Fig. 3.

MT6-MMP is released from human PMNs and is catalytically active. (A) PMNs (2 × 10⁷ cells) were allowed to sediment for 55 min at 37°C before being left untreated (lane 1) or stimulated with 4 nM PMA (lane 2) or 100 U ml⁻¹ IFN-γ (lane 3) for 20 min at 37°C. After stimulation, the media were centrifuged and both supernatants and cell pellets were collected for analysis. MT6-MMP was immunoprecipitated from the supernatants and the cell pellets were boiled in reducing 2× sample buffer. The immunoprecipitates and the cell lysates were resolved by SDS–PAGE, containing recombinant MT6-MMP (lane 4), followed by MT6-MMP detection. In (B), MT6-MMP was immunoprecipitated from the supernatants of resting or activated PMNs, resolved by SDS–PAGE and quantified by densitometry. These results are from three different blood donors and statistical significance is depicted as follows: *P < 0.05 compared with resting PMNs (Ctrl). (C) The specificity of the MT6-MMP immunoprecipitation was assessed as follows: supernatants were incubated with protein A/G beads alone (lane 1) or protein A/G beads and non-immune antibody (lane 2) and resolved by SDS–PAGE. Lane 3 shows recombinant MT6-MMP. (D) MT6-MMP was immunoprecipitated from supernatants of resting or stimulated PMNs (4 nM PMA, 100 U ml⁻¹ IFN-γ or 30 nM fMLP for 20 min at 37°C). After several washes, the immunoprecipitates were incubated with a synthetic fluorescence-quenched peptide substrate for 5 h at room temperature. Hydrolysis of the fluorogenic substrate, representing MT6-MMP catalytic activity, was monitored with a spectrofluorometer. As negative control, the substrate was incubated with protein A/G beads and a control antibody (rabbit IgG), but with no PMNs (control medium). As a positive control, whole PMN lysates were incubated with the substrate as described above. The results are shown as mean relative activity ± SEM of three independent blood donors over control medium. Statistical significance is depicted as follows: *P < 0.05 compared with unstimulated PMNs.