title: Ocular surface epithelial cells up-regulate HLA-G when expanded in vitro on amniotic membrane substrates.
abstract: OBJECTIVE
  To study the modulation of immunoregulatory genes in ocular surface epithelial cells cultured on amniotic membrane (AM).
  
  
  METHODS
  Microarray analysis was performed in a conjunctival epithelial cell line (CCL20.2) expanded on denuded AM. Among the genes that were upregulated by an AM substrate compared with collagen-coated dishes, the fetal nonclassic major histocompatibility complex molecule, HLA-G, was found to be the only immunoregulatory gene up-regulated by more than 2.5-fold. Because CCL20.2 is contaminated by HeLa cells, expression of HLA-G mRNA was confirmed in primary-cultured limbal (LE) and conjunctival epithelial (CE) cells by reverse transcriptase-polymerase chain reaction (RT-PCR), semiquantitative real-time PCR, immunocytochemistry, and Western blot analysis. A functional assay was performed using an HLA-G-transfected K-562 human erythroleukemia cell line.
  
  
  RESULTS
  Freshly dissociated limbal epithelial cells express HLA-G mRNA; however, protein levels were low. Western blots and immunocytochemistry showed that both LE and CE cells upregulated the HLA-G protein when cultured on collagen-coated dishes and on AM. HLA-G mRNA levels were significantly higher in CE cultured on AM compared with collagen. Natural killer (NK) cell-induced cell lysis of an HLA class 1-negative K-562 human erythroleukemia cell line was slightly reduced when transfected with LE-derived HLA-G mRNA.
  
  
  CONCLUSIONS
  CE and LE cells express functional HLA-G when expanded ex vivo, which may affect inflammation and immune reaction when transplanted to the ocular surface.
authors.affiliation: Cornea Center, Tokyo Dental College, Chiba, Japan.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Higa
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
authors.forename: Jun
authors.initials: J
authors.lastname: Shimazaki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
mesh_terms: D000650:Amnion / Q000502:physiology*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D018920:Coculture Techniques; D003228:Conjunctiva / Q000166:cytology*; D004847:Epithelial Cells / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006680:HLA Antigens / Q000235:genetics* / Q000378:metabolism; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger