We, therefore, cloned a 1335 bp fragment of the CCR2 promoter using the sequence described by Yamamoto and colleagues. This fragment was then subcloned into the mammalian e pression together vector pGL3 upstream of the luciferase gene, generating the pGL3 1335 construct. In addition to the sequences upstream of the TATA bo , pGL3 1335 included 115 bp of the 5UTR, which contains the two tandem C EBP repeats that are thought to be necessary for the basal e pression of the CCR2 gene. Subsequently, we transfected this construct into the THP 1 cells using DEAE de tran and either left the cells untreated, or treated them with PMA, or PMA plus ionomycin for 48 hours in the presence or absence of staurosporine. Cells were then lyzed and assayed for transcriptional activity.

Our results showed that the pGL3 1335 construct, itself, gave a 13 fold induction over the background construct lacking the CCR2 promoter. Fur thermore, both PMA and PMA plus ionomycin strongly abrogated this transcriptional activity suggesting that the dual signal transduction path ways activated by PMA and PMA plus ionomycin medi ated regulation of CCR2 e pression at the level of transcription. In the presence of staurosporine, inhibition of CCR2 promoter activity mediated by PMA, but not PMA plus ionomycin, was abrogated. Thus, these data indicate that the PMA mediated inhibition of CCR2 promoter activity is ultimately regu lated by one or more staurosporine sensitive transcription factors.

Treatment with IFN and M CSF produces a similar differentiation phenotype to that seen with PMA and ionomycin The above results reflect a phenotype induced by pharma cologic agents and we ne t wanted to ensure that this phe notype is applicable to physiologic agents also. To that end, THP 1 cells treated with IFN plus M CSF have GSK-3 already been shown to promote monocyte maturation, although it has yet to be confirmed that these agents reg ulate CCR2 e pression at the level of transcription. Initially, though, we wanted to demonstrate that mono cytes treated with IFN plus M CSF showed changes in morphology similar to that observed with freshly isolated monocytes. After 48 hours treatment with IFN plus M CSF, monocytes became adherent and increased in size similar to that observed for freshly isolated monocytes in culture. PMA treated monocytes also underwent similar changes in morphology. Furthermore, flow cytometric studies revealed that monocytes treated with either IFN plus M CSF or PMA strongly upregulated the macrophage matu ration markers CD11b, CD36 and CD68. Sim ilar results were observed for cells treated with PMA plus ionomycin.