Fimbriae from Porphyromonas gingivalis Induce Chemiluminescence Response of Macrophages in

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1 Note Bifidobacteria Microflora Vol. 11 (1), 39-43, 1992 Fimbriae from Porphyromonas gingivalis Induce Chemiluminescence Response of Macrophages in a Different Manner to Its Lipopolysaccharide Emiko ISOGAI,1 Hiroshi ISOGAI,2 Nobuhiro Fujii,3 Kimiharu HIROSE,1 Hitomi WAKIZAKA,1 Hiroko MIURA1 and Fuminobu YOSHIMURA4 Department of Preventive Dentistry, Higashi Nippon Gakuen University, 1Ishikari- Tobetsu 1757, Hokkaido , 2Division of Animal Experimentation and 3Department of Microbiology, Sapporo Medical College, Sapporo 060 and 4Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Chikusa, Nagoya 464 (Received for publication, January 27, 1992) Abstract Fimbriae from Porphyromonas gingivalis were found to induce an early chemiluminescence response of mouse peritoneal macrophages. A significant dose-dependent increase in the response was observed. The response induced by the fimbriae was different from that induced by the lipopolysaccharide (LPS). Viable P. gingivalis whole cells stimulated macrophages and the chemiluminescence response cojoined the two responses induced by fimbriae and LPS. These observations suggest that P. gingivalis fimbriae may be involved in the pathogenesis of adult periodontal disease via the triggering of reactive oxygen intermediates by macrophages in the disease in a different manner to its LPS. Key words : P. gingivalis; fimbriae; chemiluminescence The Gram-negative anaerobic organism Porphyromonas (formerly Bacteroides) gingivalis has been implicated in the etiology of human periodontal disease (14). P. gingivalis has fimbriae on its cell. surface (12). Yoshimura et al (16) characterized chemical, morphological and immunological properties of the purified fimbriae. However, the function of fimbriae was not fully understood. Recently we demonstrated fimbriae-associated bacterial adhesion by using monoclonal antibodies (8). Although a number of studies have shown that LPS from P. gingivalis has a potent ability to stimulate macrophages (6, 7), little is known about the function of the fimbriae with respect to monocytes/macrophages. Hanazawa et al (5) showed that P. gingivalis fimbriae activate macrophages and subsequently induce gene expression and production of interleukin-1 (IL-1) in the cells. In this study, we examined the chemiluminescence response of macrophages induced by the fimbriae. We showed that P. gingivalis fimbriae induce a chemiluminescence response in different manner to its LPS. 39

2 40 E. ISOGAI et al Peritoneal macrophages were obtained from BALB/c mice 8 to 10 weeks of age by the method previously described (7). The cells were suspended (5 ~ 106 cells/ml) in RPMI 1640 (ph 7.4) supplemented 10% fetal calf serum, L-glutamine (2 mm), sodium pyruvate (1 mm), and HEPES (20 mm) without phenol red. The same buffer was used for luminol solution and stimulant solution. One hundred ƒêl of macrophage suspension and 10 ƒêl of luminol solution (2 mg/ml) were mixed After 10 min, 10 ƒêl of stimulant in various concentrations was added and the chemiluminescence response was measured by luminescence analyzer (Biolomat LB 9500 Berthold, Germany).., P. gingivalis 381 was grown anaerobically at 37 C in GAM broth (Nissui Co., Japan). Fimbriae (16) and lipopolysaccharide (7) were extracted from P. gingivalis 381 and purified by the methods previously described, respectively. In brief, cells were harvested by centrifugation and suspended in 20 mm tris-hydrochloride (ph 7.4) containing 0.15 M NaCl and 20 mm MgCl2 by repeated pipetting. The suspension was agitated by stirring for 30 min. The bacterial washings were obtained as the supernatant after centrifugation at 8000 ~g for 20 min. The supernatants were treated by ammonium sulfate (40% saturation). The precipitated protein was collected by centrifugation and suspended in 20 mm Tris-hydrochloride (ph 8.0). The sample was dialyzed, centrifuged at 8000 ~ g for 20 min, applied to column of DEAE-Sepharode CL-6B, and eluted with a linear gradient of 0 to 0.3 M NaCl in 20 mm Tris-hydrochloride (ph 8.0). The fractions eluted at 0.15 M NaCl were pooled, concentrated by ammonium sulfate preparation, and dialyzed against 3 mm sodium bicarbonate (ph 8.0). The purity was assessed by the stained SDSpolyacrylamide gel, and at least 98%. For LPS extraction, the fimbriae-removed bacterial cells were collected by centrifugation at 10,000 ~ g for 15 min. The bacterial pellet was resuspended in distilled water. The LPS was extracted by the hot-phenol method and purified by the method previously described (7). The final product contained 28% carbohydrate (as neutral sugar), 16% lipid and 8% protein. They were used for stimulant in the chemiluminescence assay. Viable P. gingivalis 381 (a fimbriate strain, 2 ~ 108/ml, 10 ƒêl) was used as stimulant for the chemiluminescence assay. Fimbriae from P. gingivalis induced a chemiluminescence response of macrophages of BALB/c mice. Figure 1 shows a typical pattern of chemiluminescence. The first peak (peak 1) was observed at 1.1 }0.8 (mean }SD) min and the second peak (peak 2) was observed at 18.8 }1.5 min, after stimulation with the fimbriae. The chemiluminescence pattern in macrophages stimulated with the fimbriae was different from that in the cells stimulated with LPS; LPS induced "peak 2" as a main peak but fimbriae induced peak 1 as a main peak. Viable cells of P. gingivalis 381 also stimulated macrophages and the response cojoined two responses induced by the fimbriae and LPS. Peak 1 level was 3,024 }563 (mean }SD) and peak 2 level was 2,672 }318 (mean±sd), respectively. Peak positions were similar to those of fimbriae- or LPS-stimulation; peak 1:1.1 }0.6, peak 2: 21.5 }1.8. Table 1 shows the peak count of chemiluminescence of the macrophages induced by fimbriae and LPS. A significant dose-dependent increase in the response was observed.

3 MACROPHAGES AND FIMBRIAE OF P. GINGIVALIS 41 Fig. 1. Pattern of chemiluminescence response to fimbriae (10, Đg) and LPS (1 Đg) from P. gingivalis 381. Viable cells (2 ~ 108/ml, 10 Đl) of P. gingivalis also induce chemiluminescence response (...). The arrow indicates the start of stimulation. Table 1. Chemiluminescence response of macrophages to fimbriae and LPS from P. gingivalis 381 a ND, not done. Destruction and alteration of periodontal connective tissues in periodontal disease are thought to result from both direct action of bacterial products and the indirect action of inflammatory responses induced by bacterial infection (13). Macrophages have a wide range of biological functions related to immune response and connective tissue metabolism. In particular, oxygen radicals have been considered as possessing bactericidal properties but also, because of their toxicity, to cause tissue damage during inflammation. The generation of reactive oxygen

4 42 E. ISOGAI et al intermediates by macrophages seems to be of great importance and is linked to their cytocidal (2, 9), immuno-regulatory (11) and probably to other activities (3, 10). The present study has shown that fimbriae have a potential activity to stimulate macrophages in a different manner to LPS. Various responses such as cytokine production could be induced in macrophages stimulated with fimbriae. Actually, it has been reported that thymocyte-activating factor is produced by human gingival fibroblasts (1) after stimulation of fimbriae from P. gingivalis (4). Furthermore, the fimbriae strongly induced gene expression and production of IL-1 in the macrophages (5). They discussed that the fimbriae induced a marked increase in the c-myc mrna level (a macrophage activation marker) in mouse macrophages 1 hr after the treatment. In contrast, chemiluminescence response was observed in mouse macrophages 1.1 min after the treatment. Such a different stage of macrophage activation was present and the oxygen metabo - lism before the increase of c-myc gene expression may be important for a mechanism (s) for regulation of transcription of the gene. Viable P. gingivalis 381 stimulated macrophages and the chemiluminescence response cojoined the two responses by fimbriae and LPS. Various components were present on the bacterial surface. Capsular polysaccharides and membrane proteins may stimulate macrophages. Therefore, the chemiluminescence response by whole cells could reflect the distribution and content of various cell surface components. P. gingivalis strains with or without fimbriae have been clinically isolated (12, 15). Our data showed that fimbriated P. gingivalis stimulated macrophages with higher potency than a non-fimbriated strain. The structure of bacterial cell surface is important in the host response and the presence of fimbriae may contribut e to the pathogenicity via macrophage activation. REFERENCES (1) Charon, J.A., T.A. Luger, H.E. Mergenhagen, and J.J. Oppenheim Increased thymocyte-activating factor in human gingival fluid during gingival inflammation. Infect. Immun.38 : (2) Freeman, B.A., and J.D. Crapo Biology of disease: free radicals and tissue injury. Lab.I nvest. 47: (3) Grygelwski, R.J., R.M. J. Palmer, and S. Moncada Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: ( 4) Hanazawa, S., K. Hirose, Y. Ohmori, S. Amano, and S. Kitano Bacteroides gingivalis fimbriae stimulate production of thymocyte-activating factor by human nfect. Immun. 56: gingival fibroblast.i (5) Hanazawa, S., Y. Murakami, K. Hirose, S. Amano, Y. Ohmori, H. Higuchi, and S. Kitano Bacteroides (Porphyromonas) gingivalis fimbriae activate mouse peritoneal m acrophages andi nduce gene expression and production of interleukin-1. Infect. Immun. 59: (6) H anazawa, S., K. Nakada, Y. Ohmori, T. Miyoshi, S. Amano, and S. Kitano Functional role of interleukin-1 in periodontal disease: induction of interleukin -1 production by Bacteroides gingivalis lipopolysaccharide in peritoneal macrophages from C3H/HeN and C3H/HeJ mice.i nfect. Immun. 50 :

5 MACROPHAGES AND FIMBRIAE OF P. GINGIVALIS 43 (7) Isogai, H., E. Isogai, N. Fujii, K. Oguma, W. Kagota, and K. Takano Histological changes and some in vitro biological activities induced by lipopolysaccharide from Bacteroides gingivalis. Zentralbl. Bakteriol. Hyg. A269: (8) Isogai, H., E. Isogai, F. Yoshimura, T. Suzuki, W. Kagota, and K. Takano Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae. Archs Oral Biol. 33: (9) Nathan, C.F., H.W. Murray, M.E. Wiebe, and B.Y. Rubin Identification of interferon-ć as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158: (10) Neill, M.A., W.R. Henderson, and S.J. Klebanoff Oxidative degeneration of leukotriene C4 by human monocytes and monocyte-derived macrophages. J. Exp. Med. 162: (11) Novogrodsky, A., A. Ravid, A.L. Rubin, and K.H. Stenzel Hydroxyradical scavengers inhibit lymphocyte mitogenesis. Proc. Natl. Acad. Sci. 79: (12) Okuda, K., J. Slots, and R.J. Genco Bacteroides gingivalis, Bacteroides asaccharolyticus and Bacteroides melaninogenicus subspecies: cell surface morphology and adherence to erythrocytes and human buccal epithelial cells. Curr. Microbiol. 6: (13) Page, R.C., and H.E. Schroeder Current status of the host response in chronic marginal periodontitis. J. Periodontol. 52: (14) Slots, J The predominant cultivable microflora of advanced periodontitis. Scand. J. Dent. Res. 85: (15) Suzuki, Y., F. Yoshimura, K. Takahashi, H. Tani, and T. Suzuki Detection of fimbriae and fimbrial antigens on the oral anaerobe Bacteroides gingivalis by negative staining and serological methods. J. Gen. Microbiol. 134: (16) Yoshimura, F., T. Takasawa, M. Yoneyama, T. Yamaguchi, H. Shiokawa, and T. Suzuki Fimbriae from the oral anaerobe Bacteroides gingivalis; physical, chemical, and immunological properties. J. Bacteriol. 163: