A and PMA plus ionomycin mediated the downregulation of CCR2 through inhibition of CCR2 specific gene transcription. Moreover, else physiological treatment of THP 1 monocytes with two known differentiation factors, IFN and M CSF, also pro moted a differentiation phenotype essentially identical to that observed using pharmacologic stimuli. These data indicate that the activation of several intracellular signal ing pathways selectively regulate the e pression of CCR2 during monocyte maturation into macrophages. Materials and methods Cell lines The THP 1 human monocytic cell line was grown in RPMI 1640 medium containing 10 % fetal calf serum, 100 U ml penicillin and 100 g ml streptomycin. The cells were main tained in culture at 37 C and 5% C02.
Typically, cells were stimulated with 50 nM phorbol myr istate acetate or 1 nM PMA plus 1 M ion omycin in the presence or absence of the PKC inhibitor staurosporine. Isolation and culture of human peripheral blood monocytes Peripheral blood mononuclear cells were iso lated from freshly prepared leukopacks that were between 2 4 hours old. Briefly, 20 ml of blood from leukopacks were diluted using PBS and layered over 15 ml of Ficoll Paque PLUS. Cells were then centrifuged at 400 g for 20 min utes at room temperature. After this time, PBMCs were collected from the interphase and washed with PBS and centrifuged at 150 g for 10 minutes. Monocytes were further isolated from PBMCs using Percoll gradient centrifugation as previ ously described. Lipid staining of the monocytes revealed that their purity was greater than 90%.
Finally, the cells were resuspended and cultured at 106 ml in RPMI 1640 supplemented with 10% autologous serum, penicillin and streptomycin. Cloning the CCR2 promoter A 1335 bp fragment of the promoter from the hCCR2 gene was cloned into the pGL3 vector using sequences determined by Yamamoto and colleagues. This construct, termed pGL3 1335, contained the tandem C EBP sites plus 1220 bp of the promoter sequence 5 of the transcriptional start site. The 5 primer contained a restriction site for kpnI, while the 3 primer contained a HindIII site. Each primer started with a 2 bp GC rich clamp. The full primer sequences used are as follows The genomic PCR was performed using an annealing tem perature of 55 C and an e tension tempera ture of 72 C, 30 cycles of PCR were performed.
RNA isolation and RT PCR Total RNA was isolated using TRIzol and by following the manufacturers instructions. Briefly, cells were Entinostat lyzed in TRIzol and then mi ed with chloro form. The lysate was then centrifuged Belinostat buy to separate RNA, DNA and protein. Total RNA, which is contained in the upper aqueous phase was recovered and mi ed with iso propanol to precipitate the RNA. The RNA was finally washed in 75% ethanol to remove impurities and dis solved in water. 5 g of RNA prepared in this way was then taken and DNase treated to remove further enzymatic contamina tion, before being reverse transcribed to cDNA using a ProSTAR First Strand RT PC