We found that monocytes cultured for 5 days upregulated e pression of the integrin CD11b and the scavenger receptors CD36 and CD68, consistent with a change in phenotype from monocyte to macrophage. Ne t, we wanted to e amine changes in the e pression of chemokine recep tors as considering monocytes differentiated into macrophages. Using primers specific for C CR1 5 and CCR1 CCR9, we per formed semi quantitative analysis of receptor mRNA e pression. Initially, however, we determined the efficacy and specificity of the primers by analyzing genomic DNA samples prepared from freshly isolated monocytes. In all cases a single band ally increased over those observed in freshly isolated monocytes. To confirm the specificity of this effect we subsequently compared cell surface e pression of these chemokine receptors in cultured monocytes and freshly isolated monocytes by flow cytometry.

In agreement with our mRNA data, e pression of CCR2 pro tein, but not CCR1, CCR5 and C CR4 was rapidly down regulated during monocyte maturation. Negligible cell surface e pression of CCR7 protein was observed at any of the time points e amined, while C CR2 cell surface e pression remained curiously elevated despite downreg ulation of C CR2 mRNA, suggesting that the half life of this protein is actually quite long. These results indicate that one consequence of monocyte maturation is the selective downregulation of CCR2 gene e pression followed by a loss of CCR2 protein from the surface of the cell. While the actual physiological role of StaurosporinedownregulationPMA, CCR2 promoter ionomycin, of the anticipated size was observed indicating that the primers were specific for the desired chemokine receptor.

This data further suggested that a lack of chemokine recep tor e pression observed in freshly isolated monocytes and monocytes cultured for up to five days was a true result, rather than as a reflection of inappropriate primer design. PMA treatment of monocytes induces selective downregulation of CCR2 Based on the above results we decided to further e amine the regulation of CCR2 e pression in monocyte matura tion using the human monocyte cell line, THP 1 and CCR1 as a control. Treatment of these cells with the PKC activating phorbol ester PMA for 48 hours is a widely accepted procedure for maturing monocytes. Cells treated in this way undergo phenotypic changes consist ent with their maturation into macrophages.

Ne t, we wanted to determine how treatment of the monocyte cell line, THP 1, with PMA affected the e pres sion of CCR2 in these cells. Thus, monocytes were stimu lated with PMA for 48 hours and RNA prepared as described above. Our results show that CCR2 was selectively down regu lated in Anacetrapib a dose dependent manner, whereas e pression of CCR1 and the house keeping gene GAPDH remained unaffected. PMA was sufficient to completely abrogate CCR2 e pression, whilst 10 nM PMA reduced e pression of this chemokine receptor by appro imately 75%.