Insulin-like growth factor 1-induced interleukin-1 receptor II overrides the activity of interleukin-1 and controls the homeostasis of the extracellular matrix of cartilage

OBJECTIVE: We examined the effect of the insulin-like growth factor 1 (IGF-1)/IGF receptor I (IGFRI) autocrine/paracrine anabolic pathway on the extracellular matrix (ECM) of human chondrocytes and the mechanism by which IGF-1 reverses the catabolic effects of interleukin-1 (IL-1). METHODS: Phenotypically stable human articular cartilage cells were obtained from normal cartilage and maintained in culture in alginate beads for 1 week to reach equilibrium of accumulated cell-associated matrix (CAM) compounds. Levels of CAM components aggrecan and type II collagen (CII) and levels of intracellular IGF-1, IL-1alpha, and IL-1beta and their respective plasma membrane-bound receptors IGFRI, IL-1 receptor I (IL-1RI), and the decoy receptor IL-1RII were assayed using flow cytometry to investigate the relationship between the autocrine/paracrine pathways and the homeostasis of ECM molecules in the CAM. The effects of IGF-1 on the expression of IGF-1, IL-1alpha, and IL-1beta and their respective receptor systems, the aggrecan core protein, and CII were determined by flow cytometry. RESULTS: Cause-effect relationship experiments showed that IGF-1 up-regulates the levels of IGF-1, IGFRI, aggrecan, and CII in the CAM. No effects on the expression of IL-1alpha and IL-1beta and their signaling receptor IL-1RI were observed. However, IGF-1 was able to reverse IL-1beta-mediated degradation of aggrecan and the repression of the aggrecan synthesis rate. Interestingly, levels of aggrecan and CII in the CAM strongly correlated not only with IGF-1, but also with IL-1RII, which acts as a decoy receptor for IL-1alpha and IL-1beta. This suggests that IGF-1 and IL-1RII may cooperate in regulating ECM homeostasis. Additional experiments demonstrated that IGF-1 up-regulated IL-1RII, thereby overriding the catabolic effects of IL-1. CONCLUSION: These findings reveal a new paradigm by which IGF-1 influences chondrocyte metabolism, by reversing the IL-1-mediated catabolic pathway through up-regulation of its decoy receptor.     

Dependence of interleukin‐1–induced arthritis on granulocyte–macrophage colony‐stimulating factor

Objective

To determine whether granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and macrophage CSF (M‐CSF or CSF‐1) are involved in the methylated bovine serum albumin/interleukin‐1 (mBSA/IL‐1)–induced arthritis model.

Methods

Following systemic injection, IL‐1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM‐CSF and M‐CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL‐1 can enhance GM‐CSF and M‐CSF production, we reasoned that they might be playing a part in IL‐1–mediated arthritis. GM‐CSF–deficient (GM‐CSF−/−) and M‐CSF–deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL‐1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM‐CSF and M‐CSF, and to the M‐CSF receptor. Numbers of macrophages (Mac‐2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine‐positive) cells.

Results

GM‐CSF−/− mice and normal mice treated with anti–GM‐CSF antibody did not show IL‐1–induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL‐1–induced disease, but blocking antibody to M‐CSF or to the M‐CSF receptor failed to diminish disease in normal mice.

Conclusion

GM‐CSF is involved in the IL‐1–induced arthritis that follows mBSA injection; M‐CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM‐CSF, and possibly M‐CSF, in the proinflammatory actions of IL‐1.

     

Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

Objective

To investigate the role of furin‐like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation.

Methods

Bovine nasal cartilage was stimulated to resorb with the addition of interleukin‐1α (IL‐1α)/oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec‐RVKR‐CH₂Cl, or selective matrix metalloproteinase 1 (MMP‐1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using ³H‐acetylated collagen and gelatin zymography, respectively.

Results

The addition of Dec‐RVKR‐CH₂Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP‐2, suggesting that furin‐like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP‐1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP‐13 resulted in a 50% blockage of collagen release. The addition of Dec‐RVKR‐CH₂Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin‐activated enzymes in the pathways leading to proteoglycan degradation.

Conclusion

Furin‐like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP‐13, which can be activated by furin‐processed membrane‐type 1 MMP‐1, appears to be a major collagenase involved in collagen degradation induced by IL‐1α/OSM. Furin‐like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.

     

Insulin‐like growth factor‐I and the liver

Insulin‐like growth factors (IGFs) play an essential role in growth and development, as well as in the overall cellular regulation and metabolism in the human body. In chronic liver disease, IGF levels are decreased, and the circulating levels correlate to the extent of hepatocellular dysfunction. Patients with cirrhosis are characterised by a variety of metabolic disturbances, including nutritional and metabolic complications such as insulin resistance, malnutrition, osteopenia and hypogonadism, all related to IGF‐I deficiency. The complex process of hepatic fibrogenesis and the systemic consequences in cirrhosis are only partly understood. Disruption of the growth hormone (GH)–IGF‐I axis seems to be closely associated with the development of liver disease, and treatment with recombinant human IGF (rhIGF)‐I has been shown to halt, and even reverse, the fibrotic degeneration. IGF‐I in itself has a strong antifibrotic effect that acts directly through the GH/IGF system and indirectly by the regulation of hepatoprotective and profibrogenic factors. It is most likely that IGF‐I deficiency contributes to the diverse metabolic complications of cirrhosis. At present, liver transplantation remains the only efficient treatment of cirrhosis, and thus new methods of managing the disease are called for. RhIGF‐I supplementation and IGF‐I gene therapy may represent future perspectives of treatment.     

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

Objective

Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) and its soluble receptor (sIL‐6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL‐6 plays a role in proteoglycan loss caused by a combination of injury and TNFα.

Methods

Injured or uninjured cartilage disks were incubated with or without TNFα and/or IL‐6/sIL‐6R. Additional samples were preincubated with an IL‐6–blocking antibody Fab fragment and subjected to injury and TNFα treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme‐linked immunosorbent assay to determine chondrocyte production of IL‐6.

Results

In bovine cartilage samples, injury combined with TNFα and IL‐6/sIL‐6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL‐6/sIL‐6R, injury plus TNFα exposure up‐regulated chondrocyte production of IL‐6, but incubation with the IL‐6–blocking Fab significantly reduced proteoglycan degradation.

Conclusion

Our findings indicate that mechanical injury potentiates the catabolic effects of TNFα and IL‐6/sIL‐6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFα appeared partly due to endogenous IL‐6, since GAG loss was partially abrogated by an IL‐6–blocking Fab.

     

Insulin‐like growth factor‐II: its role in metabolic and endocrine disease

Insulin‐like growth factor‐II (IGF‐II) is a widely expressed 7·5 kDa mitogenic peptide hormone. Although it is abundant in serum, understanding of its physiological role is limited compared with that of IGF‐I. IGF‐II regulates foetal development and differentiation, but its role in adults is less well understood. Evidence suggests roles in a number of tissues including skeletal muscle, adipose tissue, bone and ovary. Altered IGF‐II expression has been observed in metabolic conditions, notably obesity, diabetes and the polycystic ovary syndrome. This article summarizes what is known about the actions of IGF‐II and its dysregulation in metabolic and endocrine diseases. The possible causes and consequences of dysregulation are discussed along with the implications for diagnostic tests and future research.     

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.

     

Intraarticular injection of heparin‐binding insulin‐like growth factor 1 sustains delivery of insulin‐like growth factor 1 to cartilage through binding to chondroitin sulfate

Objective

Insulin‐like growth factor 1 (IGF‐1) stimulates cartilage repair but is not a practical therapy due to its short half‐life. We have previously modified IGF‐1 by adding a heparin‐binding domain and have shown that this fusion protein (HB‐IGF‐1) stimulates sustained proteoglycan synthesis in cartilage. This study was undertaken to examine the mechanism by which HB‐IGF‐1 is retained in cartilage and to test whether HB‐IGF‐1 provides sustained growth factor delivery to cartilage in vivo and to human cartilage explants.

Methods

Retention of HB‐IGF‐1 and IGF‐1 was analyzed by Western blotting. The necessity of heparan sulfate (HS) or chondroitin sulfate (CS) glycosaminoglycans (GAGs) for binding was tested using enzymatic removal and cells with genetic deficiency of HS. Binding affinities of HB‐IGF‐1 and IGF‐1 proteins for isolated GAGs were examined by surface plasmon resonance and enzyme‐linked immunosorbent assay.

Results

In cartilage explants, chondroitinase treatment decreased binding of HB‐IGF‐1, whereas heparitinase had no effect. Furthermore, HS was not necessary for HB‐IGF‐1 retention on cell monolayers. Binding assays showed that HB‐IGF‐1 bound both CS and HS, whereas IGF‐1 did not bind either. After intraarticular injection in rat knees, HB‐IGF‐1 was retained in articular and meniscal cartilage, but not in tendon, consistent with enhanced delivery to CS‐rich cartilage. Finally, HB‐IGF‐1 was retained in human cartilage explants but IGF‐1 was not.

Conclusion

Our findings indicate that after intraarticular injection in rats, HB‐IGF‐1 is specifically retained in cartilage through its high abundance of CS. Modification of growth factors with heparin‐binding domains may be a new strategy for sustained and specific local delivery to cartilage.

     

Elevated expression of interleukin‐7 receptor in inflamed joints mediates interleukin‐7–induced immune activation in rheumatoid arthritis

Objective

To evaluate the expression and functional ability of the high‐affinity interleukin‐7 receptor (IL‐7Rα) in patients with rheumatoid arthritis (RA).

Methods

Expression of IL‐7Rα and IL‐7 was determined in synovial tissue from RA patients and was compared with that in synovial tissue from patients with undifferentiated arthritis (UA) and osteoarthritis (OA). IL‐7Rα expression on CD4 T cells, CD19 B cells, and CD14 monocyte/macrophages from RA synovial tissue, synovial fluid, and peripheral blood was also assessed. The proliferative capacity of IL‐7Rα^bright and IL‐7Rα^dim/− T cells was measured. In addition, we examined IL‐7R blockade with soluble human IL‐7Rα (hIL‐7Rα) in the prevention of immune activation of peripheral blood mononuclear cells.

Results

We found significantly higher IL‐7Rα expression in RA and UA synovial tissue than in OA synovial tissue, and the level of IL‐7Rα expression correlated significantly with the levels of CD3 and IL‐7 expression. CD4 T cells from RA synovial fluid and synovial tissue strongly expressed IL‐7Rα. A substantial percentage of B cells and macrophages from RA synovial fluid and synovial tissue also expressed IL‐7Rα, although less prominently than T cells. We found that peripheral blood IL‐7Rα^bright T cells that did not express FoxP3 were highly proliferative as compared with IL‐7Rα^dim/− T cells that did express high levels of FoxP3. Soluble hIL‐7Rα inhibited IL‐7–induced proliferation and interferon‐γ production by mononuclear cells from RA patients.

Conclusion

Our data suggest that enhanced expression of IL‐7Rα and IL‐7 in RA patients contributes significantly to the joint inflammation by activating T cells, B cells, and macrophages. The inhibition of IL‐7R–mediated immune activation by soluble hIL‐7Rα further indicates an important role of IL‐7Rα in inflammatory responses in RA, suggesting IL‐7Rα as a therapeutic target for immunotherapy in RA.

     

Detection of nitrotyrosine in aging and osteoarthritic cartilage: Correlation of oxidative damage with the presence of interleukin‐1β and with chondrocyte resistance to insulin‐like growth factor 1

Objective

To determine whether oxidative damage to cartilage proteins can be detected in aging and osteoarthritic (OA) cartilage, and to correlate the results with the local production of interleukin‐1β (IL‐1β) and the responsiveness of isolated chondrocytes to stimulation with insulin‐like growth factor 1 (IGF‐1).

Methods

The presence of nitrotyrosine was used as a measure of oxidative damage. Histologic sections of knee articular cartilage, obtained from young adult and old adult cynomolgus monkeys, which develop age‐related, naturally occurring OA, were evaluated. Each cartilage section was graded histologically on a scale of 0–7 for the presence of OA‐like changes, and serial sections were immunostained using antibodies to nitrotyrosine and IL‐1β. Chondrocytes isolated and cultured from cartilage adjacent to the sections used for immunostaining were tested for their response to IGF‐1 stimulation by measuring sulfate incorporation in alginate cultures. For comparison with the monkey tissues, cartilage sections from human tissue donors and from tissue removed at the time of OA‐related joint replacement surgery were also immunostained for nitrotyrosine and IL‐1β.

Results

The presence of nitrotyrosine was associated with aging and with the development of OA in cartilage samples from both monkeys and humans. All sections that were highly positive for IL‐1β also showed staining for nitrotyrosine. However, in a few sections from older adult monkeys and humans, nitrotyrosine was present but IL‐1β was absent, suggesting that some age‐related oxidative damage is independent of IL‐1β. In chondrocytes that were isolated from monkey cartilage positive for nitrotyrosine or IL‐1β, the response to stimulation with IGF‐1 was significantly reduced. In some samples from older adult monkeys, IGF‐1 resistance was seen in cells isolated from tissue that did not stain for nitrotyrosine or IL‐1β.

Conclusion

Oxidative damage due to the concomitant overproduction of nitric oxide and other reactive oxygen species is present in both aging and OA cartilage. This damage can contribute to the resistance of chondrocytes to IGF‐1 stimulation, but it is unlikely to be the sole cause of IGF‐1 resistance in these chondrocytes.