Sickle Red Blood Cell Induced Adhesion of Neutrophils to Endothelial Cells and Biologic Correlates of Leukocyte Activation

In sickle cell disease (SCD), any event that slows the passage of red blood cells (RBCs) in the microcirculation can promote hemoglobin polymerization, red cell sickling, and vaso-occlusion. Homozygous (SS) RBCs have a sticky surface and, compared to normal (AA) RBCs, attach readily to both endothelial cells and other blood cells. Furthermore, the adhesive function of SS RBCs is up-regulated by the stress hormone epinephrine, resulting in significant vaso-occlusion and increased mononuclear leukocyte adhesion to the vascular endothelium in a mouse model. We therefore postulated that direct interaction between SS RBCs and neutrophils may also lead to activation and adhesion of neutrophils to endothelial cells. In order to explore whether SS RBCs can induce adhesion of neutrophils to endothelial cells independent of exogenous cytokine stimulation, neutrophils were isolated from healthy donors using density gradient media and then labeled with fluorescent dye. Fluorescent neutrophils were then co-incubated with epinephrine-treated SS or sham-treated (untreated) SS, AA or no added RBCs (control) at 37°C. Graduated height flow chambers were used to quantify adhesion of neutrophils to human umbilical vein endothelial cells (HUVECs) grown on glass slides coated with gelatin. We then also examined markers of hematologic status and endothelial activation to determine if any of these were correlated with the ability of SS RBCs to induce neutrophil adhesion. Enzyme linked immunosorbent assays (ELISAs) were used to quantify inflammatory markers (IL-1B, IL-6, IL-8), soluble L selectin (sL-SEL) and soluble P selectin (sP-SEL) in the plasma of the SS patient samples used in adhesion assays. We found that neither sham-treated nor epi-treated AA RBCs (n=3) significantly increased neutrophil adhesion to HUVECs, while both sham-treated and epinephrine-stimulated (epi) SS RBCs significantly increased neutrophil adhesion to endothelial cells (n=17, 42.0% adhesion for epi-SS RBCs vs 20.0% for no RBCs and 27.2% for untreated SS RBCs, with p values as shown in Figure 1). ELISA immunoassays confirmed elevated levels of sP-SEL and sL-SEL in SS patients, and levels of sL-SEL correlated with total leukocyte count (p=0.005). However, neither these biomarkers nor leukocyte count correlated with the ability of patient RBCs to stimulate normal leukocyte adhesion. Levels of IL-1B, IL-6 and IL-8 also did not correlate with the ability of patients' RBCs to stimulate neutrophil adhesion. However, we also found that HbF levels correlated inversely with sL-SEL levels (P=0.014), suggesting that HbF not only helps prevent RBC sickling but also reduces leukocyte activation. Other hematologic markers (Hb level, platelet count, reticulocyte count, LDH) also did not correlate with the ability of epi-SS RBCs to induce neutrophil adhesion. Therefore, we conclude that SS RBCs are able to stimulate neutrophil adhesion, especially under conditions that activate the adhesion receptors responsible for RBC-neutrophil interaction. In addition, we found that sL-SEL was increased in the presence of higher WBC counts, while it was inversely related to HbF levels. Taken together, these data suggest that the effect of HbF on SS RBCs reduces leukocyte activation, and this may occur in part through reduction of SS RBC-leukocyte interaction.