Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation

Figure 5

The effect of priming on apoptosis induced by heparin. (a) Representative histograms of flow cytometry showing the percentage of apoptotic cells after incubation with 25 U/mL heparin for 30 min: HD PMNLs with IC-FITC and IC-PE (1), HD PMNLs with IC-FITC and anti-CD11b-PE antibody (2), HD PMNLs with Annexin-V-FITC and IC-PE (3), and HD PMNLs with Annexin-V-FITC and anti-CD11b-PE antibody (4). (b) Apoptosis in NC PMNLs (●) after 15 min stimulation with increasing concentrations of PAF. Cells were preincubated with PAF, washed, and subjected to 30 min incubation with 25 U/mL heparin. Apoptosis was determined by flow cytometer analysis, using Annexin-V commercial kit as described in Section 2. Data are expressed as percentage of apoptotic PMNLs; . In addition, the percentage of apoptotic NC PMNLs that bind heparin was also measured after preincubation of NC separated PMNLs with PAF and anti-CD11b antibodies (■), before heparin incubation. The boxes and whiskers presentations represent apoptosis in HD PMNLs incubated with 25 U/mL heparin for 30 min, with and without incubation with anti-CD11b antibodies. (c) Apoptosis in NC PMNLs (upper left panel 1) and 15 min after stimulation with 10⁻⁶M PAF (lower left panel 2). These cells were treated with 25 U/mL heparin for 30 min (panels 3 and 4). Apoptosis was evaluated by the TUNEL method (FITC green staining). Nuclei were stained with Hoechst reagent (blue staining). The percent of apoptotic NC PMNLs that bind heparin was measured after preincubation of the PMNLs with anti-CD11b antibodies (red staining) (panels 5 and 6) or IC-PE antibodies (panels 7 and 8) before incubation with heparin. Quantification of apoptosis: over 300 cells were counted, from at least three independent experiments.

(a)

(b)

(c)