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Blood, Vol. 113, Issue 21, 5254-5265, May 21, 2009
Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and β2 integrin–dependent cell clearance program
Blood Maugeri et al.
113: 5254
Supplemental materials for: Maugeri et al
Files in this Data Supplement:
- Table S1. TRAP-6 induces platelet but not neutrophil activation (PDF, 17.8 KB)
- Figure S1. Assessment of platelet adhesion and internalization by flow cytometry (JPG, 80.6 KB)
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(A) lymphocytes, monocytes and neutrophils were identified within the CD45+ population by their side scatter (left panel, y axis). The expression of CD14 and CD66b defines the neutrophil and the monocyte clusters, respectively (right panels). (B) neutrophils were incubated in absence and presence of resting and activated platelets. Platelet adhesion to neutrophils was assessed by the expression of the CD61 platelet antigen. Platelet internalisation by neutrophils was assessed by the increase of the expression of the CD61 antigen after permeabilization, a treatment that allows the mAb to penetrate the plasma membrane.
- Figure S2. Expression of P-selectin, exposure of phosphatidylserine and Mac-1 upregulation in activated cells (JPG, 11.3 KB)
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The top profile depicts phosphatidylserine exposure by activated (filled profile) versus resting (open profile) platelets (83.3±3.9% vs 8.3±1.7% FITC-Annexin A5+ platelets). The middle profile depicts P-selectin expression of activated (filled profile) versus resting (open profile) platelets (51.0±1.4% vs 4.3±1.5 %). The bottom profile depicts the expression of the active isoform of Mac-1 on neutrophils exposed to activated (filled profile) or resting (open profile) platelets (47.7±2.5% FITC-327c+ cells vs 7±1.2%).
- Figure S3. P-selectin–, β2integrin- and phosphatidylserine-dependent clearance of activated platelets in whole blood (JPG, 44.6 KB)
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The percentage of neutrophils with adherent (filled bars) and internalised (grey bars) platelets (panel A), their MPO (open bars) content and the concentration of soluble released (grey bars) MPO (panel B) were evaluated after the interaction of neutrophils with platelets in whole sodium citrated blood, in the absence of TRAP-6 (resting platelets, TRAP-6 −) or in the presence of TRAP-6 (activated platelets, TRAP-6+) . The assay was performed either adding TRAP-6 alone or adding simultaneously recombinant Annexin 5A to block the recognition of phosphatidylserine, the anti P-Sel mAb to block P-selectin or the Mac-1 mAb to block the β2 subunit binding site. Results are expressed as mean ± SEM of 5–10 different experiments. *** P < 0.0001, * P < 0.005, significantly different from control.
- Figure S4. The phagocytosis of platelets in independent of the cyclooxigenase pathway (JPG, 24.7 KB)
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Neutrophils and platelets were derived from healthy donors before or four hours after aspirin ingestion (500 mg). Neutrophils were then challenged with autologous activated platelets and the percentage of neutrophils with adherent (filled bars) and internalised (grey bars) platelets evaluated. Adhesion and phagocytosis are expressed as mean ± SEM of 3 independent experiments.
- Video 1. Neutrophils swiftly and effectively phagocytose activated platelets in suspension (AVI, 38.2 MB)
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Neutrophils (5 × 106/mL) were challenged with red-emitting platelets pre-labeled with the BCECF-AM intracellular dye, activated with TRAP-6 (1 × 109/mL, see Methods for details). Time lapse images were acquired at 37°C. Images are from one experiment representative of five.
- Video 2. Neutrophils do not phagocytose resting platelets (AVI, 42 MB)
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Neutrophils (5 × 106/mL) were challenged with red-emitting resting platelets pre-labeled with the BCECF-AM intracellular dye, (1 × 109/mL, see Methods for details). Time lapse images were acquired at 37°C. Images are from one experiment representative of five.
- Video 3. Neutrophils swiftly and effectively phagocytose adherent activated platelets under arterial shear stress (AVI, 34.1 MB)
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Platelets (3 × 107/mL) were adhered to glass and interaction with neutrophils (5 × 106/mL) studied in a shear stress of 2 dynes/cm2 for 2 minutes. The chamber was then perfused with medium alone for further 10 minutes at 20 dynes/cm2. Adherent neutrophils were observed by contrast phase video microscopy (see Methods for details). Images are from one experiment representative of five.
- Video 4. Hindrance with phosphatidylserine recognition by Annexin AV does not influence adhesive interactions between neutrophil and platelets under arterial shear stress (AVI, 34.3 MB)
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Platelets (3 × 107/mL) were adhered to glass and treated with Annexin AV (15 µg/ml). Interaction with neutrophils (5 × 106/mL) was studied in a shear stress of 2 dynes/cm2 for 2 minutes as described above. Images are from one experiment representative of five.
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