IMP Sebum Freso y Viejo as Piel Montagna 13

8/3/2019 IMP Sebum Freso y Viejo as Piel Montagna 13

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H I S T O L O G Y A N D C Y T O C H E M I S T R Y O F H U M A N S K I NI X. THE Dm ~m UT~ON Ot No N- SP~ . c ~I c EST~a AS~S*

BY WILLIAM MONTAGNA, PH.D.(From Arnold Biological Laboratory, Brown University, Pro Ven ce)

P ~s 3 To 5(Received for publicat ion, September 8, 1954)

INTRODUCTION

A b u n d a n t n o n - s p e ci f ic es t e ra s e a c t i v i t y c a n b e d e m o n s t r a t e d i n h u m a n s k i n,b y t h e m e t h o d o f N a c h l a s a n d S e l i g m a n ( 5 ). I n t h e e p i d e r m i s t h e e n z y m e i sc o n c e n t r a t e d i n a s t r o n g l y r e a c t i v e b a n d j u s t a b o v e t h e s t r a t u m g r a n u l o s u m( F i g . I ) . Th i s s t r i k i n g m o r p h o l o g i c a l e n t i t y i s p e c u l i a r t o t h e g e n e r a l e p i d e r m i sa n d i s n o t f o u n d i n t h a t o f t h e p a l m ( F i g . 2 ). E n z y m e a c t i v i t y i s p r o n o u n c e di n t h e o u t e r s h e a t h o f h a i r f o l l ic l e s ( F i g . 5 ) , a n d i n t h e s e b u m o f s e b a c e o u sg l a n d s ( F i g s . 6 a n d 7 ) . Th e a x U l a r y a p o c r i n e s we a t g l a n d s a r e v e r y s t r o n g l yr e a c t iv e b u t t h e e c c r i n e g l a n d s e v e r y w h e r e i n t h e s k i n s h o w o n l y t r a c e s o f t h ee n z y m e ( F i g s . 9 to I I ) .

Materials and MethodsThe biopsy specimens used in th is investigation were collected from the scalp, the back, thechest, the palm, and the axilla of human volunteers. Each specimen was removed without,

anesthesia with a high-speed rotary biopsy punch, 5 ram. in diam eter. Those from the scalp,back, chest, and palm were collected from healthy males about 25 yea rs old; those from theaxilla were collected from two females 24 an d 34 y ears old. T he p ieces were fixed 24 hours inchilled, 10 per cent neutral formaldehyde and eventually sectioned with the freezing micro-tome at 10 to 15 #. Th e sections were hardened for 2 hours in 10 per cent formaldehyde, Theywere then washed thoroughly in water, defatted in acetone for 5 minutes, returned to dis-tilled water, and finally placed in the incubation mixture described b y Nachlas and Seligman(5), using a-naphth yl acetate as the su bstrste (2). The incubation mixture was prepared bydissolving 20 rag. of c~-naphthyl aceta te in 0.5 ml. acetone, a nd then a dding to it 20 ml. of 0.Iit phosphate buffer at pH 7.4 (6). The solution was stirred, and when its cloudiness disap-peared, 20 rag. of naphthanil diazo blue B w as added to it. The solution was filtered before it* This work was supported in part by a gr ant from the U nited States Public Health Serv-ice, RG 2125 C4.I Although Naehla s and Seligman (5) used acetone as a fixative because "alm ost comp leteinactivation was produced by exposure of the tissue for 24 hours to formaldehyde (I0 per cent). ." in the experience o f this investigator the results obtained with the two fixatives wereessentially similar. Formaldehyde has the advantage of being a better fixative for the main-tenance of tissue integrity. It has been noted moreover th at strong enzyme ac tivity is preservedeven in tissues deliberately left in formaldeh yde for ! week.

13J. B Io~ Ys lc, AND BIoCn'2~. C~TOL., 195~, Vol. 1, No, I


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14 F _ . S ~ S E S I N H U MA N S K INw a s u s e d . A f t e r t h e s e c t io n s h a d b e e n i n c u b a t e d a t r o o m t e m p e r a t u r e f r o m 5 t o 3 0 m i n u t e s,t h e y w e r e w a s h e d i n d i s t il l e d w a t e r a n d m o u n t e d i n g l y c e r i n j e l ly .

OB SER VATI ONSThe epidermis of the genera l skin has a modera te amount of demonst rable

enzyme ac t ivi ty in the basa l layer and in the lower two or three ce l l s of theMalpighian layer . The reac t ion fades in the upper ce l l s and i s comple te ly ab-sen t f rom the g ranu la r l aye r . Jus t ab ove t he g ranu la r l aye r i s a v e ry s t rong lyreact ive , uninterrupted band (Fig. 1) . This band rests above the granularl aye r and be low the s t r a tum corneum, bo th o f wh ich a re un reac t i ve . In t heepidermis of the pa lm, the reac t ion i s confined a lmost ent i re ly to the lowerthree o r four layers of ce ll s of the M alpighian lay er (Fig. 2) . There i s no st ron glyreac t i ve band above t he g ranu la r l aye r ; t he s t r a tum luc idum i s weak ly r eac t i veand the granular layer and the st ra tum corneum are unreact ive .In the pi losebaceous canal of ha i r fol lic les , enzyme ac t iv i ty i s ident ica l w i ththat of the surface epidermis (Fig. 8) . The ce ll s of the outer sh eath a bou nd inenzyme ac t ivi ty a l l the way to the t ip of the matr ix (Fig. 3) . Enzyme react ionin th e rest of the foll ic le i s scant . The ce ll s in the dermal papi l la conta in reac-t ive granules. In rest ing hai r foll ic les , the outer sheath h as on ly a mod era teamoun t o f enzyme reac t i on . Tha t pa r t o f t he ou t e r shea th which fo rms theepithel ia l capsule around the ha i r c lub, however , conta ins i sola ted c lusters ofvery s t rongly reac t ive ce l ls (Fig . 5) . The peg of ce ll s underneath the capsule ,which mainta ins conta c t wi th the derm al papi lla , the so ca lled "hai r germ ",is weakly reac tive . D uring catagcn, whe n th e bulb of the ha i r foll ic le begins todegenerate, in preparation for the establishment of a quiescent foll icle, i ts cellsare par t icular ly r ich in esterases (Fig. 4) . The ce l l s of the dermal papi l la re-main ac t ive dur ing ca tagen b ut a re re la t ive ly unreact ive in quiescent foll ic les .The indi fferent per iphera l sebaceo us ce l ls and those ce ll s which a re und er-going sebaceo us transf orm ation are rich in non-specific esterases (Figs. 6 and7). Th e m a tu re cel ls , howeve r , and t he newly fo rmed sebum in t he midd le o fthe ac ini a re unreact ive . The old sebum in the ducts and in the pi losebaceouscanal , however , i s very st rongly reac t ive (Fig. 7) . The hai r shaf t and i t s sur-rounding, f ragmen t ing internal sheath emerge f rom the fol licle ba th ed in ester-ase-rich sebum (Fig. 8).

Eccr ine sweat glands show a weak to modera te reac t ion. The reac t ion i sst rongest in the "d ark " ce ll s (3); the c lear cel ls show only a t race of i t (Fig . 9) .The t ransi t ion segm ent of the duct show s scant enzym e react ion whi le the restof the duct i s negat ive .

The axi l la ry apocrine sweat glands have st rong esterase ac t ivi ty . Th e enzym eis confined to the u pper p ar t o f the epi the l ia l ce ll s; the basa l p ar t an d th e myo -epi the l ia l ce l l s a re unreact ive (Figs. 10 and 11) . The st rength of the reac t ionin the epi the lia l ce ll s i s dependent u pon the am ount of secre t ion mater ia l s tored


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WILLIAM MONTAGNA 15

in the cel l s . The cel l s wi th the most secret ion mater ial have the s t rongest en-zym e r eac t ion (F i g. 10 ) . The duc t s show on l y a t r ace o f r eac t ion .In the connect ive t i ssue, enzyme act iv i ty i s found in the endothel ial cel l s of

capi l lar ies around the epidermal appendages and in the f ibroblast s . Mast cel l si n t he t e l a subcu t anea show var i ab le num ber s o f r eac t i ve g ranu les .

DISCUSSIONOne of the pecu l iar it ies of sk in surface l ip ids i~ tha t they c ontain large qua n-

t i t ies of f ree fa t ty ac ids (7) . Since the chief sources of thes e l ip ids are the seba-ceous g l ands and t he l i p i d p roduc t s o f kera t i n i za t i on , t he f a t t y ac i d s mus tor ig inate f rom these l ip ids . I f , as has been s tated , non-speci f ic es terases hy-drolyze esters of shor t and long chained fat ty acids wi th var ious alcohols ( 5 ) ,then the f ree fat ty acids in surface lip ids could be released through the act ionof these enzym es. The localization of the enzym e in the epidermis and in th esebum seems t o be par t i cu l a r l y s t r a t eg i c fo r such ac t i on . The band ou t l i nedby the enzy me in the epiderm is is a morphological en t i ty which coincides wi tht he s t r a t um l uc i dum and wi t h a l ayer r ich i n p ro t e i n -bound su l fhyd ry l g roups(4) . However , t he s t r a t um l uc i dum p roper i n t he ep i dermi s o f t he pa l m showsonly a negligible reaction for non-specific esterases. Therefore, the esterase-r ich layer in the general sk in cannot be considered to be the s t ratum lucidum,and pe rhaps deserves a nam e o f i t s own .

In co n t r as t w i t h t he t ween met hod o f Gomor i (1) , wh i ch demo ns t r a t es on l yt r aces o f es te r ase ac t i v i t y i n human sebaceous g l ands , t he p resen t t echn i queshows abun dan t enzyme ac t i v i t y . Enzym e ac t i v i t y i s a l so found i n t he i nd if fe r-en t seba ceous cell s and in th e cel l s which a re s tor ing se bace ous l ip ids . T hema t u re an d deca y i ng sebaceaus ce ll s, and t he new sebu m i n t he cen t e r o f t heacinus show no enzym e react ion . I t i s surpr is ing , therefore, to f ind s t rong en-zyme ac t i v i t y i n t he sebum i n t he excre t o ry duc t and i n t he p i l o sebaceouscana l. S i nce t he es t e rases i n the sebum mus t b e deri ved f rom t he sebaceouscel l s , one might speculate that when the cel l s break down they release some-th ing which inhib i t s enzyme act iv i ty . The act ion of such a presumpt ive inhib i -t o r mus t , however , be sho r t l ived, o r add i t i ona l subs t ances i n t he duc t m us tovercome t he ac t i on , because , t he o l d sebum abounds i n es t e rase ac t i v i t y .

Since apocr ine sweat g lands in the axi l la s tore large amounts of l ip ids~ theabundan t enzyme wh i ch t hey con t a i n cou l d be t i ed up wi t h f a t t y hyd ro l y s i s .Enzyme act iv i ty i s par t icular ly s t rong in cel l s which have v is ib le secret ionp roduc t s i n t he cy t op l asm. Th i s p rope r t y sharp l y separa t es t he apocr ine f romt he eccr ine g lands , wh i ch show on l y modera t e t o w eak enzym e r eac ti on .

The eccr ine sweat g lands possess scanty enzyme act iv i ty , and the local iza-t ion of the act iv i ty i s conf ined to th e " da rk" cel ls , in cont ra st to alkaline phos-phatase , which i s local ized pr imar i ly in the "clea r" cel ls . Th e "c lear" and "d ark "cel ls , then , appe ar to be tw o dis t inct typ es of secretory cell s .


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1 6 E S T E R A S E S I N H U M A I ~ S K I N

S U M M A R YI . I n t h e ep id ermi s n o n - sp ec i f ic e s t e r ase ac t i v i t y o u t l in es a s t ro n g ly r eac t i v e

b a n d b e t w e e n t h e s t r a t u m g r a n u l o s u m a n d t h e s t r a t u m c o r n e u m . I n t h e e p i -d e rmi s o f t h e p a lm, t h e re i s n o su ch es t e r ase - r i ch b an d .

2 . T h e o u t e r s h e a t h o f a c t i v e h a i r f ol li c le s h a s s t r o n g e n z y m e a c t i v i t y . T h ed eg en era t i n g h a i r b u lb i n ca~g en fo l li c les i s v e ry s t ro n g ly r eac t i v e , an d c lu s t e r so f ce l l s a ro u n d t h e h a i r c l u b i n q u i escen t fo Uid es a r e r i ch i n en zy me ac t i v i t y .

3 . S t ro n g en zy m e ac t i v i t y i s f o u n d i n y o u n g seb aceo u s cel ls , wh i l e d ecay in gs e b a c e o u s ce il s a n d n e w l y f o r m e d s e b u m a r e u n r e a c t i v e . O l d s e b u m , h o w e v e r ,i s v e r y i n t e n s e l y r e a c t i v e .

4 . O n l y t h e " d a r k " c e ll s o f e c c r in e sw e a t g l a n d s sh o w a r e a c t i o n ; t h e " c l e a r "ce i l s a r e n eg a t i v e .

5 . Th e ce i l s o f ax i l l a ry ap o cr in e g lan d s ab o u n d i n en zy m e.BIBLIOGRAPHY

I. Gomori , G. , Arch . Path . , 1946 , 41~ 121.2. Gomori, G., Microscopic Histochemistry, Chicago, University of Chicago Press,1952.3 . M ontagna, W., Chase, H. B . , and Lobi tz , W. C. , J r . , J. Inv. DermatoL, 1953, 20,415.4. M ontagna , W., Eisen, A. Z., Rade mac her, A. H., and Chase, H. B., Y. Inv. Der-matol., 19 54, 23, 23.5 . N achlas , M. M., and Seligman, A. M . , Y . N at . Cancer Ins t . , 1949, 9, 415.6 . Pearse, A. G. E. , His tochemist ry . Theoret ical and Applied, Lon don , J . and A.Churchill, Ltd., 1954.7 . Rothm an, S . , Physio logy and Biochem ist ry o f the Sk in , Chicago , Univers i ty o fChicago Press, 1954.

EXPLANATION OF PLATESPLATE 3

FIG. 1. Epidermis from the axil la. Reactive granules are present in the lower ceilsof the Malpighlan layer. An estemse-rich band is to be noted above the granularlayer. X 560.FIG. 2 . Epidermis o f the palm. Enzym e react ion i s demonst rab le in the lowercells of the M aipighian layer. The re is, however, no strongly reactive band above th egranular layer , a ind icates the s t ratum co meu m and b the s t ratum iucidum. 60 .FIG. 3. Estera se ac tivity in the bulb of an ac tive hair foll icle from the chest . Strongenzyme act iv i ty i s ev iden t in the ou ter sheath . With the excep t ion of the cone of b lackcells around the dermal papilla (P) which contain melanin, all dark colored materialrepresen ts enzyme act iv i ty . X 60.FIG. 4. Catag en hair foll icle from the chest . Th e degene rating cells of the bulb showvery s t rong enzy me act iv i ty . The dermal pap i l la (P) also con tains react ive cel ls . 270.


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THE JOURNAL OFBIOPHYSIC~ AND BIOCHEMICALCYTOLOGY

PLATE 3VOL. 1

(Montagna: Esterases in huma n skin)


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PLATE 4FIC. 5. Hair club from the chest. The epithelial capsule contains clusters of veryreactive cells. X 270.Fro. 6. Sebaceous glands from the axilla. X 270.Fro. 7. Sebaceous gland from the scalp. Degenerating cells and new sebum are un-

reactive; the old sebum, on the other hand, is strongl y reactive. X 270.Fro. 8. Pilosebaceous canal conta ining strongly reactive sebum. This was found ina specimen from the axilla. X 270.


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T H E J O U R N A L O FBIOPHYSICAL AND BIOCHEMICALC Y T O L O G Y

P L A T E 4VOL. 1

(Mon tagna : Es te ra ses in human sk in)


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PLATE 5F IG . 9 . E n z y m e a c t i v i t y i n th e e c c r i n e s w e a t g l a n d s o f t h e a x i l l a . T h e " d a r k " c e l ls

s h o w t h e s t r o n g e s t r e a c t i o n . X 5 6 0.F I o . 1 0 . A b u n d a n t r e a c t i o n p r e s e n t i n a x i l l a r y a p o c r i n e g l a n d . X 5 6 0.F I G . 11 . M o d e r a t e r e a c t i o n e v i d e n t i n t h e a p i c a l p o r t i o n o f t h e c e ll s o f t h e a p o c r i n e

g lands . X 560 .


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THE JOURNAL OFBIOPHYSICAL AND BIOCHEMICALCYTOLOGY

PLATE 5VOL. 1

(Montagna: Esterases in human skin)

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IMP Sebum Freso y Viejo as Piel Montagna 13