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. 2001 Apr;85(4):444–449. doi: 10.1136/bjo.85.4.444

Suppression of interleukin 1α and interleukin 1β in human limbal epithelial cells cultured on the amniotic membrane stromal matrix

A Solomon 1, M Rosenblatt 1, D Monroy 1, Z Ji 1, S Pflugfelder 1, S Tseng 1
PMCID: PMC1723909  PMID: 11264135

Abstract

AIMS—Amniotic membrane (AM) transplantation reduces inflammation in a variety of ocular surface disorders. The aim of this study was to determine if AM stroma suppresses the expression of the IL-1 gene family in cultured human corneal limbal epithelial cells.
METHODS—Human corneal limbal epithelial cells were cultured from limbocorneal explants of donor eyes on plastic or on the AM stroma. Transcript expression of IL-1α, IL-1β, IL-1 receptor antagonist (RA), and GAPDH was compared with or without addition of lipopolysaccharide to their serum-free media for 24 hours using RNAse protection assay (RPA). Their protein production in the supernatant was analysed by ELISA.
RESULTS—Expression of IL-1α and IL-1β transcripts and proteins was significantly reduced by cells cultured on the AM stromal matrix compared with plastic cultures whether lipopolysaccharide was added or not. Moreover, expression of IL-1 RA by cells cultured in the lipopolysaccharide-free medium was upregulated by AM stromal matrix. The ratio between IL-1 RA and IL-1α protein levels in AM cultures was higher than in plastic cultures.
CONCLUSIONS—AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1α and IL-1β. These data may explain in part the effect of AM transplantation in reducing ocular surface inflammation, underscoring the unique feature of the AM as a substrate for tissue engineering.



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Figure 1  .

Figure 1  

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(A) Ribonuclease protection assay of RNAs extracted from cultured human corneal epithelial cells. Cells were switched to a serum-free medium with or without 10 µg/ml LPS. Samples were hybridised with riboprobes to IL-1α, IL-1β, IL-1 RA, and GAPDH, followed by treatment with ribonuclease and inactivation with proteinase K. Protected RNA fragments were precipitated and separated on a 6% polyacrylamide Urea-TBE sequencing gel. Numbers in parentheses show the size in base pairs. (B) Graphical presentation of the relative amounts for IL-1α, IL-1β, and IL-1RA mRNAs when corrected for the different amounts of GAPDH mRNA in the same sample. A significant increase in the amount of IL-1α and IL-1β mRNAs was observed following the treatment with LPS in plastic cultures, whereas these amounts were significantly decreased in AM cultures for IL-1α (*p=0.029) and had a marginally significant decrease for IL-1β (**p=0.054). In contrast, the amount of IL-1RA mRNA was significantly upregulated in non-stimulated cells cultured on the AM (***p=0.0002). Data are expressed as the mean (SEM) from five different experiments based on primary cultures from five different donor corneas, respectively.

Figure 2  .

Figure 2  

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Protein amounts of IL-1α (A), IL-1β (B), and IL-1RA (C) measured by ELISA in conditioned media of human limbal epithelium cultured on plastic or AM, and collected after 24 hours in a serum-free medium with or without LPS. (A) A significant decrease of IL-1α protein was noted in LPS stimulated cells on AM (*p=0.009). (B) A significant decrease of IL-1β protein was noted in LPS stimulated and non-stimulated cells on AM (*p=0.017). (C) No change was noted in IL-1RA protein. (D) The IL-1RA/IL-1α ratio was increased in LPS stimulated cells on AM (*p=0.008). Data are expressed as the mean (SEM) from five independent experiments using five different donor corneas.

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