Research Article
Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation
Figure 2
Dose-dependent effect of heparin on PMNL apoptosis and priming. (a) Representative histograms for apoptosis of HD PMNLs without heparin (1) and with 100 U/mL heparin (2) based on Annexin-V-FITC and PI binding. Cell populations: Annexin-V−/PI−, Annexin-V+/PI−, and Annexin-V+/PI+ were regarded as alive, early apoptotic, and late apoptotic or necrotic, respectively. (b) Early apoptosis (Annexin-V+/PI−) in NC PMNLs without stimulation (●) and after 15 min stimulation with 1 μM PAF (■) and HD PMNLs (▲). Cells were preincubated with PAF, washed, and incubated with increasing concentrations of heparin for 30 min. Apoptosis was determined by flow cytometer analysis, using a commercial kit for detection of Annexin-V as described in Section 2. Data are expressed as percentage of apoptotic PMNLs; . for HD at 25, 50, and 100 U/mL heparin versus no heparin. for NC pretreated with 1 μM PAF incubated with 100 U/mL heparin versus no heparin. for NC at 100 U/mL heparin versus no heparin. (c) Early and late (total) apoptosis (sum of Annexin-V+/PI− and Annexin-V+/PI+) in NC PMNLs without stimulation (●) and after 15 min stimulation with 1 μM PAF (■) and HD PMNLs (▲). for HD at 25, 50, and 100 U/mL heparin versus no heparin. for NC pretreated with 1 μM PAF incubated with 25, 50, and 100 U/mL heparin versus no heparin. for NC at 100 U/mL heparin versus no heparin. (d) Superoxide release from NC PMNLs without stimulation (●) and after 15 min stimulation with 1 μM PAF (■) and HD PMNLs (▲). Following preincubation with PAF, cells were washed and incubated with increasing concentrations of heparin for 30 min. The rate of superoxide release was determined after cells activation with 0.32 × 10−7 M PMA. The changes in optical density were monitored at 549 nm continuously in the presence of 0.08 mM cytochrome C. Data are represented by nmoles/106 cells/10 min; . for HD at 25, 50, and 100 U/mL heparin versus no heparin. for NC pretreated with 1 μM PAF incubated with 25, 50, and 100 U/mL heparin versus no heparin. for NC at 25, 50, and 100 U/mL heparin versus no heparin.
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