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

ABSTRACT: 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.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.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.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.

SUBMITTER: Solomon A

PROVIDER: S-EPMC1723909 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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

Solomon A A Rosenblatt M M Monroy D D Ji Z Z Pflugfelder S C SC Tseng S C SC

The British journal of ophthalmology 20010401 4

<h4>Aims</h4>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.<h4>Methods</h4>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 c ...[more]

PMID: 11264135

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Project description:PurposeTo propose an improved stem cell-based strategy for limbal stem cell deficiency (LSCD) treatment.DesignExperimental randomized or parallel-group animal study.SubjectsFifty adult male New Zealand white rabbits.MethodsHuman limbal stem/progenitor cells (LSCs) and limbal stromal stem/progenitor cells (LSSCs) were cultured in serum-free conditions and further differentiated into corneal epithelial cells and keratocytes, respectively. All cell types were characterized with lineage-specific markers. Gene expression analysis was performed to identify the potential function of LSSCs in corneal regeneration. Two LSCD models of rabbits for transplantations were used: transplantation performed at the time of limbal and corneal epithelial excision (LSCD model) and transplantation performed after clinical signs were induced in an LSCD model (pLSCD model). The pLSCD model better mimics the pathologic changes and symptoms of human LSCD. Rabbit models received LSC or LSC plus LSSC treatment. Corneal epithelial defects, neovascularization, and opacity were assessed every 3 weeks for 24 weeks. ZsGreen-labeled LSSCs were used for short-term tracking in vivo.Main outcome measuresRates of corneal epithelial defect area, corneal neovascularization and opacity scores, graft survival rate, and immunofluorescence staining of specific markers.ResultsBoth LSC transplantation and LSC plus LSSC cotransplantation effectively repaired the corneal surface in the LSCD model. These 2 strategies showed no significant differences in terms of graft survival rate or epithelial repair. However, corneal opacity was observed in the LSC group (in 3 of 8 rabbits), but not in the LSC plus LSSC group. Notably, when treating LSCD rabbits with distinguishable stromal opacification and neovascularization, cotransplantation of LSCs and LSSCs exhibited significantly better therapeutic effects than transplantation of LSCs alone, with graft survival rates of 87.5% and 37.5%, respectively. The implanted LSSCs could differentiate into keratocytes during the wound-healing process. RNA sequencing analysis showed that the stromal cells produced not only a collagen-rich extracellular matrix to facilitate reconstruction of the lamellar structure, but also niche factors that accelerated epithelial cell growth and inhibited angiogenesis and inflammation.ConclusionsThese findings highlight the support of stromal cells in niche homeostasis and tissue regeneration, providing LSC plus LSSC cotransplantation as a new treatment strategy for corneal blindness.

Dataset Information

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

Publications

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

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