Induction of matrix metalloproteinase-1 by tumor necrosis factor-α is mediated by interleukin-6 in cultured fibroblasts of keratoconus

Abstract

Inflammatory molecules and matrix metalloproteinase (MMPs) have been found over-expressed in the tear film of patients with keratoconus. However, the mechanistic link between inflammatory molecules and MMPs in the pathogenesis of keratoconus remains still elusive. Therefore, we investigated the effect of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) on MMP-1 expression and used IL-6 antibody (IL-6 Ab) to examine the role of IL-6 on TNF-α mediated regulation of MMP-1 in fibroblasts of normal cornea and keratoconus. Real-time polymerase chain reaction, Enzyme-linked immunosorbent assay, and Western blot data demonstrated that MMP-1 and IL-6 were expressed in fibroblasts of normal cornea and keratoconus. Levels of MMP-1 and IL-6 were significantly higher in keratoconus than normal cornea. TNF-α treatment led to a significant increase in IL-6 levels. IL-6 treatment induced MMP-1 synthesis in normal cornea and keratoconus. TNF-α increased MMP-1 expression in a dose- and time-dependent manner and this response was completely inhibited by the IL-6 Ab. In conclusion, these results indicate that fibroblasts of keratoconus shows increased levels of IL-6 and MMP-1 gene and protein expression and IL-6 mediates the TNF-α-induced MMP-1 expression.

Keywords: Corneal fibroblasts; inflammatory cytokine; keratoconus; matrix metalloproteinases (MMPs).

Figure 1

Effect of TNF-α on MMP-1 mRNA expression and protein synthesis in fibroblasts of normal cornea (n = 3) and keratoconus (n = 3). Cells were incubated for 12 h in the presence of various concentrations of TNF-α and stimulated with the absence or presence of 1 ng/mL TNF-α for the indicated times intervals. Total RNA and cell-culture supernatants were then collected. (a and d) Levels of MMP-1 mRNA measured by real-time PCR from total RNA. (b, e, and f) Representative Western blot for MMP-1 protein in cell-culture supernatants. (c and g) Quantitative analysis of MMP-1 protein levels by Image-Pro Plus. The results are presented as mean ± standard deviation of three independent experiments. KC: keratoconus, NC: normal cornea. Cells without TNF-α treatment were used as control. * P < 0.05, compared with their own control group; ^#P < 0.05, KC control group vs. NC control group

Figure 2

Levels of IL-6 in fibroblasts of normal cornea (n = 3) and keratoconus (n = 3) cultured with TNF-α. Cells were treated with various TNF-α concentrations for 12 h or incubated with the absence or presence of 1 ng/mL TNF-α for various incubation times. Total RNA and cell-culture supernatants were then collected. (a and c) IL-6 mRNA levels determined by real-time PCR from total RNA. (b and d) IL-6 protein in supernatants measured using a human IL-6 ELISA kit. Values are presented as mean ± standard deviation of three independent experiments. KC: keratoconus, NC: normal cornea. Cells without TNF-α treatment were used as control. *P < 0.05, compared with their own control group; ^#P < 0.05, KC control vs. NC control

Figure 3

Effect of IL-6 on MMP-l levels in fibroblasts of normal cornea (n = 3) and keratoconus (n = 3). Cells were treated with IL-6 for 12 h at the indicated concentration and incubated in the absence or presence of 25 ng/mL IL-6 for various times. Total RNA and cell-culture supernatants were then collected. (a and d) MMP-1 mRNA expression detected by real-time PCR from total RNA. (b, e, and f) Representative Western blot for evaluation of MMP-1 protein in cell-culture supernatants. (c and g) Quantitative analysis of MMP-1 protein levels using Image-Pro Plus. Data are expressed as mean ± standard deviation of three independent experiments. KC: keratoconus, NC: normal cornea. Cells without IL-6 incubation were used as control. *P < 0.05, compared with their own control group; ^#P < 0.05, KC control vs. NC control

Figure 4

Effect of TNF-α and IL-6 on the levels of MMP-1. Cells were cultured with 1 ng/mL TNF-α or 25 ng/mL IL-6 individually and jointly for 12 h. Total RNA and cell-culture supernatants were then collected. (a) The mRNA levels of MMP-1 determined by real-time PCR from total RNA. (b) Representative Western blot for MMP-1 protein in cell-culture supernatants. (c) Quantitative analysis of MMP-1 protein levels by Image-Pro Plus. Values are expressed as mean ± standard deviation of three independent experiments. KC: keratoconus (n = 3), NC: normal cornea (n = 3). *P < 0.05, compared with cells stimulated by TNF-α or IL-6 individually

Figure 5

Expression of MMP-1 in fibroblasts of normal cornea (n = 3) and keratoconus (n = 3) under both TNF-α and IL-6 Ab. Cells were treated with 1 ng/mL TNF-α and 40 ng/mL IL-6 Ab during 12 h. Total RNA and cell-culture supernatants were then collected. (a) IL-6 protein detected in cell-culture supernatants by ELISA. (b) IL-6 mRNA expression from total RNA determined by real-time PCR. (c) Representative Western blot for MMP-1 protein in cell-culture supernatants. (d) Quantitative analysis of MMP-1 protein levels by Image-Pro Plus. Results are presented as mean ± standard deviation of three independent experiments. KC: keratoconus, NC: normal cornea. Cells without IL-6 Ab or TNF-α treatment were used as control. *P < 0.05, TNF-α + IL-6 Ab vs. control