Induction of proMMP-9 during maturation and polarization of human macrophages. (A) Morphological changes during monocytes differentiation into macrophages. Freshly isolated monocytes were incubated in the presence of M-CSF for 7 days. Note a gradual change in the morphology from rounded monocytes to elongated cells, first noticeable on day 3 and representing almost all M0 macrophages by day 7. Images were acquired using an Olympus CKX-41 microscope equipped with Olympus U-LS30-3 video camera and Infinity Capture software and processed using Adobe Photoshop. Objective lens, ×20. (B) Kinetic analysis of proMMP-9 production during macrophage maturation and polarization. (Left) Gelatin zymography was conducted on serum-free (SF) medium conditioned by 2 × 10⁴ human monocytes cultured in the presence of M-CSF for 7 days and then polarized into M1- or M2 macrophages. The position of 92-kDa proMMP-9 monomer and ∼200-kDa homodimer is indicated on the right. Human recombinant proMMP-9 was loaded to provide the means of quantification of proMMP-9 production. (Right) Zymographic analysis of granule contents released by 1 × 10⁴ neutrophils. The position of 92-kDa proMMP-9 monomer, 125-kDa NGAL heterodimer, and ∼200-kDa homodimer is indicated on the right. Human recombinant proMMP-9 (3 ng per lane) was run to estimate the MMP-9 load in neutrophils. (C) Immunofluorescent analysis of MMP-9 expression in isolated neutrophils and monocytes and mature and polarized macrophages. Macrophages were differentiated from monocytes cultured on coverslips, whereas purified neutrophils and monocytes were directly placed on coverslips after cell isolation. The cells were fixed and then stained for human MMP-9 with mAb 8-3H (green). Cell nuclei were contrasted with 4′6 diamidino-2-phenylindole (blue). Images were acquired using a Carl Zeiss AxioImager M1m microscope equipped with AxioVision Re.4.6 software and processed using Adobe Photoshop. Objective lens, ×40.