Altered Functional Properties of Eosinophils In Sickle Cell Anemia and Effects of Hydroxyurea Therapy

Abstract 2656

The pathophysiology of sickle cell anemia (SCA) is now recognized to include a chronic inflammatory condition that contributes to the recurrent episodes of vaso-occlusion that characterize this disease. The vaso-occlusive process is a multi-step process that involves different cell types, such as red blood cells, reticulocytes, activated endothelial cells, platelets and leukocytes. In vivo transgenic animal studies indicated that the neutrophil appears to be the most important leukocyte in vaso-occlusion in previous transgenic animal in vivo studies; however, eosinophils (EOs) may also participate in this phenomenon in humans. EOs are found in significantly elevated numbers in sickle cell disease individuals and, in static adhesion assays, demonstrate augmented adhesive properties suggesting an activated state of these cells. However, the role of EOs in the pathophysiology of SCD and the effect of hydroxyurea (HU) therapy on the functional properties of these cells are not well understood. The aim of this study was to evaluate EO adhesion, migration and degranulation in SCA patients and the effect of HU therapy on these cells. EOs were purified from the peripheral blood of healthy controls (CON) (n≥10), steady-state SCA patients (n≥12) and SCA patients on HU therapy (n≥15), using Percoll gradient separation, followed by immunomagnetic sorting. EO adhesion was determined by static adhesion assays and degranulation was evaluated by measurement of eosinophil peroxidase activity. Migration of EOs was determined in 96-multiwell chemotaxis chamber assays. Plasma levels of Eotaxin-1, 2 and RANTES, important eosinophil attracting chemokines, were determined by ELISA. The absolute number of EOs in the peripheral blood of SCD patients not treated with HU was significantly higher compared to control individuals (0.504±0.09 vs 0.188±0.04; P=0.01, respectively). SCA patients taking HU presented significantly lower numbers of EOs (0.200±0.05, P = 0.01). Basal adhesion of EOs from SCA patients was significantly higher than in healthy controls (22.8±2.6 vs 13.7±1.7%, respectively, P = 0.004). Furthermore, the EOs from SCA patients on HU therapy demonstrated a significantly lower adhesion to fibronectin (12.5±2.0%, P=0.002). Spontaneous EO chemotaxis was significantly increased in SCA patients, compared to healthy controls (12.9±1.56 vs 6.97±0.8 × 10⁵/ml, respectively, P=0.009). Interestingly, the chemotatic response of EOs isolated from SCA patients taking HU was also higher than that of control EOs (12.1± 1.9 × 10⁵/ml). EO chemotaxis in response to RANTES, Eotaxin and IL5 was significantly augmented in all groups compared to spontaneous chemotaxis (RANTES: CON: 15.3± 2.3; SCA: 19.9±3.0; SCAHU: 25.0±4.7; Eotaxin: CON: 18.7±3.0; SCA: 24.5±4.2; SCAHU: 24.0±5.2; IL5: CON: 13.4±2.4; SCA: 18.8±3.0, SCAHU: 16.6±4.5, P<0.05). Baseline eosinophil peroxidase release was higher in SCA EOs, compared to CON EOs (0.4±0.04 vs 0.2±0.02, respectively, P=0.001), but patients on HU presented a lower EOs degranulation than SCA not on HU (0.19±0.03, P=0.003). Plasma levels of Eotaxin-1 and RANTES were significantly higher in SCA individuals, compared to CON individuals (124±7; 1057±125 vs 78±7; 540±50.2 pg/ml, P=0.0002; P=0.0009, respectively). HU therapy was not associated with any change in Eotaxin-1 and RANTES plasma levels (117.9±9.7; 1175±126.2 pg/ml, respectively). Eotaxin-2 plasma levels did not vary among groups (485±80.8, 450±34, 398±47.8 pg/ml for CON, SCA and SCAHU, respectively). EOs of patients with SCA demonstrate a higher capacity for spontaneous migration, stimulated migration and degranulation. Therapy with HU is associated with reduced adhesion and degranulation of EOs in these patients, but had no effect on the chemotactic ability of these cells or chemokine levels. The presence of relative eosinophilia along with altered functional properties in SCA patients warrants further investigation of the role of EOs in the vaso-occlusive process. In particular, complications associated with allergic reactions, such as acute thoracic syndrome and asthma in SCA children, in which EO adhesion and degranulation may be more important, should be studied. The characteristics of the effect of HU on EO also suggest that SCA events in which EO chemotaxis plays a role are likely to respond poorly to HU therapy, and should lead to the development of alternative therapeutic strategies.