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

Kazunari Higa, Shigeto Shimmura, Jun Shimazaki, Kazuo Tsubota

Research output: Contribution to journalArticle

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

Fingerprint

HLA-G Antigens
Amnion
Up-Regulation
Epithelial Cells
Leukemia, Erythroblastic, Acute
Messenger RNA
Collagen
Cell Line
Western Blotting
Immunohistochemistry
Genes
In Vitro Techniques
Microarray Analysis
Major Histocompatibility Complex
Reverse Transcriptase Polymerase Chain Reaction
HeLa Cells
GTP-Binding Proteins
Natural Killer Cells
Real-Time Polymerase Chain Reaction
Neutrophils

Keywords

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

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Ocular surface epithelial cells up-regulate HLA-G when expanded in vitro on amniotic membrane substrates. / Higa, Kazunari; Shimmura, Shigeto; Shimazaki, Jun; Tsubota, Kazuo.

In: Cornea, Vol. 25, No. 6, 07.2006, p. 715-721.

Research output: Contribution to journalArticle

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N2 - 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.

AB - 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.

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