α4β1 integrin engagement up-regulates MMP-9 in B-CLL cells. (A) B-CLL cells (2 × 106) from 4 different patients were suspended in RPMI, 0.1% FCS and incubated on wells coated with 0.5% BSA, 10 μg/mL FN-H89, or 10 μg/mL VCAM-1. Some cells were pretreated for 30 minutes with anti-α4 or -α5 mAbs (10 μg/mL) and then incubated in the presence of the Abs. After 24 hours, the conditioned media were concentrated 50 times and analyzed by gelatin zymography. Values for MMP-9 production (arbitrary units) represent the average of all 6 samples studied; basal levels of MMP-9 on BSA were normalized to 100. (B) B-CLL cells from one patient were incubated on wells coated with the indicated concentrations of FN-H89 or VCAM-1, and after 24 hours analyzed as explained. (C) B-CLL cells from 2 patients (5 × 105/100 μL) were incubated on the upper chamber of Transwell filters coated with resting or TNF-α–activated HUVECs, and in the presence or absence of the indicated mAbs. After 24 hours, the conditioned media were concentrated 5 times and analyzed by gelatin zymography. Values for MMP-9 production (arbitrary units) represent the average of the 2 samples studied. Error bars indicate standard deviation. * P < .05; ** P < .01.
