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Objective: Our goal was to test the hypothesis that specific integrin receptors regulate chondrocyte biosynthetic response to dynamic compression at early times in 3D gel culture, during initial evolution of the pericellular matrix, but prior to significant accumulation of further-removed matrix. The study was motivated by increased use of dynamic loading, in vitro, for early stimulation of tissue engineered cartilage, and the need to understand the effects of loading, in vivo, at early times after implantation of constructs., Methods: Bovine articular chondrocytes were seeded in 2% agarose gels (15x10(6)cells/mL) and incubated for 18 h with and without the presence of specific integrin blockers (small-molecule peptidomimetics, function-blocking antibodies, and RGD-containing disintegrins). Samples were then subjected to a 24-h dynamic compression regime found previously to stimulate chondrocyte biosynthesis in 3D gel as well as cartilage explant culture (1 Hz, 2.5% dynamic strain amplitude, 7% static offset strain). At the end of loading, proteoglycan (PG) synthesis ((35)S-sulfate incorporation), protein synthesis ((3)H-proline incorporation), DNA content (Hoechst dye 33258) and total glycosaminoglycan (GAG) content (dimethyl methylene blue (DMMB) dye binding) were assessed., Results: Consistent with previous studies, dynamic compression increased PG synthesis and total GAG accumulation compared to free-swelling controls. Blocking alphavbeta3 abolished this response, independent of effects on controls, while blocking beta1 abolished the relative changes in synthesis when changes in free-swelling synthesis rates were observed., Conclusions: This study suggests that both alphavbeta3 and beta1 play a role in pathways that regulate stimulation of PG synthesis and accumulation by dynamic compression, but through distinct complementary mechanisms., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation., Design: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo., Results: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin., Conclusion: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.

IL-1-activated chondrocytes express a large number of genes which contribute to cartilage degradation. The signaling pathways activated in response to IL-1 in these cells are not well-defined. We examined the effects of IL-1 and other stimuli on the mitogen activated protein kinase (MAPK) pathways in rabbit articular chondrocytes. We demonstrate that IL-1 activates three MAPKs, ERK, JNK and p38, in a time and dose-dependent manner. Activation is maximal by 15 minutes and returns to baseline levels by 1 hour. Maximal activation of ERK and p38 occurs with 1 ng/ml IL-1 whereas activation of JNK requires 10-fold higher levels. In contrast to IL-1, the PKC activator, PDBu preferentially activates ERK while TNF alpha preferentially activates JNK. LPS and TGF beta fail to stimulate any of the kinases examined. These results suggest that activation of the various MAPK pathways is important in the response of chondrocytes to IL-1, cytokines and growth factors.

Objective: To compare the alterations in proteoglycan metabolism in antigen induced arthritis and polycation induced arthritis and to determine the involvement of interleukin-1 (IL-1) in the cartilage degradation that occurs in these models of rheumatoid arthritis (RA)., Methods: The time course for loss of proteoglycan into the synovial fluid (SF) and inhibition of proteoglycan synthesis, as well as depletion of articular cartilage proteoglycan content, was compared in rabbit antigen arthritis and polycation arthritis. The ability of recombinant IL-1 receptor antagonist IL-1ra to block the acute cartilage loss at 24 h in these models was investigated, compared to its ability to block the cartilage breakdown induced by direct administration of IL-1 in rabbits., Results: Initial loss of cartilage proteoglycan was accompanied by release of high levels of glycosaminoglycan (GAG) into the SF and decrease in proteoglycan synthetic rates in both antigen and polycation induced arthritis SF GAG rapidly returned to control levels, while proteoglycan synthesis and cartilage proteoglycan content remained depressed, suggesting that the inhibition in proteoglycan synthesis prevented recovery to normal levels. GAG loss from the cartilage into the SF in response to IL-1 injection, as well as other effects of IL-1 challenge, was blocked in a dose dependent manner by IL-1ra administered either intraarticularly (ED50 = 160 ng) or intravenously (iv) (ED50 = 0.09mg/kg). In the antigen induced arthritis model, IL-1ra (20 mg/kg, iv -2h) inhibited GAG release by 40%, whereas in polycation induced arthritis no inhibition was observed even with repeated administration of high doses of inhibitor., Conclusion: These studies suggest that sustained depression of proteoglycan synthesis may be responsible for the chronic depletion of articular cartilage proteoglycan in the antigen and the polycation model of RA. However, while IL-1 may play a role in the initial breakdown of articular cartilage in antigen induced arthritis, it does not appear to be involved in polycation induced arthritis in the rabbit.

Rheumatoid arthritis and osteoarthritis are characterized by an early depletion of cartilage proteoglycans, which leads to a decrease in cartilage compressibility and, eventually, to a loss of joint function. Interleukin-1, which is thought to have a role in mediating this loss of proteoglycans in arthritis, induces an acute depletion of proteoglycans from articular cartilage following intra-articular injection in rabbits. As the structure and metabolism of proteoglycans are known to change with age, my laboratory investigated the effect of age on depletion and recovery of proteoglycans in response to interleukin-1 in the rabbit. Loss of cartilage proteoglycans induced by interleukin-1 was less severe in immature animals, increased until the age of sexual maturity, and then remained constant. The rate of recovery and compensatory overshoot in the rate of proteoglycan synthesis following challenge with interleukin-1 was more rapid in immature animals and may have been responsible for the quicker return of the cartilage proteoglycan content to control levels in younger animals. With multiple exposures to interleukin-1 at time intervals too short for recovery to occur, smaller amounts of interleukin-1 induced loss of proteoglycans, and the proteoglycan content and the rate of synthesis remained depressed longer after treatment had stopped. The decreased ability of mature cartilage to replace proteoglycans rapidly after exposure to cytokines would increase the probability of subsequent inflammatory episodes before recovery is complete; this may result in increased susceptibility of adult cartilage to proteoglycan depletion.

Interleukin-1 beta (IL-1) increases the synthesis of prostaglandins as well as the expression of synovial fluid phospholipase A2 (PLA2) mRNA and activity by chondrocytes. In order to examine the potential involvement of cyclooxygenase in the induction of prostaglandins by IL-1, the effect of IL-1 on rabbit articular chondrocyte expression of cyclooxygenase enzymes was investigated. By Northern analyses, mRNA for cyclooxygenase-2 (COX-2) was found to be constitutively expressed, and its expression was increased in cultures treated for 24 h with IL-1 (100 ng/ml). Cyclooxygenase-1 mRNA was not detected in control or IL-1-treated cultures. IL-1 caused a concentration-dependent increase in steady-state levels of COX-2 message as assessed by slot-blot analyses. Half-maximal induction of the COX-2 message levels was estimated to require 1.2 ng/ml IL-1. In duplicate slot blots probed with a cDNA for synovial fluid PLA2, half-maximal induction of PLA2 mRNA was estimated to require 0.15 ng/ml IL-1, approximately 10-fold less IL-1 than required for COX-2. In addition, maximal increase in COX-2 message levels was only 3-fold as compared with a 13-fold induction of PLA2. No change in message levels for the intracellular protein, actin, was found between control and experimental cultures. Cycloheximide enhanced COX-2 transcript levels, but did not modulate IL-1 induction of COX-2. Actinomycin D did not inhibit IL-1 augmentation of COX-2 mRNA levels; whereas, induction of PLA2 mRNA levels was completely inhibited, implicating transcriptional mechanisms in the induction of PLA2 but not COX-2 mRNA. These data demonstrate that chondrocyte expression of COX-2 and synovial fluid PLA2 are differentially modulated by IL-1.

The synthesis of an 88-kDa gelatinolytic enzyme, identified as a zymogen of matrix metalloproteinase (proMMP)-9, was induced in the primary culture of rabbit articular chondrocytes by cotreatment with recombinant interleukin 1 beta (rIL-1 beta) and the protein kinase C (PKC) agonists, phorbol 12,13-dibutyrate (PDBu) or mezerein. Negligible 88-kDa gelatinolytic activity was produced by unstimulated cells or cells treated with a PKC activator alone at concentrations up to 100 ng/ml, and only a modest induction occurred with rIL-1 beta alone at concentrations of 1-100 ng/ml. However, when these cells were treated with a PKC activator in the presence of IL-1 beta (1 ng/ml), induction was striking, with enzymic activity detectable at a concentration as low as 1 ng/ml of mezerein or 10 ng/ml of PDBu. Rabbit chondrocytes in culture constitutively produced the zymogen of MMP-2 (proMMP-2) and its production was not altered by treatment with IL-1 beta or PKC agonists alone or in combination. Recombinant tumor necrosis factor alpha (rTNF alpha) did not substitute for IL-1 beta in inducing proMMP-9 in the presence of PKC activators, nor was the combination of IL-1 beta or TNF alpha alone effective. These data indicate that rabbit articular chondrocytes have a potential to synthesize and secrete proMMP-9 under certain biological and pathological conditions but that the expression of proMMP-9 is differently regulated from that of other MMPs.

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.

A cDNA that codes for human rheumatoid synovial fluid phospholipase A2 (PLA2) hybridized with a RNA of the same size (900 base pairs) isolated from rabbit articular chondrocytes (RAC). Treatment of RAC with 100 ng/ml recombinant human interleukin-1 beta (IL-1) for 24 hours caused a 13-fold increase in mRNA for this PLA2. Timecourse studies demonstrated that maximal induction of PLA2 mRNA occurred by 16 hours post addition of IL-1 (100 ng/ml). Augmentation of RAC PLA2 mRNA levels was dose-dependent; half-maximal induction was estimated to require 0.15 ng/ml IL-1. Actinomycin D inhibited IL-1 effects on PLA2 mRNA levels. Coordinate effects of IL-1 on RAC PLA2 activity were observed with respect to time and dose dependence as well as actinomycin D sensitivity.

Interleukin-1 (IL-1) is a cytokine produced by a number of connective-tissue and inflammatory cells which has been shown in organ culture to stimulate the breakdown of cartilage proteoglycans and inhibit their synthesis. Intraarticular injection of human recombinant IL-1 beta into the knee joints of rabbits induced a dose-related decrease in cartilage proteoglycan content and increased infiltration of cells into the synovial fluid. Following a single intraarticular injection, the loss of proteoglycan was maximal at 3 days. By 7 days, proteoglycan content began to return toward control levels. IL-1 also resulted in a dose-related decrease in the ability of cartilage to synthesize new proteoglycan as measured by 35S incorporation. These in vivo effects of IL-1 on articular cartilage closely reflect those effects observed in vitro in organ culture and are consistent with the hypothesis that IL-1 may play a role as a mediator of the loss of cartilage in some arthritic diseases.