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Suppression of interleukin-10 release from human periodontal ligament cells by interleukin-1β in vitro
Archives of Oral Biology 45 (2000) 179±183
www.elsevier.com/locate/archoralbio
Short communication
Suppression of interleukin-10 release from human periodontal ligament cells by interleukin-1b in vitro J. Deschner a, B. Arnold b, A. Kage c, B. Zimmermann d, V. Kanitz b, J.-P. Bernimoulin b,* a Department of Operative Dentistry and Periodontology, University of Cologne, Germany Department of Periodontology, ChariteÂ, Humboldt University Berlin, FoÈhrer Str. 15, 13353 Berlin, Germany c Institute of Chemistry and Biochemistry, ChariteÂ, Humboldt University Berlin, Germany d Institute of Anatomy and Institute of Clinical Pharmacology and Toxicology, Free University of Berlin, Germany b
Accepted 7 September 1999
Abstract Periodontitis is characterized by an in¯ammatory process induced by periodontopathogenic bacteria in the subgingival plaque. Periodontal in¯ammation can be enhanced by both an increase of in¯ammatory stimulators, e.g. interleukin (IL)-6, and a decrease of in¯ammatory inhibitors, e.g. IL-10. The amount of IL-1b is known to be increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites compared to healthy sites. This in vitro study sought to clarify whether IL-1b (1 ng/ml) has a regulatory eect on the release of these two cytokines from human periodontal ligament (PDL) cells. PDL cells derived from healthy premolars were grown in the presence and absence (control) of IL-1b. The concentration of IL-6 and IL-10 in the supernatants was assessed by enzyme-linked immunosorbent assay after 48 h of culture. PDL cells incubated with IL-1b released signi®cantly ( p < 0.05) higher amounts of IL-6 and signi®cantly ( p < 0.01) smaller amounts of IL-10 compared to control. These results give further support to the observation that IL-1b can increase the IL-6 secretion from PDL cells. Moreover, they provide original evidence that PDL cells secrete IL-10, which can be suppressed by IL-1b. It is concluded that PDL cells can function as accessory immunoin¯ammatory cells amplifying the in¯ammatory process in periodontitis and, thereby, contributing to periodontal breakdown. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Periodontal ligament; Interleukin-1b; Interleukin-10; Periodontitis; Cell culture; Cytokine
Periodontitis is characterized by an in¯ammatory process, which is induced by periodontopathogenic bacteria in the subgingival plaque. The subgingival bacteria and their products stimulate host cells to
Abbreviations: IL, interleukin; MHC, major histocompatibility complex; PBS, phosphate-buered saline; TNF, tumour necrosis factor. * Corresponding author. Tel.: +49-30-45062632; fax: +4930-45062931.
release numerous in¯ammatory mediators, such as prostaglandins and cytokines (Page, 1991; BirkedalHansen, 1993). The periodontal in¯ammation can be enhanced by both an increase of in¯ammatory stimulators, e.g. IL-6, and a decrease of in¯ammatory inhibitors, e.g. IL-10. The proin¯ammatory IL-6 is synthesized by a variety of dierent cell types and represents one of the major physiological mediators of the acute-phase reaction (for review, see Hirano et al., 1990; Van Snick, 1990; Wolvekamp and Marquet,
0003-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 9 ) 0 0 1 2 0 - X
180
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
Fig. 1. Concentration of IL-6 in the supernatants of human periodontal ligament (PDL) cells in the presence [IL-1b (+)] and absence [IL-1b (ÿ)] of 1 ng/ml IL-1b after 48 h of culture.
1990). The immunosuppressive IL-10 was found in the supernatants of both type 1 helper and type 2 helper T-cell clones and inhibits their antigen-speci®c proliferation and cytokine production (De-Waal-Malefyt et al., 1991; 1993; Del-Prete et al., 1993). Human IL-10 is also a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration (Jinquan et al., 1993). In monocytes, IL-10 inhibits the production of IL-1, IL-6 and TNF-a (DeWaal-Malefyt et al., 1991), antagonizes the chemokine production (Berkman et al., 1995; Marfaing-Koka et al., 1996) and suppresses the upregulation of IL-1 receptor genes and class II MHC antigens (De-WaalMalefyt et al., 1991; Dickensheets and Donnelly, 1997). In polymorphonuclear leucocytes, IL-10 inhibits the production of IL-1a, IL-1b, IL-8 and TNF-a (Wang et al., 1994). In periodontitis, pathogenic bacteria in the subgingival plaque cause a subepithelial in®ltration by in¯ammatory cells, which produce and release IL-1b (Matsuki et al., 1993; Takeichi et al., 1994). This cytokine can also be produced by epithelial cells and gingival ®broblasts (Lundqvist et al., 1994; Agarwal et al., 1995; Hillmann et al., 1995). The amount of IL-1b is increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites compared to healthy sites
(Honig et al., 1989; Hou et al., 1995; Tsai et al., 1995). Little is known about the eects of IL-1b on human periodontal ligament cells. This in vitro study sought to clarify whether IL-1b has a regulatory eect on the release of IL-6 and IL-10 from human periodontal ligament cells. Cells from the middle third of the root surface of healthy premolars were used. The premolars were obtained from ®ve patients (one tooth per patient) who had to undergo regular orthodontic treatment. Informed consent was obtained from all patients. After extraction, the premolars were washed twice with Hank' salt solution (Biochrom, Berlin, Germany). The periodontal tissue was then removed with a surgical scalpel. The coronal and apical portions of the root were not used in order to avoid contamination by cells of the gingiva, nerves, and blood vessels. The tissue was minced and then cultured as explants in culture ¯asks (Nunc, Wiesbaden, Germany) containing Dulbecco's minimal essential medium supplemented with 15% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, non-essential amino acids, 500 mg/ml glucose and 50 mg/ml ascorbic acid (all from Biochrom) under 5% CO2 in air at 378C. After con¯uence, the cells were detached with 0.05% trypsin in PBS (Biochrom) and
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
181
Fig. 2. Concentration of IL-10 in the supernatants of human periodontal ligament (PDL) cells in the presence [IL-1b (+)] and absence [IL-1b (ÿ)] of 1 ng/ml IL-1b after 48 h of culture.
subcultured in culture ¯asks. The cultures were used between the fourth and sixth passage. The ligament cells were detached from the culture ¯ask by 0.05% trypsin in PBS and centrifuged at 800 rev/min for 10 min; 4 106 cells were seeded in every tissue well (Nunc) containing 2 ml medium (see above). Cells were grown for 48 h at 378C in a humidi®ed atmosphere containing 5% CO2 in air in the presence or absence of recombinant human 1 ng/ml IL-1b (Biochrom) during the entire culture period. The viability of the cells was controlled and followed by trypan blue staining (Biochrom). No dierences could be found between ligament cells incubated with IL-1b and control. The concentration of IL-6 in the supernatants was assessed by enzyme-linked sandwich immunosorbent assay after 48 h of culture. Samples were incubated in antihuman IL-6 monoclonal antibody (Endogen, Woburn, U.S.A.)-coated 96-well microtitre plates (Nunc) overnight at 48C. A second monoclonal IL-6 antibody (Endogen), which was biotin-labelled, was added and incubation continued for 1 h. This step was followed by a 1-h incubation with streptavidin±peroxidase (Sigma, St. Louis, U.S.A.). Peroxidase activity was developed with tetramethylbenzidine (Fluka, Neu Ulm, Germany) and the optical density of each well
was measured at a wavelength of 450 nm. The concentration of IL-10 (monoclonal capture antibody and biotin-labelled detection antibody from Endogen) in the supernatants was measured in the same way as described for IL-6. For statistical analysis, the data from all experiments (six experiments per tooth) were taken together (n = 30) and a non-parametric test (Mann±Whitney) was performed to ®nd signi®cant dierences ( p < 0.05 and p < 0.01) between cells incubated with IL-1b and controls. Data were processed with the statistical software package SPSS (SPSS Inc., Chicago, U.S.A.). After 48 h of culture, we measured the concentration of IL-6 in the supernatants of the periodontal ligament cells in the presence and absence (control) of IL-1b. Cells incubated with IL-1b released signi®cantly ( p < 0.05) higher amounts of IL-6 than control, as shown in Fig. 1. After 48 h, we also determined the concentration of IL-10 in the supernatants of the ligament cells. Cells incubated with IL-1b released signi®cantly ( p < 0.01) smaller amounts of IL-10 than control, as shown in Fig. 2. Several investigations have shown that the amount of IL-1b is increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites (Honig et al., 1989; Hou et al., 1995; Tsai et al., 1995). In addition
182
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
to its proin¯ammatory activity, IL-1b can stimulate bone resorption and inhibit bone formation and thereby lead to bone loss (Stashenko et al., 1987; Lerner, 1991; Linkhart and MacCharles, 1992). In periodontal ligament cells, IL-1b can decrease the alkaline phosphatase activity (Takeshita et al., 1992) and increase the collagenase activity (Ohshima et al., 1994), and therefore might be involved in the attachment loss. Human periodontal ligament cells incubated with TNF-a lose their osteoblastic-like characteristics, which are important for maintaining mineralized tissue homeostasis, and acquire functions of accessory immunoin¯ammatory cells synthesizing IL-1b, IL-6 and IL-8 in response to lipopolysaccharides (Quintero et al., 1995). To clarify the role of periodontal ligament cells in the in¯ammatory process, we here investigated the release of a major proin¯ammatory (IL-6) and a signi®cant immunosuppressive (IL-10) cytokine from these cells in the presence and absence of IL-1b. IL-6 is one of the major physiological mediators of the acute-phase reaction (for review, see Hirano et al., 1990; Van Snick, 1990; Wolvekamp and Marquet, 1990). As expected, IL-1b caused an increase of the IL-6 release from the periodontal ligament cells, which has been reported before (Shimizu et al., 1992). It is also in agreement with that investigation that the cells in our experiments displayed a basal secretion for IL6. IL-10 plays a key part in suppression of the in¯ammatory process and a decrease of this immunosuppressive cytokine could amplify the local in¯ammation process in periodontitis. In our investigation, periodontal ligament cells incubated with IL-1b released signi®cantly smaller amounts of IL-10 than control. These results provide evidence for the ®rst time that periodontal ligament cells secrete IL-10, which can be suppressed by IL-1b. Moreover, these results give further support to the observation that IL-1b can increase the secretion of IL-6 from the ligament cells. Taking together the results from these experiments, we conclude that IL-1b changes the cells of the periodontal ligament into accessory immunoin¯ammatory cells amplifying the in¯ammatory process in periodontitis and, thereby, contributing to periodontal breakdown.
References Agarwal, S., Baran, C., Piesco, N.P., Quintero, J.C., Langkamp, H.H., Johns, L.P., Chandra, C.S., 1995. Synthesis of proin¯ammatory cytokines by human gingival ®broblasts in response to lipopolysaccharides and interleukin-1 beta. J. periodont. Res. 30, 382±389. Berkman, N., John, M., Roesems, G., Jose, P.J., Barnes, P.J., Chung, K.F., 1995. Inhibition of macrophage in¯amma-
tory protein-1 alpha expression by IL-10. Dierential sensitivities in human blood monocytes and alveolar macrophages. J. Immun. 155, 4412±4418. Birkedal-Hansen, H., 1993. Role of cytokines and in¯ammatory mediators in tissue destruction. J. periodont. Res. 28, 500±510. Del-Prete, G., De-Carli, M., Almerigogna, F., Giudizi, M.G., Biagiotti, R., Romagnani, S., 1993. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-speci®c proliferation and cytokine production. J. Immun. 150, 353±360. De-Waal-Malefyt, R., Haanen, J., Spits, H., Roncarolo, M.G., te-Velde, A., Figdor, C., Johnson, K., Kastelein, R., Yssel, H., de-Vries, J.E., 1991. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-speci®c human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J. exp. Med. 174, 915±924. De-Waal-Malefyt, R., Yssel, H., de-Vries, J.E., 1993. Direct eects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Speci®c inhibition of IL-2 production and proliferation. J. Immun. 150, 4754±4765. Dickensheets, H.L., Donnelly, R.P., 1997. IFN-gamma and IL-10 inhibit induction of IL-1 receptor type I and type II gene expression by IL-4 and IL-13 in human monocytes. J. Immun. 159, 6226±6233. Hillmann, G., Hillmann, B., Geurtsen, W., 1995. Immunohistological determination of interleukin-1 beta in in¯amed human gingival epithelium. Archs oral Biol. 40, 353±359. Hirano, T., Akira, S., Taga, T., Kishimoto, T., 1990. Biological and clinical aspects of interleukin 6. Immunol. Today 11, 443±449. Honig, J., Rordorf-Adam, C., Siegmund, C., Wiedemann, W., Erard, F., 1989. Increased interleukin-1 beta (IL-1 beta) concentration in gingival tissue from periodontitis patients. J. periodont. Res. 24, 362±367. Hou, L.T., Liu, C.M., Rossomando, E.F., 1995. Crevicular interleukin-1 beta in moderate and severe periodontitis patients and the eect of phase I periodontal treatment. J. clin. Periodont. 22, 162±167. Jinquan, T., Larsen, C.G., Gesser, B., Matsushima, K., Thestrup-Pedersen, K., 1993. Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL8-induced CD4+ T lymphocyte migration. J. Immun. 151, 4545±4551. Lerner, U.H., 1991. Bradykinin synergistically potentiates interleukin-1 induced bone resorption and prostanoid biosynthesis in neonatal mouse calvarial bones. Biochem. biophys. Res. Commun. 175, 775±783. Linkhart, T.A., MacCharles, D.C., 1992. Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism. Endocrinology 131, 2297±2305. Lundqvist, C., Baranov, V., Teglund, S., Hammarstrom, S., Hammarstrom, M.L., 1994. Cytokine pro®le and ultrastructure of intraepithelial gamma delta T cells in chronically in¯amed human gingiva suggest a cytotoxic eector function. J. Immun. 153, 2302±2312. Marfaing-Koka, A., Maravic, M., Humbert, M., Galanaud,
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183 P., Emilie, D., 1996. Contrasting eects of IL-4, IL-10 and corticosteroids on RANTES production by human monocytes. Int. Immunol. 8, 1587±1594. Matsuki, Y., Yamamoto, T., Hara, K., 1993. Localization of interleukin-1 (IL-1) mRNA-expressing macrophages in human in¯amed gingiva and IL-1 activity in gingival crevicular ¯uid. J. periodont. Res. 28, 35±42. Ohshima, M., Otsuka, K., Suzuki, K., 1994. Interleukin-1 beta stimulates collagenase production by cultured human periodontal ligament ®broblasts. J. periodont. Res. 29, 421±429. Page, R.C., 1991. The role of in¯ammatory mediators in the pathogenesis of periodontal disease. J. periodont. Res. 26, 230±242. Quintero, J.C., Piesco, N.P., Langkamp, H.H., Bowen, L., Agarwal, S., 1995. LPS responsiveness in periodontal ligament cells is regulated by tumor necrosis factor-alpha. J. dent. Res. 74, 1802±1811. Shimizu, N., Ogura, N., Yamaguchi, M., Goseki, T., Shibata, Y., Abiko, Y., Iwasawa, T., Takiguchi, H., 1992. Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells. Archs oral Biol. 37, 743±748. Stashenko, P., Dewhirst, F.E., Rooney, M.L., Desjardins,
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L.A., Heeley, J.D., 1987. Interleukin-1 beta is a potent inhibitor of bone formation in vitro. J. Bone Miner. Res. 2, 559±565. Takeichi, O., Saito, I., Tsurumachi, T., Saito, T., Moro, I., 1994. Human polymorphonuclear leukocytes derived from chronically in¯amed tissue express in¯ammatory cytokines in vivo. Cell Immun. 156, 296±309. Takeshita, A., Niu, Z.G., Hanazawa, S., Takara, I., Higuchi, H., Katayama, I., Kitano, S., 1992. Eect of interleukin-1 beta on gene expressions and functions of ®broblastic cells derived from human periodontal ligament. J. periodont. Res. 27, 250±255. Tsai, C.C., Ho, Y.P., Chen, C.C., 1995. Levels of interleukin1 beta and interleukin-8 in gingival crevicular ¯uids in adult periodontitis. J. Periodontol. 66, 852±859. Van Snick, J., 1990. Interleukin-6: an overview. Annu. Rev. Immunol. 8, 253±278. Wang, P., Wu, P., Anthes, J.C., Siegel, M.I., Egan, R.W., Billah, M.M., 1994. Interleukin-10 inhibits interleukin-8 production in human neutrophils. Blood 83, 2678±2683. Wolvekamp, M.C., Marquet, R.L., 1990. Interleukin-6: historical background, genetics and biological signi®cance. Immunol. Lett. 24, 1±9.
www.elsevier.com/locate/archoralbio
Short communication
Suppression of interleukin-10 release from human periodontal ligament cells by interleukin-1b in vitro J. Deschner a, B. Arnold b, A. Kage c, B. Zimmermann d, V. Kanitz b, J.-P. Bernimoulin b,* a Department of Operative Dentistry and Periodontology, University of Cologne, Germany Department of Periodontology, ChariteÂ, Humboldt University Berlin, FoÈhrer Str. 15, 13353 Berlin, Germany c Institute of Chemistry and Biochemistry, ChariteÂ, Humboldt University Berlin, Germany d Institute of Anatomy and Institute of Clinical Pharmacology and Toxicology, Free University of Berlin, Germany b
Accepted 7 September 1999
Abstract Periodontitis is characterized by an in¯ammatory process induced by periodontopathogenic bacteria in the subgingival plaque. Periodontal in¯ammation can be enhanced by both an increase of in¯ammatory stimulators, e.g. interleukin (IL)-6, and a decrease of in¯ammatory inhibitors, e.g. IL-10. The amount of IL-1b is known to be increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites compared to healthy sites. This in vitro study sought to clarify whether IL-1b (1 ng/ml) has a regulatory eect on the release of these two cytokines from human periodontal ligament (PDL) cells. PDL cells derived from healthy premolars were grown in the presence and absence (control) of IL-1b. The concentration of IL-6 and IL-10 in the supernatants was assessed by enzyme-linked immunosorbent assay after 48 h of culture. PDL cells incubated with IL-1b released signi®cantly ( p < 0.05) higher amounts of IL-6 and signi®cantly ( p < 0.01) smaller amounts of IL-10 compared to control. These results give further support to the observation that IL-1b can increase the IL-6 secretion from PDL cells. Moreover, they provide original evidence that PDL cells secrete IL-10, which can be suppressed by IL-1b. It is concluded that PDL cells can function as accessory immunoin¯ammatory cells amplifying the in¯ammatory process in periodontitis and, thereby, contributing to periodontal breakdown. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Periodontal ligament; Interleukin-1b; Interleukin-10; Periodontitis; Cell culture; Cytokine
Periodontitis is characterized by an in¯ammatory process, which is induced by periodontopathogenic bacteria in the subgingival plaque. The subgingival bacteria and their products stimulate host cells to
Abbreviations: IL, interleukin; MHC, major histocompatibility complex; PBS, phosphate-buered saline; TNF, tumour necrosis factor. * Corresponding author. Tel.: +49-30-45062632; fax: +4930-45062931.
release numerous in¯ammatory mediators, such as prostaglandins and cytokines (Page, 1991; BirkedalHansen, 1993). The periodontal in¯ammation can be enhanced by both an increase of in¯ammatory stimulators, e.g. IL-6, and a decrease of in¯ammatory inhibitors, e.g. IL-10. The proin¯ammatory IL-6 is synthesized by a variety of dierent cell types and represents one of the major physiological mediators of the acute-phase reaction (for review, see Hirano et al., 1990; Van Snick, 1990; Wolvekamp and Marquet,
0003-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 9 ) 0 0 1 2 0 - X
180
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
Fig. 1. Concentration of IL-6 in the supernatants of human periodontal ligament (PDL) cells in the presence [IL-1b (+)] and absence [IL-1b (ÿ)] of 1 ng/ml IL-1b after 48 h of culture.
1990). The immunosuppressive IL-10 was found in the supernatants of both type 1 helper and type 2 helper T-cell clones and inhibits their antigen-speci®c proliferation and cytokine production (De-Waal-Malefyt et al., 1991; 1993; Del-Prete et al., 1993). Human IL-10 is also a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration (Jinquan et al., 1993). In monocytes, IL-10 inhibits the production of IL-1, IL-6 and TNF-a (DeWaal-Malefyt et al., 1991), antagonizes the chemokine production (Berkman et al., 1995; Marfaing-Koka et al., 1996) and suppresses the upregulation of IL-1 receptor genes and class II MHC antigens (De-WaalMalefyt et al., 1991; Dickensheets and Donnelly, 1997). In polymorphonuclear leucocytes, IL-10 inhibits the production of IL-1a, IL-1b, IL-8 and TNF-a (Wang et al., 1994). In periodontitis, pathogenic bacteria in the subgingival plaque cause a subepithelial in®ltration by in¯ammatory cells, which produce and release IL-1b (Matsuki et al., 1993; Takeichi et al., 1994). This cytokine can also be produced by epithelial cells and gingival ®broblasts (Lundqvist et al., 1994; Agarwal et al., 1995; Hillmann et al., 1995). The amount of IL-1b is increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites compared to healthy sites
(Honig et al., 1989; Hou et al., 1995; Tsai et al., 1995). Little is known about the eects of IL-1b on human periodontal ligament cells. This in vitro study sought to clarify whether IL-1b has a regulatory eect on the release of IL-6 and IL-10 from human periodontal ligament cells. Cells from the middle third of the root surface of healthy premolars were used. The premolars were obtained from ®ve patients (one tooth per patient) who had to undergo regular orthodontic treatment. Informed consent was obtained from all patients. After extraction, the premolars were washed twice with Hank' salt solution (Biochrom, Berlin, Germany). The periodontal tissue was then removed with a surgical scalpel. The coronal and apical portions of the root were not used in order to avoid contamination by cells of the gingiva, nerves, and blood vessels. The tissue was minced and then cultured as explants in culture ¯asks (Nunc, Wiesbaden, Germany) containing Dulbecco's minimal essential medium supplemented with 15% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, non-essential amino acids, 500 mg/ml glucose and 50 mg/ml ascorbic acid (all from Biochrom) under 5% CO2 in air at 378C. After con¯uence, the cells were detached with 0.05% trypsin in PBS (Biochrom) and
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
181
Fig. 2. Concentration of IL-10 in the supernatants of human periodontal ligament (PDL) cells in the presence [IL-1b (+)] and absence [IL-1b (ÿ)] of 1 ng/ml IL-1b after 48 h of culture.
subcultured in culture ¯asks. The cultures were used between the fourth and sixth passage. The ligament cells were detached from the culture ¯ask by 0.05% trypsin in PBS and centrifuged at 800 rev/min for 10 min; 4 106 cells were seeded in every tissue well (Nunc) containing 2 ml medium (see above). Cells were grown for 48 h at 378C in a humidi®ed atmosphere containing 5% CO2 in air in the presence or absence of recombinant human 1 ng/ml IL-1b (Biochrom) during the entire culture period. The viability of the cells was controlled and followed by trypan blue staining (Biochrom). No dierences could be found between ligament cells incubated with IL-1b and control. The concentration of IL-6 in the supernatants was assessed by enzyme-linked sandwich immunosorbent assay after 48 h of culture. Samples were incubated in antihuman IL-6 monoclonal antibody (Endogen, Woburn, U.S.A.)-coated 96-well microtitre plates (Nunc) overnight at 48C. A second monoclonal IL-6 antibody (Endogen), which was biotin-labelled, was added and incubation continued for 1 h. This step was followed by a 1-h incubation with streptavidin±peroxidase (Sigma, St. Louis, U.S.A.). Peroxidase activity was developed with tetramethylbenzidine (Fluka, Neu Ulm, Germany) and the optical density of each well
was measured at a wavelength of 450 nm. The concentration of IL-10 (monoclonal capture antibody and biotin-labelled detection antibody from Endogen) in the supernatants was measured in the same way as described for IL-6. For statistical analysis, the data from all experiments (six experiments per tooth) were taken together (n = 30) and a non-parametric test (Mann±Whitney) was performed to ®nd signi®cant dierences ( p < 0.05 and p < 0.01) between cells incubated with IL-1b and controls. Data were processed with the statistical software package SPSS (SPSS Inc., Chicago, U.S.A.). After 48 h of culture, we measured the concentration of IL-6 in the supernatants of the periodontal ligament cells in the presence and absence (control) of IL-1b. Cells incubated with IL-1b released signi®cantly ( p < 0.05) higher amounts of IL-6 than control, as shown in Fig. 1. After 48 h, we also determined the concentration of IL-10 in the supernatants of the ligament cells. Cells incubated with IL-1b released signi®cantly ( p < 0.01) smaller amounts of IL-10 than control, as shown in Fig. 2. Several investigations have shown that the amount of IL-1b is increased in gingival tissues and in the gingival crevicular ¯uid from in¯amed sites (Honig et al., 1989; Hou et al., 1995; Tsai et al., 1995). In addition
182
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183
to its proin¯ammatory activity, IL-1b can stimulate bone resorption and inhibit bone formation and thereby lead to bone loss (Stashenko et al., 1987; Lerner, 1991; Linkhart and MacCharles, 1992). In periodontal ligament cells, IL-1b can decrease the alkaline phosphatase activity (Takeshita et al., 1992) and increase the collagenase activity (Ohshima et al., 1994), and therefore might be involved in the attachment loss. Human periodontal ligament cells incubated with TNF-a lose their osteoblastic-like characteristics, which are important for maintaining mineralized tissue homeostasis, and acquire functions of accessory immunoin¯ammatory cells synthesizing IL-1b, IL-6 and IL-8 in response to lipopolysaccharides (Quintero et al., 1995). To clarify the role of periodontal ligament cells in the in¯ammatory process, we here investigated the release of a major proin¯ammatory (IL-6) and a signi®cant immunosuppressive (IL-10) cytokine from these cells in the presence and absence of IL-1b. IL-6 is one of the major physiological mediators of the acute-phase reaction (for review, see Hirano et al., 1990; Van Snick, 1990; Wolvekamp and Marquet, 1990). As expected, IL-1b caused an increase of the IL-6 release from the periodontal ligament cells, which has been reported before (Shimizu et al., 1992). It is also in agreement with that investigation that the cells in our experiments displayed a basal secretion for IL6. IL-10 plays a key part in suppression of the in¯ammatory process and a decrease of this immunosuppressive cytokine could amplify the local in¯ammation process in periodontitis. In our investigation, periodontal ligament cells incubated with IL-1b released signi®cantly smaller amounts of IL-10 than control. These results provide evidence for the ®rst time that periodontal ligament cells secrete IL-10, which can be suppressed by IL-1b. Moreover, these results give further support to the observation that IL-1b can increase the secretion of IL-6 from the ligament cells. Taking together the results from these experiments, we conclude that IL-1b changes the cells of the periodontal ligament into accessory immunoin¯ammatory cells amplifying the in¯ammatory process in periodontitis and, thereby, contributing to periodontal breakdown.
References Agarwal, S., Baran, C., Piesco, N.P., Quintero, J.C., Langkamp, H.H., Johns, L.P., Chandra, C.S., 1995. Synthesis of proin¯ammatory cytokines by human gingival ®broblasts in response to lipopolysaccharides and interleukin-1 beta. J. periodont. Res. 30, 382±389. Berkman, N., John, M., Roesems, G., Jose, P.J., Barnes, P.J., Chung, K.F., 1995. Inhibition of macrophage in¯amma-
tory protein-1 alpha expression by IL-10. Dierential sensitivities in human blood monocytes and alveolar macrophages. J. Immun. 155, 4412±4418. Birkedal-Hansen, H., 1993. Role of cytokines and in¯ammatory mediators in tissue destruction. J. periodont. Res. 28, 500±510. Del-Prete, G., De-Carli, M., Almerigogna, F., Giudizi, M.G., Biagiotti, R., Romagnani, S., 1993. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-speci®c proliferation and cytokine production. J. Immun. 150, 353±360. De-Waal-Malefyt, R., Haanen, J., Spits, H., Roncarolo, M.G., te-Velde, A., Figdor, C., Johnson, K., Kastelein, R., Yssel, H., de-Vries, J.E., 1991. Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-speci®c human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression. J. exp. Med. 174, 915±924. De-Waal-Malefyt, R., Yssel, H., de-Vries, J.E., 1993. Direct eects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Speci®c inhibition of IL-2 production and proliferation. J. Immun. 150, 4754±4765. Dickensheets, H.L., Donnelly, R.P., 1997. IFN-gamma and IL-10 inhibit induction of IL-1 receptor type I and type II gene expression by IL-4 and IL-13 in human monocytes. J. Immun. 159, 6226±6233. Hillmann, G., Hillmann, B., Geurtsen, W., 1995. Immunohistological determination of interleukin-1 beta in in¯amed human gingival epithelium. Archs oral Biol. 40, 353±359. Hirano, T., Akira, S., Taga, T., Kishimoto, T., 1990. Biological and clinical aspects of interleukin 6. Immunol. Today 11, 443±449. Honig, J., Rordorf-Adam, C., Siegmund, C., Wiedemann, W., Erard, F., 1989. Increased interleukin-1 beta (IL-1 beta) concentration in gingival tissue from periodontitis patients. J. periodont. Res. 24, 362±367. Hou, L.T., Liu, C.M., Rossomando, E.F., 1995. Crevicular interleukin-1 beta in moderate and severe periodontitis patients and the eect of phase I periodontal treatment. J. clin. Periodont. 22, 162±167. Jinquan, T., Larsen, C.G., Gesser, B., Matsushima, K., Thestrup-Pedersen, K., 1993. Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL8-induced CD4+ T lymphocyte migration. J. Immun. 151, 4545±4551. Lerner, U.H., 1991. Bradykinin synergistically potentiates interleukin-1 induced bone resorption and prostanoid biosynthesis in neonatal mouse calvarial bones. Biochem. biophys. Res. Commun. 175, 775±783. Linkhart, T.A., MacCharles, D.C., 1992. Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism. Endocrinology 131, 2297±2305. Lundqvist, C., Baranov, V., Teglund, S., Hammarstrom, S., Hammarstrom, M.L., 1994. Cytokine pro®le and ultrastructure of intraepithelial gamma delta T cells in chronically in¯amed human gingiva suggest a cytotoxic eector function. J. Immun. 153, 2302±2312. Marfaing-Koka, A., Maravic, M., Humbert, M., Galanaud,
J. Deschner et al. / Archives of Oral Biology 45 (2000) 179±183 P., Emilie, D., 1996. Contrasting eects of IL-4, IL-10 and corticosteroids on RANTES production by human monocytes. Int. Immunol. 8, 1587±1594. Matsuki, Y., Yamamoto, T., Hara, K., 1993. Localization of interleukin-1 (IL-1) mRNA-expressing macrophages in human in¯amed gingiva and IL-1 activity in gingival crevicular ¯uid. J. periodont. Res. 28, 35±42. Ohshima, M., Otsuka, K., Suzuki, K., 1994. Interleukin-1 beta stimulates collagenase production by cultured human periodontal ligament ®broblasts. J. periodont. Res. 29, 421±429. Page, R.C., 1991. The role of in¯ammatory mediators in the pathogenesis of periodontal disease. J. periodont. Res. 26, 230±242. Quintero, J.C., Piesco, N.P., Langkamp, H.H., Bowen, L., Agarwal, S., 1995. LPS responsiveness in periodontal ligament cells is regulated by tumor necrosis factor-alpha. J. dent. Res. 74, 1802±1811. Shimizu, N., Ogura, N., Yamaguchi, M., Goseki, T., Shibata, Y., Abiko, Y., Iwasawa, T., Takiguchi, H., 1992. Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells. Archs oral Biol. 37, 743±748. Stashenko, P., Dewhirst, F.E., Rooney, M.L., Desjardins,
183
L.A., Heeley, J.D., 1987. Interleukin-1 beta is a potent inhibitor of bone formation in vitro. J. Bone Miner. Res. 2, 559±565. Takeichi, O., Saito, I., Tsurumachi, T., Saito, T., Moro, I., 1994. Human polymorphonuclear leukocytes derived from chronically in¯amed tissue express in¯ammatory cytokines in vivo. Cell Immun. 156, 296±309. Takeshita, A., Niu, Z.G., Hanazawa, S., Takara, I., Higuchi, H., Katayama, I., Kitano, S., 1992. Eect of interleukin-1 beta on gene expressions and functions of ®broblastic cells derived from human periodontal ligament. J. periodont. Res. 27, 250±255. Tsai, C.C., Ho, Y.P., Chen, C.C., 1995. Levels of interleukin1 beta and interleukin-8 in gingival crevicular ¯uids in adult periodontitis. J. Periodontol. 66, 852±859. Van Snick, J., 1990. Interleukin-6: an overview. Annu. Rev. Immunol. 8, 253±278. Wang, P., Wu, P., Anthes, J.C., Siegel, M.I., Egan, R.W., Billah, M.M., 1994. Interleukin-10 inhibits interleukin-8 production in human neutrophils. Blood 83, 2678±2683. Wolvekamp, M.C., Marquet, R.L., 1990. Interleukin-6: historical background, genetics and biological signi®cance. Immunol. Lett. 24, 1±9.