M2 polarization of human macrophages downregulates TIMP-1 expression, resulting in production of highly angiogenic TIMP-1–deficient proMMP-9. (A) (Upper) Production of proMMP-9 and TIMP-1 by monocytes and mature and polarized macrophages. Neutrophil releasate from 5 × 103 cells and SF medium conditioned by 2 × 104 monocytic cells was analyzed for MMP-9 and TIMP-1 by western blotting under nonreducing conditions. Following transfer, the membrane was cut into 2 portions, which were probed for MMP-9 (top) and TIMP-1 (bottom). Human recombinant proMMP-9 and TIMP-1 were run at the indicated amounts to provide means for quantification of protein production. Positions of the 92-kDa monomer, 125-kDa heterodimer, and ∼200-kDa homodimer of proMMP-9 and 28-kDa TIMP-1 are indicated on the left. Position of molecular weight markers in kilodaltons is indicated on the right. Bar graph, comparative analysis of angiogenesis-inducing capacity of monocytic cell-conditioned media. SF CM from monocytic cells and neutrophil releasate was incorporated at volumes providing equal amounts of proMMP-9 (3 ng per collagen onplant). From 4 to 6 embryos, each grafted with 6 collagen onplants, were analyzed per variable in 5 independent experiments. Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with no-cell control (NC, 1.0). **P < .01; ***P < .0001. (B) (Upper) Analysis of the levels of TIMP-1 naturally complexed with proMMP-9 produced by monocytes and mature and polarized macrophages. ProMMP-9 was purified by affinity chromatography from neutrophil releasate and SF medium conditioned by monocytes or different types of macrophages. Western blot analysis of isolated proMMP-9 preparations was conducted under reducing conditions to determine the amount of TIMP-1 complexed with distinct proMMP-9 preparations. Note that the preparation of neutrophil proMMP-9 is completely devoid of TIMP-1 and that reduction led to collapsing of all neutrophil proMMP-9 forms to the 92-kDa monomer species. Positions of purified proMMP-9 monomer and the complexed 28-kDa TIMP-1 are indicated on the left. Positions of molecular weight markers are indicated on the right. Bar graph, stoichiometric ratio of proMMP-9 to TIMP-1 in purified preparations of proMMP-9 from monocytes and macrophages was calculated based on standard amounts of recombinant proMMP-9 and TIMP-1, which were run in parallel in additional lanes of the same gel (supplemental Figure 1C). ***P < .0001. (C) Gene expression analysis of MMP9 and TIMP1 during IL-4–mediated M2 polarization. M0 macrophages were introduced to 20 ng/mL IL-4, and mRNA was extracted from the cells at the indicated time points. Gene expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction relative to the levels of the corresponding gene (1.0) before addition of IL-4 (0 hours). Presented are data from 1 of 4 independent experiments run in triplicates. ***P < .0001. (D) Western blot analysis of MMP-9 and TIMP-1 secretion during IL-4–mediated M2 polarization. M0 macrophages were introduced to IL-4 for the indicated time (hours) and then switched to SF conditions. Following a 48-hour incubation, the samples of CM were analyzed for MMP-9 and TIMP-1 by western blotting. (E) IL-mediated regulation of MMP-9 and TIMP-1 in M2 macrophages. M0 macrophages were introduced to 20 ng/mL of human IL-4, IL-10, and IL-13, or left nontreated (NT). Following a 24-hour induction, the cells were switched to SF medium, and CM as collected after 48 hours. CM was analyzed for MMP-9 and TIMP-1 by western blotting. Samples of CM were normalized to equal number of cells and processed by SDS PAGE on 4% to 20% gels under reducing conditions. After transferring of separated proteins, the membrane was cut horizontally, and the upper potion probed with anti–MMP-9 antibody and the lower portion with anti–TIMP-1 antibody. The position of the molecular weight markers is indicated on the right. (F) Angiogenic potential of IL-activated macrophages. Angiogenic potency of CM from M0 macrophages induced by different ILs was analyzed in the CAM assy. Data are from 1 of 3 independent experiments, each involving from 4 to 6 chick embryos with 5 to 6 collagen onplants. Bar graph presents means ± SEM of fold changes in the levels of angiogenesis compared with SF control (1.0). **P < .005 and ***P < .0001 in comparison with nontreated (NT) macrophages.
