Increased leukocyte adhesion to vascular endothelium contributes to vaso-occlusion in sickle cell disease. Since nitric oxide bioavailability is decreased in sickle cell disease and nitric oxide may inhibit leukocyte adhesion, we investigated whether stimulation of NO-signaling pathways can reduce the adhesive properties of neutrophils from sickle cell disease individuals (sickle cell diseaseneu). sickle cell diseaseneu presented greater adhesion in vitro to both fibronectin and ICAM-1 than control neutrophils. Co-incubation of sickle cell diseaseneu with the nitric oxide-donor agents, sodium nitroprusside and dietheylamine NONOate (DEANO), and the guanylate cyclase stimulator, BAY41-2272, all significantly reduced the increased adhesion to fibronectin/ICAM-1. Oxadiazolo[4,3-a]quinoxalin-1-one, a guanylate cyclase inhibitor, reversed sodium nitroprusside/DEANO-diminished adhesion to fibronectin, implicating cGMP-dependent signaling in this mechanism. Interestingly, intracellular cGMP was significantly higher in neutrophils from sickle cell disease individuals on hydroxyurea (sickle cell diseaseHUneu). Accordingly, sickle cell diseaseHUneu adhesion to fibronectin/ICAM-1 was significantly lower than that of sickle cell diseaseneu. Agents that stimulate the nitric oxide/cGMP-dependent pathway may have beneficial effects on leukocyte function if used in these subjects.

A role for leukocytes in sickle cell vaso-occlusive crisis is becoming increasingly recognized. Neutrophil counts are higher in sickle cell patients and neutrophils from these patients demonstrate increased adhesion to endothelial monolayers under certain circumstances. The effects of selected cytokines on the adhesion mechanisms of normal neutrophils and neutrophils from sickle cell anaemia patients (SCA neutrophils) were investigated. Neutrophils were separated from the blood of homozygous (HbSS) SCA patients and healthy controls. Following pre-incubation (25 min, 37°C) of the cells with cytokines, the adhesion of the cells to fibronectin (FN)-coated plates (20 µg/ml) was determined (60 min, 37°C, 5% CO₂). Basal adhesion of normal and SCA neutrophils to FN was not statistically different. Pretreatment of normal neutrophils with either IL-6 (10–100 pg/ml), GCSF (1– 10 ng/ml) or IL-8 (1–100 ng/ml) had no significant effect upon their adhesion to FN. In contrast, SCA neutrophil adhesion to FN was increased significantly following pre-incubation with IL-6, G-CSF and IL-8 (p < 0.01). RANTES (1–100 ng/ml) had no significant effect on either normal or SCA neutrophil adhesion to FN. Flow-cytometric analyses demonstrated that IL-8 (10 ng/ml) significantly augments CD11b (Mac-1 integrin subunit) expression on SCA neutrophils, but not normal neutrophils. IL-6 and G-CSF (10 pg/ml and 10 ng/ml, respectively), however, had no effect on SCA neutrophil adhesion molecule expression. In conclusion, SCA neutrophil adhesion mechanisms may increase in the presence of certain cytokines, in vivo, and this activation may contribute to the physiopathology of sickle cell disease.

Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.

Summary Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP‐dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti‐inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue‐specific elevation of cGMP, we studied the gene expressions of cGMP‐specific PDEs (−1A, −5A and −9A) in the reticulocytes and neutrophils of healthy controls, steady‐state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34+‐derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73‐6691, significantly increased production of the γ‐globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.

The significance of the leukocyte in sickle cell disease (SCD) pathophysiology is becoming increasingly recognised; we sought to examine whether the chemotactic properties of neutrophils of SCD individuals may be altered and, further, to better understand the signalling events that mediate altered SCD neutrophil function. Adhesion to immobilised fibronectin (FN) and chemotaxis of control and SCD neutrophils were assessed using in vitro static adhesion assays and 96-well chemotaxis chamber assays. Adhesion assays confirmed a significantly higher basal adhesion of SCD neutrophils to FN, compared with control neutrophils. Chemotaxis assays established, for the first time, that SCD neutrophils demonstrate greater spontaneous migration and, also, augmented migration in response to IL-8, when compared with control neutrophils. Co-incubation of SCD neutrophils with KT5720 (an inhibitor of PKA) abrogated increased basal SCD neutrophil adhesion, spontaneous chemotaxis and IL-8-stimulated chemotaxis. Stimulation of SCD neutrophils with IL-8 also significantly augmented SCD neutrophil adhesion to FN with a concomitant increase in cAMP levels and this increase in adhesion was abolished by KT5720. Interestingly, the adhesive properties of neutrophils from SCD individuals on hydroxyurea therapy were not significantly altered and results indicate that a reduction in intracellular cAMP may contribute to lower the adhesive properties of these cells. Data indicate that up-regulated cAMP signalling plays a significant role in the altered adhesive and migratory properties in SCD neutrophils. Such alterations may have important implications for the pathophysiology of the disease and the cAMP-PKA pathway may represent a therapeutic target for the abrogation of altered leukocyte function.