Yuko Ujiie1, Takeo Karakida2, Yasuo Yamakoshi2, Tomoko Ohshima3, Kazuhiro Gomi4, Shinichiro Oida2. 1. Department of Periodontology, Tsurumi University School of Dental Medicine, Japan. Electronic address: ujiie-y@tsurumi-u.ac.jp. 2. Department of Oral Biochemistry, Tsurumi University School of Dental Medicine, Japan. 3. Department of Oral Microbiology, Tsurumi University School of Dental Medicine, Japan. 4. Department of Periodontology, Tsurumi University School of Dental Medicine, Japan.
Abstract
OBJECTIVE: Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DESIGN: Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. RESULTS: The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. CONCLUSION: We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.
OBJECTIVE:Human gingival epithelium is continuously exposed to bacteria and acts as the first line of defense in periodontal tissues. It is crucial to maintain healthy, non-inflamed gingival tissue to avoid gingivitis and periodontitis. The purpose of this study was to investigate the influence of IL-4 in human gingival fibroblasts (hGF) on the activation of osteoclasts. DESIGN: Two hGF samples were obtained from two healthy patients, and one was collected from a commercially available resource. The hGFs were cultured, and conditioned medium of hGF (hGF-CM) was stocked at -80°C. The mRNA was isolated from the hGF cultures and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of suppressive osteoclastogenetic mediators, such as interleukin (IL)-4, osteoprotegerin (OPG), IL-10, IL-27, and IL-33. The hGF-CM was used to investigate the inhibitory function of mouse macrophages supplemented with either glutathione S-transferase-Receptor activator of NF-kB ligand (GST-RANKL), human recombinant (rh)IL-4, or rhOPG but not a combination. Differentiation of osteoclasts was examined by tartrate resistant acid phosphatase (TRAP) staining and TRAP assay. The suppressive role of IL-4 was assessed by neutralizing IL-4 antibody in the TRAP assay. RESULTS: The hGF-CM reduced both TRAP positive staining and activity in a dose-dependent manner. IL-4 and OPG mRNA expressions were expressed in hGF-CM from three different donors but that of IL-10, IL-27, or IL-33 was not detected. In the RAW264 culture, rhIL-4 and rhOPG reduced TRAP positive staining as well as activity in a dose-dependent manner. Moreover, addition of neutralizing antibodies for IL-4 reduced the inhibitory effect of conditioned medium from gingival fibroblasts in the RAW264 culture. CONCLUSION: We concluded that hGF potentially contained suppressive mediators, such as IL-4 and OPG, for osteoclastogenesis. Moreover, we confirmed that the differential inhibition of osteoclast is caused by OPG as well as IL-4 in hGF-CM.
Authors: Carolyn G J Moonen; Sven T Alders; Hetty J Bontkes; Ton Schoenmaker; Elena A Nicu; Bruno G Loos; Teun J de Vries Journal: Front Immunol Date: 2018-07-25 Impact factor: 7.561