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Porphyromonas gingivalis-Epithelial Cell Interactions in Periodontitis

Groupe de Recherche en Écologie Buccale, Faculté de médecine dentaire, Université Laval, Quebec City, Quebec, Canada, G1K 7P4

Correspondence: ^* corresponding author, Daniel.Grenier{at}greb.ulaval.ca

	ABSTRACT

TOP ABSTRACT (I) INTRODUCTION (II) ADHESION TO EPITHELIAL... (III) INTERNALIZATION BY... (IV) EPITHELIAL CELL RESPONSES (V) CONCLUSIONS REFERENCES

 
Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.

Key Words: periodontitis • epithelial cell • Porphyromonas gingivalis • adhesion • invasion

	(I) INTRODUCTION

TOP ABSTRACT (I) INTRODUCTION (II) ADHESION TO EPITHELIAL... (III) INTERNALIZATION BY... (IV) EPITHELIAL CELL RESPONSES (V) CONCLUSIONS REFERENCES

 
Periodontal disease is a complex multifactorial disorder involving Gram-negative anaerobic bacteria and host cell interactions, the combined effects of which lead to the destruction of tooth-supporting tissue. More specifically, periodontitis results from chronic inflammation of the gingiva and occurs by its spread into the deeper structures of the periodontium, leading to progressive destruction of periodontal tissues, including the alveolar bone (Williams, 1990). Approximately 15% of the population is affected by severe forms of the disease, which, if untreated, may result in tooth loss and systemic complications (American Academy of Periodontology, 1996). In addition, periodontitis has been associated with cardiovascular disease and pre-term delivery of low-birthweight infants (Teng et al., 2002). The progression of periodontitis is episodic, with active and inactive phases of tissue destruction, which reflects the opposing actions of bacterial challenges and host immune responses. The intimate interactions between periodontopathogens and host cells have become the subject of intensive investigations.

Porphyromonas gingivalis is a Gram-negative black-pigmented strict anaerobic bacterium that has been implicated as a major etiologic agent in the development and progression of periodontitis, more particularly, the chronic form (Lamont and Jenkinson, 1998; Holt et al., 1999). P. gingivalis produces a broad array of potential virulence factors involved in tissue colonization and destruction as well as in host defense perturbation (Holt et al., 1999). P. gingivalis is in close contact with the epithelium in periodontal pockets in vivo (Noiri et al., 1997) and can invade various cell lines, including epithelial cells (Sandros et al., 1994; Lamont et al., 1995; Belton et al., 1999; Rudney et al., 2001), endothelial cells (Deshpande et al., 1998; Dorn et al., 2000), and fibroblasts (Amornchat et al., 2003). The gingival epithelium is a stratified squamous epithelium that is an interface between the external environment, which is exposed to bacterial challenges, and the underlying periodontal tissue. The basal layer of the gingival epithelium is separated from and attached to the connective tissue by the basement lamina. The gingival epithelium can be divided into oral, sulcular, and junctional epithelia, based on their architecture. The sulcular epithelium, which extends from the oral epithelium to the gingival sulcus facing the teeth, and the junctional epithelium, which mediates the attachment of teeth to gingiva, are not keratinized, in contrast to the oral gingival epithelium. The sulcular and the coronal margins of the junctional epithelium are in close contact with bacteria in the gingival sulcus and appear to be crucial sites with regard to the development of periodontal diseases. During periodontitis, loss of connective tissue attachment and bone resorption associated with the formation of periodontal pockets is related to the pathologic conversion of the junctional and the sulcular epithelium to a pocket epithelium. Invasion of mammalian epithelial cells is an important strategy developed by pathogenic bacteria to evade the host immune system and cause tissue damage. Gingival epithelial cells are the primary physical barrier to infections by periodontopathogens in vivo. While the epithelium was previously thought to be passive, Dale (2002) proposed a new perspective, assigning an active role to the epithelium in the host response to bacterial infections. The epithelium reacts to bacterial challenges by signaling host responses and integrating innate and acquired immune responses. This review focuses on the current understanding of host epithelial cell-P. gingivalis interactions in the pathogenesis of periodontitis.

	(II) ADHESION TO EPITHELIAL CELLS

TOP ABSTRACT (I) INTRODUCTION (II) ADHESION TO EPITHELIAL... (III) INTERNALIZATION BY... (IV) EPITHELIAL CELL RESPONSES (V) CONCLUSIONS REFERENCES

 
There is a strong correlation in vivo between the number of bacteria attached to the periodontal epithelium and the severity of the inflammation (Vaahtoniemi et al., 1993). The capacity of P. gingivalis to attach to a variety of squamous human epithelial cell lines in vitro has been reported by several investigators. P. gingivalis adheres to human primary gingival epithelial cells (Isogai et al., 1988; Lamont et al., 1992; Weinberg et al., 1997; Yilmaz et al., 2002) as well as to epithelial cell lines, such as KB cells (epidermoid carcinoma) (Duncan et al., 1993; Sandros et al., 1993; Huard-Njoroge et al., 1997; Delcourt et al., 1998), HEp-2 cells (laryngeal origin) (Nakagawa et al., 2002a), HeLa cells (cervical carcinoma), and Ca9-22 cells (gingival carcinoma) (Watanabe et al., 1992; Hamada et al., 1994).

Adhesion of P. gingivalis to host cells is multimodal (Lamont and Jenkinson, 1998) and involves a variety of cell-surface and extracellular components, including fimbriae, proteases, hemagglutinins, and lipopolysaccharides (LPS) (Cutler et al., 1995). Among the large array of virulence factors produced by P. gingivalis, the major fimbriae (FimA), as well as cysteine proteinases (gingipains), contribute to the attachment to and invasion of oral epithelial cells via different receptors (Weinberg et al., 1997; T Chen et al., 2001). Adhesion and subsequent invasion of epithelial cells by P. gingivalis are likely critical in the pathogenesis of periodontitis, especially during the initial stages of infection.

Roles of FimA in Adhesion to Epithelial Cells
P. gingivalis major fimbriae FimA is considered a critical determinant for the colonization of the oral cavity by this microorganism. The major fimbriae FimA is composed of a subunit protein (fimbrillin) with a molecular mass ranging from 41 to 45 kDa, depending on the strain (Lee et al., 1991). The gene coding for fimbrillin (fimA) is present in a single copy in the chromosome and is monocistronic (Dickinson et al., 1988; Hamada et al., 1994). Amino acid sequence analysis has revealed no significant homology with fimbrial proteins from other bacteria, indicating that P. gingivalis produces a unique class of fimbriae (Dickinson et al., 1988). The fimA gene is present in all fimbriated strains of P. gingivalis so far examined and is absent in afimbriated strains (Holt et al., 1999). Several groups of investigators have provided clear evidence to support the key role of P. gingivalis major fimbriae FimA in adhesion to and invasion of many types of mammalian cells, including epithelial cells (Isogai et al., 1988; Njoroge et al., 1997; Sojar et al., 1999). FimA-deficient mutants of P. gingivalis have been constructed, and all have an attenuated capacity to adhere to and invade epithelial cells (Njoroge et al., 1997; Weinberg et al., 1997; Umemoto and Hamada, 2003). Invasive strains of P. gingivalis carrying a FimA mutation are non-invasive in a tissue culture invasion model and have a significantly reduced ability to cause disease in mice following oral inoculation (Malek et al., 1994). In addition, synthetic peptides analogous to the fimbrillin sequence (Lee et al., 1991) and antibodies directed against fimbriae significantly inhibit the capacity of P. gingivalis to adhere to and invade epithelial cells (Isogai et al., 1988; Njoroge et al., 1997; Dorn et al., 2000; Sojar et al., 2002). The allelic variations in fimA observed among strains of P. gingivalis result in fimbrial diversity in terms of the sizes and N-terminus amino acid sequences of the proteins (Dickinson et al., 1988). The terminal region corresponding to amino acid residues 49 to 90 of the fimbrillin protein has been identified as the potential epithelial cell-binding domain of P. gingivalis fimbriae (Sojar et al., 1999). fimA genes encoding fimbrillin (FimA) can be grouped into six variants (types I to V and Ib) on the basis of their nucleotide sequences (Hamada et al., 1994; Nakagawa et al., 2002b).

Functional differences in P. gingivalis FimA variants with regard to the adhesion to and invasion of human epithelial cells have been the focus of recent investigations. A type II FimA strain (HW24D1) was found to adhere to and invade significantly more epithelial cells than strains with the other known fimA genotypes (fimA types I, III, IV, and V) (Nakagawa et al., 2002a; Amano et al., 2004). Interestingly, recombinant type II FimA (rFimA) protein adheres to and is internalized by human epithelial HEp-2 cells more efficiently than other rFimA types and accumulates around the nucleus (Nakagawa et al., 2002a). The adhesion and internalization of P. gingivalis with type II FimA are inhibited by anti-FimA type II antibodies (Nakagawa et al., 2002a). In contrast, Dorn et al.(2000) did not observe any correlation between invasiveness and specific FimA type in P. gingivalis.

Electron microscopic analyses revealed that, while epithelial-cell-adhering strains of P. gingivalis have abundant peritrichous fimbriae on their surfaces, poorly adhering strains such as W50 and W83 possess very few fimbriae and are sparsely covered with short fimbriae-like structures, referred to as minor fimbriae (Watanabe et al., 1992). Like some of the naturally occurring non-adhering P. gingivalis strains, FimA-deficient mutants are devoid of classic fimbriae and produce short fimbriae-like structures that do not react with anti-FimA antibodies (Hamada et al., 1994; Hamada et al., 1996; Arai et al., 2000). Little research has been done on the role of P. gingivalis minor fimbriae in adhesion to epithelial cells. Recently, Umemoto and Hamada (2003) demonstrated the importance of the mfa1 gene, which codes for the minor 67-kDa fimbriae (Hamada et al., 1996), in binding to and invasion of gingival epithelial cells by P. gingivalis. Using the homologous recombination technique, they constructed fimA (MPG1), mfa1 (MPG67), and double-knock-out (MPG4167) mutants of strain ATCC 33277. Consistent with previous reports, these authors showed that FimA-deficient mutant MPG1 has a reduced ability to bind to epithelial cells. They also observed that inactivation of the mfa1 gene results in an increased binding ability of mutant MPG67 compared with the wild-type strain, suggesting that mfa1 gene mutation may cause changes in cell-surface properties. The mutant MPG67 was shown to exhibit numerous long fimbriae in its surface, and to adhere to human epithelial cells by forming larger clumps by auto-aggregation than did the wild-type strain. In contrast, the capacity of the double-knock-out mutant MPG4167 to adhere to gingival epithelial cells is completely abolished. In addition, all three mutants have a decreased ability to invade gingival epithelial cells, suggesting that both minor Mfa1 (67 kDa) and major FimA (41 kDa) proteins contribute to the ability of P. gingivalis to invade epithelial cells.

Integrins as Epithelial Cell Cognate Receptors for FimA
Integrins are a super-family of heterodimeric transmembrane molecules made up of diverse non-covalently-bound - and β-chains. The nature of the β-chains defines the family of integrins, and both - and β-chains contribute to the binding of ligands. 2β1, 3β1, 6β1, and 6β4 integrin subunits are expressed by gingival epithelial cells (Hormia et al., 1992; Del Castillo et al., 1996; Thorup et al., 1997). Integrins are involved in cell-extracellular matrix and cell-cell interactions and function as host cell receptors for microbial adhesins. For instance, 5β1 integrin can act as a receptor for the integrin-binding proteins of Yersinia spp., Shigella flexneri, Bordetella pertussis, and Pseudomonas aeruginosa (Watarai et al., 1996; Roger et al., 1999; Ishibashi et al., 2001). More attention is also being paid to the involvement of the target adhesin-receptors expressed on human epithelial cells in the attachment of P. gingivalis fimbriae.

Weinberg et al.(1997) first identified a 48-kDa surface protein on gingival epithelial cells that binds fimbriated P. gingivalis but not afimbriated strains. These authors suggested that the 48-kDa protein may function as a cognate fimbriae receptor and hypothesized that the interaction between fimbriae and this protein may be the first step in a signaling process that mediates the uptake of the bacteria into the host cells. Yilmaz et al.(2002) recently reported that there is a physical association between P. gingivalis rFimA protein and the β1 integrin and 5β1 integrin heterodimers expressed on gingival epithelial cells. Moreover, the adhesion of type II rFimA-coupled microspheres to HEp-2 cells and the adhesion of P. gingivalis cells (type I FimA) to gingival epithelial cells are significantly reduced by anti-5β1 integrin and anti-β1 integrin antibodies, respectively. The fact that binding inhibition is not completely abolished suggests that there are additional receptors for fimbriae. Nevertheless, antibodies against Vβ3 integrin as well as RDG (arginine-aspartic acid-glycine) peptide have a negligible effect on fimbriae adhesion to epithelial cells (Nakagawa et al., 2002b). A recent study pointed to the participation of host neuraminic acid and glucuronic acid in P. gingivalis adherence to KB oral cells (Agnani et al., 2003). The addition of either carbohydrate in a soluble form caused a significant decrease in P. gingivalis adhesion to KB cells. However, these authors did not identify cadherins, cellular adhesion molecules (CAM), or β1, β3, and V integrins as potential receptors that mediate P. gingivalis binding to epithelial cells. Carbohydrate chains on epithelial cell membrane glycolipids have been reported to act as receptors for P. gingivalis (Hellström et al., 2004). This study also identified a β1 integrin, independent of the RGD binding motif of integrin, as a cognate receptor that mediates P. gingivalis fimbriae attachment to epithelial cells.

Fimbriae have been implicated in P. gingivalis internalization by gingival epithelial cells (Weinberg et al., 1997). Numerous studies have revealed that ligand binding to integrins initiates a signal transduction cascade that coordinates and regulates a variety of cellular responses that induce the uptake of bacteria by host cells (Rankin et al., 1992; Rosenshine et al., 1992) (see "Internalization" section). Antibodies directed against β1 integrin 2 inhibit the invasion of gingival epithelial cells by P. gingivalis by up to 94% (Yilmaz et al., 2002). However, the internalization into gingival epithelial cells of a fimbriae-deficient mutant was not completely blocked by the anti-β1 integrin antibodies. The authors suggested that fimbriae-integrin interactions initiate one pathway that leads to P. gingivalis internalization, and that there may be other fimbriae-independent pathways that promote the uptake of bacteria.

Weinberg et al.(1997) reported that fimbriae bind to more than one epithelial cell receptor. Two major components with molecular masses of 50 kDa and 40 kDa bind with high affinity to P. gingivalis fimbriae (Sojar et al., 2002). The 50-kDa protein corresponding to cytokeratin was identified as an epithelial cell ligand for native fimbriae.

Roles of Gingipains in Adhesion to Epithelial Cells
P. gingivalis is an asaccharolytic bacterium that produces and releases a large array of proteolytic enzymes that play essential roles in the growth of this bacterial species. Among these enzymes, trypsin-like proteinases, called gingipains, have been purified and characterized, and their functions and pathological roles in periodontitis have been extensively investigated over the past decade (Potempa et al., 1995; Genco et al., 1999; Nakayama, 2003). Gingipains are responsible for most of the extracellular and cell-bound proteolytic activities produced by P. gingivalis. Three different genes code for arginine-X (Arg-gingipain A and B [rgpA and rgpB]- and lysine-X (Lys-gingipain [kgp])-specific cysteine proteinases, which occur in multiple forms due to proteolytic processing of the initial polypeptides (Potempa et al., 1995; Potempa and Travis, 1996).

Gingipains contribute to the virulence potential of P. gingivalis in a multifactorial way, especially by influencing the binding of the bacterium to host tissues. These proteinases may play a role in binding to host cells, either by binding to a cognate receptor or by exposing cryptitope receptors. P. gingivalis strains with high levels of trypsin-like protease activity (Arg-gingipain activity) adhere better to human epithelial cells than do strains with lower levels of such activity (Grenier, 1992). The mature forms of Arg-gingipain A and Lys-gingipain possess a catalytic domain and three or four hemagglutinin/adhesin (HA) domains (HA1 to HA4) linked by strong non-covalent bonds (Potempa et al., 1995; DeCarlo and Harber, 1997). The HA domains of Arg-gingipain A and Lys-gingipain share a high degree of homology (over 97%) and have been implicated in the adherence of P. gingivalis to gingival epithelial cells (T Chen et al., 2001; Chen and Duncan, 2004). Chen and Duncan (2004) provided additional evidence for the involvement of gingipain adhesin domains in the binding of P. gingivalis to epithelial cells. They showed that antibodies against the recombinant adhesin domain of Arg-gingipain block the attachment of native gingipain adhesins to epithelial cells (HEp-2) and inhibit the adherence of P. gingivalis to epithelial monolayers. Furthermore, Scragg et al.(2002) have suggested that the adhesin domain is involved in the nuclear targeting of P. gingivalis W50 proteinases in epithelial cells. More recently, Rautemaa et al.(2004) reported that the P. gingivalis thiol proteinase localizes near the perinuclear region in the cytoplasm of periodontal epithelial cells.

The catalytic domains of gingipains, and, more specifically, Arg-gingipains A and B, can modulate P. gingivalis binding to epithelial cells (T Chen et al., 2001). Chen et al. (T Chen et al., 2001) proposed that while the attachment of P. gingivalis to epithelial cells is mediated by Kgp and RgpA gingipain HA domains from Kgp and RgpA, detachment of bacterial cells is mediated by RgpA and RgpB catalytic activities. Gingipain catalytic activities may thus enhance the binding of P. gingivalis to host cells by a mechanism, previously described by Gibbons et al. (Gibbons, 1989; Gibbons et al., 1990), in which hidden segments of cell adhesion molecules, referred to as ’cryptitopes’, are exposed following enzymatic degradation of host matrix proteins.

Gingipains have been shown to play important physiological roles, more particularly in controlling the expression of virulence factors and the stability and/or processing of extracellular and cell-surface proteins (Kadowaki et al., 1998). Both subunits of the two types of fimbriae are regulated by proteolytic processing involving Rgp and Kgp. Rgp processes the precursor form of fimbrillin to the mature form FimA and is involved in fimbrial formation (Onoe et al., 1995; Xie et al., 2000). This is supported by the fact that a double rgpA/rgpB-deficient mutant possesses very few fimbriae on its cell surface (Nakayama et al., 1996; Weinberg et al., 1997).

Other Components Involved in Adhesion to Epithelial Cells
The binding of P. gingivalis to epithelial cells is a multimodal process involving several bacterial cell-surface structures that may act in concert to allow for binding to host cells. Chandad and Mouton (1995) and Du et al.(1997) provided evidence that HA-Ag2—which possesses antigenic, structural, and functional similarities with P. gingivalis fimbriae—may be a bacterial ligand involved in the binding of P. gingivalis to epithelial cells. Glycosyltransferase, which is coded for by the gtfA gene, also has a role in the binding of P. gingivalis to epithelial cells (HEp-2) (Narimatsu et al., 2004). A gtfA-deficient mutant without mature fimbriae had a reduced ability to auto-aggregate and attach to epithelial cells as well as several extracellular matrix proteins, including type I collagen, laminin, and fibronectin. However, the expression of FimA protein and mRNA in the mutant was not altered. From these observations, the authors suggested that the P. gingivalis GtfA is required for a sugar transfer reaction in fimbriae formation, and that GtfA plays an essential role in auto-aggregation and binding to epithelial cells. Consistent with these findings, Duncan et al.(1996) previously showed that the overexpression of an open reading frame (ORF) for a putative glycosyltransferase can enhance binding to the cells.

Capsular polysaccharides protect bacterial cells from the host immune system. However, the presence of a capsule may also interfere with the initial step of bacterial binding to epithelial cells. Recently, Dierickx et al.(2003) demonstrated that unencapsulated P. gingivalis strains adhere significantly more than do their encapsulated variants to epithelial cells from the periodontal pockets of patients with periodontitis. This observation was also reported for various human pathogens, including Klebsiella pneumoniae, Neisseria meningitidis, and Haemophilus influenzae (St Geme and Falkow, 1992; Virji et al., 1995; Sahly et al., 2000). The capsule of P. gingivalis, unlike its fimbriae, which make the cell surface hydrophobic (Watanabe et al., 1992), lowers the hydrophobicity of the bacterial surface (van Winkelhoff et al., 1993). This suggests that bacterial surface hydrophobicity contributes to their capacity to adhere to epithelial cells. Encapsulated strains of P. gingivalis that are virulent in a mouse model can be classified into six serogroups (K-antigen types; K1 to K6) based on their capsular antigens (Laine and van Winkelhoff, 1998). Correlations between FimA type and capsular antigen type have been established by Amano et al.(1999): K1 for type IV FimA; K2, K3, and K5 for type II FimA; K4 for type II FimA; and K6 for type Ib FimA. Interestingly, P. gingivalis strains belonging to the K4 serogroup and those with type II FimA both adhere significantly better to epithelial cells than do the other K-antigen and FimA types (Nakagawa et al., 2002a; Yilmaz et al., 2002; Dierickx et al., 2003).

	(III) INTERNALIZATION BY EPITHELIAL CELLS

TOP ABSTRACT (I) INTRODUCTION (II) ADHESION TO EPITHELIAL... (III) INTERNALIZATION BY... (IV) EPITHELIAL CELL RESPONSES (V) CONCLUSIONS REFERENCES

 
P. gingivalis can be internalized by primary cultures of gingival epithelial cells (Lamont et al., 1992, 1995), oral epithelial cell lines (Duncan et al., 1993; Sandros et al., 1993), and multilayered pocket epithelial cells (Sandros et al., 1994). P. gingivalis has also been observed in gingival epithelial cells in vivo (Noiri et al., 1997; Rudney et al., 2001). The binding of P. gingivalis to epithelial cells induces the formation of membrane invaginations that surround and engulf the bacteria (Lamont et al., 1992; Lamont and Jenkinson, 2000; Houalet-Jeanne et al., 2001). The invasive process occurs within 20 minutes, with large numbers of bacteria localized in the perinuclear region (Belton et al., 1999; Houalet-Jeanne et al., 2001; Park et al., 2004). Once inside the cells, P. gingivalis remains viable and is capable of multiplying (primary and KB cells) and surviving for prolonged periods (Papapanou et al., 1994; Lamont et al., 1995; Madianos et al., 1996; Houalet-Jeanne et al., 2001; Yilmaz et al., 2003). Recent studies have shed considerable light on the mechanisms involved in P. gingivalis-epithelial cell interactions. P. gingivalis has developed strategies to ensure survival in host cells and elicit host responses that result in tissue destruction. Invaded epithelial cells are thought to provide a protective environment for the microorganisms. The mechanisms P. gingivalis uses to internalize into host cells are similar to those described for invasive enteric pathogens (Lamont and Jenkinson, 1998). Many recent studies have focused on P. gingivalis’ interactions with and invasion of epithelial cells. We used a three-dimensional (3-D) engineered human oral mucosa model, in which epithelial cells interact with fibroblasts in the lamina propria, to demonstrate, for the first time, that P. gingivalis can migrate through the basement membrane and reach the underlying connective tissue (Fig. 1B), which is consistent with previous in vivo observations of P. gingivalis in periodontal connective tissue (Saglie et al., 1988; Andrian et al., 2004). Ultrastructural analyses showed that the infiltrating bacteria penetrate beneath the superficial cell layer and are internalized within multilayered gingival epithelium, as well as at the junction between the stratified epithelium and the lamina propria (Fig. 1A). No visible histological changes were observed in that junction in the 3-D model when a gingipain-null mutant was used, thus providing additional evidence for a critical role of gingipains in tissue destruction.

View larger version (77K): [in this window] [in a new window]   Figure 1. P. gingivalis invasion of a three-dimensional (3-D) engineered human oral mucoasa model. (A) Structural modifications to the 3-D engineered human oral mucosa model following a P. gingivalis ATCC 33277 infection. (a) Uninfected control model; (b) P. gingivalis ATCC 33277-infected model. Scale bars, 50 µm. (B) Transmission electron micrograph of P. gingivalis in a multilayer of epithelial cells and in the underlying connective tissue of a 3-D engineered human oral mucosa model. These Figs. are from Andrian et al.(2004), and are reprinted with permission.

The invasive process of pathogenic bacteria is frequently associated with signaling activities involving MAPKs. MAPKs are serine-threonine protein kinases that play a central role in transmitting the signals from a diverse group of extracellular stimuli to the nucleus, controlling many cell-signaling responses, including cell proliferation and differentiation, stress responses, apoptosis, and cell cycles (Robinson and Cobb, 1997). The MAPK superfamily includes the stress-activated protein kinase c-Jun N-terminal (JNK), the extracellular signal-regulated kinase (ERK), and the p38 MAP kinase (Robinson and Cobb, 1997). Phosphorylation of MAPKs results, in many cases, in subcellular translocation and subsequent activation of diverse substrate proteins, including transcription factors such as nuclear transcriptional factor (NF-B), other kinases, and cytoskeletal proteins. P. gingivalis specifically activates JNK and down-regulates the extracellular signal-regulated kinase ERK1/2 in human gingival epithelial cells, whereas p38 and NF-B are not activated (Watanabe et al., 2001). JNK activation is related to bacterial invasion, whereas the inhibition of ERK1/2 activity is likely mediated by internalized bacteria, a phenomenon that possibly prevents the activation of NF-B. Specific inhibitors of MEK1/2, which is the upstream regulator of ERK1/2 activation, do not affect the invasion rate of bacteria. Exposure of epithelial cells to Rho family guanosine-5'-triphosphatase (GTPase)-specific inhibitor toxin B does not prevent JNK phosphorylation, suggesting that stimulation of JNK may occur at a step subsequent to GTPase activation. These results indicate that internalization of P. gingivalis is independent of MEK/ERK1/2 signaling pathways. Other evidence points to the involvement of MAPK responses in the P. gingivalis invasion process, since bacteria rendered non-invasive by heat or sodium azide treatments (Lamont et al., 1995; Belton et al., 1999) do not disrupt MAPK responses.

Internalization of P. gingivalis is correlated with tyrosine phosphorylation of eukaryotic cells. Genistein, a tyrosine kinase inhibitor, strongly impairs P. gingivalis internalization by epithelial cells, suggesting the involvement of tyrosine phosphorylated proteins in signal transduction during invasion (Sandros et al., 1996; Watanabe et al., 2001). A 43-kDa eukaryotic cell protein corresponding to MAPK has been identified as a target for protein tyrosine phosphorylation (Sandros et al., 1996). P. gingivalis fimbriae interactions with its cognate receptor β1-integrin on the surfaces of gingival epithelial cells can initiate signal transduction that may lead to bacterial uptake by epithelial cells (Lamont et al., 1995; Belton et al., 1999; Yilmaz et al., 2002). When fimbriae bind to integrin, downstream signaling events—including the tyrosine phosphorylation of a 68-kDa focal adhesion signaling component (paxillin) and the activation of a focal adhesion tyrosine kinase (FAK)—have been observed (Yilmaz et al., 2002). Paxillin and FAK are believed to play an important role in an integrin-mediated signal transduction cascade that regulates adhesion, survival, proliferation, differentiation, and migration (Clark and Brugge, 1995). In mammalian cells, the phosphorylation of paxillin and FAK leads to the activation of other specific signaling molecules, thus promoting the assembly of focal adhesion complexes subsequent to integrin activation (Clark and Brugge, 1995). Immunofluorescence staining has been used to demonstrate that there is a significant recruitment of phosphorylated paxillin and FAK from the cytosol to the cell periphery to form focal adhesion complexes. Following prolonged exposure of epithelial cells to P. gingivalis, the focal adhesion complexes dissociate, and the paxillin and FAK are redistributed into the cytoplasm, mainly to the perinuclear area. This is correlated with the localization of bacterial cells in the perinuclear region (Yilmaz et al., 2003).

The formation of integrin-associated focal adhesion cytoskeletal proteins is associated with actin microfilament and microtubule cytoskeletal re-organization (Clark and Brugge, 1995). Increasing numbers of reports have highlighted the involvement of both actin microfilaments and microtubule cytoskeleton re-arrangements during the P. gingivalis invasion process, which may promote the invaginations of the membrane that bring the bacteria into the host cells (Lamont et al., 1995). P. gingivalis is unable to enter epithelial cells that have been treated with microtubule polymerization inhibitors (noco-dazole and colchicine) and a microfilament inhibitor (cytochalasin D) (Lamont et al., 1995). Immunofluoresence analyses have revealed that the invasion of P. gingivalis via integrin contact induces the nucleation of actin filaments, which form thin filamentous microspike-like structures and long stable filaments distributed throughout the cell. A significant disassembly and nucleation of the actin and microtubule filamentous network after extended periods of infection have also been noted (Yilmaz et al., 2003). Together, these results suggest that bacterial receptors and phosphotyrosine-dependent intracellular signaling trigger an internalization process involving a re-arrangement of the cytoskeleton.

Increased intracellular calcium concentrations in epithelial cells have been associated with invasion by P. gingivalis. Following contact with epithelial cells, P. gingivalis causes a transient increase in Ca²⁺ in the cells, resulting from the release of calcium ions from thapsigargin-sensitive intracellular stores (Izutsu et al., 1996). Belton et al.(2004) reported that P. gingivalis induces oscillations in nuclear and cytoplasmic spaces by activating a Ca²⁺ influx through Ca²⁺ channels in gingival epithelial cells. The fluctuation in cytosolic calcium ions, which are important mediators of eukaryotic signaling, may initiate a cascade of intracellular responses that mediate cytoskeletal remodeling. Yilmaz et al.(2002) suggested that the integrin receptor initiates one of the pathways by which cell signal transduction may activate cytoskeletal elements. The molecular signaling events that occur during the invasion of gingival epithelial cells by P. gingivalis are illustrated in Fig. 2.

View larger version (31K): [in this window] [in a new window]   Figure 2. Current model of P. gingivalis interactions with gingival epithelial cells. Interactions between P. gingivalis fimbriae, gingipains, and other potential adhesins with various epithelial cell-surface receptors (PAR-1/2, TLR2, integrins) lead to the activation of epithelial cell signaling pathways and the modulation of gene expression. The entry of P. gingivalis into epithelial cells is associated with the phosphorylation/dephosphorylation of signaling molecules such as MAP kinases, the modulation of calcium influx, and the re-arrangement of the cell cytoskeleton. Interactions of fimbriae with integrins initiate down-stream signaling events, including the formation of focal adhesion molecules such as FAK/paxillin. The intracellular localization of gingipains can interfere with the pathways of the focal adhesion molecules FAK/paxillin and MAP kinase. This model has been adapted from a model proposed by Lamont and Jenkinson (1998). See text for references. Abbreviations: Ca++, calcium; ERK, extracellular signal-regulated kinase; GTP, guanosine triphosphate; IkB, inhibitory factor; ILβ, interleukin; JNK, c-Jun N-terminal; MAPKKK, mitogen-activated protein kinase kinase kinase; MEK, extracellular signal-regulated kinase activator kinase; NF-B, nuclear transcriptional factor; PAR, protease-activated receptor; RAS, small-GTPase; and TLR, Toll-like receptor.

Expression of Virulence Factors by P. gingivalis during Entry into Epithelial Cells
A crucial part of bacterial pathogenicity involves the expression, in the host cell cytoplasm, of virulence factors that interfere with and alter host processes (Thanassi and Hultgren, 2000). When P. gingivalis enters into contact with epithelial cells, it secretes a novel set of proteins that may have intracellular effector activities (Park and Lamont, 1998). Park and Lamont (1998) identified, in P. gingivalis, a contact-dependent protein secretion pathway similar to that required for the translocation of proteins, one that mediates the entry of invasive enteric pathogens into host cells (Zierler and Galan, 1995). However, the P. gingivalis-dependent extracellular secretion pathway has not yet been characterized. The attachment of P. gingivalis to the epithelial cell surface leads to the secretion of FimA, homologs of a phosphoserine phosphatase, and polysaccharide biosynthetic enzymes (Park and Lamont, 1998; W Chen et al., 2001). In contrast, the secretion of gingipain cysteine proteinases is inhibited following brief contact with primary cultures of gingival epithelial cells, whereas prolonged contact with epithelial cells induces an increased secretion of Lys-gingipain (Park and Lamont, 1998; Agnani et al., 2000). Temporally, up-regulation of the expression of P. gingivalis genes, which are essential for maintaining cellular function and viability, is also induced following P. gingivalis contact with HEp-2 human epithelial cells (Hosogi and Duncan, 2005). Increased expression of genes involved in oxidative stress—including the superoxide dismutase (sod), alkyl hydroxide reductase (ahpCF), thioredoxin peroxidase (tpx), and thioredoxin reductase (trxB) genes, which are involved in the detoxification of reactive oxygen species (ROS) and peroxides—has been reported. Heat-shock genes involved in maintaining protein stabilization and cellular functions—including groEL, dnaK, and htpG—are also expressed by P. gingivalis.

Novel factors have been identified that play a role during P. gingivalis entry into gingival epithelial cells. These include a metallo-endopeptidase (PepO), a cation-transporting ATPase, and an ATP-binding cassette (ABC) transporter (Ansai et al., 2003; Park et al., 2004). The independent inactivation of each gene has revealed that most mutants display an indistinct microvillus formation of the actin cytoskeleton and are poorly internalized compared with parent strains (Park et al., 2004). Park et al.(2004) have suggested that internalization-defective mutants may not induce the formation of actin stress fibers, suggesting a role for these proteins in the induction of host cytoskeletal responses. Once inside the epithelial cell, P. gingivalis releases outer membrane vesicles (Sandros et al., 1994; Houalet-Jeanne et al., 2001). The proteolytic activity of these extracellular structures may be responsible for the degradation of host proteins.

	(IV) EPITHELIAL CELL RESPONSES

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In addition to providing a physical barrier against invading pathogens, epithelial cells play an important role in innate host immune defenses. Interactions between P. gingivalis and epithelial cells lead to the activation of several complex signaling cascades, which ultimately regulate the transcription of target genes that encode effectors and regulators of the immune response. Effectors of the innate immune system, pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), and antimicrobial peptides are up-regulated and may have a direct impact on disease progression and the inflammation processes.

Cell-surface Modifications and Apoptosis
While gingival epithelial cells containing internalized P. gingivalis exhibit morphological changes such as cell rounding and detachment from the substratum (Lamont et al., 1992; Belton et al., 1999), they do not undergo apoptosis and maintain their physiological integrity for extended periods (Nakhjiri et al., 2001). P. gingivalis gingipains have been implicated in morphological changes to gingival epithelial cells (Johansson and Kalfas, 1998) through the degradation of cell adhesion molecules, including occludin, E-cadherin, beta-1 integrin (Katz et al., 2000), ICAM-1, vascular cell adhesion molecule-1, and very late antigen-1 (Wang et al., 1999; Tada et al., 2003). We have demonstrated that P. gingivalis LPS can potentiate syndecan-1 shedding from the gingival epithelial cell surface by exploiting host shedding signaling pathways. We have also showed that gingipains contribute to the release of syndecan-1 from the gingival epithelial cell surface (Andrian et al., 2006). By shedding the syndecan-1 ectodomain, P. gingivalis may modulate the activation of host effectors and disrupt cell-cell interactions mediated by syndecan-1 (Andrian et al., 2005). P. gingivalis blocks camphotecin-mediated apoptosis of epithelial cells, up-regulates anti-apoptotic molecule Bcl-2 expression, and down-regulates pro-apoptotic molecule Bax expression (Nakhjiri et al., 2001). Unlike what has been observed with primary gingival epithelial cells, P. gingivalis gingipains induce cell detachment and apoptosis in KB oral cells through the cleavage of N-cadherin and β1-integrin (Z Chen et al., 2001). While the signaling pathways mediated by gingipains are not fully understood, β-integrin may be involved in inducing apoptosis (Yilmaz et al., 2002; Sheets et al., 2005). β-integrin is expressed on gingival and junctional epithelial cells (Hormia et al., 1990) and acts as an adhesin receptor for fimbriae (Yilmaz et al., 2002). Integrins are associated with numerous survival pathways, including those leading to apoptosis (Matter and Ruoslahti, 2001).

Membrane-bound mucin 1 (MUC1) is a component of the non-immune host defense system and mediates the attachment of bacteria to host cells (Lillehoj et al., 2001). MUC1 activates anti-apoptotic pathways in rat fibroblasts (Raina et al., 2004). MUC1 is ubiquitously expressed on oral epithelial surfaces and may provide protection against bacterial infections (Offner and Troxler, 2000). An increased expression of MUC1 has been observed in KB oral cells stimulated with P. gingivalis whole cells, but not with P. gingivalis LPS (Li et al., 2003). Li et al.(2003) reported that pro-inflammatory cytokines—including IL-1β, IL-6, TNF-alpha, and IFN-gamma—up-regulate MUC1 expression, leading to an over-expression of MUC1 on the cell surface. These results suggest that MUC1 plays a role in host defenses. However, the mechanism by which MUC1 participates in host defenses and in the maintenance of cell integrity is not known.

Cytokine Production
P. gingivalis induces a strong pro-inflammatory cytokine response in gingival epithelial cells in vitro, which is correlated with the adhesive/invasive potential of P. gingivalis (Njoroge et al., 1997; Sandros et al., 2000). Kesavalu et al.(2002) reported that P. gingivalis induces pro-inflammatory cytokine expression in an in vivo murine calvarial model. They showed that P. gingivalis induces different levels of cytokine expression, the highest being TNF-, followed by IL-1β and IL-6. Sandros et al.(2000) reported that the binding of P. gingivalis to the surfaces of epithelial cells results in an increased secretion of IL-1, IL-8, IL-6, and TNF-. P. gingivalis fimbriae and LPS can also up-regulate IL-1β, IL-6, IL-8, TNF-, and monocyte chemoattractant protein-1 (MCP-1) gene expression and protein synthesis in gingival epithelial cell lines (Njoroge et al., 1997; Sandros et al., 2000). Fimbriae use Toll-like receptor 2 (TLR2), which is predominantly expressed in human gingival epithelial cells, as a co-receptor to induce cell activation and IL-8 expression (Asai et al., 2001). LPS mediates cytokine release in many cell lines, including monocytes and gingival fibroblasts (Wilson et al., 1996), and acts as an agonist and antagonist of p38 MAPK activation (Darveau et al., 2002). However, LPS-mediated receptor activation on epithelial cells remains to be better-characterized. Kusumoto et al.(2004) have reported that P. gingivalis component(s) distinct from fimbriae and LPS can induce IL-8 and MCP-1 production through the activation of TLR2 and NF-B in human gingival epithelial cells. They suggested that polysaccharidic components have a role in this induction.

While pro-inflammatory chemokine IL-8 is up-regulated in oral and gingival epithelial cells following challenge with several periodontopathogens—including A. actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, and Prevotella intermedia (Yumoto et al., 1999; Han et al., 2000)—P. gingivalis inhibits IL-8 expression and secretion by gingival epithelial cells following an extended period of infection (Madianos et al., 1997; Darveau et al., 1998; Huang et al., 1998, 2001, 2004). This may inhibit neutrophil transepithelial migration and accumulation in infection sites (Madianos et al., 1997), thereby allowing P. gingivalis to escape host defense mechanisms and survive for long periods in periodontal tissue. Unlike the IL-1β response, which is strongly correlated with the adhesive and invasive potential of P. gingivalis, IL-8 up/down-regulation is independent of the invasive property (Huang et al., 2001). P. gingivalis may inhibit IL-8 accumulation at two levels: (i) IL-8 degradation by proteinases, and (ii) IL-8 regulation by unidentified factor(s) (Darveau et al., 1998; Mikolajczyk-Pawlinska et al., 1998; Zhang et al., 1999; Huang et al., 2001). The regulation of IL-8 expression is dependent on the activation of the NF-B, MAPK p38, and MEK/ERK pathways (Huang et al., 2004). Pre-treatment of P. gingivalis with heat or proteases enhances IL-8 mRNA induction, suggesting that proteinaceous components are involved in IL-8 gene regulation. Fimbriae activate NF-B and up-regulate IL-8 expression via TLR2 (Asai et al., 2001). While Kusumoto et al.(2004) reported that P. gingivalis components induce IL-8 up-regulation and NF-B activation via TLR2, proteinase and heat treatments are ineffective in preventing the induction. In contrast, down-regulation of IL-8 mRNA by viable P. gingivalis involves the MEK/ERK, but not the NF-B or MAPK p38 pathway (Huang et al., 2004). Huang et al.(2004) suggested that the up-/down-regulation of IL-8 may involve MEK/ERK pathways that may be regulated by different factors. P. gingivalis cysteine proteinases may disrupt multi-signaling pathways, including those leading to the activation of MAPKs and NF-B, as has been reported for the Yersinia YopJ protein (Palmer et al., 1999), suggesting that gingipains may be responsible for the induction of MEK/ERK regulation (Watanabe et al., 2001). P. gingivalis gingipains mediate IL-8 up-regulation in gingival epithelial cells and cause proteolysis of focal adhesion molecules such as paxillin and FAK (Hintermann et al., 2002; Chung et al., 2004).

P. gingivalis gingipains may play a pivotal role in the evasion of host defenses by disrupting cytokine signaling networks. Gingipains cleave and degrade most pro-inflammatory cytokines, including IL-1β (Fletcher et al., 1997), IL-6 (Banbula et al., 1999), TNF- (Calkins et al., 1998), and IL-8 (Mikolajczyk-Pawlinska et al., 1998; Zhang et al., 1999). Interestingly, RgpB activates the protease-activated receptors (PAR) PAR-1 and PAR-2 on the KB cell surface and induces an increase in intracellular calcium levels, resulting in an up-regulation of IL-6 secretion (Lourbakos et al., 2001). Moreover, gingipains can inactivate the effector molecules of the innate and acquired immune systems (Lamont and Jenkinson, 1998; Imamura, 2003) and therefore contribute to the progression of periodontal diseases.

Similarly, P. gingivalis down-regulates the expression of intercellular adhesion molecule-1 (ICAM-1) by gingival epithelial cells and degrades secreted ICAM-1 (Huang et al., 2001). Both IL-8 and ICAM-1 are responsible for the accumulation and activation of neutrophils in the epithelium (Sugiyama et al., 2002). P. gingivalis also down-regulates the expression of 4 genes related to host innate immunity—including IL-1β, IL-8, macrophage protein-alpha 2, and migration inhibitory factor-related protein-14—in gingival epithelial cells (Huang et al., 2004).

Antimicrobial Peptide Production
Antimicrobial peptides have emerged as potential participants in host defenses at mucosal surfaces. Antimicrobial peptides are small, endogenous, polycationic molecules that constitute a ubiquitous and significant component of innate immunity. Most of these peptides exert their antimicrobial activity by interacting with the bacterial cell membrane, leading to the disorganization of the bilayer and resulting in pore formation (Brogden, 2005). Among the antimicrobial peptides expressed by gingival epithelial cells, calprotectin and β-defensin have been reported to provide protection against P. gingivalis infections. Elevated calprotectin levels have been detected in gingival crevicular fluid (GCF) from patients with periodontitis (Nakamura et al., 2000). The expression of calprotectin by gingival epithelial cells enhances resistance to P. gingivalis infections and is correlated with reduced invasion and binding of P. gingivalis to epithelial cells (Nisapakultorn et al., 2001). Calprotectin is a cytosolic calcium-binding protein with broad-spectrum antimicrobial activity (Steinbakk et al., 1990). Calprotectin may kill or inhibit P. gingivalis growth within epithelial cells, or may interfere with the internalization process. The antimicrobial peptide human β-defensin (hBD), which is found primarily in association with gingival epithelial surfaces, may restrict intracellular bacterial replication and prevent the physical destruction of host cells. Three human β-defensins—hBD-1, hBD-2, and hBD-3—are expressed in gingival epithelial cells. While hBD-1 expression is constitutive in human epithelial cell, hBD-2 and hBD-3 expression is modulated by pro-inflammatory mediators or bacterial products (Krisanaprakornkit et al., 1998; Dale et al., 2001; Diamond et al., 2001). Exposure of primary cultures of gingival epithelial cells to P. gingivalis increases the expression of hBD-2 (Chung et al., 2004). Gingipains are directly involved in the regulation of hBD-2 in cultured gingival epithelial cells (Chung et al., 2004). hBD-2 expression uses several signaling pathways. Gingipains can activate one pathway through the PAR-2 receptor host-signaling pathway, which is dependent on phospholipase C and calcium influx (Krisanaprakornkit et al., 2003; Chung et al., 2004). The regulation of hBD-2 expression also involves the MAPK and NF-B signaling pathways (Krisanaprakornkit et al., 2002). Neither LPS nor fimbriae appear to be involved in hBD-2 induction by gingival epithelial cells (Chung et al., 2004).

Modulation of MMP Secretion
P. gingivalis can advance into deeper epithelial layers (Papapanou et al., 1994) via a paracellular pathway through the degradation of epithelial cell-cell junctional proteins (Katz et al., 2000). However, the intercellular spread of P. gingivalis has not been observed. P. gingivalis also penetrates gingival tissue in vitro and in vivo to initiate tissue destruction (Saglie et al., 1988). P. gingivalis degrades basement membrane proteins in a reconstituted basement membrane model, which indicates that it may be able to penetrate the connective tissue (Andrian et al., 2004). The capacity of P. gingivalis to breach epithelial cell integrity may allow it to migrate into deeper tissues, which, in turn, may lead to tissue destruction mediated by both bacterial and host proteinases. Stimulated gingival epithelial cells can produce different proteolytic enzymes that contribute to the degradation of intracellular and extracellular host proteins. MMPs, which are zinc-dependent neutral proteinases, are implicated in tissue remodeling and cell migration during the normal turnover of periodontal tissue, and may also be involved in the pathophysiology of periodontitis (Uitto et al., 2003). There is a positive correlation between the presence of high levels of specific MMPs (MMP-1, -8, -9, -13) and the severity of periodontitis (Tervahartiala et al., 2000). Cultured gingival epithelial cells can produce collagenase and gelatinase activities, including collagenase-1 (MMP-1), collagenase-3 (MMP-13), gelatinase A (MMP-2), gelatinase B (MMP-9), and matrilysin (MMP-7), as well as chymotrypsin-like activities (Uitto et al., 2003). Epithelial cells also express MMPs in vivo (Tervahartiala et al., 2000; Uitto et al., 2003). These enzymes have the potential to play an important role in the tissue destruction observed in periodontitis, since they can degrade extracellular matrix proteins, including collagen types I, III, and IV, fibronectin, tenascin, elastin, entactin, and proteoglycans. MMP secretion can be induced in cultured gingival epithelial cells by several cytokines as well as by bacterial components such as lipopolysaccharide and phospholipase C (Birkedal-Hansen et al., 1993; Ding et al., 1995; Sorsa et al., 2004). The regulation of MMP-9 production by gingival epithelial cells is disrupted following contact with P. gingivalis (Fravalo et al., 1996; DeCarlo et al., 1997, 1998), a phenomenon that may interfere with extracellular matrix repair and re-organization (Grayson et al., 2003). Purified gingipain proteinases of P. gingivalis up-regulate MMP-8 and MMP-3 expression in rat mucosal epithelial cells (DeCarlo et al., 1998) and activate latent forms of MMPs such as MMP-1, -3, and -9 (DeCarlo et al., 1997).

	(V) CONCLUSIONS

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The outcome of the molecular cross-talk between bacteria and host cells has major implications for health and disease. P. gingivalis has developed adaptive strategies to invade gingival epithelial cells and overcome the protective defense mechanisms of epithelial cells. P. gingivalis adheres to and invades epithelial cells by targeting specific host receptors, modulating host signaling events, and deregulating the host cytokine network. P. gingivalis-epithelial cell interactions result in the disruption of tissue homeostasis and the structural and functional integrity of gingival epithelial cells, which may contribute to bacterial persistence and the progression of chronic manifestations of periodontal diseases. Future studies are now focusing on understanding the signaling events that culminate in the epithelial invasion processes of P. gingivalis. A 3-D mucosal model has enabled us to study the interactions between epithelial cells and the underlying connective tissue during P. gingivalis infections. We and other groups have used human gingival fibroblasts/polymorphonuclear leukocytes and epithelial cell/macrophage cell co-culture models to gain a more comprehensive view of the regulation of local immune mechanisms (Nemoto et al., 2000; Bodet et al., 2005). Further studies involving microbial consortia and cell co-culture models will lead to a better understanding of the complexity of the pathogenic process of mixed infections such as periodontitis.

	ACKNOWLEDGMENTS

 
This work was supported by the Canadian Institutes of Health Research.

Received for publication June 16, 2005. Accepted for publication November 8, 2005.

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Journal of Dental Research, Vol. 85, No. 5, 392-403 (2006)
DOI: 10.1177/154405910608500502


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