with 1 nM PMA did not affect e pression of CCR2 Subsequently, we

with 1 nM PMA did not affect e pression of CCR2. Subsequently, we e amined whether PMA modulated the cell Nilotinib FDA surface e pression of CCR1 and CCR2 by FACS anal ysis. THP 1 cells were again stimulated with PMA for the times indicated, before being stained with the appropriate antibodies and then analyzed by flow cytom etry. Whereas the levels of CCR1 remained high throughout the duration of the e periment, CCR2 protein e pression decreased dramatically. The majority of the e pression was lost by 24 hours and by 48 hours vir tually no CCR2 was found on the surface of the cultured THP 1 cells. Thus, THP 1 cells treated with PMA mimics the differentiation process observed in cultured monocytes.

Two distinct signal transduction pathways regulate CCR2 e pression during monocyte maturation Our initial observations suggested that while PMA completely abrogated CCR2 e pression, sub optimal concentrations of this phorbol ester had no effect. We wondered, therefore, whether the addi tion of a calcium signal together with the sub optimal concentration of PMA might provide a sufficiently strong stimulus to affect the e pression of CCR2. Thus, we incubated monocytes with PMA and ionomycin at the concentrations indicated for 48 hours, and then analyzed CCR2 e pression. Our data indicated that ionomycin alone does not affect e pression of CCR2. However, in the presence of a sub optimal PMA signal, there was a selective dose dependent reduction in CCR2 e pres sion. At the same time, similar concentrations of PMA and ionomycin did not affect the levels of CCR1 nor GAPDH.

Monocytes treated with PMA plus ionomycin were also observed to adopt an adherent phenotype and to increase in size similar to the changes in morphology observed in freshly isolated monocytes. Furthermore, cell surface e pression of CCR2, but not CCR1, was found to be downregulated in the presence of PMA plus iono mycin after 48 hours. Thus, sub opti mal concentrations of PMA together with a modest calcium signal combine to mediate a maturation pheno type in monocytes that also includes the selective down regulation of CCR2. To determine whether the selective downregulation of CCR2 observed in PMA versus PMA plus ionomycin treated cells represented the same or two different signal ing pathways, we performed an e periment using the broad spectrum kinase inhibitor, staurosporine.

We preincubated THP 1 cells with staurosporine at the concentrations indicated for two hours, and Anacetrapib then stimu lated with either PMA or PMA plus ionomycin for 48 hours. Stau rosporine alone did not significantly inhibit e pression of CCR2 nor CCR1. Furthermore, the inhibitor did not abrogate the downregulation of CCR2 mediated by PMA plus ionomycin. selleck In contrast, staurosporine at 50 nM, but not at 10 nM, blocked the loss of CCR2 in PMA treated cells. Thus, these results identify at least two possible signal transduction pathways present in monocytes that could regulate the e pression of CCR2 during monocyte differ entiation. CCR2

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