Oral Diseases (2015) 21, 667–673 doi:10.1111/odi.12334 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes J Kido1, Y Bando2, M Bando1, Y Kajiura1, Y Hiroshima2, Y Inagaki1, H Murata1, T Ikuta1, R Kido3, K Naruishi1, M Funaki2, T Nagata1 1
Department of Periodontology and Endodontology, Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima; 2Clinical Research Center for Diabetes, Tokushima University Hospital, Tokushima; 3Department of Anatomy and Cell Biology, Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima, Japan
OBJECTIVE: YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. SUBJECTS AND METHODS: The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. RESULTS: YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. CONCLUSIONS: GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis. Oral Diseases (2015) 21, 667–673 Keywords: YKL-40; gingival crevicular fluid; periodontitis; diabetes
Correspondence: Jun-ichi Kido, Department of Periodontology and Endodontology, Institute of Health Biosciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan. Tel: +81 88 633 7344, Fax: +81 88 633 7345, E-mail: kido.jun-ichi@ tokushima-u.ac.jp Received 24 January 2015; revised 16 February 2015; accepted 25 February 2015
Introduction YKL-40 is a glycoprotein with a molecular weight of approximately 40 kDa and is named from three amino acids of tyrosine (Y), lysine (K), and leucine (L) at the Nterminal (Hakala et al, 1993; Rathcke and Vestergaard, 2009). YKL-40 is also called chitinase-3-like protein 1 (CHI3L1) and human cartilage glycoprotein 39 (HC-gp39) and strongly binds to chitin and heparin, but lacks chitinase activity (Hakala et al, 1993; Shackelton et al, 1995; Rehli et al, 1997; Rathcke and Vestergaard, 2009). A variety of cell species including neutrophils, macrophages, chondrocytes, synovial cells, osteoblastic cells, endothelial cells, and cancer cells express and secrete YKL-40 (Rehli et al, 1997; Volck et al, 1998; Connor et al, 2000; Ceuninck et al, 2001; Nishikawa and Millis, 2003; Johansen et al, 2006; Rathcke and Vestergaard, 2006, 2009). YKL40 expression increases in macrophages and vascular muscle cells during their differentiation (Shackelton et al, 1995; Rehli et al, 1997) and is observed in vivo in macrophages of inflammatory tissues with atherosclerosis (Boot et al, 1999) and of small cell lung cancer tissue (Junker et al, 2005), in peripheral blood mononuclear cells and synovium in patients with rheumatoid arthritis (Baeten et al, 2000), and in ductal breast cancer cells (Johansen et al, 2006). YKL-40 induces the proliferation of chondrocytes, fibroblasts, and synovial cells, increases proteoglycan synthesis by chondrocytes (Ceuninck et al, 2001; Recklies et al, 2002), and inhibits the differentiation of mammary epithelial cells (Scully et al, 2011) and angiogenesis in tumor tissues (Johansen et al, 2006; Shao, 2013). In mice in which the CHI3L1 gene was knocked down, antigeninduced Th2 response was inhibited and epithelial YKL40 rescued Th2 responses, suggesting that YKL-40 plays a role in the immune system (Lee et al, 2009). YKL-40 appears to be associated with inflammation, tissue remodeling, immune responses, and angiogenesis considering some of its functions. The level of YKL-40 in body fluids and tissues increases in some diseases with inflammation and in
GCF YKL-40 level in periodontitis and DM
J-i Kido et al
668
cancers. Serum YKL-40 level in patients with clinically active rheumatoid arthritis was found to be higher than that of clinically inactive patients and was decreased by steroid treatment (Johansen et al, 1999); its level in patients with coronary artery disease and atherosclerosis was elevated and was associated with cardiovascular mortality (Kucur et al, 2007; Kastrup et al, 2009; Rathcke and Vestergaard, 2009). High levels of YKL-40 in cerebrospinal fluid from patients with meningitis and in the serum of psoriasis patients with endothelial dysfunction and children with severe asthma were reported (Østergaard et al, 2002; Konradsen et al, 2013; Erfan et al, 2015). Serum YKL-40 level was elevated in patients with some tumors including breast cancer, colorectal cancer, glioblastoma, ovarian cancer, small cell lung cancer, prostate cancer, and melanoma (Johansen et al, 2006). Rathcke and Vestergaard (2006, 2009) and Rathcke et al (2006) reported that plasma YKL-40 level in patients with type 2 diabetes mellitus (T2DM) was significantly higher than that of non-DM individuals. A high level of YKL-40 in plasma from patients with T2DM was associated with fasting plasma glucose, but not with body mass index (Nielsen et al, 2008). Elevated YKL-40 level in serum was independently associated with the ratio of urinary albumin to creatinine and albuminuria (Røndbjerg et al, 2011) and predicted an early stage of nephropathy and mortality in patients with T2DM (Lee et al, 2012; Persson et al, 2012). These reports suggest that YKL-40 is a marker of inflammation and YKL-40 level is associated with DM and its complications. Gingival crevicular fluid (GCF) contains many proteins and peptides (Kido et al, 2012), which are possible markers for periodontitis and other diseases (Kido et al, 1999; Ozmeric, 2004; G€ um€ ußs et al, 2013; Kajiura et al, 2014). Few GCF components have been investigated as diagnostic markers for periodontitis in patients with DM. Glycated albumin (GA) in blood reflects a condition of glycemic control for several weeks and has been used as a marker for DM (Koga and Kasayama, 2010). GA was contained in GCF as well as blood, and the GA level in GCF from patients with DM was significantly higher than that of non-DM individuals, but did not change in GCF collected from periodontitis sites (Kajiura et al, 2014), suggesting that GCF GA may be a screening marker for diabetes mellitus, but not periodontitis. The purpose of this study was to compare the YKL-40 levels in GCF samples collected from subjects with or without periodontitis and DM and to investigate whether GCF YKL-40 is a useful marker for periodontitis in patients with DM.
Materials and methods Subjects and clinical examinations This study was performed from February 2012 to March 2014 with the approval of the Ethics Committee of Tokushima University Hospital (approval No. 442 and 1327) in accordance with the Helsinki Declaration of 1975, as revised in 2000. One hundred and twenty-one subjects (69 males and 52 females, mean age: 62.0 10.9) gave written informed consent after listening to an explanation of this study. They were patients with periodontitis and T2DM, as well as healthy subjects without either disease. All subjects were non-smokers and had no Oral Diseases
cancers and systemic inflammatory diseases including pneumonia, rheumatoid arthritis, psoriasis, and inflammatory bowel disease and no history of antibiotic therapy within 3 months. The participants were classified into four groups: diabetes mellitus without periodontitis (DM, n = 24), chronic periodontitis without DM (CP, n = 38), periodontitis with DM (DM-P, n = 30), and non-periodontitis, and non-DM (healthy: H, n = 29). One to two GCF samples were collected from the subjects, and the numbers of GCF samples in the H, DM, CP, and DM-P groups were 34, 25, 41, and 32, respectively, with a total of 132. Their characteristics are shown in Table 1. Probing pocket depth (PD), bleeding on probing (BOP), gingival index (GI), and bone loss were used for a clinical diagnosis of periodontitis. After clinical periodontal examination in full-mouth of all participants, patient with periodontal diseases was defined as a subject who showed that a rate of periodontal pockets (PD of more than 4 mm) was more than 30%. Periodontitis sites were selected from periodontal sites with PD of more than 4 mm, GI score of more than 1, bone loss of more than a third of tooth root length, and BOP positive or BOP negative in patients with periodontal diseases. Healthy individuals and patients with DM were defined as the subjects who had no periodontal pockets. Non-periodontitis sites were selected from periodontal sites with PD of
ORIGINAL ARTICLE
YKL-40 level in gingival crevicular fluid from patients with periodontitis and type 2 diabetes J Kido1, Y Bando2, M Bando1, Y Kajiura1, Y Hiroshima2, Y Inagaki1, H Murata1, T Ikuta1, R Kido3, K Naruishi1, M Funaki2, T Nagata1 1
Department of Periodontology and Endodontology, Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima; 2Clinical Research Center for Diabetes, Tokushima University Hospital, Tokushima; 3Department of Anatomy and Cell Biology, Institute of Health Bioscience, The University of Tokushima Graduate School, Tokushima, Japan
OBJECTIVE: YKL-40 is a chitin-binding glycoprotein, the level of which increases in inflammatory diseases, diabetes mellitus (DM), cardiovascular diseases, and tumors. Gingival crevicular fluid (GCF) contains many proteins and markers of periodontitis. The purpose of this study was to investigate YKL-40 level in GCF from patients with periodontitis and DM and the association between YKL-40 level and chronic periodontitis (CP) or DM. SUBJECTS AND METHODS: The subjects were 121 patients with DM, CP, DM and periodontitis (DM-P), and healthy subjects (H). GCF was collected using paper strips after the sites for GCF collection were clinically evaluated for probing depth (PD), gingival index (GI), and bleeding on probing (BOP). YKL-40 in GCF was identified by Western blotting, and its level was determined by ELISA. RESULTS: YKL-40 was contained in GCF samples from H, DM, CP, and DM-P sites, and its levels (amount and concentration) in CP and DM-P were significantly higher than those in H and DM. GCF YKL-40 level significantly correlated with PD and GI, and its level in BOP-positive sites was significantly higher than that in BOP-negative ones. CONCLUSIONS: GCF YKL-40 level was elevated in periodontitis, but not DM. YKL-40 in GCF may be an inflammatory marker for periodontitis. Oral Diseases (2015) 21, 667–673 Keywords: YKL-40; gingival crevicular fluid; periodontitis; diabetes
Correspondence: Jun-ichi Kido, Department of Periodontology and Endodontology, Institute of Health Biosciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima 770-8504, Japan. Tel: +81 88 633 7344, Fax: +81 88 633 7345, E-mail: kido.jun-ichi@ tokushima-u.ac.jp Received 24 January 2015; revised 16 February 2015; accepted 25 February 2015
Introduction YKL-40 is a glycoprotein with a molecular weight of approximately 40 kDa and is named from three amino acids of tyrosine (Y), lysine (K), and leucine (L) at the Nterminal (Hakala et al, 1993; Rathcke and Vestergaard, 2009). YKL-40 is also called chitinase-3-like protein 1 (CHI3L1) and human cartilage glycoprotein 39 (HC-gp39) and strongly binds to chitin and heparin, but lacks chitinase activity (Hakala et al, 1993; Shackelton et al, 1995; Rehli et al, 1997; Rathcke and Vestergaard, 2009). A variety of cell species including neutrophils, macrophages, chondrocytes, synovial cells, osteoblastic cells, endothelial cells, and cancer cells express and secrete YKL-40 (Rehli et al, 1997; Volck et al, 1998; Connor et al, 2000; Ceuninck et al, 2001; Nishikawa and Millis, 2003; Johansen et al, 2006; Rathcke and Vestergaard, 2006, 2009). YKL40 expression increases in macrophages and vascular muscle cells during their differentiation (Shackelton et al, 1995; Rehli et al, 1997) and is observed in vivo in macrophages of inflammatory tissues with atherosclerosis (Boot et al, 1999) and of small cell lung cancer tissue (Junker et al, 2005), in peripheral blood mononuclear cells and synovium in patients with rheumatoid arthritis (Baeten et al, 2000), and in ductal breast cancer cells (Johansen et al, 2006). YKL-40 induces the proliferation of chondrocytes, fibroblasts, and synovial cells, increases proteoglycan synthesis by chondrocytes (Ceuninck et al, 2001; Recklies et al, 2002), and inhibits the differentiation of mammary epithelial cells (Scully et al, 2011) and angiogenesis in tumor tissues (Johansen et al, 2006; Shao, 2013). In mice in which the CHI3L1 gene was knocked down, antigeninduced Th2 response was inhibited and epithelial YKL40 rescued Th2 responses, suggesting that YKL-40 plays a role in the immune system (Lee et al, 2009). YKL-40 appears to be associated with inflammation, tissue remodeling, immune responses, and angiogenesis considering some of its functions. The level of YKL-40 in body fluids and tissues increases in some diseases with inflammation and in
GCF YKL-40 level in periodontitis and DM
J-i Kido et al
668
cancers. Serum YKL-40 level in patients with clinically active rheumatoid arthritis was found to be higher than that of clinically inactive patients and was decreased by steroid treatment (Johansen et al, 1999); its level in patients with coronary artery disease and atherosclerosis was elevated and was associated with cardiovascular mortality (Kucur et al, 2007; Kastrup et al, 2009; Rathcke and Vestergaard, 2009). High levels of YKL-40 in cerebrospinal fluid from patients with meningitis and in the serum of psoriasis patients with endothelial dysfunction and children with severe asthma were reported (Østergaard et al, 2002; Konradsen et al, 2013; Erfan et al, 2015). Serum YKL-40 level was elevated in patients with some tumors including breast cancer, colorectal cancer, glioblastoma, ovarian cancer, small cell lung cancer, prostate cancer, and melanoma (Johansen et al, 2006). Rathcke and Vestergaard (2006, 2009) and Rathcke et al (2006) reported that plasma YKL-40 level in patients with type 2 diabetes mellitus (T2DM) was significantly higher than that of non-DM individuals. A high level of YKL-40 in plasma from patients with T2DM was associated with fasting plasma glucose, but not with body mass index (Nielsen et al, 2008). Elevated YKL-40 level in serum was independently associated with the ratio of urinary albumin to creatinine and albuminuria (Røndbjerg et al, 2011) and predicted an early stage of nephropathy and mortality in patients with T2DM (Lee et al, 2012; Persson et al, 2012). These reports suggest that YKL-40 is a marker of inflammation and YKL-40 level is associated with DM and its complications. Gingival crevicular fluid (GCF) contains many proteins and peptides (Kido et al, 2012), which are possible markers for periodontitis and other diseases (Kido et al, 1999; Ozmeric, 2004; G€ um€ ußs et al, 2013; Kajiura et al, 2014). Few GCF components have been investigated as diagnostic markers for periodontitis in patients with DM. Glycated albumin (GA) in blood reflects a condition of glycemic control for several weeks and has been used as a marker for DM (Koga and Kasayama, 2010). GA was contained in GCF as well as blood, and the GA level in GCF from patients with DM was significantly higher than that of non-DM individuals, but did not change in GCF collected from periodontitis sites (Kajiura et al, 2014), suggesting that GCF GA may be a screening marker for diabetes mellitus, but not periodontitis. The purpose of this study was to compare the YKL-40 levels in GCF samples collected from subjects with or without periodontitis and DM and to investigate whether GCF YKL-40 is a useful marker for periodontitis in patients with DM.
Materials and methods Subjects and clinical examinations This study was performed from February 2012 to March 2014 with the approval of the Ethics Committee of Tokushima University Hospital (approval No. 442 and 1327) in accordance with the Helsinki Declaration of 1975, as revised in 2000. One hundred and twenty-one subjects (69 males and 52 females, mean age: 62.0 10.9) gave written informed consent after listening to an explanation of this study. They were patients with periodontitis and T2DM, as well as healthy subjects without either disease. All subjects were non-smokers and had no Oral Diseases
cancers and systemic inflammatory diseases including pneumonia, rheumatoid arthritis, psoriasis, and inflammatory bowel disease and no history of antibiotic therapy within 3 months. The participants were classified into four groups: diabetes mellitus without periodontitis (DM, n = 24), chronic periodontitis without DM (CP, n = 38), periodontitis with DM (DM-P, n = 30), and non-periodontitis, and non-DM (healthy: H, n = 29). One to two GCF samples were collected from the subjects, and the numbers of GCF samples in the H, DM, CP, and DM-P groups were 34, 25, 41, and 32, respectively, with a total of 132. Their characteristics are shown in Table 1. Probing pocket depth (PD), bleeding on probing (BOP), gingival index (GI), and bone loss were used for a clinical diagnosis of periodontitis. After clinical periodontal examination in full-mouth of all participants, patient with periodontal diseases was defined as a subject who showed that a rate of periodontal pockets (PD of more than 4 mm) was more than 30%. Periodontitis sites were selected from periodontal sites with PD of more than 4 mm, GI score of more than 1, bone loss of more than a third of tooth root length, and BOP positive or BOP negative in patients with periodontal diseases. Healthy individuals and patients with DM were defined as the subjects who had no periodontal pockets. Non-periodontitis sites were selected from periodontal sites with PD of
