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250 Annals of the Rheumatic Diseases 1994; 53: 250-255

The effects of Tenidap on induced Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from proliferation of human synovial fibroblasts in vitro

D L Mattey, E Evans, P T Dawes

Abstract (EGF),2 acidic and basic fibroblast growth Objectives-Tenidap, a new anti- factors (FGFs),3 4 tumour necrosis factor alpha rheumatic agent, is a lipoxygenase and (TNF),5 as well as combinations of inhibitor, and is reported (for example, interleukin-1 and transforming to inhibit the production and action of growth factor 0).6 The major source of these interleukin 1 (IL-1). Since eicosanoids, factors is believed to be leukocyte populations, IL-1, and other cytokines may influence particularly mononuclear phagocytes, which the growth of fibroblasts in the joint infiltrate the rheumatoid joint during synovium the study was carried out to inflammation. determine the effects of Tenidap on cyto- Synovial fibroblasts are likely to play an kine induced proliferation of these cells in important role in the inflammatory process vitro. since they have been shown to produce various Methods-Cell cultures derived from mediators, including cytokines,7 prostag- patients with a variety of rheumatic landins,8 and proteolytic such as diseases were cultured in different collagenase and stromelysin.' 9 They may also concentrations ofTenidap sodium, with or orchestrate the responses of other cells within without IL-1, tumour necrosis factor the synovium and may affect the survival of alpha (TNF), IL-6, basic fibroblast growth lymphocytes and other haemopoietic cells factor (bFGF), or transforming growth within the joint.1" factor beta (TGF,). Cell proliferation was The proliferation of synovial fibroblasts may measured using a crystal violet colouri- be affected not only by factors produced within metric assay. E2 levels in the joint but also by various anti-inflammatory culture supernatants were measured by drugs and second-line agents administered to radioinmmunoassay. patients with RA. Since most patients with RA Results-Tenidap at concentrations above are on such agents it is difficult to assess the 10 pug/ml inhibited cell growth, while at role of individual growth promoting (or 1-25-5 ,ug/ml there was a small but inhibiting) factors produced in vivo. For http://ard.bmj.com/ significant increase in proliferation example, non-steroidal anti-inflammatory compared with controls. A further drugs (NSAIDS) inhibit production of increase in growth was obtained when such as (PGE2) cells were incubated with Tenidap + IL-I, which is generally inhibitory to fibroblast cell TNF or bFGF, and this was significantly growth. It has been shown that treatment with higher than in the presence of any cyto- the NSAID indomethacin increases synovial kine alone. Stimulation of IL-1 induced fibroblast cell growth in vitro in the presence on October 2, 2021 by guest. Protected copyright. growth by Tenidap was reduced by of interleukin-1 i or TNF." Furthermore IL-I addition of high levels of exogenous PGE2 and PDGF act synergistically to stimulate (100 ng/ml) although growth was still synovial fibroblast proliferation in the presence higher than in IL-I alone. of indomethacin.' Other cyclo-oxygenase Conclusions-Depending on concentra- inhibitors and glucocorticoids have similar tion, Tenidap may inhibit or stimulate enhancing effects on IL- 1 induced fibroblast synovial fibroblast growth. Our results proliferation."l In contrast, the second line suggest that augmentation of growth by agent D-penicillamine causes suppression of low concentrations cannot be explained by human skin fibroblasts in the presence of inhibition of PGE2 production alone. physiological levels of copper sulphate.'2 This may stimulate cell inhibition was attributed to the of Staffordshire Tenidap directly production Rheumatology Centre, growth or may block other fibroblast hydrogen peroxide in the presence of these The Haywood, factors which are involved in control of agents. Thus drugs which affect the production Burslem, Stoke on cytokine induced proliferation. of growth inhibitory (or growth promoting) Trent, United Kingdom factors may have long term effects on D L Mattey (Ann Rheum Dis 1994; 53: 250-255) proliferation and/or survival of synovial E Evans fibroblasts, and may have significant effects on P T Dawes cellular interactions within the joint. The Correspondence to: a of such effects are Dr D L Mattey Proliferation of synovial fibroblasts is clinical consequences poorly Staffordshire Rheumatology recognised feature ofrheumatoid arthritis (RA) understood at present. Centre, are a novel The Haywood, although it is still unclear which factors Tenidap sodium, anti-inflammatory Burslem, responsible for driving this phenomenon. drug which has combined inhibitory activity Stoke on Trent, Many growth factors/cytokines have been against the cyclooxygenase and 5-lipoxygenase United Kingdom. derived pathways,'3 14 is reported to inhibit the Accepted for publication implicated, including platelet growth 10 December 1994 factor (PDGF),' epidermal growth factor production of IL-1, IL-6 and TNF'5 16 and the The effects of Tenidap on cytokine induced proliferation of human synovial fibroblasts in vitro 251

action of IL-1.1 It also inhibits the release of Mes, pH 6-0. The plates were shaken at room activated neutrophil collagenase. 18 Since temperature for 20 minutes and excess dye was inhibition of eicosanoids and cytokines may removed extensive with deionised by washing Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from have significant effects on synovial fibroblast water. Plates were air-dried before solubili- growth, we have investigated the effects of sation ofthe bound dye in 100 [LI of 10% acetic Tenidap, alone or combined with various acid. The optical density (OD) of the dye cytokines, on proliferation of these cells in extracts was measured directly in the plates vitro. using a Titertek Multiskan Plus Mk II micro- plate reader at a wavelength of 590 nm. Quad- ruplicate determinations were made at each Materials and methods experimental point. Blank values were 1) CELLS obtained from quadruplicates of cell free wells Human synovial tissue was obtained, with on each plate which were stained in an iden- Ethical Committee approval, from patients tical way to those containing cells. Experiments undergoing surgery. Synovial fibroblasts were with different cell cultures showed a direct released from the tissue by digestion of syno- correlation between OD and cell numbers (not vium with 2 mg/ml collagenase (Type 1A, shown). The effects of Tenidap +/- cytokines Sigma). Skin fibroblasts were obtained from on cell growth were represented by plotting cell slices of epidermis/dernis from healthy volun- numbers (calculated from OD values) against teers. We established three normal skin fibro- drug concentration. blast cell cultures and 17 synovial fibroblast cell The percentage growth relative to untreated cultures from patients with a variety of controls was calculated as follows:- rheumatic conditions. Cells were maintained in % growth = Dulbecco's Modified Eagles Medium OD (test/day 7) -OD (day 0) 100 (DMEM) with 10% foetal calf serum (FCS), X glutamine, penicillin and streptomycin (com- OD (control/day 7) - OD (day 0) plete culture medium, CCM). Experiments Day 0 = cells after 48 hours serum starvation were carried out on cells at various passage Day 7 = cells after 48 hours serum numbers (2-30). Most experiments were starvation + 7 days test or control performed on cells between passage 2 and 10. medium Tenidap sodium was dissolved in DMSO, at a concentration of 10 mg/ml before dilution in 2) PROLIFERATION ASSAY culture medium. Indomethacin was prepared We used a crystal violet colourimetric tech- in a similar way. The following cytokines were nique to examine the growth of fibroblasts in used alone or in combination with Tenidap: 96 well plates.'9 Cells were seeded at a density IL-1 alpha (human recombinant, 0X1-10 ng/ of 5 X 103 cells/well in CCM. Cells were ml), TNF alpha (human recombinant, 0X1-10 cultured for three days in CCM, followed by ng/ml), IL-6 (human recombinant, 0*5-5 ng/ http://ard.bmj.com/ 48 hours of serum starvation in DMEM with ml), basic FGF (human recombinant, 0X1-10 0-2% foetal calf serum to take cells into ng/ml), TGF beta (from human platelets, quiescence (Go). After serum starvation, 0X1-5X0 ng/ml). TGF beta was obtained from experiments were carried out either in CCM or Sigma. All other cytokines were from British serum free medium (SFM) consisting of Biotechnology Ltd. DMEM with insulin (25 ,ug/ml), transferrin (25 ,ug/ml) and (25 ng/ml) on October 2, 2021 by guest. Protected copyright. (Sigma), plus 0-1% bovine serum albumin 3) PGE2 MEASUREMENT (BSA). Test medium (for example, SFM + PGE2 was measured by a standard radio- Tenidap) was added to the cells which were immunoassay (Amersham) on cell free super- then incubated for a further seven days. In natants from the 96 well growth assay plates. preliminary experiments cells were examined Supernatants were stored at -70°C before use. for growth at various time points. We found that cells continued to proliferate over a seven day period and that although increases in STATISTICAL METHODS growth were measurable within two to three The Wilcoxon test for matched pairs (non days they were far more marked after seven parametric data) or the paired t test (parametric days, as were differences between treatments. data) was employed to determine the signifi- After culture the medium was removed and the cance of the difference in cell growth between cells were fixed for one hour at room tempera- fibroblast cultures treated with or without ture with 3.5% formaldehyde in phosphate Tenidap. All statistics were calculated using the buffered saline (PBS). One plate was always Number Cruncher Statistical System. fixed after serum starvation and before addition of test medium to give the baseline value (day 0). Internal standardisation of each assay was Results carried out by seeding an additional plate with CELL PROLIFERATION single, double and multiple amounts of starting (1) Tenidap cell numbers. Plates were washed three times by A range of concentrations of Tenidap sodium submersion in deionised water. (0a 1-80 pug/ml) were tested in our proliferation After air-drying (approx two hours) the cells assays. In most cell cultures, Tenidap above 10 were stained by addition of 100 RI of a 0-1% ,ug/ml in SFM inhibited cell proliferation. At solution of crystal violet dissolved in 200 mM higher concentrations (>20 ,ug/ml) Tenidap 252 Mattey, Evans, Dawes

20 caused gradual detachment of cells over the seven day period of the assay. Trypan blue

staining revealed that many cells were no Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from longer viable. In SFM, adherent cell numbers were reduced by 50% over seven days in the 15 presence of 20-25 pug/ml Tenidap (fig 1), and more than 80% of detached cells were dead on 0 0 day 7 (not shown). In CCM an equivalent 0 result was achieved by 45-55 pug/ml (fig 1). x 10 Cells cultured in SFM containing 0-5% instead 0 of 0 lI% BSA were more resistant to Tenidap toxicity with a 50% reduction in numbers occur- C.) ------ring at approximately 80 >g/ml (not shown). In SFM, a significant increase (p<0 002) in proliferation of synovial fibroblasts was noted in response to Tenidap at 1 25-5 pg/ml (fig 1, table 1). The ability of Tenidap to stimulate cell proliferation did not appear to be related to type of disease or cell passage number were 0 r-----i although there variations in the growth o 10 20 30 40 50 60 70 80 response. More than half the cell cultures Tenidap concentration (,ug/rnl) examined (9/17) showed a significant increase Figure 1 Typical seven day growth responses ofsynovialfibroblasts in SFM (0) and in growth. Similar results were obtained with CCM (m) in the presence ofdifferent concentrations of Tenidap. An increase ita growth is cells cultured in CCM. seen over the concentration range 1 25-5 ,ug/ml with a sharp decrease in growtth at 10 ,g/ml in SFM and 20 ug/ml in CCM. Each point represents the mean (SEM) of nine experiments carried out in quadruplicate. Dotted line represents cell numbers at day 0. (2) Tenidap + IL-I alpha All cell cultures but one (16/17) demonstrated significantly increased growth in response to Table 1 Effect of Tenidap (5 ,g/ml) on the seven day growth ofsynovialfibroblasts In the IL-1 alpha (1 ng/ml) in presence ofdifferent cytokines SFM or CCM (table 1). In the presence of IL-I alpha + Tenidap (5 Treatment N Mean %growth (SEM) povalue ,ug/ml) there were further marked increases in without Tenidap with Tenidap growth (table 1). This included cell cultures that showed little or no growth response to SFM 28 100 134(8-6) < IL-I (1 ng/ml) 37 215(22) 304(32) <:0.0001** Tenidap alone. The one cell culture that failed TNF (5 ng/ml) 21 190(21) 258(33)

bFGF (IO ng/ml) 11 137(10) 186(13) <:0.02* increased growth upon addition of Tenidap. http://ard.bmj.com/ TGFB (5 ng/ml) 5 137(17) 156(31) >o01 + The proliferative response to IL-I + Tenidap N = number of experiments. was found in synovial fibroblasts from patients * p value obtained from Wilcoxon Test for matched pairs. with a number of rheumatic diseases including ** p value obtained from paired t test. + not significant. RA, OA, AS and pigmented villonodular synovitis. A significant increase was also seen in the proliferation of normal skin fibroblasts

and those from a patient with scleroderma on October 2, 2021 by guest. Protected copyright. 20 - (data not shown). Maximum growth was obtained in the presence of 5 jig/ml Tenidap (fig 2). Similar results were obtained with cells cultured in IL-1 + CCM although the 15 maximum growth response occurred at 10 jig/ml in some cases (not shown).

0 0 0 (3) Indomethacin + IL-I alpha x Indomethacin was similar to Tenidap in u0 10* c causing an increase in cell proliferation which ______was enhanced in the presence of IL-1 (fig 3). The maximal IL- I + indomethacin induced stimulation was usually less than the maximal 5 increase seen in the presence of IL- 1 + Tenidap. The addition of indomethacin (-(10 ,ug/ml) to IL- 1 alpha + Tenidap (5 pLg/ml) caused no further stimulation ofcell growth. At higher concentrations (5 0 jig/ml and above) indomethacin reduced the proliferative 40 0 10 20 30 50 60 70 80 response to IL-1 + Tenidap. Tenidap concentration (pLg/ml

Figure 2 Typical seven day growth response ofsynovialfibroblasts iin SFM + IL-I (1 ng/ ml) and different concentrations of Tenidap. Growth is significandy iincreased in 5 ,g/ml (4) Addition ofPGE2 Tenidap but is sharply reduced at 20 ,ug/ml and above. Each point re the mean (SEM) ofsix experiments carried out in quadruplicate. Dotted epresents Tenidap may augment IL-1 alpha induced numbers at day 0. proliferation by blocking eicosanoid synthesis, The effects of Tenidap on cytokine induced proliferation of human synovial fibroblasts in vitro 253

This suggests that other factors apart from PGE2 are important in limiting IL-1 alpha

induced growth. Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from

(5) Tenidap + other cytokines TNF alpha (5 ng/ml) in SFM induced significant growth increases (table 1). Growth was increased further by addition of Tenidap. x 11-6 (5 ng/ml) caused little or no change in cell proliferation (compared with SFM alone) in the cell lines tested (table 1). Addition of 5 5- ,ug/ml Tenidap to IL-6 containing cultures increased cell growth to levels equivalent to those seen in Tenidap alone. Basic FGF (10 ng/ml) in SFM stimulated small but significant increases in synovial fibroblast proliferation and further increases were seen upon addition of Tenidap (table 1). The growth increases seen in the presence of or 0 10 20 30 40 50 60 70 80 TGFP TGF,3 + Tenidap were not statistically Indomethacin concentration (,ug/ml) significant. Figure 3 Effect ofindomethacin on the seven day growth ofsynovialfibroblasts in SFM with (0) or without (9) IL-I (1 ng/ml). A small increase in IL-I inducedproliferation was seen at 1 ,ug/ml indomethacin. Each point represents the mean (SEM) ofsix experiments carried out in quadruplicate. Dotted line represents cell numbers at day 0. PGE2 PRODUCTION The amounts of PGE2 in culture media after seven days of treatment with cytokines in the thus preventing the production of growth presence or absence of Tenidap (5 ,ug/ml) are inhibitory which act as a shown in table 2. Maximum PGE2 production negative feedback mechanism on IL-I induced occurred in media containing IL-1 (1 ng/ml). stimulation. Since PGE2 is normally produced High levels were also found in TNF treated by synovial fibroblasts in response to IL-1 and cultures but little PGE2 was detected in cells is reported to have growth inhibitory cultured with IL-6 or SFM alone. Little or no properties, we added this prostaglandin at PGE2 was detectable in cultures containing various concentrations (3-100 ng/ml) to cell Tenidap. cultures with IL-1 alpha (1 ng/ml) + Tenidap

(5 ,ug/ml). PGE2 at 3 ng/ml showed no http://ard.bmj.com/ inhibitory effect on growth. At higher Discussion concentrations of PGE2, the growth We have shown that Tenidap sodium at augmented by Tenidap was reduced. However, concentrations between 1 25-5 pug/ml en- even at 100 ng/ml the growth response was still hanced the proliferation of synovial fibroblasts higher than in SFM + IL-1 alpha alone (fig 4). in vitro. Tenidap alone in SFM or CCM increased cell growth. Furthermore, cell

proliferation induced by IL-1, TNF, or bFGF on October 2, 2021 by guest. Protected copyright.

20 - was significantly increased in the presence of Tenidap. In addition Tenidap increased synovial fibroblast proliferation in the presence ofIL-6. The ability ofTenidap to stimulate cell proliferation was not related to the type of I 15 - disease or cell passage number. Increases in cell growth were also obtained with skin fibroblasts 0 0 from normal subjects and scleroderma patients x (unpublished observations). .0 Tenidap induced proliferation of fibroblasts 10 - E in vitro was dependent on drug concentration. Little or no effect on cell proliferation was seen at <1-25 while >10 was 0 ,ug/ml ,ug/ml growth

5 - Table 2 Effect ofcytokines ± Tenidap on PGE2 production by synovialfibroblasts in vitro Treatment PGE2 (pg/mi) * SFM 40-98 I I 1, 'i k li K 14 N N I L NI SFM + Ten <0-1 0- I ,& '\.NI ; 4 ; . N, IL-I (1 ng/ml) 2013-22 0 0 0 3-125 6-25 12-5 25 50 100 IL-1 +Ten <0-1 TNF (5 ng/ml) 1316-43 PGE2 concentration (ng/mI) TNF + Ten 1-04 Figure 4 Growth response ofsynovialfibroblasts in Tenidap IL-I and different IL-6 (5 ng/ml) 32-32 IL-6 + Ten <0-1 concentrations ofPGE2. Cells were culturedfor seven days in SFM ( ,), SFM + Tenidap (5 ,g/ml) (0), SFM + Tenidap + IL-I (0) with different concentrations ofPGE2. Data * The data represent the mean values of 3 separate experiments are representative ofexperiments carried out on three different cell cultures in quadruplicate. on 3 cell cultures. Error bars represent the SEM. Dotted line represents cell numbers at day 0. All experiments carried out in SFM. 254 Mattey, Evans, Dawes

inhibited. At higher concentrations (>15 ,ug/ Tenidap. In our experience the variations in ml) Tenidap was toxic to cells in SFM growth between cell cultures were not related

although the toxicity was significantly reduced to the type of disease or severity of disease in Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from by incubating cells in SFM containing higher the patients from which they were obtained. levels of BSA, or in medium with 100% FCS. Moreover, although most cell cultures This may be explained by increased binding of exhibited slower growth with increasing Tenidap to protein under these conditions. passage number, some cell cultures still More than 99% of Tenidap is reversibly bound showed significant proliferation at passage to human plasma proteins over the range of numbers >25. Even in slower growing cells at 1-100 tg/ml. (Pfizer Central Research). high passage numbers (>20) Tenidap caused It has been shown previously that indome- significant augmentation of cytokine induced thacin enhances IL-1 and TNF induced growth. proliferation of synovial fibroblasts in the We found in both serum free and serum presence of serum,"1 probably through containing media that Tenidap stimulated cell inhibition of PGE2 production. We have proliferation with and without cytokines. It confirmed this using a range of indomethacin should be pointed out that our serum free concentrations in SFM. However, the maximal medium contained some protein (0X1% BSA) growth stimulation produced in the presence of as well as transferrin, and the pro- IL-I + indomethacin was less than the gression factor, insulin, which may act syner- maximal stimulation by IL-I + Tenidap. gistically with other growth factors.23 Indeed, Furthermore, high levels of PGE2 (100 ng/ml) we have found that insulin synergises with IL- 1 only caused partial inhibition ofthe cell growth to increase growth of synovial fibroblasts induced by Tenidap + IL- 1. Exogenous PGE2 (unpublished observations). In contrast to added at concentrations equivalent to those many previous studies we used an assay that found in IL-I treated cultures (2-3 ng/ml) had reflects the actual change in cell numbers. little or no effect on the growth of cells in Although [3H] thymidine incorporation is Tenidap + IL-1. Recent work by Taylor et al6 widely considered to correlate with cell growth, has questioned the concept that PGE is an this type of assay can produce misleading important growth inhibitor in synovial results since increased thymidine uptake does fibroblasts since cytokine combinations which not always correlate well with increases in cell stimulated high levels of PGE production also number.3 24 25 Also, the full effect ofa particular caused increased proliferation. Our results cytokine on cell growth may be underestimated suggest that augmentation of synovial by [3H] thymidine incorporation which is fibroblast proliferation by Tenidap cannot be usually carried out over a relatively short time explained by inhibition of PGE2 production period (24-48 hours). Release of other growth alone. This suggestion is reinforced by the factors from the cytokine treated cells may

demonstration that Tenidap is able to augment occur at some time after initial stimulation so http://ard.bmj.com/ synovial fibroblast proliferation in SFM alone that the eventual increase in fibroblast numbers or in the presence of cytokines such as bFGF results from an indirect autocrine mechanism.26 and IL-6 which do not stimulate PGE2 In our study synovial fibroblasts continued to production.20 21 In contrast to our results with proliferate over the seven day culture period. Tenidap, a previous study showed that Although changes in cell number were indomethacin had no potentiating effect on measurable after two to three days, differences

bFGF induced synovial proliferation.20 between treatments were more apparent after on October 2, 2021 by guest. Protected copyright. There have been conflicting reports on the seven days. effects of IL-1 on fibroblast proliferation with The consequences of stimulating synovial some studies showing little or no stimulatory fibroblast growth in vivo are still unclear. It is effects' 3 6 22 while others have shown generally assumed that increased proliferation significant stimulation."l 12 However, a number of these cells is associated with the progression of these studies have used different fibroblast of RA, with evidence from histomorphological types such as human lung fibroblasts22 or studies showing a close temporal relationship human foreskin fibroblasts,'2 and often only between rapid proliferation of fibroblast-like one cell line was examined. We agree with cells of the synovium and articular cartilage Remmers et a13 that conflicting results from destruction.27 However, while it is clear that different studies may be due to differences in synovial fibroblasts have the potential to cell type, cell passage number and the type of produce many pro-inflammatory agents they growth assay used. Clearly, caution is called for may also be involved in anti-inflammatory in the interpretation of these studies and their processes, including tissue repair. For example, relationship to fibroblast proliferation in the IL-1 will promote synthesis and release of both synovial joint. collagenase and its inhibitor (tissue inhibitor of To try and overcome some of these metalloproteinase, TIMP), as well as ECM difficulties we examined a large number of cell components such as collagen and fibronectin.28 cultures (n = 17) from patients with a variety of Thus increased proliferation of synovial rheumatic conditions, and growth was fibroblasts in vivo may not be damaging per se, investigated over a range of passage numbers. rather it is alterations in their state of activation There were certainly variations in the total and changes in the balance between amount of growth between cell cultures and production of pro- and anti-inflammatory between passage numbers (data not shown), mediators which are important. although overall there was a clear demon- In vitro studies have indicated that Tenidap stration of enhanced growth in the presence of inhibits the production of IL-1 and TNF in 255 Mattey, Evans, Dawes

human peripheral blood mononuclear cells by 11 Gitter B D, Labus J M, Lees S L, Scheetz M E. at Characteristics of human synovial fibroblast activation by 70-80% concentrations slightly lower than IL-1 3 and TNF. Immunol 1989; 66: 196-200. those clinically achievable (20 ,Lg/ml).29 It also 12 Matsubara T, Hirohata K. Suppression ofhuman fibroblast proliferation by D-Penicillamine and copper sulfate in Ann Rheum Dis: first published as 10.1136/ard.53.4.250 on 1 April 1994. Downloaded from inhibits the activity ofIL-I in T cell activation'7 vitro. Arthr Rheum 1988; 31: 964-72. and down regulates IL-I expression in 13 Moilanen E, Alanko J, Asmawi M Z, Vapaatalo H. CP- 66,248 a new anti-inflammatory agent is a new potent human chondrocytes.30 The results from our inhibitor of leukotriene B4 and synthesis in study indicate that Tenidap at low human polymorphonuclear leucocytes in vitro. Eicosanoids 1988; 1: 35-39. concentrations (up to 10 ,ug/ml) does not 14 Blackburn W D, Heck L W, Loose L D, Eskra J D, Carty inhibit the mitogenic activity of IL-1 alpha, T J. Inhibition of 5-lipoxygenase product formation and polymorphonuclear cell degranulation by Tenidap TNF alpha or bFGF on human synovial sodium in patients with . Arth Rheum fibroblasts, although at possible therapeutic 1991; 34: 204-210. 15 Otterness I G, Bliven M L, Downs J T, Natoli E J, Hanson levels (15-20 Lg/ml) it does inhibit growth D C. Inhibition of interleukin I synthesis by tenidap: a even in the presence of cytokines. Our data new drug for arthritis. Cytokine 1991; 3: 277-83. 16 Sipe J D, Bartle L M, Loose L D. Modification of pro- suggest that the concentration of Tenidap inflammatory cytokine production by the antirheumatic achieved in vivo in the local environment of the agents Tenidap and . J Immunol 1992; 148: 480-4. joint, and the availability of bioactive cytokines 17 McDonald B, Lowe L, Rosenwasser L J. The influence of may be critical factors in the effect ofthis agent a novel arachidonate inhibitor, CP-66,248 on the production and activity of human monocyte IL-1. Arth on synovial fibroblast proliferation. Rheum 1988; 31(Suppl 4: S17, Abst). 18 Blackburn W D, Loose L D, Heck L W, Chatham W W. Research was funded by the Haywood Rheumatism Research Tenidap, in contrast to several available non steroidal and Development Foundation, and Pfizer Ltd. We would like anti-inflammatory drugs, potently inhibits the release of to thank Pfizer for providing the Tenidap. activated neutrophil collagenase. Arth Rheum 1991; 34: 211-16. 19 Kueng W, Silber E, Eppenberger U. Quantification of cells cultured on 96 well plates. Anal Biochem 1989; 182: 1 Kumkumian G K, Lafyatis R, Remmers E F, Case J P, Kim 16-19. S J, Wilder R L. 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The streptococcal cell wall arthritis. Growth factors 1990; 2: effect of lymphokines and monokines on fibroblast 179-88. growth. J Leuk Biol 1990; 47: 312-20. 4 Hamerman D, Taylor S, Kirschenbaum I, et al. Growth 23 Rosengurt E. Signal transduction pathways in mitogenesis. factors with heparin binding affinity in human synovial In: Waterfield MD, ed. Growthfactors. London: Churchill fluid. Proc Soc Exp Biol and Med 1987; 186: 384-9. Livingstone, 1989: 513-28. 5 Thornton S C, Por S B, Penny R, Richter M, Shelley L, 24 Elias L. Stimulation by tumor necrosis factor of HL-60 Breit S N. Identification of the major fibroblast growth thymidine salvage pathway metabolism dissociated from factors released spontaneously in inflammatory arthritis as proliferation.J Cell Physiol 1988; 136: 95-102. platelet derived growth factor and tumour necrosis factor- 25 Ho C-K, Ou B-R, Hsu M-L, Su S N, Yung C-H, Wang S-Y. alpha. Clin Exp Immunol 1991; 86: 79-86. 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