Figure 2.
Figure 2. Proinflammatory macrophages express high levels of Plg-RKT in vitro and in vivo. (A) MFIs of Plg-RKT on human unpolarized macrophages (M0), human macrophages polarized with LPS (Sigma Aldrich) and IFN-γ (Thermo Fisher Scientific) [M(LPS+IFN)] and human macrophages polarized with IL-4 (Thermo Fisher Scientific) and IL-13 (Santa Cruz, Dallas, TX) [M(IL-4+IL-13)] (n = 5). Unpolarized and polarized macrophages were stained with anti–Plg-RKT mAb and analyzed on a Novocyte flow cytometer (ACEA Biosciences). (B) Plg-RKT mRNA levels in human unpolarized macrophages (M0), human macrophages polarized with LPS and IFN-γ [M(LPS+IFN)], and human macrophages polarized with IL-4 and IL-13 [M(IL-4+IL-13)] (n = 3). (C) Unpolarized and polarized macrophages were seeded into Costar Transwell Permeable Supports (5.0-µm pore size; Corning) in complete culture medium consisting of RPMI 1640 containing 20% fetal bovine serum (Biochrome Millipore, Berlin, Germany). Cells were incubated for 1 hour at 37°C, 5% CO2 in a humidified incubator. Cells were either preincubated for 30 minutes with anti–Plg-RKT mAb (140 nm) or with vehicle. Thereafter, plasminogen (0.2 µM; Roche) or vehicle was added or cells were left without the addition of either mAb or plasminogen as control. Then, the medium was removed, replaced by phosphate-buffered saline, pH 7.4, containing the respective additions or no additions, and inserts were transferred into wells containing complete culture medium. Cells were allowed to migrate for 3 hours at 37°C. Quantification of migrated cells was performed as described for monocytes in the legend to panel E of Figure 1 (n = 5). (D-E) Representative images of immunohistochemical staining of human carotid artery sections (D; n = 15) and adipose tissue sections (E; n = 8) stained for CD80, Plg-RKT, and plasminogen. Carotid plaque samples were obtained as described.30 Visceral fat tissue was obtained from 8 patients undergoing bariatric bypass surgery.31 Tissue samples were fixed in 4% formaldehyde and embedded in paraffin. Sections of human carotid artery plaques were stained with hematoxylin and eosin, and sections of human carotid artery plaques and of adipose tissue samples were stained with an anti-CD80 antibody as a marker for M(LPS+INF) macrophages (rabbit polyclonal, dilution 1:100; Santa Cruz), anti–Plg-RKT mAB (dilution 1:500), an antiplasminogen antibody (goat polyclonal; dilution 1:100; Santa Cruz), and a respective isotype control antibody for the anti–Plg-RKT mAb (dilution 1:250; eBiosciences, San Diego, CA) and normal goat IgG as control antibody for the antiplasminogen antibody (dilution 1:750; R&D Systems, Minneapolis, MN). Afterward, sections were incubated for 18 hours with secondary antibodies (anti-rabbit polyclonal, labeled with Cy3; Biolegend; anti-goat polyclonal, labeled with Dylight 650 and anti-mouse polyclonal labeled with FITC, dilution 1:200; both Abcam). Slides were scanned on a fully automated system (TissueGnostics, Vienna, Austria) with a ×20 objective, acquired with tissueFAX software and analyzed with tissueQuest software. CD80 is depicted in red; Plg-RKT is depicted in green, and plasminogen is depicted in yellow. White arrows in the merged panel mark cells staining positive for CD80, Plg-RKT, and Plg. Cells with MFI >100 for CD80 were defined as CD80 high-expressing cells and therefore as proinflammatory; cells with MFI <100 were defined as anti-inflammatory. MFIs for Plg-RKT and plasminogen were then measured for the 2 defined CD80 high- and low-expressing cells, respectively, and compared. Values are displayed as MFIs (A), ΔCt (B), or fold change vs control (C) ± standard error of mean. Statistical significances were calculated using analysis of variance and Tukey’s post hoc test when >2 groups were compared (A-C) or Student t tests (unpaired) for comparison of 2 groups (D,E). DAPI, 4′,6-diamidino-2-phenylindole.

Proinflammatory macrophages express high levels of Plg-RKT in vitro and in vivo. (A) MFIs of Plg-RKT on human unpolarized macrophages (M0), human macrophages polarized with LPS (Sigma Aldrich) and IFN-γ (Thermo Fisher Scientific) [M(LPS+IFN)] and human macrophages polarized with IL-4 (Thermo Fisher Scientific) and IL-13 (Santa Cruz, Dallas, TX) [M(IL-4+IL-13)] (n = 5). Unpolarized and polarized macrophages were stained with anti–Plg-RKT mAb and analyzed on a Novocyte flow cytometer (ACEA Biosciences). (B) Plg-RKT mRNA levels in human unpolarized macrophages (M0), human macrophages polarized with LPS and IFN-γ [M(LPS+IFN)], and human macrophages polarized with IL-4 and IL-13 [M(IL-4+IL-13)] (n = 3). (C) Unpolarized and polarized macrophages were seeded into Costar Transwell Permeable Supports (5.0-µm pore size; Corning) in complete culture medium consisting of RPMI 1640 containing 20% fetal bovine serum (Biochrome Millipore, Berlin, Germany). Cells were incubated for 1 hour at 37°C, 5% CO2 in a humidified incubator. Cells were either preincubated for 30 minutes with anti–Plg-RKT mAb (140 nm) or with vehicle. Thereafter, plasminogen (0.2 µM; Roche) or vehicle was added or cells were left without the addition of either mAb or plasminogen as control. Then, the medium was removed, replaced by phosphate-buffered saline, pH 7.4, containing the respective additions or no additions, and inserts were transferred into wells containing complete culture medium. Cells were allowed to migrate for 3 hours at 37°C. Quantification of migrated cells was performed as described for monocytes in the legend to panel E of Figure 1 (n = 5). (D-E) Representative images of immunohistochemical staining of human carotid artery sections (D; n = 15) and adipose tissue sections (E; n = 8) stained for CD80, Plg-RKT, and plasminogen. Carotid plaque samples were obtained as described.30  Visceral fat tissue was obtained from 8 patients undergoing bariatric bypass surgery.31  Tissue samples were fixed in 4% formaldehyde and embedded in paraffin. Sections of human carotid artery plaques were stained with hematoxylin and eosin, and sections of human carotid artery plaques and of adipose tissue samples were stained with an anti-CD80 antibody as a marker for M(LPS+INF) macrophages (rabbit polyclonal, dilution 1:100; Santa Cruz), anti–Plg-RKT mAB (dilution 1:500), an antiplasminogen antibody (goat polyclonal; dilution 1:100; Santa Cruz), and a respective isotype control antibody for the anti–Plg-RKT mAb (dilution 1:250; eBiosciences, San Diego, CA) and normal goat IgG as control antibody for the antiplasminogen antibody (dilution 1:750; R&D Systems, Minneapolis, MN). Afterward, sections were incubated for 18 hours with secondary antibodies (anti-rabbit polyclonal, labeled with Cy3; Biolegend; anti-goat polyclonal, labeled with Dylight 650 and anti-mouse polyclonal labeled with FITC, dilution 1:200; both Abcam). Slides were scanned on a fully automated system (TissueGnostics, Vienna, Austria) with a ×20 objective, acquired with tissueFAX software and analyzed with tissueQuest software. CD80 is depicted in red; Plg-RKT is depicted in green, and plasminogen is depicted in yellow. White arrows in the merged panel mark cells staining positive for CD80, Plg-RKT, and Plg. Cells with MFI >100 for CD80 were defined as CD80 high-expressing cells and therefore as proinflammatory; cells with MFI <100 were defined as anti-inflammatory. MFIs for Plg-RKT and plasminogen were then measured for the 2 defined CD80 high- and low-expressing cells, respectively, and compared. Values are displayed as MFIs (A), ΔCt (B), or fold change vs control (C) ± standard error of mean. Statistical significances were calculated using analysis of variance and Tukey’s post hoc test when >2 groups were compared (A-C) or Student t tests (unpaired) for comparison of 2 groups (D,E). DAPI, 4′,6-diamidino-2-phenylindole.

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