Figure 1.
Monocyte subsets and Plg-RKT. (A) Peripheral blood mononuclear cells were isolated from leukapheresis chambers obtained from healthy thrombocyte donors using lymphocyte separation medium 1077 (PromoCell, Heidelberg, Germany) and used for either monocyte subset isolation employing anti-CD45, anti-CD14, and anti-CD16 antibodies or for generating macrophages as described.15,26 Gating strategy for human monocyte subsets in whole blood is shown. The relative amounts of cells per subset were in accordance with published data, CM: 76% ± 5%, IM: 5% ± 2%, NCM: 19% ± 7%.15 (B) Mean fluorescent intensities (MFIs) of Plg-RKT on human monocyte subsets (n = 12). Monocytes in freshly drawn blood were stained with anti-CD14 antibody (PerCP/Cy5.5, Clone HCD14), anti-CD16 antibody (APC-Cy7, Clone 3G8), anti-CD45 antibody (Pacific Blue, Clone HI30), and anti–Plg-RKT mouse monoclonal mAb7H1, prepared and characterized in our laboratory.6,7 Samples were analyzed with a Novocyte Flow cytometer (ACEA Biosciences, San Diego, CA). (C) Plg-RKT mRNA levels in human monocyte subsets. Cells were sorted based on CD14 and CD16 expression into CMs, IM, and NCMs using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ), and afterward, RNA was isolated for quantitative polymerase chain reaction. Primer sequences for Plg-RKT were “tgg​aac​cct​ttt​aga​aag​aat​ga” and “ttg​gca​gct​gca​att​tac​tc” (Universal Probe Library, #73; Roche, Basel, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization (n = 3). (D) Plasminogen binding to human monocyte subsets in the presence or absence of anti–Plg-RKT mAb (clone7H1, developed and characterized in our laboratory)6,7 or EACA. PBMCs were stained with fluorochrome-labeled antibodies against CD45, CD14, and CD16 and incubated with FITC-labeled plasminogen (0.5 µM; n = 9).6 Fluorescence was measured with a Novocyte Flow cytometer. To test specificity of plasminogen binding, samples were additionally incubated with EACA (0.2 M; Sigma Aldrich, St. Louis, MO; n = 9) or with anti–Plg-RKT mAb (140 nM; n = 3). (E) Cell migration of human monocyte subsets in the presence or absence of plasminogen. Sorted IMs were seeded into inserts of Costar Transwell Permeable Supports (5.0-µm pore size; Corning, New York, NY) in RPMI 1640 containing 1% bovine serum albumin (both from Sigma Aldrich). Cells were incubated in total for 1 hour at 37°C, 5% CO2 in a humidified incubator. After 15 minutes, cells were either preincubated with anti–Plg-RKT mAb (280 nM; filled triangles, n = 6), isotype control antibody (280 nM; BD Biosciences; open squares, n = 3), or uPA aminoterminal fragment (50 ng/mL; Sekisui Diagnostics, Lexington, MA; filled diamonds, n = 4), or after an additional 30 minutes either EACA (0.2 M; open diamonds, n = 5) or aprotinin (2 µM; Sigma Aldrich; half-filled diamonds, n = 3) was added. Then plasminogen (0.4 µM; Roche) was added. Control cells were incubated for the times indicated above in vehicle only (n = 5). The number of migrated cells under this condition was set at 1, and values under experimental conditions are expressed as x-fold migration compared with control. As additional controls, cells were treated with anti–Plg-RKT mAb (280 nM; open triangles, n = 5) in the absence of plasminogen. Then, inserts were transferred into wells containing complete culture medium containing N-formyl-met-leu-phe (100 nM; Sigma Aldrich) as a chemoattractant. Cells were allowed to migrate for 3 hours at 37°C. Cells on the bottom side of the membrane were fixed with 4% paraformaldehyde and washed with phosphate-buffered saline. Membranes were then cut out and mounted on slides with Prolong Gold Antifade mounting medium (Thermo Fisher Scientific, Waltham, MA) and analyzed on a Zeiss Axio Imager fluorescence microscope. For calculating cell migration, cells on 5 randomly selected microscopic fields (magnification ×40) of the insert membrane were counted for every condition. Plasminogen-dependent migration was not exhibited by CMs or NCMs (CM: 0.9-fold vs control; NCM: 1.1-fold vs control). $P < .05 as compared with control; #not significant as compared with control; *P < .05, **P < .01, ***P < .001, as compared with plasminogen only; §not significant as compared with plasminogen only. (F) MFIs of uPAR on human monocyte subsets (n = 4). Cells were stained with anti-uPAR antibody (phycoerythrin; Clone VIM5; BioLegend, San Diego, CA). (G) Gating scheme for murine monocyte subsets in whole blood. Mouse monocytes were isolated from whole blood, and subsets were gated employing anti-CD11b and anti-Ly6C antibodies29 (Biolegend) using a Novocyte flow cytometer (ACEA Biosciences). (H) MFIs of Plg-RKT in murine monocyte subsets (n = 8; female, n = 4; male, n = 4). Whole blood samples were stained with anti-CD11b (allophycocyanin; Clone M1/70, BioLegend), anti-Ly6C (Phycoerythrin; Clone HK1.4, BioLegend), and anti–Plg-RKT antibody mAb.6,7 (I) Plasminogen binding to murine monocyte subsets in whole blood from female Plg-RKT+/+ wild-type (WT) mice and Plg-RKT−/− mice incubated with FITC-labeled plasminogen (0.5 µM; n = 3). Plg-RKT gene targeted mice (8-10 weeks of age) were backcrossed 10 generations into the C57Bl/6J background. (All animal experiments were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.) (J) MFIs of Plg-RKT cell surface expression on murine male and female monocyte subsets. Whole blood samples were stained with anti-CD11b, anti-Ly6C, and anti–Plg-RKT as described in the legend to panel H (n = 8; 4 male and 4 female). (K) Ly6Chigh and (inset) Ly6Clow monocytes recovered in the peritoneal lavage of female Plg-RKT+/+ WT mice and Plg-RKT−/− mice either untreated (triangles) or 72 hours following intraperitoneal (IP) injection with thioglycollate (circles), as used previously in our laboratory7,8 (without thioglycollate treatment: n = 6 mice per group; with thioglycollate treatment: n = 5 mice per group). (L) Ly6Chigh and (inset) Ly6Clow monocytes recovered in the peritoneal lavage of male Plg-RKT+/+ WT mice and Plg-RKT−/− mice either untreated (triangles) or 72 hours following IP injection with thioglycollate (circles) (without thioglycollate treatment: n = 6 mice per group; with thioglycollate treatment: n = 5 mice per group). (M) Gating scheme for murine monocyte subsets in peritoneal lavage 72 hours following IP injection with thioglycollate. Mouse monocytes were isolated from peritoneal lavage and gated employing anti-CD11b and F4/80 antibodies (Biolegend) using a Novocyte flow cytometer (ACEA Biosciences). Values are displayed as MFIs (B,D,F, and H-J), delta cycle threshold (ΔCt) (C), fold change vs control (E), or total cell number (K,L) ± standard error of mean. Red symbols are used for CMs; blue symbols are used for IMs, and green symbols are used for NCMs for human monocytes (A-F). Blue symbols are used for Ly6Chigh, and red symbols are used for Ly6Clow murine monocytes (G-M). Statistical significances were calculated using analysis of variance and Tukey’s post hoc test when >2 groups were compared (B-F,I,K-L), and values of P < .05 were considered significant and are provided in the respective panels. Student t tests (unpaired) was used for comparison of 2 groups (H,J) and values of P < .05 were considered significant and are provided in the respective panels. FSC, forward scatter; n.s., not significant; SSC, sideward scatter.

Monocyte subsets and Plg-RKT. (A) Peripheral blood mononuclear cells were isolated from leukapheresis chambers obtained from healthy thrombocyte donors using lymphocyte separation medium 1077 (PromoCell, Heidelberg, Germany) and used for either monocyte subset isolation employing anti-CD45, anti-CD14, and anti-CD16 antibodies or for generating macrophages as described.15,26  Gating strategy for human monocyte subsets in whole blood is shown. The relative amounts of cells per subset were in accordance with published data, CM: 76% ± 5%, IM: 5% ± 2%, NCM: 19% ± 7%.15  (B) Mean fluorescent intensities (MFIs) of Plg-RKT on human monocyte subsets (n = 12). Monocytes in freshly drawn blood were stained with anti-CD14 antibody (PerCP/Cy5.5, Clone HCD14), anti-CD16 antibody (APC-Cy7, Clone 3G8), anti-CD45 antibody (Pacific Blue, Clone HI30), and anti–Plg-RKT mouse monoclonal mAb7H1, prepared and characterized in our laboratory.6,7  Samples were analyzed with a Novocyte Flow cytometer (ACEA Biosciences, San Diego, CA). (C) Plg-RKT mRNA levels in human monocyte subsets. Cells were sorted based on CD14 and CD16 expression into CMs, IM, and NCMs using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ), and afterward, RNA was isolated for quantitative polymerase chain reaction. Primer sequences for Plg-RKT were “tgg​aac​cct​ttt​aga​aag​aat​ga” and “ttg​gca​gct​gca​att​tac​tc” (Universal Probe Library, #73; Roche, Basel, Switzerland). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization (n = 3). (D) Plasminogen binding to human monocyte subsets in the presence or absence of anti–Plg-RKT mAb (clone7H1, developed and characterized in our laboratory)6,7  or EACA. PBMCs were stained with fluorochrome-labeled antibodies against CD45, CD14, and CD16 and incubated with FITC-labeled plasminogen (0.5 µM; n = 9).6  Fluorescence was measured with a Novocyte Flow cytometer. To test specificity of plasminogen binding, samples were additionally incubated with EACA (0.2 M; Sigma Aldrich, St. Louis, MO; n = 9) or with anti–Plg-RKT mAb (140 nM; n = 3). (E) Cell migration of human monocyte subsets in the presence or absence of plasminogen. Sorted IMs were seeded into inserts of Costar Transwell Permeable Supports (5.0-µm pore size; Corning, New York, NY) in RPMI 1640 containing 1% bovine serum albumin (both from Sigma Aldrich). Cells were incubated in total for 1 hour at 37°C, 5% CO2 in a humidified incubator. After 15 minutes, cells were either preincubated with anti–Plg-RKT mAb (280 nM; filled triangles, n = 6), isotype control antibody (280 nM; BD Biosciences; open squares, n = 3), or uPA aminoterminal fragment (50 ng/mL; Sekisui Diagnostics, Lexington, MA; filled diamonds, n = 4), or after an additional 30 minutes either EACA (0.2 M; open diamonds, n = 5) or aprotinin (2 µM; Sigma Aldrich; half-filled diamonds, n = 3) was added. Then plasminogen (0.4 µM; Roche) was added. Control cells were incubated for the times indicated above in vehicle only (n = 5). The number of migrated cells under this condition was set at 1, and values under experimental conditions are expressed as x-fold migration compared with control. As additional controls, cells were treated with anti–Plg-RKT mAb (280 nM; open triangles, n = 5) in the absence of plasminogen. Then, inserts were transferred into wells containing complete culture medium containing N-formyl-met-leu-phe (100 nM; Sigma Aldrich) as a chemoattractant. Cells were allowed to migrate for 3 hours at 37°C. Cells on the bottom side of the membrane were fixed with 4% paraformaldehyde and washed with phosphate-buffered saline. Membranes were then cut out and mounted on slides with Prolong Gold Antifade mounting medium (Thermo Fisher Scientific, Waltham, MA) and analyzed on a Zeiss Axio Imager fluorescence microscope. For calculating cell migration, cells on 5 randomly selected microscopic fields (magnification ×40) of the insert membrane were counted for every condition. Plasminogen-dependent migration was not exhibited by CMs or NCMs (CM: 0.9-fold vs control; NCM: 1.1-fold vs control). $P < .05 as compared with control; #not significant as compared with control; *P < .05, **P < .01, ***P < .001, as compared with plasminogen only; §not significant as compared with plasminogen only. (F) MFIs of uPAR on human monocyte subsets (n = 4). Cells were stained with anti-uPAR antibody (phycoerythrin; Clone VIM5; BioLegend, San Diego, CA). (G) Gating scheme for murine monocyte subsets in whole blood. Mouse monocytes were isolated from whole blood, and subsets were gated employing anti-CD11b and anti-Ly6C antibodies29  (Biolegend) using a Novocyte flow cytometer (ACEA Biosciences). (H) MFIs of Plg-RKT in murine monocyte subsets (n = 8; female, n = 4; male, n = 4). Whole blood samples were stained with anti-CD11b (allophycocyanin; Clone M1/70, BioLegend), anti-Ly6C (Phycoerythrin; Clone HK1.4, BioLegend), and anti–Plg-RKT antibody mAb.6,7  (I) Plasminogen binding to murine monocyte subsets in whole blood from female Plg-RKT+/+ wild-type (WT) mice and Plg-RKT−/− mice incubated with FITC-labeled plasminogen (0.5 µM; n = 3). Plg-RKT gene targeted mice (8-10 weeks of age) were backcrossed 10 generations into the C57Bl/6J background. (All animal experiments were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.) (J) MFIs of Plg-RKT cell surface expression on murine male and female monocyte subsets. Whole blood samples were stained with anti-CD11b, anti-Ly6C, and anti–Plg-RKT as described in the legend to panel H (n = 8; 4 male and 4 female). (K) Ly6Chigh and (inset) Ly6Clow monocytes recovered in the peritoneal lavage of female Plg-RKT+/+ WT mice and Plg-RKT−/− mice either untreated (triangles) or 72 hours following intraperitoneal (IP) injection with thioglycollate (circles), as used previously in our laboratory7,8  (without thioglycollate treatment: n = 6 mice per group; with thioglycollate treatment: n = 5 mice per group). (L) Ly6Chigh and (inset) Ly6Clow monocytes recovered in the peritoneal lavage of male Plg-RKT+/+ WT mice and Plg-RKT−/− mice either untreated (triangles) or 72 hours following IP injection with thioglycollate (circles) (without thioglycollate treatment: n = 6 mice per group; with thioglycollate treatment: n = 5 mice per group). (M) Gating scheme for murine monocyte subsets in peritoneal lavage 72 hours following IP injection with thioglycollate. Mouse monocytes were isolated from peritoneal lavage and gated employing anti-CD11b and F4/80 antibodies (Biolegend) using a Novocyte flow cytometer (ACEA Biosciences). Values are displayed as MFIs (B,D,F, and H-J), delta cycle threshold (ΔCt) (C), fold change vs control (E), or total cell number (K,L) ± standard error of mean. Red symbols are used for CMs; blue symbols are used for IMs, and green symbols are used for NCMs for human monocytes (A-F). Blue symbols are used for Ly6Chigh, and red symbols are used for Ly6Clow murine monocytes (G-M). Statistical significances were calculated using analysis of variance and Tukey’s post hoc test when >2 groups were compared (B-F,I,K-L), and values of P < .05 were considered significant and are provided in the respective panels. Student t tests (unpaired) was used for comparison of 2 groups (H,J) and values of P < .05 were considered significant and are provided in the respective panels. FSC, forward scatter; n.s., not significant; SSC, sideward scatter.

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