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Periodontology 2000, Vol. 43, 2007, 1440 Printed in Singapore.

All rights reserved

2007 The Authors. Journal compilation 2007 Blackwell Munksgaard

PERIODONTOLOGY 2000

The role of T cells in periodontal disease: homeostasis and autoimmunity


E R I C A G E M M E L L , K A Z U H I S A Y A M A Z A K I & G R E G O R Y J. S E Y M O U R

It is now over 30 years since the seminal paper of Ivanyi and Lehner (106) suggested a fundamental role for T cells in periodontal disease. It is well established that the development of gingivitis is identical to the development of a delayed-type hypersensitivity reaction and that the so-called stable lesion is essentially a T-cell-mediated response. On the other hand, numerous studies have shown that periodontitis is predominantly a B-cell response with T cells having an immunoregulatory role. Recently however it has been shown that, albeit in mice, the periodontopathic organism Porphyromonas gingivalis down-regulates well over a thousand genes in CD4-positive T cells while up-regulating only about 30 genes. The role of autoimmunity has also been revived with the demonstration of both anti-collagen and anti-heatshock protein 60-reactive T cells in the gingival tissues. In addition, the so-called T regulatory or Treg cells have been demonstrated in periodontal tissues. These cells are thought to have a central role in the control of autoimmunity. The question therefore arises as to the role of T cells in periodontal disease. This article will explore the concept that because P. gingivalis is turning off T-cell genes it may be the mechanism that enables a balance or stability to be reached between the plaque biolm and the host, i.e. the role of T cells may be homeostatic rather than defensive or destructive. With respect to autoimmunity in periodontal disease the article will put forward the concept that this is an important and normal component of chronic inammation. A possible scenario in this context could be that because chronic inammation is dened as the simultaneous presence of destruction and repair, as the tissue is destroyed, i.e. the collagen is broken down by the various proteases, it must then

be removed and the amino acids must be made available for re-use by the broblasts. The production of anti-collagen antibodies could then opsonize the broken down collagen fragments and enhance their phagocytosis by broblasts, hence facilitating the re-cycling process. Such a scenario would suggest that the role of autoimmunity and therefore the controlling mechanisms are an essential part of homeostasis. Similarly, anti-heat-shock protein reactions in chronic inammation could be the means of removing damaged or destroyed cells locally and again the role of T cells is homeostasis.

Introduction
It is well established that periodontal disease results from the interaction of the hosts defense mechanisms with microorganisms in the dental plaque biolm. Since the 1990s, biolms containing complexes including P. gingivalis, Fusobacterium nucleatum, Tannerella forsythia, and Treponema denticola have been related to clinical measures of periodontal disease, particularly pocket depth and bleeding on probing (215). Notwithstanding these observations, it has also been shown that there is a high degree of volatility with respect to the numbers of these organisms over time, such that it would appear that they are more widespread in the community than previously thought (42). Indeed, it is now recognized that many people carry the organisms without manifesting disease progression (42). In this context, it is clear that most people are in balance with their biolm for most of the time and it is only when this balance is disturbed that disease results. Such disturbances may involve changes in the relative amounts of the respective cytokines or may involve

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The role of T cells in periodontal disease

Fig. 1. Diagrammatic representation of the cytokine balance in periodontal disease. Moving the fulcrum to the right even though there is no change in the relative amounts of cytokines, favors the formation of a progressive lesion (A). Moving the fulcrum to the left again with no change in the relative amounts of cytokines this time favors the development of a stable lesion (B).

changes in the controlling cytokines without changes in their respective amounts. These changes would be akin to moving the fulcrum either to the left or right as shown in Fig. 1. Such shifts could occur as a result of environmental inuences leading to an opportunistic increase in the numbers of organisms, a depression of the hosts defense mechanisms or indeed both. The innate susceptibility of the host therefore reects the interplay between the bacteria, the hosts immune system, and environmental factors (41, 199).

T cells in periodontal disease


In 1965, Brandtzaeg and Kraus (26) demonstrated the presence of immunoglobulin-producing plasma cells in the gingival tissues of patients with periodontal disease. This was the rst direct evidence that adaptive immune mechanisms play a role in the pathogenesis of periodontal inammation. It was not until 1970, however, that Ivanyi and Lehner (106) using

peripheral blood lymphocyte transformation assays highlighted a role for cell-mediated immunity in periodontal disease. Since then, immunohistological studies have supported the concept that the response to plaque bacteria is immunological in nature. An experimental gingivitis study established that a T-cell/macrophage lesion identical to a delayed hypersensitivity reaction (178) occurs within 48 days of plaque accumulation (204) and that this was synonymous with the early lesion of Page and Schroeder (171) and with the putative stable lesion (200). The expression of human leukocyte antigen DR and DQ by the inltrating T cells further suggested that these cells are activated but the lack of CD25 expression would indicate that they are not proliferating locally within the tissues (204). In contrast, other early studies demonstrated that the periodontitis lesion involved predominantly B cells and plasma cells (128, 137, 181, 198, 202, 204) as well as a decrease in the CD4 : CD8 ratio (39, 170, 220, 234). The study by Ivanyi and Lehner (106) was the rst to report possible suppression of cell-mediated immunity in advanced periodontitis subjects. The induction of lymphocyte suppression by a number of periodontopathic bacteria including P. gingivalis, Actinobacillus actinomycetemcomitans, Treponema denticola, Capnocytophaga ochracea, and F. nucleatum (71, 205207, 218) was then demonstrated. In addition, T cells extracted from periodontal diseased tissues were reported to have a reduced ability to respond in an autologous mixed lymphocyte reaction (39) supporting the suggestion of a suppression of cell-mediated responses in periodontitis. Seymour et al. (203) demonstrated a lack of interleukin-2 (IL-2) production by unstimulated T cells extracted from adult periodontitis patients and suggested this to be a reason for the failure of these cells to undergo spontaneous proliferation (38). In support of this study, a reduced production of IL-2 and/or IL-2 receptor (IL-2R) expression by T cells in patients with a reduced autologous mixed lymphocyte reaction has been shown (116). Furthermore, the autologous mixed lymphocyte reaction has been reported to return to normal following periodontal therapy (222), supporting the concept that the suppressive effect of plaque bacteria may be fundamental in the conversion of a stable lesion to a progressive lesion. In recent years, herpesviruses have been associated with destructive periodontal disease and in this context it is well established that these and other enveloped viruses induce a cellular immune response, and an increase in CD8 T cells. In addition,

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EpsteinBarr virus promotes the proliferation of B cells and it has been postulated that this response to the periodontal presence of viruses could explain the observed immunohistological changes and predominance of B cells in the progressive lesion (reviewed in Ref. 213). The reduced functional capacity of peripheral blood mononuclear cells from periodontitis patients with respect to their spontaneous proliferative response compared with the response of cells from healthy subjects has recently been revisited in a study by Emingil et al. (60). However, because of the complex etiology of periodontal disease, answers to why periodontopathic bacteria induce their effects have not yet been explained adequately. It is nevertheless possible to speculate that the role of T cells in the stable lesion is one of homeostasis, i.e. of maintaining the balance between the host and the biolm, and it is when the balance shifts in favor of the suppressive effect of the bacteria that disease progression occurs. T cells are involved in nearly all immunoregulatory interactions both in vivo and in vitro (184) and a delicate balance between effector and regulatory subsets is required for immune homeostasis (183). The production of appropriate cytokines in response to infection is necessary for the development of protective immunity (76). T helper type 1 (Th1) cells increase the ability of macrophages to kill intracellular and extracellular pathogens and also mediate delayed-type hypersensitivity reactions (186). Furthermore, there is evidence that T cells are involved in the recruitment and activation of neutrophils at the site of infection (31, 46). Neutrophil activation has been shown to require direct contact with T cells and is independent of cytokine release (262). Therefore in the stable lesion, activation of neutrophils may be crucial in keeping the infection under control. It has been suggested that a strong innate immune response in the gingival tissues leads to the production of IL-12, which in turn leads to a Th1 response (76). The presence of natural killer cells in gingival tissues has also been demonstrated (255) and may be signicant in the establishment of a Th1 response. The production of interferon-c then enhances the phagocytic activity of both neutrophils and macrophages and hence containment of the infection. It is also well established that the herpesviruses promote the expression of Th1 cytokines, such that containment of any viral inuence would also favor a Th1 response (reviewed in Ref. 213).

In contrast, the B-cell nature of the progressive lesion (197) suggests either an increase in the production of Th2 cytokines or a decline in the production of Th1 cytokines. In other words, it suggests a shift in the balance towards Th2. Gramnegative bacteria associated with disease progression have long been cited as being polyclonal B-cell activators (reviewed in Refs 201 and 238), as has EpsteinBarr virus (213). Therefore, as a result of environmental factors causing growth of the plaque biolm, and hence an increase in the suppressive effect of the bacteria, the biolm may circumvent the protective function of neutrophils and, concomitant with an increase in bacteria-derived polyclonal B-cell activators, this may in turn lead to the activation of B cells and their migration into the lesion (72). Further proliferation of these B cells could be induced by a local EpsteinBarr virus infection (213).

Cytokine proles in periodontal disease


Studies over the past decade or so have supported the hypothesis that Th1 cells are associated with the stable lesion and Th2 cells are associated with disease progression (5, 16, 67, 72, 126, 143, 175, 182, 209, 240). However, other studies have reported a predominance of Th1-type cells or reduced Th2 responses in diseased tissues (54, 191, 232). Recently, the involvement of both Th1 and Th2 cells in periodontal disease in humans (19, 68, 78, 113, 158, 179, 248, 257) and in a mouse model (77, 84) has been suggested. However, while showing cytokine patterns reecting both subsets in inamed gingival tissues, Yamamoto et al. (257) conceded that a predominant expression of Th2 cytokines could contribute to the induction of high B-cell responses in local disease sites. Indeed, in recent years several reports have established that Th2 responses are associated with periodontitis. One of these studies demonstrated that peripheral blood mononuclear cells from patients with so-called early-onset periodontitis (probably what is now recognized as aggressive periodontitis) expressed reduced interferon-c protein and decreased interferon-c and IL-2 messenger RNA in response to mitogens, indicating reduced Th1 responses, while higher IL-5 and granulocytemacrophage colony-stimulating factor were produced by cells from chronic periodontitis patients, suggesting increased Th2 responses (209). More signicantly, an investigation of the activity of

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The role of T cells in periodontal disease

P. gingivalis cysteine proteases found that these gingipains hydrolyzed IL-12, thereby reducing IL-12induced interferon-c production by CD4 cells. It was concluded that inactivation of IL-12 by the gingipains would therefore favor Th2 cells with disease progression (261). Signicantly, lower levels of IL12p70 produced by peripheral blood cells from periodontitis patients after stimulation with Escherichia coli lipopolysaccharide have also been reported (65). Whether E. coli or P. gingivalis induces the same response with respect to IL-12, or whether plaque bacteria, in particular those associated with periodontitis, down-regulate the systemic response as found in the early studies two to three decades ago is not certain. The results of the early studies could, however, be explained by the down-regulation of IL12 and therefore of Th1 responses and hence IL-2mediated proliferation. Finally, the numbers of immunoglobulin G4-positive (IgG4+) B cells in the gingival tissues have been shown to increase relative to IgG2+ cells with increasing inammation, indicating the inuence of IL-4 and Th2 responses and decreased interferon-c and Th1 responses in large inltrates in periodontal disease (86). In 1995, a study showed that the majority of CD4 clones established from gingival tissues and raised non-specically using mitogen and IL-2 had Th2 phenotypes producing high levels of IL-4 and low levels of interferon-c. However, the majority of CD8 clones produced equal amounts of IL-4 and interferon-c (248). A further study demonstrated two subsets of CD8 clones, one of which produced high levels of interferon-c but no IL-4 or IL-5 (Th1) and mediated cytolytic activity. The other subset produced high levels of IL-4 together with IL-5 and displayed no cytotoxicity but could suppress the proliferative response of cytotoxic CD8 T-cell clones. It was concluded that CD8 T cells might participate in the local response by suppressing interferon-cproducing cells and favoring humoral immune responses (249). A higher level of intracellular IL-4 produced by peripheral blood CD8 cells from highly susceptible patients with severe periodontitis has since been shown, further supporting a role for CD8 cells in periodontitis together with a shift towards a type 2 function (174). However, Teng (236) recently played down a role for CD8 cells in periodontal disease by concluding that this subset does not participate directly in periodontal disease destruction during disease progression. It was suggested that these cells not only produce important cytokines for both innate and adaptive immune responses but also participate in lysis of bacteria-infected or

bacteria-damaged tissues and cells. Obviously with the increased numbers of CD8 cells relative to CD4 cells in progressive lesions, the determination of the functions of this subset is paramount in understanding the pathogenesis of periodontal disease. It is likely that different T-cell subsets predominate at different stages of disease and the inability to determine disease activity clinically is a major limitation in all studies (168). However, it remains clear that the balance of cytokines in inamed periodontal tissues is what determines whether the disease remains stable or leads to progression and tissue destruction (168). In this context, the control of Th1 and/or Th2 expression is fundamental to understanding the immunoregulatory mechanisms in chronic periodontitis.

B cells in periodontal disease


There is no doubt that T cells play a fundamental role in periodontal disease. They are the dominant cell type in the cell-mediated (macrophage/lymphocyte) response and are necessary for both specic antibody production and polyclonal B-cell activation, which has been cited as being signicant in the pathogenesis of periodontal disease (20, 32, 53, 104, 141, 169). However, polyclonal activators do not activate all B cells. Approximately 30% of B cells may be stimulated with different antigens acting on different subpopulations. Further, the antibodies produced will most likely be of low afnity and the memory component may not be induced (238). While B-cell activation in periodontitis could be the result of nonspecic polyclonal activation and/or specic induction of sensitized B cells, IgG production by gingival cells from patients with adult periodontitis has been shown to be reduced in comparison with peripheral blood B cells from the same patients and this distinctiveness of the local B-cell response has been suggested to be the result of the unique combination of T cells in the gingival tissues of patients with periodontal disease (135). The inability of specic antibodies to eliminate the causative organisms of periodontal disease could be the result of a number of factors, including poor antigenicity of the virulence determinants and elicitation of antibodies with poor anti-bacterial properties (212). High titers of specic antibodies to P. gingivalis (147, 254) and A. actinomycetemcomitans (63, 129, 147, 190) have been demonstrated in the serum and gingival crevicular uid of periodontal disease subjects, although the reports with respect to

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disease activity are conicting (15, 56, 157). Studies on immunodominant antigens of P. gingivalis (43, 124, 176) and A. actinomycetemcomitans (29, 30, 57, 62) have also shown different patterns of immunoreactivity. The production of anti-P. gingivalis antibodies with different avidities in various forms of periodontal disease has been suggested to reect the quality of the humoral response, which may affect progression of the disease (153). Anti-A. actinomycetemcomitans (242) and anti-P. gingivalis antibodies (154) with higher avidities have been suggested to confer greater resistance to continued or repeated infection. Non-protective low avidity anti-P. gingivalis antibodies may be incapable of effectively mediating a variety of immune responses (131, 251). During the chronic phase of the disease, the antibody response has been suggested to be generally protective, facilitating bacterial clearance and arresting disease progression (166). An increased capacity of serum to opsonize P. gingivalis has been shown to be a distinctive feature in patients with past destructive periodontal disease (254). This may be because of the ability of anti-P. gingivalis protease antibodies, which occur late in periodontitis infections, to block the anti-opsonizing activity against C3 and IgG (45). Opsonic IgG antibodies to A. actinomycetemcomitans, which may facilitate neutrophil-mediated phagocytosis and be protective against this periodontopathic organism, have also been demonstrated (13, 242). Repeated infection with A. actinomycetemcomitans has been shown to elicit an anti-leukotoxin antibody that protects neutrophils from the leukocidal activity of the leukotoxin (242). Therefore, if specic antibodies with high avidity and protective IgG subclasses to immunodominant antigens are formed, the infection may be cleared and the disease will not progress. However, polyclonal B-cell activation by periodontopathic bacteria and the production of non-specic and/or low-avidity specic antibodies may not clear the infection. Continued B-cell activation leads to the production of high levels of IL-1 resulting in tissue destruction. Dendritic cells may provide signals that initially activate T cells, while B-cell presentation may allow for further activation and clonal expansion of these already activated cells. An allogeneic mixed leukocyte reaction was used to show that activated B cells could act as potent antigen-presenting cells in the presence of P. gingivalis or A. actinomycetemcomitans, resulting in the production of high levels of interferon-c and minimal IL-5 (139). Although other cytokines such as IL-4 were not measured in this

study, there is evidence that B cells direct Th2 CD4+ T cells whereas macrophages activate Th1 cells (reviewed in Ref. 22) providing support for the concept of antigen-specic T-cell and B-cell activation leading to Th2 responses in the B-cell periodontitis lesion.

Inhibition of innate immunity in periodontitis


In the gingival sulcus, neutrophils form a barrier between the epithelium and the plaque biolm (9), which in most cases prevents bacterial invasion of the epithelium and underlying connective tissue (96). Both P. gingivalis and A. actinomycetemcomitans have been reported to be capable of invading epithelial cells and even the connective tissues in diseased sites (64, 125, 192). Signicantly, P. gingivalis has been shown to have direct effects on the host innate immune responses (reviewed in Ref. 48). The migration of neutrophils from the circulation into the tissues has been shown to be inhibited by suppressing the expression of the neutrophil-binding adhesion molecule E-selectin on endothelial cells (47) and by blocking neutrophil transmigration through oral epithelium by inhibiting the epithelial cell production of the chemokine IL-8 (138). A mouse model also demonstrated inhibition of neutrophil phagocytosis of immune serum-opsonized P. gingivalis (85), possibly as a result of the cleavage of complement and immunoglobulins by the P. gingivalis proteases that prevent opsonization and subsequent neutrophil killing of the bacteria (133, 193, 194, 221). A. actinomycetemcomitans produces a protein that inhibits neutrophil chemotaxis and hydrogen peroxide production (7, 8) as well as a cytolytic leukotoxin which lyses susceptible target cells including neutrophils, monocytes and T cells (142, 227, 228). Interestingly, unlike P. gingivalis and A. actinomycetemcomitans, a recent mouse model study demonstrated that T. forsythia, which is also implicated in periodontal disease progression, did not affect neutrophil migration into T. forsythia-induced lesions (89) and therefore may not inhibit innate immune responses. Macrophages too may be targeted for inhibition by P. gingivalis. P. gingivalis has been reported to stimulate the production of IL-1b by B cells rather than monocytes and, as discussed below, to affect macrophage migration by inhibiting the production of one of the major monocyte/macrophage chemokines, monocyte chemoattractant protein-1 (79, 80).

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T-cell suppression in periodontitis


Cytokines
We have recently used microarray analysis to show that in BALB/c mice, P. gingivalis has a powerful down-regulatory effect on splenic CD4 and CD8 cells. Only ve and 28 genes respectively were up-regulated in the two subpopulations and in contrast, close to 1200 genes were down-regulated in each subset. P. gingivalis had separate effects on the two sub-populations with overlap being limited to 20%. While most of the down-regulated genes were concerned with metabolism, some coded for immune response proteins. These included several chemokine-like factors, chemokine ligands, and chemokine receptors, suggesting interference with the recruitment of selected leukocytes or their binding (Table 1, Ref. 87). As stated above, P. gingivalis inhibits macrophage migration in humans by inhibiting the production of monocyte chemoattractant protein-1 (79). Furthermore, decreased levels of the neutrophil chemoattractant IL-8 and monocyte chemoattractant protein-1 have been demonstrated in umbilical vein endothelial cells challenged with P. gingivalis (118). These studies are in contrast to a recent report that found a more frequent and higher expression of monocyte chemoattractant protein-1 and its receptor CCR4 in gingival biopsies taken from patients with chronic periodontitis, although these ndings were relative to biopsies from patients with aggressive periodontitis (69) not to control healthy or gingivitis tissue sections. However, P. gingivalis gene expression for colony-stimulating factor 1 receptor, which regulates the proliferation and differentiation of monocyte/macrophage precursors (252), was also down-regulated (87). Stimulatory DNA including lipopolysaccharide has been shown to stop the growth of murine marrow-derived macrophages proliferating in colony-stimulating factor 1 by downmodulating surface colony-stimulating factor 1 receptor expression (196). No increase in macrophage numbers and little evidence of macrophage activation have been demonstrated in advanced periodontitis compared with minimally inamed tissues (35), suggesting that macrophages may have protective effects in the stable lesion which are abrogated in the advanced destructive lesion by P. gingivalis. Another gene down-regulated by P. gingivalis codes for the cytokine receptor IL-17R (87). IL-17Rdecient mice have a defect or display a signicant delay in neutrophil recruitment into infected sites

resulting in susceptibility to infection (114), which may account partly for the reported inhibition of neutrophils in the P. gingivalis-induced lesion in mice (75). In contrast to the study in mice, IL-17 expression has been shown to be up-regulated in human periodontitis tissue (165). This nding was supported by the gene expression prole of T-cell clones established from periodontitis patients where 51% of gingival T-cell clones expressed IL-17 compared with only 11% of peripheral blood T-cell clones (105). In addition, stimulation of peripheral blood mononuclear cells by P. gingivalis antigen enhanced not only transcription but also translation of the IL-17 gene (165). As IL-17 is capable not only of inducing IL-6 in gingival broblasts but also of enhancing the humoral immune response as well as the inammatory response, the balance between the production of IL-17 and expression of its receptor further reects the fact that cytokines cannot be studied in isolation and that it is the balance of cytokines that is fundamental in disease expression. Further genes down-regulated in the microarray study included those which code for myeloperoxidase, peptidoglycan recognition protein, CD14, tolllike receptor 1 (TLR1) and toll interacting protein (87). Myeloperoxidase is produced by macrophages and neutrophils and participates in the respiratory antimicrobial defense system (151). Peptidoglycan recognition proteins, which are also critical for innate immune responses, bind peptidoglycan in bacterial cell walls and are involved in the killing and degradation of cell wall components and the initiation of host defense reactions (144). CD14 is expressed mainly by macrophages and plays a central role in innate immunity as a receptor for bacterial lipopolysaccharide (61). Lipopolysaccharide also stimulates the innate immune response through TLR4. TLR1 has been found to be physically associated with TLR4 and has been demonstrated to have the capacity to abrogate TLR4 signaling and thus prevent innate responses to lipopolysaccharide (217). TLR1 has also been shown to associate with TLR2 to recognize native mycobacterial lipoprotein as well as other lipopeptides (233). A recent study reported that the Th1 cytokines interferon-c and granulocyte macrophage colony-stimulating factor enhanced TLR1 expression while the Th2 cytokine IL-4 downregulated TLR2 expression in monocytes and dendritic cells (123). Furthermore, toll-interacting protein has been demonstrated to co-immunoprecipitate with TLR2 and TLR4 and may be an important constituent of both the TLR2 and TLR4 signaling pathways (28). Overall, these results support other data

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Table 1. Gene expression changes in Porphyromonas gingivalis-activated CD4 and CD8 cells
Gene title Cytokines, chemokines, related factors, cytokine receptors Chemokine-like factor Chemokine-like factor super family 3 Chemokine-like factor super family 7 Chemokine (C-C motif) ligand 2 Chemokine (C-C motif) ligand 9 Chemokine (C motif) receptor 1 Chemokine (C-C motif) receptor 2 Chemokine (C-C motif) receptor 3 Chemokine (C-X-C motif) receptor 6 Colony-stimulating factor 1 receptor Colony-stimulating factor 2 receptor, a, low-afnity (granulocyte-macrophage) Interferon-c receptor 2 Interleukin-1b Interleukin 1 family, member 9 Interleukin-1 receptor-associated kinase 4 Interleukin 2 receptor c chain Interleukin 6 receptor a Interleukin 6 signal transducer Interleukin 7 receptor Interleukin 10 receptor b Interleukin 16 Interleukin 17 receptor Interleukin 17 receptor B Interleukin 18 Interleukin 18 receptor accessory protein Transforming growth factor b1 Transforming growth factor b-induced Transforming growth factor b receptor II Ig binding/B-cell immunity CD19 antigen CD22 antigen CD79A antigen (immunoglobulin-associated a) CD79B antigen Fc receptor, IgE, high afnity I, c polypeptide Fc receptor, IgE, low afnity II, a polypeptide Fc receptor, IgG, high afnity I Cd19 Cd22 Cd79a Cd79b Fcer1g Fcer2a Fcgr1 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + + ) + + + + + ) + ) Cklf Cklfsf3 Cklfsf7 Ccl2 Ccl9 Xcr1 Ccr2 Ccr3 Cxcr6 Csf1 Csf2ra Ifngr2 Il1b Il1f9 Irak4 Il2rg Il6ra Il6st Il7r Il10rb Il16 Il17r Il17rb Il18 Il18rap Tgfb1 Tgfbi Tgfbr2 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + ) ) ) ) + + + + ) + ) + ) + ) ) + ) + + + ) + + ) + ) + + + + + ) ) ) + + ) + ) + ) + + ) + + ) ) + + ) + Gene symbol CD4 CD8 CD4 CD8

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Table 1. Continued
Gene title Fc receptor, IgG, low afnity Iib Fc receptor, IgG, low afnity III Similar to immunoglobulin c-2a heavy chain Immunoglobulin heavy chain 4 (serum IgG1) Immunoglobulin heavy chain 6 (heavy chain of IgM) Immunoglobulin heavy chain (gamma polypeptide) Immunoglobulin j chain variable 21 (V21) /// Anti-c-erbB-2/ p185 monoclonal antibody SER4 heavy chain variable region Immunoglobulin kappa chain variable 32 (V32) immunoglobulin lambda chain, variable 1 T cell immunity CD2 antigen (cytoplasmic tail) binding protein 2 CD3 antigen, f polypeptide CD8 antigen, a chain CD8 antigen, b chain 1 T-cell receptor b, variable 13 T-cell receptor b, variable 13 /// Similar to TCRBV7S1 T-cell receptor b, variable 13 /// T-cell receptor b, joining region T-cell receptor c, variable 4 Antigen presentation/major histocompatibility complex b2-microglobulin CD1d1 antigen CD86 antigen ICOS ligand Histocompatibility 2, class II antigen A, a Histocompatibility 2, class II antigen A, b1 Histocompatibility 2, class II antigen E a Histocompatibility 2, class II antigen E b Histocompatibility 2, class II, locus DMa Histocompatibility 2, class II, locus Mb1 /// histocompatibility 2, class II, locus Mb2 Histocompatibility 2, class II, locus Mb2 Histocompatibility 2, O region a locus Histocompatibility 28 Human leukocyte antigen-B associated transcript 8 Ia-associated invariant chain Histocompatibility 2, T region locus 23 /// RIKEN cDNA C920025E04 gene histocompatibility 2, T region locus 24 B2m Cd1d1 Cd86 Icos H2-Aa H2-Ab1 H2-Ea H2-Eb1 H2-DMa H2-DMb1 /// H2-DMb2 H2-DMb2 H2-Oa H28 Bat8 Ii H2-T23 /// C920025E04Rik H2-T24 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + ) + + + + + + + + + + + ) ) + ) ) + ) + ) ) ) + + ) ) + ) + + Cd2bp2 Cd3z Cd8a Cd8b1 Tcrb-V13 Tcrb-V13 Tcrb-V13 /// Tcrb-J Tcrg-V4 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + ) + ) + + + + + + + + + Gene symbol Fcgr2b Fcgr3 Igh-1a Igh-4 Igh-6 Ighg Igk-V1 Igk-V32 Igl-V1 CD4 ) ) + + ) ) ) ) ) CD8 ) ) ) ) ) ) ) ) ) CD4 + + ) ) + ) ) ) ) CD8 + + ) ) + + + + +

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Table 1. Continued
Gene title Innate immunity Complement component 1, q subcomponent binding protein Complement component 1, q subcomponent, a polypeptide Complement component 1, q subcomponent, b polypeptide Complement component 1, q subcomponent, c polypeptide Complement component 3 Complement component 5, receptor 1 Complement component 6 CD14 antigen Myeloperoxidase Peptidoglycan recognition protein 1 Toll interacting protein Toll-like receptor 1 Extracellular matrix and cell adhesion molecules a disintegrin and metalloprotease domain 8 a disintegrin and metalloprotease domain 10 a disintegrin and metalloproteinase domain 17 a disintegrin and metalloproteinase domain 19 (meltrin b) Integrin a4 Integrin a6 Integrin aL Integrin aM Integrin aV Integrin aX Integrin b2 Integrin b2-like Integrin b4 binding protein Matrix metalloproteinase 9 Procollagen, type III, a1 Procollagen, type XIV, a1 Syndecan 3 Tissue inhibitor of metalloproteinase 2 Intercellular adhesion molecule 2 Platelet/endothelial cell adhesion molecule 1 Selectin, lymphocyte (l-selectin) Adam8 Adam10 Adam17 Adam19 Itga4 Itga6 Itgal Itgam Itgav Itgax Itgb2 Itgb2l Itgb4bp Mmp9 Col3a1 Col14a1 Sdc3 Timp2 Icam2 Pecam1 Sell ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + ) ) ) ) + ) ) + + ) ) ) + + + + + ) ) ) ) + + + + + + + ) ) + + + ) ) ) ) ) + + + C1qbp C1qa C1qb C1qg C3 C5r1 C6 Cd14 Mpo Pglyrp1 Tollip Tlr1 ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + + + + ) + + + + ) ) ) ) + ) ) + ) ) ) ) + + Gene symbol CD4 CD8 CD4 CD8

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Table 1. Continued
Gene title Selectin, platelet (p-selectin) ligand Vascular cell adhesion molecule 1 Bone metabolism Osteoclast stimulating factor 1 Wound healing Elastase 1, pancreatic Elastase 2 Elastase 3B, pancreatic
Reproduced from Gemmell et al. (87).

Gene symbol Selpl Vcam1

CD4 ) )

CD8 ) )

CD4 + +

CD8 + +

Ostf1

Ela1 Ela2 Ela3b

) ) )

+ + +

) ) )

) ) )

demonstrating that P. gingivalis evades host innate immune responses. P. gingivalis also down-regulated a number of genes encoding cytokines or cytokine receptors in T cells (87). These included the genes for IL-1b, IL-1 family 9 and IL-1R-associated kinase 4 (IRAK-4). IL-1 is a principal mediator of inammatory responses acting on many cell types and is itself produced by many different cells, including macrophages, endothelial cells, B cells, broblasts, epithelial cells, astrocytes, and osteoblasts in response to microorganisms, bacterial toxins, complement components or tissue injury (52). One of the most important actions of IL-1 is its induction of other cytokines (164). IL-1 is the most potent known inducer of bone demineralization (219) as well as of major changes in the connective tissue matrix (180). However, while P. gingivalis depressed the gene for IL-1b in T cells, it has, as stated above, been shown to induce an increased percentage of peripheral blood B cells from periodontitis patients to produce IL-1b compared with macrophages (73). Since macrophages are not a dominant feature of the advanced lesion (35) and suppressed cell-mediated immunity is associated with advanced periodontitis (25, 106), B cells may be the major source of IL-1 in periodontitis. One of the major effects noted in the microarray study apart from suppression of T-cell function, was the shift away from Th1 responses (87). IRAK is recruited to the IL-1R complex on IL-1 stimulation and is required for optimal transduction of IL-1-induced signals (136). However, more interestingly, interferon-c production and Th1 cell proliferation have been shown to be severely impaired in IRAK-4decient mice (223). P. gingivalis also down-regulated the gene coding for interferon-c receptor 2 in CD8

cells. T cells in mice that are decient in this receptor were found to have a defect in Th1 differentiation leading to lower amounts of interferon-c in response to antigen challenge, with an alteration in interferonc-induced immunoglobulin class switching in B cells (134). A study on the effect of tobacco smoke on human peripheral blood cells by microarray analysis found a signicant depression in the gene for interferon-c receptor 2 as well as that for chemokine receptor 2 (188), so that a subject with P. gingivalisinduced periodontitis who was also a smoker may well have reduced interferon-c responses. Two other genes down-regulated by P. gingivalis included IL-18 and IL-18R accessory protein (IL-18Rap) (87). IL-18 is a member of the IL-1 family (90) and the IL-18Rap is required for IL-18 signaling (24). IL-18 can act with IL-12 to promote the generation of interferon-c-producing Th1 cells (160), suggesting again that P. gingivalis promotes the down-regulation of this T-cell subset. However, another study reported higher concentrations of IL-18 in gingival biopsies from sites with a probing depth >6 mm compared with healthy sites although IL-12 concentrations were signicantly lower (108). This combination would also lead to reduced Th1 responses. Yet another study reported a higher production of IL-18 by whole blood cells from patients with periodontitis although the stimulus was E. coli lipopolysaccharide (65). Although this study also showed reduced IL-1b in response to E. coli, the lipopolysaccharides from this bacterium and from P. gingivalis appear to have differential effects. The gene coding for the IL-10Rb was down-regulated by P. gingivalis in CD4 cells (87), suggesting an inability of these cells to respond to IL-10. IL-10 has many biological functions including the limitation

23

Gemmell et al.

and termination of inammatory responses and regulation of proliferation and differentiation of immunocompetent cells, including T and B cells and antigen-presenting cells (reviewed in Ref. 6). The nonresponsiveness of T cells to P. gingivalis has recently been shown, although peripheral blood B cells did proliferate and high levels of IL-10 were produced by monocytes. IL-10 itself could induce B-cell proliferation and it was suggested that P. gingivalis may play a role in polyclonal B-cell activation associated with periodontal disease via a macrophage-dependent IL-10 route (34). However, as previously stated, macrophages are most likely not activated in periodontitis lesions (35), suggesting the possible importance of the lack of this immunosuppressive cytokine in periodontitis lesions. However, one of the important effects of IL-10 is its direct inhibitory effects on interferon-c production (49) and in humans, IL-10 promotes Th2 responses while suppressing delayed-type hypersensitivity reactions and other Th1-cell-mediated responses (103). While the microarray study indicated that CD4 cells would not respond to IL-10 because of the down-regulation of the IL-10Rb gene, the down-regulation of the other genes, including IRAK-4, interferon-c receptor 2, IL18 and IL-18Rap, would still suggest a swing away from Th1 responses. The microarray study also demonstrated the downregulation by P. gingivalis of the gene encoding the IL-2Rc chain in CD8 cells (87). The IL-2Rc is shared by receptor complexes used by IL-2 and other cytokines including IL-4, IL-7, IL-9 and IL-15, all of which are involved in lymphocyte development and/ or activation. IL-2Rc is physically associated with JAK3 tyrosine kinase (which was also down-regulated in CD4 cells), and this molecular pair may be considered to be the trigger of the signaling cascades. X-linked severe combined immunodeciency in humans is caused by mutations in the IL-2Rc gene that result in absent T cells and non-functional B cells (10). Prevotella intermedia strains have been shown to not only inhibit T-cell and B-cell proliferation in response to mitogens and antigens, but to inhibit IL-2R expression on T cells (208). The down-regulation of the IL-2Rc chain may well support studies demonstrating a lack of IL-2 production and reduced IL-2R expression by T cells in patients with a reduced autologous mixed lymphocyte reaction (116, 203). P. gingivalis also induced the down-regulation of the genes coding for transforming growth factor-b1 and the transforming growth factor-b2 receptor (87). Transforming growth factor-b is a pleiotropic cytokine with transforming growth factor-b receptors

being expressed on most cell types (88); down-regulation of the cytokine and its receptor would have wide-ranging effects (87). A mouse model in which transforming growth factor-b was blocked specically in T cells, demonstrated that T-cell homeostasis requires transforming growth factor-b signaling (88). Hence, the ability of P. gingivalis to down-regulate the genes coding for this cytokine further illustrates the dynamic balance that may be occurring between the plaque biolm and the host. Another study in mice showed that transforming growth factor-b is important for B-cell development and that B-cell progenitors are differentially affected according to their stage of differentiation (112). Transforming growth factor-b is also involved in all aspects of wound healing entailing inammation, re-epithelialization, matrix formation, and remodeling (3). It is produced locally at the site of resorption of bone and has been shown to initiate new bone formation (33). The down-regulation of transforming growth factor-b in T cells conrms an earlier study which demonstrated that more transforming growth factor-b may have been produced by peripheral blood mononuclear cells in culture in the absence of stimulatory bacteria (72).

Surface membrane antigens


As well as down-regulating a number of cytokinerelated genes, P. gingivalis also down-regulated several genes encoding proteins which directly affect T-cell function (87). These included CD2-binding protein 2 expression. This protein binds to a site within the cytoplasmic region of CD2 and overexpression of the isolated protein enhances IL-2 production on cross-linking of CD2 in Jurkat T cells (163). The cell adhesion molecule CD2 is expressed by T cells and recognized by CD48 in mice and CD58 (lymphocyte function-associated antigen-3; LFA-3) in humans (253). CD2CD48 interactions have been shown to be critical for the production of sufcient IL-2 and interferon-c to induce the differentiation of CD8+ cells into functional cytotoxic cells (155). The CD2 molecule is expressed on the majority of murine lymphocytes and plays a regulatory role in antigenspecic responses via the T-cell receptor (167, 243). The absence of CD2 on murine T-cell receptor a/b+ T cells has been shown to co-segregate with nonresponsiveness (243). CD2+ T cells have been demonstrated in the gingival tissues of patients with chronic periodontitis (91) although a lower percentage of positive cells has been reported compared with peripheral blood T cells (74). Taken together, these

24

The role of T cells in periodontal disease

results suggest an increase in non-responsive T cells in the gingival tissues as a result of the down-regulation of the CD2 binding protein 2 by P. gingivalis in periodontitis. Yet again, they also indicate a swing away from the Th1 cytokines IL-2 and interferon-c. The T-cell receptor is a complex composed of the antigen-binding heterodimer (a/b or c/d chains) and a signal transducing complex consisting of the CD3 dimers (CD3 c/e and CD3 d/e) and the T-cell receptor f homodimer (95). This T-cell receptorCD3 complex plays a key role in antigen recognition, T-cell activation, and triggering antigen-specic responses (23). The microarray study demonstrated that the gene coding for CD3f was down-regulated by P. gingivalis (87). T cells from patients with systemic lupus erythematosus display T-cell receptor f chain aberrations as well as defective IL-2 production (162), suggesting another association between reduced IL-2 production and P. gingivalis-induced periodontitis. The cell surface glycoprotein CD8 functions as a co-receptor with the T-cell receptor for interaction with major histocompatibility complex class I molecules (50) CD8 exists as homodimers of a polypeptide chains and heterodimers of a and b chains and while the CD8a chain binds to major histocompatibility complex class I, the function of CD8b, for which there are two genes, CD8b1 and CD8b2, is not as clear (51, 161). Gemmell et al. (87) demonstrated down-regulation of the genes for CD8a and b1 chains in both CD4 and CD8 cells. It has recently been shown that CD8 is expressed on CD4 cells in rats and these cells helped in primary humoral responses and produced mainly Th1 cytokines (115). Therefore, it follows that in periodontitis, Th1 cytokines produced by helper T cells expressing CD8 may be decreased. However, a down-regulation of CD8 responses induced by P. gingivalis is at odds with reports showing a decrease in the CD4 : CD8 ratio in periodontitis biopsies compared with peripheral blood and healthy/gingivitis tissue (170, 234).

(21). Adhesion molecules are therefore important factors in determining the homing of leukocyte subsets as well as imparting other functional roles such as differential cytokine production. The genes encoding endothelial cell adhesion molecule-2 and several integrins were down-regulated in mice by P. gingivalis, particularly in the CD8 cells (87). Integrins mediate the migration of leukocytes through the extracellular matrix and induce signals that direct different cellular signaling pathways (44). Interestingly, the b2 integrin subunit of LFA-1 (CD18) was down-regulated in CD8 cells, which would result in the abrogation of the LFA-1/endothelial cell adhesion molecule-2 pathway. Overall these results may indicate a role for P. gingivalis in down-regulating Th2 cytokines.

Antigen presentation
As already stated, P. gingivalis and A. actinomycetemcomitans are capable of invading epithelial cells (64, 192). Keratinocytes expressing class II molecules have been reported in inamed sites of gingival tissues (204), suggesting that in humans, gingival keratinocytes can present antigens to the underlying lymphocytes (189). An animal model has only recently shown that rat gingival epithelial cells treated with interferon-c and A. actinomycetemcomitans express major histocompatibility complex class II molecules and the co-stimulatory molecule CD80 and can stimulate A. actinomycetemcomitans-specic CD4 cells to proliferate (145). However, it is also possible that Langerhans cells in the gingival epithelium (82, 109) may also present P. gingivalis antigens. Furthermore, different dendritic cell subsets have been demonstrated in the gingival tissues and have been shown to associate with clusters of CD4 cells, suggesting that antigen presentation to T cells may occur in the connective tissues (109). While peptide antigens are recognized in an major histocompatibility complex-restricted manner, lipidcontaining antigens such as lipoprotein and lipopolysaccharide are recognized in a CD1-restricted manner. Cells expressing all isoforms of CD1, namely CD1a, b, c, and d, are present in periodontitis tissue and the total expression of CD1 is equivalent to that of CD83, a marker of mature dendritic cells. This suggests the potential importance of lipid antigens in chronic periodontitis (2). It is however, becoming apparent that T cells themselves can present antigen. Resting cattle CD4 cells from calves immunized with ovalbumin or respiratory syncytial virus were found to proliferate in

Cell adhesion molecules


Leukocytes migrate into the tissues from the peripheral circulation and the rst step in this migration is binding to endothelial cell adhesion molecules. These endothelial cell adhesion molecules are expressed on endothelial cells as well as on leukocytes and a recent report showed that while LFA-1/ endothelial cell adhesion molecule-1 binding resulted in the production of IL-10, LFA-1/endothelial cell adhesion molecule-2 binding induced a stronger secretion of the Th1 cytokine tumor necrosis factor-a

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response to c/d T cells pulsed with antigen-presenting cells expressing high levels of major histocompatibility complex class II molecules and synthesizing the co-stimulatory molecule CD80 (40). Another study showed similar results using circulating porcine c/d T cells. Peripheral blood from ovalbumin-immunized pigs depleted of all conventional antigen-presenting cells were able to proliferate in response to antigen and this response was abolished after depletion of c/d T cells and anti-major histocompatibility complex class II or anti-CD4 antibodies (230). An earlier study in humans demonstrated that tetanus antigen-specic T-cell clones pre-incubated with antigen followed by irradiation could present antigen and initiate proliferation by autologous cloned T cells. Again this antigen presentation was abrogated by treatment with anti-human leukocyte antigen-DR or anti-tetanus antibodies. Autologous peripheral blood resting T cells or phytohemagglutinin-activated T-cell blasts could not present antigen to responder cloned T cells (37). More recently, it has been reported that human T cells can acquire large quantities of major histocompatibility complex class II molecules from various types of antigen-presenting cells in an antigen-independent manner and this required direct cell-to-cell contact and interaction of adhesion molecules. The newly acquired major histocompatibility complex class II molecules were capable of presenting antigen to T helper cells, suggesting that T cells interact with other T cells to regulate immune responses by presenting major histocompatibility complex class II peptide complexes obtained from nearby antigen-presenting cells (241). A further study showed that lamina propria T cells from actively inamed inammatory bowel disease mucosa expressed large amounts of major histocompatibility complex class II molecules and CD86 and could stimulate allogeneic naive peripheral blood T-cell proliferation. This process was reduced by the addition of IL-10. It was suggested that this interaction between T cells could contribute to the perpetuation of inammation (59). The study by Gemmell et al. (87) showed that mRNA for certain class I and II molecules and the Ia-associated invariant chain were down-regulated in both CD4 and CD8 cells. The H-2 major histocompatibility complex of mice encodes two functional proteins, Aa Ab (A) and Ea Eb (E) (226). Efcient loading of class II molecules with peptides requires the invariant chain and the class II-like molecule H2-M (now H2-DM) (122). Cells from mice devoid of the invariant chain show aberrant transport of major histocompatibility complex class II molecules,

leading to reduced levels of class II complexes at the surface which do not have the typical compact conformation indicative of tight peptide binding (246). Because of the physical association of H2-O with H2-M and the co-localization in class II+ vesicles, it has been suggested that H2-O may have a related function in peptide exchange (173). Generally, downregulation of these genes would lead to impairment of antigen presentation functions. However, the extent of T-cellT-cell interaction in the gingival tissues is at this stage unclear. The suggestion of impairment of antigen presentation functions in P. gingivalis-induced periodontitis was also reected in the down-regulation of the genes for CD86 (a ligand of the major co-stimulatory CD28) and inducible co-stimulatory molecule ligand (a member of the CD28 family). Activation of T cells leading to cytokine production requires a signal transduced through the T-cell receptor as well as a second signal transduced by a co-stimulatory molecule (70). CD28 is the major co-stimulatory signal receptor for T cells and its natural ligands are CD80 (B7-1) and CD86 (B7-2), which are expressed either constitutively or after activation on antigen-presenting cells (1). Inducible co-stimulatory molecule, a member of the CD28 family, is expressed by activated T cells and binds with its ligand, which is constitutively expressed on B cells. Interaction leads to differentiation of B cells to plasma cells (127), suggesting that antigen-specic activation of B cells in periodontitis may be suppressed by P. gingivalis. Activated monocytes, B cells and dendritic cells express B7 molecules (111). Macrophages constitutively express low levels of CD86, CD80 being induced after treatment with interferon-c. CD86 expression is also low on B cells until activation, which induces a rapid up-regulation of these molecules (107). The percentage of CD86+ B cells and macrophages has been shown to be signicantly higher than the percentage of CD80+ macrophages in gingival tissue sections (81). Another study showed that CD86 was up-regulated mostly on B cells isolated from periodontitis lesions and that a number of periodontopathic bacteria including P. gingivalis up-regulated CD86 on B cells in vitro (139). It is now becoming clear that T cells also express co-stimulatory molecules. Activated human peripheral blood T cells, CD4 and CD8 clones and natural killer clones have been shown to express B7 molecules (11). Anti-CD28 antibodies or CTLA4-Ig fusion protein have been demonstrated to inhibit the proliferation of specic T-cell clones in response to T cells acting as antigen-presenting cells or

26

The role of T cells in periodontal disease

proliferation of peripheral blood mononuclear cells in a primary allostimulation with activated T cells as stimulator cells. B7 was also found to be expressed on subsets of freshly isolated activated CD4 or CD8 cells from some individuals infected with human immunodeciency virus or from others with autoimmune diseases, although T cells from healthy individuals did not express B7 (256). Hirokawa et al. (98) suggested that B7 molecules functioned as co-stimulatory molecules on T cells and played a role in interactions between T cells leading to clonal expansion of activated T cells. Another study has shown that memory CD4 cells express CD86 while naive CD4 cells do not, although the positive memory cells co-stimulated the naive T cells with anti-CD3 antibodies to induce IL-2 production. The naive cells were shown to express CD86 after co-stimulation with CD86 and T-cell receptor ligation (92). A mouse model showed that when T-cell donors from CD80/ CD86-decient mice were used for allogeneic transplant recipients, graft-vs.-host disease accelerated compared with wild-type T cells. On the other hand, T cells from CD86 transgenic mice that overexpressed CD86, mediated the reduced graft-vs.-host disease mortality. This study demonstrated the importance of T-cell-associated B7 molecules as negative regulators of immune responses (235). The signicance of down-regulation of CD86 on T cells in periodontal disease is currently unknown.

Autoimmunity: natural killer T cells


Mouse CD1d1 is a member of the CD1 family of evolutionarily conserved major histocompatibility complex antigen-like molecules (110). Unlike the classical major histocompatibility complex products that bind peptides, mouse and human CD1d molecules present glycolipid antigens such as a-galactosylceramide to CD1d-restricted natural killer T cells. The use of CD1d knockout mice has demonstrated an inability to clear metastatic tumors and a-galactosylceramide was able to inhibit disease in diabetes-prone non-obese diabetic mice, suggesting a critical role for CD1d-dependent T cells in various disease conditions (102). Another study showed that CD1 expression increased on antigen-presenting cells in Listeriainfected mice and the use of anti-CD1 antibody reduced transforming growth factor-b2 levels while increasing IL-12 and interferon-c at disease onset. The results pointed to a regulatory role for CD1-reactive cells in the immune response to Listeria (224).

Mouse natural killer 1 cells constitute a subset of T-cell receptor ab+ T cells that express natural killer surface receptors. They are thought to play an immunoregulatory role because of their ability to secrete IL-4 within minutes of primary activation (17), although another report demonstrated a rapid production of both IL-4 and interferon-c in mice after treatment with a-galactosylceramide (211). A unique feature of their T-cell receptor repertoire is the expression of an invariant T-cell receptor chain, Va14-Ja281 in mice (18) and Va24-JaQ in humans (177). A recent immunohistological study examined natural killer T cells in periodontal disease and found that the frequency of Va24-JaQ T-cell receptorexpressing T cells was higher in periodontitis lesions than in gingivitis tissues or peripheral blood. The natural killer T cells also appeared to associate with CD1d+ cells and it was suggested that these cells play a regulatory role in periodontal disease (258). Autoimmunity has been suggested to be a feature of periodontal disease. Cross-reactivity of human heat-shock protein 60 and P. gingivalis GroEL, which is a bacterial homologue, has been shown in periodontal disease (66, 225). Heat-shock protein 60-specic, as well as P. gingivalis cross-reactive, T cells have also been demonstrated to accumulate in periodontitis lesions (259). Taken together these data suggest that both a humoral and a cell-mediated specic immune response to heat-shock protein 60 may be important in the disease process. Additionally, anti-collagen type l and lll antibodies have been demonstrated in the gingivae of periodontitis patients (99) and collagen type 1-specic T-cell clones have been identied in inamed tissues of periodontitis patients (248). The study by Yamazaki et al. (258) suggests that an immune response to autoantigens such as collagen type I or heat-shock protein 60 may be well controlled by natural killer T cells. A relationship between a deciency in natural killer T cells and autoimmune diseases has been cited in mice (150, 231). An impairment of the subtle balance could be involved in the pathogenesis of periodontal disease (Fig. 2) The results however, did show increases of natural killer T cells in periodontitis, suggesting a functional role for these cells and because of their ability to secrete rapid amounts of cytokines, they may inuence the T helper cytokine response. In support of the above ndings (258), CD1d is the most prominent CD1 molecule in periodontitis and the CD1d-expressing cells increased with increasing number of invariant natural killer T-cell inltration (2). It is becoming clear that both bacterial and

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Indirect mechanism
TLRs

Microbial components (lipopolysaccharide, lipoteichoic acid, etc.) cytokines

Dendritic cell

Increased synthesis of these molecules CD1d Endogenous glycolipid

NKT cell

cytokines

B
Direct mechanism
Microorganisms Dendritic cell microbial glycolipids

CD1d CD1 reactive T cell (NKT ells)

cytokines

Fig. 2. Putative roles of autoimmune response and regulatory mechanisms in susceptible patients and non-susceptible patients. In susceptible patients (A), infection and subsequent inammation result in the up-regulation of auto-antigens such as heat-shock protein 60 and collagen type I and the activation of auto-reactive T and B cells specic to those antigens. Although regulatory T cells are induced in the lesion, their number and function may not be sufcient to control immune pathology. In non-susceptible patients (B), on the other hand, the autoimmune response stimulates scavenger cells to take up the degenerated self-components resulting in acceleration of the tissue repair process. These mechanisms may be well controlled by regulatory T cells. Consequently, tissue integrity is maintained and the stable lesion can be seen.

CD1 glycolipid autoantigens microbial glycolipids

endogenous glycolipids presented by CD1d can be recognized by human natural killer T cells but in some instances, the recognition of bacteria by natural killer T cells may be indirect (146) (Fig. 3). One study suggested that Salmonella infection may lead to an altered environment, where natural killer T cells are stimulated by inammatory cytokines in combination with CD1d-mediated presentation of autologous ligands, induced in response to infection (27). This scenario could also be applicable to chronic inam-

Fig. 3. Indirect and direct mechanisms of CD1d-reactive natural killer T-cell (NKT) activation. Microbial components such as lipopolysaccharide, lipoteichoic acid and other lipopeptides can be recognized by TLRs resulting in increased synthesis of endogenous glycolipid. Natural killer T cells recognize endogenous glycolipids presented by CD1d. Natural killer T cells are also stimulated by inammatory cytokines induced in response to infection (A). Microbial glycolipid can also be directly presented by CD1d molecule, resulting in the activation of natural killer T cells (B).

matory periodontal diseases in which inammatory cytokines are up-regulated. However, down-regulation of CD1d in murine T cells by P. gingivalis has been demonstrated (87) implying that even though there may be higher numbers of natural killer T cells in periodontitis, these may not be functional if T-cellnatural killer

28

The role of T cells in periodontal disease

T-cell interactions were a feature of periodontal disease. In addition to invariant natural killer T cells, other phenotypes of regulatory T cells, possibly CD4+ CD25+ regulatory T cells, Th3 and Tr1 inltrate and may play roles in periodontal disease (159). The role of autoimmunity in chronic inammation is still not clear. It is possible that autoimmunity is a feature of all chronic inammatory processes. In this context it has been known for many years that gingival broblasts are able to phagocytose collagen such that anti-collagen antibodies may facilitate this phagocytosis and hence the removal of broken down collagen. At the same time an anti-heat-shock protein response may enhance the removal of dead and dying cells such that these autoimmune responses may be a natural part of chronic inammation. Control of these responses would therefore be essential, hence the increase in regulatory natural killer T cells in periodontal tissues. This concept further illustrates that the role of T cells in periodontal disease may be one of immune homeostasis. Further studies are clearly needed to test this hypothesis and to determine the role of regulatory T cells in periodontal inammation.

Bone loss
Do T cells have a role in tissue destruction in periodontal disease? It has been known for some time that cells of the immune system can inuence bone cell function (132). Osteoclasts share a common origin with cells of the macrophage/monocyte lineage and respond to and produce cytokines that regulate cells of this lineage. Osteoblasts originate from bone marrow stromal stem cells of mesenchymal origin and have the capacity to produce factors that inuence the lineage development of bone marrow cells (132). Receptor activator of nuclear factor-jB ligand (RANK-L), which is also known as osteoprotegerin-L (OPG-L), regulates osteoclast differentiation and function (229). The receptor for RANK-L/OPG-L is RANK (4) and a variety of cells produce a decoy receptor OPG, which when released by cells, binds RANK-L/OPG-L to prevent activation of RANK (210). While these factors have potent effects on osteoclast development, they also have regulatory effects on immune cell function (132). Results of a study on RANK-L/OPG-L-decient mice showed that this factor is critical in T-cell maturation and T cells in these mice showed poor induction of cytokines such as interferon-c, IL-2, and IL-4 in response to anti-CD3 and anti-CD28 (119). Increased

concentrations of RANK-L and decreased concentrations of OPG have been reported in the gingival crevicular uid from periodontitis patients compared with control subjects and the ratio of RANK-L/OPG was also signicantly higher, suggesting that these two factors contribute to alveolar bone destruction in periodontal disease (152). A similar nding was reported in another study with levels of RANK-L being higher in active sites that were probably associated with tissue destruction compared with inactive sites (244). Interestingly, human gingival broblasts were shown to express OPG rather than RANK-L and OPG mRNA expression and production by gingival broblasts was augmented by lipopolysaccharide stimulation. Supernatants of lipopolysaccharide-stimulated broblasts reduced the numbers of tartrate resistant acid phosphatasepositive cells generated by monocytes cultured in the presence of RANK-L and macrophage colony-stimulating factor, suggesting the inhibition of monocyte-derived osteoclasts via an OPG pathway (156). Gingival broblasts therefore may not play a role in bone resorption in periodontal disease. Another study by Liu et al. (130) supported the nding of higher levels of RANK-L and lower levels of OPG in advanced periodontitis. More signicantly, RANK-L mRNA was expressed mainly by inammatory lymphocytes and macrophages as well as proliferating epithelium in the vicinity of inammatory cells. Although both soluble and membrane-bound RANKL can be produced by activated T cells (119), the frequency of RANK-L mRNA-positive gingival T-cell clones was low but variable compared with the high and constant frequency of other cytokines such as interferon-c and transforming growth factor-b1 (105). This may reect disease activity of the sites from where the cells were extracted or the susceptibility of the patients from whom the tissues were obtained. CD4 knockout mice, but not CD8 knockout mice, lose less alveolar bone in response to oral P. gingivalis infection than immunocompetent mice of the same genetic background, suggesting that CD4 cells may contribute to bone resorption (14). Experiments in non-obese diabetic/severe combined immunodecient (NOD/SCID) mice transplanted with human peripheral blood lymphocytes from periodontitis patients and orally challenged with A. actinomycetemcomitans also showed that human CD4 cells but not CD8 cells or B cells were able to mediate alveolar bone destruction (237). This study also showed that A. actinomycetemcomitans stimulated the production of OPG-L by CD4 cells, while inhibition of OPG-L

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Gemmell et al.

function using OPG diminished bone destruction and reduced the numbers of periodontal osteoblasts after bacterial challenge. The authors acknowledged that OPG-L produced by CD4 cells may not be the sole inducer of increased alveolar bone destruction in periodontal disease because injection of soluble OPG for 4 weeks did not completely block this bone destruction in vivo. An OPG-L-independent induction of osteoclast differentiation has been reported (117). This study found that osteoclasts could be generated from mouse bone marrow cells and this was not inhibited by OPG. As already stated, IL-1 has a major role in bone resorption in periodontal disease and both IL-1 and tumor necrosis factor-a have been reported to regulate the balance of OPG-L and OPG (100, 101) thereby contributing to bone destruction independently of cell-mediated immunity (237). In this respect, P. gingivalis was shown to down-regulate the gene expression for osteoclast stimulating factor 1 as well as for IL-1b in CD4 cells (87), suggesting that P. gingivalisinduced T cells may not play a role in the bone resorptive process in periodontal disease. However, even though B cells were shown not to contribute to A. actinomycetemcomitans-induced bone resorption in mice (237), as discussed earlier, P. gingivalis induced IL-1b production by B cells rather than monocytes (73). Therefore, with increased numbers of B cells associated with disease progression, a link between B cells and alveolar bone destruction in human periodontitis may be more than possible.

Connective tissue matrix destruction


Connective tissue remodeling occurs in growth and development and is regulated by the interplay of cellcell and cellmatrix interactions involving the production of enzymes, activators and inhibitors and cytokines and growth factors (185). Proteinases such as the metalloproteinases (MMPs) are key enzymes in tissue degradation and this family of neutral proteases is part of a larger class of metalloproteinases that include the ADAMS (disintegrin and metalloproteinase domain) (187). MMPs produced by resident cells including broblasts, macrophages, and epithelial cells and also inammatory cells have been cited as playing roles in tissue destruction in periodontal disease (148, 195, 260) and are regulated by tissue inhibitors of metalloproteinases (TIMP). It has been suggested that tissue destruction in disease processes may be the result of an imbalance

of metalloproteinases over tissue inhibitors (185). The concentration of TIMP-1 in the saliva of patients with periodontitis has been shown to be lower than in the saliva of healthy subjects, while collagenase activity was higher. Initial therapy resulted in reciprocal changes of TIMP-1 and collagenase levels. Furthermore, collagenase in the healthy subjects consisted mainly of procollagenase whereas in diseased patients, active collagenase was found predominantly (94). Also, greater collagenase activity was found in the gingival crevicular uid of periodontitis patients than in the uid of control subjects and the collagenase was demonstrated to be derived mostly from neutrophils (245). MMP9 produced by neutrophils was shown to be prominent not only in the crevicular uid but also in the gingival tissue samples from patients with periodontitis (214, 250). Latent MMP2 and MMP9 are expressed in the gingival tissues of patients with periodontitis; active MMP2 was only detected in tissues associated with clinical disease (121) although another study suggested a role for MMP9 in the gingival epthelial response to periodontal infection (213, 214). Yet another study suggested a role for both MMP2 and MMP9 in tissue destruction in periodontitis (140) while Seguier et al. reported increased amounts of the active form of MMP9 in gingival tissue specimens of periodontitis patients (195). Recently it has been reported that P. gingivalis and A. actinomycetemcomitans supernatants can activate MMP2 in human periodontal ligament cells, although while A. actinomycetemcomitans induced a reduction in TIMP2 secretion, there was no change in the level of TIMP2 in the presence of P. gingivalis supernatants (239). Although direct T-cell-derived metalloproteinases have not been reported, our microarray study showed that MMP9 and TIMP2 were down-regulated in CD4 cells (87). The microarray study also demonstrated a downregulation of the gene for procollagen type III in CD4 cells. Whether or not the same is true for broblasts remains to be determined. Both collagen type III and bronectin have been shown to be greatly diminished in inamed gingiva (247), indicating a case for P. gingivalis-induced type III collagen deciency in periodontitis. Transmission electron microscopy of biopsies from patients with rapidly progressive periodontitis and from adult periodontitis patients demonstrated the almost complete destruction of collagen types l and lll in areas with leukocyte inltration while collagen types V and Vl were dominant (97). Another study correlated the number of inammatory cells with the area of collagen bers

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The role of T cells in periodontal disease

that was decreased in gingival connective tissues of patients with severe periodontitis compared with healthy controls. Second, increases in the amounts of MMP1, MMP2, MMP3, and MMP9 and the active form of metalloproteinase 9 were correlated with the number of CD22+ B cells and CD68+ cells, which would include macrophages and T-cell intracellular antigen 1-positive cells (195), implicating T cells in the production of metalloproteinases. However, while P. gingivalis may down-regulate metalloproteinase 9 in T cells, metalloproteinase production by other cells, including B cells, may be increased in periodontitis.

Susceptibility to periodontal disease


It is clear that variations in disease occur in individuals who harbor the same periodontal pathogens in their dental plaque as well as in patients in whom the bacterial composition varies (235). While periodontopathic bacteria and the inammation they provoke are essential for disease progression, environmental risk factors such as tobacco smoking, psychosocial stress, and systemic diseases such as diabetes modify the host response and may be major determinants of the enormous variation in susceptibility (172). It also appears that genetic factors may determine susceptibility or resistance to periodontal disease (12, 93). Results from family studies suggested that environmental factors might be major determinants of variation in periodontitis, although twin studies indicated that both environmental and genetic factors inuenced disease progression (149). Interestingly, although genetic predispositions to periodontitis may involve defects other than those of the immune response, including defects in collagen, cementum, and epithelium (93), genetic control of the immune response in periodontal disease is signicant because of the importance of immunity in disease progression. In this respect, an animal model study has shown that the CD4 cytokine response to P. gingivalis depended on the H-2 haplotype (84), indicating a strong genetic inuence on T-cell immunity to this periodontopathic bacterium. To understand the extent to which the variation in cytokine responses in periodontal disease may be attributed to genetic determinants, genetic polymorphisms in cytokine genes have become an area of research. Polymorphisms in the IL-1 cluster have been a focus of attention since Kornman et al. (120)

demonstrated that genotype-positive individuals had a 20-fold increased risk of developing severe periodontitis after the age of 40 than those individuals who are genotype negative (120). A recent microbiological study of IL-1-positive and IL-1-negative adults showed that signicantly higher counts of bacteria, including T. forsythia, P. gingivalis, T. denticola as well as others, were detected in periodontal pockets >6 mm in depth in genotype-positive subjects than in individuals who were genotype negative (216), suggesting that there may be a correlation between a tendency of genotype-positive people to harbor these bacteria in their plaque. In a 5-year longitudinal study on the progression of periodontal disease, Cullinan et al. (41) showed that while there was no direct effect of the IL-1 genotype on disease progression there were interactive effects between the IL-1 genotype, the presence of P. gingivalis and disease and between IL-1 genotype, smoking, and disease such that the IL-1 genotype could be considered as a secondary risk factor for periodontal disease. This study was the rst to show the interactions between bacteria, genetics, and environment in the progression of periodontal disease. In their review on cytokine gene polymorphisms in periodontal disease Taylor et al. (235) suggested that apart from IL-1 polymorphisms, a role for cytokine gene polymorphisms and susceptibility to chronic periodontitis have not been established, although understanding cytokine regulation and immune regulation in periodontal disease remains of major concern.

Conclusion
Despite over 40 years of research into the immunology of periodontal disease the role of T cells remains an enigma. It is clear from the data obtained from the recent microarray study that in BALB/c mice, P. gingivalis suppresses the T-cell response in a number of ways including down-regulating the expression of genes which affect the T-cell receptor CD3 complex, CD2 binding protein 2 and CD8 expression (87). The down-regulation of genes coding for a number of cytokines and/or cytokine receptors suggests a swing away from Th1 responses. Although a concomitant up-regulation of genes encoding Th2 cytokines was not demonstrated, the overwhelming results of this study demonstrated that down-regulation of both CD4 and CD8 cells could also lead to suppression of help for the antigen-specic B-cell response in periodontitis if P. gingivalis was a major dental plaque constituent. These results have led to

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the development of the hypothesis that T cells have a fundamental role in maintaining immune homeostasis in the presence of the plaque biolm. In this context it can be seen to be in the best interest of the biolm and of the host if a balance can be reached. The T-cell response can therefore be considered to be the default response where activation is balanced with suppression. It is when this balance is disturbed that disease progression occurs. This hypothesis is based on ndings using a mouse model of P. gingivalis infection. However, mice do not develop periodontal disease and the P. gingivalisinduced immune response is a simple response to one albeit signicant periodontopathic bacterium. In humans, dental plaque is a complex biolm. Subgingival plaque, which resides in a more protected location than supragingival plaque, is not subject to the same degree of intraoral abrasion or salivary host defense mechanisms and as with biolms in general, is very resistant to removal (48). It is becoming clear that clusters of bacteria occur (215) and each bacterium in the cluster is likely to affect the responses induced by the others and vice versa. In this respect a number of animal models have shown modulation of the immune response by co-immunization with two periodontal bacteria. Mice infected with P. gingivalis together with F. nucleatum had signicantly larger skin lesions than those infected with P. gingivalis alone while active immunization with P. gingivalis protected against challenge with both organisms (58). Chen et al. (36) reported that immunization of A. actinomycetemcomitans together with P. gingivalis resulted in rst- and second-degree lesions compared with rst-degree lesions only, which followed immunization with A. actinomycetemcomitans alone. The serum anti-P. gingivalis response was higher in mice injected with both organisms than in mice injected with only P. gingivalis although this was not observed with the anti-A. actinomycetemcomitans antibody response. Another study showed that although the levels of anti-F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti-P. gingivalis and anti-F. nucleatum antibodies, although at lower levels than the other groups immunized with bacteria respectively (83). Furthermore, the inhibition of neutrophil phagocytosis of immune serum-opsonized P. gingivalis was shown to be modulated by the presence of anti-F. nucleatum antibodies, while anti-P. gingivalis

antibodies induced an inhibitory effect on the phagocytic response to F. nucleatum (85).These studies highlight the complex often synergistic responses with co-infection (55), which may have relevance to the multibacterial infection found in human periodontal disease. The role of autoimmunity in chronic inammation is also of major interest. In this context it can be postulated that autoimmunity is a critical and integral part of chronic inammation in that it enhances the removal of collagen by enhancing broblast phagocytosis of protease-digested collagen fragments as well as the removal of destroyed or dying cells. Control of this process by regulatory T cells then becomes fundamental and again if there is a disturbance in this homeostatic mechanism enhanced tissue destruction could result.

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