Hydrogen peroxide suppresses the proteoglycan synthesis of intact articular cartilage

The effects of oxygen derived metabolites on intact murine patellar cartilage were investigated. Hydrogen peroxide was found to suppress the chondrocyte proteoglycan synthesis as measured by 35SO4 incorporation. Superoxide or hydroxyl radicals were not involved in chondrocyte damage. Autoradiography showed a severe inhibition of 35SO4 uptake by the chondrocytes in the central area of the patella. Histological examination did not show acute effects on the morphology of the affected cartilage. When kept in tissue culture up to 96 h, patellae exposed to hydrogen peroxide still showed considerable suppression of 35SO4 uptake and occasional cell death was observed. These data support the concept that cartilage damage, as a result of inflammation, may be related to the generation of toxic oxygen metabolites.     

Interactions of synovial fluid immunoglobulins with chondrocytes.

OBJECTIVE. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. METHODS. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with 125I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by 51Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. RESULTS. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF. Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. CONCLUSION. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.     

An experimental model for hydrogen peroxide-induced tissue damage. Effects of a single inflammatory mediator on (peri)articular tissues

Hydrogen peroxide is receiving increasing attention as a mediator of tissue damage during inflammation. To evaluate its destructive potential in vivo, we devised a model in which hydrogen peroxide is, initially, the sole mediator of tissue damage. Glucose oxidase, which was made cationic to obtain good retention in tissue, was injected intraarticularly in mouse knee joints. This enzyme produces hydrogen peroxide, using endogenous glucose as a substrate. The local production of hydrogen peroxide induced drastic vascular damage, as measured by 99mTc uptake and leakage of 125I-albumin. The chondrocyte proteoglycan synthesis was severely inhibited, as measured by 35SO4 incorporation. Histologic examination showed impressive inflammatory and degenerative changes, including periarticular infiltration, chondrocyte death, subchondral erosions, and muscle necrosis. Intraarticular administration of catalase could inhibit these vascular effects and cartilage damage. Systemic administration of ebselen, a synthetic glutathione peroxidase-like compound, provided partial protection. Indomethacin and piroxicam were not effective in the acute phase. We think this model is useful both for testing drugs that are purported to act as scavengers of hydrogen peroxide and for studying chronic destructive processes.     

Role of cellular superoxide dismutase against reactive oxygen metabolite-induced cell damage in cultured rat hepatocytes.

Reactive oxygen metabolites have been reported to be important in the pathogenesis of ischemia/reperfusion-induced and alcohol- and drug-induced liver injuries. We investigated the role of superoxide dismutase, cellular and extracellular, in preventing reactive oxygen metabolite-induced cytotoxicity in cultured rate hepatocytes. Cells were exposed to reactive oxygen metabolites enzymatically generated by hypoxanthine-xanthine oxidase. Cytotoxicity was quantified by measuring 51Cr release from prelabeled cells and lactate dehydrogenase release. Reactive oxygen metabolites caused dose-dependent cytotoxicity. Good correlation was found between the values for 51Cr and lactate dehydrogenase release. Reactive oxygen metabolite-induced cell damage was reduced by catalase but not by superoxide dismutase. Cellular superoxide dismutase and catalase activities were not increased after incubation with exogenous superoxide dismutase and catalase for up to 5 hr. Pretreatment with diethyldithiocarbamate inhibited cellular superoxide dismutase activity without inhibiting other antioxidants such as catalase, glutathione, glutathione reductase and glutathione peroxidase and sensitized cells to reactive oxygen metabolite-induced cytotoxicity. We conclude that hydrogen peroxide is an important mediator in hypoxanthine-xanthine oxidase-induced cell damage and that superoxide dismutase plays a critical role in cellular antioxidant defenses against hypoxanthine-xanthine oxidase-induced cytotoxicity in cultured rat hepatocytes in vitro.     

Basic FGF mediates an immediate response of articular cartilage to mechanical injury

The extracellularly regulated kinase (ERK), one of the three types of mitogen-activated kinases, was rapidly activated after cutting porcine articular cartilage either when maintained as explants or in situ. Cutting released a soluble ERK-activating factor from the cartilage, which was purified and identified by MS as basic fibroblast growth factor (bFGF). Experiments with neutralizing Abs to bFGF and an FGFR1 tyrosine kinase inhibitor showed that this growth factor was the major ERK-activating factor released after injury. Treating cartilage with the heparin-degrading enzyme heparitinase also caused release of bFGF, suggesting the presence of an extracellular store that is sequestered in the matrix and released upon damage. Basic FGF induced the synthesis of a number of chondrocyte proteins including matrix metalloproteinases 1 and 3, tissue inhibitor of metalloproteinases-1, and glycoprotein 38, which were identified by MS. The strong induction of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 suggests that bFGF could have a role in remodeling damaged tissue.     

Interactions of synovial fluid immunoglobulins with chondrocytes

Objective. To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. Methods. SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with ¹²⁵I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by ⁵¹Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. Results. Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF. Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. Conclusion. Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.     

Transient chondrocyte nonresponsiveness to insulin-like growth factor-1 upon H2O2 exposure is not related to IGF receptor damage.

We have shown that for anatomically intact murine cartilage, insulin-like growth factor-1 (IGF-1) is the major anabolic stimulus. Using an experimental arthritis model, we found that cartilage from an arthritic joint could not be stimulated in vitro with IGF-1. This nonresponsiveness was not caused by a generalized disturbance of chondrocyte metabolism since forskolin, an activator of adenylate cyclase, could stimulate cartilage from arthritic joints. To investigate whether hydrogen peroxide may cause IGF-1 nonresponsiveness, we exposed normal murine cartilage to H2O2 in vitro as well as in vivo. We found that cartilage, in which chondrocyte proteoglycan synthesis was inhibited due to H2O2 action in vitro, showed a normal response to IGF-1 after 24-h tissue culture. A time dependent but full recovery was found. In contrast, cartilage which was longterm exposed to H2O2 in vivo after injection of amidated glucoseoxidase (aGO) showed only a moderate IGF-1 response. This lack of total recovery was not due to chondrocyte death or to retained aGO producing extra H2O2 during tissue culture. Further studies with isolated bovine chondrocytes revealed that H2O2 did not damage the IGF-1 receptor. Binding of radiolabelled IGF-1 to H2O2 treated chondrocytes was unimpaired. Our data indicate that H2O2 inhibits chondrocyte proteoglycan synthesis via a mechanism not related to disturbance of IGF-1 signalling. Transient chondrocyte IGF-1 nonresponsiveness found after H2O2 exposure is not related to IGF receptor damage, and contrasts with the complete nonresponsiveness found in arthritic cartilage.     

An experimental model for hydrogen peroxide–induced tissue damage. Effects of a single inflammatory mediator on (peri)articular tissues

Hydrogen peroxide is receiving increasing attention as a mediator of tissue damage during inflammation. To evaluate its destructive potential in vivo, we devised a model in which hydrogen peroxide is, initially, the sole mediator of tissue damage. Glucose oxidase, which was made cationic to obtain good retention in tissue, was injected intraarticularly in mouse knee joints. This enzyme produces hydrogen peroxide, using endogenous glucose as a substrate. The local production of hydrogen peroxide induced drastic vascular damage, as measured by ^99mTc uptake and leakage of ¹²⁵I-albumin. The chondrocyte proteoglycan synthesis was severely inhibited, as measured by ³⁵SO₄ incorporation. Histologic examination showed impressive inflammatory and degenerative changes, including periarticular infiltration, chondrocyte death, subchondral erosions, and muscle necrosis. Intraarticular administration of catalase could inhibit these vascular effects and cartilage damage. Systemic administration of ebselen, a synthetic glutathione peroxidase-like compound, provided partial protection. Indomethacin and piroxicam were not effective in the acute phase. We think this model is useful both for testing drugs that are purported to act as scavengers of hydrogen peroxide and for studying chronic destructive processes.     

Update on the biology of the chondrocyte and new approaches to treating cartilage diseases

Osteoarthritis (OA) is a joint disease that involves degeneration of articular cartilage, limited intraarticular inflammation manifested by synovitis and changes in the subchondral bone. The aetiology of OA is largely unknown, but since it may involve multiple factors, including mechanical, biochemical and genetic factors, it has been difficult to identify unique targets for therapy. Chondrocytes, which are the unique cellular component of adult articular cartilage, are capable of responding to structural changes in the surrounding cartilage matrix. Since the initial stages of OA involve increased cell proliferation and synthesis of matrix proteins, proteinases and cytokines in the cartilage, laboratory investigations have focused on the chondrocyte as a target for therapeutic intervention. The capacity of the adult articular chondrocyte to regenerate the normal cartilage matrix architecture is limited, however, and the damage becomes irreversible unless the destructive process is interrupted. Current pharmacological interventions that address chronic pain are insufficient and no proven disease-modifying therapy is available. Identification of methods for early diagnosis is of key importance, since therapeutic interventions aimed at blocking or reversing structural damage will be more effective when there is the possibility of preserving normal homeostasis. At later stages, cartilage tissue engineering with or without gene therapy with anabolic factors will also require therapy to inhibit inflammation and block damage to newly repaired cartilage. This review will focus on experimental approaches currently under study that may lead to elucidation of effective strategies for therapy in OA, with emphasis on mediators that affect the function of chondrocytes and interactions with surrounding tissues.     

Osteoarthritis. Emerging evidence for cell interactions in the breakdown and remodeling of cartilage

Interactions among the cells of joint components appear to contribute to the dynamic events associated with breakdown of the cartilage matrix and remodeling of the articular surface in osteoarthritis. The mediator interleukin-1 may be one of the stimuli that induce release of hydrolytic factors from synovial membrane cells and chondrocytes, which contribute to the degradation of collagen and proteoglycans in the cartilage matrix. The remodeling process is associated with chondrocyte proliferation and vascular and cellular ingrowth from subchondral bone with osteophyte formation. It is postulated that growth factors isolated from cartilage and bone may contribute to repair and remodeling of the articular end of the joint.     

Sequential changes of parathyroid hormone related protein (PTHrP) in articular cartilage during progression of inflammatory and degenerative arthritis

OBJECTIVE: To investigate immunolocalisation of parathyroid hormone related protein (PTHrP) in two sequential models of experimental cartilage damage (inflammatory and degenerative) in order to elucidate differences in chondrocyte response to the disease. METHODS: Immunohistochemistry with a polyclonal rabbit antiserum to the N-terminal domain of PTHrP was used to detect this protein in two different rabbit models sharing progressive cartilage damage: antigen induced arthritis (AIA) and osteoarthritis (OA) secondary to partial medial meniscectomy. Cartilage specimens from early (2 days in AIA; 8 weeks in experimental OA) and late (3 weeks in AIA; 52 weeks in OA) disease were compared. RESULTS: Cell and matrix PTHrP staining in early AIA and OA was similar to that in controls. Late AIA cartilage showed a significant decrease in PTHrP positive cells and in the cartilage matrix. In contrast, at late OA stages, distinct PTHrP positivity was detected in proliferating cell clones, as assessed by proliferating cell nuclear antigen staining around cartilage damaged areas. CONCLUSION: PTHrP staining of hyaline articular cartilage shows a different pattern during progression of each type of arthritis: an overall decrease associated with the inflammatory disease, and an increase in the proliferating chondrocyte clones with degenerative arthritis.     

Role of iron and glutathione redox cycle in acetaminophen-induced cytotoxicity to cultured rat hepatocytes

The aims of this study were to investigate the roles of iron as a catalyst in reactive oxygen metabolite-mediated cellular injury and of the endogenous antioxidant defenses against acetaminophen-induced cytotoxicity in cultured rat hepatocytes. Hepatocytes were isolated and cultured from either 3-methylcholanthrene-treated or untreated rats. Cytotoxicity was evaluated by measuring⁵¹Cr and lactate dehydrogenase release. Acetaminophen caused dose-dependent cytotoxicity in 3-methlycholanthrene-treated, but not untreated, cells. There was a good correlation between⁵¹Cr and lactate dehydrogenase release values. Pretreatment with both diethyl maleate, which covalently binds glutathione as catalyzed by glutathione-S-transferase, and bis(chloroethyl)-nitrosourea, an inhibitor of glutathione reductase, enhanced acetaminophen-induced cytotoxicity. Inhibition of endogenous catalase activity by pretreatment with aminotriazole did not affect acetaminophen-induced cellular damage. Addition of exogenous catalase failed to protect against acetaminophen-induced cytotoxicity. Preincubation with both deferoxamine, a ferric iron chelator, and phenanthroline, a ferrous iron chelator, diminished acetaminophen-induced cytotoxicity. These results indicate that iron is crucial in mediating acetaminophen-induced cytotoxicity and that the glutathione redox cycle, but not catalase, plays a critical role in the endogenous defenses against acetaminophen-induced cellular damage in cultured rat hepatocytesin vitro.This work was supported by the Medical Research Service of the Veterans Affairs.     

Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species.

Nitric oxide, NO, which is generated by various components of the immune system, has been presumed to be cytotoxic. However, NO has been proposed to be protective against cellular damage resulting during ischemia reperfusion. Along with NO there is often concomitant formation of superoxide/hydrogen peroxide, and hence a synergistic relationship between the cytotoxic effects of nitric oxide and these active oxygen species is frequently assumed. To study more carefully the potential synergy between NO and active oxygen species in mammalian cell cytotoxicity, we utilized either hypoxanthine/xanthine cell cytotoxicity, we utilized either hypoxanthine/xanthine oxidase (a system that generates superoxide/hydrogen peroxide) or hydrogen peroxide itself. NO generation was accomplished by the use of a class of compounds known as "NONOates," which release NO at ambient temperatures without the requirement of enzyme activation or biotransformation. When Chinese hamster lung fibroblasts (V79 cells) were exposed to hypoxanthine/xanthine oxidase for various times or increasing amounts of hydrogen peroxide, there was a dose-dependent decrease in survival of V79 cells as measured by clonogenic assays. However, in the presence of NO released from (C2H5)2N[N(O)NO]-Na+ (DEA/NO), the cytotoxicity resulting from superoxide or hydrogen peroxide was markedly abrogated. Similarly, primary cultures of rat mesencephalic dopaminergic cells exposed either to hydrogen peroxide or to hypoxanthine/xanthine oxidase resulted in the degradation of the dopamine uptake and release mechanism. As was observed in the case of the V79 cells, the presence of NO essentially abrogated this peroxide-mediated cytotoxic effect on mesencephalic cells.     

Induction of cartilage damage by overexpression of T cell interleukin-17A in experimental arthritis in mice deficient in interleukin-1

OBJECTIVE: To examine the capacity of T cell interleukin-17A (IL-17A; referred to hereinafter as IL-17) to induce cartilage damage during experimental arthritis in the absence of IL-1. METHODS: Local IL-17 gene transfer was performed in the knee joint of IL-1-deficient mice and wild-type controls during streptococcal cell wall (SCW)-induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS-4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP-mediated cartilage damage. In addition, systemic anti-IL-1alpha/beta antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL-17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion. RESULTS: During SCW-induced arthritis, local T cell IL-17 gene transfer turned this acute, macrophage-driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL-1 dependency of cartilage PG depletion was fully abrogated when IL-17 was locally overexpressed in the joint. Moreover, local IL-17 gene transfer increased MMP expression without the need for IL-1, although IL-1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL-17 was overexpressed in the knee joints of mice with collagen-induced arthritis, anti-IL-1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion. CONCLUSION: These data show the capacity of IL-17 to replace the catabolic function of IL-1 in cartilage damage during experimental arthritis.     

Experimental arthritis in C57black/6 normal and beige (Chediak-Higashi) mice: in vivo and in vitro observations on cartilage degradation.

Mice with the beige mutation are known to be deficient for polymorphonuclear leucocyte (PMN) elastase and cathepsin G and can therefore be used as a model for protease dependence of tissue destruction in inflammatory conditions. The in vitro and in vivo effect of PMN activation on cartilage damage in C57black/6 normal and beige mice was measured. In vitro it was found that stimulation of normal PMNs with chemotactic peptide caused degradation of articular cartilage matrix owing to an elastase dependent mechanism; PMNs of beige mice did not induce degradation of cartilage. In vivo, using zymosan induced arthritis, which is a model characterised by a PMN-rich infiltrate and exudate, no significant differences were found between the two strains with respect to (a) joint oedema formation as measured by technetium-99m uptake; (b) matrix degradation as measured quantitatively and with histology; (c) chondrocyte proteoglycan synthesis as measured by radiosulphate uptake. At day 28 after induction of arthritis, when inflammation is waning, no differences in end stage irreversible damage to joint tissues were found. The relevance of these observations to the supposed role of PMN derived neutral proteases in joint inflammation is discussed.     

Target cell lysis mediated by concanavalin A-triggered human neutrophils

Summary Human neutrophils, triggered by Concanavalin A, were cytotoxic against chicken red blood cell targets as determined by the ⁵¹Cr release method. The cytolysis increased with the effector: target ratio, reaching optimal levels when 2–4 neutrophils were available for each chicken red blood cell. The target cell lysis required an optimal release of highly reactive oxygen by-products by neutrophils, since neutrophils from a patient with chronic granulomatous disease failed to exhibit any cytolytic activity. Superoxide dismutase, catalase and inhibitors of heme-containing peroxidases (azide and cyanide) significantly inhibited the neutrophil-mediated cytotoxicity. Together these results indicate that superoxide anion and the myeloperoxidase-hydrogen peroxide system are simultaneously involved in the target cell injury by Concanavalin A-triggered neutrophils.     

Aging,articular cartilage chondrocyte senescence and osteoarthritis

The incidence of osteoarthritis (OA), the disease characterized by joint pain and loss of joint form and function due to articular cartilage degeneration, is directly correlated with age. The strong association between age and increasing incidence of osteoarthritis (OA) marks OA as an age related disease. Yet, like many other age related diseases, OA is not an inevitable consequence of aging; instead, aging increases the risk of OA. Articular cartilage aging changes that may lead to articular cartilage degeneration include fraying and softening of the articular surface, decreased size and aggregation of proteoglycan aggrecans and loss of matrix tensile strength and stiffness. These changes most likely are the result of an age related decrease in the ability of chondrocytes to maintain and repair the tissue manifested by decreased mitotic and synthetic activity, decreased responsiveness to anabolic growth factors and synthesis of smaller less uniform aggrecans and less functional link proteins. Our recent work suggests that progressive chondrocyte senescence marked by expression of the senescence associated enzyme beta-galactosidase, erosion of chondrocyte telomere length and mitochondrial degeneration due to oxidative damage causes the age related loss of chondrocyte function. New efforts to prevent the development and progression of OA might include strategies that slow the progression of chondrocyte senescence or replace senescent cells. This revised version was published online in July 2006 with corrections to the Cover Date.