T E JOURNAL OF INVESTIGATIVE DERMATOLOGV, 65:191 - 200, 1975 Vol. 65, No.1 c:pyright @ 1975 by The Williams & Wilkins Co. Printed in U.S.A .

STUDIES OF THE MECHANISM OF EPIDERMAL INJURY BY A ST APHYLOCOCCAL EPIDERMOL YTIC

KIRK D. WUEPPEH , M.D.' , RO BERT L. DIMOND, M.D.t, AND DENNIS D. KNUTSON , M.D.:/: D epartment of Dermatology, Medical School Division, Un iversity of Oregon Health Sciences Center, Portland, and Oregon Regional. Primate Research Center, Beaverton, Oregon

E xperimental anima l models of the two forms of toxic epidermal necrolysis have been reviewed : a murine model of staphylococcal-induced epidermolysis and a ha mster model of graft-versus-host disease. In t he former, a protein , epidermolysin , has been purified and characterized. The exotoxin has a molecular weight of approximately 30,000 and causes a split beneath the granular layer. It is effective at 3 x 10- 12 moles. Epidermolysin does not require an intact complement system for its action since BloD2 mice deficient in C5 or mice injected with the decomplementing agent in cobra factor were susceptible to its epidermolytic effects. Neither are immunocompetent thymocytes required for the action of the toxin since ha irl ess, athymic adult (nu/nu) mice are susceptible. A few reports of epi­ dermolysis due to an exotoxin of group I have a ppeared. This toxin is antigenically different from the exotoxin of group II organisms. A model of drug- induced toxic epiderma l necrolysis has been described in hamsters, but the toxic principle released from sensitized lymphoid cell s has not yet been characteri zed .

Toxic epidermal necrolysis is one of t he m ost eases have been linked with bullous impetigo and a dramatic examples of epidermal injury. In t he staphylococcal scarl atiniform syndrome. A similar past, careful histopathologic examination has entity has been reported in adults with compro­ helped to cl arify t he causes. More recently, the mised immune function or overwhelming staphylo­ development of an experimental animal model a nd coccal septicemia; t hus this syndrome and its t he availability of a highly purified epidermolytic clinical variants extend over a wide age group. toxin , epidermolysin, have enabled us to explore In 1956, Lyell described a skin erupt ion which the pat hogenesis of the process and to attempt to closely resembled scalding and postulated that determine how the toxin exerts its selective action "some circulating toxin specificall y damages t he on the epidermis. epidermis and results in its necrosis" [IJ. He call ed In this paper we will review t he epidermolytic t he resulting syndrome toxic epidermal necrolysis form of toxic epidermal necrolysis, focusing on t he (TEN), defining necrolysis as " a neo logism coin ed protein exotoxin(s} elaborated by some phage to combine t he clinical appearance of epidermol­ group II Staphylococcus aureus which are responsi­ ysis with t he histologic observation of epidermal ble for the epidermal injury. necrosis" [2 J. Cases of t his syndrome had been previously CLINICAL BACKGROUND reported under various na mes [3 J, but it is still For nearly a century, physicians have observed , most commonly referred to in English as Lyell's particularly in neonates or young children, the syndrome, toxic epidermal necrolysis, or the abrupt onset of blistering a nd exfoliative epider­ scalded-skin syndrome. mal diseases which have been called by such In 1878 Ritter von Rittershain reported 297 cases names as " dermatitis exfoli ativ a neonatorum," of n eonates suffe ring from a skin eruption he " toxic epidermal necrolysis," or "erythrodermie named dermatitis exfoli ativa neonatorum (DEN) bullo use avec epidermolyse." Recently these dis- (4 J. In 1961 Frain-Bell and Koblenzer suggested a r elationship' between t he DEN of von Ritters hain Supported, in part, by PHS Training Grants 5TOl AM a nd the TEN of Lyell [5 J. Koblenzer later reviewed 05300 and AM 05512, and a gra nt from the Derm atology t he li terature and presented cases of his own to Foundation. establish t his relationship convincingly [6 ]. S. * Recipient of Resea rch Career Development Award aureus was first isolated from a patient with DEN 5K04 AM 40086 04. in 1898 [7 Although vo n Rittershain did not t Trainee, PHS Training Grant 5TOl AM 5300. Present J. address: Oregon Reg ional Primate Research Ce nter, associate DEN with infection, several authors have Beaverton, Oregon 97005. since establi shed it as a staphylococcal disease :/: Present address: Department of Dermatology, Mayo [8- 13 J. Clinic, Roc hester, Minnesota 55901. On the other hand , TEN appeared to be a Reprint req uests to: Dr. K . D. Wu epper, Department of Dermatology, Medical School Division, University of multifactorial disease. In 30 of the 128 Cases Oregon Health Sciences Ce nter, Portland, Oregon 97201. reviewed by Lyell in Britain in 1967, a staphylococ- 191 192 WUEPPER. DIMOND. AND KNUTSON Vol. 65. NO .1 cal infection seemed to be the predominant fea­ bullae or peeling, postulated a circulating toxin ture, in 36 a drug was implicated, in 28 a miscel­ arising from the site of infection, often the phar­ lany of diseases coexisted, and in 34 there was no ynx, ears, conjunctiva, or skin elsewhere. Arbuth_ clue to the cause [14). In the 28 cases of miscellane­ nott and co-workers suggested, erroneously as it ous disease, drugs were often given and may have turned out, that the delta hemolytic toxin was been responsible for the complex ity. Lyell felt that responsible since, in England, most of the strain many of the children in the idiopathic group were of' staphylococci isolated from cases of TEN pro- suffering from an undiagnosed staphylococcal in ­ duced the delta toxin [28 J. . fe ction; all of t he cases of staphylococcal origin Our knowledge of this disease advanced consid_ were children under lG years of age . erably when in 1970 Melish and Glasgow [29 J In the same year, Lowney et al reported 10 of injected group II staphylococci that had beell their own cases and reviewed 139 published case isolated from cases of staphylococcal TEN, bullous reports [1 5 J. They toc> observed that the staphylo­ impetigo, or staphy!ococcal scarlatiniform erup, coccal cases occurred in children whereas the tlOn mto newborn mice and epidermolysis enSued drug-related cases occurred in both children and The experimental lesion s developed from the fo r: adu~ts. The mortality rates among the patients mation of a Nikolsky sign to widespread peelin!> differed-7% among the 1- to 5-year-old group, 44% Histologic sections were charact.eri zed by the sa~~ among the patients over 6. kind of cleavage plane wi thin the epidermis at the Staphylococci cultured from cases of TEN had level of the stratum granulosum that is found ill already been id ent ifi ed as organisms of phage human patients. Since the organisms could not b ~ group II, usuall y type 55 or 71 [16,1 7J, and had cultured from the sit.e of the peeling or even seel) been associated with Ritter's disease [9- 13 J and histologicall y near this area, these workers hypoth, bullous impetigo (l1,18- 20J as well as with a esized that the products of the staphylococci wer~ scarl atiniform eruption (21- 24 ]. In 1970, Melish being disseminated from the site of infection and and Glasgow proposed that dermatitis exfoliativa were causing lesions elsewhere. They dev.eloped all neonatorum, staphylococcal TEN in children, expertmental modelll1. which they established that staphylococcal scarl et ['ever, and bullous impetigo group II staphylOCOCC I are the etIO logiC agents Of were clin ical manifestations of infection wi th staphylococcal TEN. phage group II staphylococci and represented a spectrum of diseases wi th a sin gle etiology [2 5 ]. Hamster Model of Grant-versus-Host TEN

HISTOPATHOLOGIC OBSERVATIONS In 1968, Billingham and Streilein developed all animal model for studyin g the drug- induced vari. Before the pathoge nesis of TEN co uld be under­ ety of TEN (30], and Streilein discussed that stood, the patholog ic changes which accompany work at the Symposium in 1969 [31). They ob. the disease had to be described. In his first case, served that graft-versus-host (GVH) reactions ill Lyell reported that the pathology was confined to t he hamster resulted in an explosive, ne c r oti~ the epid ermis where necrosis of the epidermal ce ll s epidermolys is with large sheets of intact epidermis resulted in blister formation between the epidermis peeling from the affected animals. Cutaneous reac, and dermis; in one case, however, the necros is was. tions are characteristic of other animal and humal\ more superficial and formed an intraepidermal cases of GVH, but the TEN-like picture seems tQ spli t [1]. Koblenzer observed an intraepidermal be peculiar to the hamster. TEN, however, has split in the children he studied (6); Lown ey et al been associated with a GVH reaction in humans also observed an intraepid ermal split in small (32,33 J and has occasionally complicated G\ I{ children with staphylococcal infection but a sub­ reactions in rats [34 J and monkeys [35 J. Whe\) epidermal split in the older children [1 5). While sensitized lymphoid cells from donors of either agreein g that the intraepidermal split was typical parental strain are injected in tracutaneo usly Ll1 t ~ of staphylococcal TEN, Lyell postul ated that the adult F I hybrid hamsters, the GVH reaction nonstaphylococcal cases had the dee per split [26 ]. including TEN, occurs. Streilein showed that th; Thus, by 1969 staphylococcal TEN was generall y skin is not the antigenic stimulus for this reaction regarded as being histologically and, in so me and hypothesized that when the leukocytes of th, respects, clinically different from nonstaphylococ­ hybrid are attacked by the injected lymphoid cel\ ~ cal TEN. of the parents, a pharmacologi cally active macro.1 EXPERIMENTAL MODELS OF EPIDERMAL molecular agent is released which damages the NECROLVSIS epidermis [31]. He supported this hypothesis b\'1 finding that serum obtained during the acute G ~ Murine M odel of Staphy lococcal TEN reaction was cytotoxic toward suspensions of epi.: Evidence that a circulating toxin is implicated dermal cells grown in vitro. In t his connection,\ in staphylococcal TEN is now firmly established. serum obtained from one of the patients with GVH In 1967, both Lowney et al [15) and Samuels [27) compli cated by TEN produced cytotoxic effects in! suggested that staphylococcal TEN resul ts from vitro against epithelial cells [3'6]. Therefore, in th( some diffusible toxic product of the staphylococci. drug- induced form of TEN there is some eviden ' Lowney et al [15], who observed that only rarely to support Lyell 's hypothesis that a c ir c ul atil~ were staphylococc i cultured from the site of the toxin d amages the epidermis. July 1975 STAPHYLOCOCCAL EPIDERII10LYTIC TOX IN 193

THE STAPHYLOCOCCAL EXOTOXIN, EPlDERMOLYSIN in Pevikon, fractions containing epid ermolysin were pooled, dialyzed, and chromatographed on Isolation and Pu.rification carboxymethyl 8ephadex C-50 in phosphate buffer, From the supel'll atant of cultures from certain pH 6 (Fig. 2). Proteins were elu t ed from the cation p hage group II strains of S. aureu.s, Arbuthnott, exchanger by a sodium chl oride gradient . Epider­ Kent, Lyell, and Gemmell .[37 ] and ~apra l and molysin eluted as the major protein peak and was M iller [38 ] isolated and partially punfted a factor measured by bioassay in newborn mi ce. Its purity whic h caused epidermolysis in newbol'll mice. was ascertain ed in polyacrylamide gel electropho­ Later, M elish, Glasgow, and Turner [39 J, Kondo, resis (Fig. 3). Sakura i, and Sarai [40J, and Dimond and Wuepper [41 ] purified this extracellular prod uct which has Characterization of EpidermolYsin been called "exfoliatin" [38 J or "epid ermolytic When stored at 4°C or -20°C, epid ermolysin re­ toxin" [37 ]. S in ce the primary histologic finding in mains stable for several months . It is remarkably this m odel, as in human patients, is a lysis within heat stable and resists heating to 60°C for 1 hI' or to t he epidermis whi ch is characterized by a separa­ 100°C for 20 min [38,40J, but boiling for longer t ion of the cells rather than by profuse scaling 01' periods leads to decreased actiVity [38,40 J. exfoliation , we believe the toxin is epidermolytic The toxin is said to be stable in buffers from pH and have proposed that it be called "epider- 5.0 to 9.0 but precipitates below pH 4.0 [39,41J. molysin" [41 J. The substance is not irreversibly denatured at this Some workers have prod uced tox in by growin g pH, however, since its activity is restored when it the bacteria in semisolid nutrient agar under 20% is resolubilized in acetate buffer containing sodium CO [37] or in yeast extract-trypticase. soy broth dodecyl sulfate [41 J. 2 under 10% CO 2 [38,40]; others have 1Il0cuiated The electrophoretic behavior of epidermolysin is Medium 199 in dialysis sacs and implanted them like that of a beta globulin [39,41 J: it sediments at . within the rabbit peritoneum [39,41J for a few days. approximately 38 in gradients of sucrose [39 ], and Obvious differences in the quantity of toxin pro­ its isoelectric point was found by two laboratories duced by various strains have been reported [38]. to be 6.7 to 7.0 [39,42] but 4.0 to 4.5 by two others Simple schemes for purifyin g toxin include ad­ [38,40). sorption chromatography to hydroxylapatite [37 ], The molecular weight of epidermolysin has been isoelectric fo cusing in polyampholytes [39,42 J, and investigated by several methods. By gel permea­ chromatography upon diethylaminoethyl cellulose tion chromatography on 8ephadex G-50, results of [40] or Sephadex [38 J, or carboxymethyl Sephadex 23,500 [38 ], and 24, 000 [40] have been obtained. after block electrophoresis in Pevikon [41 ]. Al­ Electrophoresis in aC l'ylamide gels containing so­ though epid ermolysin is effectively separated by dium dodecyl sulfate have given results of 28,600 in these procedures from most proteins in culture one laboratory (Fig. 4) (41 ) and 33,000 in another supernatants, the a-hemolytic toxin is extremely [41J. By analytical ultracentrifugation, we have simil ar in size and charge behavior. The a­ obtained a value of 32,500, assuming epidermolysin hemolytic toxin and epidennolysi n are separable to be a globul ar protein with a partial specific after electrophoresis in polyacrylamide gels [40, volume of 0.74 [41J . Evidence tb at the toxin is a 41 ] or isoelectric fo cusing [39,42 ]. protein has been provided by the digestion of We performed zone electrophoresis at the isoe­ epidermolysin with proteolytic enzymes; when lectric point of t he a -toxin , pH 9.0, as our first step measured by bioassay, its activity was reduced or in purification [41 J and obtained good separations destroyed by incubation wi th pronase [38 J, trypsin of t hese two proteins (Fig. 1). After electrophoresis [38,39 ], or pepsin [30 ].

2 0

1.8 1800

1. 6

14

I• 12 r 360 • oS 10 ~ ~ ! 280 ~ 08 ~ ~ ~ 200 " C.6 t\:" :§ OA

02

-5 10 15 20 15 30 35 H Seg men l Numbe r (+) F IG. 1. Zone electrophores is of Staphylococcus aureus (strain EV) cul ture filtrate in Pevikon containing 0.05 M Tris buffer, pH 9. 194 WUEPPER, DIMOND, AND KNUTSON Vol. 65, NO.1

1000 " 10 ";> 9 S? 800 • e "~ ~ ~ 7 " 600 ~ '"~ :% 6 ~ .::-. & 5 .:::~ (l Oot " ~ ~" .(l ~ 100~

, 5 '0 ,5 20 15 30 ~5 40 Fractio n Number FIG. 2. C hr o~atography of epid ermolysin , recovered from zone electrophoresin in Pevikon, uP.on carboxymethyl Sephadex C-50 111 phosphate buffer 0.01 M, pH 6. Adsorbed prote1l1s were eluted with a gradient o( 0. 2 M NaCI in the phosphate buffer.

A B c D 20 (-) 5 10 '>, <;)..... 4 '" 3 ~ EPIDERMOLYSIN ~ I '- 2 ~ :::. ~" ~

0.5 '----0-'.-2--0--L4---0J..6---oL.e----'1 0

Rela tive Mob ility FIG. 4. Acry lamide gel electrophoresis in sodium do. decyl sulfate (SDS) of staphylococcal epidermolysin relative to marker protein s of known molecular weights The marker protein s were horse heart cytochrome C (1) : cytochrome C dlmer (2) , ovalbum1l1 (3), bovine albumin monomer (4) and dim er (5). The molec ular we ight of epidermolysin 28,600.

(+) the activity of epidermolysin. When tox in was F IG. 3. Polyacrylamide gel electrophores is of crude recovered in supernatants, it was again measured staphylococcal culture fil trate (A), fractions recovered by bioassay [38,44 ]. Because of different methodol. from zone electrophoresis containing a-toxin (8) and ogies, the unit of epidermolysin measured by thi epidermolysin (C), and, finally, hi ghl y puri fied epid er­ method has not been standardized and therefore molysin recovered from CM Sephadex chromatography (D). resists co mparison by t he various investigators. Kapral and Miller [38] injected 0.02 ml of toxin Epidermolysi n is immunogenic, and precipitat­ in I-day-old mice and defined an exfoli atin g do e ing antibod ies to the protein have been raised in (ED,o) as that which gives a positive Nikolsky sign rabbits (Fig. 5) [40,41,43 ]. These antibod ies, pas­ in 50% of the group after 3 hr. Having injected 0.1 sively administered to neonatal mice, neutrali ze ml in mice 1 to 5 days of age which had been the effect of toxin in vivo [43]. The toxin appears randomized, Melish and her co-workers defined to be a single polypeptide chain, not associated an exfoli atin g unit (EU) as t he rec iprocal of th1 with a subunit structure. It migrates as a single dilution which causes exfoli ation in at least one of moiety in acrylam ide gels after it has been reduced each group of three mice after 2 hr [39 ]. Finally, by dithiothreitol with SDS [41,42 ]. Kondo et ai, who injected 0.05 ml in mice or unstated age, defin ed their unit (EU) as th! Measurement of Epidermolysin reciprocal of the minimum dose whi ch gives 8 The bioassay, first described by Melish and positive Nikolsky sign in 100% of t he group afteri Glasgow (29 ), rema ins the on ly functional test for hr [40 J. July 1975 STAP HYLOCOCCAL EPIDERMOL't'TIC TOXIN 195

TAllLE I. Effect of mouse age on th e epidermo/ysin bwassav

Time (min) for Epidermolysin injected N ikol s k~' sign" (I'g in 0.02 m!) I day' 3 days 5 days

2.6 15 20 25 0.65 50 60 70 0. 16 75 110 145

" Mean of duplicate determinations.

h Age of recipient mice, in days.

re?ently, hairless adul t mice [45,46 ], epil ated adult ml?e (47], and, most importantly, adult human bell1 g~ have been show n to be susceptible (47 ). It is there/ore not surprising that in the past t wo years seve:al cases of TEN caused by group II staphylo­ COCC I have been reported in adults [48-54 ]. MUltiple M olewlar Forms of Epidermolysin . Epidermolysin is found in mUl t iple molecul ar FIG. 5. Antigenic distinct.ion of staphylococcal a-toxin (arms [39-41). In our preparations fractions ob­ and e pidermolysin by double diffusion in agar. Antigens were placed in wells indicated by E (highly purified tained from P evikon e l ectrop h o r ~s i s contained epide r molysin) or A (a-toxin , Burrowes Well come); anti­ only one or two minor substan ces which were sera were p laced in well s a (antibody to a-toxin ), b examined by polyacrylamide ge l electrophoresis (a nt ibody to epidermolysin ), and c (a 1:1 mixt ure of a (PAGE) and were at first thought to be contami­ an d b). nants . When these were subjected to chromatogra­ We have subsequently investigated the dose­ phy upon CM-Sephadex, two peaks of protein were response in mice of specified age after t he injection s~e n. Epidermolytic activi ty was measured by of known quan tities of epidermolysin in a vo lume bIO assay 111 both peaks, and each protein had the of 0.02 ml (44). Part of the data is given in Table 1 sa.me apparent molecular weight (28,600) as deter­ to show how important the age of the recipient is mll1ed by acrylamide gel electrophoresis in SDS. on t he time for a locali zed Nikols ky sign to become To determine whether we were dealing with elec­ evident. We have observed a linear response be­ trophoretic va ri ant of the toxin [41], we performed tween 15 and 90 min in I -day-old mice when the electrophoresis in PAGE on a fraction which con­ injected dose was between 2.6 and 0.1 f.L g of tain ed about equal amoun ts of each moiety. The epidermolysin. Presumably, a major cause of these ge ls were cut. t he proteins were eluted in buffer, differences in mice of various ages is the t ime ~ nd ~~o distinct zo nes of epidet'molysin were req uired for diffusion of the toxin from its subcuta­ IdentIfied by bioassay. T he two substances were neous depot to the epidermis. immunochemically identical. Whether these dif­ It is generall y agreed that less than 1 f.L g of ferent species of epidermolysin res ul ted fi·om aene epidermolysin causes a positive Nikolsky sign in duplication or postsynthetic alteration of the ~e n e newborn m ice. In our la boratory, a pprox im ately product has not yet been determined. 100 ng (about 3 x 10- 12 moles) cau. e a positive .M el ~ s h et al (39 ) suggested that e pidermolysin ikolsky sign 90 min after s ubcutaneous injection eXIsts lI1 polymeric forms with differe nt molecular in a I-day-old mouse; these results indicate the weights. Kondo et al (40) described four sub­ potency of this agent. This is the same general stances, obtained from strain Zm of S . aur eus, range of biologic effectiveness of histamine, brady­ phage group II , type 55/71 , which had exfoliatina kinin, or t he anaphylatoxin fragments deri ved activity but different specific activitie, . Each fro m serum complement co mponents. of these separable moieties had th e same mo­ Neit her clinical nor investigative studies have l e~ ul a r weigtJt and was neutra li zed by antibody fu lly exploited the measurement of epidermolys in raIsed against one of them (40 ). antigen even though such measurement of epid er­ molysin or of antibody to epidermolysin by sensi­ (rom Group ! Staphy lococci tive immunochemical techniques could probably Recently, the production oran exfoliating princi­ be useful for diagnostic purposes or for defining in­ ple by staphylococcal strain s not belonain a to dividuals at risk. phage group II has been reported [40~55~56 ) . To date, a mong newborn rats, rabbits, chickens, M cClosky described a patient w ith a scar­ mice, and human patients, only the last two latiniform eruption and necrotizing fasciitis from respond to the tox in (38). SomeLhing a bout Lhe which S. aureus, phage type 85, was isolated. The maturation of mouse and huma n epidermis organ ism produced epidermolysis in newborn seemed to account for the fa ct that only newborn mice 155 1. Of the 52 isolates which were phage mice and children were susceptible [37,39). More typed in a large group of children with toxic 196 WUEPPEH, DIM OND, AND K NUTSON Vol. 65, No. i epidermal necroiysis by Rasmussen, only one was ant iserum to epidermolysin a nd did not precipitate group I, type 29; the remaining 51 were identifi ed ~i t h antibody to epidermolysin by double diffu sioll in phage group Il [56 ]. In agar. Kondo et al recovered group I S. aureus from 1 patient with TEN and from 3 others wi t h bullous Genetic Co ntrol of Epidermolysin Synthesis im petigo [40 ]; a ll fo ur organisms were sensit ive to phage type 79. T hese authors showed that t hese Whether the e.pidermol yt ~ c toxin is under the strains produced a s ubstance which had epider­ genetic control of the b act~ n a, the bacte ri o ph ag~ moly t ic activity in newborn mice and also showed or plasmlds that reS Id e In the bacteria, POse I that t he epidermolytic prin cipl e differed from a not.her problem . Som e bacteri al toxin s, e.g., di ph~ epidermolysin since it was not neutralized by an then a tOX in, stap l:y l oco~ca l , strep, tococcal erythroge l11 c tOXin , and probably staphy lococcal alpha toxin , are under the control of ' bacteri ophage that h as lysogenized the bacteri'l [57]. Plasmids are extrachromosomal genetic e l ~q ments composed of double-stranded D NA, wh ic~ may repli cate independently of bacterial chrom(), som es; probabl y they are adventitious inhabitant of the cells and occur in all staphylococcal ce ll ~~ Best established a re t he pl as m. i~ s which contai\; genetIC Illtormat.lO n for synthesIzlllg penicillinasl\ but other plasmIds are known to confer resista n (!' to tetracycl in.e and other a.n tibiotics . The ability IJ~ staphylOCOCC I to synt heSIze delt.a ~ox in is al 0 t hought to be controll ed by a plasmId [58J. SOnte recent work by Rogolsky et al [59 ] and Warren et I [60 1 has suggested that the genetic control ~ f F IG. 6. The ep idermis of a 3-day-old mouse 60 min after the injection of ep id er molysin. Cleavage occurs below the epidennolysin synt hesis is not exerted by a b act~ , granular ce ll laye r of the epide rmis (Gl and the follicul ar nophage but rather by the extrachromosomt\\ infun dibulu m (F ) (x 1,700). genes of a plasmid.

FIG. 7. Keratinocytes in the plane of cleavage 45 min afte r ep id ermolysi n inject ion. Peripheral cyto plas m (*l edematous, des moso mes (single arrows) appea r in tact, and the intercellular spaces (doub le arrows) are not dilat ( x 24,430). July 1975 ST APHYLOCOCCAL EP IO EIlMOLYTIC TOXI N 197 Ultrastructural Studies of Epidermolysis lected to give a positive Nikolsky sign 45 min after injection. No attempt was made, however, to Electron microscopic studies of staphylococcal abrade the skin or to create a plane of cleavage so T EN in the mouse were first reported by Lilli­ that the ul trastructural changes could be evalu­ bridge, Melish and Glasgow [61). who suggested ated in situ. The spec imens obtained from norm al that the initial event was the disappearance of control mice and from t hose injected with epider­ what they called "extracellular bubbles" between molysin were identical at 15 and 30 min ; in those the cells of the stratum granulosum followed by the obtained at 45 min , earl y changes had appeared widening of the in tercellular spaces and the spli t­ along the pl ane between the granular and malpigh­ ting of the . They also suggested that ian layers. In the cells adjacent to thi s zo ne, a the " bubbles" are ana logous to Odland bodies marked swelling of' the peripheral cytopl a m be­ (lam ellar granules, me mb.rane coatin? granules, tween desmosomal attachments was observed , but ker a tinosomes) and con tam proteolytic enzy mes the width of the in terce llular spaces remained which are released through t he action of the toxin normal (Fig. 7). By 60 and 75 min, the in tercellular and then act on adjacen t des moso mes to produce spaces had widened along the zo ne of cleavage. in t erdesm osoma l cleavage. Init iall y in tact, t he desmosomal attachments sub­ In our ult rastructural studies, the lamell ar gran­ sequently separated through the in terd es moso mal ules in bot h the co ntrol and toxin-treated animals con tact zo ne (Fi g. 8); those which formed the cleft rem ained intact and were norm a ll y and abun­ border had numerous microv illi and half desmo­ dantly distributed between the cells of the granular somes along their free surfaces (Fig. 9) . Still later, layer [62) . Wherever we observed ~ pid e r~ a l cleav­ some cell membranes were disrupted, and the age t he cleft followed a plane Immediately be­ alterations in cell orga nelles suggested cytolysis. neath the lowest granul ar cells except in sections Elias et al have reported similar observati ons in con taining developing hair follicles where the specimens obtain ed from human patients (47 ]. clea vage plane passed beneath the granular ce ll The fact that the cells separated before the leakage layer of the folli cul ar in fundibulu~ ~ Fi g. 6): of horseradish peroxidase or thorium dioxide into After epidermolysin had been ll1,l ected ll1 to a the separatin g cells suggests that se paration oc­ su b c u t aneous site on th e back, specimens were curred before cytolysis [63 ). He also showed that removed a t 15-min intervals. The dose was se- surface coats of keratin ocytes stainable by ru -

F IG . 8. T he lowermost gra nula r ce t.J (G,) and a cell rrom t he ma lpighi an layer (C,) 60 min after the injection or epi­ de rmolytic toxin. Swelling of the pel'l pheral cytopl a m (* ) IS prominent, intercellular spaces (Ie) are wide, a nd desmo­ !Gmes have started to separate (arrows). A keratohyaline granule ( KH) marks the granula r cell ( x 18,950). 198 WUEPPER, DIMOND, AND KNUTSON Vol. 65, NO.1 Although there is no evidence that epider_ molysin is an enzyme, a prototype of those en­ zymes which release epidermal cells fr om one another is trypsin (64), which cleaves polypept ide chains at carboxy terminal lysine or arginine. F or example, trypsin cleaves the major glycoprotein of erythrocyte membranes (glycophorin) and thus releases much of t he carbohydrate moiety into the nuid phase [65 ]. We h ave been unable to inhibit epidermolysin with trypsin inhibitors from soy beans or lim a beans or by the tissue trYPsin inhibitor Trasylol (41). Additional evidence is available from the fact that few enzymes can withstand boiling to the same degree as epider_ molysin . Finally, we have preli minary evidence F,G. 9. Part of an epidermal cell borderin g t he epider­ mal cleft (C) 75 min after the injection of epidermolysin . that, unlike most enzymes, epidermolysin binds The nucleus (N) and tonofilaments (TF) are intact. fir~ly to t h ~ cell surface of ke:atinocytes. Whe)) Microvilli (arrows) and half desmosomes (*l are presen t e~ldermo l ys m or bovill e albumll1 wa~ conjugateq along the free cell surface (x 34,000). With the.fluorochrome dy~ Fluorescamme and incu, bated With cyrostat sectIOns or mouse skin, Fluo, TABLE II. Effect of epidermolysin in. selec ted strain.s of rescamine- epidermolysin was seen to bind at th~ mice surface of keratinocytes [41] . Strain tested (nQ.) Treat- C3' C5' Epider- There is . a lso no evidenc~. that epidermolysit) menta molys is',' exerts a. duect CytotoxIc. eff~ct. We considereq B10/D2 Saline Norma Normal 616 whet her It could exert an md,rect cytotoxic effect new line (6) by activating serum comp!emenL If the terminal 810/D2 CoF Low Low 6/6 components C5- 9 were activated 111 the vi cinity Of new line (6) kerat inocytes,. m e mbra~ e da mage wi th d ec re ase~ 810/0 2 Saline Normal None 4/4 cellu.lar adhes lv e r~e ss might em; ~e. Th~ ~ poss ibili t~. old line (4) was Il1vestlgated 111 newborn mI ce defi CIent in C~ 810/D2 CoF Low None 4/4 (BIO/D2 "old" ) or intact in C5 (BIO/D2 " new") I old line (4\ addition, groups of t h ~se mice were treated ~ i t~ Nude None NO" NO 2/2 the decomplementll1g factor 111 co bra venom (CoF) (athym ic) (12) which depletes C3 and C5 via the alternative (pro perdin) pathway of cOl~np l ement activation. Con)' " Saline or cobra venom factor (Cordis) 1 U/gm in ­ plement c~mponents d e pe~ld e n t upon C5, or C3 h; jected intraperitoneall y 24' nr prior to epidermolysin. the case of CoF -treated anImals, were not required " Mouse C3 and C5 determined by double diffusion in for epidermolysin action (Tab. II). agar. A third possible mechanism of epidermolysi\ "Number positive/number tested. action is its effec t on epidermal Iysosomes, which "NO, not done. might lead to the release of constituents that 1il'e injurious to the cell membrane. thenium red were not removed by epidermolysin, A purely biophysical effect of epidermolysi an indication that cleavage cannot be attributed to could alter. e \ ec tro st~t i c c h ~ l:ge ~ between cells an ~ t he removal of stainable surface acid mucopo\ysac­ lead to theIr separatlOn by fnct lonal forces. Such & charides. ?iscrete cleavage plane be~eat h the g r~ n. ul a r l ay~ \ IS not seen, lor example, WIth chaotroplc Ions ofth MECHANISM OF' ACTIO N OF EPIDERMOLYSIN Hofmeister series which exert their effect at th' T o learn more about the norma l process of junction of the dermis a nd epidermis [66 ]. Nevel: keratinocyte adhesiveness and to determine how the less , this hypothesis deserves to be ex plored. epidermolysin so dramatically disrupts this normal Despite mounting ev id ence for 01' against the~e function of epidermis, we initiated a study or its four hypotheses, the mechanism of action of elll, mechanism of action. We considered four major dermolysin remains elusive. We are, however, con hypotheses: (a) epidermolysin exerts an enzymatic fident that future investigations will not only p activity specific for an epidermal substrate which duce data on the biochemical basis for e pi d ~1 is requ ired for normal cell adherence; (b) epider­ molysin action but lead to improved method ~ molysin has a cytotoxic effe ct by which it disrupts studying other bullous diseases and to a b ett ~' cellular fun ction directly or activates a humoral understanding of t he normal epidermis. mechanis m which leads to cytolysis by an indirect action (c) epidermolysin labilizes lysosomal en­ zymes and thus leads to autolysis; (d) epider­ We are indebted to Mrs. Cinda Lobitz. Mrs. Judi! \ Pedersen, Ms. J oyce Beemll n. Il nd Ms. Yvette Fruti i!{ mo lysin exerts a biophysical effe ct which disrupts for skillful technical assistance. Dr. Marvin Rittenb", normal cytoadherence. kindly supplied the antiserum to mouse C5. 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