The Initial Lesion

The initial lesion is typically said to develop within 2 to 4 days of accumulation of plaque at a site that was otherwise plaque-free and at which there was no inflammation evident microscopically. However, this situation is probably never encountered in reality, and the gingival tissues always have characteristics of a low-grade chronic inflammatory response as a result of the continual presence of the subgingival biofilm. In other words, the initial lesion corresponds to the histologic picture that is evident in clinically healthy gingival tissues. This low-grade inflammation is characterized by dilation of the vascular network and increased vascular permeability, permitting the neutrophils and monocytes from the gingival vasculature to migrate through the connective tissues toward the source of the chemotactic stimulus—the bacterial products in the gingival sulcus. Upregulation of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in the gingival vasculature facilitates the migration of neutrophils from the capillaries into the connective tissues. Increased leakage of fluid from the vessels increases the hydrostatic pressure in the local microcirculation, and as a result, GCF flow increases. Increased GCF flow has the effect of diluting bacterial products and also potentially has a flushing action to remove bacteria and their products from the crevice, although given the nature of the bacterial biofilm, it is likely that only planktonic (free-floating) bacteria are removed in this way.

The Early Lesion

The early lesion develops after about 1 week of continued plaque accumulation and corresponds to the early clinical signs of gingivitis. The gingiva are erythematous in appearance as a result of proliferation of capillaries, opening up of microvascular beds, and continued vasodilation.¹⁰⁴ Increasing vascular permeability leads to increased GCF flow, and transmigrating neutrophils increase significantly in number. The predominant infiltrating cell types are neutrophils and lymphocytes (primarily thymic lymphocytes [T-cells]),¹³⁵ and the neutrophils migrate through the tissues to the sulcus and phagocytose bacteria. Fibroblasts degenerate, primarily via apoptosis (programmed cell death), which increases the space available for infiltrating leukocytes. Collagen destruction occurs, resulting in collagen depletion in the areas apical and lateral to the junctional and sulcular epithelium. The basal cells of these epithelial structures begin to proliferate to maintain an intact barrier against the bacteria and their products, and as a result the epithelium can be seen proliferating into the collagen depleted areas of the connective tissues (Figure 21-2).¹⁵¹ As a result of edema of the gingival tissues, the gingiva may appear slightly swollen, and accordingly, the gingival sulcus becomes slightly deeper. The subgingival biofilm exploits this ecologic niche and proliferates apically (thereby rendering effective plaque control more difficult). The early gingival lesion may persist indefinitely or may progress further.

Figure 21-2 Histologic appearance of gingivitis. A series of photomicrographs illustrating gingivitis (H&E). In all cases, the tooth would be to the left side of the image. Low magnification of the gingiva (A) demonstrates hyperplastic junctional and sulcular epithelium with a dense inflammatory cell infiltrate in the adjacent connective tissue. Medium magnification of the epithelial-connective tissue interface (B) shows numerous intraepithelial inflammatory cells along with intercellular edema. The connective tissue contains dilated capillaries (hyperemia), and there is a dense inflammatory cell infiltrate. High magnification (C) shows neutrophils and small lymphocytes transiting the sulcular epithelium.

The Established Lesion

The established lesion roughly corresponds with what clinicians would refer to as chronic gingivitis. The progression from the early lesion to the established lesion depends on many factors, including the plaque challenge (the composition and quantity of the biofilm), host susceptibility factors, and risk factors (both local and systemic). In the initial work by Page and Schroeder, the established lesion was defined as being dominated by plasma cells.¹³¹ In human studies, reports have suggested that plasma cells predominate in established gingivitis in older subjects,⁵¹ whereas lymphocytes predominate in younger individuals, although the relevance of these findings is not clear.^23,51 What is clear from all the studies is that there is a significant inflammatory cell infiltrate in established gingivitis that occupies a considerable volume of the inflamed connective tissues. Large numbers of infiltrating cells can be identified adjacent and lateral to the junctional and sulcular epithelium, around blood vessels, and between collagen fiber bundles.²² Collagen depletion continues, with further proliferation of the epithelium into the connective tissue spaces. Neutrophils accumulate in the tissues and release their lysosomal contents extracellularly (in an attempt to kill bacteria that are not phagocytosed), resulting in the further tissue destruction. Neutrophils are also a major source of MMP-8 (neutrophil collagenase) and MMP-9 (gelatinase B), and these enzymes are produced in large quantities in the inflamed gingival tissues as the neutrophils migrate through the densely packed collagen fiber bundles to enter the sulcus. The junctional and sulcular epithelium form a pocket epithelium that is not firmly attached to the tooth surface and that contains large numbers of neutrophils and is more permeable to the passage of substances into or out of the underlying connective tissue. The pocket epithelium may be ulcerated and is less able to resist the passage of the periodontal probe, so bleeding on probing is a common feature of chronic gingivitis. It is important to remember that these inflammatory changes are still completely reversible if effective plaque control is reinstituted.

The Advanced Lesion

The advanced lesion marks the transition from gingivitis to periodontitis. This transition is determined by many factors, the relative importance of which is, at present, unknown but includes the bacterial challenge (both the composition and the quantity of the biofilm), the host inflammatory response, and susceptibility factors, including environmental and genetic risk factors. Histologic examination reveals continued evidence of collagen destruction (extending now into the periodontal ligament and alveolar bone). Neutrophils predominate in the pocket epithelium and the periodontal pocket, and plasma cells dominate in the connective tissues. The junctional epithelium migrates apically along the root surface into the collagen depleted areas that develop below it to maintain an intact epithelial barrier. Osteoclastic bone resorption commences, and the bone retreats from the advancing inflammatory front as a defense mechanism to prevent spread of bacteria into the bone (Figure 21-3). As the pocket deepens, plaque bacteria proliferate apically into a niche, which is very favorable for many of the species that are regarded as periodontal pathogens. The pocket presents a protected, warm, moist, and anaerobic environment with a ready nutrient supply, and since the bacteria are effectively outside the body (not withstanding that they are in the periodontal pocket), they are not significantly eliminated by the inflammatory response. Thus a cycle develops in which chronic inflammation and associated tissue damage continue; the tissue damage mainly being caused by the inflammatory response, yet the initiating factor, the biofilm, is not eliminated. Destruction of collagen fibers in the periodontal ligament continues, bone resorption progresses, the junctional epithelium migrates apically to maintain an intact barrier, and as a result, the pocket deepens fractionally. This makes it even more difficult to remove the bacteria and disrupt the biofilm through oral hygiene techniques, and thus the cycle perpetuates.

Figure 21-3 Histologic appearance of periodontitis. A photomicrograph of adjacent demineralized teeth with the interproximal gingiva and periodontium in situ (H&E, low magnification). The root of the tooth on the right is coated with a layer of dental plaque/calculus, and there is attachment loss with the formation of a periodontal pocket. The periodontium is densely inflamed and there is alveolar bone loss producing a triangular-shaped defect; vertical bone loss. The base of the pocket is apical to the crest of the alveolar bone and is termed an infrabony periodontal pocket.

(From Soames JV, Southam JC: Oral pathology, ed 4, Oxford, 2005, Oxford University Press.).

Lipopolysaccharide

Lipopolysaccharides (LPS) are large molecules composed of a lipid component (lipid A) and a polysaccharide component. They are found in the outer membrane of gram-negative bacteria, they act as endotoxins (LPS is frequently referred to as endotoxin), and they elicit strong immune responses in animals. LPS is highly conserved in bacterial species, which reflects its importance in maintaining the structural integrity of the bacterial cells. Immune systems in animals have evolved to recognize LPS via toll-like receptors (TLRs), a family of cell surface molecules that are highly conserved in animal species from Drosophila (a genus of fruit flies) to humans, reflecting their importance in innate immune responses. TLRs are also present in lower animals and are in fact more varied than in higher species.²⁶ TLRs are cell surface receptors that recognize microbe-associated molecular patterns (MAMPs), which are conserved molecular structures located on diverse pathogens. TLR-4 recognizes LPS from gram-negative bacteria and functions as part of a complex of cell surface molecules, including CD14 and MD-2 (also known as lymphocyte antigen 96). Interaction of this CD14/TLR-4/MD-2 complex with LPS triggers a series of intracellular events, the net result of which is increased production of inflammatory mediators (most notably cytokines) and the differentiation of immune cells (e.g., dendritic cells) for the development of effective immune responses against the pathogens. It is particularly interesting to the periodontist that the pathogen Porphyromonas gingivalis has an atypical form of LPS and is recognized by both TLR-2 and TLR-4.^38,45

It is important to remember that a component of gram-positive cell walls, lipoteichoic acid (LTA), also stimulates immune responses, although less potently than LPS. LTA signals through TLR-2. Both LPS and LTA are released from the bacteria present in the biofilm and stimulate inflammatory responses in the tissues, resulting in increased vasodilation and vascular permeability, recruitment of inflammatory cells by chemotaxis, and release of proinflammatory mediators by the leukocytes that are recruited to the area. LPS in particular is of key importance in initiating and sustaining inflammatory responses in the gingival and periodontal tissues.

Bacterial Enzymes and Noxious Products

Plaque bacteria produce a number of metabolic waste products that contribute directly to tissue damage. These include noxious agents, such as ammonia (NH₃) and hydrogen sulfide (H₂S), and short-chain carboxylic acids, such as butyric acid and propionic acid. These acids are detectable in GCF and are found in increasing concentrations as the severity of periodontal disease increases. These substances have profound effects on host cells (e.g., butyric acid induces apoptosis in T-cells, B-cells, fibroblasts, and gingival epithelial cells).^94,95,164 The short-chain fatty acids may aid P. gingivalis infection through tissue destruction and may also create a nutrient supply for the organism by increasing bleeding into the periodontal pocket. The short-chain fatty acids also influence cytokine secretion by immune cells and may potentiate inflammatory responses after exposure to proinflammatory stimuli such as LPS, interleukin-1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α).¹²²

Plaque bacteria produce proteases, which are capable of breaking down structural proteins of the periodontium such as collagen, elastin, and fibronectin. Bacteria produce these proteases to digest proteins and thereby provide peptides for bacterial nutrition. Bacterial proteases disrupt host responses, compromise tissue integrity, and facilitate microbial invasion of the tissues. P. gingivalis produces two classes of cysteine proteases that have been implicated in periodontal pathogenesis. These are known as gingipains and include the lysine specific gingipain Kgp and the arginine specific gingipains RgpA and RgpB. The gingipains can modulate the immune system and disrupt immune-inflammatory responses, potentially leading to increased tissue breakdown.¹³⁷ Gingipains can reduce the concentrations of cytokines in cell culture systems⁷ and they digest and inactivate TNF-α.²⁵ The gingipains can also stimulate cytokine secretion via activation of protease-activated receptors (PARs). For example, RgpB activates two different PARs (PAR-1 and PAR-2), thereby stimulating cytokine secretion¹⁰⁷ and both Rgp and Kgp gingipains stimulate IL-6 and IL-8 secretion by monocytes via activation of PAR-1, PAR-2 and PAR-3.¹⁸²

Microbial Invasion

Microbial invasion of the periodontal tissues has long been a contentious topic. In histologic specimens, bacteria, including cocci, filaments, and rods, have been identified in the intercellular spaces of the epithelium.⁵⁰ Periodontal pathogens such as P. gingivalis and Aggregatibacter actinomycetemcomitans have been reported to invade the gingival tissues,^30,76,146 including the connective tissues.¹⁴⁵ Fusobacterium nucleatum can invade oral epithelial cells, and bacteria that routinely invade host cells may facilitate the entry of noninvasive bacteria by coaggregating with them (Figure 21-4).⁴⁷ It has also been shown that A. actinomycetemcomitans can invade epithelial cells and persist intracellularly.⁴⁹ The clinical relevance of these findings is unclear, however. Some investigators have suggested that tissue invasion by subgingival bacteria is an active process, whereas others have considered it to be an artifact, or simply a passive translocation process.

Figure 21-4 Invasion of epithelial cells by Fusobacterium nucleatum. In both images, a single epithelial cell is shown, being penetrated by invading F. nucleatum bacteria (3-4 bacteria are evident in A and 1 bacterium is evident in B). The ruffled surface of the epithelial cells (multiple small fingerlike projections, much smaller than the F. nucleatum bacteria) is likely to be an artifact. In B, F. nucleatum may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly as evidenced by the single coccoid bacterium (Streptococcus cristatus) that has coaggregated with the F. nucleatum bacterium as it penetrates the epithelial cell.

(Images courtesy Dr. A.E. Edwards, Dr. J.D. Rudney, and Dr. T.J. Grossman, Bath University, United Kingdom, and the University of Minnesota.).

The reports of bacteria present in the tissues have sometimes been used to justify the use of antibiotics in the treatment of periodontitis as a means of attempting to eliminate those organisms that are located in the tissues and that are therefore “protected” from mechanical disruption by root surface debridement. It has also been reported that bacteria in the tissues represent a “reservoir for reinfection” after nonsurgical management. However, until the clinical relevance of bacteria being present in the tissues is better defined, it is inappropriate to make clinical treatment decisions (e.g., whether to use adjunctive systemic antibiotics) on this premise alone.

Fimbriae

The fimbriae of certain bacterial species, particularly P. gingivalis, may also play a role in periodontal pathogenesis. P. gingivalis fimbriae stimulate immune responses, such as IL-6 secretion,^96,127 and the major fimbrial structural component of P. gingivalis, FimA, has been shown to stimulate nuclear factor (NF)-κB and IL-8 in a gingival epithelial cell line via TLR-2.⁵ Monocytes are also stimulated by P. gingivalis FimA, secreting IL-6, IL-8, and TNF-α.⁴⁸ P. gingivalis fimbriae also interact with complement receptor-3 (CR-3) to activate intracellular signalling pathways that inhibit IL-12 production mediated by TLR-2 signalling.⁶⁶ This may be of clinical relevance as IL-12 is important in activating natural killer (NK) cells and CD8⁺ cytotoxic T-cells, which themselves may be important in killing P. gingivalis–infected host cells such as epithelial cells. Indeed, blockade of the CR-3 receptor promotes IL-12–mediated clearance of P. gingivalis and negates its virulence.⁶⁶ Bacterial fimbriae are therefore important in modifying and stimulating immune responses in the periodontium.

Bacterial Deoxyribonucleic Acid and Extracellular Deoxyribonucleic Acid

Bacterial deoxyribonucleic acid (DNA) stimulates immune cells via TLR-9, which recognizes hypomethylated CpG regions of the DNA.⁹² CpG sites are regions of DNA at which a cytosine nucleotide is found next to a guanine nucleotide (separated by a phosphate molecule, which links the C and G nucleotides together, hence “CpG”). Extracellular DNA (eDNA) is likely to play a role in the development and structure of the biofilms formed by oral bacteria and has been identified as an important component of the matrix in a number of bacterial biofilms.^167,194 eDNA is derived from the chromosomal DNA of bacteria in biofilms, and the majority of eDNA is released after bacterial cell lysis.^2,176 However, there is also evidence that eDNA secretion may occur from bacterial cells by mechanisms that are independent of cell lysis.^68,140 The significance of this finding is not yet clear, but such “donated” DNA may be used by bacterial species as a means of increasing genetic diversity (if taken up by other bacteria), thereby contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response. Thus eDNA may function as a source of genetic information for naturally transformable bacteria in the biofilm¹⁸⁹ and/or as a stimulus for host immunity. Little is known about the role of eDNA in oral biofilms, however. It has been demonstrated that DNA isolated from P. gingivalis, A. actinomycetemcomitans, and Peptostreptococcus micros stimulates macrophages and gingival fibroblasts to produce TNF-α and IL-6 in a dose-dependent manner, and therefore immune stimulation by bacterial DNA from subgingival species could contribute to periodontal pathogenesis.¹²³

Cytokines

Cytokines play a fundamental role in inflammation and are key inflammatory mediators in periodontal disease.¹⁵⁹ They are soluble proteins and act as messengers to transmit signals from one cell to another. Cytokines bind to specific receptors on target cells and initiate intracellular signalling cascades resulting in phenotypic changes in the cell via altered gene regulation.^17,174 Cytokines are effective in very low concentrations, are produced transiently in the tissues, and primarily act locally in the tissues in which they are produced. Cytokines are able to induce their own expression either in an autocrine or paracrine fashion and have pleiotropic effects (i.e., multiple biologic activities) on a large number of cell types. (Autocrine signalling means that the autocrine agent, in this case cytokines, binds to receptors on the cell that secreted the agent, whereas paracrine signalling affects other nearby cells.) Simply, cytokines bind to cell surface receptors, trigger a sequence of intracellular events that lead ultimately to the production of protein by the target cell, which alters that cell’s behavior, and could result in, for example, increased secretion of more cytokines in a positive feedback cycle leading to inflammation.

Cytokines are produced by a large number of cell types, including infiltrating inflammatory cells such as neutrophils, macrophages, and lymphocytes, and also by resident cells in the periodontium, including fibroblasts and epithelial cells.¹⁷⁰ Cytokines signal, broadcast, and amplify immune responses and are fundamentally important in regulating immune-inflammatory responses and in combating infections. However, they also have profound biologic effects that lead to tissue damage in chronic inflammation, and prolonged and excessive production of cytokines and other inflammatory mediators in the periodontium leads to the tissue damage that characterizes the clinical signs of the disease. For example, cytokines mediate connective tissue and alveolar bone destruction through the induction of fibroblasts and osteoclasts to produce proteolytic enzymes (i.e., MMPs) that break down structural components of these connective tissues.¹²

There is significant overlap and redundancy between the function of individual cytokines, and cytokines do not act in isolation, but rather in flexible and complex networks that involve both proinflammatory and antiinflammatory effects and that bring together aspects of both innate and acquired immunity.⁸ Cytokines play a key role at all stages of the immune response in periodontal disease. Among the most studied (and probably the most important) cytokines in periodontal pathogenesis are the proinflammatory cytokines IL-1β and TNF-α. Both of these cytokines play a key role in the initiation, regulation, and perpetuation of innate immune responses in the periodontium, resulting in vascular changes and migration of effector cells such as neutrophils into the periodontium as part of a normal immune response to the presence of subgingival bacteria.⁵⁷

Prostaglandins

The prostaglandins (PGs) are a group of lipid compounds derived from arachidonic acid, a polyunsaturated fatty acid found in the plasma membrane of most cells. Arachidonic acid is metabolized by cyclooxygenase-1 and -2 (COX-1 and COX-2) to generate a series of related compounds called the prostanoids, which includes the PGs, thromboxanes, and prostacyclins. PGs are important mediators of inflammation, particularly prostaglandin E₂ (PGE₂), which results in vasodilation and induces cytokine production by a variety of cell types. COX-2 is upregulated by IL-1β, TNF-α, and bacterial LPS, resulting in increased production of PGE₂ in inflamed tissues. PGE₂ is produced by various types of cells and most significantly in the periodontium by macrophages and fibroblasts. PGE₂ results in induction of MMPs and osteoclastic bone resorption and has a major role in contributing to the tissue damage that characterizes periodontitis.

Matrix Metalloproteinases

MMPs are a family of proteolytic enzymes that degrade extracellular matrix molecules such as collagen, gelatin, and elastin. They are produced by a variety of cell types, including neutrophils, macrophages, fibroblasts, epithelial cells, osteoblasts, and osteoclasts. The names and functions of key MMPs are shown in Table 21-1. The nomenclature of MMPs has been based on the perception that each enzyme has its own specific substrate, for example, MMP-8 and MMP-1 are both collagenases (i.e., they break down collagen). However, it is now appreciated that MMPs usually degrade multiple substrates, with significant substrate overlap between individual MMPs.⁷⁰ The substrate-based classification is still used, however, and MMPs can be divided into collagenases, gelatinases/type IV collagenases, stromelysins, matrilysins, membrane-type metalloproteinases, and others.

TABLE 21-1 Classification of Matrix Metalloproteinases

MMPs, Matrix metalloproteinases; MT, membrane type.

Adapted from Hannas AR, Pereira JC, Granjeiro JM, et al: Acta Odontol Scand 65:1-13, 2007.

MMPs are secreted in a latent form (inactive) and are activated by the proteolytic cleavage of a portion of the latent enzyme. This is achieved by proteases, such as cathepsin G, produced by neutrophils. MMPs are inhibited by proteinase inhibitors, which have antiinflammatory properties. Key inhibitors of MMPs found in the serum include the glycoprotein α₁-antitrypsin and α₂-macroglobulin, a large plasma protein produced by the liver that is capable of inactivating a wide variety of proteinases. Inhibitors of MMPs that are found in the tissues include the tissue inhibitors of metalloproteinases (TIMPs), which are produced by many cell types; the most important in periodontal disease is TIMP-1.¹⁸ MMPs are also inhibited by the tetracycline class of antibiotics, which has led to the development of sub-antimicrobial formulation of doxycycline as a licensed systemic adjunctive drug treatment for periodontitis that exploits the anti-MMP properties of this molecule (see Chapter 48).

Interleukin-1 Family Cytokines

The IL-1 family of cytokines comprises at least 11 members, including IL-1α, IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-33.

IL-1β plays a key role in inflammation and immunity, is closely linked to the innate immune response, and induces the synthesis and secretion of other mediators that contribute to inflammatory changes and tissue damage. For example, IL-1β stimulates the synthesis of PGE₂, platelet-activating factor (PAF), and nitrous oxide (NO), resulting in vascular changes associated with inflammation, increasing blood flow to the site of infection or tissue injury. IL-1β is mainly produced by monocytes, macrophages, and neutrophils and also by other cell types such as fibroblasts, keratinocytes, epithelial cells, B-cells, and osteocytes.⁴⁰ IL-1β increases the expression of ICAM-1 on endothelial cells and stimulates secretion of the chemokine CXCL8 (which is IL-8), thereby stimulating and facilitating the infiltration of neutrophils into the affected tissues. IL-1β also synergizes with other proinflammatory cytokines and PGE₂ to induce bone resorption. IL-1β has a role in adaptive immunity, regulates the development of antigen-presenting cells, such as dendritic cells, stimulates IL-6 secretion by macrophages (which in turn activates B-cells), and has been shown to enhance antigen-mediated stimulation of T-cells.¹³ GCF concentrations of IL-1β are increased at sites affected by gingivitis⁷³ and periodontitis,⁹⁸ and tissue levels of IL-1β correlate with clinical periodontal disease severity.¹⁶⁵ Studies in experimental animals have shown that IL-1β exacerbates inflammation and alveolar bone resorption.⁸⁷ It is clear from the multiplicity of studies that have investigated this cytokine that IL-1β plays a fundamental role in the pathogenesis of periodontal disease.⁹⁰

IL-1α is primarily an intracellular protein that is not normally secreted and therefore is not usually found in the extracellular environment or in the circulation.⁴³ Unlike IL-1β, biologically active IL-1α is constitutively expressed and likely mediates inflammation only when released from necrotic cells, acting as an “alarmin” to signal the immune system during cell and tissue damage.¹⁶ The precise role of IL-1α in periodontal pathogenesis is not well defined, although studies have reported elevated IL-1α levels in GCF and gingival tissues in patients with periodontitis.¹³⁹ IL-1α is a potent bone resorbing factor involved in the bone loss that is associated with inflammation.¹⁷² It is possible that the measured IL-1α in gingival tissues represents intracellular IL-1α that has been released from damaged or necrotic cells. It is probable that IL-1α plays a role in periodontal pathogenesis, possibly as a signalling cytokine (signalling tissue damage) and contributing to bone resorptive activity.

IL-1Ra has structural homology to IL-1β, and binds to the IL-1 receptor (IL-1R1). However, binding of IL-1Ra does not result in signal transduction, therefore IL-1Ra antagonizes the action of IL-1β.⁴² IL-1Ra is important in regulating inflammatory responses and can be considered to be an antiinflammatory cytokine. IL-1Ra levels have been reported to be elevated in the GCF and tissues of patients with periodontal disease, suggesting a role in immunoregulation in periodontitis.¹⁴²

IL-18 interacts with IL-1β and shares many of the pro-inflammatory effects of IL-1β. It is mainly produced by stimulated monocytes and macrophages.⁶³ There is increasing evidence to suggest that IL-18 plays a significant role in inflammation and immunity. IL-18 results in proinflammatory responses, including activation of neutrophils.¹⁰¹ It is chemoattractant for T-cells,⁸⁸ and it interacts with IL-12 and IL-15 to induce interferon gamma (IFN-γ), thereby inducing T-helper (Th1) cells, which activate cell-mediated immunity.¹⁹⁷ Interestingly, in the absence of IL-12, IL-18 induces IL-4 and a Th2 response, which regulates humoral (antibody-mediated) immunity.¹⁹⁸ There is very limited direct evidence for a role of IL-18 in periodontal pathogenesis. Oral epithelial cells secrete IL-18 in response to stimulation with LPS,¹⁴³ and a correlation between GCF IL-18 levels and sulcus depth has been reported.⁸² IL-18 levels have been reported to be higher than those of IL-1β in patients with periodontitis, suggesting that IL-18, along with IL-1β is predominant in periodontitis lesions.¹²⁹ Since IL-18 has the ability to induce either Th1 or Th2 differentiation, it is likely to play an important role in periodontal disease pathogenesis.¹³⁰

Interleukin-6 and Related Cytokines

The cytokines in this group, which include IL-6, IL-11, leukemia-inhibitory factor (LIF), and oncostatin M, share common signalling pathways via signal transducers glycoprotein (gp) 130.⁷⁴ IL-6 is the most extensively studied of this group and has pleiotropic proinflammatory properties.⁸⁶ IL-6 secretion is stimulated by cytokines such as IL-1β and TNF-α and it is produced by a range of immune cells, including T-cells, B-cells, macrophages, and dendritic cells, as well as resident cells such as keratinocytes, endothelial cells, and fibroblasts.¹⁸⁶ IL-6 is also secreted by osteoblasts and stimulates bone resorption and development of osteoclasts.^81,93 IL-6 is elevated in the cells, tissues, and GCF of patients with periodontal disease.^56,103 IL-6 may have an influence on monocyte differentiation into osteoclasts and a role in bone resorption in periodontal disease.¹²⁸ IL-6 also has a key role in the regulation of proliferation and differentiation of B-cells and T-cells (in particular the Th17 subset).⁸⁶ IL-6 therefore has an important role in periodontal pathogenesis, although less than that of IL-1β or TNF-α.

IL-6 also has many activities outside the immune system, for example, the cardiovascular system and the nervous system. It has an important role in hematopoiesis and in signalling the production of C-reactive protein in the liver. Furthermore, IL-6 stimulates T-cell differentiation and function and is important in the regulation of the balance of T-cell subsets, especially the activation of Th17 cells (a subset of T-cells that produce IL-17), and the balance with regulatory T-cells (T_reg cells).¹⁴

Prostaglandin E₂

The cells primarily responsible for PGE₂ production in the periodontium are macrophages and fibroblasts. PGE₂ levels are increased in the tissues and in GCF at sites undergoing periodontal attachment loss. PGE₂ induces the secretion of MMPs and osteoclastic bone resorption and contributes significantly to the alveolar bone loss seen in periodontitis. PGE₂ release from monocytes from patients with severe or aggressive periodontitis is greater than that from monocytes from patients who are periodontally healthy.^55,125 A large body of evidence has demonstrated the importance of PGE₂ in periodontal pathogenesis, and given that prostaglandins are inhibited by nonsteroidal antiinflammatory drugs (NSAIDs), researchers have investigated the use of NSAIDs as potential host-response modulators in the management of periodontal disease.^191,192 However, daily administration for extended periods is necessary for the periodontal benefits to become apparent, and NSAIDs are associated with significant unwanted side effects, including gastrointestinal problems, hemorrhage (from impaired platelet aggregation resulting from inhibition of thromboxane formation), and renal and hepatic impairment. NSAIDs are therefore not indicated as adjunctive treatments for the management of periodontitis.

The prostaglandins, including PGE₂, are derived from the COX pathway of arachidonic acid metabolism. There are two main isoforms of the COX enzyme, COX-1 and COX-2. COX-1 is constitutively expressed and has antithrombogenic and cytoprotective functions. COX-2 is induced after stimulation with various cytokines, growth factors, and LPS. Inhibition of COX-1 by nonselective NSAIDs results in the majority of the unwanted effects associated with NSAID usage such as gastrointestinal ulceration and impaired hemostasis. Induction of COX-2 results in the production of elevated quantities of prostaglandins, such as PGE₂, and therefore inhibition of COX-2 by NSAIDs that selectively inhibit COX-2 results in a reduction of inflammation without the unwanted effects commonly seen after long-term NSAID use. Preliminary studies in animal models showed that selective COX-2 inhibitors slowed alveolar bone loss,^15,78 and human studies confirmed that prostaglandin production in the tissues was modified.¹⁸⁷ However, in a dramatic and unfortunate development, the selective COX-2 inhibitors were later identified to be associated with significant and life-threatening adverse events, resulting in several of these drugs being withdrawn from the market.⁴⁶ The selective COX-2 inhibitors cannot therefore be considered as adjunctive treatments for periodontal disease.

Matrix Metalloproteinases

MMPs are a family of zinc-dependent enzymes that are capable of degrading extracellular matrix molecules, including collagens.^18,144 MMPs play a key role in periodontal tissue destruction and are secreted by the majority of cell types in the periodontium, including fibroblasts, keratinocytes, endothelial cells, osteoclasts, neutrophils, and macrophages. In healthy tissues, MMPs are mainly produced by fibroblasts, which produce MMP-1 (also known as collagenase-1), and these have a role in the maintenance of the periodontal connective tissues. Transcription of genes coding for MMPs is upregulated by cytokines such as IL-1β and TNF-α.¹⁰⁸ MMP activity is regulated by specific endogenous tissue inhibitors of metalloproteinases (TIMPs) and serum glycoproteins such as α-macroglobulins, which form complexes with active MMPs and their latent precursors.¹⁴¹ TIMPs are produced by fibroblasts, macrophages, keratinocytes, and endothelial cells and are specific inhibitors that bind to MMPs in a 1 : 1 stoichiometry.⁷⁰ MMPs are also produced by some periodontal pathogens, such as A. actinomycetemcomitans and P. gingivalis, but the relative contribution of these bacterially-derived MMPs to periodontal pathogenesis is small. The great majority of MMP activity in the periodontal tissues derives from infiltrating inflammatory cells.

In healthy periodontal tissues, collagen homeostasis is a controlled process that is mediated extracellularly by MMP-1 (expressed by resident cells, primarily fibroblasts) and intracellularly by a variety of lysosomal acid–dependent enzymes. In inflamed periodontal tissues, excessive quantities of MMPs are secreted by resident cells and the large numbers of infiltrating inflammatory cells, particularly neutrophils, as they migrate through the tissues. As a result, the balance between MMPs and their inhibitors is disrupted, resulting in breakdown of the connective tissue matrix,^18,175 and leading to the development of collagen depleted areas within the connective tissues, as described earlier. Neutrophils are key infiltrating cells in periodontitis that accumulate in large numbers in inflamed periodontal tissues (see Figure 21-2). Neutrophils have evolved to respond rapidly and aggressively to external stimuli, such as bacterial LPS, and they release large quantities of destructive enzymes very rapidly. The predominant MMPs in periodontitis, MMP-8 and MMP-9, are secreted by neutrophils⁶² and are very effective at degrading type 1 collagen, the most abundant collagen type in the periodontal ligament.¹¹⁰ MMP-8 and MMP-9 levels increase with increasing severity of periodontal disease and decrease after treatment.^61,62,85 The prolonged and excessive release of large quantities of MMPs in the periodontium leads to significant breakdown of structural components of the connective tissues, contributing to the clinical signs of disease.

MMPs play a fundamental role in connective tissue homeostasis and also disease pathogenesis and possess a wide range of biologic effects that are relevant in periodontitis (Table 21-3). MMPs are important in alveolar bone destruction and are expressed by osteoclasts, which also express cathepsin K. Cathepsin K is a lysosomal cysteine protease that is mainly expressed in osteoclasts and plays a key role in bone resorption and remodelling. This enzyme can catabolize collagen, gelatin, and elastin and can therefore contribute to the breakdown of bone and cartilage.

TABLE 21-3 Biologic Activities of Selected MMPs Relevant to Periodontal Disease

MMPs, Matrix metalloproteinases; MT, membrane type; ECM, extracellular matrix.

Adapted from Hannas AR, Pereira JC, Granjeiro JM, et al: Acta Odontol Scand 65:1-13, 2007.

MMPs are critical for osteoclast access to the resorption site, particularly MMP-9 and MMP-14. MMP-14 is located in the ruffled border of osteoclasts, and osteoblasts and osteocytes (but not osteoclasts) express MMP-13, which is present in resorption lacunae, and functions to remove collagen remnants left over by osteoclasts.⁷⁰ MMPs also contribute to osteoclast recruitment and activity, by releasing cytokines and RANKL (see later section). MMPs are also important in osteoblastic bone formation, including MMP-2, MMP-9, MMP-13, and MMP-14. MMP-14 also contributes to normal bone homeostasis, and MMP-14–activated transforming growth factor beta (TGF-β) inhibits osteoblast apoptosis.

Increased understanding of the importance of MMPs in periodontal pathogenesis has led to the development of systemic drug therapies to modulate the host inflammatory response by inhibiting MMP levels. Doxycycline has been used for this indication, at sub-antimicrobial doses (20 mg twice daily) that have no antibiotic effect but do demonstrate an anticollagenase effect. Doxycycline, like all the tetracyclines, possesses the ability to downregulate MMPs, and this was recognized as representing a novel treatment strategy for the management of periodontitis. The sub-antimicrobial formulation (20 mg twice daily) has been shown to inhibit collagenase activity in the gingival tissues and GCF of patients with chronic periodontitis,⁶¹ and a large number of clinical trials have now confirmed the clinical benefits of using this formulation of doxycycline as an adjunct to periodontal therapy.¹³⁸ Host-response modulation as a treatment concept for periodontitis is discussed further in Chapter 48.

Chemokines

Chemokines are cytokine-like molecules that are characterized by their chemotactic activity. This activity gave rise to the term chemokine (i.e., they are chemotactic cytokines). Chemokines orchestrate leukocyte recruitment in physiologic and pathologic conditions,²⁰ therefore are important in periodontal pathogenesis, resulting in chemotactic migration of neutrophils through the periodontal tissues toward the site of the bacterial challenge in the periodontal pocket.¹⁶² Chemokines play a key role in neutrophil recruitment and recruitment of other adaptive and innate immune cells to the site of immune and inflammatory responses. The chemokines are divided into two sub-families according to structural similarity, the CC and CXC sub-families.¹⁶⁰ The chemokine, CXCL8, which is more familiarly known as IL-8, has been demonstrated to be localized in the gingival tissues in areas of plaque accumulation, in the presence of neutrophilic infiltration,¹⁷⁷ and has also been found in GCF.¹¹¹ Interaction between bacteria and keratinocytes results in upregulation of IL-8 and ICAM-1 expression in the gingival epithelium and the development of a chemotactic gradient of these molecules in the gingiva, which stimulates neutrophil migration into the epithelial layers and the gingival sulcus.^178,179 Similar chemotactic gradients are also present in the gingiva of periodontally healthy individuals, which suggests a role for this process in the maintenance of periodontal health, and supports the findings of infiltrating neutrophils being present even in clinically healthy tissues.¹⁷⁹

It is becoming clear that chemokines play an important role in leukocyte migration in periodontal disease. CCL2 and CCL5 (also known as regulation on activation normal T-cell expressed and secreted [RANTES]) play a role in macrophage migration, and CCL3 (also known as macrophage inflammatory protein-1α [MIP-1α]) and CXCL10 play a role in T-cell migration in inflamed periodontal tissues.¹⁶² Chemokines play important roles in immune responses, repair, and inflammation and regulate osteoclast activity by influencing myeloid cell differentiation into osteoclasts, which may be of particular importance in the context of periodontitis.

Antiinflammatory Cytokines

The balance between proinflammatory and antiinflammatory events is crucial in determining disease progression, and it is now clear that individual cytokines do not act in isolation but as part of complex networks of mediators that have different functional activities. Antiinflammatory cytokines include IL-10, TGF-β, IL-1Ra, IL-1F5, and possibly IL-1F10.

The IL-10 family of cytokines have multiple pleiotropic effects and of particular interest possess immunosuppressive properties.^32,34 IL-10 is produced by T_reg cells, monocytes and B-cells and suppresses cytokine secretion from Th1 cells, Th2 cells, monocytes, and macrophages. The role of IL-10 in periodontal disease has been minimally studied, but animal models support that IL-10 downregulates inflammatory responses. For example, IL-10 knock-out mice are more susceptible to alveolar bone loss than wild-type mice.¹⁴⁷ IL-10 is also present in GCF and periodontal tissues.⁷⁹

TGF-β is a growth factor that functions as a cytokine and has immunoregulatory roles, such as the regulation of T-cell subsets and the action of T_reg cells, and it also plays a role in repair and regeneration.¹⁹⁵ It has multifunctional roles in various cellular functions, including angiogenesis, synthesis of the extracellular matrix, apoptosis, and inhibition of cell growth. TGF-β levels are higher in the GCF and periodontal tissues of patients with periodontitis and gingivitis than those who are periodontally healthy.⁶⁹

Saliva

Saliva secreted from the three major salivary glands (parotid, submandibular, and sublingual), as well as from the numerous minor salivary glands, has an important role in maintaining oral and dental health. The action of shearing forces associated with saliva flow is important in preventing the attachment of bacteria to the dentition and the oral mucosal surfaces. Human saliva also contains numerous molecular components that contribute to host defenses against bacterial colonization and periodontal disease (Table 21-4). These components include molecules that nonspecifically inhibit formation of the plaque biofilm by inhibiting adherence to oral surfaces and promoting agglutination (e.g., mucins), those that inhibit specific virulence factors (e.g., histatins that neutralize LPS), and those that inhibit bacterial cell growth (e.g., lactoferrin) and may induce cell death.^60,97 Saliva also contains specific immunoglobulin A (IgA) antibodies to periodontal pathogens that target specific antigens and inhibit bacterial adherence. Patients with periodontal disease have elevated levels of specific IgA, as well as IgG and IgM, antibodies to periodontal pathogens. However, tooth surfaces coated with a salivary pellicle can provide attachment opportunities for plaque bacteria, thus P. gingivalis can attach to the salivary pellicle via fimbriae.

TABLE 21-4 Constituents of Saliva that Contribute to Innate Immunity

IgA, Immunoglobulin A; LPS, lipopolysaccharides.

Epithelial Tissues

The epithelial tissues play a key role in host defenses as they are the main site of initial interaction between plaque bacteria and the host and also are the site of invasion of microbial pathogens. The keratinized epithelium of the sulcular and gingival epithelial tissues not only provides a protection for the underlying periodontal tissue but also acts as a barrier against bacteria and their products.^11,152 In contrast, the unique microanatomic structure of the junctional epithelium has significant intercellular spaces, is not keratinized, and exhibits a higher cellular turnover rate. These properties render the junctional epithelium permeable, allowing for inward movement of microbes and their products and outward movement of GCF and the cells and molecules of innate immunity. Furthermore, the spaces between the cells of the junctional epithelium widen with inflammation, resulting in increased GCF flow.¹⁵²

Some species of periodontal bacteria invade host epithelial tissues; at the molecular level the processes of adhesion and invasion are coupled. Studies of the invasion of gingival epithelial cells by P. gingivalis have served as a paradigm for the study of this process; infection of host cells by P. gingivalis involves the action of proteases and cell surface fimbriae.^3,96 Invasion by P. gingivalis is initiated through signalling via interaction of bacterial components with surface integrins, PAR-1 and PAR-2 and TLR.^67,96,196 This in turn activates intracellular signalling pathways (e.g., mitogen-activated protein kinase [MAPK]) and results in the reorganization of actin filaments and microtubules and a modulation of Ca²⁺ influx. It is thought that inhibition of host cell apoptosis may facilitate survival of intracellular bacteria and that bacteria in this compartment are afforded protection from elements of the host immune response. Invasion of host cells could therefore be relevant in the spread and persistence of certain periodontal bacteria. Also, in vivo analysis and studies of a three-dimensional (3D)-engineered human oral mucosa model demonstrate that P. gingivalis can migrate through the basement membrane of epithelial layers and invade connective tissue.³ Histologic analysis reveals that, in periodontitis, epithelial cells become more rounded and tend to detach from the underlying connective tissue.¹⁵² Proteases break down cell-cell junctions in epithelial tissues by digesting transmembrane proteins and adhesion molecules (e.g., E-cadherin). Microanatomic changes associated with the onset of periodontitis, such as the widening of the spaces between the cells of the junctional epithelium and the development of the pocket epithelium, further facilitate bacterial invasion. Infection through the basement membrane and into the underlying connective tissues is facilitated by bacterial-derived proteases and host proteases derived from infiltrating neutrophils.

At the cellular and molecular level, most in vitro studies of epithelial cell responses to periodontal bacteria have been carried out in primary gingival epithelial cells or various immortalized cell lines derived from oral epithelial tissue, and these studies have provided insight into host-cell responses to periodontal bacteria.^3,67,69,196 Epithelial cells also constitutively express antimicrobial peptides (e.g., hBDs and LL-37) and the synthesis and secretion of these molecules is upregulated in response to periodontal bacteria. Neutrophils are also a source of antimicrobial peptides (α-defensins). Antimicrobial peptides are small, polycationic peptides that disrupt bacterial cell membranes and thereby directly kill bacteria with broad specificity.

The different categories of antimicrobial peptide are defined on the basis of structural homology. The α-defensins (e.g., human neutrophil peptides 1-4) are expressed by neutrophils and as such are commonly found in GCF. The hBDs, such as hBD1-3, are expressed in gingival epithelial cells, the salivary glands, and in the tongue, as well as in immune cells such as macrophages and dendritic cells; some hBDs are constitutively expressed and others are only expressed in response to cytokines and bacterial products (e.g., gingipains of P. gingivalis).^3,37 A third class of antimicrobial peptides are the cathelicidins, of which LL-37 is the only known peptide in this category expressed in human cells. LL-37 is expressed in high levels in the junctional epithelium but like the hBDs has a widespread expression pattern in the mouth and is found in the salivary glands, tongue, and leukocytes, as well as in the connective tissue. Antimicrobial peptides have more recently assumed greater importance because it is recognized that they have a wider role in regulating innate and adaptive immune responses to infection.⁴¹ Thus these molecules have chemokine-like activity, stimulating the chemotaxis of a range of leukocytes involved in innate and acquired immunity. Antimicrobial peptides also stimulate mast cell degranulation and cytokine production and likely have a role in wound healing through their effect on keratinocyte differentiation. Furthermore, there is some interest in their possible role in therapy for oral inflammatory diseases.⁴¹

Epithelial cells stimulated with bacterial components and cytokines directly produce MMPs, which contribute to loss of connective tissue. Epithelial cells also secrete a range of cytokines in response to periodontal bacteria (such as P. gingivalis, A. actinomycetemcomitans, F. nucleatum, and P. intermedia), which signal immune responses. These include the proinflammatory cytokines IL-1β, TNF-α, and IL-6, as well as the chemokines IL-8 (CXCL8) and monocyte chemoattractant protein-1 (MCP-1), which serve to signal neutrophil and monocyte migration from the vasculature into periodontal tissue.

In some (but not all) experimental systems, P. gingivalis has been shown to inhibit IL-8; it is suggested that in vivo this effect may have a temporary local immune suppression in the periodontium and facilitate the accumulation and invasion of pathogenic periodontal bacteria and the initiation of periodontitis.^39,67 P. gingivalis is an example of one periodontal pathogen with a range of virulence factors that affect host immune defenses,^67,96 as indicated in Table 21-5.

TABLE 21-5 Virulence Factors of P. gingivalis that Interact with the Immune System

IL, Interleukin; LPS, lipopolysaccharides.

Gingival Crevicular Fluid

GCF originates from the postcapillary venules of the gingival plexus. It has a flushing action in the gingival crevice but also likely functions to bring the blood components (e.g., neutrophils, antibodies, and complement components) of the host defenses into the sulcus.⁶⁵ The flow of GCF increases in inflammation, and neutrophils are an especially important element of GCF in health and disease.⁹⁰

Pathogen Recognition and Activation of Cellular Innate Responses

If plaque bacteria and their products penetrate the periodontal tissues, then specialized “sentinel cells” of the immune system can recognize their presence and signal protective immune responses. Thus macrophages and dendritic cells express a range of pattern recognition receptors (PRRs) that interact with specific molecular structures on microorganisms called microbe- associated molecular patterns (MAMPs) to signal immune responses. Thus innate immune responses are activated that provide immediate protection, and adaptive immunity is also activated with the aim of establishing a sustained antigen-specific defense. Excessive and inappropriate immune responses lead to chronic inflammation and the concomitant tissue destruction associated with periodontal disease. A glossary of terms relevant to periodontal immunobiology is presented in Table 21-6.

TABLE 21-6 Glossary of Terms Relevant to Periodontal Immunobiology

The best studied of the signalling systems involved in recognition of plaque bacteria is the interaction of bacterial LPS with TLRs: P. gingivalis, A. actinomycetemcomitans, F. nucleatum all possess LPS molecules that interact with TLR-4 to activate myeloid immune cells. However, individual species of plaque bacteria have a wide variety of MAMPs, which may interact with PRRs. Studies of P. gingivalis have served as a paradigm for investigations of host-bacteria interactions in periodontal disease at the molecular level. Thus P. gingivalis LPS signal via TLR (predominantly TLR-2), and fimbriae, proteases, and DNA from P. gingivalis are all recognized by host cells through interaction with specific PRRs. A number of nonimmune cells in the periodontium (epithelial cells, fibroblasts) also express PRRs and may recognize and respond to MAMPs from plaque bacteria.

Although the signalling pathways activated by PRRs may be diverse, in general terms they converge to elicit similar host-cell responses in the form of upregulation of cytokine secretion and in the case of antigen-presenting cells, such as dendritic cells, cell differentiation leading to enhanced signalling of the adaptive immune response. Dendritic cells also have C-type lectin receptors (e.g., mannose receptor, langerin, and DC-SIGN) which recognize glycans on pathogens but the role of these interactions in periodontal disease is not known.

Signalling of cytokine responses via PRRs influences innate immunity (e.g., neutrophil activity), adaptive immunity (T-cell effector phenotype), and the development of destructive inflammation (e.g., activation of fibroblasts and osteoclasts). A number of cytokines are particularly important in innate immune signalling, and there is now good evidence that these have a role in immune responses in the periodontium. The archetypal proinflammatory cytokine is IL-1β, which exerts its action directly by activating other cells that express the IL-1R1 receptor (e.g., endothelial cells) or by stimulating the synthesis and secretion of other, secondary mediators such as PGE₂ and NO. The effect of IL-1β is amplified via a synergistic action with other cytokines such as TNF-α. Upregulation of ICAM-1 and E-selectin on endothelial cells is central to the migration of neutrophils into the periodontium, and this is stimulated by IL-1β and TNF-α. IL-1β also stimulates the secretion of the chemokine IL-8, which stimulates neutrophil chemotaxis (see next section). IL-1β and TNF-α also activate MMP secretion from fibroblasts and osteoclasts; this may facilitate the movement of neutrophils in the connective tissues and thus protective innate responses but may also ultimately contribute to tissue destruction associated with periodontal disease, along with MMPs from neutrophils.

Other cytokines upregulated as a result of activation of PRRs include IL-6, which influences the development of a number of immune cells, including B-cells and dendritic cells, as well as stimulating osteoclast differentiation and thus bone turnover. Other cytokines provide specific signals that contribute to the development of specific CD4⁺ T-helper cell subsets (e.g., IL-4, IL-12, and IL-18) (see section on Adaptive Immunity). In addition to cytokines that activate immune responses, other cytokines are upregulated that have a role in immune regulation by suppressing the activity of other cytokines; these include IL-1Ra, IL-10, and TGF-β. Cytokines from T-cell subsets feedback and modify innate immune responses; thus IFNγ from Th1 cells activates macrophages, IL-17 from Th17 cells synergizes with IL-1β and TNF-α to reinforce inflammatory reactions, and IL-10 and TGF-β suppress immune responses. The action of many cytokines produced in the periodontium is not limited to one aspect of host immune responses (cytokines are pleiotropic); for example, it is increasingly recognized that IL-1β stimulates dendritic cell differentiation and has a role in activating Th17 cells.

Neutrophil Function

Although macrophages have phagocytic capability, neutrophils are the “professional” phagocytes critical to clearance of bacteria that may invade host tissues. Neutrophils are a feature of healthy gingival tissues, and there is a significant migration of these cells in the absence of clinical signs of inflammation through the intercellular spaces of the junctional epithelium.^90,152 This is part of a “low grade defense” against plaque bacteria and is necessary to prevent inflammation and periodontal tissue damage.¹⁵² The importance of neutrophils in maintaining periodontal health is demonstrated clinically by the observations of periodontitis in patients with neutrophil defects⁷¹ and the association of periodontitis with experimental immunosuppression in animal models.⁹⁰

Small foci of other leukocytes (lymphocytes, plasma cells, or macrophages) are also found in the healthy gingiva. A small proportion (1% to 2%) of the intercellular spaces in healthy junctional epithelium are occupied by neutrophils (and other leukocytes at various stages of differentiation); but this can increase to 30% with even modest inflammation. In the inflammatory state there are changes to the local vasculature in the gingiva: high endothelial venules develop from the postcapillary venules of the gingival plexus, which facilitates leukocyte emigration and increases flow of GCF into the pocket.¹⁵²

Neutrophils migrate from the gingival plexus to the extravascular connective tissue and then into the junctional epithelium via the basement membrane. The presence of a layer of neutrophils in the junctional epithelium forms a host defense barrier between subgingival plaque and gingival tissue. At the molecular level, the interaction of adhesion molecules (e.g., ICAM-1 and LFA-3) on endothelial and epithelial cells with β2 integrins on neutrophils facilitates neutrophil migration.

Indeed there is some evidence from immunohistochemistry for the existence of gradients of IL-8 (a “chemotactic gradient”), as well as gradients of ICAM-1, which purportedly direct the neutrophils from the vasculature into the tissues and to the junctional epithelium.¹⁷⁹ Increased leukocytes in the periodontium contribute to the disruption of the junctional epithelium by degradation of basement membrane through protease release and the action of reactive oxygen species. Increased expression of IL-8 and adhesion molecules in inflammation may be the result of direct signalling by bacterial products or by signals from cytokines (e.g., IL-1β and TNF-α) upregulated by plaque bacteria. Acute phase proteins, such as α2-macroglobulin, are increased in periodontal tissues as a result of increased vascular leakage in the inflammatory state, resulting in complement and plasmin activation, which also contributes to host defenses.

The neutrophils that infiltrate the periodontium express F_c receptors and complement receptors commensurate with their function in phagocytosis of opsonized bacteria and bacterial antigens. Significantly, the function of neutrophils is not obviously affected by the anaerobic environment within the periodontal pocket. Within the sulcus itself, 95% of the cells are neutrophils, and since neutrophils are present in both health and disease, the transmigration is thought to be a process distinct from that of GCF flow. The remaining 5% of cells are other leukocyte subtypes possibly passively carried into the sulcus by the flow of GCF. The relative proportions of the different leukocytes are highly variable, possibly reflecting the fact that their presence is the result of a passive process and/or a reflection of the natural fluctuations of different cell types during an immune response. Also, the distribution of leukocytes in periodontal tissues is not even; mononuclear cells predominate in the connective tissue and neutrophils in sulcus. This may be the result of selective mechanism mediated by specific chemokine responses and adhesion molecule interactions.

Antigen-Presenting Cells

A central element of the activation and function of T-cells and B-cells is the presentation of antigen by specialized antigen-presenting cells to T-cells and the development of a specific cytokine milieu that influences the development of T-cells with a particular effector function. Antigen-presenting cells are sentinel cells in mucosal tissues such as the periodontium. These cells detect and take up microorganisms and their antigens, after which they may migrate to lymph nodes and interact with T-cells to present antigen. The periodontium is often compared to other mucosal tissues and the skin in terms of its repertoire of immune cells, and it contains a number of “professional” antigen-presenting cells.³⁶ These include B-cells, macrophages, and at least two types of dendritic cells (dermal dendritic cells and Langerhans cells). These cells naturally express major histocompatibility complex (MHC) Class II molecules necessary for antigen presentation to cognate T-cell receptors and may take up specific antigens and transport them to local lymph nodes, thereby facilitating the activation of specific effector T-cells and the generation of an antigen-specific immune response to periodontal pathogens. Although these cells have been identified in periodontal tissues, have been shown to stimulate antigen specific T-cell responses in experimental systems, and are generally increased in periodontitis, their relative contribution to antigen presentation in vivo remains to be determined. Expression of MHC Class II molecules may be induced in other cells present in the periodontium (e.g., fibroblasts and epithelial cells), which then also take up antigen and present antigen locally in the periodontium.

It is increasingly recognized that engagement of PRRs and in particular TLRs by MAMPs from pathogenic microorganisms is not only central to signalling innate immunity in the form of cytokine upregulation but is also a critical element of activation of antigen-presenting cells and the elaboration of T-cell effector function. Thus TLR activation increases the expression of costimulatory molecules on antigen-presenting cells, which are critical in the interaction of these cells with T-cells. Also, TLR activation enhances antigen uptake and processing. Different antigen-presenting cells process and present antigens by different pathways and mechanisms, and this variation is one of the factors, along with the presence of specific combinations of cytokines, that influences the phenotype of T-cell effector function produced during specific immune responses.⁵⁹

T-Cells

There are a number of different subsets of thymic lymphocytes (T-cells) that develop in the bone marrow and thymus and migrate to the peripheral tissues to participate in adaptive immune responses. The expression of the cell surface molecules (CD4 or CD8) or particular T-cell antigen receptors (αβ or γδ) broadly defines functional T-cell subsets that emerge from the thymus. The role of T-cells in periodontal disease has been established through immunohistologic studies of disease tissues.¹⁵⁷ CD4⁺ helper T-cells are the predominant phenotype in the stable periodontal lesion, and it is thought that alterations in the balance of effector T-cell subsets within the CD4⁺ population may lead to progression toward a destructive, B-cell–dominated lesion.⁵⁹ CD4⁺ T-cell subsets are defined on the basis of their phenotypic characteristics and effector functions. The nature of the antigen-presenting cells, which present antigen to cognate T-cell receptors on T-cells, and the presence of specific combinations of cytokines and chemokines influence the nature of the CD4⁺ T-cell effector subset, which develops from naive T-cells (Figure 21-5). CD4⁺ T-cell subsets are defined by the expression of specific transcription factors and their functional characteristics are associated with their cytokine secretion profile.

Figure 21-5 CD4⁺ T-cell subsets and immune responses to microorganisms. Antigen-presenting cells (dendritic cells, macrophages, and B-cells) present specific antigen to naive CD4⁺ T-cells (Th0 cells), which then differentiate into CD4⁺ effector T-cells (i.e., Th1, Th2, Th17, and T_reg cells) and regulate different aspects of the immune response. Cytokines produced by innate cells in response to microbe-associated molecular patterns (MAMPs) signalling via pattern-recognition receptors (PRRs) influence the nature of the CD4⁺ effector T-cells that develop from activated Th0 cells. The nature of the antigen-presenting cell and the nature of the antigen itself are also factors that influence effector T-cell function. IL, Interleukin; IFNγ, interferon gamma; TGF-β, transforming growth factor beta.

The best-defined functional subsets of CD4⁺ T-cells are the Th1 and Th2 cells, and a dynamic interaction between Th1 and Th2 cells may provide an explanation for fluctuations in disease activity and progression of periodontal disease (Box 21-3). Th1 cells secrete IFNγ, which activates cell-mediated immunity (macrophages, natural killer (NK) cells, and CD8⁺ cytotoxic T-cells) against pathogenic microorganisms. Activation of macrophages promotes phagocytosis and killing of microbial pathogens, whereas NK cells and CD8⁺ T-cells are cytotoxic T-cells that kill infected host cells. Conversely, Th2 cells regulate humoral (antibody-mediated) immunity and mast cell activity through the secretion of the cytokines IL-4, IL-5, and IL-13. Thus predominance of Th2 cells leads to a B-cell response. The B-cell response may be protective, for example, by the production of specific antibodies that would serve to clear tissue infections through interaction with the complement system and by enhancing neutrophil phagocytosis. However, B-cells are also a source of proinflammatory cytokines that contribute to tissue destruction.

T_reg cells have an immunosuppressive action mediated by secretion of TGF-β and are important in preventing autoimmune disease. These cells are increased in periodontitis lesions and may therefore have a role in disease pathogenesis.¹¹⁹ A number of lines of evidence suggest that the pathogenesis of periodontal disease may involve some elements of autoimmunity.⁵⁹ For example, there is immunologic cross-reactivity between HSP60 expressed on human cells and the GroEL molecule of P. gingivalis, and specific serum antibodies and antigen-specific T-cells to these molecules have been detected in periodontal disease. Similarly autoantibodies and specific T-cells against other host (i.e., self) molecules, such as type I collagen, have been measured in periodontal disease.

Th17 cells are another recently described T-cell subset that has a proinflammatory action important in immune responses against extracellular infections mediated by the cytokine IL-17. Infections with a diverse range of pathogens have been shown to activate strong Th17 cell responses, and Th17 cells are thought to provide a substantial inflammatory response to clear microorganisms that Th1/Th2 cells have failed to eradicate. IL-17 has a number of activities in common with proinflammatory cytokines, such as IL-1β and TNF-α, and has a synergistic activity with these cytokines, particularly TNF-α. IL-17 induces proinflammatory cytokine expression (including IL-1β and TNF-α) in macrophages and stimulates chemokine expression and thereby activates neutrophil infiltration. There is increasing evidence for a role of IL-17 and Th17 cells in periodontal disease.⁵⁴ IL-17 is detected in periodontal biopsies from disease sites and levels of IL-17 are increased in sites with deep pockets and increased loss of attachment. IL-17 induces IL-6 and IL-8 secretion by gingival fibroblasts and also upregulates MMP-1 and MMP-3 in these cells. IL-17 also induces IL-1β and TNF-α secretion from macrophages and gingival epithelial cells. Significantly, in a mouse model of periodontitis (induced by P. gingivalis infection), IL-17 receptor deficiency (IL-17RA knockouts) resulted in increased susceptibility to alveolar bone loss, suggesting a protective role of IL-17 in bone homeostasis, possibly via an effect on neutrophil function.

A number of other effector CD4⁺ T-cell subsets have recently been defined based on their cytokine secretion profile: these include Th9 cells and Th22 cells. Also, T-cell subsets have been defined based on their specific anatomic location. For example, Th22 cells home to the skin in which they likely stimulate antimicrobial peptide production and differentiation of keratinocytes. Also, T follicular helper cells (T_FH) are located in germinal centers in lymph nodes in which they provide B-cell help and stimulate Ig class switching. The homing of particular T-cell subsets to specific anatomic locations is defined by the expression of specific chemokine receptors which confer responsiveness to specific chemotactic signals which are produced in those locations. However, thus far, the majority of work on these novel T-cell subsets has been carried out in mouse models and in vitro systems; their relevance to human biology in vivo and disease remains to be fully elucidated.

Cytokines produced by differentiated T-cell subsets feedback to stimulate differentiation and sustain the activity of the cells from which they are derived (i.e., in a positive feedback loop). Simultaneously, they inhibit the development of other, competing subsets. For example, IL-4 from Th2 cells inhibits the development of Th1 cells, and IFNγ from Th1 cells inhibits Th2 cells.

It is increasingly appreciated that individual CD4⁺ T-cell clones, once they have encountered antigen and differentiated in response under the influence of a specific cytokine environment, may not be terminally differentiated cells but rather there is functional flexibility between T-cell subsets and in particular within the memory T-cells population (Figure 21-6). Thus Th17 and T_reg cells can interconvert, depending on the local concentrations of IL-6, IL-23, and TGF-β.¹⁷³ Therefore a picture of functional plasticity in effector T-cells subsets associated with regulating specific immune responses to pathogens is emerging. It is thought that the plethora of functional subsets of T-cells, their anatomic location, and their ability to switch phenotype is a reflection of the requirement for effective responses against diverse pathogens, and this is certainly likely to be a highly relevant facet of immune responses in polymicrobial diseases such as periodontal disease. Other immune cells also have subsets that are defined by expression of cell surface markers and diverse functional and anatomic locations (e.g., myeloid immune cells and NK cells, but these are less well defined than CD4⁺ T-cell subsets).

Figure 21-6 Plasticity of T-cell subsets. Although Th1 and Th2 cells have a relatively stable phenotype, other T-cell subsets are functionally “plastic” and can alter phenotype under the influence of different cytokine environments. Thus Th17 cells can develop into Th1 and Th2 cells under the influence of IL-12 and IL-4, respectively. T_reg cells can convert into Th17 cells in the presence of IL-6 and IL-21. Other effector T-cell subsets with unique cytokine secretion profiles have been identified (e.g., Th9 and Th22 cells), although these may be transitory and their in vivo relevance remains to be determined. IL, Interleukin; IFNγ, interferon gamma; TGF-β, transforming growth factor beta.

Emerging evidence indicates therefore that periodontal pathogenesis involves complex interactions between a number of interacting T-cell subsets that not only modulate adaptive immune function (Th1, Th2 and T_reg cells) but also feedback to modify and enhance innate function (Th17 cells).⁵⁴ The balance of these different subsets may very well shift and alter longitudinally in periodontal disease. However, the direct relationship (if any) between different T-cell subsets and different clinical presentations remains to be determined.

Antibodies

Specific antibodies are produced in response to an increasing bacterial challenge in periodontal disease and are the endpoint of B-cell activation. Circulating antibodies may be more important than locally produced antibodies, but even so, these generally appear in a high titer but have low biologic activity so there is some doubt as to their effectiveness. Commensurate with the appearance of antibodies against plaque bacterial antigens is the appearance of differentiated plasma cells that characterize the established lesion in periodontal disease. High levels of antibodies appear in GCF (in addition to those in the circulation), and these are produced locally by plasma cells in periodontal tissues.⁹ Antibodies to periodontal pathogens are primarily IgG with few IgM or IgA types produced.

Many species of oral bacteria elicit a polyclonal B-cell response (with the consequent production of specific antibodies against those bacteria). However, these responses augment responses against nonoral bacteria and may lead to production of autoantibodies (e.g., antibodies against collagen and connective tissue proteins), which may contribute to tissue destruction in periodontal disease.^9,59 The incidence and levels of specific serum and GCF IgG antibodies are raised in chronic periodontitis, which suggests that local and peripheral generation of antibodies may be important in the immune response to periodontal pathogens. Antibodies (IgA) to periodontal pathogens are also found in saliva. Variations in the levels of specific antibodies to different species in different clinical presentations suggest differences in pathogenesis. For example, antibodies to A. actinomycetemcomitans of IgG₂ subclass predominate in aggressive periodontitis.¹⁴⁸

Other P. gingivalis molecules (fimbriae and hemagglutinin) also act as antigens. Specific antibodies are also generated by serotype specific carbohydrate antigens (e.g., capsular polysaccharide of P. gingivalis and carbohydrate of A. actinomycetemcomitans LPS). The subclass distribution of antibodies is influenced by cytokines derived from monocytes.¹⁴⁸ For example, IgG₂ production is regulated by IL-1α, IL-1β, and PGE₂ from monocytes, as well as PAF from neutrophils. PGE₂ and PAF indirectly induce Th1 responses and therefore IFNγ, which stimulates IgG₂ production. Individuals with aggressive periodontitis have monocytes that are hyper-responsive to LPS and produce elevated quantities of PGE₂.⁹ A. actinomycetemcomitans is commonly associated with aggressive periodontitis, which induces IL-12 production that regulates NK cells and Th1 cells. These cells are a source of IFNγ, which in turn regulates IgG₂.

A number of studies have reported an effect of treatment on levels of specific antibodies to periodontal pathogens. For example, plaque removal reduces the titers of antibodies to P. gingivalis and A. actinomycetemcomitans in serum, GCF, and saliva.⁹ Some studies have observed a transient increase in antibody titers after treatment, which may be due to the release of antigens into the tissue and circulation.

The significance of antibodies in periodontitis is not clear. It is not known if these antibodies have a protective function and whether they participate in disease pathogenesis. Although there is some evidence for a correlation between clinical parameters of disease and titer of specific antibodies to periodontal pathogens, other studies report an inverse correlation of antibody levels and avidity with periodontal destruction. Also, specific antibodies to periodontal pathogens are found in healthy individuals, as well as in those with periodontal disease.

Most research on the analysis of specific antibodies has focussed on antibodies to P. gingivalis, and antigens derived from this organism have been investigated as potential vaccines for periodontal disease.^124,136 Thus a significant reduction in disease progression in nonhuman primate and rodent models was observed after immunization with heat-killed P. gingivalis or antigens from P. gingivalis. Also, immunization with P. gingivalis proteases (gingipains) prevents colonization with P. gingivalis and reduces bone loss.¹²⁴