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Blood, Vol. 112, Issue 4, 1091-1100, August 15, 2008
Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion
Blood Xiao et al.
112: 1091
Supplemental materials for: Xiao et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 2.69 MB)
- Figure S1. Antibody characterization (JPG, 29.7 KB)
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(A and B) Western blot of antibodies 197.2 and 197.3 against immobilized PF4. PAGE was performed either under non-reducing (panel A) or reducing conditions (panel B). Results demonstrate that 197.2, unlike 197.3, recognizes a linear epitope on PF4. (C) ELISA studies that measure 197.2 and 197.3 binding to immobilized immobilized PF4 that is either in free-form, or when it is complexed with heparin (0.05-2U/ml). Both 197.2 and 197.3 bind PF4 in the absence of heparin. Heparin decreases 197.2 binding while it increases 197.3 binding to immobilized PF4. Data are Mean ± STD.
- Figure S2. Neutrophil shape assessed using flow cytometry (JPG, 21.7 KB)
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Flow cytometry changes in neutrophil forward- versus side-scatter plot observed upon addition of: (A) no stimulus; (B) protamine sulfate + 197.3; (C) Recombinant PF4 (rPF4) + 197.3 and (D) Human platelet PF4 (hPF4) + 197.3. Changes in cell scatter pattern are observed in (C) and (D), but not (A) and (B). rPF4 and hPF4 alone do not cause any shape change, i.e. they resemble (A) and (B) (not shown). Further, similar changes as in (C) and (D) are typically observed when human neutrophils are stimulated with other potent agonists like IL-8 and formyl peptide (fMLP).
- Figure S3. Costimulation control with non-specific immune complex (JPG, 29.3 KB)
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Experiments were performed with isolated human neutrophils under conditions identical to that described in Figure 1 of the main manuscript. 20nM IL-8 was used as a positive control. rPF4 (recombinant PF4) and BSA were applied at 1µM, and all antibodies were used at 20µg/ml. 9 min. after incubation with isolated human neutrophils, PF4 plus 197.2 immune complex resulted in cell activation while BSA plus anti-BSA complex under identical conditions did not result in significant Mac-1 upregulation. This was observed with BSA plus anti-BSA immune complex formed with both a monoclonal mouse anti-BSA Ab (mAb from Genscript, clone A00837.01(IgG1)) and with complex formed using rabbit anti-BSA polyclonal Ab (pAb from Invitrogen (IgG fraction)). * p<0.05 with respect to all other treatments. Data are presented as Mean ± STD (N=3).
- Figure S4. Mac-1 upregulation kinetics using different experiment protocols (JPG, 29.9 KB)
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Human neutrophils were either costimulated with 2µM hPF4 plus 10µg/ml 197.3 at t=0s (filled circles), 2µM hPF4 was added to neutrophil 3min prior to 10µg/ml 197.3 at t=0s (open circles), or 2µM hPF4 and 10µg/ml 197.3 were mixed together for 6min prior to neutrophil addition at t=0s (open squares). Mac-1 expression/upregulation on neutrophils was measured. Data were presented as mean ± SEM (N ≥ 3).
- Figure S5. Neutrophil activation in whole blood in response to costimulation with PF4 and anti-PF4 mAb (JGP, 34.5 KB)
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Human blood was diluted 1:5 in Hepes buffer. Phycoerythrin conjugated mouse mAb against Mac-1 (CD11b-PE) was added. A range of stimulus conditions were applied at 37°C, and neutrophil CD11b expression was monitored at the 9 min. time point, following RBC lysis with NH4Cl solution. Interleukin-8 (IL-8) was applied at 20nM, all antibodies were at 20µg/ml (197.3, 197.2 or IgG1 control), recombinant PF4 (rPF4) was added at 1µM, and heparin was at 2U/ml. 2.2 fold increase in neutrophil Mac-1 upregulation in whole blood upon costimulation with PF4 and anti-PF4 mAb could be reduced to baseline level upon addition of heparin. Data are presented as Mean ± SEM. *p<0.05 with respect to no stimulus.
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