Figure 4: ProMMP-8 and proMMP-9 share binding sites on the surface of PMNs:

In A and B, PMNs from Mmp-8^−/− x Mmp-9^−/− mice were activated for 15 min at 37°C with 10⁻⁶ M PAF followed by 10⁻⁶ M fMLP to induce surface expression of Timp-1. In A, the activated PMNs were then pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-8 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous murine proMMP-9. Cells were then washed and immunostained with rabbit anti-murine Mmp-9 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)₂-conjugated to Alexa 488 to detect bound pro and active Mmp-9. In B, the PAF- and fMLP- activated and PMNs were pre-incubated for 45 min at 4°C with or without exogenous full length murine proMmp-9 (50–400 nM). Cells were then incubated for an additional 60 min at 4°C with 100 nM exogenous full length murine proMMP-8. Cells were then immunostained with rabbit anti-murine Mmp-8 IgG, or non-immune rabbit IgG followed by goat anti-rabbit F(ab)₂-conjugated to Alexa 488 to detect bound pro and active Mmp-8. In A and B, images of immunostained cells were captured and surface-bound pro and active Mmp-9 (in A) or pro and active Mmp-8 (in B) were quantified using MetaMorph software. Data are mean + SEM (n = 150–200 cells/group). Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.001 when compared with PMNs incubated in the absence of exogenous Mmp. The results shown are representative of at least 3 independent experiments.