Anti-Apoptotic Effect, Mediated by Elevated Intracellular Cyclic AMP Levels, in Neutrophils of Sickle Cell Disease Patients.

Abstract

Sickle cell disease (SCD) is now widely regarded as a chronic inflammatory condition, characterized by high base-line leukocyte counts, alterations in inflammatory cytokine levels, vascular endothelial injury and increased red and white cell adhesiveness. We hypothesized that this inflammatory state may be exacerbated by alterations in the apoptotic process in inflammatory cells, such as neutrophils. Neutrophils were isolated from the peripheral blood of healthy controls and individuals with SCD in steady state by separation over a ficoll-paque gradient. Following washing and lysis of contaminating red cells, neutrophils were cultured in DMEM supplemented with 10% (v/v) autologous serum and antibiotics for 20 h (4 x 106 cells/ml, 37oC, 5% CO2). Apoptosis was assessed by flow cytometry using a fluorescein isocyanate-labeled recombinant annexin V antibody and propidium iodide staining. Morphological analysis confirmed the apoptotic state of cells. After 20 h in culture, the percentage of non-apoptotic neutrophils from SCD patients (SCD neutrophils; 13.34 ± 1.03 %, n=8) was significantly higher than the percentage of non-apoptotic normal neutrophils (7.68 ± 1.59, n=7; P<0.01). Apoptosis is mediated by a number of signaling pathways, with the cAMP-dependent pathway being known to have an important anti-apoptotic role in neutrophils. Accordingly, measurement of cAMP in isolated normal and SCD neutrophils by ELISA demonstrated levels of cAMP to be significantly increased in the neutrophils of SCD individuals (4.55 ± 0.38 pMol/1x 106 cells compared to 2.18 ± 0.39 pMol/1x 106 cells in normal individuals, n>14, P<0.001). Co-incubation of SCD neutrophils during 20h-culture with a cAMP-dependent protein kinase (PKA) inhibitor, KT5720 (3μM), significantly decreased the percentage of non-apoptotic cells (12.88 ± 1.49 %, decreased to 6.18 ± 0.51 %, n=6; p=0.01) to levels similar to those seen in normal neutrophil cultures, indicating that the anti-apoptotic effect seen in SCD neutrophils is probably mediated by a cAMP-PKA dependent mechanism. Interestingly, when SCD neutrophils were cultured in medium containing fetal calf serum instead of autologous serum, the number of non-apoptotic cells at 20 h of culture was not different to the number of normal non-apoptotic neutrophils (8.91 ± 2.55 % compared to 9.08 ± 2.11 %, respectively, n= 7, P>0.05). This finding may indicate that this anti-apoptotic effect may be maintained by survival factors contained in serum; indeed incubation of normal neutrophils with SCD patient serum (10 % v/v, 30 min, 37oC), but not control serum, significantly augmented intracellular cAMP levels by 117.9 ± 10.5 % (n=8, p<0.01). In conclusion, we have observed an increased survival of neutrophils from SCD individuals under culture conditions, probably mediated by an up-regulated cAMP-PKA pathway and possibly dependent on cAMP-elevating survival factors contained in the serum. Since neutrophil apoptosis is imperative for the resolution of inflammation, increased neutrophil survival may be an important contributing factor to the chronic inflammatory state that characterizes SCD.