Increase of CCL20 expression by human gingival fibroblasts upon stimulation with cytokines and bacterial endotoxin

Abstract

We have demonstrated recently that CCL20 was expressed in periodontal diseased tissues and abundant CCR6 positive T cells infiltrated in periodontally diseased tissue. However, it is uncertain which cells can elicit CCL20 production. In the present study, we examined the properties of CCL20 production by human gingival fibroblasts (HGF) culture. Here, we report that interleukin-1 beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and Escherichia coli lipopolysaccharide (LPS) can significantly induce the production of CCL20 by HGF. We found that TNF-alpha and E. coli LPS enhanced the production of CCL20 by HGF treated with IL-1beta. In contrast, interferon-gamma (IFN-gamma) dramatically diminished CCL20 production induced by IL-1beta. Moreover, we demonstrated that nuclear factor-kappaB (NF-kappaB), p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) play an important role in mediating the production of CCL20 induced by IL-1beta and TNF-alpha. On the other hand, we found that not only NF-kappaB, p38 MAPK and ERK but also c-Jun NH2-terminal kinase (JNK) are involved in CCL20 production induced by E. coli LPS. Finally, we found that HGF express CCR6, CCL20 receptor, and CCL20 induced vascular endothelial growth factor (VEGF) by HGF. Taken together, these findings that HGF will be a source of CCL20 in periodontal tissue, and the CCL20 production will be controlled by proinflammatory cytokine and bacterial LPS in periodontally diseased tissue. Thus, CCL20 by HGF might be involved in inflammatory cells infiltration, and promote the progression of periodontal disease.

Fig. 1

Induction of CCL20 mRNA by interleukin (IL)-1β and tumour necrosis factor (TNF)-α. (a) Total RNA was prepared from human gingival fibroblasts (HGF) treated without or with IL-1β (0·1–100 ng/ml) or TNF-α (0·1–100 ng/ml) as indicated. After 4 h, reverse transcription-polymerase chain reaction (RT-PCR) analysis was carried out for CCL20 and GAPDH. (b) Total RNA was prepared from HGF treated without or with IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 2, 4, 6, 12 and 24 h. RT-PCR analysis was carried out for CCL20 and GAPDH. Similar results were obtained in three repeated experiments.

Fig. 2

Release of CCL20 by human gingival fibroblasts (HGF). HGF was cultured in the absence or presence of interleukin (IL)-1β (1–100 ng/ml), tumour necrosis factor (TNF)-α (1–100 ng/ml), interferon (IFN)-γ (1–100 ng/ml), Escherichia coli lipopolysaccharide (LPS) (1–100 µg/ml), Porphyromonas gingivalis LPS (1–100 µg/ml), Staphylococcus aureus lipoteichoic acid (LTA) (1–100 µg/ml) and Streptococcus mutans LTA (1–100 µg/ml) for 24 h at 37°C. Medium was removed and assayed for CCL20 release by enzyme-linked immunosorbent assay. Data are presented as the mean ± SD (n = 3; by Student's t-test, *P < 0·05, stimulated versus unstimulated). Similar results were obtained in three repeated experiments.

Fig. 3

Effects of tumour necrosis factor (TNF)-α, Escherichia coli lipopolysaccharide and interferon (IFN)-γ on CCL20 release by interleukin (IL)-1β−stimulated human gingival fibroblasts (HGF). HGF was stimulated with IL-1β (10 ng/ml) in the presence or absence of TNF-α (0·1–10 ng/ml), E. coli LPS (0·1–10 µg/ml) or IFN-γ (0·1–10 ng/ml) for 24 h at 37°C. Medium was removed and assayed for CCL20 release by enzyme-linked immunosorbent assay. Data are presented as the mean ± SD (n = 3; by Student's t-test, *P < 0·05). Similar results were obtained in three repeated experiments.

Fig. 4

Effects of signalling pathway inhibitors the production of CCL20 production by stimulated human gingival fibroblasts (HGF). HGF was pretreated for 1 h with or without MG-132 (0·5–50 µ M), PD98052 (0·2–20 µ M), SB203580 (0·1–10 µ M), SP600125 (0·2–20 µ M) or dimethylsulphoxide (1 : 2000 dilution) as a control, and treated with interleukin (IL)-1β (10 ng/ml), tumour necrosis factor (TNF)-α (10 ng/ml) or Escherichia coli lipopolysaccharide (10 µg/ml) for 24 h at 37°C. Medium was removed and assayed for CCL20 release by enzyme-linked immunosorbent assay. Data are presented as the mean ± SD (n = 3, by Student's t-test, *P < 0·05 versus stimulated). Similar results were obtained in three repeated experiments.

Fig. 5

Release of vascular endothelial growth factor (VEGF) by human gingival fibroblasts (HGF) treated with CCL20. (a) CCR6 expression on HGF. HGF was incubated with human CCR6 mouse antibody or isotype-matched mouse IgG. Cells were then stained with fluoroscein isothiocyanate (FITC)-labelled goat antimouse IgG and analysed with flow cytometry to determine the expression of CCR6. Data are presented as the mean ± SD (n = 3; by Student's t-test, *P < 0·05). Similar results were obtained in three repeated experiments. (b) HGF was stimulated in the absence or presence of CCL20 (1–100 ng/ml) for 24 h at 37°C. Medium was removed and assayed for VEGF release by enzyme-linked immunosorbent assay. Data are presented as the mean ± SD (n = 3; by Student's t-test, *P < 0·05, stimulated versus unstimulated). Similar results were obtained in three repeated experiments.