GM-CSF and IL-4 in combination enhance TACE expression and activity in monocytes. (A) Monocytes were cultured for 6 hours in the presence or absence of GM-CSF and IL-4 in combination. mRNA expression of a battery of known sheddases and expression of their endogenous inhibitors in monocytes were analyzed by RT-PCR. (B) Cell lysates were harvested after culturing monocytes for 48 hours in the presence or absence of GM-CSF and IL-4 in combination. TACE immunoreactivity was analyzed by immunoblotting with an antibody against human TACE. (C) Monocytes from 4 different donors were cultured for 48 hours in the presence or absence of GM-CSF and IL-4 in combination or TPA. Total cell lysates were prepared, and TACE activity in the cell lysates was measured using an internally quenched fluorescent substrate for TACE in the InnoZyme TACE Activity Kit. Fluorescence intensity was measured at an excitation wavelength of 320 nm and emission wavelength of 405 nm by a fluorometer (Fluoroscan Ascent FL; Labsystems). Results were displayed in relative fluorescence units (RFU) per milligram of protein (means ± SD) according to the manufacturer's instruction.