Functional effect of proMMP-9 binding on B-CLL cell migration through Matrigel and HUVECs. B-CLL cells (5 × 105), treated or not with the indicated Abs or transfected with the indicated siRNAs, were incubated for 30 minutes with or without 10 μg/mL proMMP-9. Cells were added to the upper chamber of Transwell filters coated with either Matrigel (A) or TNF-α–activated HUVECs (B), and 150 ng/mL CXCL12 was added to the medium in the bottom chamber. After 24 hours, migrated cells were counted by flow cytometry. Average values (n = 3) represent the percentage of the total number of cells added. (C) proMMP-9 was incubated in solution with TIMP-1 (110 nM each) for 30 minutes and the complex immunoprecipitated with anti–TIMP-1 or anti-HLA (Ctl) mAbs and protein G beads. Immunoprecipitates and supernatants were analyzed by gelatin zymography. (D) B-CLL cells from a representative sample were incubated for 30 minutes with proMMP-9 alone or complexed to TIMP-1, and analyzed by flow cytometry using anti–MMP-9 pAbs. Numbers indicate MFI values for basal MMP-9 (shaded areas) or exogenously added proMMP-9 or proMMP-9/TIMP-1 (continuous lines). (E,F) B-CLL cells (5 × 105) were preincubated with or without the indicated proteins for 30 minutes at 4°C. Cells were washed and added to Transwell filters coated with Matrigel (E) or TNF-α–activated HUVECs (F), and migration was determined after 24 hours by flow cytometry. Average values (n = 3) are expressed as the percentage of the total number of cells added. T-1/pMMP-9 indicates TIMP-1/proMMP-9 complex. *P ≤ .05; **P ≤ .01.
