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KGF and FGF-10 expression in human gingival fibroblast cells by serum, pro-inflammatory cytokines and… Sanaie, Ali Reza 2000

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K G F A N D FGF-10 EXPRESSION IN H U M A N GINGIVAL FIBROBLAST C E L L S B Y S E R U M , P R O - I N F L A M M A T O R Y CYTOKINES A N D DRUGS T H A T A R E ASSOCIATED WITH GINGIVAL O V E R G R O W T H by A L I R E Z A SANAIE B.Sc, The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFLMENT OF REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biological and Medical Sciences, Faculty of Dentistry) We accept this thesis as conforming to the registered standard  T H E UNIVERSITY OF BRITISH C O L U M B I A APRIL 2000  © A l i Reza Sanaie, 2000  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 copying  of this thesis for scholarly  department  or  by  his  or  her  purposes  representatives.  may be granted It  is  by the head of my  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of  Qr^LV  D>Q  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  extensive  copying  or  my written  Abstract  Keratinocyte growth factor (KGF) and fibroblast growth factor-10 (FGF-10) are two highly homologous members of the fibroblast growth factor family that are expressed by fibroblast cells and are know to be paracrine mediators that induce epithelial cell proliferation in psoriasis, Crohn's disease, and ulcerative colitis. K G F expression is highly upregulated during wound healing and chronic inflammation. Periodontitis is a chronic inflammatory disease that has associated with it proliferation of the oral sulcular and junctional epithelium during disease onset. However, the K G F and FGF-10 expression in human gingival fibroblasts has not yet been confirmed by any study. Therefore, we examined the K G F and FGF-10 protein and gene expression using serum, pro-inflammatory cytokines (EL-la, IL-ip, IL-6, TNF-a) and drugs associated with gingival overgrowth (Cyclosporine-A (CSA), Dilantin, and Nifedipine). Northern results showed that treatment of fibroblast cells with 10.0% FBS, IL-la, IL1(3, and CSA increased K G F expression by 1.2, 1.2, 1.13, and 1.2 fold at 3 hours and by 1.6, 1.6, 1.4, and 2 fold at 6 hours, respectively. On the other hand, T N F - a and IL-6 increased the K G F expression by 1.25 and 1.2 at 3 hours but decreased to 1.16 and 1.1 at 6 hours, respectively. However, these treatments did not change FGF-10 mRNA expression levels. In addition to Northern analysis, the sandwich ELISA technique was used to investigate protein expression. Using this technique, we found that K G F protein level in human gingival fibroblasts was maximally increased by 24 hours and decreased over time, when treated with pro-inflammatory cytokines. In addition, treatment of six different gingival fibroblast cell lines (3 isolated from sites of periodontal health and 3 from chronic periodontitis) with proinflammatory cytokines and drugs associated gingival overgrowth, illustrated that pro-  ii  inflammatory cytokines and C S A treatment increased K G F protein expression in all six-cell lines similarly. However, Nifedipine and Dilantin failed to do so.  Lastly, in a combination  study, the presence of one cytokine with C S A induced K G F protein expression but no additive effect was found. The result of this study illustrated that the upregulation of K G F expression, may play an important role during epithelial proliferation in periodontitis and CSA associated gingival overgrowth.  Table of Contents  PAGE Abstract Table of Contents List of Tables List of Figures List of Abbreviations Acknowledgment  ii iv viii ix xi xiii  Chapter 1 LITRATURE REVIEW 1.1  Structure Organization of Periodontium  1  1.1.1 Introduction 1.1.2 Gingiva in Health 1.1.2.1 Epithelium 1.1.2.2 Connective Tissue 1.1.2.2.1 Connective Tissue Cellular Compartment 1.1.2.2.2 Extracellular Matrix 1.1.3 Gingiva in Disease 1.1.3.1 Initial Lesion 1.1.3.2 Early Lesion 1.1.3.3 Established Lesion 1.1.3.4 Advanced Lesion 1.1.4 Conclusion  1 1 1 3 3 4 5 5 6 6 7 7  1.2. Drug-induced Gingival Enlargement 1.2.1 Dilantin 1.2.1.1 Introduction 1.2.1.2 Tissue Changes in Dilantin-induced Gingival Overgrowth Epithelium Connective Tissue 1.2.1.3 Pathogenic Mechanism for Dilantin-induced Gingival Overgrowth 1.2.2 Nifedipine 1.2.2.1 Introduction 1.2.2.2 Tissue Changes in Nifedipine-induced Gingival Overgrowth Epithelium Connective Tissue 1.2.2.3 Pathogenic Mechanism for Nifedipine-induced Gingival Overgrowth 1.2.3 Cyclosporine-A 1.2.3.1 Introduction  9 9 9 10  11 11 11 12  13 13 13  iv  1.2.3.2  Tissue Changes in Cyclosporine-A-induced Gingival Overgrowth Epithelium Connective Tissue 1.2.3.3 Pathogenesis of Cyclosporine-A-induced Gingival Overgrowth 1.2.4 Conclusion 1.3 Cytokines 1.3.1 Pro-inflammatory Cytokines 1.3.1.1 Interleukin-l(IL-l) Receptor Level of Expression in GCF Biological Activities 1.3.1.2 Interleukin-6 (IL-6) Receptor Level of Expression in GCF Biological Activities 1.3.1.3 Tumor Necrosis Factor (TNF-a) Receptor Level of Expression in GCF Biological Activities 1.3.2 Conclusion 1.4 Fibroblast Growth Factors 1.4.1 Fibroblast Growth Factor Receptors (FGFRs) 1.4.2 Keratinocyte Growth Factor (KGF) 1.4.2.1 K G F Receptor (KGFR) 1.4.2.2 K G F Biological Effects K G F in Wound Healing K G F in mflammatory Diseases 1.4.2.3 K G F Regulation by Proinflammatory Cytokines 1.4.3 Fibroblast Growth Factor-10 (FGF-10) 1.4.4 Conclusion  14  15 16 17 17 17  19  20  22 23 25 27 27 29  31 32 35  Chapter 2 2.1  Aim of the Study  36  Chapter 3 M E T H O D S A N D MATERIALS 3.1 3.1.1 3.1.2 3.2  Cell Culture Cell Culture for Northern Experiments Cell Culture for ELISA Experiments Northern Analysis  37 37 37 38 v  3.2.1 3.2.2 3.3 3.3.1 3.3.2 3.4 3.4.1 3.4.2 3.4.3 3.5 3.5.1  R N A Preparation Northern Blotting Plasmid Preparation Cloning of Genomic cDNA Restriction Fragment cDNA Purification Southern Analysis Restriction Endonuclease Digestion of FGF-10 in Southern Southern Blotting Southern Hybridization Enzyme-linked lmmunosorbant Assay (ELISA) Staining and Measuring Cell Density  38 39 39 39 40 41 42 42 43 43 45  Chapter 4  4. 4.1 4.1.1 4.2 4.3 4.4  Results- Part I K G F Protein Determination Using Sandwich ELISA Laboratory optimization on HRP and antibody concentrations in ELISA Serum induction of K G F protein expression Southern analysis of K G F and FGF-10 cDNA probes Serum induction of K G F and FGF-10 mRNA expression  46 46 49 51 53  Chapter 5  5.  Results-Part II  5.1 5.1.1 5.2  Pro-inflammatory cytokine regulation of K G F protein expression Time point study K G F protein expression in gingival fibroblast cells isolated from sites of periodontal health and diseased Pro-inflammatory cytokine regulation of K G F and FGF-10 mRNA expression  5.3  55 57 60 63  Chapter 6  6.  Results-Part III  6.1  Cyclosporine-A, Dilantin, and Nifedipine induction of K G F protein expression  65  6.2  Cyclosporine-A. Dilantin, and Nifedipine regulation of K G F protein expression in gingival fibroblast cells isolated from sites of periodontal health and diseased Cyclosporine-A regulation of K G F and FGF-10 mRNA expression Combination (drug and cytokine) study  68 70 71  6.3 6.4  vi  Chapter 7 DISCUSSION 7.1 7.2 7.3 7.4 7.5  Periodontitis as a Chronic mflammation Serum and Pro-inflammatory Cytokine Induction of K G F Epithelial Proliferation Associated with Gingival Overgrowth FGF-10 Conclusion  74 75 78 80 82  7.6  Future Work  83  References  84  Appendices Appendix A: Appendix Appendix Appendix Appendix  99 A l : HRP and Antibody Optimization A2: Standard Curve A3: Serum Induction of K G F Protein and Relative Cell Numbers A4: Serum Induction of K G F mRNA Expression  Appendix B: Appendix B1: IL-1 a Induction of K G F Protein and Relative Cell Numbers Appendix B2: IL-1 (3 Induction of K G F Protein and Relative Cell Numbers Appendix B3: IL-6 Induction of K G F Protein and Relative Cell Numbers Appendix B4: TNF-oc Induction of K G F Protein and Relative Cell Numbers Appendix B5: Cytokines Induction of K G F Protein over 72 hours and Relative Cell Numbers Appendix B6: Cytokines Induction of K G F Protein in Fibroblasts Isolated From Sites of Healthy and Diseased Gingiva and Relative Cell Numbers Appendix B7: Cytokine Induction of K G F mRNA Expression Appendix C: Appendix C1: CS A Induction of K G F Protein and Relative Cell Numbers Appendix C2: Dilantin Induction of K G F Protein and Relative Cell Numbers Appendix C3: Nifedipine Induction of K G F Protein and Relative Cell Numbers Appendix C4: Drug Induction of K G F Protein in Fibroblasts Isolated From Sites of Healthy and Diseased Gingiva and Relative Cell Numbers Appendix C5: C S A Induction of K G F mRNA Expression Appendix C6: C S A and IL-la/IL-ip Combination Effect on K G F Protein Expression and Relative Cell Numbers  99 99 99 99 100 100 100 100 100 101 102 102 103 103 103 103 104 104 105  vu  List of Tables  Table 1:  Members of the FGF Family  Table 2:  FGF-FGFRs Specific Binding Affinity  List of Figures  PAGE Fig. 1:  Diagram of healthy and periodontal diseased gingiva  8  Fig. 2:  Structure of FGFR protein  26  Fig. 3:  Regulation of K G F by Cytokines  32  Fig. 4:  Relative relationship of FGF family members to each other 33  Fig. 5 A & B:  Optimization of Horse Radish Peroxidase (HRP) and antibody concentrations for sandwich ELISA  47  Standard curve prepared using known K G F Concentrations  48  Fig. 6:  Fig. 7 A & B:  10.0% Fetal Bovine Serum (FBS) induction of K G F expression and cell numbers in H G F  50  Fig. 8 A & B & C:  Southern analysis of K G F and FGF-10 cDNA probes  52  Fig. 9 A & B:  Serum induction of K G F and FGF-10 mRNA expression  54  Fig. 1 0 A & B :  EL-la and IL-lp induction of K G F protein expression and cell number over different concentrations in H G F  56  EL-6 and TNF-a induction of K G F protein expression and cell numbers over different concentrations in H G F  58  Pro-inflammatory cytokines (IL-1 a, IL-1 p, IL-6, TNF-a) induction of K G F protein expression and cell numbers over 72 hours  59  Variation in morphology of fibroblasts isolated from sites of periodontal health and or chronic periodontitis  61  Pro-inflammatory cytokines (IL-la, IL-1 p, IL-6, TNF-a) induction of K G F protein expression and cell numbers in gingival fibroblasts isolated from sites of periodontal health and as well as chronic periodontitis over 24 hours  62  Pro-inflammatory cytokines regulation of K G F and FGF-10 mRNA expression  64  Fig. 11 A & B:  Fig. 12 A & B:  Fig. 13 A & B & C:  Fig. 14 A & B:  Fig. 15 A & B:  Fig. 16 A & B:  Fig. 17:  Fig. 18:  Fig. 19 A & B:  Fig. 20 A & B:  Fig. 21 A & B:  Cyclosporine-A and Dilantin induction of K G F protein expression and cell numbers over different concentrations in H G F  66  Nifedipine induction of K G F protein expression and cell numbers over different concentrations in H G F  67  Cyclosporine-A, Dilantin and Nifedipine induction of K G F protein expression and cell numbers in gingival fibroblasts isolated from sites of periodontal health and as well as chronic periodontitis over 24 hours  69  Cyclosporine-A regulation of K G F and FGF-10 mRNA expression  70  Combination of Cyclosporine-A (CSA) and EL-la and IL-ip induction of K G F protein expression and cell numbers in H G F  72  Combination of Cyclosporine-A (CSA) and T N F - a and IL-6 induction of K G F protein expression and cell numbers in H G F  73  x  List of Abbreviations  aFGF:  acidic Fibroblast Growth Factor  bFGF:  basic Fibroblast Growth Factor  CSA:  Cyclosporine-A  DEPC:  Diethylpyrocarbonate  ECM:  Extracellular Matirx  ELISA:  Enzyme-linked Irnmunosorbant Assay  FB S:  Fetal B ovine S erum  FGF:  Fibroblast Growth Factor  FGFR:  Fibroblast Growth Factor Receptor  GCF:  Gingival Crevicular Fluid  HGF:  Human Gingival Fibroblast  HRP:  Horse Radish Peroxidase  ICE:  Interleukin-1P Converting Enzyme  IL-1:  Interleukin-1  IL-1R:  Interleukin-1 Receptor  IL-1RA:  Interleukin-1 Receptor Antagonist  IL-6:  Interleukin-6  IL-6Ra:  Interleukin-6 Receptor a  KGF:  Keratinocyte Growth Factor  KGFR:  Keratinocyte Growth Factor Receptor  LB:  Luria Broth  O.D.:  Optical Density  PBS:  Phosphate Buffer Saline  PMN:  Polymorphonuclear Neutrophils  TNF-oc:  Tumor Necrosis Factor-oc  TNF-R:  Tumor Necrosis Factor Receptor  TMB:  3,3,5,5-TetraMethylBensidine  Acknowledgement  I would like to thank Dr. E.E. Putnins for his excellent supervision, feedback, and for allowing me to use the facilities in his lab. His patience and words of encouragement made learning easier and more enjoyable during the last two years. I am also grateful to Dr. Douglas Waterfield, Dr. V. J. Uitto, and Dr. Colin Wiebe for providing me with their input, which helped me to bring this thesis to completion. I also would like to thank, Dr. Lari Hakkenin for providing me with different cell lines as well as his advices on improving my technical skills. Furthermore, I am very grateful to Cristian Sperantia, and Jim Firth for their technical supports. Finally, I am very grateful to my parents and Anna, who were there for me when I needed them most. In the moments of doubt, their words of encouragement allowed me to proceed further and without their support, finishing this project would not have been possible. The funding for this study was provided by The British Columbia Health Research Foundation.  xiii  J  Chapter 1 Literature Review 1.1  Structural Organization of Periodontium  1.1.1  Introduction The periodontium, which is composed of alveolar bone, root cementum, periodontal  ligament and gingiva (Hassell, 1993), is responsible for the attachment of teeth. Periodontal diseases affect selectively and collectively each of these tissues. This chapter focuses on periodontal soft tissue (epithelium and connective tissue) structural organization in health and the changes that occur with disease.  1.1.2  Gingiva in Health  1.1.2.1 Epithelium The gingival epithelium is divided into three groups, oral gingival epithelial, oral sulcular epithelial and junctional epithelium. Oral gingival keratinocytes form the epithelial component of the marginal and attached gingiva. This keratinized stratified squamous epithelium consists of four layers: Stratum basale, Stratum spinosum, Stratum granulosum and Stratum corneum (Muller-Glauser et al., 1983; Schroeder, 1981). The Stratum basale is the germinative layer. This layer is mainly composed of cuboidal cells that are attached to the underlying basal lamina via hemidesmosomes. As these cells migrate through epithelial strata, they keratinize (Sten et al., 1983). Cells occupying the stratum spinosum layer no longer synthesize basal lamina components and express increasing levels of cytoplasmic filaments in the cell. As the cells move to the stratum granulosum layer, cells become more flattened and keratinohyaline granules and enzyme containing vesicles appear (Schroeder and Theilade, 1966). As the  1  cells approach the outermost layer, the stratum corneum, they become even more flattened and depending upon the degree of keratinization, they may lose their nucleus. Cell will maintain cell-cell attachment, until they are sloughed (Hassell, 1993; Schroeder and Theilade, 1966; Schroeder et al, 1990; Sten et al, 1983). hi contrast to oral gingival epithelium, oral sulcular epithelium forms the coronal aspect of the gingival sulcus. Oral sulcular epithelium is nonkeratinized making it easier for pathogenic periodontal microorganisms and their virulence factors to penetrate the tissue (Fry and App, 1978; Caffesse et al., 1982; Schroeder and Listgarten, 1977). Oral junctional epithelial is the third component of the gingival epithelium. This tissue has a unique structure that differs in many aspects from the other oral epithelial types. It is roughly 15 to 18 cells thick at the base of the sulcus (Schroeder and Theilade, 1966) and consists of a basal and suprabasal layers (Sawaf et al., 1991). Oral junctional epithelium is attached to the hard surface of the tooth by a basal lamina and hemidesmosomes, which are produced by the specialized band of longitudinally oriented epithelial cells that are adjacent to the tooth surface (Schroeder and Listgarten, 1977). Junctional epithelium is the structure that is most directly challenged by dental plaque associated bacteria. The lack of keratinization in the oral junctional epithelium and the presence of wide intercellular gaps between cells make this tissue susceptible to direct bacterial penetration or penetration of bacterial associated virulence factors. The increased mitotic rate of the epithelium and sloughing provides limited protection and reduces the susceptibility of bacterial invasion. In addition, the presence of leukocytes in the gingival sulcus also provides protection. These leukocytes are predominantly neutrophils (91% polymorphonuclear neutrophils (PMN) and 9%> mononuculear cells) (Wilton et al., 1976).  2  They appear in small numbers in the extravascular site of connective tissue adjacent to the sulcus. Eventually, they travel through the junctional epithelium into the gingival sulcus and become one part of the gingival crevicular fluid (GCF). Further protection is provided by the flushing action of the G C F that flows through intercellular gaps. Other constituents of G C F are organic compounds such as matrix proteins, proteoglycans, carbohydrates and serum-derived factors (cytokines, complement factors, immunoglobulins) (Embery and Waddington, 1994; Klinkhammer, 1968).  1.1.2.2 Connective Tissue Connective tissue, known as the lamina propria, lies beneath the gingival epithelia. It is composed of organized collagen fibers, cells and other extracellular matrix components (Itoiz and Carranza, 1996).  1.1.2.2.1 Connective Tissue Cellular Compartment Fibroblast cells are by far the most common cells in gingival connective tissue. Gingival fibroblasts produce and maintain the extracellular components of gingival connective tissue and periodontal ligament fibroblasts produce and maintain the connective tissue components of the periodontal ligament attachment complex (Phipps et al., 1997). Fibroblasts produce structural proteins, such as collagen and elastin as well as glycoproteins and glycosaminoglycans, which makes up the ground substance of the connective tissue (Hassell, 1993). Fibroblasts also participate in tissue remodelling by producing enzymes such as matrix metalloproteinases (Birkedal-Hansen, 1988). In histological sections, gingival fibroblasts isolated from sites of periodontal health typically appear elongated and their nucleus appears rather large. However, many studies  3  have confirmed the existence of fibroblast subpopulations in the gingival connective tissue (Schor and Schor, 1987). Hakkinen and Larjava, (1992), Roberts and Morey (1985), Cho and Garant (1984) used light microscope nuclear morphometry to examine for the presence of fibroblast subpopulation in periodontal tissue. These data showed that discrete populations of cells have different nuclear sizes. Other studies revealed more significant morphological heterogeneity in actin content (Azuma et al., 1975), amount of endoplasmic reticulum (Beersten and Everts, 1977), phagocytosis of collagen fibers (Ten Cate et al., 1976), and nuclear-cytoplasmic ratio (Gould, 1983) in fibroblast cells at the same site. Additional cells make up the rest of the cellular composition of the connective tissue. These include endothelial, polymorphonuclear leukocytes, plasma and mast cells. In healthy gingival connective tissue, the number of inflammatory cells is relatively small. However, their numbers increase significantly with inflammation (Hassell, 1993). 1.1.2.2.2 Extracellular Matrix Extracellular matrix (ECM) is the intercellular substance of tissue and is mainly composed of collagenous proteins and macromolecules such as proteoglycans. Extracellular matrix regulates cellular functions by mediating signals via cell adhesion, cell-cell attachment, and binding of soluble growth factors. A predominant extracellular matrix component is collagen. Up to 65% of the connective tissue is occupied by organized collagens fibers, which provide the gingiva with a rigid structural framework (Van der Rest and Garroone, 1991; Van der Rest 1991). Most of the collagen fibers are composed of collagen types I and HI. Briefly, collagen type I proteins are organized into denser fibrils within the lamina propria and type HJ collagen proteins  4  appear to be localized in smaller fibers near the basement membrane adjacent to the epithelial junctions. Other collagen fibers, such as collagen type n , V , VI, XI, IX and XII (Huang et al., 1991; Wang et al., 1980) are also found in E C M . However, they account for a very small percentage of the total collagen fibers network (Karimbux et al., 1992). Proteoglycans are a large and diverse group of macromolecules that are widely distributed in mammalian tissues. These proteins consist of one or more sulfated polysaccharides that are covalently linked to a protein core. The role of proteoglycans in extracellular matrix includes everything from maintenance of the matrix to cell-cell interactions (Rahemtulla 1992). For example, versican is a chondroitin sulfate proteoglycan that occupies a large portion of space in the interstitial spaces and thought to influence cell migration and cell attachment (Mariotti, 1993). There are many other proteoglycan molecules, which are outside of the scope of this thesis and will not be discussed here.  1.1.3 Gingival Changes with the Onset of Disease Dental plaque is the major factor, which induces gingival inflammation. In addition to induction of gingival inflammation, dental plaque may induce changes in both the epithelial and connective tissue compartments of the periodontium. Based on clinical observation and measurements, the progression of gingivitis to periodontitis has been classically divided into four stages: initial lesion, early lesion, established lesion, and advanced lesion (Page and Schroeder, 1976).  1.1.3.1 Initial Lesion The initial lesion may be a response to the generation of chemotactic and antigenic substances in the region of the gingival sulcus. Briefly, in response to accumulation of plaque, an acute vasculitis subjacent to the inflamed area junctional epithelium develops. A  5  portion of the perivascular collagen disappears and the resultant space is filled with fluid, serum proteins and inflammatory cells. There may or may not be a slight alteration of the most coronal portion of the junctional epithelium (Schroeder and Page, 1972; Schroeder and Page, 1990; Kornman et al, 1997). 1.1.3.2 Early Lesion The accumulation of lymphoid cells develops subjacent to the inflamed areas gingival sulcus, junctional epithelium, underlying connective tissue. Cytopathic alterations in resident fibroblasts are demonstrated by a vacuolated cytoplasm and paucity of nuclear chromatin. In addition, epithelial cell proliferation into lamina propria is initiated and collagen content is reduced. It is thought that the unusual high number of lymphoid cells and the presence of increased cytokines and growth factor concentrations at the inflamed site, could be responsible for alteration to cell populations and cell behaviour (Schroeder and Page, 1972; Schroeder and Page, 1990; Kornman et al., 1997). 1.1.3.4 Established Lesion This stage is distinguished from the early lesion by high numbers of plasma cells in the inflamed area. Just like the early lesion stage, the established lesion is confined to the bottom of the epithelial sulcus and small portion of the underlying connective tissue. However, proliferation of the junctional and sulcular epithelial cells into the infiltrated connective tissue is more apparent. As the result of disruption of the attachment apparatus (hemidesmosomes), sulcular pockets are deepened (Fig. 1) and the intercellular gaps in the junctional epithelium are widened (Schroeder and Page, 1990). The loss of connective tissue, especially collagen, is noted. No bone loss occurs in the established lesion. This  6  stage of gingival disease is widespread among humans and it may not progress to an advanced lesion. 1.1.3.3 Advanced Lesion This stage is characterized predominantly by the deepened periodontal pocket, fibrosis of gingiva, alveolar bone loss and eventually an increase in tooth mobility. Collagen is broken down in gingival connective tissue and is highly reduced in the areas adjacent to the periodontal pockets. At the root surface, the acute inflammatory processes will result in complete breakdown of the collagen fibers and bone. As the destruction of the periodontal attachment continues at the root, cementum becomes exposed to the oral cavity (Fig. 1) (Kornman et al., 1997; Schroeder and Page, 1972). 1.1.4  Conclusion The periodontal soft tissue is composed of epithelium and gingival connective tissue,  where fibroblasts maintain E C M organization. Each of these periodontal components has a distinct biochemical and architectural structure, yet they function as one integrated unit. With onset of inflammation significant changes occur in the connective tissue and epithelium. The gingival connective tissue in health has few inflammatory cells but with accumulation of plaque, neutrophils, lymphocytes and plasma cells accumulate significantly in gingival connective tissue, sulcular and junctional epithelium.  7  Fig. 1: Diagram of healthy and periodontal diseased gingiva (Scanned with permission from Dr. E. E. Putnins)  8  1.2  Drug-induced Gingival Enlargement The gingiva of periodontium may be enlarged in response to various interactions  between the host and the environment. Most commonly enlargement of the gingiva is due to plaque induced inflammation. However, usages of certain drugs are associated with disproportionate and disfiguring overgrowth of gingival tissue. The anticonvulsant drug (Dilantin), calcium channel blocker (Nifedipine) and immunosuppressant drug (Cyclosporine-A) all are associated with induction of gingival overgrowth. Many studies have suggested that the drug alone is insufficient to cause gingival overgrowth and the presence of other factors such as dental plaque and inflammation are required (Hassell and Hafti, 1991). However, other studies have shown that the drugs themselves can induce gingival enlargement in some people (Hassell and Hafti, 1991; Hassell 1982). In this section, we will review the characteristics of some of the drugs that are associated with gingival overgrowth.  1.2.1  Dilantin  1.2.1.1 Introduction Meritt and Putnam in 1938 introduced clinical usage of Dilantin or phenytoin (5diphenylphenytoin) for controlling seizure disorders at a concentration of 15-25 mg/kg (serum concentration 2-25pg/ml) in patients with epilepsy (Ratanakorn et al., 1997). Due to the effectiveness and availability, phenytoin remains the first drug of choice for physicians in controlling convulsive seizure disorders. However, use of phenytoin is also associated with gingival enlargement. Nuki and Cooper (1972) suggested that both gingival inflammation and local irritants are prerequisites for gingival enlargement. In the absence of these requirements, no gingival enlargement was observed.  9  1.2.1.2 Tissue Changes in Dilantin-induced Gingival Overgrowth Epithelium The stratified squamous epithelium that covers the Dilantin induced gingival overgrowth is thick and has a thin keratinized layer. Long rete pegs, often described as acanthotic, are extended deep into the lamina propria (Butler et al., 1987; Dongari et al., 1993). Tissue culture methods have been used to investigate mechanisms involving Dilantin induced gingival overgrowth. Vittek and coworkers (1983) found that there is a positive correlation between increased formation and binding of 5-a dihydrotestosterone and the degree of gingival enlargement in patients on chronic Dilantin therapy. They suggested that the effect of this drug might be mediated via an alteration of hormonal activity (androgen) in oral epithelium (Vittek et al., 1983).  Connective Tissue In histological sections of patients with Dilantin induced gingival overgrowth the lamina propria is characterized by proliferation of fibroblasts and increase in collagen. Noncollagenous proteins such as glycosaminoglycans are also increased in the lamina propria (Butler et al., 1987; Dongari et al., 1993). Similar observations were made in vitro using cell culture techniques. Tissue culture studies have shown that phenytoin has a direct stimulatory effect on fibroblast cell's proliferation and protein synthesis, especially collagen. Shafer (1960) reported that in vitro treatment with phenytoin (5 ug/ml) increased human gingival fibroblast cells proliferation (2 fold) and collagen synthesis. Hassell and Hafti (1991), examined protein production in fibroblast cells isolated from overgrown gingival tissue using 5ng/ml of Phenytoin. They  10  reported that most of the proteins produced and released into the culture media were collagen and collagenases. However, the collagenase was mainly inactive.  1.2.1.3 Pathogenic Mechanisms for Dilantin-induced Gingival Overgrowth It is postulated that Dilantin could increase the synthesis of hydrophilic noncollagenous protein in the matrix, which would induce water to bind to these sites and cause swelling of the gingival tissue (Dahllof et al., 1984). Also, Dilantin is known to increase the production of platelet-derived growth factor by macrophages, which affects the connective tissue repair and growth (Dill et al., 1993). Even though Dilantin is not a calcium channel blocker, it could alter the transportation of calcium into fibroblasts, which could affect and alter the function of collagenous degrading enzymes (Romanos et al., 1992). Another study suggested that phenytoin could affect mast cells in tissue. As a result, mast cells will degranulate releasing heparin, histamine and hyaluronic acid in the surrounding tissue. Consequently, these substances could be taken up by fibroblasts as metabolites resulting in an increase in collagen production and accumulation (Angelopoulos, 1975).  1.2.2  Nifedipine  1.2.2.1 Introduction Nifedipine is a calcium channel blocker that prevents the calcium-dependent ATPase from degrading ATP (Butler et al, 1987) and thereby decreases the oxygen requirement at the cellular level. In the case of patients with cardiovascular conditions, Nifedipine regulates the smooth muscles to function at a lower energy level. It is routinely prescribed to patients with chronic stable angina and ventricular arrhythmias. The dosage for Nifedipine may vary from 40-80 mg/kg (serum concentration 18-1121 ng/ml) (Ellis et al., 1993). However, Nifedipine use may result in gingival overgrowth as an undesirable side effect. Although  11  many studies have investigated the role of this drug in gingival overgrowth, very little is known about the biological mechanism. Interestingly, not all the patients treated with Nifedipine develop gingival overgrowth. It is reported that only 42.5% of patients that undergo Nifedipine treatment may develop gingival overgrowth (Hallmon and Rossmann, 1999). This observation has been made also in animal models (Heijl and Sundin, 1987, Ellis et al. 1993). Two independent in vitro studies reported that Nifedipine concentrations of 7x10" to 2 x l O " M (14ng/ml to 70ng/ml) 8  7  had no effect on collagenous protein production or proliferation of the human gingival fibroblast cells (Nishikawa et al., 1991; Mckevitt and Irwin, 1995). This is suggestive that a "responder" and "non responder" fibroblast population may exist within gingiva (Henderson et al., 1997). 1.2.2.2 Tissue Changes in Nifedipine-induced Gingival Overgrowth Epithelium Many histological studies investigated changes in epithelial tissue sections of patient with Nifedipine gingival overgrowth (Van der Wall and Tuinzing 1985; Henderson et al., 1997). Those studies described the epithelial sections to exhibit parakeratosis, proliferation and elongation of long rete pegs into the underlying lamina propria. Van der Wall and coworkers (1985) also reported an increased epithelial width that accompanied the inflammation, edema, and presence lymphocytes and plasma cells. Connective Tissue In addition to the histological changes that are found in the epithelium, connective tissue changes have also been described. Fibroblast proliferation and fibrosis of the lamina  12  propria are apparent. Also, a significant increase in collagen is observed. In addition, the capillary vascularization is also increased (Barak et al., 1987; Van der Wall et al., 1985). -  1.2.2.3 Pathogenic Mechanisms for Nifedipine-induced Gingival Overgrowth It is speculated that calcium channel blockers modulate the intracellular calcium concentrations and directly or indirectly alter the calcium-dependent mechanisms of fibroblasts resulting in alteration of the collagen homeostasis (Hallmon and Rossmann, 1999). That is, by decreasing the calcium transportation into fibroblast, the activity of the collagenous enzymes decrease and less collagen is degraded. , There is, however, no information available on how Nifedipine regulates epithelial changes.  1.2.3 Cyclosporine-A 1.2.3.1 Introduction Cyclosporine-A was first isolated in 1970 as a metabolite of the fungus specie Tolypocladium inflatum. This drug is a powerful immunosuppressant, which is used increasingly for its antirejection action in organ transplant procedures. A therapeutic level of 5-20 mg/kg of body weight per day (serum concentration of 100-400 ng/ml) is usually administered (Hassell and Hefti, 1991). It is thought that the immunosuppressive activity of Cyclosporine appears to be exerted selectively on the CD4+ (helper) T-lymphocytes, inhibiting synthesis of cytokines, such as IL-2. This cytokine in particular is a key molecule in the development of an immune response (Kanitakis and Thivolet, 1990). Cyclosporine-A is an immunosuppresant with significant clinical applications, but it is associated with gingival overgrowth. Gingival overgrowth involving Cyclosporine-A was first reported by Rateitschak-Pluss and coworkers in 1983. They studied 50 kidney transplants patients, most of whom developed gingival enlargement within 4-6 weeks after  13  onset of Cyclosporine-A use. Wysocki et al. (1983) and Tyldesley and Rotter (1984) published similar findings. Several studies demonstrated children and young females have a greater risk of developing Cyclosporine-A induced gingival overgrowth (Seymour and Heasman, 1988). This may be caused by interaction between Cyclosporine-A and sex hormones (Seymour and Jacobs, 1992). On the other hand, some studies reported that gingival overgrowth does not occur in all patients consuming Cyclosporine-A (Wysocki et al. 1983; Rateitschak-Pluss et al., 1983). There is evidence that gingival overgrowth, as an adverse effect of Cyclosporine-A, could be dosage related (Wysocki et al. 1983; Rateitschak-Pluss et al., 1983). It is also suggested that this overgrowth maybe be due the sensitivity of individual patients to the drug (Wysocki et al. 1983). However, Hafti et al. (1994) and Seymour and Jacobs (1992) reported that the overgrowth was more likely to occur if the plasma concentration level of Cyclosporine-A exceeds 400 ng/ml.  1.2.3.2 Tissue Changes in Cyclosporine-A induced Gingival Overgrowth Epithelium Many studies of histological changes in epithelium caused by C S A have similar findings (Seymour et al., 1996; Pisanty et al., 1988; Tyldesley et al., 1984). The overlying stratified squamous epithelium is parakeratotic and has varying thickness. Epithelial ridges penetrate deep into the connective tissue, creating irregularly arranged collagen fiber bundles. In addition, swelling of epithelial cells and widening of intercellular gaps were also observed (Pisanty et a l , 1988). This led to the impression that Cyclosporine-A gingival enlargement is a result of accumulation of non-collagenous material and a thickening of the epithelial layer (Pisanty et al., 1988).  Connective Tissue The connective tissue is highly vascularized and an accumulation of inflammatory cells has been reported. Predominant infiltrating cell types are the plasma cell and lymphocytes (Rateitschak-Pluss et al., 1983). Most histological studies failed to report any increase in the density of fibroblast cells. However, Mariani et al. in 1993 reported an increased proportion of myofibroblast cells in immunohistochemical studies on Cyclosporine-A induced gingival overgrowth. These fibroblast cells were described as satellite or trapezoidal form, had a prominent nucleus with well dispersed chromatin and a cytoplasm with well-developed rough endoplasmic reticulum (Yamasaki et al., 1987). 1.2.3.3 Pathogenic Mechanisms for Cyclosporine-A -induced Gingival Overgrowth The homeostasis of connective tissue may serve as a target for drug induced gingival overgrowth. Recently, in vitro studies have shown that growth factors, such as plateletderived growth factor B, could be one the targets for Cyclosporine-A induced gingival overgrowth. This particular growth factor is a dimeric polypeptide that acts as a mitogen for fibroblast cells and synthesis of glycosaminoglycans, fibronectin, and collagen (Hallmon and Rossman, 1999). Plemons et al. (1996) and Nares et al. (1996) demonstrated that plateletderived growth factor B mRNA is significantly increased relative to normal controls. Iacopino et al. (1997) confirmed these results. However, many other studies have failed to elucidate the pathogenesis of Cyclosporine-A induced gingival overgrowth because it is multifactorial and only fewer that 40% of patients taking this drug develop gingival overgrowth.  15  1.2.4  Conclusion Gingival enlargement is an unwanted side effect associated with usage of three drugs:  Dilantin, Nifedipine and Cyclosporine-A. This enlargement is partly due to an increase in tissue amount and changes in connective tissue as well as epithelium. Many studies indicated (Pisanty et a l , 1988; Hassell and Hafti, 1991; Hallmon and Rossmann, 1999) that tissue enlargement is also partly due to accumulation of inflammatory cells during inflammation, which subsequently may lead to an increase in cytokine expression and more severe inflammation in that area.  16  1.3  Cytokines The complex interactions between inflammatory cells and other elements of  connective tissue are mediated by a series of low molecular weight proteins that are collectively called cytokines. Some cytokines exhibit autocrine function, (bind to the cell that produced them), where as others function in a paracrine manner (bind to distant cells and altering their behaviour or properties). Many cells in addition to those of the immune system release cytokines. Cytokines that are mainly produced by T cells are called "Interleukins" (IL) and currently 18 of them are known (Gracie et al., 1999). Many of the ELs play a role in inflammation although other cytokines, such as TNF-a, can also play an important role during inflammation (Masada et al., 1993; Rossmando et al., 1990).  1.3.1  Pro-inflammatory Cytokines Accumulation of plasma cells and lymphocytes in the periodontal tissues suggests  that cytokines participate in pathological changes to the periodontium. EL-la, 11-1(3, TNF-a and EL-6 are four of the major pro-inflammatory cytokines that have been detected in gingival crevicular fluid at high concentrations during gingival inflammation (Masada et al., 1993; Rossmando et al., 1990). This chapter will briefly review these pro-inflammatory cytokines that play a significant role in the onset and progression of periodontal diseases.  1.3.1.1 Interleukin-1 (IL-1) Among all the pro-inflammatory cytokines, EL-1 has been studied in greatest detail. So far, two members of the IL-1 family have been identified, EL-la and EL-1(3 (Dinarello, 1993). The primary translation products of EL-la and IL-ip are 271 and 269 amino acid long and correspond to molecular masses of 30.6 kDa and 30.7 kDa, respectively. EL-la is biologically active when produced but EL-1 (3 is not. In order for EL-113 to have biological 17  activity it must be activated by a cysteine protease called IL-ip converting enzyme (ICE) (Dinarello, 1994). Receptor E L - l a and EL-lp are structurally related and bind with similar affinity to the same receptor, IL-1 receptor (IL-1R). IL-1R is comprised of a 319 amino acid extracellular portion and a 21 amino acid hydrophobic transmembrane segment. When glycosylated, this protein weighs about 80 kDa (Schindler and Dinarello, 1990). IL-1 a and IL-lp are agonists and when they bind to the receptor and can elicit a broad range of cellular responses. There is also an IL-1 receptor antagonist (IL-1RA), which blocks the biological responses of the two agonists by competing for the IL-1R binding site (Dinarello, 1994). Level of Expression in G C F IL-1 a and IL-ip are usually produced in response to microbial invasion, inflammation and tissue injury. These proinflammatory cytokines are predominantly produced and released by polymorphonuclear leukocytes, but other cells such as fibroblasts and macrophages also produce and release IL-1. The concentration of IL-1 a and EL-l p in gingival crevicular fluid was measured and the result indicated that there is a correlation between the increase of IL-1 a and IL-ip concentration and the presence or absence of periodontal disease (Mathur et al., 1996; Mogi et al., 1999). Using ELISA, the concentration range of I L - l a and IL-ip in healthy periodontium is 9-74 pg/ml and 30-3,071 pg/ml, respectively (Stashenko et al, 1991; Mathur et al., 1996). However, these concentration were much higher in GCF taken from patients with periodontitis. I L - l a was increased to 50342 pg/ml and IL-lp to 108-11,695 pg/ml (Stashenko et al., 1991; Mathur et al., 1996).  18  Biological Activities Although, there are two different forms of EL-1, IL-1 a and EL-ip, their functional effects are similar (Dinarello, 1993). In vitro studies have shown that EL-1 can directly induce the production of metalloproteinases, plasminogen activators, EL-6, and prostaglandin E in gingival fibroblasts (Richards and Rutherford, 1988; Mocha et al., 1988; Bartold and 2  Haynes, 1991). In addition EL-1, especially IL-1 a, has been shown to have strong stimulation effect on bone resorption by activating osteoclasts and down-regulating bone formation (Tatakis, 1993, Alexander et al., 1994). These data suggests that local production of EL-1 by PMNs and fibroblasts could be important in connective tissue destruction, loss of attachment and progression in periodontal disease (Schindler and Dinarello, 1990; Alexander et a l , 1994, Okada and Murakami, 1998). 1.3.1.2 Interleukin-6 (IL-6) EL-6 is another pleiotropic cytokine influencing immune responses and inflammatory reactions.  Human EL-6 is a single-chain 24 kDa protein with small posttranslational  variations at the N-terminus. The protein consists of 211 amino acids with a hydrophobic signaling sequence of 24 residues (Mire-Slui and Thorpe 1998; Jablons et al., 1989). Receptor The EL-6 receptor is composed of two different glycoproteins. The first component, EL-6Ra, is a transmembrane glycoprotein (80 kDa), which has a very high affinity for EL-6 as a ligand. Other transmembrane proteins called "pgl30" will then surround the EL-6/EL-6Ra complex and initiate signal transduction (Mire-Slui and Thorpe, 1998).  19  Levels of Expression in G C F IL-6 is constitutively produced by lymphoid cells, especially T-cells (Alexander et al., 1994). This pro-inflammatory cytokine, like others, is produced in healthy periodontium but at a very low concentration (5-17 pg/ml). The IL-6 level is significantly increased in the diseased periodontium (56 pg/ml) (Mogi et al, 1999). This increase may be due to higher expression in T-cells, but fibroblasts and endothelial cells may also contribute to the increase level in inflamed tissues by increasing their own IL-6 production (Jablons et al., 1989; MireSluiand Thorpe, 1998) Biological Activities IL-6 was originally identified as a B-cell differentiation factor, and it may play an important role in antibody induction and B-cells proliferation (Fujihashi et al., 1993, Matsuki et al., 1992). Furthermore, IL-6 may also play an important role in bone resorption by stimulating osteoclasts (Mire-Slui and Thorpe, 1998). These findings imply that IL-6 in addition to E L - l a and IL-ip, plays an important role in periodontal tissue destruction. 1.3.1.3 Tumor Necrosis Factor-q (TNF-q) Human TNF-a is composed of 233 amino acids, which is modified during posttranslational processes. These posttranslational modifications (such as acylation) activate TNF-a.  The transmembrane bound TNF-a is a 26 kDa protein, whereas the  secretory TNF-a is modified to a 17 kDa protein (monomer) and is composed of 157 amino acids (Smith and Baglioni, 1987; Jones et al., 1989). After secretion usually 3 of these monomers form a bell shaped trimer as this is necessary for receptor activation.  20  Receptor T N F - a affects cells via two related membrane receptors termed TNF-R1 and TNFR2. The molecular masses of the human receptor proteins are 60 and 80 kDa, respectively. Signaling transduction occurs when two or three receptors cluster around a single T N F - a trimer resulting in activation of secondary signaling cascades (tyrosine kinases and prostaglandins) (Adam et al., 1995). The remarkable absence of homology between the intracellular regions of both TNF receptors indicates that they are involved in different signal-transduction pathway. Most of the known TNF effects, such as nuclear factor K B (NF KB) activation, EL-6 induction, and fibroblast proliferation occur by activating the smaller TNF receptor. However, T-cell activation by TNF-a appears to be mediated through the larger receptor (Mire-Sluis and Thorpe, 1998).  Levels of Expression in GCF As with EL-1, TNF-a expression level is increased in inflamed periodontal tissue (Stashenko et al., 1991) and present in the GCF of patients with gingivitis and periodontitis (Rossomando et al., 1990). Using ELISA, TNF-a concentrations in gingival crevicular fluid from healthy and diseased tissue were determined. TNF-a concentration in healthy tissue was 26 pg/ml, whereas in diseased tissue it increased to 434 pg/ml (Stashenko et al., 1991).  Biological Activities TNF-a completely distinct in structure from EL-1 but has enormous functional overlaps with it (Alexander and Damoulis, 1994). TNF-a can induce the secretion of collagenase, prostaglandin E2, and IL-6 in human fibroblasts and bone culture cells (Elias et al., 1987; Meikle et al., 1989). It has also been shown that TNF-a can induce bone resorption (Bertolini et al., 1986). These finding support the hypothesis that T N F - a could play and 21  important role in tissue destruction during periodontal diseases. This hypothesis is further supported by an in situ hybridization study that showed an increase in T N F - a mRNA expression in macrophages and T cells of gingival tissue of patients with moderate to severe periodontitis (Matsuki et al., 1992). However, with all the supporting evidence, the exact involvement of TNF-a in the pathogenesis of periodontal disease is not yet clear.  1.3.2  Conclusion Several pro-inflammatory cytokines possess bioactivities that may cause or contribute  to destruction of bone and connective tissue in periodontitis. These pro-inflammatory cytokines include IL-la, II-ip, IL-6 and TNF-a.  They are produced continuously, but in  periodontitis their concentration and expression levels are significantly increased in the GCF. In addition to the destructive potential of these pro-inflammatory cytokines, they may also stimulate active tissue repair by increasing growth factor expression levels.  22  1.4  Fibroblast Growth Factor Family Fibroblast growth factors (FGFs) are members of a family of polypeptides that are  potent regulators of cell proliferation and differentiation. FGFs are also described as members of a larger growth factor family called the heparin binding growth factors (Baird and Klagsbrun, 1991). The FGF family is currently composed of 19 members (Table 1) which demonstrate a high level of structural homology (30-60% based on the amino acid sequence) which is suggestive of a common ancestral gene. Some of the members are products of the proto-oncogenes such as int-2 (FGF-3), hst (FGF-4), FGF-5 and FGF-6 (Werner et a l , 1998). Collectively, these growth factors play an important role in tissue development and maintenance, wound healing, cell proliferation, and migration in a variety of tissues (Yamaguchi and Rossant, 1995; Galzie et al., 1997; Marchese et al., 1995; Farell et a l , 1998). The regulation of secretion of FGFs likely depends on the N-terminal signaling sequence, for example, FGF-3 to 8, -10, -15, -17, -18, and -19 have consensus sequences and are targeted for secretion. However, FGF-1, -2, -9, -11, -12, -13, -14 and -16 do not have the consensus secretory signaling sequences (Kiefer et al., 1993).  23  FGF Members  Common Name  Signal Sequence  References  FGF-1  acidic FGF, aFGF  -  FGF-2  basic FGF, bFGF  -  Gimenez-Gallego et a l , 1986 Abraham et a l , 1986  FGF-3  int-2  FGF-4  Kaposi FGF, K-FGF; hst-1  + +  FGF-5  -  FGF-6  hst-2  FGF-7  Keratinocyte Growth Factor, K G F  FGF-8 FGF-9 FGF-10  Moore etal, 1987 Delli-Bovi et al., 1987  + + +  Maries et al., 1989  Androgen Induced Growth Factor, AIGF  +  Tanakaetal, 1992  Glial Activate Factor, GAF (KGF-2)  -  Miyamoto et a l , 1993  +  Yamasaki et al., 1996  Zhanetal, 1988 Rubin etal, 1989  FGF-11  FHF-1 (FGF Homologous Factor)  -  Munoz-Sajuan et al., 1999  FGF-12  FHF-2  Smallwood et al., 1997  FGF-13  FHF-3  FGF-14  FHF-4  -  FGF-15  -  +  Mcwhiteret a l , 1997  -  Miyake et al., 1998  + +  Hoshikawa, etal., 1998  FGF-16 FGF-17 FGF-18 FGF-19  -  Hartung et al., 1997 Yamamoto et al., 1998  Ohbayashi et al., 1998 Nishimura et al., 1999  Table 1: Members of the FGF Family.  24  1.4.1  Fibroblasts Growth Factor Receptors (FGFRs) The fibroblast growth factor receptor (FGFR) family consists of at least four members  (FGFR1, FGFR2, FGFR3, and FGFR4). Alternative splicing of four FGFR genes in vertebrates produces structural variations within each of the FGFRs and a variety of ligandreceptor binding specificities (Johnson and Williams, 1993). Although structural variations among these specific receptors exist, FGFRs consist of a basic structure of an extracellular region with two or three immunoglobulins-like domains, a transmembrane region, and a cytosolic tyrosine kinase domain which is activated on ligand binding (Fig. 2). Generally, because of the cytoplasmic tyrosine kinase activity, they fall under the broad category that includes all tyrosine kinase receptors. However, the "acidic box", located between first and second immunoglobulin-like domain (Fig. 2), in FGFRs confirms uniqueness for this receptor group (Johnson and Williams, 1993). Regulation of FGFRs and their unique pattern of their expression are far from being understood. However, reports indicate that these receptors are regulated at the transcription, translation and post-translation levels (Asakai et al., 1995; Liuzzo and Moscatelli, 1996).  25  I  exon # (promoter)  NH2 signal peptide ig-1  acidic box CAM-binding domain heparin-binding region 5 and 6  tg-2 ligand binding domain  8 (B)  or  fTTl  ^alternatively spliced (confers ligand specificity) transmembrane region — — juxtamembrane region l|  9 (C)  10  11-14 kinase insert  kinase regions  14-18 19 COOH  Alternative splicing of the carboxylic half of the Ig 3 loop yields the B or C splicing variations in FGFR1-3 (Szebenyi and Fallon, 1999). Fig  2:  Structure of F G F R protein.  26  The remainder of this chapter will focus only on two specific FGF family members, namely FGF-7 (Keratinocyte Growth Factor-1, KGF) and FGF-10 (KGF-2).  1.4.2  Keratinocyte Growth Factor (KGF) Keratinocyte Growth Factor (FGF-7) was first discovered as an epithelial mitogen,  secreted by lung fibroblasts, stromal cells and some lymphoid cells (Rubin et al., 1989; Rubin et a l , 1995; Finch and Cheng, 1999). K G F cDNA encodes for a 194 amino acid protein, which includes a classical signal peptide for secretion. K G F protein was initially purified as a monomeric polypeptide with a molecular weight of 26-28 kDa. However, when this protein is expressed in bacteria, a protein with a molecular weight of 21 kDa is obtained. This molecular weight difference is due to post-translational modifications (such as N glycosylation) occurring in mammalian cells (Ron et al., 1993a). K G F has nucleotide homology to acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), FGF-3, FGF-4, and FGF-5 of 37%, 39%, 44%, 33% and 41%, respectively (Finch et al., 1989). In particular, FGF-10 shares with K G F a nucleotide homology of 60% (Emoto et al., 1997). K G F and FGF-10 are known as specific inducers of epithelial cell proliferation. This specificity is due to the expression of a KGF-specific receptor on epithelial cells (Werner et al., 1998).  1.4.2.1 KGF Receptor (KGFR) K G F elicits intracellular signaling through binding and activation of K G F receptor (KGFR). K G F R (FGFR2-IIIb) is a splice transcript variant of FGFR2 and binds K G F , FGF10 and aFGF. In 1992, Miki et al. discovered that the K G F R transcript was specific and only expressed on epithelial cells, whereas other FGFR2 transcripts were not as specific. The specificity of the K G F R was due to 49 amino acids that are in the carboxyl-terminal half of  the third Ig loop (Fig. 2) (Yayon et al., 1992). These 49 amino acids confirm ligand-binding specificity for this receptor, i.e. aFGF and bFGF bind equally well to FGFR2, however only K G F , FGF-10 and aFGF bind with a high affinity to K G F R (Table 2) (Yayon et a l , 1992). The specific expression of the K G F receptor on epithelial cells and the fact that stromal cells secrete K G F suggests that K G F is a paracrine stromal cell mediator for epithelial cells (Finch etal., 1989).  FGF1  FGF -2  FGF3  FGF4  FGF5  FGF6  FGF -7  FGF8  B Variant C Variant FGFR 2  +++  ++  +  +  -  -  -  -  +++  +++  ++  -  B Variant C Variant FGFR 3  +++  ~  ++  +  +++  ++  -  +++  B Variant C Variant FGFR 4  +++  -  ~  -  +++  +++  -  +++  -  C only  +++  +++  -  +++  -  FGF9  *FGF -10  FGFR 1  +++  ++ +  +++ +  -  ++  +++  -  N/D  +++ +  ++  N/D  ++  N/D  ++  +++  N/D  +++  +++  N/D  Table 2: FGF-FGFRs Specific Binding Affinity. Szebenyi and Fallen, 1999 * Added portion  28  1.4.2.2 K G F Biological Effects Many in vivo and in vitro studies have been published on the effects of K G F on the epithelial cells. In an in vivo study, systematic administration of K G F to rats (intravenous or subcutaneous) induced epithelial hyperplasia in mammary glands of both male and female rats (Ulich et al., 1994). Furthermore, systematic K G F administration induced epithelial hyperplasia in pancreatic ducts as well (Yi et al., 1994). R N A analysis revealed, K G F and K G F R were also expressed throughout the gastrointestinal tract of adult rats (Housley et al., 1994). A single K G F treatment in this study caused epithelial proliferation from foregut to the colon and liver. These studies provided evidence that K G F is a potent mediator of epithelial proliferation. Guo et al. (1993) studied the role of K G F in maintaining tissue homeostasis by changing K G F expression from a paracrine system to an autocrine epithelial mediator in transgenic mice. They used a human keratin promoter to actively express K G F in keratinocytes of transgenic mice. K G F over expression resulted in hyperproliferative changes in epidermal thickness and tongue epithelium. Suppression of hair follicle formation, increased salivation and smaller immature salivary glands were also observed. On the other hand, Werner et al, (1994) studied what effect blocking of the K G F receptor would have on epithelial homeostasis. This task was performed by expressing a truncated version of FGFR2blTI. Upon ligand binding, the truncated version of receptor blocked signal transduction. As a result, the skin of these transgenic mice exhibited epidermal atrophy and abnormal hair follicles. Most importantly, reepithelialization after injury was impaired.  29  KGF in Wound Healing K G F expression is upregulated during wound healing (Werner et al., 1992; Brauchel et al., 1995, Marchese er al., 1995). K G F mRNA is induced by 9 fold after 12 hours, 160 fold within 24 hours, and remained upregulated by 100 fold over the basal level by 7 day th  post-injury (Werner et al., 1992). In the same study, using in situ hybridization, they showed K G F mRNA was expressed at high levels in cells below the wound and at the wound edge, and the K G F R was expressed in the epidermis. In contrast to K G F induction in wound healing, K G F R transcription levels did not change. Topical application of K G F to wound enhanced the wound healing process (Sotozono et al., 1995, Staiano-Coico et al., 1993). Staiano-Coico et al. (1993) observed that the epidermis of the K G F treated wound was significantly thicker and exhibited acanthosis. In another study, application of lOpg/ml of human K G F accelerated corneal epithelial wound healing (Sotozono et al., 1995). The number of epithelial cells in the S-phase increased 2 fold, which suggested K G F increased proliferation of epithelial stem cells in vivo (Sotozono etai., 1995).  KGF in Inflammatory Diseases Many studies have reported that K G F is significantly upregulated at the site of inflammation (Bajaj-Elliot et al., 1997; Brauchle et al., 1996). K G F expression is significantly increased at mRNA and protein levels in specimens collected from Crohn's disease and ulcerative colitis patients (Brauchle et al., 1996). The level of K G F expression in these specimens correlated strongly to the level of inflammation in that area. This correlation was assessed histologically by analyzing the proinflammatory cytokines expression such IL-ip in the inflamed area. Therefore, K G F appears to play an important  30  role in epithelial proliferation and the presence of pro-inflammatory cytokines at the inflamed site could be one of the regulatory mechanisms for K G F expression (Farrell et al., 1996). 1.4.2.3  K G F Regulation by Proinflammatory Cytokines The regulation of K G F expression by other growth factors and pro-inflammatory  cytokines in non-oral cavity tissue has been investigated. Earlier, Brauchle et al. (1994) examined the K G F induction of human skin fibroblasts by pro-inflammatory cytokines (IL1(3, IL-6, TNF-a). They found that these pro-inflammatory cytokines induced K G F mRNA. Other in vitro studies also reported that stimulation of human skin fibroblasts with IL-6, platelet-derived growth factor-BB (PDGF-BB) or tumor necrosis factor-a (TGF-a) induced K G F mRNA expression as well. However, TGF-P and basic fibroblast growth factor (bFGF) do not induce K G F expression (Chedid et al., 1994; Weng et al., 1997; Tang and Gilchrest, 1995).  Pro-inflammatory cytokines are produced by monocytes/macrophages and PMNs that are present at sites of inflammation. In addition, local fibroblast and epithelial cells also secrete pro-inflammatory cytokines such as IL-1 (Okada and Murakami, 1999). Production of cytokines by inflammatory cells and by the local cells (fibroblast and epithelial cells) may upregulate K G F expression in the fibroblasts connective tissue fibroblasts (Fig. 3).  31  PMNs  <s»  Fibroblast  Epithelial Cells Fig. 3. Regulation of K G F by cytokines.  1.4.3  Fibroblast Growth Factor-10 ( F G F - 1 0 )  Human FGF-10 is another recently cloned FGF family member. As the evolutionary tree of human FGFs indicates (Fig. 4) FGF-10 is most closely related to K G F (Emoto et al., 1997). This similarity explains the alternative designation for FGF-10, namely KGF-2 (Miceli et al., 1999).  32  • h~FGF12 • • • • •  h-FOyi4 b-FQrl3 h-FGF15 h-FGF11 h-pari 6  • h-FGF10 h-FOT7  h-FGFl h-FOF2 h-FOF4 h-FOF6 h-FOF5 h-FGF18 h-FGF8 h-FGari7  h-FGF19  Fig. 4: Relative relationship of FGF family members to each other (Xie et al, 1999)  FGF-10 shares 60% homology (based on amino acid sequence) with K G F (Emoto et al, 1997; Hattori et al., 1997; Miceli et al., 1999). FGF-10 cDNA encodes for 208 amino acids from which approximately 40 amino acids serve as a signaling sequence. When this protein is expressed in E. coli, a major protein with the mass of 19.3 kDa is isolated (Emoto et a., 1997). This molecular mass may differ when this protein is isolated from mammalian cells because of glycosylation. FGF-10, just like K G F is described as a paracrine mediator for epithelial cell proliferation since FGF-10 mRNA is detected in the dermis and not in the epidermis of mouse-tail. Also, cultured fibroblasts and not keratinocytes express FGF-10 (Beer et al., 1997). In addition, human FGF-10 has the capability to bind with high affinity to K G F R  33  (FGFR-2bffl) (Jimenez and Rampy, 1999). Unlike K G F , FGF-10 is also capable of binding to FGFR-lbUI and FGFR-4 with the high affinity (Jimenez and Rampy, 1999).  Mouse FGF-10 protein shares approximately 92% homology to human FGF-10 protein, (Beer et al., 1997). Unlike K G F , mouse FGF-10 protein appears in the cell lysates or matrix and not in the tissue culture media (Beer et al., 1997). Addition of heparin to culture media inhibits K G F receptor binding (Bottaro et al., 1993). However, in the case of FGF-10, addition of heparin to the culture media allows two different forms of N-glycosylated FGF10 (22 kDa and 30 kDa) to be released (Beer et al., 1997). This suggests FGF-10 is either cell or matrix associated.  FGF-10 may play an important role in wound healing (Jimenez and Rampy, 1999; Tagashira et al., 1997). Tagarashi and his coworkers reported that mouse FGF-10 mRNA expression was upregulated on the first day of injury and decreased rapidly by day three. th  Topical application of FGF-10 to healing wound improved wound tensile strength by the 5 day after injury. Also, epidermal thickness and wound collagen content were significantly increased with FGF-10 topical application (Jimenez and Rampy, 1999). These observations suggest FGF-10 just like K G F may accelerate or enhance the wound healing process (Jimenez and Rampy, 1999). The regulation of FGF-10 by cytokines has not been as extensively investigated as K G F but the regulation of FGF-10 by growth factors and cytokines is known to be different from KGF. In cultured fibroblast cells, expression of FGF-10 was repressed by TGF-p, TNF-a and PDGF-BB treatment and IL-1 and E G F treatment did not induce FGF-10 expression (Beer et al., 1997). 34  1.4.4  Conclusion Fibroblast growth factors affect a wide variety of tissues. K G F and FGF-10 are two  FGF members that specifically induce epithelial cellular responses. This is due to the presence of the KGFR, which is a specific splice variant of FGFR-2b. Induction of K G F has been found in wound healing and chronic inflammations. Presence of cytokines at the site of wound healing and inflammation may upregulate K G F expression. In contrast to K G F , FGF10 is less understood. The expression and regulations of K G F and FGF-10 in periodontitis, another form of chronic inflammation, has not yet been investigated.  35  Chapter 2 2.1  A i m of the Study Periodontitis is a complex and multifactorial disease that afflicts a large number of  adults. One aspect associated with disease onset and progression is proliferation of oral sulcular and junctional epithelium. We suspect that secretion of two epithelial specific growth factors that are known to induce epithelial proliferation, Fibroblast Growth Factor 7 (FGF-7/Keratinocyte growth Factor, KGF) and Fibroblast Growth Factor 10 (FGF-10), may play a major role in this aspect of disease. The aims of this study are to examine the induction and regulation of protein and gene expression of these two growth factors in vitro. 1.  The literature to date has not yet confirmed K G F and FGF-10 expression by gingival  fibroblasts. We will first examine if serum stimulated gingival fibroblasts express K G F and FGF-10 mRNA and protein. 2.  Periodontal disease is associated with increased expression of pro-inflammatory  cytokines (EL-la, IL-1 (3, TNF-a, EL-6). Therefore, we will examine the regulation and expression of K G F and FGF-10 in gingival fibroblasts by these pro-inflammatory cytokines. 3.  Cyclosporine-A, Nifedipine and Dilantin are three drugs whose use by patients is  associated with gingival overgrowth. One of the histological findings of overgrowth tissue is gingival epithelial proliferation. We will examine if these drugs regulate K G F and FGF-10 protein and gene expression in gingival fibroblast cells.  36  Chapter 3 Methods and Materials 3.1  Cell Culture  3.1.1  Cell Culture for Northern Experiments Human gingival fibroblasts (HGF) isolated from healthy gingiva (subculture 5-15)  were plated at 5000-7000 cells per cm in 100 mm Falcon culture dishes (Falcon cat. 2  #351005) for Northern experiments. The HGF were cultured for 7-9 days in a M E M with 10.0% fetal bovine serum (FBS) with 5.0% C 0 (37°C). When the plates reached 75% 2  confluency, the cells were quiesced over-night with a M E M + 1.0% FBS. Subsequently, cultures were treated for 3 or 6 hours with either a M E M + 1.0% serum (negative control), a M E M + 10.0% FBS (positive control), 30 ng/ml I L - l a + 1.0% FBS (Endogen cat. # RIL1A-2), 30 ng/ml IL-lp + 1.0% FBS (Endogen cat. # R-IL1B-5), 5 ng/ml IL-6 + 1.0% FBS (Endogen cat. # R-IL6-10), 30 ng/ml TNF-a + 1.0% FBS (Endogen cat. # R-TNFA-10), 250 ng/ml Cyclosporine-A + 1.0% FBS (Sigma cat. # 3662).  3.1.2  Cell Culture for ELISA Experiments HGF isolated from healthy gingiva (subculture 7-13) and HGF isolated from sites of  chronic periodontitis (subculture 4-8) were used. The myofibroblast-like cells were obtained from patients with chronic periodontitis (Fig. 14) (Hakkinen and Larjava, 1992). Cells were seeded into 96 well plates (Falcon cat. # 353072) at 10,000 cells/well [in selected experiments, due to differences in proliferation rate between fibroblasts isolated from healthy and diseased sites, experiment started one day after plating]. Consequently, the HGF were cultured for 2-4 days in a M E M with 10.0% FBS. When the plates reached 75% confluency, the cells were washed with phosphate buffer saline (PBS, pH 7.4) and quiesced overnight in 37  a M E M + 1.0% FBS. Subsequently, depending upon the experimental design they were treated for either 24, 48, or 72 hours with a M E M + 1.0% serum (negative control), a M E M + 10.0% FBS (positive control), EL-la/ EL-lpV TNF-a + 1.0% FBS (5, 10, 20, 30, 50 ng/ml), EL-6 + 1.0% FBS (5,10, 30, 50ng/ml), Cyclosporine-A (10, 100, 150, 250, 500 ng/ml) + 1.0% FBS, Nifedipine (0.1, 0.5, 1.0, 2.5, 5.0 ng/ml) (ICN cat. # 151743), Dilantin (0.1, 0.5, 1.0, 2.5, 5 ng/ml) (Sigma cat. # 4505). 3.2  Northern Analysis  3.2.1  R N A Preparation After incubation periods, the cells are lysed with 1.2 ml of TRIzol Reagent (Gibco  cat. # 15596). Trizol reagent is a monophasic solution of phenol and guanidine isothiocyanate, which was used for single step R N A isolation (Chomczynski and Sacchi, 1987) at room temperature. The HGF were lysed directly in the culture plates and scraped with a sterile plastic scraper into RNase free polypropylene tubes. 300pl of chloroform was added. Samples were vigorously hand-shaken (15 seconds) and incubated at room temperature (2-3 minutes). Subsequently, samples were centrifuged (13,000 g for 25 minutes at 4°C). RNA-containing aqueous phase on the top was collected (approximately 650 pi) into RNase-free eppendorf tube. The R N A in the aqueous solution was precipitated with 400 pi of isopropanol, stored overnight at -20 °C and R N A was pelleted (13,000 g for 20 minutes at 4°C). The R N A pellet was washed with 200 pi of 80% ethanol, air-dried for 10 minutes and dissolved in 20 ul of RNase-free water (RNase free water was prepared by adding 0.5 ml of diethylpyrocabonate [DEPC]; Sigma cat. # D-5758; to 1.0 liter of water, left overnight at room temperature and autoclaved). The total R N A yield and purity were determined for each sample by spectrophotometeric analysis. A 0.7 pi aliquot of the R N A sample was added to  38  70 ul of RNase-free water and the optical density (O.D.) was measured. The R N A yield per sample on average was 35-40 ug and the purity (measured as the ratio of A260/A280) was consistently greater than 1.55. 3.2.2  Northern Blotting Purified R N A (15 pg) was prepared for loading on the gel by adding 9 pi of loading  cocktail (5 M formaldehyde and ethidium bromide 40 pg/ml) to each sample. The tubes were heated (65°C for 15 minutes) chilled on ice for at least 5 minutes. R N A was then fractionated on 1.2% agrose gels, containing 2.2 M formaldehyde and 20 m M 3-Nmorpholinolpropanesulfonic acid (MOPS, pH 7.4), at 50 volts for 1 hour and 10 minutes (Lehrach et al., 1977). The gel was washed twice with lOx SSC for 20 minutes. R N A in the gel was transferred for 3 hours onto a Hybond-N nylon membrane (0.45 pm pores, Amersham) using a Posiblot Pressure Blotter (Stratagene, CA) and the R N A was crosslinked to the membrane with U V light (2 minutes and 45 seconds). Subsequently, these membranes were hybridized with P labeled K G F , FGF-10 or G A P D H probes (BioChain 3 2  Institute Inc. cat. #01112031). 3.3  Plasmid Preparation  3.3.1  Cloning of Genomic cDNA Restriction Fragments Epicurin Coli XLl-Blue M R F Kan Super Competent Cells (Stratagene, cat. #  200248) were transfected by recombinant plasmid (pGEM-T) carrying a 0.730 kb insert of human FGF-10 cDNA (Dr. Nobuyuki Itoh from Kyoto University in Japan) or by recombinant plasmid (pT3/T7 118U, Pharmacia) carrying a 0.324 kb insert of human K G F cDNA (Dr. S. Aaronson in National Institution of Health, (NTH)). The Epicurin Coli X L l Blue M R F Kan Super Competent Cells were thawed on wet ice. To permeabilize the cells a  39  100 ul aliquot of the thawed cells were transferred to a 1.5 ml autoclaved micro centrifuge tube containing 1.7 ul of (3-mercaptoethenal. The tube was incubated on ice for 10 minutes and gently shaken every 2 minutes. Following this incubation, 1-3 pi (5-10ng) of plasmid D N A was added to the cells, gently mixed and incubated on ice for 30 minutes. The cells were heat-shocked for 5 seconds at 42 °C in a water bath and immediately transferred to ice for 2 minutes. After the heat-shocking process, 0.9 ml of Luria Broth (LB) was added to the tube and incubated inside a shaker (225 rpm) in a 37 °C water bath for 1 hour. Subsequently, using a-complementation technique and "X-gal" containing agar plates the transfected clones (containing the cDNA fragment of interest) were identified. Following visual screening, the transfected colonies (white colonies) were chosen and cultured in LB media. 100 pg/ml of ampicillin was added to prevent unwanted bacterial growth. 3.3.2  cDNA Purification After 24 hours incubation (37 °C), the glass culture tubes with media were cooled on  ice and the recombinant bacteria were pelleted with centrifugation (13,000 g for 30 seconds at 4 °C). The pellet was washed with lx STE, gently mixed and repelleted with centrifugation (30 seconds at 13,000 g at 4 °C). To begin cell lysis, the cells were washed with 200 pi of lx Solution I (containing, 0.5 M glucose, 0.1 M E D T A , 0.25 M Tris) on ice for 5 minutes and 300 pi of lx Solution H (10% 2 M NaOH and 1% SDS) at room temperature for 5 minutes. During this step bacterial D N A (attached to the bacterial wall) as well as the plasmids in bacteria were denatured. To re-nature plasmids, 300 pi of lx Solution HI (containing, 60% 5M K O A c and 11.5% of HAC) was added to the tube and incubated (5 minutes at 4 °C). The test tube was spun for 10 minutes at 13,000 g at 4 °C. 40  The clear supernatant with plasmids was removed into a fresh test tube. Phenol: Chloroform extraction technique was used to purify the plasmid from any organic residuals (RNA, low molecular weight proteins, and broken pieces of bacterial DNA). 600 pi of Phenol: Chloroform mixture (ratio of 1:1) was added to the supernatant, mixed and vortexed. After 30 minutes of centrifugation (13,000 g), the top aqueous layer was removed into a fresh tube. The aqueous solution was washed with a mixture containing 49 parts Chloroform and 1 part Isoamyl alcohol to remove residual phenol. The solution was centrifuged at 13,000 g at 4 °C for 5 minutes and the top aqueous layer was removed into a fresh tube. While in Chloroform, 20 pg/ml of ribonuclease A was added to the tube to digest RNA. The solution was vortexed, left on ice for 5 minutes, and 500 pi of isopropyl alcohol was added to solution to precipitate the plasmids at -20 °C overnight. Next day, the tube was centrifuged (4 °C for 30 minutes at 13,000 g) and the pellet was washed 2x with 70% alcohol and air dried for 10 minutes. The purified plasmid pellet was resuspended in 60 pi of T E buffer (pH 8.0). The cDNA fragments were purified from the plasmids using, restriction enzyme endonucleases digestion. Plasmids containing the K G F insert were cut with BamHI (Pharmacia cat. # 27-086803) and EcoRI (Pharmacia cat. # 27-085403) and plasmids containing the FGF-10 insert were cleaved with PstI (Pharmacia cat. # 27-088603) and Sph (Pharmacia cat. # 27-095102). The inserts werefractionatedon 1.2% agarose gel. The insert bands were cut out of the gel and purified using the Sephaglas BandPrep Kit from Pharmacia (cat. # 27-9885-01). 3.4  Southern Analysis Nucleotide analysis (Lesergene) indicated that FGF-10 has an overlapping region of  125 bp with K G F . In this overlapping region, the nucleotide homology is 40% (Fig. 8 A). 41  Therefore Southern analysis was used to determine if the probes would cross-hybridize at the selected stringency during Northern hybridization. 3.4.1  Restriction Endonuclease Digestion of FGF-10 in Southern 40 ng of the FGF-10 insert was added to a 1.5 ml micro centrifuge tube. To it, the  following were added: 1 pi of HaeHI (Pharmacia cat. # 27-0866-01), 2 pi of restriction endonuclease buffer and 7 pi of distilled H 0 to bring up the total volume to 20 pi. The 2  micro centrifuge tube was incubated at 37 °C for 5 hours. Digestion cut the FGF-10 cDNA at the 200 nucleotide producing two different fragments, a 200bp and a 530bp fragment. th  The 200bp fragment contained region of 40% homology to the K G F probe (Fig. 8 A). 3.4.2  Southern Blotting After the FGF-10 cDNA fragment was digested, the FGF-10 and K G F cDNA were  separated on a 1.2% wide-range agarose gel containing 400 pg/ml of ethidium bromide (Fig. 8 B). A 0.1 kb reference ladder (Gibco cat. 15628-019) was run on the gel, simultaneously. Each lane contained 10 ng of DNA. The gel was run at 50 volts for 1 hour and 20 minutes. Following fragmentation, the gel was depurinated with 0.25N HC1 for 15 minutes. To denature the D N A in the gel for better transfer, the gel was washed once with 0.5N NaOH and 1.5M NaCl for 15 minutes. The gel was neutralized for 30 minute in a solution (1.0M Tris pH7.5 and 1.5MNaCl). A PosiBlot pressure blotter was used to transfer the D N A onto a nylon membrane (Amercham, Hybond-N Membrane). The D N A was transferred to the nylon membrane for 2 hours. Subsequently, the D N A was cross-linked to the membrane by exposing the membrane for 2 minutes and 45 seconds to U V light.  42  3.4.3  Southern Hybridization A random primed D N A labeling kit (Boehringer Mannheim cat. # 1004760) was used  to label probes. 12 ng of purified K G F or FGF-10 cDNA fragment was added to a microcentrifuge tube and was heat denatured for 10 minutes at 96 °C in a thermocycle apparatus and quenched on ice for 5 minutes. One pi of each: dATP, dGTP, dTTP; 2 pi of reaction mixture; 5 pi of the [a P]dGTP and the appropriate volume of distilled H 0 (total volume to 32  2  19 pi) was added. Finally, 1 pi of Klenow enzyme was added and incubated in a microcentrifuge tube (37 °C for 2 hours). The nylon membranes were placed into the hybridization bags and soaked in the pre-hybridization solution (5% SDS, 50mM PIPES, 0.1 M NaCl, 5mM Na2HP04, ImM EDTA). The membranes in pre-hybridization solution were incubated for 1 hour and 30 minutes at hybridization temperature (67 °C). Subsequently, the labeled probe mixture were passed through a Sephadex column-G50 (Pharmacia Biotech cat.# 17-0855-01) to purify [a P]dGTP labeled probes. The purified probe was heat32  denatured (97 °C for 10 minutes), quenched on ice for 5 minutes and added to the hybridization bag. The membranes were hybridized over-night at 67 °C.  On the next day,  the membranes while in the hybridization bag were washed 3x with the washing solution (0.15 M of NaCl, 0.015 M Na Citrate - 2 H 0 , and 5.0% SDS) at room and hybridization 3  2  temperatures. The membranes were wrapped in All-purpose Laboratory Wrap (Fisherbrand) (Fisher Scientific cat.# 01810) and exposed to x-ray films (Kodak, B I O M A X cat. # 8701302). 3.5  Enzyme-linked Immunosorbant Assay (ELISA) The ELISA wells (96 well plates) were coated with 1.0 pg/ml of the capturing  antibody (monoclonal anti-human FGF-7 antibody; R & D cat. # MAB251) for 24 hours. The 43  wells were washed and blocked with a solution (2% Bovine Serum Albumin (BSA), 5% sucrose and 0.05% NaN3) for 1 hour. The wells were washed 3x for 3-5 minutes with washing solution (PBS, pH 7.4, and 0.05% Tween 20) at room temperature, before adding conditioned media and known K G F concentrations to create a standard curve. Conditioned media were centrifuged (13,000 g for 5 minutes) to eliminate transferring cellular debris and 200 pi of the conditioned media was added to anti-KGF monoclonal antibody coated ELISA wells and incubated at room temperature for 2 hours. In each ELISA run a standard curve was prepared. This standard curve was produced by preparing varying K G F concentrations (Upstate Biotechnology cat. # 01-118): 1, 50, 125, 250, 500, 750 pg/ml. After 2 hours, the conditioned media was discarded and ELISA wells were washed. Following this washing, 100 pi (200ng/ml; biotinylated anti-human FGF-7 antibody; R & D cat. # BAF251) of the detecting antibody was added and incubated for another 2 hours at room temperature. At the end of the incubation period, the wells were washed, lOOpl of Horse Radish Per-oxidase (HRP) Streptavidin conjugate (Zymed, Cat. # 43-8323) was added to each well (1:4000). After 20 minutes of incubation time, the wells were washed 3x with the washing solution and 100 pi of the substrate solution called 3,3,5,5-TetraMethylBensidine (TMB), ready to use, (Zymed, cat. # 00-2023) was added to each well and incubated for 15-40 minutes. The reaction was stopped (100 pi of 1 M HC1) and the 96 well ELISA plate were read at 450 nm in an optical density reader (Titertek Mutliskan).  44  3.5.1  Staining and Measuring Cell Density After collecting the conditioned media from 96 wells, the cells were washed with 100  pi of PBS and air dried for approximately 10 minutes. The cells are fixed (4% formaldehyde and 5.0% sucrose), for 25-30 minutes in the wells and washed with distilled H 2 O . The fixed cells were stained with a crystal violet stain (0.1 % crystal violet and 0.1 m M of Boric Acid) for 10-15 minutes and then the stain was removed from the wells by washing once with distilled water and then dried. Before reading the plates, the wells were washed once again with distilled water for 1 or 2 minutes, dried and dissolved in 10.0%> acetic acid (for 10 minutes) and the color was read (570 nm). In this system, the optical density (O.D.) correlated to the relative cell numbers in each well, (Kueng, et al., 1989; Makela et al., 1999).  45  Chapter 4 4  Results-Part I Refer to appendix A for graphed data.  4.1  K G F Protein Determination Using Sandwich E L I S A A sandwich ELISA technique was used to quantify the level of K G F protein  expression in conditioned media obtained from serum-stimulated gingival fibroblasts. However, certain parameters in this technique required optimization, including the dilution of Horse Radish Peroxidase (HRP) and antibody concentration determination. 4.1.1  Laboratory optimization on H R P and antibody concentrations in E L I S A Three different HRP dilutions (1:2000; 1:4000; 1:10,000) and two K G F  concentrations (10 pg/ml; 750 pg/ml) were selected in the preliminary ELISA optimization study (Fig. 5 A). As suggested by the manufacture ( R & D System), a capturing antibody concentration of 1.0 pg/ml and the secondary detecting antibody concentration of 200 ng/ml was used. At low K G F concentration (10 pg/ml) the O.D. increased in a HRP dose dependent manner, whereas with the higher K G F concentration (750 pg/ml) the absorption increased from 1:10,000 to 1:4,000. No further increase in O.D. was found with increased HRP (1:2,000). Therefore, a 1:4,000 HRP dilution was subsequently, used. After establishing the ideal HRP concentration, the concentration of capturing and detecting antibody were determined. Two concentrations of capturing antibody (0.5 pg/ml and 1.0 pg/ml) and three concentrations of detecting antibody (100 ng/ml, 150 ng/ml and 200 ng/ml) were selected in this experiment (Fig. 5 B). The K G F concentration was 750 pg/ml. The highest O.D. was obtained with 1.0 pg/ml of capturing and 200 ng/ml of detecting antibody. 46  — C h 0.25  H  0.2  H  o  o-  10 pg/ml K G F  T  750 pg/ml K G F  T 1  m 0.15-.T  h-OH  •8"  -  •  0.05 H  -  1:10000  i 1:2000  1:4000 11KI" Concentration  0.4  Tr,  I I  Detecting Antibody 1 OOng/ml  fill  Detecting Antibody 150ng/ml  H  Detecting Antibody 200ng/ml  T  T  T  0.2  1  1  • .o.. .0.-1'  0.5 ug/nil  1.0 ug/ml  Capturing Antihoil\ Coiicenlrutioii  Fig. 5 A & B. Optimization of Horse Radish Peroxidase (HRP) and antibody concentrations for Sandwich ELISA. ELISA wells were coated with appropriate concentration of capturing antibody and known concentration of KGF was added. Subsequently, appropriate concentration of detecting antibody was added. A HRP dilution was then added and followed with TMB. The color reaction was stopped with 1 N HC1 and O.D. was read at 450 nm. A. Three different HRP dilutions with two different KGF concentrations were used. 1:4000 HRP dilution produced the highest O.D. B . The optimal concentration for capturing and detecting antibody for highest O.D. was investigated. The results indicate that with highest antibodies concentrations, the highest O.D. could be achieved. *p<0.05 significant within and across the groups (Mean ± S.D.); n=4 47  We next determined if a linear standard curve could be produced using the previously established experimental parameters. Six different concentrations (1 pg/ml, 50 pg/ml, 125 pg/ml, 250 pg/ml, 500 pg/ml and 750 pg/ml) of K G F were used as ligand to produce this curve (Fig. 6). The absorption data were then graphed and the formula for the linear line was calculated (program used). This formula was used to calculate pg/ml of K G F in the conditioned media samples.  0-  200  400  600  SOO  KGF (pg/ml)  Fig. 6. Standard curve prepared using known K G F concentrations. ELISA wells were coated with 1.0 ug/ml of capturing antibody and known concentration of KGF was added. Subsequently, 0.2 ug/ml of detecting antibody was added. A 1:4,000 HRP dilution was then added and followed with TMB. The color reaction was stopped with 1 N HCL. The data was plotted and a curve was fit to the data points. (Mean ± S.D.); n=4  48  4.2  Serum induction of K G F protein expression Using our optimized sandwich ELISA technique, serum induction on K G F protein  expression was subsequently examined (Fig. 7 A). Serum treatment (10.0% FBS) of human gingival fibroblast cells increased K G F protein expression by approximately 4.0 fold over the negative control (1.0% FBS). However, 10.0% FBS treatment also induced a significant increase in cell numbers (approximately by 1.25 fold) (Fig. 7 B). Therefore, the increase of K G F concentration was partially due to an increase in the cell number in the culture plates. When the expressed K G F protein was corrected for the cell number increase, the K G F protein expression was 3.2 fold higher compared to the control.  49  6 0 0  * T  1 400''  —  o a-  2 0 0  H T  ± 1.0% FBS  10.0% FBS  Fig. 7 A & B. 10.0% Fetal Bovine Serum (FBS) induction of K G F expression and cell numbers in HGF. HGF were cultured in 96 well plates and maintained until 75% confluent; treated with media containing either 1.0% FBS or 10.0% FBS. Averaging data of six different experiments produced this figure. A. Sandwich ELISA was used to quantify the concentration of KGF in the conditioned media (24 hours). B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% acetic acid. Changes in relative cell numbers were measured by reading the O.D. (570 nm). * p< 0.05 over 1.0% FBS Control (Mean ± Standard Error of six different experiments); n=4  50  4.3  Southern analysis of KGF and FGF-10 cDNA probes Since, K G F and FGF-10 have a nucleotide homology of about 60%, we examined if  our two cDNA probes had regions of overlap using "Lesergene" (Fig. 8 A). A line drawing of K G F and FGF-10 cDNA probes was produced to outline the degree and the region of K G F and FGF-10 cDNA probe nucleotide homology (Fig. 8 A).  A region  of 125 nucleotides shared 40%> homology. However, 22 nucleotides within this region have a homology of 86.4%. Thus, a Southern analysis was performed to examine if cross hybridization between these two cDNA probes would occur at our selected stringency condition. In order to examine for any cross hybridization, a Hae HI restriction enzyme digest was performed on the FGF-10 cDNA. A 200 bpfragmentseparated the region with homology from the rest of the cDNA probe (Fig. 8 B). Southern hybridization using P labeled FGF-10 cDNA 32  cDNA  (Fig. 8 C-right) was done.  (Fig. 8 C-left) and  KGF  No cross hybridization was found between either probe.  In particular no P - K G F cDNA hybridization was found to the 200bp cDNA FGF-10 32  fragment (lane 6). Thisfragmentcontained the region of possible cross hybridization due to high nucleotide homology. Similarly, no cross-hybridization of the P-FGF-10 probe to the 32  K G F cDNA was found. Even when the film was left for extended period of time no cross hybridization was found (Fig. 8 C).  51  A,  22bp w i t h 8 6 , 4 % homology  K G F Probe FGF-10 Probe Haelll restriction site at 200th nucleotide Total overlap 125bp with 40% homology KGF 4  15.  FGF-10 5  6  7 3 0 bp 5 3 0 bp 3 2 4 bp 2 0 0 bp  c.  Fig. 8 A , B & C . Southern analysis of K G F and FGF-10 cDNA probes. KGF and FGF-10  cDNA probes share regions of homology but no cross-hybridization was found with Southern blotting. A. The line drawing, outlining the region and the degree of homology between cDNA probes (Lasergene). Haelll digest of the FGF-10 cDNA produces a 200 base pair fragment containing this region of homology. B. Ethidium bromide gel, lane 1, 4 KGF (324bp), lane 2, 5 uncut FGF-10 (730bp), lane 3, 6 Haelll cut FGF-10 (530pb and 200bp). C. Southern hybridization of filter was made. Filter from (B) was hybridized with PFGF-10 cDNA on the left and with P-KGF cDNA probe on the right. No cross hybridization between probes was found. In particular, no cross hybridization was found in 200bp FGF-10 cDNA fragment even though this contained the region of homology. 52 32  32  4.4  Serum induction on K G F and FGF-10 mRNA expression Treatment of gingival fibroblast cells with 10.0% serum induced K G F mRNA  expression at 3 and 6 hours (Fig. 9 A). However, there was no significant increase in K G F mRNA expression when treated with 1.0% FBS for 3 and 6 hours. The increase in K G F mRNA expression was 1.2 fold at 3 hours and 1.6 fold at 6 hours (Fig. 9 B). Serum treatment (10.0% FBS) did not induce FGF-10 mRNA expression (Fig. 9 A). The expression of FGF-10 mRNA was weak and required significantly longer exposure time (9 days), whereas it took only 4-5 days for K G F and only 5 hours for G A P D H .  53  KGF a4kw FGF-lO(iJkb) 3 hrs  6 hrs  c o n t r o l *#  io.o% F B S  mmm  3hrs 6 hrs  m  GAPDH(ukb) 3 hrs 6hrs  wMwm  H W  wmwm  Fig. 9 A & B. Serum induction of K G F and FGF-10 mRNA expression. Gingival fibroblast cells isolated from sites of periodontal health were maintained in aMEM containing 10.0% FBS until 75% confluent. Subsequently, cells were serum starved over night and treated with 1.0% and 10.0% FBS for 3 and 6 hours; then, RNA was extracted and purified. 15ug of RNA was separated on agarose gel and blotted to nylon membrane. The membrane was hybridized with KGF, FGF-10, and GAPDH random labeled P-cDNA probes. A. Serum induced a significant increase in KGF mRNA (2.4 kb) at 3 hours and 6 hours but there was no change in FGF-10 mRNA expression. Expression of GAPDH was unchanged in the control and treatment groups. B. KGF expression was corrected to GAPDH levels and fold increase in KGF mRNA expression over time was graphed. 10.0 % FBS increased KGF mRNA expression by 1.2 fold at 3 hours and 1.6 fold at 6 hours, respectively; whereas there was no change in KGF mRNA expression with 1.0% FBS over 6 hours. 32  54  Chapter 5 5  Results-Part II Refer to appendix B for graphed data.  5.1  Pro-inflammatory cytokine regulation of KGF protein expression Pro-inflammatory cytokine (IL-la, IL-ip, IL-6 and TNF-a) regulation of K G F  protein was examined using sandwich ELISA. IL-la (5 ng/ml, 10 ng/ml, 20 ng/ml, 30 ng/ml and 50 ng/ml) increased K G F protein expression significantly in a dose dependent manner (Fig. 10 A). However, the K G F induction curve started to plateau at 30 ng/ml. At this concentration, IL-la increased the K G F protein induction by 2.3 fold. Therefore, this concentration was selected as the "effective concentration" for the remaining experiments. IL-la did not significantly affect cell numbers (Fig. 10 A). In the case of IL-1 (3, four out of five different concentrations (10 ng/ml, 20 ng/ml, 30 ng/ml and 50 ng/ml) significantly increased the K G F protein expression in a concentration dependent manner (Fig. 10 B). At a concentration of 30 ng/ml of IL-ip, the curve plateaued. Since there was no significant difference in K G F concentration in samples treated with 30 ng/ml and 50 ng/ml of EL-ip, we selected 30 ng/ml as the effective concentration for the remaining experiments. At this concentration the K G F protein expression was 2.5 fold higher then the negative control (1.0 % FBS). No significant difference in cell numbers was found for any of the IL-ip concentrations (Fig. 10 B).  55  Fig. 10 A& B. IL-lct and IL-ip induction of K G F protein expression and cell numbers over different concentrations in HGF. HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with 1.0% FBS + IL-loc and IL-ip (0 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 30 ng/ml, 50 ng/ml). A . Sandwich ELISA was used to quantify KGF protein concentration in the condition media (24 hours). B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. IL-1 a and IL-1 f3 significantly increased KGF protein expression but did not affect cell numbers. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4 56  IL-6 increased K G F protein expression in a concentration dependent manner (Fig. 11 A) . However, the K G F induction curve plateaued at a much lower concentration (5 ng/ml). Therefore, this concentration (5 ng/ml) was chosen as the effective concentration for the future experiments. 5 ng/ml of IL-6 induced K G F protein expression by 2 fold over the negative control. There was no significant increase in cell numbers other than at concentrations of 10 and 50 ng/ml. Lastly, TNF-a, significantly increased K G F protein expression at 5-50 ng/ml (Fig. 11 B) in a concentration dependent manner except with no significant difference between the K G F protein expression at 30 ng/ml and 50 ng/ml (Fig. 11 B). Therefore, 30 ng/ml of TNFa was selected at the effective concentration for the future experiments. The K G F protein induction at 30 ng/ml was 2.7 fold higher than the negative control. A n increase in cell numbers at two concentrations (20 ng/ml and 50 ng/ml) was found but this was not observed at other concentrations. 5.1.1  Time point study The pro-inflammatory cytokine induction of K G F protein expression was studied  over 24-72 hours (Fig. 12 A & B). Treatment of human gingival fibroblast cells with the effective concentration of cytokines (IL-la 30 ng/ml, IL-lp 30 ng/ml, IL-6 5 ng/ml and TNFa 30 ng/ml) showed that K G F expression was maximally increased by 24 hours and then protein levels generally decreased over time. On the other hand, 10.0% FBS treatment maintained K G F protein expression level over 72 hours. As was previously shown, there was no significant change in the relative cell number using the effective cytokines concentrations but there was with 10.0% FBS (Fig. 12 B).  57  Fig. 11 A & B. IL-6 and TNF-a induction of K G F protein expression and cell numbers over different concentrations in HGF. HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with 1.0% FBS + IL-6 and TNF-a. A . Sandwich ELISA was used to quantify the concentration of KGF in the condition media (collected from wells after 24 hours treatment period). B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. IL-6 and TNF-a increased KGF protein expression, significantly. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4  58  Fig. 12 A & B. Pro-inflammatory cytokines (IL-la, IL-lp\ IL-6 and TNF-a) induction of K G F protein expression and cell numbers over 72 hours. HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with the effective concentration of pro-inflammatory cytokines (IL-la 30 ng/ml, IL-1 P 30 ng/ml, IL-6 5 ng/ml, and TNF-a 30 ng/ml). A. Sandwich ELISA was used to quantify the expressed KGF in the condition media collected at 24, 48, 72 hours after the treatment. B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. KGF protein expression maximally detected in conditioned media at 24 hours and then decreased over the next 2 days. KGF protein levels in the 10.0% FBS was detected maximally at 24 hours and was stable over the next 2 days. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4 59  5.2  K G F protein expression in gingival fibroblast cells isolated from sites of periodontal health and disease The pro-inflammatory cytokine regulation of K G F protein expression was also  studied in different fibroblast cell line populations (Fig. 14 A & B). Six gingival fibroblast cell lines (three isolated from sites of periodontal health and three from sites of chronic periodontitis) were treated with the pro-inflammatory cytokines. The gingival fibroblast cells isolated from sites of chronic periodontitis had different morphology than the gingival fibroblast cells isolated from sites of periodontal health. Fig 13 row A is a representation of cells that were treated with 1.0% FBS and in rows B and C cells were treated with the effective concentration of EL-la (30 ng/ml) and CSA (250 ng/ml), respectively. Gingival fibroblast cells isolated from sites of chronic periodontitis exhibited morphological consistency with myofibroblast-like cells (Hakkinen and Larjava, 1992; Yamasaki et al., 1987). They were rounder, more spread out, and seemed to have more extensions than the gingival fibroblast cells isolated from sites of periodontal health (Fig 13, left columns). On the other hand, the gingival fibroblast cells isolated from sites of periodontal health were more elongated, tightly packed and exhibited fewer cellular extensions (Fig. 13, right columns). 10.0% FBS treatment was included as the positive control. EL-la, IL-ip, IL-6 and TNF-a all induced K G F protein expression over 1.0% FBS treatment (Fig. 14 A). A l l cell lines similarly expressed K G F protein when stimulated with pro-inflammatory cytokines. No significant difference within each group or between the two groups (cells isolated from sited of periodontal health and cells isolated from sites of chronic periodontitis) was found. Other than 10.0%> FBS treatment, no significant cell number difference was found (Fig. 14 B).  60  Fig. 13 A , B & C . Variation in morphology of fibroblasts isolated from sites of periodontal health or chronic periodontitis. Fibroblasts isolatedfrompatients with chronic periodontitis (left column) and fibroblasts isolated from healthy human gingiva (right column) exhibit significant differences in morphology. Cells in row A. are treated with 1.0% FBS; B. with IL-la (30 ng/ml) and C. with Cyclosporine (250 ng/ml).  61  A.  800  10.0% F B S 600  g  400  -I  fats 2 0 0  Gingival Fibroblasts Isolated from Sites of Periodontal Health  B.  Gingival Fibroblasts Isolated from Sites of Chronic Periodontitis  IL-1 a  TNF-a  d  0.75  U  45 0.5  O  U u  0.25  H  Gingival Fibroblasts Isolated from Sites of Periodontal Health  Gingival Fibroblasts Isolated from Sites of Chronic Periodontitis  Fig. 14 A & B. Pro-inflammatory cytokines (IL-la, IL-ip, IL-6 and TNF-a) induction of KGF protein expression and cell numbers in gingival fibroblasts isolated from sites of periodontal health as well as chronic periodontitis over 24 hours. Gingival fibroblasts were cultured in 96 well plates and maintained until 75% confluent; then treated with the effective concentration of pro-inflammatory cytokines (IL-la 30 ng/ml, IL-1 (3 30 ng/ml, IL-6 5 ng/ml, and TNF-a 30 ng/ml). A. Sandwich ELISA was used to quantify the expressed KGF in the condition media (24 hours). B. The relative cell numbers in each well was determined. Cells werefixedwith 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. All cell lines similarly expressed KGF protein when stimulated with proinflammatory cytokines. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4 62  5.3  Pro-inflammatory cytokine regulation of K G F and FGF-10 mRNA expression Treatment of human gingival fibroblast cells with the proinflammatory cytokines  increased K G F mRNA expression (Fig. 15 A). I L - l a induced K G F mRNA expression 1.2 and 1.6 fold at 3 hours and 6 hours, respectively (Fig. 15 B). Similarly, EL-1 (3 induced K G F mRNA expression by 1.1 and continued to increase to 1.4 fold at 3 and 6 hours, respectively. TNF-a and IL-6 K G F mRNA expression levels increased earlier than EL-la and IL-ip. TNF-a and EL-6 induced K G F mRNA expression at 3 hours by 1.2 and 1.25 fold but the mRNA levels decreased to 1.1 and 1.16 fold at 6 hours (Fig. 15 B). Pro-inflammatory cytokine treatment did not affect the FGF-10 mRNA expression at 3 or 6 hours, (Fig. 15 A). G A P D H results were used to adjust for minor variation in mRNA loading on gel.  63  KGF (2.4 kb) 3 hrs  FGF-10 (i.7 kb)  6 hrs  Control IL-la  3 hrs  6 hrs  —  LL-ip • I TNF-a H  safe  o «  S ft  «*»  *  *•  Hi  H  A  #*  A.  WBP; :lH»i  aHB ™Hr  •  4  6 hrs  3 hrs  • • 1  -am  MHK£JHJH  IL-6 B.  (  jriHh  m Hi  mm  GAPDH i . kb)  3 hrs  M  w  6hrs  1.6 H  55  1  1.4  H  ft  o  1.2 -  I  .a  s I  ns - - ^  1.0% FBS  IL-1  a  IL-1  |3  TNF- a  IL-6  Cytokines  Fig. 15 A & B. Proinflammatory cytokine regulation of K G F and FGF-10 mRNA  expression. Gingival fibroblast cells isolated from sites of periodontal health were maintained in aMEM containing 10.0% FBS until 75% confluent. Subsequently, cells were serum starved over night and treated with proinflammatory cytokines (IL-la 30 ng/ml, IL-1 p 30 ng/ml, TNF-a 30 ng/ml, and IL-6 5 ng/ml) for 3 and 6 hours; then, RNA was extracted and purified. 15ug of RNA was separated on agarose gel and blotted to nylon membrane. The membrane was hybridized with KGF, FGF-10, and GAPDH random labeled P-cDNA probes. A. Pro-inflammatory cytokines induced a significant increase in KGF mRNA (2.4 kb) at 3 and 6 hours but there was no change in FGF-10 mRNA expression. Expression of GAPDH was consistent in control and treatment groups. B. KGF mRNA expression was corrected to GAPDH levels and fold increase in KGF mRNA expression over time was graphed. IL-la and IL-ip increase KGF mRNA expression by 1.2 and 1.13 fold at 3 hours and 1.6 and 1.4 fold at 6 hours; whereas, TNF-a and IL-6 increase KGF mRNA expression by 1.2 and 1.25 fold at 3 hours and 1.1 and 1.16 at 6 hours over 1.0% FBS 3 hours, respectively. 32  64  Chapter 6  6  Results-Part III Refer to appendix C for graphed data.  6.1  Cyclosporine-A, Dilantin, and Nifedipine regulation of K G F protein expression Cyclosporine-A, Dilantin, and Nifedipine induction of K G F protein expression was  studied in human gingival fibroblast cells. These concentrations were chosen to match the range of serum concentrations in patients with gingival overgrowth (Hassell and Hafti 1991; Hallmon et al., 1999). Cells were treated with five different concentrations of C S A (0 ng/ml, 10 ng/ml, 100 ng/ml, 150 ng/ml, 250 ng/ml, 500 ng/ml). In a concentration dependent manner C S A significantly induced K G F protein expression (Fig. 16 A). A n effective concentration of 250 ng/ml was selected for the future experiments. The relative cell numbers showed a gradual decrease with increasing C S A concentrations. However, only 100 ng/ml and 500 ng/ml was associated with a statistically significant decrease. Dilantin and Nifedipine concentration were chosen based on an in vitro study (Hassell and Hafti, 1991). Dilantin and Nifedipine treatment did not change K G F protein expression at any of the selected concentrations. These two drugs, at the concentration range that was chosen, did not change the relative cell numbers either (Fig. 16 B and Fig. 17). Therefore, they were excluded from the K G F and FGF-10 mRNA expression study. In another pilot experiment, higher concentration of Nifedipine (40-80 ng/ml) and Dilantin (10-25 ng/ml) were used. However, cell numbers were decreased significantly with increasing drug concentrations (data not shown).  65  —  —  o  CM  CSA (ng/mL)  CSA (ng/ml)  B. 400  H  300  H  0.3  P. «  s 200  •  nr  T • 1  E  D  100  0.2  *H  3  o.i H  —  Dilantin (ng/ml)  v.  Dilantin (ng/ml)  Fig. 16 A & B. Cyclosporine-A (CSA) and Dilantin induction of K G F protein expression and cell numbers over different concentrations in HGF. HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with different concentrations of CSA and Dilantin. A. Sandwich ELISA was used to quantify the concentration of KGF in the condition media (collected from wells after 24 hours treatment period). B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. CSA but not Dilantin increased KGF protein expression in gingivalfibroblastcells over 24 hours. * p< 0.05 over 1.0% FBS Control (Mean + S.D.); n=4 66  O  NilVdiph,,, . . . . , „ »  O  Nifedipine  CN  (iiy'inl)  Fig. 17. Nifedipine induction of K G F protein expression and cell numbers over different concentrations in HGF. HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with different concentrations of Nifedipine. A. Sandwich ELISA was used to quantify the concentration of KGF in the condition media (collected from wells after 24 hours treatment period). B. The relative cell numbers in each well were determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. Nifedipine treatment did not induce KGF protein expression in gingival fibroblast cells. (Mean ± S.D.); n=4  67  6.2  Cyclosporine-A, Dilantin, and Nifedipine regulation of K G F protein expression in gingivalfibroblastcells isolated from sites of periodontal health and diseased Treatment of six gingival fibroblast cell lines (three lines isolated from sites of  periodontal health and three from sites of chronic periodontitis) with Dilantin and Nifedipine did not change K G F protein expression in either group. However, C S A treatment induced K G F protein expression in the "healthy" and "diseased" fibroblast cell line groups (Fig 18 A). No difference in CSA induction between cell groups was found. No significant changes in cell numbers were found with the drug concentrations that were used (Fig. 18 B).  68  WD  600  H  400  H  I I  1.0% FBS  1:1  Cyclosporine-A  ESS  Dilantin  Eg  Nifedipine  T  a to  3  1  1 200  Gingival Fibroblasts Isolated from Sites of Periodontal Health  Gingival Fibroblasts Isolated from Sites of Chronic Periodontitis  B.  Q O  0.75  H  0.5  H  0.25  H  •  1.0% FBS  E3*  Nifedipine  Cyclosporine-A  Dilantin  sXI  S 3  Z.  13 U >  JS' "3  Gingival Fibroblasts Isolated from Sites of Periodontal Health  Gingival Fibroblasts Isolated from Sites of Chronic Periodontitis  Fig. 18 A & B. Cyclosporine-A, Dilantin, and Nifedipine induction of K G F protein expression and cell numbers in gingival fibroblasts isolated from sites of periodontal health as well as sites of chronic periodontitis over 24 hours. Gingivalfibroblastswere cultured in 96 well plates and maintained until 75% confluent; then treated with the following concentrations of drugs: Cyclosporine-A (250 ng/ml), Dilantin (5 ng/ml), Nifedipine (20 ng/ml). A. Sandwich ELISA was used to quantify the expressed KGF in the condition media collected at 24 hours after the treatment. B. The relative cell numbers in each well was determined. Cells werefixedwith 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. Cyclosporine increased KGF protein expression over 24 hours infibroblastcells isolatedfromsites of periodontal health and disease. However, Dilantin and Nifedipine did not have an effect on KGF expression over 24 hours. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4 69  6.3  Cyclosporine-A regulation of K G F and FGF-10 m R N A expression  Cyclosporine-A treatment of fibroblast cells increased K G F m R N A expression by 1.2 and 2 fold at 3 and 6 hours, respectively (Fig. 19 A & B). However, C S A did not induce FGF-10 mRNA expression. The G A P D H expression levels indicated mRNA loading was relatively consistent among the treatment groups.  A.  KGF(2.4kb) 3 hrs  Control  6 hrs  ilHI  4»  03  FGF-10(i.7kb) GAPDH(i.4kb) 3 hrs  .  6 hrs  3 hrs  **  6 hrs  Treatment Period (hrs)  Fig. 19 A & B. Cyclosporine-A regulation of K G F and FGF-10 m R N A expression.  Gingival fibroblast cells isolated from sites of periodontal health were maintained in aMEM containing 10.0% FBS until 75 % confluent. Subsequently, cells were serum starved over night and treated with CSA (250 ng/ml) for 3 and 6 hours; then, RNA was extracted and purified. 15ug of RNA was separated on agarose gel and blotted to nylon membrane. The membrane was hybridized with KGF, FGF-10, and GAPDH random labeled cDNA probes. A.CSA induced a significant increase in KGF mRNA (2.4 kb) at 3 and 6 hours but there was no change in FGF-10 mRNA expression. Expression of GAPDH was consistent in control and treatment groups. B. KGF mRNA expression was corrected to GAPDH levels and fold increase in KGF mRNA expression over time was graphed. CSA increases KGF mRNA expression by 1.2 fold at 3 hours and 2 folds at 6 hours over 1.0% FBS, respectively. 70  6.4  Combination (drug and cytokine) study In addition to investigating the individual effects of cytokines and drugs On K G F  protein expression, the effect of CSA in combination with pro-inflammatory cytokines was examined. Human gingival fibroblast cells isolated from sites of periodontal health were treated with the effective concentration of pro-inflammatory cytokines and CSA. C S A + each cytokine induced K G F protein expression in comparison to negative control (1.0% FBS) (Fig. 20 A & 21 A). However, pro-inflammatory cytokines in combination with C S A did not have an additive effect on K G F protein expression. No significant change in cell numbers was found when the fibroblast cells were treated with pro-inflammatory cytokines in combination with CSA (Fig. 20 B & 21 B).  71  A. 600  E s. to  H  •  1.0% FBS  •  10.0% FBS  B  IL-1 p  13  IL-1 a  CSA  400  200  Cytokine + CSA  Cytokine/Drug Alone  •  •  Cytokine/Drug Alone  10.0% FBS  ESI  rL.]  a  CSA  Cytokine + CSA  Fig. 20 A & B. Combination of Cyclosporine-A (CSA) and I L - l a and I L - i p induction  of K G F protein expression and cell numbers in H G F . HGF were cultured in 96 well plates and maintained until 75% confluent; then treated with CSA (250 ng/ml) and IL-la (30 ng/ml) or CSA (250 ng/ml) and IL-1 (3 (30 ng/ml). A. Sandwich ELISA was used to quantify the concentration of KGF in the condition media (collected from wells after 24 hours treatment period). B. The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. Combination of CSA and IL-la and IL-1 p did not have an additive effect on KGF protein expression. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4  72  A.  a-  400  H  a.  to o  Cytokine + CSA  Cytokine/Drug Alone  B. 0.6  1.0% FBS  10.0% FBS  IL-6  CSA  m  TNF«  Q  O u <u  0.4  H  Xi  s s  u  0.2  «  Cytokine/Drug Alone  Cytokine + CSA  Fig. 21 A & B. Combination of Cyclosporine-A (CSA) and TNF-a and IL-6 induction of KGF protein expression and cell numbers in HGF. H G F were cultured in 96 well plates and maintained until 75% confluent; then treated with CSA (250 ng/ml) and T N F - a (30 ng/ml) or C S A (250 ng/ml) and IL-6 (5 ng/ml). Sandwich ELISA was used to quantify the concentration of K G F in the condition media (collected from wells after 24 hours treatment period). The relative cell numbers in each well was determined. Cells were fixed with 4% formaldehyde, stained with 0.1% crystal violet and dissolved with 10% Acetic Acid. Changes in relative cell numbers were measured by reading the color intensity at 570 nm. Combination of CSA and TNF-a and IL-6 did not have an additive effect on K G F protein expression. * p< 0.05 over 1.0% FBS Control (Mean ± S.D.); n=4  73  Chapter 7 Discussion 7.1  Periodontitis as a Chronic Inflammatory Disease Many studies have investigated different aspects of tissue repair in patients with  chronic inflammation such as psoriases, Crohn's disease, and ulcerative colitis (Bajaj-Elliot et al., 1997; Brauchle et al., 1996; Finch and Cheng, 1999). Histological observation of tissues obtained from patients with Crohn's disease and ulcerative colitis showed accumulation of inflammatory cells as well as local cytokine production (Werner et al., 1992). These studies reported that inflammation was also associated with an increase in epithelial cell proliferation. Associated with this increase in epithelial proliferation was upregulation of K G F mRNA expression (Bajaj-Elliot et al., 1997; Brauchle et a l , 1996). K G F is a specific inducer of epithelial proliferation, which is expressed by the stromal cells (Finch et al., 1989, Galzie et al., 1997) Periodontitis is another chronic inflammatory disease that shows a similar histological appearance to the above chronic inflammatory conditions. Accumulation of dental plaque on the tooth surface will induce inflammation in the gingival connective tissue. Recruitment of inflammatory cells to the gingival connective tissue occurs in response to dental plaque accumulation (Page and Schoroeder, 1976; Mathur et al., 1996; Mogi et al., 1999; Rassomando et a l , 1990). The inflammatory cell recruitment starts by accumulation of Tcells and macrophages in the early lesion and will follow by accumulation of neutrophils and plasma cells in the established and advanced lesions (Kornman et al., 1997, Page et al., 1976).  74  Increase in the proinflammatory cytokines (EL-a, EL-1(3, IL-6 and TNF-a) at inflammatory sites are expressed in part by inflammatory cells (Page et al., 1976; Kornman et al., 1997). However, epithelial, fibroblast, and endothelial cells also contribute to this local increase in inflammatory cytokines (Stashenko et al., 1991; Mogi et al., 1999, Page et al., 1976; Kornman et al., 1997). One aspect of periodontal disease onset is proliferation of junctional and oral sulcular epithelium (Schroeder and Page, 1990). Mechanisms regulating this epithelial proliferation during disease onset are of interest. Since periodontitis is a chronic inflammatory disease and K G F is a specific inducer of epithelial proliferation in chronic inflammation and wound healing, we investigated K G F expression by gingival fibroblasts. 7.2  Serum and Pro-inflammatory Cytokine Induction of K G F K G F is expressed by fibroblasts in oropharyngeal mucosa and periodontal ligaments  (Knerner et a l , 1999; Dabelsteel et al., 1997). However, it has not yet been described whether K G F is expressed by gingival fibroblasts. Our results indicated that in vitro stimulation of human gingival fibroblast cells do in fact express K G F mRNA protein. Ten per cent FBS induced a rapid induction of K G F mRNA in human gingival fibroblasts. This may be due to presence of serum-derived growth factors. Brauchle et al. (1994) analyzed the effects of purified serum growth factors. None of the mitogens (platelet derived growth factor BB, epidermal growth factor, transforming growth factor pi) were as potent as the serum. This is suggestive that the serum-induced K G F gene activation may be a combinational effect of several factors. However, presence of serum growth factors may not be the only inducers of K G F mRNA.  Presence of high  75  number of inflammatory cells in the inflamed sites may also induce K G F expression when they express pro-inflammatory cytokines. Healthy gingiva has a very low cytokine concentration. Some of pro-inflammatory cytokine in the sulcus GCF are: I L - l a (9-74 pg/ml), LL-ip (30-3,071 pg/ml), LL-6 (5-17 pg/ml), and T N F - a (26 pg/ml). However, during inflammation the concentrations of these cytokines increase in GCF; I L - l a (50-342 pg/ml), IL-lp (108-11,695 pg/ml), LL-6 (56 pg/ml) and TNF-a (434 pg/ml) (Stashenko et a l , 1991; Mogi et al., 1999; Mathur et al., 1996). We chose cytokine concentrations that fell between the GCF described values. These concentrations were also used by other in vitro studies and they showed these cytokines induced K G F mRNA expression in foreskin fibroblast cells (Brauchle et al., 1994; Chedid et al., 1994; Weng et al., 1997; Tang and Gilchrest, 1995). The pro-inflammatory cytokines (IL-la, LL-ip, 11-6 and TNF-a) upregulated K G F mRNA and protein expression in human gingival fibroblasts. There is only one study that indicates TNF-a treatment did not induce K G F mRNA expression (Chedid et al., 1994). However, this study used a TNF-a concentration of 5 ng/ml, which was 6 times lower than what was used in majority of the studies, including ours. It is possible that this concentration was too low; therefore, no increase in K G F mRNA expression was induced. Based on the earlier observation by Brauchel and her coworkers (1994), K G F mRNA expression is upregulated as early as 60 minutes when treated with 10% FBS. Because of this early and rapid temporal expression, it was suggested that K G F is a primary response gene (Brovo et a l , 1990; Brauchle et al., 1994; Dabelsteen et al., 1997). In a wound healing study, K G F mRNA was upregulated by 9 fold at 12 hours and by 160 fold at 24 hours (Werner et al., 1992). Reconstruction of the skin epithelial barrier against pathogens and 76  virulence factors is essential to species survival. Our finding, that gingival fibroblast tissue is upregulated at 3 hours when treated with 10% FBS or pro-inflammatory cytokines, supports that K G F may function as a primary response gene that stimulates repair and reconstitution of the epithelial barrier in gingiva. In this process it protects gingival tissue from bacterial invasion. As it is mentioned before, K G F gene mRNA is highly expressed in the dermis of cutaneous wounds and in chronic inflamed tissue, which may contribute to repair of epithelial tissue (Chedid et al., 1994). It is thought that the induction of K G F gene transcription may be influenced by the action of EL-l and EL-6 (Dinarello, 1988), which are two of the major of pro-inflammatory cytokines. NF-EL-6 is a transcriptional activator that is induced by EL-6 and EL-1. The presence of two sequences similar to the consensus of NF-IL6 transcriptional activator in the K G F promoter region, suggests a possible mechanism through which the action of IL-1 and EL-6 may lead to induction of K G F gene expression (Finch etai, 1995). Fibroblast subpopulations in gingival connective tissue exist in healthy and diseased periodontal sites (Hakkinen and Larjava, 1992). The existence of subpopulations suggests that K G F expression levels may differ among fibroblast cell populations. Therefore, we examined K G F expression in two different cell populations. Three cell lines isolated from healthy periodontium and 3 cell lines from sites of chronic periodontitis were treated with pro-inflammatory cytokines. Fibroblasts isolated from diseased periodontium exhibited morphology and growth characteristics that were consistent with myofibroblast-like cells (Shor and Shor, 1987; Hakkinen and Larjava, 1992; Robert and Morey, 1985; Cho and Garant, 1984; Yamasaki et al., 1987). Pro-inflammatory cytokine treatment of these cells  77  induced K G F protein expression. However, no significant difference between the K G F protein expression among the fibroblasts isolated from healthy and diseased periodontium were found. Based on these in vitro studies we would not anticipate that the level of K G F expression by fibroblasts in healthy and diseased sites would vary. These differences can only be better understood by in situ studies, which would examine K G F expression in healthy and diseased periodontium.  7.3  Epithelial Proliferation associated with Gingival Overgrowth Histological studies of Nifedipine, Dilantin and Cyclosporine-A induced gingival  overgrowth have described changes to the connective tissue and epithelium. The essential feature of all drug induced gingival overgrowth is excessive accumulation of epithelial (deep rete ridges) and connective tissue elements, especially collagen. (Van der Wall and Tuinzing, 1985; Nery et al., 1995; Henderson et al., 1997; Seymour et al., 1996; Pisanty et al., 1988; Butler et al., 1987; Dangri et a l , 1993). On the other hand, histological studies reported that topical application of K G F on wounds consistently increased epidermal thickening and the epidermis exhibited pronounced deep rete ridges (Staiano-Coico et al., 1993; Werner, et a l , 1994). This led us to investigate the possible regulation of K G F by these drugs, which are associated with gingival overgrowth. The drug concentrations C S A (250 ng/ml), Nifedipine (20 ng/ml) and Dilantin (5 ng/ml) were chosen such that they fell within the serum range concentrations of 100-400 ng/ml, 18-1121 ng/ml, and 2-25 ng/ml, respectively (Hassell and Hafti 1991; Nishikawashi et al., 1991; Ratanakorn et a l , 1997). The G C F concentration of 202 ng/ml for Nifedipine was much higher than the reported serum concentration (Thomason et al., 1995). However, we are not aware of any reports indicating G C F concentrations of Cyclosporine-A and Dilantin. Studies have suggested, Cyclosporine-A, Dilantin and  78  Nifedipine could be concentrated in the bacterial plaque, which may act as a reservoir for the drugs (Seymour and Smith, 1991; Thomason et al., 1995; Niimi et al., 1990). Therefore, CSA and Dilantin concentration, just like Nifedipine, may be much higher in GCF than serum. Our data indicated that Cyclosporine-A increased K G F mRNA and protein expression levels whereas Nifedipine and Dilantin did not induce K G F protein expression. This suggests that K G F may not play a significant role in Nifedipine and Dilantin induced gingival enlargement or other factors are needed to induce K G F expression. In contrast to Nifedipine and Dilantin, C S A induced K G F mRNA and protein expression in a concentration dependent manner. Some authors have reported that the severity and occurrence of drug associated gingival overgrowth varies among patients, which might be due to presence of different cell subpopulation in the gingival soft tissues (Handerson et al., 1997; Wysochi et al., 1983; Rateitschak-Pluss et al., 1983). We tested this hypothesis by investigating the K G F protein expression in gingival fibroblast cell lines, isolated from healthy and diseased periodontium. C S A treatment consistently increased K G F protein expression in both cell lines. Most of the histological studies have concluded that the C S A induced gingival overgrowth is a result of accumulation and thickening of epithelial layers (Pisanty et al., 1988; Seymour et al., 1996; Niimi et al., 1990). No mechanism has yet been associated with C S A involvement in this epithelial accumulation. However, it has been recently reported that C S A could prolong the induction of the junB gene, which might lead to the persistence of high level of AP-1 activity (Lohi et al., 1994). The K G F promoter region is composed of two AP-1  79  transcriptional activator sites (Finch et al., 1995). Thus, C S A may increase K G F expression by increasing AP-1 activity. Inflammation in conjunction with C S A has been shown to induce the severity of gingival overgrowth (Hassell and Hafti, 1991; Seymour and Smith, 1991), we examined if C S A in combination with another pro-inflammatory cytokine would have an additive effect on K G F expression.  Our result showed that there was no additive effect. However, these  data do not suggest that an additive effect could not be produced in the gingival tissue. Other unidentified factors such as higher concentration of C S A that may be stored in the dental plaque could potentially play an important role.  7.4  FGF-10 FGF-10 has the highest nucleotide homology with K G F and binds with the same  affinity to the epithelial specific KGFR receptor (Yamasaki et al., 1996). Studies have indicated that FGF-10 has similar biological activities as KGF; i.e. FGF-10 mRNA is upregulated on the first day of injury (Tagarashi et al., 1997); topical application of FGF-10 to the wound will improve wound breaking strength (Jimenez and Rampy, 1999); and epidermal thickness and collagen both significantly increased with FGF-10 topical application (Jimenez and Rampy, 1999). Therefore, in addition to K G F mRNA expression, we also investigated the regulation of FGF-10 mRNA expression by pro-inflammatory cytokines and CSA. Our data indicated that FGF-10 is expressed at much lower levels in comparison to K G F and the pro-inflammatory cytokines and C S A did not affect FGF-10 expression significantly. Out of four known sizes of FGF-10 (1.7, 4.2, 4.4, 4.7 kb) (Dietmar Beer et al., 1997), we were able to identify two bands, namely 1.7 and a barely detectable 4.7 kb. This illustrates that even though FGF-10 and K G F are the closest F G F member to each  80  other and bind to the same receptor, there are differences between them. FGF-10 is not upregulated by pro-inflammatory cytokines nor CSA. These data suggest that FGF-10 would not be a significant regulator of oral sulcular and junctional epithelium proliferation.  81  7.5  Conclusion 1. Our data confirmed that human gingival fibroblast cells express K G F and FGF-10. 2. 10.0% serum treatment induced K G F mRNA and protein expression in human gingival fibroblast cells. 3. Pro-inflammatory cytokines (IL-la, IL-ip, IL-6 and TNF-a) upregulated K G F mRNA and protein expression in human gingival fibroblast cells. 4. C S A but not Dilantin or Nifedipine induced K G F mRNA and protein expression in human gingival fibroblast cells. 5. Pro-inflammatory cytokines and C S A treatment of human gingival fibroblast cells did not effect FGF-10 mRNA expression significantly. 6. The onset of proliferation of epithelial cell in periodontitis may be due to upregulation of K G F expression by pro-inflammatory cytokines.  82  7.6  Future Work 1. This study determined that K G F mRNA expression is increased in human gingival  fibroblasts in the presence of pro-inflammatory cytokines, in vitro. Based on these observations, we hypothesize that K G F mRNA expression would be induced in tissue section collected from patients with severe periodontitis. Correlating inflammation to K G F expression would support the hypothesis that cytokine expression in inflamed area would play a role in the increased levels of K G F expression. Therefore, in situ hybridization technique could be used to investigate this finding. 3. The, presence of CSA, but not Dilantin and Nifedipine, increased K G F protein expression in human gingival fibroblasts, in vitro. Therefore, it would be of interest to compare the K G F expression in CSA, Nifedipine and Dilantin specimens using in situ hybridization. 2. Further investigation by which CSA induces interacellular signalling to increase K G F expression should be pursued.  83  References 1. Abrahham, J.A., Whang, J.L., Tumolo, A. Mergia, A., Friedman, J., Gospodarowicz, D., Fiddes, J.C. 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Molecular Cell Biology. 1988: 8: 3487-3495.  98  Appendix A: Appendix A l : HRP and Antibody Optimization Sample lOpg KGF & 1:20000 HRP  O.D. 0.038  S.D. 0.001  lOpg KGF & 1:15000 HRP lOpg KGF & 1:10000 HRP  0.05  0.013  0.063  0.01  750pg KGF & 1:20000 HRP 750pgKGF& 1:15000 HRP 750pg KGF & 1:10000 HRP  0.116 0.143 0.182  0.007 0.004  Capturing Antibody Concentration  Detecting Antibody lOOng/ml  0.002 Detecting Antibody 150ng/ml  0  Detecting Antibody 200ng/ml 0.1  S.D. lOOng/ml  S.D. 150 ng/ml  S.D.200 ng/ml 0.01  0.5 ug/ml  0.216  0.256  0.278  0.03  0.014  0.003  1.0 ug/ml  0.228  0.25  0.3  0.019  0.016  0.01  Appendix A2: Standard Curve KGF (ng/ml) 0 1 50 125  Absorption @ 450 nm 0 0.026 0.076 0.131  S.D. 0 0.007 0.013 0.01  250 500  0.167 0.286 0.43  0.015 0.025 0.03  750  Appendix A3: Serum Induction of K G F Protein and Relative Cell Numbers Per-cent FBS in Media 1.0% FBS  KGF (pg/ml)  S.E.  130  10.0% FBS  516  S.E.  23  Per-cent FBS in Media 1.0% FBS  Relative Cell Numbers 0.39  0.01  31  10.0% FBS  0.47  0.014  Appendix A4: Serum Induction of K G F mRNA Expression Hours 0  1.0% FBS 1.03  10.0% FBS 1.03  3 6  1 1  1.2 1.6  99  Appendix B Appendix B l : I L - l a Induction of K G F Protein and Relative Cell Numbers IL-la (ng/ml) 0  KGF (ng/ml)  S.D.  IL-la (ng/ml)  Relative Cell Numbers  S.D.  100  25  0  0.422  0.02  5  157  4  5  0.43  0.02  10  221  37  10  0.45  0.03  20  233  19  20  0.44  0.04  30  235  25  30  0.44  0.05  50  338  51  50  0.45  0.01  Appendix B2: IL-1 (3 Induction ofKGF Protein and Relative Cell Numbers  IL-1 (3 (ng/ml)  KGF (ng/ml)  S.D.  0  100  25  5  141  10  151  20  IL-1 (3 (ng/ml) Relative Cell Numbers  S.D.  0  0.422  0.05  28  5  0.447  0.01  20  10  0.442  0.02  172  11  20  0.48  0.04  30  248  40  30  0.46  0.015  50  289  33  50  0.44  0.01  Appendix B3: IL-6 Induction of K G F Protein and Relative Cell Numbers IL-6 (ng/ml)  KGF (pg/ml)  S.D  IL-16 (ng/ml)  Relative Cell Numbers  S.D.  0  100  25  0  0.422  0.02  5  202  7  5  0.46  0.02  10  136  36  10  0.47  0.017  30  213  36  30  0.46  0.02  50  184  36  50  0.46  0.01  Appendix B4: TNF-a Induction of K G F Protein and Relative Cell Numbers TNF-a (ng/ml)  KGF (pg/ml)  S.D  TNF-a (ng/ml)  Relative Cell Numbers  S.D.  0  100  25  0  0.422  0.02  5  188  40  5  0.44  0.01  10  227  42  10  0.46  0.02  20  207  26  20  0.49  0.01  30  273  40  30  0.47  0.01  50  261  48  50  0.483  0.02  Appendix B5: Cytokines Induction of K G F Protein over 72 hrs and Relative Cell Numbers and Relative Cell Numbers KGF (pg/ml) 48 hrs 149.8  KGF (pg/ml) 72 hrs 110  (S.D.) 24 hrs  (S.D.) 48 hrs  (S.D.) 72 hrs  1.0% FBS  KGF (pg/ml) 24 hrs 242.8  22  4  27  IL-la  380.9  383.3  287.9  37  33  40  IL-IP  436.9  339.2  253.4  9  24  28  IL-6  379.5  392.2  226  29  43  41  TNF-a  292.9  229  223.7  21  35  29  10.0% FBS  539.9  530  545.9  37  36  25  Cytokines  Relative Cell Numbers 48 hrs 0.38  Relative Cell Numbers 72 hrs 0.35  (S.D.) 24 hrs  (S.D.) 48 hrs  (S.D.) 72 hrs  1.0% FBS  Relative Cell Numbers 24 hrs 0.4  0.007  0.01  0.01  IL-la  0.385  0.37  0.36  0.01  0.009  0.02  IL-ip  0.4  0.37  0.344  0.01  0.02  0.02  IL-6  0.39  0.37  0.34  0.005  0.02  0.01  TNF-a  0.41  0.4  0.35  0.009  0.02  0.01  10.0% FBS  0.5  0.51  0.51  0.01  0.02  0.02  Cytokines  Appendix B6: Cytokines Induction of KGF Protein in Fibroblasts Isolated from Sites of Healthy and Diseased Gingiva and Relative Cell Numbers IL-1 P (KGF pg/ml)  IL-6 (KGF pg/ml)  222.5  211.8  175.3  243  252  202  Test Groups  IL-1 a (KGF pg/ml)  Healthy Chronic Cases  Test Groups  IL-1 a (Relative Cell Numbers)  IL-1 p (Relative Cell Numbers)  IL-6 (Relative Cell Numbers)  Health  0.59  0.58  0.59  Chronic  0.55  0.56  0.57  TNF1.0% FBS a (KGF (KGF pg/ml) pg/ml) 194.5 51 198  62  10.0% (S.D.) ( S.D.) ( IL-6 FBS S.D.) IL-ip (KGF IL-la pg/ml) 47 670.6 30.8 47.02 21  631  10.0% 1.0% FBS TNF-a FBS (Relative (Relative (Relative Cell Cell Cell Numbers) Numbers) Numbers) 0.69 0.59 0.59 0.58  0.55  0.67  (S.D.) TNF-a  (S.D.) 1.0% FBS  (S.D.) 10.0% FBS  35  8  58  5.5  12  35  56  31  (S.D.) (S.D.) (S.D.) IL-1 a IL-1 (3 IL-6  (S.D.) (S.D.) (S.D.) T N F - a 1.0% 10.0% FBS FBS  0.04  0.04  0.04  0.04  0.04  0.04  0.02  0.03  0.04  0.03  0.03  0.03  Appendix B7: Cytokine Induction of K G F mRNA Expression Samples  3 hrs (Fold Increase)  6 hrs (Fold Increase)  1.0% FBS  1.01  1.14  IL-1 a  1.2  1.65 1.4  P  1.13  TNF-a  1.2  1.1  IL-6  1.25  1.16  IL-1  102  Appendix C: Appendix CI: CSA Induction of KGF Protein and Relative Cell Numbers CSA (ng/ml)  KGF (pg/ml)  S.D.  CSA (ng/ml)  S.D. 0.02 0.01  0  118  19  0  Relative Cell Numbers 0.29  10 100  145 166  27 25  10 100  0.28 0.275  0.009  150 250  193 267  33 26  0.2855 0.261  0.009 0.02  500  255  37  150 250 500  0.23  0.01  Appendix C2: Dilantin Induction of KGF Protein and Relative Cell Numbers Relative Cell Numbers  27  0  0.298  0.01  36  0.05  0.31  0.009  179  38  0.1  0.307  0.01  205  24  0.5  0.309  0.01  1  0.31  0.003  2.5  0.297  0.01  0.305  0.001  KGF (pg/ml)  S.D.  165  0.05  191  0.1 0.5 1  144  27  2.5  172  32  5  S.D.  Dilantin (ng/ml)  Dilantin (ng/ml) 0  191  36  5  Appendix C3: Nifedipine Induction of KGF Protein and Relative Cell Numbers Relative Cell Numbers  S.D.  27  Nifedipine (ng/ml) 0  0.298  0.01  23 37  0.05 0.1  0.291 0.281  0.01 0.04  130  39  0.5  0.298  0.02  119 193 150  15  1  35 33  2.5 5  0.31 0.311 0.305  0.008 0.01 0.01  Nifedipine (ng/ml) 0  KGF (pg/ml)  S.D.  165  0.05 0.1  120 179  0.5 1 2.5 5  103  Appendix C4: Drug Induction of K G F Protein in Fibroblasts Isolated from Sites of Healthy and Diseased Gingiva and Relative Cell Numbers CsA  Test Groups  Dilantin  Nifedipine  FBS  (S.D.) CSA  (S.D.) Dilantin  (S.D.) Nifedipine  1.0%  (S.D.)  Healthy  344  70  57  51  65  17  17  1.0% FBS 8  Chronic Cases  248  59  62  62  109  6  22  12  Test Groups  CSA (Relative Cell Numbers)  Health  0.59  0.59  0.59  Chronic Cases  0.59  0.57  0.57  (S.D.) CSA  (S.D.) Dilantin  0.58  0.04  0.04  0.03  0.04  0.56  0.04  0.03  0.02  0.02  Dilantin Nifedipine 1.0% FBS (Relative Cell (Relative Cell (Relative Cell Numbers) Numbers) Numbers)  (S.D.) (S.D.) Nifedipine 1.0% FBS  Appendix C 5 : CSA Induction of KGF mRNA Expression Time (hrs)  1.0% FBS (Fold Increase) CSA (Fold Increase)  3  1  1.5  6  1.2  2  104  Appendix C7: CSA and IL-la/IL-lB Combination Effect on K G F Protein Expression and Relative Cell Numbers Test Groups  ILla (KGF pg/ml)  IL-IP (KGF pg/ml)  CSA (KGF pg/ml)  1.0% FBS (KGF pg/ml)  Cytokine/ Drug Alone Cytokine + CSA  88.8  82  53  34  69  87  10.0% (S.D.) (S.D.) FBS (KGF IL-1 a IL-1 P pg/ml) 442  45  13  32  14  (S.D.) CSA  (S.D.) 1.0% FBS  (S.D.) 10.0% FBS  11  11  26  CSA 1.0%o FBS 10.0% FBS (S.D.) (S.D.) (S.D.) IL-1 P IL-1 a (Relative IL-1 a IL-1 P CSA (Relative (Relative (Relative (Relative Cell Cell Cell Cell Cell Numbers) Numbers) Numbers) Numbers) Numbers)  Test Groups  0.48  0.49  0.6  0.02  0.03  0.01  0.01  (S.D.) 1.0% FBS  (S.D.) 10.0% FBS  0.02  0.02  0.01  Cytokine/ Drug Alone Cytokine + CSA  0.46  0.46  0.45  0.45  Test Groups  TNF-a (KGF pg/ml)  IL-6 (KGF pg/ml)  CSA (KGF pg/ml)  1.0% FBS (KGF pg/ml)  10.0% FBS (KGF pg/ml)  (S.D.) TNFa  (S.D.) IL-6  (S.D.) CSA  (S.D.) 1.0'% FBS  (S.D.) 10.0%. FBS  Cytokine/Drug Alone Cytokine + CSA  197  219  50  431  219  30  6  14  23  24  157  161  27  27  Test Groups  Cytokine/ Drug Alone Cytokine + CSA  CSA IL-6 1.0% FBS TNF-a (Relative (Relative (Relative (Relative Cell Cell Cell Cell Numbers) Numbers) Numbers) Numbers) 0.37  0.35  0.36  0.35  0.35  0.44  (S.D.) 10.0% FBS TNF-a (Relative Cell Numbers) 0.37  (S.D.) IL-6  0.01  0.02  0.02  0.02  (S.D.) (S.D.) (S.D.) CSA 1.0% 10.0% FBS FBS  0.01  0.02  0.01  105  

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