Expression of the chemokines RANTES and MIP-1β in human vasculature and atherosclerotic plaque

Katie Jones1, Rhoda Kuc1, Sidath Katugampola2, Anthony Davenport1
1University of Cambridge, Cambridge, United Kingdom, 2Pfizer Global Research and Development, Sandwich, Kent, United Kingdom

The chemokines RANTES and MIP-1β bind with high affinity to the chemokine receptor CCR5 and are able to potently elicit a chemotactic response through this receptor. In addition to a suggested role for CCR5 in atherosclerosis in an ApoE-/- mouse model (Braunersreuther et al., 2006), CCR5 mRNA has also been detected in cultured human vascular smooth muscle cells and CCR5-like immunoreactivity (IR) found in human atherosclerotic plaque (Schecter et al., 2000). Animal models also show a localisation of RANTES and MIP-1β to atherosclerotic plaque (Lutgens et al., 2005). The aim of this study was to investigate RANTES and MIP-1β distribution, together with that of CCR5, in human vasculature.

Messenger RNA (mRNA) was extracted from the smooth muscle layer of human arteries and veins including saphenous vein, coronary artery and aorta and reverse transcribed. PCR was performed with RANTES specific primers (Yang et al., 1997), MIP-1β specific primers (Krzysiek et al., 1999) and CCR5 specific primers (Lu et al., 1999). For immunocytochemistry (ICC), 30 μm sections of human vessels including saphenous vein, normal and diseased coronary artery, mammary artery and aorta, were incubated with rabbit anti-RANTES antisera or rabbit anti-MIP-1β antisera (Abcam plc, UK) (n≥3 for each tissue) and detection was via an avidin/biotinylated enzyme complex method. For dual-labelling fluorescence ICC, slides were also incubated with mouse anti-smooth muscle α-actin (SMαA), mouse anti-von Willebrand Factor (vWF) or mouse anti-CD3 antisera (DakoCytomation, Denmark) to identify co-localisation to smooth muscle, endothelium and T-lymphocytes respectively.

PCR products corresponding to the expected size for RANTES, MIP-1β and CCR5 amplified cDNA were detected in both arterial and venous smooth muscle, indicating cellular expression of these transcripts. RANTES-like immunoreactivity (IR) and MIP-1β-like IR was detected in the smooth muscle layer and endothelial layer of all tissues tested and to atherosclerotic plaque. Expression within smooth muscle and endothelium was confirmed by co-localisation with SMαA-like IR and vWF-like IR using confocal microscopy. Additionally co-localisation of RANTES-like IR and MIP-1β-like IR was seen with CD3-like IR within atherosclerotic plaque, possibly implicating T-lymphocytes as a potential source of these chemokines in atherosclerosis.

These data demonstrate the expression of RANTES and MIP-1β mRNA as well as indicating protein expression in all vessels investigated, including atherosclerotic artery, suggesting widespread human vascular distribution. Expression of CCR5 mRNA has also been detected in a range of human vessels, complimenting the detection of CCR5 mRNA in human vascular smooth muscle cells (Schecter et al., 2000), These results, showing expression of both receptor and ligands in the vasculature, may indicate a potential paracrine role for these chemokines in vascular disease such as atherosclerosis.

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