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.
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 humanerythroleukemia 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 humanerythroleukemia 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.
Authors: Yu Mi Han; Roberto Romero; Jung-Sun Kim; Adi L Tarca; Sun Kwon Kim; Sorin Draghici; Juan Pedro Kusanovic; Francesca Gotsch; Pooja Mittal; Sonia S Hassan; Chong Jai Kim Journal: Biol Reprod Date: 2008-08-06 Impact factor: 4.285
Authors: Simon Jasinski-Bergner; Markus Eckstein; Helge Taubert; Sven Wach; Christian Fiebig; Reiner Strick; Arndt Hartmann; Barbara Seliger Journal: Front Immunol Date: 2022-02-03 Impact factor: 7.561
Authors: Elizabeth Crabtree; Liujiang Song; Telmo Llanga; Jacquelyn J Bower; Megan Cullen; Jacklyn H Salmon; Matthew L Hirsch; Brian C Gilger Journal: Sci Rep Date: 2019-12-27 Impact factor: 4.379