IFN-γ plus M-CSF promotes specific down-regulation of CCR2. (a). THP-1 cells were either untreated (lane 1, upper, middle and lower panels) or treated with 500 U/ml IFN-γ plus 5 ng/ml M-CSF (lane 2 upper, middle and lower panels) or 50 nM PMA (lane 3 upper, middle and lower panels) for 48 hours. Messenger RNA was then prepared and RT-PCR performed using primers for CCR1 (upper panel), CCR2 (middle panel) and GAPDH (lower panel). M is a 100 bp DNA ladder. Similar results were obtained in three other experiments. (b). THP-1 cells were transfected with either 5 μg of vector alone (pGL3-basic) or with 5 μg of the pGL3-1335 construct. In addition, each sample was also transfected with 2 μg of pRL-SV40 (renilla) to act as an internal control. Cells were then either left untreated or treated with either 500 U/ml IFN-γ plus 5 ng/ml M-CSF or 50 nM PMA. Subsequently, cell extracts were prepared and assayed for both luciferase and renilla activity. After normalization to the renilla control, CCR2 transcriptional activity was calculated relative to the pGL3-basic vector, which was arbitrarily assigned a value of 1. Similar results were obtained in two other experiments.