Fig. 6.

TNF-α and IL-6 altered the barrier function of corneal epithelial cells. (a) Relative transepithelial electrical resistance (TEER) of human primary corneal epithelial cells (CEP) incubated with PBS (control), TNF-α (10 ng/mL), IL-6 (10 ng/mL), or TNF-α (10 ng/mL) + IL-6 (10 ng/mL) for 24 h. ANOVA was used to determine significant differences (p < 0.0001), and Dunnett's multiple comparisons tests were used for paired comparisons between PBS and TNF-α or IL-6-treated groups. P < 0.05 was considered statistically significant. (b) CEP cells were cultured in chamber slides at 5 × 10⁴ cells per well and incubated with PBS (control), TNF-α (10 ng/mL), IL-6 (10 ng/mL), or TNF-α (10 ng/mL) + IL-6 (10 ng/mL) for 24 h. Claudin-1 and ZO-1 were detected by immunofluorescence staining. (c) CEP cells were cultured in 6-well plates at 1 × 10⁵ cells per well and incubated with PBS (control), TNF-α (10 ng/mL), IL-6 (10 ng/mL) or TNF-α (10 ng/mL) + IL-6 (10 ng/mL) for 24 h. Claudin-1 and ZO-1 were detected by western blot analysis. The relative expression levels were determined by densitometry analysis. Depicted western blots are one representative figure of three independent experiments. Results are the mean ± S.D. of three independent experiments. (d) CEP cells were seeded on transwell inserts with 0.4-μm pore size incubated with PBS (control), TNF-α (10 ng/mL), IL-6 (10 ng/mL) or TNF-α (10 ng/mL) + IL-6 (10 ng/mL) for 16 h and the levels of TNF-α, IL-6, and IL-8 in apical and basolateral compartments were determined with enzyme-linked immunosorbent assay (ELISA). ANOVA was used to determine significant differences (p < 0.0001), and Tukey's multiple comparisons tests were used for paired comparisons between TNF-α (&) or IL-6 (#) with TNF-α + IL-6-treated CEP cells. P < 0.05 was considered statistically significant. (e) The basolateral compartment media of CEP cells treated with PBS (control), TNF-α (10 ng/mL), IL-6 (10 ng/mL), or TNF-α (10 ng/mL) + IL-6 (10 ng/mL) for 16 h were collected. The basolateral compartment media were used to treat human primary retinal pigment epithelial cells for 16 h, and the levels of TNF-α, IL-6, and IL-8 were determined with ELISA. ANOVA was used to determine significant differences (p < 0.0001), and Tukey's multiple comparisons tests were used for paired comparisons between TNF-α (&) or IL-6 (#) with TNF-α + IL-6-treated retinal pigment epithelial cells. P < 0.05 was considered statistically significant.