Lydia Tsagris

Sensitivity of Anulus Fibrosus Cells to Interleukin 1β : Comparison With Articular Chondrocytes

Study Design. Anulus fibrosus cells from rabbits were grown in primary culture 1) to study their ability to produce prostaglandin E2 and Type II phospholipase A2, and to express stromelysin-1 messenger ribonucleic acid; and 2) to study the effect of interleukin 1&bgr; on this production and on proteoglycan aggregation. Objectives. To investigate the potency of anulus fibrosus cells to respond to interleukin 1&bgr; by producing degradative and inflammatory agents as compared with the potency of articular chondrocytes in the same animal. Summary of Background Data. Interleukin 1&bgr; has been implicated in the degradation of intervertebral discs. The way anulus fibrosus cells differ from articular chondrocytes in their responses to interleukin 1&bgr; remains to be established. Methods. Anulus fibrosus cells and articular chondrocytes were obtained from young rabbits, grown in primary culture, and incubated with interleukin 1&bgr;. The newly synthesized proteoglycan was measured by labeling with [35S]-sulfate. Proteoglycan aggregation was analyzed by the elution profile on Sepharose 2B columns. The contents of collagen Type II and stromelysin-1 messenger ribonucleic acid were assessed by Northern blot analysis. The Type II phospholipase A2 activity was measured using a fluorometric substrate. Prostaglandin E2 production was evaluated by radioimmunoassay. Results. Anulus fibrosus cells had 2.5-fold less Type II collagen messenger ribonucleic acid than articular chondrocytes, and interleukin 1&bgr; had no significant effect on this. Anulus fibrosus cells synthesized and secreted fourfold less proteoglycan than articular chondrocytes. Interleukin 1&bgr; reduced the anulus fibrosus content of total [35S]-sulfated proteoglycan by 35% (P < 0.01), and that of articular cells by 41% and decreased proteoglycan aggregation. Interleukin 1&bgr; induced the production of stromelysin-1 messenger ribonucleic acid in both cell types. The stromelysin-1 messenger ribonucleic acid content of anulus fibrosus cells was one half that of articular cells. Interleukin 1&bgr; increased the production of prostaglandin E2 and caused a dose-dependent secretion of Type II phospholipase A2 activity in both cell types. Its effect was 2.5-fold lower in anulus fibrosus cells than in articular chondrocytes. Conclusion. Anulus fibrosus cells can be stimulated by interleukin 1&bgr; to produce factors implicated in local degradative and inflammatory processes. This production is associated with decreased proteoglycan aggregation. Anulus fibrosus cells respond slightly less well to interleukin 1&bgr;in vitro than do articular cells.

Dual effects of 17ß-oestradiol on interleukin 1ß-induced proteoglycan degradation in chondrocytes

Objective: To determine whether 17β-oestradiol (E2) modulates interleukin (IL) 1β-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process. Methods: Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1β combined or not with 0.1–10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [35SO4]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct. Results: E2 modulated the IL1β-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1β-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1β effects. A biphasic E2 effect was also observed on IL1β-induced disaggregation of PG, 53–58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1β and E2 (0.1–10 nmol/l) did not modify IL1β-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression. Conclusion: Oestrogen concentration may have an inverse effect on IL1β stimulated proteoglycan degradation and MMP production by chondrocytes.

Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion

Group IIA secreted/synovial phospholipase A2 (GIIAPLA2) is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E2 (PGE2), the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA2, along with their chemical synthesis and results from PLA2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA2 affinities than did C1, and such predictions were confirmed by in vitro PLA2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1β-stimulated PGE2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints.