Comparative in vitro Study of Topical Ocular Anti–Allergic Eye–Drops on Human Conjunctiva–Derived Cells: Response to IFN and Toxic Effects

Abstract

Abstract: : Purpose: To investigate the various effects associated with the use of antihistamines, mast cell stabilizers or drug preparations with dual effects, whenever preserved or not, on conjunctiva–derived–cells, including inflammation–modulating as well as toxic and oxidative effects, possibly responsible for noxious actions of these drugs solutions. Methods: Conjunctiva–derived cells (Wong Kilbourne cell line) were stimulated in vitro with IFNγ (25U/ml) in order to induce the surface expression of intercellular adhesion molecule–1 (ICAM–1). 1/100 dilutions of anti–allergic eye–drops were added to the cells for 24 hours and IFNγ–stimulated cells as well as cells treated with ophthalmic preparations alone were evaluated by flow cytometry for the expression of the inflammation–related marker ICAM–1. Toxicological assays were also performed using cold light cytofluorometry: viability and apoptosis as well as ROS and O2^.– production were assessed using neutral red, Hoechst/propidium iodide, H₂–DCFDA and hydroethidine tests, respectively. Results: None of the anti–allergic eye–drops tested could reduce the basal and IFNγ–stimulated expression of the inflammatory marker ICAM–1. Conversely, the preserved eye–drops even induced higher ICAM–1 expression levels on IFNγ–stimulated cells than that generated by IFNγ alone or combined with non–preserved eye–drops, thus suggesting that BAC might enhance the IFNγ–induced surface expression of ICAM–1. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress. Conclusions: This study confirms that IFNγ has pro–inflammatory effects on human conjunctiva–derived cells and that these cells may contribute to ocular inflammation in allergic diseases. Although some anti–allergic molecules have been found to possess anti–inflammatory properties, preserved ophthalmic formulations may enhance inflammatory profiles in the presence of low inflammatory stimulus (IFNγ) and displayed toxic as well as oxidative properties on these cells. Thus, care should be taken in the formulation of the eye–drops, as preservatives might interfere with the potential anti–inflammatory properties of the drug molecule itself.

Keywords: conjunctiva • drug toxicity/drug effects