Suppression Of Fibrotic Responses In Conjunctival Fibroblasts Activated By Peripheral Blood Eosinophils By The Amniotic Membrane Matrix

Abstract

Abstract: : Purpose:Mast cells, eosinophils and fibroblasts play a key role in inflammatory and fibrotic responses in allergic inflammation. The amniotic membrane was recently found to downregulate such responses. The purpose of this study was to evaluate anti-fibrotic effects of the amniotic membrane matrix (AMM) on conjunctival fibroblasts in an in-vitro model of ocular allergic inflammation. Methods:Human conjunctival or lung fibroblasts were seeded on plastic or on the stromal side of the AMM, which was mounted on plastic inserts. Human peripheral blood eosinophils activated with lipopolysaccharide, or human umbilical cord mast cell cellular sonicates were added to sub-confluent monolayers of the cultured fibroblasts. Proliferation of sub-confluent fibroblasts grown on the AMM was assessed using [₃H]-thymidine incorporation assay. The expression of Transforming Growth Factor (TGF)-ß1 and of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) in conjunctival or lung fibroblasts was measured in conditioned media from these cultures by ELISA, and in RNA extracts by RT-PCR. Results:After 4 days in culture, [₃H]-thymidine incorporation assay indicated a markedly reduced proliferation of activated conjunctival fibroblasts when cultured on the AMM. The expression of TGF-ß1 was significantly suppressed in activated conjunctival fibroblasts cultured on the AMM compared to those cultured on plastic, while the expression of GM-CSF was suppressed in human lung fibroblasts cultured on the AMM. Conclusion:The AMM is capable of suppressing fibrotic responses in an in-vitro model of ocular allergic inflammation. These data may provide a basis for exploring matrix components in the amniotic membrane for the prevention of the fibrotic manifestations in severe allergic eye disease.

Keywords: 365 conjunctiva • 437 inflammation • 380 cytokines/chemokines