Phosphodiesterase type 4 blockade prevents platelet-mediated neutrophil recruitment at the site of vascular injury

Abstract

Objective: Platelet-neutrophil interactions play a key role in cardiovascular disease and inflammatory processes. Src family kinases mediate P-selectin glycoprotein ligand-1-Mac-1 cross talk necessary for firm platelet-neutrophil adhesion. Because Src family kinase activity can be regulated by cAMP-dependent pathways, in this work, we evaluated the role of phosphodiesterases in the signaling events that are required to sustain platelet-neutrophil interactions and neutrophil recruitment at the site of vascular injury.

Approach and results: In neutrophils exposed to P-selectin, selective phosphodiesterase 4 (PDE4) inhibition prevented Src family kinase-mediated phosphorylation of the proline-rich tyrosine kinase 2 on Tyr579/Tyr580. The effects of PDE4 inhibition required protein kinase A, likely through protein kinase A-mediated activation of COOH-terminal Src kinase, a major negative regulator of Src family kinases. PDE4, but not other phosphodiesterase inhibitors, reduced platelet-neutrophil conjugates as well as neutrophil firm adhesion on spread platelets under flow conditions. The effect of PDE4 inhibition on neutrophil adhesion was primarily mediated by downregulation of P-selectin-induced activation of Mac-1. In a murine model of endovascular injury, selective inhibition of PDE4 significantly reduced neutrophil recruitment at the site of vascular damage.

Conclusions: This study identifies PDE4 as a central node in the signaling network that mediates platelet-neutrophil adhesion and suggests that pharmacological inhibition of PDE4 may represent a novel therapeutic avenue for the treatment of cardiovascular disease.

Keywords: neutrophils; phosphodiesterase 4 inhibitors; platelets.

Figure 1. Rolipram inhibits SFK-mediated Pyk2 phosphorylation in neutrophils exposed to soluble P-selectin

Panel A. Neutrophils were pretreated with either rolipram or PP2 (both 10 μM) alone or in combination with H89 (10 μM) or DMSO for 2 minutes at 37°C and then exposed to soluble P-selectin (10 μg/ml) for 2 minutes at 37°C with constant stirring at 1000 rpm. Samples were lysed in reducing Laemmli's buffer, boiled for 10 minutes, and then diluted 1:20 with 1% Triton X-100 lysis buffer containing protease and phosphatase inhibitors. Pyk2 was immunoprecipitated with 10 μg/ml specific antibody at 4°C, overnight. Immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with the phospho-tyrosine specific antibody (PY99) or anti-Pyk2 antibodies. Bars report optical density values of phosphorylated and total Pyk2 expressed as increase over basal levels. Data are means±SEM of 3 independent experiments. *P<0.05 vs unstimulated samples, by ANOVA and Dunnet test. Panel B. Neutrophils were treated with rolipram (0.1-20 μM) or vehicle for 2 minutes at 37°C and then incubated by soluble P-selectin. Samples were prepared and separated as described above and immunoblotted with either the phospho-specific antibody recognizing phosphorylated tyrosine residues 579/580 within the activation loop of Pyk2 or anti-Pyk2 antibody. Western blot result is representative of 3 experiments. Values are means ± SEM of 3 independent experiments. * indicates statistical difference (P<0.05 by ANOVA, Dunnett test) versus control (untreated) group.

Figure 2. Rolipram promotes PKA-mediated protein phosphorylation in neutrophils interacting with soluble P-selectin

Neutrophils were treated with rolipram or/and calyculin A (10 μM) in presence of either H89 or vehicle control (DMSO) for 2 minutes at 37°C and then exposed for 2 additional minutes to soluble P-selectin. Immunoblot was first performed with an antibody that recognizes proteins phosphorylated on serines or threonines within the PKA consensus motif sequence K/RK/RXS/T followed by an anti-Csk antibody (Panel A). Alternatively samples were run in duplicates and immunoblot was performed in parallel with the antibody that recognizes proteins phosphorylated on serines or threonines within the PKA consensus motif sequence K/RK/RXS/T and by the anti-Csk antibody (Panel B). Values are mean ± SEM of 3 independent experiments. * P<0.05 by ANOVA, Dunnett test) versus control (untreated) group.

Figure 3. Neutrophil – platelet co-aggregate formation is attenuated by PDE4 inhibition

Panel A. As described in Methods, BCECF-loaded platelets were activated with thrombin receptor (PAR-1)-activating peptide (TRAP, 25 μM) and then fixed with paraformaldehyde. Neutrophils were treated with increasing concentrations of rolipram (PDE4 inhibitor), RO2017-24 (PDE4 inhibitor), zardaverine (PDE3 and 4 inhibitor), metoxyquinazoline (PDE5 inhibitor), cilostamide (PDE3 inhibitor) or vehicle for 2 minutes at 37°C. Platelets and neutrophils were incubated (10:1 ratio) and stirred at 1000 rpm for 2 minutes. Co-aggregate formation was detected by flow cytometry. The results are displayed as percent of neutrophils with attached platelets (mean ± SEM, from 5-6 independent experiments). * P<0.05 by ANOVA, Dunnett test versus control (DMSO) treatment. Panel B. Neutrophils were treated with rolipram (10 μM in the absence or in the presence of increasing concentrations of H89 (PKA inhibitor) before coincubation with platelets. The results are mean ±SEM of 3 independent experiments. * P<0.05 by ANOVA, Dunnett test versus control (H89-untreated) group.

Figure 4. Firm adhesion of neutrophils to adherent platelets under shear flow is attenuated by PDE4 inhibition

Panel A. Schematic representation of the experimental procedure. A parallel flow chamber was used to perfuse neutrophils (5×10⁶/ml) over a platelet surface at a shear stress of 2 dynes/cm² for 2 minutes. This was followed by perfusion of media for 2 min at 20 dynes/cm². The number of attached neutrophils was determined in the last 20 seconds of perfusion. Panel B. Neutrophils were pretreated for 2 minutes at 37°C with DMSO or 5-10 μM of rolipram (PDE4 inhibitor), zardaverine (PDE3 and 4 inhibitor), or cilostamide (PDE3 inhibitor). The absolute number of neutrophils remaining firm adherent were analysed in the last 20 seconds of perfusion at shear stress of 20 dynes/cm². Data are reported as percentage of control (DMSO-treated samples). Bars in the left panel are mean ± SEM from 5 independent experiments. *P<0.05 by ANOVA, Dunnett test versus DMSO-treatment. Panel C. Neutrophils were pretreated for 2 minutes at 37°C with DMSO, rolipram alone or rolipram in combination combined with H89 (both 10 μM). The absolute number of neutrophils rolling or firm adhered was analyzed in the last 20 seconds of perfusion at shear stress of 2 dynes/cm². Graphs are mean ± SEM of 3 independent experiments. *P<0.05 by ANOVA, Dunnett test versus DMSO-treatment.

Figure 5. The effect of rolipram on neutrophil recruitment to adherent platelets is mediated by inhibition of Mac-1

Bone marrow neutrophils and blood platelets were isolated from WT, Mac-1-/-or P-selectin-/- mice (N = 5). 5×10⁶ neutrophils/ml were perfused over a platelet surface at a shear stress of 2 dynes/cm² for 2 minutes. This was followed by perfusion of media for 2 min at 20 dynes/cm². Panel A. WT or Mac-1-deficient neutrophils were perfused over WT or P-selectin-/-platelets. Graphs display the total number of neutrophils interacting with the platelet surface 2 minutes after perfusion at 2 dynes/cm². Panel B. WT or Mac-1-/- neutrophils were treated with rolipram (10 μM). The number of neutrophils remaining firm adhered at shear stress of 20 dynes/cm² are displayed. Panel C. Human neutrophils were treated with rolipram (1 μM) or vehicle for 2 minutes at 37°C and stimulated with soluble P-selectin (10 μg/ml) at 37°C without stirring for 10 minutes in the presence of isotype matched PE antibody or PE-conjugated CBRM1/5, which specifically recognizes epitopes of the integrin molecule that are exposed only after activation dependent conformational changes. After stimulation neutrophils were fixed in 2% PFA, for 30 minutes, washed and analyzed by flow cytometry. Histograms are from one representative experiment. The bar graph depicts the increase in CBRM1/5 binding (mean fluorescent intensity; mean ± SEM of 3 independent experiments) over that observed in resting neutrophils .

Figure 6. Rolipram reduces neutrophil recruitment at the site of vascular injury

1 hour before injury animals were i.p. injected with either rolipram (3mg/kg) or vehicle. Injury of femoral arteries was performed in rolipram- (n=11) and vehicle-treated (n=10) mice as described in Methods. Representative cross sections (stained with hematoxylin and eosin) of femoral arteries from either vehicle or rolipram-treated mice at 1 hour after injury are shown. Bars represent the average number of leukocytes accumulated along the injured vessel (mean ±SEM). *P<0.05 by Student T test.