FIGURE 3.

MMP-9 activation by mMCP-4 in vivo and in vitro. A, neonatal WT and mMCP-4^−/− mice were injected i.d. with control IgG or pathogenic anti-mBP180 IgG. Skin samples were obtained at 4 and 24 h after IgG injection, and protein extracts (30 μg/lane) were analyzed by gelatin zymography. Both the pro- and active forms of MMP-9 were seen in skin samples of pathogenic IgG-injected WT (lanes 3 and 4) and mMCP-4^−/− mice (lanes 1 and 2) at both time points. B, MMP colorimetric assay revealed a significant reduction of active MMP-9 in the skin samples of pathogenic IgG-injected mMCP-4^−/− mice at 12 and 24 h as compared with active MMP-9 levels in the lesional skin of diseased WT mice (bars 6 and 8 versus 5 and 7). At 4 h, WT and mMCP-4^−/− mice showed compatible levels of active MMP-9 (bars 3 and 4). MMP-9^−/− mice exhibit background levels of activity at 0, 4, 12, and 24 h after injection with pathogenic antibodies (not shown). *, p < 0.01. n = 6 for each group. C, bone marrow-derived MCs (1 × 10⁶) from WT or mMCP-4^−/− mice were stimulated with recombinant C5a (50 ng/ml) at 37 °C for 1 h. The supernatants were then incubated with recombinant pro-MMP-9 (1 μg/ml) at 37 °C for 4 h. The digestion mixtures were analyzed by gelatin zymography. The supernatant of C5a-treated (lane 1) and not BSA control-treated (lane 2) MCs activated pro-MMP-9. Addition of rabbit anti-mMCP-4 antibody to the C5a-treated supernatant completely blocked MMP-9 activation (lane 3). C5a-activated mMCP-4^−/− MC supernatants do not activate MMP-9 (lane 4).