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Expression of keratinocyte integrins in acute and chronic inflammation of the human periodontium Larjava, Kirsi Haapasalmi 1997

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EXPRESSION OF KERATINOCYTE INTEGRINS IN ACUTE AND CHRONIC INFLAMMATION OF THE HUMAN PERIODONTIUM by KIRSI HAAPASALMI (LARJAVA) Licentiate of Dental Science, The University of Turku, Finland 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Oral Biological and Medical Sc iences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1997 © Kirsi Haapasalmi (Larjava), 1997 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T Epithelium plays a crucial role in protection, destruction and repair of human mucosal and epidermal t issues. Integrins compose a family of heterodimeric transmembrane glycoproteins that mediate cell adhesion and information transfer from extracellular milieu into the cytoplasm. In the present work, the expression of integrins in human mucosal and epidermal wound keratinocytes during acute (wound healing) and chronic inflammation (periodontitis) was investigated. Integrins and their l igands were localized in frozen sections using immunohistochemistry. During wound healing, expression of av(36 integrin was induced relatively late during wound healing. Maximal expression was observed in 7-day-old wounds, in which epithelial sheets had joined and granulation tissue was present. Fibronectin and tenascin, both possible ligands for avP36 integrin, were concentrated underneath the basal keratinocytes expressing this integrin. During chronic inflammation, focal loss of integrins and basement membrane components was common in periodontal pocket epithelium. In some local areas, however, upregulation of integrin expression was observed. The results suggest, that during acute inflammation, the reservoir of the keratinocyte integrins increases to correspond to new demands for keratinocyte migration and gene regulation. During chronic inflammation, however, the expression of integrins in keratinocytes is variable, suggesting focal areas of integrin up- and down-regulation that may possibly reflect the areas of healing and destruction in the periodontium. iii TABLE OF CONTENTS PAGE ABSTRACT ii TABLE OF CONTENTS iii LIST OF F IGURES vi P R E F A C E ix ACKNOWLEDGEMENTS x ABBREVIATIONS xi INTRODUCTION 1 CHAPTER ONE Review of the Literature 3 1.1. Integrins 3 1.2 Keratinocyte integrins and their ligands 5 1.3 The function of integrins with special emphasis on keratinocytes 16 1.4 Integrins and basement membrane 1 9 1.5. Epithelial wound healing 21 1.6. Keratinocyte integrins and wound healing 2 3 1.7. TGF-3 in wound healing 25 1.8. Junctional and periodontal pocket epithelia 2 6 CHAPTER TWO The aims of the study 30 CHAPTER THREE Keratinocytes in human wounds express ccvf36 integrin... 31 3.1. Introduction 31 3.2. Material and methods 32 Material 32 Immunohistochemical studies 33 3.3. Results 35 Human mucosal wound keratinocytes express ocvf36 integrin that is localized in areas of accumulation of fibronectin and tenascin 35 Wound keratinocytes in fused epidermal wounds express ccvf36 integrin 38 3.4. Discussion 42 3.5. Summary 45 CHAPTER FOUR Expression of epithelial adhesion proteins and integrins in chronic inflammation 4 7 4.1. Introduction 4 7 4.2. Material and methods 48 Material 48 Immunohistochemical studies 48 4.3. Results 49 Microscopic morphology of inflamed periodontal tissue 49 Localization of LM-1 and type IV collagen 51 • ' Localization of components of epithelial anchoring system 54 V Localization of fibronectin and tenascin 57 Distribution of 31 and a6(34 integrins in epithelial cells 57 4.4. Discussion 63 4.5. Summary 67 CHAPTER FIVE General discussion and conclusions 69 Bibliography 73 VI LIST OF FIGURES Figure 1. Keratinocyte integrin heterodimers and their known ligands. C O L = collagen; LM-5 = laminin-5; FN = fibronectin; TN = tenascin; VN = vitronectin 6 Figure 2. Ultrastructure of the basal keratinocytes and the underlying basement membrane 1 3 Table 1. Antibodies used to recognize different integrins and their l igands in these studies 34 Figure 3. Integrin ocv36 is expressed in some but not all 3-day-old human mucosal wounds. Panels A, B, D, and E, separate sections of the same specimen of open 3-day-old mucosal wound. Panel C, closed 3-day-old wound. Panel F, nonwounded area of 3-day-old wound, same section as shown in panel C. Staining of ocv integrin (panels A, B), or av36 (panels C, D, E, F) integrin complex by immunofluorescence. Arrows mark the wound site and dotted line epithelial connective tissue interphase. E, epithelium; GT, granulation tissue; CT, connective tissue 36 Figure 4. Integrin av36 and its putative ligands, fibronectin and tenascin, localize in similar areas in the 7-day-old wounds. Antibodies against av integrin (panel A), av36 integrin complex (panel B), tenascin (panel C) and fibronectin (panel D) were used 37 Figure 5. In human dermal wounds 31 integrin is expressed throughout the healing process but av and ocv36 integrins are not expressed until day 7. 1-day-old (panels A, D), 3-day-old (panels B, E) and 7-day-old (panels C, F, G) dermal wounds were stained with antibodies specific to 31 integrin (panels A, B, C), (xv integrin (panels D, E, F), and av36 integrin complex panel G). Epithelial-blood clot (C) interphase marked with dotted line in panels D and E. E, epithelium; CT, connective tissue; GT, granulation tissue 40 vii Figure 6. Expression of tenascin is upregulated in wound granulation tissue. Distribution of tenascin in 1-day-old (A), 3-day-old (B), and 7-day-old (C) human dermal wounds. Arrowheads indicate wound margin and arrows tenascin containing subepithelial connective tissue in panels A and B. E, epithelium; C, clot; CT, connective tissue; GT, granulation tissue 41 Figure 7. Hematoxylin and eosin staining of healthy (A), and chronically inflamed (B) periodontium. Gomori 's periodic acid methenamine silver staining of basement membrane zone in normal (C) and inflamed periodontal tissue (D). Arrows indicate location of basement membrane (C and D). OE; oral epithelium; GT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 50 Figure 8. Immunolocalization of LM-1 (A and C) and type IV collagen (B and D) in healthy (A and B) and in chronically inflamed periodontal tissue (C and D), respectively. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 52 Figure 9. Distinct patterns of extravascular (arrows) LM-1 (A-C) and type IV collagen (D) in inflamed periodontal tissue. Arrowheads indicate LM-1 and type IV collagen associated with vascular basement membrane 53 Figure 10. Immunostaining of type VII collagen associated with oral epithelium (OE) and with periodontal epithelium in chronic inflammation (PE) 55 Figure 11. Immunolocalization of type VII collagen (A-D) and LM-5(E, F) in healthy oral mucosa (A, C, E) and in chronically inflamed periodontal tissue (B, D, F). OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 56 viii Figure 12. Immune-localization of fibronectin (A, B) and tenascin (C, D) in healthy oral mucosa (A, C) and in chronically inflamed periodontium (B, D). Note the reduction in intensity of staining for each molecule in inflamed subepithelial connective tissue. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 59 Figure 13. Immunolocalization of 31 -integrin (A, D, G), oc6-integrin (B, E), B4-integrin (C, F) in healthy oral epithelium (A-C) and in chronically inflamed periodontal tissue (D-G). Double-immunostaining of 81-integrin (H) and type Vll-collagen (I) in inflamed periodontal tissue. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 60 Figure 14. Immunolocalization of 131 (A, D, G), a3 (B, E, H) and oc2 (C, F, I) integrins in healthy oral epithelium (A-C), and in periodontal epithelium in chronic inflammation (D-l). In inflammation, some areas of periodontal epithelium show focal loss of these integrins (D-F) whereas in others the full thickness of inflamed periodontal epithelium is stongly positive for 131, a 3 and oc2 integrins (G-l). Arrows mark epithelial-connective tissue boundary (dashed black line, panel G). Arrowheads (panel G) mark 31 staining which is comparable to normal oral epithelium, and dashed white line indicates area of epithelium that demonstrates a loss of 31 staining. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 61 Figure 15. Immunolocalization of 31 (A), a6 (B), 34 (C), type IV (D), LM-5 (E), and type VII (F) in parallel serial sections of chronically inflamed periodontal tissue. Note the strong expression of 31 integrin (A) in epithelium and also the streak-like appearance of LM-5 and type VII collagen (E,F) in connective tissue. PE; periodontal epithelium in chronic inflammation; ICT; inflamed connective tissue 62 X ACKNOWLEDGEMENTS I would like to thank Dr. Hannu Larjava and Dr. Vel i-Jukka Uitto for their guidance and support during my studies. I would like to thank you for your patience that you showed during this long period of time that it took from me to finish this program. I would like to thank Dr. Don Brunette for his suggestions and insight as a member of my research committee. I would like to thank all the co-authors in the two publications that this work has produced. In particular, I would like to acknowledge Dr. Dean Sheppard and Dr. Kenneth Yamada for their generosity for providing some of the antibodies used in this study. I am thankful for Mr. Cristian Sperantia for his technical support during this project. I would like to thank all my friends at U B C who have supported me during these years. I can not thank enough my loving husband, Hannu, for his endless love, patience and support he has given me during these years. Last I would like to greatfully acknowledge the financial support that I have received during this program from following sources: Finnish Cultural Foundation Student Clinicians of American Dental Association, Henry M. Thornton Fellowship Women Dentists of Finland Faculty of Graduate Studies, UBC, Dr. George S. Beagrie Award ABBREVIATION LM-5 laminin-5 LM-1 laminin-1 RGD arginine-glycine-aspartic acid BPA bullous pemphigoid antigen FAK focal adhesion kinase TGFB transforming growth factor 3 £. junctional epithelium 1 INTRODUCTION During healing and destruction of human epithelial tissues, cell adhesion receptors of the integrin family play an important role (Larjava et al., 1996). Integrins are responsible for interactions of epithelial cells with the extracellular matrix and to some extend for cell to cell adhesion. Integrins also convey signals from the extracellular mileu into the nucleus regulating gene expression. Alterations in the expression of integrins and cell adhesion proteins in keratinocytes during human and animal wound healing have been previously reported (Larjava et al., 1993; Gailit and Clark, 1994; Watt and Jones, 1993). Larjava and co-authors (1993) have shown that the expression of av-integrins is induced in keratinocytes of mucosal wounds. Others have confirmed the induction of this integrin subunit in healing cutaneous wounds (Juhasz et al., 1993; Cavani et al., 1993). It has not been clear, however, which 3 subunit is associated with the ocv subunit in wound keratinocytes. The av subunit can form heterodimers with multiple 3 subunits, namely with 131, 33, 35 and 36. Vitronectin receptor av35 integrin has been detected in keratinocytes in an animal wound healing model (Gailit et al., 1994). In the present study, one of the goals was to find out which 3 subunit forms a heterodimer with av subunit in different stages of human mucosal wound healing and which extracellular matrix proteins could be found colocal ized as its possible ligands. Very little is known about epithelial cell behaviour during chronic inflammation. In 2 periodontal disease, epithelial cells proliferate into inflamed connective tissue forming long epithelial extensions. The basement membrane between the epithelium and connective tissue also shows alterations (Thilander, 1968; Freedman et al., 1968; Takarada et al., 1974 a, b; Peng et al., 1986). The mechanisms of epithelial adhesion and migration lin chronic inflammation have not been adequately described. The distribution of cell adhesion receptors and proteins of developing and healthy periodontal soft and hard tissues have been examined in several animal studies (Thesleff et al., 1987; Caffesse et al., 1987; Steffensen et al., 1992), but there are no studies showing alterations of these molecules in conjunction with epithelial cells and chronically inflamed periodontal pockets. Therefore, the second goal of this study was to find out the alterations of epithelial integrins and cell adhesion proteins during the chronic inflammation of human periodontal t issues in order to elucidate the role of keratinocytes in periodontal d isease. 3 CHAPTER ONE Review of the literature 1.1. Integrins Integrins are cell surface glycoproteins that function as cell-to-cell and cell-to-extracellular matrix adhesion receptors (Albelda and Buck, 1990; Ruoslahti, 1991; Hynes, 1992). Through binding to matrix proteins integrins mediate information transfer from the extracellular milieu to the cytoplasm and nucleus, leading to alterations in cell functions and ultimately in cell behaviour (Schwartz, 1992). Integrins are known to play important role in regulating a wide range of cellular interactions during growth, development, differentiation, and immune response (Hemler, 1990; Ruoslahti, 1991; Hynes, 1992). Integrins are composed of a single a and a single 13 subunit that are non-covalently l inked to each other. At least 15 different a and 8 f3 subunits are currently known. These subunits can variously combine to form more than 20 different cell surface receptors that have distinct ligand binding specificities. It is known that some (3 integrin subunits, namely 31, 32, 33 and 37, are able to associate with multiple a subunits. Some a subunits can become paired with more than one 3, namely oc4, a6 and particularly av, which appears to be able to combine with five different 3 subunits. Both a and 3 subunits are transmembrane glycoproteins that contain a single hydrophobic transmembrane segment. Usually integrins have relatively large extracellular domains and short cytoplasmic tails. Integrins seem to share a distinctive shape containing mushroom-shaped head region that is thought to consist of both a and 3 subunits, and mediate integrin binding to ligands. Integrins have long extracellular domains that hold the head approximately 20 nm above the cell surface and penetrate through the p lasma membrane v ia hydrophobic transmembrane domains. Integrins have relatively short (15-60 amino acids in length) carboxy-terminal tails protruding into the cytoplasm. Integrin 34 serves as an exception to this rule having an unusually long cytoplasmic domain, consisting of more than 1000 amino acids (Sastry and Horwitz, 1993). Extracellular domains of both a and 3 subunits are large, over 75 kDa for 3 subunits and over 100 kDa for a subunits. The ligand binding site of the integrins is formed of the NH 2-terminal extracellular sequences of both subunits. The transmembrane organization permits integrins to bind to extracellular l igands and to transmit signals into the cytoplasm. The intracellular tails interact with cytoplasmic molecules to regulate extracellular ligand-binding affinity. These functions have been termed "outside-in" and "inside-out" signalling, respectively (Ginsberg et al, 1992). The ligand binding process in which both subunits participate, is dependent on divalent cations (calcium, magnesium, manganese). L igand binding causes clustering of the receptors that leads to cytoskeletal organization and signall ing (Miyamoto et al., 1995). The cytoplasmic tail of 3 subunit seems to be critical for the organization of actin microfilaments through its binding capacity to talin and a actinin. Hierarchy in signal transduction depends on ligand occupancy and receptor aggregation (Miyamoto et al., 1995). Integrin aggregation alone seems to 5 induce tensin and focal adhesion kinase (pp125FAK) accumulation. Aggregation and ligand occupancy leads to clustering of talin and a-actinin. A combination of these events results in accumulation of a large number of intracellular proteins. Specificity of integrin function can be attributed to the structures of both the extracellular and intracellular domains of integrins. Ligand specificity varies depending on the cell type and functional state, and a single cell can have several integrin receptors for the same ligand. Most integrins are expressed in many different cell types, but some integrin subunits are considered specific to a particular cell type, e.g. 32 integrins to leukocytes and 36 integrin to epithelial cells (Sheppard, 1996, for review). 1.2 Keratinocyte integrins and their ligands The repertoire of integrins expressed by different cell types varies greatly. Human keratinocytes are known to express at least eight integrin heterodimers that mediate cell adhesion to various extracellular matrix proteins (Figure 1). Several integrins are able to bind multiple ligands in a cell line dependent fashion. The regulation of integrin function is complicated, since a single cell can express multiple integrins with apparently similar binding specificity. Keratinocytes are specia l ized cells that cover and protect the skin and mucosa (Eckert, 1989). Epidermal and mucosal keratinocytes undergo terminal differentiation when they move from basal layer to the surface. Figure 1. Keratinocyte integrin heterodimers and their known l igands (Larjava et al., 1996). C O L = collagen; LM-5 = laminin-5; FN = fibronectin; TN = tenascin; V N = vitronectin Polarized keratinocytes attach to the basement membrane through the basal cell membrane and to adjacent keratinocytes via lateral cell membranes. In normal cornified and stratified epithelia, integrins are expressed by basal keratinocytes but are absent in differentiating superfical layers (Hertle et al, 1991; Larjava et al., 1990; Konter et al., 1989; Peltonen et al., 1989). This suggests that integrins are important 7 in keratinocyte adhesion to the basement membrane, proliferation and differentiation. During wound healing, keratinocytes must be able to interact with various components of wound bed extracellular matrix. Based on immunolocalization studies, normal human keratinocytes residing in resting epithelium express only four different integrins; cc2f31, CX3I31, oc931 and cc6G4 (DeLuca et al., 1990; Hertle et al., 1991; Konter et al., 1989; Peltonen et al., 1989, Palmer et al., 1993). Keratinocytes are shown to be activated when transferred from tissue to cell culture conditions (Grinnell, 1992). As a consequence of this process, they start to express integrins in addition to those found in vivo. Cultured keratinocytes express in addition to the integrins mentioned above, av(35, av(36 and a 5 p i integrins that are also found to be present in keratinocytes during wound healing (Larjava et al, 1993; Cavani et al, 1993; Juhasz et al, 1993). Many, but not all, integrins bind to extracellular matrix proteins containing the tripeptide arginine-glycine-aspartic acid (RGD) (Ruoslahti and Pierschbacher, 1987; Pierschbacher and Ruoslahti 1984; Pytela et al., 1985; Ruoslahti and Pierschbacher, 1986). First identified in fibronectin, it has since been shown to be a cellular recognition sequence in many extracellular matrix and platelet adhesion proteins. The conformation of the R G D site appears to determine which integrin an R G D protein or R G D peptide will bind (Ruoslahti and Pierschbacher, 1987). This tripeptide is the major binding site in fibronectin. Fibronectin is a key component of extracellular matrix and plasma, thus 8 functioning in cell adhesion, cell and embryonic differentiation, cell migration, and thrombosis (see Ruoslahti, 1988 for review). Fibronectin is an important molecule in the provisional matrix during wound repair. Fibronectin has several functional domains that permit its interactions with a remarkably wide range of cell types, extracellular matrix molecules, and cytokines. The fibronectin molecule is composed of two polypeptides, with a total molecular mass of 560 kDa. Even though fibronectin is encoded by only a single gene, it exists in a number of variant forms. These forms differ in sequence at three general regions of alternative splicing of its precursor mRNA. If alternative splicing involves cell adhesion sequences, it provides posttranscriptional mechanism for potential regulation of fibronectin cell-type specificity. Depending on the particular combination of spl iced sites, there can be 20 different variants of the human fibronectin subunit (Yamada and Clark, 1996, for review). Two separate forms of fibronectins, p lasma fibronectins, present in blood as a soluble plasma glycoprotein, and cellular fibronectin, produced by a wide variety of cell types, have been described. Cellular fibronectins vary in composition and they typically contain considerably higher proportions of alternatively spl iced sequences compared to p lasma fibronectin. The alternative splicing of fibronectin can insert or delete cell-type-specific adhesion sites. Spl iced sequences tend to be retained in cellular fibronectin, especial ly by certain embryonic and malignant cells (Vartio et al., 1987; Oyama et al., 1989) and healing wounds (ffrench-Constant et al., 1989; Brown et al., 1993). Fibronectin contains several peptide sites capable of mediating cell adhesion. 9 These are located in three general regions: the central cell-binding domain, the alternatively spl iced IIICS region, and the heparin-binding region. Most cells can adhere to the centrally located "cell binding" domain (Akiyama et al., 1985, 1994). A crucial sequence in this domain is R G D (Pierschbacher and Ruoslahti, 1984, 1987; Yamada and Kennedy, 1984). Deletion or point mutation to this sequence leads to the loss of nearly all adhesive activity (Obara et al., 1988). It is known that human keratinocytes can express four receptors that are potentially able to bind to fibronectin, namely oc5f31, oc3G1, oc831 and avG6 integrins. The classical fibronectin binding receptor is cc5B1 integrin that binds to the R G D sequence of the fibronectin molecule (Singer et al., 1988; Ak iyama et al., 1989). oc5F31 is considered to be a specia l ized receptor for fibronectin and was originally characterized on fibroblasts (Singer et al., 1988). Integrin oc5f31 mediates fibronectin-matrix assembly, cell adhesion and migration on fibronectin (Akiyama et al., 1989). Another fibronectin RGD site binding receptor in epithelial cells is the avB6 integrin (Busk et al., 1992). I36 integrin is expressed exclusively in epithelial cells, and only in a single integrin heterodimer, avR6, a receptor for the extracellular matrix proteins fibronectin (Busk et al., 1992; Weinacker et al., 1994) and tenascin (Prieto et al., 1993). The otvf36 integrin is highly expressed in the lung, skin, and kidney during organogenesis (Breuss et al, 1995). In normal epidermis or oral mucosal epithelium this receptor is absent (Breuss et al., 1995) but becomes highly expressed in response to injury or inflammation, and in malignant epithelial neoplasms (Breuss et 10 al., 1995). Neither of the main fibronectin receptors, oc5G1 or av(36 integrins, appears to be present in keratinocytes residing in the resting epithelium, but the expression of both of these integrins can be induced simply by placing epithelial cells in cell culture. Another integrin, oc8G1 can also bind to fibronectin in a RGD-dependent manner (Schnapp et al., 1995) and it has been localized in chicken epithelium but its expression in human keratinocytes is not clear. a3f31 integrin can bind to fibronectin in some cell types, but it is believed to function as a receptor for laminin-5 in keratinocytes (Carter et al., 1991; Delwel et al., 1994). In resting and wounded epithelia, keratinocytes are required to be able to interact with various collagen types. Different types of col lagens are recognized mainly by one single receptor, the a231 integrin. Unlike keratinocyte adhesion and motility on fibronectin and vitronectin, interactions with col lagens are not mediated by R G D even though collagens contain these sequences. Col lagens are extracellular matrix glycoproteins that provide structural support for the t issues and also regulate many cellular functions. Based on their genetic origin and primary structure, collagens can be divided into several groups. The basic structure of all col lagens is triple-helical conformation that is formed of three a chains. Altogether 32 a chains have been identified, each having a common repetitive motif of Gly-X-Y, which allows a chains to wrap around each other and form the triple-helical structure. A total of 19 different collagen types have been identified and characterized in vertebrates (Eckes et al., 1996, for review). Col lagens differ in their a chain structure as well as in their contents 11 of collagenous and noncollagenous regions in their chains. Col lagens have been subdivided into two main subfamil ies based on this ultrastructural organization; the fibril-forming and nonfibrillar col lagens (van der Rest and Garrone, 1992). Type IV collagen is the major col lagenous component of the basement membrane. It forms a multilayered, highly insoluble structural network that interacts with other basement membrane molecules, such as laminins, nidogen, and heparan sulphate proteoglycan (Yurchenko and Schittny, 1990). Type IV collagen belongs to a subfamily of nonfibrillar collagens, that can also be called network-forming col lagens. Type IV collagen is the component of lamina densa of the basement membranes (Figure 2). In vivo keratinocytes express oc2pi which recognizes a variety of collagens. This is also the main receptor for type IV collagen in keratinocytes. Direct interactions of keratinocytes with type IV collagen are rare but may occur e.g. during healing of blister wounds in which case keratinocytes migrate on the components of lamina densa. Keratinocytes are more likely to interact with fibrillar col lagens, such as types I, III and V collagens during healing of full thickness wounds. A lso in this case, interaction of keratinocytes with collagens is mediated via a231 integrin. One peculiar collagen type is localized as a transmembrane protein of keratinocyte cell surface. This type XVII collagen is better known as bullous pemphigoid antigen 2 (Uitto and Christiano, 1992). It appears to be interacting with a634 integrin although the functional consequences of this interaction are not known. Both a2(31 and cc3B1 integrins can serve as receptors for laminin-1 (LM-1) 12 (Elices et al., 1991; Carter et al., 1990; Adams and Watt, 1991). These integrins may act in concert to mediate cell binding to LM-1. Antibodies against a2 and a3 individually inhibit attachment to LM-1, but together the antibodies inhibit more effectively (Carter et al., 1990; Adams and Watt, 1991). Although several studies point to the conclusion that normal epidermal and mucosal keratinocytes bind to LM-1, others seem to disagree (see above, Karecla et al., 1994; Zhang et al., 1996). Zhang and co-authors (1996) showed that normal keratinocytes adhere poorly to and become non-motile on LM-1 rich matrix and it could therefore act as an endogenous stop-signal for migrating keratinocytes. They also showed that integrin oc6(31 is an important LM-1 receptor that is not normally expressed in keratinocytes but becomes an important LM-1 receptor in malignant transformation of mucosal keratinocytes. This promotes their migration on LM-1, an important process in invasion and metastasis. It is questionable if keratinocytes in vivo are even required to adhere to LM-1. LM-1 can be found in the lamina densa of the basement membranes (Figure 2). Like all other members of the laminin-family, LM-1 is made of a, p and y chains. Laminin-5 (LM-5, also known as epiligrin, kalinin or nicein) is found in the basement membrane of skin and other epithelial tissues, and it serves as a component of anchoring filaments that span through the basement membrane (Figure 2) (Carter et al., 1991; Roussel le et al., 1991). basal keratinocytes 13 anchoring filaments/ laminin-5 anchoring fibrils (type VII collagen) desmosome lamina lucida -lamina densa laminin-1, type IV collagen $ — ct6p4 integrin Q — a 2 p l , a 3 p i integrins | — BPA2, type XVII collagen Figure 2. Ultrastructure of the basal keratinocytes and the underlying basement membrane (from Larjava et al., 1996). LM-5 is a glycoprotein synthesized by keratinocytes (Carter et al., 1991). The extracellular domain of oc6p4 integrin interacts with LM-5 (Roussel le et al., 1991, Niessen et al., 1994). This integrin is known to link the basal keratinocytes to the underlying basement membrane, and that this link mainly relies on the interaction with LM-5 in the anchoring filaments (Figure 2). The functional importance of a6f54 integrin is substantiated by in vitro experiments in which antibodies against it have been shown to dissociate hemidesmosomes and lead to cell detachment (Kurpakus et al., 1991). Knockout mice lacking either a6 or 34 integrin can not form hemidesmosomes and show blistering of skin (Fassler et al., 1996). Besides playing an important function in keratinocyte cell-matrix adhesion, as d iscussed above, a2(31 and a3(31 integrins are believed to interact with each other (Symington et al., 1993). The a331 and oc2B1 integrins localize commonly at the lateral surfaces of the stationary basal keratinocytes (Hertle et al., 1991; Konter et al., 1989; Larjava et al., 1990; 1992; Peltonen et al., 1989). It has been suggested that these receptors may play roles in homotypic cell-cell interaction in epithelia, supported by findings that ct231 and a331 integrins can bind to each other (Carter et al., 1990; Symington et al., 1993) and that ot331 can bind to itself (Sriramarao et al., 1993). Some studies discount the functional importance of 31 integrins in cell-cell binding of keratinocytes (Tenchini et al., 1993). LM-5 is also recognized by a331 integrin (Carter et al., 1991). Transgenic mice lacking oc3 integrin show localized blistering of the skin (DiPersio et al., 1997). In mice and man, oc331 integrin is occasionally found at the basal surface of the basal keratinocytes suggesting that a331 integrin col laborates with oc634 integrin in LM-5 binding and they may be functionally interchangeable in some circumstances. Vitronectin (also known as serum spreading factor, S-protein and epibolin) is an abundant protein present in blood plasma, extracellular matrices and fibrin clots. Vitronectin is known to be an active cell adhesion mediator as well as playing a role in 15 cell migration and invasion (Preissner, 1991, for review). The <xvf31, ocvB3, av l lb3 and <xv35 integrins are all vitronectin receptors binding to the R G D cell attachment sequence of the protein (Cherny et al., 1993). Only av35 integrin is known to be expressed by the keratinocytes, thus being their only vitronectin recognizing receptor. It is important to note that all of these vitronectin receptors can also bind fibronectin. Tenasc ins are a family of large extracellular matrix molecules that presently include tenascins -C, -R, and -X. Tenascin expression is tightly regulated, making this protein particularly interesting. The expression of tenascin is closely associated with morphogenetic events, including embryonic induction and migration, wound healing, and tumorigenesis (Chiquet-Ehrismann, 1990; Whitby and Ferguson, 1991; Koukoulis et al., 1991; Whitby et al., 1991). One study shows that even if tenascin gene is totally disrupted in developing mice, the animals seem to develop and grow normally (Saga et al., 1992). These mice lacking tenascin yet phenotypically normal, cast doubt on the theory that tenascin plays an essential role in normal development, never the less, as mentioned earlier, tenascin is generally considered an important protein during embryonic development. It is found in developing brain and in mesenchyme associated with epithelial-mesenchymal interactions (Chiquet-Ehrismann, 1990; Thesleff et al., 1991). In adult t issues tenascin is found in the connective tissue underneath epithelium (Lightner et al., 1989) but its expression is strongly upregulated during wound healing (Mackie et al., 1988; Chuong and Chen, 1991; Whitby et al., 1991). Several integrins have been found to bind tenascin, namely oc2f31, avR6 and cx9f31 integrins (Prieto et al., 16 1993; Yokosaki et al., 1994, 1996; Bourdon and Ruoslahti, 1989). 1.3 The function of integrins with special emphasis on keratinocytes As mentioned earlier, cell behavior is dependent on the ligand-binding specificities of the integrins present on the cell surface. Characteristics of the integrins are their overlapping but nonidentical binding specificities. Several integrin heterodimers are able to bind multiple ligands depending of the cell type that is expressing them. Binding is based on protein-protein recognition, and in many cases a central element of the molecular recognition event involves only a short adhesive peptide sequence (Yamada, 1991), such as R G D (Arg-Gly-Asp) peptides in fibronectins. The types and concentrations of integrins on cell surfaces are important determinants of the specificity of binding to extracellular matrix proteins, although the activation state and spatial distribution (diffuse vs. aggregated) of the integrins also appear to be important (Yamada et al., 1996) Integrins can become important signalling molecules after ligand binding or receptor clustering. For example, integrin-mediated adhesion of cells to a variety of extracellular matrix molecules including fibronectin, vitronectin, laminin, and collagen will lead to enhanced tyrosine phosphorylation of the focal adhesion kinase (FAK) (Guan et al., 1991; Kornberg et al. 1991, 1992; Guan and Shalloway, 1992). Since this protein localizes to focal contacts and may be involved in their organization, it has been very informative as a model for transmembrane signal transduction via integrins. Other 17 signaling molecules are likely to be identified in association with integrin cytoplasmic domains. Since the ligand binding affinity (Briesewitz et al., 1995) of an integrin can be modulated by activation of a and 3 chains by various signalling molecules it has been suggested that both a and 3 cytoplasmic domains can alter receptor assembly and function and hence, cell adhesion (OToo le et al., 1994; Schwatz et al., 1995). The possible activation candidates, other than FAK, include calreticulin, a-actinin, and talin, because they have all been found to bind integrin cytoplasmic domains in vitro. Especial ly the cytoplasmic domain of integrin a subunit may function as affinity controller (OToo le et al., 1994), but 3 subunit seems to have a role in maintaining the receptor in a high-activity state (OToo le et al., 1995). The activation of the cytoplasmic domains alters the relationship of the transmembrane a and 3 domains and thus leads to conformational change and altered ligand binding affinity of the extracellular domains. The ligand binding affinity of integrins can also be modulated by divalent cations, such as calcium and manganese (Gailit and Ruoslahti, 1988). Calcium can inhibit and manganese can enhance the function of some 31 integrins by affecting the appearance of a conformation favorable to ligand binding (Bazzoni et al., 1995). Integrin a531 is suggested to contain distinct binding sites for magnesium, manganese and calcium, and their different usage modulates adhesion to fibronectin (Mould et al., 1995). The affinity of an integrin can also be modulated by co-operation with another type of cell adhesion receptor, e.g., cell surface proteoglycan (lida et al., 1995). Integrin activation and functional regulation are very complex events and remain, 18 therefore, largely unknown. Integrins are important not only in stable matrix anchorage and signalling but also in cell migration. The migratory state is found in embryonic cells as well as cells at the wound sites. The migratory phenotype is characterized by generally diffuse organization of integrins. A lso migratory keratinocytes express integrins at different activity levels (Hertle et al., 1992, Larjava et al., 1993; Cavani et al., 1993; Juhasz et al., 1993). The stationary phenotype is recognized by integrin organization into focal adhesion sites with intracellular special ized cytoskeletal complexes composed of talin, oc-actin and FAK (OToo le et al., 1994). These two distinct molecular phenotypes most likely have an important functions in wound repair, which will be d iscussed later in detail. In epithelium, integrins also function in cell differentation. Stem cells for keratinocytes appear to express the highest level of 31 integrins (Jones and Watt, 1993) . Keratinocytes with characteristics of stem cells can be isolated based on their high 31 integrin expression (Jones and Watt, 1993). During the vertical migration of the epithelial cells from the basal cell layer to the suprabasal layers the integrin expression stops (Adams and Watt, 1991; Watt and Jones, 1993). Loss of functional integrins from the cell surface precedes epithelial differentiation (Hodivala and Watt, 1994) . Integrin engagement to the ligand is believed to prevent differentiation (Watt and Jones, 1993). Fibronectin has been shown to inhibit keratinocyte differentiation by occupying integrins on keratinocyte cell surface (Watt and Jones, 1993). These 19 findings suggest that integrins have important role also in keratincyte proliferation and differentiation. New research data on keratinocyte integrins suggest that integrins are associated with the regulation of epithelial driven inflammation. Transgenic animals lacking 36 integrin show normal early wound healing but develop inflammatory lesions in the lungs and skin (Huang et al., 1996). Suprabasal 31 integrin expression is seen in many inflammatory skin diseases such as psoriasis (Watt and Hertle, 1994, lor review). Carroll and his co-workers (1995) used forced expression of 31 integrins in suprabasal layers using involucrin promoter. This resulted in skin inflammation resembling psoriasis. Both above mentioned studies indirectly suggest that integrins are associated with the regulation of inflammation that is controlled by epithelial cells. 1.4 Integrins and basement membrane The basement membrane of the skin and mucosa consists of a large number of structural molecules that form an attachment zone at the epithelial-connective tissue interface. On the epithelial side of the basement membrane, basal keratinocytes are attached to underlying basement membrane structures via hemidesmosomes. Hemidesmosomes are known to contain three major protein components, namely bullous pemphigoid antigens 1 and 2 (BPA1 and BPA2) (Jones et al., 1994; Marinkovich, 1993; Uitto and Christiano, 1992) and integrin cc6p4. Integrin ot6p4 serves as a major receptor in the hemidesmosomal structure (Jones et al, 1991; Sonnenberg 20 et al, 1991; Stepp et al, 1990) that interacts with LM-5, a component of the anchoring filaments (Roussel le et al .,1991; Niessen et al., 1994). Other proteins, such as laminin-6 and uncein, may also be present in anchoring filaments (see figure 2.) (Zeng et al., 1994). Anchoring filaments interact with underlying lamina densa, that is composed of LM-1, nidogen and type-IV collagen. Type VII collagen is the major component of anchoring fibrils that are morphologically distinct attachment structures extending from the lower portion of the basement membrane, namely lamina densa, to the papillary dermis (Sakai et al, 1986). In functional experiments in vitro antibodies against oc6p4 integrin have been shown to cause dissociation of hemidesmosomes and led to cell detachment (Kurpakus et al, 1991). Transgenic animals lacking either a6 or 134 integrins develop blisters in their skin (Fassler et al., 1996, for review). B PA2 has been sequenced and found to be a membrane anchored glycoprotein with unusual aminoterminal-intracellular domain and col lagenous extracellular domain. Therefore the protein has been termed type XVII collagen (Giudice et al, 1991; Li et al, 1993). Recently, it has been demonstrated that the aminoterminal domain of B PA2 interacts with a 6 subunit of the a6l34 integrin (Hopkinson et al., 1995). It has been suggested that interaction of type XVII collagen with ct6(34 integrin may be crucial in mucocutaneous diseases that affect epithelial-connective tissue junction such as junctional epidermolysis bullosa (Uitto and Christiano, 1992). Extracellularly, <x6R4 integrin colocal izes and interacts with LM-5 (Niessen et al., 1994; Roussel le et al., 1991). Some patients with serious 21 skin conditions like lethal junctional epidermolysis bullosa have serum autoantibodies against LM-5 (Oomloge-Hultsch et al., 1992). All mutations of this oc6(34 - LM-5 complex have also potential oral and dental implications beyond blistering since it is known that the adhesion of junctional epithelium to the tooth enamel is also mediated by a6f34 integrin (Hormia et al., 1992). A split of junctional epithelium from the tooth due to mutations in this complex could weaken the host resistance in this area and possibly lead to early onset periodontal disease. Oral findings of these patients have not been carefully reported because attention has focused on their more serious skin condition. It should be noted that blisters may also raise from mutations in type VII collagen (dystrophic epidermolysis bullosa) (Uitto and Christiano, 1992). 1.5. Epithelial wound healing Wound healing is a complex phenomenon that involves series of controlled events including: a) an inflammatory stage involving aggregation of platelets and later migration of macrophages, fibroblasts, and lymphocytes at the injury site; b) the formation of a provisional extracellular matrix, mainly composed of fibrinogen, fibrin, fibronectin, and vitronectin; c) the migration of epithelial cells from the edges of the wound, and; d) the local synthesis and secretion of a wide variety of regulating growth factors and cytokines (Pierce et al, 1989; Juhasz et al, 1993; Bennett and Schultz, 1993; Boyce, 1994). After the epithelium has been disrupted at the time of tissue injury, reepithelialization must occur as rapidly as possible in order to re-22 establish tissue integrity. This process begins within hours after injury. Epithelial cells from residual epithelial structures loose their contact with basement membrane and move quickly across the wound defect. Clark and col leagues (1982) have shown that these keratinocytes migrate on provisional matrix in the newly created wound bed. Initially, the keratinocytes laterally migrate and cover the wound by a process of migration. Later, as the reepithelialization proceeds, the proliferation of keratinocytes that feed the advancing epithelial edge, becomes more important. During wound healing, the function of keratinocytes is to rapidly cover the exposed connective tissue (Stenn and Malhorta, 1992). This process depends upon a variety of interactions between cells and the extracellular matrix. As the basal keratinocytes at the wound site are exposed to the new, provisional matrix, the phenotype of the cel ls is changed from stationary to migratory. In this process, keratinocytes detach themselves from basement membrane, migrate laterally into the wound bed and finally regenerate the basement membrane. In mucosa and in skin the migration seems to involve similar patterns although the source of migrating epithelial cells is different. In the mucosa the basal layers of the wound edge epithelium serves as a major source of the keratinocytes migrating into the wound area. In the skin, however, epithelial cells arise not only from the wound edge but also from hair follicles and sweat glands. Keratinocytes have been shown to synthesize most of the main components, such as type IV collagen, laminin and type VII collagen, that are present in the basement membrane zone (Stenn and Malhorta, 1992). During wound healing or 2 3 any other such conditions that can involve cell movement, such as cancer or some skin diseases, the interaction between epithelial cells and underlying tissues changes. Cel ls will be exposed to new extracellular matrix proteins and cytokines, which can lead to altered integrin expression of the basal cells. The ultimate goal of normal wound healing is to have epithelial cells to form new stable organized basement membrane structure that is essential for the integrity of the tissue. During wound healing, most of the components of the basement membrane zone are missing underneath migrating keratinocytes (Larjava et al., 1993). Only LM-5 appears to be deposited against the wound bed matrix by keratinocytes during migration (Larjava et al., 1993). The role of LM-5 appears critical even for healing of the blister wounds where lamina densa remains intact (Kainulainen et al., submitted). Keratinocytes in chronic aphtous ulcers that have developed a healing response fail to deposit a proper LM-5 rich matrix (Richards et al., 1996). 1.6. Keratinocyte integrins and wound healing As the epithelial cells undergo major environmental changes during the wound healing, they have to adjust their cell surface receptors for the new demands. During wound healing keratinocytes have to migrate on the provisional matrix different from their stationary matrix. This requires a change of distribution of some integrins and expression of some totally new integrins. Integrin ot6B4 is present in migratory epithelial cells although hemidesmosomes are absent. Expression of B1 integrins is 24 strongly up-regulated during wound healing (Cavani et al, 1993; Gailit et al, 1994; Hertle et al, 1992; Juhasz et al 1993; Larjava et al, 1993). Both oc2(31 and «331 integrins seem to be equally expressed. Integrin a3f31 is known to be able to bind both fibronectin and LM-5, that are both present in provisional matrix (Carter et al, 1991; Larjava et al, 1993). Integrin oc2f31 is known to be able to bind many collagens, that form contacts with migrating keratinocytes, such as type I, III, V and VI collagens. During wound healing, keratinocyte 31 integrins also localize in suprabasal cell layers of the epibolus. This may be due to transfer of nondifferentiated migrating cells into suprabasal cell layers. Migratory keratinocytes are induced to express a531 integrin that is not present in stationary cells (Larjava et al., 1993). This induction seems to start: relatively early after wounding and end shortly. Distribution of a 6 3 4 integrin change from hemidesmosomal to diffuse during cell migration (Kurpakus et al, 1991). Integrins of the av-family are absent or expressed only weakly in normal epithelia. In contrast, av-integrins have been shown to be present in wound keratinocytes (Cavani et al., 1993; Juhasz et al., 1993; Larjava et al., 1993). The partner 3-subunit associated with the av-polypeptide has been unidentified in human wounds. Migrating keratinocytes in porcine epidermis have been reported to express the av35 integrin (Gailit et al., 1994). ocv35 integrin is present in cultured keratinocytes and has been shown to mediate keratinocyte migration on vitronectin (Kim et al., 1994). Vitronectin is present in the provisional wound bed matrix and it is, therefore, reasonable to assume that av35-vitronectin interaction can also exist during wound 25 healing. 1.7. TGF-13 in wound healing In wounds proper regulatory signals are required for normal repair. Currently it is bel ieved that transforming growth factor-betas (TGF-Bs) have a central role in the wound healing process. TGF-Bs are a family of polypeptides that have multiple regulatory actions in cell growth, differentiation, and developmental processes (Sporn and Roberts, 1992; Massaque et al., 1992). Three highly homologous TGF-B genes have been identified in mammals, representing TGF-B1, TGF-B2 and TGF-B3 polypeptides. Schmid et al (1993a) showed for the first time that human keratinocytes of intact skin express TGF-33 . This growth factor seems to play an important role in epidermal maintenance (Scmid et al., 1993a). In animal studies only small amounts of TGF-B2 and TGF-B3 mRNAs had been found in keratinocytes of intact dermis (Scmid et al., 1993b). All TGF-B isoforms are found in healing wounds of animals (Kane et al., 1991; Levine et al., 1993). However, Schmid et al. (1993a) did not find any detectable levels of TGF-B2 mRNA in human wound keratinocytes. Wound fibroblasts and macrophages are known to express both TGF-B1 and TGF-B2 in cells adjacent to the wound (Obberghen-Schill ing et al., 1988; Van Waes, 1995). TGF-B1 is the major isofprm in wound keratinocytes. Induction of TGF-B1 in migrating keratinocytes is crucial for the successful re-epithelialization of skin wounds. In vitro studies have shown that TGF-B3 inhibits growth of primary human keratinocytes 26 (Graycar et al. 1989) and TGF-31 seems to stimulate keratinocyte motility by switching the cells from differentiating to regenerative phenotype (Mansbridge and Hanawalt, 1988), and by inducing their production of fibronectin (Nickoloff et al., 1988). In cultured human keratinocytes, TGF-31 has also been shown to increase the levels of mRNA for some integrins, such as a5, av and 35, that may facilitate migration of wound keratinocytes (Gailit et al., 1994, Zambruno et al., 1995). TGF-31 may also stimulate the proliferation of wound keratinocytes at the wound margins indirectly by inducing the expression of other polypeptide growth factors, e.g. platelet-derived growth factor (PDGF) (Anatoniades et al., 1991; Chen et al., 1992). 1.8. Junctional and periodontal pocket epithelia Human periodontium contains oral gingival, sulcular and junctional epithelia, the underlying connective tissue, periodontal ligament, cementum and alveolar bone. Oral gingival epithelium is continuous with the sulcular epithelium, which then apically changes to epithelium which is attached to the tooth, called junctional epithelium (JE). Based on the ultrastructure and cytochemical features the J E is a so-called simple, non-differentiating and non-keratinizing type (Nanci and Schroeder, 1996; Schroeder and Listgarten, 1997, for review). All J E cells are very much alike ultrastructurally, and characteristically all contain only few cytoplasmic filaments that are composed of keratins 5, 13, 14, and 19. Keratin 19 is a marker of simple epithelia, such as J E (Schroeder, 1981; Feghali-Assaly et al., 1994). In addition J E cells express epidermal 27 growth factor (EGF) and the intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen (LFA-3) on their surface (Crawford, 1992; Gao and Mckenzie, 1992). The turnover rate of junctional epithelium is exceptionally high. This is characterized by constantly coronally migrating daughter cells as well as high rate of exfoliating cells into sulcular fluid (Schroeder and Listgarten, 1997, for review). Proliferation appears to happen throughout the entire area of J E (Salonen, 1986). Another characteristic feature of J E is that the intercellular spaces are large and variable in s ize and there are less desmosomes than in other areas. During inflammation intercellular spaces further increase in size. In inflamed J E these enlarged spaces are increasingly occupied by transmigrating neutrophilic granulocytes and infiltrating mononuclear cells, such as macrophages and lymphocytes (Schroeder and Listgarten, 1997 for review). The free surface of J E lines the bottom of the gingival sulcus. The structure separating the J E from the tooth enamel is called internal basal lamina whereas the external basal lamina separates the junctional epithelial cells from gingival connective tissue. Internal and external basal lamina (basement membrane) differ in composit ion. The external basal lamina contains typical constituents of the BM zone, while the internal basal lamina has been reported to contain type VIII collagen and LM-5 (Salonen et al 1991, Hormia et al, in press). LM-1 and type IV collagen appear to be absent in the internal basal lamina (Hormia et al., 1992). Hemidesmosomes connect basal cells to the internal basal lamina (also called DAT-cells) (Overman and Salonen, 28 1994). Integrin oc6f34, a critical component of the hemidesmosome complex, is found in the internal basal lamina (Hormia et al, 1992). It is believed to mediate adhesion of DAT cells to the LM-5 substratum on the enamel surface (Hormia et al, in press). Cultured epithelial cell rests of Malassez are thought to resemble J E cells and are known to be able to attach on various extracellular matrix components (Uitto et al., 1992). Periodontal pocket represents a pathologically deepened gingival sulcus in which the J E undergoes pathologic changes to form periodontal pocket epithelium. These changes include proliferation or disintegration of the internal basal lamina and are characteristic for periodontal disease. Based on keratin profile pocket epithelium resembles J E (MacKenzie et al., 1993). Epithelial cell behavior is poorly characterized in chronic inflammation of the human periodontium. In inflamed tooth supporting t issues (periodontal disease), increased proliferation of surrounding epithelial cells into inflamed connective tissue stroma is commonly seen (Schroeder and Listgarten, 1997, for review). The healthy J E is transformed into periodontal pocket epithelium causing these epithelial cells to proliferate and form deep epithelial ridges into inflamed connective tissue. As a result, the relative area occupied by epithelial cell in the gingival tissue surrounding the tooth increases compared to healthy gingiva. The local immunologic activity in J E fails to prevent the bacterial intrusion and the resultant induced immunologic response that leads to release of extensive amounts of cytokines (see above) and a strong inflammatory defence reaction. This is followed by an altered extracellular connective tissue matrix that the epithelial cells can adhere and migrate along. Basement membrane, or transformed external basal lamina, between the gingival epithelium and the connective tissue also shows some alterations, with discontinuities and other morphological changes seen during chronic inflammation (Thilander, 1968; Freedman et al., 1968; Takarada et al., 1974; Takarada et al., 1974; Peng et al., 1986). Loss of several heparan sulphate proteoglycans in chronically inflamed human periodontium has also been reported (Oksala et al., 1997). Perlecan, a structural component of the basement membrane zone disappears, as well as CD44 and syndecan-1, both of which are believed to function in cell-cell and cell-extracellular matrix interactions of epithelial cells (Oksala et al., 1997). The distribution of the adhesion receptors, integrins, of the chronically inflamed human epithelial cells have not been characterized before this study. 30 CHAPTER TWO The aims of the study The specific aims of this study were: 1. To find out which member of the av integrin family is expressed during reepithelialization of healing periodontal wounds in humans. The hypothesis was that the wound keratinocytes express ccv36 integrin as their fibronectin and tenascin receptor in healing wounds. 2. To characterize the alterations that occur in the expression of integrins and their ligands in epithelial tissue of chronically inflamed human periodontium. The hypothesis was that the expression of integrins and their ligands change in periodontal pockets to make pocket epithelial cells capable of adhering and migrating on extracellular matrix modified by chronic inflammation. 31 CHAPTER THREE KERATINOCYTES IN HUMAN WOUNDS EXPRESS avG6 INTEGRIN 3.1. Introduction Several reports have shown alterations in the expression of cell adhesion receptors, integrins, and their ligands during human and animal wound healing (Gailit and Clerk, 1994; Watt and Jones, 1993, for reviews,). In vivo, integrins are expressed by basal keratinocytes but are absent in differentiating upper cell layers (Larjava et al., 1990; Konter et al., 1989; Peltonen et al., 1989; Hertle et al., 1991; Larjava et al., 1992). This suggests that integrins have an important role in keratinocyte proliferation and differentiation. Several recent reports have dealt with integrin distribution in human and animal wound healing (Hertle et al., 1992; Juhasz et al., 1993; Cavani et al., 1993; Gailit et al., 1994). Expression of 131 integrins is generally strongly up-regulated in wounds. Besides a231 and oc3(31, also oc5f31 is present in the migrating keratinocytes. Although hemidesmosomes are generally absent under the migrating cells both oc6B4 and LM-5 are present. Since also oc3f31 can bind LM-5 (Carter et al., 1991), it is conceivable that both oc3(31 and a6(34 are involved in LM-5 binding in migrating keratinocytes. Integrins of the ocv-family are not stongly expressed in healthy human epidermis or mucosa (Larjava et al., 1993). We and others have recently shown that expression of av-integrins is induced in wound keratinocytes (Larjava et al., 1993; Juhasz et al., 1993; Cavani et al., 1993). It is not clear which 3-subunit is associated with the av-32 subunit in wounds. In some models of wound healing, av35 appears to be the prominent member of the ocv-integrin family in wound keratinocytes (Gailit et al., 1994) This integrin has been shown to mediate migration of cultured keratinocytes on vitronectin substrates (Kim et al., 1994). In this section I present evidence that ocv36 is abundant in wound keratinocytes during human mucosal and dermal wound healing. Furthermore, av36 expression occurs concomitantly with the expression of tenascin in wound granulation tissue. Since ccv36 may mediate cell adhesion to both fibronectin and tenascin (Prieto et al., 1993), I propose that this binding serve as a transient adhesion mechanism for keratinocytes prior to the assembly of the organized basement membrane. 3.2. Material and methods Material Experimental human palatal mucosal wounds (full thickness) were made in volunteers in Finland. Human experimental protocol was designed because most of the antibodies available against integrin polypeptides recognize human antigens. The experimental protocol was approved by the Ethical Committee of the University of Oulu, Finland. Incisional V-shaped wounds (about 1.5 cm long, 2 mm wide and deep) were created in human palate (distal from the first premolar tooth, about 4 mm from the gingival margin). Punch biopsies (4mm) were taken from wounds on day 3 and 7, rinsed in physiological saline, cut at an acute angle to the wound, embedded in 33 Tissuetek (Miles Inc, Elkhart, IN) and immediately frozen in liquid nitrogen. Biopsies from skin wounds were obtained from volunteers after wounding on day 1, 3, and 7. Biopsy blocks were stored at -70°C until used. Spec imens were sectioned (6 u.m) in a cryostat and fixed with -20°C acetone for 5 minutes and stored at -70°C until used. Morphological analysis was performed using toluidine blue staining. Immunohistochemical studies For immunolocalization studies, sections were incubated with primary antibodies diluted in phosphate buffered saline (PBS) containing 1mg/ml bovine serum albumin (BSA) for 60 minutes, after which sections were rinsed four times in 60 minutes with the PBS /BSA solution. After rinsing, sections were incubated with affinity-purified rhodamine-conjugated secondary antibodies (dilution 1:50, anti-mouse cat. #1214608, anti-rabbit cat. #1238841, both from Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 60 minutes. Sections were rinsed and mounted using cyanoacrylate adhesive (Chemoco). Samples were examined using a Zeiss Axioskop 20 fluorescence microscope, and photographed using MC 80 Zeiss Microscope Camera . Antibodies against the following proteins were used in this study (Table 1); ccv-integrin (Mab P3G8; Wayner et al., 1991), av(35 integrin (Mab P1F6; Weinacker et al., 1994), cxv(36 integrin (Mab E7P6; Breuss et al., 1993), (36 integrin (Mab R6G9, Breuss et al., 1993) , 31 integrin (antiserum 3847, Roberts et al., 1988), tenascin (Chemicon, Temecula, CA), and fibronectin (antiserum, Chen et al., 1985). We have previously shown that P3G8 34 and P1F6 react with human integrins in immunostaining (Nikkari et al., 1995). Table 1. Antibodies used to recognize different integrins and their l igands in these studies. Integrin/ligand Ant ibody Reference oc2 117 AMAC , Inc. oc3 J143 Kantor et al. oc5 mAb BIIG2 Werb et al. cc6 mAb GoH3 Sonnenberg et al. av mAb P3G8 Wayner et al. 31 Serum, 3847 Roberts et al. 34 mAb 345-11A Kennel et al av35 mAb P1F6 Weinacker et al. 36 mAb R6G9 Breuss et al. av36 mAb E7P6 Breuss et al. Type IV mAb ICN Biomedicals Type VII mAb Chemicon Laminin Serum Risteli et al Kalinin mAb G B 3 Verrando et al Control staining was performed without the primary antibodies and with nonimmune mouse serum. No specif ic reaction was obtained with either of the controls. 35 3.3. Results Human mucosal wound keratinocytes express av36 integrin that is localized in areas of accumulation of fibronectin and tenascin. Expression of av(36 integrin was first investigated in experimental full thickness wounds of oral mucosa. Epithelial sheets showed marked migration into the wound bed on day 3, and all wounds were closed on day 7. Some of the wounds were already closed on day 3, which allowed us to study integrin distribution in fused epithelium during formation of early granulation tissue. Integrins of the 131 -family were present in normal keratinocytes of oral mucosa while av, avf35 or av(36 integrins were absent (Fig. 3F). At the edge of the migrating epithelial sheet of 3-day-old wounds, antibodies against av integrin and av(36 integrin complex were reactive in some but not all specimens (Fig.3A, B, D, E). Antibody against the avf35 integrin was nonreactive in all specimens (not shown). The leading epithelial front strongly expressed (31 integrins (not shown). In those 3-day-old wounds in which epithelial sheets were fused, av(36 integrin was present in the basal cell layer in the wound area (Fig. 3C), but absent from non-wourided site (Fig. 3F). Antibodies specific to av integrin (P3G8) and avf36 integrin complex (E7P6) reacted with basal keratinocytes in all 7-day-old wounds (Fig. 4A and B, respectively). Antibody (R6G9),. recognizing (36 polypeptide, gave staining results identical to those obtained with antibody reactive to av(36 integrin complex (E7P6) (not shown). 36 'All fJtv Figure 3. Integr in a v 6 6 is e xp r e s sed in s ome but no t all 3-day-o ld human muco sa l wound s . Panels A, B, D, and E, separate sect ions of the same specimen of open 3-day-old mucosal wound. Panel C, closed 3-day-old wound. Panel F, nonwounded area of 3-day-old wound, same sect ion as shown in panel C. Staining of av integrin (panels A, B), or av&6 (E7P6, panels C, D, E, F) integrin complex by immunofluorescence. Arrows mark the wound site and dot ted line epithelial connect ive tissue interphase. E, epithelium; GT, granulation tissue; CT, connect ive t issue. Bar 50 iL/m: A, D; bar 20 £vm: B, E, F; bar 100 ^m: C. Figure 4. Integr in av&6 and i t s pu t a t i v e l i gands , f i b r o n e c t i n and t e n a s c i n , l o ca l i ze in s im i la r a reas in the 7-day-o ld wound s . Ant ibodies against av integrin (panel A) , «vll6 integrin complex (panel B), tenascin (panel C) and f ibronectin (panel D) were used. Arrows indicate the epithelial connect ive t issue interphase. E, epithel ium. Bar 100 ^ m: A, B; bar 50 /jm: C, D. 38 We then localized the two putative ligands of av(36 integrin, fibronectin and tenascin, in 7-day-old wounds that displayed high expression of this receptor. Fibronectin was present throughout the normal connective tissue and abundantly in the wound granulation tissue matrix, in which it showed a band-like distribution around the pseudo rete ridges of fused wound epithelium (Fig. 4D). Tenascin was present strongly in subepithelial connective tissue but weakly in deeper areas of the nonaffected tissue (not shown). Expression of tenascin was intense in the granulation tissue of 7-day-old mucosal wounds (Fig. 4C). Like fibronectin, the relative amount of tenascin appeared greatest underneath the cells expressing ocvf36 integrin. Wound keratinocytes in fused epidermal wounds express av36 integrin Because cutaneous epithelium presumably has a different source of stem cells than mucosal epithelium do, we also investigated the expression of integrins and tenascin in full-thickness skin wounds. Stimulation of 131 integrin expression was observed at the wound margin as soon as day 1 after wounding, and migrating epithelium remained strongly reactive up to day 7 (Fig. 5A-C). No expression of av, 35, or 36 integrin was found in epithelial cells of nonwounded epidermis or in 1- or 3-day-old wounds (shown for av in Fig. 5D,E). In 7-day-old wounds, basal epithelial cells covering the entire wound granulation tissue reacted strongly with antibodies against both av integrin and av36 integrin complex (Fig. 5F and G, respectively) but not with antibodies to a v35 integrin (not shown). In nonwounded areas, tenascin was present only at the subepidermal connective tissue (Fig. 6A). This protein was absent from the coagulum of 1-day-old wounds and from the wound bed matrix of 3-day-old wounds (Fig. 6A-B). In contrast, the expression of tenascin was high in granulation tissue of 7-day-old wounds (Fig. 6C). F ig . 5. In human de rma l wounds ftl i n teg r in is e x p r e s s e d t h r o u g h o u t the hea l ing p r o c e s s bu t «v and avft6 in tegr ins are no t e x p r e s s e d unt i l day 7. 1-Dau old (panels A, D), 3-day-old (panels B, E) and 7-day-old (panels C, F, G) dermal wounds were stained with antibodies specific to &1 integrin (panels A, B, C), «v integrin (panels D, E, F), and av&6 integrin complex panel G). Epithelial-blood clot (E/C) interphase marked with dotted line in panels D and E. E, epithelium; CT, connect ive t issue; GT, granulation t issue. Bar 50 jL/m. 41 F ig . 6. Exp re s s i on o f t e na s c i n is up regu l a t ed in de rma l wound g ranu l a t i on t i s sue . Biopsy specimens were stained with ant ibody to tenasc in by immunofluorescence as descr ibed in materials and methods. Distribution of tenascin in 1 -day-old (A), 3-day-old (B), and 7-day-old (C) human dermal wounds. Arrowheads indicate wound margin and arrows tenascin containing subepithelial connect ive tissue in panels A and B. E, epithelium; C, clot; CT, connect ive tissue; GT, granulation t issue. Bar 100 yvn 42 3.4. Discussion We have reported previously that migrating wound keratinocytes start to express receptors of the av integrin family (Larjava et al., 1993). Several recent studies have shown the presence of av integrins in keratinocytes migrating in experimental wounds (Larjava et al., 1993; Juhasz et al., 1993; Cavani et al., 1993). The av associated 3 subunit has remained, however, unresolved. In the present study, I found that av36 is strongly expressed in wound keratinocytes. In a porcine wound model, migrating keratinocytes appear to express a v 3 5 integrin (Gailit et al., 1994). In the human mucosal wound healing model, I have not found detectable levels of avf35 integrin expression with immunofluorescence techniques. Further, I could not detect the av35 integrin in the healing epidermis of human skin. It is possible that porcine wound keratinocytes are different from human cells in their regulation of integrins. Alternatively, it is possible that the immunofluorescence method used was not sufficiently sensitive to detect minute levels of av35 in human skin. The antibody used here, was found to be reactive in immunofluorescence for av(35 integrin in human synovium (Nikkari et al., 1995). The antibody recognizes the av(35 complex. It is possible, therefore, that 35 is present in wound keratinocytes but in another conformation not reactive with this monoclonal antibody. Cultured keratinocytes have been reported to express a v35 integrin (Adams and Watt., 1991). Our results suggest that multiple av-family integrins are expressed by cultured keratinocytes since the expression of 36 was low compared to that of 4 3 total av integrin expression. It is also possible that both 135 and 31 serve as partners for av integrin subunit. Expression of av36 was detected at the tip of migrating mucosal epithelium in some occasions and appeared to occur late during wound healing, s ince specimens were often nonreactive or weakly reactive in open 3-day-old wounds but became reactive in c losed 7-day-old wounds. The age of the wound however, was less important than the stage of healing, since small 3-day-old wounds in which epithelial sheets had fused expressed av36 integrin. After seven days of healing, all wound specimens contained granulation tissue under the epithelial coverage and expressed strongly av36 integrin. These data agree with previous findings using av specific antibody (Larjava et al., 1993), and also with findings using a graft healing model in which upregulation of av occurs relatively late and persists for longer periods than induction of, for example, a531 expression (Juhasz et al., 1993). This suggests that factors involved in regulating av36 expression are released after the initial movement of wound keratinocytes has already occured. Initial assembly of hemidesmosomes and reconstruction of the basement membrane zone was observed in spec imens that were strongly reactive with a v 3 6 antibody, suggest ing that keratinocyte interaction with both basement membrane and granulation tissue matrix contributes to the upregulation of a v36 expression. Deposition of basement membrane material and assembly of hemidesmosomes may not be necessary for the induction of av36 but may be required for optimal expression 44 of avl36 in wounds. In normal tissues, expression of 36 mRNA is found exclusively in epithelia (Breuss et al., 1993). Its expression is restricted however, to kidney tubule epithelium and endometrial epithelium, while in other epithelia, e.g., in normal skin, expression of 36 mRNA is undetectable. Cultured cells use av36 as a fibronectin-binding integrin (Weinacker et al., 1994). It is possible, therefore, that wound keratinocytes use av36 for binding to fibronectin. It is puzzl ing that wound keratinocytes upregulate the expression of at least two fibronectin binding integrins. Both oc531 and ocv36 are expressed in different stages of wound healing. It is possible that ct531 is used mainly to bind cells to plasma-derived fibronectin during the early stage. av36 in contrast, could serve as a receptor for the differentially spl iced embryonic type of fibronectin produced during formation of granulation tissue (ffrench-Constant et al., 1989). There is no experimental evidence, however, suggesting that differentially spl iced fibronectins would function differently in this respect. Furthermore, both oc531 and ocv36 bind to the common R G D repeat present in all fibronectins (Busk et al., 1992). Alternatively, ocv36 and cc531 could mediate different cellular responses to fibronectin. Tenascin was present underneath the epithelium both in normal mucosa and human dermis, as described earlier (Lightner et al., 1989). Its distribution extended deeper into the connective tissue stroma in mucosa compared to dermis. Expression of tenascin was strongly upregulated during granulation tissue formation both in mucosal and skin wounds, confirming previous reports from other wound healing 45 models (Mackie et al., 1988; Chuong and Chen, 1991; Whitby et al., 1991). Clearly, multiple integrins mediate cell adhesion to tenascin (Prieto et al., 1993; Bourdon and Ruoslahti, 1989). Evidently, avf36 is also involved in recognition of the third fibronectin type repeat in tenascin, although direct data are still lacking (Prieto et al., 1993). Tenascin appears in wounds at about the same time as expression of av36 is optimal. In addition, tenascin is localized in proximity to basal keratinocytes associated with incompletely regenerated basement membrane. It is feasible therefore, that keratinocytes could utilize av36 for binding to tenascin. Basal keratinocytes express another integrin receptor, a931 , that could participate in tenascin binding (Palmer et al., 1993; Yokosak i et al., 1994) either alternatively or in conjunction with other integrins. 3.5. Summary Cell adhesion receptors of the integrin family play a major role during re-epithelialization of human wounds. We have previously documented that the expression of av family integrins is induced in keratinocytes of mucosal wounds (Larjava et al., 1993). In the present investigation, I extended these studies to determine whether av36 integrin is expressed during wound healing in humans. Mucosal and epidermal wound sections from 1- to 7-day-old wounds were used for immunolocalization of integrins and their putative ligands. Expression of avf36 integrin appeared relatively late during mucosal and dermal wound healing. Maximal expression was seen in 7-day-old 46 wounds in which epithelial sheets had fused and granulation tissue was present. Fibronectin and tenascin, both possible ligands for ocv(36 integrin, were found concentrated underneath the basal epithelial cells expressing this receptor, and the maximal expression of tenascin coincided with that of avG6 integrin. These results suggest that the expression of ocv(36 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing. 47 CHAPTER FOUR EXPRESSION OF EPITHELIAL ADHESION PROTEINS AND INTEGRINS IN CHRONIC INFLAMMATION 4.1. Introduction During the pathogenesis of periodontal disease, the J E between the tooth surface and gingival connective tissue moves apically and becomes transformed into periodontal pocket epithelium (Page and Schroeder, 1990). These epithelial cells proliferate and form epithelial ridges in inflamed connective tissue. As a result, the relative volume of gingival tissue occupied by epithelial cells increases compared to healthy tissue. This dynamic process of epithelial cell movement involves adhesion to and migration along an extracellular connective tissue matrix that is altered due to chronic inflammation. The basement membrane also shows alterations, with discontinuities and other morphological changes in chronic inflammation (Thilander, 1968; Freedman et al., 1968; Takarada et al., 1974; Peng et al., 1986). The adhesion and migration mechanisms olf epithelial cells in chronically inflamed tissues are, so far, poorly characterized. It is likely, however, that the expression of specific cell surface adhesion receptors is altered as a result of changing functional demands and the effects of bioactive substances present in inflammation (Uitto and Larjava, 1991). In this section I demonstrate alterations in the expression of (31 and (34 integrins and basement membrane zone proteins in chronic periodontal inflammation. 48 4.2. Material and methods Material. Chronically inflamed periodontal tissue spec imens were obtained from 22 subjects during extraction of periodontally involved teeth with a clinically hopeless prognosis. Spec imens were rinsed in physiological saline, placed on a cork disk in a defined orientation, embedded in Tissue-Tek II O.C.T. compound (Miles Inc. Elkhart, IN), and immediately frozen in liquid nitrogen. Samples were stored at -70°C until used. Spec imens were sect ioned (6 urn) in a cryostat. Adjacent sect ions corresponding to those used for immunohistological studies were stained with hematoxylin and eosin for histopathological and morphological analyses. Gomori 's periodic acid methenamine silver method was used to histochemically stain the basement membrane zone (Culling, 1963). Immunohistochemical studies Frozen sections (6 u,m) were fixed and stored as descr ibed in the previous work. Immunolocalization of integrins and examination of stainings were performed as described previously. Antibodies against the following integrin subunits and their ligands were used (sources and references for antibodies are given in t ab l e I): oc2, oc3, a5, a6, av, 31, f34, 135, 36, fibronectin, type IV and VII collagens, tenascin, LM-1, and LM-5. Control stainings were performed omitting the primary antibody or with nonimmune mouse serum. No specific staining was obtained with either of these control staining procedures (not shown). 49 4.3. Results Microscopic morphology of inflamed periodontal tissue Chronically inflamed tissue sections were obtained from patients suffering from advanced adult periodontal disease. Spec imens containing both relatively healthy and chronically inflamed tissue were investigated. Compar isons were always made between these two areas (Fig. 7 A and B) in the same section. Using hematoxylin-eosin staining, a heavy infiltrate of inflammatory cells was seen in close proximity to the epithelium of inflamed area. Cellular infiltrates were mainly composed of chronic inflammatory cells, i.e. lymphocytes and plasma cells (Fig. 7B). In chronically inflamed sites, nonkeratinized epithelium varied in thickness from severa l ce l l layers to long epithelial extensions into the inflamed connective tissue. We then used Gomori 's periodic acid methenamine silver method (Culling, 1963) to examine the basement membrane underlining the epithelium. This staining visualizes aldehydes generated by periodate treatment, and it can be used to evaluate the integrity of basement membrane in tissue sections. In relatively healthy areas of oral epithelium, basement membrane staining was continuous separating the oral epithelium from the underlying stroma (Fig. 7C). In contrast, in inflamed periodontal t issues only faint, discontinuous staining was observed (Fig. 7D). F igure 7. Hematoxyl in and eosin staining of healthy (A) , and chronically inflamed (B) per iodont ium. Gomor i ' s per iodic acid methenamine s i lver s ta in ing of basemen t membrane zone in normal (C) and inflamed per iodontal t issue (D) . Arrows indicate location of basement membrane (C and D). OE; oral epithel ium; CT; connect ive t issue; PE; periodontal epithel ium in chronic inflammation; ICT; inflamed connect ive t issue. Bar 50 jvm: A, B; bar 100 /jm: C, D. 51 Localization of LM-1 and type IV collagen We then examined the putative adhesion molecules for epithelial cells, LM-1 (classical laminin) and type IV collagen using immunolocalization techniques. In healthy oral epithelium, both LM-1 and type IV collagen were found in epithelial and vascular basement membranes (Fig. 8A and B). In chronically inflamed tissue, both LM-1 and type IV col lagen were still present at the epithelial basement membrane zone, although in somewhat reduced amounts (Fig. 8). Numerous blood vesse ls surrounded by LM-1 and type IV collagen were present in the inflamed connective tissue (Fig. 8C and D). In addition, anomalous localization of punctate extravascular laminin and to a lesser extent type IV collagen was found to be present in the inflamed connective tissue stroma. Visually, the total amount of basement membrane components therefore often appeared to be increased in the inflamed area due to the deposition of new vascular and extravascular basement membrane material. Using higher magnification, we were able to demonstrate several different patterns of extravascular LM-1 accumulation (Fig. 9 A-C). Clearly, LM-1 was often present surrounding blood vesse ls (Fig. 9A), but it was also found in punctate (Fig. 9B) and streak-like (Fig. 9C) distributions. Type IV collagen was also present in the stroma in a punctate pattern (Fig. 9D). Staining appeared to be specific, because only faint diffuse background staining was obtained with control antibodies (mouse nonimmune IgG or rabbit serum), or by omitting the first antibody (conjugate only) (not shown). 52 F igure 8. Immunolocalization of LM-1 (A and C) and type IV col lagen (B and D) in healthy (A and B) and in chronically inflamed periodontal t issue (C and D), respectively. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive tissue. Bar 2 0 0 yvn: A; bar 100 ^m: B, C, D. F igure 9 . Dist inct patterns of extravascular (arrows) LM-1 (A-C) and type IV col lagen (D) in inflamed periodontal t issue. Arrowheads indicate LM-1 and type IV col lagen associated with vascular basement membrane. Localization of components of epithelial anchoring system Epithelium is anchored to the underlying connective tissue via hemidesmosome structures in which anchoring filaments and anchoring fibers composed of LM-5 and type VII collagen, respectively, serve as linking elements (Rousselle et al., 1991; Keene et al., 1987). Since the oc6FJ4 integrin is believed to use some of these proteins as adhesive ligands, we compared their distributions in healthy and chronically inflamed tissues. Based on immunolocalization, type VII collagen was relatively well preserved at the basement membrane zone in chronic inflammation (Fig. 10). With higher magnification there was, however, a visible reduction of the intensity of staining in areas of chronic inflammation (compare Fig. 11C and D). In five out of eleven of the samples, type VII collagen was also found in unusual streak-like distributions in the subepithelial connective tissue stroma of inflamed tissue (compare Fig. 11C to 11D and 15F). In eight out of eleven samples the intensity of LM-5staining was generally reduced at the basement membrane zone in chronic inflammation, but in three samples it seemed to be relatively well-preserved (Fig. 11E and F). In four out of eleven samples LM-5 was found in similar streak-like patterns as type VII collagen in the connective tissue stroma of inflamed tissue (Fig. 15E and F). 55 F igure 1 0 . Immunostaining of type VII col lagen assoc iated with oral epithel ium (OE) and with periodontal epithelium in chronic inflammation (PE). F igure 1 1 . Immunolocalization of type VII col lagen (A-D) and LM-5 (E, F) in healthy oral mucosa (A, C, E) and in chronically inflamed periodontal t issue (B, D, F). OE; oral epithelium; CT; connect ive tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive t issue. Bar 100 iim: A, B, E, F; bar 30 ym: C, D. 57 Localization of fibronectin and tenascin Fibronectin and tenascin are also putative ligands for epithelial cell adhesion receptors. Fibronectin was present throughout the connective tissue adjacent to healthy oral epithelium, but it was clearly diminished in inflamed stroma (Fig. 12A and B). A fragmented and punctate staining pattern of fibronectin surrounded the inflammatory infiltrate. Tenascin was enriched in the subepithelial zone of healthy oral epithelium (Fig. 12C). In chronically inflamed tissue, tenascin was almost completely absent (Fig. 12D) and positive staining for tenascin was only seen at the basement membrane zone. Distribution of 31 and a6B4 integrins in epithelial cells Staining for 131 integrins was found around the periphery of basal keratinocytes of healthy oral epithelium (Fig. 13A). In chronic inflammation, two distinct patterns were seen. Most often, a focal loss of epithelial f31 integrins was seen in inflamed areas (Fig. 13D). Most of the samples, however, also included areas where the full thickness of epithelium (Fig. 13G) or the extending tip of it (Fig. 13H) was found to be positive for 31 integrins. In both cases, cells in the inflamed stroma showed bright staining for 31 integrins (see Fig. 13H). Immunolocalization of a231 and oc331 integrins by specific antibodies confirmed the variable staining pattern of 31 integrins observed with 31 specific antibodies (Fig. 14). Epithelial cells showed a loss of integrin expression in some areas of the section, while up-regulation was observed in others. During inflammatory stimulation, the staining pattern also changed from localization around 58 basal cells to a pattern encompassing cells in all cell layers (Fig. 14G, H, I). The transition from a nearly normal staining pattern to that of strongly induced expression was often remarkably abrupt sharp (e.g. see Fig. 14G). A continuous staining pattern was observed for a6 and 64 integrins at the basal aspect of basal keratinocytes in noninflamed areas (Fig. 13B, C). Focal losses of both a6 and 34 integrin subunits were common in epithelial cells at inflamed sites (Fig. 13E, F). Double staining for 31 integrins and type VII collagen permitted better visualization of the alterations, since the loss of integrin staining sometimes made it difficult to distinguish epithelial cells from other cells in the stroma (Fig. 13H, I). It was noted that in areas of relatively high 31 integrin expression in epithelium, also type VII collagen often became localized in streaks in the subepithelial connective tissue (Fig. 13H and I). This view was supported by findings from serial parallel sections that were stained for 31, oc6, 34, type IV collagen, LM-5, and type VII collagen (Fig. 15A-F). In areas which showed streak-like distributions of type IV and VII collagen and LM-5, all cell layers of epithelium were strongly positive for 81 integrins. In addition, staining for both <x6 and 34 integrins was relatively intense. Members of the av-integrin family were not detected in epithelia of healthy or inflamed tissue. Attempted immunolocalization with specific antibodies recognizing ocv35 and ocv36 integrins gave negative results in all samples (data not shown). 59 Figure 1 2. Immunolocalization of f ibronectin ( A , B) and tenascin ( C , D) in healthy oral mucosa ( A , C ) and in chronically inflamed periodontium (B, D ) . Note the reduction in intensity of staining for each molecule in inflamed subepithelial connect ive t issue. O E ; oral epithelium; C T ; connect ive tissue; P E ; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive tissue. Bar 100 ^m: all f igures. 60 F igure 1 3. Immunolocalization of fcl -integrin (A, D, G), a6-integrin (B, E), rl4-integrin (C, F) in healthy oral epithelium (A-C) and in chronically inflamed periodontal t issue (D-G). Double-immunostaining of &1 -integrin (H) and type Vll-collagen (I) in inflamed periodontal t issue. OE; oral epithelium; CT; connective tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive t issue. Bar 3 0 *vm: A, C, D, F, G; bar 100 ^m: B, E, H, I. 61 Figure 14 . Immunolocalization of HI (A, D, G), a3 (B, E, H) and a2 (C, F, I) integrins in healthy oral epithelium (A-C), and in periodontal epithelium in chronic inflammation (D-l). In inflammation, some areas of periodontal epithel ium show focal loss of these integrins (D-F) whereas in others the full thickness of inflamed periodontal epithelium is stongly positive for HI , u3 and a2 integrins (G-l). Arrows mark epithelial-connective t issue boundary (dashed black line, panel G). Arrowheads (panel G) mark HI staining which is comparable t o normal oral epithelium, and dashed white line indicates area of epithelium that demonstrates a loss of G1 staining. OE; oral epithelium; CT; connect ive tissue; PE; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive t issue. Bar 100 /vm. 62 Figure 1 5. Immunolocalization of &1 (A), a6 (B), S4 (C), type IV (D), LM-5 (E), and type VII (F) in parallel serial sect ions of chronically inflamed periodontal t issue. Note the strong expression of &1 integrin (A) in epithelium and also the streak-like appearance of LM-5 and type VII collagen (E,F) in connect ive t issue. PE; periodontal epithelium in chronic inflammation; ICT; inflamed connect ive t issue. Bar 100 jL/m. 63 4.4. Discussion Epithelial cell adhesion, migration and proliferation have been studied in acute inflammatory conditions such as wound healing (Stenn and Malhorta, 1992, for review). However, epithelial cell behavior in chronic inflammatory conditions is much less characterized. Periodontal disease is characterized by chronic inflammation and slowly progressive tissue loss and provides, therefore, an interesting model to study epithelial cell behavior during chronic inflammation. The epithelium undergoes alterations that lead to ulceration, degeneration, and also increased proliferation and formation of epithelial rete ridges into inflamed connective tissue stroma. Our study demonstrates that major alterations take place in the expression of epithelial adhesion molecules and their receptors in the chronic inflammation of periodontal disease. General structural alterations of the basement membrane zone in inflammatory periodontal lesions have been previously well-characterized. Several studies describe a diffuse appearance (Thilander, 1968), localized discontinuities (Freedman et al., 1968) or degradation (Takarada et al., 1974a, b) of the basement membrane facing inflamed connective tissue. Our study showed major alterations of individual adhesion molecules at the basement membrane zone. Despite extensive loss of subepithelial collagen, most of the matrix components associated with the basement membrane zone were partially retained in chronic inflammation. Generally, all components of the basement membrane zone including type IV collagen and laminins demonstrated 64 reduced staining intensity compared to normal noninflamed tissue. These proteins were, however, better preserved than stromal proteins fibronectin and tenascin, quantities of which were found to be markedly reduced in inflamed areas. In vitro experiments have shown that all of the above mentioned proteins are readily c leaved by matrix metalloproteinases (Birkedal-Hansen, 1993) which are present in high amounts in inflamed tissues (Makela et al., 1994). Fragments of fibronectin have been demonstrated directly in fluids collected from sites of chronic inflammation (Carsons et al., 1985; Talonpoika et al., 1989; Wysocki and Grinnell, 1990). Our results suggest, however, that proteins of the basement membrane may be woven into a meshwork in which protein epitopes persist although proteins are partially degraded. Focal loss of type IV collagen, LM-1 and LM-5 was observed in some samples, similar to data obtained in a previous study of type IV collagen immunolocalization in inflamed periodontal tissue (Peng et al., 1986). In many specimens, however, type VII collagen and to a lesser extent LM- 5 were found in unusual locations in inflamed stroma. It is believed that type VII collagen is synthesized by keratinocytes, although stimulus from fibroblasts seems to be needed for optimal synthesis (Regauer et al., 1990; Konig et al., 1991). It is a lso possible, however, that fibroblasts in chronic inflammation could contribute to collagen VII accumulation, since TGF-13 is able to induce type VII collagen expression in fibroblasts (Konig et al., 1992). Chronic lesions of periodontal disease contain large numbers of newly formed capillaries surrounded by basement membrane, as demonstrated in our samples 65 stained with antibodies against LM-1 and type IV collagen. These basement membrane components associated with new vessels were not found to be degraded at sites of inflammation. In fact, significant amounts of extravascular LM-1 and to lesser extent type IV collagen were found in the inflammatory sites. This atypical localization seems to be rather specif ic to chronic inflammation of periodontal d isease and is not found during acute inflammation of wound healing (Larjava et al., 1993) or during chronic inflammation of rheumatoid arthritis (Nikkari et al., 1993), where type IV collagen and laminin are found exclusively in the basement membrane area. Electron microscopic studies have earlier demonstrated the presence of extra layers of basement membrane-like material surrounding the capillaries of inflamed gingival lesions (Gavin, 1970). It is not known which cells deposit this material, but sprouting endothelial cells are candidates for this activity. In addition granulation tissue fibroblasts may be responsible, because they have been found to synthesize basement membrane components in vivo (Betz et al., 1992). Our findings point to the conclusion that during chronic periodontal inflammation, the basement membrane zone proteins type VII collagen and laminin partly replace the connective tissue stroma. These proteins may then serve as adhesive ligands for epithelial cells, granulation tissue fibroblasts and inflammatory cells. We have shown earlier that 31 integrins localize mainly at lateral borders of basal cells in normal mucosa and skin (Peltonen et al., 1989; Larjava et al., 1992; Larjava et al., 1993) and that these integrins could mediate cell-cell adhesion of 66 keratinocytes (Larjava et al., 1990; Carter et al., 1990). The hemidesmosomal integrin oc684 is exclusively localized at the basal aspect of basal cells in normal epithelia. Both of these integrin types are found in an altered distribution surrounding suprabasal cells during wound healing (Larjava et al., 1993; Hertle et al., 1992) and in psoriatic epidermis (Hertle et al., 1992; Pellgrini et al., 1992). In the present study, focal loss of integrins was a common finding in epithelium of chronically inflamed periodontal lesions. This loss may result from specific downregulation of integrin expression, since the cells in the same area seemed to deposit normal amounts of basement membrane components. There were, however, also areas in which the expression of both a231 and oc331 integrins appeared to be paradoxically markedly upregulated. Integrin expression is clearly up-regulated during wound healing (Larjava et al., 1993; Cavaniet al., 1993; Juhasz et al., 1993; Clark, 1990). Stimulated expression of 31 integrins by keratinocytes has been found in many inflammatory dermal lesions such as psoriasis (Pellegrini et al., 1992), lichen (Konter et al., 1990; Ralfkiaer et al., 1991) and cutaneous lymphoma (Ralfkiaer, 1992). In contrast, integrin expression has been reported to be relatively unchanged in dystrophic or systemic d iseases such as epidermolysis bullosa (Nazzaro et al., 1990) and systemic sclerosis (Sollberg et al., 1992). Periodontal lesions are known to contain areas with variable disease involvement as judged by varying gingival surface topography and tissue histology (Saglie et al., 1982). Cytokines are known to exert potent cell-specific effects on integrin expression (Heino, 1993; Heino et al., 1989; Milam et al., 1991). It is therefore 67 likely that integrin expression is regulated locally in pocket epithelial cells by combinations of inflammatory cytokines. 4.5. Summary Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of periodontal tissues, increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, LM-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and LM-5 were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the 31 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of 31 integrins (a2(31 and a3p i ) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for 31 integrins. No members of the av integrin family were found in any epithelia studied. Expression of the a634 integrin was high in basal cells of healthy tissue, but weak in epithelium 68 associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. 69 CHAPTER FIVE GENERAL DISCUSSION AND CONCLUSIONS Periodontal epithelia play a critical role in protection, destruction and repair of human periodontium. During optimal repair, epithelium migrates and covers the wound surface to prevent the infection and damage of vulnerable underlying connective tissue. During destruction, J E undergoes transformation to the pocket epithelium that increases in volume and changes architecture. Both scenarios, regeneration and destruction, involve active epithelial migration either in the wound provisional matrix in the case of regeneration, or in the inflamed connective tissue matrix during periodontal d isease, respectively. During the last decade information on the cell adhesion receptors and their ligands in the extracellular matrix has explosively increased. Even though there are still many unanswered questions some clear patterns have emerged from these studies. Integrins that form a large family of cell surface receptors are mainly responsible for the cell-matrix interactions. Cel ls react rapidly to the changes in their environment by relocation of exisiting integrins and expressing new integrins specifically targeting the pericellular matrix proteins. Matrix binding is a prerequisite for cell migration and proliferation of epithelial cells. During wound healing, migrating epithelial cells of the wound margin intensely express integrins binding to fibronectin, vitronectin, tenascin and col lagens. Only after epithelial confrontation and formation of new basement membrane do the cells revert to their normal integrin expression pattern. 70 Epithelial cell adhesion undergoes marked modulation during keratinocyte migration and basement membrane reconstruction during wound healing (Larjava et al., 1996, for review). Receptors that already exist in stationary cells change their cell surface distribution and probably also function. Alpha5(31 fibronectin receptor is induced and it is thought to mediate binding to serum fibronectin during the early stage of healing. At later stage of wound healing, av36 integrin could replace oc531 as the fibronectin binding receptor to transfer epithelial cells to lower affinity binding that would then allow basement membrane reconstruction. Alternatively, a v36 integrin could participate in regulation of connective tissue deposition by wound fibroblasts. Temporal and spatial expression pattern of ocv36 integrin matches with collagen deposition by wound fibroblasts that are known to crosstalk with the neighboring epithelial cells. Although this thesis deals with ccvf36 integrin, it should be remembered that other integrins are also important during wound healing. Integrins <x634 and a331 mediate keratinocyte adhesion to LM-5 that migrating cells actively deposit during wound healing. LM-5 appears to be always present under the migrating epithelial cells. LM-5 can not be providing its normal function as a constituent of the hemidesmosomes since they are missing. It is possible that proteolytic cleavage of LM-5 by gelatinase A makes it a migratory substrate for keratinocytes while intact LM-5 would then support the formation of stable anchoring contacts, the hemidesmosomes (Giannelli et al., 1997). These new data emphasize how complex the wound healing process is. Not only alterations of integrins and their functions but also 71 processing of the wound bed matrix play important roles in recruitment of epithelial cells into the wound. During wound healing, oc2(31 and oc9B1 integrins could mediate epithelial adhesion to collagen and tenascin, respectively. Mucosal keratinocyte migration seems to occur similarly to that of the epidermal keratinocytes although the source of epithelial cells is different. In the skin, epithelial cells arise not only from the edge of wound but also from hair follicles and sweat glands. In mucosa, the wound edge epithelium serves as a major source of keratinocytes that migrate into the wound bed. How the expression of integrins is modulated during healing of epithelial-hard tissue interphase during formation of long J E is not known. Without doubt cell adhesion mechanisms are instrumental for healing of mucosa and skin. Understanding of these mechanisms would contribute to our understanding of clinical treatment modalities such as guided tissue regeneration. During chronic inflammation of periodontal disease, several changes occur in the extracellular matrix molecules and integrin expression of the structural cells. While the amount of fibronectin and tenascin is largely decreased, several other basement membrane proteins are abnormally expressed in connective tissue stroma. Wide variation in integrin expression follows. In some areas a strong increase takes place in the pocket epithelium while in other sites the expression is depressed. We have only started to understand the regulatory factors of integrin expression. It is clear, however, that both composition of the pericellular matrix and a variety of cytokines are responsible for this regulation. Chronic inflammation of periodontal disease produces a variety of cytokines that potentially regulate integrin expression in epithelial cells. High concentration of interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), granulocyte colony stimulating factor (G-CSF) , and tumor necrosis factor-a (TNFa) have been found in chronic inflammation of oral mucosa (Yamamoto and Osaki, 1995). Although the role of some of these cytokines in the regulation of integrins is known in some cell types, the complex synergistic and antagonistic action of these compounds in integrin expression remains unsolved. Additional complexicity is created by altered extracellular matrix of chronic inflammation which may contribute to the regulation of expression of integrins. It is also possible, that bacterial components of the periodontal pocket directly act on epithelial cells inducing changes in their integrin expression. This new phenotype of epithelial cells could be proinflammatory and be potentially responsible for the maintenance of chronic inflammation by secretion of multitude of cytokines. 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