Interleukin-1 receptor antagonist (IL-1RA) prevents apoptosis in ex vivo expansion of human limbal epithelial cells cultivated on human amniotic membrane.
Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.
Sun
Chi-Chin
CC
Department of Ophthalmology, Chung Gung Memorial Hospital, Keelung, Taiwan.
Su Pang
Jong-Hwei
JH
Cheng
Ching-Yi
CY
Cheng
Hsiao-Fen
HF
Lee
Yun-Shien
YS
Ku
Wan-Chen
WC
Hsiao
Ching-Hsi
CH
Chen
Jen-Kan
JK
Yang
Chuen-Mao
CM
D000293:Adolescent; D000328:Adult; D000368:Aged; D000650:Amnion / Q000166:cytology*; D017304:Annexin A5 / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D066298:In Vitro Techniques; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology*; D008875:Middle Aged; D009500:Neutralization Tests; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D017304:Annexin A5; D000906:Antibodies; D017077:Culture Media, Conditioned; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D016650:Fluorescein-5-isothiocyanate