Michael A. Pratta

Interleukin-1β stimulates phospholipase A2 mRNA synthesis in rabbit articular chondrocytes

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.

Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) is induced in rabbit articular chondrocytes by cotreatment with interleukin 1β and a protein kinase C activator☆

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.

Effect of interleukin-1-β and tumor necrosis factor-α on cartilage proteoglycan metabolismin vitro

The activities of recombinant interleukin-1-β (IL-1) and recombinant tumor necrosis factor-α (TNF) on cartilage proteoglycan metabolism were compared in an organ culture system. IL-1, 1 to 100 ng/ml, and TNF, 10 to 1,000 ng/ml, increased proteoglycan degradation. The concentration-response curves were parallel. The timecourse for degradation was similar for the two cytokines during a 6 day incubation. Both cytokines inhibited the synthesis of new proteoglycan as measured by35S incorporation. The inhibition curves were parallel and concentration-related between 1 and 10 ng/ml for IL-1 and between 10 and 100 ng/ml for TNF. Maximal inhibition was 60% in the presence of IL-1 (10 ng/ml) or TNF (100 ng/ml), and plateaued at higher concentrations. IL-1 was ten fold more potent than TNF in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesis. Degradation in response to TNF, but not to IL-1, could be blocked by a polyclonal antibody to TNF. A polyclonal antibody to IL-1 could block proteoglycan breakdown in response to both cytokines suggesting that TNF may be mediating proteoglycan degradation by inducing the production of interleukin-1.

In vivo studies on the effects of human recombinant interleukin-1β on articular cartilage

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-1β into the knee joints of rabbits induced a dose-related decrase 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 by35S incorporation. Thesein vivo effects of IL-1 on articular cartilage closely reflect those effects observedin 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.

Effect of antiinflammatory drugs on human interleukin-1-induced cartilage degradation

Human monocyte IL-1 stimulated the release of proteoglycans from cartilage in organ culture in a concentration-related manner. This stimulation required protein synthesis as shown by inhibition with cycloheximide. The metal chelator, 1,10-phenanthroline, inhibited breakdown, suggesting the involvement of a metalloproteinase. Various nonsteroidal anti-inflammatory drugs (100 μM), and the corticosteroids, dexamethasone and hydrocortisone (1–10 μM), were not effective in blocking proteoglycan release. Of the disease modifying agents tested, levamisole was ineffective while the antimalarials, chloroquine (100 μM) and hydroxychloroquine (100 μM), inhibited the action of IL-1. The free- radical inhibitor SOD (5000 U/ml but not 1000 U/ml) was effective while catalase (8000 U/ml) was not. The protective effects of SOD and the antimalarials suggest that oxygen reactive species may play a role, while lack of inhibition with NSAIDs and corticosteroids indicate that arachidonic acid metabolites may not be important in this degradative process.

The effects of interleukin-1 on the expression of thrombospondin and fibronectin by rabbit articular chondrocytes

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.

Metabolism resistant isothiazolone inhibitors of cartilage breakdown

A series of 2-(arylmethyl)pyridoisothiazolones is reported that inhibit the IL-1 beta induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, non-peptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.

2,5-Diarylisothiazolone: novel inhibitors of cytokine-induced cartilage destruction

A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.

Effect of ebselen on IL-1-induced alterations in cartilage metabolism

Abstract.Objective: To evaluate the effect of the antioxidant-like anti-inflammatory agent, ebselen, on cartilage proteoglycan degradation and to determine whether its cartilage protectant activity is related to its antioxidant activity.¶Materials and Methods: Cartilage in organ culture was stimulated with interleukin-1 (IL-1), and proteoglycan degradation was assessed by measuring the amount of sulfated glycosaminoglycan released into the media, proteoglycan synthesis evaluated by [35S]-sulfate incorporation, and prostaglandin E2 (PGE2) release determined by radioimmunoassay (RIA). Glutathione peroxidase (GSH-Px) activity was evaluated in a coupled test system using NADPH/GSSG reductase as an indicator and cyclooxygenase activity was evaluated using sheep seminal vesicle prostaglandin synthase.¶Results: Ebselen caused a concentration-dependent inhibition of IL-1-stimulated proteoglycan degradation with an IC50 of 4.7u2009μM. Cartilage PGE2 release was also reduced in the presence of ebselen (IC50u2009=u20096.2u2009μM). However, at concentrations up to 100u2009μM, ebselen had no effect on the inhibition of proteoglycan synthesis by IL-1. Induction of proteoglycan breakdown was also inhibited by a sulfur analog of ebselen. This analog was devoid of GSH-Px activity and was 50-fold less potent in cyclooxygenase inhibitory activity, but was equipotent to ebselen in inhibiting cartilage degradation.¶Conclusions: Ebselen, unlike other NSAIDs, blocks cartilage proteoglycan breakdown without inhibiting proteoglycan synthesis. This effect is independent of its GSH-Px activity and its ability to inhibit cyclooxygenase and PGE2 production. Therefore, this compound may provide a new mechanism for protecting cartilage matrix from degradative factors in arthritic joints.

Inhibition of cartilage degradation by isothiazoloquinolinones

Abstract Several aryl-fused isothiazoloquinolinones were prepared and were found to inhibit IL-1β induced degradation of bovine nasal cartilage in an organ culture assay. These compounds, derived conceptually from the benzisothiazolone ring system, represent a new, non-peptide class of inhibitors of cartilage degradation.