Ocular surface epithelial cells up-regulate HLA-G when expanded in vitro on amniotic membrane substrates.
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.
Higa
Kazunari
K
Cornea Center, Tokyo Dental College, Chiba, Japan.
Shimmura
Shigeto
S
Shimazaki
Jun
J
Tsubota
Kazuo
K
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
D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D012333:RNA, Messenger