Interleukin-1β enhances the response of human articular chondrocytes to extracellular ATP

52

Bioch6nica et Biop~'~+ica Acta. 1137( 1992152-58 © 1992Elsevier Science Publishers B.V. All fights reselv,~d0167-4889/92/$05.C0

Interleukin-l~ enhances the response of human articular chondrocytes to extracellular ATP A l i s o n M . C a s w e l l a, W e n g S. L e o n g a a n d R . G r a h a m

G. Russell b

+Department of Biochemistry and Modular B ~ . ~ ; Unk'en~.'y o f ~ Le+~ CUIO and b Department of Human Metabolism and Clinical Biochem~r~; Uriiz'er~O. of Stwffield Medical School, Stwj~leld (UK)

(Received 31 January 1992) (Revised manuscript received 8Juue 1992)

Key words: Articular chondrocyte; lnterleukin-i; Purinocepmr, P2; Prnstag~andinE It has been observed that both interleukin-I (IL-I) and extracellular ATP stimulate the production of prostaglandin E (PGE) by human articular chondrocytes in monolayer culture. The combined effects of recombLo.ant human IL-I/$ and ATP were therefore studied using these cells. IL-I/] rapidly enhanced the response to a maximally effective concentration of ATP (100 pM). On continuons exposure of the ceils to the cytokine, its effect was greatest after aptnox. 24 h and tended to decline thereafter. The enhancement of the response m 100/zM ATP by lL-li~ was dose-dcpendenL Removal of IL-I/3 prior to treating the cells with IGO/J.M ATP did not affect the degree of enhancement of the response. The effect of the cytokioe ou the response to suboptimal concentrations of extracellular ATP was also tested. IL-I/~ lowered the minimum concentration of ATP required to elicit an increase in the production of PGE by human articular chondroeytas. These findings are of interest, since they indicate a synergistic interaction between a cytokine and a purinergic agonist+ Moreover, since both the sensitivity of the cells to extracellular ATP and the maximum response to this agent were enhanced, it is possible that IL- ! modulates more than one step in the process of Pz-parinoceptor-mediated stimulation of PGE production. These observations may be relevant to the pathogenesis of some forms of arthritis.

Introduction T h e r e is cousiderab!e interest in the effects of extracellular adenine nucleotides and adenosine, and cell surface receptors for these agonists (purinoceptors) have been identified in many tissues [1]. Burustock p ~ d the classification of purinoceptors as P~, most ruspGnsive to adenosine, and P2, most responsive to ATP, in 1978 [2]. Activation of P2-purinoceptors frequently results in enhanced production of prostaglandins [3-7]. W e have recently demonstrated that extracellular A T P promotes the production of prostaglandin E ( P G E ) by human articular chondrocytes in monolayer culture. The lo'~est concentration of A T P that elicited a n effect was in the range 1 - 5 / z M and maximal stimulation was observed with concentrations of A T P in the range 50-100 p.M. A t a n A T P concen-

Correspondence to: A.M. Caswell, Department of Biochemistryand Moh:cular Biolog'/,University.of Leeds, Leeds L$2 9JT, UK. Abbreviations: [Ca2*]i, cytnsolic concentration of flee calcium; Hopes, 4-(2-hydmx'yethyl-l-piperaziueethanesulphonicacid: PGE, prostaglandiu E; rhlL-IO, recombinant human interleukin-1/];TNFa, tumour necrosis factor a.

tration of 100/zM, P G E accumulated in iite medium at a constant rate for at least 4 h. A D P also elicited a response, but A M P and adenosine were virtually ineffective. The o r d e r of potency o f these agonists plus the nature of the response are consistent with the presence of P2-type purinoceptors at the surface of human articular chondrocytes [8]. The mechanism whereby extracellular A T P promotes an increase in the production of P G E by h u m a n articular chondrocytes is not knowu. However, activation of P2-purinoceptors is frequendy associated with an increase in the cytosolic concentration of free calcium ([Ca2+]i) [9-12], and phospholipase A 2 in rabbit articular chondrocytes is sensitive to calcium [13]. Hence, extracellular A T P may promote production of P G E by h u m a n articular chondrocytes via activation of phospholipase A 2 resulting from an increase in [Ca 2+ ]iE n h a n c e d production o f P G E by cultured human articular chondrocytes in response to intedeukin-1 (ILl ) has also been reported on several occasions [14-17]. In addition, IL-I has been shown to enhance the stimulation of the production of P G E by histamine in h u m a n articular chondrocytes [18]. It was establLshod that the interaction was occurring at a post-receptor

level and that histamine was acting via receptors of the H~-subclass [18]. Activation of this class of histamine receptor can also be associated with an increase in [Ca2+]i [|9]. Hence, histamine and extracellular ATP may stimulate production ot PGE in human articular chondrocytes by similar mechanisms. It was therefore of interest to examine whether IL-1 enhanced the response of these cells to extracellular ATP. Interactions between recombinant human (rh) IL-II/ and extracellular ATP were studied both at a maximally effective concentration of ATP (100 ItM) and at sub-optimal concentrations, in addition, the timecourse of the development of the interactioi, between rhlL-l~ and 100/zM ATP was studied. Some of this work has appeared in abstract form [20]. Experimental procedures

Culture zechniqw'.s. Adult human articular cartilage was removed from knee joints obtained after surgical amputation for lower limb isehaemia, or from femoral heads obtained after surgery for femoral neck fracture. Articular chondrocytcs were isolated by enzymic digestion and cultured as described previously [17,21,22]. For experimental use, confluent primary cultures were dispersed with 0.05% (w/v) trypsin/0.02% (w/v) EDTA and subcultured into 1.1 cm diameter wells (48-well plates) at a density, of approx. 3-104 cells/cm 2. in order to minimise variations between different positions on the plate, only the central 24 wells were used. The cells were maintained for a further 5-8 days prior to the commencement of experiments, to ensure the reestablishment of any cell surface components lost during the subculturing procedure. Incubation of human articular chondrocytes with ddL-l~ and ATP. Unless otherwise stated, the cells were preincubated for periods of 24-72 h with appropriate concentrations of rhIL-l/3 in minimum essential medium (Eagle) supplemented with Earle's salts, 100 U/nil penicillin and 100 p.g/ml streptomycin plus 10% (v/v) foetal calf serum. Where necessary, medium plus the cytokine was replaced every 24 h. At the end of the preincubation period, the cells were rinsed three times with serum-free Hepes-buffered minimum e~sential medium (Eagle) supplemented with Earie's salts and antibiotics, as above. Production of PGE was then monitored in the preseuc¢ or absence of ATP during a 4-h incubation of cells in this medium (0.25 ml/weli). Appropriate concentrations of rhiL-!/$ were also a~ded to the incubation medium, unless otherwise stated. A 4-h incubation period was used, because, as noted in the introduction, it had been observed previously that in the presence of 190/tM ATP, PGE accumulated at a constant rate in the medium throughout this period [8]. At the end of the incabation, media were removed and stored at - 20~C. Cell monolayers were rinsed with

phosphate-buffered saline (Dulbeceo's, without calcium and magnesium) to remove residual traces of medium. The plates were then stored at -2WC, until the protein content of the wells was measured. Measurement of prostaglandin E (PGE). PGE was measured in aliquots of incubation medium by radioimmunoassay methods in which free antigen was selectively adsorbed onto dextran-coated charcoal. Unfortunately, while these studies were being performed, the antiserum supplied by Steranti Research ceased to be commercially available. Later measurements were therefore made according to a slightly different protocol, using an antiserum supplied by Sigma. However, both methods gave similar results. In the initial studies, the assay buffer was 10 mM Fris-HCl (pH 7.4) containing 140 mM NaCI, 0.1% (w/v) gelatin and 0.02% (w/v) sodium azide and I vial of lyophilised antiserum was reconstituted in 100 ml buffer. Sample or standard (0.1 ml) was incubated overnight at 4°C with 0.1 ml [3HIPGE2 (248 Bq) and 0.1 ml antiserum. Unbound PGE was then removed by the addition of 0.2 ml ice-cold assay buffer containing 0.6% (w/v) Norit A charcoal and 0.125% (w/v) dextran. Following incubation at 4°C for 10 min, charcoal was removed by centrifugation for 15 min at 4°C at 2000 x g (ray 24.5 cm) and 0.2 mi of the supernatant was counted in 4 mi Emulsifier Safe scintillation fluid. In the later studies, the assay buffer was 10 mM sodium phosphate (pH 7.4) containing 150 mM NaCI, 0.1% (w/v) bovine serum albumin and 0.1% (w/v) sodium azide and 1 vial of lyophilised antiserum was reconstituted in 300 ml buffer. Sample or standard (0.1 ml) was incubated for 30 min at 4°C with 0_5 ml antiserum. [3H]PGE, was then added (0.1 ml, 148 Bq) and the tubes incubated overnight at 4°C. Unbound PGE was removed by the addition of ~.2 ml ice-cold assay buffer containing 1% (w/v) activated charcoal and 0.1% (w/v) dextran. The subsequent steps were essentially as described above, except that 0.7 ml of the final supernatant was counted. For both assays, results were calculated from standard curves, range: 5-2000 pg PGE2 per tube. Measurement of protein. Cell monolayers were solubilised in 1 M NaOH and protein was measured by a n~.odification of the Lowry method [23]. Bovine serum albumin (range: 4-20 p.g) was used as standard. Experimental design. Within experiments, each condition was tested in quadruplicate and the results expressed as the mean 4- standard error (S.E.). For the majority of the experiments, the increase in PGE production due to ATP and the associated standard error were then calculated. When more than one multiwell plate was used for an experiment, variation in basal production of PGE between plates was sometimes observed. Hence only results obtained from the same multiwell plate were compared statistically. The signifi-

54 TABLE 1 Effect o f coincubatkm with rhlL-ll3, and preincubation for 24 h plus cw~cubati~ on the sKmudation of the prodl~tion of PGE by human articular clumdr~, es in rr~,mlayer culture by extracellular A TP On each tissue culture plate, one group of wells was preincubated with IL-L Ceils were then incubated in the presence or absence of ATP ( 1130 /~M). IL-I was included in the medium during the incubation_+ATP in the w~lls that had been preincubated with the cytokine and also in a second group of wells. Each concentration of IL-I was tested on a separate mulfiwell plate. PGE released into the medium was measured as described in Experimental Procedures. Experimental results are presented as the mean_+S.E. (n = 4). Prcincubation (24 h)

Colncuhetion (4 h)

No No Yes

No Yes Yes

No NO Yes No No Yes

IL-I (U/mr)

pg PGE/p.g protein per 4 h No ATP

ATP (100/tM)

Change due to ATP

0.04 0.04

23.64- 3-56 30.0_+ 7.58 70.9_+ i 1.2

1314-20.1 176_+ 16.0 218_+ 18.0

107 +20.4 146 _+17.7 14,7 _+21.2

No Yes Yes

0.4 0.4

20.3_+ 2.91 124 _+10.9 95.2± 14.8

llO_+ 4.87 388_+41.6 395_+28.8

.*g9.7+ 5.67 264 +43.0 a .'~00 4-32.4 b

No Yes Yes

4.0 4.0

13.8_+ 1.96 436 +38.9 215 _+23.6

123_+10.9 843_+55.4 89i_+25.0

109 4-11.1 407 _+67.7 b 676 _34.4 b'c

a Response to ATP greater than that in the absence of 1L-I. P < 0.01. b Response to ATP grea:er :aan that in the absence of IL-I, P < 0.001. c Response to ATP greater than when IL-! was only included during the incubation with A l P , P

cance of differences between mean values was determ i n e d u s i n g o o e - w a y a n a l y s i s o f v a r i a n c e a n d t h e significance of interactions between ATP and rhlL-lp was assessed using two-way analysis of variance. Experi m e n t s w e r e p e r f o r m e d a t l e a s t t h r e e t i m e s , u s i n g cells f r o m d i f f e r e n t d o n o r s . U n l e s s o t h e r w i s e i n d i c a t e d , all experiments gave essentially the same results. Data from representative experiments are presented here. Material& R e c o m b i n a n t h u m a n I L - l f i w a s a g e n e r ous gift of Glaxo Group Research, Greenford, Middlesex. T i s s u e c u l t u r e m e d i a , e n z y m e s a n d a n t i b i o t i c s w e r e p u r c h a s e d f r o m G i b c o B R L , L i f e T e c h n o l o g i e s , PaLs-

,:

3.01.

ley, S t r a t h c l y d e , U I L F o e t a l c a l f s e r u m w a s o b t a i n e d f r o m N o r t h u m b r i a n Biologicals, C r a m l i n g t o n , N o r t h u m b e r l a n d , U K . [5,6,8,11,1~i4,15(n)-3H]Prustaglandin E 2 was supplied by Amersham International, Aylesbury, UK. Norit A charcoal was purchased from Aldrich, Gillingham, UIL Activated charcoal (untreated powder, 250-350 mesh), anti-PGE, ATP, bovine s e r u m a l b u m i n ( f r a c t i o n V , f o r p r o t e i n assay), b o v i n e ~rom albumin (essentially fatty acid free, for radioimmunoassay of PGE), dentran (approx. average molecul a r w e i g h t 7 0 0 0 0 ) a n d P G E 2 w e r e all s u p p l i e d b y Sigma, Poole, UK.

TABLE I1 Effect of the length of preincubation with rhlL-ll3 on the enhancement of the response o f human articular clumdrocytes in monolayer culture to extracellular ATP Cells were preincubated w/th IL-i for 24-72 h and were then incubated in the presence or absence of ATP (IG0 p.M). IL-I was included in the medi,m during the incubatinn_+ATP. Each concentration of IL-I was tested on a separate multiwell plate. PGE released into the medium was measured as descn'bed in Experimental Procedures. Experimental results are presented as the mean_+S.E. (n = 4). Preincubation

ILol

pg PGE//zg protein per 4~h

(h)

(U/ml)

No ATP

24 48 72

0.04

59.0 + 5.88 49-5 + 258 45.4+6.24

276 ± 39.2 277_+ 14.2 229+46.1

217 + 39.6 228 + 14.4 184+46.5

24 48 72

0,4

48.0_+ 1.83 61.6 4-_4.93 55.74-4.37

057Z35.1 440 4- 27.2 ,~444-18.6

609+35.1 378 4-27.6 a 288+_ 19.1 a

24 q8 72

4.0

319 4-25.7 128 _+10.1 84.0-+ 12.9

11894-5.86 816-+4'1.3 676_+ 34.7

8704-_26A 688-+ 50.3 a 592_+ 37.0 a

ATP (IG0 ttM)

a Response to ATP lower than when cells were preincubated with IL-i for only 24 h, ? < 0.001.

Change due to ATP

TABLE I11 Effect o f the concentration o f rhlL-l[3 on the enhancement of the response o f Imman articular chondro~.tes in monolayer cultun~ to extracelhdar ATP

Cells were preincubated with various concentrations of IL-I for 24 h and were then incubated in the presence or absence of ATP (100 /zM). IL-I was included in the medium during the incubation-+ ATP. PGE released into the medium was measured as described in Experimental Procedures. Experimental results are presented as the me~n -+S.E. (n = 4). IL-I (U/ml)

pg PGE/Itg protein per 4 h

0.04 0.4 4.0

6.61+_i.79 47.5_+9.19 13.4 -+2.13 86.8-+7.21 50.6 -+4.94 210 -+9.03

No ATP

ATP (190 pM)

Change due to ATP 40.9_+ 9.36 73.4-+ 7.31 a 159 +10.3 bx

Response to ATP greater than that in the presence of 0.04 U/ml IL- i, P < 0.05. b Response to ATP greater than that in the presence of 0.04 U/ml ILol, P < 0.001. ¢ Response to ATP greater than that in the presence of 0.4 U/ml IL-I, P < 0.01.

A n t i - P G E w a s p u r c h a s e d f r o m Steranti R e s e a r c h , St Albans, U K . All o t h e r chemicals w e r e o b t a i n e d f r o m Merck, Poole, U K a n d w e r e o f A R g r a d e o r o f the ~,~ighest purity available. Results In the majority o f the studies, A T P was u s e d at 100 p M to induce the p r o d u c t i o n o f P G E , since this conc e n t r a t i o n h a d b e e n observed t.o b e maximally effective in earlier studies [8]. T h e p r o d u c t i o n o f P G E was increased consistently b y 100 a M A T P in t h e experim e n t s desen'bed here, b u t t h e response v a r i e d f r o m

1.7-fold to 9.7-fold, d e p e n d i n g o n t h e cell culture. In a c c o r d a n c e with earlier w o r k [14-17], rhlL-1/3 also increased the p r o d u c t i o n o f P G E in these studies a n d the m i n i m u m effective c o n c e n t r a t i o n a p p e a r e d to b e in the r a n g e o f 0.4 U / m L W h e n the two a g e n t s w e r e tested together, the increase in the p r o d u c t i o n o f P G E p r o m o t e d by A T P was significantly e n h a n c e d by the addition o,r rhlL-1/3 to the incubation m e d i u m a t a c o n c e n t r a t i o n o f 0.4 o r 4 U / m l , b u t n o t w h e n the c o n c e n t r a t i o n was 0.04 U / n i l (Table I). A d d i t i o n a l p r e i n c u b a t i o n o f the cells with rhIL-1/] for 24 h f u r t h e r increased the response to A T P w h e n t h e c o n c e n t r a t i o n o f the cytokine w a s 4 U / m l , h u t the two lower c o n c e n trations h a d n o f u r t h e r effect (Table 1). O n extension o f the p r e i n c u b a t i o n p e r i o d b e y o n d 24 h, t h e e n h a n c e m e n t o f the response to A T P by 0.4 a n d 4 U / n i l r h I L - 1 / / : e n d e d to decline (Table ID. This decline w a s statistically significant in two o u t o f t h r e e experiments. I n a third experiment, a d o w n w a r d t r e n d was a p p a r e n t , b u t did not r e a c h statistical significance ( d a t a not shown). T h e r e was little c h a n g e in th~ response to A T P w h e n the cells w e r e p r e i n c u b a t e d with 0.04 U / m l rhlL-1/3 for m o r e t h a n 24 h (Table ID, which in c o n j u n c t i o n with the results p r e s e n t e d in T a b l e I suggests t h a t the cells d o not r e s p o n d to this c o n c e n t r a t i o n o f the cytoldne. A s the results p r e s e n t e d in T a b l e s I a n d il indicated t h a t 24 h p r e i n c u b a t i o n plus addition o f the cytokine to the inct.t~ation m e d i u m i n d u c e d maxim a l e n h a n c e m e n t o f the response to A T P , the t h r e e c o n c e n t r a t i o n s o f rhlL-1/3 w e r e t h e n c o m p a r e d u n d e r these conditions o n the s a m e multiwell plate. T h i s c o n f i r m e d t h a t the e n h a n c e m e n t o f t h e response to A T P w a s d o s e - d e p e n d e n t (Table liD. Finally, it w a s d e m o n s t r a t e d t h a t following p r e i n c u b a t i o n for 24 h with rhlL-1/3 at a c o n c e n t r a t i o n o f 0.4 o r 4 U / m l , t h e p r o d u c t i o n o f P G E i n d u c e d by A T P w a s similar irre-

TABLE IV Effect of remocal of rML-ll3 prior to incubation ~th ATP on the enhancement a f the response of human articular chondrocytes in monola)~er culture to ¢~traeellular A TP

On each tissue culture plate, r¢,'ogloups of wells were preincuhated with IL-I for 24 h. Cells were then incubated in the presence or absence of ATP (100 pM). IL-! was included in the medium during the incubation-+ATP in one group of wells that had been preiaeabated with the cytokine. Each concentration of IL-I was tested on a separate multiwell plate. PGE released into the medium was measured as described in Experimental Procedures. Experimental results are presented as the mean_+S.E. (n = 4). Preim'ubation (24 h)

Coineabation (4 h)

IL-I (U/ml)

pg P G E / # g protein per 4 h No ATP ATP (100 #M)

No Yes Yes

No No Yes

0.4 0.4

12.3 +2.38 11.7 +0.91 15.2 _+4.66

21.8+ 2.82 45,3_+ 7.30 50.5+ 6.07

No Yes yes

No No Yes

4.0 4.0

6.41-+2.95 34.0 _+3.14 57.,1 -+5.61

18.4_+ 2.35 144 _+12,3 173 -+16.0

Response to ATP greater than that in the absence of IL-I, P < 0.05. Response to ATP greater than that in the absence of IL-I, P < 0.001.

Change due to A'I'P 9.50+ 3.68 33.6 :l: 7.36 a 35.3 -+ 7.65 a 12.0 -4- 3.77 110 +12.7 b 116 _4-1"].0b

56 spective (~f whether the cytoldne was also added to the incubation medium (Table IV). Responsiveness to low concentrations of A T P following treatment of the cells with rhlL-1/~ was also evaluated. A s noted ezrlier, it had been observed that the lowest concentration of A T P that elicited an increase in the production of P G E was in the range 1-5 /zM [8]. This value varied somewhat between cultures, p o s s ~ l y reflecting differences in their ability to metabolise extracellular ATP. Cells were therefore incubated with A T P concentrations in the range 0 - 2 5 /zM following p r e t r e a t m e a t with rhlL-1/3 at a concentration of 0.4 or 4 U / m l . The cytokine was also a d d e d to the incubation medium. In two out of three experiments, the higher concentration of rhlL-1/3 decreased the m i n i m u m concentration of A T P required to elicit a response whereas the lower concentration was incffecti,~e (Fig. 1). Interestingly, altho,,,gh the threshold concentration o f A T P was also reduced in a third e~periment, on this occasion only the lower concentration of rhlL-1/3 was effective (data not shown).

these concentrations was effective in inducing the latter response o n each occasion. It would appear, therefore, that there is some variation between cultures in sensitivity to IL-1/3. The apparent lowering of the minimum concentration of extracellular ATP required to elicit an increase in the production of P G E may result merely from the increased responsiveness to A T P induced by the cytokine. However, the shape of the dose-response curves in the presence and absence of a n effective concentration of IL-1/3 suggests increased sensitivity of the cells to extracellalar ATP. There are several mechanisms whereby responsiveness of h u m a n art3~Jlar chundrocytes to extracellular A T P could be modulated by 1L-l/3. Firstly, the cytokine could have reduced the metabolism of A T P at the cell surface. This would decrease the lowest concentration of extracellular A T P required to elicit an increase in the production of PGE, but would not readily account for the increased response to a maximally effective concentration. Moreover, we have shown that low concentrations of extracellular ~xTP ( < 12.5 / t M ) are probably catabofised at the surface of human articular chondrocytes by the action of ecto-nucleoside triphosphate pyrophosphatase [24] and results from preliminary studies have indicated that exposure to IL-I,8 for at least 48 h is required to induce a reduction in the activity of this enzyme (unpublished data). Secondly, IL-1/3 could have altered ~he n u m b e r of P2-PUrinoceptors a n d / o r their affinity for ATP. A n increase in receptor n u m b e r would account for the enhanced responsiveness to a maximally effective concentration of extracellular ATP, whereas increased

Discussion These data demonstrate a synergistic interaction between IL-I/3 and a ma.,dmally effective concentration of A T P in the stimulation of the production of P G E by human articular chundrocytes in monolaycr culture. In addition, there was a reduction in the lowest concentration of A T P required to elicit an increase in the production of PGE. The former response was induced by both 0.4 and 4 U / m l I f - l / 3 , whereas only one of

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Fig. I. Effect of rhlL-l,B on the response of human articular chondrocytes in monolayt;rculture to low concentrations of extraccllular ATP. Cells were preincubated with IL-I for 24 h and were then incubated with a range of concentrations of ATP (0-?.5 p.M). li~l was included in the medium during the incubation +_ATP. Each concentration of IL-I was tested on a separate muhiwell plate. PGE released into the mediun, was measured as described in "ExperimentalProcedures. Results are presented as the mean±S.E. (n=4). Values ol" P represent a comparison of responses in the presence and absence of ATP: *P < 0.05, **P < 0.01, ***P < 0.O01.

affinity of the receptors for ATP would reduce the minimum concentration required to elicit a response. Ligand-binding studies will be required to assess whether either of these changes occurs, but at present there does not appear to be a suitable radiolabelled P2-purinoceptor agonist or antagonist for studies with human articular chondrocytes. [3H]Adenosine 5'(a,~-methylene)triphosphate and adenosine 5'-0-2thio[35S]diphosphate have been used as radioligands in studies of agonist binding at purinoceptors of the P2~and P2fsubelasses, respectively, in other tissues [25,26]. However, studies to date using analogues of ATP have suggested that the putative P2-purinoceptor in human articular cbondrocytes does not belong to either of these subclasses [8]. Thirdly, IL-1/] may have modulated events subsequent to the interaction of extracellular ATP with its receptor. The observed sy~.~rgism supports the view that IL-1/3 and ATP activate separate siguall~ng pathways in human articuler chondrocytes. As noted in the introduction, extracellular ATP may promote production of PGE via activation of phospholipase A 2 resulting from an increase in [Ca2+]i. IL-/3, on the other hand, is unlikely to induce changes in [Ca2+]i in these cells [27,28], but it has been shown to increase the synthesis of phospholipase A 2 in rabbit articular chondrocytes [13,29,30]. Clearly, the presence of increased icvels of this enzyme which couM then be activated by a signal resulting from the interaction of ATP with its receptor, e.g., calcium, would ac~count for the observed synergism. Moreover, a similar mechanism would account for the enhanced responsiveness to histamine induced by IL-I, noted in the introduction. However, the rapid development oftbe interaction between IL-1/$ and c~.~,ll'-'-l~r ATP, plus the apparent reduction in the minimum concentration of ATP required to elicit a reponse, suggest the involvement of additional changes. The experimental observations described here raise the question of whether other cytokines also influence the stimulation of the production of PGE by human articular chondroeytes by extraceilular ATP. Of the cytokines that may modulate the metabolism of cartilage, tumour necrosis factor a (TNFa) is of particular interest, since like IL-I, this cytokine also increases the production of PGE by human articular chondrocytes [16,31], promotes cartilage resorption [31-34] and inhibits the synthesis of proteoglycans [33,35,36]. We are currently studying the effects of T N F a and our preliminary results indicate that this cytokine does enhance the production of PGE by human articular chondrocytes induced by a maximally effectk,e concentration of extracellular ATP, but the minimum concentration of ATP required to elicit a response appears to be unchanged (unpublished data). It is also possible that other biological responses in articular and fibroeartilage may be modulated by interactions between ATP

and cytokines. We have recently demonstrated that extracellular ATP stimulates the resorption of cultured explants of bovine nasal cartilage by a mechanism that does not involve PGE [37] and are currently studying the effect of both 1L-l/3 and T N F a on this response. Our results to date indicate that both cytokines interact with ATP in inducing this response, although relattvely high concentrations of IL-1/~ are required (unpublished data). Overall, there is evidence to suggest that responses mediated by P2-porinoeeptors in articular and fihrocartilage may be generally controlled by cytokines and that a range of cytoldnes may be effective. Clearly, further studies to identify the responses involved and the cytokines that can interact with extracellular ATP should prove fruitful. Interactions between cytokines and extracellular ATP in the regulation of the metabolism of articular cartilage could be of considerable importance in understanding the pathogenesis of some forms of arthritis. The mechanisms whereby extracellular ATP arises in articular cartilage have yet to be defined, but as this tissue is not innervated it is unlikely that significant amounts will be derived from nerve endings. It is highly probable, however, that ATP will be released from articular chm, drocytes that have been damaged as a result of cheraical or mechanical stimuli. Thus, if cartilage is wea~:e.ned as a result of resorption induced by cytokiiies, ceils may become increasingly susceptible to mechanical damage leading to release of ATP and a consequent enhancement of matrix loss. Similarly, if ATP is released and induces cartilage resorption, the breakdown products could then trigger an inflammatory response and the associated production of cytokines. Clearly, these areas warrant further investigation. Acknowledgements We are grateful to the Wellcome Trust, UK, for financial support for this work, to Glaxo Group Research, UK, for their generous gift of rhlL-1/3 and to Mr. R J . Newman and his colleagues at St. James's University Hospital, Leeds for the provision of human tissue. R~n~

1 Gordon,J.L. (1986) BMchem.J. 233, 309-319. 2 Burnstock,G. (1978) in Cell Membrat~eReceptowfor Drugsand Hormones(Straub, R.W.and Bolls,L., eds.),pp. 107-118,Raven Press, New York. 3 Brown, C.M. and Burnstock,G. (1981) Eur. J. Pharmacol.69, 81-86. 4 Forslmrg, EJ., Feuerstein, G., Shohami, E. and Pollard, H.B. (1987) Proc. Natl. Acad. Sci. USA 84, 5630-5634. 5 Needleman,P., Minkes,M.S. and Douglas,J.R. (1974)Cire. Res. 34, 455-460.

6 Pearson, J.D, Slakey, L L and Gordon, J.L (i983) Biochem. J. 214, 273-276. 7 Pfeilschifter, J . Thuring. 13. and Festa, F. (1989) Eur. J. Biochem. 186, 509-513. 8 CaswelL A.M, Leong. W.S. and Russell, R.G.G. (199D Biochim. Binph~. A~ta 1074.151-158. 9 Arkhammar. P_ Hallberg, A_ Kindmark, H., Nilsson. T_ Rotsman, P. and Berggren, p . o . (1990) Biochem. J. 265, 203-211. 10 Benham, C.D. and Tsien, ILW. (1987) Nature 328. 275-278. i l Cowen, D.S. Lazarus, H.M. Shurin, S.B. Stoll, S.E. and Dnbyak. G.R. (1989) J. Chn. invesL 83, 1651-1660. 12 Halla~. TJ. and Pearson. J.D. (1986) FEBS Lett. 207, 95-99. 13 Gilman, S.C. and Chang. J. (1990)J. Rheumatol. 17, 1392-1396. 14 Bunning. R.A.D, Van Damme, J , Richardmn, H J . Hughes, D.E., Opdonakker, G , Biliau, A. and Russell R.G.G. (]986) Bh3chem. ~ . Res. Commun. 139, il50-:157. 15 Bunning, R.A.D, RusselL R.G.G. and Vaa Damme. J. (1990) Biochem. Bio~hys. Res. Commun. 166, |163-1170. I6 Campbell I.K., Piccoli, D.S. and Hamilton. .LA. (1990) Biochim. Biophys. Acta 1051. 310-318. 17 Meats, J.E., McGuire, M.B. and Russell R.G.G. (1980) Nature 286, 891-892. 18 Taylor, DJ. and W0olley. D.E. (1987) Ann. Rheum. Dis. 46, 431--435. 19 Schwartz, J.-C. (1979) Life Sci. 25, 895-912. 20 Caswell, A.M. Leong. W.S. and Russell. R.G.G. (1990) Br. J. RheumatoL 29, AI. 17. 21 ~ e l l . A.M. and Russell R.G.G. (1988) Biochim. Biophys. Acta 966, 310-317. 22 Srivastava, V.M.L, Malemud, CJ. and Sokoioff. L (1974) Connect. Ti~ue Res. 2,127-136.

23 Lov,,-y, O.H., Rosebrough, NJ.. Farr, A.L and Randall, RJ. (1951) J. Biol. Chem. 193, 265-_-'275. 2,* Cao~,velL A.M. and Russell R.G.G. (1985) Biochim. Bi~,phys. Acia 847, 40-47. 25 13o. X. and Burnstoek. G. (1989) J. Auton. Nun1. Syst. 28. 85-88. 26 Cooper, C.l~ Morris. AJ. and Harden. T.K. (1989) J. Biol. Chem. 264, 6202-6206. 27 PacificL R_ CKixellL R-, Rifas. I-, Halstead. L and Avioli. LV. (1988) J. Bone Mirror. Res. 3.107-111. 28 Sung. K.. Mendoiow. D . Georgcscu. H.I. and Evans. C.H. (1988) Biochim. Biophys. Acta 971. 148-156. 29 L~'ons-Giorda:to, B_ Davis. G.L, Galbraith, W., Pratta. M.A. and Arner, E.C. (1989} Biochem. Biophys. Res. Commun, 164, 488495. 30 Stes~ns, T.M, Chin, J.E., McGo-.~n, M, Giannaras, J. and Kerr. J.S. (1989) Agents Actions 27, 385-387. 31 Banning. R-A.D. and Russell. R.G.G. (1989) Arthritis Rheum. 32. 780-784. 32 Campbell. I.K_, Pia:olL D.S. Robert.~, M.J~ Muirden, ICD. and Hamilton, J-A. (1990) Arthritis Rheum. 33. 542-552. 33 Pratta, M.A., Di Meo, T.M. RuhL D.M_ and Arner, E.C. (1989) Agents Actions L~'7.250-253. 34 Smith. RJ~ Chin. J.E.. Sam. L.M. and Jnsten, J.M. (1991) Arthritis Rheum. 34, 78-83. 35 Tyler, J.A. (1985) Biochem. J. 227, 869-878. 36 Koh'bas. LM. and Goldberg. R.i_ (1959) Agents Actions 27, 245-249. 37 Leong. W.S~ RusselL ILG.G. and C~,w.'e~LA~M.(1990) Biochem. So~ Trans. 18. 951-95~