Hydroxyurea Reverses Increased Adhesive Properties of Neutrophils in Sickle Cell Disease.

Abstract

Neutrophils play an important role in sickle cell disease (SCD) and the vaso-occlusion that characterizes the disorder; having a central and, possibly, initiating role in the vaso-occlusive (VO) process due to their adhesion to both the vascular endothelium and red cells. Hydroxyurea (HU) therapy significantly decreases the neutrophil count in SCD, however, to our knowledge, the effects of HU therapy upon the augmented adhesive properties of these cells has not been investigated. As such, the effects of HU therapy on the adhesion of SCD neutrophils to the endothelial cell adhesion molecule, ICAM-1, were investigated utilizing an in vitro static adhesion assay. Neutrophils were isolated from peripheral blood of healthy controls, SCD individuals in steady state and SCD patients on HU therapy (SCDHU; 20–30 mg/kg/day, 3-month minimum duration) by separation over a ficoll-paque gradient. Separated neutrophils were resuspended in RPMI medium and allowed to adhere to recombinant ICAM-1-coated 96-well plates (5x106cells/ml, 30 min, 37°C, 5% CO2). Adhesion to ICAM-1 was calculated, using a standard curve, as the percentage of the original cell suspension adhered. SCD neutrophil adhesion to ICAM-1 was approx. doubled (17.09±2.99 %; n=10) compared to control neutrophils (8.89±1.10 %; n=9; P<0.05); in contrast, adhesion of neutrophils from SCDHU patients was similar to that of control cells (8.12±2.79 %; n=6; P<0.05 compared to SCD). Nitric oxide (NO) bioavailability is decreased in SCD, and HU may be a NO donor. Thus, we measured levels of the NO second messenger, cyclic guanosine monophosphate (cGMP), in subjects' neutrophils, since cGMP is an inhibitor of cell adhesion in some cell types. Cyclic adenosine monophospate (cAMP) may also be important for activating cellular adhesion, thus neutrophil cAMP was also evaluated. Whilst intracellular cGMP levels were not significantly different in SCD neutrophils compared to controls (0.110±0.022 pMol/107 cells and 0.142±0.036 pMol/107 cells, n≥11, respectively, P>0.05), cGMP levels in SCDHU neutrophils were significantly higher (0.241±0.023 pMol/107 cells, n=9, P<0.01 compared to SCD). In contrast, whilst cAMP levels are significantly increased in SCD neutrophils (4.55 ±0.38 pMol/106 cells and 2.15±0.38 pMol/106 cells, n≥14, SCD and control, respectively, P<0.001), cAMP levels were significantly lower in SCDHU neutrophils than in SCD neutrophils (3.26 ±0.46 pMol/106 cells, n=14, P<0.05). Flow cytometry demonstrated, however, that the surface expressions of the major neutrophil adhesion molecules were unaltered on SCDHU neutrophils compared to SCD neutrophils; mean expression levels of the CD11a, CD11b, CD18, CD49d and CD29 integrin subunits were unaltered (P>0.05, data not shown). Results indicate that the HU-dependent inhibition of neutrophil adhesion does not appear to be due to any change in surface integrin expression, but may be the consequence of alterations in adhesion molecule activity (affinity or avidity). Thus, we demonstrate that HU therapy significantly reduces the ability of neutrophils of SCD neutrophils to adhere to the endothelial adhesion molecule, ICAM-1. Such an inhibition of the augmented adhesive properties of SCD neutrophils may be important for the prevention of VO processes. A concomitant increase in neutrophil cGMP levels was seen following HU therapy, whilst a decrease in neutrophil cAMP levels was observed, indicating that alterations in the intracellular cyclic nucleotide (modulators of cell adhesion) balance may constitute one of the beneficial effects of HU therapy on neutrophil function.