Clin Oral Implants Res 2013 Terheyden

Hendrik Terheyden Bernd Stadlinger Mariano Sanz Annette I. Garbe Jörg Meyle Authors’ affiliations: Hendrik Terheyden, Department of Oral & Maxillofacial Surgery, Red Cross Hospital, Kassel, Germany Bernd Stadlinger, Clinic of Cranio-Maxillofacial and Oral Surgery, University of Z€ urich, Z€ urich, Switzerland Mariano Sanz, Faculty of Odontology, University Complutense of Madrid, Madrid, Spain Annette I. Garbe, Institute of Physiological Chemistry, Dresden University of Technology, Dresden, Germany Jörg Meyle, Department of Periodontology, University Gießen and Marburg, Giessen, Germany Corresponding author: Hendrik Terheyden Department of Oral & Maxillofacial Surgery, Red Cross Hospital, Hansteinstrasse 29, 34121 Kassel, Germany Tel.: 004956130865500 Fax: 004956130865534 e-mail: terheyden@rkh-kassel.de Inflammatory reaction – communication of cells Key words: adaptive immune system biofilm, bone resorption, cytokines, infection control, inflammatory reaction, innate immune system, osteoclastogenesis, periodontitis, RANKL, T lymphocytes, Th1 cells, TNFa, T-reg cells Abstract This article presents scientific background information on the animated 3D film “Inflammatory Reactions – Communication of Cells” (Quintessence Publications, ISBN 978-1-85097-231-0). Gingivitis and periodontitis are understood as the result of a coordinated action of a few clearly identified cellular players who communicate with each other via cytokines. For didactic reasons, the course of a periodontal infection is described here in four phases: (1) bacterial biofilm formation and development of a host response in the marginal periodontium, (2) innate immune response leading to gingivitis, (3) role of the adaptive immune system in attachment loss and pocket formation, and (4) down-regulation of inflammation and periodontal regeneration and repair following biofilm removal. The control of the cells is discussed as a cytokine network, which can be modulated in pro- or anti-inflammatory direction depending on the control of the bacterial infection. Degradation of soft tissue structural proteins like collagen and proteoglycans by matrix metalloproteinases and degradation of hard tissue matrix by osteoclasts are explained as an interference of the immune system with the natural equilibrium of tissue remodeling. Five mechanisms of promotion of bone loss through the influence of the immune system are described. One example is bone resorption as a consequence of the shift of the RANKL/osteoprotegerin balance by soluble RANKL synthesized by CD4+Th1 cells as well as the interference with the coupling of osteoclasts and osteoblasts through dedifferentiation of osteoblasts by TNFa. Finally, the signaling required for down-regulation of inflammatory reactions and the reasons for the incomplete regeneration after periodontal bone loss are discussed. Date: Accepted 17 March 2013 To cite this article: Terheyden H, Stadlinger B, Sanz M, Garbe AI, Meyle J. Inflammatory reaction – communication of cells. Clin. Oral Impl. Res. 00, 2013, 1–9 doi: 10.1111/clr.12176 This review article presents scientific background information on the animated 3D film “Inflammatory Reactions – Communication of Cells” (Quintessence Publications, ISBN 978-1-85097-231-0). In the film and in the present manuscript, gingivitis and periodontitis are understood as the result of a coordinated action of clearly identified cellular players, which communicate with each other. In the inflammatory and immune systems, cell communication is predominantly organized by protein, peptide, or glycoprotein messenger molecules called cytokines. Some of these cytokines (called chemokines) are specialized in directing inflammatory and immune cells to the site of infection by chemotaxis by creating concentration gradients of messengers. Messenger molecules interact with selective, chemically fitting transmembrane receptors, © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd thereby activating intracellular second messenger systems that amplify, modulate, and transport the signal to the cell nucleus, where it induces a modulation of the activity of genes and protein synthesis, hence changing the cell homeostasis. It is therefore important to understand most biological systems as cytokine networks (Kinane et al. 2011), where there is a finely balanced equilibrium of competing molecular signals and the net composition and concentration of messenger molecules will result in different cellular actions (Preshaw & Taylor 2011). Messenger molecules can be presented membrane bound to neighboring cells, soluble in the interstitial fluid in the vicinity of the releasing cell or disseminated in the form of hormones throughout the organism. Soluble receptor molecules can serve as an extracellular signal modifier or a decoy receptor and 1 Terheyden et al
Inflammatory reaction – communication of cells thus can regulate the active concentration of messenger molecules. Beside proteins, other messenger molecules include the group of eicosanoids such as the prostaglandins and leukotrienes, which are small molecules enzymatically derived from fatty acid metabolism. These molecules bind to G protein cell membrane receptors or communicate with cells in direct contact via ion channels. Another special messenger molecule present in almost all eukaryotic cells, including plants, is the gas nitric oxide (NO). It moves freely across cell membranes, has no receptor, but it is a highly reactive radical with a half-life of only a few seconds. It acts by modifying the cellular activity at cytoplasmatic level (e.g., by oxidizing iron in ironcontaining enzymes). During inflammation, NO is released by phagocytes to kill bacteria, and it is induced by nitric oxide synthase (iNOS), activated by the pro-inflammatory cytokines interferon-gamma (IFN-c) and tumor necrosis factor (TNF). Otherwise, it is downregulated by transforming growth factor-beta (TGF-b) interleukin-4 (IL-4) and IL-10. In general, an inflammatory reaction can develop in two directions, either being amplified or attenuated depending on the bacterial antigen load and properties. If amplified, the innate immune reaction is followed by an adaptive or specific immune response, associated with the loss of tissue structure to create space for the immune process. Enzymatic degradation of extracellular matrix proteins (e.g., collagen) occurs in soft tissues. Osteoclastic resorption occurs in hard tissues. The attenuation of inflammation on the other hand is associated with the regeneration of these structural hard and soft tissue components. Gingivitis and periodontitis are no exemption to this general rule. Clinically, these inflammatory states appear in different degrees of severity on a spectrum between the two antipodes of tissue destruction (abscess and ulceration) and tissue healing. The inflammatory reaction is amplified by pro-inflammatory and attenuated by antiinflammatory signals (Garlet 2010). In terms of cells, some cell types are more associated with the healing process, for example, fibroblasts, endothelial cells, or regulatory T cells, while others are typically linked to chronifying the inflammatory process and causing tissue decay. There are certain cell types supporting the healing process such as fibroblasts, endothelial cells, or regulatory T cells while others promote the aggravation of inflammation and tissue decay. Examples for the latter are the polymorphonuclear 2 | Clin. Oral Impl. Res. 0, 2013 / 1–9 leukocytes (PMN) or CD8-positive T lymphocytes (cytotoxic T cells). The contrast between pro- and anti-inflammatory cell types is reflected in bone with osteoblasts representing the anti-inflammatory side, whereas osteoclasts share many similarities with pro-inflammatory cells. The cellular synthesis products can also be categorized as anti-inflammatory proteins, such as collagen and proteoglycans, and pro-inflammatory proteins, such as collagen-degrading enzymes like matrix metalloproteinases (MMP). MMP-8 is a collagenase secreted by PMN cells (Hasty et al. 1990), and its function is to breakdown collagen in the extracellular matrix (Nagase et al. 2006). MMPs are released in inactive form and require the activation by urokinase tissue plasmin activator (uPA). In fact, MMP-8 in saliva has been reported as a marker for periodontal disease activity (Rathnayake et al. 2013). The communication signals between cells can also be categorized in pro-inflammatory, such as tumor necrosis factor-alpha TNFa and interleukin-1 and anti-inflammatory, such as interleukin-10 and growth factors like transforming growth factor-beta. On the other side of the spectrum, the associated microbiota can also be categorized depending on their interaction with the host inflammatory–immune systems in commensals, which will exert a minimal antigen reaction and usually live, when in low numbers, in symbiosis with the host, and pathogens, which on the contrary have specific virulence factors that enable them to evade the host inflammatory and immune defenses and at the same time trigger a chronic inflammatory response with the potential of promoting tissue destruction. For the sake of clarity, we have divided the involved players into pro-inflammatory and anti-inflammatory and as destructive vs. regenerative, although the real situation is far more complex. In essence, the whole process is aimed to control the infection by developing a first-line response of the innate immune system (inflammation) and, if not resolved, through a response of the adaptive immune system. In these processes, there are cells, such as macrophages, that may act in both directions depending on the cytokine environment. They would act like a cellular switch, and in the appropriate messenger environment, they can turn the destructive inflammatory reaction into tissue healing and regeneration or repair by the release of angiogenic and growth factors. Also, other cells can show some plasticity in their behavior, such as fibroblasts, which can contribute to tissue destruction, and in contrast, PMN can become a source of anti-inflammatory cytokines (Nussbaum & Shapira 2011). Decisive for cellular behavior is the bacterial antigen load and the tissue cytokine network. A key feature of periodontitis is the loss of connective tissue attachment to the tooth surface. In fact, this feature will mark the transition from a reversible chronic gingivitis to an irreversible periodontitis. The result is the apical migration of the epithelial attachment, the destruction of connective tissue and alveolar bone, and the formation of periodontal pockets. The progression of this disease may proceed very slowly or contrary very aggressively with the end result of severe attachment loss, loosening of the teeth, and eventually tooth loss. Chronic periodontal infections usually show an episodic progressive clinical course. Phases of disease progression with loss of soft and hard tissue matrix are followed by phases of disease attenuation in which the matrix of soft and hard tissue can recover (Garlet 2010). Attachment loss is in first event in the loss of structural integrity of the periodontium. This soft tissue destruction is then followed by bone loss. Degradation of the extracellular matrix of the soft tissue and degradation of bone matrix are just two sides of the same coin. Both processes are induced by a similar pro-inflammatory cytokine environment, which leads to tissue destruction, needed to allow space and access for the elements in the immune defense to the site of bacterial infection. Periodontal bone loss is not directly resorbed by bacteria, but by host cells that create a disturbed bone homeostasis, with a shift balance between bone formation and resorption, in response to the bacteria (Graves et al. 2011). The bone homeostasis is regulated at cellular level by the balanced action of two bone cell types, the osteoblast and osteoclast. It has been recently discovered that these bone cell types communicate on the basis of soluble and membrane-bound receptors and ligands. Both cell actions are coupled so that bone destruction and reconstruction may be balanced. The host inflammatory and immune responses can interfere with these communication processes and tilt the balance toward bone loss by uncoupling the two cells (Graves et al. 2011). In fact, the osteoclasts are derived from hematopoietic precursors that also give rise to immune cells and share several signaling pathways with other cells of the immune system, what implies a close interplay between skeletal and the immune system (referred to as osteoimmunology). © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd Terheyden et al
Inflammatory reaction – communication of cells A tooth has a unique situation in the mammalian biology and presents a special challenge to the immune system (Bosshardt & Lang 2005). The tooth is a non-shedding hard structure that penetrates into the ectodermal soft tissue envelope covering the organism. Unlike other appendages like hair and nails, the tooth is the only natural example of such an appendage penetrating the basal lamina of the organism’s ectodermal sheath. Usually, if such an interruption of the ectodermal integrity occurs by a foreign body, the organism would expel this foreign structure and restore the ectodermal continuity by epithelialization of the resulting wound. This process usually involves bacteria by developing a putrid inflammatory reaction, such as what happens when transcutaneous pins of external fixators are used in orthopedic surgery. The marginal gingiva, however, is a zone of tolerance for the penetration of the ectoderm. This tolerance involves specialized tissues including the epithelial and connective tissue attachment apparatus that provide a biological seal between the tooth and the gingival soft tissues. These periodontal cells, unlike other mesenchymal cells, have the embryological origin from the ectomesenchyme derived from the neural crest, what may provide this unique property to the oral tissues, as the same biological seal has been described not only for teeth but also for dental implants. The oral cavity in health has a very specific microenvironment where millions of bacteria can live in harmony with our host defense mechanisms, and health is preserved provided this bacterial–host balance is maintained by controlling the amount of bacterial load through our regular oral hygiene practices. It is therefore important to understand the cellular and molecular elements involved in the pathways from health to disease and from disease to repair and regeneration. The purpose of the film and this review paper is therefore to describe didactically these complex biological mechanisms organized in four phases. Phase 1: bacterial biofilm formation and development of a host response in the periodontium Phase 2: innate immune response leading to gingivitis Phase 3: role of the adaptive immune system in attachment loss and pocket formation Phase 4: down-regulation of inflammation and periodontal regeneration and repair following biofilm removal It is understood that allocating the innate and the adaptive immune defense processes to gingivitis and periodontitis is to a certain extent artificial, but we felt it may be a useful simplification for didactic reasons. Phase 1 bacterial biofilm formation and development of a host response in the periodontium Biofilms have been defined as “organized microbial communities characterized by a first group of colonizers being irreversibly adhered to a substrate or interphase in a wet media and the rest being embedded in a matrix composed of extracellular polysaccharides produced by the bacteria.” These bacteria in biofilm exhibit an altered phenotype, related both to its growth rate as well as its genetic expression. In nature, most microorganisms live in biofilms, which protect the individual microorganisms from detachment and isolation and other harmful physical, chemical, and biological factors. In the oral cavity, biofilm formation usually occurs adhered to tooth surfaces (dental plaque) forming a highly structured extracellular matrix of sticky and viscous nature, which prevents bacterial communities from being detached, but at the same time very porous allowing a fresh supply with nutrient liquid streams (Kolenbrander et al. 2010). The nature of the biofilm allows bacteria to evade from natural defense mechanisms as defensins and antibodies in saliva and cervical fluid. The hard tooth surface is one of the few examples in the human body that provides a non-shedding hard surface on a wet media, where bacteria can adhere and form biofilms that can develop a high degree of complexity (Bosshardt & Lang 2005; Marsh & Devine 2011). The growth and maturation of these bacterial communities will heavily depend on the ecological equilibrium within the oral cavity. Bacterial species have coevolved with the host defense mechanisms resulting in a finely balanced system (Marsh & Devine 2011). The combination of these natural host defense mechanisms with the own subject oral hygiene practices usually limits the growth and maturation of the biofilms, and these health-related microbiota coexist with a healthy oral cavity or early stages of gingivitis. This balance, however, can be disturbed by either quantitative (higher bacterial load) or qualitative (growth of pathogenic species) changes in the biofilm © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd and/or changes in the host defenses resulting in the development of disease. The biofilm formation starts when proteins that occur naturally in human saliva, such as alpha-amylase and statherin, precipitate on the tooth and form an organic layer: the so-called pellicle. This pellicle layer facilitates the initial adhesion of single bacteria to the tooth surface. These socalled first and early colonizers are usually Streptococcus sp., Actinomyces sp., Lactobacillus sp., and Candida sp., which have specific membrane receptors (adhesins) on the bacterial cell surface that recognize pellicle proteins and also by other non-specific physicochemical interactions. Once the first layers of adhered bacteria are formed, other bacteria colonize and adhere to the bacterial surface forming complex structures with the help of the extracellular matrix composed of extracellular polysaccharides produced by the bacteria. The interior of the biofilm is usually a highly organized structure, combining piles of bacterial communities (towers) forming the typical mushroom-like images with channels or circulation systems where the bacteria exchange their nutrients and genetic material. Bacteria in biofilm release vacuoles, in which bacteria exchange genes and signals, which allow them to multiply and mature within a protected space (Marsh & Devine 2011). Inside the biofilm, bacteria compete for nutrients and favorable environmental characteristics, what inhibits some species in favor of others. They also have communication systems, the so-called quorum sensing, which allow them to respond to population density changes (Platt & Fuqua 2010). This is accomplished by the release of small concentrations of bacterial molecules, such as acylhomoserine lactones (AHLs) and oligopeptides, which modify the genetic activity and adapt the biofilm phenotype to the external environment. The composition of the oral biofilm is very heterogeneous, as the oral cavity forms a habitat where up to 1000 different bacterial species may coexist (oral microbiome), although most of these species are still unknown as they are yet uncultivable. The composition of the dental biofilm usually evolves with its growth and maturation, as well as with the specific ecological environment, which may determine the physicochemical atmosphere that will favor the growth of certain bacteria in detriment of others. For example, anaerobic, gram-negative, and mobile pathogenic bacteria primarily colonize the oxygen-poor deeper layers of the older mature plaque. 3 | Clin. Oral Impl. Res. 0, 2013 / 1–9 Terheyden et al
Inflammatory reaction – communication of cells These periodontal pathogens are therefore located predominantly in the mature subgingival plaque where typically there is a basal layer containing dead and inactive cells, an intermediate layer where usually gram-negative anaerobic bacteria such as Tannerella forsythia and Fusobacteria sp. are found and a top layer in which pathogens such as Porphyromonas gingivalis are present. Outside the biofilm, a fourth layer without a clear organization usually contains mobile spirochetes depicting the typical bacterial aggregates as test tube brushes or the Cytophaga–Flavobacterium–Bacteroides cluster (CFB cluster) (Zijnge et al. 2010). These outer layers of the subgingival biofilm, containing the most pathogenic species, are spatially located in close vicinity or in contact with the gingival sulcular epithelium (Fig. 1). In fact, the most pathogenic species are those that secrete end products, such as lipopolysaccharides and toxins, and are able to invade the tissues and to trigger a strong host response. There are certain species, such as P. gingivalis or Aggregatibacter actinomycetemcomitans, with demonstrated invasiveness able to colonize gingival tissues and exerting their pathogenic activity in situ. In presence of periodontal pockets, the subgingival biofilm is well protected from the natural antibacterial defense mechanisms of the oral cavity, such as saliva and its outer layers in contact with a usually ulcerated pocket epithelium, hence in direct contact with the gingival connective tissues and blood vessels. Depending on the composition and the presence of certain species in the subgingival biofilm, the induced host response in the local tissues will be stronger and more or less efficacious, as the virulence of some of these bacteria resides in their ability to circumvent the host response (by developing a capsule that resists phagocytosis, by secreting proteins, Fig. 1. Screenshot from the film. The plaque is structurally organized: Three-dimensional biofilm with an anaerobic, gram-negative bacterial flora in the deeper layers; bacteria have virulence factors, which enable them to invade the tissues and escape the immune defense. 4 | Clin. Oral Impl. Res. 0, 2013 / 1–9 such as the gingipains, that lyse the antibodies, etc.). As the biofilm matures, it also becomes calcified and forms calculus. In a healthy status, salivary proteins hold the mineral salts in solution, thus inhibiting their spontaneous crystallization, but when biofilm grows, the minerals on the tooth surface do not have access to saliva and become insoluble starting the process of calculus formation. Once the calculus is formed, the biofilm is prevented from detachment and the apical progression of the biofilm formation is enhanced. Compared to hard surfaces, much less biofilm organization usually occurs on mucosal surfaces, as these surfaces in contrast to hard surfaces clean themselves by a continuous desquamation. The epithelial attachment is a highly specialized structure where the junctional epithelial cells strongly attach to the tooth surface by a basal membrane and hemidesmosomes, and the underlying dentogingival fiber apparatus provides a mechanical support to prevent from detaching. The antibacterial defense mechanism at this level is assured by the high regeneration and desquamation rate of the junctional epithelium and the continuous outflow of gingival fluid through the periodontal sulcus (Fig. 2). Moreover, the cells of the junctional epithelium have specific antibacterial mechanisms, as they express antibacterial proteins like human b defensin 1 and chemokines and other molecules that promote the migration of PMN toward the gingival sulcus. Among these are the intercellular adhesion molecule-1 (ICAM-1) that guides PMN toward the bottom of the sulcus and IL-8 that sends chemotactic signals to PMN, which migrate in high numbers through the junctional epithelium. These constant bacterial–host interactions require loose and repeatedly opening and closing of the intercellular contacts between the epithelial cells in the junctional Fig. 2. Screenshot from the film. Part of the natural defense system: In the gingival sulcus, junctional epithelial cells are continuously exfoliated and removed together with bacteria by the flow of crevicular fluid. and sulcular epithelia, which may allow specialized bacteria to invade the epithelium and exert their pathogenicity in situ (Bosshardt & Lang 2005). Phase 2 innate immune response leading to gingivitis Innate immunity is the first line of defense and it is by nature non-adaptive. The cells responsible for the innate immune response, such as PMN, macrophages, and dendritic cells, although carrying receptor molecules that recognize patterns specifically associated to bacteria, are not able to recognize specific antigenic determinants and therefore are not able to adapt their reaction to the specific bacteria. Polymorphonuclear leukocytes are the first and predominant cells of the innate immune system in early gingivitis lesions (Garlet et al. 2005). Recruitment of these cells has been well investigated (Silva et al. 2007). When the biofilm forms on the tooth surfaces in vicinity of the junctional epithelium, they are recognized by the cells from the innate immunity through certain molecular patterns that exclusively appear in bacteria. These are called pathogen-associated molecular patterns (PAMPs) and include lipopolysaccharide (LPS), peptidoglycans and lipoteichoic acids, N-formylmethionine, and lipoproteins. These molecules are recognized by pattern recognition receptors (PRRs) on the surface of resident PMN and macrophages. One such PRR is the Toll-like receptor (TLR) that once stimulated by the bacterial antigen induces an intracellular response through the NF-jB and mitogen-activated protein (MAP) kinase pathways inducing the cell to secrete specific proteins. The activated cells from the innate immunity system then release pro-inflammatory cytokines and co-stimulatory molecules, like tumor necrosis factor (TNF) alpha, interleukin-1 (IL-1), and the chemokine CXCL8 also called interleukin-8 (IL-8). These chemokines (IL-8) are responsible for promoting further recruitment of PMN from the blood vessels and the cytokines, such as IL-1 by inducing the expression of E-selectin on endothelial cells. E-selectin allows loose reversible attachments of PMN to the inner wall of the blood vessels. Microscopically, the PMN are fished out of the blood stream and roll along the walls of the postcapillary venules. Finally, endothelial cells express intercellular adhesion molecules (ICAMs), which firmly bind to CD11a, b, c/CD18 integrin receptors on the leukocyte surface. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd Terheyden et al
Inflammatory reaction – communication of cells ICAMs are stimulated by TNF. Tight junctions between the endothelial cells are loosened, thus allowing the passage of the PMN. Histamine being released by mast cells during acute inflammation also causes the endothelial cells to separate and increases vascular permeability. Finally, the PMN digest the basal lamina of the blood vessel by membrane-standing proteinases and thus gain access to the connective tissues. This process by which the PMN squeeze themselves through gaps in the blood vessel wall is called diapedesis. Once in the connective tissue, the PMN move along a chemotactic gradient to the site of bacterial infection. IL-8 is a chemokine specific for PMN. Other PMN chemoattractants include complement fragments C3a and C5a, arachidonic acid derivatives such as leukotriene B4 (LTB4), 12-hydroxy-eicosanotetraenoic acid (12-HETE), and platelet-activating factor (PAF). The presence of extracellular matrix that may block the PMN migration is digested by metalloproteinases (Verollet et al. 2011). Once they have reached the site of the infection, the PMN recognize bacterial antigens through their specific PRRs on their cell membrane, such as TLRs and nucleotide oligomerization domain receptors (NOD). This bacterial recognition process is highly improved in presence of specific peptides from the complement system (opsonization). The opsonizing factor C3b marks bacteria with an antigen that facilitates phagocytosis. Factor C5b also facilitates the lysing of bacteria by forming pores (membrane attack complex (MAC)), which eventually will cause the bursting of the bacterial cell (Fig. 3). Defensins, which are ring-shaped proteins secreted by epithelial cells, operate in a similar manner. PMN also express early in response to bacteria stimuli different molecules, such as defensins, permeability-increasing proteins, LL-37, lactoferrin, cationic proteins, and others that facilitate the developing of the inflammatory response (Kinane et al. 2007). Fig. 3. Screenshot from the film. Bacterial cells burst osmotically by pore formation after contact with the membrane attack complex of the complement system. With the support from the opsonins, PMN are able to detect, bind, and phagocyte the bacteria. Once in their cytoplasm, they are lysed through different substances contained inside their granules. PMN also release reactive toxic oxygen and chlorine radicals, which are not only toxic for bacteria, but also for the host tissues in the vicinity of the PMN (Nussbaum & Shapira 2011). Enzymes released from the phagolysosomes of PMN destroy the extracellular matrix of the host connective tissues, and therefore, in situations where there is massive PMN recruitment, tissue damage, pus, and abscess formation may occur. If this first line of defense mediated by the PMN is overcome by the bacterial challenge, macrophages become the second cell from the innate immune system. These cells have also membrane-bound receptors such as TLRs that recognize the bacterial PPRs, such as the lipopolysaccharides from gram-negative bacteria. They also require the presence of complement (factors C3a and C5a) and immunoglobulines (IgG) to promote the bacterial phagocytosis (opsonization). Once phagocytized, the bacteria are lysed by high reactive radicals resident in the granules within its cytoplasm. Macrophages are also very active secretory cells when stimulated by bacteria, releasing not only pro-inflammatory cytokines, such as interleukins 1-beta, IL-6, and tumor necrosis factor-alpha, but also metalloproteinases (MMPs) and other active molecules. These two consecutive lines of defense against the bacterial aggression are responsible for the inflammatory reaction typical of gingivitis. Within this reaction, the gingival connective tissue is loosened and degraded to create space for the specific immune reaction (Fig. 4). The process of extracellular matrix degradation is mediated by collagen-degrading enzymes such as cathepsin G secreted by Fig. 4. Screenshot from the film. The inflammatory reaction creates space for the immune cells by degrading soft and hard tissue matrix. The tissue balance is shifted from matrix protein synthesis to degradation. The result is a loss of structural integrity of the gingival tissues. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd PMN or by metalloproteinase-8 (MMP-8) secreted mainly by the macrophages. This loss of connective tissue can be restored if the inflammatory reaction is effective in combating the bacterial challenge. Inflammation will then lead way to tissue formation and regeneration restoring gingival health. The cells responsible to restore tissue homeostasis are also the macrophages that have a high secretory activity through the release of growth factors and molecules that stimulate fibroblasts to produce collagen and extracellular matrix. Gingivitis is therefore a reversible condition provided the bacterial challenge can be contained, mainly by the innate immunity mediated through the PMN and macrophages. Phase 3 role of the adaptive immune system in attachment loss and pocket formation Periodontitis is a chronic inflammatory disease primarily affecting the connective tissue and secondarily the alveolar bone. The inflammatory processes physiologically create space for the immune reaction against the presence of millions of bacteria residing in the subgingival biofilm, some of which possessing virulence factors that enable them to evade the host defenses. A clear example of such virulence factors are the gingipains, proteases secreted by P. gingivalis that can degrade extracellular matrix proteins of the host (Ruggiero et al. 2013), such as fibronectin and tenascin-C, what leads to detachment of host fibroblasts from the extracellular matrix. Fibroblast function depends heavily from its adhesion to the ECM via integrins (see Phase 4) and when detached, fibroblasts are committed for cell death, what results in tissue destruction and disease progression. Moreover, when pathogens are able to evade the adaptive immunity, the ineffective responses lead to the release of high number of pro-inflammatory cytokines that stimulate fibroblasts, macrophages, and epithelial cells to synthesize and release MMPs, with the resulting connective destruction and attachment loss. Clinically, the progression of periodontitis can be monitored by assessing the changes in the probing attachment levels over time. As bone resorption occurs secondarily to attachment loss, the evaluation of the changes in radiographic bone levels can also monitor the progression of periodontitis over time. Bone resorption occurs from the disturbance between bone formation and bone resorption 5 | Clin. Oral Impl. Res. 0, 2013 / 1–9 Terheyden et al
Inflammatory reaction – communication of cells that physiologically occurs in bone by the competitive action of osteoblasts and osteoclasts. This process of bone remodeling is the consequence of coupling the osteoblasts and osteoclasts in the so-called basic multicellular unit (BMU). When the action of the osteoclast predominates, the resulting effect will be net bone loss. Under physiological conditions, one key regulatory cell is the osteoblast and its resting variant, the bone lining cell, as well as its precursor, the stromal cell (Wei et al. 2005). Under the influence of parathyroid hormone, osteoblasts retract from the bone surface (Lindskog et al. 1987). Osteoclasts are large multinucleated cells derived from hematopoietic precursors of the monocyte– macrophage lineage, which originate in the bone marrow and enter the site via the blood stream. Osteoclast precursors migrate into the afflicted alveolar bone from surrounding blood vessels along an IL-8 gradient. Both osteoclasts and bone marrow stromal or their osteoblast progeny express receptors for macrophage-colony-stimulating factor (M-CSF) and receptor activator of NK-kB ligand (RANKL, also called osteoprotegerin ligand OPG-L or TRANCE), two essential cytokines for the stimulation of osteoclasts, RANKL being the key pro-osteoclastic cytokine and M-CSF contributing to its recruitment, differentiation (Crockett et al. 2011), and survival, hence regulating the amount of bone resorption (Hodge et al. 2011). RANKL is also expressed by activated T cells, what demonstrates the close relationship between the immune system and osteoclast function. Monocytes, osteoclasts, and their precursors express the osteoclast-associated (OSCAR) receptor (Barrow et al. 2011), which contains a tyrosine-based activation motif (ITAM), a typical receptor in immune-competent cells. The activation of OSCAR promotes osteoclastogenesis, thus supplementing M-CSF and RANKL as a third signal for osteoclastogenesis. The ligand of OSCAR is still unclear but there is some evidence that it is the collagen of exposed bone matrix, and this may be the mechanism how osteoclasts detect exposed bone surfaces (Barrow et al. 2011). To get in direct contact with the bone surface, osteoclasts precursors penetrate the layer of resting bone lining cells covering the bone using ICAM adhesion molecules to contact the bone lining cells (Nakahama 2010). Bone lining cells form a canopy connected with capillaries in which bone remodeling takes place (Andersen et al. 2009). Osteoclast precursors differentiate into mature osteoclasts. They attach with their membrane-bound avb3 6 | Clin. Oral Impl. Res. 0, 2013 / 1–9 integrins firmly to osteopontin protein endings, which stick out of the bone matrix (Helfrich et al. 1996; Dossa et al. 2010). Osteoblasts balance the activity of membrane-bound RANKL by action of soluble osteoprotegerin (OPG), a decoy receptor of RANKL that inhibits RANK–RANKL interaction (Fig. 5). The balance of RANKL and OPG is important for the homeostasis of bone, and it is highly influenced by the immune system. Osteoblasts have membrane receptors for hormones, such as estrogens, interleukins, prostaglandins, and other messenger molecules. Their influence results in a higher or lower rate of osteoclast differentiation and thus modulates bone resorption. For instance, IL-1 and TNF alpha promote RANKL synthesis in the osteoblasts, thus promoting osteoclast activity (Wei et al. 2005). Once osteoclasts have completed their bone destruction activity, they attract and pull osteoblasts into the resorption lacunae. This is carried out by a mutual exchange of the membrane-bound ephrin receptor/ligand system (Mundy & Elefteriou 2006; Matsuo & Otaki 2012) reminiscent of handshaking (Fig. 6). The bone lining cells clean the bottom of the resorption lacuna by their membrane-bound metalloproteinases (Everts et al. 2002) and also detach from the bone growth factors like bone morphogenetic proteins (BMPs), which are stored and trapped within the exposed bone matrix. These growth factors induce new differentiation of osteoblast precursor cells, which then differentiate into osteoblasts that deposit osteoid and bone. The resorption lacuna is refilled, and the net amount of bone is fully restored completing the coupling process. In presence of inflammation, this coupling process is altered and leads to incomplete rebuilding of bone. For example, Fig. 5. Screenshot from the film. The bone balance: osteoblasts can control osteoclast differentiation by releasing osteoprotegerin (red). It is a soluble inactivating decoy receptor for RANKL, which in turn decreases osteoclast differentiation. Under inflammatory conditions, RANKL from other sources like T helper cells can bypass this system leading to massive osteoclast differentiation and thus inflammatory bone resorption. Fig. 6. Screenshot from the film. The coupling of bone resorption and bone matrix synthesis involves osteoclasts, which attract osteoblasts by virtually pulling them into the resorption lacuna by an exchange of the ephrin receptor/ligand system. TNF alpha, by inhibiting Dickkopf-1, inhibits multiple osteoblast functions, such as WNT pathway resulting in less bone formation (Diarra et al. 2007). As discussed in the last chapter, when the innate or non-specific immunity is not able to cope with the bacterial challenge, it activates the adaptive immune system. This is accomplished by a group of cells that have specific ability to present the bacterial antigens to the immune-competent cells, the T cells. The so-called antigen-presenting cells (APCs) are exemplified by the dendritic cells, which reside in the epithelium and take up bacterial antigens, travel to the lymph nodes, and there present these antigens to naive T cells. Upon activation, CD4+ T cells develop into distinct subtypes of effector T cells depending on the cytokine profile they produce; T helper (Th) cells were traditionally divided into two main subsets (Mosmann et al. 2005). Th1 cells, which are induced by IL-12 and IFN-g, produce mainly IFN-g and IL-2 but also RANKL (see below) and are involved in cellular immunity, promoting cytotoxic CD8+ T cells and maximizing the killing efficacy of macrophages. Th2 cells, which mainly produce IL-4, IL-5, and IL-10, are involved in humoral (or antibody-mediated) immunity. These cytokines are required to stimulate B cells to clonally expand and differentiate into antibody-producing cells (immunoglobulins). A third subset of T helper cells has been recently described, the Th17 cells. They serve a function in antimicrobial immunity at epithelial/mucosal barriers and hence in periodontitis (Gaffen & Hajishengallis 2008) and produce IL-17, a potent pro-inflammatory cytokine that promotes PMN recruitment. Furthermore, IL-17 is known to enhance osteoclastogenesis via induction of RANKL expression on osteoclastogenesissupporting cells, while the expression of © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd Terheyden et al
Inflammatory reaction – communication of cells RANKL on Th17 cells may also participate in enhanced osteoclastogenesis (Sato et al. 2006). Regulatory T cells (Tregs) constitute another distinct subset of CD4+ T cells, which play an essential role in establishing dominant self-tolerance, by controlling inflammatory responses and maintaining immune and bone homeostasis (Sakaguchi 2011) as it has been shown that Tregs can suppress osteoclast formation (Zaiss et al. 2007; Kong et al. 2012). It has been recently shown that specific bacteria have the ability to stimulate specific TH cells pathways by modifying the cytokine profiles. For example, A. actinomycetemcomitans and P. gingivalis may specifically stimulate the TH2 pathways by enhancing the production of IL-4, IL-5, and IL-6 (Vernal et al. 2008, 2009) and thus promoting the antibody-mediated bacterial killing pathway. As stated earlier, the most pathogenic bacteria have intrinsic mechanisms to evade the action of antibodies and thus promote the release of pro-inflammatory cytokines with ability to promote connective tissue and bone destruction, while not being effective to destroy the bacteria. Lymphocytes predominate in chronic periodontal lesions, being mainly B cells and especially plasma cells, located subjacent to the pocket epithelium and in the central portion of the lamina propria, and their proportion increases with the progression of the disease, what demonstrates that the ineffective activity of the adaptive immune system aggravates periodontal tissue destruction (Pestka et al. 2004; Nakajima et al. 2005). There are multiple examples of cross talk between bone and the immune system, what is referred to as osteoimmunology. In response to bacterial antigens, the cells and the pro-inflammatory cytokines of the adaptive immune system promote the RANKL/ OPG system toward osteoclastogenesis and also promote osteoclast survival, which results in more and prolonged bone resorption (Kajiya et al. 2010). Moreover, the immune system may also interfere with the coupling of osteoclast and osteoblast, and consequently, the bone resorption lacuna is not completely refilled. In fact, osteoclasts originate from monocytes, which are immunecompetent cells, and dendritic cells have the plasticity to directly trans-differentiate into osteoclasts (Alnaeeli et al. 2007). A few mechanisms how the adaptive immune system acts on bone are listed below: 1. Specific T helper cells, the Th1 lymphocytes (CD4+), synthesize massive amounts of new soluble RANKL, directly activating 2. 3. 4. 5. additional osteoclasts (Kawai et al. 2000; Teng et al. 2000; Leibbrandt & Penninger 2010). This has already been shown in clinical studies (Belibasakis et al. 2011). Th2 cells produce interleukin-4, which causes B-cell stimulation and the synthesis of antibodies (Fig. 7). Antibodies may aggravate periodontal bone destruction, as they opsonize bacterial antigens and stimulate macrophages activity, which in turn activate pro-inflammatory cytokine production and osteoclast recruitment (Berglundh et al. 2002). T cells synthesize chemoattractant proteins for osteoclast precursors such as monocyte chemoattractant protein-1alpha (MCP-1alpha), also called chemokine ligand 2 (CCL2) (Silva et al. 2007; Repeke et al. 2010), RANTES (regulated and normal T cell expressed and secreted protein), also called chemokine ligand 5 (CCL5). IL-1 and TNF alpha from T cells promote osteoclastogenesis through the upregulation of RANKL (Wei et al. 2005). Clinically, this self-reinforcing inflammation may lead to serious damage of bone and soft tissue. Apart from the molecular interference with cytokines of the osteoblast/osteoclast balance, interleukin-17 from Th17 cells (Gaffen & Hajishengallis 2008) may induce bone resorption secondary to general tissue damage by massive recruitment of PMN, reaching toxic levels for marginal periodontal tissues, especially in aged individuals (Eskan et al. 2012). IL-17 polymorphism is clinically associated with the severity of periodontitis (Correa et al. 2012). The coupling of osteoclast and osteoblasts is impaired by the dedifferentiation of osteoblasts by pro-inflammatory cytokines like TNF and IL-1 derived from Th1 cells (Graves et al. 2011). Fig. 7. Screenshot from the film. B cells develop into plasma cells, releasing antibodies, which opsonize bacteriae and fuel the inflammation. The labeled bacteriae are phagocytized by macrophages, which in turn secrete pro-inflammatory cytokines. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd If the activity of both innate and adaptive immune system is able to control and eliminate the bacterial challenge, inflammation is down-regulated and the processes of regeneration and repair can occur. These processes will be discussed in the next chapter. Phase 4 down-regulation of inflammation and periodontal regeneration and repair following biofilm removal The extracellular matrix has central importance for the healing of an injured or inflamed tissue. The organic extracellular matrix contains collagen and proteoglycans, and these structural components are fundamental for withstanding the mechanical loads, like shear stress being exerted on the gingiva. On a cellular level, these proteins are also involved in fibroblast and osteoblast adhesion via integrins to RGD motifs of the glycoprotein fibronectin. Using these integrin attachment sites, cells are able to migrate by ameboid movements into the healing area. Fibroblast attachment and motility within the ECM are regulated by the balance between adhesive and anti-adhesive signals (Ruggiero et al. 2013). Furthermore, proteoglycans are needed to bind and protect growth factors, such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), from dissolution and enzymatic degradation. In states of chronic inflammation, the inhibition of proteolytic activity is important to restore tissue homeostasis. Macrophages release tissue inhibitors of metalloproteinase (TIMPs) that form complexes with the matrix metalloproteinase and block their activity. When the inflammatory status is maintained, however, this balance between matrix metalloproteinase MMPs (and other aggressive proteinases like cathepsin from inflammatory PMN cells) and TIMPs is shifted. Secondly, the pro-inflammatory cytokine environment generated by the inflammation hampers regeneration by fibroblasts. Similarly, in bone metabolism, bone degradation occurs through the imbalance of the RANKL/OPG ratio and secondly by hampering regeneration due to interference with the coupling of osteoclast and osteoblasts. The end result of tissue destruction vs. healing may be predicted by the PMN content in the lesion. Persistent neutrophils can act as perpetrators of persistent inflammation (Nussbaum & Shapira 2011) and develop abscess formation with resulting tissue destruction. The prerequisite of disease attenuation is the 7 | Clin. Oral Impl. Res. 0, 2013 / 1–9 Terheyden et al
Inflammatory reaction – communication of cells control of the bacterial infection by removal of the invading bacteria and their products (e.g., lipoproteins). This process will occur when the biofilm is eliminated either professionally or through effective oral hygiene practices or by an effective immune system able to lyse the bacteria by phagocytosis or antibody-mediated lysis. For a controlled down-regulation of the inflammatory reaction, it is important that PMN undergo controlled apoptosis instead of uncontrolled necrosis with the resulting release of aggressive enzymes and pus formation. In this controlled down-regulation of PMN activity, resolvins may play a role (Van Dyke 2011). Resolvins are end products from the COX-2 pathway composed of omega-3 fatty acid eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Hasturk et al. 2012) that interact with the recruitment of inflammatory cells (Serhan et al. 2002). In healing and regeneration, macrophages also play an important role, as they act like a cellular switch, which depending on the cytokine environment, may release pro-inflammatory mediators or on the contrary synthesize anti-inflammatory signals and growth factors. They can live under hypoxic conditions and release angiogenic factors like VEGF. They can also increase collagen production by synthesizing FGF that stimulates fibroblasts to migrate to the healing site and synthesize collagen. In the adaptive immune system, down-regulation of the inflammatory process is mediated by regulatory T cells. These specialized CD4+ cells suppress host responses such as pro-inflammatory Th1 cytokines by increasing the secretion of IL-10 and also directly by stimulating the expression of OPG and TIMPs, thus fostering the rebuilding of bone matrix (Garlet 2010). In every inflammatory process, there is always a balance between disease progression and matrix rebuilding whenever the aggression that triggers inflammation is removed or eliminated. In many of these situations, the disease attenuation results in complete tissue regeneration. In other tissue compartments, such as in teeth or in the periodontium, the matrix degradation is not completely reversible, hence resulting in repair rather than in regeneration. In periodontitis, the elimination of subgingival plaque and calculus results in repair by a long functional epithelium that restores the epithelial attachment. But this downgrowth proliferation of epithelial cells along the previously infected root surfaces prevents the formation of new connective tissue attachment. Therefore, the regeneration of the affected periodontium rarely occurs, but in a small extent at the bottom where this attachment will occur though new cementum formation and subsequently new periodontal ligament and new alveolar bone formation. This periodontal repair by long junctional epithelium may be functionally less able to withstand the bacterial challenge, as epithelial cells easily detach from the root surface and facilitate pocket re-formation and re-establishment of the pathogenic subgingival bacterial plaque. There is therefore potentiality for true periodontal regeneration, but the high turnover of epithelial cells facilitates their downgrowth along the root surface, rather than the new cementum formation and coronal growth of the new attachment, which is much slower process (Nunez et al. 2012). It is a general biological principle in mammalian tissues that an inflammatory reaction requires space in the tissues for the immune system and creates it by degradation of extracellular matrix. The involved cellular processes and cytokines are similar in soft tissue and in bone, as discussed in the present paper. However, in teeth such an inflammatory tissue damage may be less reversible than in the general organism. That explains the progressive nature of periodontitis by episodic bursts of tissue destruction and incomplete repair. In general, periodontal disease progression depends on the subject’s ability to control biofilm formation and maturation. Disease progression may depend on the specific host susceptibility, which regulates the efficacy and strength of the innate and adaptive immune responses. 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