CXC chemokine ligand 16 in periodontal diseases: expression in diseased tissues and production by cytokine-stimulated human gingival fibroblasts

Abstract

Periodontal disease is an inflammatory disorder characterized by the involvement of chemokines that are important for the recruitment of leucocytes. Several cytokines are involved in regulating levels of chemokines in periodontal disease. CXCL16 is a chemokine related to the migration of T helper 1 (Th1) cells and natural killer (NK) cells. In this study, we examined its expression in periodontal tissues. Moreover, we investigated the effects of cytokines on the production of CXCL16 by human gingival fibroblast (HGF). Reverse transcription-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry revealed that CXCL16 and its receptor, CXCR6, were expressed at the mRNA and protein levels in diseased tissues. Proinflammatory cytokines [interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma] increased the mRNA expression and release of CXCL16 in a dose-dependent manner. Moreover, treatment of HGFs with IFN-gamma in combination with IL-1beta had a synergistic effect on the production of CXCL16. On the other hand, IL-4 and IL-13 inhibited the IL-1beta-induced CXCL16 production by HGFs. Inhibitors of A disintegrin and metalloprotease (ADAM)10 and ADAM17, a recently identified protease of CXCL16, reduced the amount of CXCL16 released from HGFs. These results suggest that the CXCL16 produced by HGFs may be involved in the migration of leucocytes into inflamed tissues, and provide evidence that CXCL16 production is controlled by cytokines in periodontal disease.

Fig. 1

Reverse transcription–polymerase chain reaction (RT–PCR) analysis of CXCL16 and CXCR6 mRNA expression in diseased periodontal tissue. Total RNA was prepared from two clinically healthy gingival samples (pocket depth, 2 mm) and nine diseased gingival samples (pocket depth, 4–10 mm). The expression of CXCL16, CXCR6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in periodontal tissues was analysed by RT–PCR as described in the Methods. Bands were quantified further using National Institutes of Health image software (b,c). The results are expressed relative to GAPDH mRNA as an internal standard.

Fig. 2

CXCL16 and CXCR6 immunostaining in periodontal tissues. Immunohistochemical analysis of normal gingival tissue (a,c) and diseased periodontal tissues (b,d). Immunohistochemical staining of human periodontal tissues with anti-CXCL16 antibody (a,b), anti-CXCR6 antibody (c,d) and control antibody (e). The original magnification for each photograph was ×200.

Fig. 3

CXCL16 expression by human gingival fibroblasts (HGFs). (a) HGFs were treated with or without interleukin (IL)-1β (0·1–100 ng/ml), tumour necrosis factor (TNF)-α (0·1–100 ng/ml), interferon (IFN)-γ (0·1–100 ng/ml), IL-4 (0·1–100 ng/ml), IL-13 (0·1–100 ng/ml) and IL-10 (0·1–100 ng/ml) for 4 h. Total RNA was isolated and reverse transcription–polymerase chain reaction (RT–PCR) was carried out for CXCL16 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar results were obtained in three experiments. (b) Cells were stimulated with IL-1β (0·1–10 ng/ml), TNF-α (0·1–10 ng/ml), IFN-γ (0·1–10 ng/ml), IL-4 (0·1–10 ng/ml), IL-13 (0·1–10 ng/ml) and IL-10 (0·1–10 ng/ml) for 24 h. The expression levels of CXCL16 in the supernatants were measured with enzyme-linked immunosorbent assay. The results were calculated as the mean and standard deviation (s.d.) of one representative experiment performed in triplicate. Error bars show the s.d. of the values. *P < 0·01, significantly different from the medium.

Fig. 4

Interferon (IFN)-γ, interleukin (IL)-4 and IL-13 modulate the IL-1β-induced the release of CXCL16 by human gingival fibroblasts (HGFs). (a) HGFs were treated with IL-1β (1 ng/ml) with or without tumour necrosis factor (TNF)-α/IFN-γ (0·1, 1, 10 ng/ml), and the supernatants were collected after 24 h. The expression levels of CXCL16 in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (b) HGFs were treated with IL-1β (1 ng/ml) with or without IL-4/IL-10/IL-13 (0·1, 1, 10 ng/ml), and the supernatants were collected after 24 h. The expression levels of CXCL16 in the supernatants were measured by ELISA. Data are representative of HGFs from three different donors. The results were calculated as the mean and standard deviation (s.d.) of one representative experiment performed in triplicate. Error bars show the s.d. of the values. *P < 0·01 significantly different from the IL-1β or IFN-γ-stimulated HGFs.

Fig. 5

Effects of mitogen-activated protein kinase (MAPK) inhibitors, a PI3K inhibitor and a nuclear factor (NF)-κB inhibitor on the interleukin (IL)-1β or interferon (IFN)-γ-stimulated CXCL16 expression by human gingival fibroblasts (HGFs). Cells were preincubated with SB203580 (0·2, 2, 20 µM), PD98059 (0·2, 2, 20 µM), SP600125 (0·2, 2, 20 µM), LY294002 (0·2, 2, 20 µM) or MG-132 (0·2, 20, 20 µM) for 1 h and then incubated with IL-1β (1 ng/ml) (a) or IFN-γ (1 ng/ml) (b). After 24 h incubation, supernatants were collected and CXCL16 expression was measured by enzyme-linked immunosorbent assay. Data are representative of HGFs from three different donors. The results were calculated as the mean and standard deviation (s.d.) of one representative experiment performed in triplicate. Error bars show the s.d. of the values. *P < 0·05, **P < 0·01 significantly different from the IL-1β or IFN-γ-stimulated HGFs without inhibitors.

Fig. 6

Effects of metalloproteinase inhibitors on the release of CXCL16 from human gingival fibroblasts (HGFs). Cells were preincubated with GM6001 (0·5, 5, 50 µM) or TAPI2 (0·5, 5, 50 µM) for 1 h and then incubated with interleukin (IL)-1β (1 ng/ml) (a) or interferon (IFN)-γ (1 ng/ml) (b). After 24 h incubation, supernatants were collected and the CXCL16 expression was measured by enzyme-linked immunosorbent assay. Data are representative of HGFs from three different donors. The results were calculated as the mean and standard deviation (s.d.) of one representative experiment performed in triplicate. Error bars show the s.d. of the values. *P < 0·05, **P < 0·01 significantly different from the IL-1β or IFN-γ-stimulated HGF without inhibitors.