V o l u m e 294, n u m b e r 3, 247-251 F E B S 10474 c'~ 1991 Federation of European Biochemical Societies 00145793Y91/$3.50
D e c e m b e r 1991
E i c o s a n o i d g e n e r a t i o n f r o m a n t i g e n - p r i m e d m a s t cells b y extracellular m a m m a l i a n 1 4 - k O a g r o u p II p h o s p h o l i p a s e A2 Makoto
Murakar,3i,
Ichiro
Kudo
and
Keizo
lnoue
D e p a r t m e n t o f l~Xealth C h e m i s t r y , F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s . U n i v e r s i t y o f T o k y o . l l o n g o , B u n k y o - k u .
T o k y o 113. J a p a n
Received 26 S e p t e m b e r 1991; revised version received 19 O c t o b e r 1991 T h e e x t r a c e l l u l a r f o r m o f 1 4 - k D a g r o u p I! p h o s p h o l i p a s ¢ A2 h a s b e e n f o u n d t o a c c u m u l a t e a t v a r i o u s t y p e s o f i n f l a m m a t o r y ;ites. I n t h e p r e s e n t p a p e r , w e h a v e s t u d i e d t h e p o s s i b l e r o l e o f t h e extraca~llular 1 4 - k D a g r o u p i t p h o s p h o l i p a s ¢ A 2 it-. t h e p r o c e s s o f p r o s t a g l a n d i n p r o d u c t i o n in a c t i v a t e d r a t m a s t cells. W h e n m a s t c e l l s o b t a i n e d f r o m t h e p e r i t o n e a l c a v i t y o f r a t s w e r e s e n s i t i z e ! w i t h [ g E , c h a l l e n g e d w i t h a n t i g e n a n d t h e n e x p o s e d t o e x t r a c e l l u l a r 1 4 - k D a g r o u p I! p h o s p h o | i p a s e A 2. a p p r e c i a b l e r e l e a s e o f p r o s t a g l a n d i u D2 w a s o b s e r v e t l . G e n e r a t i o n o f p r o s t a g l a n d i n D 2 w a s d e p e n d e n t o n t h e c o n c e n t r a t i o n o f t h e p h o s p h o l i p a s e A2 a s w e l l a s t h a t o f t h e a n t i g e n , w h i l e n o a p p r e c i a b l e p r o s t a g l a n d i n D 2 g e n e r a t i o t t w a s o b s e r v e d w i t h c e l l s in t h e a b s e n c e o f t h e a n t i g e n . N o h i s t a m i n e r e l e a s e w a s o b s e r v e d u n d e r Lhe s a m e c o n d i t i o n s . P h o s p h a t i d y l c h o l i n e in m a s t c e l l m e m b r a n e s w a s a p p r e c i a b l y h y d r o l y z e d t o l i b e r a t e free a r a c h i d o n i e a c i d w h e n m a s t cell~ w e r e i n c u b a t e d w i t h 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A2 a d d e d e x o g e n o u s l y in t h e p r e s e n c e o f t h e a n t i g e n . B o t h t h e g e n e r a t i o n o f p r o s t a g l a n d i n Dz a n d t h e r e l e a ~ o f a r a c h i d o n i c a c i d w e r e r e t a r d e d b y i n h i b i t o r s sDecific t o 1 4 - k D a g r o u p I! p h o s p h o l i p a s e A2. T h u s , 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A 2 m a y f u n c t i o n in t h e p r o c e s s o f i n f l a m m a t i o n b y a c t i n g o n l g E - a n t i g e r t - p r i m e d m a s t cells, w h i c h a r e n o t f u l l y a c t i v a t e d , t o g e B e r a t e e i c o ~ a n o l d s . G r o u p I I p h o s p h o l i p a s e A2: M a s t cell; P r o s t a g l a n d i n D : : R a t
1. I N T R O D U C T I O N Several studies have implicated extracellular phcspholipase A 2 in the pathogenesis of disorders of the cardiovascular, gastrointestinal and pulmonary syst e m s , s k i n a n d c o n n e c t i v e t i s s u e s [1,2]. E x t r a c e l l u l a r phospholipase A 2 f o u n d i n fluid a t v a r i o u s sites o f inflammation, such as glycogen-induced ascitic fluid in r a b b i t s [3], c a s e i n a t e - i n d u c e d a s c i t i c f l u i d i n r a t s [4], a n d human synoviai fluid in patients with rheumatoid arthritis [5-7], has been purified and identified as a 1 4 - k D a g r o u p I ! p h o s p h o l i p a s e A 2. V a r i o u s i n f l a m m a t o r y c e l l s s u c h a s p l a t e l e t s [ 7 - 9 ] a n d n e u t r o p h i l s [10] h a v e b e e n s h o w n t o c o n t a i n 14-~-V'a g r o u p 11 p h o s p h o l i p a s e A2. T h e ; n f l a m m a t o r : •tokine% such as t u m o r n e c r o s i s f a c t o r , i n t e r l e u k i h :°~,)-I a n d I L - 6 i n d u c e d t r a n s c r i p t i o n o f the 1 4 - k D a g r o u p II p h o s p h o l i p a s e A2 g e n e i n v a r i o u s c e l l s i n c l u d i n g r a t v a s c u l a r s m o o t h m u s c l e c e l l s [! 1], r a t m e s a n g i a l c e l l s [12[, r a b b i t c h o n d r o c y t e s [13], o r h u m a n h c p a t o c y t e s [14]. A n t i - i n flammatory glucocorticoid suppressed the transcription o f 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A2 [15]. I n j e c t i o n o f
endotoxin into rats enhanced the expression of 14-kDa g r o u p I I p h o s p h o l i p a s e A 2 i n v a r i o u s o r g a n s [16]. T h e s e observations suggest that extracellular 14-kDa group lI p h o s p h o l i p a s e A2 m a y b e i n v o l v e d i n t h e p r o c e s s o f inflammation. M a s t c e l l s a r e well k n o w n f o r t h e i r men: r o l e ,r, a l l e r g i c a n d h y p e r s e n s i t i v i t y s t a t e s a n d h a v e ~,~-~n i m p i , c a t e d i n a v a r i e t y o f c h r o n i c i n f l a m m a t o r y pro,~c~: .~.~ [17,18]. C r o s s - l i n k i n g o f h i g h a f f i n i t y l g E r e c e p t o r ~ . , m a s t cell s u r f a c e s w i t h I g E a n d m u l t i v a ! e n t a n t . ~ e triggers the release of a variety of chemical mediate, s stored in granules, for example histamine, and the generation of eicosanoids, such as PGD2, which has v a s o d i l a t i n g a n d b r o n c h o c o n s t r i c t i v e a c t i v i t i e s [19], o r l e u k o t r i e n e C4 ( L T C 4 ) a n d L T B 4 . A s f o r r o d e n t c o n n e c t i v e t i s s u e m a s t cells, l y s o p h o s p h a t i d y l s e r i n e ( l y s o P S ) acts as an essential cofactor for full activation upon various stimuli including IgE-antigen system [20-22]. In the present study, we found that 14-kDa group II phosp h o l i p a s e A2 l o c a t e d o u t s i d e m a s t c e l l s w a s i n v o l v e d i n generation of PGD2 in IgE- and antigen-challenged rat p e r i t o n e a l c o n n e c t i v e tissue m a s t cells e v e n in the absence of lysoPS.
Abbreviations: P ( ] , p r o s t a g l a n d i n ; L T , l e u k o t r i e n e : r C 3 ~, r e c o m b i n a n t C 3 iv; D N P - A s c a r i s , d i n i t r o p h e n y l - c o n j u g a t e d Asearis suum; PC, p h o s p h a t i d y l c h o l i n e ; P E , p h o ~ p h a t i d y l e t h a n o t a m i n e ; PS, p h o s p h a t i dylserine; lysoPS, lysophosphatidylseyine; IL, interleukein.
2. M A T E R I A L S
AND
METHODS
2. I . . M o s t cells Correspondence address: K . l n o u e , D e p a r t m e n t o f H e a l t h C h e m i s t r y , F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e , T h e U n i v e r s i t y o f T o k y o , T-3-1 H o n g o , B u n k y o - k u , T o k y o 113. J a p a n . Fax". (81) (3) 3 8 1 8 - 3 1 7 3 .
Published b y Elsevier Science Publi,~l~rs B. If.
M a s t c e l l s t*aurity; m o r e t h a n 9 0 % ) w e r e i s o l a t e d f r o m t h e peritoneal cavity of Wistar rats ('Nippon Bio-Supply Center, Tokyo, Japan) as des~'~ibed p r e v i o u s l y [20].
:~47
Volume
294,
number
3
FEBS
2.2. P h o s p h o l i p a s e A : a n d ils s p e c i f i c inhibit¢~r.~ ~ R a t 1 4 - k D a g r o u p I ! p h o s p h o l i p a s e A~ w a s p u r i f i e d f r o m r a t p l a t e lets u s i n g a n a n t i - r a t 1 4 - k D a g r o u p II p h o s p h o l i p a s e A , m o n o c l o a a ! antibody-conjugated Sepharose column as described previously [23.24]. Preparation o f 1 4 - k D a g r o u p II 1 3 h o s p h o l i p a s e A 2 - s p e c i f i c p o l y c l o n a ' a n t i b o d y R 3 7 7 1231 a n d m o n o c l o n a t a n t i b o d y M D 7 . 1 [ 2 4 ] was described previously. Preparation of rat recombinant C3 a (rC3 0t; a p r o d u c t o f t h e E. c o i l l a c Z - r : l l C 3 d g ( c o m p l e m e n t C'~ d e g r a d i n g product [25]) gone) and Ihiclocin AI, both of which inhibited group II p h o s p h o l , . ' p a s e A , r a t h e r s p e c i f i c a l l y , a r e d e s c r i b e d e l s e w h e r e ( u n published data}_ 2.3. Trt,atmen: o f m a s t cells with p h o s p h o l i p a s e A : M a s t c e l l s w e r e s u s p e n d e d i n 10 m M T f i s - H C I b u f f e r ( p H 7 . 4 L which contained 150 mM NaCI. 3.7 mM KCL I mM CaCb, 0. 1% (w/v) glucose and 0.5% (w/v) gelatin (Sigma. St. Louis. MO) (Tris-gelatin buffer), and adjusted to ! x l0 s cells/ml. The oells were sensitized with I jug/ml mouse monoclonal anti-dinitrophenyl (DNP) lgE (Scikagaku Kogyo, Tokyo. Japan) for 30 rain at 37°C. The sensitized cells were washed, suspended in Tris-gelatin buffer at I x los cells/ml, and incub a t e d w i t h 1 4 - k D a g r o u p II p h o s p h o l i p a s e Az in the presence or a b s e n c e o f D S P - - c o n j u g a t e d A s c a r i s s u u m I D N P-Ascari,~ ( d o n a t e d b y Kissei Pharmaceutical, Matsumoto. Japzn). Alternatively, the sensitized cells were stimulated with DNP-Ascaris in the presence of 10 6 M lysoPS (Funakoshi, Tokyo, Japan). After incubation, the cells were centrifuged at 7~0 × g for 5 rain at 4°U to obtain the supernatant. PGD, released into the supernatant was measured using a PGD2 assay kit (~mersham, Buckinghamshire. UK). Histamine released into the supernatant was determined by a radioenzymatic assay using [~H]methyI-S-adenosyI-L-methionine (New England Nuclear, Boston, MA) and a crude preparation of rat ki,-lney histamine methyltransfer u s e [ 2 6 l . T h e p e r c e n t a g e r e l e a s e o f h i s t a m i n e w a s c a l c u l a t e d b y d i v i ,~i n g t h e a m o u n t in e a c h s u p e r n a t a n t b~ ~ h g t i n a p r e p a r a t i o n o f s o n i cated cells (Branson Sonifier, 20 pulses, setting 4, 50% pulse cycle). 2 . 4 . L i p i d analys£~ M a s t c e l l s (I x 10~ c e l l s / m l ) w e r e i n c u b a t e d f o r I h i n T r i s - g e l a t i n buffer cor.taining 1 gCi/ml [3H]arachidonate (Amersham), washed twice with Tris-gelatin buffer, sensitized with lgE and treated with l0 ~'g/ml 14-kDa group !! phospholipase A2 and I /dg/ml DNP-Ascaris in the presence or absence of 2 ~ggml thielocin A I for i 5 rain at 37°C. The total cellular lipids were extracted by the method of Biigh and Dyer [27] and separated by two-dimensional thin-layer chromatography on silica gel plates (Merck, Darmstadt, Germany,, which were developed with a slighl modification of the solvent s)~tem described b y E s k o a n d R a e t z I28]. B r i e f l y . t h e e x t r a c t e d l i p i d s w e r e r e c o n s t i t u t e d in a small volume of chloroform and applied to silica gel plates (approximately I x I 0 s cells per pla:e). The plales were developed first with chloroform/methanol/acetic acid (65:25: I 0, v/v). dried for 60 rain u n d e r a n a i r s t r e a m , a n d d e v e l o p e d in t h e s e c o n d d i m e n s i o n w i t h chloroform/methanol/88% formic acid (65:25:10.v/v). The spots which were visible after exposure to I, vapor were identified by comparing their positions with those of authentic standard phospholipids. The !, was then removed with an air stream, the individual spots were scraped off, and the radioactivity of each was measured.
3. R E S U L T S T h e e f f e c t o f t r e a t m e n t o f rat p e r i t o n e a l m a s t c e l l s w i t h p u r i f i e d rat 1 4 - k D a g r o u p II p h o s p h o l i p a s e A 2 o n their cellular functions were examined. When unstimulated mast cells were treated with the phospholipase A2 alone, PGD2 release into the supernatant was not augm e n t e d a p p r e c i a b l y ( F i g . I). W e t h e n e x a m i n e d t h e e f f e c t oF t h e p h o s p h o l i p a s e A z o n a n t i g e n - s t i m u l a t e d m a s t cells. No appreciable PGD2 generation was observed w h e n m a s t c e i l s w e r e first s e n s i t i z e d w i t h a n t i - D N P l g E 248
LETTERS
December
1991
5O
o
40
~
3o
o.
i O'
0
2
4
6
8
i0
P h o a p h o [ I p a a e A 2 (jJglml) F i g . I. E f f e c t o f c x t r a c e l l u l a r 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A, on PGD 2 generation by rat peritoneal mast cells. Mast cells sensitized with lgE were incubated with the indicated concentrations of 14-kDa g r o u p I I p h o s p h o l i p a , s e A 2 in t h e p r e s e n c e o f 1 ~ t g / m l D N P - , 4 s c a r i s ( Q ) , D N P - A s c a r i s p l u s I 0 6 M l y s o P S ( A ) o r in t h e i r a b s e n c e ( ~ ) f o r 15 r a i n at 37°C, and PGD 2 released into the supernatant was measured as d e s c r i b e d i n s e c t i o n 2_ T h e v a l u e s i n d i c a t e a v e r a g e s _+ S D ( n = 3 ) .
and then challenged with DNP-conjugated antigen in t h e a b s e n c e o f l y s o P S , w h i c h is a n e s s e n t i a l c o f a c t o r f o r full a c t i v a t i o n o f r a t p e r i t o n e a l m a s t c e l l s [ 2 0 - 2 2 ] . W h e n these IgE-sensitized, antigen-challenged cells were further treated with purified 14-kDa group li phosphoi'p a s e A2, c o n c e n t r a t i o n - d e p e n d e n t PGD2 generation w a s o b s e r v e d ( F i g . 1). T h e c o n c e n t r a t i o n s o f t h e e n z y m e p r o d u c i n g a n a p p r e c i a b l e e f f e c t ( r a n g i n g f r o m I t o 10 /~g/ml) were c o m p a r a b l e to those d e t e c t e d at v a r i o u s i n f l a m m a t o r y s i t e s [23,29]. P G D 2 g e n e r a t i o n w a s a l s o
A
50
50 .o
~
m
4o
30
30
20
20
! A
Q.
o
o
o.bt Antigen
o"1
i
o
(pgiml)
Fig. 2. E f f e c t o f a n t i g e n o o n c e n t r a t i o n s o n e x t r a c e l l u l a r 1 4 - k D a g r o u p !I phospholipase A2-mcdiatcd PGD2 release from mast cells. The sensitized mast cells were incubated with 14-kDa group |I phospbolipase A2 (6 ~g/ml) and the indicated concentrations of DNP-A~rcaris f o r 15 m i n a t 3 7 ° C , a n d P G D 2 ( O ) o r h i s t a L ~ i n e ( o ) rel~L~4~d i n t o t h e supernatant was measured as described in section 2. The values indic a t e a v e r a g e s _+ S D ( n = 3 ) .
V o l u m e 294, n u m b e r 3
1 3 e c e m b e r 1991
FEBS LETTERS
50
T..~.~
~
PC
50
PE
P/
in
40
~
50
40
20
T---3o
30
=0
2o
_=
l(]
45
15 5
4G
1
m a.
10
0
tO
0
5
10
Time
"15
0 0
0
cells were treated with 14-kDa g r o u p II phosphoIipas¢ A: (6 jug/ml) (~'!.-~les) o r lysoPS (10 6 M) (triangles) in the presence o f D N P - , 4 s c a r i s (l gtg/ml) for the indicated period at 37°C, a n d el,her PCiD: (closed symbols) o r histamine (open symbols) relea.~:d i,~..o the s u p e r n a t a n t was m e a s u r e d as described in section 2. T h e values indicate averages + S D (n=3). dependent on the concentration o f t h e a n t i g e n ( F i g . 2). VChen the cells were challenged with 1Mg/ml antigen and treated with more than 7.5 ~ug/ml enzyme, about 50 ng o f P G D 2 p e r 106 c e l l s w a s g e n e r a t e d , a l m o s t t h e s a m e amount as that generated by fully activated mast cells; mast cells stimulated with 1 /lg/ml antigen in the prese n c e o f 10 -6 M l y s o P S g e n e r a t e d a b o u t 5 0 n g o f P ( ~ D 2 p e r l 0 s c e l l s ( F i g . 1). I t s h o u l d b e n o t e d h e r e t h a t n o further enhancement effect of exogenous 14-kDa group II phospholipase A 2 on Pg~D 2 generation was observed with cells which were fully activated by treatment with antigen plus lysoPS. In contrast to P(JD2 generation, release of histamine Table i Effect o f 14-kDa g r o u p II p h o s p h o l i p a s e A2-specific inhibitors o n extracellular 14-kDa gro~tp II p h o s p h o ! i p a s e Aa-mediated Pg~D2 release f r o m mast cells
~Io t r e a t m e n t Phospholipas¢ A 2 + A n t i b o d y R377 (40/t/g/ml)* + rC30t (2/Jg/rnl)* -t Thielocin A i {2 gtg/ml)* + A n t i b o d y IMD7.1 (15 ~ug/ml)**
10
lS
%
I~GD2 released (ng/I O* cells)
Inhibition (%)
2.1 _ + 0 . 8 3 8 . 0 -+ 1.9
2.0 3.6 2.6 37.0
± ± ± ±
~.! 1.4 0.4 2.5
;,
,o
PA
PS
(mini
F i g . 3. T i m e - c o u r s e o f I ~ G D 2 g e n e r a t i o n b y r ~ a s t c e l l s t r e a t e d w i t h extra~llular 1 4 - k D a g r o u p I | p h o s p h o l i p a s e A 2. T h e s e n s i t i T ~ d m a s t
Treatment
S
g
100 90.5 94.7 2.6
T h e e n z y m e was p r e i n c u b a t e d with the indicated a m o u n t s o f each inhibitor for 30 min at 22°C. Then l g E - a n t i g e n - p r i m e d m a s t eel Is were treated with the inbibitor-treated enzyme (~. jug/ml) and i~GD 2 released into t h e s u p e r n a t a n t was quantified as described in Section 2. Values indicate averages _+ S D (n=3). *Sufficient c o n c e n t r a t i o n t o inhibit p h o s p h o l i p a s e A 2 activity. **Sufficient c o n c e n t r a t i o n t o inhibit the in'.eraction between p h o s p h o lipase A 2 a n d heparin.
Arachidonic
15
15
20
10
1'0
15
Acid
/
10
lb
l's
is
iols "lime
5
"io
is
(rain)
Fig. 4. Time course o f phospholipid metabolism o f [~H]arachidonatelabeled mast cells after treatment with extracei]ular 14-kDa g r o u p II p h o s p h o l i p a s e A=. T h e procedure is described in :~ection 2. Values indicate p h o s p h o l i p i d c o m p o s i t i o n (average (%) ± SE') o f each sample (n=3). from mast cells was not affected by exogenous phospholipase A2 appreciably; mast cells released only about 10~ of cellular histamine (the same as the basal level) even in the presence of 14-kDa group II phospholipase A2 ( F i g . 2). The time course of PGD2 generation in4uced by antigen plus 14-kDa group II phospholipase -~'2 w a s r a t h e r different from the one induced by mtigel, plus lysoPS. The generation of PGD2 obserxed in the presence of 14-kDa group !I phospholipase A2 progressed almost l i n e a r l y w i t h i n 1 5 r a i n ~ f t e r e n z y m e c h a l l e n g e ( F i g . 3). On the other hand, PGD2 generation by cells supplemented with antigen plus lysoPS reached a plateau approximately 5 min after antigen challerige, paralleiling histamine release. The effect of exogenous 14-kDa group I! phospholipase A2 on PGD2 generation by mast cells was abolished by pretreatment of the enzyme with 14-kDa group II phospholipase A2-specitic inhibitors, such as antib o d y R 3 7 7 , t h i e i o c i n A I a n d r C 3 r, ( F~-.ble I). A m o n o clonal antibody MDT.1, which recognizes the heparinbinding domain of rat 14-kDa group II phospholipase A2 b u t d o e s n o t i n h i b i t e n z y m e a c t i v i t y [24], s h o w e d n o 249
V o l u m e 294. n u m b e r 3
FEBS LETTERS
D e c e m b e r 1991
Table I i Phospholipid metabolism of I~H]arachidonate-labeled mast cells after treatment with extracellular 14-kDa group II phospholipase Az Phospholil)ids
Treatment with phospholipase A~
No treatment Intact cells
Antigen-treated cells
Thielocin AI ( - ) Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylserine Phosphatidylethanolamine Phosphatidic acid Free arachidonate
0.7 51.0 16.5 4.3 I'Ll 3.1 11_2
__ 0.2 _+ 0.2 _+ I_1 _+ 0.4 4- 0.1 _+ 0.1 _4-0_6
0.8 51).3 15_8 4.0 13.9 3.0 10.8
± 0.1 ± 1.5 ± 0_3 + 0.5 _+ 0.8 _+ 0.5 _+ 0.4
Thieloein
t.0 39.9 14.7 4.1 15.3 4.9 20.3
-~ 0.3 ± 2.2 _+ !.1 _+ 1.0 -+ 0.3 + 0. I _+ 2.0
1.0 50.6 15.6 4.0 13.7 3.7 I 1.6
AI
(4-)
+ 0.1 +- 1.2 + 0.0 _+ 0.4 _+ 0.0 -+ 0.2 _+ 1.0
The procedure is described in Section 2. Values indicate phospholipid composition (average (%) _+ SD) of each sample (n=3). inhibitory effect, suggesting that enzymatic activity of g r o u p 11 e n z y m e m a y b e e s s e n t i a l f o r t h e g e n e r a t i o n o f P G D ~ in t h i s r e a c t i o n . The alteration of the membrane phospholipids in mast cells by treatment with the exogenous 14-kDa g r o u p l I p h o s p h o l i p a s e A2 w a s n e x t e x a m i n e d . W h e n [3H]arachidonate-!abeled cells were exposed to 14-kDa g r o u p I1 p h o s p h o l i p a s e A 2 i n t h e p r e s e n c e o f t h e a n t i gen, o n l y the r a d i o a c t i v i t y in p h o s p h a t i d y l c h o l i n e ( P C ) decreased drastically (approximately 50% to 40%) with a c o n c o m i t a n t i n c r e a s e in r a d i o a c t i v i t y o f f r e e a r a c h i d o n a t e ( a p p r o x i m a t e l y 1 1 % t o 2 0 % ) ( F i g . 4, T a b l e I1). T h e hydrolysis of PC and thereby the increment of free arachidonate were suppressed almost completely by pret r e a t m e n t o f t h e e n z y m e w i t h its s p e c i f i c i n h i b i t o r , t h i e l o c i n A I. N o a p p r e c i a b l e h y d r o l y s i s o f P C w a s o b s e r v e d when intact mast cells were treated with the enzyme u n d e r t h e s a m e c o n d i t i o n s ( T a b l e I I ) . T h e r e f o r e , 14k D a g r o u p !I p h o s p h o l i p a s e A 2 a p p e a r e d t o h a v e h y d r o l y z e d P C in t h e m e m b r a n e o f a n t i g e n - p r i m e d m a s t cells. 4. D I S C U S S I O N In the present study, we demonstrated that mammalian 14-kDa group Ii phospholipase A 2 added exogenously to lgE-antigen-primed rat peritoneal mast cells a u g m e n t e d e i c o s a n o i d g e n e r a t i o n in t h e cells. T h e c o n centratio,~s of the enzyme required for the augmentation (I-10 ~g/ ml) were within the range detected at v a r i o u s i n f l a m m a t o r y s i t e s [22.29], i n d i c a t i n g t h a t e x t r a c e l l u l a r 1 4 - k D a g r o u p II p h o s p h o l i p a s e A z m a y c o n tribute to the progression of inflammation, especially allergic reaction, in which mast cells play important roles as effector cells, by hydrolyzing mast cell phospholipids to generate precursors of pro-inflammatory lipid mediators. 1 4 - k D a G r o u p I I p h o s p h o l i p a s e A2 w a s a b l e t o i n fluence PGD2 generation only when IgE-receptors on the surfaces of mast cells were cross-linked by multivalent antigens. The activity of several phospholipases A 2 o n c e l l m e m b r a n e s is k n o w n t o b e a f f e c t e d b y l i p i d 250
p a c k i n g in t h e o u t e r l e a f l e t o f t h e p l a s m a m e m b r a n e [30]. C r o s s - l i n k i n g o f l g E r e c e p t o r s m a y c h a n g e t h e m o l e c u l a r p a c k i n g o f l i p i d s [3 !], m a k i n g t h e m s u s c e p t i b l e t o e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s e A : . T h e s e o b s e r v a t i o n s a r e in a c c o r d w i t h o u r p r e v i o u s data, which showed that mammalian 1 4 - k D a g r o u p I1 as well as group I phospholipase A: added exogenously augmented the generation of PGE~ by HL-60 granuloc y t e s o n l y i n t h e c o - p r e s e n c e o f A 2 3 1 8 7 [32]. W e a l s o r e p o r t e d t h a t i n j e c t i o n o f p u r i f i e d 1 4 - k D a g r o u p I1 p h o s p h o l i p a s e A2 i n t o the h i n d p a w o f r a t s w i t h a d j u vant arthritis exacerbated the eOema, whereas no effect w a s o b s e r v e d i n n o r m a l r a t s [29]. T h u s , e x p r e s s i o n o f the nharmacological activity of the exogenous 14-kDa g r o t p I I p h o s p h o l i p a s e A2 m a y r e q u i r e a c e r t a i n s t a g e of ongoing inflammation induced by some other factors. A c h a n g e in t h e t r a n s m e m b r a n e distribut;on of phospholipids may be one of the features of such an a c t i v a t e d s t a t e . R e c e n t l y , B o m a l a s k i e t a l . [33] d e m o n strated that purified recombinant human 14-kDa group 11 p h o s p h o l i p a s e A 2 e l i c i t e d a d r a m a t i c i n f l a m m a t o r y , arthritogenie response when injected into the joint space of healthy rabbits, although they also stated that the e n z y m e w a s c o m p l e t e l y i n a c t i v e in t h e p a w e d e m a i n flammation assay using normal rats. Although this disc r e p a n c y m i g h t be e x p l a i n e d b y t h e d i f f e r e n c e in t h e species employed, further work must be performed to clarify the molecular mechanisms of the pro-inflammatory effect of exogenous 14-kDa group II phospholipase A2. Mammalian 1 4 - k D a g r o u p II p h o s p h o l i p a s e A z hydrolyzes phosphatidylethanolamine (PE) or phosphatid y l s e r i n e (PS) m o r e efficiently t h a n P C in a n in v i t r o assay system [2-10]. The treatment of mast cells with e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s ¢ A2 r e s u l t e d i n p r e f e r e n t i a l h ? / d r o l y s i s o f m e m b r a n e P C . I t is k n o w n t h a t P C is d i s t r i b u t e d i n t h e o u t e r l e a f l e t o f p l a s m a bilayer membrane of mammalian cells, whereas both PE and PS exist almost exclusively in the inner leaflet [34]. T h u s , o n l y P C m i g h t b e a v a i l a b l e f o r e x t r a e e l l u l a r 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A 2. A l t e r n a t i v e l y , t h e
V o l u m e 294, n u m t m r 3
FEBS
g r o u p I! e n z y m e m i g h t a c t i v a t e s o m e o t h e r c e l l u l a r phospholipase(s) A2 which liberate arachidonate from PC. We found that ~last cells expressed an arachidonate-preferential cytosolic 85-kOa phospholipase Az [35]. a e D N A c l o n e o f w h i c h w a s r e c e n t l y i s o l a t e d f r o m U 9 3 7 c e l l s [36]. a n d s h o w e d t h e p o s s i b i l i t y t h a t t h e e n zyme might play an essential role in araehidonate metabolism upon application of mast cells with immunochemical stimuli (antigen plus lysoPS) via the lgE recept o r [37]. T h e p o s s i b i l i t y t h a t e x o g e n o u s 14-kDa group II p h o s p h o l i p a s e A2 activates intracellular phospholipase A2 such as araehidonate-preferential phospholipase A z cannot be ruled out at present. LysoPS is a potentiazor of the degranulation as well as PGD2 generation by rat peritoneal mast cells [20-22]. One possible mechanism whereby exogenous t4-kDa g r o u p II p h o s p h o l i p a s e A 2 m a y e x e r t i t s e f f e c t is t o generate iysoPS by hydrolysis of membrane PS. However, this possibility can be eliminated for the following r e a s o n s : ( i ) h i s t a m i n e r e l e a s e wa:; n o t i n d u c e d b y e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p c , s e A2. i f l y s o P S w a s supplied by this exogenous enzyme, then histamine release might also be induced in the same manner as P G D ~ g e n e r a t i o n ; (ii) n o a p p r e c i a b l e h y d r o l y s i s o f P S was observed when cells prelabeled with radiolabeled arachidonate w e r e e x p o s e d t o t h e 1 4 - k O a g r o u p II phospholipase A2 in the presence of antigen: (iii) stimul a t i o n o f m a s t c e l l s w i t h a n t i g e n p l u s l y s o P S is a c c o m p a nied by a rapid and drastic breakdown of phosphatidyl i n o s i t o l ( P I ) [38], w h e r e a s n o a p p r e c i a b l e r a p i d d e c r e a s e i n r a d i o a c t i v i t y in P l w a s o b s e r v e d w i t h m a s t ceils treated with antigen and the 14-kDa group lI phosp h o l i p a s e A 2 ( F i g . 4). It c a n b e c o n c l u d e d t h a t e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s c A 2 a c t e d o n l g E antigen-primed, but not fully activated mast cells to generate eicosanoids, resulting in the progression of allergic inflammation. T h e s o u r c e o f t h e e x t r a c e l l u l a r 1 4 - k D a g r o u p II p h o s p h o l i p a s e A 2 d e t e c t e d a t i n f l a m e d s i t e s is s t i l l u n i d e n t i fied. it should be noted that activated mast cells also s e c r e t e ! 4 - k D a g r o u p I! p h o s p h o l i p a s e A 2 ( u n p u b l i s h e d data). However, the amount of the enzyme released from activated mast cells is too small (less than i ng per l06 cells) to act on IgE-antigen-primed mast cells to generate a detectable level of eicosanoids. Therefore, 1 4 - k D a g r o u p l I p h o s p h o l i p a s e A~ m i g h t b e s u p p l i e d b y ceils other than mast ceils. Aeknowled~,~ments: T h i s w o r k w a s s u p p o r t e d b y G r a n t s - i n - A i d f o r Scientific Research (Nos. 01639509, 02557090 and 03680163) from the Ministry of Education, Science and Culture of Japan_
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E i c o s a n o i d g e n e r a t i o n f r o m a n t i g e n - p r i m e d m a s t cells b y extracellular m a m m a l i a n 1 4 - k O a g r o u p II p h o s p h o l i p a s e A2 Makoto
Murakar,3i,
Ichiro
Kudo
and
Keizo
lnoue
D e p a r t m e n t o f l~Xealth C h e m i s t r y , F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s . U n i v e r s i t y o f T o k y o . l l o n g o , B u n k y o - k u .
T o k y o 113. J a p a n
Received 26 S e p t e m b e r 1991; revised version received 19 O c t o b e r 1991 T h e e x t r a c e l l u l a r f o r m o f 1 4 - k D a g r o u p I! p h o s p h o l i p a s ¢ A2 h a s b e e n f o u n d t o a c c u m u l a t e a t v a r i o u s t y p e s o f i n f l a m m a t o r y ;ites. I n t h e p r e s e n t p a p e r , w e h a v e s t u d i e d t h e p o s s i b l e r o l e o f t h e extraca~llular 1 4 - k D a g r o u p i t p h o s p h o l i p a s ¢ A 2 it-. t h e p r o c e s s o f p r o s t a g l a n d i n p r o d u c t i o n in a c t i v a t e d r a t m a s t cells. W h e n m a s t c e l l s o b t a i n e d f r o m t h e p e r i t o n e a l c a v i t y o f r a t s w e r e s e n s i t i z e ! w i t h [ g E , c h a l l e n g e d w i t h a n t i g e n a n d t h e n e x p o s e d t o e x t r a c e l l u l a r 1 4 - k D a g r o u p I! p h o s p h o | i p a s e A 2. a p p r e c i a b l e r e l e a s e o f p r o s t a g l a n d i u D2 w a s o b s e r v e t l . G e n e r a t i o n o f p r o s t a g l a n d i n D 2 w a s d e p e n d e n t o n t h e c o n c e n t r a t i o n o f t h e p h o s p h o l i p a s e A2 a s w e l l a s t h a t o f t h e a n t i g e n , w h i l e n o a p p r e c i a b l e p r o s t a g l a n d i n D 2 g e n e r a t i o t t w a s o b s e r v e d w i t h c e l l s in t h e a b s e n c e o f t h e a n t i g e n . N o h i s t a m i n e r e l e a s e w a s o b s e r v e d u n d e r Lhe s a m e c o n d i t i o n s . P h o s p h a t i d y l c h o l i n e in m a s t c e l l m e m b r a n e s w a s a p p r e c i a b l y h y d r o l y z e d t o l i b e r a t e free a r a c h i d o n i e a c i d w h e n m a s t cell~ w e r e i n c u b a t e d w i t h 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A2 a d d e d e x o g e n o u s l y in t h e p r e s e n c e o f t h e a n t i g e n . B o t h t h e g e n e r a t i o n o f p r o s t a g l a n d i n Dz a n d t h e r e l e a ~ o f a r a c h i d o n i c a c i d w e r e r e t a r d e d b y i n h i b i t o r s sDecific t o 1 4 - k D a g r o u p I! p h o s p h o l i p a s e A2. T h u s , 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A 2 m a y f u n c t i o n in t h e p r o c e s s o f i n f l a m m a t i o n b y a c t i n g o n l g E - a n t i g e r t - p r i m e d m a s t cells, w h i c h a r e n o t f u l l y a c t i v a t e d , t o g e B e r a t e e i c o ~ a n o l d s . G r o u p I I p h o s p h o l i p a s e A2: M a s t cell; P r o s t a g l a n d i n D : : R a t
1. I N T R O D U C T I O N Several studies have implicated extracellular phcspholipase A 2 in the pathogenesis of disorders of the cardiovascular, gastrointestinal and pulmonary syst e m s , s k i n a n d c o n n e c t i v e t i s s u e s [1,2]. E x t r a c e l l u l a r phospholipase A 2 f o u n d i n fluid a t v a r i o u s sites o f inflammation, such as glycogen-induced ascitic fluid in r a b b i t s [3], c a s e i n a t e - i n d u c e d a s c i t i c f l u i d i n r a t s [4], a n d human synoviai fluid in patients with rheumatoid arthritis [5-7], has been purified and identified as a 1 4 - k D a g r o u p I ! p h o s p h o l i p a s e A 2. V a r i o u s i n f l a m m a t o r y c e l l s s u c h a s p l a t e l e t s [ 7 - 9 ] a n d n e u t r o p h i l s [10] h a v e b e e n s h o w n t o c o n t a i n 14-~-V'a g r o u p 11 p h o s p h o l i p a s e A2. T h e ; n f l a m m a t o r : •tokine% such as t u m o r n e c r o s i s f a c t o r , i n t e r l e u k i h :°~,)-I a n d I L - 6 i n d u c e d t r a n s c r i p t i o n o f the 1 4 - k D a g r o u p II p h o s p h o l i p a s e A2 g e n e i n v a r i o u s c e l l s i n c l u d i n g r a t v a s c u l a r s m o o t h m u s c l e c e l l s [! 1], r a t m e s a n g i a l c e l l s [12[, r a b b i t c h o n d r o c y t e s [13], o r h u m a n h c p a t o c y t e s [14]. A n t i - i n flammatory glucocorticoid suppressed the transcription o f 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A2 [15]. I n j e c t i o n o f
endotoxin into rats enhanced the expression of 14-kDa g r o u p I I p h o s p h o l i p a s e A 2 i n v a r i o u s o r g a n s [16]. T h e s e observations suggest that extracellular 14-kDa group lI p h o s p h o l i p a s e A2 m a y b e i n v o l v e d i n t h e p r o c e s s o f inflammation. M a s t c e l l s a r e well k n o w n f o r t h e i r men: r o l e ,r, a l l e r g i c a n d h y p e r s e n s i t i v i t y s t a t e s a n d h a v e ~,~-~n i m p i , c a t e d i n a v a r i e t y o f c h r o n i c i n f l a m m a t o r y pro,~c~: .~.~ [17,18]. C r o s s - l i n k i n g o f h i g h a f f i n i t y l g E r e c e p t o r ~ . , m a s t cell s u r f a c e s w i t h I g E a n d m u l t i v a ! e n t a n t . ~ e triggers the release of a variety of chemical mediate, s stored in granules, for example histamine, and the generation of eicosanoids, such as PGD2, which has v a s o d i l a t i n g a n d b r o n c h o c o n s t r i c t i v e a c t i v i t i e s [19], o r l e u k o t r i e n e C4 ( L T C 4 ) a n d L T B 4 . A s f o r r o d e n t c o n n e c t i v e t i s s u e m a s t cells, l y s o p h o s p h a t i d y l s e r i n e ( l y s o P S ) acts as an essential cofactor for full activation upon various stimuli including IgE-antigen system [20-22]. In the present study, we found that 14-kDa group II phosp h o l i p a s e A2 l o c a t e d o u t s i d e m a s t c e l l s w a s i n v o l v e d i n generation of PGD2 in IgE- and antigen-challenged rat p e r i t o n e a l c o n n e c t i v e tissue m a s t cells e v e n in the absence of lysoPS.
Abbreviations: P ( ] , p r o s t a g l a n d i n ; L T , l e u k o t r i e n e : r C 3 ~, r e c o m b i n a n t C 3 iv; D N P - A s c a r i s , d i n i t r o p h e n y l - c o n j u g a t e d Asearis suum; PC, p h o s p h a t i d y l c h o l i n e ; P E , p h o ~ p h a t i d y l e t h a n o t a m i n e ; PS, p h o s p h a t i dylserine; lysoPS, lysophosphatidylseyine; IL, interleukein.
2. M A T E R I A L S
AND
METHODS
2. I . . M o s t cells Correspondence address: K . l n o u e , D e p a r t m e n t o f H e a l t h C h e m i s t r y , F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e , T h e U n i v e r s i t y o f T o k y o , T-3-1 H o n g o , B u n k y o - k u , T o k y o 113. J a p a n . Fax". (81) (3) 3 8 1 8 - 3 1 7 3 .
Published b y Elsevier Science Publi,~l~rs B. If.
M a s t c e l l s t*aurity; m o r e t h a n 9 0 % ) w e r e i s o l a t e d f r o m t h e peritoneal cavity of Wistar rats ('Nippon Bio-Supply Center, Tokyo, Japan) as des~'~ibed p r e v i o u s l y [20].
:~47
Volume
294,
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FEBS
2.2. P h o s p h o l i p a s e A : a n d ils s p e c i f i c inhibit¢~r.~ ~ R a t 1 4 - k D a g r o u p I ! p h o s p h o l i p a s e A~ w a s p u r i f i e d f r o m r a t p l a t e lets u s i n g a n a n t i - r a t 1 4 - k D a g r o u p II p h o s p h o l i p a s e A , m o n o c l o a a ! antibody-conjugated Sepharose column as described previously [23.24]. Preparation o f 1 4 - k D a g r o u p II 1 3 h o s p h o l i p a s e A 2 - s p e c i f i c p o l y c l o n a ' a n t i b o d y R 3 7 7 1231 a n d m o n o c l o n a t a n t i b o d y M D 7 . 1 [ 2 4 ] was described previously. Preparation of rat recombinant C3 a (rC3 0t; a p r o d u c t o f t h e E. c o i l l a c Z - r : l l C 3 d g ( c o m p l e m e n t C'~ d e g r a d i n g product [25]) gone) and Ihiclocin AI, both of which inhibited group II p h o s p h o l , . ' p a s e A , r a t h e r s p e c i f i c a l l y , a r e d e s c r i b e d e l s e w h e r e ( u n published data}_ 2.3. Trt,atmen: o f m a s t cells with p h o s p h o l i p a s e A : M a s t c e l l s w e r e s u s p e n d e d i n 10 m M T f i s - H C I b u f f e r ( p H 7 . 4 L which contained 150 mM NaCI. 3.7 mM KCL I mM CaCb, 0. 1% (w/v) glucose and 0.5% (w/v) gelatin (Sigma. St. Louis. MO) (Tris-gelatin buffer), and adjusted to ! x l0 s cells/ml. The oells were sensitized with I jug/ml mouse monoclonal anti-dinitrophenyl (DNP) lgE (Scikagaku Kogyo, Tokyo. Japan) for 30 rain at 37°C. The sensitized cells were washed, suspended in Tris-gelatin buffer at I x los cells/ml, and incub a t e d w i t h 1 4 - k D a g r o u p II p h o s p h o l i p a s e Az in the presence or a b s e n c e o f D S P - - c o n j u g a t e d A s c a r i s s u u m I D N P-Ascari,~ ( d o n a t e d b y Kissei Pharmaceutical, Matsumoto. Japzn). Alternatively, the sensitized cells were stimulated with DNP-Ascaris in the presence of 10 6 M lysoPS (Funakoshi, Tokyo, Japan). After incubation, the cells were centrifuged at 7~0 × g for 5 rain at 4°U to obtain the supernatant. PGD, released into the supernatant was measured using a PGD2 assay kit (~mersham, Buckinghamshire. UK). Histamine released into the supernatant was determined by a radioenzymatic assay using [~H]methyI-S-adenosyI-L-methionine (New England Nuclear, Boston, MA) and a crude preparation of rat ki,-lney histamine methyltransfer u s e [ 2 6 l . T h e p e r c e n t a g e r e l e a s e o f h i s t a m i n e w a s c a l c u l a t e d b y d i v i ,~i n g t h e a m o u n t in e a c h s u p e r n a t a n t b~ ~ h g t i n a p r e p a r a t i o n o f s o n i cated cells (Branson Sonifier, 20 pulses, setting 4, 50% pulse cycle). 2 . 4 . L i p i d analys£~ M a s t c e l l s (I x 10~ c e l l s / m l ) w e r e i n c u b a t e d f o r I h i n T r i s - g e l a t i n buffer cor.taining 1 gCi/ml [3H]arachidonate (Amersham), washed twice with Tris-gelatin buffer, sensitized with lgE and treated with l0 ~'g/ml 14-kDa group !! phospholipase A2 and I /dg/ml DNP-Ascaris in the presence or absence of 2 ~ggml thielocin A I for i 5 rain at 37°C. The total cellular lipids were extracted by the method of Biigh and Dyer [27] and separated by two-dimensional thin-layer chromatography on silica gel plates (Merck, Darmstadt, Germany,, which were developed with a slighl modification of the solvent s)~tem described b y E s k o a n d R a e t z I28]. B r i e f l y . t h e e x t r a c t e d l i p i d s w e r e r e c o n s t i t u t e d in a small volume of chloroform and applied to silica gel plates (approximately I x I 0 s cells per pla:e). The plales were developed first with chloroform/methanol/acetic acid (65:25: I 0, v/v). dried for 60 rain u n d e r a n a i r s t r e a m , a n d d e v e l o p e d in t h e s e c o n d d i m e n s i o n w i t h chloroform/methanol/88% formic acid (65:25:10.v/v). The spots which were visible after exposure to I, vapor were identified by comparing their positions with those of authentic standard phospholipids. The !, was then removed with an air stream, the individual spots were scraped off, and the radioactivity of each was measured.
3. R E S U L T S T h e e f f e c t o f t r e a t m e n t o f rat p e r i t o n e a l m a s t c e l l s w i t h p u r i f i e d rat 1 4 - k D a g r o u p II p h o s p h o l i p a s e A 2 o n their cellular functions were examined. When unstimulated mast cells were treated with the phospholipase A2 alone, PGD2 release into the supernatant was not augm e n t e d a p p r e c i a b l y ( F i g . I). W e t h e n e x a m i n e d t h e e f f e c t oF t h e p h o s p h o l i p a s e A z o n a n t i g e n - s t i m u l a t e d m a s t cells. No appreciable PGD2 generation was observed w h e n m a s t c e i l s w e r e first s e n s i t i z e d w i t h a n t i - D N P l g E 248
LETTERS
December
1991
5O
o
40
~
3o
o.
i O'
0
2
4
6
8
i0
P h o a p h o [ I p a a e A 2 (jJglml) F i g . I. E f f e c t o f c x t r a c e l l u l a r 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A, on PGD 2 generation by rat peritoneal mast cells. Mast cells sensitized with lgE were incubated with the indicated concentrations of 14-kDa g r o u p I I p h o s p h o l i p a , s e A 2 in t h e p r e s e n c e o f 1 ~ t g / m l D N P - , 4 s c a r i s ( Q ) , D N P - A s c a r i s p l u s I 0 6 M l y s o P S ( A ) o r in t h e i r a b s e n c e ( ~ ) f o r 15 r a i n at 37°C, and PGD 2 released into the supernatant was measured as d e s c r i b e d i n s e c t i o n 2_ T h e v a l u e s i n d i c a t e a v e r a g e s _+ S D ( n = 3 ) .
and then challenged with DNP-conjugated antigen in t h e a b s e n c e o f l y s o P S , w h i c h is a n e s s e n t i a l c o f a c t o r f o r full a c t i v a t i o n o f r a t p e r i t o n e a l m a s t c e l l s [ 2 0 - 2 2 ] . W h e n these IgE-sensitized, antigen-challenged cells were further treated with purified 14-kDa group li phosphoi'p a s e A2, c o n c e n t r a t i o n - d e p e n d e n t PGD2 generation w a s o b s e r v e d ( F i g . 1). T h e c o n c e n t r a t i o n s o f t h e e n z y m e p r o d u c i n g a n a p p r e c i a b l e e f f e c t ( r a n g i n g f r o m I t o 10 /~g/ml) were c o m p a r a b l e to those d e t e c t e d at v a r i o u s i n f l a m m a t o r y s i t e s [23,29]. P G D 2 g e n e r a t i o n w a s a l s o
A
50
50 .o
~
m
4o
30
30
20
20
! A
Q.
o
o
o.bt Antigen
o"1
i
o
(pgiml)
Fig. 2. E f f e c t o f a n t i g e n o o n c e n t r a t i o n s o n e x t r a c e l l u l a r 1 4 - k D a g r o u p !I phospholipase A2-mcdiatcd PGD2 release from mast cells. The sensitized mast cells were incubated with 14-kDa group |I phospbolipase A2 (6 ~g/ml) and the indicated concentrations of DNP-A~rcaris f o r 15 m i n a t 3 7 ° C , a n d P G D 2 ( O ) o r h i s t a L ~ i n e ( o ) rel~L~4~d i n t o t h e supernatant was measured as described in section 2. The values indic a t e a v e r a g e s _+ S D ( n = 3 ) .
V o l u m e 294, n u m b e r 3
1 3 e c e m b e r 1991
FEBS LETTERS
50
T..~.~
~
PC
50
PE
P/
in
40
~
50
40
20
T---3o
30
=0
2o
_=
l(]
45
15 5
4G
1
m a.
10
0
tO
0
5
10
Time
"15
0 0
0
cells were treated with 14-kDa g r o u p II phosphoIipas¢ A: (6 jug/ml) (~'!.-~les) o r lysoPS (10 6 M) (triangles) in the presence o f D N P - , 4 s c a r i s (l gtg/ml) for the indicated period at 37°C, a n d el,her PCiD: (closed symbols) o r histamine (open symbols) relea.~:d i,~..o the s u p e r n a t a n t was m e a s u r e d as described in section 2. T h e values indicate averages + S D (n=3). dependent on the concentration o f t h e a n t i g e n ( F i g . 2). VChen the cells were challenged with 1Mg/ml antigen and treated with more than 7.5 ~ug/ml enzyme, about 50 ng o f P G D 2 p e r 106 c e l l s w a s g e n e r a t e d , a l m o s t t h e s a m e amount as that generated by fully activated mast cells; mast cells stimulated with 1 /lg/ml antigen in the prese n c e o f 10 -6 M l y s o P S g e n e r a t e d a b o u t 5 0 n g o f P ( ~ D 2 p e r l 0 s c e l l s ( F i g . 1). I t s h o u l d b e n o t e d h e r e t h a t n o further enhancement effect of exogenous 14-kDa group II phospholipase A 2 on Pg~D 2 generation was observed with cells which were fully activated by treatment with antigen plus lysoPS. In contrast to P(JD2 generation, release of histamine Table i Effect o f 14-kDa g r o u p II p h o s p h o l i p a s e A2-specific inhibitors o n extracellular 14-kDa gro~tp II p h o s p h o ! i p a s e Aa-mediated Pg~D2 release f r o m mast cells
~Io t r e a t m e n t Phospholipas¢ A 2 + A n t i b o d y R377 (40/t/g/ml)* + rC30t (2/Jg/rnl)* -t Thielocin A i {2 gtg/ml)* + A n t i b o d y IMD7.1 (15 ~ug/ml)**
10
lS
%
I~GD2 released (ng/I O* cells)
Inhibition (%)
2.1 _ + 0 . 8 3 8 . 0 -+ 1.9
2.0 3.6 2.6 37.0
± ± ± ±
~.! 1.4 0.4 2.5
;,
,o
PA
PS
(mini
F i g . 3. T i m e - c o u r s e o f I ~ G D 2 g e n e r a t i o n b y r ~ a s t c e l l s t r e a t e d w i t h extra~llular 1 4 - k D a g r o u p I | p h o s p h o l i p a s e A 2. T h e s e n s i t i T ~ d m a s t
Treatment
S
g
100 90.5 94.7 2.6
T h e e n z y m e was p r e i n c u b a t e d with the indicated a m o u n t s o f each inhibitor for 30 min at 22°C. Then l g E - a n t i g e n - p r i m e d m a s t eel Is were treated with the inbibitor-treated enzyme (~. jug/ml) and i~GD 2 released into t h e s u p e r n a t a n t was quantified as described in Section 2. Values indicate averages _+ S D (n=3). *Sufficient c o n c e n t r a t i o n t o inhibit p h o s p h o l i p a s e A 2 activity. **Sufficient c o n c e n t r a t i o n t o inhibit the in'.eraction between p h o s p h o lipase A 2 a n d heparin.
Arachidonic
15
15
20
10
1'0
15
Acid
/
10
lb
l's
is
iols "lime
5
"io
is
(rain)
Fig. 4. Time course o f phospholipid metabolism o f [~H]arachidonatelabeled mast cells after treatment with extracei]ular 14-kDa g r o u p II p h o s p h o l i p a s e A=. T h e procedure is described in :~ection 2. Values indicate p h o s p h o l i p i d c o m p o s i t i o n (average (%) ± SE') o f each sample (n=3). from mast cells was not affected by exogenous phospholipase A2 appreciably; mast cells released only about 10~ of cellular histamine (the same as the basal level) even in the presence of 14-kDa group II phospholipase A2 ( F i g . 2). The time course of PGD2 generation in4uced by antigen plus 14-kDa group II phospholipase -~'2 w a s r a t h e r different from the one induced by mtigel, plus lysoPS. The generation of PGD2 obserxed in the presence of 14-kDa group !I phospholipase A2 progressed almost l i n e a r l y w i t h i n 1 5 r a i n ~ f t e r e n z y m e c h a l l e n g e ( F i g . 3). On the other hand, PGD2 generation by cells supplemented with antigen plus lysoPS reached a plateau approximately 5 min after antigen challerige, paralleiling histamine release. The effect of exogenous 14-kDa group I! phospholipase A2 on PGD2 generation by mast cells was abolished by pretreatment of the enzyme with 14-kDa group II phospholipase A2-specitic inhibitors, such as antib o d y R 3 7 7 , t h i e i o c i n A I a n d r C 3 r, ( F~-.ble I). A m o n o clonal antibody MDT.1, which recognizes the heparinbinding domain of rat 14-kDa group II phospholipase A2 b u t d o e s n o t i n h i b i t e n z y m e a c t i v i t y [24], s h o w e d n o 249
V o l u m e 294. n u m b e r 3
FEBS LETTERS
D e c e m b e r 1991
Table I i Phospholipid metabolism of I~H]arachidonate-labeled mast cells after treatment with extracellular 14-kDa group II phospholipase Az Phospholil)ids
Treatment with phospholipase A~
No treatment Intact cells
Antigen-treated cells
Thielocin AI ( - ) Sphingomyelin Phosphatidylcholine Phosphatidylinositol Phosphatidylserine Phosphatidylethanolamine Phosphatidic acid Free arachidonate
0.7 51.0 16.5 4.3 I'Ll 3.1 11_2
__ 0.2 _+ 0.2 _+ I_1 _+ 0.4 4- 0.1 _+ 0.1 _4-0_6
0.8 51).3 15_8 4.0 13.9 3.0 10.8
± 0.1 ± 1.5 ± 0_3 + 0.5 _+ 0.8 _+ 0.5 _+ 0.4
Thieloein
t.0 39.9 14.7 4.1 15.3 4.9 20.3
-~ 0.3 ± 2.2 _+ !.1 _+ 1.0 -+ 0.3 + 0. I _+ 2.0
1.0 50.6 15.6 4.0 13.7 3.7 I 1.6
AI
(4-)
+ 0.1 +- 1.2 + 0.0 _+ 0.4 _+ 0.0 -+ 0.2 _+ 1.0
The procedure is described in Section 2. Values indicate phospholipid composition (average (%) _+ SD) of each sample (n=3). inhibitory effect, suggesting that enzymatic activity of g r o u p 11 e n z y m e m a y b e e s s e n t i a l f o r t h e g e n e r a t i o n o f P G D ~ in t h i s r e a c t i o n . The alteration of the membrane phospholipids in mast cells by treatment with the exogenous 14-kDa g r o u p l I p h o s p h o l i p a s e A2 w a s n e x t e x a m i n e d . W h e n [3H]arachidonate-!abeled cells were exposed to 14-kDa g r o u p I1 p h o s p h o l i p a s e A 2 i n t h e p r e s e n c e o f t h e a n t i gen, o n l y the r a d i o a c t i v i t y in p h o s p h a t i d y l c h o l i n e ( P C ) decreased drastically (approximately 50% to 40%) with a c o n c o m i t a n t i n c r e a s e in r a d i o a c t i v i t y o f f r e e a r a c h i d o n a t e ( a p p r o x i m a t e l y 1 1 % t o 2 0 % ) ( F i g . 4, T a b l e I1). T h e hydrolysis of PC and thereby the increment of free arachidonate were suppressed almost completely by pret r e a t m e n t o f t h e e n z y m e w i t h its s p e c i f i c i n h i b i t o r , t h i e l o c i n A I. N o a p p r e c i a b l e h y d r o l y s i s o f P C w a s o b s e r v e d when intact mast cells were treated with the enzyme u n d e r t h e s a m e c o n d i t i o n s ( T a b l e I I ) . T h e r e f o r e , 14k D a g r o u p !I p h o s p h o l i p a s e A 2 a p p e a r e d t o h a v e h y d r o l y z e d P C in t h e m e m b r a n e o f a n t i g e n - p r i m e d m a s t cells. 4. D I S C U S S I O N In the present study, we demonstrated that mammalian 14-kDa group Ii phospholipase A 2 added exogenously to lgE-antigen-primed rat peritoneal mast cells a u g m e n t e d e i c o s a n o i d g e n e r a t i o n in t h e cells. T h e c o n centratio,~s of the enzyme required for the augmentation (I-10 ~g/ ml) were within the range detected at v a r i o u s i n f l a m m a t o r y s i t e s [22.29], i n d i c a t i n g t h a t e x t r a c e l l u l a r 1 4 - k D a g r o u p II p h o s p h o l i p a s e A z m a y c o n tribute to the progression of inflammation, especially allergic reaction, in which mast cells play important roles as effector cells, by hydrolyzing mast cell phospholipids to generate precursors of pro-inflammatory lipid mediators. 1 4 - k D a G r o u p I I p h o s p h o l i p a s e A2 w a s a b l e t o i n fluence PGD2 generation only when IgE-receptors on the surfaces of mast cells were cross-linked by multivalent antigens. The activity of several phospholipases A 2 o n c e l l m e m b r a n e s is k n o w n t o b e a f f e c t e d b y l i p i d 250
p a c k i n g in t h e o u t e r l e a f l e t o f t h e p l a s m a m e m b r a n e [30]. C r o s s - l i n k i n g o f l g E r e c e p t o r s m a y c h a n g e t h e m o l e c u l a r p a c k i n g o f l i p i d s [3 !], m a k i n g t h e m s u s c e p t i b l e t o e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s e A : . T h e s e o b s e r v a t i o n s a r e in a c c o r d w i t h o u r p r e v i o u s data, which showed that mammalian 1 4 - k D a g r o u p I1 as well as group I phospholipase A: added exogenously augmented the generation of PGE~ by HL-60 granuloc y t e s o n l y i n t h e c o - p r e s e n c e o f A 2 3 1 8 7 [32]. W e a l s o r e p o r t e d t h a t i n j e c t i o n o f p u r i f i e d 1 4 - k D a g r o u p I1 p h o s p h o l i p a s e A2 i n t o the h i n d p a w o f r a t s w i t h a d j u vant arthritis exacerbated the eOema, whereas no effect w a s o b s e r v e d i n n o r m a l r a t s [29]. T h u s , e x p r e s s i o n o f the nharmacological activity of the exogenous 14-kDa g r o t p I I p h o s p h o l i p a s e A2 m a y r e q u i r e a c e r t a i n s t a g e of ongoing inflammation induced by some other factors. A c h a n g e in t h e t r a n s m e m b r a n e distribut;on of phospholipids may be one of the features of such an a c t i v a t e d s t a t e . R e c e n t l y , B o m a l a s k i e t a l . [33] d e m o n strated that purified recombinant human 14-kDa group 11 p h o s p h o l i p a s e A 2 e l i c i t e d a d r a m a t i c i n f l a m m a t o r y , arthritogenie response when injected into the joint space of healthy rabbits, although they also stated that the e n z y m e w a s c o m p l e t e l y i n a c t i v e in t h e p a w e d e m a i n flammation assay using normal rats. Although this disc r e p a n c y m i g h t be e x p l a i n e d b y t h e d i f f e r e n c e in t h e species employed, further work must be performed to clarify the molecular mechanisms of the pro-inflammatory effect of exogenous 14-kDa group II phospholipase A2. Mammalian 1 4 - k D a g r o u p II p h o s p h o l i p a s e A z hydrolyzes phosphatidylethanolamine (PE) or phosphatid y l s e r i n e (PS) m o r e efficiently t h a n P C in a n in v i t r o assay system [2-10]. The treatment of mast cells with e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s ¢ A2 r e s u l t e d i n p r e f e r e n t i a l h ? / d r o l y s i s o f m e m b r a n e P C . I t is k n o w n t h a t P C is d i s t r i b u t e d i n t h e o u t e r l e a f l e t o f p l a s m a bilayer membrane of mammalian cells, whereas both PE and PS exist almost exclusively in the inner leaflet [34]. T h u s , o n l y P C m i g h t b e a v a i l a b l e f o r e x t r a e e l l u l a r 1 4 - k D a g r o u p I I p h o s p h o l i p a s e A 2. A l t e r n a t i v e l y , t h e
V o l u m e 294, n u m t m r 3
FEBS
g r o u p I! e n z y m e m i g h t a c t i v a t e s o m e o t h e r c e l l u l a r phospholipase(s) A2 which liberate arachidonate from PC. We found that ~last cells expressed an arachidonate-preferential cytosolic 85-kOa phospholipase Az [35]. a e D N A c l o n e o f w h i c h w a s r e c e n t l y i s o l a t e d f r o m U 9 3 7 c e l l s [36]. a n d s h o w e d t h e p o s s i b i l i t y t h a t t h e e n zyme might play an essential role in araehidonate metabolism upon application of mast cells with immunochemical stimuli (antigen plus lysoPS) via the lgE recept o r [37]. T h e p o s s i b i l i t y t h a t e x o g e n o u s 14-kDa group II p h o s p h o l i p a s e A2 activates intracellular phospholipase A2 such as araehidonate-preferential phospholipase A z cannot be ruled out at present. LysoPS is a potentiazor of the degranulation as well as PGD2 generation by rat peritoneal mast cells [20-22]. One possible mechanism whereby exogenous t4-kDa g r o u p II p h o s p h o l i p a s e A 2 m a y e x e r t i t s e f f e c t is t o generate iysoPS by hydrolysis of membrane PS. However, this possibility can be eliminated for the following r e a s o n s : ( i ) h i s t a m i n e r e l e a s e wa:; n o t i n d u c e d b y e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p c , s e A2. i f l y s o P S w a s supplied by this exogenous enzyme, then histamine release might also be induced in the same manner as P G D ~ g e n e r a t i o n ; (ii) n o a p p r e c i a b l e h y d r o l y s i s o f P S was observed when cells prelabeled with radiolabeled arachidonate w e r e e x p o s e d t o t h e 1 4 - k O a g r o u p II phospholipase A2 in the presence of antigen: (iii) stimul a t i o n o f m a s t c e l l s w i t h a n t i g e n p l u s l y s o P S is a c c o m p a nied by a rapid and drastic breakdown of phosphatidyl i n o s i t o l ( P I ) [38], w h e r e a s n o a p p r e c i a b l e r a p i d d e c r e a s e i n r a d i o a c t i v i t y in P l w a s o b s e r v e d w i t h m a s t ceils treated with antigen and the 14-kDa group lI phosp h o l i p a s e A 2 ( F i g . 4). It c a n b e c o n c l u d e d t h a t e x o g e n o u s 1 4 - k D a g r o u p II p h o s p h o l i p a s c A 2 a c t e d o n l g E antigen-primed, but not fully activated mast cells to generate eicosanoids, resulting in the progression of allergic inflammation. T h e s o u r c e o f t h e e x t r a c e l l u l a r 1 4 - k D a g r o u p II p h o s p h o l i p a s e A 2 d e t e c t e d a t i n f l a m e d s i t e s is s t i l l u n i d e n t i fied. it should be noted that activated mast cells also s e c r e t e ! 4 - k D a g r o u p I! p h o s p h o l i p a s e A 2 ( u n p u b l i s h e d data). However, the amount of the enzyme released from activated mast cells is too small (less than i ng per l06 cells) to act on IgE-antigen-primed mast cells to generate a detectable level of eicosanoids. Therefore, 1 4 - k D a g r o u p l I p h o s p h o l i p a s e A~ m i g h t b e s u p p l i e d b y ceils other than mast ceils. Aeknowled~,~ments: T h i s w o r k w a s s u p p o r t e d b y G r a n t s - i n - A i d f o r Scientific Research (Nos. 01639509, 02557090 and 03680163) from the Ministry of Education, Science and Culture of Japan_
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