Avances en Patogenesis de La e.p.page,Offenbacher.1997

Periodontology 2OW. Vol. 14, 1997, 216-248 Printed in Denmark . All rights reserved

Copyright 0 Munksgaard 1997

PERIODONTOLOGY 2000 ISSN 0906-6713

Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions ROY C. PAGE, STEVEN OFFENBACHER, HUBERT E. SCHROEDER, GREGORY J . SEYMOUR &KENNETH S. KORNMAN

Purpose and rationale

The purpose of this volume of PERIODONTOLOGY 2000 is to present the basic concepts and facts constitut- ing the current understanding of the pathogenesis of human periodontitis based on the published literature. This chapter summarizes, organizes and as- sembles the most important points in the preceding chapters and, where useful, adds new information, to further synthesize and develop new hypotheses, concepts and ideas and to make clinical appli- cations. Instead of providing original references, we cite the other chapters in this volume or sections thereof as references. Additional references are provided where new information not referenced in the other chapters is presented. Our comments are root- ed in the existing literature, but we freely speculate beyond existing data and point out areas and directions for future research.

Theoretical approach

The paradigm of the pathogenesis of periodontitis is shifting. Periodontitis is a family of related diseases that differ in etiology, natural history, disease progression and response to therapy but have a common underlying chain of events. For example, the histopathological and ultrastructural features and pathways of tissue destruction as well as healing and regeneration are very similar if not identical for all forms of periodontitis. The same basic pathological

mechanisms underlie all forms of bacterially induced periodontitis. These shared events are influenced by disease modifiers, both genetic and environmental or acquired, which may differ from one stage and form of disease to another. The clinical picture observed is a result of the complex interplay of these events and modifiers and the microbial chalfenge. The severity and rate of progression of disease feed back to influence the nature and magnitude of the microbial challenge by, for example, in- fluencing the pH and availability of oxygen and various nutrients in the periodontal pocket. This is a new basic concept that was arrived at independently by multiple observers. It provides the overall frame- work upon which this volume has been constructed (in this volume, see Fig. 1 in Page & Kornman (51) and Salvi et al. (SO)).

Various forms of periodontitis with differing clinical manifestations, natural histories and responses to treatment result from differences in the microbial etiology among groups and individuals (such as different species or combination of species) and/or factors that modify host response mechanisms or alter innate susceptibility. The latter include but are not limited to such systemic conditions as diabetes mellitus and compromised host defenses, hereditary factors and such environmental factors as cigarette smoking and stress (in this volume, see: Hart & Korn- man (23) and Salvi et al. (60)) (50). These modifying factors account for the differences observed in different periodontal conditions and they do so by inter- fering with regulation, activation or inhibition of various components of the mechanisms of host response,

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tissue homeostasis and repair. An integral and essential part of this hypothesis and an aspect well sup- ported by extensive evidence is that the components of host response that normally provide protection are the same ones that accomplish destruction.

The modifying influences may affect the age of disease onset, the patterns of observed bone and tissue destruction, the rates of disease progression, the response to various kinds of therapy and the severity and frequency of disease reoccurrence. These modifying influences, such as some hereditary factors, may endure for life or may be present or absent or vary in magnitude of effect at various times in life. For example, individuals born with leukocyte adhesion deficiency syndrome 1, in which the patients are completely deprived of neutrophil protection, manifest recurrent enduring acute infections, including severe periodontitis, when they are very young. In the late teenage years and beyond, however, the frequency of infections in these patients decreases dramatically for reasons that are not understood but are probably related to amplification of non-neutrophil-based aspects of host defense, somewhat like development of collateral circulation to heart muscle following coronary artery throm- bosis. This idea fits with the known high degree of redundancy among the components of the host defense system.

This theoretical approach to the pathogenesis of periodontitis permits a better understanding of why individuals vary greatly in disease susceptibility, clinical manifestations, rate of progression and therapeutic responsiveness. Of much greater importance, it points the way to new and novel approaches to prevention, diagnosis, treatment and prognosis. In- deed, it points towards eventual control of this family of diseases in many human populations. For example, a polymorphism in the interleukin 1 (IL-1) gene family has been identified that, when positive, enhances the probability of having severe periodontitis later in life by many times (31). Approaches to successfully controlling and preventing these diseases in individuals and in populations are now on the horizon. Day-to-day practice is changing from identifying disease presence and severity and applying measures to arrest progression to a new paradigm consisting of regenerating destroyed tissue in the individuals already affected and preventing disease and maintaining health in healthy individuals. Prevention is based on assessment of risks that enhance disease susceptibility, progression and severity by affecting common, shared events in the pathogenesis and applying measures to reduce the risk.

The microbial challenge

Periodontitis is an infectious disease

Periodontitis is an infectious disease, and the major pathogens have been identified. Bacteria are essential but insufficient to cause disease. Host factors such as heredity and environmental factors such as smoking are equally as important as determinants of disease occurrence and severity of outcome. The interaction of these factors is well demonstrated by recent research findings that demonstrate the complexity of the interactions in multifactorial diseases and include roles for specific bacteria and genetic and environmental modifymg and risk factors. Re- cent work has shown that individuals with localized juvenile periodontitis, but not generalized juvenile periodontitis, have elevated levels of immunoglobulin G2 (IgG2), which are genetically controlled (76). The data suggest that a genetically determined enhanced immune response to Actinobacillus actinomycetemcomitans assists in limiting the disease to a localized clinical expression. Smoking has been shown to further compromise the individuals with generalized disease by lowering the IgG2 response. Interestingly, smoking did not reduce IgG2 in the localized juvenile periodontitis group. The specific bacterial challenge of A. actinomycetemcomitans therefore produces very different clinical disease patterns that appear to be determined by combinations of genetic factors and smoking.

Important new findings and perspectives related to the microbial challenge are listed in Table 1. Most cases of periodontitis are caused by a small number of bacteria species. Although the subgingival flora can harbor hundreds of species, serotypes and bio- types, experts agree that, except for acute necrotizing periodontitis, Porphyrornorzas gingivalis, Bacter- oides forsythus and A. actinomycetemcomitans cause most cases of periodontitis (8). This is a major step forward for several reasons. It permits tests to be developed for detecting the presence of specific periodontopathic bacteria and provides a practical rationale for monitoring patients and normal subjects over time. As the capacity to identify early in life the individuals who are highly susceptible for development of severe periodontitis in later life is developing, monitoring the flora for these pathogens becomes doubly important. Equally important, the fact that only a few predominant species are involved in causing human periodontitis makes it more likely that a vaccine can be developed for prevention in high-risk individuals, families and popu-

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Table 1. The pathogenesis of periodontitis: new findings and new perspectives on the bacterial challenge

A limited number of specific bacteria are essential to initiate disease and fuel progression but insufficient to explain the prevalence and severity of periodontitis.

environmental factors as smoking, are major determinants of disease occurrence and severity. Subgingival microbial plaque behaves as a biofilm. Bacteria living as biofilms are difficult to eradicate by antimicrobial means and are protected from host defense mechanisms. Biofilms are effectively managed by physical disruption and removal. In periodontitis, an enormous load of gram-negative bacteria is located adjacent to the inflamed tissue. Some of the recognized periodontal pathogens have multiple genetically distinct clonal types. Some of the clonal types are probably more virulent than others. Bacterial transmission is common in family units. The recognition of bacterial transmission and the

Host factors, including heredity and such

increased ability to identify individuals at high risk for severe disease have major implications in preventing and treating periodontal diseases. Porphyromom gingivulis can impair the local neutrophil response to itself and other microorganisms in the plaque by blocking the normal adhesion molecule response.

lations and for possible treatment of recalcitrant cases.

Subgingival microbial plaque is a biofilm

Costerton et al. (9) have defined biofilm as “matrix- enclosed bacterial populations adherent to each other and/or to surfaces or interfaces”. Subgingival microbial plaque adheres tightly to the tooth root surface and manifests all the characteristics expected of biofilms (see Darveau et al. in this volume (lo), pages 12-14). In addition, in contrast to other biofilms, loosely adherent and unattached bacteria are located between the biofilm and the gingival tissue. The recent realization that subgingival microbial plaque is a biofilm and the rapidly growing body of information about the nature of biofilms in general is one of the most important conceptual advances in periodontal microbiology in many years. Although the understanding of the behavior of biofilms is still minimal, the concept explains many aspects of periodontal disease that were previously obscure.

The behavior of bacteria in biofilms is remarkably different than in planktonic cultures, and pathogenicity and virulence factor expression may be enhanced significantly. The characteristics of biofilms

Table 2. Characteristics of biofilm

Ecological communities evolved to allow the survival

The communities exhibit metabolic cooperativity. There is a primitive circulatory system. Numerous microenvironments have radically

of the community as a whole.

different pH, oxygen concentrations and electric potentials. Biofilms resist the usual host defenses. Biofilms resist systemic or local antibiotics and antimicrobial agents.

are listed in Table 2. Bacteria in biofilms may produce large numbers of proteins and other components not expressed in culture. There is evidence that they exchange information, and they build complex structures that contain a primitive circulatory system and microenvironments that vary greatly in pH, oxygen availability and nutrient availability. Electrical potential differences of more than 100 mV have been measured in biofilms (9). Almost nothing is known about the behavior of the periodontal pathogens I! gingivalis, B. forsythus and A. actinomycetemcomitans in biofilm structures. Elucidation of the structure and nature of subgingival biofilms and the behavior of periodontal pathogens residing in them provides an exciting and fertile area for future research.

The fact that plaque is a biofilm may shed light on why some bacterial species require the presence of one or more other species for survival whereas, in other cases, the presence of one given species pre- cludes the presence of another. Evidence indicates that discrete colonies of specific species may be located in intimate relation to colonies of other species and that metabolites and products may be mu- tually exchanged.

Microbial plaque is notoriously resistant to the normal host defense mechanisms. Gingival fluid, which contains complement, antibodies and all the other systems present in blood for preventing and controlling infection, flows through the periodontal pocket, continuously bathing the biofilm. Comple- ment is known to be activated, and millions of active, viable leukocytes, especially neutrophils migrate continuously into the periodontal pockets and contact subgingival plaque (see Dennison & Van Dyke in this volume (11)). Nevertheless, the bacteria survive and flourish; they spread laterally and apically along the root surface, causing tissue destruction and pocket deepening. Such behavior is understand- able in terms of the known nature of biofilms in which bacteria are protected from host defenses.

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These observations may help to explain why, in some cases, the disease progresses or reoccurs even in patients with high titers of serum and gingival crevicular fluid opsonic antibodies specific for antigens of their infecting pathogens. These observations may also dampen enthusiasm for development of vac- cines for periodontitis.

As with other biofilms, subgingival microbial plaque is extraordinarily persistent and difficult to eliminate. Because individual bacterial colonies are protected by one another and by extracellular ma- terial in which they embed themselves, they are un- usually resistant to the effects of antibiotics and other antimicrobial agents either applied locally or administered systemically. This is probably why the placement into periodontal pockets of devices that deliver enormous concentrations of antibiotics that would be expected to virtually sterilize the pocket almost always yield disappointing results. It has been well established that bacteria growing in bi- ofllms are resistant to levels of antibacterial agents several times greater than those that are inhibitory in laboratory assays. The biofilm structure may also account for the rebound in the pathogenic flora, which usually occurs quite rapidly following the use of antimicrobial agents.

Physical disruption and removal are effective ways of dealing with biofilms. This is why scaling and root planing is a remarkably effective treatment for periodontitis. It has been an essential and central component of periodontal therapy since the earliest times and remains so today. Its effectiveness relative to other forms of therapy is most certainly due to the fact that it physically disrupts and removes the microbial biofllm. Scaling and root planing is likely to remain the central component of periodontal therapy for the foreseeable future.

Periodontitis is strongly associated with major systemic diseases

There is now strong evidence that periodontitis enhances the risk for various systemic diseases, including atherosclerosis, coronary heart disease, stroke and infants with low birth weight (4,481. Subgingival biofilms on the roots of teeth are likely to be an important component of this enhanced risk. As noted above, these biofilms are recalcitrant, they reoccur following treatment and they contain numerous gram-negative bacteria that continuously shed ves- icles rich in lipopolysaccharide. A single pass of a curette in a periodontal pocket may contain up to lo8 cultivable bacteria. The biofilm load in a patient

with severe generalized periodontitis is large. If one assumes the presence of 28 teeth and considers tooth roots to be circular with an average diameter of 10 mm and an average of 5 mm of biofilm on each, the total mouth biofilm area would be about 72 cm2, or an area about the size of the back of an adult human hand. This is an enormous burden of gram-negative bacteria. Lipopolysaccharide and living bacteria from the biofilms have ready access to the connective tissues and circulation through the pocket epithelium. Bacteremia occurs during treatment by root planing (generally performed every 3- 4 months to control the disease) and even during toothbrushing and chewing (4). The subgingival biofilms may provide a major and continuous source of circulating lipopolysaccharide and even living gram-negative bacteria, which directly affect vascular endothelium and account in part for the enhanced susceptibility to various systemic diseases and the worsening of other diseases such as diabetes mellitus.

Multiple clonal types

Another major advance in the area of microbiology of periodontal diseases has been the development and application of various procedures such as the polymerase chain reaction and other DNA-based techniques to detect extremely small numbers of bacteria in complex samples and the demonstration that most species of periodontopathic bacteria such as I? gingivulis encompass a large number of genetically distinct clonal types (see Darveau et al. in this volume (lo), page 25). I? gingivulis alone may have 50-100 or more. Very little is known about which of these can cause periodontitis and which cannot. If only a small portion are pathogenic, this could account for the many studies that have failed to demonstrate a strong relationship between the presence of the putative pathogenic species and active disease and for the relatively high carrier rate in periodontally normal individuals. Major tasks for the future include identifying all the different clonal types of I? gingivulis and the other major pathogens and characterizing their pathogenicity and virulence.

Bacterial transmission

Development and application of the new DNA- based techniques has also resolved other issues, such as whether periodontopathic bacteria exist as members of the normal oral flora in very small numbers that can greatly overgrow in disease-susceptible

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individuals or whether they are transmitted from one individual to another. Several studies have shown that transmission can and does occur among individuals living in close contact, such as spouses or siblings (see Darveau et al. in this volume (lo), pages 25-26). The data indicate that when a pathogenic species is present, all members of a given family who are infected carry the same clonal type, and generally only one clonal type is present in a given pocket, patient, subject or family. The latter point, however, is not yet well documented. Nothing is known about the predominance of one clonal type over another or about differing growth requirements for the various clonal types.

These observations have major implications for preventing and treating periodontal diseases. For example, when one member of a family has periodontitis, is periodontal therapy instituted to arrest disease progression and eliminate the causative bacteria in the individual sufficient? Should all family members be tested for the pathogen or pathogens? Should all family members who are infected be treated with the goal of eliminating the pathogen from the entire family whether or not signs of periodontitis are present? In other words, is the risk of reinfection and recurrent active disease enhanced when other periodontally normal family members carry the pathogen? An approach of this sort may be specifically implicated in families in which one or more members may be highly genetically susceptible to continuing or recurrent disease, such as those with Papillon-Lefhre syndrome or any of several neutrophil abnormalities. These critical questions remain unanswered.

The unique role of l? gingivulis

Numerous potential virulence factors manifested by periodontopathic bacteria have been identified and studied. Ishikawa et al. (27) describe these in detail in this volume (pages 79-85). Among these are toxins that are lethal for leukocytes, lipopolysaccharide that can activate and perpetuate tissue destruction, products that affect immune responses and cell growth, and proteases that can destroy tissue and degrade components important in host defense such as specific antibody and complement activation products. None of these factors, however, appears to account for important aspects of the pathogenesis: for example, the very rapid overgrowth or “bloom” of such pathogenic species as €? gingivalis in the subgingival flora.

An entirely new mechanism that may account in

part for observed events was described recently. Lipopolysaccharide from Escherichia coli and most other gram-negative pathogenic species activates expression of E-selectin by vascular endothelial cells, and this triggers binding of leukocytes, especially neutrophils, and their emigration from the vessels to the site of infection. This early event triggers an acute inflammatory response and the formation of an inflammatory cell infiltrate that, under favorable conditions, can eliminate the infection. Darveau et al. (10) demonstrate in this volume (page 21) that lipopolysaccharide from P gingivalis does not have the capacity to activate E-selectin expression by endothelial cells; further, it blocks E-selectin expression by other gram-negative bacteria and their lipopolysaccharide. P gingivulis lipopolysaccharide also fails to elicit expression of IL-8 (a potent chemoattractant for neutrophils), monocyte chemoattractant protein 1 and intercellular adhesion molecule 1 expression by human endothelial cells, fibroblasts and epithelial cells. In a mouse model of inflammation, it does not elicit an immediate acute inflammatory response.

These observations suggest an unusual role for P gingivulis in establishing subgingival biofilms and in the bacterial colonization and overgrowth or bloom of specific species. By shutting down the initial step in the acute inflammatory process, I! gingivulis may not only permit its own rapid growth because of the absence of components of normal host defense but it may also make possible the establishment and growth of other species found in subgingival biofilms. These properties may be among the reasons P gingivalis is so frequently associated with active tissue destruction.

Host-parasite interactions are in biological equilibrium

The combative character of the host-parasite interaction is presumed to account for the episodic nature of periodontal disease progression. The perni- cious sloughing of lipopolysaccharide from the microbial biofilm triggers the host release of catabolic inflammatory mediators that destroy connective tissues. Since the attachment apparatus, once lost, has limited ability to regenerate spontaneously, the destructive process appears unidirectional, with greater microbial challenge resulting in more disease. At some point this destructive process usually loses momentum and subsides, presumably due to renewed host defenses, and thereby defines the epi-

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sodic nature of the disease process. One of the sim- plest potential explanations for the loss of disease momentum may be the loss of attachment itself, which temporarily moves the tissue away from the bacterial biofilm. This change in physical relation- ships would be expected to reduce the concentration of bacterial products in the tissue, perhaps sufficiently to allow the host to recover. Once disease progression is apparently stopped within the lesion, a variable period of inactivity ensues during which very limited repair occurs, only to be interrupted at some time later by yet another episode of disease activity. This process has been likened to a balance or teeter-totter, with periods of hostile microbial virulence overpowering host defenses and resulting in clinical tissue destruction. The analogy is com- pleted by a renewed host defensive antibody strategy to limit the microbial damage, tipping the balance temporarily in favor of the host.

Mathematically speaking, at a patient level a random hit or first order model seems to reasonably ap- proximate the distribution of attachment loss events that occur among all sites within a patient’s mouth. Thus, the distribution of attachment loss patterns of disease appears to mimic a random selection of an intraoral site for further disease progression. The frequency rate at which these events occur within a patient, as determined by the first-order rate constant, defines how many attachment loss episodes (or “hits”) occur within a certain time period. Patients with more severe disease appear to have a higher hit-rate rather than a more prolonged or aggressive period of disease progression at a particular site. This suggests that host defenses act locally to rapidly re-establish an equilibrium and bring the site into remission. Although deeper sites have a somewhat elevated risk of experiencing an episodic hit, the nu- merical dominance of shallower sites means that a patient with disease is more likely to experience the episode at a shallow site. Thus, the random-hit model is not unbiased toward randomness with re- gard to site selection for disease progression, yet it remains a reasonable approximation, since the deep-site bias is relatively small, as the proportion of deep sites is usually small.

These models of disease progression are based on clinical observations and are not universally ac- cepted. For example, the burst-like appearance of attachment loss may be due to the poor resolution of measurement tools. Indeed, in the last decade, data have been reported using computer-assisted probes and digital radiography, which lower the threshold at which clinical change can be detected.

Even more sensitive markers of disease, such as biochemical or microbiological markers, that are capable of detecting subclinical changes in disease status have been used. Models using these more sensitive markers have provided a different perspective on the interactive process than models based on clinical observations.

Host-microbe interactions are interdependent. The biofilm has co-evolved, maintaining a symbiotic host-parasite interaction. Not all people or all pockets are equally accommodating as hosts; for example, deep sites provide a better growth environment than do less inflamed shallow sites. This complex interaction can not be easily explained or modeled as a unidirectional process, even using multiple variables such as clinical signs, levels of microbial burden or others. We suggest that it more closely mimics a closed system, like the cardiovascular system. In fact, the very nature of the regulatory processes in the body allows the system to respond to external stimuli yet keep the body within certain boundaries. Thus the body survives external stimuli, as long as they are within certain limits, because the physiological process can convert these signals into behaviors within a closed system. Affecting one component of a closed system requires adaptive responses of many elements. For example, in the cardiovascular system blood pressure, blood volume, vessel resistance, cardiac output and stroke volume are all linked interdependently, and changes in one parameter, such as blood volume, affect most of the other components within the system. In this example, blood loss reduces pressure and output, leading to vessel constriction to raise blood pressure and stroke volume and other compensatory responses in an attempt to maintain equilibrium.

There is experimental evidence that the responses in periodontal disease behave as if they were occurring in a closed system. It has long been recognized that bone resorption is a potent trigger for new bone formation. One might expect that the equilibrium in the balance of anabolic growth factors and catabolic inflammatory mediators might regulate the stability of the periodontal tissues in the presence of a microbial challenge. During bone loss, one might expect a disequilibrium, which would manifest as an increase in inflammatory mediators and a decrease in growth factors. However, at inflamed progressing sites, both inflammatory mediators and anabolic growth factors are simultaneously elevated relative to noninflamed quiescent sites. The importance in this observation is that this trait is a characteristic of closed cyclic systems and not open-ended linear

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models. Systems that are multifactorial with complex feedback often do not have linear output patterns but frequently display episodes that occur at nonlinear intervals. Such “irregular” episodes are the result of summing many nonlinear functions. This may explain why attachment loss episodes have been difficult to predict at a site level, and that, in the future, neural networks or other nonlinear modeling approaches (13) that can predict phase shift behavior may prove to better predict the natural history and clinical course of disease. Baxt (3) and others have shown that these models are superior to traditional linear modeling approaches in predicting cardiac arrest, for example.

Further consideration of periodontal disease as a closed system model with interdependence of host factors and the biofilm can also lend insight into therapeutic approaches. Anti-inflammatory agents that are known to retard bone and attachment loss might then be expected to influence the flora, perhaps by diminishing nutrient availability, or by some other indirect mechanism not yet characterized. Periodontal surgery, which reduces the pocket depth and impairs the anaerobic ecological niche, might then be expected to have a long-term effect on the microbial biofilm properties. Mechanical debride- ment, which simultaneously lowers the local microbial burden and boosts the host local antibody response, would not only be expected to result in clinical improvement but would also be expected to re-establish a new state of equilibrium for the system. The closed behavior of the system would suggest that perturbations are compensated for to reach a state of equilibrium or homeostasis.

In complex systems with feedback, many different equilibria may be achieved; otherwise the system could not be controlled. Thus, changes in the flora, such as the acquisition of new pathogens or changes in the host such as increased inflammatory response due to stress, would both change the equilibrium and initiate a series of adaptive responses, such as gingival recession, epithelial downgrowth or fibrosis. For example, host stress that compromises neutro- phi1 clearance would permit new penetration of microbial lipopolysaccharide and antigens, which would stimulate an inflammatory response. The inflammatory process would both enhance nutrient availability to the biofilm to promote the emergence of nutrient-limited species and diminish antibody production locally. The penetration of lipopolysaccharide and bacterial antigens would re-initiate an adaptive antibody response. The renewed round of inflammatory monocytic tissue destruction would

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ensue, until the neutrophil and antibody activity could again bring the infection under control.

The pleiotropic role of IL-4 serves as a good example of how adaptive responses might occur. IL-4 induces isotype switching in plasma cells to produce IgG4, a more mature, high-avidity immunoglobulin subclass produced late in the normal maturation se- quence of antibody isotype switching. This antibody is not highly opsonic and hence would not facilitate clearance. On the other hand, IL-4 also serves to inhibit cytokine release from monocytic cells and can induce apoptosis of monocytes that were previously attracted and activated within the destructive period of the lesion. At excessive levels, this interleukin would decrease the levels of bone-resorbing cytokines and levels of matrix metalloproteinases, further reducing tissue destruction. High levels of IL- 4 also induce fibrosis and scarring - another hall- mark of the periodontal lesion. Thus, periodontal disease activity represents a joint or sequential activation of the inflammatory and the antibody components of the immune response. The organisms are affected by both sides of this response: first positively by inflammation and then negatively by antibody. This is more analogous to the interactions in a closed system. This can be compared with ecological studies of population shifts that can occur in co- existing populations of predators and prey when they are confined within a fixed territory. Saying that this process is mediated entirely by the host or entirely by the bacteria would simply propagate another decade of misunderstanding. It would be like a continuing argument about whether high blood pressure is due to high cardiac output or high vascular resistance. A new appreciation for the interactive process between the biofilm and the host can promote a concept of disease and health in which both represent a state of biological equilibrium with differing levels of metabolic activity.

Assembling the players

During the early stages of developing gingival inflammation, all the players in the pathogenesis of periodontitis are prepared for their roles in the drama that is about to unfold. Resident junctional epithelial cells and fibroblasts are notified and various leukocytes and their subsets are assembled in selected proportions and activated, and very large varieties of messenger molecules that mediate cross- talk among the cells and molecules that mediate


tissue destruction are produced (see Kornman et al. (32) in this volume). Important new findings and perspectives are listed in Table 3.

Summary of events: the process is iterative

Gingiva is a unique tissue. In contrast to other tissues, under healthy noninflamed conditions the small vessels of the subgingival plexus express adhesion molecules such as E-selectin, and a continuing stream of neutrophils exits the vessels and mi- grates through the junctional epithelium into the gingival sulcus. An estimated 530,000 leukocytes per minute (predominantly neutrophils) migrate into the oral cavity in periodontally healthy adults (55). These cells reflect a normally functioning host defense against a low level challenge, and tissue destruction does not ensue.

As the microbial challenge increases, clinically manifest inflammation of the marginal gingiva begins (63, 65). Bacterial components or their products interact with the epithelium and penetrate into the underlying connective tissue. The small blood vessels immediately deep to the junctional epithelium become inflamed, resulting in enhanced permeability and a great increase in the numbers of neutrophils that exit the vessels and migrate through the junctional epithelium and into the gingival sulcus. As this occurs, small quantities of collagen and

Table 3, The pathogenesis of periodontitis: new findings and new perspectives on the gingival tissue as a stage for the host-parasite interactions

The cellular and molecular response to the evolving bacterial challenge is iterative and involves constant adjustment and regulatory feedback. The epithelium plays an active sensing and signaling role that is involved in specific leukocyte recruitment and vascular permeability. The bacterial activation of the junctional epithelium initiates vascular endothelial responses that initially involve primarily neutrophils migrating to the sulcus. The specific leukocyte populations in the tissue are tightly regulated by cytokine and chemokine responses to bacterial products that enter the tissue and selectively activate certain cytokines and chemokines. Neutrophils do not dwell in the tissue but constitute the majority of leukocytes in the sdcus, whereas macrophages, lymphocytes and plasma cells form the majority of cells in the tissue. If the bacterial challenge is not controlled early, resident tissue cells, such as fibroblasts, may become activated by bacterial products and cytokines to participate actively in tissue destruction.

other components of the perivascular extracellular matrix are destroyed.

If the microbial challenge remains undisturbed, in a matter of days marginal gingival inflammation or gingivitis appears in most individuals. Subsequent host defense activities may contain the challenge, resulting in inflammation that is not worsening or im- proving, or the microbial challenge may overcome the host defense, allowing conditions to worsen. When this occurs, supragingival plaque extends into the gingival sulcus, disrupting the union between the most coronal portion of the junctional epithelium and the root surface. As a consequence, a shallow gingival pocket with a typical pocket epithelium forms. At this stage, there is no periodontal pocket, as there is no attachment or bone loss. The presence of the pocket epithelium, however, heralds an es- calation in the progression toward periodontitis. The pocket epithelia provide ready access for bacterial substances to shower the connective tissues and to activate the epithelial cells to express IL-8 and endothelial cells of the inflamed small vessels to enhance expression of adhesion molecules, resulting in enhanced emigration of leukocytes, edema and formation of an inflammatory cell infiltrate. The infiltrate consists of monocytes and lymphocytes including B cells and T cells of both the T-helper 1 (Thl) and Th2 phenotype and emigrating neutrophils. Cyto- kines produced by these cells and antigens from the bacteria drive differentiation of B cells to specific antibody producing plasma cells. A humoral immune response that may or may not be protective may be mounted. At any given point, the host defenses may contain the microbial challenge, or the challenge may be reduced, such as by tooth cleaning, and the process reversed.

If containment does not occur, inflammation worsens and is perpetuated, and connective tissue and bone is then destroyed (Fig. 1). Macrophages activated by lipopolysaccharide produce IL- lp , tumor necrosis factor a, matrix metalloproteinases and prostaglandin EZ. IL-Ip and tumor necrosis factor a activate resident fibroblasts to produce prostaglandin E2 and matrix metalloproteinases. Both activated cell types decrease the production of tissue inhibitors of metalloproteinases, resulting in greatly increased relative levels of matrix metalloproteinases. This destroys components of the extracellular matrix, creating space for the enlarging inflammatory cell infiltrate. The microbial biofilm may extend apically and laterally. The epithelial cells activated by lipopolysaccharide can produce matrix metalloproteinases, which can destroy attached collagen fibers

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Fig. 1. The monocyte/macrophage (M0) plays a central role in the pathogenesis of periodontitis. Lipopolysac- charide (LPS), the principal virulence component of gram-negative periodontal pathogenic bacteria, is shed from the biofihn and enters the gingival tissue. It binds to lipid-binding protein (LBP), and this complex is recognized by the CD-14 receptor expressed by the monocytel macrophage. Receptor occupancy initiates transmembrane signals that activate the cells to synthesize and secrete prostaglandins, cytokines and matrix rnetalloprot- einases (MMP). The activity of the activated cell is modulated by hereditary factors and can be suppressed by interferon y. Tumor necrosis factor a (TNFa) and IL-lP

at the apical terminus of the junctional epithelium, allowing apical extension of the epithelium, formation of additional pocket epithelium and pocket deepening. As this occurs, matrix metalloproteinases mediate clinical attachment loss and prostaglandin EZ mediates resorption of the alveolar bone (and in some cases cementum and dentin), and the gingival pocket progresses to become a periodontal pocket.

The events comprising the formation of gingival and subsequently periodontal pockets as presented above may appear unidirectional. This is not the case; they are iterative, with cross-talk between the microbial challenge and the host defenses that are called into play. At any given point in the progression

bind to cell surface receptors of resident fibroblasts, initiating signals to synthesize and secrete matrix metalloproteinase and prostaglandin E2 (PGE2). Matrix metalloproteinases mediate destruction of the extracellular matrix of the gingiva and periodontal ligament, and prostaglandin E2 mediates alveolar bone destruction. IL-ls and tumor necrosis factor a directly mediate a minor portion of bone loss. The diagram does not include proteases and inflammatory mediators produced by endothelial and epithelial cells nor by the infiltrating neutrophils which con- tribute significantly to the matrix metalloproteinase concentrations. Source: modified from Offenbacher et al. (47).

of events, host defenses may contain the challenge and arrest or reverse progression, or the challenge may overcome the defenses and enhance progression.

The junctional epithelium has sensing and signaling effects on functions

The junctional epithelium has previously been viewed as more or less a bystander that provides a barrier function or curtain separating the biofilm from the connective tissue and does little else during the development of periodontitis (see Kornman et al. (32) in this volume, pages 34-37). Most of the real action

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was thought to occur in the gingival and periodontal ligament connective tissue and alveolar bone. This now seems far from the case. The junctional epithelium is engaged even prior to the onset of clinical manifestations of gingivitis, when large numbers of neutrophils migrate through the junctional epithelium and into the mouth (63). The cells of the junctional epithelium express intercellular adhesion molecule l and IL-8, a chemoattractant specific for neutrophils and for a small subset of lymphocytes. Intercellular adhesion molecule 1 appears to be especially important to the neutrophil migration across mucosal membranes. Antibody specific for intercellular adhesion molecule 1 greatly inhibits neutrophil migration. Among the oral epithelia, neutro- phi1 migration occurs through the junctional epithelium and the pocket epithelium to which it gives rise; and initially intracellular adhesion molecule 1 expression is limited to these epithelia. However, as infiltrate size increases, the junctional] sulcular and gingival epithelia all may express intercellular adhesion molecule 1 (18).

Neutrophil migration across the epithelial barrier and the accessibility of antigen to the underlying connective tissues may be affected by intraepithelial mononuclear cells. These include cells likely to be Langerhans or dendritic cells and specific subsets of lymphocytes such as mucosal T cells and T cells having a repertoire of receptors enriched for bacterial antigens. Although the role of these cells remains obscure, their populations are greater at sites of inflammation than at noninflamed sites, suggesting that they may engage in limiting antigen penetration and in antigen recognition, processing and presentation.

Following extension of the biofilm subgingivally into the gingival sulcus, the sloughing surface of the junctional epithelium and, at a later stage, the pocket epithelium are in direct contact with both the microbial mass of the subgingival biofilm or substances such as lipopolysaccharide derived from it and the gingival connective tissue. The junctional epithelium is uniquely rich in neural elements; the detailed function of these is unclear. The cells of the junctional epithelium can “sense” the biofilm environment and respond by generating inflammatory mediators that serve as messengers, transmitting information to the connective tissues, especially the small vessels of the vascular plexus in the lamina propria. IL-8 may be particularly important in facilitating the transmigration of neutrophils and their accumulation at the surface of the biofilm. There is an increasing gradient in an apicocoronal direction

guiding neutrophils to accumulate at the surface of the biofilm. The cells express high-affinity receptors that bind chemoattractant molecules at the low concentrations that exist some distance from the biofilm and cause chemotaxis. Low-affinity receptors become occupied and activate the cells near the biofilm where concentrations are high. Similarly, neutrophils express high and low affinity receptors for the chemoattractant leukotriene B4. As the biofilm extends apically between the junc-

tional epithelium and the tooth surface, the cells become detached and additional pocket epithelium forms. The epithelial cells continue to express intercellular adhesion molecule 1 and become activated and synthesize and secrete prostaglandin EP, IL-8 and matrix metalloproteinases. These products along with inflammatory metabolites and components released by the bacteria can initiate and perpetuate the inflammatory response in the underlying connective tissues.

For reasons that remain obscure but that may be related to apical extension of the biofilm or to epithelial cytokine production, basal cells of the junctional epithelium begin to replicate and extend rete ridges into the connective tissue and to migrate apically along the tooth root. Such migration and extension can only occur when the adjacent dense collagen fibers attached to root cementum are destroyed and removed to create space. The fact that the epithelial ceus can produce matrix metalloproteinase provides a potential mechanism through which pocket formation and deepening can occur. The significance of junctional epithelium-derived matrix metalloproteinase in pocket deepening remains to be studied.

The leukocyte populations of the inflammatory infiltrate are tightly regulated

One of the major contributions of Page & Schroeder in 1976 (52) was the observation that the periodontal lesion begins as an acute inflammation predomi- nated by enhanced neutrophil emigration and formation of an inflammatory cell infiltrate that soon becomes dominated by small and medium-sized lymphocytes, mostly T cells. This infiltrate then develops into a fully evolved inflammatory infiltrate dominated by B cells and plasma cells but also containing T lymphocytes, macrophages and neutrophils. In 1976, the mechanisms governing infiltrate formation, leukocyte and subset composition and cell function were not understood. Further, in hind- sight] the structural studies that had been performed

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were inadequate, in part because the concept of disease-active and disease-inactive periodontal sites had not yet been developed, and most studies of the early lesion had been conducted on specimens from adolescents (65). Major advances have since been made in elucidating the details of leukocyte infiltration at sites of inflammation (see Kornman et al. (32) in this volume, pages 37-43) (65).

Based on information now available, the staging of the pathogenesis of periodontitis as initial, early, established and advanced lesions that was done in 1976 remains basically correct, except that the early lesion, characterized by a predominance of T lymphocytes, is not a distinctly identifiable stage, as gingival inflammation develops in adults. It is instead observed during tooth eruption in children and in the marginal gingiva of adolescents (65). In adults, neutrophils and subsequently lymphocytes and monocytes leave the circulation and accumulate in the marginal gingiva as gingivitis and subsequently periodontitis develops. This process is tightly controlled, and many of the details are now known. In- teractions occur between the leukocytes and endothelial cells of the postcapillary venules that result in diapedesis and infiltrate formation. In the initial stages this interaction may result from mast cell-derived tumor necrosis factor a leading to the upregulation of endothelial cell adhesion molecules. Mast cell stimulation could occur as a result of possible neural connections with intraepithelial Langerhans cells (78).

As inflammation develops, specific and differing populations of inflammatory cells accumulate in the connective tissue and in the sulcus or pocket. Neu- trophils do not dwell in the connective tissue, but they constitute the majority of leukocytes in the sulcus or periodontal pocket, whereas macrophages, lymphocytes and plasma cells form the majority of cells in the connective tissues. Antigen-specific memory and activated lymphocytes, aELa7 mucosal lymphocytes, y6 T-cell receptor-positive cells and CDla+ antigen-presenting cells migrate selectively into the gingival tissues, This is highly regulated and is not a result of simple unregulated migration.

Mechanisms operate that are general for leukocytes and others that confer specificity and selec- tivity on diapedesis and infiltrate composition and size (see Fig. 4 in Kornman et al. (32) in this volume). The bacterial and inflammatory products that upregulate selective sets of adhesion molecules on leukocytes and endothelial cells therefore determine the migration kinetics of specific cell subpopulations out of the vasculature and into the tissue.

In addition to the participation of specific adhesion molecules, selective migration and accumulation of leukocytes is determined by the recently discovered chemokines, a family of low-molecular- weight cytokines with potent and cell type-specific chemoattractant properties. IL-8 is specific for neutrophils and a small population of lymphocytes, monocyte chemoattractant protein 1 for monocytes and other chemokines for other leukocytes.

As inflammation worsens, the pocket epithelium becomes chemoattractant negative and neutrophil migration through the epithelium and into the pocket decreases (18). A possible decrease in the gradient of chemoattraction may also occur, with neutrophils dwelling and becoming activated within the gingival tissue. This could be an important event in the evolving capacity to destroy connective tissue.

Cell homing may also participate in infiltrate formation. There is evidence from rodents that lymph- oid cells specific for the antigens of periodontal pathogens may specifically home from the circulation to the gingival tissues. Eastcott et al. (12) sensi- tized clones of lymphocytes to antigens of A. actinomycetemcomitans and infused them into mice. When the animals were orally infected with the same microorganism, the infused cells homed to the gingiva, but homing did not occur when the mice were not infected. Whether such homing exists in humans is not known.

It is these mechanisms that select for infiltrates with differing proportions and total numbers of specific leukocytes and their subsets. Infiltrate composition and size are not static; infiltrates are dynamic and change depending on changes in the subgingival microbiota that drive infiltrate formation. The properties and concentrations of factors activating the endothelial cells and the nature and duration of receptor expression are major determinants of infiltrate composition and characteristics.

Resident tissue cells become active participants in destruction

There is another major component. One of the most significant advances in understanding the pathogenesis of periodontitis over the last two decades has been documentation that, in addition to the leukocytes of the inflammatory infiltrate, the cells residing in the normal periodontium, including fibroblasts, junctional epithelial cells and vascular endothelium (all of which function in the healthy periodontium to maintain homeostasis), can be hijacked by exposure to bacterial agents such as lipopolysacchar-

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ide, cytokines such as IL-1 or tumor necrosis factor a or prostaglandins such as prostaglandin E2 and become major participants in tissue destruction.

Well-documented events involving the gingival fibroblasts illustrate this (Fig. 2). Under conditions of health, the fibroblast genes for various collagens and tissue inhibitors of matrix metalloproteinases are turned on and functioning and the genes for matrix metalloproteinases are turned off, as assessed by measurement of various types of messenger RNA. Genes for collagens and tissue inhibitors of metalloproteinases in fibroblasts in active periodontitis lesions are turned off and those for matrix metalloproteinases are turned on. In addition, in the presence of lipopolysaccharide, fibroblasts produce their own inflammatory mediators, such as IL-lp. Further, following successful treatment, as the levels of bacterial substance such as lipopolysaccharide, and proinflammatory cytokines such as IL-lP and tumor necrosis factor a decrease, the reverse occurs. Whether the same cells convert from an anabolic to a catabolic state or whether the fibroblasts are replaced by a different phenotype is not known. It is known that, at an early stage of gingivitis, resident tissue fibroblasts are killed through what appear to be immuno- pathological mechanisms, as seen in humans and

Fibroblast / Gene Activation \ Health 8

Metalloprotein&es

I Cofiagen and other extracellular matrix; Inhibitors of metalloproteinases

Tissue remodeling, maintenance, healing, regeneration

Tissue destru&on *

Fig. 2. The role of resident fibroblasts in health and in periodontitis. In situ hybridization and other studies have shown that, in the healthy periodontiurn, the resident fibroblasts genes for collagens and other extracellular matrix components and tissue inhibitors of metalloproteinases are turned on and those for matrix metalloproteinases are turned off such that tissue remodeling, maintenance, healing and regeneration can occur. In active periodontitis, genes for tissue inhibitors of metalloproteinases and components of the normal extracellular matrix are turned off and those for matrix metalloproteinases are turned on, permitting the resident fibroblasts to play a major role in tissue destruction.

monkeys, resulting in significantly reduced populations (66, 69, 70). At subsequent stages, fibroblast population sizes are restored. Whether these cells are of the same phenotype as the cells present in the healthy tissues remains unknown.

The concept of radius of effectiveness

Gross and histological features of periodontal lesions in humans give clues as to how they may develop and function. There has been considerable debate as to the determinants of the frequency and cause of intrabony lesions. Garant & Cho (14) suggested that locally produced stimulators of bone resorption may have an effective radius of action, and Waerhaug (77) demonstrated that bone resorption occurs when microbial plaque approaches to within 0.5 to 2.0 mm of the bone surface. Based on these and other observations (62), Page & Schroeder (53) postulated a range of effectiveness of the subgingival plaque (biofilm) in generating alveolar bone loss of about 2.5 mm. They stated that “when the bone surface has been resorbed to about 2.5 mm apical or lateral to the site of the bacteria, bone loss appears to cease and bone production takes over, until it equals or surpasses resorption”. The inflammatory infiltrate also plays a role. The closer the cells of the inflammatory infiltrate are to the bone, the more osteoclasts appear and the more bone is degraded (59, 64). Tal (73) provided data supporting this hypothesis by measuring intrabony lesions in 344 interproximal areas and 117 intrabony pockets in 84 patients. He observed intrabony lesions only rarely when interdental distances were less that 2.6 mm. Tho intrabony defects on adjacent teeth were observed only when the interdental distances were greater that 3.1 mm. As Schroeder (62) pointed out, lesions with dimensions much greater that 2.5 mm are seen around single teeth not only in humans but also in dogs and other species. There is evidence that these are accounted for by the invasion of the tissue by bacteria and abscess formation.

Some people develop gingivitis and others periodontitis

For several decades periodontologists have considered the central question of how gingivitis progresses to periodontitis in the belief that the answer will provide critical clues to aid in the prevention of

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Table 4. The pathogenesis of periodontitis: new findings and new perspectives on the transition of gingivitis to periodontitis

Gingivitis is generally characterized biochemically by an increase in leukotriene B4 in the gingival crevicular fluid. This is a product of degranulating neutrophils in the sulcus.

characterized by an increase in prostaglandin &, IL-10 and tumor necrosis factor a, which represent an activation of macrophages and lymphocytes within the tissue. Children do not demonstrate a macrophage and lymphocyte response to bacterial plaque accumulation, unless they have defective neutrophil function. In young adults involved in experimental gingivitis studies, some have a neutrophil (leukotriene B,) response, and some have a macrophage and lymphocyte response. The transition from gingivitis to periodontitis may be monitored by the biochemical shift from leukotriene B4 dominance to expression of macrophage and lymphocyte activation products, such as prostaglandin El. Some individuals appear to have a very transient clinical phase in the transition from gingivitis to periodontitis. Others do not appear to undergo the transition to eriodontitis. It may therefore be practical to cgaracterize individuals as gingivitis or periodontitis patients.

Later stages of gingivitis and periodontitis are

periodontitis. Important new findings and perspectives are listed in Table 4.

Current evidence suggests that, whereas gingivitis probably represents the early stage in the natural history of initiating periodontitis, it may also exist as an independent and stable clinical entity. The microbial composition of the biofilm associated with both conditions seems to be quite similar; in fact, gingivitis- and periodontitis-associated microbes are commonly found even in adolescents. And since periodontal pathogens can be transmitted, it would appear that exposure to most periodontal pathogens within a person’s lifetime is a rather ubiquitous event, but the biofilm must be sufficiently mature for these “late-blooming’’ microbes to become resident within the biofilm. In human populations such as those in rural China (541, where oral care is minimal or nonexistent, the prevalence of most periodontal pathogens is about 90-95%. In fact, many or most of these subjects are not infected with a few pathogens but rather appear to harbor virtually all currently recognized pathogens. This is somewhat surprising, since the prevalence and severity of periodontal disease is diverse and not substantially greater than that observed in populations in industrialized countries. Since bacteria are necessary for the initiation

~~ ~ ~

of periodontitis, these findings prompt the reassess- ment of the contribution of various factors to the clinical presentation of periodontitis.

It is now recognized that multifactorial diseases, such as periodontitis and atherosclerosis, usually have initiating factors, factors that permit or block disease initiation and factors that modify clinical expression once the disease has been initiated. This biological model of multifactorial disease presents com- plications in statistical models unless one explicitly recognizes the underlying biological role of each factor. The process of disease initiation is being re-exam- ined with recognition of the multifactorial nature of periodontitis. The prominent pathogens, such as P gingiualis and B. forsythus, in adult forms of periodontitis will predictably emerge in the subgingival plaque as the natural consequence of the undisturbed maturation of the dental biofilm in individuals exposed to routine social and family interchanges.

The accumulation of microbial plaque in children and adolescents leads to an inflammatory response referred to as gingivitis, the severity of which varies among individuals. Experimental gingivitis in young adults, which is caused by the overgrowth of gram- positive species during the first 2-3 weeks following the cessation of oral hygiene, presents clinically as redness and edema that begins at a few interpapil- lary and marginal areas and generally spreads to more sites, as it slowly becomes more severe at involved sites. During this period of time neutrophil infiltration, epithelial proliferation and subepithelial capillary proliferation dominate the lesion histologically. Biochemically, the barrage of neutrophil migration into the sulcus is marked by a rise in leukotriene B4 levels in gingival crevicular fluid, a product of degranulating neutrophils (25). This reflects the neutrophils actively engaging the bacteria in the sulcus, facilitating clearance. This represents the earliest stage of the lesion that can be characterized by neutrophil and keratinocyte activation facilitating neutrophil recruitment and ‘bacterial clearance. Keratinocyte IL-8 is a potent signal for intracellular adhesion molecule expression and neutrophil recruitment. Neutrophil leukotriene B4 and complement C5a are vasoactive and sufficient for marginal inflammation with little destruction of deeper connective tissues.

As the biota shifts and becomes more gram-negative by 3-4 weeks, bleeding on probing begins to rapidly increase concomitant with increases of prostaglandin E2 in gingival crevicular fluid, a marker of lipopolysaccharide penetration and monocytic activation (25). The ulceration from bleeding

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on probing is classically considered to herald the transition from gingivitis to periodontitis. Indeed, the biochemical trigger of prostaglandin Ez also suggests that neutrophil clearance capacity has been ex- ceeded and lipopolysaccharide has penetrated into the tissues, resulting in the release of prostaglandin Ezr IL-1 and probably tumor necrosis factor a. Prostaglandin E2 increases the chemotactic potency of IL-8 by 10-100 fold (7), serving to exponentially increase neutrophil recruitment. In contrast to neutrophil enzymes that are predominantly antimicrobial in nature (lysozyme, lactoferrin, defensins and P-glucuronidase), these mediators (prostaglandin EZ, IL-1 and tumor necrosis factor a) target host cells to initiate tissue catabolism. These provide signals that trigger fibroblastic apoptosis, matrix metalloproteinase release and the expression of homing receptors for lymphocytic recruitment.

What allows sufficient lipopolysaccharide to enter the tissue and activate monocytes? One possibility is that bacterial growth would bring high levels of lipopolysaccharide in contact with the gingival pocket, resulting in the formation of more pocket epithelium, which would allow more lipopolysaccharide to enter the tissues. This assumes, however, that sufficient numbers of neutrophils are no longer available to separate the bacteria from the epithelium. Another possibility is that the emergence of organisms such as €? gingiuulis causes bacterial factors to enter the tissue and interfere with the dynamics of neutrophil flux into the sulcus (see Darveau et al. (10) in this volume, page 21). This would reduce the numbers of neutrophils in the sulcus and shift the relative balance between neutrophils and the bacteria, allowing more bacterial expansion and more lipopolysaccharide to enter the tissue. Other factors that alter neutrophil function in the sulcus probably also allow a rapid bacterial shift that overwhelms the neutrophils. These factors appear to include intrinsic neutrophil defects, acquired neutrophil modifiers such as smoking and factors that alter antibody-neutrophil activity such as genetic variations in FcyII receptors (see Hart & Kornman (23) in this volume, page 209).

The increasing levels of prostaglandin EZ, IL-1 and tumor necrosis factor a represent the beginning of a new stage in the pathogenesis that involves the response of monocytes and plasma cells. In children this stage does not generally occur, unless neutrophil function is defective, such as that seen in leukocyte adhesion deficiency syndrome 1. It also suggests that children do not respond to lipopolysaccharide challenge with a monocytic and plasma cell infiltrate as

an inflammatory response to local challenge in the same way as adults. The reason may be that, in general, the cellular response to lipopolysaccharide with resultant inflammatory mediator release is very weak in children and increases with age. This is in part due to the important role lipopolysaccharide in the gut plays in the maturation of the immune system in children, especially thymus-directed processes. Indeed, germ-free animals fail to develop normal T- cell immunity if lipopolysaccharide is not present, illustrating the key role of lipopolysaccharide as a co-evolutionary signal for the maturation of the immune system (2). This nonresponsiveness in children may in part account for the apparent resistance children have to periodontitis.

In young adults with experimental gingivitis, not all patients behave the same biochemically or clinically. The maturation of the biofilm by 4 weeks and acquisition of gram-negative anaerobes presents a challenge that escapes neutrophil clearance in some patients and results in the penetration of lipopolysaccharide. This is suggested by data showing that some experimental gingivitis patients have prostaglandin E2 levels by 4 weeks identical to those in untreated adult periodontitis. Other patients have low levels of prostaglandin E2 that have not increased from baseline. The central question then is whether these different responses both represent gingivitis. Instead it appears to represent a time-dependent es- calating response to the lipopolysaccharide burden that is absent in some patients. The magnitude of the clinical severity would be directly related to the magnitude of the inflammatory response (that is, the amount of prostaglandin EZ, IL-1 and tumor necrosis factor a produced). If gingivitis represents a neutro- phi1 lesion, then this rise in prostaglandin E2 does not represent experimental gingivitis but rather experimental periodontitis that begins with gingival inflammation, a nonprotective neutrophil response and rapid lipopolysaccharide triggering of the inflammatory mediators of periodontitis.

In nonsusceptible patients, a protective response by neutrophils and antibodies limits the extent (and severity) of attachment loss. In susceptible patients, neutrophil clearance is less protective and the extent and severity of disease are governed by the magnitude of the host inflammatory response to the microbial challenge, especially the response to lipopolysaccharide. The magnitude of the inflammatory response is driven by microbial antigens and controlled by T cells. The T-cell response up- or down- regulates the inflammatory component, plasma cell differentiation and antibody production based on

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the cytokine profile. Antibody is critical to enhance neutrophil clearance, which reduces the lipopolysaccharide and antigenic challenge. Thus, the relative balance between the inflammatory response and the protective antibody response is regulated by the mix of cytokines that is produced primarily by macrophages and T cells.

Studies of the natural history of new episodes of periodontal attachment loss show that nonsusceptible patients with existing gingivitis are not at significant risk for attachment loss (37). Susceptible patients with periodontitis are at high risk for new attachment loss (22) . Thus, established gingivitis does not appear to readily make the transition to periodontitis. Stated another way, in some susceptible individuals the gingivitis state that precedes periodontitis appears to have a very transient clinical presentation that is quickly passed along the evolving path to periodontitis. Further, all sites in these susceptible patients with periodontitis remain at risk of further attachment loss, even after treatment. In patients of this type, poor plaque control and gingival inflammation will virtually always result in further periodontal breakdown.

In the last decade new appreciation has been gained of the essential role of bacteria in initiating periodontitis, but the severity of disease is determined by the magnitude and quality of the host response to the microbial biofilm. An argument can be made that gingivitis and periodontitis share the same causal factors but represent different clinical manifestations of the same fundamental disease process due to differences in how the host responds to the microbial challenge. For example, in simplistic terms the patient plays host to the microbiota and serves to incubate the common oral flora. The biofilm follows a predictable maturation process beginning with gram-positive rods and cocci and followed by gram-negative anaerobes. Some patients serve as feeble incubators due to recurrent mechanical disruption of the dental biofilm and effective host neutrophil defenses. As a result, the natural maturation of the biofilm is stifled or perhaps ar- rested and such individuals would present clinically as gingivitis patients who may have a site or two with bleeding on probing and some loss of attachment. Even after many years of exposure from exogenous sources of periodontal pathogens, such as P gingiualis, the organisms can never really establish well enough in the environment provided by the host to emerge as dominant members of the biofilm. Thus, the patient would remain nonsusceptible.

In contrast, another patient presented with the

same microbial biofiim may serve as a more nurtur- ing and less combative incubator (see Hart & Korn- man (23) in this volume), permitting the gram-negative, black-pigmented, anaerobic species that require host-derived nutrients and growth factors such as peptides and heme to occupy the biofilm. Any exogenous exposure to periodontal pathogens in this host could cause further attachment loss. Further, this host environment will never permit stable gingivitis, since the host is more reactive to the bacterial challenge and the host conditions will drive the natural maturation of the biofilm, leading to the establishment of a lush gram-negative biota containing high levels of periodontopathic bacteria. Gingi- vitis probably preceded periodontitis in susceptible patients, but the distinction between the two conditions is probably blurred and transient at best. Most importantly, the gingivitis that is prodromal to periodontitis in this circumstance would follow a distinctly different clinical course than nonprogressing gingivitis. In this context gingivitis and periodontitis really represent two different clinical manifestations of the same pathological process. The dif- ference in clinical manifestations is due to intrinsic differences in the host that modify simultaneously both the destructive response to the bacteria as well as the natural growth and maturation potential of the biofilm (in this volume, see: Hart & Kornman (23) and Salvi et al. (60)). Perhaps the wrong question is being posed in attempting to understand the transition from gingivitis to periodontitis, since both conditions may represent the same pathogenic process with varying clinical expression in different people with differing levels of susceptibility.

If this concept is proven valid, it may have some important ramifications. Individuals susceptible to periodontitis would have a minimal or no discern- ible gingivitis phase but go rapidly into initiation of periodontitis. In such subjects, preventing gingivitis as a strategy for preventing periodontitis may not be effective. These individuals may require more ex- treme control of bacteria andlor host modulation therapy. In subjects not susceptible to periodontitis, the gingivitis phase may be so stable that preventing gingivitis is not necessary to prevent the initiation of periodontitis. In addition, therapies that block periodontal disease progression may not affect gingivitis. The effects of nonsteroidal anti-inflammatory drugs on the progression of periodontal disease may be such an example, since they are effective in preventing attachment and bone loss but have only a small effect on gingival inflammation in susceptible patients or in experimental gingivitis.

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Advances in the pathogenesis OJ periodontitis

The cytokine network and prostaglandins

Characteristics of the cytokine network

The cytokines comprise a large family of polypep- tides produced in the periodontium by infiltrating leukocytes and by resident fibroblasts, junctional epithelial cells, vascular endothelium, mast cells and bone cells. The cytokines comprise the major regulators of the immunoinflammatory response characteristic of periodontitis, and they are the major determinants of the outcome. The interleukins are a subfamily of cytokines designated interleukin- 1 (IL- 1) through interleukin-15 (IL-15). The chemokines comprise an additional subfamily of smaller peptides that serve as chemoattractants for specific leukocytes. For example, IL-8 is specific for neutrophils and monocyte chemoattractant protein 1 is specific for monocytes, whereas other chemokines are specific for other mononuclear cells. Other cytokines that are important in the immunoinflammatory response in periodontitis include interferon-y (produced by activated T cells, monocytes, natural killer cells) and transforming growth factor p (produced mostly by monocytes and macrophages). Cytokines can induce one another and, in some

Table 5. The pathogenesis of periodontitis: new findings and new perspectives on cytokines and prostaglandins

‘The cytokines, chemokines and prostaglandins appear to comprise the major regulators of the imunoinflammatory responses that characterize periodontitis. Different cytobneyt terns haw been observed in tissues from erio onhtis sites, suggesting that specific &tory patterns may be Invoked in stable and active lesions. The disease active lesions do not appear to have cytokine patterns that are characteristic of either a Thl or a ThZ pattern. The proinflammatory cytokines K - l p and tumor necrosis factor a have been strongly associated with periodontltis. I’rost@andin Ez is E primary mediator of tissue destruction in periodontitis. Prostaglandin Ez levels are elevated in iridividuals with high susceptibility for severe periodontitis. Prostaglandin Fq levels are determined by the pmstagiandin endoperoxide synthase 2 (cyclooxygenase 2) enzyme, which is W y regulated by lipopolysaccharide, and various cytokines, including most prominently IL-10 and tumor necrosis factor a.

~ __ ~ . ~~ .~ ~ ~~ ~ ~~

~__________ - _______~_

cases, such as IL-lp, perpetuate production of themselves. Important new findings and perspectives are listed in Table 5.

The cytokines act at very low concentrations generally in the range of 10-lo to lo-” M. They serve as messengers that mediate cross-talk among cells by binding to high-affinity transmembrane receptors. Some cytokines such as IL-1 and tumor necrosis factor a are proinflammatory, whereas others, such as IL-10, suppress the inflammatory response. Cytokines can act synergistically or antagonistically or be additive or subtractive. They function as a cas- cading network that manifests unusual complexity, redundancy and pleotrophism. Many of the functions of cytokines depend on concentration. These features of the network are difficult to explain but may be accounted for by the following explanation. Bacteria have very short generation times that permit frequent mutation. As a consequence, they can evolve a variety of mechanisms to avoid and ma- nipulate the host defense mechanisms. Further, whereas an encounter between the host defenses and a single species may be reasonably well defined, in periodontitis the ecosystem is exceedingiy complex, and co-evolutionary processes between diverse bacteria species and the host most likely have already produced a variety of host molecules to cope with the bacterial challenge. The complexity, redundancy and pleotrophism of the cytokine network may provide a mechanism to finely tune the host response such that it properly limits the bacterial challenge but is modulated to limit tissue destruction and allow repair.

The Thl and Th2 paradigm: the cytokine profile directs the nature of the host response and regulates connective tissue homeostasis

In the mouse, CD4+ helper T cells fall into two distinct categories designated Thl and Th2 cells based on the pattern of cytokines they produce (in this volume, see: Gemmell et al. (17), pages 113-115 and Ishikawa et al. (271, pages 90-91). It has been as- sumed that naive antigen-specific T-helper cells, designated Tho, differentiate on repeated stimulation into Thl and Th2. This concept has been ques- tioned and may not be true in humans. Thl clones are generally characterized by the production of interferon y. A predominantly Thl infiltrate is thought to result in a cell-mediated or delayed-type hyper- sensitivity response with the activation of macrophages, the production of proinflammatory cytokines, including IL-1 and tumor necrosis factor a.

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Interferon y stimulates neutrophils and appears to be essential for neutrophils to control bacterial infection. On the other hand, IL-1 and other proinflammatory cytokines are found in inflamed periodontal tissues at significantly elevated levels, and these levels return to normal following successful therapy. Monocytes from patients with severe periodontitis and those with high susceptibility produce more IL- 1 before and following stimulation than cells from periodontally normal individuals (15, 16, 58). A major role for IL-l is its direct action on the promoter region of the matrix metalloproteinase gene, resulting in enhanced matrix metalloproteinase production. Individuals susceptible to severe adult periodontitis later in life manifest a unique polymorphism in the IL-1 gene family that results in the production of approximately four times more IL- l p than individuals not having that genetic trait, and their likelihood of developing severe disease increases significantly (31). IL-6 may also be important in periodontitis, as it can induce the formation of multinucleated cells that can resorb bone.

Th2 clones are generally characterized by the production of IL-4, IL-5 and IL-6. Both Thl and Th2 cells produce other cytokines such as IL-10 and IL- 13, tumor necrosis factor a and granulocyte-macrophage colony-stimulating factor. A predominantly Th2 infiltrate is considered to drive the differentiation of B cells into antibody-producing plasma cells, which may be protective or nonprotective depending on the nature of the antibody produced. A strong Th2 response can inhibit a Thl response and vice versa. For example, IL-4 can inhibit the Thl response, and interferon y can inhibit the Th2 response. Whether a given response is Thl or Th2 or mixed is determined by several factors. These include the nature of the activating antigen, the route of antigen administration and antigen concentration, the identity of the antigen-presenting cell, major histocompatibility complex molecules and the nature of the interaction with the T-cell receptor. The nature of the antigen-presenting cell, however, may be of fundamental importance.

Whether the Thl and Th2 paradigm exists in humans as it is manifested in mice remains un- proven and controversial. This is a key question for periodontal pathogenesis since the Thl and Th2 paradigm could be a major determinant of the progression from gingivitis to periodontitis, of the transition from a stable nonprogressive periodontitis lesion to a burst of destructive activity and of refractory or recurrent disease. The Thl and Th2 paradigm has been studied by measuring the levels of various

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cytokines in gingival crevicular fluid and extracts of healthy and diseased gingival tissue, determining levels of cytokine messenger RNA in the tissue using immunocytochemical techniques or polymerase chain reaction on tissue extracts and by measuring cytokine production by mononuclear cells harvested from gingival tissue. Based on their work and published studies by others, Gemmell et al. (17) postu- late in this volume that the Thl response is associated with stable periodontal lesions, whereas a Th2 response leads to the production of nonprotective antibody and disease progression. They have further suggested that a mixed Thl and Th2 lesion in which both IL-4 and interferon y are present may, depending on the relative concentration of each cytokine, lead to the production of protective antibody. Other investigators have postulated the opposite, and still others have observed no skewing toward either Thl or Th2. The data appear to be inconclusive. The pattern of cytokine production may result from a distinct cell phenotype, a specific state of cell differentiation or a transitional state. Which, if any, of these is correct is currently unclear.

The controversial nature of the Thl and Th2 paradigm studies may result from the complexity of the experiments. Humans vary enormously in susceptibility to periodontitis and rates of disease progression. Tissue destruction appears to occur in site- specific bursts of activity that are highly variable and seem to be infrequent in most patients but common in others. There are no methods for distinguishing between disease-active and -inactive sites except by rather crude methods of assessing clinical attachment loss or alveolar bone destruction over time. These factors have not been adequately controlled nor have they been taken into account in the Thl and Th2 studies. The data appear to support the conclusion that Tho, Thl and Th2 clones participate in periodontitis, and there is little evidence that any single T-helper cell subset is associated with any specific clinical status. As stated by Gemmell et al. in this volume (17), a focus on which cytokines are present, their local concentrations and hence their biological activity may be more fruitful than continuing to focus on helper cell clonal type.

This makes it clear that the levels of various cytokines are the chief determinants of the progression or suppression of periodontitis. What remains obscure is the identity of the agents and the mechanisms that determine which cytokines or antagonists are produced, when and how long they are produced and their concentrations. Further elucidation of the details of the cytokine cascade will indicate new ap-


proaches to prevention, diagnosis and treatment of periodontitis.

Arachidonic acid metabolites are key components of the pathogenesis of periodontitis

Arachidonic acid metabolites, especially prostaglandin EL, are important mediators of the immunoinflammatory response in periodontitis (see Gem- me11 et al. (17) in this volume, pages 126-129). Al- though prostaglandins can be produced by most nucleated cells and by blood platelets, the major source in inflamed gingiva is activated macrophages and fibroblasts. Arachidonic acid is generated by the action of phospholipase A2 on cell phospholipids. It is a substrate for the cyclooxygenase enzyme systems (cyclooxygenase 1 and cyclooxygenase 2, also referred to as prostaglandin endoperoxide synthase 1 and 2) that produce a large family of prostaglandins, thromboxane and prostacycline. It is also a substrate for the lipoxygenases that give rise to the leukotrienes, the most important one of which (for periodontal disease) is leukotriene Bq.

Prostaglandins and leukotrienes are major in- itiators and perpetuators of inflammation, and prostaglandin E2 is the major mediator of pathological alveolar bone destruction (see Schwartz et al. (61) in this volume, pages 166-168). Prostaglandins, especially prostaglandin EZ, are present in gingival crevicular fluid from disease active periodontal sites at concentrations five or more times greater than in samples from known inactive sites, and whole- mouth scores for prostaglandin E2 have been reported to be a marker for concurrent or future disease activity. Successful therapy markedly decreases prostaglandin E2 levels.

Mononuclear cells from patients highly susceptible to severe periodontitis release significantly more prostaglandin E2 than do cells from resistant individuals. Release is stimulated by lipopolysaccharide and by cytokines such as IL-1p. Prosta- glandin E2 participates along with cytokines in regulating IgG production. High concentrations inhibit and low concentrations acting synergistically with IL-4 enhance IgG production. Prostaglandin E2 has major catabolic effects on gingival and periodontal ligament fibroblasts. It decreases DNA synthesis and cell growth and the production of collagen and non- collagen proteins. Prostaglandin Ez also inhibits fibroblast IL-6 production induced by TL-1 p or tumor necrosis factor a. In both experimental periodontitis in monkeys and spontaneously occurring periodontitis in dogs, a rise in prostaglandin E2 is associ-

ated with a worsening clinical status. Rising prostaglandin E2 levels can be inhibited in experimental animals and humans by systemic or topical application of nonsteroidal anti-inflammatory drugs, which also inhibit bone loss. The cytokine cascade and arachidonic acid pathways present numerous opportunities to intervene therapeutically in the progression of periodontitis (see Gemmell et al. (17) in this volume, pages 129-132).

Mechanisms of tissue destruction in periodontitis

One of the highlights of the past two decades of periodontal research is the major progress made in understanding the basic mechanisms by which bacteria present in subgingival biofilms and their products and components mediate the pathological changes characteristic of periodontitis. These pathways are now understood in sufficient detail to begin to devise treatments that alter events and outcomes. Alterations in the junctional epithelium have already been considered, and the focus now turns to pathological alterations in components of the extracellular matrix of the gingiva and periodontal Iigament and resorption of alveolar bone. Important new findings and perspectives are listed in Table 6 .

Table 6. The pathogenesis of periodontitis: new findings and new perspectives on the mechanisms of tissue destruction

Periodontitis involves the destruction of bone and connective tissues, including collagens, proteoglycans, and other components of the extraceuular matrix

4 The tissue destruction is not unidirectional, but rather is an iterative process that is constantly being adjusted by the host-bacterial interactions.

determined by the balance of matrix metalloproteinases and their inhibitors. The balance of matrix metalloproteinases and inhibitors is regulated locally by exposure to IL-la, IL-lp, IL-10, transforming growth factor p and lipopolysaccharide. Alveolar bone destruction in periodontitis is a result of uncoupling of the normally tightly coupled processes of bone resorption and bone formation. Tissue prostaglandin &, IL-1s and, to a lesser extent, tumor necrosis factor a appear to mediate bone resorption in periodontitis. IL-6 may also be involved. Circulating factors, including the steroid hormones, parathyroid hormone, calcitonin, and vitamin D3, regulate the overall bone remodeling process.

The destruction of the extracellular matrix is

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Summary of the process

The changes in the connective tissues consist predominantly of destruction of the collagens, proteoglycans and other components of the extracellular matrix. Evidence of destruction is first seen in the region of the vascular plexus just deep to the junctional epithelium. As disease worsens, the area of inflamed connective tissue enlarges and eventually comes to occupy virtually all the marginal gingiva. The inflammatory infiltrate does not generally extend into the periodontal ligament. Instead, the extracellular matrix of the ligament is destroyed as alveolar bone is resorbed. As the disease progresses, the fibrous connective tissue is replaced with a dense infiltrate of inflammatory cells, and alveolar bone is destroyed. Destruction of the extracellular matrix is generally viewed as pathological and undesirable, but it is an integral and essential part of the immunoinflammatory process since it is necessary to create space for occupation by the enlarging populations of infiltrating leukocytes. In advanced lesions of long standing, manifestations of fibrosis and the scarring characteristic of chronic inflammatory diseases can be seen histologically and clinically. Specific species of bacteria in the subgingival biofilm on the roots of the teeth ultimately initiate the destruction of the extracellular matrix and alveolar bone, and some species such as l? gingiualis produce families of potent enzymes, including col- lagenase. However, virtually all the collagenases found in periodontally diseased tissues derive from host cells, not the bacteria. The bacteria drive destruction by attracting and activating host cells that carry out the observed destruction.

Although most of the extracellular matrix of the gingiva and periodontal ligament is destroyed and variable amounts of alveolar bone resorbed in advanced periodontitis, the progress of the disease is not one dimensional or unidirectional. I t is iterative and constantly being adjusted as a result of multiple and changing microbial challenges and multiple local and systemic host defenses. Thus, in addition to the destructive activity, manifestations of attempts at regeneration and healing are also apparent in histological sections. Destruction waxes and wanes with increases and decreases in the extent of immu- noinflammation, changes in the numbers and proportions of various inflammatory cells and their subsets in the tissue and fluctuation in the levels of various cytokines, prostaglandins and effector and inhibitor molecuies such as the matrix metalloproteinases and the tissue inhibitors of matrix metallop-

roteinases. The iterative nature of the pathogenesis is reflected clinically by periods of apparent quiesc- ence of various lengths followed by bursts of destructive disease activity.

Although many details are lacking, the overall events that result in connective tissue destruction and, to a lesser extent, alveolar bone resorption are reasonably well understood. Antigenic and other components of periodontopathic bacteria, especially lipopolysaccharides, stimulate the infiltrating leukocytes and resident cells to produce cytokines and prostaglandins, and they induce production of the matrix metalloproteinases and other proteolytic enzymes and additional mediators by monocytes and macrophages, resident fibroblasts, junctional epithelium and endothelium. The infiltrating neutrophils also participate. Collectively, these molecules mediate destruction of the connective tissue and alveolar bone. An illustration of the overall functioning of these events is presented in Fig. 1.

Destruction of the extracellular matrix of the gingiva and periodontal ligament is determined by the balance of matrix metalloproteinases and their inhibitors

The matrix metalloproteinases comprise a large family of related gene products with extensive homo- logies (see Table 1 and Fig. 2 in Reynolds & Meikle (57) in this volume). These enzymes are designated currently as matrix metalloproteinases 1 through 17, and the list is still growing. They fall into classes including collagenases, gelatinases, stromelysins, ma- trilysins, metalloelastase and membrane-bound matrix metalloproteinases. All the matrix metalloproteinases are produced in latent precursor form and require activation by plasmin or plasmin-like enzymes. Their activities depend on divalent cations. Collectively, these enzymes have the capacity to degrade all the components of the extracellular matrix. In the periodontium, matrix metalloproteinases are produced by macrophages and resident tissue fibroblasts, junctional epithelial cells and neutrophils activated by bacterial substances such as lipopolysaccharide, proinflammatory cytokines and prostaglandins (Fig. 1). Because of their large numbers, the fibroblasts are a major source. Neutrophils carry a pre-made matrix metalloproteinase that can be released. Because of their continuous influx, they too are a major source. At very early stages of inflammation, neutrophil matrix metalloproteinase predominates. Fibroblasts can also express a membrane-bound matrix metalloproteinase that could

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function to provide the initial attack on intact native collagen fibers in the absence of an inflammatory infiltrate.

As might be expected for enzyme activities of high destructive potential, matrix metalloproteinase production and activity are tightly controlled and regulated. Tissue inhibitors of matrix metalloproteinases play a major role, with tissue inhibitor of metalloproteinase 1 being the most important. Tissue inhibitors of metalloproteinases bind to matrix metalloproteinases and irreversibly inhibit enzyme activity. The extent of degradative activity in the tissue is largely a function of the balance between the levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases. It has been suggested that tissue degradation only occurs at locations where the levels of tissue inhibitors of metalloproteinases are low (see Reynolds & Meikle (57) in this volume, pages 147- 149). Fibroblasts and macrophages are the major sources of tissue inhibitors of metalloproteinases as well as matrix metalloproteinases, and the nature of the message received by these cells is a major determinant of outcome.

The overall regulation of matrix metalloproteinase and tissue inhibitors of metalloproteinase production in the periodontium is complex and involves cytokines, hormones, growth factors, prostaglandins and bacterial factors such as lipopolysaccharide (see Reynolds & Meikle (57) in this volume, page 149). Gingival fibroblasts produce prostaglandin E2 but not matrix metalloproteinase following exposure to lipopolysaccharide; they produce matrix metalloproteinase following exposure to IL-lp (Fig. 1). In general, proinflammatory cytokines such as IL-lP upregulate matrix metalloproteinase and downregulate tissue inhibitors of metalloproteinase production, whereas other cytokines such as IL-10 downregulate matrix metalloproteinase and upregulate tissue inhibitors of metalloproteinase. IL- 1 p upregulation appears to occur through stimulation of nuclear factor KB proteins and thereby acts on the promoter region of the matrix metalloproteinase gene. Transforming growth factor p is especially important in downregul- ating matrix metalloproteinase and upregulating tissue inhibitors of metalloproteinase. Cell-associated IL-la is a potent stimulator of matrix metalloproteinase release by other cells. These observations demonstrate that the balance of cytokines in the gingival tissue is the major determinant of whether tissue degradation occurs or homeostasis is maintained, and IL-1s plays a key role.

There is strong evidence that IL-1-induced matrix metalloproteinases participate in degrading the

extracellular matrix in periodontitis and that tissue inhibitors of metalloproteinase, IL-10 and transforming growth factor p participate in resolving inflammation and maintaining homeostasis. Gingival crevicular fluid from sites of active disease have elevated levels of IL-lP and matrix metalloproteinase and low Ievels of tissue inhibitors of metalloproteinase, and the same is true for diseased versus healthy gingival tissue. Cultures of peripheral blood monocytes exposed to lipopolysaccharide produce IL- 1 s and matrix metalloproteinases, and cells from individuals who are highly susceptible to periodontitis are more responsive than are cells from resistant individuals. Successful treatment results in a significant decrease in leveIs of matrix metalloproteinase and IL-1p in the tissue and gingival crevicular fluid and in increased levels of tissue inhibitors of metalloproteinase.

Several approaches for treating periodontitis by decreasing the degradation of the extracellular matrix are apparent (see Reynolds & Meikle (57) in this volume, pages 152-153). The most studied of these is the use of inhibitors of matrix metalloproteinase activity based on the fact that activity depends on divalent cations. Chemically modified tetracycline and low-dose doxycycline inhibit matrix metalloproteinase activity by chelating divalent cations. Low doses prevent collagen breakdown and alveolar bone loss in animal studies and result in reduction in pocket depth and clinical attachment loss in human clinical trials. These drugs appear to work by binding enzyme-associated calcium ions, making the enzyme more susceptible to proteolysis. Hydroxamic acid derivatives also inhibit matrix metalloproteinase activity, but their usefulness in periodontitis has not been studied. The isothiazolones can inhibit car- tilage proteoglycan degradation without decreasing synthesis. These drugs appear to target the activation step of matrix metalloproteinase without being active against the active enzyme. There has been no work on the use of inhibitors of phospholipase A2, which controls the rate-limiting step in prostaglandin production.

Mechanisms of destruction of alveolar bone

Progress in elucidating details of the mechanism of bone resorption and regeneration in periodontitis has lagged behind considerably the progress related to soft tissue. Far less progress has been made in understanding the basic events accounting for alveolar bone loss in periodontitis. Certain facts are well established (see Schwartz et al. (61) in this vol-

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ume, pages 160-164). Bone loss in periodontitis is a result of an uncoupling of the normally tightly coupled processes of bone resorption and bone formation; enduring pathogenic levels of prostaglandin E2, IL-10 and, to a lesser extent, tumor necrosis factor a mediate the loss. IL-6, a cytokine that mediates the formation of multinucleated cells that resorb bone, may also be involved. It is also well documented that the activities of osteoblasts and osteoclasts and their precursor cells are highly integrated and coordinated with one another and with osteo- cytes. There appear to be many feedback loops among these cells, with osteoblasts producing factors that enhance osteoclasts and vice versa. Details of these cell-cell and matrix-cell interactions even in normal bone remain obscure. All these are regulated in an overall long-term sense by circulating factors such as steroid hormones, parathyroid hormone, calcitonin and vitamin DS. Fine tuning is achieved by events occurring locally in bone such as the release of IL-lp, prostaglandin EZ, IL-6 and various other locally produced proteins and factors such as bone morphogenic proteins, fibroblast growth factor, transforming growth factor p, platelet-derived growth factor and epidermal growth factor released from resorbing bone matrix. These enhance the attachment and replication of various bone cells and regulate their activity. Virtually nothing is known about the effect on these events of substances derived from infecting periodontopathic bacteria or the large families of cytokine and regulatory molecules produced during active disease progression. A top priority for further periodontal research must be elucidating the details of normal and abnormal bone physiology, much as has already occurred with the mechanisms of destruction and reconstitution of the extracellular matrix.

There is strong evidence that prostaglandin E2 is the major mediator of alveolar bone loss in periodontitis and that IL-lp, tumor necrosis factor a and IL-6 also play important roles (see Schwartz et al. (61) in this volume, pages 166-168). These mediators can be produced by resident fibroblasts and leukocytes in the inflammatory infiltrate, especially macrophages. They are found in the inflamed gingival tissue and gingival crevicular fluid in elevated concentrations, and these concentrations decrease following successful therapy. Inhibition of prostaglandin E2 production suppresses or blocks alveolar bone destruction in both experimental animals and spontaneously occurring periodontitis in humans. Nevertheless, details of how this group of mediators actually functions in normal bone in the periodon-

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tium and in bone destruction in periodontitis remain elusive, and they are sometimes contradictory. For example, there is evidence that prostaglandin E2 actually stimulates bone apposition in vivo in some situations. The roles of calcitonin and vitamin D3 as well as factors released locally by bone resorption that inhibit resorption and stimulate formation, such as transforming growth factor p, matrix metalloproteinase, fibroblast growth factor, and insulin-like growth factor, remain obscure.

Age has dramatic effects on bone: both men and women lose bone mass after age 25-30 years. The prevalence and severity of periodontitis begin to increase considerably after these same ages. Gender and hormone levels also appear to affect bone status. Both estrogens and androgens affect bone status, but loss of bone mass is greatly accelerated among postmenopausal women not on estrogen re- placement therapy. Nevertheless the prevalence and severity of periodontitis are greater among aging men than among aging women. The relationship between osteoporosis and periodontitis remains obscure.

Regeneration of periodontal attachment including alveolar bone, cementum and functional periodontal ligament has rapidly become a frequently attempted treatment procedure known as guided tissue regeneration. Factors known to be related to bone regeneration including bone morphogenic proteins, platelet-derived growth factor, insulin-like growth factor 1 and transforming growth factor p have to be assessed in experimental animals. The role of these factors in the mechanisms underlying regeneration and the factors involved in its success or failure remain unknown.

A wide range of potential approaches exist for al- tering alveolar bone destruction in periodontitis (in this volume, see: Schwartz et al. (61), pages 166-168 and Reynolds & Meikle (57), pages 152-153). Prosta- glandin E2 is well documented to participate in the immunoinflammatory response in periodontitis in mediating connective tissue alterations and bone resorption, and the nonsteroidal anti-inflammatory drugs effectively inhibit these activities. Neverthe- less, these drugs are not currently used in treating or preventing periodontitis. When they are administered systematically over long time periods, nephro- toxicity and other undesirable side effects can occur. There is strong evidence that their use topically in oral rinses or toothpaste is effective in slowing or ar- resting periodontitis, although more clinical trial data are needed.

The prostaglandin family of mediators is pro-


duced mainly by the cyclooxygenase 1, which is expressed constitutively. At sites of inflammation, cyclooxygenase 2, a recently discovered pathway that is not produced constitutively, can be induced by lipopolysaccharide, IL- 1 p and tumor necrosis factor a (82). Cyclooxygenase 2 is attracting considerable attention as a potential target for intervention since blocking cyclooxygenase 2 could significantly reduce the production of prostaglandins specifically at sites of inflammation and tissue destruction. Radicicol, a fungal antibiotic, inhibits cyclooxygenase 2 probably by inhibiting protein tyrosine kinase. Substrate arachidonic acid is made available by the action of phospholipase A2 on cell phospholipids. Cortico- steroids inhibit phospholipase A*, probably through lipocortin, and can thereby block the production of prostaglandins and leukotrienes. Whether any of these drugs will prove useful in controlling periodontitis remains to be seen. There is a new family of drugs known as the cytokine-suppressing anti-inflammatory drugs (see Reynolds & Meikle (57) in this volume, pages 152-153). The prototype is SKF86002. These drugs selectively inhibit the protein kinase family designated RK38, or CS binding protein. This family is activated by lipopolysaccharide, proinflammatory cytokines or physicochemical stress. Ac- tivation is required for production of IL-1, tumor necrosis factor a and probably prostaglandins. Cyto- kine-suppressing anti-inflammatory drugs strongly inhibit cyclooxygenase 2. The potential role of these new agents in treating periodontitis will undoubt- edly be an area of active investigation in the future.

The transition from a stable periodontal site to active disease progression

Prior to the 1980s, the presence of periodontal pockets was equated with active periodontitis. Sev- eral longitudinal studies conducted in the 1980s (29, 35, 36) modified this view. These studies demonstrated clearly that stable, disease-inactive as well as progressing disease-active periodontal pockets exist. Most periodontal sites (usually more than 90%) in most adult periodontitis patients are disease inactive and nonprogressing, and the occurrence of disease active sites is relatively rare and episodic. Most disease-active sites occur in a very small proportion of the patients. These observations focused attention on the nature of the transition from a stable periodontal site to active disease progression. A clear

understanding of this transition could shed considerable light on better approaches to prevention and therapy.

The transition from a stable to a disease-active site may or may not be the same as the transition from gingivitis to periodontitis. Indeed, as discussed in the previous section, gingivitis lesions and periodontitis lesions may differ considerably from the very beginning of the microbial challenge. This would not be the case for the transition from an already existing but stable periodontal pocket to a disease-active site. If periodontal disease is to progress, the microbial challenge, subgingival biofilms, must continue to be present, although in some individuals the magnitude of the challenge may be surprisingly small in quantity. In addition. there must be per- sisting inflammation and destruction of the components of the extracellular matrix manifested as loss of clinical attachment and a net loss of alveolar bone. Pockets may or may not become deeper.

Theoretically, the characteristics of disease progression may be expected to consist of the continuing presence of lipopolysaccharide and other bacterial components, an inflammatory cell infiltrate, high levels of proinflammatory cytokines including IL-1p and tumor necrosis factor a and low levels of IL-10 and transforming growth factor p, cytokines that suppress the immunoinflammatory response, low levels of tissue inhibitors of metalloproteinases and high levels of matrix metalloproteinases and prostaglandin EP. Macrophages, fibroblasts and to a lesser extent other resident and inflammatory cells activated by lipopolysaccharide and cytokines, especially IL-lf3 and tumor necrosis factor a, produce matrix metalloproteinases and prostaglandin EZ, and can maintain them at pathogenic levels. The question now becomes, what factors and events regulate and determine production and levels of these mediators? The following represents speculation on mechanisms that can explain disease progression.

Loss of the chemotactic gradient

In a stable periodontal pocket, normal host defense contains the microbial challenge. Leukocytes, mostly neutrophils, leave the small vessels and migrate through the connective tissue and pocket epithelium to form a “wall” of viable cells between the biofilm and the pocket wall (see Fig. 7 in Kornman et al. (32) in this volume). This emigration is mediated by a gradient of chemoattractant molecules consisting of IL-8 produced by the epithelial cells and peptides of the N-formyl-methionyl-leucyl-phenylalanine type

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emanating from the biofilm. The leukocytes follow this gradient and do not become activated until their low-affinity receptors became occupied by high concentrations of mediators they encounter as they reach the biofilm. Upon activation, these cells phagocytose and kill bacteria and release chemoattractants and killing agents such as oxygen intermedi- ates. Under conditions that disrupt the chemoattractant gradient, neutrophils recognize no guidance system and accumulate within the connective tissue, where they become activated and release enzymes such as matrix metalloproteinase that destroy the tissue, resulting in attachment loss and bone loss. Further, loss of the gradient can preclude containment of the microbial challenge by neutrophils and antibody and can allow lipopolysaccharide to shower the connective tissues. Gemmell et al. (18) have provided evidence for loss of the chemoattractant gradient by showing that, as inflammation worsens, pocket epithelium may become negative for intercellular adhesion molecule 1, and neutrophil migration through the epithelium and into the pocket decreases. Loss of the gradient could also result from changes in the composition of the biofilm or production of high levels of chemoattractant within the inflamed connective tissue. This hypothesis has not been proven but appears to merit further investigation.

Catabolic activities of resident cells

The resident tissue fibroblasts and junctional epithelial cells may be hijacked by the bacterial challenge and convert from their normal activities of creating and maintaining tissue homeostasis and health to destroying the structures they initially created and maintained. There is evidence for this hypothesis, as presented on pages 226-227 of this chapter. The combinations and concentrations of bacterial substances (lipopolysaccharide) and various mediators that cause the resident cells to convert from an anabolic to an catabolic state may herald the transition from a stable to a disease active lesion.

Thl and Th2 lymphocyte phenotype

The nature of the Thl and Th2 responses may be a major determinant of the progression to destructive periodontitis. Although studies aimed at determining the possible association between the Thl and Th2 lymphocyte phenotype ratios and disease status have been performed, the results are not conclusive. In the active periodontitis lesion, Th2 cells appear to

participate increasingly such that both Thl and Th2 like cells are probably involved. In this context, progression of the disease is probably related to the balance and levels of both Thl and Th2 cytokines. High levels of IL-lP and tumor necrosis factor a would drive active disease, whereas high levels of IL-10 would downregulate inflammatory cytokines, such as IL-lP production by macrophages, and hence reduce the potential for tissue destruction.

Anergy

Recent data from experimental animals suggest that anergy may be an important component of both the transition from gingivitis to periodontitis as well as progression from a stable to an active lesion. Presen- tation of antigen by nonprofessional antigen-presenting cells such as epithelial or endothelial cells, results in anergy (24, 75). The mechanism of this anergy is unknown, but it is well established that junctional and pocket epithelial cells express major histocompatibility complex class I1 antigens (67) and hence could be capable of antigen presentation, which results in anergy and possible apoptosis of the Thl cells and thereby allows cells with a Th2 cytokine profile to emerge. Endothelial cells, which in humans constitutively express major histocompatibility complex class 11, could also present antigen leading to anergy (75). In this context, regulation of the immune response may reside at the level of antigen presentation such that presentation by pro- fessional antigen-presenting cells (such as dendritic cells and macrophages) results in stimulation of destructive cytokines while presentation by nonprofessional antigen-presenting cells (such as epithelial or endothelial cells) results in anergy and no tissue destruction. There seems little doubt that levels of proinflammatory cytokines, matrix metalloproteinases and prostaglandin E2 increase greatly and tissue inhibitors of metalloproteinases decrease in tissues manifesting periodontitis relative to those with gingivitis. Additional studies need to be performed to test this concept.

Leukocyte emigration

Suppression of leukocyte diapedesis and migration may be involved in determining whether disease progression occurs. Administration of anti-neutro- phi1 serum or drugs that cause severe neutropenia to rats or dogs results in the rapid onset and progression of periodontal tissue destruction (in this volume, see: Ishikawa et al. (27) and Dennison & Van

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Dyke (11) page 67) (1). The release of soluble E-selectin by endothelial cells and the shedding of L-selectin can modulate the diapedesis of leukocytes such as neutrophils. Decreased L-selectin levels have been reported for neutrophils from gingival capillary blood from patients as compared with cells from peripheral blood (33). These differences were not observed in neutrophils from healthy subjects, suggesting a change in the selectin-mediated ability of neutrophils to interact with endothelial cells in the chronically inflamed gingiva. Bacterial species in the biofilm produce large quantities of polyamines that can inhibit neutrophil migration (79) and perhaps most importantly, I? gingiudis has been shown to block multiple aspects of the endothelial signaling process critical to local recruitment of neutrophils, which suppresses the primary host defense system protecting against the bacterial challenge. The enormous importance of neutrophils in fending off the pathogenic effects of the subgingival bioflm has been amply demonstrated (see Dennison & Van Dyke in this volume (1 I)) .

Changes in the composition of the biofilm and invasion

The transition from gingivitis to periodontitis may be related to changes in bacterial composition of the biofilm or invasion of the tissue by bacteria from the biofilm. In other words, the bloom of certain species in the flora may be causally related to the burst of destructive activity characteristically observed in periodontitis.

Environmental and acquired risk factors

A new paradigm for periodontal diseases is emerg- ing. Progress in understanding these diseases can be summarized more or less by decade. In the 1960% the major development was the demonstration that bacteria in dental plaque cause human gingivitis and periodontitis (30, 34, 38). In the 1970% specific species of predominantly gram-negative, anaerobic bacteria were associated with these diseases (28, 44, 71, 7 4 , and a wealth of evidence was presented that the immune system of people with periodontitis could recognize the antigens of these infecting bacteria (28). The 1980s saw strong documentation of the association of specific bacteria with active tissue destruction, beginning characterization of the immune

response to antigens and mitogens of the infecting bacteria and beginning elucidation of the role of cytokines and prostaglandins in the pathogenesis (19,21,49,56, 68). It was also during the 1980s that the site-specific nature of periodontitis was demonstrated (72), that the prevalence of disease, especially severe disease, was shown to be much lower than previously believed (61, and that progression of tissue destruction at specific sites was episodic and relatively infrequent (29, 35,361. Significant progress was made in elucidating the pathways of connective tissue destruction and alveolar bone resorption (5).

Prior to the 1980% periodontitis was thought to be universally prevalent in human adults by early middle age. Because of this, no thought was given to important roles for factors other than bacteria. The role and importance of bacteria in the etiology of periodontitis dominated thought in the 1970s and 1980s, and almost all preventive measures and treatments were targeted at eliminating or at least controlling the bacterial challenge. Consideration of a major role for the host was generally an afterthought and played no role in diagnostic and therapeutic de- cisions. This began to change dramatically in the 1980s with the demonstration that the prevalence of periodontitis was in fact much lower than previously thought: disease of sufficient severity to cause loss of teeth affects only about 15% of adults in most countries (6).

The 1990s are dramatically different; research has discovered that bacteria are essential but insufficient for disease, that bacteria account for a relatively small portion of the variance in susceptibility for disease expression (about 20% by some estimates, and tobacco smoking accounts for more) (20), and that hereditary factors alone can account for up to roughly 50% of the variance (42,431. This is the decade of the paradigm shift; this is the decade of the host and disease modifiers (see Fig. 1 in Page & Kornman (51) in this volume on page 10). In addition, this shift provides a new perspective on the distinctly different roles of the bacteria, the host and risk factors and indicators in the disease process.

So far this chapter has focused on the nature of the microbial challenge, assembling the cells and systems that participate in periodontitis, the role of the immune system, complement and the phagocytic cells, regulation by the cytokine network and mechanisms by which the connective tissues of the gingiva and periodontal ligament are destroyed and alveolar bone resorbed. These components of the pathogenesis are shared by all forms of periodontitis (47, 48). The next section focuses on the factors that

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influence the shared common events. Important new findings and perspectives are summarized in Table 7. In this volume, Salvi et al. (60) discuss factors that can affect shared events in the pathogenesis in a manner that hastens the onset of disease, increases its rate of progression or severity or causes the disease to be refractory to treatment or to reoccur. These include diabetes mellitus, tobacco smoking, stress, HIV infection and osteoporosis. Other factors reported in the literature are alluded to but not discussed in detail. These include several additional diseases and conditions that compromise host defense, severity of disease at baseline, age, presence of pathogenic bacteria in the oral flora, socioeconomic status, educational level, oral hygiene and frequency of visits to the dentist. Heredi- tary factors are considered in a separate section.

Diabetes mellitus

Diabetes mellitus is well documented as a risk factor for periodontal disease; diabetics have an odds ratio of about 2-3 relative to non-diabetics. Individuals with diabetes mellitus have an increased prevalence of periodontitis and respond poorly to therapy. Peri-

Table 7. The pathogenesis of periodontitis: new findings and new perspectives on the environmental and acquired risk factors

Periodontitis appears to behave as a multifactorial disease in which multiple genetic and environmental factors interact to produce the disease and modify its clinical expression. One prime example of these interactions is the IgG2 levels in early-onset periodontitis, which appear to be influenced by both genetic factors and smoking.

systemic diseases, including cardiovascular disease, diabetes and pre-term low-birth-weight delivery. Diabetes is a well-documented risk factor for periodontitis. Many diabetics manifest an upregulated monocyte phenotype that is characterized by expression of high levels of prostaglandin E2, IL-10 and tumor necrosis factor a in response to a lipopolysaccharide challenge. Diabetes is known to alter collagen metabolism and the basement membranes of small vessels. Smoking is a well-documented risk factor for periodontitis. Smoking inhibits neutrophil function, suppresses IgG2 antibody response, enhances rnonocyte release of IL-1s and may directly affect osteoblast function. Psychosocial stress may be associated with an increased severity of periodontitis. HIV infection is a risk factor for an ulcerative form of periodontitis.

Periodontitis is strongly associated with major

odontitis may be more severe in patients with diabetes of long duration and with poor metabolic control. There is evidence that diabetics with excellent metabolic control are no more susceptible to severe periodontitis than non-diabetics (see Salvi et al. (60) in this volume). The diabetic state does not appear to affect the composition of the subgingival biofilms; instead, numerous aspects and steps in the shared pathogenesis can be affected and thereby susceptibility enhanced and severity worsened (see Salvi et al. (60) in this volume, pages 181-183). Nevertheless, diabetic individuals vary substantially; the diabetic state may have multiple manifestations and conse- quent effects on periodontitis susceptibility in some individuals and none in others. This great variation may be genetically based.

The mechanisms by which diabetes enhances the risk for severe periodontitis are poorly understood, although some diabetics manifest pathological changes that may be related (see Salvi et al. (60) in this volume, pages 181-183). Neutrophil adhesion, chemotaxis, phagocytosis and killing can be impaired and the patient deprived of the major host defense against microbial challenge. Many diabetics manifest an upregulated monocyte phenotype resulting from metabolic or genetic effects. Such cells are proinflammatory in that they produce more IL- lp , tumor necrosis factor a and prostaglandin E2 both unchallenged and in response to lipopolysaccharide challenge than normal. The gingival tissues and gingival crevicular fluid contain elevated concentrations of these mediators, and their presence can elevate levels of matrix metalloproteinase, enhance tissue destruction and increase disease severity. The connective tissues in diabetics may manifest pathological change. Collagen production by fibroblasts in the periodontal ligament and gingiva may decrease and the production of gingival matrix metalloproteinase may increase. Matrix metalloproteinase activity in gingival crevicular fluid can be elevated, and gingival fibroblasts in culture synthesize less collagen than normal. The excess matrix metalloproteinase appears to derive from neutrophils. Under hyperglycemic conditions, collagen is nonen- zymatically glycosylated and its solubility and turn- over rates change. Diabetics also have impaired production of bone matrix components by osteoblasts.

A fundamental lesion is thickening of the basement membranes of small vessels with nonenzy- matic glycosylation of proteins and accumulation of deposits within the vessel wall and on the luminal surfaces. These changes narrow the vessel lumen and interfere with transport across the vessel wall.

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Patients vary greatly in the development of such lesions; they are seen most frequently in individuals having a genetic predilection, diabetes of long duration and poor metabolic control of hyperglycemia. Advanced glycation end products induce oxidant stress in periodontal tissues, and this may be related to enhanced periodontal disease. Immune regulation may also be affected. In subjects with type 1 diabetes there is a clear association with HLA-BB and B15, and about 95% of such patients manifest DR3 or DR4 or both.

Of the possibilities described 'above, the elevated levels of IL-1, tumor necrosis factor a, prostaglandin E2 and matrix metalloproteinases are likely to have the greatest influence on the common shared events in the pathogenesis of periodontitis.

Tobacco smoking

A wealth of data demonstrate that cigar tte smoking is the known environmental risk mos 9 strongly associated with periodontitis, and especially severe periodontitis (see Salvi et al. (60) in this volume, pages 183-184). Indeed, in some studies, the impact of smoking actually outweighs the effect of the pathogenic bacteria as a determinant of outcome. Smoking has marked effects on the clinical features of periodontitis. The gingival tissue tends to be hyperkeratotic and fibrotic with thickened margins and tends to manifest minimal erythema and edema relative to disease of equal severity in nonsmokers. Attachment loss is generally more severe in the anterior regions, especially on the palatal aspects of the maxillary anterior teeth. The disease is more severe and widespread relative to a nonsmoking cohort of comparable age. There may be no positive association between periodontal status and high plaque and calculus scores. Surgical and nonsurgical therapies are less effective in smokers than in nonsmokers, and the disease is more likely to recur.

Both locally and systemically induced effects on the periodontium have been described (see Salvi et al. (60) in this volume, page 185). Smoking tends to mask gingival inflammation by constricting the blood vessels of the gingiva, as well as the coronary arteries and vessels of the placenta. Smoking affects leukocyte function. The functional activity of both salivary and tissue neutrophils is suppressed. Smok- ing has major effects on immune responsiveness. A negative significant association has been shown between smoking and serum antibody levels to certain periodontal bacteria. The effect may be specific. Smoking suppresses production of the IgG2 subclass

of immunoglobulin both in periodontitis patients and periodontally normal individuals. Levels of IgG2 antibody specific for antigens of A. actinomycetem- comituns are significantly lower among smokers with adult periodontitis and rapidly progressive periodontitis than among nonsmokers with the same diseases. Since the predominant serum antibody response to the major periodontal pathogens is mostly of the IgG2 subclass, suppression of IgG2 production may be a primary mechanism through which smoking enhances susceptibility to severe periodontitis. Smoking appears to have other effects on immune regulation: T-cell subset ratios appear to be altered in smokers. Nicotine also increases the lipopolysaccharide-induced release of IL-1 p by monocytes.

Smoking may directly affect connective tissues and bone. Cytotoxic substances such as nicotine and its major metabolite, cotinine, can be detected in saliva and crevicular fluid and in serum and urine, demonstrating their systemic availability. These substances rapidly penetrate epithelium and affect fibroblasts. Smoking decreases the intestinal absorp- tion of calcium and may thereby affect osteoblast function and increase bone loss. Toxic substances from tobacco smoke can coat the root surfaces of periodontally involved teeth and interfere with post- surgical healing. Finally, there is evidence that smoking may affect the composition of the subgingival flora and enhance subgingival infection. Smoking al- ters the short-term oxidation-reduction potential in plaque and may thereby result in an increased proportion of anaerobic bacteria.

Because cigarette smoking has extraordinary effects in enhancing the susceptibility to severe periodontitis, which hinders successful therapy, smoking cessation is now becoming an important part of successful prevention and management of periodontitis.

Psychosocial stress

Clinicians have long suspected that stress is important in susceptibility to periodontitis and in response to therapy. Research is this area is still in its infancy, and documentation of an association between stress and the common forms of periodontitis is lacking. Data support an association between stress and acute necrotizing ulcerative gingivitis, and stressed acute necrotizing ulcerative gingivitis patients manifest depressed neutrophil chemotaxis and phagocytosis as well as reduced lymphocyte proliferation in response to nonspecific mitogen stimulation. Stress, distress and coping behaviors may be associ-

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ated with an increased severity of periodontal tissue destruction. Salvi et al. (60) have evaluated the data thoroughly and conclude in this volume (pages 185- 186) that, although stress is a potential risk factor, it has not been adequately evaluated.

Cellular and molecular interactions between neur- oendocrine and immune systems have been well documented (see Salvi et al. (60) in this volume, pages 186-187). Corticosteroids inhibit several inflammatory cells, including macrophages, neutrophils, eosinophils and mast cells. These are mediated by suppressing cytokines such as IL-1, IL-2, IL-3, IL- 4, tumor necrosis factor a, interferon y and granulocyte-macrophage colony-stimulating factor and by inhibiting arachidonic acid metabolism and the production of prostaglandins and leukotrienes. De- pressed immune responsiveness resulting from stress has been postulated as one of several factors involved in destructive periodontal diseases.

HlV infection

A form of periodontitis designated as necrotizing ulcerative periodontitis has been described in patients with HIV infection or AIDS. The disease is painful, rapidly destructive and frequently does not respond to treatment. The frequency of this form of periodontitis seems very low. Nevertheless, AIDS is clearly a risk factor for this form of periodontitis (see Salvi et al. (60) in this volume, pages 188-189).

The subgingival flora is similar to that seen in adult periodontitis, although Candida albicans and enteric bacterial species may be present in the subgingival biofilms. Patients may manifest decreased neutrophil chemotaxis, phagocytosis and bacterial killing and impaired Fc receptor-specific clearance. Monocyte chemotaxis may also be abnormal. The T4:T8 lymphocyte ratio and the absolute numbers of T4 helper cells may be decreased. Levels of IL- 1 p in gingival crevicular fluid are elevated. These abnormalities taken together would be expected to result in inadequate host defense against the microbial challenge and cytokine-mediated enhancement of matrix metalloproteinase-mediated connective tissue destruction and prostaglandin E-mediated alveolar bone loss.

Genetic risk factors for periodontitis

Evidence for genetic susceptibility

Genetic factors that act on and modify host responses to the microbial challenge are major deter-

minants of susceptibility to periodontitis and the rates and extent of disease progression and severity. New findings and perspectives are listed in Table 8. Evidence for genetic susceptibility comes from three sources: the association of periodontitis with specific genetically transmitted disease traits, twin studies of adult periodontitis and linkage and segregation studies of families with early-onset forms of periodontitis. Among the genetically transmitted disease traits are neutropenia, trisomy 21 and Papillon-Le- f h e syndrome, which confer abnormalities in neutrophil number or function and thereby greatly enhance susceptibility to periodontitis. Twin studies of adult periodontitis have shown greater concordance for periodontitis susceptibility between monozygotic twins than between dizygotic twins and demon.. strated that heredity accounts for about 50% of thc enhanced risk for severe periodontitis. Studies on families with early-onset periodontitis have shown one or more autosomal major gene loci linked to en-

Table 8. The pathogenesis of periodontitis: new findings and new perspective on the genetic risk factors

Periodontitis appears to behave as a multifactorial disease in which multiple genetic and environmental factors interact to produce the disease and modify its clinical expression. One prime example of these interactions may be seen in the IgG2 levels in early- onset periodontitis, which appear to be influenced by both genetic factors and smoking. Genetics has been shown to influence both early- onset periodontitis and adult forms of periodontitis. There are currently five promising candidates for a genetic influence on periodontitis. Given the critical protective role neutrophils play in periodontitis, genetic defects in neutrophil function would be expected to alter the disease. IgG2 subclass of antibody is prominent in both early-onset periodontitis and adult periodontitis. IgG2 levels are known to be genetically regulated at the G2M23 locus, which has been associated with early-onset periodontitis. IgG2 levels are also reduced in some smokers. A polymorphism in the genes coding for the FcyII receptor on neutrophils has been associated with early-onset periodontitis and with phagocytic function of neutrophils in conjunction with IgG2 antibodies. A combination of two polymorphisms in the IL-1 genes has been associated with more severe periodontitis in adults. Given the apparent role of prostaglandin E2 in periodontitis, genes controlling the prostaglandin endoperoxide synthase enzymes may be good candidates for genetic influences on periodontitis. A recent linkage analysis in early-onset periodontitis families identified a disease associated with the physical region on chromosome 9q32-33, which contains the gene for prostaglandin endoperoxide svnthase- I .

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hanced susceptibility for periodontitis (see Hart & Kornman (23) in this volume, pages 207-21 1).

Candidate genes for enhanced periodontitis

The genetic traits that may confer enhanced susceptibility to periodontitis are listed in Table 9 and discussed below (see Hart & Kornman (23) in this volume, pages 206-211).

Abnormalities in neutrophil function have been demonstrated in approximately 75% of individuals with juvenile periodontitis. Cells from about 75% of these individuals manifest suppressed chemotaxis and phagocytosis in v i m . In addition, the cells are unable to mobilize extracellular calcium normally, and intracellular signaling pathways appear to be abnormal. These abnormalities could hamper the critical role the phagocytic cells play in fending off the microbial challenge and thereby account for enhanced disease susceptibility (see Hart & Kornman (23) in this volume, pages 207-211) (26).

Serum IgG2 antibody appears to be the major serum antibody produced in response to periodontal infection. The capacity to produce IgG2 is genetically determined and varies greatly among individuals (see Hart & Kornman (23) in this volume, pages 207- 209). Studies on more than 100 families with early- onset periodontitis have demonstrated a linkage of disease susceptibility to the human leukocyte antigen region of chromosome 6 containing the immune response genes (81). Segregation analysis of such families has revealed a major locus accounting for approximately 62% of the variance for IgG2 production (39, 40). Thus, a genetically determined reduced capacity to produce IgG2 during the course of periodontal infection may in part account for the en-. hanced disease susceptibility.

The phagocytic cells, especially the neutrophils and specific antibody, comprise the major host defense against periodontal infection. These cells function by

Table 9. Genetic traits that may confer enhanced susceptibility for periodontitis

Abnormal phagocyte function Reduced capacity 10 produce IgG2. chromosome 6 hFc-y-IUIa polymorphism hmor necrosis factor a polymorphism,

Variable rnonocyte and macrophage function IL-10 polymorphism, chromosome 2q13 Prostaglandin endoperoxide synthase 1 gene,

chromosome 6

chromosome 9q32.33 - - ___. . . - - . __

accumulating at the sites of challenge by chemotaxis, where they phagocytose and kill microorganisms that have been opsonized. The major opsonin is specific antibody, which is mostly IgG2 in periodontitis patients. The phagocytic cells express a family of cell surface receptors that recognize bacteria coated with specific antibody by binding the Fc region and thereby provide the recognition mechanism for phagocytosis and killing. More than one receptor can recognize bacteria opsonized by IgGl and IgG3, but only one receptor, designated hFc-y-RIIa, recognizes bacteria coated with IgG2. This receptor is polymorphic at amino acid reside number 131, where either histidine or arginine may be present (see Hart & Kornman (23) in this volume, page 209). When the receptor is homozygous for histidine, the receptor affinity is high; when the receptor is homozygous for arginine it is low, and when the receptor is heterozygous it is of intermediate affinity. Neutrophils from individuals who are homozygous for histidine effectively phagocytose and kill bacteria opsonized by IgG2. Individuals who are homozygous for arginine or those who are heterozygous may manifest enhanced susceptibility to infections by encapsulated bacteria such as Huerno- philus infuenzue and to meningococcal disease. The same may be true for periodontitis sites, since the predominant humoral response for periodontal microbial infection is IgG2. Therefore, even though a given individual may produce high titers of high-affinity IgG2 antibody, phagocytosis and killing of infecting bacteria may not occur if the low-affinity receptor is expressed.

Monocytes and macrophages are key in the pathogenesis of periodontitis because they are activated by lipopolysaccharide and proinflammatory cytokines and can produce large amounts of tumor necrosis factor a, IL-lp, prostaglandin E2 and matrix metalloproteinase (Fig. 1). Cell responses are genetically determined and unique for individuals. Vari- ation in responsiveness among individuals is pro- found and stable over time. Blood monocytes from individuals who are susceptible to severe periodontitis release significantly more tumor necrosis factor a, I t - l p and prostaglandin E2 than monocytes from individuals who are resistant (15, 16, 45, 58). Based on these observations, Offenbacher (46) pro- posed a model for the pathogenesis of periodontitis in which susceptible individuals manifest a hyperresponsive monocyte phenotype relative to individuals who are resistant.

The proinflammatory cytokines tumor necrosis factor a and IL-1p and prostaglandins (especially prostaglandin E2) are extremely important in the

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Fig. 3. In sicu hybridization studies and analyses performed on gingival crevicular fluid and tissue extracts have shown that periodontal health is characterized by low levels of proinflammatory cytokines, prostaglandin E2 (PGE,) and matrix metalloproteinases (MMPs) and high levels of tissue inhibitors of metalloproteinases (TIMP) and cytokines that suppress the immunoinflammatory response. The opposite is observed in active periodontitis. TNF-a: tumor necrosis factor a; INF-1: interferon y; TGF- f.k transforming growth factor p.

pathogenesis of periodontitis (Fig. 3). The levels of these molecules and of matrix metalloproteinase are high at sites of active tissue destruction and low at healthy and successfully treated sites. The tumor necrosis factor a gene has been mapped to chromosome 6 , and a polymorphism exists in the gene promoter region that results in greatly increased tumor necrosis factor a production (41, 80). Individuals ex- pressing this polymorphism manifest enhanced susceptibility to certain infections. Although this polymorphism has not yet been linked directly to susceptibility to periodontitis, there is a strong possibility that such a link does exist. Similarly, the IL-1 gene family located on chromosome 2q13 is polymorphic. Individuals with one of the specific genotypes produce about four times more IL-1p than do genotype- negative individuals, and they are about 20 times more likely to develop severe adult periodontitis at ages beyond 40 years than are those who are gene negative (31). Finally, studies on more than 100 families with early-onset periodontitis have demonstrated a linkage with chromosome 9q32,33 containing the gene for cyclooxygenase 1 (prostaglandin endoperoxide synthetase 1) (81). This is the enzyme system responsible for producing prostaglandin EZ. The linkage of the gene to enhanced disease susceptibility is most likely related to enhanced levels of prostaglandin E2 production. One or more of these genetic abnormalities may account at least in part

for the hyperresponsive monocyte phenotype suggested by others (46, 47).

The pathogenesis of periodontitis: a new paradigm

Periodontitis is a family of related chronic inflammatory diseases, all of which are bacterial infections, The bacteria most well documented to cause these diseases include I! gingivalis, B. forsythus and A. acti- nornyceterncornitans (8). Other species may be involved in causing periodontitis, but they act more indirectly than these species, and other species such as enteric bacteria may be involved in special cases. Nevertheless, these species are the primary pathogens in most cases. Bacteria are necessary but insufficient to cause periodontitis. In studies appropriate for multivariate analysis, the bacterial component accounts for a relatively small proportion (roughly 20%) of the variance in disease expression. Host factors are equally or more important than the bacteria in determining disease development and outcome. The complex interplay between the bacterial challenge and innate and acquired host factors deter- mines the outcome.

We propose a new paradigm for the pathogenesis of periodontitis (see Fig. 1 in Page & Kornman (51) in this volume). This paradigm is based on advances in knowledge in three specific areas. First, the subgingival microbial flora is highly organized into biofilms and manifests the characteristics of biofilms. The bacteria are largely protected from the host defenses and are highly resistant to chemotherapeutic agents. Physical disruption by scaling and root planing is an effective treatment. Second, major advances have been made at the cellular, molecular and genetic levels in understanding the pathways and mechanisms by which the bacteria present in these biofilms initiate and perpetuate the immunoinflammatory response that destroys the connective tissue of the gingiva and periodontal ligament and resorbs the alveolar bone. It is now known that these pathways underlie and are shared by all forms of chronic marginal periodontitis. Third, although bacteria cause periodontitis and are essential for disease to occur, bacteria alone are insufficient. A susceptible host is required. Acquired and environmental risk factors such as tobacco smoking as well as genetically transmitted traits modify the shared pathways by which bacteria cause tissue destruction, and they determine disease susceptibility, onset, progression, severity and outcome. This new way of

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looking at the pathogenesis of periodontitis opens entirely new approaches to preventing and managing this family of diseases.

Although many of the details of the pathogenesis of periodontitis are complex and not yet well understood, certain aspects stand out. Perhaps most importantly, the dynamic events of pathogenesis are determined primarily by the signaling and regulating molecules that direct cellular function. Elevated levels of bacterial substances characterize periodontitis, especially active progressing lesions. The main substances are lipopolysaccharide, the proinflammatory cytokines (IL-1, tumor necrosis factor a and, to a lesser extent, IL-61, prostaglandins (especially prostaglandin E2) and the matrix metalloproteinases. In marked contrast, an absence of or very low levels of bacterial substance, especially lipopolysaccharide, the presence of cytokines that suppress the immunoinflammatory response (IL-10 and transforming growth factor J3) and tissue inhibitors of metalloproteinases combined with low levels of prostaglandin E2 and matrix metalloproteinases characterize stable and resolving periodontal lesions and sites of periodontal health (Fig. 3). These are central features of the pathogenesis shared by all forms of periodontitis. Environmental, acquired and genetic risk factors are major determinants of the presence and concentration of specific antibodies, cytokines, prostaglandins and proteases. Many of these risk factors can be modified to prevent or alter disease onset or progression or used as tests to determine disease susceptibility.

Since it was discovered in the 1960s and following years that periodontitis is infectious, therapy and prevention have been targeted almost exclusively at the bacteria and eliminating or reducing them to numbers below the threshold levels required to initiate and perpetuate disease. This approach, especially physical disruption and removal of the subgingival biofilms by procedures such as scaling and root planing, will remain a central and essential part of periodontal therapy. Nevertheless, the new paradigm modifies the traditional approach to account for host and risk factors.

The presence and concentrations of factors characterizing destructive periodontitis lesions vary greatly among individuals, and many are determined genetically. Environmental and acquired factors such as tobacco smoking also affect these factors significantly, either directly or indirectly. These observations point the way to new approaches for prevention, diagnosis and treatment. Approaches that suppress the levels of the agents that characterize active

disease and that enhance the levels of those that characterize health may be expected to be effective. Interventions that reduce the levels or activities of IL-lp, tumor necrosis factor a, prostaglandin E2 and matrix metalloproteinases and those that enhance the levels or activities of IL-10, transforming growth factor p and tissue inhibitors of metalloproteinases could serve as the basis for effective treatment. The various chapters in this volume have suggested several such approaches.

Under the new paradigm, periodontics is rapidly changing from diagnosing and treating existing disease to prevention and health promotion. Reduction of risk becomes the primary objective of intervention for individuals and populations. Identifymg the factors that place individuals and groups at enhanced risk and managing risk as a means of prevention and treatment are of ever increasing importance. Some risk factors are immutable to change; others are not. For example, the data show that smoking cessation alone can reduce the risk of severe periodontitis remarkably. The risk imposed by diabetes, although immutable, can be significantly reduced or eliminated by measures ensuring the highest level of metabolic control.

The most dramatic potential for future control or virtual elimination of periodontal disease may ema- nate from advances in understanding the role of heredity in determining susceptibility to and the severity of disease. Although this field is still in its infancy, enough is known already to glimpse the future. Sev- eral studies are linking susceptibility to early-onset periodontitis to the human leukocyte antigen region of chromosome 6, the site of the genes regulating the IgG2 humoral immune response and the production of tumor necrosis factor a. IgG2 is the major antibody class produced in response to periodontal pathogens. The data indicate that a major locus, possibly at that location, can account for 62% of the variance in IgG2 levels. If this is the case, a measure of the capacity of an individual to mount an IgG2 response could be a major indicator of disease susceptibility, severity and progression. Several genetic polymorphisms in the tumor necrosis factor gene family are known to exist. Polymorphisms in the tumor necrosis factor genes associated with susceptibility to periodontitis can be searched for and probably exist.

The discovery of polymorphisms in the genes for the hFc-y-RIIa receptor on the phagocytic cells further elucidates the possible genetic basis for susceptibility to all forms of periodontitis. This polymorphism results in the expression of receptors of

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high, low or intermediate affinity. Since the hFc-y- RIIa receptor is the only one that recognizes bacteria opsonized with IgG2, expression of the low-affinity receptor may be expected to significantly enhance susceptibility to periodontal pathogens and to severe periodontitis, regardless of the capacity of the affected individuals to produce high levels of biolo- gically effective antibody.

Similarly, the observation of linkage between susceptibility to early-onset periodontitis and the region on chromosome 9 encoding for cyclooxygenase 1 is seminal and more definitively documents the significance of prostaglandin E2 in the pathogenesis of periodontitis. This gene encodes for the constitutively produced cyclooxygenase enzyme system responsible for production of prostaglandin EZ, the major mediator of alveolar bone destruction in periodontitis. Location and characterization of the gene for cyclooxygenase 2, which is activated by IL-1 specifically in inflammatory diseases such as periodontitis, may have even more important implications.

IL-1 and especially IL-1P is unquestionably important in the pathogenesis of periodontitis. A polymorphism in the IL-1 gene family associated with high susceptibility to severe adult periodontitis at age 40 and beyond has been identified, and a laboratory test to detect the polymorphism has been developed (31). Individuals who do not smoke and test positively produce approximately 4-fold more IL- 1 p in response to lipopolysaccharide, substantially increasing the probability that they will develop severe periodontitis relative to those who test negatively. Smoking and the IL-1P gene test could identify more than 85% of the individuals with enhanced susceptibility for severe periodontitis.

These observations point the way to the future. We envision the development and availability of relatively simple, inexpensive tests for the polymorphism in the IL-lP gene family, for hFc-y-RIIa phagocyte receptor affinity and for the capacity to mount an effective IgG2 humoral immune response. We envision a time when young people can be tested and the results combined with other potential risk factors to accurately assess their risk of periodontitis. Young individuals could probably be accurately dif- ferentiated into those who are almost certain to develop periodontitis in later life and those who are not. Susceptible individuals could be monitored, for example, using DNA probes to detect early colonization by periodontal pathogens. When colonization occurs, it could easily and inexpensively be eliminated. Elimination of periodontitis as a significant

disease in some populations now seems to be possible.

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