mast cell degarnulation

7/30/2019 mast cell degarnulation

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MAST CELL DEGRANULATION AND HISTAMINE RELEASEOBSERVED IN A NEW IN VITRO SYSTEM*J;

BY DAVID LAGUN OFF, M.D. , AND EAR L P. B END ITT , M .D.(From the Department of Pathology, University of Washington, Seattle)

PLATE 52(Received for publication, May 18, 1960)

Mast cells participate in some types of inflammatory reactions involvingchanges in the microcirculation of certain tissues. Among the known vasoactivesubstances of importance, histamine has been found in mast cells (1) of severalspecies of animals and in certain species serotonin is also present (2). A varietyof substances (the formaldehyde polymer of p-methoxyphenethylmethylamine(48/80), ovomucoid, and dextran) when administered to the rat elicit aninflammatory response, cause histamine and serotonin release and the morpho-logical change of degranulation (3, 4). No clear description of the processinvolved in release of the active amines and other mast cell constituents hasyet been presented. A major obstacle has been the lack of an appropriate wayof observing the action of various agents on the structure of the mast cell andits constituents under controlled conditions. Hence, a new method of observa-tion was sought to study the mechanism of mast cell secretion in vitro.

Certain properties of Millipore filters made these suitable for use in thesystem: biological inertness, relatively high filtration rates, appropriate porediameters for retaining mast cells, and transparency following dehydrationand clearing. This paper presents a method developed for in vitro study of mastcells. The method permits observations of cell morphology and measurementsof such substances as histamine and serotonin under a variety of experimentalconditions. Application of this method has revealed significant differences inbehavior among agents known to liberate histamine and cause degranulationof mast cells in vivo. Thus, 48/80 and polymyxin B act directly on mast cells,while ovomucoid and dextran have no discernible effect on isolated mast cells.

Methods and M aterialsMillipore filters, Ty pe A A (0.8 # pore diameter), 1 inch diameter (Millipore Filte r C orpora-

tion, Watertown, Massachusetts), were used in the chimneys provided with the filters. It was* This work supported in par t by a grant- in-aid f rom United States Publ ic H eal th Service.(H-3174).

A preliminary report of this work was presented at the Histochemical Society Meetings,April, 1959. Work performed as a Uni ted States Publ ic Heal th Service Post-doctoral Researchfellow of the N at ional H ear t Ins t i tute.

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5 7 2 M AST C E L L DE GR ANUL AT I ON AND HI ST AM I NE R E L E ASEf o u n d n e c e ssa r y i n o r d e r in g t h e f i l t e r s t o sp e c i f y " sm o o th su r f a c e d . " O th e r wi se o n e o b t a in e d ,b y c h a n c e , l o t s w i th "a l l i g a to r e d " su r f a c e s wh ic h a r e u n sa t i s f a c to r y f o r m ic r o sc o p i c o b se r v a -t i o n s .O v o m u c o i d w a s p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e o f H e k t o e n a n d C o l e ( 5 ) , a n d t h es a m e l o t s w e r e u s e d a s i n p r e v i o u s s t u d i e s o f t h i s s u b s t a n c e ( 3, 4 ) . T h e d e x t r a n e m p l o y e d w a st h e 8 0 , 0 0 0 m o l e c u la r w e i g h t p o l y m e r ( C o m m e r c i a l S o l v e n ts C o r p . , N e w Y o r k ) . T h e B u r r o u g h s -W e l l c o m e & C o m p a n y , I n c . , T u c k a h o e , N e w Y o r k , k i n d l y s u p p l ie d 4 8 / 8 0 . P o l y m y x i n B w a sp u r c h a s e d f r o m C a l i f o r n i a B io c h e m ic a l C o r p o r a t i o n , L o s An g e l e s . F in a l d i l u t i o n s o f a l la g e n t s we r e p r e p a r e d i n T y r o d e ' s so lu t i o n o f a l o w c a l c iu m c o n c e n t r a t i o n ( 6 ) .

M a s t c e l l s we r e o b t a in e d f r o m th e p e r i t o n e a l c a v i t i e s o f 2 0 0 t o 2 7 5 g m . f e m a le Sp r a g u e -D a w l e y r a t s b y t h e m e t h o d a l r e a d y d e s c r i b e d ( 7 ) . T h e p e r i t o n e a l c a v i t y o f e a c h r a t w a sw a s h e d w i t h 2 0 m l . o f t h e l o w c a l c i u m T y r o d e ' s s o l u t i o n c o n t a in i n g h e p a r i n ( 1 0 / z / m l . ) . C e l lsi n t h e w a s h i n g s w e r e c o u n t e d i n a s t a n d a r d b l o o d c e l l -c o u n t i n g c h a m b e r a f t e r a p p r o p r i a t ed i l u t i o n w i th a c o u n t in g f lu id ( 8 ) . T h e r e c o v e r e d f lu id c o n t a in in g t h e c e l l s wa s d i l u t e d t o af i n a l v o lu m e o f 5 0 m l . w i th t h e T y r o d e ' s so lu t i o n . A 5 m l . a l i q u o t o f t h i s su sp e n s io n wa s t h e ni n t r o d u c e d i n t o a c h i m n e y , w i t h t h e f i l t er i n p la c e , a n d t h e f l ui d w a s p e r m i t t e d t o f lo w t h r o u g h .T h e c e l l s c o l l e c t e d o n t h e f i l t e r we r e c a r e f u l l y wa sh e d w i th a h e p a r in - f r e e T y r o d e ' s so lu t i o n .

I n o r d e r t o t e s t t h e a c t i v i t y o f d i ff e r e nt s u b s t a n c e s o n t h e c e l ls t h e p a d s s u p p l ie d w i t h t h ef i lt e r s w e r e t h o r o u g h l y s a t u r a t e d w i t h a s o l u t i o n o f a g i v e n a g e n t i n T y r o d e ' s s o l u ti o n , a n dth e f i l t e r wa s p l a c e d o n t o p o f t h e p a d . A sm a l l v o lu m e o f t h e so lu t i o n wa s a l so sp r e a d o v e rt h e t o p o f t h e f i l t e r i n o r d e r t o k e e p i t w e t . T h e p a d a n d f i l t e r w e r e t h e n p l a c e d i n a P e t r id i s h , a n d a n a t m o s p h e r e s a t u r a t e d w i t h w a t e r v a p o r w a s m a i n t a i n e d i n t h e c l o s e d d i s h b yu s i n g g a u z e sp o n g e s w e t w i t h T y r o d e ' s s o l u t i o n . T h e P e t r i d i s h w a s i n c u b a t e d f o r t h e d e s i re dt i m e a t a s e l e c te d t e m p e r a t u r e u s u a l l y 2 5 - 2 7 C . I n e a c h e x p e r i m e n t f o u r f i l te r s w e r e p r e p a r e du n d e r i d e n t i c a l c o n d i t i o n s . A t t h e c o n c l u si o n o f t h e i n c u b a t i o n t h e f i l t e rs w e r e r e m o v e d f r o mt h e i r p a d s , p l a c e d i n t h e c h i m n e y , a n d w a s h e d w i t h 5 m l . o f T y r o d e ' s s o l u t i o n . T w o o f t h ef i l te r s we r e f i x ed b y t h e a d d i t i o n t o t h e c h im n e y o f 5 m l . of 4 p e r c e n t f o r m a ld e h y d e i n wa te r .T h e o t h e r t w o f i lt e r s w e r e e a c h p l a c e d i n a s e p a r a t e 1 0 m l. b e a k e r w i t h 2 m l . o f T y r o d e ' ss o l u t i o n t h e n f r o z e n a n d t h a w e d t h r e e t i m e s . T h e f i lt e r w a s r e m o v e d , a n d t h e s o l u t i o n a s s a y edf o r h i s t a m i n e o n t h e i s o l a te d g u i n e a p i g il e u m ( 3 ) . T h e v a l u e o b t a i n e d r e p r e s e n t e d t h e h i s t a -m i n e r e m a i n i n g i n t h e c e l ls a f te r t r e a t m e n t , t h e " r e s i d u a l h i s t a m i n e . " W i t h i n t h e l i m i t s o fe r r o r o f t h e m e t h o d o f a s s a y t h i s p r o c e d u r e e x t r a c t e d a s m u c h h i s t a m i n e a s c o u l d b e e x t r a c t e dw i t h a c i d .T h e two f i l t e r s wh ic h we r e r e se r v e d f o r m ic r o sc o p i c e x a m in a t i o n we r e s t a in e d f o r 3 0 t o 6 0s e c o n d s w i t h 0 . 1 p e r c e n t a q u e o u s t o l u i d i n e b l u e a d j u s t e d t o p H 4 . 5 w i t h 0 . 1 N a c e ti c a c id ,t h e n w a s h e d i n w a t e r a n d a l lo w e d t o d r y t h o r o u g h l y i n a i r. T h e w e t , s t a i n e d f i lt e rs c o u ld b ee x a m in e d w i th t h e m ic r o sc o p e , b u t f o r t h e b e s t p r e p a r a t i o n s f i l t e r s we r e a i r - d r i e d , c l e a r e d w i thx y l en e , a n d m o u n t e d i n p e r m o u n t .

I n t h e s t e p s o u t l i n e d a b o v e , c o n s i d e r a b l e c a r e w a s r e q u i r e d i n h a n d l i n g t h e f i l t e r s . A n yc o n t a c t w i th t h e c e l l - b e a r in g su r f a c e o f t h e f i l t e r b e f o r e f i x a t i o n d i s r u p t e d t h e m a s t c e l ls .T h e r e f o r e t h e f i l t e r s we r e h a n d l e d a t t h e i r e d g e s w i th f i n e f o r c e p s a n d we r e n e v e r a l l o we d tor e s t c e l l - su r f a c e d o wn .

I n se v e r a l e x p e r im e n t s m a s t c e l l s we r e se p a r a t e d f r o m o th e r f r e e p e r i t o n e a l c e l l s b y d i f f e r -e n t i a l s e d i m e n t a t i o n i n c o n c e n t r a t e d s u c r os e , o r a l b u m i n , a s w e h a v e p r e v i o u s l y d e s c r ib e d( 7, 8 ) . T h e y we r e f i n a l l y c o ll e c t e d o n t h e M i l l i p o r e f i l t e r s f o r o b se r v a t i o n .

E X P E R I M E N T A L O B S ER V A T IO N SThe Distribution of Histamine among the Peritoneal Cel l Types.--.Since i n

m o s t o f t h e e x p e r i m e n t s m a s t c e l l s c o n s t i t u t e d o n l y 5 t o 8 p e r c e n t o f a m i x e dc el l p o p u l a t i o n , i t w a s i m p o r t a n t t o d e t e r m i n e h o w m u c h o f t h e m e a s u r e d


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D A V I D L A G U N O FF A N D E A R L P . B E N D I T T 57 3h i s t a m i n e i n t h e p e r i t o n e a l c e ll s r e p r e s e n t e d h i s t a m i n e c o n t r i b u t e d b y m a s tc e ll s. I n o r d e r t o d o t h i s m a s t c e l ls w e r e s e p a r a t e d f r o m t h e o t h e r c e l ls p r e s e n ti n t h e i n i t i a l p e r i t o n e a l w a s h us i n g c o n c e n t r a t e d s u c r o s e o r a l b u m i n ( 7 ) , ( 8) ,a n d t h e i n i t i a l p o p u l a t i o n , t h e m a s t c e l l f r a c t i o n , a n d t h e i n t e r f a c e f r a c t i o nw e r e a s s a y e d f o r h i s t a m i n e . I n T a b l e I a r e s h o w n t h e r e s u l t s o f t w o s u c h e x p e r i -m e n t s . I t c a n b e s e e n t h a t t h e r a t i o o f h i s t a m i n e p e r 1 06 m a s t c e l l s i n e a c h o ft h e p o p u l a t i o n s w i t h i n a g i v e n e x p e r i m e n t i s v e r y n e a r l y t h e s a m e . T h e s er e s u l t s i n d i c a t e t h a t w i t h i n t h e l i m i t s o f e r r o r o f t h e m e t h o d s i n v o l v e d a l l o f

T A B L E I

E x p e r i m e n t C el l f r ac t i o n

Initial washAlbumin layerInterface layerSucrose layerInterface layer

Nu m b er o f ce i ls X 1 0 6O t h e r p cr i t o -M a s t c e l l s n ea l ce i l s

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T A B L E I IHistam ine C ontent of Mast Cel ls af ter Incubat ion

T r e a t m e n t o f c e l l s

C ol le ct io n o n M il li po re f il te r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .C ol le ct io n o n M il li po re f il te r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Cen tri fugat ion twice a t 1000 x .p .~s , fo r 5 min . . . . . . . . . . . . . . . .Cen tr i fugation twice a t 1000 R.P . . fo r 5 min . . . . . . . . . . . . . . . .

I n c u b a t i o nt i m e

01 hr .01 hr .

R e s i d u a l h i s t a -mi n e / zg . / 1 0 5mas t cells

0.500.400.500.08

t h e h i s t a m i n e i n t h e o r i g i n a l p e r i t o n e a l c e l l p o p u l a t i o n i s a c c o u n t e d f o r b y t h em a s t c e l l s . T h u s a n y s i g n i f i c a n t c h a n g e i n h i s t a m i n e c o n t e n t o f t h e e n t i r e c e l lp o p u l a t i o n r e p r e s e n t s a c h a n g e i n m a s t c e l l h i s t a m i n e .

T h r o u g h o u t t h e c o u r s e o f th e s e e x p e r i m e n t s l a r g e v a r ia t i o n s w e r e o b s e r v e db o t h i n t h e n u m b e r s o f c e ll s h a r v e s t e d f r o m a s i n g l e a n i m a l a n d i n t h e h i s t a m i n ec o n t e n t o f t h e s e c e l l s . N o s a t i s f a c t o r y e x p l a n a t i o n f o r t h e s e v a r i a t i o n s h a s y e tb e e n f o u n d .

B eh a v io r o f U n t rea ted M a s t C e l l s o n th e F i l t e r . - -W h en t h e m a s t c e l l s w e r ee x a m i n e d i m m e d i a t e l y a f t e r d e p o s i ti o n o n t h e f i lt e r, t h e y a p p e a r e d i n t a c t(F ig . 1 ) . A n o cca s io n a l ce l l sh o w ed lo s s o f g ran u les . F o l lo w in g in cu b a t io n fo ra s l o n g a s 1 h o u r i n T y r o d e ' s s o l u t io n t h e r e w a s l i t t le c h a n g e i n t h e i r a p p e a r -


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5 7 4 MAST CELL DEGRANULATIONAND HISTAMINE RELEASEa n c e . F u r t h e r e v i d e n c e o f t h e s t a b i l i t y o f t h e c e ll s w a s s ee n i n t h e h i s t a m i n ed e t e r m i n a t io n s w h i c h d e m o n s t r a t e d o n l y 2 0 p e r c e n t lo s s o f h is t a m i n e f r o mt h e c e l ls i n t h e p e r i o d o f i n c u b a t i o n , i n c o n t r a s t t o t h e i n s t a b i l i t y o f t w i c ec e n t r i f u g e d c e l ls w h i c h l o s t 8 4 p e r c e n t o f t h e i r h i s t a m i n e o n s u b s e q u e n t i n c u b a -t i o n ( T a b l e I I ) .

1 2 0 i8 0 ~0 ' 4 0 8 0 '

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0.0 5 0.5 5.0 50 50 04 8 8 0 C o n c . / z g ./ m I .

BTEXT-FIG. 1. Histamine loss from m ast cells incubated in 48/80. (A) Effect of time. 48/80,20/zg ./ml. (B) Effect of concentration of 48/80, cells incubated 30 minutes. Values are ex-pressed as per cent of control cell histamine extractable from the treated cells.

T A B L E I I IHistamine Release

Incubation media

T y ro de 's so lu tio n . . . . . . . . . . . . . . . . . . . . . . . . . . . .48/80 50 ~g./ml..D ex tran 1 m g ./ m l . . . . . . . . . . . . . . . . . . . . . . . . . .O vo mu co id 1 m g ./ ml . . . . . . . . . . . . . . . . . . . . . . . . I

Supernatant0 .31 .00 .30 .2

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1.10 .41. 21. 5

Total1 .41 .41. 51. 7

* Average of determinations on two separate filters.C e l ls i s o l a te d b y t h e s u c r o s e p r o c e d u r e s p o n t a n e o u s l y s p i l le d t h e i r g r a n u l e s

w h e n s u s p e n d e d i n T y r o d e ' s s o l u t i o n a n d t h e n c o l l e c t e d o n t h e f i lt e rs . I nc o n t r a s t t h e c e l l s i s o l a t e d b y t h e a l b u m i n p r o c e d u r e r e t a i n e d t h e i r i n t a c tm o r p h o l o g y ( F i g . 6 A ) .

I n V i t ro E ffec ts o f 48 /80 , Po lymy x in B , Ovomucoid, and Dex tra n . - - Wh en4 8 / 8 0 w a s a d d e d t o t h e i n c u b a t i o n f l u i d i n a p p r o p r i a t e c o n c e n t r a t i o n s , w i d e -s p r e a d d e g r a n u l a t i o n o f m a s t c e l ls w a s e v i d e n t ( F i g . 4) , a n d h i s t a m i n e r e l ea s ew a s r e a d i l y d e m o n s t r a b l e . T h e h i s t a m i n e l o s s o f c e ll s t r e a t e d w i t h 4 8 / 8 0 w a sm e a s u r e d o v e r a r a n g e o f t i m e i n t e r v a l s , a n d t h e a c t i o n o f th i s a g e n t w a s f o u n dt o b e r a p i d ( T e x t -f i g . 1 A ) . T h e h i s t a m i n e r e l e a s e d a t v a r y i n g c o n c e n t r a t i o n s


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D A V I D L A G U N O F F A N D E A R L P . B E N D I T T 57 5o f 4 8 / 8 0 w a s a l so m e a s u r e d ( T e x t -f i g . 1 B ) ; a b o u t 5 0 p e r c e n t o f t h e h i s t a m i n ew a s r e le a s e d a t a c o n c e n t r a t i o n o f 5 . 0 g g . / m l . W h e n h i s t a m i n e w a s m e a s u r e di n t h e s u p e r n a t a n t f l u i d a l l t h e h i s t a m i n e , w i t h i n t h e l i m i t s o f th e a s s a y m e t h o d ,l i b e r a t e d f r o m t h e c e l ls w a s a c c o u n t e d f o r i n t h e s u p e r n a t a n t f l u id ( T a b l e I I I ) .

T A B L E I VH i s t a m i n e L o s s f r o m S e p a ra t e d M a s t C e ll s

I n c u b a t i o n m e d i aR e s i d u a l h i s t a m i n e i.tg./lO5m a s t c e l ls

E x p e r i m e n t 1 E x p e r i m e n t 2T y r o d e ' s s o l u t io n c o n t r o l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . 0 0 . 54 8 / 8 0 2 0 ~ t g ./ m l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . 2 0 . 0 6

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T E x T - F I o . 2 . H i s t a m i n e l o s s f r o m m a s t c e l l s i n c u b a t e d o n M i l l ip o r e f il t e rs w i t h d i f f e r e n ta g e n t s . V a l u e s a r e e x p r e s s e d a s p e r c e n t o f c o n t r o l c e ll h i s t a m i n e e x t r a c t a b l e f r o m t h e t r e a t e dce l l s .

I n o r d e r t o d i s c o v e r w h e t h e r o r n o t 4 8 / 8 0 w a s a c t i n g vi a t h e o t h e r c e l l sp r e s e n t i n t h e p e r i t o n e a l c e l l p o p u l a t i o n , t h e c o m p o u n d w a s t e s t e d w i t h m a s tc e l l s s e p a r a t e d f r o m t h e o t h e r c e l l c o n s t i t u e n t s b y s e d i m e n t a t i o n i n c o n c e n -t r a t e d a l b u m i n . 4 8 / 8 0 w a s f o u n d t o b e a s e f f e ct i v e w i t h s e p a r a t e d m a s t c e l lsa s w i t h t h e w h o l e p e r i to n e a l c e l l p o p u l a t i o n ( T a b l e I V ) , ( F ig . 6 B ) .

P o l y m y x i n B i n a c o n c e n t r a t i o n o f 5 0 ~ g . / m l . o f i n c u b a t i n g f l u i d b e h a v e dl i k e 4 8 / 8 0 . O v o m u c o i d a n d d e x t r a n w e r e w i t h o u t e v i d e n t e f f e c t o n t h e m a s tcel l s in the in vi tro s y s t e m i n c o n c e n t r a t i o n s o f 1 m g . p e r m l . T h e s e f in d i n g sa r e i l l u s t ra t e d a n d s u m m a r i z e d i n F i g s. 1 to 5 a n d T e x t - f ig . 2 . W h e n f r e s h r a t


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576 M A S T C EL L D E G R A N U L A T I O N A N D H I S T A M I N E R E L E A S Es e r u m w a s a d d e d i n a c o n c e n t r a t i o n o f 5 0 p e r c e n t t o t h e i n c u b a t i o n m e d i u mc o n t a i n i n g o v o m u c o i d , d e g r a n u l a t i o n o f m a s t c e l ls o r h i s t a m i n e r e l ea s e d i dn o t o c c u r .

D I S C U S S I O N

I n e a r l y a t t e m p t s t o s t u d y m a s t c e ll s a n d t h e m o d e o f a c t i o n o f v a ri o u sagen t s on t hese ce l l s i n i so l a t ed sys t ems , we enco unt e r ed cons i de rab l e d i f f i cu l ty .T h e c e l ls f r a g m e n t e d e a s i l y , a n d l a r g e a m o u n t s o f h i s ta m i n e w e r e l i b e r a t e ds p o n t a n e o u s l y f r o m c e ll s s u s p e n d e d i n v a r i o u s m e d i a . A s b o e - H a n s e n a n d G l i c kf o u n d t h a t 3 5 p e r c e n t o f m a s t c e l l s i s o l a t e d b y t h e s u c r o s e g r a d i e n t m e t h o di n c o n t r o l p r e p a r a t i o n s s h o w e d d a m a g e ( 9 ) . O f e v e n m o r e s i g n i f i c a n c e i s t h ef a c t t h a t 4 8 / 8 0 c a u s e d n o e v i d e n t d e g r a n u l a t i o n o f m a s t c e l ls u n d e r t h e i rc o n d i t i o n s o f o b s e r v a t i o n . T h e r e a s o n s f o r t h is a p p e a r t o b e i n p a r t a c o n s e -q u e n c e o f r e p e a t e d c e n t r i f u g a t i o n a n d i n p a r t a c o n s e q u e n c e o f t h e i n c r e a s e df r a g i l i ty o f c e ll s a f t e r e x p o s u r e t o v e r y h y p e r t o n i c s u c r o s e so l u ti o n s . T h e v e r yprocess o f smear i ng and d ry i ng o f ce l l s on g l a s s s l i des i t s e l f causes d i s t o r t i ona n d d i s r u p t i o n s o t h a t v a l i d o b s e r v a t i o n s o f c h a n g e s i n c e l l m o r p h o l o g y a r ep r e v e n t e d . T h u s w e w e r e l e d t o s e a r c h fo r a n e w m e t h o d o f o b s e r v in g c h e m i c a land morpho l og i ca l changes i n mas t ce l l s .

M i l l i pore f i l t e r s , o r i g i na l l y dev i sed fo r t he co l l ec t i on o f bac t e r i a and o t he rpa r t i c l e s f rom f l u i ds , have been used be fore t o co l l ec t ce l l s f r om body f l u i dsfo r cy t o l og i c exami na t i on i n cance r d i agnos i s ( 10 , 11) . T he f i l t e r s hav e a l sob e e n e m p l o y e d i n t i ss u e c u l t u r e ( 12 ) a n d i n e x p e r i m e n t s r e q u i r i n g in vivoseques t r a t i on o f f o r e i gn ce l l s ( 13) . As shown i n our expe r i ment s , t he f i l t e r s a r ea l so use fu l f o r t he co l l ec t i on and s t u dy o f pe r i t onea l m as t ce ll s. T he ce l ls ont he M i l l i pore f i l t e r s a s we l l a s be i ng r ead i l y v i sua l i zed i n t he l i gh t mi c roscopem a y b e p r o c e s s e d f o r e l e c tr o n m i c r o s c o p y ( 1 4 ). T h e u s e o f f i l te r s f o r q u a n t i t a -t i ve measurement s o f r ad i oac t i ve t r ace r s i n ce l l s i s a l so f eas i b l e (15) .

T h e e n z y m e t h e o r y o f m a s t c e l l d e g r a n u l a t i o n h a s b e e n r e v i v e d i n t h e r e p o r t so f H o g b e r g a n d U v n i i s ( 1 6 - 1 8 ). O n e o f t h e p r o b l e m s i n i n t e r p r e t i n g t h e i r w o r ki s t ha t wh ol e ti s sues , r a t m esen t e r i e s , were u t i l i zed ; t h i s makes un ce r t a i n t hes i te o f a c ti o n o f a n y a g e n t u n d e r i n v e s t i g a t i o n . I n s u c h a c o m p l e x ti s s u e o rm i x e d c e ll p o p u l a t i o n i t c a n n o t b e a s s u m e d t h a t b e c a u s e a n e f f ec t o n m a s t c e ll si s o b s e r v e d t h e a g e n t o r c o n d i t i o n s e m p l o y e d a r e d i r e c t l y a f f e c t i n g t h e m a s tce ll s. I n t h i s s i t ua t i on i t ma y we l l be t ha t an ag en t w hi ch causes m as t ce l ld e g r a n u l a t i o n i s o p e r a t i n g f i r s t u p o n a n o t h e r t i s s u e c o n s t i t u e n t t o p r o d u c e as u b s t a n c e t h e n c a p a b l e o f a l te r i n g m a s t c e l ls .

W e h a v e s t u d i e d m a s t c e l ls is o l a te d f r o m t h e i r u s u a l t i s su e e n v i r o n m e n t , a n di n a l i m i t e d n u m b e r o f e x p e r i m e n t s , f r e e o f o t h e r c e ll t y p e s . U n d e r t h e s e c o n d i -t i ons ma s t ce l l s depo s i t ed on t he su r f ace o f a M i l l i pore f i l te r r e sponde d t o 48 / 80i n th e s a m e w a y t h a t t h e y d o in vivo i n t h e d o r s a l s k in o f r a t f e e t . P o l y m y x i nB p r o d u c e d a s i m il a r re s p o n s e, b u t n o r e s p o n s e t o o v o m u c o l d o r d e x t r a n c o u l d


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DAVID LAGUNOFI~ AND EARL P. BENDITT 577b e e l i c i t e d e v e n w i t h c o n c e n t r a t i o n s w h i c h e a s i l y p r o d u c e o b s e r v a b l e a l t e r a -t i ons in vivo ( 4 ). A d i f f e re n c e b e t w e e n t h e a c t i o n o f 4 8 / 8 0 a n d o v o m u c o i d o nm a s t c e ll s h a s b e e n o b s e r v e d u n d e r q u i t e d i f f e r e n t c i r c u m s t a n c e s : G o t h a n dc o w o r k e r s ( 19 ) h a v e c o n f i r m e d a n d e x t e n d e d t h e f i n d i n g s o f A d a m k i e w i c z a n dL a n g l o i s (2 0 ) t h a t i n s u li n g r e a t l y e n h a n c e s d e x t r a n a n d o v o m u c o i d a c t i v i t yw i t h o u t a f f e c ti n g 4 8 / 8 0 a c t i v i t y .

S e v e r a l r e a so n s c a n b e c o n s i d e r e d t o a c c o u n t f o r t h e d i f fe r e n c e i n a c t i o n o ft h e s m a l l e r b as i c c o m p o u n d s , 4 8 / 8 0 a n d p o l y m y x i n B , a n d t h e l a r g e r m o l e c u le s ,o v o m u c o i d a n d d e x t r a n : ( a ) F r e e m a s t c e l ls i n t h e p e r i t o n e a l c a v i t y m a y d i f f e ri n t r i ns i ca l l y i n t he i r r ange o f r e spons i veness f rom t he mas t ce l l s o f t he dor sa ls k i n o f t h e f o o t ; ( b) D e x t r a n a n d o v o m u c o i d m a y n o t a c t d i r e c t l y o n m a s t c e l lsb u t a c t , o r a r e a c t e d u p o n , e l se w h e r e in t h e t is s u e s o r c ir c u l a t in g p l a s m a p r o -d u c i n g a s u b s t a n c e w h i c h c a n t h e n i n d u c e m a s t c e ll c h a n g e s ; ( c ) A p a r t o f t h em e c h a n i s m o f t h e m a s t c e ll re s p o n s e to o v o m u c o i d a n d d e x t r a n i s p r o h i b i t e di n so m e u n d e t e r m i n e d w a y b y t h e in vitro c o n d i t io n s . A t p r e s e n t , e x p e r i m e n t a ld a t a d o n o t p e r m i t a c l e a r c h o i c e a m o n g t h e p o s s i b il it ie s . P r e l i m i n a r y a t t e m p t st o e x t r a c t f r o m t h e d o r s a l s k i n o f r a t f e e t fo l lo w i n g o v o m u c o i d t r e a t m e n t , as u b s t a n c e c a p a b l e o f d e g r a n u l a t i n g m a s t c e l l s h a v e b e e n u n s u c c e s s f u l , a n da d d i t i o n o f f re s h r a t s e r u m t o t h e i n c u b a t i o n m e d i a a s i n d i c a t e d d i d n o t m a k eo v o m u c o i d e f f ec t iv e .

T h e p r o b l e m o f m a s t c e l l s e c r e ti o n h a s b e e n i n v e s t i g a t e d i n vitro r e c e n t l y i nt wo o t he r l abora t o r i e s . Arche r (21 , 22) emp l oyed ce ll s s epa ra t ed i n concen-t r a t e d a l b u m i n . H e f o u n d 4 8 / 8 0 a n d a n u m b e r o f o t h e r a g e n t s a b l e t o r el e a s eh i s t a m i n e f r o m m a s t c e l ls in vitro, b u t i f m o r e t h a n 4 5 m i n u t e s e l a p s e d b e t w e e nt h e c o l l e c t io n o f ce ll s a n d i n c u b a t i o n w i t h t h e h i s t a m i n e r e le a s in g a g e n t t h ec e l l s b e c a m e r e f r a c t o r y . A r c h e r w a s f u r t h e r a b l e t o d e m o n s t r a t e t h a t e x t r a c t so f s eve ra l t i s sues were capab l e o f r e l eas i ng h i s t ami ne . He pos t u l a t e s t ha t ana r g i n i n e - c o n t a i n i n g p e p t i d e w h i c h h e f o u n d i n t h e e x t r a c t o f s p l e e n w a s t h ea c t i v e s u b s t a n c e .

S i n c e A r c h e r ' s s t u d y w a s p u b l i s h e d , U v n / i s a n d T h o n ( 2 3 ) h a v e r e p o r t e di nves t i ga t i ons ca r r i ed ou t w i t h mas t ce l l s i so l a t ed by a me t hod i n whi ch f i co l l ,a p o l y s a c c h a r i d e - li k e c o m p o u n d , i s s u b s t i t u t e d f o r t h e a l b u m i n u s e d b y A r c h e ra n d b y u s . C e l ls s o o b t a i n e d a r e u n r e s p o n s i v e t o 4 8 / 8 0 a f t e r w a s h i n g i n s e r u m -f r e e so l u t io n s , a r e s u l t d i f f e r in g f ro m o u r s . T h i s m a y p e r h a p s b e e x p l a i n e d b yt he e f f ec t s o f fi co ll on t he ce l ls o r by t he t i me whi ch e l ap sed i n co l l ec t i ng t hece l l s . W hen se rum was used i n a l l phases o f t he ce l l s epa ra t i on p rocedure t hee f f e ct i v en e s s o f 4 8 / 8 0 w a s c o m p a r a b l e t o t h a t f o u n d i n o u r e x p e r i m e n t s .

T h e r e s u l t s o f t h e t h r e e s t u d i e s c a r ri e d o u t s o f a r i n d i c a t e t h e i m p o r t a n c e o fth e in vitro s t u d y o f m a s t c e l l s e c r e t i o n i n t h e a n a l y s i s o f t h e m e c h a n i s m b yw h i c h m a s t c e ll s r e s p o n d t o a v a r i e t y o f s ti m u l i . A n a t t r a c t i v e h y p o t h e s i s m a yb e c o n s t r u c t e d f r o m t h e a v a i l a b l e d a t a a s f o l lo w s : 4 8 / 8 0 a n d p o l y m y x i n B a r em e m b e r s o f a c la s s o f a g e n t s w h i c h c a n a c t d i r e c t l y o n t h e m a s t c e ll t o p r o d u c e


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578 MAST CEL L DEGRANULATIONAND H ISTAMIN E RELEASEg r a n u l e s e c r e t i o n a n d h i s t a m i n e r e l e a s e , w h i l e a n o t h e r c l a s s o f c o m p o u n d sr e p r e s e n t e d b y d e x t r a n a n d o v o m u c o i d a c t a t a n u n i d e n t i fi e d s i t e i n t h e t is s u e st o r e l e a se a c o m p o u n d o f t h e d i r e c t - a c t i n g t y p e . T h e i d e n t i fi c a t i o n o f a n i n t e r -m e d i a t e c o m p o u n d p a r t i c i p a t i n g i n t h e c h a i n o f e v e n t s i n v o l v e d in t h e a c t i o no f o v o m u c o i d a n d d e x t r a n i n r a t s w o u l d s e r v e t o v a l i d a t e t h i s h y p o t h e s i s .

SUMMARY

A m e t h o d i s d e s c r i b e d b y w h i c h m a s t c e l l s h a r v e s t e d f r o m t h e r a t p e r i t o n e a lc a v i t y c a n b e d e p o s i t e d o n t h e s u r f a c e o f a M i l l i p o r e f il t e r w i t h o u t g r o s s i n j u r y ,p e r m i t t i n g o b s e r v a t i o n o f m o r p h o l o g i c a l a n d c h e m i c a l c h a n g e s i n d u c e d b y av a r i e t y o f a g e n t s.

T h e s e i s o l a t e d p e r i t o n e a l m a s t c e l l s r e s p o n d t o 4 8 / 8 0 a n d t o p o l y m y x i n Bb u t n o t t o d e x t r a n o r o v o m u c o i d w i t h d e g r a n u l a t i o n a n d h i s t a m i n e r e l e a se .T h u s f o u r a g e n t s w h i c h in v ivo a p p e a r t o h a v e s i m i l a r a c t i v i t i e s h a v e b e e n f o u n db y m e a n s o f in vitro a n a l y s i s t o o p e r a t e b y a t l e a s t t w o d i f f e r e n t m e c h a n i s m s .

BIBLIOGRAPHY1. Ri ley , J . F . , The Mast Cel ls , E . S . L iv ingston L td . Ed inburgh , 1959 .2 . Bend i t t , E . P . , Wong , R . L . , Arase, M. , and Roeper , E . , 5 -Hydroxy tryp taminein ma st cel ls, Proc. Soc. Exp. Biol. and Med. , 1955, 90, 303.3 . Bend i t t , E . P . , Bader , S . , and Lam, K. B . , S tud ies o f the mechan ism of acu tevascu lar react ions to in ju ry . I . The re la t ionsh ip o f mast cel ls and h is tamine to

the p roduct ion o f edema b y ovomuco id in ra ts , Arch. Path., 1955, 60, 104.4 . Row ley , D. A. , and Bend i t t , E . P . , 5 -H ydrox y tryp ta mine and h is tamine as med i-ato rs o f the vascu lar in ju ry p roduced by agen ts which damage mast cel ls inr a t s , J . E xp . M ed . , 1956, 108, 399.5 . Hek toen , L . , and Co le , A . G. , The p ro teins o f egg whi te : The p ro te in in egg whi teand thei r re la t ionsh ip to the b lood p ro teins o f the domest ic fowl as determinedby the p recip i t in react ion , J. Infect. Dis. , 1928, 42, 1.6 . Dalgliesh, C. E. , Toh, C. C. , and Work, T. S. , Fractionation of the smooth muscles t imulan ts p resen t in ex t racts o f gast ro in tes t inal t ract : Iden t i f icat ion o f 5 -hydro xy tryp tam ine and i ts d is t inction f rom substance P, J. Physiol. , 1953,120, 298.7 . Bend i t t , E . P . , and Arase, M. , An enzyme in mast cel ls wi th p roper t ies l ikech y m o t ry p s in , J . E xp . M ed . , 1959, 110, 451.8 . Lagunoff, D . , and B end i t t , E . P . , 5 -H ydroxy tryp toph ane decarboxy lase act iv i tyin r a t m as t ce l l s , Am. J. Physiol. , 1959, 196, 993.9. Asboe-Hansen, G. , and G lick, D. , Influence of various agents on m as t cells iso-la ted f rom ra t per i toneal f lu id , Proc. Soc. Exp. Biol. and Med. , 1958, 98, 458.10. Seal, S. H. , A me thod for conce ntrating c ancer cells suspended in large qua ntitiesof fluid, Cancer, 1956, 9, 866.

11. Del Vecchio, P. R. , DeWitt, S. H. , Borelli , J. I . , Ward, J. B. , Wood, T. A. , Jr. ,and Malrugren, R. A. , Application of Millipore fil tration technique to cytologicmater ia l , J. N at. Cancer Inst. , 1959 , 9.% 427.


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DAVID LAGUNOFF AND EAR L P. BENDITT 57 912. Grobstein, C., Trans-fi l ter indu ction of tubules i ll mouse metanephrogenic mesen-chyme , Exp. Cell Research, 1956, 10, 424.13. Prehn, R . T . , Algire , G. H. , and Weaver , J . M . , The di ffusion chamb er in homo-gra ft research, Transplant Bull., 1955, 2, 147.14. Strunk, S., and Lagunoff, D., unpublished observations.15. Lagunoff, D., Calhoun, R., and Benditt , E. P., Proc. Soc. Exp. Biol. and Med.,1960, 104, in press.16. Hogberg, B., and Uvn~s, B., The Mechanism of the disruption of mast cells pro-duced by compound 48/80, Acta Physiol. Scand., 1957, 41, 345.17. H ogberg, B ., and U vn~ts, B., Inh ibitor y action of al l icin on degran ulation of mastcells produ ced by compound 48/80 , histamin e l iberator from ascaris, lecithinaseA, and ant igen, Acta Physiol. Scand., 1958, 44, 157.18. Uvn~s, B., The mechanism of histamine l iberation, J. Pha rm. and Pharmacol.,1958, 10, 1.19. Goth, A. , Nash, W. L . , Nagler , M . , and Holma n, J . , Inh ibi t ion of his tamine

release in experim ental diabetes, Am. J. Physiol. , 1957, 190, 25.20. Adamkiewicz, V. W., and Langlois, Y., Sensit ization by insulin to the Dextran"anaphyl ac t o i d" r eac ti on , Canad. J. Biochem., 1957, 35, 251.21. Archer, G. T., Release of histamin e from m ast cells by t issue extracts, Nature,1958, 182, 726.22. Archer, G. T., The release of histamine from the mast cells of the rat , Aust. J.Exp. Biol. , 1959, 37, 333.23. U vn ~, B. , and Tho n, I . L . , I sola t ion of "biologically intac t" m ast cell s, Exp. CellResearch, 1959, 18, 512.


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58 0 MAST CEL L DEGRANULATION AND HISTAMINE RELEASE

E X P L A N A T I O N O F P L A T E 5 2A p p e a r a n c e o f p e r i t o n e a l m a s t c e l ls i n c u b a t e d 1 h o u r o n M i l l i p o r e f il t e r s t h e n f i x e d

i n 4 p e r c e n t f o r m a l d e h y d e a n d s t a i n e d w i t h 0 . 1 p e r c e n t a c i d i c a q u e o u s t o l u i d i n eb lue . X 680 .F I G . 1 . I n c u b a t i o n i n T y r o d e ' s s o l u t i o n d e m o n s t r a t i n g i n t a c t m a s t c e l l s .F I G . 2 . I n c u b a t i o n i n o v o m u c o i d , 1 m g . / m l , d e m o n s t r a t i n g i n t a c t m a s t c e l ls . O n e

c e l l s h o w s s o m e d e g r a n u l a t i o n .F I 6 . 3 . I n c u b a t i o n i n d e x t r a n , 1 m g . / m l , d e m o n s t r a t i n g i n t a c t m a s t c e l ls .F I 6 . 4 . I n c u b a t i o n i n 4 8 / 8 0 5 0 #g . /ml . d e m o n s t r a t i n g d e g r a n u l a t i o n .F I G . 5 . I n c u b a t i o n i n p o l y m y x i n B 5 0 / z g ./ m l . d e m o n s t r a t i n g d e g r a n u l a t i o n .F I G S. 6 A a n d 6 B . A p p e a r a n c e o f p e r i t o n e a l m a s t c e l l s c o l l e c t e d b y s e d i m e n t a t i o n

i n a l b u m i n s o l u t i o n .F I G . 6 A . A f t e r 1 h o u r i n c u b a t i o n o n M i l l i p o r e fi l t e r i n T y r o d e ' s s o l u t i o n .F I 6 . 6 B . A f t e r 1 h o u r i n c u b a t i o n u n d e r t h e s a m e c o n d i t i o n s b u t i n 4 8 / 8 0 5 0 / ~ g . / m l .


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THE JOURNAL OF EXPERIMENTAL MEDICINE VOL. 112 PLATE 52

(Lagunoff and Bendl tt: Ma st cell degranu lation and histamine release)

mast cell degarnulation

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