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

Abstract

PURPOSE.To investigate the immune response of human conjunctival epithelium to hyperosmolar stress. METHODS. 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 Ͻ 0.05). RESULTS. HLA-DR expression increased in DE subjects with respect to control (% mean Ϯ SD, respectively, 46.16 Ϯ 7.2 vs. 7.48 Ϯ 1.14, P Ͻ 0.0001) and exhibited significantly high correlations with tear osmolarity values (r ϭ 0.614; P Ͻ 0.0001). In vitro experiments showed a progressive increase in HLA-DR expression as the osmolarity of the medium was increased from 6.75 Ϯ 1.16 (% mean Ϯ SD) in iso-osmolarcultured cells to 9.96 Ϯ 1.37 and 12.94 Ϯ 4.04 in cells cultured in, respectively, 350 and 400 mOsm/L (P Ͻ 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. CONCLUSIONS. 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;52:5488 -5496)

Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro

“…We used a conjunctiva-derived cell line for studying inflammatory markers induced by hyperosmotic and toxic stresses, regarding the expression of CCL2, IL-8, IL-6, MIF and ICAM-1 and the osmoprotectant element NFAT5 in order to exhibit the active participation of Hyperosmolarity is now considered a predictive parameter of dry eye assessment and a good diagnosis criterion for the disease [37,40] , showing better correlation with symptoms than standard clinical dry eye signs [41,42] . Recently, hyperosmolarity at the epithelial cell level has been proposed as a potent inducer of inflammatory events and the increased expression of inflammatory markers has been shown to exhibit high correlations with tear osmolarity values in dry eye patients [7] . At the same time, BAK has been demonstrated to induce dry eye disease by causing tear film instability and hyposecretion.…”

“…The corneal tissue, highly innervated and transparent (mandatory for visual function), has already been studied in response to hyperosmotic stress [6] . However, only a few in vitro and in vivo hyperosmolarity studies have been conducted on conjunctival cells [7][8][9] in comparison to corneal cells [10][11][12][13][14][15] . Instead, many in vivo models of dry eye disease have been built to assess the inflammatory consequences of dry eye syndrome [16,17] .…”

“…These findings have been also confirmed in animal [17] and in vitro models [10] of dry eye disease. Several studies showed that the expression of immune-related antigens such as human leukocyte antigen (HLA-DR) and the intercellular adhesion molecule (ICAM)-1 are increased in the conjunctival cells of impression cytology specimens in dry eye patients [21,22,24] as well as on human conjunctival epithelium in hyperosmolar conditions [7] .…”

Quantitative Analysis of Tear Film Fluorescence and Discomfort During Tear Film Instability and Thinning

“…36 Many have speculated that tear thinning leads to increases in hyperosmolarity, 14,16,32 providing a chemical stimulus for surface polymodal nociceptors 11 and increasing the inflammatory response. 10,12,37 Recently, we showed, using a combination of imaging and psychophysical and cell biology methods, that tear film hyperosmolarity may transiently spike during eye opening trials, perhaps reaching levels as high as 800 to 900 mOsm/ kg. 15 Thus, given tear film evaporation, thinning and turnover rates, [38][39][40] it appears reasonable to attribute the slow increases in discomfort during trials for subjects 1 and 2 ( Fig.…”

Biomarkers of Ocular Surface Disease Using Impression Cytology

“…As well as assessing morphological changes in squamous metaplasia, IC can be used to quantify inflammatory markers, such as the MHC Class II antigen HLA-DR. HLA-DR was found to be induced by exposure of conjunctival cells to hyperosmolarity [24]. HLA-DR is thus considered a proinflammatory marker, and it is specifically upregulated in patients with severe DED.…”