To investigate the immune response of human conjunctival epithelium to hyperosmolar stress.
Tear osmolarity was measured with TearLab Osmolarity System (OcuSense) in 15 normal subjects and 25 dry eye (DE) patients; conjunctival imprint cytology samples were obtained at the nasal bulbar area. Subconfluent primary human conjunctival epithelial cells (pHCECs) and human conjunctival organ cultures (hCOCs), both cultured in iso-osmolar medium (305 mOsm/L), were exposed for 24 hrs to media with progressively higher osmolarity, with or without the ion channel inhibitor ruthenium red (RuR). Human leukocyte antigen (HLA)-DR expression was evaluated by immunocytochemistry, on imprints from subjects, on pHCECs, on formalin fixed-paraffin embedded hCOCs, and by RT-PCR Statistical evaluation was performed by applying the unpaired Student's t-test, as well as Spearman's rho and Pearson's r correlation coefficients (significance p<0.05).
HLA-DR expression increased in DE subjects with respect to control (% mean±SD respectively 46.16±7.2 vs. 7.48±1.14, p< 0.0001) and exhibited significantly high correlations with tear osmolarity values (r 0.614, p<0.0001). In vitro experiments showed a progressive increase in HLA-DR expression as the osmolarity of the medium was increased from 6.75±1.16 (% mean±SD) in iso-osmolar-cultured cells to 9.96±1.37 and 12.94±4.04 in cells cultured in, respectively, 350 and 400 mOsm/L (p<0.05). A stepwise progressive increase was also found in hCOCs. Results were confirmed by RT-PCR. RuR significantly reduced HLA-DR expression in hyperosmolar-cultured cells
Data from complementary techniques demonstrate that extracellular hyperosmolarity induces HLA-DR overexpression in human conjunctival epithelial cells in both DE patients and in vitro cell culture models.
Invest Ophthalmol Vis Sci. 2011 Apr 15. [Epub ahead of print]
Versura P, Profazio V, Schiavi C, Campos EC.
Ophthalmology Unit, University of Bologna, Italy.