title: Hyperosmolar stress upregulates HLA-DR expression in human conjunctival epithelium in dry eye patients and in vitro models.
abstract: OBJECTIVE
  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 < 0.05).
  
  
  RESULTS
  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.
  
  
  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.
authors.affiliation: Ophthalmology Unit, University of Bologna, Bologna, Italy. piera.versura@unibo.it
authors.forename: Piera
authors.initials: P
authors.lastname: Versura
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Profazio
authors.forename: Costantino
authors.initials: C
authors.lastname: Schiavi
authors.forename: Emilio C
authors.initials: EC
authors.lastname: Campos
mesh_terms: D000328:Adult; D002478:Cells, Cultured; D003228:Conjunctiva / Q000378:metabolism*; D015352:Dry Eye Syndromes / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006684:HLA-DR Antigens / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012430:Ruthenium Red / Q000737:chemistry; D012462:Saline Solution, Hypertonic / Q000494:pharmacology*; D013666:Tears / Q000378:metabolism*
chemical_list: D006684:HLA-DR Antigens; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D012430:Ruthenium Red