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FEB S Letters 376 (1995) 164-166 FEB S 16337

Th e ph orb ol 12-myris tate 13- acetate (PM A )- indu ced oxidat ive burst in ratper i toneal neutrophi l s i s increased by a 0 .1 mT (60 Hz) magnet ic f ie ld

S a s h w a t i R o y a, Y a s u k o N o d a a, V a l o r ie E c k e r t b , M a r e t G . T r a b e r a, A k i t a n e M o r P ,R o b e r t L i b u r d y b, L e s t e r P a c k e r a '*

aDepartment of Molecular and Cell Biology, 251 LS A, University of C alifornia, Berkeley, CA 94 720-3200, USAbLawrence Berkeley National Laboratory, Life Sciences Division, Berkeley, C A 94 720, USARece ived 13 Octo ber 1995; revised vers ion received 23 Oct ober 1995

Abstr ac t M agne t i c fi el ds (M F ) m ay af fe c t b i o l og i c a l sys te m sby increasing free radical concentrations . To test this , we havei nve s t i gate d whe the r l ow fr eque nc y (60 Hz ) l ow i n te ns i ty (0 .1 r oT )M F c a n m o d u l a t e t h e p h o r b o l 1 2 - m y r i s t a t e 1 3 - a c e t a t e ( P M A )induced respiratory burst in pr imed rat per i toneal neutrophi l s ,fol lowed in real t ime using the dye 2' ,7' -dichlorofluoresc in(D CF H) , w hi ch r e ac t s wi th f r e e rad i ca l -de r ive d ox i dants suc h asH2 02 (whi c h i s for m e d fr om the d i sm utat i on o f supe r oxi de ) tobeco me 2',7' -dichlorofluorece in (D CF ), a highly f luorescent com -pound . In the pr e se nc e o f the M F , a 12 .4% i nc r e ase in the f luor es -c e nc e s i gnal was obse r ve d i n P M A-s t i m ul ate d ne utrophi ls (n = 5 ,P < 0 .02 , 18 pa i r s o f m e asur e m e nts ) . W e be l i e ve th i s r e pr e se ntsthe f ir s t e xpe r i m e nta l ob se r vat i on o f M F i n fl uenc ing e ve nts i n -volving free radical spec ies generated during s ignal transductionin l iving cells.K e y w o r d s . M a g n e t i c f i e l d s ; F r e e r a d i c a l s ; N e u t r o p h i lr e s p i r a t o r y b u r s t

I. IntroductionA v a r i e t y o f b i o l o g i c a l e f f e c ts h a s b e e n a s s o c i a t e d w i t h e x p o -

s u r e t o l o w f r e q u e n c y e l e c t r o m a g n e t i c f i el d s ( E M F ) , a n d h a v eb e e n p r o p o s e d t o b e d u e , a t l e a s t i n p a r t , t o m a g n e t i c f i e l d( M F ) - i n d u c e d i n c r e a s e s i n f re e r a d i c a l c o n c e n t r a t i o n s [ 1 ,2 ]. M Fe f f e c t s o n t h e r a t e o f r a d i c a l p a i r ( R P ) r e c o m b i n a t i o n i s at h e o r e ti c a l ly s o u n d m e c h a n i s m b y w h i c h M F i n t e ra c t w i t h b io -l o g i c a l s y s t e m s [ 2 ] , a n d i s w e l l - e s t a b l i s h e d i n v i t r o i n o r g a n i c -b a s e d m e d i a [ 3 ,4 ]. M F i n c r e a s e t h e p r o b a b i l i t y t h a t a f r e e r a d -i c a l p a i r w i l l r e m a i n i n t h e t r i p l e t c o n f i g u r a t i o n ( b y d e c r e a s i n gi n t e r s y s t e m c r o s s i n g ) , t h u s i n c r e a s i n g t h e p r o b a b i l i t y t h a t t w of r e e r a d i c a l s w i ll e s c a p e w i t h o u t t e r m i n a t i o n . B e c a u s e f e w e rt e r m i n a t i o n s o f r a d i c a l p a i r s o c c u r , t h e o v e r a l l c o n c e n t r a t i o no f r a d ic a l s i n c r e a s e s, a n d a p o t e n t i a t i o n o f f r e e - r a d i c a l i n d u c e de f f e c ts i n b i o l o g i c a l s y s te m s m a y b e e x p e c t e d [ 2] ; b o t h t i m e -v a r y i n g a n d s t a t ic m a g n e t i c f i e l d s c a n p a r t i c i p a t e i n s u c h i n te r -a c t i o n s [ 5 ] .

W e e v a l u a t e d w h e t h e r l o w f r e q u e n c y ( 6 0 H z ) , l o w - i n t e n s i t y(0 .1 r o T ) M F c a n m o d u l a t e t h e P M A i n d u c e d r e s p i ra t o r y b u r s to f fr e e r a d ic a l g e n e r a t i o n i n p r i m e d r a t p e r i t o n e a l n e u t r o p h i l s .T h e g e n e r a t i o n o f r e a c t iv e o x y g e n s p e c i e s ( R O S ) i n c l u d i n g s u -p e r o x i d e ( O ~- ) b y t h e p la s m a m e m b r a n e N A D P H o x i d a se o fn e u t r o p h i l s i s a m a j o r m e c h a n i s m o f b a c t e r i a l k i l li n g [6 ] . T h eO ~ - t h u s f o r m e d e n z y m a t i c a l l y d i s m u t a t e s t o h y d r o g e n p e r o x -i d e ( H 2 0 2 ) t h r o u g h c a t a l y s is b y s u p e r o x i d e d i s m u t a s e , o r i s

*Cor r es pond ing au tho r .

c o n v e r t e d t o o t h e r r e a c t i v e o x y g e n i n t e r m e d i a t e s s u c h a s h y -p o c h l o r i t e b y t h e m y e l o p e r o x i d a s e r e a c t i o n [ 7] .

T h e n e u t r o p h i l r e s p i r a t o r y b u r s t o f o x i d a n t s w a s a s sa y e d i nr e a l -t i m e u s i n g 2 ' , 7 '- d i c h l o ro f l u o r e sc i n d i a c e t a t e ( D C F H - D A ) ,w h i c h d i f f u s e s i n t o c e l l s , w h e r e i t i s c l e a v e d b y n o n - s p e c i f i ce s t e r a se s t o f o r m 2 ' , 7 ' - d i c h l o r o f l u o r e s c i n ( D C F H ) , w h i c h r e-a c t s w i t h f r ee r a d i c a l - d e r i v e d o x i d a n t s ( e . g. H 2 0 2 , f o r m e d f r o mt h e d i s m u t a t i o n o f O ~ -) a n d i s c o n v e r t e d t o t h e h i g h l y f l u o re s -c e n t c o m p o u n d 2 ' ,7 ' - d ic h l o r o fl u o r e s ce i n ( D C F ) . H e n c e , ac h a n g e i n t h e f l u o r e s c e n t s i g n a l i n t h e p r e se n c e o f a M F w i l li n d i c a t e a n i n f lu e n c e o f M F o n o x i d a n t g e n e r a t i o n [ 8] .

2 . M a t e r i a l s a n d m e t h o d s2.1. Preparation of prim ed neutrophilsF or each ba tch o f neu t roph i ls , tw o S p rague D aw ley male r a ts ( 200 -250 g b .w.) were anes thet ized with ether and injected intraper i toneally( i .p . ) with 30 ml of s ter i le 2% casein dissolved in phosphate-bufferedsal ine, pH 7.4 (PBS). The rats were al lowed to rec over and were sacr i-f iced 12-14 h af ter in ject ion. Pr imed neutrophils were recovered f romthe pe r i tonea l cav i ty by l avage w i th 60 ml o f w arm (37C) P BS . P e r i to -nea l b leed ing w as avo ided . T he ce ll s w ere poo led fo l low ing cen t r i f uga-t ion for 5 min at 200 x g , washed three t im es with ice-cold PBS, resus-pended in a vo lume o f 1 -2 ml o f i ce -co ld PBS , and kep t on i ce p r io rto the as say . T h i s p rocedu re p roduces ce l l p r epara t ions w h ich con ta in80 95% neutrophils [9 ,10] .2.2. Respiratory burst assay with the DCFH-DA during exposure to0.1 mT (60 H z) electric f ieldsA s tock s o lu t ion o f D C F H -D A (10 r aM ) in e thano l w as p r epared ,pu rged w i th n i t r ogen and s to r ed a t - 20 C . N eu t roph i l s ( 106 ce ll s/ml )w ere added to the r eac t ion mix tu re (10 /~M D CF H -D A in P BS ) to af ina l vo lume o f 1 .036 ml . The r eac t ion mix tu re , w i thou t P M A , w asmixed in a cuve t t e , then p laced in a the rm o jacke ted cuve t t e -ho lde r andb rough t to 37C in a com pu te r - con t ro l l ed , LS -5B P erk in -Elm er LS -5Bdua l mon och rom ato r s pec t ropho to f luo r imete r . The t empera tu r e o f th i scuve t t e w as main ta ined a t 37 + 0.05C th roughou t . F luo res cence w asrecorde d us ing two d if ferent excitat ion waveleng ths (Ex, 480 or 502 nm;Em 530 nm) . P M A (50 nM ) w as added to the cuve t t e , and r eco rd ingsw ere begun . A n a pp rox ima te 10 (~200 s lag t ime w as no t i ced be fo re anexponential increase in f luorescence was observed.

The cuv ette was or was not exposed to a 0 .1 mT (rms) s inusoidal 60H z m agne t i c f i eld genera ted by a H e lm ho l tz co i l o r i en ted s o the app l i edmagnetic f ield vector was paral lel to the long axis of the cuvette . The60 H z s igna l w as p rov ided f rom a s t andard s igna l genera to r and f edunampl i f i ed in to the H e lm ho l tz co i l. The M F t r ea tm en ts and co n t ro l(no M F ) t r ea tmen ts in f ive s epara te exper imen ts w ere s equen t i a l lyal ternate d ove r a to tal of 18 runs . F or contro l exposures the coil wasno t ene rg ized and the 60 H z m agne t i c f ie ld in tens i ty a long the long ax i s(ver tical axis ) of the cuvette , as well as for the two oth er or tho gon alaxes , was

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S . R o y e t a l . /F E B S Le t t e r s 3 76 ( 1 9 9 5 ) 1 6 4 - 1 6 6 16 5u m e o f ~ 1 0 m m i n l en g t h a n d 4 m m i n d i a m e t e r a n d w a s u s e d f o rm e a s u r e m e n t s a l o n g t h e c u v e t t e lo n g - a x is . T h e t r a n v e r s e p r o b e ( m o d e lF T G 1 - 0 4 1 5 ) i n t e g r a t e s o v e r a r e c t a n g u l a r -s h a p e d a r e a o f ~ 1 0 m m 4 m m .2 .3. S ing le ce l l measur ements o f ox idan t genera t ionA D e l t a S c a n s y s t e m ( P h o t o n T e c h n o l o g ie s I n c . N e w B r u n s w i c k , N J )w a s u s e d f o r s i ng l e -c e l l f l uo r e s c e nc e pho t om e t r y . Th i s s ys t e m ha s ac o m p u t e r - c o n t r o l l e d , d u a l e x c i t a t i o n m o n o c h r o m a t o r c o u p l e d v i a af i be r - op t i c l i gh t gu i de t o a N i kon D i a pho t i nve r t e d mi c r os c ope . C e l l sw e r e t r e a te d i n a te s t t u b e a t 3 7 C w i t h D C F H - D A a n d t o P M A a sde s c r i be d a bove f o r t he c on t r o l c uve t t e e xpe r i me n t s , t he n t r a ns f e r r e di n t o a 35 mm t i s s ue c u l t u r e p l a t e a nd ma i n t a i n e d a t 37 C us i ng a f o r c e dw a r m a i r c u r t a i n ( S a ge I n s t . , W hi t e P l a i n s , N Y ) a nd a he a t i ng s t a ge( Me d i c a l S ys t e ms C or p . , G r e e nva l e , N Y ) . S i ng l e c e l ls w e r e v i s ua li z e da t 100 x u s i ng a t he r mo s t a t t e d ob j e c t i ve (37 C ) a nd f l uo r e s c e nc e w a sm o n i t o r e d u s i n g t w o e x c i t a t io n w a v e l e n g t h s (E x 48 8 n m a n d 5 0 2 n m ;N i ko n d i c h r o i c c ube w i t h l ong - pa s s F I T C f i lt e r ) f o r a s i ng l e c e l l ove rt i me . D ur i ng t he s ing le -c e ll m e a s u r e m e n t s , da t a a c qu i s i t i on w a s pa us e dbr i e fl y t o r e - pos i t i on t he ma s k i ng ba r r i e r s t o t he pho t om e t e r ( t he s t a gew a s n o t m o v e d ) t o m o n i t o r t h e a r e a i m m e d i a t e l y a d j a c e n t t o , a n dw i t h i n one c e ll d i a me t e r , o f t he o r i g i na l c el l. D a t a a c qu i s i t i on w a spa us e d a f i na l t ime t o r e - pos i t i on t he o r i g i na l c e ll f o r c on t i nue d f l uo r e s -c e nc e mon i t o r i ng . T he s t a t i c ma gne t i c fi e ld ve c t o r s ( 3 o r t hog ona l a xe s)a t t he pos i t i on o f t he ce ll s on t he mi c r os c ope s t a ge i mme d i a t e l y a bovet he 100 x ob j e c t i ve w e re a pp r ox i m a t e l y 0 . 02, 0 . 06 a n d 0 . 08 r oT . Thea m bi e n t ma gn e t i c fi el d s, w i t h a l l e qu i p me n t on , i n t he s a me o r t h ogo na lp o s i t i o n s w e r e < 0 . 0 2 m T .2 .4. S ta t is t ic a l analysisThe r e s u l t s o f f ive g r oups o f e xpe r i me n t s w e r e a na l yz e d . O n 5 s e pa -r a t e o c c a s io n s n e u t r o p h i l s w e r e o b t a i n e d ; t h e n u m b e r o f p a i r s o f + M Fs t i mu l a t e d r e s p i r a t o r y bu r s t f o r e a c h e xpe r i me n t w e r e n = 3 , 5 , 5 , 2 a nd3 f o r a t o t a l o f 36 s e pa r a t e obs e r va t i ons . The f l uo r e s c e nc e w a s me a s -u r e d c o n t i n u o u s l y f o l lo w i n g a d d i t i o n o f P M A . T h e d a t a f r o m t h e f i rs t2 0 0 s o f ' l a g ti m e ' w e r e d i s re g a r d e d a n d t h e d a t a f r o m t h e r e m a i n i n gt i me po i n t s ( e ve ry 6 s up t o 500 s ) w e r e f i tt e d t o a n e xpon e n t i a l c u r veu s i n g a c o m p u t e r g r a p h i c s p r o g r a m ( D e l t a G r a p h P r o f e s s i o n a l 3 . 5 ;D e l t a P o i n t I nc . ) . The c o r r e l a t i on c oe f f i c ie n t s w e r e >0 . 9 f o r a l l o f t he36 s a mpl e s . The s l ope s o f t he f i t t e d c u r ve s o f t he pa i r e d c ompa r i s onso f + M F a n d - M F w e r e c o m p a r e d u s i n g a c o m p u t e r s t at i st i cs p r o g r a m( S u p e r A n o v a ; A b a c u s C o n c e p t s ; A N O V A 1 - w i th i n f a c to r ) . T h e a v e r -a g e v a lu e f o r + M F a n d f o r - M F w a s c a lc u l a te d f o r e a c h d a y ' s e x p e r-i m e n t s ; t h e n t h e se p a i rs ( + M F a n d - M F ) w e r e u s e d t o e v a lu a t e th ee f f e c t o f MF on r e s p i r a t o r y bu r s t ( de g r e e s o f f r e e dom = 4 ) .

1000

A 8 0 0 -~ 0 ~O . ~ _e . t - = 6 0 0 -0I t . . . I t . . .o 4 0 0 -

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. . . - / , , , ,- .

o oo oo o

o o-o -

. . - . . . -

-M F

I0 2 0 0 4 1~ 0 6 0 0T i m e ( s e c )

F i g . 1 . R e a l -t i m e a s s a y o f o x i d a n t g e n e r a t i o n f o l lo w i n g P M A i n d u c e dr e s p i r a t o r y bu r s t i n ne u t r oph i l s . The f l uo r e s c e nc e s igna l o f D C F i n t hep r e s e nc e o r a bs e nc e o f MF ( 0 .1 mT, 60 H z ) . C on d i t i on s a r e de s c r i be di n s e c t i on 2 . O ne r e p r e s e n t a t i ve e xpe r i me n t o f 18 pa i r s i s s how n .

o 1 0g 8 -O"E- - ~ = 6 -Q ~ 4 ~

I f ~ I t1 2 3 4 5 ove ra l lE x p e r i m e n t1 + M F [ ~ - M F

F i g . 2 . R a t e o f c ha nge i n t he f l uo r e sc e nc e s i gna l o f D C F i n r a t pe r i t o -n e a l n e u t r o p h i l s s t i m u l a t e d w i t h P M A o v e r t i m e i n t h e p r e s e n c e o ra bs e nc e o f MF ( 0 . 1 mT, 60 H z ) . The me a n o f e a c h da y ' s e xpe r i -me n t s + S . D . , a s w e ll a s t he ove r a l l me a n + S . D . , a r e s how n , n = 3 , 5 ,5 , 2 , a nd 3 f o r da ys 1 t h r ough 5 , re s pe c ti ve l y .

3 . R e s u l t s

V a r i o u s d o s e s ( 0 . 0 1 - 5 0 0 n M ) o f P M A w e r e te s t e d in t h er e a c t i o n m i x t u r e i n t h e a b s e n c e o f ex t e r n a l ly a p p l i e d M F . T h eD C F f l u o r e sc e n c e s ig n a l i n c r e a s e d w i t h i n c r e a s i n g P M A u p t o5 n M ; n o f u r t h e r i n c r e a s e s w e r e o b s e r v e d w i t h g r e a t e r d o s es o fP M A ( d a t a n o t s h o w n ) . A 1 0 - fo l d g r e a t e r f i n al c o n c e n t r a t i o no f P M A w a s u s e d in a l l M F e x p e r i m e n t s t o p r o d u c e a m a x i -m u m o x i d a t i v e b u r s t. T h e r e f o r e , t h e e ff e ct s o f M F o n o x i d a t i v eb u r s t o b s e r v e d i n o u r e x p e r i m e n t s a r e n o t d u e t o v a r i a t i o n s i nt he a m o u n t o f P M A a d d ed .

F i g . 1 i l l u s t r a t e s a t y p i c a l ti m e c o u r s e f o r D C F f l u o r e s c e n c es i g n a l f o l l o w i n g a c t iv a t i o n w i t h P M A ( 5 0 n M ) i n t h e a b s e n c eo r p r e s e n c e o f a 0 .1 m T ( 6 0 H z ) M F . W e o b s e r v e d a 1 2 .4 %( P < 0 . 0 2 ) i n c r e a s e i n t h e e x p o n e n t i a l r a t e o f i n c r e a s e i n t h ef l u o r e s c e n c e s i g n a l i n t h e p r e s e n c e o f t h e M F ( F i g . 2) .

T o d e t e r m i n e t h e l o c a t i o n o f o x i d a n t s w h i c h i n t e r a c t e d w i t hD C F H , w e p e r f o r m e d m e a s u r e m e n t s i n si n g le c el ls . W h e n f l u o -r e s c en c e d e t e c t i o n w a s f o c u s s e d u p o n a s i n g le n e u t r o p h i l , aP M A - i n d u c e d i n c r e a s e in f l u o re s c e n c e w a s o b s e r v e d . H o w e v e r ,t h e f l u o r es c e n c e o f t h e b u f f e r i m m e d i a t e l y a d j a c e n t t o t h e c e l l( w i t h i n o n e c e ll d i a m e t e r ) w a s s i m i l a r to b a c k g r o u n d f l u o re s -c e n c e p r i o r t o P M A s t i m u l a t i o n ( F i g . 3) . T h e s e d a t a s h o w t h a tt h e e v e n t s r e c o r d e d i n t h e se e x p e r i m e n t s w e r e i n t r a c e l l u la r i no r i g i n .

I n c o n t r o l e x p e r i m e n t s , t h e e f fe c t o f M F o n t h e r e a c t i o n o fD C F a n d H 2 0 2 a l o n e w a s d e t e rm i n e d . T h e e s t e r f o r m o f D C F( D C F H - D A ) c a n n o t r e a c t w i t h H 20 2 ; th e r ef o r e, h o m o g e n a t e so f n e u t r o p h i l s w e r e p r e p a r e d a s a s o u r c e o f e s t e ra s e a c t i v i ty t oh y d r o li z e t h e D C F H - D A t o D C F . U s i n g a c o n c e n t r a t i o n o fH 2 0 2 o f 1 0 / z M , w h i c h p r o d u c e d a n o p t i m u m r a t e o f f l u o re s -c e n c e, w e c a r r i e d o u t t h r e e e x p e r i m e n t s i n t h e p r e s e n c e o f t h eh o m o g e n a t e i n t h e ab s e n c e o r p r es e n c e o f t h e s a m e M F u s e di n o u r i n t a c t n e u t r o p h i l e x p e r i m e n t s . T h e r e w a s n o d i f f e re n c e

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166 S. Roy et al . /FE BS Letters 376 (1995) 164-166

3 1= = 2 -O

1

Single-cel l , 100xPMA induced respiratoryburst. 37C Re tur n tosam e cell

B j onUfferl?d~ahiSeeI I ]200

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m

i i0 400 600 800 1000T i m e ( s e c o n d s )

Fig . 3 . Real t ime recording of oxidant generat ion fo l lowing PMA in-duced respi ra tory b urst in neut rophi ls . The fluorescence s ignal of DC Fis shown for a s ingle cell (0-800 s and 900-1000 s) and for the bufferin the imm ediate v ic in i ty of the cel l (wi th in one cel l d iameter; 800-900s). The two l ines represent f luorescence prod uced fro m tw o exci ta t ionwavelengths, 480 and 502 nm.

i n t h e r a t e o f f lu o r e s c e n c e i n c r e a s e i n t h e p r e s e n c e o r a b s e n c eo f M F ( d a t a n o t s h o w n ) , i n d i c a t i n g t h a t M F d o n o t a f f e ct th er e a c ti o n o f H 20 2 a n d D C F a l o ne .

I n a d d i t i o n , n o s i g n i f ic a n t d i f f er e n c e s w e r e o b s e r v e d i n t h ep r e s e n c e o r a b s e n c e o f t h e M F f r o m n e u t r o p h i l s t h a t w e r e n o ti n d u c e d w i t h P M A ( d a t a n o t s h o w n ).4 . D i s c u s s i o n

T h i s s t u d y d e m o n s t r a t e s a s t a t i s t i c a l l y s i g n i f i c a n t 1 2 .4 % i n -c r e a s e i n t h e e x p o n e n t i a l r a te o f f l u o re s c e n c e i n t h e p r e s e n c eo f M F d u r i n g th e P M A - i n d u c e d r e s p i ra t o r y b u r s t o f p r i m e d r a tn e u t r o p h i l s . T h i s i n c r e a s e d p r o d u c t i o n o f f l u o r es c e n c e s u g g e st st h a t a g r e a t e r c o n c e n t r a t i o n o f r a d ic a l s a c c u m u l a t e d i n t h ep r e s e n c e o f M F . T h e d y e D C F H r e a c ts w i t h a v a r ie t y o f r e a c -t i v e o x y g e n s p e c i e s w h i c h a r e e i t h e r f r e e r a d i c a l s ( e . g . p e r o x y n i -t r i t e ) o r d e r i v e d f r o m f r e e r a d i c a l s ( e .g . H 2 0 2 , w h i c h i s f o r m e df r o m t h e d i s m u t a t i o n o f O ~ -) .

I n c r e a s e d o x i d a n t g e n e r a t i o n i n o u r P M A - i n d u c e d r a t p e r i -t o n e a l n e u t r o p h i l s i n t h e p r e s e n c e o f M F m a y b e d u e t o a ni n c r e a s e d l i f et i m e ( a n d h e n c e c o n c e n t r a t i o n ) o f f r e e r a d i c a l s i nt h e c e l ls d i r e c t ly c a u s e d b y t h e M F . S u c h a n e f f e c t h a s b e e no b s e r v e d i n c h e m i c a l s y s t e m s [ 1 1 - 1 3 ], i n c l u d i n g c h e m i c a l s y s-t e m s i n w h i c h t h e b i o l o g i c a l ly i m p o r t a n t v i t a m i n E ( t o c o p h e r -o x y l ) r a d i c a l w a s s h o w n t o b e a f f e c te d b y M F [ 14 ]. R e c e n t l y ,M F h a v e b e e n o b s e r v e d to m o d u l a t e i n t e r s y s t e m c ro s s i n g ra t e so f f r e e r a d i c a l d r i v e n e n z y m e r e a c t i o n s ( e . g. B t 2 e t h a n o l a m i n ea m m o n i a l y as e ) [ 15 ]. N e v e r t h e l es s , a l t e r n a t i v e e x p l a n a t i o n sc a n n o t b e e l im i n a t e d b y o u r r e su l t s; f or e x a m p l e , M F m a ya f f ec t a c t i v a t i o n o f t h e r e s p i r a t o r y b u r s t b y P M A . I n d e e d , C a 2+i s a s e c o n d m e s s e n g e r i n P M A - i n d u c e d n e u t r o p h i l o x i d a t i v e

b u r s t [ 16 ], a n d e l e c t r o m a g n e t i c f i el d s h a v e b e e n s h o w n t o h a v ee f f ec t s o n i n t r a c e l l u l a r c a l c iu m i n c r e a s e s i n r a t t h y m o c y t e s a n dh u m a n T - l y m p h o c y t e s [ 1 7, 18 ]. A n M F e f fe c t o n C a 2+ s e e m su n l i k e ly , h o w e v e r , a s t h e c o n c e n t r a t i o n s o f P M A u s e d w e r eg r e a t e n o u g h t o c a u s e a s a t u r a t i n g c h a n g e i n C a > , b u t e f f e ct so n o t h e r s t e p s i n t h e p a t h w a y f r o m a c t i v a t i o n to r e l e a se o fr e a c t iv e o x y g e n s p e ci e s c a n n o t b e r u l e d o u t .

B e n e f ic i a l e f fe c ts o f M F i n c o r t i c o s t e r o i d - i n h i b i t e d i n te s t i n a lw o u n d h e a l i n g h a v e b e e n r e p o r t e d [ 19 ]. T h i s m a y b e p a r t i a l l yd u e t o t h e i n c r e a s e d c o n c e n t r a t i o n o f f r ee r a d i c a l s / o x i d a n t sp r o d u c e d d u r i n g t h e r e s p i r a t o r y b u r s t o f n e u t r o p h i l s a t t h ew o u n d s i te , w h i c h w o u l d i n c r e a s e b a c t e r i a l k i ll i n g a n d t h u sa c c e l e ra t e w o u n d h e a l i ng . T h i s f i n d i n g is i n a c c o r d a n c e w i t ho u r o b s e r v a t i o n o f a n M F - i n d u c e d i n cr e a s e i n th e n e u t r o p h i lr e s p i r a t ir y b u r s t .

O u r f i n d in g s d e m o n s t r a t e a n i n t e r ac t i o n b e t w e e n M F - a n dP M A - i n d u c e d o x i d a n t g e n e r a t i o n i n r a t p e r i t o n e a l n e u t r o p h i l s .W e b e l ie v e th i s r e p r e s e n t s t h e f ir s t o b s e r v a t i o n o f M F i n f l u e n c -i n g e v e n t s i n v o l v i n g fr e e r a d i c a l s p e c i es g e n e r a t e d d u r i n g s i g n a lt r a n s d u c t i o n i n l i v i n g c el ls .

Acknowledgements. Su p p o r t ed b y N IH (G M 2 7 34 5 ) an d , i n p a r t , b y t h eOffice of Energy M anage me nt , Ut i l i ties System Division , of the DOE,unde r cont ra ct DE-AC03-76SF00098 (R.L. , V.E.). The author s thankD rs . E r i c W i t t an d Jo h n M ag u i re fo r t h e i r v a l u ab l e co mm en t s an dsuggestions.

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