Epidermolysis Bullosa Dystrophica-Recessive: a Possible Role of Anchoring Fibrils in the Pathogenesis

REPORTS

EPIDERMOLYSIS BULLOSA DYSTROPHICA-RECESSIVE: A POSSIBLE ROLE OF ANCHORING FIBRILS IN THE PATHOGENESIS

ROBERT A. BRIGGA MA N, M.D., AND CLA YTON E. WHE ELER, JR. , M.D. Department of Dermatology, The University of North Carolina Schoo l of Medicine, Chapel Hill, North Caro lina

The purpose of t his study was to define the ultrastructura l defects and pathogenesis of e pidermolysis bullosa dystrophica- recessive (EBD- R ). The only consisten t ultrastructural alteration found in EBD- R was an a bsence of anchoring fibrils. In many specimens of n on blistered , nontraumatized E BD- R skin, absence of anchoring fibrils was t he only ultrastructura l a bnormali ty observed . The possibility that lack of anchoring fibrils was a secondary change result ing from previous bli stering and scarring was eliminated by our o bservation that anchoring fibrils were consistently absent in the never previously blistered s kin of two newborns wi th E BD- R. In experimentally traumatized skin, the epidermis and d ermis separated in the region of the epidermal- derma l junction normall y occupied by a n choring fibrils. Basal la mina and derma l microfibril bundles appeared to be norma l. Using recom binant grafts, we demonstrated t hat a nchoring fibrils were not fo rmed by EBD- R d ermis when combined wi th E BD- R epidermis or normal epidermis. Anchoring fibrils were formed when normal dermis was co mbined wi th norma l and EBD- R epidermis. These s tudies indicate that the defect in EBD- R resides in the dermis and that the defect may be associated with impa ired formation of anchoring fibrils.

Epid ermolysiS bullosa dystrophica- recessive, pharynx, esophagus, and anal canal are involved. termed epidermolyse bullouse polydyspl asique by In t hese sites, scarring leads to mouth and tongue Touraine [1 ], congenita l, ge nera li zed , subletha l deformities and fr equent esophageal stenosis. Im epidermolysis bullosa dystrophica by Gedde-Da hl pa i'red growth, anemia, fr equent cutaneous infec [2 ] and d ermolytic bullous dermatosis- recessive by tion, and den tal a bnorma li ties are common . Pear son [3], is an autosomal recessive disease T he purpose of t hi s paper is to define the which begin s at birth or soon thereafter and pat hologic defect in epidermolysis bullosa dys pursu es a n unrelenting course throughout li fe , trophica-recessive (E BD-R) and to co nsider its occasio n a lly terminating fatall y at an earl y age. pathogenesis. Pearson [4 ] found a consistent ult ra The clinical defect is a fa ilure of epiderma l- derma l structural separation in the dermis just beneath the adherence resul ting in blisters and subsequent basal lamina (basemen t membrane) . In addition, erosio n s which involve t he entire skin surface wi t h he poin ted ou t a marked reduction of fine fibers a predilection for sites of frequent tra uma, part icu just beneath the basal lamina (subsequently iden larly t h e neck, shoulders, elbows, knees, buttocks, tifi ed as anchoring fibrils) and varying degrees of hands, a nd fee t . Dystrophic scarring, a ha llmark of co llagen degradation. Subsequent reports on the the disease, is most severe in sites of recurrent ultrastructura l abnorma li ty in EBD- R have pre blister ing a nd leads to a vari ety of functional sen ted a more confused pi cture. Vogel and Schny impair m en ts including mitten-like epiderma l en de l' [5,6] observed separation beneath the basal casemen ts of t he hands and feet, disfi gurement, la mina in so me patients and in the in termembra and jo int contractures. Ha ir and nails are co m- ' nous space in others. Kobayasi [7] fo und blisters monly lost. Mucous membranes of the ora l cavi ty, located in the in termembranous space in one patien t wi th recessive epidermolysis bullosa . From Manuscript received Dece mber 26, 1974; in rev ised the clinical descript ions in these patien ts, it is fo rm March 5, 1975 ; accepted for publication March 10, diffi cul t to obtain a cl ear view of the diseases being 1975. studied . It is possible that severa l types of epider T hi s stud y was supported by Resea rch Grant 2 ROl AM 10546 and Derm atology Training Grant 5 ROl AM molysis bullosa we re lumped together as EBD- R. 5298 from the National Institutes of Health, and Grant It is a lso possible that EBD- R is actuall y a group of RR 46 fr om the General Clinical Research Centers different diseases whi ch have a similar clinical Branch of the Division of Resea rch Resour ces, U. S. appearance. Public Health Service. Reprint requests to : Dr. R. A. Bri gga man, Department In the presen t study, we sha ll presen t evidence of Dermatology, The Uni ve rsity of North Caro lina Sc hoo l t hat the most consistent and often the only ul tra of Medicine, Chapel Hill , North Carolina 275 14. structUl" a l defect noted in EBD- R is an absence of 203 204 BRiGGAMAN AND WHEELEIl Vol. 65, NO. 2 anchoring fibr il s. Using a system in which anchor was chosen for biopsy. These were away from any exist' ing fibri ls norma ll y re-form in recombin a nt grafts blisters or obvious scarring, although it should be no~n~ grown for a period of time on the chorioallantoic t hat t he skin of older patients had a crinkled, atroPh~c membrane of embryonated chicken eggs [8], we appea.!·a nce even In these areas. In two cases (patients Iii sha ll present evidence t hat formation of these a nd EO), biopSies were obtatned soon al ter birth f completely normal-appearing skin which had never structures may be impaired in EBD- R a nd that ;Olll blI' stere d prevIo . us I y. B"l opsles were d one under 10een I the defect resides in the EBD- R dermis. lidocain e anesthesia using a scalpel to obta in a t~a MATERIALS AND METHODS sp li~ - thickness skin specimen. Special care was taken :~ aVOid traumatizing t he blOP.sy sites. Sen a l biopsies wer~ Patient, done over a 3-year penod 111 patient Bl a nd a 2-v The patients comprising this study constitute a clini period inpatients Sl, S2, H1,H 2, and H3. Biopsies ~:~: call y homogenous group which conforms to the clinical also obtalne? trom multiple Sites, II1ciudlng t high , abdo_ features of EBD- R described above. All patients have a men, back, lor~a rm , and upper a r~l to determine whether generalized bullous disorder resulting in severe dys pathologiC dll/erences cou ld be found III different an trophic scarring. Famili al cases consistent with an auto tomic sites. a- somal recessive mode of inheritance make up the bulk of M inimal frictional trauma. An area of skin free r the patient group. obvious blisters and scarring was subjected to minirn 0 Familial cases. Family B is of Turkish extraction with trauma insufficient to produce an overt blister a 1 2 of 5 siblings involv ed. No other family members are epidermal- dermal separation. Five to 10 strokes with Or affected. The parents are second co usins. Patient Bl is an ordinary pen~iJ eraser with li ght application of pr essu~: 18-year-old male of small stature who has mitten-li ke were used, al ter whlGh the area was blopsied as previ enclosures of the hands and feet and severe esophageal ously described. . stenosis. Patient B2 is a 7-year-old male who has simi lar M echanically separated epidermis and dermis p but milder involvement. lions of split-thickness skin specimens obtained ~s ~r. Family S is a Negro family in which 2 of 5 siblings are scribed above were placed in tissue culture mediu e· involved. No other fami ly members are affected. Patient (Medium 199 with 10% fetal calf serum) and imm ed i ate;~ Sl is a 13-year-old male of small stature with severe transported to t he laboratory (lapsed time less t han 5 mitten-like enclosures of the hands and feet, Ilexure min) .. Under a. dissecting microscope, epidermis and contractures of the elbows and knees, and esophageal dermiS were easd y separated USIng Jeweler's forceps a d stenosis. Patient S2 is an ll-year-old female of small di secting needles to tease the components apart. Th~ stature manifesting severe scarring about the face, scar specimens will be referred to hereafter as " mecha nicall , ring alopecia of the scalp, scarring of the conjunctivae separated" epidermis and dermis. ~ with ectropion of the lower lids, and esophageal stenosis. Family H is a Caucasian fa mily in which 3 of 4 siblings Recombination Studies Usi /t{{ Normal and Abnormal are involved. Other members of the family are normal. Epidermis and Dermis Patient HI is a 21-year- old male with very severe Preparation of skin component,. An area of skin free or dystrophic scarring on his arms, legs, and face. Patient obV IOUS bltsters or scarrIng was chosen on patients with H2 is a 20-year-old j'')male with esophageal stenosis and EBD-R. The site was prepared with t incture of iodine severe mitten-like enclosures of both hands and feet. and. alcohol and then anesthetized with lidocaine con. Patient H3 is a 19-year-old male who has similar al taInIng ep\llephnne. A splt t-thlckness sheet of skin mea. though slightly milder involvement than hi s siblings. He suring approx imately 1.5 by 3- 6 em was removed using a also has esophageal stenosis. sterile Castroviejo dermatome set to cut at 0.4 mm. The Sporadic cases. Patient J C is a 20-year-old Caucasian gratt sites were dressed With sterde petrolatum-impreg. male with severe dystrophic scarring of the extremities, nated ga.uze and wrapped with flexible gauze dressing. In mitten-like enclosures of the hands and feet, scarring of our patients, healIng was rapid and comparable t the conjunctivae with ectropion of the lower lids, severe normal co ntrol subjects. No instance of secondary infec~ fl exural contractures of the elbows and knees, and lI on or other compli cation at the graft sites was noted esophageal stenosis. The spli t-t.hickness sheet of skin was placed in M ediun; Patient IH was a I -month-old Caucasian female with 199 With 10% fetal calf serum added and transported to extensive blisters on t he arms, legs, back, chest, and t he laboratory . I ~o l ated epidermiS and dermis \Ve~ groin . Oral blisters and erosions were also present. She prepared as descnbed above by mechal1lcally stripping was observed from birth. Although extensive areas of the epidermis a nd dermis apart. Separated sheets of blisters and erosions were noted, the patient also had epIdermiS and dermiS were cut Into pieces approximatel\' many areas whi ch were never involved with blisters and 8 mm in diameter for grafting. . erosions. This patient died out of the hospital at 6 months Split-thickness sheets of skin were obtained from the of age of unknown causes. No autopsy was obtained. thigh or buttock of norm al healthy subjects in a manner Patient EO is a newborn Caucasian male foll owed identical to that described above. The skin was placed in since birth. An extensive area of erosion was noted at 0.4% trypsin solution (Difco 1:250) at 4°C. for approxi. birth on the right leg. Subsequently, blisters and erosions mately 1 hr after which the skin was transferred to Cold have occurred in the oral cavity, face, arms, chest, left Medium 199 with 30% fe tal calf serum to inactivate the leg, and abdomen where subjected to minimal trauma. enzyme, and the epidermis and dermis were separated. Large areas of normal-appearing skin which had not been Preparation and cultivation of recombinant graft . previously blistered were also present. Parents are first Recombin ant grafts were co nstructed as indicated in cousins. Figure 1 using all possible recombinations of normal and Source of Skin for Ultrastructural Studies in EBD- R abnormal epidermis and dermis as follows: (1) normal Patients epidermis with normal dermis, (2) EBD- R epidermis with EBD- R dermis, (3) EBD- R epidermis with normal Nontraumatized, nonblistered epidermolysis bullooa dermis, and (4) normal epidermis with EBD- R dermis. skin. An area of skin, usually on the abdomen or thigh, The dermal co mponent of each recombinant was inverted Au.g. 1975 EPIDERMOLYS IS BULLOSA DYSTROPH ICA-RECESSIVE 205

MAL SKIN ~ TRYPSI N ~ E a n choring filaments [11,12]. The basal la mina is a NOR ~~--;;-;C-~ D con t inuous e lectron -opaque layer approximately EPIDERM OLYSIS ",~"",. ..." , -..,,,,.".,,,,•.,., ....,..,.. .•~ • •,., . ~ ~ E 30 to 35 n anom eters (nm) in t hickness. In t h e BULLOSA Seporor 1on ~~ 0 d ermis s ubjacen t to the basal la mina, three differ en t fibrillar compon ents are seen: (1) coll agen E ~ EPIDERMIS fibers, (2) a n choring fibrils (specia l fibrils of t h e o ~ DERMIS(INVERT ED) dennis) a nd (3) d e rmal microfibril bundles (Fig. CAM RECO MBINANT 2). Anchoring fibrils have a c har acteristic m orphol-

F IG. 1. Diagra m showing procedures of epidermal- der mal separation and preparation ot recomblllants . . f m i ts norma l position in order to present a freshl y cut ;~rma l surface at the. new epidermal- derm al j~n ct i o n . Inversion of t he dermis ehmll1a(;ed the possibility t hat dermal structures present at the former epiderma l- der- a.l junction could be carned over II1 to the new m idermal- dermal in terface, thereby compli cating inter· epeta t ion of formation of new structures. The various prcom binants were then grafted to the chorioall antoic re em brane (CAM) of embryonated chi cken eggs as de· m ribed previously [8,9 J. After periods of cultivation on ~~e CAM, t he recombinants \~e r e harvested and e~am . ed by ti ght and electron microscopy. A total of 119 Incom binants were done using sk in components from 5 r~bjects with EBD- R (patients Bl, S I, S2, H 2, and H3). ~f these, 14 were recombin ants of EBD- R epidermis and EBD- R dermis. Forty·six were recombinants of EBD- R pidermis and norma l dermis, 69 were recombinants of e rmal epidermis and EBD- R dermis, and 30 were ~~ntrol recombinants co mposed of norm al epidermis and derm is. Only the recombmant grafts whi ch were Judged by light micro ~co Pl c cri teri a [9J to be we ll mainta ined after periods of growth on the C~ M were I~clud ed .111 the wdy. This group of 88 recombll1ant grafts constituted ~he material for t he recombination position of the study. Most r ecombinants were examined after 8 or 9 days' growth on t~e CAM . In three expe~ im e n ts, grafts were harvested after 3, 5, 7, and 9 days cul tivatIOn on the CAM in order to study t he sequence of formation of structureS at the epidermal- derm al in terface.

Electron Microscopy Specimens for electron mi croscopic examination were either fixed directly in 1% phosphate-buffered osmium or prefixed in 4% phosphate-buffered paraform a ld ehyde and postfixed in osmium. All specim ens were em bedded in Epan, cut wi t h a d ia mond knife, and examined in a JEM T-7 electron microscope. RESULTS

Epidermal- Dermal Junction Ln NormaL Human Skin The epidermal- d ermal junction in n orma l a dult human s kin before any manipulation s or cult iva-' tion is s h own in Figure 2 (10]. T he plasma m e m brane of an epidermal basal cell forms t he m ore superficial portion of t he junction . Along t he plasma membra n e a re seen focal e lectron-den se thickenings termed hemidesm osom es into whic h tonofilaments of t he basal cell converge. On t h e dermal s ide of t he plasma m e mbrane is the e lec tron-lucent intermembranous space separating t h e FIG. 2. Epidermal- dermal junction of normal human plasma membra n e from t he b asal la mina. Fine skin . Anchoring fibrils (AF) are seen beneath the basal lamina (BL). A dermal microfibril bundle (DMB) is seen fi laments can be seen traversing t h e intermembra extending from the basal lamina deep into the dermis. noUS space, particula rly in t h e area beneath he m i Coll agen fibers (e) (x 39,000). Calibration bar 1.0 desmosomes. These fila m en ts have been called micrometer. 206 BIHGGAMAN AND WHEELER Vol. 65, NO.2 ogy consisting of an asymmetri c, transverse No differences were noted in biopsies of n on. banded cen tra l area with fil a mentous or bra nched t ra umatized , nonblistered skin from patients with portions at eit her end extending superficia ll y to t he EBD- R who were biopsied seri a ll y over a period of basal lam ina a nd deep into t he dermis 113,14]. 3 years (B1) a nd 2 years (Sl, S2, H I , H2, a nd H 3). Aggregates or parallel-a rranged bundles of fin e All biopsies howed t he features described a bove fibrils measuring approx imately 10 nm in dia meter indicating t hat t he nature of t he pathologic d efect (microfibri ls) a re attached at one end directly to does not vary with time in a ma nner which can be t he undersurface of the basal la mina. These bun demonstrated on ultrastructura l examination . dles m ay course relatively long distances in to t he Skin fr om a variety of different a natomic region deeper dermis. These fibrils are simila r to the was examined to determine whether differences microfibrils found at t he periphery of elastic fibers could be found in t he pat hologic defect in different 115]. Kobayasi [16] a nd Rodrigo a nd Pereira [17J regions. All regions which we examined (thigh have suggested t hat t hey are part of t he elastic abdomen , back, forearm , upper arm ) showed simi: tissue system of skin. There is no accepted term for lar ultrastructura l a lterations conform ing to the t hem. In t his paper the term, dermal microfibril description above. bundles, wi ll be used. Previously unblistered EBD- R shin trom new. borns. Specim ens of skin from 2 newborn infants Studies on the Ultrastructural Pathologic Defect in with EBD- R were exam ined. Areas of skin which EBD- R had never been bli stered previously were examined Nontraumatized, nonblistered EBD- R skin. to look for primary pathologic a lterations. This The epidermis a nd dermis were attached in most would eliminate possible secondary changes which specimens of skin fr om nontra umatized, nonblis might have resul ted from previous bli stering and tered EBD- R skin (Fig. 3). The most striking a nd subsequen t healing .a nd scarring. Examination of consisten t abnorma li ty noted was a n a bsence of t he skin showed a pi cture identical to t hat de. a nchoring fibrils. Rarely an ill -defined fibril was scribed above. Anchoring fibrils we re absen t subja. seen in t he usua l position of a nchoring fibrils cent to t he basal la mina. Occasionall y incipieni subjacent to t he basal lamina . T hese lacked t he epiderma l- dermal separation was seen in a plan ~ cha racteri stic cen tra l cross- ba nding a nd peripheral just below t he basal la mina. fa nlike fibrilla r a rray. We have never seen normal ExperimentaLLy induced blistering an d a nchoring fibrils in the skin of patients wit h epidermal-dermal separation. Minimal frictional EBD- R. The basal lamina a nd other structures trauma: Specimens of skin purposefull y exposed to comprising the epiderma l- derma l junction superfi mild fri ctiona l t ra uma insuffi cient to produce cia l to the basal la mina a ppeared to be norm a l. clinicall y obvious bli stering in vivo commonly Basal la mina was well preserved as a n essentia ll y showed areas of frank and incipient epidermal. continuous e lectron-dense la mina of fa irly uniform dermal separation . As indicated previously, sepa. t hi ckness (Fig. 4). In t he a rea of hemidesmosomes, ration occurs in a pla ne immediately subjacent to a nchoring filaments were seen t raversing t he elec the basal lamina. Figure 6 shows separation of the tron-Iu qmt in terme mbra nous space. Dermal con epid ermis a nd derm is wit h t he basal la mina and nective tissue fibers were ge nera ll y in tact. Collagen attached dermal material fo rming the rooC of t he fibers of norma l diameter a nd cha racteristic perio blister cavity, and in tact coll agen fibers; microfi. dicity on transverse section were regula rl y seen in bril bundles. a nd other dermal elements forminIJ this .naterial. S ince most of the coll agen fibers the base. 6 were cut on oblique section, t he possibility that M echanicall y se pa rated epidermis a nd dermi . so me of t he fibers were a bnorma l could not be S kin specim ens removed fr om patients wi th eliminated. However, significant areas of col EBD- R were separated with ease into isolated lagenolysis were not seen in speci mens of non b li s epid erma l a nd derma l components. All patients tered , nontraumatized skin . Derma l microfibril demonstrated thi s finding usin g skin fr om a variety bundles were seen extending from t he basal la mina of different skin regions. Patients biopsied seriall;. deep in to t he dermis. Small aggregates and bun over a period of time up to 3 years persistentl;. dles of microfibrils and occasiona l individua l mi showed this defect. Although 5 to 10 min usuall;. crofibrils were noted beneath the basal lamina elapsed fr om actual biopsy to sepa ration. thi: (Fig. 3) . period was not requ ired sin ce severa l specimens In some specimens, areas of incIpIent were separated immediately after remova l froll! epiderma l- derma l sepa ration were evident, proba the patient. One can draw an analogy between thi bly resulting from unavoidable trauma sustained in vitro separation a nd t he in vivo Nikolsky si gl1~ during t he biopsy procedure (Fig. 5). Sepa ration We believe t hat these observations a re of impor. a lways occurred in t he pla ne just below t he basal tance since they demonstrate that the patholocrir la mina in t he position normally occupied by an abnormality in EBD- R, the fa ilure of the epider. choring fibr ils. Frequently, a variable a mount of m is a nd derm is to adhere properl y to one another, dermal materi a l rema ined attached to the under is present in appa rently normal skin of the, surface of t he basal la mina, but other times, patients. We will stress this poin t again later. separa tion left t he basal la mina essentia ll y bare. M oreover, t he abnorma li ty occurs in a ll skin reo Aug. 1975 EPIDEHMOLYSIS BULLOSA DYSTHOPHICA- HECESS IVE 207

FIG. 3 . Epidermolysis bullosa dystrophica. Nonblistered, non traumatized area. Basal lamina (BL) is intact. Anchoring fibrils are absent. Dermal microfibril bundle (DMB) a nd individual microfibrils (M) are seen beneath the basal lam ina. Coll agen fibers (C) on longitudinal and cross-section appear normal (x 31,500). Calibration bar 1.0 micrometer. gions and is persistent wi thout significant varI a consistently in a plane just under the basal lamina. tions over long periods of time. As observed in Figure 7, the undersurface of Ultrastructura l examination of mechanically mechanically 'separated epidermis co nsists of basal separa ted epiderm is and derm is offers t he opportu lamina, sometimes bare or sometimes with a thin nity to determine t he level at which the separation layer of attached dermal material. The co mposi occurred. In a ll specimens, separa tion occurred tion of dermal material could not be clearly de- 208 BRIGGAMAN AND WHEELER Vo l. 65, No. 2

losa were combined wit h dennis from t he same patient. At the time of grafting, t he epiderma l- der_ mal interface of t hese recombinants consisted of t he intact basal lamina (Fig. 8) apposed to the inverted surface of t he dermis. Because basal lamina was present in t hese recombinants prior to cultivation, no commen t on basal lamina forma tion can be made. Anchoring fibrils were not

FIG. 4. Epidermolysis bullosa dystrophi ca. Intact epiderm al- dermal junction from a nonblistered , non traumatized area. Anchoring fibrils are absent from their normal location beneath the basal lamina (BL). Co llagen fibers (C) are present (x 32,000) . Ca libration bar 0.5 micrometer.

FIG . 5. Epidermolysis bu ll osa dystrophica. Incipient epid ermal- dermal separation (SEP) has occurred in the plane beneath the basal lamina (BL) normall y occupied by anchoring fibrils. Co ll agen fibers (C) (x 20,000). Calibration bar 1.0 micrometer. . F IG . 6. Epidermolys is bullosa d ~st r op hi ca. Minimal fnctlOnal traum a. Complete separatIO n of epidermis and dermis has occu rred form in g a blister cavity (CA V) fined . All stru ctures superficial to the basal lamina Basal lam in a and attached dermal material (D) form th~ roof of the cavity, and in tact collage n fibers (Cl. dermal appeared norm a l. microfibril bundle (DM), and ~ther dermal elements form the base (x 22,500). CalibratIOn bar 1.0 micrometer. Recombination Studies Using Normal and EBD- R Epidermis and Dermis Recombinants co mposed of normal epidermis and dermis. Basal lamina and anchoring fibrils re-formed in recom binants of norma l viable epider mis and d ermis as previously described [8 ). Basal lamina was seen first at about day 3 of cultivation . It appeared first under hemidesmosomes, and extended from t his site to form a con t inuous electron-dense lamina in many areas by days 7 to 9 of cultivation. Anchoring fibrils were first noted underlying basal lamina, particularly under or near hemidesmosomes at 5 to 7 days. In some 7 a reas of the recombinants, anchoring fibrils in creased in number during cultivation to approach the number found in norma l skin. F IG. 7. Epidermolys is bullosa dystrophi ca. Mechani. Recombinants co mposed of abnormal epidermis ca lly separated epidermis. The undersurface consists of intact basal lamina. All structures superficial to the basal and abnormal dermis. In these recom binants, lamin a appear norm al (x 22,500). Ca libration bar 1.0 epidermis from patients with epidermolysis bul- mi crometer. Aug. 1975 EPIDERMOLYS IS BULLOSA DYSTROPHICA - RECESSIVE 209 mal epidermis isolated by cold trypsinization was co mbined wi th dermis from a patien t wi th EBD- R T he plasma membrane of the epidermal basal cell was a pposed to the inverted dermis. No basal la mina or an choring fibrils were present prior to cul tivation. During cul tivation, basal lamina re formed to produce con tinuous electron-dense lam ina in m any areas (Fig. 9). An choring fibrils were not seen in these recombinations. Recombinants co m posed of EBD- R epidermis and normal dermis. In t hese recombinants, t he new epiderma l- derma l interface consisted of the basal la mina of the EBD- R epidermis a pposed to t he inverted surface of the norma l dermis. Aga in , no commen t can be made regarding the formation of basal la mina during cul tivation since basal la mina was present prior to cultivation. Anchoring fibrils were absent in the recombinants at the time of grafting. After periods of cul tivation, anchoring fibrils were fo und underlying the basal lamina (Fig. 10). T hese an choring fibrils appeared to attach at one end to the undersurface of the basal la mina, exhibited a cen tral cross- banded region and a deep fanlike fibrillar array extending into the dermis. They appeared first at days 5 to 7 of cult ivation and increased in number during cul ti vation. T he number of anchoring fibrils fo und after day 9 of cul tivation in these recombinants was less t han in recombinants composed of norma l epider mis and dermis. Alt hough fe wer in number, there is no doubt that anchoring fibrils of characteri stic m orphology re-formed in recombinants of EBD- R epiderm is and norma l derm is. T herefore, normal dermis can correct t he disease abnormali ty (i.e., a bsence of anchoring fibrils) when cul tured in com bination wi th abnorma l epidermis. Ultrastruc tural evidence is thus. provided that the site of this disease resides in t he derma l co mponent.

FIG. 8. Recombinant composed of epidermolysis bul· DI SCUSSION losa e pidermis and epidermolys is bullosa dermis. An· cho r i n ~ fibrils a re a bsent. Basal .lamina and a ll more Role of Anchoring Fibrils in the Pathogenesis of superficial structures 01 t he Junction a ppear norma l (x 19 000) . Calibration bar 1.0 micrometer. EBD- R 'FIC. 9. Recombinant co mposed of norma l epid ermis We postulate that an absence of anchoring fibrils and e pidermolysis bullosa dermis. Basal lamina reo is t he primary stru ctura l defect in EBD- R leading for med during cul t ivation. Anchoring fibrils are a bsent from t h eir normal posit ion beneath t he basal lamina ( x to disruption of the structura l in tegri ty of the 25 000). Calibration bar 0.5 micrometer. epiderma l- d erma l junction (epiderma l- derma l FIG . 10. Recombinant co mposed of epidermolysis bul· separation) a nd subsequent bli ster formation. In los a epidermis and norma l dermis. Anchoring fibrils (AF ) m any specimens of non blistered , non traumatized re-for med during cul t ivation on the CAM ( x 35,000). ' Cali b r ation bar 0.5 micrometer. EBD- R skin, a bsence of anchoring fibrils was the only ul trastructura l abnorma li ty observed . T he possibility that lack of anchoring fibrils was a present in these recom bin ants before or at the tim e secondary cha nge resul ting from previous blister of grafting a nd none was noted during peri ods of ing and scarring was eliminated by our observation cultivation up to 9 days. The a ppeara nce of the that a nchoring fibrils were consistent ly absent in epid erm al-dermal junction in these recombinants the newel' previously blistered skin of 2 newborns duplicated the appearance of t he natura l disease with EBD- R. In ex perimentall y traumatized skin, state, i.e., the anchoring fibrils were a bsent. Basal t he epiderm is and derm is separa ted in the region of lamin a and a ll more superfi cial structures of the the epidermal- dermal junction norm a ll y occupied junction a ppeared norm a l. by anchoring fibrils. Us ing recom binants of norm a l Recombinan ts co mposed of normaL epidermis and EBD- R epid ermis and dermis cultured on the and EBD- R dermis. In t hese recombinants, nor- CAM, we demonstrated that a nchoring fibrils were 210 BRIGGAMA N AND WHEELE R Vo l. 65, N o.2 not formed by EBD- R dermis when combined with demonstrated destruction of collagen fibers in t h e EBD- R epidermis or normal epidermis. An choring skin of epidermolysis bullosa dystrophica. Some fibrils were formed when norm a l dermis was com times, coll agenolysis was extensive, leaving only bined wi th normal and EBD- R epidermis. The amorphous debris in the space under the basem en t only consisten t a lteration fo und in our experiments membrane. Following this lead, E isen [20 J showed was an absence of anchoring fibrils. We, t herefore, an elevation of co ll agenase activity that was high regard the resul ts of this work as evidence t hat t he est in the blistered areas but a lso elevated in defect in EBD- R is so mehow associated wi t h an apparen tly norma l, nonbJistered skin . Lazarus [21) abnormali ty of anchoring fibrils. confirmed this observation of elevated collagen ase We agree wi th Pearson's finding of collagen activi ty in the blistered skin bu t found normal degradation in EBD- R [3, 4). Al though co ll agen levels of activity in nonblistered skin . He suggested degrad ation may be very extensive, particularly in that the elevated co ll agenase activity in E BD- R or near preexisting blisters, epidermal- dermal sep might be a secondary event because of t he norma l aration can occur in EBD- R in t he absence of activity in non blistered skin. Coll agenase activity a pparent coll agen degradation (Figs . 3, 4) . would be expected to be elevated in nonblistered Dermal mi crofibril bundles a ppear normal in skin if co ll agenase played a prima ry role in t h e EBD- R (Figs. 2, 3). Alt hough no attempt was pathoge nesis. Recently, Bauer, Gedde-Dahl, an d m ade to quan titate t heir numbers, they seem to be E isen [22 ] measured human skin co ll agenase u ti encountered wi t h norma l fr equency in EBD- R li zing a radioimmunoassay technique in patien ts skin. T he nature of these fibrils is controversial. An with EBD- R. Tissue levels of human skin collagen _ association of dermal microfibril bundles wi t h der ase ~e r e elevated . in both a ffected and clinically mal elastic system has been proposed [1 6,17) and unaff ected skll1 of these patients, mdlcating t h a t is interesting in light of the previous suggestion t he enzy me could be 'playin g a primary role in t he that EBD-R is an abnormali ty of elastic fib ers pathogenesis of the disease. [18, 19 ). E lastic fibers were thought to be decreased If human skin coll agenase plays a prima ry role in on light microscopic examination of E BD- R skin the pathogenesis of EBD- R, the enzyme should be [1 8 J. expected to produce damage to the epiderma l- d er_ Basal la mina and the more superfi cia l stru ctures mal junction id entical to t he observed ul trastruc_ co mposin g the epidermal- dermal junction are re tural pathology of EBD- R, namely, preferen tia l markabl y unaffected in E BD- R. Norm al-appear destruction of anchoring fibrils, varyin g degrees of ing basal la mina is consistent ly observed in t he co ll agen degeneration, and complete sparing of t he roof of EBD- R blisters and mechanicall y se parated basal lamina and dermal mi crofibril bundles. U n E BD- R skin (Fig. 6) . T he plasma mem brane of fo rtunately, it has not bee n possible to in vestigate epidermal basal cell s and hemidesmosomes are the effects of human skin coll agenase on stru ctu re also una ffected . T he in termem branous space and at the epidermal-dermal junction. Studies of t he the fin e fil a ments (anchoring fil a ments) whi ch effect of bacteri al collagenase on the epiderma l_ cross the space, particul arl y subjacent to hem id es dermal junction do not correlate well wi th t h e mosomes; are likewise normal. ultrastructura l pathology of E BO- R. K ahl a n d The abse nce of anchoring fibrils in EBD- R Pearson [23 ) found t hat bacteri al coll agenase yields important information a bout the bi ologic wh en injected in to the skin of rabbits, produced function of t hese fibril s. T he fa ilure of collagen degeneration and loss of anchoring fibrils epiderm al- dermal adherence and the severe blis bu t also destruction of the basal lamina whi ch i ~ tering seen in EBD- R patients is impressive evi well preserved in EBD- R, even in areas of severe dence of the importance of anchoring fibrils in t he coll agen degeneration. attachment of epidermis and dermis. T he term , We have observed that epidermal- dermal anchorin g fibril, appears to be a ve ry appropriate se paration may be seen in EBD- R in t he absence of one in view of our findings. overt co ll agen degeneration wi t h t he only stru c_ tura l defect bein g a lack of anchoring fibrils. It is possible and even likely that co ll agenase or other Pathogenic M echanii;ms: Destruction Vi; Impaired proteases may be active in t he dermis without Formation overt co ll agenolysis seen on electron microscopy. Two possible mechani sms have bee n proposed to Nevertheless, fu ture stud ies in thi s area will have ex pl ain the pathoge nesis of EBD- R: (1 ) norma ll y to account for t he preferent ial destruction of an for med structures whi ch are necessary for the choring fibrils compared to coll agen. attachment of epidermis and dermis are da maged An a ltern ative pathogeni c mechani sm is t hat or destroyed, or (2) t hese structures are not formed EBD- R may resul t fr om impa ired form ation of or, if formed, are defective so that separation of structures at t he epiderma l- d erma l junction. epidermis and dermis resul ts fr om either their Usin g a system in which anchoring fibrils normally abse nce or improper fun ction. re- form in recombinant gra fts grown on the chick E vidence along several lines has been presented CAM , we have presented ev idence that anchorin<1 in support of the first possibility t hat norma ll y fibrils do not re-form as expected when EBD-R formed structures required for attachmen t are dermis is present as a co mponent of the graft. V\ e destroyed by the disease process. Pearson [4] have prev iously demonstrated that anchoring fi - Aug . 1975 EPIDERMOL YS IS BULLOS A D YST RO PHIC- RECESSIVE 211 brils a re proba bly of derma l Ori gIn since their bullosa dystrophica. Dermatologica 131:81-82, forma tion is dependen t upon the derma l compo 1965 7. Kobayasi T: Dermo-epiderma l junction in the reces ne n t of the recombinant [8]. The resul ts of our sive type of epidermolysis buJlosa. An electron recom bin a tiOl~ stu?ies can be int e rpr e te ~. as evi mi croscope study. Acta Derm Ve nereol (Stockh) dence of impa u'ed (orma tIOn of anchorIng fIbrIls by 47:57- 59, 1967 EBD- R d ermis, a lthough our studies do not ex 8. Bri ggaman RA, Dalldorf FG, Wheeler CE Jr: Forma tion of ori gin of basal lamina and anchoring fibrils clude other possibilities. This is the first study in in adult human skin. J Cell BioI 51:384- 395, 1971 which the possibility of impa ired forma tion finds 9. Briggaman RA , Wheeler CE Jr: Epiderma l- dermal experimen tal support. interactions in adult human skin: role of dermis in How specific is the absence of anchoring fibrils epiderma l m a in tena nce. J Invest Dermatol 51: 454- 465, 1968 for EBD- R? Anton-Lamprecht and Schnyder re 10. Briggaman RA, Wheeler C E Jr: The epiderma l- der cen t ly reported that anchoring fibrils were absent ma l junction. J Invest Derma tol 65 :000- 000, 1975 in a p atien t wi th dominantly inheri ted epidermol 11 . Kobayasi T : An electron microscope study on the ysis bullosa dystrophica (EBD- D) [24 ]. We, too, dermo-epidermal junction. Acta Derm Venereol (Stockh) 41: 481- 490, 1961 have found that a nchoring fibrils are missing in 12. Hashimoto K, Lever W: An ultrastructural study of EBD- D (unpublished observations) . In 3 patients ce ll junctions in pemphigus vulgaris. Arch Derma fro m a large kindred of EBD- D, anchoring fibrils tol 101 :287- 298, 1970 wer e a bsent in blistered skin and in the scarred 13. P a lade G, Farquhar M: A specia l fibril of t he dermis. J Cell BioI 27:215- 224 , 1965 area s which resulted fr om previous blistering, but 14. Swanson JL, Helwig E : Special fibrils of human were present in uninvolved skin. This is different dermis. J Invest Dermatol 50:195- 199, 1968 fro m E BD- R where anchoring fibrils are a bsent 15. Greenlee T , Ross R, Hart man J: The fin e structure of even in never previously blistered skin. Anchoring elastic fi bers. J Cell Bioi 30: 59- 71, 1966 16. Kobayasi T : Electron microscopy of elastic fibers fib rils are present in other forms of epidermolysis and dermal membra ne in normal human skin. bullosa, including epidermolysis bullosa simplex Acta Derm Venereol (Stockh) 48:303- 312, 1968 and j unctional bullous epidermatosis (Berli tz's 17. 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