Detection and characterization of extracellular phospholipase A2 in pleural effusion of patients with tuberculosis

Life Sciences, Vol. 49, pp. Printed in the U.S.A.

1095-1102

Pergamon

DETECrlON AND CHARACTERIZATION OF EXTRACELLULAR PHOSPHOLIPASE A2 IN OF PATIENTS WITH TUBERCULOSIS

Press

PLEURAL EFFUSION

Suk Hwan Baekl, Kiyoshl Takayamaz, [chlro Kudo2, kelzo Inoue2, Hyun Woo Lee3, Jun Young Do3 and Hyeun Wook ChangI 1 Department of B1ochemlstry, College of Pharmacy, Yeungnam University, Gyongsan, Korea 2 Department of Health Chemistry, Faculty of Pharmaceutical Science, The Unlverslty of Tokyo, Hongo, Japan 3 Department of Internal Medicine, College of Medlclne, Yeungnam (Recelved

in flnal

Unlverslty, Gyongsan,

Korea

form July 31, 1991)

Summary Extracellular phosphollpase A2 activity has been pleural requiring

fluid

of

protein

patients

w~th t u b e r c u l o s i s

and has a pH optimum o f 10 0

This

enzyme

ldentlfled xn is

a calcium

The enzyme was i n h i b i t e d

by the active s l t e - d l r e c t e d hlstld]ne reagent, p -bromophenacyl bromide Ionic and non-lonlc detergents, or the sulfhydry] reagent d l t h l o t h r e l t o l , caused loss of enzyme actlvlty When substrate s p e c l f l c l t y was tested uslng 2-[l-14C]llno]eoyl phospholiplds as substrates, phosphatldylethanolamlne was the best substrate, followed by phosphatldy]serlne and phosphatldylchollne rhls phosphollpase Az showed hlgh a f f i n i t y for heparin, and was recognlzed by a monoclonal antlbody ralsed against phospho]ipase A2 from human synovlal fluid These flndings suggest that an extracellular phosphollpase A2, which may belong to the 14K group phosphollpase A2 family, exlsts Ln the pleural fluld of patients with tuberculosls Phospholipase A2 is an ubtquztous enzyme found in both exocrlne and endocrxne secretions (I-3). and which is ,n~o[ved in generatlon of varlous prolnflammatory chemical medlators such as elcosanolds and platelet-actlvatlng factor (PAF) (4-6) Because of the potent effect of these l,pid mediators on a variety of tissues, they have been implicated as major contributors to the process leading to inflammation and tlssue injury High levels of extracellular phosphollpase A2 associated with inflammatory processes in man and animal models have been extensively reported elsewhere (7-]0) Although the orlgln of this extracellular phosphollpase A2 has not been clarlfled, various stlmull have been shown to inltlate its release (11.26.27) Platelets (11). monocytes and polymorphonucleocytes have been reported to respond to speclflC stimuli by extrace]]u]ar release of lysosomal phosphollpase Az (12) We have characterized and purlfled extracellu]ar phosphollpase A2 from inflammatory exudate of rats (13) and from human syr,ovlal fluld (8) Pleural effusion occur in systemlc illnesses, such as systemic lupus erythematosus (SLE) and rheumatoid dlsease, and may result from injury (14) The presence 0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc

1096

PLA2

of e l c o s a n o l d s and p l e u r a l e x u d a t e In in an animal model s t u d y , we d e t e c t e d

in T u b e r c u l o s z s

Vol.

49, No.

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1991

a c u t e - p h a s e r e a c t a n t s has been o b s e r v e d in c a r r a g e e n a n - i n d u c e d a r a t model (15) We have r e c e n t l y r e p o r t e d t h a t p l e u r a l e x u d a t e showed a p p r e c i a b l e a c e t y l h y d r o l a s e a c t i v i t y (16) In t h e p r e s e n t e x t r a c e l l u l a r p h o s p h o l i p a s e ~2 in p l e u r a l e f f u s i o n o f p a t i e n t s

with t u b e r c u l o s i s M a t e r i a l s and Methods Materials sham, U k

[ 1 - 1 4 C ] L i n o l e i c a c t d and [ 1 - 1 4 C ] a c e t a t e were p u r c h a s e d from Amer-

Normal mouse Ig G was p u r c h a s e d from Sigma

Preparation

E

of Labeled S u b s t r a t e

was p r e p a r e d a s d e s c r i b e d p r e v , o u s l y (17)

Coll

[ 1 4 C ] p h o s p h a t i d y l e t h a n o l a m l n e (PE)

The s p e c i f i c a c t i v i t y

was a d j u s t e d to 300

cpm/nmol 1 - A c y l - 2 - [ 1 - 1 4 C ] l l n o l e o y l - s n - g l y c e r o - 3 - p h o s p h o c h o l i n e , 1 - a c y l - 2 - [ 1 - 1 4 C ] l l n o l e o y l - s n - g l y c e r o - 3 - p h o s p h o e t h a n o l a m t n e and 1 - a c y l - 2 - [ 1 - 1 4 C ] l i n o l e o y l - s n - g l y c e r o 3 - p h o s p h o s e r i n e were s y n t h e s i z e d a s d e s c r i b e d p r e v i o u s l y (18) Their specific a c t i v i t y was a d j u s t e d to 300 cpm/nmol by r e s p e c t i v e d i l u t i o n ~ l t h u n l a b e l e d egg y o l k phosphatldylchollne,

egg yolk

p h o s p h a t l d y l e t h a n o l a m i n e and egg y o l k

phosphatidyl-

s e r i n e p r e p a r e d from egg yolk p h o s p h a t l d y l c h o l i n e by t r a n s p h o s p h a t l d y l a t i o n (19) P r e p a r a t i o n o f Enzyme Source

T h i r t e e n t u b e r c u l o s i s p a t i e n t s were s e l e c t e d from

Yeungnam U n i v e r s i t y H o s p i t a l The c r i t e r i a f o r t h e d i a g n o s i s of e x u d a t i v e p l e u r a l e f f u s i o n were t e s t e d with b i o c h e m i c a l , b a c t e r i o l o g i c a l , c y t o l o g i c and p a t h o l o g i c studies C e i l s and d e b r i s were removed from p l e u r a l f l u i d of t h e p a t i e n t s by c e n t r l f u g a t l o n i m m e d i a t e l y a t 4°C. and then pooled and s t o r e d a t -20°C u n t i l u s e d During s t o r a g e , no s i g n i f i c a n t d e c r e a s e of p h o s p h o l l p a s e A2 a c t i v i t y was o b s e r v e d

assay of

Assay o f P h o s p h o l I p a s e A2 Fhe s t a n d a r d i n c u b a t i o n s y s t e m (250 ill) f o r t h e of p h o s p h o l i p a s e A2 c o n t a i n e d lO0 mM glyclne-NaOH (pH 10 0), 6 mM CaC12, 20 l g

protein

and 50 nmol of 2 - [ 1 - 1 4 C ] l l n o l e o y l

PE

The r e a c t i o n was c a r r i e d

out at

37°C f o r 40 mln, t h e n s t o p p e d by adding 1 25 ml of D o l e ' s r e a g e n t (20) Radioactive f r e e f a t t y a c i d s r e l e a s e d were e x t r a c t e d by the method d e s c r i b e d p r e v i o u s l y (18) Analytical Procedures P r o t e i n c o n c e n t r a t i o n was measured with a P i e r c e p r o t e i n a s s a y k i t (21) Bovine Serum ~lbumln s e r v e d as the p r o t e i n s t a n d a r d Results C e l l - f r e e f l u i d o f e x u d a t l v e p l e u r a l e f f u s i o n c o l l e c t e d from 13 t u b e r c u l o s i s p a t i e n t s was a s s a y e d f o r p h o s p h o l l p a s e a c t i v i t y a t b a s i c pH u s i n g [ 1 4 C ] a c y l - l a b e l e d

E. c o l l PE a s a s u b s t r a t e To d e m o n s t r a t e t h e p o s i t i o n a l s p e c i f i c i t y o f t h e p l e u r a l p h o s p h o l I p a s e more c o n c l u s i v e l y , pooled f l u i d was i n c u b a t e d w i t h e i t h e r 1-[1-14C] p a l m l t o y l - 2 - a c y l - s n - g l y c e r o 3-PE or 1 - a c y l - 2 - [ 1 - 1 4 C ] l l n o l e o y l - s n - g l y c e r o - 3 - P E The a n a l y s i s o f r a d i o a c t i v e p r o d u c t s by t h i n l a y e r chromatography r e v e a l e d t h e f o r m a t i o n of l y s o PE from 1 - [ 1 - 1 4 C ] p a l m . t o y [ PE or f a t t y a c i d from 2 - [ 1 - 1 4 C ] l l n o l e o y l PE ( F i g 1) These f i n d i n g s i n d i c a t e t h a t the p l e u r a l p h o s p h o l t p a s e ts of t h e "Az" t y p e When m e a s u r e d u n d e r o p t i m a l c o n d i t i o n s with v a r i o u s p h o s p h o l l p i d s d i f f e r i n g in t h e i r p o l a r head g r o u p s , PE was t h e b e s t s u b s t r a t e , followed by PS and PC with the p h o s p h o l l p a s e Az from p o s t hepar~n S e p h a r o s e CL-6B column c h r o m a t o g r a p h y , the observed specific activities were 310 (PE). 13 2 (PS) and 7 0 (PC) nmol/mln/mg protein

Vol.

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1991

PLA2 in T u b e r c u l o s i s

(A)

1097

(B)

A

Y

E:

40

20

60 mcubatton

Fig. Time

course

and

positional

20

40

60

fluid

phosphollpase

time(mm) 1

specificity

of

pleural

A2

activity. The r e a c t i o n m i x t u r e (250 #1) c o n t a i n e d 100 m~t glycine-NaOH (pH 1 0 . 0 ) , 6 mM CaC12, 40 ~g o f p r o t e i n and 50 nmol o f e i t h e r 2 - [ 1 - 1 4 C ] l l n o l e o y l PE (A) or 1 - [ 1 - 1 4 C ] p a l m t o y l PE (B). R e a c t i o n s were c a r r i e d o u t a t 37°C, r e a c t i o n p r o d u c t s were e x t r a c t e d w i t h B l i g h and D y e r ' s s o l v e n t (31) and t h e n a n a l y z e d by t h l n l a y e r c h r o m a t o g r a p h y (Merck S i l i c a 60) u s i n g a s o l v e n t s y s t e m o f c h l o r o f o r m m e t h a n o l - w a t e r (65 35 8 4) r a d i o a c t i v e l y s o PE ( 0 ) . radioactive free f a t t y aczd ( • ) .

Factors affecting

t h e p h o s p h o l , p a s e A2 a c t i v i t y

The e f f e c t of pH on h y d r o l y s i s

o f PE by t h e p l e u r a l enzyme was d e t e r m i n e d over a pH range of 3 5-10 5 The a c t i v i t y was d e t e c t e d a t b a s i c pH and showed an optimum a t pH 10 0 ( F i g 2) The p h o s p h o l i p a s e A2 a c t i v i t y showed an a b s o l u t e r e q u i r e m e n t f o r the p r e s e n c e o f Ca 2÷ ( F i g 3) Addition

of

EDTA to

maximum a c t i v i t y

the

reaction

mixture suppressed the a c t i v i t y

was o b t a i n e d a t 6 mM Ca2.

completely

The

Other d i v a l e n t c a t i o n s (6 mM Ba 2÷, Sr 2÷

and Mg2÷) were u n a b l e to r e p l a c e the Ca 2÷ ( d a t a not shown) The a c t i v i t y of t h e c r u d e enzyme was a f f e c t e d by d e t e r g e n t T h i s enzyme was f u l l y a c t t v e on PE in t h e a b s e n c e of d e t e r g e n t or in t h e p r e s e n c e of l e s s than 0 02~ sodium d e o x y c h o l a t e , b u t above t h i s c o n c e n t r a t i o n t h e a c t , v , t y d e c r e a s e d in a d o s e - d e p e n d e n t manner A d d i t i o n o f T r i t o n X-IO0 (0 O1 - 0 2~) to the r e a c t i o n m i x t u r e i n h i b i t e d t h e a c t i v i t y

(Ftg

4)

The e f f e c t of monoclonal a n t i b o d y HP-1 on the a c t i v i t y o f p l e u r a l f l u i d p h o s p h o l l p a s e A2 was examined Four monoclonal a n t i b o d ] e s with d i f f e r i n g r e a c t l v i t l e s were r a x s e d a g a i n s t human s y n o v l a l f l u i d p h o s p h o l l p a s e A2 (25) One of them, HP-I i n h i b i t e d t h e p l e u r a l f l u i d p h o s p h o l l p a s e A2 a c t l v t t y in a d o s e - d e p e n d e n t manner , 50~ i n h i b i t i o n was o b s e r v e d with 0 5 ~g of t h e a n t i b o d y (Fig 5) The e f f e c t s o f chemical r e a g e n t s on t h e a c t i v i t y o f p l e u r a l f l u i d p h o s p h o l t p a s e Az a r e shown in Table 1 The enzyme was ] n b l b l t e d by p - b r o m o p h e n a c y l bromide ( p - B P B ) which i s known to be an a c t , r e s i t e - d i r e c t e d r e a g e n t f o r a l a r g e number o f A2 p h o s p h o l l p a s e s (22) The a c t i v i t y was a l s o l n h l b ] t e d by d l t h l o t h r e ] t o l , wh]ch

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0.5

E I=

O

E ,.,.,

¢,,I

_,J o.

4

A v

A

5

6

7

8

9

10

pH Fig

2

The pH dependence o f p l e u r a l f l u i d p h o s p h o l i p a s e Az A reaction mxture c o n t a l m n g the c r u d e enzyme (20 ~g o f p r o t e i n ) , 50 nmol o f 1 - a c y l - 2 - [ 1 - t 4 C ] l i n o l e o y l - s n - g l y c e r o - 3 - p h o s p h o e t h a n o l a m i n e and 6 mM CaC12 zn a t o t a l volume o f 250 ~l was i n c u b a t e d f o r 40 m n a t 37°C The b u f f e r s used were 100 mM sodium a c e t a t e b u f f e r w i t h i n a pH range o f 3 5-5 O, 100 mM T r i s - m a l a t e , pH 5 0-7 O, I00 mM Tris-HC1. pH 7 0-9 0 and 100 mM glyclne-NaOH pH 9 0-10 5

0.5 E E E =k,

(N

0 @ m .--I Q.

5

EDTA

0

2

4

6

Calc,um F~g

8

10

(mM)

3

E f f e c t o f Ca 2÷ 1on on the a c t z v l t y o f p h o s p h o l l p a s e A2 A r e a c t i o n mixture c o n t a l m n g t h e crude enzyme (20 ~g), 50 nmol o f 1 - a c y l - 2 - [ 1 - 1 4 C ] l l n o l e o y l - s n g l y c e r o - 3 - p h o s p h o e t h a n o l a m n e , 100 mM glyclne-NaOH (pH 10 O) and v a r i o u s c o n c e n t r a t i o n s o f CaClz or 5 mM EDTA in a t o t a l volume o f 250 ~l was i n c u b a t e d f o r 40 m n a t 37"C

Vol. 49, NO. 15, 1991

PLA2 in Tuberculosis

1099

c a u s e s s p l i t t i n g and r e d u c t i o n of d i s u l f i d e b r i d g e s , thus d e s t a b i l i z i n g the t h r e e dimensional c o n f o r m a t i o n No l n h , b l t l o n was observed with e i t h e r lodoacetamide (a s p e c i f i c SH r e a g e n t ) , or p h e n y l m e t h y l s u l f o n y l f l u o r i d e (a s e r l n e e s t e r a s e I n h i b i t o r )

E C

0.5 O

E P >

O4

0 a> t~

.J a.

v

T 01

A v !

02

detergent(w/v, %) Fig. 4 Effect of non-ionlc (Triton X-IO0, • ) and anlonlc detergent (sodlum deoxycholate, O) on the a c t i v i t y of phosphollpase A2 The crude enzyme (20 #g) was incubated with 50 nmol of 1-acyl-2-[1-1aC]llnoleoyl-sn-glycero-3-phosphoethanolamine *n the presence of various concentrations of the detergents

D{scusslon E x t r a c e l l u l a r p h o s p h o l l p a s e A2 has been d e t e c t e d tn experimental animals with c o n d l t , o n s such as experimental d e l a y e d - t y p e h y p e r s e n s i t i v i t y , a l l e r g i c u v e l t i s and e n d o t o x l n shock (23) Inflammatory dLseases in humans, such as g r a m - n e g a t i v e s e p t i c shock, rheumatoid a r t h r i t i s and p s o r L a s l s (24), are a l s o a s s o c i a t e d with high e x t r a c e l l u l a r p h o s p h o l l p a s e h2 a c t , v l t y (23) The p r e s e n t study i s the f i r s t to c h a r a c t e r , z e b l o c h e m l c a l l y the p h o s p h o l , p a s e A2 p r e s e n t ~n p l e u r a l f l u i d o f p a t i e n t s with t u b e r c u l o s i s This phosphol~pase A2 was s e n s i t i v e to Ca 2÷ c o n c e n t r a t i o n , and was o p t i m a l l y a c t i v e a t pH l0 0 The (.a 2÷ requirement f o r optimal a c t i v i t y was s i m i l a r to t h o s e o f A2 p h o s p h o l l p a s e s from o t h e r sources, such as c a s e l n a t e - I n d u c e d r a t p e r i t o n e a l exudate (13) and human synovlal f l u i d (8) Both p - b r o m o p h e n a c y l bromide and d l t h l o t h r e i t o l were i n h i b i t o r y When s u b s t r a t e s p e c ~ f l c l t y was t e s t e d u s i n g 2 - [ 1 - 1 4 C ] l I n o l e o y l p h o s p h o l , p l d s as s u b s t r a t e , p h o s p h a t l d y l e t h a n o l a m I n e was t h e b e s t substrate Sodium d e o x y c h o i a t e inhlbLted the enzyme a c t i v i t y a t a c o n c e n t r a t i o n h i g h e r than 0 02~. Recently, we establlshed four monoclonal ant]bodles against e x t r a c e l l u l a r phospholipase Az found in syno~lal f l u i d of patients wlth rheumatold arthr]~lS (25) One of them, H P - I ,nh,bltcd pleura] f l u i d phosphollpase A2 a c t i v l t y in a dose-dependent manner, 50~ ,nhJbltlon belng observed wlth 0 5 ~g of the antibody Normal mouse Ig G dld not inhlb{t the a c t l v l t y , on the contrary, i t apparently enhanced the enzyme a c t l v i t y when added in the same amount These findings indicate

ii00

PLA2

in T u b e r c u l o s i s

Vol.

49, No.

15, 1991

A v

100

A

o~

50

.J

------o 0

I

I

l

10

20

30

antibody ( p g ) F~g

5

E f f e c t o f m o n o c l o n a l a n t i b o d y (HP-1) on t h e a c t i v i t y o f p h o s p h o l l p a s e A2 c r u d e enzyme (160 ~g) was i n c u b a t e d w i t h t h e i n d i c a t e d amount o f HP-1 ( O )

The and

n o r m a l mouse I g G ( Q ) i n 200 #1 o f 0 2~ BSA-TBS f o r 1 h a t room t e m p e r a t u r e A f t e r i n c u b a t i o n , a l i q o u t was t a k e n and e x a m i n e d f o r p h o s p h o l t p a s e A2 a c t i v i t y The r e s u l t s a r e e x p r e s s e d a s p e r c e n t a g e s o f t h e a c t i v i t y o f t h e enzyme i n c u b a t e d without

antibody. TABLE 1

Effect

of

Various

Agents

on

the

Activity

Phosphollpase Treatment

Concentration

No a d d i t i o n Dlthlothreitol p -Bromophenacyl bromide iodoacetamlde Phenyimethylsulfonyl fluoride

1 0 3 0

(mM)

of

Human

Pleural

Fluid

A2 Remaining acttvlty

(~)

100 29 5 27 8

3 0

84 0

3 0

96 5

The c r u d e enzyme (20 #g) wa'~ p r e J n c u b a t e d w i t h v a r i o u s a g e n t s i n 100 mM g l y c l n e - N a O H (pH 10 0), c o n t a i n i n g 6 mM CaC12 in a t o t a l volume o f 250 #1 f o r 15 mln a t 37°C The r e a c t i o n was s t a r t e d by a d d i n g 50 nmol o f 1-acyl-2-[1-14C]llnoleoyl-sn-glycero-3-phosphoethanolamlne a s a s u b s t r a t e and was c o n t i n u e d

f o r 40 mln a t 37°C

Vol.

49, No.

15,

1991

PLA2

in T u b e r c u l o s i s

Ii01

an antlgenlc slmllarlty between human pleural fluld and synovlal fluid A2 p h o s p h o l l p a s e s S i m i l a r i t y was a l s o shown in the bloehemleal f e a t u r e s o f the two enzymes The p l e u r a l f l u l d p h o s p h o l l p a s e A2 thus may a l s o belong to the 14K group enzyme famlly (25) Varlous prosnflammatory medlators are known to the cellular release of phosphollpase A2, s u c h as thrombln ( l l ) , I L - I and tumor necrosis factor (TNF) (26-27) Although the cellular orlgln of pleural phosphollpase A2 may be derlved from some inflammatory c e l l s , such as granuloeytes or ]ymphocytes (8) In fact, pleural flu,d generally contains a total leucocyte count of 5000/~I wlth a predomlnance of lymphocytes ( 2 8 ) The presence of more than 90-95~ lymphocytes Is suggest,re of tuberculosls pleurlsy (28-29) Thus the source of pleura] fluid phosphol*pase A2 may be inflammatory c e l l s , although it is also posslble that phosphollpase A2 is actively transported from the blood stream into the pleural space to genarate hlgher concentratlons in pleura] f]uld Various elcosanolds have been detected in rat experlmental models of pleurisy (15, 30) Thus phosphollpase A2 secreted into the pleural space may play a role in the generatlon of these inflammatory mediators Acknowledgements This work was supported by a g r a n t

from the korea S c i e n c e and E n g i n e e r i n g

Foundation (KOSEF 893-0412-013-2) References 1 2 3 4

E A DENNIS, ]n the Enzymes (P D Boyer, ed ) 16, p 307-353 Academic p r e s s , New York (1983) A TU, Venoms p 23-63 Wiley New York (1977) H VAN DEN BOSCH, Blochlm BIophys Acta 604, 191-246 (1980) H VAN DEN BOSCH, In phosphollp~ds p 313-358 E l s e v i e r Biomedical P r e s s , Amsterdam

5 6 7 8

(1982) M WAITE, J L i p i d Res 26, 1379-1388 (1985) 0 H ALBERT and F SNYDER, J Blol Chem 258, 97-102 (1983) P VADAS, E STEFANSKI and W PRUZANSKI, L i f e Scl 36, 579-583 (1985) S HARA, I KUDO, H W CHANG, k MATSUTA, T MIYAMOTOand K INOUE, J

9

105, 395-399 (1989) H W CHANG, I KUDO, S

lO II

HARA, k

KARASAWAand k

INOUE, J

B1ochem

Blochem

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