Induction of Caspase-Independent Apoptosis by Sickle Cell Disease (SCD) Serum in Non-SCD Leukocytes

Increased white cell counts are associated with augmented mortality and morbidity in sickle cell disease (SCD) and leukocytes play a central role in the vaso-occlusive process of sickle cell disease by adhering to the vascular endothelium and participating in mechanisms of oxidative stress and inflammation. The relevance of neutrophil death to the pathogenesis of inflammatory disease is now recognized, since alterations in the apoptotic processes of leukocytes may affect their cellular function and inflammatory processes. As such, this study investigated the effect of serum from control individuals (CON), SCD patients in steady state (SCD) and SCD patients on hydroxyurea therapy (SCDHU; 20–30 mg/kg/day HU) on the apoptotic processes of neutrophils (Neu). Neu were separated from whole blood of CON individuals using a ficoll-paque gradient; serum was also separated and filtered through 0.22μm ultra-filters. Pools of serum were prepared by mixing serum from 10 individuals from each subject group. Neutrophils (4×10⁶ cells/ml) were cultured in the presence of pooled filtered serum (10% v/v) for 16h (37°C, 5%CO₂) in DMEM. Apoptosis was evaluated by detection of annexin V binding, and viability was determined by MTS assay. CON Neu cultured in the presence of pooled SCD serum demonstrated a significantly higher percentage of apoptotic cells (67.6 ± 4.3%; n=4) compared to Neu cultured with CON serum (52.3 ± 3.6%; n=4; P<0.01 comp. SCD, paired t-test) or SCDHU serum (55.4 ± 4.2%; n=4; P<0.01 comp. SCD). In contrast, Caspase-3 activity was not significantly different in CON Neu following 16h of culture in the presence of CON and SCD serum (2.46±0.13 and 2.50±0.19-fold increased caspase-3 activity at 16h compared to basal activity, respect., n>5; P>0.05); Neu cultured with SCDHU serum demonstrated a higher caspase-3 activity (4.23±0.70-fold increased activity, n=8, P>0.05 Wilcox. matched pairs), but this increase was not statistically different to that of Neu cultured with CON serum. Viability did not differ significantly following 16h-culture of CON Neu with CON, SCD, or SCDHU serum (0.70±0.05; 0.80±0.06; 0.86±0.08 OD_490nm, respect., n>9, P>0.05); furthermore co-incubation of the Neu with serums in the presence of the programmed necrosis inhibitor, Necrostatin (10μM), had no significant effect on cell viability (0.80±0.05; 0.83±0.05; 0.86±0.05 OD_490nm, for CON, SCD and SCDHU respect., n>9, P>0.05), indicating that SCD serum does not appear to induce a process of necroptosis in Neu. Neutrophil death in SCD, like other inflammatory diseases, appears to be regulated by a complex network of both intracellular death and survival signaling pathways that may be modulated by extracellular stimuli. We demonstrate, under the conditions described herein, that filtered SCD serum can induce an apoptotic process in non-SCD neutrophils that does not appear to be mediated by alterations in caspase-3 activity. High levels of circulating TNF-α, a cytokine that has been previously found elevated in the serum of our population of SCD patients, are known to elicit neutrophil death via a caspase-independent mechanism; likewise, intracellular reactive oxygen species (ROS) production also induces caspase-independent neutrophil apoptosis. Interestingly, activity levels of superoxide dismutase (SOD), were found in significantly lower levels in SCD serum compared to CON serum (data not shown), suggesting that SCD serum may have a greater capacity to induce ROS production in neutrophils. Notably, serum from SCDHU individuals did not stimulate increased apoptosis in control neutrophils, indicating a reduction in the serum factor(s) that elicit apoptosis in these cells. As such, Neu may be subject to both anti-and pro-apoptotic stimuli in the circulation of SCD individuals. Since alterations in Neu apoptotic processes may have significant effects on Neu function and number, in turn contributing to inflammatory processes and cellular damage at sites of inflammation, studies to understand the complex balance of these mechanisms in SCD are necessary.