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Organotypic keratinocyte cultures on de-epithelialized tongue mucosa Hildebrand, H. Christopher 2000

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O R G A N O T Y P I C K E R A T I N O C Y T E C U L T U R E S O N DE-EPITHELIALIZED TONGUE MUCOSA by H. C H R I S T O P H E R HILDEBRAND Dr. Med. Dent., The University of Frankfurt, Germany 1994 A T H E S I S S U B M I T T E D IN PARTIAL FULFILLMENT O F T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Oral Biological and Medical Sciences) W e accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A September 2000 © H. Christopher Hildebrand, 2000  U B C Special Collections - Thesis Authorisation F o r m  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e requirements f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  The U n i v e r s i t y o f B r i t i s h Vancouver, Canada  1 ofl  Columbia  9/28/00 4:31 P M  ii ABSTRACT Organotypic cultures have been used to study epithelial cell behavior for many years. The aim of this study was to develop an organotypic culture method that better mimics the three-dimensional morphology of interdigitating rete ridges and connective tissue (CT) papillae and conserves the basement membrane zone (BMZ). Bovine tongue mucosa was chosen for the raft donor and incubated with cold 1 M sodium chloride solutions for 4 days to separate the epithelial tissue from the BMZ and underlying C T matrix. Level of separation was studied by electron microscopy (EM) and immunohistochemical staining of several integrin subunits, laminin-1 and - 5 , type IV and V l l collagen, tenascin, fibronectin, vitronectin and heparan sulfate proteoglycan. After the separation, IHC staining and E M observations suggest a separation through the lamina lucida. Rafts were used for growth of organotypic cultures of human keratinocytes. Three different keratinocyte cell lines were cultured submerged for 6 days after which the cultures were raised to air-liquid interface and maintained for up to 40 days. Sections were prepared for routine histology, E M and IHC staining. Histomorphology varied significantly between the cell lines. Sometimes more than 15 cell layers resembling normal epithelial histology were present. Keratinocytes in these raft cultures were found to express P4 integrins against the preexisting BMZ. In addition, human keratinocytes deposited their own matrix molecules, particularly tenascin, laminin-1 and - 5 . Expression of integrin subunits and differentiation markers revealed some differences from normal tissue, which depended on the cell line. The ultrastructure of the B M Z including hemidesmosomes was similar to the normal dermal-epidermal  iii junction. This culture model seems to mimic normal epithelium including the BMZ and is potentially useful for multiple applications for studies on epithelial cell behavior in vitro.  iv  TABLE OF CONTENTS ABSTRACT  II  TABLE OF CONTENTS  IV  LIST OF TABLES  VI  LIST OF FIGURES  VII  ACKNOWLEDGEMENTS  IX  ABBREVIATIONS  X  INTRODUCTION  1  CHAPTER ONE-REVIEW OF THE LITERATURE  2  1. l BOVINE TONGUE MUCOSA 1.2 KERATINS 1.3 INVOLUCRIN 1.4 EPITHELIAL INTEGRINS 1.5 T H E BASEMENT MEMBRANE ZONE ( B M Z )  1.5.1 1.5.2 1.5.3 1.5.4  Plasma membrane and Hemidesmosomes Lamina Lucida Lamina Densa Sublamina Densa  1.6 MUCOSA SEPARATION 1.7 KERATINOCYTE CULTURE MODELS  /. 7. / Culture methods  2 4 7 8 11  12 15 16 18 20 23  23  CHAPTER TWO-AIM OF THE STUDY  35  CHAPTER THREE-MATERIAL AND METHODS  36  3.1 SUBSTRATE TISSUE PREPARATION, SEPARATION 3.2 LOCALIZATION OF THE SPLIT 3.3 CULTURE  36 37 38  3.3.1 Cells 3.3.2 Culture media 3.3.3 Culture process  CHAPTER FOUR-RESULTS 4.1 TONGUE MUCOSA  4.1.1 Histology 4.1.2 Immunohistochemical staining of bovine tongue mucosa 4.2 SEPARATION  4.2.1 Immunohistochemistry for cold salt separated tissue 4.2.2 Electronmicroscopy 4.3 CULTURES  38 38 39  42 42  42 45 50  51 51 54  4.3.1 HaCaT. 4.3.2 HGK 4.3.3 NHEK. 4.3.4 Immunostaining 4.4 HaCaT TEM.  54 56 58 60 72  CHAPTER FIVE-DISCUSSION  74  5.1 BOVINE MUCOSA  74  V  5.2  SEPARATION TECHNIQUE  76  5.3  CULTURE MODEL  77  C H A P T E R SIX-CONCLUSIONS  88  REFERENCES  90  vi LIST O F T A B L E S Table 1: Keratin distribution in different epithelia  6  Table 2: Integrin subunits and combinations....  8  Table 3: Antibodies and working concentrations  41  VII  LIST OF F I G U R E S Fig 1: (A) HE staining of unseparated bovine tongue, showing filiform papillae. Note distinct anterior and posterior cell lines and anterior and posterior keratin layer (arrow). (B) IHC for T N , location at the anterior C T core and C T papillae Bar 200 um 44 Fig. 2: HE staining and IHC staining of bovine tissue before and after cold salt separation: (A, D, G) unseparated BMZ; (B, E, H) connective tissue; (C, F, I) epithelium; (A-C) HE; (D-F) integrin (J 1; (G-l) integrin (54. Bar 200 um  46  Fig. 3: IHC staining of bovine tissue before and after cold salt separation: (A, D, G) unseparated BMZ; (B, E, H) connective tissue; (C, F, I) epithelium. (A-C) type IV collagen; (D-F) type VII collagen; (G-l) H S P G . Bar 200 um 47 Fig 4: Unseparated bovine tongue: (A) HE staining of filiform papillae; (B) IHC staining K14; (C) integrin a3; (D) K10, anterior cell line of filiform papillae; (E) Involucrin, reaction in posterior cell line and posterior cornified layers. Bar 200 urn 48 Fig. 5: Scanning electron microscopy after separation showing the formerly attached surfaces facing up: (A, C) epithelium; (B,D) connective tissue  49  Fig 6: Transmission electron micrographs of bovine tongue BMZ, (ct= connective tissue, e=epithelium), (A) before separation, (C,D) partial separation with basal cell remnants, note persisting hemidesmosome and anchoring filaments (arrowhead); (D) lamina densa covered lamina propria completely separated from epithelium (magnification 365000x) 53 Fig. 7: HE staining of HaCat keratinocyte cultures:(A, C) 5 day culture; (B, D) 18 day culture. Bar 200 um 55 Fig 8: HE staining of H G K cultures, 14 days after air exposure. (A) H G K fill all voids between C T papillae but show limited stratification, note nuclear polymorphism. (B) higher magnification, some palisading of basal cells but no distinct spinous or granular cells. Bar 200 um 57 Fig 9: HE staining of 14 day old N H E K culture. (A) section showing both para- and orthokeratotic areas. (B) higher magnification of stratum granulosum with keratohyalin granules. (C) Non-keratinzed culture section. Note saw-tooth like rete ridges (arrow) and areas devoid of nuclei. Bar 200 um 59 Fig 10: IHC staining of HaCaT cultures, E C M : (A) T N , control tissue without cells; (B) TN of HaCaT culture; (C) H S P G , (D) CVII; (E) CIV; (F) LM-5; G+H varying expression of LM-1. Bar 200 um 61  Vlll  Fig 11: IHC staining of HaCaT cultures, integrin subunits: (A) B1; (B) R4; (C) B6;  (D) av; (E) a2, (F) a3, Bar 200um  62  Fig 12: IHC staining of HaCaT cultures, differentiation markers: (A) K14, (B) involucrin; (C) K10, 5 day old culture, arrowhead: non-reactive basal cells; (D) K10, 18 day old culture. Bar 200 um 63 Fig 13: IHC staining of H G K cultures, E C M : (A) CIV; (B) CVII; (C) T N ; (D) LM-1; (F) H S P G ; (G) LM-5. Bar 200 um 65 Fig 14: IHC staining of H G K cultures, integrin subunits: (A+B) B4; (C) B1; (D) cc3;  (E) av; (F) B6. Bar 200 um  66  Fig 15: IHC staining of H G K cultures, differentiation markers: (A) K14; (B) K10; (C) Involucrin. Bar 200 um 67 Fig 16: IHC staining of N H E K cultures, E C M : (A) T N , wide diffuse band at the BMZ; (B) CIV; (C) CVII; (D) H S P G ; (E) LM-1; (F) LM-5. Bar 200 um 69 Fig 17: IHC staining of N H E K cultures, Integrin subunits: (A) B4; (B) B1; (C) a2; (D) a3; (E) av; (F) B6. Bar 200 um 70 Fig 18: IHC staining of N H E K cultures, differentiation markers: (A) K14; (B) K10; (C) involucrin. Bar 200 um 71 Fig 19: T E M of the BMZ of 20 day old HaCat culture on de-epithelialized lamina propria (ct), (A) note outer dense plate (arrow) and anchoring fibrils (AF), 28000x; (B) hemidesmosomes and anchoring filaments (arrowheads), 82000x 73  ix  ACKNOWLEDGEMENTS  I would like to thank Dr. Hannu Larjava for his creativity in providing and guiding this interesting topic.  I would like to acknowledge Dr. Colin Wiebe's preliminary efforts in establishing the basics for this culture model and sharing information and his experience.  I would like to thank Mr. Christian Sperantia for his entertaining and patient technical support and Dr. Lari Hakkinen for sharing his knowledge and providing prompt solutions and new ideas.  I am thankful to Dr. Edward Putnins and Dr. Doug Waterfield for their valuable input as members of my research committee.  I thank my classmates Dr. Nazanin Narani and Dr. Priscilla Walsh for sharing the load and helping out during the last three years.  Finally, I would like to thank my wife Nadja for her patience, understanding and neverending support.  ABBREVIATIONS BM basement membrane BMZ basement membrane zone BP bullous pemphigoid BPA(G) bullous pemphigoid antigen BSA bovine serum albumine CIV type IV collagen CVII type V l l collagen CT connective tissue DED De-epidermized dermis DMEM Dulbecco's modified Eagle's medium ECM extracellular matrix figure Fig FN fibronectin HE Hematoxylin / Eosin HGK Human gingival keratinocyte HSPG heparan sulfate proteoglycan IHC Immunohistochemistry INV involucrin K keratin LM-1 laminin 1 LM-5 laminin 5 MSBM Minimally supplemented basal medium N(H)EK Human epidermal keratinocytes ORS Outer root sheath PBS phosphate buffered saline SEM scanning electron microscopy TEM transmission electron microscopy TN tenascin VN vitronectin  1  INTRODUCTION  Organotypic cultures are three-dimensional tissue cultures used to reconstruct a tissue or organ in vitro, the objective being to allow the cells to exhibit as many properties of the original organ as possible (Parenteau, 1994). Organotypic cultures have been used for studies of cell behavior, differentiation, drug effects, and cellmatrix interactions. Common techniques use an artificial collagen-fibroblast matrix, which have just recently shown an organized basement membrane zone (BMZ) including hemidesmosomes, but usually lack interdigitating rete ridges. The epidermal side of de-epidermized human dermis has been successfully used as culture matrix and these cultures differ from cultures using reticular dermis (Regnier etal., 1981). This indicates a significant influence of the basement membrane in providing a substratum for attachment and growth of keratinocytes. The purpose of this study was to develop an organotypic culture using a papillary matrix with a preexisting B M Z to create an epithelial culture with interdigitating rete ridges and connective tissue papillae in vitro.  2  C H A P T E R ONE-Review of the Literature  1.1 Bovine tongue mucosa Tongue is an organ that derives from the first to fourth branchial arch with the anterior 2/3 (corpus linguae) derived from the first arch. A V-shaped groove called sulcus terminalis marks the joint to the posterior third (radix linguae) (Schiebler and Schmidt, 1987) Tongue mucosa is a rather tough mucous membrane (tunica mucosa), which is connected to striated muscle by a thin layer of connective tissue (Trautwein and Fiebiger, 1952). The subepithelial connective tissue (lamina propria) blends with the connective tissue of the muscle (epimysium) (Banks, 1974). The epithelial-connective tissue interface is highly corrugated with connective tissue papillae projecting into the epithelium separating epithelial rete ridges (rete pegs). Stem cells in lingual epithelium are based on the rete ridge structure and both connective tissue papillae and filiform papillae occur at the junction of two or more epithelial clones (Seddon etal., 1992). The dorsal surface (dorsum linguae) contains various lingual papillae whereas the ventral surface is smooth. Most abundant are filiform papillae, which are inclined posteriorly. They have mechanical properties and consist of a connective tissue core with an epithelial cover characterized by a heavy stratum corneum (Trautmann and Fiebiger, 1952). In most species the connective tissue core does not project beyond the epithelium. In ruminants the connective tissue core gives rise to several secondary papillae (Trautmann and Fiebiger, 1952, Stinson and Calhoun, 1976).  3 Bovine filiform papillae are fully keratinized and composed of a hard keratin core derived from a dominant posterior cell line and an outer sheath of soft keratin derived from a thinner anterior cell line (Steflik ef al., 1983). The posterior cell line lacks keratohyalin granules (KHG) whereas the anterior cell line contains basophil and eosinophil K H G . Larger and longer filiform papillae are termed conical papillae (Schiebler and Schmidt, 1987), which are located caudally of the circumvallate papillae (Chamorro etal., 1994). Fungiform papillae are mushroom shaped structures with a nonkeratinized epithelium. They have gustatory function and contain taste buds (Banks, 1974). All of the taste buds are located at the apical surface (Davis ef al., 1979). A prominent connective tissue core with secondary papillae projects into a rather thin epithelium. Circumvallate papillae are located at the sulcus terminalis and contain numerous taste buds in their lateral walls (Trautmann and Fiebiger, 1952, Dasgupta ef al., 1990, Davies et al., 1979). These large papillae do not project beyond the epithelial surface and are surrounded by a deep furrow. Serous glands open into the base of the furrow and cleanse this area (Banks, 1974). Foliate papillae are located at the lateral tongue surface but are absent in ruminants (Trautmann and Fiebiger, 1952). However, Chamorro et al. (1986) found foliate papillae in their S E M study in bovine tongue. In a follow-upstudy the same group also observed lenticular papillae (de Paz Cabello ef al., 1988). The dorsal tongue is covered by stratified squamous epithelium with varying degrees of keratinization (Stinson and Calhoun, 1976). The degree of keratinization also varies between species (Schiebler and Schmidt, 1987). In humans the dorsal  4 surface (dorsum linguae) is covered by keratinized epithelium in specific areas, whereas the ventral surface is completely nonkeratinized as indicated by the cytokeratin pattern (Sawaf et al, 1990). Based on morphological criteria, however, there has been controversy on the keratinization of tongue epithelia. It has been described as nonkeratinized for all parts (Coujard et al., 1980, Poirier and Ribadeau-Dumas, 1984), keratinized for the complete dorsum (Provenza and Seibel, 1986), the ventral surface may be keratinized (Brightman, 1984) to filiform papillae are keratinized and the remainder nonkeratinzed (Milaire, 1980). Interpapillary regions resemble cheek mucosa and contain no stratum granulosum whereas papillary cells contain keratohyalin granules (Plackova and Skach, 1975). In other species keratinization pattern can be quite distinct from humans. Mouse tongue epithelium is higly keratinzed on ventral and dorsal surfaces (Rentrop ef al., 1986). For Middendorf goose tongue epithelium a very thin and indistinct keratinized layer and strongly keratinized conical papillae were reported (Iwasaki etal., 1997).  1.2 Keratins Keratins are a group of probably more than 20 related proteins that are responsible for the formation of intermediate filaments (IF) in epithelial cells (Rentrop et al., 1986). Intermediate filaments are the primary component of the keratinocyte cytoskeleton and thread in bundles throughout the cytoplasm from a perinuclear 'cage' of filaments spreading to desmosomes and hemidesmosomes (Holbrook, 1994). Intermediate filaments (diameter of about 10 nm) provide structural resilience (Morley and Lane, 1994). Suprabasal cells may contain twice the amount of keratin  5 than basal cells. According to their charge properties keratins are divided into subclasses of basic to neutral type II keratins and acidic type I keratins (Rentrop ef al., 1986). In vivo keratins polymerize from heterodimers of one type I with one type II keratin (Hatzfeld and Weber, 1990). Basal epidermal cells express at least one basic and one acidic subunit, which is different from the keratin pair of suprabasal cells (Rentrop ef al., 1986). Keratins are furthermore classified according to their molecular weight determined by gel electrophoresis or numbered by the catalogue of Moll etal. (1982). Keratins are useful markers of differentiation because their expression is both region- and differentiation-specific (Dale etal., 1990). They may also be used for determining the origin of specific tissues (Zhang and Oakley, 1996). Specific keratin pairs are generally expressed in different types of epithelium with the lowest weight present in simple and glandular epithelium, intermediate size keratin in stratified epithelia and the highest weight keratins in keratinized epithelia (Dale etal., 1990). Keratinized epithelia express K1/2 and K10/11 whereas nonkeratinzed epithelia express K4 and K13; basal cells of all oral epithelia express K5/14 (Dale et al., 1990, Morley and Lane, 1994). K20 is found in Merkel cells and tissue of endodermal origin (Zhang and Oakley, 1996). About 8 % of basal cells express K1/10 suggesting that the onset of terminal differentiation occurs in human epidermis in a subpopulation of keratinocytes, which are still located in the basal layer (Regnier etal., 1986). K16 is expressed in suprabasal keratinocytes at epidermal wound edges (Paladini et al., 1996).  6 Type of epithelium Type 1 Keratin Type II Keratin Simple epithelia K7, K8 K18, K20 Basal stratifyable cells K5 K14, (K17, K19) Stratified mucosal K6 K16 regenerative Stratified non-cornifying K4 K13 mucosal Stratified corneal K3 K12 Stratified cornifying K2, K1 K9, K10, K11 epithelia Table 1: Keratin distribution in different epithelia (modilFied from Morley and Dale, 1994) Gene expression of keratins does not necessarily coincide with the presence of these proteins. While only selected suprabasal cell columns in buccal mucosa epithelium express K1/10, proteins m R N A for those keratins are present in basal, parabasal, and lower prickle layers, showing a much wider expression than that of their proteins (Bloor et al., 1996). This indicates that a switch in keratin expression may occur rapidly in the presence of an adequate stimulus. Dorsal tongue epithelium shows a rather complex keratin expression in association with papillae. In an immuno-histological study by Sawaf et al. (1990) interpapillary, ventral and lateral surfaces of human tongue did not express terminal differentiation keratins including K1, K2, K10, K11, but are positive for K4 and K13 in all suprabasal cells. Furthermore basal cells of all surfaces were positive for K19. In the posterior of keratinized filiform papillae suprabasal cells were positive for K1-2 and K10-11, basal cells and cornified cells were negative, whereas in the anterior part, only scattered suprabasal cells gave reactions. K4 and K13 were positive in both anterior and posterior suprabasal cells. Different observations are made in bovine tongue mucosa showing a positive reaction for K4 and K13 in interpapillary regions and K10 and K11 present in a narrow zone of one or two cell layers at the  7 boundary between the papillary core and the anterior column (Heid et al., 1988). K19 was restricted to basal cells.  1.3 Involucrin Involucrin is a protein expressed in differentiating keratinocytes and is a major component of the cornified cell envelope (Dale et al., 1990). The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum, which is made by cross-linking precursor proteins by a membrane-associated transglutaminase (Baden etal., 1987). Antibodies directed against envelopes from bovine epidermis react with human involucrin (Kubilus etal., 1987). Different stratified squamous epithelia, whether they bear a stratum corneum or not, express involucrin which is not present in the deepest epithelial cells but appears in the course of their outward migration (Banks et al., 1981). Both buccal and palatal mucosa express involucrin (Reibel et al., 1989) with only the basal cells staining negative in buccal mucosa whereas in palatal mucosa both basal and parabasal cells of the rete ridges are stained negative in immunohistochemistry. Involucrin is a useful marker of differentiation in cell culture but it is not useful in distinguishing between keratinized and non-keratinized epithelium (Dale et al., 1994).  8 1.4 Epithelial Integrins Integrins are heterodimeric transmembrane glycoproteins composed of noncovalently linked a and fi subunits. Integrins connect the extracellular matrix to the cytoskeleton and transmit signals in both directions (Brakebusch ef al., 1997). Currently 8 IS and 24 a subunits have been identified which form at least 24 different aS heterodimers (Brakebusch etal., 1997), some of which are listed in the table below.  p1  P2  P3  P4  P5  p6  P7  P8  a1  Yes  a2  Yes  a3  Yes  a4  Yes  a5  Yes  a6  Yes  a7  Yes  a8  Yes  a9  Yes  aD  -  Yes  aL  -  Yes  aM  -  Yes  cxV  Yes  "  Yes  Yes  Yes  Yes  aX  -  Yes  •  •  •  •  allb  -  Yes  •  •  •  alELb  •  *  •  •  -  -  -  -  -  -  Yes  -  Yes  -  -  Yes  •  Table 2: Integrin subunits and combinations."Yes" represent that this a/p dimer has been found, - represents that it is not found (yet) (modified from Kosten, 1997)  9 More recently the integrin heterodimer a10B1 was discovered in basal keratinocytes of normal epidermis (Favre ef al., 1997), in chondrocytes (Camper ef al., 1998) and several other tissues (Lehnert etal., 1999). In oral mucosa the a2, a3, a6, p i , and p4 subunits are highly expressed in normal epithelium, and there is weaker, more variable expression of a5 and av (Jones ef al., 1993, Larjava etal., 1993). a9 pairs with p i and was detected by immunohistochemistry in airway epithelium, in the basal layer of squamous epithelium, and in smooth muscle, skeletal muscle, and hepatocytes (Palmer etal., 1993, Wang etal., 1995). In gingiva, palatal and buccal mucosa it is expressed by basal cells and immediate suprabasal cells at rete ridges (Hakkinen etal., 2000). Expression of all subunits is the highest in the basal cell layer of normal epithelium, but extensive staining above the basal layer can also be observed, particularly in the floor of the mouth and the lateral margin of the tongue (Jones ef al., 1993). a2B1 is located primarily at the lateral surfaces basal keratinocytes (Roberts, 1994) suggesting a role in cell-cell interactions. <x3R1 is located pericellular and a6R4 is a hemidesmosome component located at the basal cell surface (Stepp et al, 1990.) (see below). In cultured keratinocytes av forms dimers with Q>5 (Adams and Watt, 1991), B1 (Koivisto etal., 1999) and S6 (Breuss etal., 1995). p6 expression is restricted to epithelia and is up-regulated with morphogenetic events, tumorigenesis, and epithelial repair (Breuss etal., 1995) Integrins a5S1, avB6 and avB5 are not routinely expressed in keratinocytes resting on intact basement membrane (Zambruno et al., 1995). However, Jones et al. (1993) detected av in the basal layer of cheek, floor of the mouth, lateral tongue and hard palate, although  10 weaker and more variable than al,  3 and 6 and p i and OA. av was also expressed  in normal ovarian epithelium where it was co-localized with S3 (Carreiras et al., 1996). avp3 is also present in angiogenic vascular tissue (Brooks ef a/.,1994). From wound healing studies it was suggested that the expression of avp6 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing (Larjava et al., 1993, Haapasalmi etal., 1996). Cultured keratinocytes usually express avp5, a 5 p i , avB1 and avp6 (Marchisio ef al., 1991, Juhasz etal., 1993, Koivisto etal., 1999). In adherent cultured cells integrins localize to focal contacts and p i associates with vinculin, actin, talin and aactinin (Watt and Hertle, 1994). B1 is also suggested to bind to collagen IV during skin BM assembly (Fleischmajer et al., 1998). Focal adhesions are distinct from hemidesmosomes and do not contain a6p4 (Carter ef al., 1990b). Epithelial cells use a 2 p i as a collagen receptor and a 3 p i as a laminin-5 (LM-5) receptor (Carter ef al., 1990a, Hakkinen ef al., in press). It was proposed that a 3 p i binds the laminin5/6 complex as part of the interhemidesmosomal BM attachment (Burgeson and Christiano, 1997). a 5 p i binds to FN and a 9 p i binds to TN (Larjava et al., 1996). Ligands for avp5 include V N and for avp6 FN and T N (Clark ef al., 1996). During development of the kidney, lung, and skin, p6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin, but is expressed in several types of carcinoma (Breuss ef al., 1995). avG>6 integrin was also expressed in 4 1 % of leukoplakia specimens, and 8 5 % of lichen  11 planus samples, but in none of the tissues with inflammatory hyperplasia, chronic inflammation or normal tissue (Hamidi etal., 2000). More recently ccvp6 could not be detected in keratinized or non-keratinized normal oral tissues (Hakkinen et al., 2000). It was suggested that <xvp6 affects cell spreading, migration and growth during reorganization of epithelia in development, tissue repair, and neoplasia (Breuss ef al., 1995). The expression of avfi6 integrin is strongly and specifically upregulated by transforming growth factor-B1 (TGFR1) (Zambruno et al., 1995, Koivisto ef al., 1999), promoting adhesion, spreading and motility of HaCaT keratinocytes on fibronectin (Koivisto etal., 1999). avB1 (Mungeref al., 1998) and avB6 (Munger ef al., 1999) have been demonstrated to bind the latent form of TGFQ>. avfi6 expressing cells are capable of activating latent TGF-B1 (Munger ef al., 2000).  1.5 The Basement membrane zone (BMZ) Basement membranes are thin layers of a specialized extracellular matrix that form the supporting structure on which epithelial and endothelial cells grow on, which do not only provide a mechanical support and divide tissues into compartments, but also influence cellular behavior (Paulsson, 1992). Basement membrane components are involved in cell migration and influence stratification and differentiation (Fine, 1994). A vast number of B M Z components are synthesized by the basal keratinocytes including type IV, V and Vll collagen, laminins 5 and 6, heparan-sulfate-proteoglycan and hemidesmosomal plaque proteins (Burgeson and  12 Christianson, 1997). Fibroblast contribute nidogen, additional laminins, type I, III and IV collagen (McKee, 1996). The epidermal B M Z consist of 4 major structural components: the basal cell plasma membrane, the lamina lucida, the lamina densa, and the sublamina densa zone (sub-basal lamina), which contains anchoring fibrils (Katz, 1984).  1.5.1 Plasma membrane and Hemidesmosomes In skin and mucosa a columnar shaped basal cell rests on the basement membrane. On E M images the plasma membrane of keratinocytes is corrugated and pinocytotic vesicles are frequently observed (Katz, 1984). The plasma membranes of basal cells are joined to the lamina densa of the basement membrane by hemidesmosomes (Holbrook, 1994). Hemidesmosomes are present in stratified squamous, transitional, and pseudo-stratified epithelium but not in the gut (Jones et al, 1994). Hemidesmosomes resemble structurally half a desmosome, which join epithelial cells, but their chemical composition is distinct (Jones ef al., 1994). Hemidesmosomes have an inner (cytoplasmic-related) and outer (membrane-related) region. The submembranous plate underlies the hemidesmosome within the lamina lucida. Anchoring filaments extend through that plate to the lamina densa (Holbrook, 1994). Anchoring fibrils extend from the lamina densa and insert in the papillary dermis where they associate with basement membrane-like structures, known as anchoring plaques (Uitto and Pulkkinen, 1996), or loop back to the lamina densa (Holbrook, 1994).  13 Hemidesmosomes have been shown to consist of at least four distinct components, including bullous pemhpigoid antigen 1 and 2 (BPAG), plectin, and integrin cc6G4. The primary protein of hemidesmosomes is B P A G (Holbrook, 1994). B P A G s were localized in hemidesmosomes in bovine tongue mucosa and epithelial cells in culture using autoantibodies from patients with bullous pemphigoid (Klatte ef al., 1989). There are at least two distinct B P A G s . B P A G 1 (BPA230, HD2) is a 230 kD polypeptide that is localized intracellularly both in vivo and in vitro (Westgate et al., 1985). B P A G 2 (BPA180, HD4) is a transmembranous collagenous protein of 180kD. The extracellular domain of B P A G 2 contains Gly-X-Y repeats indicative of its collagen-like nature (Giudice etal., 1991) and is therefore also termed collagen XVII (Li et al., 1993). This collagenous domain may also extend across the lamina lucida (Burgeson and Christiano, 1998). BPA180 is suggested to play a role in recruitment of BPA230 to hemidesmosomes and may regulate the location of BPA230 by direct interaction with the protein (Nievers et al., 1999). BPA230 is important for the formation of the inner plaque and provides adhesion to keratin filaments (Burgeson and Christiano, 1998). Plectin (HD1, IFAP= intermediate filament associated protein) is a 500 kD protein and is located at the intracellular hemidesmosomal plaque. It may directly associate with the cytoskeleton and also with hemidesmosomal integrin a6G4 (Burgeson and Christiano, 1998). IFAP 300, which is similar but not identical to plectin, is present in hemidesmosomes and desmosomes, co-localizes with BPA230 but extends deeper into the cytoplasm and binds IF, serving as a linker protein between keratin and desmosomes and hemodesmosomes (Skalli et al., 1994).  14 Integrin a6S4 is another important component of hemidesmosomes which is not only an integral structural part but also serves in transduction of cell signals from the E C M to the interior of the cell and may therefore play a role in the organization of the cytoskeleton, proliferation, apoptosis and differentiation (Borradori and Sonnenberg, 1999). The B4 subunit has a very long cytoplasmic domain, which binds both plectin and BPA180 and is therefore essential for the formation of hemidesmosomes (Nievers et al., 1999). The extracellular domain of Integrin a6&4 binds to laminin-5 (LM-5) connecting hemidesmosomes to the anchoring filaments of the lamina lucida (Burgeson and Christiano, 1998). Binding of LM-5 or stimulation with epidermal growth factor (EGF) or insulin results in tyrosine phosphorylation of the B4 subunit, which is a regulatory process for assembly and disassembly of hemidesmosomes as well as for cell motility (Nievers et al., 1999). LM-5 may bind directly to type VII collagen (Rouselle et al., 1997) or form a complex with nidogen (Dziadek and Timpl, 1985). Nidogen effectively promotes the formation of a ternary complex between laminin and collagen IV as well as a ternary complex between laminin and proteoglycans (Aumailley etal., 1993). High-affinity binding of nidogen to laminins involves a single binding site on the laminin gamma 1 chain and is thus a property shared by almost all laminin isoforms. This mediates the connection of laminins to the collagen IV network, perlecan and other proteins, which is considered to be an essential step in the stabilization of basement membranes (Mayer ef al., 1998). Epithelial cells also attach directly to the basement membrane via integrin a3R1 which binds intracellularly to actin and to E C M lamininsl, 5, 6 and 7 (Burgeson and Christiano, 1997).  15  1.5.2 Lamina Lucida The lamina lucida is an electron-lucent area of 20-40 nm thickness and appears amorphous except for the areas directly underlying hemidesmosomes (Katz, 1989). In this area anchoring filaments traverse from hemidesmosomes to the lamina densa. A major component in the lamina lucida is laminin. However, laminin is not limited to the lamina lucida but may reside within different compartments of the dermo-epidermal junction (Fine, 1994). Numerous isoforms of laminin have been isolated and many different terms have been used for identical molecule. The classical laminin, an 800 kD glycoprotein, is composed of 3 distinct polypeptide chains and plays a role in epithelial cell attachment (Terranova et al., 1980). Anchoring filaments extend from the basal keratinocytes' hemidesmosomes to lamina densa, thus traversing the lamina lucida. Anchoring filaments are thin thread-like structures of 2-4 nm thickness (McKee 1996). The major component of anchoring filaments is a member of the laminin family, laminin-5 (previously named kalinin, epiligrin, nicein, BM600) (Rousselle etal., 1991, Marinkovich et al, 1992). Laminins are the most abundant structural noncollagenous glycoproteins ubiquitously present in basement membranes. They are multidomain molecules constituting a family of possibly more than 50 members (Aumailly and Rousselle, 1999). Some members such as laminins 5, 6 and 10 are specific for the basal lamina present under stratified epithelia. The laminin family consists of a group of heterotrimers of various combinations of three chains, a , I3>, and y, which are synthesized and secreted by keratinocytes. Laminin-6, previously  16 named k-laminin ,is restricted to anchoring filament-containing basement membranes (Marinkovich et al., 1992). Laminin-6 shares similarities with two other lamina lucida-located laminins, laminin-1 and laminin-5 (Marinkovich etal., 1992). Laminin expression is tissue- and development-specific and certain laminin chains are synthesized by both menschymal and epithelial cells (Aumailley and Rousselle, 1999). Laminin 5 is capable of binding to laminin 6, collagen V l l , nidogen, and the extracellular domain of BPA180 (Reddy et al., 1998) as well as the B4 subunit. Distinct domains of laminin do not only mediate cell attachment but also influence cellular proliferation, differentiation and motility via their integrin receptors (Paulsson, 1992). Also associated with anchoring filaments is a novel protein, ladinin, which serves as autoantigen in the linear IgA disease (Uitto and Pulkinnen, 1996).  1.5.3 Lamina Densa The lamina densa is an electron-dense amorphous area, which is approximately 3060 nm wide (Katz, 1989). It has been demonstrated that the lamina densa is of epidermal origin and that anchoring fibrils at least in part originate from the dermis (Briggaman etal., 1971). Type IV Collagen (CIV) is one of the major components of skin B M Z (Fine, 1994). Type IV collagen comprises a family of collagen isoforms that lack the fibrillar and banded appearance of other collagens and retains its telopeptide nonhelical portion resembling procollagen (Katz, 1984). Heterogeneity described for CIV may reflect variations in the structural arrangement of basement membranes and therefore in  17 their functional properties (Desjardin et al., 1996). It has been proposed that the retention of the procollagen-specific portion prevents the alignment into fibrils (Stanley etal., 1982). CIV forms a netlike structure and it has been ultrastructurally localized in the lamina densa of epidermal B M Z (Yaoita etal., 1978). Due to short nonhelical segments CIV shows an anomalous sensitivity to proteolytic enzymes (Schuppan ef al., 1980, Timpl et al., 1978). It has been demonstrated that CIV consists of two distinct chains, oc1 (IV)- and cc2 (IV)-chains (Ristelli etal., 1980). A CIV macromolecule is composed of two a1 and one a2 chains. However, CIV composition of other chains has been reported (Tanaka et al., 1997) and several different genes for CIV were located (Kuhn 1995 for review). At the N-terminus a short 26 kD domain, called 7S-collagen, is linked to a short nontriple helical segment, which is connected to long triple helical domain (Ristelli ef al., 1980). The 7s-collagen segment is more resistant to collagenase degradation due to overlapping and disulfide bonds of 7S domains of 4 molecules (Stanley ef al., 1997). A model by Timpl etal. (1981) proposes that four CIV molecules cross-link at the 7 S region while two molecules join at the globular domains (NC1) forming a net like structure. Both surfaces of the lamina densa are lined by strongly anionic heparan sulfate proteoglycan (HSPG) (Stanley etal., 1979). Heparan sufalte proteoglycans are members of cell surface proteins with long carbohydrate chains of repeating disaccharide units (Hardingham and Fosang, 1992). H S P G s are large molecules with equal amounts of protein and covalently link heparan sulfate. H S P G is a normal constituent of basement membranes that presumably plays an important role in organization of basement membrane components and that also may  18 determine the permeability of basement membranes to acidic molecules (Hassell et al., 1980). Other size molecules of H S P G have been isolated and may vary with the origin of the basement membrane (Katz, 1984). The core protein of H S P G resides in the lamina densa and lesser amounts are present within the lamina lucida and beneath the lamina densa, coating both occasional anchoring filaments and anchoring fibrils as well as collagen in the papillary dermis (Fine, 1994). H S P G s of the basement membrane include perlecan, syndecan and more recently discovered collagen XVIII (Halfter etal., 1998). Other non-collagenous components of the lamina densa include KF-1 antigen which was located by Breathnach et al. (1983) and LDA-1, which is detectable not only within the dermal-epidermal junction but also within dermal vascular and appendageal basement membranes (Fine and Gay, 1986). Nidogen (entactin) is another ubiquitous self-aggregating B M Z component of 100150 kD molecular weight that binds strongly to laminin (Timpl et al., 1983). At the electron microscopic level, in intact skin, entactin was primarily localized to the lamina densa and adjacent upper papillary dermis, but is also detectable within the lamina lucida and in close apposition to overlying hemidesmosomes (Horiguchi et al., 1989). 1.5.4 Sublamina Densa The sublamina densa (lamina fibroreticularis) is the area subjacent the lamina densa and contains anchoring fibrils, microfibrillar bundles and collagen fibers (Katz, 1989). Anchoring fibrils extend from the lamina densa into the underlying connective tissue and either loop back or extend into anchoring plaques containing  19 collagen IV (Keene et al., 1987). However, with post-embedding labeling Shimizu et al. (1997) suggested that most (> 9 0 % ) , if not all, of the anchoring fibrils in human skin do not extend perpendicularly into the dermis, but instead originate and terminate in the lamina densa, forming individual semicircular loops that constitute a network of anchoring fibrils. On their course through the sublamina densa zone anchoring fibrils loop around collagen fibers. Collagen fibers with a typical fiber periodicity consist of randomly oriented singles fibers in the sublamina densa and are not arranged in bundles unlike collagen fibers in the deeper dermis (Briggaman and Wheeler, 1975). Anchoring fibrils contain type VII collagen (CVII), which forms antiparallel dimers of 2 triple helices (Bruckener-Tuderman et al., 1999). One triple helix is composed of 3 a1-chains and is interrupted 19 times by short noncollagenous sequences, which may provide flexibility to the molecule (BruckenerTudermann et al., 1999). Anchoring fibrils also contain other proteins, such as AF1 and A F 2 , which decorate anchoring fibrils (Goldsmith and Briggaman, 1983), and GDA-J/F3, which is localized at the insertion points at the lamina densa and is synthesized both by keratinocytes and skin fibroblast (Gayraud et al., 1997). Type VII collagen may bind to type I and IV collagen, laminin-5 and fibronectin (Chen et al., 1997). Both epithelial cells and mesenchymal cells are capable of producing type VII collagen (Bruckner-Tuderman et al., 1999) and collagen VII synthesis of epithelium grown on devitalized human dermis deprived of BM is stimulated by T G F R 2 (Konig and Bruckner-Tuderman, 1994). Cultured keratinocytes may also synthesize collagen types I, III, and V (Prunieras etal., 1983). Fibronectin is a large protein and is present throughout the upper dermis and also in the lamina lucida (Fine, 1994), although it is not considered a "true" BM component.  20 Fibronectin possesses multiple binding sites for E C M and cell receptors. There is a soluble dimeric plasma fibronectin and a multimeric cellular fibronectin. Another E C M component located at the B M Z is tenascin. Tenascin is a large hexameric glycoprotein that is abundantly expressed at epithelial-mesenchymal interaction sites during embryogenesis, but has a rather restricted distribution in adult tissues (Hakkinen etal., 2000). In normal oral tissues immunoreactivity can be observed as a delicate line under the epithelium, whereas in hyperkeratotic lesions, inflammation, Candida infection, dysplasia and carcinoma tenascin expression is enhanced and may extend deeper into the lamina propria (Tiita ef al., 1994). Increased expression of tenascin observed in those pathologic conditions was assumed to be of epithelial origin (Tiita et al., 1994). Tenascin is also prominent under the leading edges of wound margins and throughout the granulation tissue, but absent in scar tissue (Mackie et al., 1988). 1.6 Mucosa separation Various techniques and purposes for epidermal-dermal (epithelial-connective tissue) separation have been reported. Dermo-epidermal separation is especially useful for immunofluorescence differentiation between hemidesmosomal antigens and those of the lamina densa or sub-lamina densa zone (see Willsteed etal., 1991 for review). Furthermore substrate sensitivity is increased for certain antigens after splitting (Willsteed at al., 1990). A commonly used agent for detachment of viable epithelial cells is trypsin. Trypsinization at 4°C for 1-2h causes a separation within the lamina lucida (Jensen and Mottet 1970). A n increase in lysosomes and blebbing of the basal cell  21 cytoplasm with basal lamina fragments remaining attached between the blebs was observed. After separation by cold trypsinization anchoring filaments are absent from the dermis and epidermis but hemidesmosomes and tonofilaments of the basal cells remain intact (Briggaman etal., 1971). Basal lamina and sub-basal fibrillar elements also remain intact (Briggaman and Wheeler, 1975). However, cold trypsinization is also capable of producing a high-level intraepidermal split at the level of spinous and granular layer (Skerrow, 1980), after incubation with 0 . 2 5 % trypsin for 2h and more advanced at 5h. In the same study the incubation with trypsin at 37°C for 2.5h resulted in detachment of basal and spinous cells, increased membrane fluidity and cytoplasmic protrusions. A more rapid and reliable proteolytic separation agent is thermolysin at 4°C for 1 h (Walzer et al., 1989). The same group also reported a split between basal and suprabasal cells with trypsin. However, intraepidermal splits were also observed with thermolysin by another group (Willsteed at al., 1991). Dispase is another proteolytic enzyme that induces dermo-epidermal separation (Kitano and Okada, 1983) but destroys the lamina densa (Stenn etal., 1989). Scaletta and MacCallum (1970) reported a split in the lamina lucida of oral mucosa after 50-60 minute incubation in 20 mM EDTA at 37°C They described intact desmosomes and hemidesmosomes, shrinkage of basal cells with widening of the intercellular spaces and also observed solitary basal cells remaining attached to the connective tissue in occasional experiments. An intact lamina densa covered the connective tissue, which showed increased spaces between collagen fibrils. It was concluded that divalent cations have a principal role in maintaining the integrity of the BMZ, especially hemidesmosomal filaments. Swelling and loosening of the  22 basal lamina which remained attached to the mesenchyme has also been observed (Goel and Jurand, 1968). Incubation with 1mM EDTA in buffered saline for 2h at 37°C resulted in easy mechanic separation of mouse ear skin in a study by Harris et al. (1980). The epithelium remained viable and proliferation and metabolic activity could be assessed. Mackenzie and Hill (1981) used 3mM EDTA for 2h to induce separation of mouse ear skin and tongue mucosa resulting in functional integrity of the tissues, which were used for recombination and grafting. Scaletta ef al. (1978) used 1 M NaCI solution for 96 h at 4°C before mechanical dermo-epidermal separation of human skin. The basal lamina was attached to the dermis, which remained ultrastructurally unaltered and resembled the split induced by chelating agents. NaCI treatment is supposed to remove soluble connective tissue components (Karasek and Charlton, 1971). Hemidesmosomes remain intact at the epidermal surface (Willsteed et al, 1991). Lower concentrations of NaCI also induced a split within the lamina lucida (Karpati ef al., 1991). The sub-basal dense plate (SDP) with a wreath of anchoring filaments remained on the epidermal side of the split adjacent to the hemidesmosomal part of the plasma membrane of basal keratinocytes. De-epidermization of human skin was also achieved by incubation in warm P B S for various lengths of time, which induces activation of proteases (Regnier ef al., 1981). However, prolonged incubation in P B S has been shown to destroy the normal antigenicity of the B M Z (Danno ef al., 1982). A purely mechanical induction of DEJ is the application of continuous suction, which results in a separation within the lamina lucida (Kiistala and Mustakallio, 1967,  23 Saksela et al., 1981). The split resembles that of junctional epidermolysis bullosa (Christiano and Uitto, 1996). Laminin, and fibronectin immunostaining show a reaction with both the base and the roof of the blister whereas type IV and III collagen are present only at the base (Saksela ef al., 1981). Woodley et al. (1983) applied cold trypsinization, cold salt, warm saline and suction blister for DEJ separation and observed a split through the lamina lucida with all 4 methods. Only cold trypsinization resulted in loss of immunoreactivity for H S P G . B P A type IV and V collagen and laminin remained unaltered for all methods. Only B P A remained with the epidermis while the lamina densa covered the dermis as observed by electron microscopy. It was hypothesized that trypsin degrades the H P S G protein core. Other DEJ separation methods include heat separation (Ohata et al., 1995), sodium thiocyanate (Diazef al., 1977), and thermolysin (Hybinette etal., 1999). Dithiothreitol pretreatment results in separation between the lamina fibroreticularis and lamina densa zone (Epstein etal., 1979, Osawa and Nozoka, 1995). Most of the techniques decribed above have been applied on skin or mucosa with relatively shallow rete ridges.  1.7 Keratinocyte culture models  1.7.1 Culture methods Cultured epidermis is already used for a number of applications ranging from a permanent skin replacement to organotypic culture models for toxicity testing and basic research (Parenteau et al., 1991). Various approaches have been applied for  24 culturing human epidermal keratinocytes. Cultures can be composed of whole skin, whole epidermis or can be seeded as isolated cells. Several types of matrices have been used to culture cells on, which can either be viable or non-viable. Another factor that can be modified indefinitely is the culture medium and its components, including presence of serum, calcium ions, retinoids, vitamin D3 and phorbol esters (see Leigh and Watt, 1994 for review). Presence of calcium ions is essential for the formation of desmosomes and stratification (Leigh and Watt, 1994). Complete absence of retinoids results in hyperkeratosis of keratinocytes cultures on collagen/fibroblast lattice (Regnier etal., 1990). Cells can be cultured submerged in medium or exposed to air, which has a major influence on differentiation (Regnier ef al., 1981). Organ cultures  Attempts to culture keratinocytes can be followed back to the late 19 century when th  Ljunggren (1897) cultured excised human skin in ascites fluid. Such organ cultures have limitation as cells remain viable only for short periods and keratinocytes become overgrown by fibroblasts (Leigh and Watt, 1994). Currently major application for organ cultures are studies of skin adnexal structures (Leigh and Watt, 1994). However, the 3 week survival and limited degeneration of porcine skin culture was reported when the medium is defined, skin is attached and grown at the air-liquid interface (Chapman etal., 1989). In an organ culture exposed to air detachment and necrosis occurred at day 14 (Capon ef al., 1998). However, this organ culture was found more suitable for studying cryolesions than reconstituted  25 skin (Capon etal., 1998). Cultures of human skin exposed to air may also be suitable for wound healing studies (Jansson etal., 1996). Explant cultures  A s opposed to organ cultures explants of whole skin are placed on dishes coated with collagen-fibronectin or vitrogen-fibronectin mixture (Morris and Fischer, 1994). Studies on human explant cultures have been reported as early as 1967 (Manojlovic and Hienz, 1967). Epidermal outgrowth can be observed by four days with stratification greatest close to the explants, while fibroblast outgrowth is rare (Morris and Fischer, 1994). Human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin, resulting in maturation into distinct basal, squamous, granular, and keratinized layers (Freeman ef al., 1976). A s the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantify cell growth. This experiment was compared to explant cultures on deepidermized dermis by Regnier et al. (1981) who observed beginning of degeneration of the original explants grown on pig skin at 3 weeks, and in general absence of hemidesmosomes and basal lamina in the outgrowth. Explants on deepidermized dermis lacked palisading of basal cells, showed cornified cells in the second layer and hemidesmosome formation of the outgrowing epithelium including thin filaments crossing the lamina lucida. Successful explant cultures of mouse cells (Halprin etal., 1979) have also been described. Excised wound skin specimens grown at the air liquid interface were used for wound healing experiments showing  26 accelerated re-epithelialization and expression of K1 and K10 after transplantation of keratinocyte suspension (Moll ef al., 1998). Rheinwald and Green cultures  It was suggested that epithelial cells in general may not be independent cell types and that their poor cultivability may be due to failure to provide suitable fibroblast support (Rheinwald and Green, 1977). Fibroblasts may provide required growth factors and extracellular matrix (Alitalo etal., 1982). Rheinwald and Green (1975a) seeded single cell suspensions on irradiated 3T3 embryonic mouse fibroblasts, which resulted in suppression of fibroblast overgrowth and expansion of clonal keratinocyte colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium (6-8 layers) in which the dividing cells are confined to the lowest layer(s). Fibroblasts appeared to be a strict requirement for keratinization and an important though less strict requirement for cell growth (Rheinwald and Green, 1975 b). However, the fibroblast function can be carried out by medium harvested from 3T3 cultures. Morphological differentiation of RheinwaldGreen cultures is not complete as no distinct granular or cornified layer are present (Leigh and Watt, 1994), which becomes more normal after grafting onto mice or humans. 3T3 cells detach from the surface as keratinocyte colonies grow and they are washed away with successive media changes (Hansbrough, 1992). The addition of hydrocortisone makes the colony morphology more orderly and distinctive, and maintains proliferation at a slightly greater rate (Rheinwald and Green, 1975b). By adding epidermal growth factor (EGF) to the medium the lifetime of cultures could  27 be expanded from 50 to 150 generations (Rheinwald and Green, 1977). E G F seems to delay senescence of the cells by maintaining them in a state further removed from terminal differentiation. This effect is revealed by a greater ability of the cells to survive subculturing and to initiate new colonies, but not necessarily by an increased growth rate (Rheinwald and Green, 1977). Recombination cultures  Epidermis and dermis may be recombined with its original or a different mesenchyme after separation. Recombination of chick embryo epidermis opposed to a chick tendon resulted in the formation of a new basal lamina (Jensen and Mottet, 1971). After trypsin separation and recombination of viable dermis and epidermis, basal lamina starts to be formed next to hemidesmosomes after three days and anchoring fibrils start to form on day 5 (Briggaman ef al., 1971). The formation of basal lamina still occurred with non-viable dermis but no anchoring fibrils were formed. Epidermis, deprived of its basement membrane is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, muscle tissue, or a film of fibrous type I collagen, but not when recombined with heat (100 degrees C) killed dermis (Bard and Sengel, 1984). Cultures on ECM or "dermal equivalent"  In order to create a more in wVo-like condition cultures are grown on a more physiologic substrate, mostly collagen gels and are exposed to air, resulting in improved architecture but not normal expression of keratins (Fusenig, 1994).  28 Keratinocytes were cultured on type I collagen gels as early as 1971 (Karasek and Charlton, 1971). When suspensions of epidermal cells are seeded onto collagenfibronectin coated dishes they may stratify after confluence (Morris and Fischer, 1994). However, the structural organization and formation of distinct strata is generally poor in cultures on collagen gel (Fusenig, 1994). Differentiation is improved by adding a dermal fragment underneath the gel (Mackenzie and Fusenig, 1983). Both living and dead dermis was used which exerted similar effects on the keratinocytes. Cultures could be maintained for up to three weeks and degeneration at two weeks could not be prevented by replacing the dermal tissue (Mackenzie et al., 1993). Good patterns of growth and morphology, and appropriate expression of cytoplasmic and cell surface differentiation markers, were found only in specimens grown in association with dermal elements. It was concluded that diffusible factors of dermal origin facilitate epithelial growth and differentiation (Mackenzie, etal., 1993). Another approach to study the effect of mesenchymal epithelial interactions and to improve structural organization and correct expression of differentiation markers are co-cultures of keratinocytes on collagen-embedded fibroblasts (Bell etal., 1979). The matrix can either be free-floating and contracted or attached and noncontracted. A s fibroblasts secrete collagenases the matrix will sooner or later become disintegrated, which can be prevented by collagenase inhibitors or addition of hydrocortisone (Fusenig, 1994). Another problem is fibroblast migration and direct contact with epithelial cells, which can be delayed by using the antipodal culture system (Fusenig, 1994). Epithelial cells can either be seeded as suspensions or placed as explants onto a collagen-fibroblast matrix (Coulomb etal.,  29 1986). Epidermal growth is enhanced when the collagen matrix had previously been reorganized by fibroblasts, and is greatest when living fibroblasts persist in this matrix (Coulomb et al., 1989). Differentiation features were not significantly affected by the presence of human sublethally irradiated fibroblasts (Limat et al., 1989) and cultured cells exposed to air on a synthetic membrane formed all layers of keratinized stratified epithelium in the absence of fibroblasts (Noel-Hundson et al., 1995). Comparing keratinocytes from trunk skin and foreskin, differences observed in situ persisted in recombinant cultures and transplants along with the expression of keratin K13 (typical for foreskin in situ) irrespective of the type of mesenchyme (Boukamp ef al., 1990). It was concluded that regulation of keratinocyte growth and differentiation is mesenchyme-dependent, but that differences between epidermis of different body sites are also controlled by intrinsic programs. When cultures of human oral keratinocytes are transplanted on mice they maintain some of their original features (Lindberg and Rheinwald, 1990). It was concluded that regional differentiation of the oral epithelium is based on an intrinsic specialization of regional keratinocyte stem cells. In earlier studies major differences between epidermis grown on dermal equivalent and epidermis in vivo were reported, including lacking cuboidal shape of basal cells, non-polarized distribution of B P A and laminin, immediate suprabasal expression of involucrin, and unusual keratin expression (Asselineau etal., 1986). However, more recently in keratinocyte-fibroblast cultures from gingival, Tomakidi et al. (1999) demonstrated similar histomorphology, normal expression of differentiation specific keratins and keratinocyte-type integrins when compared to the original tissue. Initially, integrin av was expressed throughout the whole epithelium but limited to  30 basal aspect of cells after 2 weeks, and later interruption of the band-like av integrin immunolocalization at the subepithelial site. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells) expression of all typical differentiation markers was present in the reconstituted epithelium, though with different localization as compared to normal epidermis (Smola et al., 1993): keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia, but normalization of localization and proliferation occurred after transplantation onto nude mice. Involucrin, K1 and K10 are also expressed in basal and suprabasal cells 8h after wounding of fully differentiated keratinocyte/fibroblast-cultures (Garlick and Taichman, 1994). Despite normal tissue architecture reconstructed epidermal tissues show abnormal immunohistochemical staining of keratins, involucrin and fillagrin (Fusenig, 1994). Keratin 14, restricted to basal cells in vivo, can be observed through most of the epithelium and gene expression also shows extending immunolocalization (Tomakidi etal., 1997). In an oral mucosa model histoarchitecture and keratin expression normalized at 3 weeks with K5 expression restricted to basal cells and K4 and K13 located suprabasally, polarized integrin expression of basal and parabasal cells and linear subepithelial codistribution of CIV, LM-1 and LM-5 (Tomakidi etal., 1998). Presence of dermal cells in the matrix seems to have a major influence on the production of B M Z components. Epidermal cells grown on plastic or on collagen gels showed poor differentiation, lack a structured BMZ, and incomplete expression and deposition of B M constituents (Bohnert etal., 1986). However, upon reimplantation in vivo, differentiation was normalized, expression of B M components  31 complete and a structured B M reformed. When cultures on collagen gel were similarly associated with dermal mesenchyme in vitro, epidermal differentiation and expression of B M components were almost normalized, but a structured B M was absent (Bohnert ef al., 1986). However, a later study under similar conditions showed an orderly structured BMZ (Smola et al, 1998). When keratinocytes were grown on collagen matrix with and without fibroblast only those with mesenchymal cells showed deposition of Collagen IV and laminin-1, whereas laminin-5 and nidogen were expressed both in mono and co-cultures at early stages (Smola etal., 1998). At 3 weeks a band-like pattern of collagen Vll was observed at the BMZ in co-cultures, only. The ultrastructural organization normalized at 3 weeks, including the formation of hemidesmosomes, a lamina lucida and densa, and anchoring fibrils. The epithelium-matrix interface was straight and did not mimick the rete ridge-connective tissue papillae pattern of skin or oral mucosa. Fleischmajer ef al. (1998) described BMZ-component synthesis and formation of hemidesmosomes, anchoring filaments, anchoring fibrils and a lamina densa in keratinocyte-fibroblast colcultures. Fibroblast cultured alone also synthesized CIV, LM-1, perlecan and nidogen but no BM assembly occurred. Delayed expression of K1 and K10, protracted expression of K14 and early onset of involucrin suggest that homeostasis in these cultures is not yet established (Fusenig, 1994). Applied modifications of the ECM/lattice composition include addition of chondroitinsulfate (Boyce etal., 1990), a gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate  32 proteoglycan (Tinois etal., 1984), and type III collagen mesh gels, resulting in a corrugated dermo-epidermal interface (Lillie etal., 1988). The introduction of immortal cell lines may facilitate the immediate culture but they may have lost their differentiation capacity or aberrantly express differentiation markers in the absence of mesenchymal support as demonstrated in HaCat keratinocytes (Ryle etal., 1989). HaCaT keratinocytes are spontaneously transformed human epidermal cells that are virtually immortal and nontumorigenic (Boukamp et al., 1988). They are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells which is strongly modulated by environmental conditions (Ryle etal., 1989). K1 and K10, were expressed independently in conventional submerged cultures which was not strictly correlated with the degree of stratification (Ryle ef al., 1989). When transplanted onto nude mice HaCaT cultures show normal histomorphology, suprabasal expression of K1 and K10, generally basal expression of K14 and integrin R1 and synthesis of collagen IV, 'classical' laminin, laminin-5 and collagen Vll at the B M Z (Breitkreutz ef al., 1998). HaCaT cells in organotypic cocultures on top of collagen gels containing human dermal fibroblasts formed dysplastic epithelium within 1 week, but developed a well structured and differentiated squamous epithelium with a parakeratotic stratum corneum at 3 weeks (Schoop et a/.,1999). After 1 wk, keratins 10 and 16, involucrin, and transglutaminase were expressed in suprabasal layers, and formed a nearly complete basement membrane including hemidesmosomes and anchoring fibrils in 3 weeks.  33 Cultures on reticular dermis and de-epidermized dermis (DED)  Prunieras ef al. (1979) first described the cultivation of cell suspensions on deepidermized dermis. Regnier et al. (1981) removed the epidermis by warm incubation in P B S for 4-7 days and the dermis was sterilized by antibiotic treatment or gamma-irradiation. Human epidermal keratinocytes were seeded both on deepidermized dermis and on reticular pig skin and immediately grown at the air-liquid interface. Migration into "pits and holes of the uneven papillary dermis", stratification with granular layers and flattened cornified cells as well as complete formation of hemidesmosomes and desmosomes were observed in the cultures on deepidermized dermis. On reticular pig skin, however, few cells attached resulting in very poor cultures. It was concluded that type IV collagen of the B M Z provides a better substrate for cell attachment, growth and differentiation than reticular dermis or glas/plastic surfaces. B P A is expressed at the basal pole of basal cells after 7 days and K1/K10 fill all suprabasal layers (Regnier et al., 1990). This reconstructed epidermis is similar to epidermis in vivo in respect to differentiation, undulated dermal-epidermal interface and ultrastructure. The undulating surface of acellular dermis acts as a template and organizes seeded keratinocytes into a rete ridge-like pattern (Medalie etal., 1997). A decrease in K1/10 expression and thickening of the stratum corneum can be observed at increased concentrations of vitamin D3, while retinoic acid treatment resulted in absence of K1/K10 and a parakeratotic stratum corneum (Regnier ef al., 1990). Involucrin and transglutaminase expression are similar to in vivo epidermis (Regnier and Darmon, 1989). Stratification also occurs in submerged cultures on DED including keratohyaline synthesis and cornification, but epidermal morphogenesis is greatly improved when  34 cultures are exposed to air and also express the 67 kD keratin (K1) (Regnier et al., 1986). The presence of a basal lamina may have direct inductive effects or indirect effects by acting as a filter when cultures are exposed to air and fed from below. Human keratinocyte cultures grown at the air-liquid interface on de-epidermized dermis at 37 degrees C in serum- and EGF-containing medium showed hyperproliferation followed by rapid senescence (Gibbs et al., 1997). Low temperature resulted in prolonged life-span of the culture, whereas culturing at 37° C increased the rate of differentiation without affecting the rate of proliferation. Involucrin and transglutaminase were abnormally expressed irrespective of the culture conditions. Cultures on DED were also applied for keratinocytes from hair follicle, carcinoma cells and other pathological keratinocytes (Regnier ef al., 1990). Closer in vivo resemblance was achieved by integration of Langerhans cells and melanocytes (Regnier ef al., 1997).  35  C H A P T E R TWO-Aim of the study  Several well defined organotypic cultures models have been described previously. However, in any model attempts were never made to use bovine de-epidermized tongue mucosa as a substrate. Moreover the authors failed to investigate integrin expression and E C M expression in systems using DED. A distinct feature of bovine mucosa lamina is the extreme interdigitation of long rete ridges and connective tissue papillae combined with compartments of highly varying degrees of differentiation. Aim of this study was: 1. to create an organotypic keratinocyte culture that mimicks interdigitating rete ridges and C T papillae on a preserved BMZ. The hypothesis was that the highly irregular surface may create an ideal substratum for the formation of distinct epithelial compartments. 2.  to compare cultures of different cell lines and compare their pattern of integrin expression and differentiation. The hypothesis was that cell specific differences will be expressed despite identical culture conditions.  36  C H A P T E R THREE-Material and Methods  3.1 Substrate tissue preparation, separation 4 bovine tongues from freshly sacrificed cows (2-3 years old) were used. The mucosa from pre-sulcular dorsal surface was harvested 2-3 hrs after sacrifice. Mucosal specimens from anterior (tip), middle and posterior tongue were excised using a Bard-Parker #15 blade. Samples were also taken from the lateral tongue border at the transition from dorsal to ventral surface. Muscle tissue was removed as much as possible. Tissue pieces of approximately 1 X 0.5 X 0.25 cm size were incubated with the following agents and conditions: a) 1 M NaCI aequous solution, 4°C, 96h. b) 0.04% EDTA, 4°C, 2h c) 0 . 4 % EDTA, 4°C, 2h d) 0 . 4 % EDTA, 4°C, 24h e) P B S , 37°C, 72-96h f)  0 . 2 5 % trypsin, 4°C, 18h.  The epithelium was then separated from the connective tissue (CT) with forceps. Unseparated control specimens from the three dorsal and lateral regions immediately after excision were embedded in Histo Prep (Fisher Scientific, New Jersey, NY), snap frozen in liquid nitrogen and stored at -80° C for frozen sections. The same was done with split epithelium and C T specimens. Fresh C T specimens were disinfected by washing in 7 0 % ethanol for 1 minute and then washed in  37 D M E M (Life technologies, Grand Island, NY) for 1 h. Specimens were either stored in D M E M at -80° C or directly used for cultures.  3.2 Localization of the split 5-7 um thick frozen sections were made from all samples (Reichert-Jung Frigocut N 2800). The specimens were mounted on silane (Sigma, St. Louis, MO) coated slides and fixed in acetone for 5 minutes at -20° C before being stored at -80° C. Slides were stained with H E or used for immunohistochemistry (IHC), using the primary antibodies listed in table 1 and methods described previously (Larjava etal., 1993). Briefly, slides were incubated with the primary antibody diluted in PBS/BSA (1 mg/ml) overnight and then washed with PBS/BSA and incubated with secondary rhodamine-conjugated antibody (mouse/rabbit, 1:50, Boehringer Mannheim Biochemicals, Indianapolis, IN) for 1 h. Non-immune serum and PBS/BSA only for primary incubation served as negative controls. Cover slides were mounted after washing with PBS/BSA and air drying. Slides were examined and photographed with a Zeiss Axioskop 20 fluorescence microscope (Zeiss, Jena). Several specimens of epithelium and C T were prepared for scanning electron microscopy (SEM) using routine protocol. Glutaraldehyde-fixed unseparated mucosa, epithelium and C T were prepared for T E M . Samples were impregnated with osmiumtetraoxide, uranylacetate, alcohol-dehydrated and embedded in Epoxyresin (Epon 812). 500 nm thick sections were cut with a glass knife (Microtome MT 6000, Sorvall) and stained with tolouidine blue/boric acid. 80 nm sections were  38 made with a diamond knife, contrasted with uranylacetate and lead-citrate, and viewed and photographed with a transmission electron microscope (Philips 300).  3.3 Culture 3.3.1 Cells Human HaCat keratinocyte cell lines from frozen stock were cultured in 25 c m  2  plastic flasks (T25, filter caps,Falcon) in D M E M (changed every 2 days) until confluent. Human epidermal keratinocytes (NHEK) and human gingival keratinocytes (HGK) from frozen stock were cultured in 25 c m plastic flasks (T25, 2  filter caps, Falcon) in K G M (Clonetics, San Diegeo, CA) (changed every 2 days) until 8 0 % confluent. The H G K used derived from an immortal human gingival keratinocyte cell line with a partially triploid chromosome set and were isolated for studies on matrix metalloproteinase expression (Makela, 1998). 3.3.2 Culture media Culture media were prepared by a modification of a protocol described by Parenteau (1994). Minimally supplemented basal medium (MSBM), contained a 3:1 mix of D M E M and Ham's F12, supplemented with 5ug/ml insulin, 5 ug/ml transferrin, 10" M ethanolamine, 10" M phosphorylethanolamine, 20 pM 4  4  triiodthyronine, 0.4ug/ml hydrocortisone, 5.3x10" selenious acid, 0.18 mM adenine, 8  gentamycin/streptomycin/amphotericine B (Life Technologies, Grand Island, NY), and 5 % FBS. M S B M was used for 6 days for submerged culturing. It was then replaced by a cornification medium (CM). C M contained 1:1 of D M E M and Ham's  39 F12, same supplements as M S B M and 2 % serum. C M was used for another 6 days. C M was replaced by a maintenance medium (MM) after another 6 days. MM was same as C M but serum level reduced to 1%.  3.3.3 Culture process Keratinocytes were trypsinized, counted and seeded onto the connective tissue side formerly attached to the epithelium with 200,000 cells in 10ul medium per piece. After 2 h for attachment the specimens were placed on the permeable membrane of an insert-culture well system (Organogenesis Inc, Canton). The surface of the C T substrate was raised to the air liquid interface 6 days after seeding, leaving the keratinocytes exposed to air and fed only from below. Supply with medium was maintained through the insert membrane. The medium was changed every 2  n d  day  for the entire culturing process. Samples from HaCaT cultures from 5 to 40 days after raising to air liquid interface were stained with a vital dye (PT #6402A, Promega) and snap frozen in liquid nitrogen. Specimens were stored at -80° C until sectioned and stained with HE or prepared for IHC. H G K and N H E K culture samples were examined 14 days after air exposure (total culture time 20 days) and stained for IHC as described above. Frozen sections of human gingiva and bovine tongue served as (positive) controls, sections incubated with non-immune serum or P B S served as negative controls. A minimum of 3 sections for each raft and antibody was stained. HaCat cultures from 20 days after air exposure were prepared for T E M as described above.  40  C T substrates without cells were cultured under the exact same conditions to serve as a control. Control specimens were embedded for frozen sections together with culture samples for direct comparison.  Antigen  Tenascin (TN) Laminin-1 (LM-1) Laminin-5 (LM-5)  T y p e IV C o l l a g e n ( C I V ) T y p e VII C o l l a g e n (CVII) Heparan-sulfate-proteoglycan (HSPG) Integrin f i 6  Clonal type  Monoclonal, BC24 Monoclonal, G B 3 Monoclonal, 1924  Polyclonal, P S 057  Dilution  Reference/Source  1:400  S i g m a , St. L o u i s , M O  1:30  V e r r a n d o era/., 1 9 8 7  1:100  1:100  Chemicon, Temecula Monosan, Uden, N L  Monoclonal, 1345 Monoclonal  1:50 1:25  Chemicon, Temecula  Monoclonal, G6B1  1:10  H u a n g era/., 1 9 9 8  Integrin av  Monoclonal, L230  1:10  H o u g h t o n era/., 1 9 8 2  Integrin a2  Monoclonal, M A B 1950Z  1:10  Chemicon, Temecula, CA  Integrin a3  Monoclonal, M A B 1952  1:100  Chemicon, Temecula, CA  Integrin a 5  Monoclonal, M A B 1986  1:100  Chemicon, Temecula, CA  Integrin avfJ5  Monoclonal, M A B 1961  1:100  Chemicon, Temecula, CA  Integrin p1  Polyclonal, 3847  1:500  R o b e r t s era/., 1 9 8 8  Integrin p 4  Monoclonal, A054  1:400  Gibco BRL, Gaithersburg, M D  Keratin 1 0  Monoclonal, LL002  1:100  S e r o t e c Ltd., O x f o r d , England  Keratin 14  Monoclonal, M A B 3230  1:100  Chemicon, Temecula, CA S i g m a , St. L o u i s , M O  K e m e n y etal., 1 9 8 8  Involucrin  Monoclonal, S Y 5  1:100  Vitronectin  Monoclonal,  1:100  N i k k a r i era/., 1 9 9 5  Fibronectin  Polyclonal, F3648  1:500  S i g m a , St. L o u i s , M O  Fibronectin E D A  Monoclonal, MAS521  1:100  Harlan Sera-Lab, Loughborough, England  Table 3: Antibodies, working concentrations, and sources  42  C H A P T E R FOUR-Results 4.1 Tongue mucosa 4.1.1 Histology HE staining of bovine dorsal tongue mucosa showed a thick stratified sqamous epithelium (Fig 1, A). Pronounced C T papillae protrude into the epithelium but do not extend above its surface. There are over 30 cell layers between rete ridges and the epithelial surface. There was a distinct basal cell layer, but no granular cell layer. A thin amorphous eosinophilic layer covered the interpapillary surface. On sagittal sections numerous filiform papillae are notable based on a prominent connective tissue core (Fig 1, A). The C T core of filiform papilla is located posterior to the center of filiform papillae and has several secondary papillae. It does not extend beyond the epithelial surface. Filiform papillae are bent posteriorly and an anterior and posterior cell line composing the papilla can be distinguished. The connective tissue separating the epithelium from the muscular tissue is relatively thin and rich in cells and vessels. Inflammatory cells were not observed. There was no principal difference between the three investigated areas, except for epithelial thickness. Epithelium from the middle sections appeared thinnest. No papillae other than filiform were observed. Lateral tongue samples generally resembled interpapillary dorsal areas. However, the epithelium was much thinner, less than half the thickness. There were no surface papillae and C T papillae were shorter. The epithelial compartments were  identical to interpapillary dorsal epithelium lacking a stratum granulosum and stratum corneum.  44  Fig 1- (A) HE staining of unseparated bovine tongue, showing filiform papillae, distinct anterior and posterior cell lines and separation between anterior and posterior keratin layer (arrow). (B) IHC staining for T N , location at anterior C T and C T papillae. Bar 200 um  45  4.1.2 Immunohistochemical staining of bovine tongue mucosa IHC for H S P G , CIV and CVII resulted in a linear staining of the B M Z (Fig. 3). Furthermore blood vessels showed a positive reaction for CIV and H S P G . T N was located at the B M Z of the tip of CT-papillae and the connective tissue core of filiform papillae only. In association with the C T core of filiform papillae T N was located at the anterior wall lining the B M Z of cells that form the keratin core and at the upper third of C T papillae (Fig 1, B). Antibodies to human LM-1 and LM-5 were not reactive (not shown). FN and V N were localized throughout the C T (not shown). Integrin I3>1 was located pericellular of basal and occasionally suprabasal cells and reacted with cells and vessels in the C T (Fig 2, D). Integrin B4 was present at basal poles of basal cells, resulting in a linear expression at the B M Z (Fig 2, G). Subunit cc3 was located pericellular in basal and suprabasal cell (Fig 4, C). Antibodies to integrins a2, a5, av, av&5 or B6 were not reactive (not shown). K14 was located mostly at basal cells of the epithelium and scattered in some suprabasal layers (Fig.4, B). K10 was generally absent but an intense reaction could be observed in the anterior cell line just below the keratin core of filiform papillae (Fig 4, D). Involucrin was expressed in the posterior cell line associated with filiform papillae and also in the posterior keratin core (Fig 4, A). A less intense expression was observed in some anterior cells.  46  Fig 2 HE and IHC staining of bovine tissue before and after cold salt separation: (A, D, G) unseparated BMZ; (B, E, H) connective tissue; (C, F, I) epithelium; (A-C) HE; (D-F) integrin p1; (G-l) integrin p4. Bar 200 um  Fig. 3: IHC staining of bovine tissue before and after cold salt separation: (A, D, G, J) unseparated BMZ; (B, E, H, K) connective tissue; (C, F, I, L) epithelium. (A-C) type IV collagen; (D-F) type VII collagen; (G-l) H S P G ; (J-L) T N . Bar 200 um  48  Fig 4: Unseparated bovine tongue, IHC staining: (A) Involucrin, reaction in posterior cell line and posterior cornified layers, minor reaction some cells of the anterior cell line; (B) K14, (C) integrin a3; (D) K10, reaction in the anterior cell line of filiform papillae. Bar 200 um  Fig 5: Scanning electron microscopy after separation showing the formerly attached surfaces facing up: (A,C) epithelium; (B,D) connective tissue.  50  4.2 Separation After incubation with various agents the tissues could be separated with forceps requiring various degrees of force. Specimens incubated in warm P B S completely disintegrated upon application of any force and were discarded. The microscopic separation was evaluated on HE stained specimens. Trypsin and EDTA incubation resulted in an intraepidermal split in all cases (not shown): Tissue prepared in cold salt mostly showed a clean separation between epithelium and C T (Fig 2, B+C). Multiple cross sectioned collapsed C T papillae that appear unattached to the lamina propria are "floating" above the CT. Mesenchymal cells are still visible within the CT. Intensely stained basal cells remained with the epithelium and former location of C T papillae appear as voids. Some cold salt separated specimens revealed clusters of epithelial remnants or interrupted layers attached to the C T (not shown). This was observed more frequently in tissues from the base or tip of the tongue. Therefore only tissues from the middle of the tongue dorsum were used from here on.  51  4.2.1 Immunohistochemistry for cold salt separated tissue IHC with various anti-integrin- or anti-ECM-antibodies (Table 3) was applied for further determination of the location of separation and possible changes in antigenicity due to the separation process. H S P G , CIV and CVII were observed as a linear staining at the surface of the C T and were negative for the epithelium (Fig 3) TN was scarcely present at isolated locations of the C T surface (Fig 3, K). Staining for LM-1 and LM-5 remained negative (not shown). FN and V N were present only in the C T compartment and remained unaltered (not shown). The epithelium did not stain for any E C M components. Basal cells of the detached epithelium maintained the staining pattern for integrin R1 and Q>4 as seen before the separation (Fig 2. D-G).  4.2.2 Electronmicroscopy S E M examination (Fig. 5) confirmed findings of HE and IHC stainings, showing a complex C T surface free of epithelial cells. Multiple connective tissue papillae of various length and diameter were seen without evidence of epithelial cell remnants. C T papillae are mostly collapsed and folded. The surface of each C T papilla demonstrated multiple folds and grooves. On the lateral surface of the C T the reticular structure of the lamina propria could be observed. The epithelial part showed the basal cell surface with rete ridges. Smaller "holes" indicate the previous location of C T papillae and larger ones the previous location of  52 C T cores of filiform papillae. The surface of the epithelium revealed small irregularities and indentations. T E M observation of unseparated specimens demonstrated normal ultrastructural appearance of the BMZ (Fig 6, A), including hemidesmosomes, anchoring filaments, increased thickness and density of the lamina densa subjacent to hemidesmosomes and anchoring fibrils in the sublamina densa. Intermediate filaments appeared to be associated with the cytoplasmic hemidesmosomal plaque. After separation an unaltered, amorphous lamina densa covers the C T surface (Fig 6, D). Occasional epithelial remnants were observed in some areas (Fig 6, B+C). When the separation was incomplete hemidesomosomes with anchoring filaments could still be observed, which indicates that they have not been dissolved during the incubation process. Compared to unseparated specimens there are no ultrastructural changes in deeper areas of the lamina propria. These observations suggest a separation through the lamina lucida.  53  Fig 6: Transmission electron micrographs of bovine tongue BMZ. (ct= connective tissue, e=epithelium), (A) before separation, (C,D) partial separation with basal cell remnants, note persisting hemidesmosome and anchoring filaments (arrowhead); (D) lamina densa covered lamina propria completely separated from epithelium (magnification 365000x).  54  4.3 Cultures 4.3.1 HaCaT Culture rafts remained vital as indicated by vital dye staining until end of the experiment after 40 days. HaCat cells migrated into irregularities of the C T surface filling out all empty spaces at 5 days (Fig 7, A+C). A highly corrugated epithelial C T interface was formed, resembling rete ridges and C T papillae. Collapsed and folded C T papillae appear as round or ovoid structures surrounded by epithelium. Stratification of keratinocytes was observed 5 days after raising the culture to the air-liquid interface. Some morphologic differentiation could also be seen with flattening of cells in the superficial layers (Fig 7). Maximum stratification was observed from day 18 on with up to 15 cell layers (Fig 7, B+D). The epithelium appeared well organized and differentiated with a distinct basal layer. There was a tendency towards vertical orientation of basal cell nuclei and basal cell palisading. No major changes occurred in the period from 18-40 days. Epithelial migration was seen at the lateral borders of some rafts. The migrating layer usually consisted of 23 cell layers (not shown). In none of these cultures was there a stratum granulosum or stratum corneum. There were no changes evident in the CT. Occasionally HaCaT cells grew over the unseparated bovine epithelium. Original bovine epithelium could be easily distinguished by its different morphology and lack of chromophilia. When this occurred stratification was reduced remarkably (not shown).  55  Fig. 7: HE staining of HaCaT keratinocyte cultures: (A,C) 5 day culture; (B, D) 18 day culture. Bar 200 um.  56  4.3.2 H G K  H G K cultures resemble HaCaT cultures in principle regarding the rete ridge like pattern, but show less stratification and some flattening of the most superficial cells only (Fig 8). H G K cultures also lacked a stratum granulosum and corneum. The number of cell layers was much smaller and were comparable to early HaCat cultures. Rarely were more than 5 layers above a C T surface observed. The degree of organization also appears less than in HaCaT cultures. There was a big variety in nuclear shape, size and density. H G K also showed signs of epithelial migration at raft margins. The surface of H G K cultures shows a thin eosinophilic line with mostly flattened dense nuclei.  57  Fig 8: HE staining of H G K cultures, 14 days after air exposure. (A) H G K fill all voids between C T papillae but show limited stratification, note nuclear polymorphism. (B) higher magnification, some palisading of basal cells but no distinct spinous or granular cells. Bar 200 um  58  4.3.3  NHEK  N H E K cultures in general show the complete histomorphology of a keratinized stratified squamous epithelium. There is a basal layer with columnar or cuboidal basal cells, a few layers of spinous cells, one or two layers of granular cells and a distinct keratin layer mostly free of nuclei (Fig 9). Keratohyalin granules were present in the granular cell layer (Fig 9, B). However, some cultures or culture sections lack a stratum granulosum and corneum (Fig 9, C). Instead a fine eosinophilic line covers the surface. The rete ridge pattern of N H E K cultures seems to be distinct from the original tongue epithelium, HaCaT or H G K cultures. The tips of rete ridges have more of a saw-tooth like appearance rather than being rounded. Some regions in the stratum spinosum had an almost amorphous appearance lacking nuclei (Fig 9, C).  59  Fig 9: HE staining of 14 day old N H E K culture. (A) section showing both para- and orhtokeratotic areas. (B) higher magnification of stratum granulosum with keratohyalin granules. (C) Non-keratinzed culture section. Note saw-tooth like rete ridges (arrow) and areas devoid of nuclei. Bar 200 um  60  4.3.4 Immunostaining Negative controls (PBS/BSA instead of primary antibody) showed no specific reaction. Control C T substrate, cultured parallel without cells and sectioned/stained simultaneously with cultures, remained generally unaltered during the culture process. CIV, CVII were still located at the BMZ. H S P G showed the same pattern but with reduced intensity at the C T surface compared to split mucosa before culturing, whereas vessels within the C T showed similar intensity as before the culturing process. Negative reactivity for LM-1 and LM-5 persisted. TN was expressed in a few areas of the BMZ as a line at the former B M Z surface (Fig 10, A). FN was distributed throughout the C T and most abundant at the BMZ. V N showed weak scattered reactivity remote from the former BMZ (not shown). HaCaT  In general there was no difference between young (5 days) and more mature (> 18 days) cultures in respect to E C M and integrin expression. IHC of HaCaT (Fig 10) cultures demonstrated positive staining at the B M Z for type IV and VII collagen and H S P G . Antibodies to LM-1 and LM-5, that were not reactive with bovine tissue, decorated the B M Z in the cultures, which suggests that HaCaT cells were able to deposit B M Z components onto the existing bovine BMZ (Fig 10, F-G). TN was present in a wide band at the BMZ (Fig 10, B). Basal aspects of basal keratinocytes expressed integrin p4 (Fig 11, B). Integrin subunits av, G1 and 66 were present throughout all layers of the epithelium with clusters of increased intensity (Fig 11, C+ D). Integrins a2 and a3 were also located pericellular in all layers (Fig 11, E+F), but no a5 or avB5 was observed (not shown).  Fig 11: IHC staining of HaCaT cultures, integrin subunits: (A) B1; (B) B4; (C) G6; (D) av; (E) a2, (F) a3, Bar 200um  Fig 12: IHC staining of HaCaT cultures, differentiation markers: (A) K14, (B) involucrin; (C) K10, 5 day old culture, arrowhead: non-reactive basal cells; (D) K10, 18 day old culture. Bar 200 um  64  K14 was expressed in all epithelial layers at all stages, slightly more intense in basal cells (Fig 11, A). K10 appeared in mostly suprabasal layers at day 5 after air exposure but was present in all layers after 18 days (Fig 11, C+D). A few basal cells showed no expression of K10 at day 18. Suprabasal expression was more intense than in basal layers. Staining for involucrin showed the most intense reaction at the epithelial surface, but was also visible in all other layers. HGK IHC Mature H G K cultures demonstrated CIV, CVII, H S P G , LM-1, LM-5 and TN at the BMZ (Fig 13). Expression of LM-1, LM-5 and TN again was new and distinct from control tissue. TN expression was different from HaCaT cultures demonstrating only a thin line under the basal cells (Fig 13, C). Integrin B4 was present at the BMZ as a line in some cultures or showed a diffuse positive reaction throughout all layers (Fig 14, A+B). Integrin subunits a3, B1, B6 and av demonstrated a diffuse expression in all layers, whereas a2, a5 and avS5 staining showed no reaction (not shown). K14, K10 and involucrin were expressed in all layers (Fig 15). A slightly increased expression of K14 in basal cells and and increased intensity of involucrin at the surface can be observed.  Fig 13: IHC staining of H G K cultures, E C M : (A) CIV; (B) CVII; (C) TN; (D) LM-1; (F) H S P G ; (G) LM-5. Bar 200 um  Fig 14: IHC staining of H G K cultures, integrin subunits: (A+B) IS4; (C) B1; (D) a 3 ; (E) av; (F) B6. Bar 200 um  67  Fig 15: IHC staining of H G K cultures, differentiation markers: (A) K14; (B) K10; (C) Involucrin. Bar 200 um  68  NHEK IHC  In mature N H E K cultures, CIV, CVII, H S P G , LM-5 and LM-1 were located at the BMZ (Fig 16). T N was distributed at the B M Z as a wide band (Fig 16, A), comparable to that seen in HaCat cultures. Integrin subunits Q>4 was present at basal poles of basal cells and B1 was located pericellular of basal cells (Fig 17, A+B). Subunits oc2 and ct3 were located pericelluar of basal cells and some suprabasal cells (Fig 17, C+D), a5 was limited to the periphery of basal cells, only (not shown). ccvR5 was not reactive (not shown). Integrins av and B6 also demonstrated a pericellular expression around basal and some suprabasal cells, which was most intense at the tips of rete ridges (Fig 17, E+F). K10 was present in the whole cytoplasm of all cell layers, though weaker in basal cells (Fig 18, B). K14 showed strongest expression in basal cells but was also expressed in all other layers (Fig 18, A). Involucrin staining was most intense at the superficial layer but was also visible around the cell membranes of deeper layers (Fig 18, C).  Fig 16: IHC staining of N H E K cultures, E C M : (A) T N , wide diffuse band at the BMZ; (B) CIV; (C) CVII; (D) H S P G ; (E) LM-1; (F) LM-5. Bar 200 um  Fig 17: IHC staining of N H E K cultures, Integrin subunits: (A) R4; (B) B1; (C) a2; (D) cc3; (E) av; (F) S6. Bar 200 |jm  71  72  4.4 HaCaT T E M Toluidine Blue stainings of ultrathin sections used for T E M showed no signs of old epithelium. Ultrastructural observations of the B M Z in mature HaCaT cultures demonstrated basal cells plasma projections interdigitating with basal lamina protrusions. Furthermore a normal appearance of a lamina lucida, lamina densa and numerous hemidesmosomes were seen (Fig 19, A). Hemidesmosomes opposed areas of increased thickness and density in the lamina densa. Anchoring filaments were present in the lamina lucida subjacent to hemidesmosomes. Althought the lamina densa was mostly continuous some interruption could be observed. Collagen fibers could be discerned in deeper C T areas. There were some areas with anchoring fibrils in the sublamina densa zone. Hardly any keratin filaments could be discerned in association with hemidesmosomes. Hemidesmosomal submembranous dense plates could be seen in some areas (Fig 19, A, arrow).  73  Fig 19: T E M of B M Z of 20 day old HaCat culture on de-epithelialized lamina propria (ct), (A) note submembraneous dense plate (arrow) and anchoring fibrils (AF), 28000x; (B) hemidesmosomes and anchoring filaments (arrowheads), 82000x  74  C H A P T E R FIVE-Discussion 5.1 Bovine mucosa The histology of bovine tongue mucosa observed in this study is in agreement with previous findings (Trautwein and Fiebiger, 1952, Steflik etal., 1983). Expression of K14 in basal layers, absence of K10 in interpapillary areas, absence of K10 in basal and cornified papillary areas, but strong expression in the posterior cell line of filiform papillae was described in human tongue (Sawaf et al, 1990). However, Heid etal. (1988) located K1/10 in the anterior parapapillary column of bovine tongue filiform papillae, which is in agreement with observations made in this study. The histomorphology of human and bovine papillae itself is rather distinct which may explain the conflicting results. Involucrin expression was prominent in the posterior cell line of filiform papillae, but hardly in the remaining epithelium. Hohl et al. (1995) observed involucrin in tongue epithelium only in association with filiform papillae. Currently, there are no comprehensive studies on bovine tongue integrin expression or distribution of E C M components. Human oral tissues have been studied in detail and the results may be applicable to the bovine species. Expression of integrin R4 and B1 in bovine tongue mucosa appears similar as described for human epidermis (Watt and Hertle 1994). cc5, av, &6 and R5 were absent as reported for human oral mucosa (Zambruno ef al., 1995). However, a5, av and Q>5 may be variable and expression was observed in human lateral margins of tongue mucosa and cheek (Jones et al., 1993). No avB5 was observed in our study. Integrin a 3 was located in basal and several suprabasal cell layers whereas antibody to integrin a2 was not reactive. Distribution of integrin a 3 is in agreement  75 with previous in vivo observations and reflects its role for cell-cell adhesion (Watt and Hertle, 1994). Lack of reactivity of integrin cc2 antibody may be attributed to bovine specific antigenicity when antibodies against human a2 integrin is used, as it is normally expressed in human oral mucosa (Jones etal., 1993). E C M components CIV, CVII, H S P G were expressed at normal location at the BMZ. TN, which is expressed as a thin line in normal oral epithelium (Tiitta et al., 1994, Hakkinen et al., 2000), was present only in specific areas such as the B M Z at tips of C T papillae and in the anterior C T core of filiform papillae. This selected distribution may indicate a special role linked to mechanical function, proliferative organization within the overlying epithelium or epithelial-mesenchymal interaction as reported previously (Sloan ef al., 1990). They observed a limited expression in gingiva but a more uniform distribution in the basement membrane of oral mouse lining epithelium including ventral tongue. This observation was not made in this study but rather confirms the finding that tenascin was restricted to the connective tissue of specialized papillary areas in the dorsal lingual mucosa. Furthermore, immunoreactivity for TN in human control gingiva was presented as wide band subjacent to the epithelium (not shown) which differs from previous observations (Sloan ef al., 1990, Tiitta ef al., 1994) but is in agreement with other studies (Tucker etal., 1991, Becker etal., 1993). An increased TN presence was also associated with fungiform and circumvallate papillae (Mistretta and Haus, 1996). Negative reactivity for antibodies against LM-1 and LM-5 suggests high species specificity for the used antibodies and differences in antigenicity between bovine and human laminins. Anti-LM-1 and-5- antibodies used with human gingiva resulted in positive reaction at the BMZ (not shown). Mere absence of laminins seems very  76 unlikely as they constitute integral structural components of the B M Z and their absence results in severe loss of epidermal-dermal cohesion (Aumailley and Krieg, 1996). Furthermore the presence and normal distribution of laminin in tongue epithelium has been reported in sheep (Mistretta and Haus, 1996) in intestinal mucosa (Lohi et al., 1996, Perreault etal., 1998) and laryngeal mucosa (Nehrlich ef al., 1998). In conclusion there are no unusual findings related to bovine histology, integrin pattern or E C M distribution. However, selective T N location at the anterior connective tissue core has not been described in detail previously. The highly distinct differentiation pattern associated with filiform papillae is especially interesting as two cell lines, that eventually both cornify when forming the spine of filiform papillae, have a different expression of K10 and involucrin.  5.2 Separation technique The separation techniques used for this model have been described previously with different tissues. The intraepithelial separation seen with cold trypsinization is similar to observations made by Skerrow (1980) but different from results described by Jensen and Mottet (1970) and Woodley etal. (1983). Separation attempts with EDTA failed in this study despite prolonged incubation periods. A possible explanation for different outcomes with E D T A and trypsin pretreatment might be the epithelial thickness. The tongue mucosa used here is considerably thicker than previously used epithelia like mouse ear skin (Harris ef al., 1980), mouse tongue (Mackenzie and Hill, 1981) or human oral mucosa (Scaletta and MacCallum,1970).  77 Epithelial thickness as a limiting factor was indicated by differences between tongue areas where thickness varied. Long C T papillae, and large C T cores with secondary papillae interdigitating with rete ridges result in an increased surface area for epithelial anchorage, which is an additional factor complicating separation over such an extended area. Torn rete ridge tips remaining interspersed between C T papillae could be observed in E T D A and trypsin split samples. Another difference might be tissue sample size. Subjectively the mechanical separation appeared more difficult with large pieces. However, these factors seem to play less of a role with NaCI incubation. This might be due to the long incubation period, where the increased resistance to agent penetration due to thickness and large surface area is overcome with time. For mucosa split by salt it is certain that the split occurred within the lamina lucida, which is in agreement with previous observations (Willsteed at al., 1990, Scaletta et al., 1978, Karpati etal., 1991). E C M components investigated remained unaffected as judged by IHC using this separation method. This confirms and adds to findings by Woodleyetal. (1983). We conclude that NaCI induced split is the most reliable and results in minimal morphologic or biochemical alterations which is in agreement with conclusions by Willsteed etal. (1991). 5.3 Culture model The methods applied in this study resulted in an organotypic culture of stratified squamous epithelium. De-epithelialized tongue mucosa appears to have similar properties as DED with comparable culture histomorphology as described  78 previously (Regnier ef al., 1981). One difference between DED and BM covered lamina propria of bovine tongue is the length of C T papillae. Collapsed and embedded C T papillae may interfere with an orderly organization of epithelial layers as cells leave the basal layer and differentiate but then encounter mesenchymal tissue again on their ascent to the surface. Instead of having one basal layer in a vertical direction there might be several seen on one section. The interpretation of sections are complicated by showing a two-dimensional image of a truely threedimensional structure. The direction of migration from the basal cell layer may therefore be not only vertical towards the surface but also horizontally. Interpretation of immunolocalized integrins and differentiation markers is also compromised as cells, although located close to the surface, may actually be basal cells on a C T papilla. Another difference is the separation technique and therefore the biochemical composition of the substrate in DED cultures. Alterations in antigenicity have been described with P B S incubation (Danno etal., 1982). However Regnier ef al. (1981, 1990) have not comprehensively assessed the distribution of E C M components. Finally, the keratinization of the original tissue was different. Whereas the epidermis is clearly keratinized the majority of tongue mucosa is not, but has an inhomogenous keratinization pattern due to filiform papillae. It was not possible to determine whether the C T cores of filiform papillae had specific influence on culture appearance, as they could not be identified any longer in the culture samples. The presence of connective tissue from selected keratinized areas might be of interest to study the influence of epithelial-mesenchymal interaction on differentiation. It has been demonstrated that in vivo the underlying C T determines whether epithelium becomes keratinized or not (Karring et al., 1975, Mackenzie and Hill, 1984) and that  79 the C T determines histodifferentiation (Squier and Kammeyer, 1983). However, transplanted epithelium may maintain its original morphology (Billingham and Silvers, 1967). There may be separate connective tissue influences on epithelial architecture and cyto-differentiation and there is a regionally-related variation in the competence of epithelia to respond to these influences (Mackenzie and Hill, 1984). In our experiments it could be observed that cultures of the same cell line under identical conditions could produce either keratinized or non-keratinized epithelium. There are two variables in this culture model that could account for this observation: (1) differences between certain areas within the substrate (CT cores of filiform papillae) and (2) variation in the fluid level. Substrate pieces were not uniformly thick. Therefore the level of different rafts within one well and even within one raft could differ significantly resulting in different degrees of air exposure. Air exposure has a significant effect on terminal differentiation (Regnier et al., 1981). All other parameters in this model were controlled and equal for all cultures. It is possible that non-keratinzed areas reflect an earlier stage in time and might become keratinzed later. Another difference from the in vivo situation is the absence of viable cells in the substrate. After 4 days incubation in 1M NaCI it can be assumed that none of the original cell remain alive. This was confirmed by absence of any reaction in the lamina propria after vital dye staining. A viable dermal substrate might be necessary for the formation of a basal lamina of explant outgrowth (Woodley etal., 1980), although in recombination culture a basal lamina was formed on devitalized dermis (Briggaman et al., 1971). More recent studies have shown that a living dermal substrate is neither required for normal histomorphology, keratinization (Mackenzie and Fusenig, 1993, Noel-Hundson etal., 1995) or  80 formation of a basement membrane (Tinois et al., 1991). In the absence of living dermal components features of the cell lines seem to determine histomorphology. Only N H E K were capable of producing a cornified layer. The transformation seems to limit the potential for keratinization as both HaCaT and H G K are derived from keratinizing tissue, i.e. epidermis and gingiva. The potential for keratinization of HaCaT is indicated by the expression of K10 in this model and keratinization has been demonstrated in vitro (Schoop et al., 1999) and by grafting of HaCaT cultures onto mice (Breitkreutz et al., 1998). Regarding keratinization of HaCaT cultures viable mesenchymal cells might be the key difference between co-cultures and our model. The role of cell origin for differentiation is also fundamental. Cells from "nonkeratinized gingival tissue" ("gingival sulcus tissue") produced a non-keratinized culture on fibroblast-collagen lattice and expressed differentiation markers of nonkeratinized epithelium (Tomakidi etal., 1998, 1999). Under similar culture conditions epidermal keratinocytes and HaCaT cells produce a keratinized culture (Fusenig 1994, Schoop etal., 1999). In recombination cultures of epithelium and connective tissue of different origin a significant modulation in the expression of differentiation markers was paralleled by similarly directed changes in the architecture of the heterotransplanted tissues, thus indicating that both morphogenesis and cytodifferentiation of certain adult epithelia can be influenced by extrinsic mesenchymal factors (Schweizer et al., 1998). Type of mesenchymal cells may play a crucial role in differentiation in vitro (Limat etal., 1994). However recombination studies by Boukamp ef al. (1990), transplantion of oral keratincytes to mouse skin (Lindberg and Rheinwald, 1990) and combined recombination and grafting  81 experiments (Mackenzie and Hill, 1981) imply that intrinsic programs also determine differentiation. In general it appears that diffusible factors from a living organism may determine differentiation. Yet, intrinsic programs may take control, especially in a culture situation. H G K did not produce cultures with normal histomorphology, having only a few cell layers without distinct compartments. Furthermore they showed irregular shape of nuclei and hyperchromasia. Abnormal stratification and aberrant integrin expression was seen in other transformed keratinocytes grown on collagen/fibroblast lattice (Kaur and Carter, 1992). Mature H G K at best resembled early HaCaT cultures. The poor histodifferentiation, nuclear polymorphism and aberrant diffuse integrin expression may be attributed to their spontaneous transformation and partially triploid chromosome set. Histological features and integrin expression pattern of this cell line have not been described previously. The examined differentiation markers in this study were limited. However, it is clear that their expression is not normal and rather distinct from bovine tongue and human gingiva, which served as control (not shown). Suprabasal expression of K14 and presence of K10 in basal layers occurred in all cultures. Expression of K14 in all layers was described in cultures grown in porcine skin, but K10 was limited to suprabasal cells (Matouskova etal., 1998). Suprabasal expression of the basal cell keratin K14 in epidermis has been associated with dermatofibromas (Stoler etal. 1989). In non-keratinized gingiva K14 gene expression is detectable in the basal cell compartment but showed extending immunolocalization, which was similar to gingival in vitro cultures (Tomakidi et al., 1997). Cultures of gingival junctional epithelium demonstrated a diffuse suprabasal expression of K14 (Papaioannou et  82 al., 1999). HaCaT and N H E K cultures expressed K14 most intensely at basal cells, which was slightly reduced in suprabasal areas. These observations are different from transplanted HaCaT and N E K cultures where K14 is mostly restricted to basal cells and K10 to suprabasal cells (Breitkreutz et al., 1998). With the limited cell layers in H G K cultures no difference could be observed between basal and suprabasal staining signal intensity. Expression of K1, which pairs with K10, has been reported in cultures on DED (Regnier ef al., 1986). K10, typically expressed suprabasally in keratinized epithelium, was present throughout all layers and all cultures, whether they were keratinized or not. This implies that cells maintained characteristics from the tissues they originated from, which were all keratinized. Yet, HaCaT and H G K lacked some induction to keratinize which was obviously not required for NHEK. In 5 day old HaCaT cultures some basal cells did not stain for K10, but virtually all stained after 20 days. This might indicate that differentiation progresses with time through all compartments. Although a similar culture technique was applied, K1/K10 was located only in suprabasal cells cultured on DED (Regnier etal., 1990). Different observations may be based on culture media composition or the fact that Regnier ef al. (1981, 1990) exposed the cultures to air immediately after seeding. Involucrin expression was again distinct from bovine tongue mucosa and gingiva. In normal tissue involucrin is not present in the deepest epithelial cells but appears in the course of their outward migration. However, involucrin is expressed in cultures of keratinocyte from various origins in immediate suprabasal layers (Banks ef al., 1981). Matouskova etal., (1998) reported involucrin and transglutaminase expression in the granular and horny layer. Normal expression of involucrin and late  83 differentiation markers was also reported for culture of N E K and O R S cells (Limat et al., 1991). In our study, only the most superficial layers of gingiva and posterior cells of filiform papillae demonstrated involucrin, whereas all cultures showed varying degrees of reactivity in all compartments. Superficial reaction was the strongest, but some staining was observed also in basal layers. N H E K cultures revealed an involucrin distribution closest to normal with intense staining at the surface and rather weak expression in basal cell layers. Involucrin synthesis appears to occur prematurely in the other two cell lines under the applied culture conditions. This does not seem to be time dependant as there was no difference between 5 day and 18 day old HaCaT cultures. In general these findings indicate the onset of differentiation is accelerated. This does not appear to be reflected by morphology in HaCaT cultures, but in N H E K cultures where cells start to flatten rather close to the basal layer. Integrin distribution by HaCaT and H G K was abnormal except for the OA subunit. Yet, some H G K cultures even showed abnormal generalized distribution for IJ4. Integrin subunit av, found in normal epithelium by some investigators (Jones et al., 1992) but not by others (Zambruno ef al., 1995), and G6 were present in all layers of HaCaT and HGK. a3 and B1 were present throughout all layers in HaCaT and H G K cultures, but only in HaCaT cultures as a distinct pericelluar distribution could be observed regularly. The same pattern was observed for a2 in HaCaT cultures but was absent in HGK. Suprabasal expression of a3 and a2 subunitis can be induced in vivo by retinoids (Hakkinen ef al., 1998). A s no specific signal was seen in either culture for avB5 it seems likely that avR6 heterodimers were formed, although it  84 cannot be ruled out that av61 heterodimers were present. Absence of av65 also coincides with the limited presence of its E C M ligand V N in all cultures. However, av65 was observed in other culture systems (Adams and Watt, 1991, Marchisio ef al., 1991). Transplanted HaCaT cultures show normal integrin expression of 61, 64, a2, a3, a6, and only weak expression of av mostly at epithelial margins (Breitkreutz etal., 1998). In contrast to normal skin (Klein etal., 1990) an abnormal polarized distribution was observed for a2, a3 and 61 in epidermal keratinocyte and outer root sheath cell cultures on a collagen I matrix (Limat and Klein, 1993). N H E K cultures showed integrin expression closest to normal, which is similar to observations in other culture systems. Both a2, a3, a5, 61, av and 66 were located pericellular of basal cells. Normal integrin expression of a2, a3, a6 and 61 was observed in cultures of N E K and O R S cells on fibroblast/collagen lattice (Limat ef al., 1995). Expression of a561, av66, av61 and av65 was observed in cultured keratinocytes (Marchisio ef al., 1991). This is similar to our findings except for the absence of av65 in our model. Cultures on pig skin demonstrated integrin 61 located at basal cells (Matouskova etal., 1998). Continuous normalization of integrin expression and polarized basal cell expression for av, which became interrupted at 3 weeks was described in gingival cultures (Tomakidi et al, 1999). It might be that N H E K cultures, which were terminated at 14 days, would progress in the same fashion. Integrin av66 or/and av61 was likely to be formed as av65 was absent and av, 66 and 61 colocalized pericellular of basal cells. The 66 subunit has not been addressed in organotypic culture models. The integrin av66, which has a restricted distribution in normal epithelium, augments the proliferation of epithelial  85 cells both in collagen gels and in vivo (Dixit etal., 1996). Presence of avR6 also coincides with abundant expression of it's ligands TN (Prieto et al., 1993) and FN (Weinacker et al., 1994) in HaCat and N H E K cultures. All three different cultures demonstrated the deposition of LM-5, T N and various degrees of LM-1. A s immunoreactivity may be modified or masked by other components and further altered in artificial in vitro systems (Fusenig, 1994), controls without cells were examined at the same time and same experiment. Changes in controls were limited to reduction of V N and reduction of H S P G at the surface. This was a subjective observation without measurements. No unmasking or expression of components previously not present occurred. It has been well established that epithelial cells are capable of producing all major B M Z components, which has also been demonstrated in various co-cultures (Tomakidi ef al., 1998, Tomakidi et al, 1999, Breitkreutz et al, 1998, Fleischmajer etal., 1993). Mesenchymal cells are also capable of BM-ECM secretion (McKee, 1996). The BM underlying the epithelium of the intestine is generally believed to be of both epithelial and mesenchymal origin. In the intestine TN and decorin are of sole mesenchymal origin (Perreault ef al., 1998). In our culture model only keratinocytes were present to deposit E C M and TN was clearly a product of epithelial cells as there were no vital mesenchymal cells and no TN expression changes in cultured controls occurred. At the leading edge of excisional wounds or explant cultures TN is not expressed but appears at later stages under the wound bed (Latijnhouwers etal., 1996). However, Mackie etal. (1988) and Hakkinen etal. (2000) localized TN isoforms under migrating epithelial cells. There are only limited reports on TN in organotypic cultures. In a keratinocytefibroblast coculture T N was expressed throughout the dermis (Fleischmajer ef al.,  86 1993). A s living fibroblasts were present they seem to be part of the source for TN. In chick embryo skin recombination cultures T N was expressed with other E C M components but not in 13 day old embryo skin (Akimoto etal., 1992). Expression of other E C M components in our culture system is likely and is indicated by expression of all investigated components under the epithelial edges of the margins. However, as migrating epithelial cells could not be observed on regular basis and the termination of the original B M Z could not be well identified it cannot be certainly distinguished between a positive signal of "old" and new E C M . Deposition of B M Z components by HaCaT cells has been demonstrated in transplants (Breitkreutz et al., 1998). When HaCaT cells are injected into athymic mice they form cysts with peripheral deposition of B M Z components (LM-5, CIV, CVII) (Kainulainen etal., 1998). Expression of LM-1 varied in HaCaT cultures and was rather weak in NHEK. Signal strength in HaCaT seems to increase with culture time but this observation was not conclusive. LM-5 was clearly expressed in all cultures, which is furthermore reflected by the formation of anchoring filaments in the lamina lucida as judged by TEM. Not only are major B M Z components secreted, they are also shown to form an ultrastructurally normal B M Z both in vitro (Woodley et al, 1980, Regnier et al., 1981, Regnier etal., 1990, Tomakidi etal., 1999, Schoop etal., 1999, Stark etal., 1999) and when transplanted (Breitkreutz et al. 1998). When the lamina densa is present it seems to be utilized and integrated into the new BMZ. Our E M observations imply that hemidesmosomes either form opposed to specific, thickened and denser lamina densa regions or are capable of modifying them in this fashion. A s both CVII and anchoring fibrils appear to be present before and after culturing it cannot be  87 concluded whether new ones have been formed. Formation of anchoring fibrils was observed at one week in transplanted cultures (Breitkreutz etal., 1998) and around explant cultures (Woodley et al., 1980). In both cases a viable dermal substrate is present which was shown to be essential for anchoring fibril formation (Briggaman and Wheeler, 1975). Although epidermal cells are capable of CVII secretion it seems unlikely that new anchoring fibrils were formed in our cultures. Although N H E K ultrastructure was not assessed it seems very likely that they closely resemble that of HaCaT, i.e. normal tissue, as all other features of N H E K cultures were as close or even closer to normal than in HaCaT. Yet, the final evidence remains to be presented. N H E K cultures appear closest to normal, but HaCaT cultures may benefit from their low maintenance, rapid growth and so far undetermined life span in our model. HaCaT cultures remained vital and histologically unaltered for up to 40 days after raising the culture to the air liquid interface. The majority of studies on organotypic cultures are limited to a few weeks. No attempts to grow organotypic cultures for prolonged time periods are currently known. The significant time span of our HaCaT cultures may be extended even further. This may be attributed to the immortality of HaCaT (or transformed HGK) or/and the presence of deep rete ridges, which may harbour increased numbers of stem cells. This remains to be confirmed by prolonged experiments and proliferation essays. An extended longevity of an organotpypic culture expands its application to more time requiring experiments and long-term observations. N H E K may progress quicker in their terminal differentiation, limiting their lifespan, which is below that of O R S cultures (Limat et al., 1991). Yet, the terminal lifespan remains to be explored with our culture approach. Transformed  88 H G K produce rather poor histomorphology and show many abnormalities in keratin and integrin expression. However, these cultures may still be valuable for studying and comparing various pathologic conditions. Again, they expand rapidly and their lifespan limit is yet to be explored.  C H A P T E R SIX-Conclusions  The applied separation technique produced a basement membrane covered C T substrate. The B M Z components appear to remain relatively unaltered during this procedure. The preexisting basement membrane of the C T substrate supports the formation of a stratified squamous epithelium of keratinocyte cultures. B M Z components appear to remain relatively intact during the study period, except for the reduction of V N and weakening of H S P G in control substrate without living cells . Weakening of H S P G was not observed at the B M Z of cultures and an increase in LM-1 and -5 and TN can be based on cellular deposition. Secretion of other basement membrane components appears likely and is indicated by positive staining of areas where cells migrate. N H E K and HaCaT rafts resemble normal epithelial histology, but keratinization occurred only in N H E K cultures. Normal ultrastructural appearance of the B M Z in keratinocyte cultures was observed previously and confirmed in our model. Presence of integrin p4 together with ultrastructural observations provides evidence for the formation of hemidesmosomes. Cornification and keratohyalin granules were described previously in cultures on DED, which could be observed in N H E K cultures only. The differentiation pattern of this model was only partially addressed, but it can be  89 concluded that it differs from in vivo situations. Integrin expression in N H E K cultures was close to normal except for presence of integrin subunits av and B6. HaCaT cultures show upregulation of several subunits throughout all cell layers. H G K show a rather disorganized histomorphology and aberrant integrin expression. In summary, the N H E K culture model on top of bovine tongue basement membrane appears to closely resemble normal tissue structure and may be used for multiple applications in studying epithelial cell biology.  90  REFERENCES Adams JC, Watt F M : Expression of p i , p3, p4, and p5 integrins by human epidermal keratinocytes andnon-differentiating keratinocytes. J Cell Biol. 115: 829-41, 1991 Akimoto Y, Obinata A, Endo H, Hirano H: Immunohistochemical study of basement membrane reconstruction by an epidermis-dermis recombination experiment using cultured chick embryonic skin: induction of tenascin. J Histochem Cytochem. 40: 1129-37. 1992 Alitalo K, Kuismanen E, Myllyla R, Kiistala U, Asko-Seljavaara S, Vaheri A; Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells. 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