Aim: Non-infectious posterior uveitis is an immune-mediated retinal disease. Migration of circulating monocytes through the retinal endothelium, with differentiation to macrophages, is the critical event, since macrophages mediate the damage that leads to vision loss. Development of effective treatment relies on delineating disease mechanisms in in vitro human models. Our aim was to determine the impact of retinal endothelial transmigration on human monocyte differentiation in vitro.

Methods: Boyden chamber assays were used to simulate human monocyte migration across retinal endothelium. 1 x 10⁶ human peripheral blood monocytes (PBMCs) from four donors, isolated by negative selection, were cultured for 24 hours on a 0.3 cm² PET membrane with 3 µM perforations, coated with bovine collagen type 1 and seeded with 6 x 10⁵ immortalized human retinal endothelial cells (HRECs). Cell surface expression of monocyte (CD14⁺, CD16^+/-), M1 (CD80⁺, CD64⁺) and M2 (CD200R⁺, CD209⁺) macrophage markers was determined by flow cytometry (BD FACSCanto II). Monocytes in both apical (non-migrated) and basal (migrated) chambers were immunophenotyped 24 hours post-culture and compared to fresh monocytes and cultured monocytes without HRECs.

Results: Human monocytes migrated across retinal endothelial cell monolayers in small numbers (range = 10⁴ – 10⁵ cells). CD14 expression was significantly higher in cells cultured with HREC contact. CD16 expression was not highly expressed, and varied with donor and culture conditions. There were no significant differences in macrophage markers, with CD64 moderately expressed under all culture conditions, and CD80, CD200R and CD209 expressed on small populations and with low expression in some donors.

Conclusions: Based on our preliminary results, PBMCs migrate across non-stimulated, human retinal endothelial monolayers and migration does not impact M1 versus M2 polarisation, based on CD80/CD64 and CD200R/CD209 surface expression.