Expression of the fetal HLA-G molecule by ocular surface epithelium is enhanced when expanded on amniotic membranes

Research Project

Project/Area Number	15591879
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Ophthalmology
Research Institution	TOKYO DENTAL COLLEGE
Principal Investigator	SHIMAZAKI Jun Tokyo Dental College, Department of Dentistry, Associate Professor, 歯学部, 助教授 (40170930)
Co-Investigator(Kenkyū-buntansha)	TSUBOTA Kazuo Tokyo Dental College, Cornea center, Chief of Research, 角膜センター, 教授(研究部長) (40163878)
Project Period (FY)	2003 – 2004
Project Status	Completed (Fiscal Year 2004)
Budget Amount *help	¥3,500,000 (Direct Cost: ¥3,500,000) Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000) Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Keywords	Amniotic membrane / HLA-G / corneal coniunctival epitherial cells / Anti-inflammatory / immune response / Cytotoxic assay / 細胞障害性試験 / IFN-γ
Research Abstract	PURPOSE. To investigate the expression and function of HLA-G in limbal and conjunctival epithelial cells when expanded on amniotic membrane(AM). METHODS. A conjunctival epithelial cell line (CCL20.2) was used to screen mRNA expression of HLA molecules when cultured with or without amniotic membrane. Expression of HLA-G mRNA in primary cultured limbal and conjunctival epithelial cells was measured by RT-PCR and real-time PCR. Immunocytochemical staining and western blot analysis was done to confirm HLA-G protein expression. The HLA class I negative K-562 human erythroleukaemia cell line was transfected with limbal epithelium-derived HLA-G mRNA for functional analysis using an NK cell-lysis assay. RESULTS. Microarray analysis revealed increased expression of HLA-G, and decreased expression of other HLA genes when CCL20.2 cells were cultured on AM rather than plastic. Limbal and conjunctival epithelial cells were also shown to express HLA-G by RT-PCR, immunocytology and western blots. Semi-quantitative real-time PCR showed a significant increase in HLA-G expression in conjunctival epithelial cells when cultured on AM. Although expression of HLA-G in limbal epithelial cells did not change with AM substrate alone, a more than 2-fold greater upregulation was observed following the addition of IFN-γ when cultured on AM. NK cell-induced cytolysis of K-562 cells was slightly inhibited by transfection of HLA-G. CONCLUSION. Conjuctival and corneal epithelial cells express functional HLA-G when expanded ex vivo, which is enhanced by using an AM substrate and by IFN-γ stimulation.