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The importance of laminin 5 in the dermal–epidermal basement membrane
Journal of Dermatological Science 25 Suppl. 1 (2000) S51 – S59 www.elsevier.com/locate/jdermsci
The importance of laminin 5 in the dermal–epidermal basement membrane Toshio Nishiyama a,*, Satoshi Amano a, Makoto Tsunenaga a, Kuniko Kadoya b, Akira Takeda b, Eijiro Adachi c, Robert E. Burgeson d a Shiseido Life Science Research Center, Yokohama, Japan Department of Plastic and Reconstructi6e Surgery, Kitasato Uni6ersity School of Medicine, Sagamihara, Japan c Department of Molecular Morphology, Kitasato Uni6ersity Postgraduate Medical School, Sagamihara, Japan d The Cutaneous Biology Research Center, Massachsetts General Hospital and Har6ard Medical School, Charlestown, MA, USA b
Abstract The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal – dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal–dermal junction is essential to stability of the skin. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Laminin 5; Basement membrane; Anchoring complex; Dermal – epidermal junction; Keratinocyte sheet; Transplantation
1. Introduction The basement membrane at the dermal – epidermal junction has been proposed to have many functions. As described later, its most obvious * Corresponding author. Tel.: +81-45-7884111; fax: + 8145-7887251. E-mail address: [email protected] (T. Nishiyama).
function is to tightly link the epidermis to the dermis. Another obvious function of the basement membrane is to determine the polarity of the epidermis and to provide a barrier to epidermal migration. Once the basement membrane has been assembled, the epidermal cells recognize the surface adjacent to the basement membrane as the basal surface. Stratification of the epidermis proceeds, with the proliferating cells remaining at-
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tached to the basement membrane and the daughter cells migrating into the upper layers [1 – 3]. It is assumed that the basement membrane influences the epidermal differentiation, to maintain the proliferative state of the basal layer. Under normal circumstances, basement membrane prevents the direct contact of epidermal cells with the dermis. However, following injury that penetrates or disrupts the basement membrane, the epidermal cells lose their contact with the basement membrane, and come in contact with naked dermis. Under these conditions, the epithelial cells modify their behavior to cover and close the wound. These behavioral changes include the upregulation of proteolytic enzymes and other changes that accompany conversion to a migratory phenotype. Other functions of the basement membrane derive from the positioning of the structure between the epidermal cells and the dermal cells. The epidermis and the dermis never function independently [4–6]. Instead, normal skin homeostasis requires the constant passage of signals back and forth between the two cell types. In general, these signals are small molecules made in one compartment that are thought to diffuse to the opposite compartment. These signals must cross the basement membrane. Components of the basement membrane can selectively facilitate or prevent the passage of these signals. In some cases, the signaling molecules are stored by the basement membrane and only released if the basement membrane is destroyed. The basement membrane between the epidermis and the dermis contains unique structures that maintain the attachment of the epidermis. The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis [7]. One of the key components of the anchoring complex is laminin 5 [8]. Past studies have shown that laminin 5 is essential to epidermal attachment, as mutations in the genes encoding the laminin 5 chains underlie the severe blistering phenotype of Herlitz’ junctional epidermolysis bullosa [9,10]. It is clear that laminin 5 constitutes the anchoring filaments and binds the transmembrane
hemidesmosomal integrin a6b4, which is known to be the receptor of laminin 5. With regard to binding of laminin 5 with either components of the basement membrane or of the papillary dermis, it has recently been elucidated that: (1) laminin 5 directly binds type VII collagen which forms the anchoring fibrils that insert into the papillary dermis [11]; and (2) laminin 5 forms a covalent complex with laminin 6 or 7 and this laminin 5–6/7 complex interacts with type IV collagen in the basement membrane through nidogen [12]. It was hypothesized that laminin 5 is a major contributor to epidermal–dermal stability. One corollary to this hypothesis is that laminin 5 might improve epidermal attachment in a variety of healing processes or clinical situations where epidermal–dermal attachment and basement membrane formation might be compromised. Therefore, the effect of laminin 5 during the cell culture of a skin equivalent model, and during keratinocyte sheet grafting has been investigated. The results strongly suggest that laminin 5 promotes epidermal attachment by increasing the rate of basement membrane formation.
2. Materials and methods
2.1. Purification of laminin 5 Human keratinocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The spent medium was filtered to remove cells. EDTA, PMSF, and N-ethylmaleimide were then added to final concentrations of 5 mM, 50, and 50 mM, respectively, to inhibit endogenous protease activity. To remove non-specific binding by fibronectin, the medium was first passed through gelatin-Sepharose (Pharmacia Biotech AB, Uppsala, Sweden) and then applied to an anti-laminin 5 affinity column in which 6F12, an anti-b3 chain monoclonal antibody, was conjugated to CN-activated Sepharose (Pharmacia Biotech AB, Uppsala, Sweden). Laminin 5 was eluted with 1 M acetic acid, followed by dialysis against phosphate-buffered saline (PBS). The purified laminin
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5 was composed primarily of the fully processed form, a3 (165 kDa), b3 (140 kDa) and g2 (105 kDa) [13,14].
2.2. Human keratinocytes and fibroblasts Human epidermal keratinocytes and dermal fibroblasts were isolated from human foreskin or skin sections discarded during plastic surgery using methods described in previous papers [13,14].
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prevent non-specific binding of antibodies. The sections were incubated with antibody in a humidified chamber for 1 h at 37°C. They were then incubated with biotinylated rabbit anti mouse IgG for 10 min and with avidin–biotin– peroxidase complex for 5 min in a humidified chamber. The sections were soaked for 5 min in 0.02% diaminobenzidine in PBS (pH 7.4) supplemented with 0.005% H2O2. They were counterstained with hematoxylin, dehydrated, cover-slipped, and finally examined under a light microscope.
2.3. Skin equi6alent model Human keratinocytes were grown in a modified serum-free keratinocyte growth medium (KGM) containing 0.03 mM Ca2 + . Human dermal fibroblasts were grown in DMEM with 10% FBS. Skin equivalents were prepared according to the modified method described in a previous paper [13]. The keratinocytes were cultured on top of a dermal equivalent consisting of type I collagen and fibroblasts in a three-dimensional gel [13], and the culture was raised to an air-liquid interface. The medium for the skin equivalent culture was prepared from a 1:1 mixture of KGM and DMEM supplemented with 10 % FBS and adjusted to a final concentration of 1.8 mM Ca2 + . The medium was changed every other day. The keratinocytes formed a stratified squamous epithelium within 7 days. The skin equivalents were subsequently cultured in the same medium supplemented with laminin 5 from day 7 through day 14. The medium containing laminin 5 was also changed every other day. The skin equivalents were processed for biochemical or morphological analysis on day 14 to evaluate the effect of exogenous laminin 5 on basement membrane formation.
2.4. Immunohistochemistry of skin equi6alents Fresh skin equivalents were snap-frozen in O.C.T compound (Sankyo Miles). Cryostat sections (6 mm) were cut, mounted on albumincoated slide glasses, and air-dried. They were rehydrated with PBS, and incubated with normal rabbit serum diluted with PBS at 1:5 ratio to
2.5. Preparation of keratinocyte sheets for grafting assay Human keratinocytes were cultured on a 3T3 feeder layer in keratinocytic growth medium prepared according to the method reported by Rheinwald and Green [15]. The keratinocytes formed sheet structure of several cell layers and were detached from the culture dish using dispase (Godoshuseki, Tokyo) by incubation at 37°C for 2.5 h. Sheets were washed twice with Hank’s solution and then cut into 1.5× 1.5 cm2 fragments. MEIPAC (Aterocollagen sheet, Meiji Seika, Tokyo) was used as a carrier for keratinocyte sheets. For group 1, laminin 5 was added to the surface of the basal keratinocyte sheets (diluted with DMEM to provide a final concentration of 1 mg/cm2). As a control (group 2), DMEM alone was added to the sheets. The sheets were incubated at 37°C for 15 min. After incubation, the sheets were washed twice with Hanks solution and then grafted.
2.6. Grafting procedure On the day of the experiments, the nude mice or rats were anesthetized with sodium pentobarbital. Skin flaps of 1.5 cm2 were elevated and fullthickness skin defects were made in the backs. The cultured human keratinocyte sheets were grafted onto the panniculus carnosus. The sheets of group 1 (mice, n= 12; rats, n=15) and group 2 (mice, n=12; rats, n= 15) were grafted. Silicone gauze was applied over the grafted sheets and the
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skin flap was replaced. The margin was then sutured with nylon.
2.7. Assessment of take rate and histological analysis The take rate was assessed by measurement of the area of existing epithelium 7 days after grafting. The wounds were photographed and the areas of epithelialization were quantitated. The rate of graft take was shown as a percentage of the original size of the grafting sheet. Excised grafts were fixed by the AMeX method [16]. After incubation with acetone, methyl benzoate, and xylene, the pieces of grafts were incubated with paraffin for 3 h at 60°C. These embedded tissues were used for immunohistochemical examination.
3. Results
3.1. Deposition of basement membrane components in cultured skin equi6alents Keratinocytes were cultured on top of dermal equivalents formed from type I collagen and fibroblasts raised to an air–liquid interface. As observed by light microscopy, the keratinocytes formed a stratified squamous epithelium within 7 days with the presence of basal, spinous, granular, and corneal cell layers. Immunohistochemical analysis showed that type IV collagen and the a3 chain of laminin (contained in laminins 5, 6 and 7) were deposited on the dermal–epidermal inter-
2.8. Transmission electron microscopy Skin equivalents and grafted skins were fixed by microwave irradiation [17] in Zamboni’s fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 7.4) and immersed in the same solution for 2 h on ice. The specimens were rinsed with 0.1 M phosphate buffer (pH 7.4) and processed as follows. Each sample was rinsed in 0.1 M phosphate buffer (pH 7.4), post-fixed with 1% osmium tetroxide, immersed in 1% tannic acid, dehydrated and embedded in Epon 812. Ultrathin sections of Epon-embedded sample were stained with 2% uranyl acetate and Raynolds’ lead citrate. The sections were examined with an electron microscope (Hitachi model H7100, Hitachi, Tokyo).
2.9. Antibodies The monoclonal antibodies (mAbs) BM-165 and 6F12 recognize a3 and b3 chains of laminin 5, respectively [18]. Anti-human type IV collagen mAb JK-199 [19] and anti-type VII collagen mAbs NP-32 and NP-185 [20] have been previously described. Laminin 1 pAb (catalog number L-9393), anti-vimentin pAb (catalog number V4630), and anti-keratin mAbs (catalog numbers C-8663 and C-7284) were purchased from Sigma (St. Louis, MO).
Fig. 1. Immunoperoxidase micrographs of type IV and VII collagens and laminin 5 in skin equivalents. Type IV (a,d) and VII collagens (b,e) and a3 chain of laminin (c,f) were found in the dermal – epidermal interface in 14-day skin equivalents. Specific staining for type IV collagen (a) and type VII collagen (b) in the skin equivalents supplemented with laminin 5 was observed more strongly than in control cultures (d,e). Specific staining for laminin 5 was restricted to the dermal – epidermal junction although the skin equivalents were cultured in laminin 5-supplemented medium (c). The bar represents 50 mm.
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Fig. 2. Electron micrographs showing the dermal–epidermal interface of skin equivalents cultured for 14 days in the medium without supplemental laminin 5. The lamina densa was rarely observed at the dermal–epidermal interface, the site in skin where lamina densa would be most expected. Banding fibrils are closely apposed to the basal surface of keratinocytes. The bar represents 100 nm.
face area in 14-day skin equivalents in the absence of exogenous laminin 5 (Fig. 1d,f). Only traces of type VII collagen were observed extracellularly at the dermal–epidermal junction and these were primarily restricted to areas adjacent to basal keratinocytes (Fig. 1b,e). Specific staining for type IV and VII collagens in skin equivalents supplemented with laminin 5 (Fig. 1a,b) was more intense than that in the control (Fig. 1d,e). Specific staining for the laminin a3 chain was also restricted to the dermal – epidermal junction even when skin equivalents were cultured in laminin 5-supplemented medium (Fig. 1c).
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membrane and the subepithelial dense layer, were observed in the skin equivalents supplemented with laminin 5 than in controls. Most laminae densae were connected to the hemidesmosomelike structures of basal keratinocytes by anchoring filaments traversing the lamina lucida, but a lamina densa was not found between these hemidesmosome-like structure (Fig. 3). By morphometrical analysis the averaged length of lamina densa per 1 mm of the dermal–epidermal interface was found to be increased by 180, 230, and 520% of control culture values when the skin equivalents were cultured in the medium supplemented with laminin 5 at the concentration of 1, 5, and 20 mg/ml, respectively (data not shown).
3.3. Impro6ement of keratinocyte sheet grafting with laminin 5 At 7 days postgrafting, attached and surviving keratinocyte sheets were observed on the transplanted area. The graft take was measured as the area of epithelialization in both nude mice and nude rats, the area of epithelialization being larger in group 1 (laminin 5-treated) than in group 2 (control). The results were: (A) nude mice: 58.59 21.9% in group 1, 38.39 30.5% in group 2; (B) nude rats: 53.1921.9% in group 1, 35.39 22.5%
3.2. Basement membrane formation in cultured skin equi6alents Little lamina densa was observed in the 14-day cultured skin equivalents in the absence of exogenous laminin 5. Close inspection revealed banding fibrils apposed to the basal surface of keratinocytes (Fig. 2). In contrast, focal areas of the lamina densa along the subepidermal surface were observed in 14-day cultured skin equivalents supplemented with laminin 5 (Fig. 3). More hemidesmosome-like structures, such as electron dense plaques along the inner leaflet of the cell
Fig. 3. Electron micrographs showing the dermal – epidermal interface of skin equivalents cultured for 14 days in medium supplemented with laminin 5 (5 mg/ml). Discontinuous laminae densae (arrowheads) were observed under the basal keratinocytes. The lamina densa is connected to hemidesmosomes of basal keratinocytes by anchoring filaments traversing the lamina lucida. The bar represents 100 nm.
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in group 2. Statistical analysis indicated that the difference was significant in both groups. At 7 days postgrafting, type IV collagen stained more strongly at the dermal – epidermal junction in group 1 than in group 2 by immunohistochemical staining (Fig. 4a,d). Alpha 3 chain of laminin also stained more strongly at the dermal – epidermal junction in group 1 than in group 2, as did type VII collagen (Fig. 4c,d and b,e). The effect of laminin 5 upon the ultrastructural organization of the basement membrane was examined by electron microscopy as shown in Fig. 5. At day 3 after grafting, the lamina densa of group 1 (laminin 5-treated, Fig. 5B) was more continuous than that of group 2 (control, Fig. 5A). This shows that early morphological organization of basement membrane components is caused by pretreatment with laminin 5. At day 7 after grafting, electron-dense plates characteristic of hemidesmosomes were observed in both groups and no significant differences in the continuity of the basement membrane were observed (data not shown).
4. Discussion The present study demonstrates that laminin 5 has the potential to accelerate formation of basement membrane in cultured skin equivalents. Addition of exogenous laminin 5 increased the length of lamina densa, which was recognized by an anti-type IV collagen antibody, in a concentration-dependent manner [13]. It was also found that laminin 5 increased the frequency of hemidesmosomal structures at the dermal – epidermal junction and that most laminae densae were associated with the hemidesmosomal structures. Regardless of the concentration of laminin 5 supplemented in the culture medium, the morphological sequence of the lamina densa formation in skin equivalents is unchanged and is as follows: (1) the appearance of hemidesmosomal structures along the inner leaflet of the subepidermal cell membrane of basal keratinocytes; (2) the focal appearance of the lamina densa with anchoring filaments in apposition to the hemidesmosomes; (3) the lateral elongation of the lamina densa [21].
The observation is consistent with the morphological sequence in embryos and young animals wherein the lamina densa also develops first in apposition to the hemidesmosomes [22]. From these results, it appears likely that the hemidesmosomal structures facilitate formation of the basement membrane by functioning as an initiation site in skin equivalents and that the increments of hemidesmosomal structures gained by the addition of laminin 5 accelerate the assembly of basement membranes in skin equivalents. Immunohistochemical analysis of laminin 5 showed that laminin 5 deposition was restricted to the dermal–epidermal junction and did not occur at any sites in the collagen gel or within the epidermis, even though laminin 5 was added to the culture medium. Basal keratinocytes are known to express a3b1 and a6b4 integrins [23– 25], which are known to be specific receptors for laminin 5 [26,27]. It is possible that exogenously added laminin 5 might bind to basal keratinocytes through laminin 5-specific integrins, a6b4 and a3b1, and then might be accumulated beneath these basal cells. The accumulation might accelerate hemidesmosome formation and might function as a nucleus for lamina densa formation. The present observations strongly suggest a unique role for laminin 5 in basement membrane formation in skin equivalents. Since purified and exogenously applied laminin 5 has the ability to bind keratinocytes to substrates and to enhance formation of the basement membrane, this molecule might have clinical utility. In this study, one also focused on the role and influence of laminin 5 in the take of grafted cultured human keratinocyte sheets and the formation of the basement membrane in vivo. Laminin 5 plays an important part in the adherence of epidermis to dermis in normal skin and the present study demonstrated that exogenous application of laminin 5 improves transplanted keratinocyte sheet attachment to the granulation tissue in wound beds, acting as a biological ‘glue’ [28]. The results suggest that the increase in successful keratinocyte sheet transplantation is due to both increased cell attachment and increased basement membrane formation. Therefore, laminin 5 may be clinically useful for the treat-
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Fig. 4. Deposition of type IV collagen (a,d), type VII collagen (b,e), and a3 chain of laminin (c,f) at the dermal – epidermal junction in immunostaining at 7 days post grafting. It is stained more strongly at the dermal – epidermal junction in group 1 (a – c) than in group 2 (d – f). The bar represents 100 mm. Fig. 5. The effect of laminin 5 on ultrastructural organization of basement membrane. At day 3 after grafting, the lamina densa of group 1 (A) is more continuous as compared to group 2 (B). The bar represents 0.5 mm.
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ment of severe burns either by partially or wholly overcoming the problem of keratinocyte graft stability, or in combination with dermal grafting methods. It is believed that these observations justify further studies of the efficacy of exogenous laminin 5 to increase keratinocyte graft take to human burn patients.
5. Conclusion In vitro and in vivo evidence indicates that laminin 5 participates in several essential functions in skin biology. From published reports [8,11,12] and the present studies, it was concluded that the function of laminin 5 is as follows. Laminin 5 has two functional domains which can bind integrins of basal keratinocytes and type VII collagen, forming anchoring fibrils, and the anchoring complex provides an essential link between the epidermal basal cells and the papillary dermis. This role of laminin 5 is essential to the resistance of the epidermis from external stress, such as frictional stress. Laminin 5 also accelerates the assembly of basement membranes at the dermal–epidermal junction. Therefore, laminin 5 is a key component not only for strongly linking the epidermis to the dermis, but also for the repair or regeneration of the basement membrane at the dermal–epidermal junction. An intact basement membrane at the dermal – epidermal junction is essential to the viability of the skin and participates in the selective flow of communication between the dermis and the epidermis.
References [1] Bohnert A, Hornung J, Mackenzie IC, Fusenig NE. Epithelial-mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes. Cell Tissue Res 1986;244:413 – 29. [2] Watt FM. Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis. J Cell Biol 1984;98:16–21. [3] Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 1987;84:2302–6.
[4] Finch PW, Rubin JS, Miki T, Ron D, Aaronson SA. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 1989;245:752 – 5. [5] Hirai Y, Takebe K, Takashina M, Kobayashi S, Takeichi M. Epimorphin: a mesenchymal protein essential for epithelial morphogenesis. Cell 1992;69:471 – 81. [6] Nishiyama T, Tsunenaga M, Akutsu N, Amano S, Nakayama Y. Role of dermal – epidermal communication on regulation of skin structure and function. In: Tagami H, Parrish JA, Ozawa T, editors. Skin: Interface of a Living System. Amsterdam: Elsevier BV, 1998:119 – 37. [7] Nishiyama T, Amano S, Burgeson RE. Structure and molecular assembly of the dermal – epidermal attachment complex in skin. Connect Tissue 1998;30:213 – 7. [8] Rousselle P, Lunstrum GP, Keene DR, Burgeson RE. Kalinin: an epithelium specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol 1991;114:567 – 76. [9] Pulkkinen L, Christiano AM, Airenne T, Haakana H, Tryggvason K, Uitto J. Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nat Genet 1994;6:293 – 8. [10] Aberdam DF, Galliano J, Vailly J, Pulkkinen L, Bonifas J, Christiano AM, Trygvasson K, Uitto J, Epstein EJ, Ortonne JP, Menneguzzi G. Herlitz’s junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for gamma 2 subunit of nicein/kalinin (LAMININ-5). Nat Genet 1994;6:299 – 304. [11] Rousselle P, Keene DR, Ruggiero F, Champliaud MF, Rest M, Burgeson RE. Laminin 5 binds the NC-1 domain of type VII collagen. J Cell Biol 1997;138:719 – 28. [12] Champliaud MF, Lunstrum GP, Rousselle P, Nishiyama T, Keene DR, Burgeson RE. Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. J Cell Biol 1996;132:1189– 98. [13] Tsunenaga M, Adachi E, Amano S, Burgeson RE, Nishiyama T. Laminin 5 can promote assembly of the lamina densa in the skin equivalent model. Matrix Biol 1998;17:603 – 13. [14] Amano S, Nishiyama T, Burgeson RE. A specific and sensitive ELISA for laminin 5. J Immunol Methods 1999;224:161 – 9. [15] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331 – 4. [16] Sato Y, Mukai K, Watanabe S, Goto M, Shimosato Y. The AMeX method. A simplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining. Am J Pathol 1986;125:431 – 5. [17] Mizuhira V, Hasegawa H, Notoya M. Microwave fixation and localization of calcium in synaptic vesicles. J Neurosci Methods 1994;55:125 – 36.
T. Nishiyama et al. / Journal of Dermatological Science 25 (2000) S51–S59 [18] Rousselle P, Aumailley M. Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors. J Cell Biol 1994;125:205–14. [19] Kino J, Adachi E, Yoshida T, Nakajima K, Hayashi T. Characterization of a monoclonal antibody against human placenta type IV collagen by immuno-electroblotting, antibody-coupled affinity chromatography, and immuno-histochemical localization. J Biochem 1988;103:829 – 35. [20] Sakai LY, Keene DR, Burgeson RE. Type VII collagen is a major component of anchoring fibrils. J Cell Biol 1986;103:1577– 86. [21] Hirone T, Taniguchi S. Basal lamina formation by epidermal cells in cell culture. In: Berstein IA, Seiji M, editors. Biochemistry of Normal and Abnormal Epidermal Differentiation. Tokyo: Tokyo Univ. Press, 1980:159–69. [22] Osawa T, Nozaka Y. Mouse epidermal basement membrane fixed by microwave irradiation, with special reference to half desmosomes. Connect Tissue 1992;23:65–70. [23] Hertle MD, Adams JC, Watt FM. Integrin expression during human epidermal development in vivo and in vitro. Development 1991;112:193–206.
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[24] Marchisio PC, Bondanza S, Cremona O, Cancedda R, De Luca M. Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes. J Cell Biol 1991;112:761 – 73. [25] Watt FM, Kubler MD, Hotchin NA, Nicholson LJ, Adams JC. Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions. J Cell Sci 1993;106:175 – 82. [26] Delwel GO, de Melker AA, Hogervorst F, Jaspars LH, Fles DL, Kuikman I, Lindblom A, Paulsson M, Timple R, Sonnenberg A. Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms. Mol Biol Cell 1994;5:203 – 15. [27] Niessen CM, Hogervorst F, Jaspars LH, de Malker AA, Delwel GO, Hulsman EH, Kuikman I, Sonnenberg A. The a6b4 integrin is a receptor for both laminin and kalinin. Exp Cell Res 1994;211:360 – 7. [28] Takeda A, Kadoya K, Shioya N, Tsunenaga M, Nishiyama T, Amano S, Burgeson RE. Pretreatment of human keratinocyte sheets with laminin 5 improves their grafting efficiency. J Invest Dermatol 1999;113:38 – 42.
The importance of laminin 5 in the dermal–epidermal basement membrane Toshio Nishiyama a,*, Satoshi Amano a, Makoto Tsunenaga a, Kuniko Kadoya b, Akira Takeda b, Eijiro Adachi c, Robert E. Burgeson d a Shiseido Life Science Research Center, Yokohama, Japan Department of Plastic and Reconstructi6e Surgery, Kitasato Uni6ersity School of Medicine, Sagamihara, Japan c Department of Molecular Morphology, Kitasato Uni6ersity Postgraduate Medical School, Sagamihara, Japan d The Cutaneous Biology Research Center, Massachsetts General Hospital and Har6ard Medical School, Charlestown, MA, USA b
Abstract The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal – dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal–dermal junction is essential to stability of the skin. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Laminin 5; Basement membrane; Anchoring complex; Dermal – epidermal junction; Keratinocyte sheet; Transplantation
1. Introduction The basement membrane at the dermal – epidermal junction has been proposed to have many functions. As described later, its most obvious * Corresponding author. Tel.: +81-45-7884111; fax: + 8145-7887251. E-mail address: [email protected] (T. Nishiyama).
function is to tightly link the epidermis to the dermis. Another obvious function of the basement membrane is to determine the polarity of the epidermis and to provide a barrier to epidermal migration. Once the basement membrane has been assembled, the epidermal cells recognize the surface adjacent to the basement membrane as the basal surface. Stratification of the epidermis proceeds, with the proliferating cells remaining at-
0923-1811/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0923-1811(00)00142-0
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tached to the basement membrane and the daughter cells migrating into the upper layers [1 – 3]. It is assumed that the basement membrane influences the epidermal differentiation, to maintain the proliferative state of the basal layer. Under normal circumstances, basement membrane prevents the direct contact of epidermal cells with the dermis. However, following injury that penetrates or disrupts the basement membrane, the epidermal cells lose their contact with the basement membrane, and come in contact with naked dermis. Under these conditions, the epithelial cells modify their behavior to cover and close the wound. These behavioral changes include the upregulation of proteolytic enzymes and other changes that accompany conversion to a migratory phenotype. Other functions of the basement membrane derive from the positioning of the structure between the epidermal cells and the dermal cells. The epidermis and the dermis never function independently [4–6]. Instead, normal skin homeostasis requires the constant passage of signals back and forth between the two cell types. In general, these signals are small molecules made in one compartment that are thought to diffuse to the opposite compartment. These signals must cross the basement membrane. Components of the basement membrane can selectively facilitate or prevent the passage of these signals. In some cases, the signaling molecules are stored by the basement membrane and only released if the basement membrane is destroyed. The basement membrane between the epidermis and the dermis contains unique structures that maintain the attachment of the epidermis. The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis [7]. One of the key components of the anchoring complex is laminin 5 [8]. Past studies have shown that laminin 5 is essential to epidermal attachment, as mutations in the genes encoding the laminin 5 chains underlie the severe blistering phenotype of Herlitz’ junctional epidermolysis bullosa [9,10]. It is clear that laminin 5 constitutes the anchoring filaments and binds the transmembrane
hemidesmosomal integrin a6b4, which is known to be the receptor of laminin 5. With regard to binding of laminin 5 with either components of the basement membrane or of the papillary dermis, it has recently been elucidated that: (1) laminin 5 directly binds type VII collagen which forms the anchoring fibrils that insert into the papillary dermis [11]; and (2) laminin 5 forms a covalent complex with laminin 6 or 7 and this laminin 5–6/7 complex interacts with type IV collagen in the basement membrane through nidogen [12]. It was hypothesized that laminin 5 is a major contributor to epidermal–dermal stability. One corollary to this hypothesis is that laminin 5 might improve epidermal attachment in a variety of healing processes or clinical situations where epidermal–dermal attachment and basement membrane formation might be compromised. Therefore, the effect of laminin 5 during the cell culture of a skin equivalent model, and during keratinocyte sheet grafting has been investigated. The results strongly suggest that laminin 5 promotes epidermal attachment by increasing the rate of basement membrane formation.
2. Materials and methods
2.1. Purification of laminin 5 Human keratinocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The spent medium was filtered to remove cells. EDTA, PMSF, and N-ethylmaleimide were then added to final concentrations of 5 mM, 50, and 50 mM, respectively, to inhibit endogenous protease activity. To remove non-specific binding by fibronectin, the medium was first passed through gelatin-Sepharose (Pharmacia Biotech AB, Uppsala, Sweden) and then applied to an anti-laminin 5 affinity column in which 6F12, an anti-b3 chain monoclonal antibody, was conjugated to CN-activated Sepharose (Pharmacia Biotech AB, Uppsala, Sweden). Laminin 5 was eluted with 1 M acetic acid, followed by dialysis against phosphate-buffered saline (PBS). The purified laminin
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5 was composed primarily of the fully processed form, a3 (165 kDa), b3 (140 kDa) and g2 (105 kDa) [13,14].
2.2. Human keratinocytes and fibroblasts Human epidermal keratinocytes and dermal fibroblasts were isolated from human foreskin or skin sections discarded during plastic surgery using methods described in previous papers [13,14].
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prevent non-specific binding of antibodies. The sections were incubated with antibody in a humidified chamber for 1 h at 37°C. They were then incubated with biotinylated rabbit anti mouse IgG for 10 min and with avidin–biotin– peroxidase complex for 5 min in a humidified chamber. The sections were soaked for 5 min in 0.02% diaminobenzidine in PBS (pH 7.4) supplemented with 0.005% H2O2. They were counterstained with hematoxylin, dehydrated, cover-slipped, and finally examined under a light microscope.
2.3. Skin equi6alent model Human keratinocytes were grown in a modified serum-free keratinocyte growth medium (KGM) containing 0.03 mM Ca2 + . Human dermal fibroblasts were grown in DMEM with 10% FBS. Skin equivalents were prepared according to the modified method described in a previous paper [13]. The keratinocytes were cultured on top of a dermal equivalent consisting of type I collagen and fibroblasts in a three-dimensional gel [13], and the culture was raised to an air-liquid interface. The medium for the skin equivalent culture was prepared from a 1:1 mixture of KGM and DMEM supplemented with 10 % FBS and adjusted to a final concentration of 1.8 mM Ca2 + . The medium was changed every other day. The keratinocytes formed a stratified squamous epithelium within 7 days. The skin equivalents were subsequently cultured in the same medium supplemented with laminin 5 from day 7 through day 14. The medium containing laminin 5 was also changed every other day. The skin equivalents were processed for biochemical or morphological analysis on day 14 to evaluate the effect of exogenous laminin 5 on basement membrane formation.
2.4. Immunohistochemistry of skin equi6alents Fresh skin equivalents were snap-frozen in O.C.T compound (Sankyo Miles). Cryostat sections (6 mm) were cut, mounted on albumincoated slide glasses, and air-dried. They were rehydrated with PBS, and incubated with normal rabbit serum diluted with PBS at 1:5 ratio to
2.5. Preparation of keratinocyte sheets for grafting assay Human keratinocytes were cultured on a 3T3 feeder layer in keratinocytic growth medium prepared according to the method reported by Rheinwald and Green [15]. The keratinocytes formed sheet structure of several cell layers and were detached from the culture dish using dispase (Godoshuseki, Tokyo) by incubation at 37°C for 2.5 h. Sheets were washed twice with Hank’s solution and then cut into 1.5× 1.5 cm2 fragments. MEIPAC (Aterocollagen sheet, Meiji Seika, Tokyo) was used as a carrier for keratinocyte sheets. For group 1, laminin 5 was added to the surface of the basal keratinocyte sheets (diluted with DMEM to provide a final concentration of 1 mg/cm2). As a control (group 2), DMEM alone was added to the sheets. The sheets were incubated at 37°C for 15 min. After incubation, the sheets were washed twice with Hanks solution and then grafted.
2.6. Grafting procedure On the day of the experiments, the nude mice or rats were anesthetized with sodium pentobarbital. Skin flaps of 1.5 cm2 were elevated and fullthickness skin defects were made in the backs. The cultured human keratinocyte sheets were grafted onto the panniculus carnosus. The sheets of group 1 (mice, n= 12; rats, n=15) and group 2 (mice, n=12; rats, n= 15) were grafted. Silicone gauze was applied over the grafted sheets and the
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skin flap was replaced. The margin was then sutured with nylon.
2.7. Assessment of take rate and histological analysis The take rate was assessed by measurement of the area of existing epithelium 7 days after grafting. The wounds were photographed and the areas of epithelialization were quantitated. The rate of graft take was shown as a percentage of the original size of the grafting sheet. Excised grafts were fixed by the AMeX method [16]. After incubation with acetone, methyl benzoate, and xylene, the pieces of grafts were incubated with paraffin for 3 h at 60°C. These embedded tissues were used for immunohistochemical examination.
3. Results
3.1. Deposition of basement membrane components in cultured skin equi6alents Keratinocytes were cultured on top of dermal equivalents formed from type I collagen and fibroblasts raised to an air–liquid interface. As observed by light microscopy, the keratinocytes formed a stratified squamous epithelium within 7 days with the presence of basal, spinous, granular, and corneal cell layers. Immunohistochemical analysis showed that type IV collagen and the a3 chain of laminin (contained in laminins 5, 6 and 7) were deposited on the dermal–epidermal inter-
2.8. Transmission electron microscopy Skin equivalents and grafted skins were fixed by microwave irradiation [17] in Zamboni’s fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer pH 7.4) and immersed in the same solution for 2 h on ice. The specimens were rinsed with 0.1 M phosphate buffer (pH 7.4) and processed as follows. Each sample was rinsed in 0.1 M phosphate buffer (pH 7.4), post-fixed with 1% osmium tetroxide, immersed in 1% tannic acid, dehydrated and embedded in Epon 812. Ultrathin sections of Epon-embedded sample were stained with 2% uranyl acetate and Raynolds’ lead citrate. The sections were examined with an electron microscope (Hitachi model H7100, Hitachi, Tokyo).
2.9. Antibodies The monoclonal antibodies (mAbs) BM-165 and 6F12 recognize a3 and b3 chains of laminin 5, respectively [18]. Anti-human type IV collagen mAb JK-199 [19] and anti-type VII collagen mAbs NP-32 and NP-185 [20] have been previously described. Laminin 1 pAb (catalog number L-9393), anti-vimentin pAb (catalog number V4630), and anti-keratin mAbs (catalog numbers C-8663 and C-7284) were purchased from Sigma (St. Louis, MO).
Fig. 1. Immunoperoxidase micrographs of type IV and VII collagens and laminin 5 in skin equivalents. Type IV (a,d) and VII collagens (b,e) and a3 chain of laminin (c,f) were found in the dermal – epidermal interface in 14-day skin equivalents. Specific staining for type IV collagen (a) and type VII collagen (b) in the skin equivalents supplemented with laminin 5 was observed more strongly than in control cultures (d,e). Specific staining for laminin 5 was restricted to the dermal – epidermal junction although the skin equivalents were cultured in laminin 5-supplemented medium (c). The bar represents 50 mm.
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Fig. 2. Electron micrographs showing the dermal–epidermal interface of skin equivalents cultured for 14 days in the medium without supplemental laminin 5. The lamina densa was rarely observed at the dermal–epidermal interface, the site in skin where lamina densa would be most expected. Banding fibrils are closely apposed to the basal surface of keratinocytes. The bar represents 100 nm.
face area in 14-day skin equivalents in the absence of exogenous laminin 5 (Fig. 1d,f). Only traces of type VII collagen were observed extracellularly at the dermal–epidermal junction and these were primarily restricted to areas adjacent to basal keratinocytes (Fig. 1b,e). Specific staining for type IV and VII collagens in skin equivalents supplemented with laminin 5 (Fig. 1a,b) was more intense than that in the control (Fig. 1d,e). Specific staining for the laminin a3 chain was also restricted to the dermal – epidermal junction even when skin equivalents were cultured in laminin 5-supplemented medium (Fig. 1c).
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membrane and the subepithelial dense layer, were observed in the skin equivalents supplemented with laminin 5 than in controls. Most laminae densae were connected to the hemidesmosomelike structures of basal keratinocytes by anchoring filaments traversing the lamina lucida, but a lamina densa was not found between these hemidesmosome-like structure (Fig. 3). By morphometrical analysis the averaged length of lamina densa per 1 mm of the dermal–epidermal interface was found to be increased by 180, 230, and 520% of control culture values when the skin equivalents were cultured in the medium supplemented with laminin 5 at the concentration of 1, 5, and 20 mg/ml, respectively (data not shown).
3.3. Impro6ement of keratinocyte sheet grafting with laminin 5 At 7 days postgrafting, attached and surviving keratinocyte sheets were observed on the transplanted area. The graft take was measured as the area of epithelialization in both nude mice and nude rats, the area of epithelialization being larger in group 1 (laminin 5-treated) than in group 2 (control). The results were: (A) nude mice: 58.59 21.9% in group 1, 38.39 30.5% in group 2; (B) nude rats: 53.1921.9% in group 1, 35.39 22.5%
3.2. Basement membrane formation in cultured skin equi6alents Little lamina densa was observed in the 14-day cultured skin equivalents in the absence of exogenous laminin 5. Close inspection revealed banding fibrils apposed to the basal surface of keratinocytes (Fig. 2). In contrast, focal areas of the lamina densa along the subepidermal surface were observed in 14-day cultured skin equivalents supplemented with laminin 5 (Fig. 3). More hemidesmosome-like structures, such as electron dense plaques along the inner leaflet of the cell
Fig. 3. Electron micrographs showing the dermal – epidermal interface of skin equivalents cultured for 14 days in medium supplemented with laminin 5 (5 mg/ml). Discontinuous laminae densae (arrowheads) were observed under the basal keratinocytes. The lamina densa is connected to hemidesmosomes of basal keratinocytes by anchoring filaments traversing the lamina lucida. The bar represents 100 nm.
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in group 2. Statistical analysis indicated that the difference was significant in both groups. At 7 days postgrafting, type IV collagen stained more strongly at the dermal – epidermal junction in group 1 than in group 2 by immunohistochemical staining (Fig. 4a,d). Alpha 3 chain of laminin also stained more strongly at the dermal – epidermal junction in group 1 than in group 2, as did type VII collagen (Fig. 4c,d and b,e). The effect of laminin 5 upon the ultrastructural organization of the basement membrane was examined by electron microscopy as shown in Fig. 5. At day 3 after grafting, the lamina densa of group 1 (laminin 5-treated, Fig. 5B) was more continuous than that of group 2 (control, Fig. 5A). This shows that early morphological organization of basement membrane components is caused by pretreatment with laminin 5. At day 7 after grafting, electron-dense plates characteristic of hemidesmosomes were observed in both groups and no significant differences in the continuity of the basement membrane were observed (data not shown).
4. Discussion The present study demonstrates that laminin 5 has the potential to accelerate formation of basement membrane in cultured skin equivalents. Addition of exogenous laminin 5 increased the length of lamina densa, which was recognized by an anti-type IV collagen antibody, in a concentration-dependent manner [13]. It was also found that laminin 5 increased the frequency of hemidesmosomal structures at the dermal – epidermal junction and that most laminae densae were associated with the hemidesmosomal structures. Regardless of the concentration of laminin 5 supplemented in the culture medium, the morphological sequence of the lamina densa formation in skin equivalents is unchanged and is as follows: (1) the appearance of hemidesmosomal structures along the inner leaflet of the subepidermal cell membrane of basal keratinocytes; (2) the focal appearance of the lamina densa with anchoring filaments in apposition to the hemidesmosomes; (3) the lateral elongation of the lamina densa [21].
The observation is consistent with the morphological sequence in embryos and young animals wherein the lamina densa also develops first in apposition to the hemidesmosomes [22]. From these results, it appears likely that the hemidesmosomal structures facilitate formation of the basement membrane by functioning as an initiation site in skin equivalents and that the increments of hemidesmosomal structures gained by the addition of laminin 5 accelerate the assembly of basement membranes in skin equivalents. Immunohistochemical analysis of laminin 5 showed that laminin 5 deposition was restricted to the dermal–epidermal junction and did not occur at any sites in the collagen gel or within the epidermis, even though laminin 5 was added to the culture medium. Basal keratinocytes are known to express a3b1 and a6b4 integrins [23– 25], which are known to be specific receptors for laminin 5 [26,27]. It is possible that exogenously added laminin 5 might bind to basal keratinocytes through laminin 5-specific integrins, a6b4 and a3b1, and then might be accumulated beneath these basal cells. The accumulation might accelerate hemidesmosome formation and might function as a nucleus for lamina densa formation. The present observations strongly suggest a unique role for laminin 5 in basement membrane formation in skin equivalents. Since purified and exogenously applied laminin 5 has the ability to bind keratinocytes to substrates and to enhance formation of the basement membrane, this molecule might have clinical utility. In this study, one also focused on the role and influence of laminin 5 in the take of grafted cultured human keratinocyte sheets and the formation of the basement membrane in vivo. Laminin 5 plays an important part in the adherence of epidermis to dermis in normal skin and the present study demonstrated that exogenous application of laminin 5 improves transplanted keratinocyte sheet attachment to the granulation tissue in wound beds, acting as a biological ‘glue’ [28]. The results suggest that the increase in successful keratinocyte sheet transplantation is due to both increased cell attachment and increased basement membrane formation. Therefore, laminin 5 may be clinically useful for the treat-
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Fig. 4. Deposition of type IV collagen (a,d), type VII collagen (b,e), and a3 chain of laminin (c,f) at the dermal – epidermal junction in immunostaining at 7 days post grafting. It is stained more strongly at the dermal – epidermal junction in group 1 (a – c) than in group 2 (d – f). The bar represents 100 mm. Fig. 5. The effect of laminin 5 on ultrastructural organization of basement membrane. At day 3 after grafting, the lamina densa of group 1 (A) is more continuous as compared to group 2 (B). The bar represents 0.5 mm.
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ment of severe burns either by partially or wholly overcoming the problem of keratinocyte graft stability, or in combination with dermal grafting methods. It is believed that these observations justify further studies of the efficacy of exogenous laminin 5 to increase keratinocyte graft take to human burn patients.
5. Conclusion In vitro and in vivo evidence indicates that laminin 5 participates in several essential functions in skin biology. From published reports [8,11,12] and the present studies, it was concluded that the function of laminin 5 is as follows. Laminin 5 has two functional domains which can bind integrins of basal keratinocytes and type VII collagen, forming anchoring fibrils, and the anchoring complex provides an essential link between the epidermal basal cells and the papillary dermis. This role of laminin 5 is essential to the resistance of the epidermis from external stress, such as frictional stress. Laminin 5 also accelerates the assembly of basement membranes at the dermal–epidermal junction. Therefore, laminin 5 is a key component not only for strongly linking the epidermis to the dermis, but also for the repair or regeneration of the basement membrane at the dermal–epidermal junction. An intact basement membrane at the dermal – epidermal junction is essential to the viability of the skin and participates in the selective flow of communication between the dermis and the epidermis.
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