THE EPIDERMAL-DERMAL JUNCTION 79 the Morphologic Sequence of Events of the Re-For Mation of Hemidesmosomes (Fig

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THE EPIDERMAL- DERMAL JUNCTION

ROBERT A. BRIGGA MAN , M .D ., AN D CLAYTON E. WH EELER. JR., M .D . Department of Dermatology, The University of N orth Carolina Schoo l of M edicine, Chapel H ill , North Carolina

Ultrastructurally, the epiderma l- dermal junction is composed of four component areas: (1) the basal cell plasma membrane with its specialized attachment dev ices or hemidesmo somes, (2) an electron-lucent area, the lamina lucida, (3) the basal lamina, and (4) the sub-b asal lamina fibrous components, including anchoring fibrils, dermal microfibril bundles, and coll agen fibers. The light microscopic " basement membrane" comprises only the s ub-basal lamina fibrous zone. Other cell types, including melanocytes and M erkel cell s, are also found at the epidermal- dermal junction. Structures at the junction deri ve their origin from t he epidermis and dermis: the basal lamina is primarily of epidermal origin , t he anchoring fibrils of dermal origin . The junction serves t he following functions: (1) epidermal- dermal adherence, (2) mechanical support fo r the epidermis, and (3) a barrier to the exchange of cells and of some large molecules across the junction.

The structures situated at the junction between ing fibers" (4], " pre-elastic fibers" [5 ], and "elas the epidermis and dermis constitute an anatomic tofibrils" [6], extend perpendicularl y toward the functional unit, the epidermal- dermal junction. interface . In co mbined PAS- orcein-stained speci The purpose of this paper is to review the current mens, these fibers pass into the PAS-pos itive information on its morphology, development, for basement membrane. They stain positively with mation, composition, and function. Selected path elastic tissue stain but are more lig htly stained ologic alterations of the junction will be consid than the coarse elastic fibers of the reticul ar ered, particularly those which reveal information ,dermis. Silver (reticulum) stains reveal a zone of about its structure and function . The reader is fin e silver-positive fibers in the papillary dermis referred to several excellent earlier revi ews of this immediately under t he epidermis which is also su bject [1 - 3 ). referred to as t he light microscopic " basement membrane. " It corresponds to the P AS-positive STRUCT URE AT THE EPIDERMAL- DERMAL JUNCTION " basement membrane" in localization. By means Ligh t M icroscopy of immunoflu orescen t antibody techniques, immu prominent undulations of t he boundary zo ne noglobulins wi th specific localization to the base between t he epidermis and the dermis form the ment membrane zone can be found in t he serum dennal papillae- rete ridge pattern, whose specifi c and the skin lesions of patients with bullous fe at ures are characteristic of different regions of pemphigoid a nd the skin of patients wi th lupus t he skin surface. Special stains aid in the definition erythematosus [7]. of this zone by light mi croscopy (Fig. 1) . Periodic The prevailing view of the epidermal- dermal acid- Schiff stain demonstrates a thin, uniform, junction at the li ght microscopic level is t hat it is a nonfibriJIar area of intense staining at the feltwork of reticulum (silver-positive) fibers and epidermal- dermal interface which is sometimes probably elastic fibers embedded in a neutral called the light microscopic or PAS-posit ive "base glycoprotein (PAS-positive) matrix and containing ment membrane." In the papillary dermis, moder an tige nic materi als which in teract wi th " ba ement ately coarse elastic fibers (orcein stain) form a membrane" antibodies. felt work running roughly parallel to the in terface, Electron Microscopy but separated by a narrow distance. From this fe ltwork, fin e fibers, variously called "fin e connect- When viewed by electron mi croscopy [8- 10 ], a high level of structural organi zation which is not appreciated by light mi croscopy is seen at t he This study was supported by Research Grant 2 ROI epidermal- dermal junction. T his structure can be AM 10546 and Dermatology Training Grant 5 ROl AM divided in to four components: (1) basal cell plasma 05298 from the N ational Instit utes of H ealt h, a nd Grant RR 46 from the Genera l Clinical Resea rch Centers membrane with its special attachment devices, Branch of t he Division of Research Resources, U. S. hemidesmosomes, (2) t he lamina lucida (o r inter Public H ealth Service. membranous space), (3) the basal lamina (basal R eprint requests to: Dr. R. A. Briggama n, Depa rtment membrane, basement membrane, lamina densa, of D ermatology, School of M edicine, The Univers ity of North Carolina at Chapel Hill , Chapel Hill , North adepidermal membrane) , and (4) the sub-basal Carolina 27514. lamina fibrous elements composed of at least three 71 72 BRiGGAMAN AND WIiEELER Vol . 65, No. I

the sub-basal dense plaque into the basal laminl\ (Figs. 7, 8). These filaments were first describecl by Kobayasi (10,19] and termed "anchoring fila. ments" [1 6,20]. Randomly oriented filaments resembling anchoring filaments can be seen in the lamina lucida away from hemidesmosom es, but PAS ELASTIC RETICULUM they are much less numerous in this latter location In the area of the epidermal- d erma l junctiol; FIG. 1. Period ic acid - Schiff (PAS), elastic, and reticu lum staining properties of the epidermal- dermal junction. where adjacent epidermal basal cells meet, the lamina lucida and the intercellular space between adjacent basal cells are contiguous even though different types: anchoring fibrils, bundles of fibrils resembling microfibrils, and sin gle collagen fibers some evid ence indicates that they have d ifferent (Figs. 2- 6). staining and permeability properties. Both ru. Basal cell plasma membrane-hemidesmosomes . the.nium ~ed [1 6] and pemphigus antibody [211 The dermal surface of the basal cell plasma mem stam the Illtercellular space, but not the lamina brane is irregula rl y convoluted with interdigitating lucida. In addition, la nt hanum readily p e rme ate ~ cytoplas mic projections a nd derma l invaginations the intercellular space but not the lamina lucid ~ (Fig. 2); seldom is it nat for m ore than a short [22]. These dlfferences suggest that the two differ distance. These convolutions are not the same as in composition but what the differences are has not the dermal papill ae- rete undulations seen at the yet been determined. ligh t microscopic level. The plasma membra ne Basal lamina. The basal lamina is a continUOl! itself is approxim ately 7 to 9 nanometers (nm) electron-dense layer which has a granular amOr, thick and is composed of three asymmetrical phous appearance in Epon-embedded specimens layers: a thicker internal leaflet which abuts the but is fibrillar in Vestopal specimens. This m ay cytopl asm ; a n intermediate, more electron-lucent indicate a two-phase system, i.e., fibrillar compo. zone; and a relatively thin external leaflet (Figs. 7, nents e mbedded in an amorphous granular mate. 8). Pinocytotic vesicles occur frequently a long the ri a l. True breaks or gaps of the basal lamina are plasma membrane (Fig. 5) which is studded at comparatively rare in normal skin and must be intervals with electron-dense thickenings or he mi distinguished from areas of tangential section desmosomes (basal attachment plaques, basal which obscure the orderly laminated structures dense bodies) (Figs. 2-4). The ultrastructure of the described above. The basal lamina varies in thick, hemidesmosome has been examined repeatedly ness and density particularly beneath t he hemides, over the years [11- 17), the most recent studies mosomes where it is t hicker and more dense. Th ~ indicating that it is similar to but not identical reduplication of the basal lamina, which is com, with t he desmosome. An electron-opaque thicken m an in normal skin (Fig. 6), may result from th ~ ing, t he "attachment pl aque," approximately 20 to remodeling of the epidermal- dermal junction a 40 nm thick, is present on the cytoplasmic surface basal cells are released to migrate toward th ~ of the internal leaflet of the plasma membrane surface and from the subsequent re-formation of (Figs. 7, 8). On sections cut exactly perpendicular the basal lamina beneath another basal eel! which to the plasma membrane, tonofilaments radiate takes up residence at the same location. Since the toward the attachment plaque but actually termi basal la mina is a durable structure [23 ], the olq n ate in a relatively electron-dense area separated epidermal- d ermal junction, including the basal from the plaque by a narrow, relatively electron lamina, a nchoring fibrils, and other fibrillar ele, lucent zone. On obli que sections, the tonofilaments ments, m ay persist for some time after the forma. seem to fu se with the attachment plaque. The tion of a new junction. Partially destroyed basal external lean et of the plasma m embrane can be lamina can a lso be seen in some areas. Reduplica, seen on high resolution electron micrographs as a tion of the basal lamina is also seen beneath fine line (M line) on the outer surface of the attach melanocytes at the junction (Fig. 9). ment plaque [1 5,17 ]. Sub-basalla.mina fibrous elem ents. Three types Lamina lu cida . The lamina lucida or intermem of fibrous structures found beneath the basal branous space is a n electron-lucent zone which lamina will be discussed : (1) anchoring fibrils, (2) separates the plasma membrane from the basal microfibrils arranged in bundles, and (3) collagen lamina (Figs. 2- 8) . In most areas it is amorphous. fibers. However, adjacent to t he hemidesmoso mes, an Anchoring fibrils (special fibrils of the dermis) ' electron-dense line is frequently seen which has were first observed in amphibian skin [24] and been call ed the sub-basal dense plaque (the periph later in human skin as well as in the mucous eral density [1 8 ] and Haftplat or H line [15,17]) . membranes of the mouth, vagina, and cervix Although this layer is similar in pos ition to the [25,26 J. Their unique structure enables them to be intercellular contact layer of the regular desmo some-, it is not certain whether it is identical. Also in the region of hemidesmosomes, fine fil amen ts • Anchoring fibrils should not be confused with the anchorin g filament described earlier or with lhe anchor· t raverse the lam in a luc ida perpendicularly from ing filament bundles which will be mentioned later in the outer leaflet of the plasma membra ne th rough this secL ion. July 1975 THE EPIDERMAL- DERMAL JUNCTION 73

FIG. 2. Epidermal- derm al junction. Irregul arly convoluted in terface between epidermis (E) and dermis (D ). Anc horing fibril s (AP). Derm al mi crofibril bundle (DME) extending from basa l lami na (EL) dee p in to dermis (arrows) ( x 10,000). Ca li bration bar 1000 nm. separated from other sub-basal fibrous elements Another characteristic sub-basal lamina fibrous (Figs. 2- 4) . Their cross- banded midportion has an structure consists of bundles of fine fibrils which irregular periodicity different from any known insert directly into the basal lamina, usuall y in the form of collagen. The superficial end of the fibril region of basal cell protrusions into the dermis terminates in and meshes with the fabric of the (Figs. 2-4). These bundles course perpendicularly basal lamina; the distal end extends into the or obliquely from the epidermal- dermal junct ion dermis. Sometimes the distal ends of adjacent deep in to the dermis and sometimes can be fol anchoring fibri ls form an in terlocking meshwork in lowed for very long distances (Fig. 2). On high the dermis beneath the basal lamina (Fig. 4). magnification electron micrographs, the individual Occasionally, anchoring fibrils loop beneath coll a fibrils are approximately 8 to 11 nm in diameter gen fibers and reattach to the basal la mina a short and possess a regular beaded periodicity of 12 to 17 distance laterally (Fig. 3a). Their distribution nm on longitudinal section and a hollow tubular along the basal lamina is not uniform ; for example, profile on cross-section (Figs. 3b, 4). These features they are more numerous in the areas beneath the are shared with the microfibrils which are fo und at hemidesmosomes where t he basal lamina is more the periphery of elastic fibers [27- 29 ) and have led dense t han at other sites. to the specul ation that these bundles are an 74 BRIGGAMAN AND WHEELER Vol. 65, NO . 1 extension of the dermal elastic tissue system [19, of them, it will be used for the time being in this 30 J. Although to our knowledge no one has ob paper. served the connection of these microfibril bundles Coll agen fibers with a typical coll agen fiber to the typical elastic fibers, they may be analogous periodicity are the third fibrous component in the to the perpendicularly arranged brush work of sub-basal lamina location. They are randomly fibers observed on orcein-stained skin viewed by oriented, single fibers, but unlike the co llagen light microscopy. Similar fibril bundles have bee n fibers of the deeper dermis, are not arranged in observed in embryonic chick skin where they have bundles (Figs . 2-4). Neither do they attach directly been call ed anchoring filament bundles [31,32 ]. to the basal lamina but so metimes lie so close to it These structures may be related to the previously that they are contained within a loop of anchoring described oxytalan fiber [33-35J; so far, there is no fibrils (Fig. 3a) . ge nerally agreed upon term for them. Since dermal In summary, the fine structure of the epider. microfibril bundle is a reasonably good description mal-derma!- junction shows a highly organized

FIG. 3. a: Epidermal- dermal junction. Hemidesmosome (H). Lamina lucid a (LL). Coll agen (e). Anchoring fibril (AP) form a meshwork beneath basal lamina (BL). Loop of anchoring fibers connecting to basal lamina (arrow). Dermal microfibril bundle (DMB) extends from basal lamina in to dermis ( x 49,700). Calibration bar 500 nm. b: Insertion of dermal microfibril bundle (DMB) into basal lamina (BL) ( x 60,000). Cali bration bar 500 nm. I1 July 1975 THE EPIDERMAL- DERMAL J UNCTION 75

FIG. 4. Sub-basal lamina fibrous components of epidermal- dermal junction. Anchoring fibril (arrow) showi ng mid-zone asymmetri ca l cross-banding. Interlocking meshwork of anchoring fibrils (AF). Dermal microfibril bundle (DMB). Some microfibrils on cross-section ex hibi t holl ow tubular profile (x 86,300). Ca li bration bar 500 nm.

FIG. 5. Pinocytotic vesicles (PV) along basal ce ll plasma membrane at epid erm al- dermal junction (x 95,000) . Calibration bar 500 nm. arrangement of membranes and lamellae, includ ing the plasma membrane, lamina lu cida, and basal lamina, which are connected to tonofila FIG. 6. Reduplication of basal lamina (arrow) (x ments ins ide the basal cell at hemidesmosomes, 67,700). Calibration bar 500 nm. laced across by a nchoring filaments, and anchored to the dermis by dermal microfibril bundles and brane" which correlates with the 0.5- to l.O-micron anchoring fibrils. PAS or sil ver reactive li ght microscopic "basement Correlation of light and electron microscopic membrane" is seen with electron microscopy. T he " basement membrane." No con t inuous "mem- basal lam ina seen by electron microscopy is more 76 BRIGGAMAN AND WHEELER Vol. 65, No. I lamina was less than that seen with lead or uranyl stains. The lamina lu cida was entirely free of silvet particles, but the sub-basal lamina fibrous ele. ments, particul arl y the anchoring fibrils, showeq heavy contrast. In these studies, the PAS reactiv. ity and argy rophilia observed with light micros. copy correlated better with the sub-basal lamina band of fibrils and fibrill ar material than with the basal lamina, plasma membrane, or lamina lucida. The ultrastructural locali zation of "basement membrane " antibodies observed by immunofluo. rescent techniques has not been clearly defined. In lupus erythematosus and bullous pemphigOid, human immunoglobulins are found ult rastructur. ally at the epidermal- dermal junction with peroxi. dase-conjugated and ferritin-conjugated antibody methods [41- 45). However, authori t ies disagree about whether immunoglobulin localization ex. hibits preferential affinity for specific structures at the junction and about which structures are in. volved. In lupus erythematosus, several investigator FIG. 7. Diagram of hemidesmosome. Tonofilaments [41-43) have fo und no preferential immunoglobu. (TF) . Attachment plaque (AP) . Plasma membrane (PM) lin locali zation to specifi c structures. Sub-basal with a thicker inner leaflet and thinner outer lea!l et. Sub-basal dense plaque (SBDP). La mina lucida (LL). fibrous com ponents, including anchoring fibril Anchoring fil aments (A). Basal lamina (BL) which is and coll agen fibers, were heavily labeled . In one more dense beneath the hemides moso me. An choring study [42), immunoglobulins were also found fibril (AP) show in g proximal fanlike fibrous portion within the basal lamin a, the lamina lucida, and which fuses with basal la mina cross-banded mid-zone and d ist al fib rous portion extending in to dermis. covering t he outer leaflet of t he pl asma membrane, particularly subjacent to the hemidesmosomes, but not in another (43 ]. On the other hand, Wanger than 10 tim es thinner than that seen by light and Hashimoto [44 ] found specifi c basal laminll microscopy. What then is the ultrastructural local immunoglobulin localization in lupus erythema. ization of the light microscopic " basement mem tosus, but few other structures at the epidermal, brane" and with what fine structures does it dermal junction were labeled. These difference correlate? may merely represent variations in methods but Using an electron microscopic modification of they may also result from different antigen spec. t he periodic acid proced ure on scalp skin and ificities or antibody-binding ~ h aracter i st i cs (i.e., gingiva, Swift and Saxton [36 ] found a heavy immunoglobulins specifica lly bound to antigens at deposit ion of silver grain s which indicated periodic the junction or nonspecificaJly deposited immune acid reactivity in the sub-basal lamin a fibrous complexes [42]) in various patients with lupu al:ea, particul arly over t he anchoring fibrils. Other erythematosus. More work will be required t() structures at the junction (i.e., plasma membrane, cl arify these issues. lam in a lu cid a, and basal lamina) were sparsely labeled. Several groups have confirmed this obser vation [37,38 ]. When skin and glomerular "basement mem brane" were compared wit h a technique similar to that above, the latter was heavily labeled, an indication of the periodic acid reactivity of the electron microscopic glomerular basemen t mem brane itself [39 ). Anchoring fibrils and other sub epidermal fibrous elements are not present in the glomerular basement membrane area; this may indicate an essent ial difference in the skin and glomerul ar "basement membrane." To find the ultrastructural correlation of the " basement membrane" revealed by the reticulum FIG. 8. Hemidesmosome shown at high magnification, stain , Berger and Hundeiker [40 ) investigated the Compare with diagram in Figure 7. TOllofilaments (TF) . argy rophilic properties of the epiderm al- dermal Attachment plaque (APj. Plasma membrane (PM). Lamin a lucida (LL) . Anchoring fi laments (arrow). Basal junction wi th silver salt impregnation of the skin. la mina (BL). Anchoring fibrils (AF) ( x 100,000l . Cali· T he contrast in the pl asma membrane and basal bration bar 250 nm. July 1975 THE EPIDERMAL-DERMAL JUNCT ION 77

FIG. 9. Melanocyte-derm al junction. Melanocyte (M) wi th adj acent epiderm al basal ce ll s (EBG). Reduplication of basal lamina (arrow) (x 10,000 ). In bullous pemphigo id , Wanger and H ashimoto fo und basal lamina labeling which they regarded as specific since fe w other structures of t he junc tion were labeled [44 ]. M odification of the junctional zone in areas of melanocytes. Frequently part of the cell surface of mela n ocyt es abuts the junctiona l zone. The melanocyte- dermal junction is similar to the FIG. 10. Melanocyte-·derm al junction. Diagram show in g details of dense pl ate. Dense pl ate (DP). Plasma epidermal- dermal junction described above. Fig membrane (PM). Lamin a lu ci da (LL). Anchorin g fil a ures 9- 11 show the melanocyte- dermal junction to ments (A). Basal lami na (BL). consist of t he plasma mem brane of t he melanocyte along which are hemidesmosome-like thickenings, mal basal cells. Anchoring fibrils can be seen a lamina lucida, a cont inuous basal lamina, and beneath the basal lamina in the area of melano occasional sub-basal lamina fibrous elements. T he cytes, but they are more sparse in thi s location. term dense plate has been retained for the hem Merkel cells are also present at the epidermal idesm osome-like structures along the plasma dermal junction [46 ]. membrane of melanocytes [9, 45 ] which consist of a cytoplasmic- dense zone abutting the internal leaf COMPOSIT IO N OF STRUCTURES OF T HE let of the plasma membrane. In t hese areas, EPIDERMAL- DERMAL JUNCTIO N anchoring fil aments course through the lamina No direct information on the chemical composi lucida from the outer leafl et of the plasma mem tion of the cutaneous basal lamina, anchoring brane to the basal lamina. However, the sub-basal fibrils, or lamin'a lucida is available because it is den se plaques seen in the lamina lucida beneath impossible to isolate and purify these components t h e hemidesmosomes are absent fro m the from their position between the chemically tough melanocyte-dermal junction. Intracytoplasmic fil epidermis and dermis. This might be a fruitful area aments corresponding to the tonofilaments of basal for fu ture research. cells can be seen in melanocytes, but they do not converge on the cytoplasmic electron-dense area. E MBRY ONIC DEVELOPMENT The basal lamina seems to be less dense and The fo llowing description of the development of thinner beneath melanocytes than beneath epider- the fine structures of the epidermal- dermal junc- 78 BRIGGAMAN AND WHEELER Vol. 65, No. I tive germinative and periderm layers, a continuous basal lamina can be discerned at the epider. mal- mesodermal junction. The basal lamina is thin (20 nm in diameter), homogeneous, anet granul ar, and is separated from the plasma mem. brane by a uniform lam ina lucida approximately 30 to 40 nm thick. Fibrillar elements lie beneath the basal lamina, some of which fuse with the basal lamina and sometimes seem to pass through intI) the lamina lucida. These basal fibrils lack thl! distinguishing characteristics of coll agen, anchor. ing fibr ils, or microfibrils. After pleurostratification of the epid ermis (9th to 12th week), the structures of t he adUlt epidermal- dermal junction begin to appear, but the details of their formation are not well workeQ F IG. 1] . High magnification electron micrograph of out . At about the same tim e, hemidesmosomes melanocyte dermal junction. Dense plate (DP) ( x 96,000). Calibration bar 500 nm. appear along the plasma membrane, sparse at first but progressively more numerous. Almost simulta. neously, anchoring fibrils appear and progressively Plll -' ·· ··' · ·-···~'·-··--·· ~ ...... -. increase in numbers. At approximately the 16th :;.:. ~ ~ -:.,. -.....::. ••~ ...: -: ~ j ... . ' , - .';:,;- ~: • • • week, the structures at the epidermal- dermal junc. tion have full y developed adult characteristics.

FORMATION AND ORIGIN OF STRUCTURES AT THl\ ~ EPIDERMAL- DERMAL JUNCTI ON Hemidesmosomes ~ Krawczyk and Wilgram [49] studied the in vivo re-formation of hemidesmosomes in suction. HJJL induced subepidermal blisters. This involved the 3a in teraction between epidermal. basal ce ll s which J migrated from the edge of the blister and preexist. ing basal lam in a which formed the blister base. Jl[ lli. lli! 3b ) ) 1* t --. . """1".1 'l"""f11~1"1"":'1 1 ':"":"1i ~II"':"'III""'O"""A-. --PfIl Flc. 12. Embryonic development of epidermal- dermal 1 : :'~: , :! ; : ;. : .~ . \ B l junction. (1): Stingle-Iayered ectoderm stage. E ctoderm :: .. ". :J:.. :: ..:: .. : .. :' .'.:: ...... :::<:/,:::< .. :.:'.:,::,::: plasma membrane (PM) devoid of hemidesmosomes. Fibrill ar materi al beneath plasma membrane. (2): Stage of bilamina r epidermis with germinative and periderm layers . Basal lamina (BL) present separated from plasma membrane (PM) by lamina lucida. Sub-basal fibrill ar material. (3): Stage of pleurostratification of epidermis . 2 30 shows early formation of hemidesmosomes (H) and anchoring fibril s (AF) . 3b shows latter stage approaching adult condit ion.

tion in human embryonic skin is derived from the embryonic studies of Breathnach [47] and Ha 3 shimoto et al [48]. The sequence of events is presented diagrammaticall y in F igure 12. At t he stage of the single-layered ectoderm, the ectoder mal- mesodermal junction is composed of t he ecto dermal plasma membrane devoid of any hem ides mosome-like modifications. On the mesodermal side of the plasma membrane, a layer of relatively 4 dense fibri llar material lies in direct apposition to the plasma membrane in so me areas and separated FIG. 13 . Re-formation of hemidesmosome. (1): Forma. from it by an irregu lar electron-lucent space in tion of anchoring filaments (A) . P lasma membralll others. Except for the plasma membrane, none of (PM) . Basal lamina (8L). (2): Formation of sub-basal l these structures co rresponds to the morphology of dense plate (SBDP). (3): Formation of attachmenl plaque (AP) . (4): lnse rLion of Lonofilaments (TF) in area the adult epidermal- dermal junction. of attachment plaque. (Redrawn from Krawczyk and By t he stage of bi lamin ar epidermis with defini- Wilgram.) July 1975 THE EPIDERMAL-DERMAL JUNCTION 79 The morphologic sequence of events of the re-for mation of hemidesmosomes (Fig. 13) occurs in four DAY 0 1lL .U1.H 1 ill. stages: (1) the extension of fine fi la ments (anchor ing fi laments) from the external leaflet of the plasma membrane to the basal lamina, (2) the ~ 0 , 0, condensation of these filaments which produces an electron-dense line (sub-basal dense plaque) sepa rated from the parallel to the plasma membrane, (3) t he formation of an electron-dense attachment plaque a long the internal leaflet of the plasma DAY 3 Ju JlL 1lL ~M ill ~ 'tf~ oI membrane, and (4) the insertion of tonofil aments 'l1ic """' BL ~~' into the area of the attachment plaque. The time ~ sequence of these events was brief, measured in ~ hours. T he formation of hemidesmosomes and 0-' desmosomes followed the same sequence of events ° and thus again suggested the basic sim il arity of these attachment structures. DAY 5 Basal Lamina and Anchoring Fibrils The formation of basal lamin a in adult mamma lian skin during wound healing has been demon strated with the electron microscope by Odland and Ross [50) and Croft and Tarin [51 ). The newly formed basal lamin a was thought by these authors to arise from the epidermis, but this assumption co uld not be verified because viable dermal cells, wh ich might have contributed to basal lamina formation, were present in the vicinity of the healing wound. Briggaman, Dalldorf, and Wheeler [5 2) studied the formation and origin of basal lamina and FIG. 14 . Formation of basal lamina and anchoring fibrils. On day 0, basal lam in a and anchorin g fibrils anchoring fibrils in adul t human skin (Fig. 14). absent at new epidermal- dermal junction. Hemidesmo Epidermis and dermis were separated by cold somes (I!). Coll agen (C). On day 3, basal lamina- like trypsinization. Viable epidermis and viable in material (BL) beneath hemidesmosomes. On day 5, verted dermis were recombined and grafted to anchoring fi laments (A) seen between basal lamin a (BL) and hemidesmosome. An choring fibrils (AF) present. On chorioall antoic mem brane of em bryonated chicken day 7, basal lamina and anchoring fib rils increased. eggs fo r different periods up to 10 days. Before grafting, basal lamin a and anchorin g fibrils were absent from the freshly trypsinized epidermis, but supported the hypothesis of epid ermal origin. An hemidesmosomes and tonofil aments of the basal choring fibrils did not form in recombinants con cells remained intact. Basal lamina and anchorin g taining freeze-thawed dermis; this indicated that fibrils were also absent from the freshly cut in dermal viability was required for anchoring fibril verted surface of the dermis. Three days after formation and supported the theory of dermal grafting, basal lamina began to form immediately origin of the anchoring fibrils. next to hemidesmosomes of the epidermal basal FUNCTIONS cell at t he epidermal- dermal in terface. From the 5th to the 7th day after grafting, basal lam in a Three functions can be conceived for the became progressively more dense and in many epidermal- dermal junction: (1) attachment of epi areas extended to become continuous at the dermis and dermis or epidermal- dermal adher epidermal- dermal interface. On day 5 of cultiva ence, (2) mechanical support for epidermis, and (3) tion, anchoring fibrils appeared first in grafts of barrier to t he transfer of material and cells across epidermis and viable dermis and became progres the junction. sively more numerous thereafter. To determine whether the origin of the basal lamina and anchor Epidermal-Dermal Adherence ing fibrils was epidermal or dermal, the dermis was There is strong evidence that the epidermal-der rendered nonviable by repeated freezing and thaw mal junction serves to attach the epidermis and ing 10 times and by recombination with viable dermis. Indeed, it seems self-evident that the epidermis. The fact that basal lamin a formed as structures at the junction must function to attach readily in these recombinants of epidermis with the epidermis and dermis. When several experi freeze-thawed nonviable dermis as with viable mental and pathologic condit ions associated with dermis indicated that dermal viability was not epidermal- dermal separation were analyzed, they essential for the formation of basal lamina and gave some insight into this function and its impor- 80 BRIGGA MA N AN D WH EELEH Vol. 65, No. I tance. In several of these condit ions, selective structural defects at t he junction ena bled us to correlate the failure of epidermal- dermal adher ence wit h a sp ecific structural defect. Cold First, let us consider those conditions which Trypsin resul t in separation in the sub-basal lamina area. One of the mechanobullous diseases, epidermolysis x X bullosa dystrophica-recessive, provides an " experi o ~, ment of nature" in which we have recently doc u # mented an a bsence of anchoring fibrils [53 J. In Figure 15, an choring fibrils are a bsent from the epiderm al- derm al junction; this is t.rue even in t he never previously blistered skin of a newborn wi t h this disease. The basal la mina and other structures EBHL ~ at the junction appear to be completely normal. Suction Dermal microfibril bundles are also present . In the X ex perim entall y tra umatized skin of t hese patients, the epidermis and dermis separated in an area which is normally occupied by an choring fibrils. ff T he failure of epidermal- derm al adherence and FIG. 16. Epiderm al-derm al se parati on. In lamina lu. the severe blisterin g that resulted is impressive cid a betwee n pl as ma membrane and basal lamin a. Cold evidence of th e importance of anchoring fibrils in trypsin . Epiderm olysis bullosa herediLari a letali s (EBHL). Suction. the attachment of epidermis and dermis. In view of these findings, t he term anchoring fibril appears to produce separation in the lamina lucid a between be highl y appropriate. the plas ma membrane and basal lamina (Fig. 16), Bactel ial coll agenase induces epidermal- derm al Cold trypsinization (crude trypsin Di fco 1: 250 at se paration and blistering after injection into the 4°C for 1- 2 hr) causes the lamina lucida to separate skin (54 ). T his enzy me pro duces coll agen degrada [52,56 ]. After trypsinization, the la mina lucida h as tion and destroys t he anchoring fibrils and basal a vacant appearance which is fr equen tly associated lamina (Fig. 15). Thus, t he latter may be collage wi th widening of the space or frank epidermal- d er. nous or have a collagenous component. But t hi s m al separation . In the region of hemidesmosom es, assumption should be made with caution because the anchoring fil aments are spa rse or absent; ir " the preparations of collagenase used in these stud present, they are sometimes attenuated. The at. ies contain ed other prot eases which might be tachment pl aque portion of the hem ide m OS01l) ~ responsible for destroying the anchoring fibrils and rema ins unaltered . Cytoplasmic bl ebs bound by basal la mina (55 ]. pl asma membrane are frequently enco un tered on Next let us consid er several conditions which the epidermal side of the separation (Fig. 17aL Basal lamina and sub-basal fibrillar elemen ts are in tact. The selectivity of t his procedure is rem ark. able considering the number of bioactive enzy mes p·resent in this preparation (57 J. Al though the agent responsible for "cold trypsinization" has not EB D-R been identified, a selective structura l defect can be de mons tra ted which result.s in the loss of epiderm al- derm al adherence (i. e., dissolution of t he lamina lucida). Another "experiment of nature," which is man i. fested by separation in t he lamina lucid a [58 ), i epidermolysis bullosa hereditari a letalis (junc. tional bullous epidermatosis) (Fig. 16). When the epidermis is mechanically stri pped away fr om the dermis in thin spli t-thickness pieces of skin frolll Bacterial these patients, the level of separat. ion is clearly Collagenase revealed . Figure 17b is an electron micrograph of separated epiderm is in which the under surface of the epidermis consists of plasma membrane except at hemidesmosomes where anchoring fil a ments and tufts of basal lamina remain attached . Again, FIG. 15. Epiderm al- derm al se parati on. Sub -basa l plasma membrane-bound cytoplasmic blebs pro. lamin a area. Epiderm olysis bullosa dystrophica-recessiv·e ject from the under surface of the epidermis. Intact (EBD -R). An.choring fibrils abse nt. Bacteri al co ll age n ase. Co ll age n, basa l lamina and anchoring fibrils de basal lamina and sub-basal fibrillar elements can stroyed. be seen at the former epidermal- dermal junction 01 July 1975 T HE EPlDEHMAL- DEHMAL JUNCTION 81

... . .' ) <~ ~ : B ") , .)

. 'b

F IG. 17. a: Cold trypsin separated epidermis. Cytoplasmic bl ebs (8) with enclosed hemidesmosomes. Anchoring filaments, basal la mina a nd anchoring fib rils absent ( x 20,500). Calibration bar 500 nm. b: Epidermolysis bull osa hereditari a letali s. Mecha ni cally se pa rated epid ermis. Anchoring filaments with attached portion of basal lamina beneath hemidesmosomes (arrow). Cytoplasmi c bleb (8) ( x 66,000). Cali bration bar 500 nm .

separated dermis. T hese findings suggest that suction press u r~ and ski n temperature. Ad herence impairment of epidermal- dermal adherence oc decreases cont inuously with increasing tempera curs eit her in t he lam ina lu cida or in the attach ture rangin g from 20°C to 50°C [61 ]. At 50°C, the men t of an chorin g filaments and basal lamina. epid ermis and dermis can be mechanicall y sepa The application of continuous suction to the skin rated with relative ease [62 ), an indication of for a period of time produces epid ermal- dermal virtua ll y complete loss of epiderm al- derm al adher separation in the lamin a lucid a [59,60 ) similar to ence at this temperature. This suggests that a that in epidermolysis bu ll osa hered itari a letali s hi ghly viscous bond is pre ent in the lamin a lucida (EBHL) (Fig. 16). Epiderm al- dermal adherence which constitutes the weakest link in the struc measured by the blistering ti me depends upon tures of the junction which are normally in volved 82 BRIGGA MAN AND WHEELER Vol. 65, NO.1 in epidermal- dermal attachment . We suggest that molecular-weight materi al, t he epidermal- derma l a defective bond in the lamina lucida is responsible junction does present a barrier to th eir diffusion . for EBHL. Thorotrast is retained beneath the basal lamina where it seems to accumulate after subepiderm al Mechanical Support injection . Only sma ll a mounts of thorotrast eros t he basal lam ina [68 ). E xcept for t he studies Another putative function of the epidermal- der men tioned a bove, li ttle is known a bout the charac. mal junction is to prov ide mechanical support for teristics of t he "junctional ba rrier" regarding m o. t he epid ermis. Some evid ence that the basal la m lecula r size, solubility properties, and other physi. in a may provide this support is obtained by cal characteristics of the m ateria ls which are studying t he behavior of basal ce ll pl asma mem excluded by t he barrier. This may have som e brane when the basal la mina is presen t or lacking. practical significance for the pathogenesis of sk in. In epidermolysis bullosa dystrophica-recessive, the diseases, part icularly t hose involving an immune mechanicall y separated epidermis always had m echan ism where it is important to know wheth er basal la mina covering its under surface. In this various classes of immunoglobulins or immune condition, no bleb formation of the plasma mem complexes cross this "barrier. " brane was seen when t he basal la mina was present. T he extent to which t he structures at t he T he conditons whi ch produced sepa ration in t he epiderm al- derma l junction represent a barri er to lamina lu cida also produced a pl asm a mem brane t he movement of cell s has not been clearl y defin ed. devoid of basal lam in a. In t his sit uation, cytoplas The fact that cells can cross the junction is weI! mic blebbing was frequently encountered . We established. Migration of " foreign cells" into t he interpret t hese observations to indicate t hat t he epidermis (exocytosis) occurs in various patho[ogit basal la mina stabili zes the typically unstabl e conditions. Wolff has provided electron micro. plasma membrane which undergoes prominent scopic documentation of int1 ammatory and n eo. blebbing in the a bsence of basal .l a mina. plastic cell s in t he process of penetrating t he P lasma membrane blebbing is a reversible proc junction [69 ). T he sequence of events is: (1) ess. Seen init ially in recom binant grafts containing disintegration of th e basal lamina in an area trypsin-separated epidermis, it gradually disap adjacent to but not necessarily in contact wi th t he pears d uring cul t ivation as basal la mina re-forms penetrating cell , (2) fo rmation of a gap in t he [52 ). Under ex perimen tal conditions in t issue cul junction and between adjacent basal cells, (3) t ure, mechani cal support and stabili zation of t he penetration by in vading cell or cells, and (4) basal cell layer can be provided by materi a ls other subsequent closure of a gap by tongue-like projec. t han basal la mina; for example, by Millipore fil ter tions of basal cells on e it her s ide. The process [63 ), co ll agen ge l [64 ), or glass [65 ). However, the seems to invo lve some mechanism for destruct ion basal la mina probably serves this .function in vivo. of an area of the junction by the invading cell perhaps by t he loca'l release of proteolytic enzy m e; Barr ier Function [70). The fact that exocytosis of inflammatory To what extent do t he st ructures at t he and neoplastic cell s occurs does not mean t hat epidermal- dermal junction act as a barrier to the there is no barrier to cell movement across the passage of materi als in to and out of t he epidermis epiderma l- dermal junction . These observation across the junction? We will consid er this q uestion can coex ist wi th the fact that a barri er does exist in t hree areas: sm all molecul es,. large molecul es, and t hat only cells wi t h a mechanism for breaking and cell s. down t he barrier can effect passage t hrough it. No evidence exists that t he b asal lamina or any The proposed junctiona l ba rrier to cell move. oiher junctiona l structures are a barrier to t he ment across the junction m ay be involved in d iffusion of water, electrolytes, and other low cutaneous carcinogenesis. A cardinal criteri on for molecul a r-we ight materi a ls. Indeed it would be invasive carcinoma is in vasion of the underlying diffi cult to im agin e a "junctional barrier" to these stroma. Disruption of the barrier at t he epithelial, materi als since it is generall y agreed t hat the stroma l junction might be expected to fl:ic ilitate epidermis depends upon t he dermal blood supply transit ion from preinvasive to invasive carcinoma. fo r its nutrit ion . Ultrastructural changes have been reported at the Several studies have demonstrated in quali ta epitheli al- connective tissue junction during exper. t ive terms t he passage of large-molecul ar-weight imental induction of skin t umors wit h carcinogenic materia ls across the epidermal- derm al junction . chemicals and in preinvasive and invasive carci. Horseradish peroxidase, a water-soluble protein nom a 0 [' t he human oral e pithelium and ectocervix of a pproximately 40, 000 molecular weighi, readily [71- 75 ). P reinvasive changes consisted of focal crosses the basal la mina and lamina lucid a and breaks in t he basal lamina t hrough which epider . . passes in to t he upper epidermis via t he interce llu m al basal cell s protruded in to t he underl ying lar spaces [66,67). Lantha num (22 ) and ruthenium dermis (Fig. 18). Separation and wi dening of the red [1 6 ) a lso cross the epiderma l- dermal junction la mina lucid a occurred where hemidesmosomes a nd a ppear within the epidermis. However, t here is were less numerous or a bsent (Fig. 18). Increased some evidence that in the case of still -larger- basal la mina material was a lso present in some July 1975 T HE EPIDEHM Al.-DERM AL JUNCTION 83 Yea rbook of Dermatology. Edited by F Malkinson, R Pearson . Chicago, Year Book, 1973, pp 5-27 8. Pea e DC: T he basement membrane: substratum of x histological order and complex ity, P roceed ings of the 4th Intern ational Congress of Electron M icros copy. Berlin, Spri nger- Ve rl ag, 1960, pp 139- 155 9. Odland GF: The fine strucjure of the interrelation ship of cell s in the human epidermis. J Biophys Biochem Cytol 4:529- 538, 1958 10. Kobayasi T : An electron microscope study on the derm a-epidermal junction. Acta Derm Venereal (Stockh) 41 :481- 49 1, 1961 FIG. 18. Alterations of epiderma l- dermal junction as· sociated wit h preinvasive carcinoma. Upper (eft: CytQ' 11. Farquh ar MG, Palade GE: J unctional complexes in plasmic protrusion through gap in basal la mina. Upper various epitheli a. J Cell BioI 17:375- 412, 1963 12. Farquhar MG, Palade G: Cell junctions in amphib right : Separation and widening of lamina lucida where ian skin . J Cell Bioi 26:263- 29 1, 1965 hemidesmosomes are absent. L ower: Reduplication of 13. Kelly DE: Fine structure of desmosomes, hem id es basal lamina. mosomes and an adepidermal globul ar layer in developin g new epidermis. J Cell BioI 28:51- 72, areas of the junction, either as multil ayered du 1966 plication of t he basal lam in a similar to that seen 14. Brody I: An electron microscopic study of the junc occasion all y in normal skin (Fig. 18) or as atypical tional and regul ar desmosomes in normal human epid ermis. Acta Derm Ve nereal (Stockh ) flocculent deposits of basal lamina-like m ateri al at 48:290- 302, 1968 t he junction. Changes associated wi th invasive 15. Rupee M, Hoffmeister 1-1 : Sur Ultrastrukt ur der carcinom a were more advanced. Basal lamina was Halbdesmosomen. Derm atolog ica 138:453- 458, completely a bsent from extensive areas of the 1969 16. Hashimoto K, Lever W: An ultrastructural. tudy of junction and was associated wi th prominen t cyto cell junctions in pemphigus vul garis. Arch Derma· plasmic protrusion in to the connective tissue. In tol 101:287- 298, 1970 one study, cytoplasm ic protrusions were thought to 17. Komura J , Ofuji S: Ultrastructure of half form free "vesicles" which ruptured and released desmosomes fi xed only in glutaraldehyde. Derma tologica 144:35- 39, 1972 their con tents into surrounding connective tissue. 18. Stern IB : E lectron mi croscopi c observations of oral Vesicular rupture was correlated with destruction epi thelium. 1. Basal cell s and basement mem of t he focal areas of adjacent connective tissue [71 ). brane. Periodontics 3:224-238, 1965 T his observation is in teresting in light of the 19. Kobayasi T : E lectron microscopy of elastic fibers and dermal membrane in normal human skin. Acta demonstration of collagenase activi ty in vari ous Derm Venereol (Stockh ) 48:303- 312, 1968 tumors, including cutaneous squa mous cell carci 20. S usi FR, Belt WD, Kell y JW: Fine structure of nom a [76 ]. One can only speculate on the role of fibrillar complexes associated wi th t he basement these changes in cutaneous carcinogenesis. T he mem brane in human oral mucosa. J Ce ll BioI 34:686- 690, 1967 observed alteration of the epidermal- dermal junc 21. 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