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P u r i f i c a t io n o f r a t c u t a n e o u s m a s t c e l lsw i th P e r c o l l de ns i ty c e n tr i fug a t i o nH . H a c h i s u k a , M . K u s u h a r a , M . H i g u c h i, K . O k u b o , a n d Y . S a s aiCutaneous Biology Unit , D epa rtme nt of Dermatology, K urum e Universi ty School of Medicine, 67, Asahimachi, Ku rum e, 830, Japa n

Summary. The skin i s the major si te on anaphylaxis ,and cutaneous m ast ce l l s have an important role in i tsreact ions. Th e i solat ion and puri ficat ion of rat cu-taneous mast ce i l s are descr ibed here . Rat abdominalskin was digested with col lagenase and hyaluronidase ,and centr i fuged with Percol l . The buoyant densi ty ofcutaneous m ast ce l l s was high, and re lat ive ly pure mastce l l s were obtained. The puri ty of cutaneous m ast ce l l swas 7 .4% -I- 2 .4% be for e and 50 .0% 6 .4% af te rPe rco ll dens ity centrifugation; periton eal m ast cells re-vealed 5.8% 1 .3% purity before and 61.0% 10.6%purity a fter the same procedure. Th e i solated cutaneouscel l s re leased 21.3% 3 .8% histamine and the peri to-ne a l mast c e l l s r e l ease d 55 .5% 3 .8% hi s tamine uponst imulat ion wi th 10 ~g /m l c ompound 48 /80 . The sefindings suggest that there are functional subsets ofconnect ive t i ssue m ast ce i l s .K e y wor ds: M ast c e ll i so la ti on - Skin - P e r e o l l

R a t m a s t c e ll s h a v e b e e n d i v i d e d i n t o t w o m a j o r s u b -p o p u l a t i o n s a c c o r d i n g t o t h e c y to c h e m i c a l l y d e te c t -a b l e p re s e n c e o r a b s e n c e o f g l y c o s a m i n o g l y c a n s : c o n -n e c t i v e t i s s u e m a s t c e l l s a n d m u c o s a l m a s t c e l l s [ 4 ] .T h e f o r m e r a r e f o u n d i n s k i n , m u s c l e a n d t h e p e r i t o -n e a l c a v i t y , w h i l e t h e l a t te r , w h i c h a r e d e v o i d o fg l y c o s a m i n o g l y c a n s , a r e f o u n d i n m u c o s a o f th e g a s -t r o i n t e s t i n a l t r a c t . D i f f e r e n c e s i n f u n c t i o n b e t w e e nt h e m m a y b e r e f l e c t e d i n d i f f e r e n c e s i n t h e i r s t i m u l if o r o n t o g e n y a n d m a t u r a t i o n , c y t o c h e m i s t ry , a n da b i l i t y t o r e s p o n d t o a c t i v a t o r s a n d m o d u l a t o r s o fm e d i a t o r s e c r e t i o n [ 4 , 14 ]. T o i d e n t i f y t h e r a t m a s t c e l ls u b s e t s , a n a l t e r n a t i v e a p p r o a c h i s t o e x a m i n e t h ed i s t ri b u t i o n o f g ra n u l e p r o t e i n a s e s . A c c o r d i n g t oOffpr in t reques ts to : Dr. Hiroshi Ha chisuk a (address see abov e)

i m m u n o h i s t o c h e m i c a l c h a r a c t e ri s ti c s , r a t m a s t c el lp r o t e i n a s e I ( R M C P I ) f r o m c o n n e c t i v e t is s u e m a s tc e ll s a n d R M C P I I f r o m m u c o s a l m a s t c el ls a r e m a s tc e ll s u b s e t s f e a s i b l y d i s ti n g u i s h a b l e b y t h e i r s e r i n e p r o -t e i n a s e c o n t e n t [ 6 ] .

T o e x a m i n e t h e f u n c t i o n o f m a s t c e ll s, m a n y i n v es -t i g a t o r s h a v e a t t e m p t e d t o i s o l a t e a n d p u r i f y t h e mf r o m v a r i o u s o r g a n s , i n c l u d i n g p e r i t o n e u m [3 , 5 , 9 , 17 ,2 0 ] , i n t e s t i n a l m u c o s a [ 1 7 ] , l u n g [ 1 6 ] , a n d s k i n [ 2 ] . R a tp e r i t o n e a l m a s t c el ls h a v e b e e n u s e d f o r e v a l u a t i o n o fa n t i a n a p h y l a c t i c a g e n t s [8 , 1 1 - 1 3 ] . S i n c e s k i n i s t h em a j o r s it e o n a n a p h y l a x i s , i t h a s b e e n a s s u m e d t h a t i tp u r i fi e s m a s t c el ls . T h e p r e s e n t s t u d y w a s u n d e r t a k e nt o i s o la t e c u t a n e o u s m a s t c e ll s f r o m r a t a b d o m i n a ls k i n .

M ate r ia l s and me thodsEnz ym e d ispers ion o f ra t cu taneous mas t ce l lsWistar strain ra ts of both sexes weighing abou t 200 g each wereused for the experiment. R at cutaneous ma st cells were dispersedenzymically in a manner similar to that previously describedfor dispersion of cells from rat skin [1]. The animals w ereanesthetized with ether and kil led by exsanguination. The ab-dom inal skin weighing abo ut 5 g was dissected and dermal fatwas removed with scissors. After washing in Tyrodc's HEPESbuffer, the t issue was then finely chopped into fragments ofabou t 1 mm 3. It was incubated (4 h, 37~ in a Ha nks solutioncontaining 1 mg/ml collagenase (Type I , Sigma, St . Louis, Mo.)and 1 mg /ml hyaluronidase (Type I , Sigma) buffered w ith10 m M HEPE S pH 7.3, and supplemented wi th 10% fetal cal fsert lrn. The incubation mixture was gently gassed throughoutwith a mix ture of air (95 %) and CO2 (5%). After 4 h incubation,the tissue was stirred with a m agnetic stirrer (45 min, 37~C). Thecell and t issue suspension was fi l tered throu gh g auze, then thecells were recovered by cen trifugation (5 rain, 150 g, 4~C) andwashed once in ice-cold Tyro de's buffer. Finally, the cells wereresuspended in Tyrod e's buffer and passed thro ugh a nylon m eshto rem ove debris.I so la t ion o f ra t per i tonea l ce l l sThe animals were anesthetized with ether and kil led by ex-sanguination. Ha nks solution containing 0.1% bovine serum

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Fig. 1. Buoyant density of nucleated cells, red blood cells, and mast cells on rat skin. Rat abdominal skin was digested with collagenaseand hyaluronidase , and cell suspension was centrifuged with Percoll discontinuous gradient . Cells were identified with Blutstan stain.S tra igh t l ine shows the buoyant density of each fraction. Buoyant density was monitored by density marker beads

albumi n and 10 units/ml heparin was injected into the peritonealcavity. After 90 s, the exudate was collected by pipette, andwashed twice with Tyrode' s solution by centr ifugation at 200 gfor 5 min at 4~ Then, pellets were resuspended in Tyrode' ssolution. Viability of cutaneous and peritoneal mast cells wasalways greater than 95%, as determined by trypan blue dyeexclusion.Perco l l cen tr i fuga l e lu tr ia t ion o f mas t ce ll sIsolation of mast cells was done using a me thod similar to thatpreviously described [5]. Percoll (colloidal silica polyvinyl-pyrrolidone) and density marker beads were obtained fromPharmacia Fine Chemicals (Uppsala, Sweden). Isotonic Percollsolutions were prepared by dissolving nine parts of Percoll withone part of a tenfold concentrated Hanks solution. Coloreddensity marker beads were used for calibration of Percoll gradi-ents and for checking the equilibrium conditions. An aliquot of0.75 ml of the cell suspension in Hanks solution was added to3.5 ml Percoll isotonic solution, resulting in a final density of1.110. Then, 0.5 ml Hanks buffer was layered on top of thesolut ion and centrifuged at 125 g for 15 rain. The two uppermostmilliliter were removed together with 0.5 ml Hanks solutionfor washing the wall of the test tube. The remaining volumecontain ing the mast cells was washed twice in Hanks solut ion.His tamine concen tra t ion assayCompound 48/80-induced h istamine release was studied usingthe following procedure. Aliquots of 0.9 ml of peritoneal cellsuspension (ca. 1 x l04 mast cells) were preincubated for 10 minat 37~ t00 gg/mt compound 48/80 (N-methyl-p-methoxy-

phenylaminewith formaldehyde; Sigma) was added in a volumeof 0.1 ml, followed by incubat ion at 37~ for 10 rain. Thereaction was stopped by dipping into ice-cold water. The cellswere sedimented by centrifugation at 400 g for ~0 min at 4~and the supernatants were collected. The pellets were resus-pended in 1 ml Tyrode's solution, heated at 100~ for 5 rain torelease their residual histamine and centrifuged at 400 g for 10rain. The aliquots from these supernatants were assayed for theirhistamine content using reverse phase high-performance liquidchromatography (HPLC) [19J. The HPLC system (Toyo Soda,Tokyo, Japan) consists of a CCPM multipump, an FS-8000spectrophotofluorometer, a TSKgel ODS-120T reversed phasechromatography column, an AS-48 auto-sampler, and a Chro-matocorder 11. The fluorescence intensity was monitored at theemission wavelength of 450 nm with the excitat!on wavelengthset at 350 nm. The results were expressed as ~ percentage ofreleased histamine by the total histamine, and each histaminerelease represents the average of the means _+ SD (n = 6). Stat-istical analyses were performed using Student 's t-test.M i c r o s c o p ic e x a m i n a t i o nSlides for the microscopical examinat ion of the dispersed cellswere prepared using Cytospin 2 (Shoadon, Cheshire, England);they were fixed in absolute methanol and stained with Giemsasolution.R e s u l t sThe d iges t ion of one ra t abdominal sk in with eol-lagenase and hyaluronidase y ie lded approximately7 x ]06 nu cle ated cells.

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F i g . 2 . B u o y a n t d e n s i t y o f r a t c u t a n e o u s m a s t c e il s . R a t c u -t a n e o u s celt suspension was layered on each density o f Percoll,and cells we re recovered from the low er portion of the tubes.Most of the c ontaminated cells were red blood cells. Cells w ereidentified with Blutstan stain

T h e b u o y a n t d e n s i ty o f c u t a n e o u s m a s t c el ls w a ss t u d i e d i n P e r c o l l c o n t i n u o u s c e n t r i f u g a t i o n . A t2 0 ,0 0 0 g f o r 9 0 r a i n 8 m l i s o t o n i c P e r c o U s o l u t i o n w a sc e n t r i f u g e d w i t h d e n s i t y m a r k e r b e a d s : T h e n 1 m l o fe n z y m i c a l l y d i s p e rs e d c u t a n e o u s C e ll s u s p e n s i o n w a sl a y e r e d a n d c e n t r i f u g e d a t 4 0 0 g f o r 3 0 r a in a t 4 ~A l i q u o t s o f 0 .5 m l w e r e a s p i r a t e d a n d w a s h e d t h r e et i m e s w i t h H a n k s s o l u t io n . S i nc e i d e n t i f ic a t i o n o f u n -s t a ine d ce l l s is d i f f i cu l t , t 0 I -tl o f ce l l su sp ens ion wa ss m e a r e d i n a g l a ss s l id e p r e c o a t e d w i t h m e t h y l e n e b l u ea n d c r e s y l v i o l e t ( B l u t s ta n , D a i i c h i P u r e C h e m i c a l ,T o k y o , J a p a n ) . M a s t c e l l s , n u c l e a t e d c e l l s , a n d r e db l o o d c e l l s w e r e e a s i l y d i f f e r e n t i a t e d a n d c e l l n u m b e r sw e r e c o u n t e d m i c r o s c o p i c a l l y w i t h i n a h i g h - p o w e rf i el d (4 0 x 1 0). T h e b u o y a n t d e n s i t y o f r a t c u t a n e o u sm a s t c e l ls i s s h o w n i n F i g . 1 . T h e u p p e r f r a c t i o n , w h i c hw a s o f l o w b u o y a n t d e n s i ty , s h o w e d i n c re a s e d n u m -b e r s o f n u c l e a t e d c e l ls a n d r e d b l o o d c e l ls . S e v e n o rl a t e r f r a c t i o n s , h i g h e r t h a n 1 . 1 0 0 , r e v e a l e d r e l a t i v e l yp u r e m a s t c e l l s i n e a c h f r a c t i o n .S i m i la r ly , t h e b u o y a n t d e n s i t y o f c u t a n e o u s m a s tc e l l s w a s m o n i t o r e d u s i n g P e r c o l l d i s c o n t i n u o u sc e n t r i f u g a t i o n . C u t a n e o u s c e ll s w e r e m i x e d w i t h 5 m lP e r c o l l - H a n k s s o l u t i o n o f v a r y i n g d e n si ti e s a n dl a y e r e d w i t h 0. 5 m l H a n k s s o l u t i o n . A f t e r c e n t r i f u g a -t i o n a t 1 25 g f o r 15 m i n a t 4 ~ t h e u p p e r I m l o f t h es o l u t i o n s w a s d i s c a r d e d a n d t h e r e m a i n d e r w a s h e dt h r e e ti m e s w i t h H a n k s s o l u t io n . E a c h f r a c t i o n w a ss t a i n e d w i t h B l u t s t a n , a n d t h e p u r i t y o f m a s t c e l lsc o u n t e d . A s s h o w n i n F i g . 2 , th e h i g h e r d e n s i t y o f

H. H achisuka et al.: Purification of cutaneous mast cellsTable 1. Compo und 48/80-induced histamine release from p eri-toneal and cutaneous mast cells

H i s t a m i n e r e l e a s e ( % )Unpuri f ied Pur i f ied

Perito nea l m ast cells 61.0 _+ 8.3 55.5 + 3.8Cuta neou s m ast cells 28.0 + 4.5 21.3 +_ 3.8Each m ast cell was purified by Percoll and stimulated with 10 g g/ml com pound 48/80. The histamine release was expressed as apercentage of released histamine by the total histamine. Noneof the differencesbetween the treatment groups was statisticallysignificant

P e r c o l l - H a n k s s h o w e d a h i g h e r p u r i t y o f m a s t c e ll s.C o n t a m i n a t e d c e ll s w e r e m o s t l y r e d b l o o d c e ll s. S i n cet h e t o t a l n u m b e r s o f m a s t c e l ls w e r e r e d u c e d d u r i n gt h e p u r i f i c a t i o n p r oc e s s , t h e b u o y a n t d e n s i t y o f P er c o llwa s se t a t 1 .110.T h e p r o c e d u r e f o r p u r if i c a t i o n o f r a t c u t a n e o u sm a s t c e l l s w a s c a r r i e d o u t a s d e s c r i b e d i n M a t e r i a l sa n d m e t h o d s . C u t a n e o u s a n d p e r i t o n e a l m a s t c e l l sw e r e i s o la t e d a n d p u r i f i e d f r o m t h e s a m e a n i m a l s , a n dt h e p u r i t y o f m a s t c e ll s w a s c o m p a r e d w i t h e a c h t is s u e .Befo re Perco l l f r ac t i on a t i o n , 7 .4 % __+ 2 .4% o f nuc l ea t -e d c e l l s w e r e r e g a r d e d a s c u t a n e o u s m a s t c e l l s , w h i l e5 . 8 % +_ 1 . 3 % w e r e c o n s i d e r e d p e r i t o n e a l t o b e m a s tc e l l s . A f t e r P e r c o l l c e n t r i f u g a t i o n , a m o n g t h e n u c l e a t -e d c e l ls t h e p u r i t y o f c u t a n e o u s m a s t c e ll s r e a c h e d5 0 . 0 % + 6 . 4 % , w h i l e th e p u r i t y r e a d i n g fo r p e r i t o n e a lm a s t c e ll s w a s 6 1 . 0 % + 1 0 . 6 % .F o r t h e s t u d y o f m a s t c e ll s e c re t io n , h i s t a m i n e -l i b e ra t i n g c o m p o u n d 4 8 / 8 0 w a s u s e d a s a s e c r e ta g o g u ei n u n p u r i f i e d a n d p u r i f i e d m a s t c e l l s u s p e n s i o n s .T a b l e I s u m m a r i z e s t h e re s u lt s . C o m p o u n d 4 8 /8 0 - in -d u c e d h i s t a m i n e r e le a s e f r o m u n p u r i f i e d a n d p u r i f i e dm a s t c e l l s w a s s l i g h t ly r e d u c e d d u r i n g P e r c o l l f r a c t i o n -a t i o n . R e l e a s e o f h i s t a m i n e f r o m p e r i t o n e a l m a s t c e l lsw a s a b o u t t w o f o l d h ig h e r t h a n t h a t o f c u t a n e o u s m a s tcel l s .

Discuss ionC o l l a g e n a s e a n d h y a l u r o n i d a s e d i g e s t io n o f r a t a b -d o m i n a l s k i n h a s b e e n s h o w n t o d i s p e rs e i n t r a d e r m a ln u c l e a t e d c e l l s , i n c l u d i n g f i b r o b l a s t s , c a p i l l a r y e n d o -t h e l i a l c e l l s , a n d m a s t c e l l s . T h i s p r o c e d u r e h a s a na l m o s t n e g l i g i b le e f f e c t o n h i s t a m i n e r e l e a s e b y p e r i t o -n e a l m a s t c e l ls , a n d t h e m a s t c e ll s o b t a i n e d r e s p o n d e dw e l l t o v a r i o u s s t i m u l i [ 1 ] . T h e r e f o r e , w e a t t e m p t e dt o f u r t h e r p u r i f y r a t c u t a n e o u s m a s t c e l l s . F o r t h ei s o l a t i o n o f m a s t c e l ls , s e v e r a l m e d i a h a v e b e e n i n t r o -d u c e d : b o v i n e s e r u m a l b u m i n [ 1 8 ], F i c o ll s o l u t i o n[10 , 20 ], M et r i z am ide [21 ] , an d Perco l l [5 ].

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H. Hac hisuk a et al .: Purification of cutaneous ma st cells 361P e r c o l l ( c o l l o i d a l s i l i c a p o l y v i n y l p y r r o l i d o n e ) i s a

u n i q u e d e n s i t y g r a d i e n t m e d i u m d e s i g n e d to m a i n t a i np h y s i o l o g i c a l c o n d i t i o n s t h r o u g h o u t c e n t r i f u g a t io ne x p e r i m e n t s . I t h a s l o w o s m o l a r i t y a n d v i s c o s it y , a n dc a n b e m i x e d w i t h v a r i o u s p h y s i o l o g i c a l s o lu t i o n s. I na d d i t i o n , d e n s i t y g r a d i e n t s a r e s e l f - g e n e r a t e d d u r i n gh i g h - s p e e d c e n t r if u g a t i o n . U s i n g t h is m e d i u m , w eh a v e p r e v i o u s l y r e p o r t e d t h a t g u i n e a p i g e p i d e r m a lc e l l s s u c c e s s f u l l y s e p a r a t e i n t o t h r e e f r a c t i o n s w h i c hc o r r e s p o n d r e l a t i v e l y t o t h e i r a r r a n g e m e n t i n v i v o[7, 15].

W i t h P e r c o t l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , t h er a t c u t a n e o u s m a s t c e l ls l o c a t e a t h i g h e r d e n s i t y f r a c -t i o n s , e s p e c i a ll y a t b u o y a n t d e n s i t i es o f 1 . 1 00 o r a b o v e .I n t h o s e f r a c ti o n s , c o n t a m i n a t e d c e ll s a r e m a i n l y r e db l o o d ce ll s. D i s c o n t i n u o u s f r a c t i o n a t i o n w i t h P e r c o l lc e n t r i f u g a t i o n s h o w s t h a t p u r i t y o f m a s t c el ls i n cr e a se sa t h i g h e r f r a c t i o n s . S i n c e c o n t a m i n a t e d c e l ls a r em o s t l y r e d b l o o d c e l l s , a s s u m e d t o h a v e n o i n f l u e n c eo n m a s t c e ll s, w e t o o k t h e b u o y a n t P e r c o ll d e n s i ty a t1 . 10 0 f o r t h e d i c o n t i n u o u s g r a d i e n t . T h i s d e n s i t y f o rr a t c u t a n e o u s m a s t c e l ls i s t h e s a m e a s t h a t f o r p e r i t o -n e a l m a s t c e l l s [ 5 ] . B l u t s t a n o r G i e m s a s t a i n i n g o fc y t o c e n t r i f u g e d s m e a r s s h o w s t h e p u r i f i e d c u t a n e o u sm a s t c el ls to b e m o r p h o l o g i c a l l y i n t a c t T r y p a n b l u ed y e e x c l u s i o n r e v e a l s a v i a b i l i t y g r e a t e r t h a n 9 5 % .

R e c e n t l y , B e n y o n e t a l. [2 ] h a v e r e p o r t e d t h e i s o -l a t io n a n d p u r i f i c a ti o n o f c u t a n e o u s m a s t c e ll s f r o mh u m a n f o r e sk i n . T h e y u s e d c o l l a g e n a se a n d h y a l u r o n i -d a s e d i g e s t i o n t o d i s p e r s e o f c u t a n e o u s m a s t c e ll s, a sB a r r e t t e t a l . [ 1 ] a n d w e h a v e d o n e . P r e t r e a t m e n t w i t ht h e s e e n z y m e s s h o w e d n e g l i g i b l e e f f e c t s f o r v i a b i l i t ya n d h i s t a m i n e r e l e a s e f r o m v a r i o u s s t i m u l i [1 ]. B e n y o ne t a l. [2 ] p u r i f i e d h u m a n c u t a n e o u s m a s t c e ll s t o 8 0 % ,h o w e v e r , o n l y 1 6 % o f t h e to t a l m a s t c e ll s w e r e r e -c o v e r e d , a n d 5 0 % p u r i t y w a s r e c o v e r e d f r o m 5 3 % o ft o t a l m a s t c e ll s. T h u s , t h e p u r i t y a n d r e c o v e r y o f t h er a t c u t a n e o u s m a s t c e ll s i n o u r e x p e r i m e n t a r e a l m o s tt h e s a m e a s r e p o r t e d b y B e n y o n e t a l . [ 2 ] .

F o r c o m p a r i s o n o f r a t c u t a n e o u s a n d p e r i t o n e a lm a s t c e ll s, p e r i t o n e a l m a s t c e ll s w e r e a l s o p u r i f i e da c c o r d i n g t o E n e r b / i c k a n d S e v e n s s o n [5 ]. C o m p o u n d4 8 / 8 0 w a s u s e d t o c o m p a r e t h e h i s t a m i n e - r e l e a s i n ga c t i v i t y o f th e s e m a s t c e l ls . O u r r e s u l ts i n d i c a t e t h a tc u t a n e o u s m a s t c e ll s a r e le ss r e ac t i v e t o c o m p o u n d4 8 / 8 0 , a n d t h i s o b s e r v a t i o n i s c o n s i s t e n t w i t h a p r e -v i o u s r e p o r t [ 1 ]. T h e s e f i n d i n g s s u g g e s t s t h a t t h e r e a r ef u n c t i o n a l s u b s e t s o f c o n n e c t i v e t i s s u e m a s t c e l ls .

I n s u m m a r y , t h e p r e s e n t s t u d y p r o v i d e s a s i m p l em e t h o d f o r th e i s o l a ti o n a n d p u r i f ic a t i o n o f r a tc u t a n e o u s m a s t c e ll s. B e c a u s e o f th e a p p a r e n t h e t e r o -g e n e i t y o f m a s t c e l ls f r o m d i f f e r e n t s it e s, i t m a y b ei m p o r t a n t t o u s e c u t a n e o u s m a s t c e ll s i n th e e v a l u a t i o no f a n t i a n a p h y l a c t i c d r u g s . A l s o , t h e a v a i l a b i l i t y o f

p u r i f i e d c u t a n e o u s m a s t c e l l s m a y f a c i l i t a t e f u r t h e rs t u d y o f t h e r o l e o f m a s t c e ll s i n s k i n a l l e rg y .

R e f e r e n c e s

1. B arrett KE , Ali H, Pearce FL (1985) Studies on ihistaminesecretion fro m enzym ically dispersed cutaneous ma st cellsof the ra t. J Invest Derm atol 84:2 2- 252. Benyon RC, L owm an MA, Church M K (1;987) Hu manskin mast cells: their dispersion, purification, and secretorycharacter ization. J Immu nol 138:86 1-86 73. Cou tts SM, Nchring R E Jr, Jariwala N U (1980) Purificationof rat peri toneal ma st cells: occupation of IgE receptors byIgE prevents loss of the receptors. J Immunol 124:2309 - 23154. Enerb/ick L (1981) The gut m ucosal mas t cell . Mo nog rAllergy 17 : 2225. Enerbfick L, Sevensson I (1980) Isolation of rat peri tonealma st cells by centrifugation on density gradients of Percoll.J Im m uno l Methods 39:135 - 1456. G ibson S, Miller HR P (1986) Ma st cell subsets in the ratdist inguished immunohistochemically by their content ofserine proteinases. Imm uno logy 58 : 101 - 1047. Hachisuka H, Nom ura H, Sasai Y, Shiotsuki K, Yokoy amaMM (19 86 ) Changes of intracellular fluidky duringkeratinocyte differentiation measu red by: fluorescencepolarization. Cell Tissue Res 246 : 5 57 - 5608 . Hach i suka H , Nom ura H , S akamot o F , M or i O , Okubo K ,Sasai Y (1988) Effect of antianaphylactic agents on sub-stance-P induced histamine release from rat peri toneal ma stcells. Arch D erm atol Res 280:158 - 1679. He iman AS, Crews FT (1985) Characterization of the effectsof phorbol es ters on ra t mast cel l secret ion. J Immunol

134: 5 4 8 - 5 5510. Ishizaka T , K6nig W, K urata M , M auser L , I shizaka K(1975) Immu nologicprop erties of ma st cells from rats infect-ed with Nippostrongylus brasiliensis. J Irnmunol 115:1078-108311. Ko da A, N agai H, W atanabe S , Yanagihar a Y, Saka motoK (1976) Inhibition of hypersensitivity reactions b y a newdrug, N(3",4"-dimethoxycinnamoyl) anthran~lic acid (N-5').J Al lergy Cl in Imm unol 57:3 96-4 0712. Kus ner EJ, Dub nick B, Herzig DJ (1973) The inhibit ion bydisodium cromoglycate in vitro o f anaphylaCtically inducedhistamine release from rat peri toneal m ast cells. J PharmacolE xp T her 184 : 41-4 613. Martin U, R 6m er D (1978) The pharmacological prope r-t ies on a ne w, orally active antianaphylacfic com pou nd:ketotifen, a benzocy cloheptathiophen e. Arzneim ittelforsch28 : 77 0- 78214. Newlands GF, Hunt ley JF , Mi l ler HR (1984) Conco mitantdetection ofm uc osa l mast cells and eosinophils in the intes-t ines of normal and Nippostrongylus-immune rats. A re-evaluation of histochemical and immunocyt0chemical tech-niques. Histochemistry 81:585-58915. Sasai Y, Hach isuka H, Mo ri O, No m ura H (1984) Separa-tion of keratinocytes by density gradient centrifugation forDN A cytofluorometry. Histochemistry 80:1 33--13 616. Schleimer RP, Macglashan DW Jr, Peters SP, Pinckard R N,Adkinson N F Jr, Lichtenstein LM (1986) Characterizationof inf lamma tory mediator release f rom pur if ied hum an lungmast cel ls . Am Rev R espi r Dis 133 : 61 4- 61717. Shan ahan F, D enbu rg JA, Fox J, Bienenst,ock J, Befu s D(1985) Ma st ce ll heterogeneity: effects Of neuroentericpeptides on histamine release. J Immunol 135:1331-1337

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362 H. Hachisuka et al.: Purification of cutaneous mast cells18. Sullivan TJ, Parker KL, Stenson W, Parker CW (1975)Modulation of cyclic AMP in purified rat mast cells. Jhnmunol 114:1473-147919. Tsuruta Y, Kohashi K, Ohkura Y (1978) Determination ofhistamine in plasma by high-speed liquid chromatography.J Chromatogr 146: 49 0- 49320. Uvn/is B, Thon I-L (1959) Isolation of"biologically intact"

mast cells. Exp Cell Res 18: 512 - 520

21. Yurt RW, Leid RW Jr, Spragg J, Austen KF (1977)Immunologic release of heparin from purified rat peritonea lmast cells. J Immunol 118 : 1201 - 1207

Received December 24, 1987