Enzymatic activity and distribution of phospholipase A2 in human cartilage

Life Sciences, Vol. 48, pp. 2457-2462 Printed in the U.S.A.

ENZYMATIC

Pergamon Press

ACTIVITY AND DISTRIBUTION OF P H O S P H O L I P A S E IN HUMAN CARTILAGE.

W. Pruzanski,

E. Bogoch,

E. Stefanski,

A2

M. Wloch and P. Vadas

I n f l a m m a t i o n Research Group, Departments of M e d i c i n e and Surgery, The W e l l e s l e y Hospital, University of Toronto, Ontario, Canada (Received in final form April 12, 1991) SUMMARY Extracellular phospholipase A2 (PLA2) with .proi n f l a m m a t o r y activity has recently been a l s c o v e r e a in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human s y n o v i u m and articular cartilage were found to contain large q u a n t i t i e s of the enzyme. In rheumatoid arthritis (RA), PLA 2 activity in synovium, superficial and deep layers of articular cartilage was 20± 14 (SEM), 168± 62 and 533±176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49±11, 569±109 and 1709±243 nmol/min/mg protein, all s i g n i f i c a n t l y higher (p<.01) than in RA. Nasal septal c a r t i l a g e c o n t a i n e d much less PLA2, 19±5.6. PLA 2 in h u m a n a r t i c u l a r and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA 2 c o n c e n t r a t i o n in articular cartilage may imply that, at least in part, cartilage is the source of PLA 2 in the joint space. Since RA cartilage and synovium have less PLA 2 activity than the corresponding OA tissues, additional sources of PLA 2 in RA synovial fluids are implicated. I n f l a m m a t i o n occurs to a variable extent in most types of arthritis. W h e r e a s in some arthritides inflammation is an early and p r o m i n e n t process that leads to the d e s t r u c t i o n of articular s t r u c t u r e s (i), in the others, milder and less p e r s i s t e n t bursts of i n f l a m m a t o r y activity complicate d e g e n e r a t i v e changes (2). Often, inflammation involves several articular tissues which interact in a complex manner (3). The etiologic agents of inflammation have not been elucidated, however several pathogenic factors including i n t e r l e u k i n - i (IL-I), tumor necrosis factor (TNF), eicosanoids, and toxic oxygen radicals have been identified (4,5). The a r a c h i d o n i c acid cascade plays an important role in i n f l a m m a t i o n (6). H i g h activity of phospholipase A 2 (PLA2) , the enzyme that initiates that cascade, has recently been found in the synovial fluids in inflamed joints (7,8) and in the sera (9) of patients w i t h active arthritis. It has also been shown that injection of soluble e x t r a c e l l u l a r PLA 2 induces inflammatory changes in the skin (i0) joints (ii) and paws (12) of experimental animals and 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc

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that serum PLA 2 levels correlate to the intensity of inflammation in r h e u m a t o i d and juvenile chronic arthritis (9,13). A l t h o u g h the p r o i n f l a m m a t o r y activity of soluble PLA 2 has been well documented, its sources have not been defined. PLA 2 was found to be synthesized and released from human synoviocytes (14,15), rat chondrocytes. Synoviumwasfound to contain modest q u a n t i t i e s of PLA 2 (20) and its mRNA transcript (21). We and others have reported in preliminary form that human a r t i c u l a r cartilage contains soluble PLA 2 (22,23). Herein, we report that human articular, but not nasal, cartilage has very high PLA 2 activity, by far exceeding other known articular sources of this enzyme and that there are differences in the c o n c e n t r a t i o n of cartilage-derived PLA 2 in r h e u m a t o i d versus osteoarthritis. Materials

and Methods

Fresh cartilage was obtained from rheumatoid and o s t e o a r t h r i t i c patients at the time of joint surgery or from p a t i e n t s u n d e r g o i n g nasal septoplasty. Fibrous and synovial tissues were dissected free and cartilage was w a s h e d twice with Hanks' b a l a n c e d salt solution (HBSS) without Ca 2+ and Mg 2+. One mm thick superficial layer of the articular cartilage was d e t a c h e d and cut into 1-2 mm fragments. The deep layer of c a r t i l a g e (1-2 mm thick) was detached and similarly prepared. The fragments of articular and nasal septal cartilage were d i g e s t e d s e q u e n t i a l l y with 0.25 Bactotrypsin (Difco) at 37 ° C for 30 min. and with 0.2% w/v solution of collagenase IA-S (Sigma) at 37 ° C for 90 min. The digests were washed 3 times in HBSS, r e s u s p e n d e d in the same buffer, homogenized on ice in a Brinkmann h o m o g e n i z e r for 3 min., and then sonicated w i t h a Vibracell s o n i c a t o r for 5 min and centrifuged at 15000 g for 60 min at 4 ° C. The supernatants were aspirated and tested (in triplicate) for PLA 2 activity. Fresh synovial tissue obtained at the time of joint surgery was washed several times in HBSS w i t h o u t Ca 2+ or Mg 2+ after removal of fat and connective tissue. After weighing, the tissue was minced, digested sequentially as d e s c r i b e d above, c e n t r i f u g e d at 300g for 10 min at 24°C, washed twice w i t h HBSS, h o m o g e n i z e d on ice and sonicated. The sonioates were spun at 2300 g for 60 min at 4 ° C, and the supernatants were tested in t r i p l i c a t e for PLA 2 activity. One unit of PLA~ activity is d e f i n e d as the hydrolysis of 1% of the total E. coli p h o s p h o l i p i d (corresponding to 0.056 nmoles of E. coli p h o s p h o l i p i d substrate) for 30 min at 37 ° C. For partial purification of PLA2, the sonicate supernatants w e r e adjusted to pH 5.0 with 0.i N HC1. The supernatants were a p p l i e d to C M S e p h a d e x C50 column equilibrated with 0.1 M acetate buffer, pH 5.0. Elution was carried out at 4 ° C s e q u e n t i a l l y w i t h 0.i M acetate buffer pH 5.0; 0.5 M acetate buffer, pH 5.0 and 2M NaCl in 0.2 M Tris HCI buffer, pH 8.5. The eluates were a s s a y e d in triplicate for protein concentration using Bio-Rad p r o t e i n assay reagent (Bio-Rad, Richmond, VA) and for PLA 2 activity. The active fractions were pooled and c o n c e n t r a t e d in 0% polyvinylpyrrolidone. PLA 2 activity was tested at various c o n c e n t r a t i o n s of Ca 2+ EDTA, NaCl and Tris buffer and for pH d e p e n d e n c e as described in detail (24). Enzymatic h y d r o l y s i s of 1 - p a l m i t o y l - 2 - (14C) - p a l m i t o y l - 3 - p h o s p h a t i d y l c h o l i n e by PLA 2 was

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Phospholipase A 2 in Human Cartilage

d e t e r m i n e d as described (25). Statistical analysis was done c o r r e l a t i o n coefficient test.

using

Student's

t

2459

test

and

Results P h o s p h o l i p a s e A 2 activity was assessed in superficial and deep layers of cartilage and in synovial tissue in o s t e o a r t h r i t i s and r h e u m a t o i d arthritis, and in nasal septal c a r t i l a g e (Table i). The specific activity of PLA 2 was lowest in nasal septal c a r t i l a g e and highest in osteoarthritic articular cartilage. Deep layers of cartilage invariably contained more PLA 2 than the s u p e r f i c i a l ones. Superficial and deep layers of OA cartilage, as well as OA synovium all contained significantly h i g h e r PLA2 a c t i v i t y than the corresponding constituents from r h e u m a t o i d joints. Comparison of PLA.2 activity in grossly d a m a g e d vs g r o s s l y normal cartilage in the same OA joint showed that superficial layers had similar PLA 2 activities, w h e r e a s PLA 2 specific activity in deep cartilage was higher in g r o s s l y abnormal areas (data not shown). PLA 2 activities in sonicates of superficial and deep OA and RA c a r t i l a g e and in nasal septal cartilage were c h a r a c t e r i z e d w i t h r e s p e c t to the effects of pH, calcium requirements, and effect of NaCI concentration and Tris buffer. All p r e p a r a t i o n s of a r t i c u l a r cartilage behaved identically. The o p t i m u m PLA 2 a c t i v i t y was found at pH 7.0 - 7.5, 7 mM Ca 2+, 50 - i00 mM NaCI and i00 m M T r i s HCI buffer. No further enhancement was observed in 200-500 mM Tris HCI buffer. PLA 2 activity was u n d e t e c t a b l e in the presence of 2 mM EDTA. Nasal cartilage PLA 2 showed identical behaviour, except that only 66%'ioss of a c t i v i t y was noted in 2 mM EDTA. Examination of the enzymatic a c t i v i t y of OA and RA cartilage PLA 2 using synthetic substrate showed sn-2 specificity. (Table 2) TABLE 1 ACTIVITY

OF PHOSPHOLIPASE

Tissue

n

A 2 IN A R T I C U L A R

TISSUES

Phospholipase A 2 Range Mean±SEM nmoles/min/mq protein

R h e u m a t o i d Arthritis Synovium s.cartilage + d. cartilage +

23 9 9

3-55 14-608 31-1569

20±14 168±62 533±176

0steoarthritis Synovium s.cartilage d. c a r t i l a g e

27 26 26

5-268 33-2765 191-4257

49±11 569±109 1709±243

14

3-82

19±5.6

Nasal

seDtal

+ s - superficial,

d - deep.

The significant differences were: superficial OA vs s u p e r f i c i a l RA cartilage p< 0.005; deep OA vs RA c a r t i l a g e p< 0.001; OA deep vs. superficial, p<0.01; RA deep vs s u p e r f i c i a l , < p 0.05; s y n o v i u m O A v s RA p< 0.02.

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A2

in Human Cartilage

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TABLE 2 ENZYMATIC

HYDROLYSIS

ENZYME

OF 1-PALMITOy~-2-( 14 C ) - P A L M I T O Y L - 3 PHOSPHATIDYLCHOLINE FRACTION OF TOTAL P R O D U C T LYSO-PC

BUFFER

CROTALUS V E N O M PLA 2 O S T E O A R T H R I T I C CARTILAGE PLA 2 R H E U M A T O I D CARTILAGE PLA 2

FATTY ACIDS

(%)

(%)

27.8 3.5 4.1 9.5

72.2 96.5 95.9 90.5

Discussion E x t r a c e l l u l a r phospholipase A 2 has recently been p u r i f i e d from synovial fluid and sequenced (27,28,29). Since its serum level c o r r e l a t e d to disease activity in adult r h e u m a t o i d and juvenile arthritis (9,13) and intraarticular injection of p u r i f i e d PLA 2 caused marked synovitis in experimental animals (Ii), it was concluded that the soluble PLA 2 in synovial fluid plays an important role in the inflammatory process in arthritis

(30).

Several sources of PLA 2 that could potentially contribute to the accumulation of this enzyme in the joint space have been identified. Human synovial tissue (19) and cultured synovial cells (14,15) have been found to synthesize and release PLA 2 to the medium, yet the rate of synthesis and release was variable and often low. Cytokine-stimulated fetal rat calvarial o s t e o b l a s t s were also found to release e x t r a c e l l u l a r PLA 2 (16). Rabbit, rat and human chondrocytes have been found to secrete PLA 2 (18,19,31,32,33,34,35). IL-I and T N F - s t i m u l a t e d rabbit and rat chondrocytes exhibited increased intracellular and e x t r a c e l l u l a r PLA 2 activities (32,33,35). A number of cartilage-derived enzymes have long been objects of intense interest (36,37,38). Cathepsin D has been identified in human articular cartilage (36,37,38). It was noted that o s t e o a r t h r i t i c cartilage contains more cathepsin D than normal cartilage and that in the individual osteoarthritic joint, d e g e n e r a t e d areas express more cathepsin D, acid and alkaline phosphatase activities than grossly normal areas (38,39). R h e u m a t o i d and osteoarthritic articular cartilage was also found to c o n t a i n and release collagenase and proteoglycanase. Release of these enzymes from cartilage was significantly enhanced b y I L 1 and s u p p r e s s e d by dexamethasone (40). I L - l - s t i m u l a t e d release of g l y c o s a m i n o g l y c a n a s e from the explants of bovine nasal cartilage was blocked by cycloheximide. T h e r e f o r e enzymatic p r o c e s s e s in the cartilage are dependent upon active p r o t e i n synthesis (41,42). Our study shows that human articular cartilage contains large q u a n t i t i e s of calcium dependent, neutral active PLA 2. P r e l i m i n a r y characterization showed that this PLA 2 is similar, if not identical, to synovial fluid PLA 2- This would confirm and extend the observation that rabbit articular cartilage

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t r a n s c r i b e s a gene encoding for PLA 2 of the synovial fluid type (43) Interestingly, articular cartilage c o n t a i n e d s u b s t a n t i a l l y h i g h e r specific activities of PLA 2 than nasal septal cartilage. Since nasal septal PLA 2 was only partly inhibited by EDTA, the p r e s e n c e of both calcium dependent and calcium independent PLA 2 in the nasal septal cartilage cannot be ruled out. Comparative studies have shown that OA cartilage contained s i g n i f i c a n t l y more PLA 2 than RA cartilage. This was noted in both superficial and deep layers of cartilage. Since erosive changes in the s u p e r f i c i a l layers of cartilage may lead to the leak or d i f f u s i o n of low m o l e c u l a r weight proteins such as PLA2, it is of interest that OA superficial layers had, in fact, higher PLA 2 content than c o r r e s p o n d i n g RA cartilage. These observations suggest that there is either lower synthesis and/or facilitated leakage of PLA 2 from R A c a r t i l a g e thereby contributing to higher R A s y n o v i a l fluid levels of PLA 2. The relative contribution of cartilagea s s o c i a t e d PLA 2 to articular inflammation is not known, but it may be an important source of the p r o i n f l a m m a t o r y enzyme in the joint space. Acknowledgment S u p p o r t e d by the Council of Canada

Arthritis

Society

and

the

Medical

Research

References i.

5. 6. 7.

E.D. HARRIS, JR., Textbook of Rheumatoloqy, W.N. Keley, E.D. Harris, Jr., S. Ruddy, C.B. Sledge, (eds), 886-915, W.B. Saunders Co. Philadelphia (1985). K.D. BRANDT, Textbook of Rheumatoloav, W.N. Keley, E.D. Harris, Jr., S. Ruddy, C.B. Sledge, (eds), 1432-1448, W.B. Saunders Co., Philadelphia, (1985). J.J. STEINBERG, J.R. HUBBARD, C.B. SLEDGE, Adv. Inflamm Res 1_!1215-241 (1986). P.L. KILIAN, I.G. OTTERNESS, Therapeutic a D D r o a c h e s to i n f l a m m a t o r y diseases. A.J.Lewis, N.S. Doherty, N.R. Ackerman, (eds), 14-16, Elsevier, New York, (1989). W.P. AREND, J-M.DAYER, Arth. Rheum. 3__33305-315 (1990). J.T O'FLAHERTY, Lab. Invest. 4_/7314-329 (1982). P. VADAS, W. PRUZANSKI, E. STEFANSKI, Life Sci. 3 6 5 7 9 - 5 8 7

8.

W. PRUZANSKI,

2. 3. 4.

(1985). P. VADAS,

E STEFANSKI,

J. Rheumatol.

1_/2211-

216 ( 1 9 8 5 ) . 9. i0. ii. 12.

W. PRUZANSKI, E.C. KEYSTONE, C. BOMBARDIER, K. SNOW, B. STERNBY, P. VADAS, J. Rheumatol. 15 1351-1355 (1988). P. VADAS, W. PRUZANSKI, V. FORNASIER, J. Invest. Dermatol. 8-6380-383 (1986). P. VADAS, W. PRUZANSKI, J. KIM, V. FORNASIER, Am. J. Pathol. 1 3 4 8 0 7 - 8 1 1 (1989). B.S.VISHWANATH, A.A. FAWZY, R.C. FRANSON, I n f l a m m a t i o n 12

549-561 13.

(1988).

E.SILVERMAN, E.STEFANSKI,

W.

P.

PRUZANSKI,

VADAS,

Arthr.

R.

LAXER,

Rheum.

K. ALBIN-COOK, 3__00 (Suppl) S-127

(1987). 14. 15.

S.C. GILMAN, E. MOCHAN, J. UHL, P. ZEIGLER, J. CHANG, Arthr. Rheum. 3 1 1 2 6 - 1 3 0 (1988). P. VADAS, W.PRUZANSKI, J. KIM, H. JACOBS, E. STEFANSKI, Arthr. Rheum. 28, $90 (1985).

2462

16.

Phospholipase A 2 in Human Cartilage

Vol. 48, No. 25, 1991

33.

P. VADAS, W. PRUZANSKI, A. SOS, A, MELCHER, H. JACOBS, T.CHEONG, Arthr. Rheum. 30 (Suppl) S-65 (1985). P. VADAS, W. PRUZANSKI, Lab Invest. 55 391-404 (1986). S.C. GILMAN. J. Rheumatol.14 1002-1007 (1987). W. PRUZANSKI, E, BOGOCH, E. STEFANSKI, M. WLOCH, P. VADAS, J. Rheumatol. 17 1386-1391 (1990). W. PRUZANSKI, Personal observation. J. SEILHAMER, P. VADAS, W. PRUZANSKI, S. PLANT, E. STEFANSKI, L. JOHNSON, Therapeutic approaches to i n f l a m m a t o r y diseases. A.J. Lewis, N.S. Doherty, N.R. A c k e r m a n (eds), 129-136, Elsevier, New York (1989). W.PRUZANSKI, E.BOGOCH, E. STEFANSKI, P. VADAS, Bull. Soc. Clinic Biol. 5_/3 49 (1988). E. VIGNON, P. MATHIEU, P. LOUISOT, J. VILAMITJANA, M.F. HARMAND, M. RICHARD, J. Rheumatol (6) (Suppl 18) 35-38, 1989. E.STEFANSKI, W. PRUZANSKI, B. STERNBY, P. VADAS, J. Biochem. I00 1297-1303 (1986) P. VADAS, W. PRUZANSKI, E. STEFANSKI, B. STERNBY, I n f l a m m a t i o n 14 173-183 (1990). F.E. CROXTON, Dover Publications Inc. New York (1959). J.J. SEILHAMER, W. PRUZANSKI, P. VADAS, S. PLANT, J.A. MILLER, J.KLOSS, L.K. JOHNSON, J. Biol. Chem. 264 53355338 (1989). R.M. KRAMER, C.HESSION, B. JOHANSEN, G. HAYES, P. MCGRAY, E.P.CHOW, R. TIZARD, E.B. PEPINSKY, J. Biol. Chem. 264 5768-5775 ( 1989). R.L. HEINRIKSON, E,T, KRUEGER, P.S. KEIM, J. Biol. Chem. 252 4913-4921 (1977). W. PRUZANSKI, P. VADAS, J. Rheumatol. 15 1601-1603 (1988). S.C. GILMAN, J. Rheumatol. 14 1002-1007 (1987). S.C. GILMAN, P.R. BERNER, J. CHANG, Agents A c t i o n s 2_!1 345347 (1987). P. SUFFYS, F. VAN ROY, W. FIERS, FEBS Letters 232 244-248

34.

J. CHANG,

17. 18. 19. 20. 21.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

(1988). 1287 35. 36. 37. 38. 39. 40. 41. 42. 43.

S.C.

GILMAN,

A.J.

LEWIS,

J. Immunol.

136 1283-

(1986).

T.M. STEVENS, J.E. CHIN, M. MCGOWAN, J. GIANNARAS, J.S.KERR, Agents Actions, 27 36-38 (1989). D.S. HOWELL, Arthr Rheum 18 167-177 (1985). A.I. SAPOLSKY, D.S. HOWELL, J.F. WOESSNER, JR., J clin. Invest. 5_/3 1044-1053 (1974). S.Y. ALI, L. EVANS, Fed. Proc. 32 1494-1498 (1973). A.I. SAPOLSKY, R.D. ALTMAN, J.F. WOESSNER, D.S. HOWELL, J. Clin. Invest 52 624-633 (1973). M. SHINMEI, T. KIKUCHI, K. MASYDA, Y. SHIMONYRA, Drugs 35 (Suppl. i) 33-41 (1988). J.R. HUBBARD, J.J. STEINBERG, M.S. BEDNAR, C.B. SLEDGE, J. Orthop. Res. 6 180-187 (1988). R.J. SMITH, N.A. ROHLOFF, L.M. SAM, J.M. JUSTEN, M.R. DEIBEL, J.C. CORNETTE, Inflammation 13 367-382 (1989). B. LYONS-GIORDANO, G.L. DAVIS, W. GALBRAITH, M.A. PRATTA, E.C. ARNER, Biochem. Biophys. Res. Comm. 164 488-495 (1989).