Porphyromonas gingivalis Fimbriae Stimulate Bone Resorption In Vitro

INFECTION AND IMMUNITY, JUly 1994, p. 3012-3016 0019-9567/94/$04.00+0

Vol. 62, No. 7

Copyright C 1994, American Society for Microbiology

Porphyromonas gingivalis Fimbriae Stimulate Bone Resorption In Vitro YASUHIRO KAWATA, SHIGEMASA HANAZAWA,* SHIGERU AMANO, YUKIO MURAKAMI, TOSHITSUGU MATSUMOTO, KOHJI NISHIDA, AND SHIGEO KITANO Department of Oral Microbiology, Meikai University School of Dentistry, Keyakidai, Sakado City, Saitama 350-02, Japan Received 4 October 1993/Returned for modification 8 November 1993/Accepted 6 April 1994

Our previous study demonstrated that Porphyromonas gingivalis fimbriae induce the expression of interleukin-1, a potent bone-resorbing cytokine, in macrophages. This demonstration suggested to us the possibility that the fimbriae may stimulate bone resorption via the generation of an inflammatory cytokine(s). The present study was performed to test this suggestion. The bone-resorbing activity was evaluated by measuring the area of resorption lacunae on bone slices incubated with calvarial bone cells taken from 14-day-old mouse embryos. Fimbriae at 0.5 ,ug of protein per ml stimulated the bone-resorbing activity significantly, and the effect was dose and treatment time dependent. Since it is well known that interleukin-1 and granulocyte macrophage colony-stimulating factor induce differentiation of osteoclast lineage cells, we examined the involvement of these cytokines in fimbria-stimulated bone resorption. Fimbria-stimulated bone resorption was abolished significantly by antisera against both cytokines. We observed by Northern (RNA) blot assay that both cytokine genes were markedly expressed in the fimbria-treated calvarial bone cells. Our present data demonstrate that P. gingivalis fimbriae stimulate bone resorption in vitro. Adult periodontal disease is one characterized by inflammation of the gingiva and resorption of alveolar bone into the periodontal tissues. We (11-13, 20) previously demonstrated that Porphyromonas gingivalis fimbriae induced gene expression and production of some inflammatory cytokines in human gingival fibroblasts and mouse peritoneal macrophages. Ogawa et al. (21) also have shown that synthetic peptides of the fimbrial protein induced significant production of interleukin-6 (IL-6) and tumor necrosis factor alpha by human peripheral mononuclear cells. Since many investigators (2, 10, 22-24) have demonstrated that inflammatory cytokines such as IL-1 (M.W. kDa.)

and tumor necrosis factor alpha are potent stimulators of bone resorption, induction of inflammatory cytokine production by P. gingivalis fimbriae prompted us to explore the possibility that the fimbriae may be involved in the pathogenesis of the alveolar bone loss observed in adult periodontal disease. The mechanism of alveolar bone resorption in periodontal disease has not yet been well defined. However, knowledge of it is very important for understanding the pathogenesis of P. gingivalis in the disease. Many studies (e.g., reference 19) have demonstrated that differentiation of osteoclast lineage cells

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(jg protein/ml) (gg protein/ml) FIG. 2. Inducing effect of P. gingivalis fimbriae on appearance of TRAP-positive cells and bone-resorbing activity of mouse embryonic calvarial bone cells. Calvarial bone cells (5 x 105 cells per bone slice) prepared from 14-day-old mouse embryos were inoculated onto bone slices and the slices were incubated in FCS (10%)-containing a-MEM with or without various doses of the fimbriae. After 7 days, TRAPpositive cells (a) and the resorbed area (b) on the bone slices were measured. The results are expressed as the means ± SE of four replicate cultures. Three identical experiments, independently performed, gave similar results.

21.514.3-_FIG. 1. SDS-PAGE of P. gingivalis fimbriae. (a) Silver stain for protein; (b) silver stain for LPS. Arrows show the positions of proteins used as apparent molecular weight markers (103).

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Corresponding author. Fax: 081-492-87-6657. 3012

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FIG. 3. Scanning electron micrograph of P. gingivalis fimbria-induced bone resorption lacunae excavated by mouse embryonic calvarial bone cells. Calvarial bone cells (5 x 105 cells per bone slice) prepared from 14-day-old mouse embryos were inoculated onto bone slices, and the slices were incubated in FCS (10%)-containing cx-MEM with or without the fimbriae (4 ,ug of protein per ml). After 7 days, the slices were examined with a scanning electron microscope for the presence of resorption lacunae.

and activation of mature osteoclasts are regulated by the cooperation among systemic and local cytokines. Therefore, it is possible that P. gingivalis may stimulate resorption of alveolar bone via a network of inflammatory cytokines produced by periodontal tissues triggered by the fimbriae, as described above. We (3) recently devised an assay system that assesses bone-resorbing activity, one involving the incubation of calvarial bone cells derived from 14-day-old mouse embryos on devitalized bone slices. This novel assay system is useful for assessment of the effect of cytokines and hormones on differentiation of osteoclast lineage cells and activation of mature osteoclasts. Using this assay, we performed the present study to test the assumption that P. gingivalis fimbriae may stimulate bone resorption via a network of cytokines produced by certain host cells. P. gingivalis ATCC 33277 fimbriae were prepared and purified from the cell washing solution according to the method of Yoshimura et al. (25), as described previously (11). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) for the fimbrial preparation was performed as de-

scribed previously (14). As shown in Fig. 1, the fimbrillin monomer of the purified fimbriae was observed on gels of SDS-PAGE as a single band having a molecular weight of 43,000; however, lipopolysaccharide (LPS) contamination of the fimbrial preparation was not detected by silver staining. The protein content of the fimbriae was measured by the method of Bradford (4). Cells producing monoclonal antibody to P. gingivalis ATCC 33277 fimbriae were obtained by a hybridoma technique. This monoclonal antibody recognizes the fimbrillin having a molecular weight 43,000 with high specificity. The culture supernatant of the monoclonal antibody-producing cells was used to examine the specificity of fimbria-stimulated bone resorption. The bone resorption assay was described in detail in our previous study (3). In brief, ICR mouse embryos at the age of 14 days (CLEA Japan, Tokyo, Japan) were dissected, and their calvariae were harvested. The calvariae were digested at room temperature for 30 min in phosphate-buffered saline (pH 7.2) containing 0.1% bacterial collagenase (Sigma, St. Louis, Mo.), 0.05% trypsin (Difco Laboratories, Detroit, Mich.), and 4 mM

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FIG. 4. Kinetics of P. gingivalis fimbria-induced bone-resorbing activity of mouse embryonic calvarial bone cells and inhibitory effect of monoclonal antibody on bone-resorbing activity. (a) Calvarial bone cells (5 x 105 cells per bone slice) prepared from 14-day-old mouse embryos were inoculated onto bone slices, and incubated in FCS (10%)-containing a-MEM with or without the fimbriae (4 p.g of protein per ml). After the selected incubation times, the resorption areas on the bone slices were measured. (b) Calvarial bone cells were cultured under the culture conditions described above. Bone cells incubated with the fimbriae were cultured for 7 days in the presence or absence of culture supernatant (1/50 dilution) of cells producing monoclonal antibody to the fimbriae. The results are expressed as the means SE of four replicate cultures. Three identical experiments, independently performed, gave similar results. ±

EDTA. The digested calvarial bone cells, at a density of 5 x 105 cells per 15 ,ul of a-Eagle's minimum essential medium (a-MEM; Flow Laboratories) containing 10% fetal calf serum (FCS; Flow Laboratories), were placed on a bovine femoral bone slice (4 by 4 mm) in each well of a 24-well flat-type Falcon plastic plate. After the bone cells had been incubated for 60 min, they were cultured for the desired times in 1 ml of a-MEM with or without the fimbriae or test samples. At selected times, the bone slices were scraped with a rubber policeman to remove cells to enable visualization of the bone surface. The scraped bone slices were dehydrated with ethanol and sputter coated with gold. Then the bone slices were examined with a T200 scanning electron microscope (Japan Electronics Co., Tokyo, Japan). The resorbed area on the bone slices was traced onto polyethylene film and then measured with a Shonic graphic analyzer (Showa Denko Co., Tokyo, Japan). Results were expressed as means ± standard errors

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FIG. 5. P. gingivalis fimbriae induce IL-1,B and GM-CSF gene expression in mouse embryonic calvarial bone cells. Calvarial bone cells (2 x 107) prepared from 14-day-old mouse embryos were inoculated in 90-mm plastic dishes with FCS (10%)-containing a-MEM with or without the fimbriae (4 ,ug of protein per ml). After 7 days, the calvarial bone cells were washed, and then the washed cells (2 X 107) were incubated in serum-free oa-MEM with or without the fimbriae (4 jig of protein per ml). Total RNA was prepared 5 h after the initiation of fimbrial treatment. Northern blot analysis was performed with IL-1,B, GM-CSF, and ,-actin cDNAs as probes.

(SE) of four replicate cultures. Significant differences were determined by one-way analysis of variance. Tartrate-resistant acid phosphatase (TRAP)-positive cells among the calvarial bone cells incubated on bone slices were detected as described previously (3). TRAP-positive cells were counted under a light microscope, and the results were expressed as the means ± SE of four replicate cultures. Total cellular RNA was extracted by the guanidine isothiocyanate procedure (6). Northern (RNA) blot analysis was done as described previously (15). Primer-extended cDNA probes labeled with 5'-[a-32P]dCTP (Amersham Japan, Tokyo, Japan) for mouse IL-1lB (provided by T. Hamilton), mouse granulocyte macrophage colony-stimulating factor (GM-CSF; American Type Culture Collection), and 1-actin (Oncor, Gaithersburg, Md.) were used for hybridization to total cellular RNA. 13-Actin was used as an internal standard for quantification of total mRNA on each lane of the gel. Goat anti-mouse IL-1lB antiserum was obtained from R&D Systems, Minneapolis, Minn. The 50% neutralization dose of the IL-1,B antiserum was 1 jig/ml. Rat anti-mouse GM-CSF antiserum was from Oncogene Scientific Inc., Manhasset, N.Y. The 50% neutralization dose of the GM-CSF antiserum was 0.5 ,ug/ml. Cross-reactions with other cytokines were not observed in either antiserum. We first examined the effect of the fimbriae on osteoclastic bone resorption by calvarial bone cells from 14-day-old mouse embryos. Figure 2a shows that the fimbriae increased, in a dose-dependent fashion, the number of cells positive for TRAP, a marker enzyme of osteoclasts, among the calvarial bone cells. Also, as shown in Fig. 2b, significant bone-resorbing activity was elicited by treatment with the fimbriae at 0.5 jig of protein per ml, and the effect was dose dependent (0.5 to 4 ,ug of protein per ml). However, TRAP-positive cells and bone-

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incubation for 7 days, the fimbria-stimulated bone resorption was measured. As shown in Fig. 6, the fimbria-stimulated bone resorption was markedly inhibited by treatment with either anti-IL-1l3 or anti-GM-CSF antiserum. These results strongly suggest the possibility that both cytokines produced by the bone cells in response to the fimbriae contribute to the

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gingivalis fimbria-induced bone-resorbing activity. Calvarial bone cells (5 x 105 cells per bone slice) prepared from 14-day-old mouse embryos were inoculated onto bone slices, and the slices were incubated in FCS (10%)-containing a-MEM with or without the fimbriae (4,ug of protein per ml). Anti-IL-1 antiserum (20 ,ug/ml) or antiGM-CSF antiserum (1,ug/ml), respectively, was simultaneously added to the cultures. After 7 days, the resorbed areas on the bone slices were SE of four measured. The results are expressed as the means replicate cultures. Three identical experiments, independently performed, gave similar results. * P < 0.01 versus no antiserum. on P.

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resorbing activity were not observed when the calvarial bone were left untreated. Figure 3 shows scanning electron microscope profiles of bone slices incubated with the calvarial bone cells in the absence or presence of the fimbriae. Bone resorption lacunae on the bone slice were clearly observed when the bone cells were incubated with the fimbriae. However, such lacunae were not detected on those incubated in the absence of the fimbriae. Next we examined the kinetics of the stimulatory effect of the fimbriae on bone-resorbing activity of the mouse embryonic calvarial bone cells. The fimbriae (4 ,ug of protein per ml) were added to the cell cultures on the bone slices. After incubation for 7 days, the area of bone resorption lacunae was measured. As shown in Fig. 4a, the stimulatory effect of the fimbriae on bone resorption activity increased in a treatment time-dependent fashion. We examined the specificity of the fimbria-stimulated bone resorption by using a monoclonal antibody against the fimbriae. Figure 4b shows that the fimbria-stimulated bone resorption was completely inhibited by the monoclonal antibody. Our previous report (12) showed that P. gingivalis fimbriae induce IL-lp expression in mouse macrophages. Since many recent studies (5, 9, 16, 18) have demonstrated that IL-1 and GM-CSF act as regulatory factors for differentiation of osteoclast lineage cells, we examined by Northern blot assay whether the fimbriae induce expression of IL-10 and GM-CSF genes in the calvarial bone cells. The calvarial bone cells were treated with the fimbriae (4 ,ug of protein per ml), and total RNA was prepared 5 h after initiation of the fimbrial treatment. Then the gene expression of both cytokines was measured. Figure 5 records the results. Although IL-lp and GM-CSF genes were not expressed in the untreated calvarial bone cells, significant gene expression of both cytokines was induced in the fimbria-treated cells. Finally, using anti-mouse IL-13 and GM-CSF antisera, we examined whether IL-1,B and GM-CSF are involved in fimbriastimulated bone resorption. The calvarial bone cells were incubated in culture medium supplemented with fimbriae (4 ,ug of protein per ml) in the absence or presence of anti-mouse IL-lp antiserum or anti-mouse GM-CSF antiserum. After cells

fimbria-stimulated bone resorption. Recently, some interesting studies (7, 8) have shown that gnotobiotic rats immunized with P. gingivalis fimbriae were protected against periodontal tissue destruction by the bacterium. Although the mechanism of alveolar bone resorption in adult periodontal disease has not been yet demonstrated, this report strongly suggests that the fimbriae are involved in the pathogenesis of alveolar bone in the disease. Here we showed that the fimbriae markedly stimulate bone resorption in vitro via the action of IL-13 and GM-CSF produced by calvarial bone cells triggered with fimbriae. Thus, our present study suggests a functional role for the fimbriae in the initiation and development of resorption of alveolar bone in adult periodontal disease. P. gingivalis fimbriae had both dose- and culture timedependent effects on bone-resorbing activity of calvarial bone cells derived from mouse embryos. The stimulatory effect was mediated significantly by IL-1,B and GM-CSF produced by the calvarial bone cells, as evidenced by the results shown in Fig. 5 and 6. The stimulatory effect of the fimbriae was not due to LPS contamination because monoclonal antibody against the fimbriae completely inhibited the stimulatory effect, and LPS in the fimbrial preparation was not detectable by silver staining. Further, stimulation of inflammatory cytokine production by several kinds of cells has been demonstrated by treatment of the cells with synthetic peptides of the fimbrial protein (21). Also, in the case of Escherichia coli fimbriae, recent reports (1, 17) indicated that the S and P fimbriae induced IL-6 and IL-8 production by renal epithelial and carcinoma cells. These reports confirm the role of fimbriae as inducers of inflammatory cytokine production. In conclusion, P. gingivalis fimbria-stimulated bone resorption may be regulated by a network of inflammatory cytokines

produced by calvarial bone cells triggered by fimbriae.

IL-1,

We express our gratitude to T. Hamilton for providing mouse cDNA. This work was supported by a Grant-in-Aid for Scientific Research (04671107, 05454490) from the Ministry of Education, Science, and Culture of Japan. REFERENCES 1. Agace, W., S. Hedges, U. Andersson, J. Andersson, M. Ceska, and C. Svanborg. 1993. Selective cytokine production by epithelial cells following exposure to Escherichia coli. Infect. Immun. 61:602-609. 2. Amano, S., S. Hanazawa, K. Hirose, Y. Ohmori, and S. Kitano. 1988. Stimulatory effect on bone resorption of interleukin-1-like cytokine produced by an osteoblast-rich population of mouse calvarial cells. Calcif. Tissue Int. 43:88-91. 3. Amano, S., S. Hanazawa, Y. Kawata, K. Ohta, H. Kitami, and S. Kitano. 1992. An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors. J. Bone Miner. Res. 7:321-328. 4. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 5. Chambers, T. J. 1988. The regulation of osteoclastic development and function. Ciba Found. Symp. 136:92-107. 6. Chirgwin, J. M. A., A. E. Przbyla, R. J. MacDonald, and W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. 7. Evans, R. T., B. Klausen, and R. J. Genco. 1992. Immunization with fimbrial protein and peptide protects against Porphyromonas

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