GM-CSF plus IL-4 induce TIMP-3 expression during differentiation of monocytes toward DCs. Freshly isolated monocytes (1 × 106 cells) were cultured in the presence of 1000 U/mL GM-CSF and 500 U/mL IL-4 for 5 days to generate imDCs, with 50 ng/mL TNF-α or 1 μg/mL LPS added for another 2 days to induce the DC maturation. (A) The TIMP-3 expression was assayed by quantitative RT-PCR in monocytes (Mo) and the cells after the induction for the indicated days. (B) Monocytes (Mo) and imDCs (DC) were stained with 4,6-diamidino-2-phenylindole (blue) and FITC-conjugated mAb (green) against TIMP-3 and then examined by confocal laser scanning microscopy system (Carl Zeiss LSM 710) with a Plan-Apochromat 63×/1.4 oil objective lens at room temperature. Images were processed using ZEN 2009 software (Carl Zeiss) and Adobe Photoshop CS5 (Adobe Systems). (C) Quantitative RT-PCR analysis of TIMP-3 mRNA in TNF-α–matured DCs and LPS-matured DCs, with imDCs as a control. Data are the mean ± SD of 3 independent experiments. *P < .05.
