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First published online 1 June 2004
doi: 10.1242/jcs.01149

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The soluble D2D3_88-274 fragment of the urokinase receptor inhibits monocyte chemotaxis and integrin-dependent cell adhesion

Federico Furlan^1,*, Simone Orlando^1,*, Carlo Laudanna², Massimo Resnati¹, Veronica Basso¹, Francesco Blasi^1,*, and Anna Mondino^1,*

¹ Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, Milan, 20132, Italy
² Division of General Pathology, Department of Pathology, University of Verona, Verona, 37129, Italy

View larger version (21K): [in a new window]   Fig. 1. D2D388-274 inhibits MCP-1 induced monocyte migration. (A) Human monocytes were obtained from the peripheral blood of healthy volunteers using Ficoll and Percoll gradients. Cell migration in response to the indicated concentration of D2D388-274 (), D2D3 () and D1 () was determined in a Boyden chamber as described in Materials and Methods. As control, cells were allowed to migrate in response to 2 nM MCP-1 (). The number of cells migrating in the absence of chemoattractant was taken as 100%. (B) Monocytes or (C) THP-1 cells were left either untreated or stimulated for 15 minutes with D2D388-274 (), D2D384-274 (), D2D3 () and D1 () as indicated. Thereafter, the ability of the cells to migrate in response to 2 nM MCP-1 was determined. The number of cells migrating towards MCP-1 was taken as 100% and the values of cells migrating after pretreatment with D2D388-274 were calculated relative to this. Each point represents the mean±s.d. of three independent experiments.

View larger version (21K): [in a new window]   Fig. 2. D2D388-274 stimulation does not induce CCR2 heterologous desensitization. (A) MCP-1 binding to CCR2 was measured on human monocytes and CCR2-overexpressing CHO cells by the addition of 1 nM 125I-MCP-1 (see Materials and Methods). The percentage of control specific binding is indicated in the figure. (B) Monocytes and THP-1 cells were loaded with 1 mM Fura-2 for 30 minutes at 37°C and then stimulated with 1 µM MCP-1 or 1 µM D2D388-274. Fluorescence output was measured at 340 nm in a fluorescence spectrophotometer. The results were confirmed in two different experiments. (C) THP-1 cells were pretreated with serum-free medium (med), 10 nM D2D388-274 (D2D3) or 10 nM uPA (uPA) for 30 minutes and then stimulated with 100 nM MCP-1 for the times indicated (x-axis). At the end of the stimulation, cells were fixed, permeabilized and stained with FITC-labeled phalloidin. The amount of bound phalloidin-FITC was evaluated by flow cytometry analysis and its increase in percent, compared to the content of F-actin detected in unstimulated cells, is shown in the figure (y-axis). The data in A and C represent the mean±s.d. of three experiments. Each experiment was performed in triplicate for each stimulus.

View larger version (16K): [in a new window]   Fig. 3. D2D388-274 inhibits ß2-integrin-dependent monocyte migration. (A) THP-1 cells were pretreated for 15 minutes at 37°C with increasing concentrations of D1 (), D2D3 (gray ), D2D388-274 () or D2D388-274 (). (B, C) THP-1 cells were pretreated for 15 minutes at 37°C with increasing concentrations of MMK-1 and ATF. After pretreatment, cells were tested for their ability to migrate in response to fMLP (A,B,C), RANTES (A) or MCP-1 (B,C). The number of cells migrating towards the different chemokines was taken as 100%, and the values of cells migrating after pretreatment with pretreating reagents were calculated relative to this. (D) THP-1 cells were incubated with either neutralizing anti-ß2 mAb or with an isotype-matched control-mAb. Thereafter the cells were allowed to migrate in response to MCP-1 (2 nM). The data in A, B, C and D represent the mean±s.d. of three experiments. Each experiment was performed in triplicate for each stimulus.

View larger version (24K): [in a new window]   Fig. 4. D2D388-274 inhibits chemokine-induced monocyte-adhesion. Rapid adhesion of monocytes to fibrinogen-coated slides was assessed as described in Materials and Methods. Adherent cells were counted in four independent 0.2 mm2 fields by using video-imaging software. Triplicates were analyzed for each condition. The data are the mean±s.d. of triplicate evaluation (a total of twelve 0.2 mm2 fields). (A) Monocytes were stimulated with MCP-1 (2 µM), fMLP (50 µM) or the indicated amounts of D2D388-274 and MMK-1 (MMK) and allowed to adhere for 3 minutes. (B) Monocytes were left untreated (-) or pretreated for 15 minutes with D2D388-274 (1 µM), MMK (50 µM), D1 (1 µM), D2D3 (1 µM) or full-length suPAR (1 µM). The ability of the cells to adhere to fibrinogen-coated slides was determined following stimulation with MCP-1 (black bars) or fMLP (white bars). As control, D2D388-274 was added to the cells simultaneously with MCP-1 or fMLP stimulation (Ctr-MIX). Gray bar, basal adhesion. (C) For titration: pretreatment for 15 minutes with suPAR at concentrations as indicated. Gray bar, basal adhesion (CTR); black bars, MCP-1 (1 µM). (D) Monocytes were left untreated (-) or preincubated with either anti-FPRL-1 Ab FPRL-1) or control Ig (Ctr Ig) for 15 minutes on ice after which they were treated for an additional 15 minutes at 37°C with medium (-), 1 µM D2D388-274, or 50 µM MMK. The ability of the cells to adhere to fibrinogen-coated slides was then determined following stimulation with MCP-1 (black bars) or fMLP (white bars). Gray bar, basal adhesion. (E) Monocytes were incubated with medium (-), the Hck inhibitor PP2 (10 nM), the PI3K inhibitor LY (25 µM) and the p38 inhibitor SB (1 µM) for 30 minutes at 37°C. Thereafter, half of the cells were treated with D2D388-274 (1 µM) for an additional 15 minutes at 37°C. Following this, all cells were stimulated with MCP-1 (2 µM) and allowed to adhere to fibrinogen-coated slides for 3 minutes. Gray bar, basal adhesion.