Abstract
Purpose:
Evaluation of an exposure or treatment effect on immortalized human meibomian gland epithelial cells can provide insight into meibomian gland function in health and disease. It has been reported that 13-cis retinoic acid (Isotretinoin, e.g. Accutane) therapy for acne is associated with meibomian gland dysfunction (MGD) as well as alterations to meibomain gland cells in culture. The purpose of this series of experiments is to further characterize the influence of 13-cis retinoic acid on the meibomian gland cells.
Methods:
Immortalized human meibomian gland epithelial cells were cultured in both serum free media and serum containing media for 24 hours prior to addition of 13-cis retinoic acid to encompass both the growth phase and differentiation. Lipid production was confirmed by staining with fluorescent LipidTOX, cell permeability and presumed death was shown by Ethidium homodimer-1 (EthD-1) staining, and cell membranes were observed with Cell Mask Orange Plasma Membrane stain. 13-cis retinoic acid was applied at various concentrations; 0.01µM-5µM. Azithromycin was added to 13-cis retinoic acid exposed cells. Cells were observed and imaged at 24hrs, 7 days or at both time points post exposure using an Olympus IX71 inverted microscope and/or a DeltaVision deconvolution microscope at 100x magnification. IL-1β concentrations were determined by ELISA.
Results:
Meibomian gland epithelial cells were adversely affected at all concentrations of 13-cis retinoic acid after 7 days in both growth and differentiated state and increased cell permeability was observed as early as 24 hours post treatment. Differential lipid staining between the cell membrane and presumed meibomian lipids was observed with the Cell Mask Orange Plasma Membrane stain and LipidTOX. Modulation of inflammatory marker IL-1β secretion was observed at all concentrations of 13-cis retinoic acid after 48hrs, and with pre- and post- azithromycin treatment. These changes were seen with just one single treatment as compared to a systemic medication, which would utilize continued dosing over an extended period of time.
Conclusions:
The toxicity of 13-cis retinoic acid exposure to meibomian gland lipid producing cells, even at low concentrations, could play a role in the development of MGD for users of this medication. Our data suggests that this damage could potentially be modified.
Keywords: 486 cornea: tears/tear film/dry eye •
557 inflammation •
599 microscopy: light/fluorescence/immunohistochemistry