Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration

Abstract

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood-retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-gamma)- and/or tumour necrosis factor-alpha (TNF-alpha)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.

Fig. 1. (a)

Time–response curves to a range of phorbol myristate acetate (PMA) doses established a threshold effect, using 10⁻⁹ m PMA (▾). Four hours post PMA treatment was determined to be optimum to produce a significant differential effect over dimethyl sulphoxide (DMSO)-treated ECV304 monolayers (□) versus PMA @5 × 10⁻⁹ m (•) or 5 × 10⁻¹⁰ m (▴) (P = 0·025). (b) Comparison of the resistances of the triamcinolone acetonide (TA)-treated cells (▴) with control cultures (□) showed that the transepithelial resistance (TER) was markedly higher in TA-treated cultures from day 12 to the conclusion of the experiment. Treatment with 10⁻⁹ m PMA at day 9 resulted in reduced resistance from 4 h post-treatment (▪). Additionally, treatment with 10⁻⁹ m PMA followed by TA (10⁻⁶ m) resulted in a significant increase in resistance compared with PMA-treated cultures (•). Each data point represents the mean TER of five Transwells (n = 5) ± s.e.m.

Fig. 2

Unstimulated ECV304 cells constitutively expressed moderate levels of ICAM-1 (▪) and MHC-I (□) compared with the isotype antibody controls. Similar levels of expression were detectable in cells treated with dimethyl sulphoxide (DMSO)/methanol or triamcinolone acetonide (TA) alone. Phorbol myristate acetate (PMA)-activated cells had significantly (P = 0·0003) up-regulated ICAM-1 expression after 72 h in culture, an approximately four-fold increase over unstimulated levels of expression. After 24 h initial exposure to PMA cells were additionally exposed to TA (10⁻⁶ m) for a further 48 h; treatment of PMA-stimulated cells with TA significantly reduced detectable levels of ICAM-1 expression (PMA versus PMA + TA, P = 0·045). AU, Arbitrary units. MHC-I levels were not significantly modulated by either TA or PMA treatment. Similarly, after 24 h initial exposure to PMA cells were additionally exposed to TA (10⁻⁶ m) for a further 48 h; treatment of PMA-stimulated cells with TA did not significantly modulate levels of MHC-I expression.

Fig. 3

Representative FACS histograms showing ECV304 expression of (a) ICAM-1 and (b) MHC-I for: a, IgG control; b, mock-treated; c, phorbol myristate acetate (PMA) + triamcinolone acetonide (TA); and d, PMA. (c) ICAM-1 for: a, mock-treated; b, IFN-γ and tumour necrosis factor-alpha (TNF-α); c, IFN-γ; d, PMA; and e, TNF-α. PMA treatment significantly up-regulated ICAM-1 expression while subsequent treatment with TA significantly reduced ICAM-1 expression. Treatment of PMA-stimulated cells with TA did not significantly modulate levels of MHC-I expression. PMA had a similar effect to other proinflammatory cytokines; IFN-γ produced equivalent levels of ICAM-modulation to PMA, while TNF-α produced less modulation than PMA. IFN-γ and TNF-α in combination produced most pronounced up-modulation of ICAM-1 expression.

Fig. 4

(A) On coverslips, resting ECV304 cells showed cell membrane-localized ICAM-1 immunostaining. (B) On coverslips staining with isotype control (non-immune immunoglobulin) revealed insignificant levels of non-specific binding to ECV304 cells. (C) On coverslips, phorbol myristate acetate (PMA) stimulation promoted hypertrophy and the formation of long processes in ECV304 cells was associated with more intense ICAM-1 expression at the plasma membrane. (D) On coverslips, subsequent treatment of PMA-stimulated cells with triamcinolone acetonide (TA) resulted in condensation of the ECV304 cell morphology associated with smaller cell size and reductions in processes. (E) On coverslips, ZO-1 immunofluorescent staining revealed the ECV304 cells to form an integrated monolayer with high levels of immunoreactivity expressed at the cell margins and occasional punctate accumulations of staining (arrowheads). (F) A high power electron micrograph showing details of a zonule occludentes junction (small arrow) and a maculae adherens (large arrow). (G) An electron micrograph illustrating that ECV304 cells form a continuous layer with apposing cells joined by both desmosomes (maculae adherens) and tight junctions (ZO). Both types of junction occurred at the apices of the cells (arrows indicate cell boundaries).