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Blood, Vol. 113, Issue 3, 714-722, January 15, 2009
A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells
Blood Shibamiya et al.
113: 714
Supplemental materials for: Shibamiya et al
Files in this Data Supplement:
- Figure S1. Effect of poly I:C on the expression of mRNA for cytokines and other hemostasis-related genes in EC (JPG, 78.9 KB)
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Cells were stimulated with poly I:C (10 µg/ml) or TNFα (25 ng/ml) for 2 h and then mRNA was isolated for RT-PCR analysis. For each gene the size of the amplification product and the number of cycles used is indicated in the figure. Poly I:C induces the gene expression for various cytokines but apart from TF and TM all the other hemostasis-related genes were not affected.
- Figure S2. Effect of inhibitory antibodies directed to TF on the influence of poly I:C on EC (JPG, 27.6 KB)
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Cells were stimulated with poly I:C (10 µg/ml) for 5 h and then a TF inhibitory antibody (MabVIC7) or control antibody (5 µg/ml) was added for 30 min before the activitiy of TF was determined by using chromogenic substrate assays for FXa generation (top panel) or plasma clotting time (bottom panel). Results are presented as relative enzyme activity or clotting time; mean ± SEM of triplicate wells. Anti-TF antibody can block TF activity in an FXa generation assay as well as in the plasma clotting assay.
- Figure S3. Effect of poly I:C on TF activity in endothelial cells and monocytes (JPG, 51.3 KB)
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Endothelial cells (top) or monocytes (bottom) were stimulated with poly I:C in complex with liposomes or in the naked form as well as LPS (1 µg/ml, only on monocytes) for 5 h and the TF activity was determined by using chromogenic substrate assays for FXa. Results are presented as relative enzyme activity; mean ± SEM of triplicate wells. Poly I:C over a range of concentrations in the naked form or in complex with liposomes stimulates endothelial cells but not monocytes. In EC cells more poly I:C was internalized in the presence of liposomes compared to naked poly I:C as demonstrated by immunostaining with the dsRNA-specifc antibody J2 (data not shown).
- Figure S4. Effect of poly I:C on TF and TM activity and expression in vascular smooth muscle cells (JPG, 40.5 KB)
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(A) Cells were stimulated with poly I:C or poly dI:dC (50 µg/ml) or LPS (100 ng/ml) + serum (2% vol/vol) for 5 h and the activities of TF (top panel) and TM (bottom panel) were determined by using chromogenic substrate assays for FXa and APC generation respectively. Results are presented as relative enzyme activity; mean ± SEM of triplicate wells. (B) After stimulation of cells, Western blot analysis was performed with the indicated antibodies. Poly I:C does not influence TF or TM expression or activity in VSMC. TNFα did not influence TF or TM expression and activity in VSMC.
- Figure S5. Effect of inhibitory antibodies directed to TNFα and IL-1β on the influence of poly I:C on EC (JPG, 55.9 KB)
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EC were pretreated with inhibitory antibody to TNFα (5 µg/ml) (topl) or IL-1 β (5 µg/ml) (bottom). Cells were then stimulated with poly I:C (10 µg/ml) or the respective cytokine (25 ng/ml) for 5 h and the activity of TF was determined by using chromogenic substrate assays for FXa generation. Results are presented as relative enzyme activity; mean ± SEM of triplicate wells. The effect of poly I:C is not mediated via the induction of TNFα or IL-1 β. NeutralizingIL-1βantibody: AB-201-NA, neutralizing TNFα antibody: AF-201-NA and control Ig Gwere from R& D Systems.
- Figure S6. Effect of poly I:C and LPS on blood cell parameters (JPG, 63.1 KB)
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PBS, poly I:C (250 µg/mouse) or LPS (50 µg/mouse) (n = 8) was applied through the tail vein in a volume of 100 µl. After 12 h EDTA-blood was collected and blood cell analysis was performed on a SysmexKX-21 Hematologyanalyzer (Norderstedt, Germany). Erythrocyte counts were not significantly different between any of the groups. Poly I:C and LPS significantly decreased leukocyte counts in both WT and TLR3-/-mice but there was no difference between the strains. LPS significantly decreased platelet counts in WT and TLR3-/-mice but there was no difference between the strains.
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