Cytokines differentially regulate ICAM-1 and VCAM-1 expression on human gingival fibroblasts

Abstract

The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on human gingival fibroblasts (HGF) may be important for migration and retention of inflammatory cells in periodontally diseased tissue. This study aimed to assess which cytokines regulate ICAM-1 and VCAM-1 expression on HGF. Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma enhanced both ICAM-1 and VCAM-1 expression on HGF. Interleukin (IL)-1beta mainly up-regulated ICAM-1 expression. On the other hand, IL-4 and IL-13 enhanced only VCAM-1 expression on HGF. IL-10 did not modulate both ICAM-1 and VCAM-1 expression. Transforming growth factor (TGF)-beta1 enhanced ICAM-1 expression. However, TGF-beta1 inhibited the VCAM-1 expression induced by TNF-alpha or IL-4. Both ICAM-1 and VCAM-1 expression by HGF was inhibited by nuclear factor-kappaB (NF-kappaB) activation inhibitor (MG-132). Mitogen-activated protein kinases (MAPK) inhibitors did not influence ICAM-1 expression induced by TNF-alpha. Interestingly, VCAM-1 expression was enhanced by MEK inhibitor (PD98059) and c-Jun NH2-terminal kinase (JNK) inhibitor (SP600125). These results mean that the balance of cytokines in periodontally diseased tissue may be essential for control of ICAM-1 and VCAM-1 expression on HGF, and the balance of ICAM-1 and VCAM-1 expression might be important for regulation of leucocytes infiltration and retention in periodontally diseased tissue.

Fig. 1

Induction of ICAM-1 and VCAM-1 by cytokines. (a)HGF were treated without or with IL-1β (0·1–100 ng/ml), TNF-α (0·1–100 ng/ml), IFN-γ (0·1–100 ng/ml), IL-4 (0·1–100 ng/ml) and IL-13 (0·1–100 ng/ml) for 4 h. Total RNA was isolated, and RT-PCR analysis was carried out for ICAM-1, VCAM-1 and GAPDH. Similar results were obtained in three repeated experiments. (b) Cells were stimulated with IL-1β (0·1–100 ng/ml), TNF-α (0·1–100 ng/ml), 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 24 h. Cell surface ICAM-1 and VCAM-1 expression was analysed by flow cytometry. These results expressed mean fluorescence intensity (ICAM-1) or percentage of positively stained cells (VCAM-1). Data are presented as the mean ± SD of three experiments.

Fig. 2

IL-4 and IL-13 synergizes with TNF-α for VCAM-1. Cells were stimulated with IL-1 β (1 ng/ml), TNF-α (1 ng/ml), IFN-γ (1 ng/ml) or E.coli LPS (1 µg/ml) and 0, 0·1, 1 or 10 ng/ml of (a) IL-4 or (b) IL-13 for 24 h. Cell surface VCAM-1 expression was analysed by flow cytometry. These results express percentage of positively stained cells. Data are presented as the mean ± SD of three experiments. *P < 0·05 (compared with medium-only control).

Fig. 3

TGF-β1 differently modulates ICAM-1 and VCAM-1 expression on HGF. (a) HGF were stimulated with TGF-β1 (0·1–100 ng/ml) for 24 h. (b) HGF were stimulated with indicated concentrations of TGF-β1 in combination with IL-1 β (1 ng/ml), TNF-α (1 ng/ml) or IL-4 (1 ng/ml) for 24 h. ICAM-1 and VCAM-1 expression on HGF was analysed by flow cytometry. These results expressed mean fluorescence intensity (ICAM-1) or percentage of positively stained cells (VCAM-1). Data are presented as the mean ± SD of three independent experiments. *P < 0·05; **P < 0·01 (compared with medium-only control or between two groups).

Fig. 4

Effects of NF-κB inhibitor on TNF-α stimulated ICAM-1 and VCAM-1 expression by HGF. Cells were preincubaled with MG-132 (0·5–50 µM) for 1 h and then incubated with TNF-α (1 ng/ml). After a 24 h incubation, ICAM-1 and VCAM-1 expression was analysed by flow cytometry. These results expressed mean fluorescence intensity (ICAM-1) or percentage of positively stained cells (VCAM-1). Data are presented as the mean ± SD of three independent experiments. **P < 0·01 (between two groups).

Fig. 5

Effects of MAPKs inhibitors on TNF-α stimulated ICAM-1 and VCAM-1 expression by HGF. Cells were preincubated with SB203580 (0·2–20 µM), PD98052 (0·2–20 µM) or SP600125 (0·2–20 µM) for 1 h and then incubated with TNF-α (1 ng/ml). After a 24 h incubation, (a) ICAM-1 and (b) VCAM-1 expression was analysed by flow cytometry. These results expressed mean fluorescence intensity (ICAM-1) or percentage of positively stained cells (VCAM-1). Data are presented as the mean ± SD of three independent experiments. *P < 0·05 (compared with TNF-α stimulated HGF without MAPKs inhibitor).