Porphyromonas gingivalis 67-kDa fimbriae induced cytokine production and osteoclast differentiation utilizing TLR2

FEMS Microbiology Letters 229 (2003) 49^55

www.fems-microbiology.org

Porphyromonas gingivalis 67-kDa ¢mbriae induced cytokine production and osteoclast di¡erentiation utilizing TLR2 Hiroko Hiramine, Kiyoko Watanabe, Nobushiro Hamada, Toshio Umemoto



Department of Oral Microbiology, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan Received 28 July 2003; received in revised form 30 September 2003; accepted 9 October 2003 First published online 5 November 2003

Abstract Porphyromonas gingivalis, a major etiological agent of adult periodontitis, has two distinctly different types of fimbriae on the cell surface. The major fimbriae, which consist of a 41-kDa fimbrillin of P. gingivalis ATCC 33277, have been known to induce inflammatory cytokine production in murine peritoneal macrophages. In this study, we examined the effects of the minor fimbriae of P. gingivalis, composed of a 67-kDa fimbrillin, on cytokine production in murine peritoneal macrophages and the ability to induce osteoclast differentiation. Murine peritoneal macrophages were stimulated with P. gingivalis 67-kDa minor fimbriae for 24 h, then the levels of interleukin (IL)-1L, tumor necrosis factor (TNF)-K and IL-6 production were determined by enzyme-linked immunosorbent assay (ELISA). To estimate osteoclast differentiation, mouse osteoclast precursors were placed on dentine slices, and cultured with or without P. gingivalis 67-kDa minor fimbriae for 7 days. P. gingivalis 67-kDa minor fimbriae clearly induced IL-1L, TNF-K and IL-6 production in mouse macrophages. Furthermore, pit formations on the dentine slices were significantly extended when the osteoclast precursors were incubated with P. gingivalis 67-kDa minor fimbriae. Pretreatment with anti-Toll-like receptor 2 (TLR2) antibody significantly inhibited IL-1L, TNF-K and IL-6 induction (P 6 0.05) in mouse macrophages and pit-forming activity of osteoclast precursor cells stimulated with P. gingivalis 67-kDa minor fimbriae. These results suggest that P. gingivalis 67-kDa minor fimbriae may provoke host inflammatory response and be involved in periodontal tissue breakdown. ? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Porphyromonas gingivalis ¢mbria; Peritoneal macrophage; Osteoclast; Toll-like receptor 2

1. Introduction Porphyromonas gingivalis is an obligatory anaerobic Gram-negative coccobacillus that has been associated with periodontal destruction in humans [1]. The representative features of adult periodontitis are in£ammation in the gingival tissue and resorption of alveolar bone in the periodontal tissues. With regard to P. gingivalis, certain virulent factors including ¢mbriae have been known to be involved in the development of periodontal tissue breakdown. P. gingivalis possesses two distinctly di¡erent types of ¢mbriae in terms of their size and antigenicity on the cell surface [2,3], namely a 41-kDa protein (FimA) and a 67kDa protein (Mfa1) in strain ATCC 33277 [4,5]. The 41kDa ¢mbriae of P. gingivalis appear as long ¢lamentous

* Corresponding author. Tel./Fax : +81 (46) 822-8867. E-mail address : [email protected] (T. Umemoto).

structures and have been reported to induce in£ammatory cytokines in human gingival ¢broblasts and murine peritoneal macrophages as well as to promote the adherence of the organism to host tissues [6,7]. Recently, the 67-kDa ¢mbriae were revealed to have the capability of inducing interleukin (IL)-1K, IL-1L, IL-6 and tumor necrosis factor (TNF)-K in murine macrophages [8]. Moreover, inoculation into the oral cavity of the 67-kDa ¢mbriae-de¢cient mutant strain, MPG67 (mfa1), resulted in a signi¢cant reduction of alveolar bone loss in rat compared to the wild-type strain ATCC 33277 [9]. However, the role of P. gingivalis 67-kDa ¢mbriae in periodontal destruction and the mechanism of bone resorption evoked by the ¢mbriae have not been well de¢ned. In£ammatory cytokines have been reported to induce osteoclast formation and activation [10,11]. Bacterial components of P. gingivalis, such as lipopolysaccharide (LPS), have also been demonstrated to have the ability to activate osteoclast formation indirectly by stimulating osteoblasts or in£ammatory cells as a consequence of inducing in£am-

0378-1097 / 03 / $22.00 ? 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(03)00788-2

FEMSLE 11281 25-11-03

50

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

matory cytokines. Among in£ammatory cytokines, IL-1 and TNF-K are thought to be pivotal factors in bone remodeling both in vitro and in vivo and IL-6 has been observed in in£amed periodontal tissues [12,13]. To illuminate the pathogenicity of the 67-kDa ¢mbriae, we examined whether the 67-kDa ¢mbriae induced osteoclast di¡erentiation and activation using a coculture of C57BL/6N mouse bone marrow cells and the MC3T3G2/PA6 system. Toll-like receptors (TLRs) have recently been identi¢ed as important signal-transducing elements of receptor complexes in recognizing microbial infection [14], and were mammalian proteins homologous to Drosophila Toll [15,16]. Ten members of the TLR families have been reported, and among them, both TLR2 and TLR4 have been found to be involved in responses to bacterial LPS, leading to the expression of proin£ammatory cytokines IL-1, IL-6, IL-8 and TNF-K [17^19], a process which is mediated via an MyD88-dependent intracellular signaling pathway that causes translocation of nuclear factor (NF)UB. Recently, it was demonstrated that TLR2 played an important role as a receptor of various Gram-positive bacterial components, such as peptidoglycan and lipoproteins [17,20^25]. There are some lines of evidence indicating that bacterial ¢mbriae or pili also utilize TLRs to activate host cells and P. gingivalis 41-kDa ¢mbriae have the capability of activating human gingival epithelial cells through TLR2 [26,27]. In this study, we examined the e¡ects of P. gingivalis 67-kDa minor ¢mbriae on in£ammatory cytokine production from murine peritoneal macrophages and induction of osteoclast di¡erentiation; and the roles of TLR2 in host cell responses were investigated.

8.0, containing 0.15 M NaCl and 10 mM MgCl2 and subjected to ultrasonication with a 3-mm microtip using a 20W pulse setting with a 50% duty cycle for 5 min in an ice bath. The supernatant of the sonic extract was centrifuged at 10 000Ug for 30 min at 4‡C and subjected to 40% ammonium sulfate saturation by the stepwise addition of ammonium sulfate. The precipitated protein was collected by centrifugation at 10 000Ug for 30 min at 4‡C, suspended in a minimum volume of 20 mM Tris bu¡er, pH 8.0, and dialyzed against the same bu¡er. The dialysate sample containing most of the ¢mbriae was subjected to further puri¢cation on a diethylaminoethyl (DEAE) Sepharose CL-6B column (1.5 by 20 cm) equilibrated with 20 mM Tris bu¡er, pH 8.0. The column was washed with 20 mM Tris bu¡er and then eluted with a linear gradient of 0^0.3 M NaCl. The protein content of the fractions was measured by ultraviolet (UV) light adsorption at 280 nm, and the presence of ¢mbrial structures was determined by electron microscopy. The fractions were analyzed by sodium dodecyl sulfate (SDS)^12.5% polyacrylamide gel electrophoresis (PAGE), and those containing ¢mbriae were pooled and concentrated by ammonium sulfate precipitation and dialyzed against 5 mM Tris bu¡er, pH 8.0. This concentrated pool was termed 67-kDa ¢mbriae. 2.3. Mice C3H/HeN and speci¢c pathogen-free BALB/c female mice were obtained from Nihon Clea Laboratories Animal Center (Tokyo, Japan). Experimental protocols for animal handling were approved by the Institutional Animal Care Committee of Kanagawa Dental Collage. 2.4. Preparation of murine peritoneal macrophages

2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this experiment were P. gingivalis ATCC 33277 and a ¢mA mutant, MPG1 [1]. Bacteria were grown anaerobically (85% N2 , 10% H2 , and 5% CO2 ) at 37‡C for 18 h in brain heart infusion broth (BHI; Difco Laboratories, Detroit, MI, USA) supplemented with 5 mg of yeast extract, 5 Wg of hemin, and 0.2 Wg of menadione per ml. For maintenance of the ¢mA mutant strain, MPG1, erythromycin was added to the media at 10 Wg ml31 . 2.2. Isolation and puri¢cation of the 67-kDa ¢mbriae from P. gingivalis The 67-kDa ¢mbriae of P. gingivalis were prepared from MPG1 cells by a modi¢cation of the method used by Yoshimura et al. [2]. P. gingivalis MPG1 from 4.0 l of BHI culture was centrifuged at 8000Ug for 25 min and the pelleted cells were suspended in 20 mM Tris bu¡er, pH

The peritoneal macrophages from C3H/HeN mice (6^10 weeks old) were induced with 3 ml of thioglycolate medium (Difco Laboratories, Detroit, MI, USA) and peritoneal exuded cells were collected 5 days after an irritative injection in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with L-glutamine, 10 mM HEPES, 100 U of penicillin G per ml, 100 Wg of streptomycin per ml, and 0.05 mM 2-mercaptoethanol. 2.5. Cytokine induction and inhibition assays Murine peritoneal macrophages were incubated in serum-free RPMI 1640 medium (1U106 cells ml31 ) in 24well culture plates at 37‡C for 1 h in a humidi¢ed atmosphere of 5% CO2 in air, then the culture was washed three times with RPMI 1640 medium to remove non-adherent cells. The culture supernatant was then replaced with fresh RPMI 1640 medium with or without various concentrations of P. gingivalis 67-kDa ¢mbriae and Escherichia coli LPS F583 (Sigma, St. Louis, MO, USA), which was prepared using the phenol^chloroform^petroleum

FEMSLE 11281 25-11-03

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

ether extraction procedure, and cultured in triplicate for 24 h. In some experiments designed to block cytokine induction, the cells were pretreated with mouse antibody against 5 Wg ml31 of TLR2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37‡C for 1 h before adding the test specimens. After incubation for 24 h, the supernatants were collected and stored at 380‡C until the assay for IL1L, IL-6 and TNF-K production. The levels of IL-1L, IL-6, and TNF-K production in the samples were determined using an enzyme-linked immunosorbent assay (ELISA) kit system from Genzyme-Techne (Minneapolis, MN, USA) and the results were determined using a standard curve prepared for each assay. 2.6. Cell line In order to estimate the di¡erentiation and the bone absorbing activity of osteoclasts stimulated with the 67kDa ¢mbriae, the MC3T3-G2/PA6 (PA6) cell line was used in this experiment. The cell line was obtained from RIKEN Cell Bank (Tsukuba, Japan) and the cells were maintained in K-MEM (K-minimum essential medium ; Flow Laboratories, McLean, VA, USA) with 20% fetal calf serum at 37‡C in 5% CO2 to prepare osteoclast precursors. 2.7. Pit formation assay and inhibition assay The dentine slices (6 mm in diameter, 0.3 mm in thickness) were prepared with an ethanol-cooled diamond saw (Isomet 2000; Buehler, Lake Blu¡, IL, USA) [2]. The slices were cleaned ultrasonically in 70% ethanol, and one slice was then placed in each well of a 48-well tissue culture plate (Sumitomo Bakelite, Tokyo, Japan). Osteoclast precursors were prepared as follows. In brief, BALB/c mouse bone marrow cells were cocultured with MC3T3-G2/PA6 in K-MEM containing 2% Type.collagen (NittaGelatin, Osaka, Japan), 10% fetal bovine serum

51

(FBS), macrophage colony-stimulating factor (M-CSF), RANKL, dexamethasone and 1K,25(OH)2 D3 for 7 days in 100-mm diameter dishes (Corning, NY, USA). After removing non-adherent cells by washing, adherent cells were harvested and recultured as mouse osteoclast precursors with M-CSF, RANKL, dexamethasone, 1K,25(OH)2 D3 and P. gingivalis ¢mbriae or E. coli LPS on dentine slices in 48-well culture plates for a further 7 days. In some experiments designed to block pit formation, the cells were pretreated with mouse anti-TLR2 (0.02 Wg ml31 for blocking) at 37‡C for 1 h in 5% CO2 in air before adding the test specimens. After incubation, the cells were stripped by ultrasonication in 0.25 M NH4 OH and the dentine slices were immersed in hematoxylin to observe the resorption pits formed by osteoclasts under microscopy. Each dentine slice was measured using the Olympus image analysis system (Olympus Co., Tokyo, Japan). The results are expressed as the mean Q S.D. of triplicate cultures.

3. Results 3.1. Induction of cytokine production by P. gingivalis 67-kDa ¢mbriae The e¡ects of puri¢ed 67-kDa ¢mbriae of P. gingivalis on the induction of in£ammatory cytokines, IL-1L, IL-6 and TNF-K, were studied in peritoneal macrophages from C3H/HeN mice at various culture periods. The cells were stimulated with 1 Wg protein ml31 of 67-kDa ¢mbriae or 0.1 Wg ml31 of E. coli LPS. The 67-kDa ¢mbriae were capable of inducing signi¢cant levels of IL-1L, IL-6 and TNF-K in C3H/HeN macrophages for 24 h (Table 1 and Fig. 2). The levels were observed to decline after a 24-h incubation, therefore, murine macrophages were stimulated with test specimens for 24 h throughout the experiments. The 67-kDa ¢mbriae induced a comparable

Table 1 Induction of cytokine production in murine peritoneal macrophages by P. gingivalis 67-kDa ¢mbriae Pretreatment

P. gingivalis 33277 67-kDa ¢mbriae 67-kDa ¢mbriae+anti-TLR2 67-kDa ¢mbriae+IgG control E. coli F583 LPS LPS+anti-TLR2 Medium only Medium+anti-TLR2 Medium+IgG control

Dose (Wg ml31 )

5 10 5

5 5 5

Cytokine level (pg ml31 ) IL-6

TNF-K

941.2 Q 28.9 509.9 Q 92.9* 477.5 Q 23.5* 967.0 Q 5.9

1409.9 Q 77.0 839.2 Q 124.1 706.4 Q 16.1* 1513.8 Q 74.8

856.2 Q 31.9 776.8 Q 8.3 14.9 Q 0.8 30.3 Q 0.9 43.9 Q 4.8

967.9 Q 172.0 890.2 Q 4.1 47.3 Q 1.2 84.0 Q 3.6 75.9 Q 14.0

Murine peritoneal macrophages were preincubated with anti-TLR2 for 1 h and then stimulated with 1 Wg ml31 of P. gingivalis 67-kDa ¢mbriae or 0.1 Wg ml31 of E. coli LPS. Cytokines produced were determined by ELISA. The results are expressed as the mean Q S.D. of triplicate cultures. *Signi¢cantly di¡erent from values of untreated control (P 6 0.01).

FEMSLE 11281 25-11-03

52

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

Fig. 1. SDS^PAGE analysis of the puri¢ed ¢mbrial proteins from P. gingivalis. The crude extract was precipitated with 40% saturated ammonium sulfate, and then the precipitate was suspended in 20 mM Tris^HCl (pH 8.0). The suspension was dialyzed and the dialysate was applied to a DEAE Sepharose CL-6B ion exchange column. The proteins were then electrophoresed on an SDS^12.5% polyacrylamide gel. The purity of each protein was determined by Coomassie brilliant blue staining. Lane 1, 67-kDa protein ; lane 2, 41-kDa protein.

amount of IL-1L and IL-6 to E. coli LPS, whereas a rather higher level of TNF-K was produced by stimulation with the ¢mbrial protein (Table 1 and Fig. 2). The ¢mbrial protein was also e¡ective in boosting production of these in£ammatory cytokines in LPS non-responsive C3H/HeJ peritoneal macrophages in which LPS from E. coli had no e¡ect on it (data not shown). 3.2. Inhibitory e¡ect of antibody against TLR2 on cytokine production by P. gingivalis 67-kDa ¢mbriae The 67-kDa ¢mbriae at a concentration of 1 Wg ml31

induced signi¢cant IL-1L, IL-6 and TNF-K in the supernatants of murine peritoneal macrophages. To determine whether TLR2 of mouse macrophages was responsible for the production of IL-1L, IL-6 and TNF-K by the 67-kDa ¢mbriae, mouse peritoneal macrophages were precultured with anti-TLR2 (5 Wg ml31 ) antibody at 37‡C for 1 h in 5% CO2 in air before adding the 67-kDa ¢mbriae. After incubation for 24 h, the culture medium was then subjected to cytokine assay. As shown in Table 1 and Fig. 2, anti-TLR2 antibody signi¢cantly inhibited IL-1L (44.4% inhibition), IL-6 (45.9%), and TNF-K (40.5%) production, induced by the 67-kDa ¢mbriae (P 6 0.01). The cytokine induction by ¢mbrial protein was dose-dependently inhibited with anti-TLR2 antibody (data not shown) while control antiserum did not have any inhibitory e¡ect. On the other hand, anti-TLR2 did not block the cytokine production by E. coli LPS. 3.3. Pit formation assay Next, we examined the ability of 67-kDa ¢mbriae of P. gingivalis to activate bone resorption using osteoclast precursor cells. BALB/c mouse bone marrow cells were cocultured with MC3T3-G2/PA6 in K-MEM containing 2% Type.collagen, 10% FBS, M-CSF, RANKL, dexamethasone and 1K,25(OH)2 D3 for 7 days to prepare osteoclast precursors. After incubation, the osteoclast precursors were stimulated with 67-kDa ¢mbriae or E. coli LPS for a further 7 days and the area of pit formation was measured. As shown in Fig. 3, 67-kDa ¢mbriae apparently developed pit formation absorbed by osteoclast precursors on the dentine slices. The e¡ect of ¢mbrial protein in promoting pit-forming activity was three-fold larger than that of non-stimulating control (Fig. 4). A similar result was obtained in osteoclast precursors stimulated with E. coli LPS.

Fig. 2. Inhibitory e¡ects of antibody against mouse TLR2 on IL-1L production of C3H/HeN murine peritoneal macrophages stimulated with P. gingivalis 67-kDa ¢mbriae or E. coli LPS. The cells were preincubated with anti-TLR2 and then stimulated for 1 h with either 1 Wg ml31 of 67-kDa ¢mbriae or 0.1 Wg ml31 of E. coli LPS. IL-1L production was inhibited by addition of 5 Wg ml31 of anti-TLR2 when cells were stimulated with P. gingivalis 67kDa ¢mbriae. Results are presented as mean Q standard deviation of triplicate determinations. *Signi¢cantly di¡erent between the groups with and without the test specimens (P 6 0.01).

FEMSLE 11281 25-11-03

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

53

Fig. 3. Photographs of pit formation on the dentine slices stimulated by P. gingivalis 67-kDa ¢mbriae or E. coli LPS. Mouse bone marrow cells were cocultured with MC3T3-G2/PA6 in K-MEM containing 2% Type.collagen, 10% FBS, M-CSF, RANKL, dexamethasone and 1K,25(OH)2 D3 for 7 days in 100-mm diameter dishes to separate adherent cells and non-adherent cells. Then, adherent cells were harvested and recultured as mouse osteoclast precursors with M-CSF, RANKL, dexamethasone, 1K,25(OH)2 D3 and P. gingivalis 67-kDa ¢mbriae (A), E. coli LPS (B), control (C) on dentine slices in 48-well culture plates for 7 days. Bars = 100 Wm.

3.4. Inhibitory e¡ect of antibody to TLR2 on pit formation by P. gingivalis 67-kDa ¢mbriae To determine the role of TLR2 on the bone absorbing activation by P. gingivalis 67-kDa ¢mbriae, mouse osteoclast precursors were pretreated with anti-TLR2 for 60 min at 37‡C before adding the ¢mbriae. After a subsequent 7-day incubation, the slices were immersed in hematoxylin to stain the resorption pits formed by osteoclasts. Pretreatment with anti-TLR2 antibody blocked enlargement of the pit area promoted by 67-kDa ¢mbriae stimulation up to 54.4% of that compared with the original stimulation (Fig. 4). Likewise, the inhibitory e¡ect was observed in dentine slices cultured with E. coli LPS (16.5% inhibition), whereas no e¡ect was observed in control non-stimulated cultures (Fig. 4).

4. Discussion P. gingivalis has been predominantly isolated from subgingival plaques and is thought to be a major etiological agent in adult periodontitis. The ¢mbriae of this organism are known to be a potent virulent factor concerned with adherence to periodontal tissues and modulation of the host immune response. Hamada et al. (1996) have found the presence of two di¡erent kinds of ¢mbriae on the cell surface of P. gingivalis ATCC 33277: the 41-kDa major subunit protein encoded by the ¢mA gene and the 67-kDa component encoded by the mfa1 gene. The ¢mA mutant, MPG1, was observed to lack the long ¢lamentous structures, however, possessed short ¢mbrial structures on the cell surface in electron microscopic study. In the present study, the 67-kDa ¢mbriae were isolated from MPG1 and subjected to further puri¢cation on a DEAE Sepharose CL-6B column. The fractions collected were observed as single band on SDS^PAGE (Fig. 1) and we con¢rmed that no LPS contamination was detected by silver staining (data not shown). We report here that the highly puri¢ed 67-kDa ¢mbriae of P. gingivalis stimulated mouse immune

response, in terms of IL-1L, IL-6 and TNF-K production, and activated the pit-forming ability of osteoclast precursor cells. The 67-kDa ¢mbriae evidently induced IL-1L, IL-6 and TNF-K production from C3H/HeN murine peritoneal macrophages at a concentration of 1 Wg ml31 (Fig. 2 and Table 1) and the levels were comparable to those stimulated with E. coli LPS (0.1 Wg ml31 ). These cytokines play important roles in various in£ammatory phases ; IL-1 is a mainly macrophage-derived mediator and plays a central role, such as in phylogenic activity, in in£ammatory reactions, whereas IL-6 is a multifunctional cytokine and referred to as B-cell stimulatory factor 2 and hepatocyte stimulating factor. These cytokines are also known to stimulate osteoclastic bone resorption in a direct or indirect manner [6,7]. Roodman et al. reported that both IL-1 and TNF-K were potent stimulators of bone resorption in vitro and in vivo [11,28]. Numerous studies have provided evidence indicating that bacterial LPS activated bone resorption of osteoclasts by inducing in£ammatory cytokines, however, few reports

Fig. 4. The e¡ect of antiserum against TLR2 on pit formation by osteoclast precursors on coculture of mouse bone marrow cells with MC3T3G2/PA6. Osteoclast precursors were cocultured with P. gingivalis 67kDa ¢mbriae or E. coli LPS (E), and both P. gingivalis 67-kDa ¢mbriae or E. coli LPS, anti-TLR2 (F). The total area per dentine slice was signi¢cantly decreased by adding anti-TLR2 to the cultures of P. gingivalis 67-kDa ¢mbriae or E. coli LPS (P 6 0.01).

FEMSLE 11281 25-11-03

54

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

demonstrated the role of ¢mbriae or pili in osteoclastogenesis. Therefore, we examined the stimulatory e¡ect of P. gingivalis 67-kDa ¢mbriae on pit formation, which represents bone resorption by osteoclasts. In the assay, we used osteoclast precursors as target cells di¡erentiated from mouse bone marrow cells, which were cocultured with MC3T3-G2/PA6 in culture medium containing RANKL and M-CSF. RANKL is a recently identi¢ed member of the TNF ligand family [29] and M-CSF is essential in the development of osteoclasts in vivo and in vitro [30]. These factors are known to support the formation of osteoclast-like multinuclear cells from their precursors [29]. As shown in Figs. 3 and 4, stimulation with 67kDa ¢mbriae obviously enlarged the pit-formed area compared with non-stimulated control. Kawata et al. [31] reported that P. gingivalis 41-kDa ¢mbriae stimulated bone resorption in vitro using mouse calvarial bone cells. In a recent study, Umemoto et al. [9] demonstrated a role of P. gingivalis 67- and 41-kDa ¢mbriae in inducing alveolar bone loss in an orally infected rat model with P. gingivalis 67-kDa ¢mbriae- and 41-kDa ¢mbriae-de¢cient mutant strains, MPG67 and MPG1, respectively. As a result, the level of alveolar bone loss in rats infected with MPG1 was higher than that in rats infected with MPG67, suggesting that 67-kDa ¢mbriae might be more essential for bone resorption. Our result indicates that the 67-kDa ¢mbriae are capable of enhancing the bone-resorbing activity of osteoclasts by inducing in£ammatory cytokines. Pretreatment of C3H/HeN murine peritoneal macrophages with anti-TLR2 antibody (5 Wg ml31 ) signi¢cantly inhibited the production of cytokines induced by P. gingivalis 67-kDa ¢mbriae, however, no e¡ect was observed in macrophages with E. coli LPS (Fig. 2 and Table 1). The 67-kDa ¢mbrial protein was also capable of boosting IL-1, IL-6 and TNF-K production in LPS non-responsive C3H/ HeJ murine peritoneal macrophages, whereas E. coli LPS had no e¡ect (data not shown). It was recently demonstrated that TLR4 plays an important role in LPS-mediated immune response as a receptor of bacterial LPS and lipid A [25]. C3H/HeJ mice were shown to have an inactivating point mutation within the signal-transducing domain of the Tlr4 gene [25]. It has been reported that P. gingivalis LPS stimulated cytokine production in C3H/HeJ mouse cells and there has been evidences that TLR2 was involved in the LPS signaling pathway of this organism [32]. In the puri¢ed 67-kDa ¢mbrial sample, however, the trace of LPS was not detected by Toxicolor LS-50M set (Seikagaku Kougyo Co, Tokyo, Japan). Meanwhile, TLR2 is known to play an important role in the signaling of various bacterial components, such as peptidoglycan and lipoteichoic acid. Ogawa et al. reported that P. gingivalis 41-kDa ¢mbriae induced IL-6 production via human TLR2 on human monocytes [33]. Our result strongly suggests that TLR2 is involved in in£ammatory cytokine production by 67-kDa ¢mbriae of P. gingivalis in murine macrophages.

In order to determine the role of TLR2 in the activation of osteoclasts by the ¢mbrial protein, osteoclast precursors were pretreated with anti-TLR2 antibody. Interestingly, osteoclast precursors were more susceptible to anti-TLR2 antibody ; pit formations stimulated with 67-kDa ¢mbriae were blocked by anti-TLR2 at 0.02 Wg ml31 , which had no e¡ect on negative control. The inhibitory e¡ect was observed on the pit-forming activity of E. coli LPS. Takami et al. examined the role of TLRs during osteoclast di¡erentiation and found clear evidence that osteoclast precursors prepared from mouse bone marrow cells expressed murine TLR1^TLR9, however, only TLR2 and TLR4 were expressed in osteoclasts [34]. There are some lines of evidence indicating that both TLR2 and TLR4 are involved in responses to bacterial LPS, leading to the expression of proin£ammatory cytokines IL-1, TNF-K, IL-6 and IL-8 [17^19]. This result suggests that bacterial LPS may be able to activate osteoclastogenesis through TLR2 and TLR4. We examined inhibitory e¡ects of anti-TLR4 antibody on pit formation and obtained the evidence that anti-TLR4 (0.1 Wg ml31 ) did not signi¢cantly block pit formation induced by 67-kDa ¢mbriae but by E. coli LPS (data not shown). As a consequence, the 67-kDa ¢mbriae of P. gingivalis stimulate murine macrophages utilizing TLR2 to lead to in£ammatory cytokine production and osteoclast activation and are implicated in the development of periodontal disease.

References [1] Ha¡ajee, A.D. and Socransky, S.S. (1994) Microbial etiological agents of destructive periodontal diseases. Periodontal 5, 78^111. [2] Handley, P.S. and Tipler, L.S. (1986) An electron microscope survey of the surface structures and hydrophobicity of oral and non-oral species of the bacterial genus Bacteroides. Arch. Oral Biol. 31, 325^ 335. [3] Lamont, R.J. and Jenkinson, H.F. (1998) Life below the gum line: pathogenic mechanism of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev. 62, 1244^1263. [4] Hamada, N., Watanabe, K., Sasakawa, C., Yoshikawa, M., Yoshimura, F. and Umemoto, T. (1994) Construction and characterization of a ¢mA mutant of Porphyromonas gingivalis. Infect. Immun. 62, 1696^1704. [5] Hamada, N., Sojar, H.T., Cho, M.I. and Genco, R.J. (1996) Isolation and characterization of a minor ¢mbria from Porphyromonas gingivalis. Infect. Immun. 64, 4788^4794. [6] Hanazawa, S., Murakami, Y., Hirose, K., Amano, S., Ohmori, Y. and Higuchi, H. (1991) Bacteroides (Porphyromonas) gingivalis ¢mbriae activate mouse peritoneal macrophages and induce gene expression and production of interleukin-1. Infect. Immun. 59, 1972^1977. [7] Hanazawa, S., Hirose, K., Ohmori, Y., Amamo, S. and Kitano, S. (1988) Bacteroides gingivalis stimulate production of thymocyte-activating factor by human gingival ¢broblasts. Infect. Immun. 56, 272^ 274. [8] Hamada, N., Watanabe, K., Arai, M., Hiramine, H. and Umemoto, T. (2002) Cytokine production induced by a 67-kDa ¢mbrial protein from Porphyromonas gingivalis. Oral Microbiol. Immunol. 17, 197^ 200. [9] Umemoto, T. and Hamada, N. (2003) Characterization of biologically active cell surface components of a periodontal pathogen. The

FEMSLE 11281 25-11-03

H. Hiramine et al. / FEMS Microbiology Letters 229 (2003) 49^55

[10] [11] [12]

[13]

[14] [15] [16] [17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

roles of major and minor ¢mbriae of Porphyromonas gingivalis. J. Periodontol. 74, 119^122. Manolagas, S.C. and Jilka, R.L. (1992) Cytokine, hematopoiesis, osteoclastogenesis, and estrogens. Calcif. Tissue Int. 50, 199^202. Roodman, G.D. (1992) Role of cytokines in the regulation of bone resorption. Calcif. Tissue Int. 53, 94^98. Gowen, M., Wood, D.D. and Russel, R.G. (1985) Stimulation of the proliferation of human bone cells in vitro by human monocyte products with interleukin-1 activity. J. Clin. Invest. 75, 1223^1229. Canalis, E. (1986) Interleukin-1 had independent e¡ects on deoxyribonucleic acid and collagen synthesis in cultures of rat calvariae. Endocrinology 118, 74^81. Medzhitov, R. and Janeway, C. (2000) The Toll receptors family and microbial recognition. Trends Microbiol. 10, 452^456. Kopp, E.B. and Medzhitov, R. (1999) The Toll-receptor family and control of innate immunity. Curr. Opin. Immunol. 11, 13^18. Wright, S.D. (1999) Toll, a new piece in the puzzle of innate immunity. J. Exp. Med. 189, 605^609. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K. and Akira, S. (1999) Toll-like receptor 4 (TLR4)dependent mice are lyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749^3752. Kirschning, C.J., Wesche, H., Merrillayres, T. and Rothe, M. (1998) Human Toll-like receptor-2 confers responsiveness to bacterial lipopolysaccharide. J. Exp. Med. 188, 2091^2097. Yang, R.B., Mark, M.R., Gray, A., Huang, A., Xie, M.H., Zhang, M., Goddard, A., Wood, W.I., Gurney, A.L. and Godowski, P.J. (1998) Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signaling. Nature 395, 284^288. Chuang, T.H. and Ulevitch, R.J. (2000) Cloning and characterization of a subfamily of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur. Cytokine Netw. 11, 372^378. Chuang, T.H. and Ulevitch, R.J. (2001) Identi¢cation of hTLR10 : a novel human Toll-like receptor preferentially expressed in immune cells. Biochim. Biophys. Acta 1518, 157^161. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sto, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K. and Akira, S. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740^745. Medzhitov, R., Preston-Hurburt, P. and Janeway Jr., C.A. (1997) A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388, 394^397. Takeuchi, O., Kaufmann, A., Grote, K., Kawai, T., Sanjo, H., Copeland, N.G., Gillbert, D.J., Jenkins, N.A., Takeda, K. and Akira, S.

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

55

(1999) TLR6: a novel member of an expanding toll-like receptor family. Gene 231, 59^65. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Hu¡el, C.V., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B. and Beutler, B. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085^2088. Frendeus, B., Wachtler, C., Hedlund, M., Fischer, H., Samuelsson, P., Svensson, M. and Svanborg, C. (2001) Escherichia coli P ¢mbriae utilize the Toll-like receptor 4 pathways for cell activation. Mol. Microbiol. 40, 37^51. Asai, Y., Ohyama, Y., Gen, K. and Ogawa, T. (2001) Bacterial ¢mbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2. Infect. Immun. 69, 7384^7395. Roodman, G.D., Ibbotson, K.J., MacDonald, B.R., Kuehl, T.J. and Mundy, G.R. (1985) 1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow. Proc. Natl. Acad. Sci. USA 82, 8231^ 8237. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami, A., Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Takahashi, N. and Suda, T. (1998) Osteoclast di¡erentiation factor is a ligand for osteoprotegrin/TRANCE/RANKL. Proc. Natl. Acad. Sci. USA 95, 3597^3602. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M.T. and Martin, T.J. (1999) Modulation of osteoclast di¡erentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 20, 345^357. Kawata, Y., Hanazawa, S. and Amano, S. (1994) Porphyromonas gingivalis ¢mbriae stimulate bone resorption in vitro. Infect. Immun. 62, 3012^3016. Hirschfeld, M., Weis, J.J., Toshchakov, V., Salkowski, C.A., Cody, M.J., Ward, D.C., Qureshi, N., Michalek, S.M. and Vogel, S.N. (2001) Signaling by Toll-like receptor 2 and 4 agonists results in di¡erential gene expression in murine macrophages. Infect. Immun. 69, 1477^1482. Ogawa, T., Asai, Y., Hashimoto, M. and Uchida, H. (2002) Bacterial ¢mbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors. Eur. J. Immunol. 32, 2543^2550. Takami, M., Kim, N., Rho, J. and Choi, Y. (2002) Stimulation by Toll-like receptors inhibits osteoclast di¡erentiation. J. Immunol. 169, 1516^1523.

FEMSLE 11281 25-11-03