P orphyromonas gingivalis L ipopolysaccharide S ignaling in G ingival F ibroblasts

Abstract

Periodontal disease is the major cause of adult tooth loss and is commonly characterized by a chronic inflammation caused by infection of oral bacteria. Porphyromonas gingivalis (P. gingivalis) is one of the suspected periodontopathic bacteria and is frequently isolated from the periodontal pockets of patients with chronic periodontal disease. The lipopolysaccharide (LPS) of P. gingivalis is a key factor in the development of periodontitis. Gingival fibroblasts, which are the major constituents of gingival connective tissue, may directly interact with bacteria and bacterial products, including LPS, in periodontitis lesions. It is suggested that gingival fibroblasts play an important role in the host responses to LPS in periodontal disease. P. gingivalis LPS enhances the production of inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) in gingival fibroblasts. However, the receptor that binds with P. gingivalis LPS on gingival fibroblasts remained unknown for many years. Recently, it was demonstrated that P. gingivalis LPS binds to gingival fibroblasts. It was also found that gingival fibroblasts express CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation primary response gene 88 (MyD88). P. gingivalis LPS treatment of gingival fibroblasts activates several intracellular proteins, including protein tyrosine kinases, and up-regulates the expression of monocyte chemoattractant protein-1 (MCP-1), extracellular signal-regulated kinase 1 (ERK1), and signal-regulated kinase 2 (ERK2), IL-1 receptor-associated kinase (IRAK), nuclear factor-κB (NF-κB), and activating protein-1 (AP-1). These results suggest that the binding of P. gingivalis LPS to CD14 and TLR4 on gingival fibroblasts activates various second-messenger systems. In this article, we review recent findings on the signaling pathways induced by the binding of P. gingivalis LPS to CD14 and Toll-like receptors (TLRs) in gingival fibroblasts.

Introduction

Periodontal disease consists of a group of infections that lead to inflammation of the gingiva and destruction of periodontal tissues and, in severe cases, are accompanied by the loss of alveolar bone with eventual exfoliation of the teeth (Haffajee and Socransky, 1994). The oral cavity is home to a menagerie of bacterial species. Over 300 different bacterial species have been cultivated from human subgingival plaque samples (Moore and Moore, 1994). Only some of these bacteria, either alone or in combination, have periodontopathic potential and can initiate periodontal diseases when a critical concentration is reached. Overgrowth of periodontal pathogens may result from a deficiency in the host defense system or modification of the subgingival environment (Marsh, 1989).

Some Gram-negative rods have been called “periodontal-disease-associated bacteria”, and they have received much attention (Slots and Genco, 1984; Dzink et al., 1985; Moore and Moore, 1994). Porphyromonas gingivalis (P. gingivalis), an oral black-pigmented Gram-negative bacterium, is mostly found in deep periodontal pockets and especially in sites with active disease (van Winkelhoff et al., 1988). Since Genco's group (Mouton et al., 1981) first reported that patients with adult periodontitis had a higher level of immunoglobulin G (IgG) antibodies against P. gingivalis than control individuals, many investigators have reported that patients with periodontitis have elevated antibody levels against sonicates of P. gingivalis in the serum and gingival crevicular fluid. Furthermore, Holt et al. (1988) demonstrated that P. gingivalis is a pathogenic bacterium of periodontal disease in monkeys.

P. gingivalis possesses bioactive materials such as cytoplasmic membranes, peptidoglycans, outer membrane proteins, lipopolysaccharide (LPS), capsules, and fimbriae on their cell surface (Offenbacher, 1996). These materials induce excessive production of cytokines and may modulate the cytokine network in periodontal tissues (Genco and Slots, 1984). Several cytokines are involved in inflammatory as well as immunological responses, and are designated as inflammatory cytokines. P. gingivalis LPS has been considered to be an important pathogenic component in the initiation and development of periodontal disease (Hamada et al., 1994; Tobias et al., 1997), because bacterial LPS is known to be a potent simulator of inflammatory cytokine production and bone resorption. One target of LPS is the gingival fibroblasts, which play an important role in the remodeling of periodontal soft tissues. Gingival fibroblasts may also function as a regulator of the cytokine network in periodontal tissues, because they produce several kinds of cytokines when stimulated by inflammatory cytokines or bacterial cell components (Takada et al., 1991b). Furthermore, at the tissue level, disease state is a result of the inflammatory reaction and immune response toward periodontopathic bacteria. Inflammatory cytokines have been extensively studied, and their presence in the periodontal tissues of periodontitis patients and their etiological correlation with periodontitis have been well-demonstrated (Takada et al., 1991b; Murakami and Okada, 1998). Recent interesting studies have demonstrated that CD14 (Wright et al., 1990; Dentener et al., 1993; Ziegler-Heitbrock and Ulevitch, 1993; Haziot et al., 1996) and Toll-like receptors (TLRs) (Medzhitov et al., 1997; Chaudhary et al., 1998; Rock et al., 1998), a class of LPS receptors, play important roles in the signal transduction in LPS-responsive cells. These results suggest that LPS may be involved in inflammatory reactions in various tissues via CD14 and/or Toll-like receptors. Therefore, it is important to elucidate whether human gingival fibroblasts express LPS receptors on their cell surface. In this review paper, we summarize recent findings on the LPS receptors on gingival fibroblasts and P. gingivalis LPS signaling.

Cytokine Network in Gingival Fibroblasts Induced by Porphyromonas gingivalis Lipopolysaccharide

(1) The lipopolysaccharide (LPS) of Porphyromonas gingivalis

Endotoxins and LPS of Gram-negative bacteria are well-known initiators of inflammation at both the local and systemic levels (Rietshelet al., 1994). Although the endotoxins of Gram-negative bacteria comprise a diverse group of substances, there are certain conserved components in their protein structures. Fig. 1A shows a diagram of the cell wall of a Gram-negative bacterium (Rietschel and Brade, 1992). The LPS molecules are located on the exterior face of the outer membrane. The most conserved portion of the protein structure is the lipid A. The lipid A moiety is typically strongly conserved within a bacterial genus, although there is often heterogeneity in the number of secondary fatty acids present. The lipid A is linked through the core region to the O-specific side-chain, which is typically heterogeneous in length and quite variable in structure from one bacterial strain to the next, and provides most, if not all, of a bacterium's antigenic signature. A study with chemically synthesized lipid A showed that the endotoxic activity of LPS is derived from the lipid A moiety (Rietschel et al., 1994). The structure of Gram-negative bacterial LPS and lipid A itself includes a group of hydrophobic fatty acid tails and, at the minimum, a hydrophilic head group of phosphorylated sugars. Consequently, LPS should have some amphipathic character. Lipid A and LPS typically strongly aggregate in solution (Brandenburg et al., 1993). A sub-stoichiometric number of ethanolamine residues provide convenient sites for derivation.

Many components of P. gingivalis have been reported to be antigenic (Ishikawa et al., 1997; Wang et al., 1998, 1999b, e,g). The sera of patients with periodontitis are positive for antibodies against various structural components of P. gingivalis, including the outer membrane protein, capsule, and fimbriae, and for antibodies against biologically active bacterial products, including LPS, hemagglutinin, and trypsin-like protease. LPS is absorbed into the root surfaces and gingival tissues of patients with periodontal disease.

The LPS of P. gingivalis differs from that of other Gram-negative bacteria in that the protein structure of P. gingivalis LPS lacks heptose and 2-keto-3-deoxyoctonate, and P. gingivalis LPS shows very little endotoxic activity in classic endotoxin assays, although it is significantly mitogenic (Mayrand and Holt, 1988). In general, the endotoxic activity of P. gingivalis LPS is very low compared with that of the LPSs isolated from enterobacteria (Ogawa, 1994). On the other hand, other reports suggested that P. gingivalis LPS is a potent inducer of various biological responses, such as bone resorption, polyclonal B-cell activation, inhibition of bone formation, and fibroblast proliferation (Mayrand and Holt, 1988; Oido et al., 1999). Other studies have investigated the activation of monocytes-macrophages by P. gingivalis LPS. Macrophages treated with P. gingivalis LPS secrete smaller amounts of tumor necrosis factor alpha (TNF-α) and prostaglandin E₂ than do macrophages treated with standard LPS preparations (Ogawa, 1994). In addition, other studies have reported that P. gingivalis LPS may induce monocytes-macrophages to secrete TNF-α (Shapira et al., 1994, 1998). On the other hand, one study reported that P. gingivalis LPS cannot induce the expression of adhesion molecules (Reife et al., 1995), while other studies reported that it can induce local tissue necrosis (Amar et al., 1996; Shapira et al., 1996). From these reports, it is reasonable to hypothesize that the potency of LPS preparations from P. gingivalis in inducing a biological response depends on the nature of the tested response, the strain of P. gingivalis used, and, possibly, the method of LPS preparation.

Two research groups (Ogawa, 1993; Kumada et al., 1995) demonstrated that purified P. gingivalis lipid A exhibits a phosphorylation and acylation pattern different from that of enterobacterial lipid As. Interestingly, they found that the structure of P. gingivalis lipid A has the same pattern at the beta (1-6)-linked glycocyamine disaccharide as the enterobacterial lipid As, but that the acyl group is variable. Since the structure of lipid A is heterogeneous (Brandenburg et al., 1993), it is believed that there are no contradictions in the above reports. Furthermore, it has been reported that a chemically synthesized lipid A of P. gingivalis, like natural lipid A, possesses very low endotoxicity, in contrast to Escherichia coli type (E. coli) synthetic lipid A (Ogawa et al., 2000). P. gingivalis lipid A has a structure distinctly different from that of enterobacterial lipid As. Namely, it has been reported that there is no 4-O-phosphoryl group in the lipid A backbone of Bacteroides fragilis (Hofstad et al., 1993) and B. intermedius (Johne et al., 1988). In addition, P. gingivalis LPS and its lipid A caused agglutination of rabbit erythrocytes (Ogawa, 1994). In contrast, other groups have reported that P. gingivalis LPS has no hemagglutinating activity (Kirikae et al., 1986). These findings suggest that P. gingivalis LPS and lipid A possess unique chemical structures. Interestingly, it was demonstrated that natural lipid A induced mitogenic responses in C3H/HeJ, a cell line that has a low response to LPS (Ogawa, 1994), and activated peritoneal macrophages and gingival fibroblasts of LPS-hyporesponsive C3H/HeJ mice (Ogawa et al., 2000). Thus, P. gingivalis LPS, which is unique due to its endotoxic activities, is a key factor in the development of periodontitis.

(2) Gingival fibroblasts as immunocompetent cells

The most common cell in the periodontal connective tissue is the fibroblast (Hassell, 1993). The role of this cell is to produce structural connective tissue proteins such as collagen and elastin, as well as glycoproteins and glycosaminoglycans that comprise the ground substance in periodontal connective tissue. Normally, periodontal fibroblasts produce and modify the extracellular matrix and play a role in maintaining tissue integrity and homeostasis (Hefti, 1993). Fibroblasts are capable of phagocytosing foreign objects and ingesting cross-linked collagen and play a critical role in the wound-healing process. Thus, gingival fibroblasts are the most abundant cells in periodontal tissue and are responsible for the synthesis and degradation of connective tissue. These cells secrete a variety of immunoregulatory cytokines and chemical mediators (Murakami and Okada, 1998). Several lines of evidence have revealed that cytokines play important roles not only in tissue homeostasis, but also in the pathogenesis of many infectious diseases. Cytokines play a primary role in tissue homeostasis and are constitutively expressed by resident cells composing the tissue. Upon stimulation with physiological and pathological stimuli in vitro, gingival fibroblasts secreted a variety of cytokines and chemical mediators (Murakami and Okada, 1998). On the other hand, in diseased states, cytokines may be secreted not only by resident cells, but also by locally infiltrating immunocompetent cells. In infectious diseases, invasion of the host tissue by bacteria or their products frequently induces a wide variety of inflammatory and immunopathologic reactions. Namely, both microbial factors and the host immune system have been implicated in the etiology of the chronic oral inflammatory disease, periodontitis. P. gingivalis LPS stimulates host cells including macrophages and fibroblasts to produce cytokines (Takada et al., 1991b). In vitro studies have shown that these cytokines, in turn, directly or indirectly activate the host cells involved in the immune-inflammatory processes.

Gingival fibroblasts respond to stimulants such as LPS, interleukin (IL)-1, IL-6, IL-8 and TNF-α. For example, microbial factors such as LPS and cytokines such as IL-1 and TNF-α induce IL-6 mRNA expression in fibroblasts (Agarwal et al., 1995). It has been reported that the effects of IL-1 and TNF-α are more involved in the regulation of IL-6 by fibroblasts rather than the direct effects of bacterial LPS (Kent et al., 1998). It has also been reported that the gingival fibroblasts isolated from diseased tissue produced a larger amount of IL-6, both constitutively and after induction, than those isolated from healthy tissue (Kent et al., 1999). It has also been suggested that one potential function of antibodies in the local gingival environment is to modify the ability of P. gingivalis or to stimulate a potentially destructive pro-inflammatory response such as the production of IL-1β and IL-6, or to interfere with the ability of P. gingivalis to negate normal host mechanisms (Steffen et al., 2000). Under inflammatory conditions, gingival fibroblasts contributed to the inactivation of circulating TNF-α through the preferential induction and shedding of TNF receptor (Ohe et al., 2000). Gingival fibroblast-lymphocyte interactions up-regulate the cytokine network in inflammatory gingival tissues (Murakami and Okada, 1997; Nemoto et al., 2000). On the genetic level, P. gingivalis DNA and its palindromic internal CpG motifs stimulate IL-6 expression in gingival fibroblasts by stimulating NF-κB binding, and this bacterial DNA may function as a virulence factor of the organism in periodontal disease (Takeshita et al., 1999). Interestingly, cultured gingival fibroblasts that are stimulated with LPS from Porphyromonas species produce IL-1, IL-6, and IL-8 (Takada et al., 1991a; Tamura et al., 1992; Sakuta et al., 1998). In addition, the lipid-A-associated proteins (LAPs) from P. gingivalis stimulate the production of IL-6 in gingival fibroblasts (Shapira et al., 1998). We demonstrated, in vitro, that upon stimulation with P. gingivalis LPS, gingival fibroblasts produced cytokines such as IL-6, which in turn activated osteoclasts (Wang et al., 1999f). We also found that IL-10 inhibited the production of IL-6 in P. gingivalis LPS-stimulated gingival fibroblasts (Wang et al., 1999d). From these findings, it is reasonable to speculate that gingival fibroblasts are one type of immunoregulatory cell in periodontal tissue.

Putative periodontopathic microbiota such as P. gingivalis are capable of not only directly attacking and destroying the host tissue, but also communicating with host cells such as gingival cells and stimulating them to produce host inflammatory mediators and cytokines. These cytokines are multifunctional and exert their effects in a paracrine and autocrine fashion to modulate inflammatory and immune responses of gingival fibroblasts. A possible scenario would be that LPS-stimulated gingival fibroblasts are regulated by a network of inflammatory cytokines. These findings suggest a cascade characterized by localized chronic inflammation that accompanies bone resorption.

Porphyromonas gingivalis Lipopolysaccharide Receptors on Gingival Fibroblasts

(1) The role of membrane CD14 (mCD14) and soluble CD14 (sCD14)

The CD14 molecule, which is primarily expressed on macrophages, was reported to bind with LPS and mediate LPS-induced cell activation (Wright et al., 1990; Ulevitch and Tobias, 1995). CD14 was the first protein to be identified as an LPS receptor for initial bacterial recognition (Wright et al., 1990). Many lipid-containing molecules—including LPS, microbial lipoproteins, walls of Streptococcal molecules, and Tuberculosis lipoarabinomannan—can bind with CD14 (Pugin et al., 1994). Two forms of the CD14 molecule have been identified. One is the glycosylphosphatidylinositol (GPI)-anchored membrane CD14 (mCD14), and the other is the soluble form of CD14 (sCD14), which lacks the GPI structure (Bazil et al., 1989). Furthermore, LPS molecules bind via their lipid A moiety to an LPS-binding protein (LBP), a glycoprotein found in normal and acute-phase serum, and this greatly enhances the sensitivity of monocytes/macrophages and neutrophils to LPS (Corradin et al., 1992; Hailman et al., 1994). The CD14 expressed on the surfaces of monocytes/macrophages and neutrophils functions primarily as a receptor for the complex of LPS and LBP (Wright et al., 1990; Dentener et al., 1993; Ziegler-Heitbrock and Ulevitch, 1993). sCD14 facilitates LPS-induced activation of endothelial and epithelial cells, which do not express mCD14 (Frey et al., 1992). Namely, when LPS is shed from the surface of the bacterium, LPS binds to a membrane-bound or the soluble form of CD14, a binding that is mediated by the serum protein, LBP. The presence of the LPS/CD14/LBP complex at the membrane results in cellular activation of LPS-responsive cell types (Wright et al., 1990). CD14 expression is correlated with increased sensitivity of many cell types to LPS and other microbial molecules in their ability to activate downstream signaling events and cytokine production (Wright et al., 1990, 1995; Ulevitch and Tobias, 1995). Since CD14 does not traverse the membrane into the cytoplasm, due to the GPI anchor, it cannot mediate LPS signaling events alone, but may require a co-receptor to activate intracellular signaling pathways (Pereira et al., 1997). Despite an inability to signal, CD14-deficient mice are hyporesponsive to LPS, suggesting that CD14 plays a critical role in this process (Haziot et al., 1998). These observations are consistent with the role of CD14 in cellular activation and cytokine production.

Studies undertaken by us (Wang et al., 1996, 1998) and others (Watanabe et al., 1996; Hiraoka et al., 1998; Sugawara et al., 1998) have shown inconsistent results on CD14 expression in gingival fibroblasts. Fig. 2A shows that LPS binds to human gingival fibroblasts, and that the fluorescein isothiocyanate (FITC)-LPS binding to human gingival fibroblasts was significantly abrogated by anti-CD14 monoclonal antibody (Wang et al., 1998). Other studies found that gingival fibroblasts do not express CD14 on the surface or at the mRNA level (Hayashi et al., 1996; Kent et al., 1998, 1999). Several reports support both sets of observations (Sugawara et al., 1998). It has been shown that fibroblasts from many tissues, such as the lung, skin, and periodontium, are heterogeneous and that these cells can be separated into subsets on the basis of morphology, size, and function (Phipps et al., 1997). It has also been reported that the gingival fibroblasts of the papillary and reticular layers of the lamina propria of attached human gingiva differ in morphology and in the production of migration-stimulating factor (Irwin et al., 1994), and that CD40 expression in gingival fibroblasts is correlated with its phenotype and function (Dongari-Bagtozoglou et al., 1997). These findings suggest that fibroblast cells are not a homogenous cell population, and that there is wide variation among fibroblast cells with respect to form, proliferation rate, expression of membrane markers, function, and other characteristics. Furthermore, gingival fibroblasts heterogeneously express CD14 (Sugawara et al., 1998), IL-10 receptor (Wang et al., 1999d), and Toll-like receptor (Wang and Ohura, 2001; Wang et al., 2001b), and can be separated into several populations. On the other hand, sCD14 mediates the LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) in gingival fibroblasts (Hayashi et al., 1996). Interestingly, the sera of periodontitis patients contained an increased level of sCD14 compared with normal controls (Hayashi et al., 1999). From these reports, it appears that gingival fibroblasts isolated from inflamed tissue may express higher levels of these receptors than gingival fibroblasts isolated from non-inflamed tissue.

(2) The discovery of Toll-like receptors (TLRs)

In 1997, Janeway's group reported the successful cloning of the human homologue of Toll (human Toll/TLR4), the first member of the Toll-like receptors (TLRs) family identified in humans (Medzhitov et al., 1997). To date, nine human TLRs have been cloned (Chaudhary et al., 1998; Rock et al., 1998; Hemmi et al., 2000). Over ten members of the TLR family have been identified in humans and mice. Additional interest in TLRs came from the finding that these receptors participate in the intracellular signaling initiated by Gram-negative bacterial LPS. Although CD14 has been known for many years to bind with LPS and as the major receptor for LPS on macrophages, monocytes, neutrophils, and gingival fibroblasts, it is a GPI anchor protein, lacks a membrane-bound domain and intracellular domain, and cannot send signals into the cells (Wright et al., 1990; Ulevitch and Tobias, 1995). Therefore, a genuine receptor that sends signals into the cells probably exists, and TLRs have recently received much attention as possible candidates for LPS receptors (Ulevitch, 1999).

Several TLRs have been identified on blood cells and monocytes-macrophages based on their homology to the Drosophila Toll protein (Medzhitov et al., 1997; Rock et al., 1998). TLRs are also involved in the defensive response to fungal infection (Belvin and Anderson, 1996; Lemaitre et al., 1996). There are remarkable structural and functional similarities between the Drosophila Toll-mediated and mammalian IL-1 receptor-mediated signalings (Medzhitov et al., 1997; O'Neill and Greene, 1998). The intracellular portion of Drosophila Toll shares sequence homology with that of the mammalian IL-1 receptor. In addition to their cytoplasmic tails, the newly identified receptors share repeating leucine-rich motifs in their extracellular region. In the past few years, two of the mammalian TLRs, TLR2 and TLR4, have been shown to mediate LPS responsiveness in in vitro transfection systems (Kirschning et al., 1998; Chow et al., 1999; Shimazu et al., 1999; Yang et al., 1999; Matsuguchi et al., 2000). Although TLR2 is capable of mediating LPS signals in vitro, its role as an LPS receptor in vivo has been questioned as a result of the recent findings that two mouse strains (C3H/HeJ and C57BL10/ScCr) that exhibit impaired ability to respond to many types of LPS have different mutations in the TLR4 gene (Poltorak et al., 1998; Qureshi et al., 1999). Also, mice with disruption of the TLR4 gene, but not those with disruption of the TLR2 gene, have phenotypes similar to those of LPS-hyporesponsive strains (Takeuchi et al., 1999). TLR2-deficient macrophages are hyporesponsive to Staphylococcus aureus peptidoglycan and mycoplasmal lipopeptide, while TLR4-deficient macrophages do not respond to Gram-positive lipoteichoic acid (Takeuchi et al., 2000). Moreover, the induction of TNF-α production in peritoneal macrophages by LPS was inhibited by anti-TLR4 antibody (Akashi et al., 2000). TLR4 alone, however, is not capable of sensing and signaling the presence of LPS (Kirschning et al., 1998; Shimazu et al., 1999). Another molecule, MD-2, which is physically associated with TLR4, is required for LPS recognition (Shimazu et al., 1999). The TLR4-MD2 complex thus serves as the LPS recognition molecule. These findings suggest that TLR4 is the dominant receptor for at least some types of LPS, whereas TLR2 is not required for LPS recognition. In contrast, it has been suggested that TLR2 mediates signals from other bacterial components, including lipoteichoic acid, peptidoglycan, and lipoproteins/lipopeptides (Brightbill et al., 1999; Hirschfeld et al., 1999; Schwandner et al., 1999; Yoshimura et al., 1999). From these findings, it is concluded that TLR2 and TLR4 on gingival fibroblasts recognize different bacterial cell-wall components: TLR2 mediates the response to peptidoglycan and lipoprotein, while TLR4 mediates the response to LPS and lipoteichoic acid.

We demonstrated that the binding of P. gingivalis LPS to TLR4 on gingival fibroblasts activates second-messenger systems (Wang et al., 2000a,b). Fig. 2B shows that LPS binds to human gingival fibroblasts, and that the FITC-LPS binding to human gingival fibroblasts was significantly abrogated by anti-TLR4 antibody (Wang et al., 2000b). Another group reported that gingival fibroblasts express TLR2 and TLR4, and that stimulation with P. gingivalis LPS increased their levels of expression (Tabeta et al., 2000). Thus, although these results suggested that TLRs are a signaling component of a cellular receptor for LPS, CD14 transgenic mice that overexpressed human CD14 were highly responsive to LPS (Ferrero et al., 1993). More recently, it has been reported that P. gingivalis LPS does not signal through TLR4, and that the signaling through TLR2 and the signaling through TLR4 differed quantitatively and qualitatively in murine macrophages (Hirschfeld et al., 2001). Therefore, it is important for the relationship among TLR2, TLR4, and CD14 on gingival fibroblasts to be understood. Further studies are needed to elucidate the differences among these receptors.

(3) The pathway of Porphyromonas gingivalis lipopolysaccharide (LPS) signaling via CD14 and Toll-like receptor-4 (TLR4)

Several signaling pathways have been implicated downstream of the binding of microbial ligands such as LPS to their receptors on gingival fibroblasts. In fact, the regulation of the expression of many immunomodulatory genes requires more than one signaling pathway. The binding of LPS with CD14 induces the transient activation of several protein kinases such as protein kinase C, protein tyrosine kinases, and mitogen-activated protein kinase (MAPK) (Weinstein et al., 1992; Dong et al., 1993; Stefanova et al., 1993; Shapira et al., 1994). The phosphorylation of intracellular proteins is essential for the LPS-induced functional activation of monocytes-macrophages (Dong et al., 1993; Shapira et al., 1994). LPS induced the tyrosine phosphorylation of 42- and 44-kDa proteins, which corresponded to p42 (ERK2) and p44 (ERK1) MAPK, in monocytes/macrophages (Wright et al., 1992). Ligation of LPS enzymatically activates ERK2 and ERK1, and this activation correlates with the increases in its tyrosine phosphorylation status (Weinstein et al., 1992; Dong et al., 1993). We (Wang et al., 1998) and another group (Watanabe et al., 1996) demonstrated that CD14 mediates the signal pathway of P. gingivalis LPS in gingival fibroblasts. P. gingivalis LPS activated tyrosine kinase in human gingival fibroblasts via CD14, leading to MCP-1 gene expression through the transcription factors, nuclear factor-κB (NF-κB) and activating protein-1 (AP-1) (Watanabe et al., 1996). Fig. 3 shows that engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins, including ERK1 and ERK2, and these events were suppressed by the anti-CD14 antibody (Wang et al., 1998). Thus, the binding of P. gingivalis LPS to CD14 on human gingival fibroblasts activates protein tyrosine phosphorylation of several intracellular proteins. These results suggest that CD14 is a binding site for LPS on the cell surface and is involved in the LPS-induced cellular activation of gingival fibroblasts. In addition to myeloid cells, endothelial cells which do not express CD14 also respond to LPS via the release of numerous pro-inflammatory mediators and the expression of various adhesion molecules in a serum-dependent manner (Haziot et al., 1993; Pugin et al., 1993). In contrast to myeloid cells in which the membrane CD14 is a receptor for LPS, the soluble form of CD14 and LBP found in the serum are involved in the LPS-induced response in endothelial cells (Haziot et al., 1993; Pugin et al., 1993). This notion led us to hypothesize that LBP is not involved in the recognition of LPS by CD14 on gingival fibroblasts, unlike that in myeloid and endothelial cells in which LBP-mediated binding of LPS occurs via CD14. Interestingly, we found that the signal-transducing pathway of P. gingivalis LPS in gingival fibroblasts is mediated by CD14 in the absence of serum (Wang et al., 1998).

Toll activates Dorsal, a Drosophila homolog of the inhibitor of nuclear factor-κB (IκB). The Drosophila serine/threonine kinase Pelle is highly homologous to IL-1 receptor-associated kinase (IRAK) (Muzio et al., 1997). Thus, the intracellular domains of TLRs are similar to the intracellular domain of IL-1 receptor (IL-1R), and this region is referred to as the Toll/IL-R domain. The binding of a ligand with IL-1R induces dimerization of the receptor, recruitment of an accessory protein, binding of an adaptor protein (MyD88), IRAK, followed by the phosphorylation of a TNF-receptor-associated factor (TRAF6) (Muzio et al., 1997; Wesche et al., 1997; Adachi et al., 1998; Burns et al., 1998). TGF-β-activated kinase (TAK-1) was shown to mediate NF-κB activation in IL-1-stimulated cells (Ninomiya-Tsuji et al., 1999). Activation of TAK-1 subsequently results in the activation of NF-κB-inducing kinase (NIK), followed by the activation of I-κB kinase (IKK), phosphorylation of IκB, and its dissociation from NF-κB (O'Neill and Greene, 1998). Thus, the signal transduction pathway activated by IL-1 in mammalian cells is highly similar to the one described for Toll. On the other hand, MyD88 is a bifurcating point in TLR4-signaling. TLR-signaling activates two branching pathways proximal to the intracytoplasmic domain of TLR4. The first is the MyD88-dependent pathway, which subsequently activates IRAK, TRAF6, and NF-κB, as mentioned above. MyD88 is associated not only with the cytokine receptor for IL-1 and IL-18, but also with various TLRs, including TLR4 (Medzhitov et al., 1998; Muzio et al., 1998). This pathway is essential for the induction of cytokines. The second pathway is an MyD88-independent pathway that cannot activate IRAK. Although the molecules involved are not known, this pathway can also activate NF-κB with delayed kinetics (Swantek et al., 2000). This NF-κB activation cannot lead to cytokine induction. In addition to the NF-κB signaling pathway, other signaling pathways have been linked to the TLRs signaling pathway. Overexpression of the dominant-active form of TLR4 activates not only NF-κB, but also AP-1 and Jun N-terminal kinase (JNK) (Medzhitov et al., 1997; Wesche et al., 1997; Muzio et al., 1998). In fact, NF-κB and AP-1 are prominently involved in the regulation of many pro-inflammatory and immunomodulatory genes (Plevy et al., 1997; Brightbill et al., 1999, 2000). The MAPK kinase pathway is also activated in response to microbial ligands, possibly downstream of TLRs activation as well. The MAP kinase-kinase-kinase (MAPKKK), TAK1, appeared to be involved in LPS-induced NF-κB activation downstream of TLR2 and TLR4 in a transfected cell line and in the murine macrophage cell line RAW264.7 (Chan et al., 1998). TAK1 is a bifurcating point in the MAP cascade and the IKK cascade. AP-1 is activated by the MAP kinase pathway, and NF-κB is activated by the IKK pathway. MyD88 knockout experiments in mice suggested that while MyD88 is essential for the responses to LPS, MyD88 is not required for activation of the MAP kinase and IKK pathways. There is growing evidence that the binding of a ligand to TLRs leads to the activation of several different signaling pathways that contribute to the specificity of target gene activation and cellular responses.

With respect to the TLRs signaling pathway in gingival fibroblasts, we demonstrated the TLR4-mediated signaling pathway induced by P. gingivalis LPS in gingival fibroblasts (Wang and Ohura, 2001; Wang et al., 2001b). Namely, we showed that LPS binds to human gingival fibroblasts (HGFs), and that HGFs express TLR4 and MyD88. P. gingivalis LPS-induced IL-1 production in HGFs was inhibited by anti-TLR4 antibody. P. gingivalis LPS treatment of HGFs activated several intracellular proteins, including protein tyrosine kinases (Fig. 4), and up-regulated the expression of IRAK, NF-κB, and AP-1; these events were suppressed by anti-TLR4 antibody (Fig. 5 ). Our findings suggest that the binding of P. gingivalis LPS to TLR4 on HGFs activates various second-messenger systems.

On the other hand, it has been reported that when macrophages (Wright et al., 1990; Nishijima et al., 1994) and gingival fibroblasts (Wang et al., 1998) were treated with a high concentration of LPS, the LPS activated certain receptors directly without the involvement of LBP and CD14. This report may have significance for TLR4 on gingival fibroblasts.

Based on our and others' findings, we show the hypothetical model of P. gingivalis LPS signaling via CD14 and TLR4 in gingival fibroblasts in Fig. 6.

A View of Therapy for Periodontal Disease

Regarding the role of local etiologic factors in periodontal disease, dental plaque and its associated bacteria that populate periodontal pockets release LPS and other bacterial products into the sulcus. These substances affect both the immune cells in the connective tissue and osteoblasts. These products may induce the production of local cytokines in immune cells. CD14 and TLRs play an important role as triggers for LPS-induced biological responses in monocytes/macrophages, neutrophils, and gingival fibroblasts. The components of the LPS signal transduction pathways via CD14 or TLRs are potential targets for the development of medical therapies for the prevention and treatment of periodontal disease. The following are potential methods that may be used in the therapy for periodontal disease: (1) stopping the binding of LPS to immunocompetent cells, or (2) stopping the pathways induced by the binding of LPS to TLR4 and CD14.

It has been reported that anti-CD14 antibody inhibits the development of P. gingivalis-LPS-induced periodontitis in mice (Wang and Ohura, 2000). We suggested that IL-10 inhibits the inflammatory response via the IL-10 receptor in P. gingivalis LPS-initiated periodontal disease (Wang et al., 1999d). Several studies have explored the clinical implication of IL-10 in patients with immune and inflammatory disease (Ishida et al., 1993, 1994; Kuhn et al., 1993). One study showed that patients with systemic lupus erythematosus (SLE) disease and rheumatoid arthritis (RA) had higher titers of IL-10 than did normal controls (Ishida et al., 1994). Conversely, some patients with common variable immunodeficiency and with inflammatory bowel diseases showed lower titers of IL-10 in their sera than did healthy controls (Kuhn et al., 1993). Another study demonstrated that most bacterial endotoxin shock patients had lower levels of serum IL-10 than did cardiogenic shock patients (Ishida et al., 1993). Furthermore, it is possible that the biological function of IL-10 affects not only HGFs but also other immunocompetent cells in inflammatory periodontal tissues. Therefore, the use of IL-10 may help prevent or improve the treatment of periodontitis. It is possible that these cytokines may participate in the tissue remodeling which occurs during the active phase of periodontal destruction. Thus, it is possible that IL-10 has a biological function not only in gingival fibroblasts but also in other immunocompetent cells in inflamed periodontal tissues.

On the other hand, certain salivary proteins provide important defense functions against bacteria, fungi, and viruses (Tenovuo, 1989). Some of these proteins are antibacterial, since they control both the established flora and act against invading pathogens (Nishikata et al., 1991; Wang et al., 1999a,c, 2001b; Kanehira et al., 2000). Furthermore, we have examined the effects of salivary proteins such as mucins, secretory IgA, lactoferrin, peroxidase, amylase, lysozyme, and histatin on P. gingivalis LPS activity. These salivary proteins bound to P. gingivalis LPS and reduced the level of LPS activity (Wang et al., 2001a). Salivary proteins may be useful for preventing or improving the treatment of periodontitis. Interestingly, a Toll protein may improve DNA-based vaccines, as suggested in a recent paper (Modlin, 2000).

Conclusion

In this review, we summarized the recent findings on CD14 and TLRs as P. gingivalis LPS receptors on gingival fibroblasts. The receptors for LPS on gingival fibroblasts and receptor activation are still controversial. Understanding the cellular interactions and characteristics of P. gingivalis LPS signaling in gingival fibroblasts is important for elucidating the inflammatory process that exists in periodontal disease.

Therefore, if we could clarify the mechanism of P. gingivalis LPS signaling in gingival fibroblasts, the prevention and therapy for periodontitis could be much improved. Thus, there is a potential for exciting further studies in this field.

Figure 1.

Endotoxins reside in the outer membrane of Gram-negative bacteria. The cut reveals the structure of the two membranes that envelop the bacterium (Rietschel and Brade, 1992).

Figure 2.

Effects of anti-CD14 monoclonal antibody (A) and anti-Toll-like receptor 4 monoclonal antibody (B) on the binding of FITC-labeled LPS to human gingival fibroblasts. Human gingival fibroblasts were incubated with an unlabeled control antibody (CT), FITC-labeled LPS (LPS binding), or anti-CD14 monoclonal antibody with FITC-labeled LPS (LPS+anti-CD14 mAb) and anti-Toll-like receptor 4 monoclonal antibody with FITC-labeled LPS (LPS+anti-TLR4 mAb). The cells were analyzed by flow cytometry (Wang et al., 1998, 2000b).

Figure 3.

Analysis of protein phosphorylation by Western blot analysis. Human gingival fibroblasts were stimulated with LPS from Porphyromonas gingivalis (P-LPS) in the presence or absence of anti-CD14 monoclonal antibody (anti-CD14 mAb). (A) The cells were subjected to Western blotting with anti-phosphotyrosine monoclonal antibody. (B) The immunoprecipitate was analyzed with anti-mitogen-activated protein kinase (MAPK: ERK2 and ERK1) kinase monoclonal antibody (Wang et al., 1996, 1998). The lanes are as follows: non-stimulated cells (lane 1), LPS-stimulated (lane 2), anti-CD14 mAb (lane 3), and both LPS and anti-CD14 mAb (lane 4).

Figure 4.

Analysis of protein phosphorylation by Western blot analysis. Human gingival fibroblasts were stimulated with LPS from Porphyromonas gingivalis (P-LPS) in the presence or absence of anti-Toll-like receptor 4 (TLR4) monoclonal antibody (anti-TLR4 mAb). (A) The cells were subjected to Western blotting with anti-phosphotyrosine monoclonal antibody. (B) The immunoprecipitate was analyzed with anti-IL-1 receptor-associated kinase (IRAK) monoclonal antibody (Wang et al., 2000a,b).

Figure 5.

Porphyromonas gingivalis LPS induces NF-κB and AP-1 expression in human gingival fibroblasts. The cells were stimulated with LPS from Porphyromonas gingivalis (P-LPS) in the presence or absence of anti-TLR4 monoclonal antibody (anti-TLR4 mAb). Gel mobility shift assay was performed with a biotin-labeled probe [NF-κB (A) or AP-1 (B)] (Wang et al., 2000a,b).

Figure 6.

A hypothetical model of Porphyromonas gingivalis LPS signaling via CD14 and TLR4 in gingival fibroblasts. The diagram shows the signaling cascade from the binding of the signaling complex to CD14 or TLR4 to the activation of NF-κB and AP-1. P.g. LPS, Porphyromonas gingivalis lipopolysaccharide; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response gene 88; IRAK, IL-1 receptor accessory protein kinase; TRAF6, TNF receptor-associated factor; TAK1, transforming growth factor-β-activated kinase1; TAB1, TAK1 binding protein1; MAPKK, map kinase kinase; AP-1, activating protein-1; JNK/p38, Jun N-terminal kinase/p38; NIK, NF-κB-inducing kinase; IKK, inhibitor of nuclear factor-κB; NF-κB, nuclear factor-κB.

Footnotes

Acknowledgements

We thank Profs. Yoshinori Kuboki, Hiroshi Tani, Katsuhide Sonoda (Hokkaido University), Yusuke Kowashi (Health Sciences University of Hokkaido), and Dr. Junzo Tanaka (National Institute for Research in Inorganic Materials) for their instruction. We are grateful to Drs. Kensuke Miyake, Sachiko Akashi (Saga Medical School), and Katsuaki Sato (The Institute of Medical Science, The University of Tokyo) for their helpful discussions. We also thank Miss Hiroko Shintani and Mr. Syuhei Usui for assistance in the preparation of this manuscript. Some of the work reported in this review manuscript was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, and by a grant from the Yamasaki Foundation for the Promotion of Dental Research (C-9601).

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P orphyromonas gingivalis L ipopolysaccharide S ignaling in G ingival F ibroblasts –CD14 and T oll-like R eceptors