i

THE J OURNAL m- I NVEST IGATIVE DEIlMATOLOGY, 65:259- 27 1, 1975 Copyrigh t © 1975 by The Williams & Wilkins Co.

Vol. 65 , No. 3 Print ed in U.S.A .

REVIEW ARTICLE

James H. Herndon, Jr. , M.D. Review Article Editor

L YSOSOMES AND THE SKIN

GERALD S. LAZARUS , M.D. · , VICTOR B. HATCHER , PH.D. , AND NORMAN LEVINE , M .D.

D epartments of M edicine and Biochemistry, Albert Einstein College of M edicine, and DiVl:sion of Dermatology, Department of M edicine, Mont efiore Hospital and Medical Center, Bron.x , New York

The importance of lysosomes in cutaneous phys­iology was a ppreciated early. The studies of Fell and Mellanby [1) on vitamin A induction of mucouS meta plas ia in chick skin and the invest iga­tions of Weissman and Fell [2] on lysoso mal labilization by ultraviolet light , were landmarks in our understanding of lysoso mal phys iology. Subse­quently lysosomes have been shown to be impor­tant in keratini zation , pigmentat ion , and seba­ceous secretion. The lysosomal system also partici­pates in numerous pathologic processes in skin incl uding epidermal phagocytosis, inflammat ion, and neoplasia.

DEFI NIT ION OF LYSOSOMES

Lysosomes were first d escribed in 1955 by deDuve et a l [3]. They are cytop lasmic organe lles which contain hydrolytic enzymes that are capable of digesting many tissue constituents . Most diges­tion occurs intracellul a rly , but hydrolytic enzymes can be secreted by exocy tosis, into the extracell ul ar com partment where they may also act on extracel­lular macromolecul es (Fig. 1) .

Lysosomal enzymes are produced in the rough endoplasmic reticulum (6] and are then trans­ported to a specialized region of the smooth endo­plasmic reticulum located at the inner surface of the Golgi stack (GERL) (7]. Here these proteins are concentrated a nd packaged into membrane­bound primary lysosomes. Primary lysosomes can fuse with organell es containing substrates for di­gest ion ; the s ingle membrane-bound vacuole which conta ins hydrolases and substrate is shown

Manuscript received December 18, 1974; in revised form April 11, 1975; accepted for publicat ion Apri l 14, 1975 . .

This work was supported by gra nts from the National Institute of Arthritis, Metabolism, a nd Digestive Dis­eases (IRO I AM 17370 01), t he National Institute of Heart and Lun g (HL 16387 01) , and a grant from the Syntex Corporation to Dr. Norma n Levine.

* Senior Investi gator, The Arthritis Foundation U.S.A. Reprint requests to: Dr. G. S. Lazarus, Division of

Dermatology, Duke University Medical Center, Durham, Nort h Carolin a 27708.

259

as a secondary lysoso me or digestive vacuole (Fig. 1) .

The substrate conta ining organell es can origi­nate by several distinct mechanisms . Heterophagy is a process by which t he cell can engulf foreign materia l into h eterophagoso m es (Fig . 1) by either phagocytosis, which is the uptake of larger, insolu­ble substances, or pinocytosis, which is the inges­tion of sma ller soluble materia l. The two processes are known collectively as endocytosis. The fusion of a heterophagosome with a primary lysosome produces a digestive vacuole .

Autophagy is a met hod by which the cell can sequester part of its own cytoplas m in auto­phagosom es for digestion (Fig. 1). In this way, the ce ll is able to rid itse lf of damaged constituents. Fusion of the autophagic vacuole with a primary lysoso me res ults in the formation of a digestive vacuole.

The membrane that delimits the digestive vacu­ole is ideally su ited for cellular economy. Large macromolec ules readily enter the lysosome by heterophagy or autophagy but they are unabl e to diffuse from the vac uole because of their size. After extensive enzymic digest ion, the breakdown prod­ucts of proteins, carbohydrates , nucleic ac ids, mucopolysaccharides , and glycoproteins are s mall enough to pass through the lysosomal membrane where they may be used in biosynthetic processes in other parts of the cell (8]. .

A residual body or telolysosome is formed when substances in the digest ive vacuole a re incom­pletely digested so that they are too large to pass through the lysosomal membrane. When these organell es have no further demonstrable hydrolytic enzyme activity, t hey are ca ll ed postlysosom es (Fig. 1) . The residual body can be extruded from the cell by the process of exocytosis or it may rema in within the cytoplasm of the cell .

MORPHOLOGI C EVIDENCE FOR THE PRESENCE OF LYSOSOMES IN SK IN

In this section we will discuss the morphologic evidence for the presence of lysosomes in the

260 LAZARUS, HATCHER, AND LEVINE

seque .. ,o,;otn r.~

Endoplasmic :~ • :: space '===='

'\. AUlophogosame /

P,;mo,y Iysosomes ~

!~ ~ & \ s,\>Je lIocUOles ~ va .o,e.

/ ~\ :~~ <':~t~ @ .~. ~.~

~ .w Elco" (f'e~ Residual Postlysosome ~ <1o,y l,sOSO \. bod, IiP\

C-J @~ TeIOI YSOS~~m; ~

.~~" y'

~ ° c · , I o S

FIG. 1. Sc hematic representat ion of lysosomal func­tions. (From Vaes [4] modified from Jacques 15 J with perm ission of the authors)

epiderm is and its appendages. T he connect ive t issue cells of t he dermis, s uch as fibroblasts, endotheli al cell s, histiocytes, mast cells , etc., are not unique to the s kin and their lysoso mes have been adequately reviewed e lsewhere [9 ].

The presence of lysoso mes in t he epidermis was initia ll y suggested fo llowing the demonstrat ion of t he lysosoma l hydrolase, ac id phosphatase, in t he granular layer [10] . Subsequen t ly, acid phospha­tase [11- 14] and ary l sulfatase [15] were localized in vacuoles in ep idermal cells . Structures meeting morphologic criteria for lysosomes were obse rved in t he basal epidermal cells of gu inea pigs and humans (Fig. 2). T hese structures regularly con­tained acid phosphatase (F ig. 3) . Ac id phosph atase was a lso locali zed to t he small juxtanuclear Golgi cisternae of t he basal and s pinous cell s and in membrane-coating gran ul es (F ig. 2c). Typical ac id phosphatase-conta ining lysosomes h ave been fo und in epidermal Langerhans cells and in mel­anosomes (see section on Pigmentation).

Cathepsin D, t he major lysosomal acid protein­ase responsible for protein degradation at ac id pH, has been locali zed immunocytochem ically in chi ck epidermal cell cultures [16] a nd rabbit skin [1 7 ]. Immunofluorescent and immunoautoradiograph ic techniques (Fig. 4) have been used to demonstrate punctate loca lizat ion of catheps in D in t he basal I ayer of t he epiderm is and a diffuse distribution of the enzyme in the granul ar layer . Catheps in D has also been found in hair fo llicles , sebaceous glands, a nd mesenchymal cells of t he derm is. These stud­ies suggest that ep idermal lysosomes may be involved in protein catabolism.

Vol. 65, No.3

BIOCHEM ICAL STUDIES OF LYSOSOMAL ENZYMES IN SKIN

Preparation of Lysosomal Fractions from Skin

Typical lysosoma l hydrolases have been found i homogenates of whole s kin [18- 26 ]. T he specif·

n

activity of these enzymes is highest in the lysos~~ ~al fr~ct i on [18,27,28]. T~ese hydrolases are a lso found. IJ1 t he supernatant fract ion , suggest ing that the vlgoro:,s treatment necessary to homogenize sklJ1 a lso disrupts lysoso mal organell es. Epidermal homogenates con tain lysosoma l enzymes with higher specifi c activit ies t han whole skin [27-30 J.

Cathepsin D

Cathepsin D is a lysoso mal acid proteinase which degrades hemoglobin at ~H 3 and proteogly_ can at pH 5 [31,32 ]. The use of a potent monospe_ c ific inhibitory ant iserum to pure catheps in D in t issue cult ure and a utolytic experiments has dem_ onstrated t hat this enzy me is important in extra_ cellu lar matrix degradation [33 ]. Cathepsin D has been positively identified in extracts of rabbit skin [34 ] and in huma n skin where approx imately lOo/c of the total enzyme was found in the epiderm is. t Antiserum to rabbit catheps in D was capable of quantitatively prec ipitat ing t he enzyme from ex­tracts of whole rabb it skin. The removal of cathep_ s in D from the crude skin extract resulted in a l most complete loss of proteolytic act ivity agai nst hemoglob in below pH 5 (Fig. 5) [34]. Autolysis experi men ts util izing i m m unoinh i bition of cathep_ s in D demonstrated t hat t his enzyme was a lso respons ibl e for degradation of st ructural s kin pro­tein at ac id pH [34 ]. 1m munocytologic studies in rabbit skin demonstrate t hat t he enzyme is local ­ized in epiderm is, ep idermal appendages, and fibrob lasts [17] (see morphologic evidence above).

Cathepsin D levels increase dramat ica lly at times of remodelin g in chi ck skin [35,36 ]. This enzyme, as well as neutral proteinase, a lso in- ' c reases dramatica lly in ex perimental inflamma_ tion induced by turpentine in rabb it skin (see Lysosomes and Cutaneous Inflammation).

Cathepsin D may playa major role in in tracellu_ lar protein digestion . Dingle et a l [37] incubated rabbit pulmonary a lveolar macrophages with ca­t hepsin D ant iserum and were able to a rrest the intracellul ar digest ion of IgG , hemoglob in , a nd proteoglycan. The antiserum-treated cells devel­oped giant vacuoles wh ich conta ined un degraded s ubstrate; t he cells remai ned viable and when t he a ntiserum was removed t heir morphology returned to normal . T hese results indicated t hat cathepsin D co uld be inhibited within t he cell and t hat t his ac id hydrolase pl ayed a major role in protein catabolism. The locali zation of cathepsin D to basal epidermal cells [1 7 ], coupled with t heir known phagocytic fun ction [38], suggests t hat

t Levine N, Hatcher VB, Lazarus GS: Unpublished res ul ts.

Sept. 1975 LYSOSOMES AND T H E SK I N 261

FIG. 2. So me morphologic var iants of epidermal lysosomes in guinea-pig epidermis. a: Single membrane-limited bodies (L) conta inin g granul e material of moderate electron density. MT, microtubul es. b: Single membrane- limi ted body (L) exhibi t ing a granu lar matrix a nd concen tric lamellae. c: A mul t ivesicu lar body (MVB), two single membrane- limi ted organelles (L) with a granu lar in terior a nd lamellar substructure (arrow), and several membrane­coating granules (OB, Odland bod ies) . d: A single membrane-limited vacuole containing ves icles and nonidentifi­able g ranular materia l ( x 49,500). (From Wolff and Schreiner [14])

F IG_ 3 . .Qifferent patterns of ac id phosphatase distribu­tion within epiderm al lysoso mes. The e lectron-dense material with in the s ingle membrane-limited bodies (Ll marks acid phosphatase act ivity (a , b, X 22 ,000; C, X 43 ,000). (F rom Wolff a nd Schreiner [1 4])

catheps in D may be of importance in t he intracel­lular hydrolysis of macromolecules in ep idermis. Cathepsin D may be involved in both extracellular degradation in s kin , and then after endocytos is of partially degraded macromolecules it may partic i­pate in intracellular digestion of protein or prote in polysaccha ride within t he vacuoles of the lysoso­mal system.

Cathepsin B,

Catheps in B, is a t hiollysoso mal proteinase (39) wh ich degrades coll agen [40 j , proteoglyca~ [41 j , and benzoylarginine 2-napthylamide at pH 6 [42). The enzyme has been found in homogenates from skin of rat, cat, guinea pig, hog, and human [43] . Fraki and Hopsu-Havu [44 ] have partially purified human s kin cathepsin B, and it appears that its properties are al most identi ca l to t he purified human liver enzyme [45,46). Using an extremely sens it ive radioassay technique, we have been able to isolate cathepsin B , from both separated epider­mis and dermi .t P reliminary studies suggest that t his lysosomal proteinase may be of great impor­tance in cutaneous catabolic processes.

Neutral Proteinases

Neutral proteinases of sk in are operative at physiologic pH. They a re presumed to be lysoso-

262 LAZARUS, HATCHER, AND LEVINE Vol. 65, No.3

. FIG. 4. Loca lizat ion of ca~heps in D in rabb it skin by immunofluorescent and autoradiographic techniques . a: Ski;­mcubated with control prelmmune sheep serum fo llowed by fluorescem-Iabeled rabbit a ntlsheep serum. K. kerati a utofluorescence;. C, co ~l agen autofluorescence ( x 400). b: Skin incuba ted with sheep. ant i rabbit cathepsin D seru;: foll owed by rabbi t antlsheep serum ( x 400). intense green lluorescence IS present 111 the cells of the ha ir follicle (a.rrow). c: Rabb it skll1 sebaceous gla nd stamed Wi t h 3H-labeled pre.lmmune s heep serum ( x 400). d: Skin incubated With shee p antlrabblt cathepsm D 3H-l abeled serum demonstrat mg loca lI zat ion of the enzyme 111 an epidermal adnexal structure (x 400) . (From Lazarus a nd Poole [17])

0 '40 1:: ........ ~030

lJ.J

<l

0 ·20

0 ·10

- ........ -2 3 4 5

pH

FIG. 5. The effect on hemoglobin degradation of crude skin extracts (0--0) a nd skin extracts depleted of cathepsin D by immunoprecipitation with spec ifi c anti ­body (e - - - e) . (From Laza rus and Dingl e [34])

mal but their exact intracellular loca li zation has not yet been documented . Neutral proteinases have been extracted from s kin of a va riety of species by high -ionic-strength buffers [47- 56; for complete reviews see 57,58 ]. In our la boratory we have isolated at least four neutral proteinases in human skin . These enzymes have been partia lly

purified and at least three a re inhibited by diiso_ propyl fluorophosphate.

A possible role for these enzymes has been s uggested followin g extract ion and purification of a neutra l proteinase from rabbit skin which was ' capable of degrading skin protein and inducing an inflammatory res ponse [59] . This low-molecular_ weigh t proteinase degraded album in and hemoglo­bin but not collagen or benzoylarginine p-nitroani­lide (BANA). Injection of the enzyme into rabbit ski n resulted in t he production of a 3-cm wheal within 15 min. After 18 hr the skin was tw ice as t hick as sk in injected with heat-inact ivated en­zyme (Fig 6A). Histologic examination of t he tissue reveal ed marked edema, poly morphonuclear leu­kocytic infil tration (Fig 6B), and dermal-epider_ mal separation below the basement membrane (Fig 6e). Separation occu rred even in areas where there was no infil tration by inflammatory cells. These results suggest that the enzyme is capable of initiating an infl ammatory reaction in skin.

The locali zat ion of neutral proteinases in skin has received so me study. Wells and Babcock [60] suggested that the neutral proteinase from human skin was localized exclusively in the epidermis whereas Ya mura and Cormia [61] found the en­zyme in both dermis and epidermis. Studies in our

Sept . 1975

A

B

FIG. 6. Effect of in traderm al injection of partia lly purified neut ra l proteinase into rabbit skin. A: Macro­scopic appearance of rabbit skin removed 18 hr afte r injection of neutral proteinase (below) or heat-inac­tivated enzyme (above). B: Micrograph ( x 4(0) of a wax-embedded sect ion of skin after t he int radermal inject ion of neutral proteinase stained with hell1atoxylin and eos in . An acute granu locyt ic infiltration with necro­sis is seen . C: Micrograph (x 400) of a ski n section from t h e periphery of the les ion that followed injection of sk in proteinase. There is dermal- epidermal separation t hat appears independent of leukocyte infiltration. (From Lazarus and Barrett [59 j)

laboratory on sepa rated skin t suggest t hat protein- . ases a re located in both t he epidermis and derma l co nnect ive tissue.

Inhibitors of Enzymes in Skin

One of t he major problems in the ident ification and isolation of skin enzymes had been t he pres­ence of inhibitors [51,62 ). One of the major protein ­ase inhibitors in homogenates of whole s kin , which necessitates extract ion with high-ionic-strength buffers for enzyme activation , appears to be iX ,­

antitrypsin [63 ). Fraki and Hopsu-Havu identified the protein immunologically and a lso found t hat

LYSOSOMES AND THE KIN 263

extracts of sk in had very li tt le Q2-macroglobulin. The latter protein appears to be capable of inhibit­ing almost a ll known t issue proteinases [64- 67 ).

ROLE OF LYSOSOMES IN CUTANEOUS PHYS IOLOGY

Epidermal Phagocytosis

Epidermal cells are capable of phagocytosis a nd digestion of exogenous material using t he lysoso­mal system. Nordquist et a l [68) injected ferr it in in traderma lly and observed t hat t he marker en ­tered the epidermis a nd loca li zed in vacuoles containing ac id phosphatase. Wolff a nd co-workers [69,70 ) injected t horotrast and peroxidase into t he dermis of gu inea pig a nd found that t he markers were rapidly endocytosed by epidermal cells; the phagosomes contai ning t he marker were stored within the vac uola r apparatus of t hese cells un ti l they underwent keratinization.

Epidermal cells are a lso capable of phagocytos­ing particulate material (38 ). Injection of latex beads into a subepidermal blister, produced by s uct ion, permitted direct contact between latex particles a nd regenerating epidermis. Wolff and Konrad [38 ] found t hat latex particles were av idly ta ken up by t he regenerating epit heli um a nd incorporated in to phagosomes. These phagocyt ic vacuoles moved toward t he nuclear zone of t he cell , a nd fusion of phagosomes with each other a nd with primary Iysosomes was observed. The uptake mechanism was s ize dependent since large beads (0.8 m icron) were taken up individua lly whereas s mall beads (0 .109 micron) were taken up in groups . The ingested beads did not interfere with kerati nization a nd th e latex was incorporated into horny cells and eliminated by desquamation. These elegant experiments demonstrate that ep i­t helium is phagocytic and that classic heterophagy occurs in mammalian epidermis.

Pigmentation

Melanosomes are transferred to keratinocytes by a highly speciali zed form of epidermal phagocyto­s is [71 ,72 ). Consequent ly one could designate t he melanoso me within a keratinocyte as a phagosome and predict t hat it should fuse with a primary lysosome to form a secondary lysosome. S upport for t his notion is provided by the observations t hat melanosomes within keratinocytes a re located within vac uo les con ta inin g ac id phosphatase ['73- 76) a nd th at injection of ferritin into t he skin resul ts in localization of t his molecule within melanosome complexes in keratinocytes [74,76,77). Wolff and Honigsmann (76) concl us ively demon ­strated that mel anosome complexes in kerat ino­cytes were secondary Iysosomes. They induced secondary lysosomes in gu inea-pig skin with thoro ­trast and observed fusion of these tracer-marked vacuoles with melanosomes by electron micr~s­copy. The end product was a vac uole containing mela nin , t horotrast, and ac id phosphatase (Fig. 7). Keratinocytes are also capable of phagocytosing

264 LAZARUS, HATCHER , AND LEVINE

FIG. 7. In te ract ions of two heav ily labeled melanosome co mplexes which conta in melanosomes in various stages of melanization (M). The complexes 1 a nd 2 have fu sed at t he point ma rked by t he arrow and a melanosome (as terisk) is a bout to cross over from I to 2 (gu in ea -pig ep idermis inj ected wi t h thorotrast, x 48,000). (From Wolff [77])

melanosomes independent of the ir interaction with melanocytes. Wolff [77] and Wolff et al [78 ] injected melanosomes into subepidermal suction blisters in guinea pig and found that keratinocytes avidly endocytosed these organell es.

A possible explanation for racia l co lorat ion was offered by Szabo et a l [79] when they found that Caucasoids, American Indians, and Monogoloids had groups of two or more melanosomes s ur­rounded by a membrane within kerat inocytes , whereas Negroids and Australian aborig ines had single, la rger, disc rete melanosomes distributed within the cytoplas m of kerati nocytes. A possible mechanis m for t he variation in distribution of melanosomes within keratinocytes in d ifferent races was suggested by studies of epidermal phago­cytosis using different s ized lat ex beads [38,80 ]. Large latex beads (0 .8 JLm) were taken up s in gly whereas s mall beads (0.1 JLm) were taken up in groups. These results suggested that uptake of pa rticles by kerati nocytes was s ize dependent and that the difference in dist ribution of melanosome particles between Negro id and Caucaso id reflected the size of the melanosome synthes ized. Furth er support for this hy pothes is was presented by Wolff et al [78 ] in their studies on pigment donation in vivo. These invest igato rs obtained large melano­somes from anagen hair of C57BL/6J mice (l.3 X 1 JLm) and small melanosomes from pigmen ted B16 mouse mel anoma (0.5 JLm x 0.3 /-Lm) and injected these part icles into a suction blister in guinea-pig skin. As expected, the large melanoso mes were taken up s ingly whereas the small melanosomes were taken up in groups.

M elanin is rarely seen above the basal ep idermal layer in Caucaso ids [74]. T his lac k of melanin in t he s uperficia l layers of epidermis is probably related to catabolism of melanoprote in by lysoso-

Vol. 65 , No . 3

mal proteinases. M elanosomal prote in is digested by lIver lysosome preparations at pH 5 [81) whereas ' 4C-dopa- labeled melanin was not de­graded by lIver lysosome preparations under s imi­lar conditIOns. The a ut hors s uggested from these d ata that lysoso mal protell1ases a re not capa bl e of degradll1g the melanin moiety. Before th is concl u­sion can be accepted it is necessa ry to incubate aut.hentic melanin with epidermal lysosome prepa­ratIOns ~v~r. a wI.de ran ?e of conditions to rul e o u t the poss ibIli ty o.f an ep idermal melanin -degrading lysosomal protelI1ase.

Keratinization

Th~ production of .. the stratum corn eum and espeCiall y Its unique fIbrous protein, kerati n , con­stitutes one of the mos~ drama~ic examples of programm ed catabolIs m 111 al l of biology. Lyso­somes probably play a s ignificant rol e in this complex process . In almost all cells studied to date lysoso mes playa vita l role in intracellul a r di ges~ tlOn and cellul ar divi sion (for rev iew of t he ro le of lysoso mes in cell divis ion, see [82) and Proteases and BIOlogica l Control:j:). It seems reasonable to assume that the lysoso mal system pl ays a simil ar :ole in ~he basa l cells and is thus indirectly 1I1volved 111 the compl ex process of keratin produc­tion. A more direct rol e in the process of keratiniza_ tion may be pl ayed by the membrane-coat inu granul e ~hich is a modified lysosomal organell e~ These unique lamellar granules conta in s ignificant amounts of ac id phosphatase [14). . The membra ne-coat in g granules are extruded 1I1tO the extracellul a r space immediately a bove t he granula r layer and may account for the observation that there is diffuse sta ining for ac id phosphatase [10,12], ary l s ulfatase [15 ], and catheps in D [17] at this level of the epidermis . The exact role of t he lamella r membrane-coat ing granules is unknown but it is intriguing to s pecul ate that these organ -' ell es are responsible for degradat ion of the kerati­nocyte glycocalyx. The fina l and most dramatic catabol ic event in keratinization occurs immedi­ately adjacent to th e granular layer. There is a sudden loss of cell organelles such as nuclei mitochondria , and ribosomes in the stratum cor~ neum. Occasional " intermediate cells" demon­strating partial degradation are seen but auto­phagic vacuoles and other manifestations of au­tophagy are not usually observed [9]. The mecha ­nis m of this phenomenon is totally unknown but the activation of the lysosomal system could playa role in th is final step in keratinization.

Vitamin A and K eratinization

Vitamin A, a potent lysosomallabi li zer [83], has profound effects on keratinization [84 ]. Hypovi-

:j: Abstracts of papers presented at t he meet ing on Proteasesand Biological Cont rol, September 10- 15 , 1974, Cold S pring H a rbor Laboratory , Cold Sprin g Harbor, New York.

Sept. 1975

taminos is A resul ts in hyperkeratos is, follicul ar p lugging, and kerat ini zing sq uamo us meta pl asia of

, the sweat glands and m ucous mem bra nes of t he trac h eobronchia l and geni tourin a ry t ract [85]. T h ese observat ions suggest t hat vitamin A has an " antikerati ni zing effect" on epithelium [86 ]. S up­por t for t his notion was presen ted by Fell and Mell a nby [1] when t hey demonstrated t hat t h e ad di t ion of excess vitamin A to tissue cul t ures of chic k epi t helium prevented keratinization and in ­duced mucous metaplas ia. Vitamin A primarily affected the basa l layer where t he cells beca me d isoriented , developed in tercellul ar lac unae, lost t h eir fil a ments , demonstrated mitochondria l en­large m ent, a nd eventually a ppeared to sec rete m u c u s [87 ]. Removal of t he t issue from excess vita min A resul ted in revers ion of t he basa l cell s to nor mal kera tini zing ep it heliu m.

T h e effect of vi ta min A on skin va ri es; rodent skin is less respons ive t han chick s kin a nd mature epith elium is less affected t han embryonic tissue [84]. Al t hough mucous meta plas ia cannot gener ­a lly be induced in rodent epidermis by vitamin A, significant changes in t he epi t helium do occur . H istologica lly t here is hy perpl as ia, acant hos is, in c r eased mi totic fi gures, and pa rakeratos is [88 ]. The o bservat ion t hat mi tot ic rate in skin treated w it h vi ta min A is increased may ex pl a in t hese histolog ic observa tions [88,90 ]. The effect of vita­m in A on mitot ic activity is dose dependent ; very h igh doses of t he v ita min , in certa in selected syste m s , actually diminish mitotic rate [88 ]. Sev­eral p ossibl e mec hanis ms have been s uggested to expl a in t he effect of v itamin A in epiderm al turnover. DeLuca and Wolf [91 ] observed inhibi ­tion of a mino-acy l t ransfer RNA synthetase in vitam in A deficiency. This enzy me links spec ific amino ac ids to t rans fer R NA pr ior to rough endo­pl asm ic reticulu m prote in synthes is . T his effect of vitamin A coul d d irect protein syn thes is a nd con sequen t cell divis ion. A second poss ibili ty migh t be t hat vi ta min A induces lysoso mal prolif­eration which acce lerates cell di vis ion. Dingle et a l [33] h ave shown that vitamin A is ca pable of induc ing lysoso mal proliferation and increas ing the concentrat ion of lysoso mal prote inases in cul ­t u res of chick lim b bone rudiment. We have fo und that t he concent rat ion of t he lysoso mal prote in ase cath e ps in D § is increased in ra bbi tskin cul t ured in the presence of ret inoic ac id . Cell cul t ure experi ­ments suggest t ha t lysoso mal hydrolases are in ­vol ved in t he process of cell d ivis ion [92,t ] and thei r inhibi t ion in lerfe res wi t h cell div is ion ,t t u­morigenes is [93,:1= 1, and phytohemagglutinin,

, induced ly mphocyte t ransformation [94 ]. Conse­quently, it is possible that vita min A increases epidermal t urnover by in creas in g t he concent ra­t ion a nd release of lysoso mal hydrolases.

Another effect of vitam in A on skin is dec reased cohes ion of t he strat um co rneum [90 ]. T his obser-

§ Laza rus GS , Din gle J T : Unpublished data

LYSOSOMES AND T HE SKI N 265

vat ion has been documented electron microscopi ­cally by fi nding s ign ifican t widening of t he in ter­cellula r lacunae [87- 93,95 ]. The most reasona ble expl anation for t his morphologic change is in­creased formation an d la bili zation of lysoso mal hydrolases in t he upper gra nula r layer. Mem brane­coa ting granul es have been shown to be lysosome­like organell es which are secreted in to th e in tercel­lula r space [1 4 ] and severa l lysoso mal hydrolases have been observed in t he upper granular layer as well [1 2, 14,17 ]. Consequent ly it is in t rigu ing to s pecul ate t hat vita min A could increase t he con­cen t rat ion of extracell ul ar lysosom al enzymes by hy pert rophy and lea kage of t he mem brane-coating granul es which t hen degrade t he ext racellul ar " cement substance," caus ing widening of t he in­tercellula r lacunae and loose packin g of kera tin .

Sebaceous Secretion

The a utolyt ic process of sebaceous sec retion has been in ves tigated in rat sebaceous glands by Brandes et a l [96 ] us ing electron microscopic and histochemical techniques. T hese workers fo und maturing sebaceous cells conta ined numerous lyso­somes which enl arged and rupt ured immediately before t he sebaceo us cell d is integrated. After cytol­ysis occurred , disc rete lysosomes were no longer vis ibl e, but staining for ac id phosphatase pers isted in t he cyto pl as m . S imila r res ul ts in macaques were obta in ed by Bell [97 ] who a lso noted that t he ac id phosphatase-conta ining ves icles conta ined crysta l­line inclus ions with a period from 55 to 105 A. P rogra mmed a utolys is a lso appears to occur in ra bbi t sebaceous glands . Lazarus and P oole [17 ] fo und t hat t he lysosomal proteinase catheps in D could be identified by immunofluorescent tec h­nique in t he periphery of sebaceous glands (Fig 4). These data suggest t hat lysosomal prol ife ration a nd consequent ru pture plays a pri mary role in t he progra mmed autolysis which is t he cha racterist ic of holoc rin e sec ret ion.

RO LE OF LYSOSOMES IN CUTANEOUS PATHOPHYS IOLOGY

Lysosomal Storage Diseases

Lysoso mal storage diseases a re characteri z~d by a primary defect in a lysoso mal hydrolase (for complete rev iew see [98 J) . Remarkably, few of these diseases have clinical cutaneous mani fes tat ions

. even t hough they can be di agnosed by study of skin-derived cells. Two exceptions a re Fabry's disease a nd t he Chediak-Higas hi synd ro me.

Fabry's Disease

Fa bry 's disease is an X-linked d isorder charac­teri zed by t he genet ic in activation of one of several !'I-galactos idase enzymes or isoenzy mes [99 ], which resul ts in in t ralysosomal acc umulat ion of cera mide trihexos ide and di galactosyl cera mide . Clinica lly t here a re characteristi c angiokeralomas on the lower t runk and upper t highs. Histologically t hese

266 LAZARUS, HATCHER , AND LEV INE

lesions demonstrate epidermal thinning and di­lated capillaries with glycolipid endothelia l in­clusions in the upper papillary dermis. The clinica l severity of t he disease corre lates with the number of zebra and concentric lamellar type inclusions seen in dermal fibroblasts and endothe lia l ce lls lIDO). Similar lesions occur throughout the body and account for the ophthalmologic, renal, neu­rologi c, and cardiovascu lar complications. The disease usually is fata l by the fifth decade but re­cent experiments with enzyme replacem ent [101, 102) in Fabry's disease may provide techniques by which patients with a variety of lysosomal storage diseases can be treated.

eM.diak-Higashi Syndrome Chediak-Higash i syndrom e is an autosomal

recess ive disease cha racterized by pale sk in which sunburns eas ily , b lue-gray ha ir color , decreased uveal pigment, and frequen t severe infections [103 ). Patients have anemia , leukopenia, and they frequent ly develop a malignant lymphoma. The hallmark of t he disease is large cytoplasmic inclu­sions which presumably a re lysoso mes [104 ). The beige mouse [105 ] and the Aleut ian mink [106, 107] are animal models of the human syndrome.

The mechanism responsible for t he formation of these giant granules is not known but studies of phagocytos is in granulocytes by Stossal et al [108] may provide an explanation. These workers fo und that t he rate of bacterial phagocytosis and the product ion of hydrogen peroxide , which is neces­sary for bacteri a l killin g, were normal in patients with Chedia k-Higashi syndrome. The phagolyso­somes contained peroxidase and J3-glucuronidase but not alka line phosphatase. These .data sug­gested tha t in Chediak-Higashi syndrome . certain granulocyte granules were unable to fuse with the phagocytic vacuole because of a defect inmem­brane fusion. Prieur et al [109 ] found that t ubul ar cells from t he kidn ey of the beige mouse could not degrade exogenous horseradish within t he giant gran ules as quickly as normal a nimals . These investigators interpreted their findings as s uggest­ing t hat t here was a defect in protein degradation ; an a lternative interp::etation might be t hat t here was a bl ock in fu sion between the phagocyt ic vacuole and the lysosome conta ining the en­zyme(s) necessa ry for catabolis m of the perox idase molecule. Another piece of ev idence suggest ing that Chediak-Higas hi granul es have ab norm al membranes is the observation that giant granul es in leukocytes sta in for ac id phosphatase much more ra pidly t han adjacent norm al-a ppearing granules [110] . Pigment dilution which is second­ary to abnorma l fusion of melanosomes into giant granules may be another consequence of t his geneti c defect in membranes [111].

The Effect of Light on The Sk in

Weissma n and Dingle [112 ] demonstrated that irradiat ion of a lysosome -rich fract ion from rat

Vol. 65, NO .3

liver resulted in lea kage of acid protein ase frorn these organ ell es. Pretreatment of the rats with hydrocort isone dec rea~e.d ligh t- induced lea kage of protell1 ase. The pOSS ibili ty that ltght could ruptur cutaneous lysosomes was first s uggested by the experiments of Weissman and Fell [113 ]. Irrad i a~ tion of feta l rat skin with light i~ the 300-nm r a n ge resulted 111 nec ros Is of ep idermiS a nd dermis. Th addition of hydrocortisone to t he cul t ure mediu~ mod ified the response to irrad iat ion so t ha t treated cultures demonstrated reta rded cell breakdown decreased vesicu lat ion, less diso rgan iza tion of th~ dermal connective t iss ue, and acce lerated produc_ tion and different iation of n ew .~ pid ermis. The aut hors aSCrIbed t he protect ive effects of co rt ico_ steroids to stabili zat ion of lysoso mes which de­creased the leakage of proteolytic enzymes.

J ohnson [114] confirmed the observat ion t h at li ght induced lysosomal rupture in s kin when h e studi ed the concent rat ion of ac id phos phatase in mouse ea r skin after irradiat ion wi th ul t rav iol et light. Ears that had been irradiated with 320-nrn light had 30% less acid phosphatase when ex_ tracted than ears that had been irradiated through plate glass. He suggested from these data tha t light, below 320-nm wave length, induced lyso_ somal rupture in the skin and that the acid phos_ phatase released from the tissue was inactivated or carried away in the blood. Th is phenomenon was somewha t specifi c for lysosomal la bilization and did not simply reflec t leakage secondary to ce ll death because irrad ia ti on did not dep lete th e tissue of histochemica lly demonstrable succinic dehydrogenase a,t the sa me t ime that acid phos_ phatase was markedly reduced [115 ]. Light- in_ duced lysosomal lab ili zation of human foreskin epidermis can be blocked by app lication of th e sunscreen, 10% sulisobenzone [116].

The mechanis m of lysoso mal lab ili zat ion by light is in completely understood, but t he innova_ tive studies of Allison and Young [117 ) with· photosensitizers have defined promising areas far invest igat ion . Cells and t iss ues ca n be damaged by t he combin ation of a photosens iti zin g agent , light of the a ppropriate wavelength, and oxygen . Us u ­a lly t he action spectrum for photosens it ization is simila r to t he absorption s pectrum of the photosen_ siti zer [118 ]. Allison noted that photosens it izers such as uroporphyrin , anthracene, and vital dyes were concentrated in lysoso mes. S ince t hese su b _ stances a re capab le of ca us in g cyto lys is by disrup_ tion of plasma membranes , tec hniques had to be developed to demonstrate t hat disruption of lyso_ somes by photosens it izers was of primary impor_ tance in cell destruction. A number of different cell types in cul ture were incubated with various pho­tosensitizers in the dark fo llowed by an additional 30-min incubat ion in fresh medium without t h e photosensi t izer. The photosensiti zers were found only in t he lysoso mes , and damage secondary to cell -membrane binding of the photosensit izer cou ld be avoided by keeping the cells in t he dark

Sep t. 1975

a nd washing t hem. Irra diat ion of t he cells wi t h lig h t of the appropriate wavelength res ul ted ini ­t ia lly in increased permeabili ty of t he lysoso mes, as m easured by increased ab ili ty to sta in fo r lysosom al acid phosphatase, foll owed by cell d eath. S uc h data s uggested t hat lysoso mal disrup ­tion was of prima ry im porta nce in li ght- mediated cell d a mage. Confirmation of this hy pothes is has been provided by H awkins a nd co-workers [119 ]. T hese worker demonstrated t hat lysosoma l ru p­t ure was t he prima ry even t in cell death produced by t h e photosens it ize r acri d ine ora nge. T his was esp ec ia ll y convincing s ince in t he sa me study t hese in vest igators showed t hat lysoso mal rupt ure was seconda ry to cell death in studi es utili zing meta­b o lic poisons and complement-suffi cient a ntice ll membra ne antiserum .

C linically, co mmon s unburn may be an excell ent mod e l of li ght- induced lysoso ma l dis rupt ion caus­in g clinica l d isease. One hour a ft er ul t rav iolet irra dia tion t he epidermis con ta ins numerous vac u­oles [1 20] which a ppea r s imila r to t he ac id phos­phatase-con ta ining auto phagic vac uoles seen in p h ototox ic reactions induced by methoxsa len [1 4 ]. T he immediate eryt hema assoc iated wit h ul t ravio ­let irradiation , may be related in part to dis rupt ion of lysoso m es of endothel ia l cells wi t h release of chemical med iators [117]. The delayed erythema could be, in par t, secondary to diffus ion of protein­as es from the epidermis seconda ry to lysoso mal dis ruption . A prime candidate for such a mediator, in a ddition to prostaglandin , would be neutra l p rote in ase which is ca pable of caus ing vasodil ata­tion , capilla ry permeabili ty, a nd chemotax is [59, 121 ].

L ysosom es and Cutaneous Inflammation

Application to t he s kin of ca nt ha ridin , a lysoso­mal l a bili zer, produces an int rae pidermal acan­t h o lyt ic blister [1 22 J. Seven hours after a ppl ication of t h e drug, bliste r fluid conta ins signi ficantly h ig h e r concent rat ions of ac id phos phatase a nd cath e ps in t han serum . T his elevat ion of ext racellu ­lar lysosom a l hydrolase is not assoc iated wit h leukocyte infil t rat ion , which suggests t hat leakage of e p idermal lysoso mes might be res pons ible for t he e n zy me eleva tions. Histologica lly, pa rt icul ate ac id phosphatase sta ining is reduced in 4 to 6 hr whereas lactate dehydrogen ase and succ inate de- . hydrogenase are not reduced fo r 8 to 10 hr [1 23 ]. T hese findin gs suggest t ha t lysoso mes containing acid phosphatase a re the structures which a re affe cted earli est by cant haridin. Granulocytes en ter t he epidermis 12 hr a ft er a pplication of can t h aridin a nd proba bly acco un t , in par t , for furthe r elevations of lysosom al hydrolases in blister fl uid . The mecha nis m for leukocyte infil t rat ion could be t he elaboration of a chemotactic lysoso­mal proteinase from t he epidermis s imil a r to the neu t r a l prote inase of rabbit [59,121] (Fig. 8 ). We have discovered severa l new neut ra l proteinases in

LYSOSO MES AN D TH E SKI N 267

EPIDER MAL IN JU RY

• LYSOSOMAL" CHE MOIATIC

PROTEI NA SES

INfLAMMATORY - CELL HYD RO LASES

EPIOER MAL HYDRDLASES

I INHIBillON BY LEAKAGE Of / . ,. MACROCLOBULIN AND

I ., · AN 1I1RYPSIN ~ _____ _ ~ ... ~~a Oafl 'd

\O\'lfY---VESSEL

F IG. 8 , Sc hemat ic representat ion of t he effects of dis ru ption of epiderma l lysosomes on t he skin , (From Lazarus a nd Hatc her 158 ])

huma n skin which appear to be capable of caus ing chemotaxis. T hese proteinases might a lso be cap­able of inducing chemotaxis by cleaving comple­ment, fibrin ogen, Hage ma n factor, or ka llikre in [1 24 ].

Lysoso me la bili zat ion , t herefore, might t rigger infl a mm ation and cause a biphas ic increase in lysoso mal protein ase level in the t iss ue (Fig. 8). An epidermal insul t coul d da mage cells which would resul t in leakage of lysoso mal hydrolase in to t he tissue . T he e enzy mes m ight cause t issue nec ros is and coul d a lso act as chemotactic agen ts. Infl a m ­matory cells would t hen infil t rate t he d ermis a nd sec rete t heir catabolic enzy mes [1 25], which , when added to endogenous s kin hydrolases, would pro­duce t issue necros is. This biphas ic phenomenon has a lready been observed in t urpen t ine granu ­lom a in rabbi t skin [1 26 ]. The infl a mmatory cata­bolic process is usuall y self- limi ted s ince infl a m ­mation is associated wit h increased vascul ar permea bili ty and lea kage of serum prote inase in ­hibi tors such as a,-mac roglobulin a nd aI-ant it ryp­s in in to the t issue. a.-Macroglobulin is ca pable of inhibi t ing a ll tiss ue prote inases stud ied to date, and it could prevent hydrolase-mediated t issue destruction as well as prote in ase- related chemo­taxis. T his self-limited inf1 a mmatory cascade may expla in how epidermal injury inst igates a n infl a m­m atory res ponse involving t he ent ire s kin .

This cascade of events is speculat ive and under m any circumstances lysoso ma l enzy me release is a secondary phenomenon . However, t here is a natu­ra lly occurring disease, lupus erythematosus, which could possibly correspond to the model of infla mmation just proposed . The wavelength of light which induces skin lesions in lupus is 290-320 nanom eters [1 27, 128 ], which is exactly the wavelength range which labili zes lysosomes [117]. Consequen t to irradiation there is da mage to the basal cells and the derma l ca pillaries. The da m­aged basal cells appear to be the source of the cir­culating DNA in li gh t- induced exacerbations [129 ],

268 LAZARUS, HATCHER , AND LEVINE

and t h ey could a lso release prote inases which cau se t issue necrosis and induce inflam m a ti on . The damaged d ermal vesse ls leak immune com­plexes (DNA a nd a n t i-DNA immunoglobulin) a nd complement which d e p osit at t he d e rm a l- ep i­d e rma l basement m e mbrane [30]. This entire li gh t­indu ced process can be inh ibited by sunscree ns which protec t aga inst t he wave le n gth of ligh t t hat induces lysoso mal lab ili zat io n. Obvio us ly, t h e re are oth er explanat ions for t his rat h e r complex process; however, t his hypoth es is is being tested in o ur la boratory a nd if it is correct it might provide new therape u t ic m o daliti es.

There are oth er infl a mm atory dermatoses t h at demonstrate m orph ologic c h a n ges in lysoso m es. Prose a nd co-wo rkers [131] reporte d lysoso mal proli fe rat io n in t h e granular a nd co rnea l layer in 3 of 6 babies with atopic derm a ti t is. Fr ic hot a nd Zelic kso n [1 32 ] confirmed t his observation a nd noted that , in contact dermatitis, ac id phosph a­tase-contain ing vac uoles had poorly defin ed limit­in g m em bra nes . Membrane m orpho logy return ed to n orm a l coi n c ident with clin ical improvem e nt. Akopian et a l [133 ] observed a n inc rease in lysoso­m a l hydro lase activi ty in basa l a nd pri ckle cells in pemphigus v ul ga ris , which s u ggested to th ese authors t hat release o f lysoso m a l e n zymes was res pons ibl e for destruction of in tere pide rmal ce ­ment s ubstance which ca used intraep iderm a l blis­ter for m at ion . The clinical s tudies enume rated above lend further s upport to t h e hy pothes is that lysosomes a re of s ignificant impo rta nce in a vari ety of in!1 a mm atory c utan eous diseases .

L ysoso mes proba bly p lay a s ign ifi cant ro le in s u c h diverse phys io logic functio ns of the sk in as cellula r div is io n , kerat inization , a nd sebaceo us secret io n . T h ere is in creas in g evidence t hat lyso­som es a lso function in numero us path ophys io logic ci rc umstan ces as wel l. S u c h di verse processes as li gh t react ions, ep iderma l infl a mm ation , a nd n eo­plas ia may b e associated w ith lysosom a l eve n ts. Further study of these organell es may not o nly furnis h in s igh ts into disease m ec h a nis m but could d e fin e a r eas for pharmacologic manipu lation whic h would profoundly inn uence t iss u e cata bol­Is m.

The aut hors a re deeply indebted to D r. Kl aus Wolff who provided num erous elect ron micrographs and manu ­scripts which were of tremendous value in t he prepa ra­t ion of this paper. Ms. Alyce Mill er provided superb assista nce in the prepa rat ion of the manuscript and Ms. June Agu iar provided ex pert technical ass ista nce. Mr. Barry S hapiro prepared the di agra ms.

REFERENCES

1. Fell HB, Mell a nby E: Metaplasia produced in c ultures of chic k ectoderm by high vitamin A. J Phys iol 119: 470-488, 1953

2. Weiss man G, Fell HB: The effect of hydrocortisone on the response of feta l rat s kin in cul t ure to ul trav iolet irradiation. J Exp Med 116: 365- 380, 1962

3 . deDuve C, Pressman BC, Geanetta R. Watt ia ux R, Appelmans F: In t racellula r distribution patterns

Vol. 65, No . 3

of enzymes in rat- li ve r t iss ue . Bioc he m J 60:604- 617 , 1953

4. Vaes G: Digest ive ca pac ity of lyso omes, Lysoso mes a nd Storage Disease . Edited by HG H ers, F Va n Hoof. New York, London . Academ ic, 1973. pp 43- 47

5. J acques P: Ho meostat ic regul ators. Ciba Founda_ tion y mpos ium , 1969, pp 180- 193

6 . Cohn ZA, Fedorko ME: The form a tion and fate of lysoso mes, Lysoso mes, vol I. Edited by JT Dingle HB Fe ll. Amsterd am, North Holl and. 1967 pp' 43- 63 '

7. Nov ikolT AB: Enzyme ul trastru cture and loca li za_ tion in neurons , Th e Neuron. Edited by H Hyd en Amsterdam , E lsevier. 1967 . pp 2.55- 318 .

8. Cohn ZA, Fedorko ME: The format ion a nd fate of lysosomes , Lysoso mes, voll. Edited by JT Dina le HB Fe ll. Amsterdam , No rth Holl and . 1967. b pp 44- 45

9. Breathnach AS: An Atlas of th e Ultras tructure of Hum a n S kin. London, Churchill . 197 1

10. Eisen AZ, Arndt .KA. Cla rk WH Jr: T he ultras tru c_ t ural locali zat ion 01 aC id phos phatase in hum a n ep idermis. J In ves t Derma tol 43:319- :326 , 1964

11. Diengdoh JV: Th e demonstra t ion of lysoso mes in mouse sk in . Q J Med 105:73- 78 , 1964

12. Olson RL, Nordquist RF: Ultra microscopic locali za_ tion of ac id phos phatase in human ep id ermis. J In vest Derm atol 46:431- 435, 1966

13. Rowden G: Ultrastructu ra l s tudies of keratini zed ep ithel ia of the mouse. J In ves t Derm atol 49: 181-197, 1967

14. Wolff K, Schreiner E: Epidermal Iysosomes elec_ tron m icroscopic-cytochemical studies. Arch Derma tol 101: 276- 286, 1970

15. Olson RL, Nordquist R, Everet t MA: Ultrast ruc_ t ura l loca li zat ion of a ryl sulfa tase in human e pid ermi s. Acta Derm Venereal (Stoc kh ) 48:556- 562, 1968

16. Poole AR, Dingle JT, Barrett AJ: Th e immunocyto_ chemical demonstra tion of catheps in D. J Histo­chem Cytoc hem 20: 261- 265 , 1972

17. Laza rus GS, Poole AR: Immunocytochemicallocali_ za tion of calheps in D in rabbit s kin. Arch Derm a_ to l (in press) 1975

18. Wynn CH, Iqbal M: Isolat ion of rat skin lysoso me a nd a compa rison with liver a nd spleen lyso­somes. Biochem J 98: 10p, 1966

19 . Ockerm a n PA: Acid hydrolases in hu man s kin. Acta Derm Venereal (Stockh) 49:139- 141, 1969 .

20. Steigerwald J C, Bartholo mew BA: The assess ment of lysoso mal glycos id ase in norm al skin . Biochim Biophys Acta 321: 256- 26 1, 1973

21. Ockerm an PA: Acid hydro lases in skin a nd plasm a in ga rgoylism. Defi ciency in /3-ga lactos idase in s kin. Clin Chim Acta 20: 1- 6, 1968

22. Goldberg MH , Cotli er E , Fichenscher LG, Kenyon K, Enat R, Borowsky SA: Mac ul a r cherry red spot, corneal cloudin g and a-galac tosidase defi­c iency. Clini ca l, biochemica l and electron micro­scopic study of a new a utosoma l recess ive storage disease. Arch In tern Med 128:387- 398, 1971

23 . Clausen J , Melchoir J C, Perregard P: E nzymic differentiation between different ty pes of Tay­Sachs d isease of s imilar clini cal appearance. Eur Neurol 7:56- 64, 1972

24 . Lev ine B, Fajerm an K, J acoby NM: Mucopolysac_ charidos is. Proc R Soc Med 65: 339-341, 1972

25. Ockerman PA, Kohlin P: Tiss ue ac id hydrolase act iviti es in Gaucher's disease. Scand J Clin Lab Invest 22:62- 64 , 1962

26. Ger ick JE: Hunter's sy ndrome: beta-ga lactos idase defi ciency in skin . N En gl J Med 280:799-802, 1969

27. Dicken CH, Dec ker RH: Biochemical evi dence for t he presence of Iysoso mes in the epidermis . J In vest Dermatol 47 :426- 431, 1966

Sep t. 1975

28. Ohkawara A, Ha lprin KM , Taylor JR, Lev ine V: Acid hydrolases in the human epidermis . Br J Derma tol 83:450- 459, 1972

29. Braun-Fa lco 0 , Ruper M: Die Verieilung der sauren Phospha tase be i norm a ler und psoriatischer Ver­hornung (E ine electronenmikroskopisch cyto­c h em isc he Un t e rs uchun g). Dermato log ica 134: 225-242, 1967

30. Mishima Y: Lysoso mal and non·lysosomal ac id phosphatase activity of the human skin (abstr) . J Cell BioI 23: 122A, 1964

3 1 . Barrett AJ: Cathepsin D. Purifi ca tion of isoenzymes from hum a n a nd chicken liver. Biochem J 117:601- 607. 1970

32 . Barrett AJ : Pu rifi ca t ion and propert ies of cathepsin D from liver of ch icken, rabbi t a nd man , Tissue Proteinases. Edi ted by AJ Barrett, JT Dingle . Amsterdam , North Holl and , 1971, pp 109- 133

33. Dingle JT, Barrett AJ, Weston PD: Cathepsin D. Cha racte rist ics of immunoinhibi t ion and the con­firma tion of a role in cartil age breakdown. Bio­chern J 123 :1 - 13, 1971

34 . Lazarus GS , Dingle JT: Catheps in D of rabb it skin : an immunoenzy mic study . J Invest Derm atol 62:1 1- 66 , 1974

35. W oessner JF: Coll agen remodel ing in chick s kin embryogenes is, Chemistry a nd Molec ul a r Biology of t he Intracellul a r Matr ix. Edited by LA Ba lazs. New York , Academic , 1970, pp 1663-1669

36. W oess ner J F: Cathepsin D: En zy mic properties a nd role in connective t issue breakdown . Tiss ue Pro­teinases . Edited by AJ Barrett, JT Dingle . Am­sterda m, North Holl and, 1971 , pp 291-311

37 . Dingle JT, Poole AR, Lazarus GS , Barrett AJ : Tmmunoinh ibition of in t racellul a r protein diges­tion in macrophages . J Exp Med 137 :11 24- 1141 , 1973

38. Wolff K, Konrad K: Phagocytos is of latex beads by epiderm al kerat inocytes in vivo. J Ultrast ruct Res 39:262- 280, 1972

39. Hartley BS : Proteolyt ic enzy mes. Annu Rev Bio­chern 29:45- 72, 1960

40. B url eigh MC, Barrett AJ , Lazarus GS: Cat heps in B" a lysoso mal enzyme that degrades native collagen. Biochem J 137 :387- 398, 1974

41. Morrison R IG. Barrett AJ, Din gle JT, Prior DC: Catheps in B, and D. Action on huma n cartilage proteog lyca ns. Bioc h im Biop hys Acta 302:4 11- 419, 1973

42. Barre tt AJ: A new assay for ca t heps in B, and other thiol prote in ases. Anal Biochem 47:280- 293, 1972

43 . Jensen CT , Hops u-Havu VK : Proteolyt ic enzy mes in the s kin . n. A co mpa ra t ive st udy of s kin homogena tes of fi ve ma mma li an spec ies. Acta Derm Venereol (Stockh ) 49:468- 479, 1969

44. F ra ki JE , Hopsu·Havu VK : Human s kin proteases : fract ionation and cha racte ri za tion. Arch Derm a­tol Forsc h 243 :52- 66, 1972

45. Barrett AJ: Human catheps in B,. Purifi ca tion a nd so me properties of the enzy me. Bioc hem J 131:809- 822. 1973

46. Starkey PM , Barrett AJ: Hum a n cathepsin B, inhibition by a,- macroglobulin a nd other serum prote ins . Bioc hem J 131:823- 831, 1973

47. B eloff A. Peters RA: Observations upon therma l burns: the influence of moderate tempera ture burns upon a proteinase of skin. J Phys iol 103:461- 476, 1945

48 . BeloIT A, Peters RA : The protein ase of skin. J Physiol 103:2p-3p, 1945

49. Fruton JA: On the proteolytic enzymes of anima l tissue. V. Peptidases of s kin , lun g and serum . J Bioi Chem 166: 72 1- 738, 1946

50. Nev ill e·Jones D, Peters RA : Further observation on the proteolyt ic enzy mes in rat skin. Bioc hem J 43:303- 308. 1948

51. Martin CJ , Axelrod AE: The proteolyt ic enzy me

LYSOSOMES AND THE SKIN 269

system of skin . I. Extraction and act ivation. J BioI Chem 224:309- 321, 1957

52. Ma rtin CJ , Axelrod AE: The proteolyt ic enzy me system of s kin. II. The cha rac terization of ester­ase activ ity. Hiochim Hiophys Acta 26:490- 501, 1957

53 . Ma rti n CJ , Axelrod AE: The proteolyt ic enzyme system of s kin . III. Purification of proteinase C and its separat ion from an inhib itor. Biochim Biophys Acta 27 :52- 62, 1958

54. Ma rtin CJ , Axelrod AE: The proteolytic enzyme system of s kin . IV . The purification of proteinase A. Biochim Biophys Acta 27 :532- 538, 1958

55 . Martin CJ , Axelrod AE: The proteolyt ic enzy me syste m of skin . V. The A,-esterase. Biochim Biophys Acta 30:79- 87, 1958

56. Gol ubow J , Martin CJ , Axelrod AE: Proteolytic enzy me syste m of skin . VI. Enzy me patterns in various animal s pecies . Proc Soc Exp Bioi 100:142-146, 1960

57. Hopsu·Havu VK, J a nsen CT: Proteolyt ic enzymes in the skin . I. A cri t ical review of t he li terature. Acta Derm Venereol (Stoc kh ) 49:458-467 , 1969

58 . Laza rus GS , Hatcher VB: Lysoso mes a nd t he s kin , Lysosomes, vol IV . Edited by JT Dingle, R Dean. Amsterda m , North Holl and (in press)

59. Lazarus GS, Ba rrett AJ: Neutra l proteinase of rabbit s kin . An enzy me capable of degrading s kin protein and inducin g an infl ammatory res ponse. Biochim Biophys Acta 350:1 - 12, 1974

60. Well s GC, Babcock C: Epidermal protease. J Invest Dermatol 21:459- 462, 1953

61. Yamura T, Cormia FE: Studies on huma n s ki n protease. J Invest Dermatol 37: 121- 124, 1961

62. Beloff A: The skin protease inhibit ing factor of plas ma . Biochem J 40:108- 115, 1946

63. Fraki JE, Hopsu-Havu VK: Human s kin proteases, part ia l purification and characteri zation of a protease inhibitor. Arch Der mato l Forse h 243: 153- 163, 1972

64. Ba rrett AJ , Starkey PM: T he interact ion ofa,-mae­roglobulin with proteinases . Characterist ics and specific ity of the reaction and a hypothesis con­ce rning its molecular mecha nis m. Biochem J 133:709-724, 1973

65 . Starkey PM: Binding a nd inhibition of catheps in B, by a , -m ac roglobulin. Bioche m Soc Trans 1: 381- 382, 1973

66 . Ha rpel PC: Stud ies on human pia ma a,-m acro­glob ulin en zy me interact ions . J Exp Med 138:508- 521, 1973

67. Werb Z, Burleigh M, Barrett AJ , Sta rkey PM: T he in te raction of a,- macroglobulin wi t h proteinases . Binding and inh ibition of ma mmalian coll age­nases and other meta l prote inases . Bioc hem J 139:359- 368, 1974

68. Nordquist RE, Olson RL, Everett MA: The trans· port , uptake and storage of ferr it. in in hum an ep idermis . Arch Dermatol 94:482- 490, 1966

69 . Wolff K, Sc hrein er E: Aufn ahme, intrazellul ii rer Transport und Abba u exogenen Proteinen in K erat in ocyten. Arch Klin Exp Derma to l 235:203- 220, 1969

70. Wolff K, Honigs mann H: Perm eability of the epi­dermis and the phagocyt ic act ivity of kera t ino­cytes. Ult rast ructura l st udies with thorot rast as a ma rker. J Ult rast ruct Res 36: 176- 190, 1971

71. Kla us SN: Pigment trans fer in mamm a li an epider­mis. Arch Derm atol 100 :756- 762, 1969

72. Prun ieras M: In te ract ion between keratinocytes a nd dendritic ce ll s. J Invest Derm atol 52: 1- 17, 1969

73. Hori Y, Toda K , Patha k MA , Cla rk WH Jr, Fitzpa­t ri ck TB: A fin e ul t rastruct ure study of the huma n epiderm a l melanoso me complex a nd its ac id phosphatase act ivity . J Ultrastr Ll ct Res 25:109- 199, 1968

74. Olson RL, Nordquist J , Everett MA : The role of

270 LAZARUS, HATCHER , AND LEVINE

epiderm a l lysosomes in melanin physiology. Br J Dermatol 83: 189-199, 1970

75. Wolff K , Sc hreiner E: Melanoso mal ac id phos ph a­tase . Arch Dermatol Forsch 421:255-272 , 1971

76 . Wolff, K , Honigsmann H: Are melanosome co m­plexes lysoso mes? J Invest Dermatol 59:170- 176, 1972

77. Wolff K: Melanocyte keratinocyte interactions in vivo; the fate of melanosom es. Yale J Bioi Med 46:384 - 396, 1974

78. Wolff K , Jimbow K, Fitzpat rick TB: Experimental p igment donation in vivo. J Ultrastruct Res (in press) 1975

79. Szabo G, Gerald AB , Pathak MA, Fitzpat rick TB: Racial differences in the fate of mela noso mes in huma n epidermis. Nature (Lond) 222: 1081- 1082, 1969

80. Wolff K , Konrad K: M elanin pigmentation: an in -vivo model for studies of melanoso me kinetics within kerat inocytes . Science 174:1034- 1035, 197 1

81. Otaki N, Seiji M: Degradat ion of mela nosomes by lysosomes. J Invest Dermatol 57:1- 5, 1971

82. Pool e AR: Tumor lysosomal enzy mes a nd invasive growt h, Lysosomes, vol III. Edited by JT Dingle. Amsterdam, North Holland, 1973, pp 303-307

83. Din gle JT: Studies on the mode of action of excess of vitamin A. III. Release of a bound protease by the act ion of vitamin A. Biochem J 79:509- 512, 1961

84. Logan WS: Vitamin A and keratinization . Arch Dermatol 105:748-753, 1972

85. Wolbach SB, Howe PR: Tissue changes following deprivation of fat solubl e vitamin A. J Exp Med 42:753- 778, 1925

86. Ha rris LJ , Innes JRM, Griffith AS : On t he patho­genes is of av itaminosis A: vita min A as the anti -kerat inizing factor. Lancet 2:614- 617, 1932

87. Fitton-Jackson S, Fell HB: Epidermal fin e structure in embryonic chicken skin during atyp ical differ­entiation induced by vita min A in culture. Dev BioI 7:394-419, 1963

88 . Sherm a n BS: The effect of vitamin A on epitheli al mitosis in vitro and in vivo. J Invest Derm atol 37:469-480, 1961

89. Christophers E , Braun-Falco 0: Mechanis ms of pa rakeratos is. Br J Dermatol 82:268- 275, 1970

90. Kligman AM , Fulton JE Jr, P lewig G: Topica l vitamin A acid in acne vulgaris. Arch Derm atol 99:469-476, 1969

91. DeLuca L, Wolf G: Vitamin A and protein sy nthesis in mucous me mbran e. Am J Clin Nutr 22: 1059- 1062, 1969

92. Schn ebli HP, Burger MM: Selective inhibi t ion of growth of transformed cells by protease inhibi­tors. Proc Natl Acad Sci USA 69:3825- 3827, 1972

93 . Troll W, Klassen A, Janoff A: Tumorigenesis in mouse skin: inhib ition by synth et ic inhibi tors of proteases. Sc ience 169:1211- 1213, 1970

94. Grayzel A, Hatcher VB, Lazarus GS: Protease act ivity of normal and PHA stimul ated human ly mphocytes. Cell Immunol (in press) 1975

95. Sagami S , Kita no Y: E lect ron microscopic study of the effect of vitamin A on t he differenti ation of reconstructed embryonic skin. Br J Dermatol 83:565-571, 1970

96. Brandes 0 , Bertini F, Smith EW: Role of Iysosomes in cellul ar lyt ic processes . II . Cell death during holoc rine sec ret ion in sebaceous glands . Exp Mol Pat hoi 4:245-265, 1965

97. Bell M: A com parat ive st udy of sebaceous gland ultras tructure in sub-huma n primates. J Cell Bioi 49:932- 936 , 1971

98. Hers HG , Van HofTF (eds): Lysoso mes and Storage Diseases. New York, Academic , 1973

99. Kent JA , Carton 0: Fabry's disease, Lysoso mes a nd Storage Diseases . Edited by HG Hers, F Van H ofl. New York , Academic, 1973, pp 357- 381

100. Van Mull en PG: Ultrastruct ure of lipid bodies and

101.

102.

103.

104.

105.

106.

107 .

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

11 8.

119.

120.

121.

122 .

Vol . 65, No.3

lysosomes in the sk in in Fabry's disease. Arch Belg Dermatol Sy phili gr 27:41 - 49, 1972

Mapes CA, Anderson RL, Swel ley CC, Desnick RJ Krivi t W: Enzyme replacement in Fabry's di ;_ ease in in born error of metabolism. SCience 169:987- 989, 1970

Desnick RJ, Allen KY, Simmons RL, Naja ri a n JS Krivit W: Treatment of Fabry 's disease: correc: t ion of the enzy mic deficiency by renal transpla n _ tation. J La b Clin Med 78:989- 990, 1971

Fitzpatrick TB, Mihm MC Jr: Abnormaliti es of the mela nin pigmentary. syste m, Dermatology in Genera l Med lcll1 e. Edited by TB Fitzpatrick KA Arndt, WH Cla rk Jr, AZ Eisen , EJ Van Scott JB Vaughan . New York, McGraw- Hill , 1971,' pp 1591- 1635

Windhorst DB, Zelickson AS, Good RA: Chediak_ Higashi sy ndrome: heredita ry giga ntis m of cyto­plas mi c organell es . Science 151:8 1- 83, 1966

Oliver C, Essner E: Distribution of a nomalo u s lysoso mes in the beige mouse : a homa logue of Chedia k- Higashi syndrome. J Hi tochem Cyto_ chern 21:218- 228, 1973

Lutzner MA, Tierney JH , Bend itt EP: Gi ant "ran_ ules and widespread cyto pl as mi c incl us ions" in a genetic sy ndrome of Aleutian mink. La b In vest 14:2063- 2079, 1966

Windhorst DB. White JG , Zelickson AS, ClaWson CC, Dent PB, Poll ara ' B, Good RA: The Ch Ef_ diak- Higashi anomaly and the Al eutia n t ra it in mink: homologous defects of lysoso mal st ructure Ann Y Acad Sc i 155:818-846, 1968 .

Stossal TP, Root RK, Vaughan M : Ph agocytosis in chronic granulomatous disease a nd the Chediak_ Hi gashi syndrome . Engl J Med 286:120-123 1972 '

Prieur OJ , Davis we, Padgett G: Defect ive function of renal lysoso mes in mi ce with the Chediak_ Higashi syndrome . Am J PathoI 67 :227- 240, 1972

White JG: T he Checliak- Higashi syndrom e: a possi _ ble lysoso mal disease. Blood 28: 143- 156, 1966

Windhorst DB, Zelickson AS , Good RA: A human pigment dilution based on hfrita bl e s ubcellul ar st ructu ra l defect . The Chediak- Hi gashi syn­drome. J Invest Dermatol 50:9- 18. 1968

Weissman A, Dingle JT: Release of lysoso ma l pro_ tease by ul t raviolet irradiation and inhibi ted by hydrocortisone. Exp Cell Res 25 :207- 210, 1961

Weissman G, Fell HB: T he effect of hydrocortisone on t he response of feta l rat skin in cul ture to ultrav iol et irradiat ion. J Exp Med 116:365- 380 1962 '

J ohnson BE: Ultraviolet radiation a nd lysoso mes in s kin. Nature (Lond) 219:1258- 1259, 1968

J ohnson BE. Daniels F Jr: Lysoso mes and t h e reactions of sk in to ultraviolet irradiation. J Inves t Dermatol 53:85- 94, 1969

Fand 1: The protect ive effect of a sunscreen UPOn the lysoso mes of ultraviolet irradiated sk in . Der_ matologica 144:237- 247, 1972

Allison AE, Magnus IA. Young MR: Role of lyso_ somes and of cell membra nes in photosens it iza_ t ion . Nature (Lond ) 209:874- 878, 1966

Allison AE, Young MR: Vital s tain ing and fluore _ cence microscopy of Iysosomes, Lysosomes, vol II. Edited by JT Din gle, HB Fell. Amsterd a m, North Holl and , 1969, pp 600-628

Hawkins HK , Ericsson JLE, Biberfeld P , Trump BF: Lysosomes a nd ph agoso me sta bility in lethal cell injury. Am J Pat hoi 68:255- 288, 1972

Nix TE Jr, Nordquist RE, Scott RJ , Everett MA: Ultrast ructura l chan ges induced by ultraviolet light in hum an epidermis: basal and spinou layers . J In vest Derm atol 45 :52- 64 , 1965

Lazarus GS: Neutral prote in ase of rabbit skin (abstrl. Clin Res 2l :480. 1973

Smith JG Jr, Burk PG, ' Rosett 1', Church F:

Sept . 1975

Enzy me 111 bl ister f1ui d. La b Invest 16:247- 253 , 1967

123. Burk PG , S mith JG Jr: T he h i toc hemistry 0 (' can t h a radin aca nth olys is (abstrl. C lin Res 15:248, 1967

124. Ruddy S, G igli I, Austen KF: The co mplement sys tem in man. N En gl J Med 287:489- 495, 1972

125. Zuri e r RB, Weiss man G, Hollste in S , Ka mmerma n S , Hsin HT: Mec hanism 0 (' lysoso mal enzy me re lease ('rom hum a n leukocytes. J Clin Inves t 53:297 - 309 , 1974

126 . Laza rus GS: The role 0(' neutra l prote inase a nd catheps in 0 in turpen t in e- indu ced inf1 a mm at ion . J In vest Derm atol 62 :367- 371, 1974

127 . Baer RL, Ha rber LC: Photobiology 0 (' lupus ery­t he ma tosus. Arch Dermatol 92:124 - 128, 1965

128. Epstein JH , Tuffa nelli DL, Dubois EL: Li ght sensi­tivit y and lupus ery t hematosus. Arch Dermatol

L VSOSOMES AN D THE SKIN 271

91 :483- 485 , 1965 129. Tan EM: Antibodies to deoxy ribonucl e ic ac id irra­

diated with ultrav iol et light: detection by precipi ­t in s a nd immun o flu oresce n ce. Science 161: 1353- 1354,1968

130 . La ndry M , Sa ms WM Jr: Systemic lupus e ry­t hematosus. Studi es of t he ant ibod ies bound to s kin. J Clin In ve t 52: 187 1- 1880, 1973

131. Prose P H , Sedlis E , Bigelow M: The d emonst rat ion o f lysoso mes in t he diseased s kin of infants with infa nt il e ecze ma. J In vest Derm atol 45:448- 457 , 1965

132. Fri chot BC ITT, Zelickson AS: Steroids, lysosomes a nd derm atitis . Acta Derm Venereol (Stoc kh ) 52:311 - 319, 1972

133. Akopian AT, P ozdnia kon OL, Ava kia n AA: 0 polo li zosom v patogeneze puzyrchatki . Vestn Derm a­to l Venerol 46: 15- 19, 1972