The finding that IL-1Ra and TNF mAb promoted integrative meniscal repair in an inflammatory microenvironment suggests that intraarticular delivery of IL-1Ra and/or TNF mAb may be useful clinically to promote meniscal healing following injury.     

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.

     

Proteomic analysis by two‐dimensional electrophoresis to identify the normal human chondrocyte proteome stimulated by tumor necrosis factor α and interleukin‐1β

Objective

To determine the intracellular proteome of normal human chondrocytes stimulated with interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) and to ascertain differences in the protein expression patterns of these 2 cytokines.

Methods

Normal human knee cartilage chondrocytes were incubated for 48 hours without stimulation or stimulated with IL‐1β (5 ng/ml) or with TNFα (10 ng/ml). For each culture condition, protein extracts from 4 normal subjects were pooled and resolved using 2‐dimensional electrophoresis. Protein spots were visualized with Sypro stain, and qualitative and quantitative analyses were performed using PDQuest software. Protein spots were then identified by mass spectrometry, using matrix‐assisted laser desorption ionization−time‐of‐flight/time‐of‐flight technology.

Results

We identified 37 spots by mass spectrometry (MS) or by MS/MS, corresponding to 35 different proteins. In IL‐1β–stimulated chondrocytes, IL‐1β was found to modulate 22 proteins, as compared with unstimulated chondrocytes. All of these proteins except connective tissue growth factor (CCND2) were up‐regulated. Proteins involved in cellular metabolism and energy (23%) that were up‐regulated or induced by IL‐1β included nicotinamide phosphoribosyltransferase, long‐chain fatty acid–coenzyme A ligase 4, δ‐aminolevulinic acid dehydratase, triosephosphate isomerase, and an isoform of glyceraldehyde‐3‐phosphate dehydrogenase. In TNFα‐stimulated chondrocytes, TNFα was found to modulate 20 proteins, as compared with unstimulated chondrocytes. All of these except chitinase 3–like 1 (cartilage glycoprotein 39), proteasome activator complex subunit 2, and G3PDH, were up‐regulated. Eighteen proteins were differently modulated by IL‐1β and TNFα. Of these, 45% were related to metabolism.

Conclusion

IL‐1β and TNFα induce different profiles of intracellular protein expression in healthy human chondrocytes. Most of the proteins that are differently regulated are proteins that are implicated in the generation of cellular energy and in glycolysis.

     

ADAMTS‐9 is synergistically induced by interleukin‐1β and tumor necrosis factor α in OUMS‐27 chondrosarcoma cells and in human chondrocytes

Objective

To compare induction of the aggrecanases (ADAMTS‐1, ADAMTS‐4, ADAMTS‐5, ADAMTS‐8, ADAMTS‐9, and ADAMTS‐15) by interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) in chondrocyte‐like OUMS‐27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.

Methods

OUMS‐27 cells were stimulated for different periods of time and with various concentrations of IL‐1β and/or TNFα. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL‐1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real‐time polymerase chain reaction and Northern blotting. ADAMTS‐9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL‐1β–stimulated OUMS‐27 cells was investigated.

Results

IL‐1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL‐1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL‐1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS‐9 protein was also induced in IL‐1β–stimulated OUMS‐27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up‐regulation in OUMS‐27 cells.

Conclusion

ADAMTS9 is an IL‐1β– and TNFα‐inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS‐9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS‐9 may have a pathologic role in arthritis.

     

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.

     

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.

     

Interleukin‐18 enhances monocyte tumor necrosis factor α and interleukin‐1β production induced by direct contact with T lymphocytes: Implications in rheumatoid arthritis

Objective

At sites of inflammation, T cells exert pathologic effects through direct contact with monocyte/macrophages, inducing massive up‐regulation of interleukin‐1 (IL‐1) and tumor necrosis factor α (TNFα). We examined the regulatory effects of IL‐18 on monocyte activation by direct contact with T lymphocytes in rheumatoid arthritis (RA).

Methods

Activated T cells were isolated from RA synovial fluid. Resting T cells and monocytes were isolated from peripheral blood mononuclear cells. RA synovial T cells or phytohemagglutinin (PHA)–stimulated T cells were fixed by paraformaldehyde and then cocultured with monocytes at a ratio of 4:1. Levels of TNFα, IL‐1β, IL‐10, and IL‐18 were measured by enzyme‐linked immunosorbent assay. Expression of adhesion molecules, IL‐18 receptor, and TNF receptors was analyzed by flow cytometry. Expression of NF‐κB p65, phosphorylated IκBα, and phosphatidylinositol 3‐kinase (PI 3‐kinase) p110 was analyzed by Western blotting.

Results

IL‐18 dose‐dependently enhanced the production of IL‐1β and TNFα, but not IL‐10, by monocytes following contact with RA synovial T cells or PHA‐prestimulated T cells. NF‐κB inhibitors N‐acetyl‐L ‐cysteine and Bay 11‐7085 and PI 3‐kinase inhibitor LY294002 inhibited the enhancing effects of IL‐18, but MAPK p38 inhibitor SB203580, ERK inhibitor PD98059, and JNK inhibitor SP600125 did not. Increased levels of NF‐κB in the nucleus, phosphorylated IκB, and PI 3‐kinase were confirmed in monocytes cocultured with PHA‐prestimulated T cells, and the levels were further increased by stimulation with IL‐18. Neutralizing antibody to IL‐18 inhibited monocyte activation induced by direct contact with PHA‐prestimulated T cells. Via cell–cell contact, PHA‐prestimulated T cells increased autocrine production of IL‐18 by monocytes, which was mediated by activation of the NF‐κB and PI 3‐kinase pathways, and up‐regulated the expression of the IL‐18 receptor in monocytes. IL‐18 up‐regulated the expression of the TNF receptors vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) on monocytes. Blocking the binding of the TNF receptors VCAM‐1 or ICAM‐1 on monocytes to their ligands on stimulated T cells suppressed the IL‐18–enhanced production of TNFα and IL‐1β in monocytes induced by contact with PHA‐prestimulated T cells.

Conclusion

IL‐18 augments monocyte activation induced by contact with activated T cells in RA synovitis, which is dependent on activation of the NF‐κB and PI 3‐kinase pathways. IL‐18 up‐regulates the expression of the TNF receptors VCAM‐1 and ICAM‐1 on monocytes, which mediate the enhancing effects of IL‐18 on T cell–monocyte contact.

     

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.

     

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.

     

Calcitonin therapy in postmenopausal osteoporosis: effects on serum levels of interleukin‐1, interleukin‐6 and tumor necrosis factor‐α

Background: It has been suggested that the effects of calcitonin (CT) therapy for senile and postmenopausal osteoporosis were due to its modulatory effects on bone‐related cytokines. A significantly increased release of IL‐1, IL‐6 and TNF‐α, which have bone resorptive effects, has been reported in osteoporotic patients. Aim: In this study we investigated the effects of CT therapy on the levels of IL‐1, IL‐6 and TNF‐α. Method: Forty postmenopausal osteoporotic women were included to the study. The first group consisting of 20 patients were given 100 IU CT subcutaneously and 1000 mg elemental calcium for 15 consecutive days. The second group or the control group also consisting of 20 patients were only given 1000 mg elemental calcium and both of the groups were not allowed to take any other medication. Results: In the first group the mean serum TNF‐α level significantly decreased from 16.9 ± 24.2 pg/mL to 8.6 ± 13 pg/mL after 1500 IU CT therapy (P < 0.05). The control group's mean serum level of IL‐1, IL‐6 and TNF‐α did not reveal any statistically significant differences (P > 0.05). Conclusion: Our results suggest that CT therapy for osteoporosis may partially be due to its inhibitory effects on TNF‐α, and probably IL‐1. However, further in‐vitro and ex‐vivo studies are needed to clarify this hypothesis.     

Detection of tumor necrosis factor α but not tumor necrosis factor β in rheumatoid arthritis synovial fluid and serum

Synovial fluids from 6 of 12 patients with rheumatoid arthritis (RA) and from 3 of 11 patients with reactive arthritis contained measurable levels of tumor necrosis factor α (TNFα). Seven of 12 sera from RA patients contained TNFα, while only 1 of those from reactive arthritis patients was positive. Gamma-inter-feron was detected in the synovial fluids and sera of only the RA patients. Tumor necrosis factor β was not detected in any sera or synovial fluids. RA patients with detectable TNFα had higher erythrocyte sedimentation rates and synovial fluid leukocyte counts.     

Successful tumor necrosis factor α blockade treatment in therapy‐resistant sarcoidosis

Sarcoidosis is a multisystemic disorder histologically characterized by a noncaseating granulomatous inflammatory process. The etiology remains unclear, but tumor necrosis factor α seems to play a crucial role. Herein we describe a patient with severe sarcoidosis involving the lung and liver. Various treatment regimens with azathioprine, methotrexate, cyclophosphamide, and pentoxifylline failed to control the disease. Therefore, salvage therapy with infliximab was commenced. Arthritis and pulmonary and liver involvement improved. We were then able to taper the corticosteroid treatment to a lower‐dose regimen with no need for additional immunosuppressive treatment. To our knowledge, this is the first reported case of successful treatment of multiorgan sarcoidosis that was previously resistant to conventional therapy.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.