Some reports have suggested that CCL2 could be involved in the early stages of CCR2 protein down modulation, while other studies indicate that the differentiation proc ess itself, is a major factor in the selective loss of CCR2 gene e pression. Numerous cytokines are known to be involved in monocyte activation and differentiation, now among them M CSF and IFN. M CSF is a lin eage specific hematopoetic growth factor that stimulates monocyte differentiation. The c fms proto onco gene encodes a high affinity receptor for M CSF and it has been shown that THP 1 cells e press this protein and that it is up regulated during differentiation. How ever, cells stimulated with M CSF alone for 48 hours did not lose e pression of CCR2.

Conversely, IFN alone, which is constitutively e pressed by monocyte lineage cells and which promotes matura tion of monocytes to macrophages, did significantly reduce e pression of CCR2, although the cells did not become adherent and neither did they change their mor phology. Interestingly, IFN has been demonstrated to up regulate levels of M CSF in mono cytes during maturation and when both IFN and M CSF were added, THP 1 cells did become adherent, changed their morphology and selectively lost CCR2, but not CCR1 all of which are characteristics of the mono cyte differentiation phenotype. These results are in keep ing with the studies published by Tangirala and colleagues, who reported similar phenomena in THP 1 cells. In addition, our studies also demonstrated that the regulatory effects mediated by IFN plus M CSF occurred at the level of transcription, where a significant down regulation in CCR2 promoter activity was observed.

Moreover, in the presence of staurosporine, IFN plus M CSF was unable to down regulate levels of CCR2. This result probably reflects the fact that IFN signals e ten sively through the JAK STAT pathway, and studies have suggested that staurosporine can block phosphorylation of Janus kinases. In addition, we have found two putative binding sites in the CCR2 promoter for STAT transcription factors which would further support the contention that these transcription factors may be impor tant in the regulation of IFN mediated downregulation of CCR2. Conclusion This study demonstrates that e pression of the chemokine receptor CCR2 is e quisitely correlated with monocyte maturation.

Freshly isolated monocytes e press high lev els of both CCR2 RNA and protein, whereas monocyte derived macrophages e press neither CCR2 RNA nor pro tein. Conversely, levels of the closely related chemokine receptor CCR1 remained stable and elevated throughout monocyte maturation. An analysis of the biochemical and molecular mechanisms underlying the regulated e pres sion of CCR2 revealed Anacetrapib the e istence of several signaling pathways that selectively down modulate CCR2 gene e pression during monocyte differentiation. this e pres sion was largely regulated at the level of transcription.