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Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes

¹ Division of General Internal Medicine, Department of Internal Medicine, University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria
² Aventis Behring Pharma, Marburg, Germany

View larger version (16K): [in a new window]   Fig. 1. Direct chemotactic effects of immunopurified AT on monocytes and lymphocytes. Chemotaxis experiments were performed in modified Boyden chambers using nitrocellulose micropore filters. Monocyte chemo-attractant protein 3 (MCP-3) for monocytes (left panel) and interleukin-8 (IL-8) for lymphocytes (right panel) were used as positive control chemotactic stimuli. Results are given as the mean±s.e.m. of the migration index, which is the ratio of the distance of migration (µm) towards a chemotactic agent and the distance (µm) towards the medium. Similar results were obtained using AT concentrate (data not shown). Multiple group comparison was performed after Kruskal-Wallis (p=0.0074 for lymphocytes, p=0.0001 for monocytes) followed by Mann-Whitney U-test (*, p<0.05). n=8 for lymphocytes, n=5 for monocytes.

View larger version (16K): [in a new window]   Fig. 2. Deactivation of chemokine-induced migration of lymphocytes (A) and monocytes (B) by AT. Experiments were performed in modified Boyden chambers using nitrocellulose micropore filters. Cells were preincubated with varying AT concentrations for 20 minutes followed by washing. Then chemotaxis of lymphocytes (A) toward Rantes [10 ng/mL] or IL-8 [1 nmol/L] and of monocytes (B) toward Rantes [10 ng/mL] and monocyte chemoattractant protein 3 (MCP-3) [10 nmol/L] was monitored. Results are given as the mean±s.e.m. of the migration index, which is the ratio of the distance of migration (µm) towards attractant and the distance (µm) towards the medium. *, p<0.05, Mann-Whitney U-test versus medium preincubation after multiple group comparison by Kruskal-Wallis test; lymphocytes: p=0.0197 toward Rantes; p=0.0163 toward IL-8; monocytes: p=0.0104 toward Rantes; p=0.0038 toward MCP-3; n=5.

View larger version (20K): [in a new window]   Fig. 3. Effects of heparinase and chondroitinase treatment on AT-induced chemotaxis. Heparinase I or chondroitinase was added to monocytes or lymphocytes (37°C/5%CO2). After an incubation period of 50 minutes, cells were washed twice and chemotaxis experiments were performed. AT [1 U/mL] served as chemoattractant. Data are expressed as chemotaxis index, which is the ratio between directed and undirected migration. Random migration was 45±3.3 µm for monocytes and 56±2.1 µm for lymphocytes (mean±s.e.m.). For statistical analysis the Mann-Whitney U test was used (*, p<0.05) after the Kruskal-Wallis test (p<0.01); n=3.

View larger version (1K): [in a new window]   Fig. 4. Effect of blocking of the heparin binding site on AT by SR90107A, a synthetic pentasaccharide, on AT-dependent PBMC migration. Coincubation of AT with different concentrations of SR90107A for 20 minutes at room temperature was performed before testing direct lymphocyte (A) and monocyte (B) chemotaxis towards AT. Chemotaxis experiments were performed in modified Boyden chambers. As a control attractant, Rantes was coincubated with SR90107A. Results are given as the mean±s.e.m. of the migration index, which is the ratio of the distance of migration towards AT and the distance towards the medium. Multiple group comparison was performed after Kruskal-Wallis (p=0.0153 for lymphocytes, p=0.0085 for monocytes) followed by Mann-Whitney U-test (*, p<0.05); n=4.

View larger version (18K): [in a new window]   Fig. 5. Effect of thrombin on AT-dependent leukocyte migration. Chemotaxis of PBMC was tested in modified Boyden chambers. The chemotaxis index, which is the ratio between directed and undirected migration of cells, is used to express the data. (A) To test effects of thrombin on AT-induced deactivation of PBMC chemotaxis toward Rantes [10 ng/mL], cells were preincubated in the presence of AT with or without thrombin for 20 minutes. Mann-Whitney U (*, p<0.05) versus control after Kruskal- Wallis (p<0.01); n=3. (B) To test whether thrombin induces cell migration, PBMC were allowed to migrate toward thrombin [100 µU/mL to 1 U/mL]. Mann-Whitney U test (*, p<0.05) versus medium after Kruskal-Wallis (p<0.01); n=3. (C) Cells preincubated with various concentrations of AT followed by washing and migration toward thrombin [10 mU/mL]. Kruskal Wallis test, p>0.05; n=3.

View larger version (23K): [in a new window]   Fig. 6. FACS and RT-PCR analyses of syndecan-4 in lymphocytes and monocytes. (A) FACS analysis of anti-syndecan-4 mAb binding to lymphocytes and monocytes. Fluorescence analysis used a FACScan Flow cytometer, and a histogram of PE-fluorescence is shown. Cells were preincubated with either 10 µg/mL isotype-matched control mouse IgG (thin line) or 10 µg/mL anti-syndecan-4 mAb (bold line) and stained with PE-conjugated streptavidin. (B) Syndecan-4 mRNA in endothelial cells, lymphocytes and monocytes. 1 µg of total RNA from each sample was reverse transcribed into cDNA and amplified for the syndecan-4 gene using PCR. Syndecan-4 is represented by the 453 base pair product.

View larger version (18K): [in a new window]   Fig. 7. Effects of syndecan-4 antibodies on AT-induced chemotaxis of monocytes and lymphocytes. Preincubation with antibodies to syndecan-4 core protein (D-16) and syndecan-4 ectodomain (5G9) was performed for 20 minutes (37°C/5% CO2). After washing, cells were allowed to migrate toward AT [1 U/mL] or Rantes [10 ng/mL] in modified Boyden chambers using nitrocellulose micropore filters. Isotype-matched IgG served as control. Response of monocytes (A) and lymphocytes (B) is expressed using the chemotaxis index (mean±s.e.m.), which is the ratio between directed and undirected migration of cells. The distance of undirected migration was 68±4.9 µm for monocytes and 57±9.5 µm for lymphocytes (n=4). Statistical analysis was performed using the Mann-Whitney U test (*, p<0.05) versus medium after Kruskal-Wallis (p<0.01), n=4. Chemotaxis toward Rantes remained unaffected after pre-treatment with syndecan-4 antibodies (C). Kruskal-Wallis (p>0.1)

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