Pro-inflammatory Cytokine Release From RPE Is Linked to S100B and RAGE Signalling

Abstract

Purpose: : Pro-inflammatory pathology is central to many retinal vascular and neurodegenerative diseases. RPE are known to express the pro-inflammatory receptor for advanced glycation endproducts (RAGE) and this study has sought to determine if S100B, a defined ligand for RAGE, can evoke expression of inflammatory cytokines and key growth factors by RPE.

Methods: : Human RPE cell lines were grown to confluency and exposed to S100B alone (5µM, 0.5µM, 0.05µM, 0.005µM) or in combination with IFNγ (50U/ml) for 24 hours. Protein and mRNA expression of a wide range of pro-inflammatory cytokines were monitored by commercial ELISA and real-time RT-PCR. VEGF expression was also monitored. ARPE-19 were engineered to over-express human full-length RAGE using a GFP tagged pcDNA 3.1 RAGE over expressing vector .Transfected RPE (RAGE++) and wild-type (WT) RPE were then exposed to ligand treatment and analysis. In all cases the experiments were conducted a minimum of 3 times.

Results: : S100B treatment induced significant alterations in the cytokines TNF-alpha (p<0.01), IL6 (p<0.05), IL8 (p<0.01), and MCP-1 (p<0.05). S100B induced upregulation of VEGF and anti-VEGF antibody (Lucentis®) treatment prevented this. MCP-1 and MIF protein expression was down-regulated on exposure to Lucentis® (p<0.001). RAGE over-expression significantly increased S100B-induced expression of VEGF and IL1alpha when compared to WT RPE, although this trend was reversed for IL1 beta and MCP-1 (p<0.001).

Conclusions: : The current study shows that S100B has a significant biological effect on RPE in culture, causing an enhanced expression of various growth factors and cytokines. S100B binds to RAGE and in RPE this receptor appears to modulate important pro-inflammatory responses.

Keywords: retinal pigment epithelium • inflammation • cytokines/chemokines