Figure 5: Immunoreactive and active levels of Mmp-8 and Mmp-9 are markedly reduced on the surface of Timp-1^−/− PMNs:

In A and B, PMNs from WT, Timp-1^−/−, Mmp-8^−/−, and Mmp-9^−/− mice were activated for 15 min at 37°C with 10⁻⁶ M PAF followed by 10⁻⁶ M fMLP. The cells were then fixed and immunostained for surface-bound Mmp-8 (in A) or surface-bound Mmp-9 (in B) using antibodies that detect both pro and active forms of these Mmps. Surface levels of Mmp-8 or Mmp-9 were quantified as described in Methods and expressed as a percentage of the amount of Mmp-8 or Mmp-9 respectively, that were expressed on the surface of activated WT PMNs. Data are mean + SEM from 3–4 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs. In C-E, PMNs from WT, Timp-1^−/−, and Mmp-8^−/− and or Mmp-9^−/− mice were activated and fixed as described in A. Equal numbers of cells from each genotype were pre-incubated for 1 h at 37°C in triplicate with and without 1–10-phenanthroline (a non-selective MMP inhibitor). Cells were then incubated at 37°C with: 1) type I-collagen conjugated to quenched FITC for 18 h (in C); 2) gelatin conjugated to quenched FITC for 6 h (in D); or 3) McaPLGLDpaAR (a quenched fluorogenic peptide substrate which is cleaved by both Mmp-8 and Mmp-9) for 3 h (in E). Mmp-mediated cleavage of each substrate was quantified in cell-free supernatant samples by fluorimetry as the 1,10-phenanthroline-inhibitable cleavage of the substrate, as described in Methods. The data are expressed as mean + SEM % cleavage of the substrate by WT cells from 4–5 separate experiments. Data were analyzed using a One-Way ANOVA followed by pair-wise testing with two-tailed Student’s t-tests. Asterisk indicates P < 0.01 compared with WT PMNs or the group indicated.