Hyperosmolar Stress Upregulates HLA-DR Expression in Human Conjunctival Epithelium in Dry Eye Patients and In Vitro Models

Abstract

To investigate the immune response of human conjunctival epithelium to hyperosmolar stress. Tear osmolarity was measured 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 hours 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 primary human conjunctival epithelial cells, 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. Ruthenium red 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.