Integrin and basement membrane normalization in mouse grafts of human keratinocytes – Implications for epidermal homeostasis

Differentiation (1997) 61:195–209

© Springer-Verlag 1997

O R I G I NA L A RT I C L E

&roles:Dirk Breitkreutz · Hans-Jürgen Stark Nicolae Mirancea · Pascal Tomakidi Heinrich Steinbauer · Norbert E. Fusenig

Integrin and basement membrane normalization in mouse grafts of human keratinocytes – Implications for epidermal homeostasis &misc:Accepted in revised form: 7 October 1996

&p.1:Abstract Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains α2, α3, β1 and α6, while β4 already showed a preferential basal location; (2) de novo expression of α5 and αv; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular α2β1 and α3β1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin α6β4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidaD. Breitkreutz (✉) · H.-J. Stark · H. Steinbauer · N.E. Fusenig Division of Carcinogenesis and Differentiation, 0240, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany N. Mirancea Romanian Academy, Institute of Biology, Bucharest, Romania P. Tomakidi Department of Orthodontics and Dentofacial Orthopedics, Dental School, University, Heidelberg, Germany&/fn-block:&dy

tion. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.

Introduction In stratified squamous epithelia morphogenesis and formation of distinct cell compartments is associated with specific patterns of cell surface molecules, which mediate cell-cell and cell-matrix recognition and adhesion. Among those, integrins are considered, apart from their role in heterotypic cell-cell interactions, to be the major players in cell matrix interactions and thus also responsible for structural tissue organization (for review see [37]). Furthermore, integrins are supposed to function as sensors for the extracellular environment and for mechanical forces activating cellular signal transduction systems which might in turn influence turnover and processing of extracellular matrix [40, 65, 77]. Integrins form obligatory dimers of one α- and a genetically distinct β-chain, and in normal adult human epidermis they are represented by α2β1, α3β1 and α6β4, which are all restricted to the cells in the proliferating basal layer. While α2β1 and α3β1 are mostly located at the lateral and apical membrane of basal cells [15, 20, 43], α6β4 is concentrated at the basal pole facing the basement membrane [13, 68]. Within the cell, the β1-complexes are linked to the microfilament system by association with other proteins such as talin, vinculin and α-actinin [11, 48]. In contrast, α6β4 is chiefly an integral component of the hemidesmosomes [13, 68] and thus associated with keratin filaments via bullous pemphigoid antigen-1 (BPAG-1, 230 kDa [70]). The principal extracellular ligand of α6ß4 is laminin-5, also known as kalinin, a

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building block of the anchoring filaments in the basement membrane zone ([14, 51, 52, 61], new laminin nomenclature [10]). Thus, one postulated integrin function is to transmit mechanical forces by connecting the cytoskeletal network of keratinocytes with the exoskeleton of the connective tissue. Whether the β1 complexes contribute to intercellular adhesion in intact epidermis is less clear [72]. On the other hand, there is strong evidence that they participate in the maintenance of basal cell functions, presumably by preventing terminal differentiation, according to previous studies on cultured keratinocytes [35] and transgenic mice [12]. This is also compatible with the observation that in hyperplastic states, such as psoriatic epidermis, integrin-positive areas are expanded, including both β1-integrins and α6β4 [20, 32]. Last but not least, the striking surface redistribution of integrins in the early phase of wound healing and in keratinocytes with ‘unrestricted’ proliferation in vitro underlines their role in the dynamic processes of tissue remodeling [16, 20]. Growth of normal epidermal keratinocytes and epithelial cells in general depends on interactions with ligands of the extracellular matrix (ECM). Strong interference with that, for example by suspending keratinocytes in semisolid medium, induces principal steps of terminal differentiation leading to rapid cell death, such as cornified envelope formation and subsequent hydrolytic processes [25, 35, 60, 75, 76]. There is strong evidence by now that one trigger for the involved internal signaling is the loss of receptor occupancy by the ECM ligands [62, 74, 79]. Apart from the the constitutively expressed integrins α2β1, α3β1 and α6β4, additional ones are induced in keratinocytes, for example αvβ5 and α5β1, during growth in vitro and serial passaging, comparable to wounded epidermis [19, 20, 50]. While this enables the cells to recognize a large variety of ligands including soluble matrix components in the serum such as fibronectin and vitronectin, they produce their own ligands, a major one being laminin-5 [14, 51]. For most of the in vitro studies with isolated keratinocytes on artificial substrates or in suspension, the question remains as to how far the results can be extrapolated to the in vivo situation. Furthermore, for such studies in the human organism there are many limitations mostly for ethical reasons. Here we have investigated the changes in integrin distribution in transplants of human keratinocytes onto nude mice at different stages of epidermal tissue reconstruction. In order to evaluate the relevance of this transplantation system as model for wound-healing-associated regenerative processes and epidermal homeostasis in humans, we have studied the successive events in the change from an activated and initially hyperproliferative to an apparently balanced tissue state by analyzing proliferative activity, differentiation as well as synthesis and assembly of basesement membrane in relation to the spatial distribution of integrins. To our best knowledge, this represents the first reported direct comparison of all these parameters in a human epidermal regeneration model.

Methods Cell culture and transplantation Normal human keratinocytes were isolated from trypsin-separated epidermis of adult skin and mainly used in their third passage as described by Smola et al. [66]. In brief, keratinocytes from frozen stocks were seeded on irradiated fibroblast feeder layers and grown in FAD medium with 5% fetal calf serum (FCS) and further supplements. According to this protocol, fibroblasts were obtained from human skin explant cultures, subcultured four times in Dulbecco’s modified Eagle’s (DME) medium containing 10% FCS and used at passages 5–7. Generally, epidermal cells were transplanted as preformed epithelia on native type-I collagen gels (from mouse tail tendon) onto nude mice [3, 4] using the transplantation chamber described previously [5, 6] with slight modifications [26, 28]. In more recent experiments keratinocytes were seeded on collagen gels with living fibroblasts embedded in the gels [47, 66] and these organotypic cocultures were transplanted giving rise to a comparable epidermal morphology. On average, there was a slightly better take rate and prolongation of survival time of the grafts using this technique, which was therefore the method of choice for the proliferation studies. In brief, organotypic cocultures were incubated for 1 day before grafting using an assembly device of concentric teflon rings, which holds the collagen gels in place (Combi Ring Dish, CRD [55] purchased from Renner, Dannstadt, Germany) as published in detail elsewhere [27]. At indicated times transplants were dissected en bloc, embedded in Tissue Tec (Lab. Tec. Prods., Naperville, Il, USA) and frozen in liquid nitrogen vapor. Histology Transplants were fixed in 3.7% formaldehyde (phosphate buffer, pH 7.4) for 24 h, embedded in 3% agar to prevent separation at the epithelial-matrix junction and again fixed for additional 24 h. Specimens were dehydrated, embedded in paraffin and 5-µm sections were stained with hematoxylin and eosin. Transmission electron microscopy Specimens were fixed in 2.5% glutaraldehyde (0.05 M cacodylate buffer, pH 7.2) for 2 h at room temperature, postfixed in 2% OsO 4 (25 mM cacodylate buffer) for 1 h, incubated for 18 h in 1% aqueous uranyl acetate, dehydrated and embedded in Epon 812. Ultrathin sections were collected on copper grids, coated with Piloform F (Walter Hert, Munich, FRG), stained with uranyl acetate and lead citrate, and examined on a Zeiss EM 109 electron microscope. Indirect immunofluorescence microscopy Frozen 6-µm sections were mounted on 3-aminopropyl-thriethoxy-silane-coated slides, air dried and processed as outlined elsewhere [71]. In brief, the specimens were fixed for 2–5 min in methanol-acetone (for BPAG-1, see below, in pure acetone) at –20°C, rehydrated with phosphate-buffered saline (PBS) and incubated with the primary antibodies for 2 h at room temperature or overnight at 4°C. In case of double labelling the two antibodies were applied either sequentially or simultaneously. Incubation with the fluorochrome-labelled secondary antibodies was for 15–30 min. Antibodies against individual keratins (described in [6, 71]), as far as not commercially available, were generous gifts from Dennis Roop (Houston, USA), Irene Leigh (London, UK), and Tung-Tien Sun (New York, USA) and rabbit anti-loricrin from Daniel Hohl (Lausanne, Switzerland). Monoclonal antibodies from mouse recognizing involucrin were kindly provided by Fiona Watt (London, UK), integrin α2, α3, αv and β1 by Eberhard Klein and Eva Bröcker (Würzburg, FRG), α5 (also commercially avail-

197 Table 1 Specification of monoclonal antibodies&/tbl.c:& Antigen

Mab Clone

Reference

Collagen type VII

LH7.2

[45, 46]

Integrins α2 α3 α5 α6 αv β1 β4

A1-43 J143 SAM1 GoH3 (Rat) LV230 AJ2 439.9B (Rat)

[42] [43, 73] [16] [69] [43] [43] [21]

Involucrin Keratins K1 K10 (+1) K5 K14 K16

SY5

[76]

LLO17 Ks8.60 AE-14 LH8 LLO25

[38] [36] [49] [57] [38]

Laminin-5 α3 β3

BM165 K140

[52] [52]

&/tbl.: able, Immunotech, Hamburg, FRG) by Giovanna Zambruno (Rome, Italy), type VII collagen by Irene Leigh, monoclonals from rat against α6 by Arnoud Sonnenberg (Amsterdam, The Netherlands), against β4 by Stephen J. Kennel (Oak Ridge, Tenn., USA) and Rita Falcioni (Rome, Italy). Rabbit and goat antibodies against ‘classical’ laminin (laminin-1; possibly reacting with other isoforms sharing the α1-, β1- or τ1-chain) and type IV collagen were generously provided by Jean-Michel Foidart (Liège, Belgium), against mouse and human nidogen by Rupert Timpl (Martinsried, FRG), bullous pemphigoid antigen-1 (against a peptide of BPAG-1, the 230-kDa component) by John Stanley (Bethesda, MD, USA), laminin-5 (or isoforms sharing α3 or β3) as well as mouse monoclonals against the laminin subunits α3 (165-kDa) and β3 (140-kDa) by Robert Burgeson (Charlestown, Mass., USA) and Peter Marinkovich (Stanford, Calif., USA). The clone specifications of monoclonal antibodies including references are listed in Table 1. Mouse monoclonal antibodies against the laminin α1chain (clone III) and rabbit antibodies against fibronectin and tenascin were purchased from Telios (San Diego, Calif., USA), fluorochrome conjugated secondary antibodies from Biotrend (Cologne, FRG), Dianova (Hamburg, FRG) and Sigma (Munich, FRG). Sections were examined and photographed on a Leica DMBRE/RD photomicroscope. In some cases (indicated in the figure legends) there was intense ‘non-specific’ staining in the cornified epithelial layers or deposits from the wound exudate on top. This was usually more pronounced when applying monoclonal and secondary anti-mouse antibodies at earlier and intermediate time points. In part this may reflect a reaction with debris of grafted cells or host leukocytes as suggested by the strong nuclear staining (Hoechst) in the top layer. Proliferation assays Animals were injected i.v. with 100 µl solution of 5-bromodeoxyuridine (BrdU) and deoxycytidine (both 65 mM; Sigma, St. Louis, USA) in phosphate buffered saline 2 h before sacrifice. Nuclear incorporation of BrdU in DNA replicating cells was detected on cryostat sections with a monoclonal antibody against BrdU (Partec, Arlesheim, Switzerland) after 15 min fixation in acetone and 10 min incubation with 2N-HCL, both at RT [59]. Total nuclear staining was achieved by bisbenzimide (5 µg/ml; Hoechst, Germany) added to the secondary antibodies. This procedure still allowed the simultaneous detection of some tissue markers by dou-

ble staining, such as several ECM- or BM-proteins, keratins K1, K10 and the integrin β4-chain, although with varying intensity, whereas the reaction with other integrin antibodies for example was completely abolished.

Results Restoration of epidermal morphology and differentiation in transplants Transplantation of mouse and human keratinocytes on compatible host connective tissue provides optimal conditions for the restoration of the epidermal phenotype including the synthesis of respective intra- and extracellular structural proteins such as specific keratins, other differentiation markers and basement membrane components [2, 4–7, 47]. Also in the transplant series described herein, using cells precultured on a collagen matrix, the tissue architecture accomplished between 1 and 2 weeks was largely normal and closely resembled human interfollicular epidermis as shown in Fig. 1. This was obviously achieved in spite of the collagen gel separating the epithelium and the host connective tissue. Furthermore, there was no striking difference between epithelial grafts either on cell-free or fibroblast-containing collagen gels, except that the overall take rate and long-term viability considerably improved with fibroblasts. A compartment of mostly cuboid basal cells was clearly distinguishable at day 7 by labelling with antibodies recognizing keratins K5 [49] and K14 ([57], Fig. 1B), presumably in their native configuration, giving virtually the same staining pattern as in normal epidermis. Accordingly, there was a strong suprabasal expression of keratins K1 and K10 (Fig. 1C, D) and involucrin ([76], Fig. 1E) at this stage, as well as of the late differentiation markers filaggrin (Fig. 1F) and loricrin ([34], see below Fig. 1H) in upper regions corresponding to the marked granular layer seen by histology. Thus, regular differentiation patterns were established quite early (Fig. 1B-F) and there was only a moderate further accumulation, especially of late markers, in older transplants (compare Fig. 1G, H). Evolution of basement membrane structures By gross morphology there was no major change of epidermal structure between days 7 and 14 other than a further increase of horny layers. However, the formation of an ultrastructurally defined basement membrane (BM) and anchoring structures between the epithelium and connective tissue proceeded more slowly. Before day 7 electron microscopy revealed only few cell-membraneassociated structures resembling hemidesmosomes with extracellular dense plaques underneath (not shown). On day 7 extended stretches of lamina densa had become apparent (Fig. 2A) with some fine bridging structures representing anchoring filaments (Fig. 2B). At 2 and 3 weeks the basal poles of keratinocytes were regularly decorated with hemidesmosomes and the lamina densa

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Fig. 1A–H Epidermal phenotype of keratinocyte transplants. Histology at day 12 (A) and epidermal differentiation visualized by indirect immunofluorescence at day 7 (B–F) and day 22 (G, H). Basal cell compartment (B) indicated by keratin K14 staining; differentiation markers keratin K10 (C, D, G) [in (D) higher antibody dilution and counterstaining with anti-BPAG1, double exposure], involucrin (E)

and corresponding (double stain) filaggrin (F), and loricrin (H). CM, collagen matrix; dotted lines and arrows in (C, D) indicate epithelialmatrix border zone. Note that especially at earlier stages as seen in B there is frequently a marked reaction in the top layers which may be either ‘non-specific’ or in some cases due to cellular debris or wound fluid deposits. All same magnification, scale bar, 50 µm&ig.c:/f

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Fig. 2A–D Ultrastructural assembly of basement membrane (BM) zone, showing discontinuous basal lamina at day 7 (A), at higher magnification (B) showing numerous hemidesmosomes. Continuous lamina densa at day 14 (C) and day 21 (D), arrows depict anchoring filaments and arrowheads anchoring fibrils. (A, B) BK, basal keratinocytes, CM, collagen matrix; scale bars, 1000 nm (A), 250 nm (B, D), 500 nm (C)&ig.c:/f

was largely continuous (Fig. 2C, D). The anchoring filaments spanning the lamina lucida became more defined and, particularly at 3 weeks, anchoring fibrils could also be clearly distinguished, showing a typical cross-striation pattern as described in epidermis [1, 22, 54]. The earliest extracellular matrix component continuously lining the lower epithelial surface was laminin-5 (kalinin [61]; new nomenclature [10]) at day 1, followed by nidogen [23], which gave a similar staining pattern by

immunofluorescence at day 4 (Fig. 3A–C). While the laminin-5 reaction was limited to the epithelium, nidogen also stained blood vessels in the host tissue (Fig. 3C [23, 51]). The specificity of the polyclonal laminin-5 antibodies was further confirmed by monoclonal antibodies against the laminin-5 subunits α3 (not shown) and β3 (Fig. 3B, D). Staining for the major BM constituents laminin-1 (Fig. 3F) and type IV collagen (compare Fig. 6A) was still relatively weak and partly diffuse on day 4 in contrast to blood vessels (Fig. 3F’; described below). For the sake of clarity we refer to laminin-1 and -5 as the major epidermal forms here, being aware that other isoforms should be present as well, but presumably at very low levels (for a detailed comment on immune detection see the next paragraph). Also the hemidesmosomal component bullous pemphigoid antigen 1 (BPAG1, the 230-kDa protein [70]) was barely detectable at this stage. All these BM constituents revealed a distinct linear demarkation of the epithelium after 1 week (Fig. 3D–G) becoming equally prominent including BPAG-1 (compare Fig. 1D). In a few cases there was some diffuse staining of the collagen matrix below, which disappeared by 11 or 14 days concomitant with increasing staining of the BM zone. Another phenomenon, not studied here in much detail, was the abundance of the extracellular matrix (ECM) molecules tenascin and fibronectin at the graft site and in the supporting collagen matrix at early stages (not shown). In particular, tenascin presumably promotes the migratory activity of the host connective tissue cells as well as keratinocytes (for a review, see [17]) and possibly the ingrowth of capillaries and blood vessels, showing an intense BM-staining below the epithelium (compare Fig. 3C, F’ and Fig. 6F, G). After 11–14 days the intensity of the reaction for laminin-1, type IV collagen and perlecan (BM type heparansulfate proteoglycan; not shown) at the junctional zone did not increase further and was comparable to the quite constant levels of laminin-5 (as shown in Fig. 5E’) and nidogen. This was estimated by comparing the reaction of vessels and septae around striated muscle within the host connective tissue providing a reliable internal standard (Fig. 3C, F’) and by staining with the individual BM-antibodies at various time points in parallel. Aparently, there was no striking difference in the time course when the reaction with antibodies against individual laminin subunits [10, 51] such as the α1 chain was compared with laminin-1 (composition α1, β1, τ1) and α3 or β3 with laminin-5 (α3, β3, τ2), respectively. As annotated for the reaction with polyclonal antibodies, this does not exclude the presence of other laminin isoforms sharing these subunits such as laminin-6 (α3, β1, τ1), which has been found associated with laminin-5 [10]. For that reason laminin-1 and -5 could be slightly overestimated, but for the presumably low abundance of the other forms this should not change the overall picture of the proceeding BM-assembly. In contrast to the other BM-constituents, type VII collagen, considered a late marker of epidermal anchorage [45, 46, 54], was very

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Fig. 3A–K Time course of deposition of BM-components. Early expressed components at BM-zone (left column, A–E): reaction against laminin-5 day 1 (A) and laminin-5 β3 chain day 4 (B) and day 7 (D), nidogen day 4 (C; inset: reaction of muscle tissue and blood vessels) and day 7 (E). Delayed deposition (right column, F–K) of laminin-1-positive components day 4 (F; F’, blood ves-

sels) and day 11 (H; increased staining), collagen IV day 7 (G), and collagen VII showing at day 11 (I) focal staining (arrowheads; arrows, BM-zone) and at day 22 (K) a continuous lining. For ‘non-specific’ staining in top layers (B, D, I) compare legend of Fig. 1. Lower magnification in inset of (C), (F, F’) and (G), scale bars, 50 µm&ig.c:/f

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Fig. 4A–I Restoration of α6β4 and β1-integrin patterns. Left (A–E): partial pericellular α6- (A) and polar β4-location (B) facing the BM-zone at day 4. Proceeding normalization of α6 at d7 (C) and day 14 (D) eventually collocating with β4 (E, day 22). Gradual restriction of β1-integrins (F–I) from extended basal-su-

prabasal location at day 4 (F) and day 7 (G) to basal pericellular location at day 11 (H), corresponding laminin-5 double staining (H’) of somewhat lower intensity than usual (compare Fig. 5E’), and day 22 (I). All except E same magnification, scale bars, 50 µm&ig.c:/f

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Fig. 5A–H Normalizing patterns of integrin α-subunits. Corresponding normalization of α2β1 (A–C) and α3β1 (D–F) integrins from day 4 (A, D) to day 7 (B, E) and day 11 (C, F), α3 chains showing an initially more distinct surface distribution then α2. Double staining in (E’) for laminin-5 (L5), corresponding to α3

(E), is representative in intensity also for later stages. Integrin chain αv at day 4 (G) and day 7 (H), decreasing with epidermal normalization. The staining within the collagen matrix for α2 and αv (similar to β1 in Fig. 4F) at day 4 demarcates in part activated human fibroblasts. All same magnification, scale bar, 50 µm&ig.c:/f

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faint at day 7 and formed some distinct patches around 2 weeks but no continuous line before 3 weeks (Fig. 3I, K), which paralleled its late ultrastructural assembly in anchoring fibrils (compare Fig. 2 and [54]). In general, grafts on collagen without fibroblasts revealed similar kinetics of synthesis and deposition of BM components as far as was examined (e.g. laminin-1, laminin-5, collagen IV) and of BPAG-1 by the grafted keratinocytes. Apparently, the effect of the host connective tissue at the graft site was sufficient for BM formation and the human fibroblasts were not required in this regard. Redistribution of integrins – Correlation to other tissue markers Under regular culture conditions all integrins present in human epidermis were found in keratinocytes, consistent with previous reports [13, 15, 19, 50]. Accordingly, integrins α2β1 and α3β1 decorated mainly cell-cell contacts, while α6β4 was seen at the basal pole in a patchy pattern facing the surface of the substratum but also in cell-free areas as presumable footprints of migrating cells. Virtually no integrin staining was observed in cells harbouring differentiation markers, such as keratins K1 and K10, which were mainly in a suprabasal position (data not shown). In transplants at early phases of epithelial assembly until day 4, the α6 integrin chain was present at the epithelial-matrix interface in large amounts, but there was also marked, mainly pericellular, α6 staining within the epithelium. Thus, in contrast to normal epidermis, part of the α6-chain was obviously not associated with β4, which was more restricted to the developing BM zone (Fig. 4A, B). At day 7 α6 showed a strong reaction at the interface, but still decorated the lateral surface of some basal cells (Fig. 4C), while β4 was exclusively located at the BM zone. Similar to α6, in early transplants the staining for β1 integrins (α2β1, α3β1) was diffuse and extended to suprabasal cell layers, besides a variably intense reaction at the matrix interface (Fig. 4F). After 1 week, the β1 distribution became more restricted and showed the highest concentration in the basal or the lowermost two layers (Fig. 4G). Normalization of the β1integrins and α6β4 continued with time, reaching an epidermis-like configuration between 11 and 14 days (Fig. 4D, H). This was accompanied by a slight decrease in intensity which apparently correlated to the normalizing composition of the BM components. The final tissue consolidation with a well-defined BM was underlined by strictly pericellular β1-location in the basal layer (Fig. 4I; see also below, α2 and α3), clearly contrasting to suprabasal keratins, and perfect coalignment of α6 and β4 at the basal pole (Fig. 4D, E) together with BPAG-1 (compare Fig. 1D). The question arose as to whether there were differences between the β1-family members α2β1 and α3β1, that would provide some hints as to differences in func-

tion in the course of epidermal reconstruction. From days 4 to 7 the distribution of α2 was more diffuse with a slightly higher concentration at the epidermal collagen interface (Fig. 5A, B), while α3 showed right from the beginning a more pronounced pericellular staining, which gradually diminished towards upper cell layers (Fig. 5D). Furthermore, confinement of α3 to the basal layer apparently occurred faster, to a large part colocalizing with laminin-5 (Fig. 5E, E’; day 7). With time, both α2 and α3 became relatively more concentrated at the lateral surface of basal cells, suggesting that the β1-integrins were largely replaced at the basal pole by α6β4 during the formation of more stable basal cell-matrix contacts (Fig. 5C, F; day 11, corresponding to Fig. 4H). Additional integrins have been previously found in activated keratinocytes, induced in response to wounding or during maintenance in culture, for example α5β1 and αvβ5 [16, 19, 20, 31, 44, 75]. Accordingly, the αv chain was prominent during the initial phase of tissue restoration (Fig. 5G, day 4) resembling roughly the α3 pattern but decreasing with the proceeding normalization (Fig. 5H, day 7). Beyond day 7 αv usually vanished but was still seen in hyperplastic areas of older grafts, mainly at the lower epithelial margins. Preliminary data showed epithelial staining at this stage for the human β5chain but not β3 (αvβ3 is expressed in newly formed capillaries [9]), confirming the presence of αvβ5 dimers in our transplants as reported for epidermis [43, 50, 75], while the other forms, αvβ1 and αvβ6 [80], might be expressed as well. The reaction for the α5 chain (α5β1) was rather weak, diffusely associated with the matrix interface, and only clearly visible very early, up to day 4 (not shown, [16]). Finally, typical of early stages (day 4) was a pronounced, but variable reaction within the graft-supporting matrix, particularly when using mouse monoclonal antibodies against α2, αv and β1 (Figs. 4F, 5A, G). This indicates an activated state of the human fibroblasts, although in some samples it also correlated with the degree of host cell infiltration at the graft site. Changes in proliferation pattern with grafting time For the kinetics of proliferative activity, the keratinocyte transplants with human skin fibroblasts (embedded in the collagen support, see Methods) were examined at various times. In transplants without fibroblasts, proliferation was more variable and generally less. Labeled nuclei (BrdU incorporation) were detected as early as 1 day after grafting, but usually epithelia were only two to three layers thick within the first 3 days and therefore difficult to analyze. As shown in Fig. 6A, on day 4 there were numerous replicating cells, in some areas reaching up to 30% of basally located cells and to a large part being in a suprabasal position, which was underlined by double staining with antibodies against type IV collagen or laminin-1. The relatively weak BM-reaction seen in these assays indicated that at the earlier phases of BM-assembly

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Fig. 6A–G Establishment of regular basal cell restricted proliferation. Proliferation (BrdU-label, red or orange nuclei) is initially high and not restricted to the basal cell layer as seen in (A) but starts to normalize already in some areas (B), both day 4. The lower rate is by and large maintained throughout as shown for day 7 (C), day 11 (D, E), day 14 (F), day 22 (G) and day 28 (H), the

proliferating cells being strictly associated with the BM-zone. In F the BM-zone is tilted and therefore appears as a broader band. All anti-BrdU stain, counterstaining for laminin-1 (A, D, G, F) and collagen IV (B, E, H), blue nuclear stain (Hoechst) in (C; no BM stain) and (E). All except E same magnification, scale bars, 50 µm

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(up to day 11; Fig. 6A–E) the major components were not only present in lower amounts, but were also more susceptible to the HCl-treatment necessary for the BrdU detection (compare Fig. 3F–H). In some areas of the 4day transplants, there was already a marked reduction of proliferating cells, becoming more restricted to the basal position (Fig. 6B), as a first sign that the transition towards tissue balance was beginning. The continuing normalization became apparent on days 7 and 11 by fewer and strictly basally located labeled cells (Fig. 6C–E), which corresponded to the marked lining by BM material shown before (Fig. 3G, H). By and large a moderate proliferation rate within this range was maintained throughout the further observation period (days 14, 22 and 28; Fig. 6F–H). Occasionally even at the later times we saw clusters of positive cells which were notwithstanding strictly aligned at the BM zone (not shown). There was no obvious correlation between local variation of mitotic activity and the degree of vascularization or endothelial proliferation within the graft bed (Fig. 6F–H). Taken together, the restoration of the regular proliferation pattern within the 2nd week was closely related to a normal distribution of integrins, progressing BM assembly and expression of epidermal differentiation markers. One clear exception to normality was the constant suprabasal expression of keratin K16 (together with K6 according to protein analysis [6]) apparently reflecting a persisting irritation at the graft site rather than a permanent hyperproliferative state (compare discussion in [71]). The K16 staining patterns (not shown) were virtually indistinguishable from those of K1 and K10.

Discussion In this keratinocyte transplantation model a distinct time course of epidermal tissue organization, proliferation, integrin localization in correlation to basement membrane formation and onset of differentiation was observed, which corresponds to the changes during healing of deep human skin wounds. Since the keratinocytes studied interact through a collagen-type-I matrix with nude mouse connective tissue, this provides a reproducible starting point from which to study the sequence of events following epithelial wound closure. The initial activated state of epithelial assembly (culminating around day 4) was signified by (i) numerous proliferating cells, also in a suprabasal position, (ii) strong and expanded expression of most of the constitutive epidermal integrin chains (α2, α3, β1, α6), (iii) de novo synthesis of αv and α5 integrins, and (iv) the presence of basal-cell-specific keratins in virtually all layers, while (v) the epithelial matrix interface was largely depleted of most basement membrane (BM) components, but strongly marked by laminin-5 and nidogen. During the following normalization phase (i) the proliferation rate dropped dramatically after day 4 and (ii) between days 7 and 11 epithelial polarity was restored, indicated

by the strictly basal pericellular distribution of β1-integrins (reduced to normal levels) and distinct α6β4 lining of the basal-matrix interface. (iii) This coincided with the downregulation of αv and α5, and (iv) regular epithelial location of differentiation markers. (v) The classical BM components (e.g. laminin-1, type IV collagen) became equally prominent, corresponding to distinct ultrastructural BM structures, while the late appearance of type VII collagen indicated the completion of epidermal reconstruction in the final consolidation phase. Regeneration of human epidermis has been studied in suction blisters or in surgical wounds [16, 32, 75] and alternatively, for the similar physiology, in pig skin wounds [29]. Our data reflect by and large the processes induced by surgical wounding where BM-structures have been destroyed, and the time-course of normalization was comparable in both situations, being completed after 2–3 weeks. Similarly, in those cases mainly the integrin chains α2, α3, α6 and β1 expanded through the regenerating hyperplastic epidermis and there was strong de novo expression of αvβ5 and moderate induction of α5β1 [16, 20, 31, 44], but also a transiently apolar β4distribution in the migrating epithelial tongue [16]. Accordingly, similar but long-lasting activated integrin patterns were found in psoriatic lesions representing a chronical hyperplastic situation [20, 32], and in turn forced suprabasal integrin expression induced a psoriatic phenotype in transgenic mice [12]. On the other hand, in the suction blister experiments, where the detached epidermis including BM-structures remained largely intact, the integrin activation pattern was delayed and most strikingly affected α6 [32]. This may reflect merely a hyperplastic reaction or a response to eventual BM-remodeling. Cell interactions with extracellular matrix (ECM) molecules are indispensible for the growth of normal epidermal keratinocytes, like other epithelial cells [53, 62, 79]. The large panel of integrins constitutively expressed in epidermis or induced (for example) in culture [19, 20, 50, 75] enables the keratinocytes to recognize a large variety of ligands. This includes circulating matrix components such as fibronectin and vitronectin, but also ligands produced by the keratinocytes themselves, particularly the epidermis-specific laminin-5 (kalinin [14, 51, 52]). Thus, in our cell transplants the basal surface revealed a strong laminin-5 reaction already 1 day after grafting, followed by nidogen becoming comparably intense by day 4. At this early stage there were still only small amounts of laminin-1, type IV collagen and bullous pemphigoid antigen-1 (BPAG-1) detectable like during initial epithelial wound coverage [29]. The possibility that other laminin isoforms may be detected as well by the antibodies used shall not be discussed here in detail because of their assumed low abundance (for further comments, see Methods and Results). High-affinity receptors for laminin-5 are the integrins α3β1 and α6β4, and both were concentrated at the lower epithelial surface of the 4-day grafts in addition to variable pericellular staining (Figs. 4 and 5D). On the other hand, α3β1,

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as a promiscous receptor, should bind to type I collagen in the grafts, like α2β1 which recognizes various collagens. Whereas α6β4 [13] and α3β1 [41] seem to have stabilizing and possibly antimigratory properties, α2β1 is apparently involved in cell movements on collagen matrices such as BM-denuded dermis. Preferential binding to type I collagen would also explain the initially higher concentration of α2β1 at the epithelial base (Fig. 5A, B). Accordingly, in vitro keratinocyte migration on collagen-coated filters was completely inhibited by blocking antibodies against α2 and β1 [64]. The actual contribution to cell-cell binding, as implied by the apicolateral β1-integrin distribution and homeotypic interactions between integrin-transfected cells and integrin-coated surfaces [72], should be low in comparison to cadherin- and desmosome-mediated adhesion. Instead, their major function then is probably to cooperate as membrane organizers with growth regulators, as discussed below. The initially larger extension of α6staining, compared to β4, and its longer lasting partially lateral dislocation from the basal pole, may indicate the presence of some α6β1, which binds laminin-1 with a higher affinity than laminin-5 [33, 69] and is associated, in contrast to α6β4, with the microfilament system, like all the other integrins. The β4-subunits may be preferentially degraded [30] and recycling of internalized integrins has been reported as well, however so far only for intact α6β4 complexes [56]. Of the two other integrins, α5β1 and αvβ5, induced in activated keratinocytes [19, 20, 43, 50, 75] the classical fibronectin receptor α5β1 is expressed in epidermal keratinocytes during fetal development and at early stages of wound healing [16, 75, 80]. In the transplants there was only very faint staining at the matrix interface, while fibronectin accumulated directly underneath the epithelium as in experimental wounds [16] implying predominant interactions via α3β1 or perhaps αvβ1. The αv reaction was more pronounced, at early stages largely resembling the α3 distribution, and later closely correlated to a hyperplastic state. As a possible functional link, recently the plasminogen activator-receptor complex has been found to be associated with αvβ5 [58], possibly modulating keratinocyte adhesiveness. Accordingly, αvβ5 or other αv-complexes (αvβ1; αvβ6 [80]) may participate in tissue remodeling by facilitating intra-epithelial cell movements. The expanded distribution of α6- and β1-integrins at day 4 corresponds to the enlarged basal cell compartment according to the proliferative activity (BrdU-label) but also other basal cell markers such as keratins K5 and K14. Functional β1-integrins are supposed to stabilize the basal cell status, in part by counteracting the onset of differentiation [35, 75]. Therefore, β1 could well be an indicator of the actual proliferative potential and elevated β1-levels have been proposed as one criterion for an epidermal subpopulation harbouring putative stem cells [39]. In any case, the major steady state function of β1integrins should be to maintain basal cell properties by gating the necessary signals to this compartment. Such

cooperation of integrins and growth factors (e.g. plateletderived growth factor PDGF or epidermal growth factor EGF) and direct interaction with growth factor receptors has been reported (for a review, see [37, 62, 79]). Furthermore, integrin-mediated interactions between ECM and the cytoskeleton may provide guidance for the transport of receptors to distinct cell surface domains. In addition, our data suggest that a concerted action of the β1 integrins and α6β4 determines the positional control of functional basal cells. The stability of cell-matrix interactions apparently increases with α6β4-mediated contacts [13], which is in line with the gradual accumulation of BM-components and improving ultrastructure we observed. Concomitantly, α3β1 seemed to be largely replaced by α6β4 both competing for laminin-5 binding. If the assumption is correct that the β1-distribution spatially defines the compartment of proliferation-competent cells in concert with attachment via α6β4 [74], the downregulation of these integrin functions should be involved in the process of regaining tissue normalization, which includes the timely onset of differentiation. Although not evaluated at the protein level, our results imply a reduction, particularly of the β1- and αv-integrins with proceeding BM formation. The effect could be mediated by the steadily accumulating ligands such as laminin-1 and type IV collagen, but also by the changing ligand ratios, for example fibronectin and laminin-5 becoming relatively less prominent. The downregulation of integrin function could be accomplished at the level of expression, translation or processing as reported for the mammary gland [18]. Furthermore, misplaced integrin subunits may in part represent inactive forms prone to be turned over [35, 74]. This should be the case for those β1-complexes, which show at intermediate stages (around day 7) some overlap with early differentiation markers (e.g. keratin K1 and K10) in the first suprabasal layer. Functional inactivation would also explain why proliferation is normalized considerably faster than the integrin patterns. One major advantage of our model is the ability to work with authentic human epithelia, while avoiding individual variability in patients. It also provides an option to test the effects of different matrices and their biocompatibility, and also the supporting effects of additional dermal resident cells. Other cells forming surface epithelia can be applied as well [47], as can corresponding immortalized [4] and tumorigenic cells [6, 8, 63], for study of their specific matrix interactions. The importance of integrins in this scenario should be testable by blocking or activating functions either by injecting specific antibodies and small peptides with respective recognition sites [37, 62, 78] or by molecular engineering of cells introducing regular, mutated or recombinant genes for individual integrins [12, 30]. Taken together, our data support the view of a dual task of integrins, first to mediate mechanical force and second to determine the status of keratinocyte competence for responding to signals. Thus, α6β4 as a strong and stable anchor of the basal cells is supposed to pro-

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vide tissue polarity and spatial information directing the subsequent steps of differentiation, while α2β1 and α3β1 should mainly contribute to initial but reversible cell adhesion to various ECM components [13, 15] still allowing cell movements [64]. In their function as structure-linked informational devices these integrins seem to guard the basal cell properties by actively blocking cell entry into the terminal differentiation pathway [24, 35, 53, 62, 75] independently of the presence of positive or negative growth regulators. To be effective during epidermal regeneration after wounding, when BM structures are disrupted, this has to be in tune with the molecular setting of the stepwise remodeling ECM. Among the classical wound factors transforming growth factor beta (TGF-β) isoforms stimulate the synthesis of various integrins and also ECM components in keratinocytes [29, 67, 80]. After the initial activation, the β1-integrins may become largely dispensable for matrix adhesion as soon as stable attachment on a defined BM is established. The actual fine-tuning seems to be accomplished by the differing affinities of the individual matrix-receptors for the various ligands changing with tissue regeneration. This is further modulated by the transiently higher concentration of components such as fibronectin and tenascin, which may have less or even anti-adhesive properties (particularly tenascin [17]). To complete normalization, feedback mechanisms have to be activated, adjusting and eventually downregulating expression, surface location and functionality of integrins in concert with the changes in the ligand make-up of the ECM and the spectrum of diffusible factors. On that basis it can be postulated that the integrins in the lower epidermis have a dual switch function interactive with the ECM below and with the differentiating compartment above, thus representing a crucial element for establishment and maintenance of the proliferating compartment as well as the regular onset of terminal differentiation in tissue homeostasis. Acknowledgements Our special thanks for providing specific antibodies are due to Drs. Daniel Hohl, Irene Leigh, Dennis Roop, Tung-Tien Sun and Fiona Watt (differentiation markers, keratins), John D. Aplin, Eva Bröcker, Rita Falcioni, Martin Hemler, Stephen J. Kennel, Eberhard Klein, Vito Quaranta, Arnoud Sonnenberg and Giovanna Zambruno (integrins), Robert Burgeson, JeanMichel Foidart, M. Peter Marinkovich, John Stanley and Rupert Timpl (components of basement membrane zone). The excellent technical assistance of Gabi Blaser and Anke Wollschläger is gratefully acknowledged as well as the valuable contributions made by Drs. Petra Boukamp, Alain Limat, Hans Smola and Gabi Thiekötter concerning cell culture, design and performance of the transplantation experiments. Last but not least we would like to thank the photo department for expert work and Martina Kegel for typing and stylistic improvements.

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