MMP-9 and heparanase in inflamed sites are decreased when neutrophil accumulation is attenuated. (A-B) Immunoblotting for MMP-9 in supernatant harvested from nonstimulated or fMLP-stimulated neutrophils. Immunized footpad serves as positive control. (B) Immunized footpad sections were examined for MMP-9 colocalization with neutrophils. (C) Inflamed footpad sections were immunostained to assess MMP-9 expression in NIMP-R14- and control rat IgG-treated mice. (D) Immunoblotting for MMP-9 in immunized footpads from NIMP-R14- or control rat IgG-treated mice. (E) Quantification of pro-MMP9 and MMP9 levels by immunoblotting. (F) Immunoblotting for heparanase in supernatant harvested from nonstimulated or fMLP-stimulated neutrophils. Spleen served as positive control. (G) Immunized footpad sections were examined for heparanase colocalization with neutrophils. (H) Inflamed footpad sections were immunostained to examine heparanase expression in NIMP-R14- and control rat IgG-treated mice. (I) Immunoblotting for heparanase in immunized footpads from NIMP-R14- or control rat IgG-treated mice. (J) Quantification of heparanase levels by immunoblotting. Each lane represents 1 individual mouse. Glyceraldehyde-3-phosphate dehydrogenase is used as a loading control. All immunoblots are representative of 2 independent experiments (n = 6-8). For all immunoblots, lanes were run on the same gel, although some lanes were noncontiguous. Quantification in panels E and J is pooled from these samples (n = 6-8). All images are representative of 5 independent experiments (n = 5). All scale bars represent 50 μm.