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

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-1alpha, IL-1beta, 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-1alpha and IL-1beta 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-1alpha protein levels in AM cultures was higher than in plastic cultures.

Conclusions: AM stromal matrix markedly suppresses lipopolysaccharide induced upregulation of both IL-1alpha and IL-1beta. 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.

Figure 1

(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

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.