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Annals of the Rheumatic Diseases, 1989; 48, 372-381 Evaluation of the effects of antiarthritic drugs on the secretion of proteoglycans by lapine chondrocytes using a novel assay procedure SIMON COLLIER AND PETER GHOSH From the Raymond Purves Research Laboratories (The University of Sydney) at the Royal North Shore Hospital of Sydney, St Leonards, NSW 2065, Australia SUMMARY A new method is described for separating free 35SO4- from 35SO4 labelled groteoglycans synthesised by rabbit articular chondrocytes cultured in the presence of excess 4 The procedure uses the low solubility product of barium sulphate to remove, by precipitation, free 35SO4- from culture medium. Optimum recovery of 35so4 labelled proteoglycans was achieved after papain digestion to release 35SO4-glycosaminoglycans, and addition of chondroitin sulphate before the precipitation step. Using this assay, we studied the effect of six drugs-indomethacin, diclofenac, sodium pentosan polysulphate, glycosaminoglycan polysulphate ester, tiaprofenic acid, and ketoprofen-on the secretion into the medium of labelled proteoglycans by lapine chondrocytes. The six drugs were tested at 0< 1, 1, 10, 50, and 100 I.g/ml over four consecutive 48 hour culture periods. A consistent concentration-response pattern was found for the four non-steroidal anti-inflammatory drugs (NSAIDs) studied. Generally they inhibited proteoglycan secretion at 50 and 100 [ig/ml but had no effect at lower concentrations. Inhibition of secretion was strongest with indomethacin and diclofenac at 50 and 100 ig/ml. In contrast with the NSAIDs studied, the two sulphated polysaccharides (sodium pentosan polysulphate and glycosaminoglycan polysulphate ester) at low concentrations increased proteoglycan secretion by chondrocytes, with maximal stimulation occurring at 1 [ig/ml. Sodium pentosan polysulphate, but not glycosaminoglycan polysulphate ester, showed inhibitory activity at 50 and 100 pg/ml. Key words: non-steroidal anti-inflammatory drugs, sodium pentosan polysulphate, glycosamino- glycan polysulphate ester, diclofenac, indomethacin, ketoprofen, tiaprofenic acid. Non-steroidal anti-inflammatory drugs (NSAIDs) cartilage metabolism, but it is often difficult to still represent the mainstay of therapeutic manage- assess the validity of such claims because of the ment of osteoarthritis. It is now recognised, how- limitations of the methods used. Ideally the influence ever, that such drugs, although effective at relieving of a drug on cartilage cells should be monitored at the symptoms of the disease, may do little to improve different drug concentrations, over different time the condition, and in some instances may contribute periods, and under carefully controlled conditions, to its progression (see review by Burkhardt and but in many cases these criteria are not fully Ghosh ). In recent years new NSAIDs and other observed. The most convenient means of assessing drugs have become commercially available which the effects of drugs on chondrocytes is by in vitro are reported to be free of deleterious effects on assays. Although this approach has its shortcomings, it can provide a primary screening system, which can Accepted for publication 18 October 1988. be followed up by animal experiments. Measurement Correspondence to Associate Professor Peter Ghosh, Raymond of or Purves Research Laboratories (The University of Sydney) at the proteoglycan production by cartilage explants Royal North Shore Hospital of Sydney, St Leonards, NSW 2065, chondrocyte monolayers generally involves pulsing Australia. the cultures with 35so-- to radiolabel the newly 372 Antiarthritic drugs and the secretion ofproteoglycans 373 synthesised proteoglycans. This method, however, 80 U/ml gentamicin (David Bull Laboratories Pty requires the subsequent separation of the resulting Ltd, Rydalmere, NSW, Australia), 20 mM HEPES 35 o4 labelled proteoglycans from the large amounts (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic of unincorporated 35SO4 -. This may be achieved by acid; Sigma Chemical Company, St Louis, MO, removal of the free 35so-- by gel filtration chro- USA), and incubation was continued at 37°C for matography of each sample,2 exhaustive dialysis of three hours with occasional shaking. The digest was individual samples,3 or papain digestion and cetyl- washed three times with 15 ml volumes of Ham's pyridium chloride precipitation of the 35S04-glycos- F12 medium plus 10% FCS (F12-FCS), the cells aminoglycans.4 These methods are time consuming were counted with a haemocytometer, and 2X 106 and tedious and, in the latter method in particular, viable cells in 12 ml F12-FCS were plated into 75 cm2 the number of steps involved could result in tissue culture flasks (Miles Laboratories, Naperville, significant cumulative experimental errors. IL, USA). A preparation from one rabbit typically In the present study we describe a rapid, simple, yielded approximately 1Ox 106 chondrocytes. The and reproducible method for separating free 4 medium was changed after three days and subse- from 3 SO4 labelled proteoglycans in chondrocyte or quently every two to three days. Confluence was cartilage cultures. The efficiency of this method reached by the chondrocyte cultures in six to eight allows for comprehensive comparative experiments days. of the effects of drugs on the production of proteoglycans by chondrocytes under a variety of PREPARATION OF TEST CULTURES conditions. We used this method to compare the Six drugs were tested for their effects on proteo- effects of six currently available antiarthritic agents, glycan secretion by articular chondrocyte cultures. each at five concentrations, on the total proteoglycan They were glycosaminoglycan polysulphate ester secretion of cultured rabbit articular chondrocytes, (Luitpold Werk, Munich, FRG), sodium pentosan measured for four consecutive 48 hour culture polysulphate (Benechemie, Munich, FRG), indo- intervals. methacin (Merck Sharp and Dohme Australia Pty Ltd, Granville, NSW, Australia), diclofenac sodium Materials and methods (Ciba Geigy Australia Ltd, Pendle Hill, NSW, Australia), ketoprofen (May and Baker Australia ANIMALS Pty Ltd, West Footscray, Victoria, Australia), and New Zealand white rabbits (6-8 weeks old) of either tiaprofenic acid (Roussel Pharmaceuticals Pty Ltd, sex were obtained from the University of Sydney Castle Hill, NSW, Australia). animal house, Castle Hill, NSW, Australia. Medium was decanted from confluent chondrocyte cultures, cells were washed with two 5 ml volumes of PREPARATION OF PRIMARY CHONDROCYTE PBS, and then 3 ml of 0-2% trypsin, 0-1% edetic CULTURES acid (Ajax Chemical Company, Sydney, Australia) Rabbits were killed by intracardiac injection of in PBS was added, and cultures were incubated at pentobarbitone (May and Baker Australia, Pty Ltd, 37°C for two to three minutes until the cells West Footscray, Victoria, Australia). Both hind legs detached. The cells were aspirated and washed in were removed, and, under aseptic conditions, the three 15 ml volumes of F12-FCS, then counted, and knee joints were opened and the articular cartilage the concentration adjusted to 2xi05 cells/ml. The was shaved off the femoral condyles, the tibial cell suspension was dispensed into the wells (105 plateaux, and the patellae. The shavings were cells/well in 0.5 ml) of 24-well plates (Flow Labora- transferred to a 15 ml plastic centrifuge tube tories, McLean, Va, USA). At this plating density (Corning Laboratory Science Company, Stone, the cells had attached to form confluent monolayers Staffordshire, UK) containing phosphate buffered after an overnight incubation. The medium was saline (PBS). After washing with two 15 ml volumes changed and each well received 450 ,ul of F12- of PBS the fragments were resuspended in 15 ml of FCS containing 4-3 x 106 disintegrations per minute 0-25% trypsin (1:250, Gibco Laboratories, Grand 4 (92-5 kBq/well) (New England Nuclear Island, New York, USA) in PBS and incubated at Corp, Bedford, MA, USA). The various drugs were 37°C for one hour. The trypsin solution was aspirated dissolved and appropriately diluted in serum free and replaced with 15 ml of a solution of 0*2% Ham's F12 medium and delivered to the wells in collagenase (type 1A, Sigma Chemical Company, 50 RI volumes, to give final volumes in the wells of St Louis, MO, USA) in Ham's F12 medium (Flow 500 Rl and the desired final drug concentrations of 0, Laboratories, McLean, Va, USA) containing 10% 0-1, 1, 10, 50, and 100 ,ug/ml. Four replicate wells fetal calf serum (FCS) (Commonwealth Serum were used for each drug concentration. Sufficient Laboratories, Parkville, Victoria, Australia), F12-FCS plus 35SO4 - was prepared in a single batch 374 Collier, Ghosh for the experiment to ensure that all wells were 4 (185 kBq/ml) for 48 hours. The medium was exposed to an equal concentration of 35so4- at removed and concentrated to approximately 5 ml each medium change. Medium was changed and using an Amicon YM10 membrane (Amicon Corp, fresh drug solutions were added after two, four, and Danvers, MA, USA). The sample was then applied six days of culture. At these medium changes and on to a 1 6x90 cm Sepharose CL2B column (Pharmacia day 8 when the experiment was terminated the South Seas Pty Ltd, Sydney, NSW, Australia), exhausted medium was pipetted from each well into which had been precalibrated with DNA (VO) and tubes and stored for assay. Na235SO4 (Vt). The column was eluted with 0.5 M sodium acetate (pH 6.8) at a flow of 10 ml/h and 35S04-PROTEOGLYCAN ASSAY fractions (3.0 ml) monitored