Expression of RAGE on Neutrophils in Response to Hyperglycemia

Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of pro-inflammatory responses. RAGE is a member of the immunoglobulin superfamily of cell surface molecules with a ligand repertoire, including AGE. Several RAGE variant proteins have been detected in human cells. Treatment of cultured human aortic endothelial cells with RAGE ligands induces the expression of RAGE transcripts. However, the induced expression of RAGE in neutrophils is currently unknown. Objective: The aim of this study was to elucidate the mechanism of hyperglycemia-induced expression of RAGE in neutrophil-like HL-60 cells. Methods: In order to study the prolonged effects of hyperglycemia on neutrophils, the promyelocytic HL-60 cell line was differentiated into neutrophil-like cells in 1.25% DMSO in RPMI1640 supplemented with 10% FBS for 6 days. After differentiation, cells were exposed to normal (5.5 mM) or high (25 mM) glucose concentrations. AGE was then added at a concentration of 5 μg/ml. Expression of RAGE at the mRNA and protein levels was quantified by Real-time PCR (Applied Biosystems, CA) and Western blotting, respectively. Results: Both Variant-1 and -2 RAGE mRNA was detected in HL-60 cells by Real time PCR analyses, but expression of variant-1 was 10 times higher than variant-2. Expression of variant-1 RAGE mRNA in HL-60 cells was AGE concentration-dependent and increased significantly in high glucose conditions compared to normal glucose (p<0.01). This observation was further confirmed at protein level where RAGE expression was significantly higher in AGE treated cells incubated in high glucose. Conclusions: These findings suggest that RAGE expression in neutrophils is upregulated by the AGE ligand, but only under conditions of hyperglycemia. Further, the variant-1 form of RAGE appears to be upregulated in diabetic conditions. Supported by USPHS Grants DE15566 and RR00533.