Leukocyte Apoptosis and Inflammation in Iron-Overloaded Patients with Sickle Cell Disease or β-Thalassemia: A Mechanism for Increased Stroke and Disease Severity in Sickle Cell Disease.
Authors: Walter Patrick B. , Killilea David W. , Fung Ellen B. , Lui Annie , Madden Jacqueline , Porter John , Evans Patricia , Ames Bruce N. , Vichinsky Elliott , Harmatz Paul 
Published: April 2020

Abstract
Sickle cell disease (SCD) is a hemoglobinopathy characterized by micro-vascular hypoxia-reperfusion, inflammation and leukocytosis. Studies in SCD have shown that leukocytosis is a strong predictor of stroke and disease severity. It is known that leukocytosis and inflammation contribute to increased leukocyte-endothelial adhesion and vasoocclusive events. Leukocytosis or increased leukocyte number is determined by the balance between cell death programs (apoptosis) and proliferation. In this study, we examine markers of apoptosis and proliferation in SCD as compared to thalassemia (a hemoglobinopathy that is vasculitis negative) and a control group. Methods: Markers of leukocyte turnover, inflammation and free iron (NTBI, non-transferrin bound iron) were compared in 11 patients with SCD (7M, 13 ± 4 yrs), 18 with thalassemia (7M, 24 ± 9 yrs) and 10 disease-free controls (5M, 27 ± 12 yrs). All SCD and thalassemia patients were healthy and event free in the previous 4 months. Blood was obtained fasting and prior to RBC transfusion; and plasma, serum and cells were separated by centrifugation. The pro-apoptotic markers nucleosome protein (DNA laddering) and Bax (initiator of mitochondrial permeability) and inflammatory marker, high-sensitivity C Reactive Protein (hsCRP) were determined by ELISA. NTBI (DNA damage inducing, pro-apoptotic) was measured by HPLC. Plasma levels of cytokines (anti-apoptotic, proliferation stimulating) were determined by using a multiplex bead-based immunoassay. Plasma lactate dehydrogenase (LDH), a marker of hemolysis, was also measured because hemolysis products have been shown to inhibit apoptosis. Results: Leukocyte number and absolute neutrophil count were 1.8 and 2.2 fold higher in SCD compared to thalassemia (P&lt;0.01). Evidence for decreased apoptosis in SCD included reduced nuleosome protein as compared to thalassemia and reduced Bax as compared to controls. In addition, LDH was significantly increased 1.9 fold in SCD relative to thalassemia (p=0.012). In contrast, NTBI was 2 fold higher in thalassemia than SCD and correlated with nucleosome protein (R=0.45; p=0.013). Finally, high levels of the cytokines IL-6 and IL-10 in SCD relative to thalassemia may be stimulating leukocyte proliferation and survival. These cytokines and hsCRP are also positively correlated with leukocyte number. Conclusions: These preliminary findings demonstrate that the balance of apoptosis vs. proliferation favors delayed cell death and enhanced cell proliferation in SCD, leading to increased circulating leukocytes. These changes may be related to hemolysis, increased levels of inflammatory cytokines, and lower amounts of NTBI in SCD. This leukocytosis increases the potential for vasoocclusive events that contribute to stroke, acute chest syndrome or renal tubule damage.