Inhibition of Increased Sickle Neutrophil Adhesion to Fibronectin and ICAM-1 by a Nitric Oxide Donor.

Abstract

Leukocytes play an important role in sickle cell disease (SCD); increased numbers of leukocytes are associated with increased SCD morbidity and mortality and leukocytes participate significantly in the vaso-occlusive process. Nitric oxide (NO), a signaling gas involved in vaso-relaxation and platelet aggregation regulation, has recently been implicated as important in SCD pathophysiology. NO plasma bioavailability is thought to be decreased in SCD, and NO therapy has been proposed for SCD and vaso-occlusive crises treatment. The possible consequences of decreased NO bioavailability in SCD are innumerous and have yet to be fully explored. As NO is an important inflammatory mediator and may be important for the inhibition of leukocyte adhesion and migration mechanisms, we compared nitric oxide metabolites and cGMP levels (second messenger for NO) in neutrophils from normal subjects and SCD patients (SCD neutrophils) and then compared to the adhesive properties of these cells and observed the effect of NO donating agents on this adhesion. Neutrophils were isolated from whole blood by separating on a Ficoll gradient. NO metabolites/cGMP level were measured in neutrophil extracts using specific assays. Levels of nitric oxide metabolites in SCD neutrophils did not differ signicantly from those in normal neutrophils (17.34 ± 7.02%, n=10; 16.30 ± 7.00%, n=8; respectively). Furthermore, levels of cGMP (0.11 ± 0.02 pMol/1x107 neutrophils, n=9) in SCD neutrophils were not significantly different to those observed in normal neutrophils (0.142 ± 0.036 pMol/1x107 neutrophils, n=7; p>0.05). Cell adhesion to fibronectin and ICAM-1 was compared using static adhesion assays. Neutrophils from SCD patients demonstrated a significantly greater adhesion to FN-coated plates (FN= 20mg/ml) than normal neutrophils (18.73 ± 6.24%, n=18; 13.52 ± 5.15%, respectively, n=15; p=0.016). Similarly, neutrophils from SCD patients demonstrated significantly higher adhesion to ICAM-1 (10mg/ml) when compared to normal neutrophils (19.51 ± 9.02%, n=13; 10.98 ± 3.95%, respectively n=9; p=0.025). Co-incubation of SCD neutrophils with a nitric oxide donor, sodium nitroprusside (SNP,10mM) abolished their increased adhesion to both FN and ICAM-1 to levels similar to those of normal neutrophil adhesion (16.53 ± 2.27 %, reduced to 12.61 ± 1.98%, n=8; p= 0.002 for fibronectin and 19.12 ± 3.00% reduced to 16.49 ± 2.60, n=8; p=0.009 for ICAM-1). In contrast, SNP did not affect normal neutrophil adhesion to either FN (11.59 ± 1.02% and 12.67 ± 1.35%, following incubation n=11; p>0.05) or ICAM-1 (12.33 ± 0.25% to 11.95 ± 0.62%, n= 5; p> 0.05) significantly. Although NO bioavailability may be decreased in the plasma of SCD patients due to NO sequestration by cell-free hemoglobin, our data indicate that NO dynamics are not significantly altered in the leukocytes of patients in SCD, as indicated by levels of NO metabolites and the major second messenger for NO, cGMP, are not significantly different in sickle cell neutrophils. We speculate that increased neutrophil adhesive properties in SCD may not be the consequence of decreased NO bioavailability, rather, other factors such as altered cytokine levels may mediate increased cell adhesion. However, whilst increased adhesion appears not to be mediated by NO-dependent mechanisms, an NO donor significantly reduced increased SCD neutrophil adhesion to both FN and ICAM-1, indicating that NO and NO donating drugs may also benefit SCD patients by reducing increased leukocyte adhesion, a mechanism important for the vaso-occlusive process.