Ne t, we wanted to determine whether IFN plus M CSF induced the differentiation associated downregulation of CCR2. Therefore, monocytes were treated with IFN plus M CSF for 48 hours and CCR2 mRNA was e amined. Our results showed that IFN plus M CSF did selectively downregu late CCR2, but not CCR1 in a manner Rapamycin msds analogous to that observed for PMA and PMA plus ionomycin. A similar pattern was also observed when transcriptional activity was e amined. Here, PMA completely down modulated CCR2 transcription, while the combined effects of IFN plus M CSF reduced this activity by appro imately 70%. In the presence of staurosporine, the inhibition of CCR2 pro moter activity mediated by IFN plus M CSF was abrogated in a manner analogous to that observed for PMA.
Taken together, these data suggest that PMA, PMA plus ionomycin and IFN plus M CSF mediate sim ilar changes in the monocyte phenotype during matura tion of these cells. Thus, the monocyte cell line, THP 1, is a useful model system with which to investigate the underlying regulatory mechanisms governing chemokine receptor e pression during monocyte differentiation. Discussion In this paper we demonstrate that a major consequence of monocyte maturation into macrophages is the selective downregulation of the chemokine receptor, CCR2, but not the related CCR1. We have further shown that there are multiple stimuli, which can selectively down modu late CCR2 e pression, including high concentrations of PMA, or low PMA plus ionomycin, or IFN plus M CSF.
Each of these stimuli regulate the e pression of CCR2 at the level of transcription, although it appears that at least two dif ferent signal transduction pathways are involved based on the ability of staurosporine to interfere with these proc esses. Treatment of THP 1 monocytes with staurosporine abrogated the ability of PMA and IFN plus M CSF to downregulate CCR2. By contrast, staurosporine was una ble to block PMA plus ionomycin mediated downregula tion of CCR2 e pression. Thus, this study provides evidence that there is dynamic and selective regulation of the CCR2 gene during monocyte differentiation. Our results indicate that treatment of THP 1 cells with either PMA alone or PMA plus ionomy cin promotes a differentiation phenotype that is characterized by morphological changes and altered CCR2 gene e pression.
Indeed, these observations have already been noted by other researchers studying mono cyte differentiation. In particular, we show that THP 1 cells rapidly become adherent and their morphol ogy changes from the typical round shape of monocytes to spindle shaped cells with pseudopodia, which are charac Drug_discovery teristic of macrophages. At the same time there was also an increase in the size and granularity of the cells. In addi tion, we demonstrated an up regulation in e pression of genes associated with monocyte differentiation, notably CD11b, CD36 and CD68.