Lysosomes and the Skin

REVIEW ARTICLE

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

LYSOSOMES 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, Montefi ore Hospital and Medical Center, Bron.x , New York

The importance of lysosomes in cutaneous phys as a secondary lysosome or digestive vacuole (Fig. iology was a ppreciated early. The studies of Fell 1) . and Mellanby [1) on vitamin A induction of The substrate containing organell es can ori gi mucouS meta plasia in chick skin and the investiga nate by several distinct mechanisms . Heterophagy tions of Weissman and Fell [2] on lysosomal is a process by which t he cell can engulf foreign labilization by ultraviolet light, were landmarks in material into h eterophagoso mes (Fig. 1) by either our understanding of lysoso mal physiology. Subse phagocytosis, which is the uptake of larger, insolu quently lysosomes have been shown to be impor ble substances, or pinocytosis, which is the inges tant in keratinization, pigmentat ion, and seba tion of sma ll er soluble material. The two processes ceous secretion. The lysosomal system also partici are known coll ectively as endocytosis. The fusion pates in numerous pathologic processes in skin of a heterophagosome with a primary lysosome incl uding epidermal phagocytosis, inflammation, produces a digestive vacuole. and neoplasia. Autophagy is a met hod by which the cell can sequester part of its own cytoplasm in auto DEFINIT ION OF LYSOSOMES phagosom es for digestion (Fig. 1). In this way, the cell is able to rid itself of damaged constituents. Lysosomes were first d escribed in 1955 by Fusion of the autophagic vacuole with a primary deDuve et a l [3]. They are cytoplasmic organelles lysosome results in the formation of a digestive which contain hydrolytic enzymes that are capable vacuole. of digesting many tissue constituents. Most diges The membrane that delimits the digestive vacu tion occurs intracellularly , but hydrolytic enzymes ole is ideally suited for cellular economy. Large can be secreted by exocy tosis, into the extracell ul ar macromolec ules readily enter the lysosome by com partment where they may also act on extracel heterophagy or autophagy but they are unabl e to lular macromolecules (Fig. 1) . diffuse from the vacuole because of their size. After Lysosomal enzymes are produced in the rough extensive enzymic digestion, the breakdown prod endoplasmic reticulum (6] and are then trans ucts of proteins, carbohydrates, nucleic acids, ported to a specialized re gion of the smooth endo mucopolysaccharides, and glycoproteins are s mall plasmic reticulum located at the inner surface of enough to pass through the lysosomal membrane the Golgi stack (GERL) (7]. Here these proteins where they may be used in biosynthetic processes are concentrated a nd packaged into membrane in other parts of the cell (8]. . bound primary lysosomes. Primary lysosomes can A residual body or telolysosome is formed when fuse with organell es containing substrates for di substances in the digestive vacuole are incom gestion; the s ingle membrane-bound vacuole pletely digested so that they are too large to pass which contains hydrolases and substrate is shown through the lysosomal membrane. When these organell es have no further demonstrable hydrolytic Manuscript received December 18, 1974; in revised enzyme activity, they are call ed postlysosomes form April 11, 1975; accepted for publication April 14, (Fig. 1) . The residual body can be extruded from 1975. . the cell by the process of exocytosis or it may i This work was supported by grants from the National rema in within the cytoplasm of the cell . Institute of Arthritis, Metabolism, and Di gestive Dis eases (IRO I AM 17370 01), t he National Institute of Heart and Lung (HL 16387 01) , and a grant from the MORPHOLOGIC EVIDENCE FOR THE PRESENCE OF Syntex Corporation to Dr. Norman Levine. LYSOSOMES IN SK IN * Senior Investi gator, The Arthritis Foundation U.S.A. Reprint requests to: Dr. G. S. Lazarus, Division of Dermatology, Duke University Medical Center, Durham, In this section we will discuss the morphologic Nort h Carolina 27708. evidence for the presence of lysosomes in the 259 260 LAZARUS, HATCHER, AND LEVINE Vol. 65, No.3

BIOCHEM ICAL STUDIES OF LYSOSOMAL ENZYMES IN SKIN n seque .. ,o,;ot r.~ Preparation of Lysosomal Fractions from Skin Endoplasmic :~ • :: Typical lysosomal hydrolases have been found i space '====' n '\. AUlophogosame / homogenates of whole s kin [1 8- 26 ]. T he specif· P,;mo,y Iysosomes ~ activity of these enzymes is highest in the l ysos~~ !~ ~ ~al fr~ct i on [1 8,27,28]. T~ese hydrolases are a lso & \ s,\>Je lIocUOles ~ found. IJ1 t he supernatant fraction , suggesting that va . o,e. / ~\ :~~ <':~ t~ the vlgoro:,s treatment necessary to homogenize @ .~. ~.~ sklJ1 also disrupts lysosomal organell es. Epidermal homogenates contain lysosomal enzymes with ~ higher specific activit ies t han whole skin [27-30 J. .Elcw" Residual Postlysosome o (f'e~ Cathepsin D ~ <1o,y l,sOSO \. bod, IiP\ Cathepsin D is a lysosomal acid proteinase C-J @~ which degrades hemoglobin at ~H 3 and proteogly_ TeIO I YSOS~~m; ~ can at pH 5 [31,32 ]. The use of a potent monospe_ .~~" ~ cific inhibitory antiserum to pure cathepsin D in y' °,c · t issue cult ure and a utolytic experiments has dem_ I o onstrated t hat this enzyme is important in extra_ S cellular matrix degradation [33 ]. Cathepsin D has been positively identified in extracts of rabbit skin [34] and in huma n skin where approximately lO o/c FIG. 1. Schematic representation of lysosomal func of the total enzyme wa s found in the epiderm is. t tions. (From Vaes [4] modified from Jacques 15 J with Antiserum to rabbit cathepsin D was capable of permission of the authors) quantitatively precipitating t he enzyme from ex tracts of whole rabbit skin. The removal of cathep_ epidermis and its appendages. T he connective s in D from the crude skin extract resulted in t issue cells of t he dermis, s uch as fibroblasts, al most complete loss of proteolytic activity against endotheli al cell s, histiocytes, mast cells, etc., are hemoglobin below pH 5 (Fig. 5) [34]. Autolysis not unique to the s kin and their lysosomes have experi men ts util izing i m m unoinh i bition of cathep_ been adequately reviewed elsewhere [9]. sin D demonstrated t hat t his enzyme was a lso The presence of lysosomes in t he epidermis was respons ibl e for degradation of structural s kin pro initiall y suggested fo ll owing the demonstration of tein at acid pH [34 ]. 1m munocytologic studies in t he lysosomal hydrolase, acid phosphatase, in t he rabbit skin demonstrate t hat t he enzyme is local granular layer [10] . Subsequent ly, acid phospha ized in epidermis, epidermal appendages, and tase [11 - 14] and aryl sulfatase [1 5] were localized fibroblasts [17] (see morphologic evidence above). in vacuoles in epidermal cells . Structures meeting Cathepsin D levels increase dramaticall y at morphologic criteria for lysosomes were observed in times of remodeling in chick skin [35,36 ]. This t he basal epidermal cells of guinea pigs and enzyme, as well as neutral proteinase, a lso in- ' humans (Fig. 2). T hese structures regularly con creases dramaticall y in experimental inflamma_ tained acid phosphatase (Fig. 3) . Ac id phosph atase tion induced by turpentine in rabbit skin (see was also locali zed to t he small juxtanuclear Golgi Lysosomes and Cutaneous Inflammation). cisternae of t he basal and s pinous cell s and in Cathepsin D may playa major role in intracellu_ membrane-coating gran ul es (Fig. 2c). Typical acid lar protein digestion. Dingle et al [37] in cubated phosphatase-conta ining lysosomes h ave been rabbit pulmonary alveolar macrophages with ca fo und in epidermal Langerhans cells and in mel t hepsin D antiserum and were able to a rrest the anosomes (see section on Pigmentation). intracellular digestion of IgG, hemoglobin, a nd Cathepsin D, t he major lysosomal acid protein proteoglycan. The antiserum-treated cells devel ase responsible for protein degradation at acid pH, oped giant vacuoles which contained un degraded has been locali zed immunocytochemically in chi ck s ubstrate; t he cells remained viable and when t he epidermal cell cultures [1 6] a nd rabbit skin [1 7 ]. a ntiserum was removed t heir morphology returned Immunofluorescent and immunoautoradiographic to normal. T hese results indicated t hat cathepsin techniques (Fig. 4) have been used to demonstrate D co uld be inhibited within t he cell and t hat t his punctate localization of cathepsin D in t he basal acid hydrolase pl ayed a major role in protein I ayer of t he epidermis and a diffuse distribution of catabolism. The locali zation of cathepsin D to the enzyme in the granular layer . Cathepsin D has basal epidermal cells [1 7 ], coupled with t heir also been found in hair fo llicles, sebaceous glands, known phagocytic function [38], suggests t hat a nd mesenchymal cells of t he dermis. These stud ies suggest that epidermal lysosomes may be t Levine N, Hatcher VB, Lazarus GS: Unpublished involved in protein catabolism. res ul ts. Sept. 1975 LYSOSOMES AND T H E SKI N 261

FIG. 2. Some morphologic variants of epidermal lysosomes in guinea-pig epidermis. a: Single membrane-limited bodies (L) conta ining granule material of moderate electron density. MT, microtubules. b: Single membrane-limited body (L) ex hibiting a granular matrix a nd concentric lamellae. c: A mult ivesicu lar body (MVB), two single membrane-limited organelles (L) with a granular interior a nd lamellar substructure (arrow), and several membrane coating granules (OB, Odland bod ies) . d: A single membrane-limited vacuole containing vesicles and nonidentifi able g ranular material ( x 49,500). (From Wolff and Schreiner [14])

Cathepsin B, Cathepsin 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] . F IG_ 3 . .Qifferent patterns of acid phosphatase distribu Fraki and Hopsu-Havu [44 ] have partially purified tion within epidermal lysoso mes. The electron-dense human s kin cathepsin B, and it appears that its material with in the s in gle membrane-limited bodies (Ll properties are al most identical to the purified marks acid phosphatase activity (a , b, X 22 ,000; C, X human liver enzyme [45,46). Using an extremely 43,000). (From Wolff a nd Schrein er [1 4]) sensit ive radioassay technique, we have been able to isolate cathepsin B , from both separated epider cathepsin D may be of importance in t he intracel mis and dermi .t P reliminary studies suggest that lular hydrolysis of macromolecules in epidermis. t his lysosomal proteinase may be of great impor Cathepsin D may be involved in both extracellular tance in cutaneous catabolic processes. degradation in skin , and then after endocytosis of partially degraded macromolecules it may partici Neutral Proteinases pate in intracellular digestion of protein or protein polysaccharide within t he vacuoles of the lysoso Neutral proteinases of skin are operative at mal system. physiologic pH. They are presumed to be lysoso- 262 LAZARUS, HATCHER, AND LEVINE Vol. 65, No.3

. FIG. 4. Locali zation of ca~ h e p s in D in rabbit skin by immunofluorescent and autoradiographic techniques . a: Ski; mcubated with control prelmmune sheep serum fo ll owed by fluorescem-Iabeled rabbit antlsheep serum. K. kerati autofluorescence;. C, co ~l age n autofluorescence ( x 400). b: Skin incubated with sheep. anti rabbit cathepsin D seru;: foll owed by rabbit antlsheep serum ( x 400). intense green lluorescence IS present 111 the cells of the hair follicle (a.rrow). c: Rabbit skll1 sebaceous gland stamed Wi t h 3H-labeled pre. lmmunes heep serum ( x 400). d: Skin incubated With shee p antlrabblt cathepsm D 3H-labeled serum demonstratmg localI zation of the enzyme 111 an epidermal adnexal structure (x 400) . (From Lazarus and Poole [17])

purified and at least three are inhibited by diiso_ propyl fluorophosphate. A possible role for these enzymes has been 0'40 s uggested followin g extraction and purification of a ...... 1:: neutral proteinase from rabbit skin which was ' capable of degrading skin protein and inducing an ~030 lJ.J inflammatory res ponse [59] . This low-molecular_

extracts of skin had very little Q2-macroglobulin. A The latter protein appears to be capable of inhibit ing almost a ll known t issue proteinases [64- 67 ).

ROLE OF LYSOSOMES IN CUTANEOUS PHYSIOLOGY Epidermal Phagocytosis Epidermal cells are capable of phagocytosis and digestion of exogenous material using t he lysoso mal system. Nordquist et al [68) injected ferrit in intradermally and observed t hat t he marker en tered the epidermis and locali zed in vacuoles containing acid phosphatase. Wolff and co-workers [69,70) injected t horotrast and peroxidase into the dermis of gu inea pig and found that the markers were rapidly endocytosed by epidermal cells; the B phagosomes co ntaining t he marker were stored within the vacuolar apparatus of t hese cells until they underwent keratinization. Epidermal cells are also capable of phagocytos ing particulate material (38 ). Injection of latex beads into a subepidermal blister, produced by s uction, permitted direct contact between latex particles and regenerating epidermis. Wolff and Konrad [38 ] found t hat latex particles were avidly taken up by the regenerating epitheli um and in corporated in to phagosomes. These phagocytic vacuoles moved toward the nuclear zone of the cell , and fusion of phagosomes with each other a nd with primary Iysosomes was observed. The uptake mechanism was size dependent since large beads (0.8 micron) were taken up indivi dua lly whereas s mall beads (0 .109 micron) were taken up in groups. The ingested beads did not interfere with keratinization and th e latex was incorporated into horny cells and eliminated by desquamation. These elegant experiments demonstrate that epi FIG. 6. Effect of in tradermal injection of partiall y t helium is phagocytic and that classic heterophagy purified neutral proteinase into rabbit skin. A: Macro occurs in mammali an epidermis. scopic appearance of rabbit skin removed 18 hr after injection of neutral proteinase (below) or heat-inac Pigmentation tivated enzyme (above). B: Micrograph ( x 4(0) of a wax-embedded section of skin after t he int radermal Melanosomes are transferred to keratinocytes by injection of neutral proteinase stained with hell1atoxylin a highly speciali zed form of epidermal phagocyto and eosin . An acute granulocytic infiltration with necro sis [71 ,72 ). Consequent ly one could designate t he sis is seen. C: Micrograph (x 400) of a skin section from t h e periphery of the les ion that followed injection of skin melanosome within a keratinocyte as a phagosome proteinase. There is dermal- epidermal separation t hat and predict that it should fuse with a primary appears independent of leukocyte infiltration. (From lysosome to form a secondary lysosome. S upport Lazarus and Barrett [59 j) for this notion is provided by the observations that melanosomes within keratinocytes are located laboratory on separated skint suggest that protein- . within vacuoles con taining acid phosphatase ases a re located in both the epidermis and dermal ['73 - 76) a nd th at injection of ferritin into the skin connective tissue. res ults in localization of this molecule within melanosome complexes in keratinocytes [7 4,76,77). Inhibitors of Enzymes in Skin Wolff and Honigsmann (76) conclusively demon One of the major problems in the ident ification strated that melanosome complexes in keratino and isolation of skin enzymes had been the pres cytes were secondary Iysosomes. They induced ence of inhibitors [51,62 ). One of the major protein secondary lysosomes in guinea-pig skin with thoro ase inhibitors in homogenates of whole s kin, which trast and observed fusion of these tracer-marked necessitates extraction with high-ionic-strength vacuoles with melanosomes by electron mi c r~ s buffers for enzyme activation , appears to be iX , copy. The end product was a vacuole containing antitrypsin [63 ). Fraki and Hopsu-Havu identified melanin, thorotrast, and ac id phosphatase (Fig. 7). the protein immunologically and also found that Keratinocytes are also capable of phagocytosing 264 LAZARUS, HATCHER , AND LEVINE Vol. 65, No . 3

mal proteinases. M elanosomal protein is digested by lIver lysosome preparations at pH 5 [81) whereas ' 4C-dopa-labeled melanin was not de graded by lIver lysosome preparations under s imi lar conditIOns. The a ut hors s uggested from these d ata that lysosomal protell1ases are not capa ble of degradll1g the melanin moiety. Before th is concl u sion can be accepted it is necessary to incubate aut.hentic melanin with epidermal lysosome prepa ratIOns ~ v~r. a wI.de ran?e of conditions to rule o u t the possibIlity o.f an epidermal melanin-degrading lysosomal protelI1ase.

Keratinization Th~ production of .. the stratum corn eum and espeCiall y Its unique fIbrous protein, keratin, con FIG. 7. Interactions of two heavil y labeled melanosome stitutes one of the mo s~ drama~i c examples of co mplexes which contain melanosomes in various stages programmed catabolI sm 111 all of biology. Lyso of melanization (M). The complexes 1 a nd 2 have fu sed at somes probably pl ay a significant rol e in this t he point ma rked by t he arro w and a melanosome complex process . In almost all cells studied to date (as terisk) is a bout to cross over from I to 2 (gu in ea-pig epidermis injected wi t h thorotrast, x 48,000). (From lysosomes pl aya vital role in intracellular di ges~ Wolff [77]) tlOn and cellul ar division (for rev iew of the role of lysoso mes in cell divis ion, see [82) and Proteases melanosomes independent of their interaction with and BIOlogical Control:j:). It seems reasonable to melanocytes. Wolff [77] and Wolff et al [7 8 ] assume that the lysoso mal system pl ays a similar injected melanosomes into subepidermal suction :ole in ~he basal cells and is thus indirectly blisters in guinea pig and found that keratinocytes 1I1volved 111 the complex process of keratin produc avidly endocytosed these organell es. tion. A more direct rol e in the process of keratiniza_ A possible explanation for racial co loration was tion may be pl ayed by the membrane-coatinu offered by Szabo et al [79] when they found that granule ~hich is a modified lysosomal organe ll e~ Caucasoids, American Indians, and Monogoloids These unique lamellar granules contain significant had groups of two or more melanosomes s ur amounts of acid phosphatase [14). rounded by a membrane within keratinocytes, . The membra ne-coatin g granules are extruded whereas Negroids and Australian aborigines had 1I1tO the extracellular space immediately above t he single, la rger, discrete melanosomes distributed granular layer and may account for the observation within the cytopl asm of keratinocytes. A possible that there is diffuse staining for acid phosphatase mechanis m for t he variation in distribution of [10,12], aryl s ulfatase [1 5 ], and cathepsin D [17] at melanosomes within keratinocytes in d ifferent this level of the epidermis . The exact role of t he races was suggested by studies of epidermal phago lamellar membrane-coatin g granules is unknown cytosis using different sized lat ex beads [38,80 ]. but it is intriguing to s pecul ate that these organ-' Large latex beads (0 .8 JLm) were taken up sin gly ell es are responsible for degradation of the kerati whereas small beads (0.1 JLm) were taken up in nocyte glycocalyx. The final and most dramatic groups. These results suggested that uptake of catabolic event in keratinization occurs immedi particles by keratinocytes was size dependent and ately adjacent to the granular layer. There is a that the difference in distribution of melanosome sudden loss of cell organelles such as nuclei particles between Negroid and Caucasoid refle cted mitochondria, and ribosomes in the stratum cor~ the size of the melanosome synthesized. Further neum. Occasional " intermediate cells" demon support for this hy pothes is was presented by Wolff strating partial degradation are seen but auto et al [7 8 ] in their studies on pigment donation in phagic vacuoles and other manifestations of au vivo. These investigators obtained large melano tophagy are not usually observed [9]. The mecha somes from anagen hair of C57BL/6J mice (l.3 X 1 nism of this phenomenon is totally unknown but JLm) and small melanosomes from pigmented B16 the activation of the lysosomal system could playa mouse mel anoma (0.5 JLm x 0.3 /-Lm) and injected role in this final step in keratinization. these particles into a suction blister in guinea-pig Vitamin A and K eratinization skin. As expected, the large melanoso mes were taken up sin gly whereas the small melanosomes Vitamin A, a potent lysosomallabili zer [83], has were taken up in groups. profound effects on keratinization [84 ]. Hypovi- M elanin is rarely seen above the basal epidermal layer in Caucasoids [74]. T his lack of melanin in :j: Abstracts of papers presented at t he meeting on Proteasesand Biological Cont rol, September 10- 15, 1974, t he s uperficial layers of epidermis is probably Co ld S pring H a rbor Laboratory, Co ld Spring Harbor, related to catabolism of melanoprotein by lysoso- New York. Sept. 1975 LYSOSOMES AND T HE SKIN 265

taminos is A resul ts in hyperkeratosis, follicul ar vat ion has been doc umented electron microscopi p lugging, and keratinizing squamous metapl asia of cally by fi nding sign ificant widening of the in ter , the sweat glands and m ucous mem bra nes of the cellula r lacunae [87- 93,95 ]. The most reasonable trach eobronchial and genitourinary tract [85]. ex pl anation for this morphologic change is in T h ese observations suggest t hat vitamin A has an creased formation an d labilization of lysosomal " antikeratinizing effect" on epithelium [86 ]. Sup hydrolases in the upper granular layer. Mem brane por t for t his notion was presented by Fell and coating granules have been shown to be lysosome Mella nby [1] when t hey demonstrated t hat the like organell es which are secreted in to th e in tercel addit ion of excess vitamin A to tissue cul t ures of lular space [1 4 ] and several lysosomal hydrolases chic k epit helium prevented keratinization and in have been observed in the upper granular layer as duced mucous metaplasia. Vitamin A primarily well [1 2, 14,17 ]. Consequently it is intriguing to affected the basal layer where t he cells became s pecul ate that vitamin A could increase the con d isoriented, developed in tercellular lacunae, lost cent ration of extracell ul ar lysosomal enzymes by t h eir fil a ments, demonstrated mitochondrial en hy pertrophy and leakage of t he mem brane-coating largem ent, a nd eventually a ppeared to secrete granules whi ch t hen degrade t he extracellular m u c u s [87 ]. Removal of t he t issue from excess " cement substance," causing widening of the in vitamin A res ul ted in reversion of t he basal cells to tercellular lacunae and loose packing of keratin. nor mal keratinizing epit helium. T h e effect of vi tamin A on skin vari es; rodent Sebaceous Secretion skin is less responsive than chick skin and mature The autolytic process of sebaceous secretion has epith elium is less affected than embryonic tissue been in ves tigated in rat sebaceous glands by [84]. Al t hough muco us metapl asia cannot gener Brandes et al [96 ] usin g electron microscopic and a lly be induced in ro dent epidermis by vitamin A, histochemical techniques. T hese workers fo und significant changes in t he epi t helium do occur. maturing sebaceous cells contained numerous lyso H istologicall y t here is hy perpl asia, acant hos is, somes which enl arged and rupt ured immediately in c r eased mi totic fi gures, and parakeratosis [88 ]. before the sebaceous cell d isintegrated. After cytol The o bservation that mitotic rate in skin treated ysis occurred, discrete lysosomes were no longer wit h vi tamin A is increased may expl ain these visible, but staining for acid phosphatase persisted histologic observations [88,90 ]. The effect of vita in t he cytopl asm . S imila r res ul ts in macaques were m in A on mitotic activity is dose dependent ; very obtain ed by Bell [97 ] who also noted th at the acid h igh doses of t he vitamin, in certain selected phosphatase-containing ves icles contained crystal system s, actuall y diminish mitotic rate [88 ]. Sev line incl usions with a period from 55 to 105 A. eral p ossible mec hanis ms have been s uggested to P rogra mmed autolysis also appears to occur in expl a in t he effect of vitamin A in epidermal rabbit sebaceous glands. Lazarus and P oole [1 7 ] turnover. DeLuca and Wolf [91 ] observed inhibi fo und t hat t he lysosomal proteinase cathepsin D tion of a mino-acyl transfer RNA synthetase in could be identified by immunofluorescent tech vitamin A deficiency. This enzy me links specific nique in t he periphery of sebaceous glands (Fig 4). amino ac ids to t ransfer R NA prior to rough endo These data suggest that lysosomal prol ife ration pl asmic reticulum protein synthesis . T his effect of a nd consequent ru pture plays a primary role in t he vitamin A co ul d direct protein synthes is and progra mmed autolysis which is t he characteristic con sequen t cell division. A second possibility of holoc rin e secretion. migh t be t hat vitamin A induces lysoso mal prolif eration which accelerates cell di vision. Dingle et al RO LE OF LYSOSOMES IN CUTANEOUS [33] h ave shown that vitamin A is capable of PATHOPHYSIOLOGY induc in g lysosomal proliferation and increasin g Lysosomal Storage Diseases the concentration of lysosomal proteinases in cul t u res of chick limb bone rudiment. We have fo und Lysoso mal storage diseases are c h aracte ri z~d by that t he co ncent ration of the lysosomal protein ase a primary defect in a lysosomal hydrolase (for cath e psin D § is increased in rabbitskin cul t ured in complete review see [98 J) . Remarkabl y, few of these the presence of retinoic acid . Cell cul ture experi diseases have clinical cutaneo us manifes tations ments suggest t hat lysoso mal hydrolases are in . even t hough they can be diagnosed by study of vol ved in the process of cell d ivision [92,t ] and skin-derived cells. Two exceptions are Fabry's their inhibit ion in lerfe res wi t h cell division,t t u disease a nd the Chediak-Higashi syndro me. morigenesis [9 3,:1= 1, and phytohemagglutinin, Fabry's Disease , induced ly mphocyte t ransformation [94 ]. Conse quently, it is possible that vitamin A increases Fabry's disease is an X-linked disorder charac epidermal turnover by in creasin g the concent ra teri zed by the genetic in activation of one of several tion a nd release of lysosomal hydrolases. !'I-galactosidase enzy mes or isoenzy mes [99 ], which Another effect of vitam in A on skin is dec reased resul ts in in t ralysosomal accumulation of cera mi de cohesion of t he stratum co rneum [90 ]. T his obser- trihexoside and di galactosyl cera mide. Clinicall y there are characteristic angiokeralomas on the § Laza rus GS, Din gle J T : Unpublished data lower t runk and upper thighs. Histologicall y t hese 266 LAZARUS, HATCHER , AND LEVINE Vol. 65, NO .3 lesions demonstrate epidermal thinning and di liver resulted in leakage of acid protein ase frorn lated capillaries with glycolipid endothelial in these organell es. Pretreatment of the rats with clusions in the upper papillary dermis. The clinical hydrocort isone d ec re a~e.d light-induced leakage of severity of the disease correlates with the number protell1 ase. The pOSS ibility that ltght co uld ruptur of zebra and concentric lamellar type inclusions cutaneous lysosomes was first s uggested by the seen in dermal fibroblasts and endotheli a l cells experiments of Weissman and Fell [11 3 ]. Irr ad i a~ lIDO). Similar lesions occur throughout the body tion of fetal rat skin with light i~ the 300-nm r a n ge and account for the ophthalmologic, renal, neu resulted 111 nec ros Is of epidermiS a nd dermis. Th rologic, and cardiovascular compli cations. The addition of hydrocortisone to the cul t ure m e diu~ disease usually is fatal by the fifth decade but re modifi ed the response to irradiation so t hat treated cent experiments with enzyme replacement [101 , cultures demonstrated retarded cell breakdown 102) in Fabry's disease may provide techniques by decreased vesiculation, less disorganization of th~ which patients with a variety of lysosomal storage dermal connectiv e t iss ue, and accelerated produc_ diseases can be treated. tion and different iation of n ew .~ pid e rmis. The aut hors aSC rIbed t he protective effects of cort ico_ eM.diak-Higashi Syndrome steroids to stabilization of lysosomes which de Chediak-Higashi syndrome is an autosomal creased the leakage of proteolytic enzymes. recessiv e disease characterized by pale skin which J ohnson [114] co nfirmed the observation t h at sunburns easily, blue-gray ha ir color , decreased li ght induced lysosomal rupture in skin when h e uv eal pigment, and frequent severe infections studied the concentration of acid phos phatase in [103 ). Patients have anemia, leukopenia, and they mouse ear skin after irradiat ion wi th ult raviolet frequently develop a malignant lymphoma. The light. Ears that had bee n irradiated with 320-nrn hallmark of t he disease is large cytoplasmic inclu light had 30 % less acid phosphatase when ex_ sions which presumably are lysosomes [104 ). The tracted than ears that had been irradiated through beige mouse [105 ] and the Aleut ian mink [1 06, 107] plate glass. He suggested from these data that are animal models of the human syndrome. light, below 320-nm wavelength, induced lyso_ The mechanism responsible for t he formation of somal rupture in the skin and that the acid phos_ these giant granules is not known but studies of phatase released from the tissue was inactivated phagocytosis in granulocytes by Stossal et al [108] or carried away in the blood. This phenomenon may provide an explanation. These workers fo und was somewhat specifi c for lysosomal labilization that t he rate of bacterial phagocytosis and the and did not simply reflect leakage secondary to production of hydrogen peroxide, which is neces cell death because irradia ti on did not deplete th e sary for bacterial killing, were normal in patients tissue of histochemica ll y demonstrable succinic with Chedia k-Higashi syndrome. The phagolyso dehydrogenase a,t the same time that acid phos_ somes contained peroxidase and J3-glucuronidase phatase was markedly reduced [115 ]. Light-in_ but not alkaline phosphatase. These .data sug duced lysosomal labili zation of human foreskin gested that in Chediak-Higashi syndrome. certain epidermis can be blocked by appli cation of th e granulocyte granules were unable to fuse with the sunscreen, 10% sulisobenzo ne [116]. phagocytic vacuole because of a defect inmem The mec hanism of lysosomal labili zation by brane fusion. Prieur et al [109 ] found that t ubular li ght is in co mpletely understood, but t he innova_ cells from t he kidney of the beige mouse co uld not tive studies of Allison and Young [11 7 ) with· degrade exogenous horseradish within t he giant photosensitizers have defined promising areas far granules as quickly as normal a nimals. These investigation . Cells and t iss ues can be damaged by in vestigators interpreted their findings as suggest t he combin ation of a photosensitizin g agent, light in g t hat there was a defect in protein degradation; of the a ppropriate wavelength, and oxygen . Usu an alternative interp::etation might be t hat t here ally the action spectrum for photosensit ization is was a bl oc k in fu sion between the phagocytic similar to the absorption s pectrum of the photosen_ vacuole and the lysosome conta ining the en sitizer [118 ]. Allison noted that photosensit izers zyme(s) necessary for catabolism of the perox id ase such as uroporphyrin , anthracene, and vital dyes molec ul e. Another piece of ev idence suggestin g were concentrated in lysoso mes. S in ce these su b _ that Chediak-Higas hi granules have abnormal stances are capable of causin g cytolys is by disrup_ membranes is the observ ation that giant granules tion of plasma membranes, techniques had to be in leukocytes stain for acid phosphatase much developed to demonstrate that disruption of lyso_ more rapidly t han adjacent normal- a ppearing somes by photosensit izers was of primary impor_ granules [110] . Pigment dilution which is second tance in cell destruction. A number of different cell ary to abnormal fusion of melanosomes into giant types in cul ture were in cubated with various pho granules may be another consequence of t his tosensitizers in the dark fo llowed by an additional genetic defect in membranes [111]. 30-min in cubation in fresh medium without t he photosensitizer. The photosensitizers were found The Effect of Light on The Skin onl y in the lysosomes, and damage secondary to Weissman and Dingle [11 2 ] demonstrated that cell -membrane binding of the photosensit izer irradiation of a lysosome-rich fraction from rat could be avoided by keeping the cells in the dark Sep t. 1975 LYSOSOMES AN D THE SKI N 267 a nd washing t hem. Irradiation of t he cells wi t h EP IDER MAL IN JU RY lig h t of the appropriate wavelength res ul ted ini t ia lly in increased permeability of t he lysosomes, as m easured by increased ability to stain fo r lysosom al acid phosphatase, foll owed by cell d eath. S uc h data s uggested t hat lysosomal disrup • LYSOSOMAL" tion was of prima ry im portance in li ght- mediated CHE MOIATIC EPIOER MA L HYDRDLASES cell d a mage. Confirmation of this hy pothesis has PROTEI NA SES been provided by H awkin s a nd co-workers [11 9 ]. T hese worker demonstrated t hat lysosomal rup t ure was t he prima ry event in cell death produced INf LAM MATORY - CELL HYD RO LA SES by t h e photosensit ize r acri d ine ora nge. T his was I IN HIBillON BY LEAKA GE Of esp ecia ll y convincin g s ince in t he same study t hese / . ,. MA CROCLOBU LIN AN D I ., · AN 1I1RYPSIN in vestigators showed t hat lysosomal rupt ure was ~ ______~... ~~a Oafl 'd \O\'lfY--- seconda ry to cell death in studies utilizing meta VESSEL b o lic poisons and co mplement-sufficient a nticell membra ne antiserum. F IG. 8, Schematic representation of t he effects of C linically, co mmon s unburn may be an excell ent disruption of epidermal lysosomes on t he skin , (From Lazarus and Hatcher 158 ]) mod e l of li ght-induced lysosoma l disrupt ion caus in g clinical disease. One hour after ul t raviolet irra dia tion t he epidermis contains numerous vacu huma n skin which appear to be capable of causing oles [1 20] which a ppear s imilar to t he acid phos chemotaxis. T hese proteinases might also be cap phatase-containing autophagic vacuoles seen in able of inducing chemotaxis by cleaving comple p h ototoxic reactions induced by methoxsalen [1 4 ]. ment, fibrinogen, Hagema n factor, or ka llikrein T he immediate erythema associated wit h ul t ravio [1 24 ]. let irradiation, may be related in part to disrupt ion Lysosome la bilization , t herefore, might trigger of lysosom es of endothel ia l cells wi t h release of infla mmation and cause a biphasic increase in c hemical med iators [11 7]. The delayed erythema lysosomal protein ase level in the t issue (Fig. 8). An could be, in part, secondary to diffusion of protein epidermal insult coul d da mage cell s which would ases from the epidermis seconda ry to lysosomal resul t in leakage of lysosomal hydrolase into t he dis ruption . A prime candidate for such a mediator, tissue. T he e enzy mes m ight cause t issue necrosis in a ddition to prostaglandin, would be neutral and coul d a lso act as chemotactic agents. Infla m p rote in ase which is capable of caus in g vasodilata matory cells would t hen infilt rate t he dermis a nd tion , capillary permeability, a nd chemotaxis [59, secrete t heir catabolic enzy mes [1 25], which, when 121 ]. added to endogenous s kin hydrolases, would pro duce t issue necrosis. This biphasic phenomenon L ysosom es and Cutaneous Inflammation has a lready been observed in t urpent ine granu lom a in rabbit skin [1 26 ]. The infla mmatory cata Application to t he s kin of cant ha ridin, a lysoso bolic process is usuall y self- limited s ince infla m mal la bilizer, produces an int raepidermal acan mation is associated wit h increased vascular t h olytic blister [1 22 J. Seven hours after a ppl ication permeability and leakage of serum protein ase in of t h e drug, blister fluid conta ins signi ficantly hibitors such as a,-macroglobulin a nd aI-ant it ryp h ig h e r concent rations of acid phos phatase a nd sin in to the t issue. a.-Macroglobulin is capable of cath e psin t han serum. T his elevation of ext racellu inhibit ing a ll tissue proteinases studied to date, lar lysosom a l hydrolase is not associated wit h and it could prevent hydrolase-mediated t issue leukocyte infil t ration, which suggests t hat leakage destruction as well as protein ase-related chemo of e p idermal lysosomes might be respons ible for taxis. T his self-limited inf1 a mmatory cascade may t he e n zy me elevations. Histologicall y, pa rt icul ate expla in how epidermal injury instigates a n infla m acid phosphatase staining is reduced in 4 to 6 hr m atory res ponse involving t he ent ire s kin. whereas lactate dehydrogenase and succ inate de- . This cascade of events is specul ative and under hydrogenase are not reduced fo r 8 to 10 hr [1 23 ]. m any circumstances lysosoma l enzy me release is a T hese findings suggest t hat lysosomes containing secondary phenomenon . However, t here is a natu acid phosphatase are the structures which are ra lly occurring disease, lupus erythematosus, affe cted earliest by cant haridin. Granulocytes which could possibly correspond to the model of en ter t he epidermis 12 hr after a pplication of infla mmation just proposed . The wavelength of can t h aridin a nd proba bly account, in part , for light which induces skin lesions in lupus is 290- furthe r elevations of lysosom al hydrolases in blister 320 nanom eters [1 27, 128 ], which is exactly the fl uid. The mecha nis m for leukocyte infil t ration wavelength range which labilizes lysosomes [11 7]. could be t he elaboration of a chemotactic lysoso Consequent to irradiation there is da mage to the mal proteinase from t he epidermis s imila r to the basal cells and the derma l capillaries. The da m neu t r a l proteinase of rabbit [59,121] (Fig. 8). We aged basal cells appear to be the source of the cir have discovered severa l new neut ral protein ases in culating DNA in li gh t- induced exacerbations [129 ], 268 LAZARUS, HATCHER , AND LEVINE Vol. 65, No . 3 and t h ey could a lso release prote inases which of enzymes in rat-li ver t issue. Bioc he m J cau se t issue necrosis and induce inflamm a tion . 60:604- 617 , 1953 4. Vaes G: Digestive capacity of lyso omes, Lysosomes The damaged d ermal vessels leak immune com a nd Storage Disease. Edited by HG Hers, F Va n plexes (DNA a nd a n t i-DNA immunoglobulin) a nd Hoof. New York, London . Academic, 1973. pp complement which d ep osit at t he d erma l- epi 43 - 47 d erma l basement m e mbrane [30]. This entire li gh t 5. J acques P: Homeostatic regul ators. Ciba Founda_ tion y mpos ium, 1969, pp 180- 193 induced process can be inhibited by sunscreens 6. Cohn ZA, Fedorko ME: The formation and fate of which protect against t he wavele n gth of ligh t t hat lysoso mes, Lysoso mes, vo l I. Edited by JT Dingle induces lysosomal labilization. Obvio us ly, t h ere HB Fell. Amsterd am, North Holl and. 1967 pp' are oth er explanations for t his rath er complex 43 - 63 ' 7. Nov ikolT AB: Enzyme ul trastru cture and locali za_ process; however, t his hypoth esis is being tested in tion in neurons, The Neuron. Edited by H Hyd en o ur la boratory a nd if it is correct it might provide Amsterdam, E lsevier. 1967 . pp 2.55 - 318 . new therapeu t ic m o dalities. 8. Cohn ZA , Fedorko ME: The formation and fate of There are oth er infla mmatory dermatoses t h at lysosomes, Lysoso mes, vo ll. Edited by JT Dinale HB Fell. Amsterdam, No rth Holl and. 1967. b pp demonstrate m orph ologic ch a n ges in lysosom es. 44 - 45 Prose a nd co-workers [131] reporte d lysosomal 9. Breathnach AS: An Atlas of the Ultrastructure of prolife ration in t h e granular a nd cornea l layer in 3 Human S kin. London, Churchill . 197 1 of 6 babies with atopic derma tit is. Fric hot a nd 10. Eisen AZ, Arndt.KA. Clark WH Jr: T he ultrastruc_ t ural locali zation 01 aC id phos phatase in huma n Zelic kson [1 32] confirmed t his observation a nd ep idermis. J Invest Dermatol 43:319- :326 , 1964 noted that, in contact dermatitis, acid phosph a 11. Diengdoh JV: The demonstra tion of lysosomes in tase-containing vacuoles had poorly defined limit mouse skin . Q J Med 105:73- 78 , 1964 ing m embra nes. Membrane m orphology returned 12. Olson RL, Nordquist RF: Ultra microscopic locali za_ tion of acid phos phatase in human epid ermis. J to n orma l coin c ident with clinical improvem ent. In vest Dermatol 46:4 31- 435, 1966 Akopian et a l [1 33 ] observed a n increase in lysoso 13. Rowden G: Ultrastructural studies of keratinized m a l hydrolase activity in basal a nd prickle cells epithel ia of the mouse. J Invest Dermatol 49: 181- in pemphigus v ulgaris , which s u ggested to these 197, 1967 14. Wolff K, Schreiner E: Epidermal Iysosomes elec_ authors t h at release of lysosom a l en zymes was tron m icroscopic-cytochemical studies. Arch res pons ible for destruction of intere pidermal ce Derma tol 101: 276- 286, 1970 ment s ubstance which caused intraepiderma l blis 15. Olson RL, Nordquist R, Everett MA: Ultrastruc_ ter form ation . The clinical studies enumerated t ural locali zation of aryl sulfatase in human epidermis. Acta Derm Venereal (Stockh ) above lend further s upport to t h e hy pothes is that 48:556- 562, 1968 lysosomes a re of s ignificant importance in a variety 16. Poole AR, Dingle JT, Barrett AJ: The immunocyto_ of in!1 a mmatory c utaneous diseases. chemical demonstration of cathepsin D. J Histo L ysosomes proba bly play a s ign ificant role in chem Cytoc hem 20: 26 1- 265 , 1972 17. Lazarus GS, Poo le AR: Immunocytochemicallocali_ s u c h diverse phys iologic functions of the skin as zation of calhepsin D in rabbit skin. Arch Derma_ cellula r div is ion , keratinization , a nd sebaceous to l (in press) 1975 secretion . T h ere is increasing evidence t hat lyso 18. Wynn CH, Iqbal M: Isolation of rat skin lysosome som es a lso function in numerous path ophysiologic and a comparison with li ver and spleen lyso somes. Biochem J 98: 10p, 1966 circ umstan ces as well. S u ch diverse processes as 19 . Ockerma n PA: Acid hydrolases in human skin. Acta ligh t reactions, epiderma l infla mmation , a nd n eo Derm Ve nereal (Stockh) 49:139- 141, 1969 . plasia may b e associated with lysosom a l even ts. 20. Steigerwald J C, Bartholomew BA: The assess ment Further study of these organell es may not only of lysosomal glycos id ase in normal skin . Biochim Biophys Acta 321: 256- 26 1, 1973 furnis h ins igh ts into disease m ech a nis m but could 21. Ockerman PA: Acid hydrolases in skin and plasma d e fin e a r eas for pharmacologic manipulation in ga rgoylism. Defi ciency in /3-ga lactosidase in whic h would profoundly inn uence t issu e catabol skin. Clin Chim Acta 20: 1- 6, 1968 Is m. 22. Go ldberg MH, Cotlier E, Fichenscher LG, Kenyon K, Enat R, Borowsky SA: Macul ar cherry red The aut hors are deeply indebted to D r. Kl aus Wolff spot, corneal clouding and a-galactosidase defi wh o provided numerous electron micrographs and manu ciency. 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