Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: possible involvement of oxygen free radicals

Homocysteinaemia is an independent risk factor for atherosclerosis. The dev elopment of atherosclerosis involves monocyte chemoattractant protein 1 (MC P-1)-mediated monocyte recruitment to the lesion site. The action of MCP-1 is mostly via its interaction with MCP-1 receptor (CCR2), which is the majo r receptor for MCP-1 on the surface of monocytes. The objective of the pres ent study was to investigate the effect of homocysteine on CCR2 expression in human THP-1 monocytes. Cells were incubated with various concentrations of homocysteine for 6, 12, 24 and 48 h. The expression of CCR2 mRNA was det ermined by nuclease protection assay and the CCR2 protein was measured by W estern immunoblotting analysis. The binding of MCP-1 to CCR2 as a functiona l receptor on the monocyte surface was determined by flow cytometry. Homocy steine (0.05-0.2 mM) significantly enhanced the expression of CCR2 mRNA (12 9-209 % of the control) and CCR2 protein (up to 183 % of control) in these cells after 24 h of incubation. Stimulation of CCR2 expression was associat ed with a parallel increase in the binding activity of CCR2 (129-191 %, of control) as well as an enhanced chemotactic response of homocysteine-treate d monocytes. Further investigation revealed that the levels of superoxide w ere significantly elevated in cells incubated with homocysteine for 12-48 h . The addition of superoxide dismutase, a scavenger of superoxide, to the c ulture medium abolished the stimulatory effect of homocysteine on CCR2 expr ession as well as the binding activity of the receptor. The stimulatory eff ect of homocysteine on the expression of CCR2 mRNA and the levels of CCR2 p rotein was also observed in human peripheral blood monocytes. In conclusion , the present study has clearly demonstrated that homocysteine stimulates C CR2 expression in monocytes, leading to an enhanced binding activity and ch emotatic response. Homocysteine-induced superoxide formation might serve as one of the underlying mechanisms for this effect.