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Basal Detachment of the Epidermis Using Dispase: Tissue Spatial Organization and Fate oflntegrin a6/34 and Hemidesmosomes
Yves Poumay, Isabelle H. Roland, Michele Leclercq-Smekens, and Robert Leloup Departement d'Histologie-Embryologie, Facultes Universitaires Notre-Dame de la Paix, Namur, Belgium
Dispase has been utilized to produce basal detachment of the epidermis of human skin biopsies and to study the consequences induced afterwards during incubations of the detached tissue. Spatial reorganization of the epidermis is observed under these conditions and is characterized by disappearance of the typical basal keratinocyte layer. Immunofluorescent labelings reveal upward migration of several cells exhibiting the basal phenotype between suprabasal differentiating keratinocytes and demonstrate progressive intracellular expression of hemidesmosomal components: the integrin a6p4 and two plaque components, the 230-kDa bullous pemphigoid antigen and HD1, a SOO-kDa protein. Using electron microscopy and immunogold techniques,
Members of a large family of cell surface receptors, namely the integrins, involved in cell-cell interactions as well as in cell adhesion to the extracellular matrix, are thought to determ.ine the organization of the epidermal tissue into proliferating
and differentiating compartments [1- 4]. Integrins are heterodimers composed of two transmembrane glycoproteins, one a and one p subunit, whose various combinations in different cell types give rise to receptors with specific binding capacities for one or severalligands, certain ligands also being bound by different integrins [5]. In epidermal keratinocytes, the integrins a2pl, a3pl, aSpl, a6p4, and avpS are predominantly expressed by basal cells but become non-functional, and progressively disappear in differentiating cells [2 - 4,6,7]. Thus, as potential tissue spatial organizers, integrins may be involved in development [8] and wound healing [9]' or in pathologic situations such as tumor invasion [10] and psoriasis [9].
In the epidermal tissue, the basal cell is polarized. Apical and lateral surfaces adhere mainly via desmosomes to neighboring basal and suprabasal cells, while the basal surface adheres to the basement membrane zone (BMZ) mainly via hemidesmosomes. Desmosomes and hemidesmosomes are both anchoring sites for keratin intermediate filaments; immunologic and molecular biologic studies suggest that they are unrelated junctions with fortuitous structural similarity [11,12]. The polarity of basal keratinocytes is also apparent from studies showing that, versus PI integrins, a6p4 is concentrated
Manuscript received March 1, 1993; accepted for publication July 28, 1993.
Reprint requests to: Yves Poumay, Departement d'Histologie-Embryologie, Facultes Universitaires Notre-Dame de la Paix, 61, Rue de Bruxellcs, B-5000 Namur, Belgium.
we demonstrate that the hemidesmosome-containing basal membrane domains enter the cell cytoplasm after detachment of the epidermal tissue. Partial recycling of internalized hemidesmosomal components is also suggested. Our findings illustrate the processing of released hemidesmosomes in detached basal keratinocytes and suggest some heterogeneity between basal cells migrating towards a supra basal position and those remaining in the basal layer. These results suggest that the dispase-detached epidermis is a self-remodeling tissue in which basal keratinocytes' and tissue's polarities observed in the anchored epidermis are progressively changing. Key words: keratinocyte/internalization/bullous pemphigoid antigen/HD1. ] Invest Dermatol1 02: 111-117, 1994
on their basal pole [4,6] and located in hemidesmosomes in association with the 230 kDa bullous pemphigoid antigen (BPA) in vivo [13 ,14] and in vitro [15,16] .
Several other proteins have been localized in hemidesmosomes. The 230-kDa BPA is present in the cytoplasmic plaque [11]. whereas the 180-kDa BP A, recognized also by the serum of some bullous pemphigoid patients, is a collagenous transmembrane protein [17J. Kalinin/epiligrin/nicein is localized in anchoring filaments [18-20], and HDI is a SOO-kDa protein also found in the cytoplasmic plaque [21], as is another polypeptide of200 kDa [22].
The detachment of basal keratinocytes from the underlying structures by separation at their basal surface occurs in several blistering skin diseases (e.g.,junctional forms of epidermolysis bullosa, bullous pemphigoid) and is also experimentally produced by dispase [23] . Artificial denno-epidermal separation by means of chemical or mechanical treatment is also frequently utilized for diagnostic purposes or for investigation of epithelial-mesenchymal interactions at the BMZ [9,24-27].
Because of its likely organizing function in the epidermal tissue [14], it is of interest to investigate the fate of integrin a6p4 and associated hemidesmosomes after detachment of the epithelium. Results obtained with detached keratinocyte cultures suggest that the tissue's spatial organization is lost after release from the culture substrate [28], maybe due to internalization of hemidesmosomes [16]. Furthermore, the expression of integrins in the roof of suction blisters revealed a special suprabasal distribution of a6p4 with intracellular staining in addition, suggesting a loss of the tissue's and tne cell 's polarities [9]. In this study, we present evidence that experimental detachment of epidermis with dispase induces internalization of a6p4-containing junctions. This event precedes disruption of tissue polarity and redistribution of hemidesmosomal components a6p4, 230-kDa BPA, and HD1.
0022-202X/94/S06.00 Copyright © 1994 by The Society for Investigative Dermatology, Inc.
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112 POUMA Y ET AL
MATERIALS AND METHODS
Skin Samples and Separation Technique Biopsies were taken from adult skin obtained after plastic surgery and transported in phosphate-buffered saline (PBS) prior to treatment with dispase. Dispase II (Boehringer Mannheim) was dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM) at a concentration of2.5 mg/ml [23]. The lower part of the dermis was removed and small pieces (around 1 cm2 of area) were floated, epidermis up, on the surface of the dispase solution at 37· C in a humidified 5% CO2 atmosphere for 90 min. Then, the epidermis was separated from the dermis with forceps.
Incubation of Detached Epidermis After detachment, the epidermis was rinsed twice in PIlS and incubated stratum corneum up in medium made of Ham's F12 and DMEM (1: 3) at 37"C in a humidified 5% CO2 atmo-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
r
sphere for 0 - 24 h. In some experiments, inhibition of protein synthesis was b produced by the adclition oflO,uM anisomycin (Sigma) and/or 5,uM cycloheximide (Sigma).
Classical Histology Skin samples and incubated detached epidermis were fixed in 40/0 formaldehyde in PIlS at room temperature for 30 min and embedded in paraffin. Sections (5 ,urn) were prepared perpendicular to the epidermal surface and stained with hemalum and erythrosin.
Antibodies The primary antibodies used were mouse monoclonal DH12 againstj31 integrin subunit (donated by J.-]. Cassiman, University ofLeuven [2]), rat monoclonal GoH3 against a6 integrin subunit (donated by A. Sonnenberg, University of Amsterdam [29]), mouse monoclonal DE-KI0 against cytokeratin 10 (purchased from Organon Teknika [30)), rat monoclonal 439-9B against j34 integrin subunit (donated by S.]. Kennel, Oak Ridge National Laboratory [31]), human bullous pemphigoid autoantibody against 230-kDa BPA (condensed immunoglobulin fraction) [11], mouse monoclonal 121 against HDl [21] (both donated by K. Owaribe, Nagoya University), mouse monoclonal Ki-67 against a human antigen expressed in proliferating cells (purchased from Dako [32]), and mouse monoclonal GB3 against kalinin/epiligrin/nicein (purchased from Sera-Lab [20]).
Immunofluorescent Labeling Specimens of skin and incubated detached epidermis were coated in O.C.T. Tissue-Tek (Miles) and immediately frozen in a methanol bath cooled with dry ice. Frozen perpendicular sections of tissues were cut at 5 )lm in a Microrn HM 500 OM cryostat at - 20 · C, air-dried, and stored at - 80· C until use. Sections were fixed for 30 min in a 40/0 formaldehyde PBS solution, washed in 0.1 M glycine, and blocked in PBS containing 0.1 % bovine serum albumin (BSA) and 0.02% Triton X-I00. For labeling ofKi-67 antigen, fixation was performed in cold acetone (-20· C), and Triton X-l00 was omitted from the blocking solution. Incubations with the antibodies diluted in the blocking solution were performed in a humidified chamber at room temperature for 60 min and were followed by three 15-min washes in the same solution. Fluorescein isothiocyanate (FlTC)-conjugated anti-rat (Dako) or anti-mouse (high species-specificity; Amersham) and, for dual labeling, biotinylated anti-rat (high species-specificity; Amersham) were used as secondary antibodies and incubated as described above. For dual immunofluorescent labeling of a6 subunit and cytokeratin 10, GoH3 and De-KiO antibodies were incubated together, as were the relevant secondary conjugated antibodies, and a third incubation was performed with Texas Red-conjugated streptavidin (Amersham). Sections incubated either without primary antibodies or with irrelevant antibodies produced in same species were used as controls.
lmmunogold Labeling of the Basal a6 Subunit After incubation in dispase, skin samples were chilled to 4 ·C, and epidermis was separated from the dermis with forceps. Experiments with cultured keratinocytes have shown that basal integrin a.6j34 remains on the cell surface under these conditions [16], so the labeling procedure was performed at 4·C. Samples of detached epidermis were rinsed in calcium (1 mM)- and magnesium (1 InM)-containing PBS. BSA (0.1 %) was then added, and the tissues were washed three times for 10 min in this blocking PBS, incubated for 60 min with the primary antibody (a 1: 10 dilution of culture supernatant of clone GoH3 was prepared in the blocking PIlS), and washed in the blocking PBS. Label ing for electron microscopy was performed with 10 nm gold-labeled goat anti-rat IgG (Amersham) diluted at 1 : 20 in PBS and followed by three washes as described above. The gold-labeled epidermis was fixed immediately (0 h) or rapidly transferred into the incubation medium (Ham's F12: DMEM, 1: 3) at 37 ·C and further incubated at this temperature for 1- 24 h before fixation with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), post-fixation in 1% osmium tetroxide, and embedding in epon. Ultra-thin sections were counterstained with uranyl acetate and lead citrate and examined with a Phillips EM 301 electron microscope.
c
'.;
o " ... ~_ -o. ~~-:: • •
Figure 1. Histology of (a) intact s ~in biopsy and (b,c) clispase-detached epidermis. Skin samples were (a) fixed immediately or incubated in dispase to separate the epidermis from the dermis before (b) immediate fixation of the detached epidermis or incubation of this tissue for (e) 8 h before fixation. The rete ridges (arrowheads) present in intact skin (a) are still observed after the detachment of the epidermal tissue (b) , but these characteristics disappear during the incubation of the detached epidermis and, simultaneously, the released basal layer is progressively replaced by flattened cells (e). Bar, 100,um.
RESULTS
Classical Histology The four layers (basal layer, stratum spinosum, stratum granulosum, and stratum corneum) observed in intact skin (Fig la) were recovered after detachment of the epidermis by dispase, and rete ridges were still present on the released basal surface (Fig Ib) . After 8 h of incubation, basal cells became flattened, and rete ridges disappeared (Fig le).
Immunofluorescent Study of Tissue Spatial Organization Because polarized organization is perturbed in dispase-detached keratinocyte cultures [28], we performed immunolabeling of integrin subunits and keratin 10 as basal [3,4,6,7] and suprabasal [301 markers, respectively, and the spatial organization of detached epidermis was analyzed during incubations for several hours. In intact skin, integrins of the PI-subfamily were expressed on the whole periphery of basal cells, whereas the 0'6 subunit was concentrated facing the BMZ (Fig 2a,b). The expression of keratin 10, revealed by FITC on sections labeled for 0'6 expression with Texas Red, was typically located in the suprabasallayers (Fig 2c). Due to unusually strong fluorescence, this FITC fluorescence was sometimes slightly visible through Texas Red-specific filters (Fig 2b).
Immediately after detachment with dispase, the expressions of basal and suprabasal markers were unchanged (data not shown).
VOL. 102, NO. 1 JANUARY 1994 EPIDERMAL DETACHMENT USING DISPASE 113
Figure 2. Immunofluorescence of integrin PI subunit (a,d,g) and dual immunofluorescence of Texas Re~-labeled integrin a6 subunit (b,e,h) and FITC-labeled keratin 10 (c/,i). In intact skin, the epidermal integrins are expressed in the basal layer; (a) pl-mtegrms are expressed on basal keratinocytes' periphery, whereas (b) a6 is polarized along the BMZ (arrowheads). Keratin 10 (c) is typically expressed in suprabasallayers only. After detachment with dispase (d), PI is still expressed on basal cells' periphery, but (g) it is progressively detected in cells located suprabasally after prolonged incubation of the detached epidermis. On the other hand, when the detached tissue is incubated for 1 h, (e) the expression of a6 (arrowheads) becomes p~tchy in the ventral cytoplasm of basal cells, (f) at some distance from the keratin-IO-expressing suprabasal cells. After 8 h of incubation (h), the expreSSIon of a6 has become mainly pericellular with occasional strongly fluorescent intracellular spots (arrowheads) in cells sometimes located suprabasally and (i) surrounded by keratin-lO-positive differentiating cells. Bar, 50 11m.
However, a 1-h incubation of the detached epidermis induced patchy labeling of the a6 subunit (Fig 2e), whereas expressions of Pl (Fig 2d) and keratin 10 (Fig 2J) were preserved. During prolonged incubations (8-24 h) (Fig 2g-i and data not shown), a few cells expressing integrin subunits progressively moved upwards in the tissue and were recovered in suprabasal positions. Simultaneously, flattened cells covered the detached basal epidermal surface and also expressed integrins but not keratin 10.
Interestingly, the pattern of a6 expression was concomitantly depolarized (Fig 2e,h). After 8 h (Fig 211) of incubation, the labeling of the a6 subunit showed fluorescent intracellular spots, particularly in suprabasal cells exhibiting the basal phenotype, and some fluorescence was also redistributed to the whole periphery of these cells. Meanwhile, the pt subunit was observed only on the periphery of the cells (Fig 2g). The upward migration is also responsible for holes observed in the suprabasal distribution of keratin 10, 8 h after the detachment of the epidermis by dispase (Fig 2i).
To determine whether cells migrating towards the suprabasal layers still exhibited other basal phenotype characteristics, a proliferation marker, Ki-67 antigen, was examined by immunofluorescence. Immediately after detachment of the epidermis (Fig 3a),
Ki-67 was located mainly in the cytoJ'lasm of the basal cells, as has been described in squamous epithelia l32]. During the incubation of the detached epidermis, the Ki-67 expression was preserved in basal cells that were moving upward (Fig 3b).
Immunofluorescent Study of Hemidesmosomal Components Previous observations of dispase-detached keratinocyte cultures have revealed that the integrin a6p4 located in hernidesmosomes was internalized under these conditions (16). As expression of the a6 subunit during incubation of the detached epidermis also suggested. internalization, other hemidesmosomal components were labeled 111 the same tissues. Analysis of P4 expression (Fig 4a,d,g) indicated a pattern totally similar to the a6-labeling using GoH3 antibody (Fig 2b,e,h), suggesting that a6p4 subunits remained associated.
Two hundred thirty-kilodalton BPA and HOt plaque components and kalinin/epiligrin/nicein were concentrated along the BMZ in intact skin (Fig 4a-c and data not shown). After detachment with dispase, no staining of kalinin/ epiligrin/nicein could be obtained with the GB3 antibody on the released epidermal tissue (data not shown), whereas the cytoplasmic plaque'S hemidesmoso-
114 POUMAY ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 3. Immunofluorescence of the Ki-67 antigen. Ki-67 is illustrated (a) in the epidermis immediately after basal dispase detachment, and (b) after 24 h of lncubation of the detached tissue. After 24 h of incubation, several cells located suprabasally express this proliferation marker typical of the basal phenotype. Bar, 50 j.lm.
mal components were recovered with a6p4 in keratinocytes. One hour after detachment by dispase, the expressions of 230-kDa BPA and HD 1 became patchy along the ventral cytoplasm of basal cells (Fig 4d - j), suggesting that there was a progressive invagination of hemidesmosomes. When the incubation was prolonged (8-24 h) , intracellular fluorescent spots containing these hemidesmosomal components were present (Fig 4g- i). According to dual immunofluorescent experiments, all the hemidesmosomal components were co-localized within these spots in cells migrating suprabasally (data
not shown). On the other hand, outside the spots, these components were dissociated: a6p4 redistrihuted towards the plasma membrane (Fig 4g), whereas the 230-kDa BPA dispersed into the cytoplasm (Fig 4h) and HDl very faintly deposited near the plasma membrane (Fig 4i).
Interestingly, the addition of protein synthesis inhibitors did not alter the redistribution of hemidesmosomal molecules in the detached epidermis (data not shown). This argues that no de /lOVO
synthesis is required to produce the observed pattern.
Figure 4. Immunofluorescence ofhemidcsmosomal components: integrin P4 subunit (a,d,g) , 230-kOa BPA (b, e,h) , and HOt (c,!,i). In intact skin (a,b,c), the labeling of each component produces a linear stain ing at the BMZ. This staining (d,eJ) becomes patchy in the ventral cytoplasm of the basal cells after t h of incubation of the detached tissue. After 8 h (g) of incubation, the expression of the P4 subunit becomes pericellular with strongly fluorescent intracellular spOtl; ncar the nucleus of cells found most frequently in suprabasal position. These intracellular spots are also revealed by staining (h) of the 230-kOa BPA and (i) of H01. The BPA is then additionally expressed scattered in the cytoplasm of the positive ce lls (II). E, epidermis; D, dermis; Bar, 50 Jim.
VOL. 102, NO.1 JANUARY 1994 EPIDERMAL DETACHMENT USING DISPASE 115
Figure 5. Immunogold labeling of basal integrin a6 subunit. The labeling was done at 4·C (a) on detached epidermis, then the labeled tissue was (b) incubated at 37"C for 1 h or (c,d) for 8 h before fixation and processing for electron microscopic observation. CollOIdal gold particles, (a) initially concentrated over detached hemidesmosomes, (b) reveal the invagination of basal membrane domains containing these junctions, after incubation of the detached tissue and (c) sometimes show persistent labeled hemidesmosomes concentrated in special stmctures found after 8 h of incubation near the nucleus (N). In the same time, (d) the label is also recovered in intercellular spaces or in small vesicles (arrowh ead) fusing with the plasma membrane, two localizations never encountered before. Bars, 0.25 flm .
Imntunogold Surface Labeling of the a6 Subunit The a6 subunit appeared to be present extracellularly on the epidermal basal surface after detachment, as it was labeled at 4 · C by the GoH3 antibody. When the labeled tissue was fixed immediately after the labeling procedure (0 h), gold particles were found exclusively on the basal surface and concentrated over hemidesmosomes (Fig 5a), as shown previously by others [13]. However, when the labeled epidermis was incubated at 37 °C for 1 h prior to fixation, basal membrane domains containing hemidesmosomes were observed invaginating into the ventral cytoplasm of the basal cells (Fig 5b), suggesting that internalization of the cell membrane was responsible for the patchy immunofluorescent labeling of hemidesmosomal components under these conditions. When the incubation was prolonged to 8 h, gold particles were seen in intracellular vacuoles or sometimes concentrated between aligned membranes exhibiting hemidesmosomal structures (Fig 5c). These structures may correspond to the spots where hemidesmosomal components seemed to be intracellularly concentrated by immunofluorescence. Simultaneously, the label was seen in small vesicles located near the plasma membrane of suprabasally located keratinocytes, and
around these cells in the intercellular space (Fig 5d). These observations suggest partial recycling of the internalized integrin a6p4, which could explain its redistribution Onto the whole cell surface, as was also observed by immunofluorescence, 8 h after detachment.
DISCUSSION
Studies of epidermal detachment stimulate many interests in epidermal biology and pathology. The epidermis can be experimentally separated from the dermis at the BMZ by chemical treatment with 1 moljl sodium chloride solution (24 - 26J, enzymatic treatment with thermo lysin [25), cold trypsin [26), dispase [27], or mechanical treatment by negative pressure, which produces suction blisters [9,25,26]. Dispase induces denno-epidermal separation at the lamina densa, probably by enzymatic destruction of the rype IV collagen [27] . It seems therefore to produce a cleavage plane localized in the lamina lucida, as no labeling of the detached epidermis was observed with the GB3 monoclonal antibody. Following release of the epi-
116 POUMAY ET AL
dermis from the dermis, we have observed, as in our previous observations on detached cultured keratinocyte sheets [28], that epidermal cells spatially reorganize at 37°C. Keratinocytes of the basal phenotype are then located suprabasal.ly, intermingled. with diff~rentiating cells. These results concur with recently pubhshed studies of the detached epidermis of suction blisters; one day after detachment, basal cells are also revealed supra basally by studies of integrin expression [9]. Whereas these investigators hypothesized that ~his uncommon localization of basal cells could result from mecharucal forces used to establish the blisters [9]' we present evidence that basal cells are moving upwards progressively after detachment. During this process, a few basal keratinocytes establish new cell-cell contacts to surround themselves with neighboring cells, while others become flattened and cover the basal surface. These findings are reminiscent of differences between different basal keratinocytes: increased cell proliferation is observed in deep compared to shallow rete ridges [33]' and the integrin a3p1 has been recently observed concentrated on the basal surface of basal cells in deep rete ridges, while found at intercellular contacts in shallow ones [34]' In dispase-detached epidermis, the disappearance of rete ridges could be a consequence of tissue spatial reorganization. However, as this reorganizing phenomenon is absent from the attached epidermis, it is likely that alterations in the adhesive properties of cells occur in detached tissues.
The internalization of integrin a6p4 appears to be linked to these alterations. Indeed, we and Hertle and colleagues [9] observed the presence of intracellular bright fluorescent a6 and P4 subunits in cells of detached epidermis. We demonstrate here, as in cultured keratinocytes [16], that integrin a6p4-containing basal membrane domains, corresponding to hemidesmosomes, are specifically internalized at 37°C after basal detachment. This internalization process is likely to explain the previously reported absence of hemidesmosomes in cultured [35] and non-cultured [25,27] detached basal keratinocytes. It is furthermore relevant to notice that, on the other hand, hemidesmoscimes seem to be ultrastructurally recovered in basal keratinocytes detached by procedures performed at 4 ° C [16,25]. The importance of temperature on the presence or absence of hemidesmosomal junctions in detached keratinocytes surely has been underestimated in the past, but interestingly, the process of hemidesmosome internalization together with its dependence on temperature has already been reported in trypsinized basal keratinocytes [36] and in a different cultured cell type after dispase treatment [11]. Thus, one may wonder whether internalization of released hemidesmosomes is a generalized phenomenon and could be controlled by mechanisms identical or similar to those involved in the internalization of released desmosomes, another intermediate filament-anchoring junction [37]?
Our ultrastructural observations and the redistribution of internalized fluorescent labeling of a6p4 to the entire cell surface, even in presence of protein synthesis inhibitors, suggest partial recycling of the internalized integrin a6p4 in detached basal keratinocytes. This concurs with previous experiments performed on cultured cells [16] and with the reported ability of several integrins, including a6p4, to circulate between the cell surface and intracellular localizations [38] . Recycling of a6p4, but also of other hemidesmosomal components, might be of particular interest to allow re-establishment of adhesion structures when new contacts with the extracellular matrix occur, as in wound healing [14,39].
Our study raises several questions regarding the role of basal cell polarity and anchorage in the spatial organization of the epidermis and regarding dynamic features of hemidesmosomes and associated components, particularly their specific internalization when anchorage at the BMZ is disrupted. It seems important to consider the detached epidermis as a self-remodeling tissue rather than as histologically stable. To conclude, we wish to stress the significance of the features reported in this work for investigators studying experimental, but also pathologic, situations of basal epidermal detachment.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
The occasional technical assistance provided by R. Deom, N . Fievet, F. Herphelill, atld M.-F. Six is gratifully acknowledged. We also thallk D. Vall Acker alld M. Lombet for the photographic artwork alld Dr. B. Bietifa it for providillg s~ill specimens. Special thallks are addressed to Drs. A. Sonllellberg (Amsterdam), S.]. Kennel (Oak Ridge) ,]'-J. Cassilllall (Lelwell), alld K. Ol/laribe (Nagoya) Jar their killd gift of antibodies.
This work was supported by a grallt Jrom the Aide Socialc aux Grands Brflles atld by a grantfrom the Fa lids Nationa l de la Recherche Scielltijique (FNRS) to YP.
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EPIDERMAL DETACHMENT USING DISPASE 117
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