Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin

Abstract

Background: Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis.

Design and methods: Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays.

Results: Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils.

Conclusions: The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.

Figures

Figure 1.

Adhesion of control and SCD neutrophils to non-stimulated (basal) and TNF-α-stimulated HUVEC. Neutrophils (2x106 cells/mL) from control (n=13) or SCD patients (n=16) were allowed to adhere to HUVEC for 30 min at 37°C, 5% CO2. Results are expressed as percentage of cells adhered (median and range). ***P<0.001, TNF-α-stimulated HUVEC compared to basal; Wilcoxon’s matched pairs test. #P<0.05, Median values differ significantly for SCD and to control cells; Mann-Whitney test.

Figure 2.

Neutrophil adhesion to HUVEC in the presence of integrin-specific blocking monoclonal antibodies. Neutrophils (2x106 cells/mL) from controls (A, n=6) or from SCD individuals (B, n=8) were allowed to adhere to HUVEC for 30 min at 37°C, 5% CO2, in the presence or absence of integrin-blocking monoclonal antibody, as indicated. *P<0.05; **P<0.01; ***P<0.001, compared to basal adhesion; Friedman’s test, followed by Dunn’s multiple comparison between selected groups and basal adhesion.

Figure 3.

Neutrophil adhesion to TNF-α-stimulated HUVEC in the presence of integrin-specific blocking monoclonal antibody. Neutrophils (2x106 cell/mL) from control individuals (A, n=4) or from SCD patients (B, n=5) were allowed to adhere to TNF-α-stimulated HUVEC for 30 min at 37°C, 5% CO2, in the presence or absence of integrin-blocking monoclonal antibody, as indicated. *P<0.05; **P<0.01; ***P<0.001, compared to basal adhesion; Friedman’s test, followed by Dunn’s multiple comparison between selected groups and basal adhesion.

Figure 4.

Adhesion of control (A) and SCD (B) neutrophils to HUVEC in the presence of simvastatin. Neutrophils from control individuals (n=8) or SCD patients (n=11) were allowed to adhere to HUVEC or to TNF-α-stimulated HUVEC cells in the presence or not of 1 μM simvastatin (SIM) or 1 μM simvastatin and 1 mM L-NAME (SIM+L-N, n=6). ***P<0.001, TNF-α compared to basal adhesion; ••P<0.01 compared to TNF-α alone; ≈≈P<0.01 compared to TNF/SIM; Friedman’s test, followed by Dunn’s multiple comparison.

Figure 5.

Effect of simvastatin on TNF-α-stimulated expression of ICAM-1 (A) and VCAM-1 (B) on HUVEC. HUVEC were stimulated, or not, with TNF-α (10 ng/mL; for 3 h) following their pre-incubation (or not) with 1 μM simvastatin (Sim). Surface ICAM-1 and VCAM-1 expression was determined by flow cytometry using an anti-CD54 phycoerythrin antibody and anti-CD106 fluorescein isothiocyanate, respectively. Data are expressed as mean MFI±SEM (n=4) for ICAM-1 (A) and % positive cells ± SEM (n=4) for VCAM-1 (B). *P<0.05; **P<0.01, ***P<0.001, compared to basal adhesion. ##P<0.01 compared to TNF-α alone. Repeated measures analysis, followed by Bonferroni’s test.

Figure 6.

Effect of simvastatin on spontaneous and IL8-stimulated neutrophil chemotaxis. Spontaneous chemotaxis (Spont) and IL-8 (100 ng/mL)-stimulated chemotaxis of neutrophils from control (n=6) and SCD individuals (n=6) was measured following the pre-incubation (or not) of neutrophils with simvastatin (SIM) (1 μM). ***P<0.001, compared with spontaneous chemotaxis; #P<0.05 compared with IL-8-stimulated control neutrophil chemotaxis; +P<0.05, compared with IL-8-stimulated SCD neutrophil chemotaxis; Friedman’s test, followed by Dunn’s multiple comparison.