Ocular surface epithelial cells up-regulate HLA-G when expanded in vitro on amniotic membrane substrates

Kazunari Higa, Shigeto Shimmura, Jun Shimazaki, Kazuo Tsubota

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9 Citations (Scopus)

Abstract

PURPOSE: 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. CONCLUSION: 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.

Original languageEnglish
Pages (from-to)715-721
Number of pages7
JournalCornea
Volume25
Issue number6
DOIs
Publication statusPublished - 2006 Jul 1

Keywords

  • Amniotic membrane
  • Conjunctival epithelium
  • Cornea immune responses
  • Corneal epithelium
  • HLA-G

ASJC Scopus subject areas

  • Ophthalmology

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