Objectives Patients with APS are at increased risk of thromboembolism. Neutrophils have been shown to play a role in inducing thrombosis. We aimed to investigate differences in neutrophil subpopulations, their potential of activation and neutrophil extracellular trap (NET) formation comparing high and low-density neutrophils (HDNs/LDNs) as well as subpopulations in patients with APS and controls to gain deeper insight into their potential role in thrombotic manifestations in patients with APS. Methods HDNs and LDNs of 20 patients with APS and 20 healthy donors were isolated by density gradient centrifugation and stimulated. Neutrophil subpopulations, their activation and NET release were assessed by flow cytometry. Results LDNs of both groups showed higher baseline activation, lower response to stimulation (regulation of activation markers CD11b/CD66b), but higher NET formation compared with HDNs. In patients with APS, the absolute number of LDNs was higher compared with controls. HDNs of APS patients showed higher spontaneous activation [%CD11b high: median (interquartile range): 2.78% (0.58–10.24) vs 0.56% (0.19–1.37)] and response to stimulation with ionomycin compared with HDNs of healthy donors [%CD11b high: 98.20 (61.08–99.13) vs 35.50% (13.50–93.85)], whereas no difference was found in LDNs. NET formation was increased in patients’ HDNs upon stimulation. Conclusion HDNs and LDNs act differently, unstimulated and upon various stimulations in both healthy controls and APS patients. Differences in HDNs and LDNs between patients with APS and healthy controls indicate that neutrophils may enhance the risk of thrombosis in these patients and could thus be a target for prevention of thrombosis in APS.

No abstract

Background: Neutrophils are a heterogeneous population of leukocytes, which can be subdivided into high and low density neutrophils (HDNs/LDNs). They are known to fight intruders with different mechanisms, including the release of neutrophil extracellular traps (NETs), which have also been associated with thrombosis. Risks of infection and thrombosis increase with age. Differences in neutrophil subpopulations and functionality have been shown in various disease states, but investigations in healthy subjects and their dependence on age are lacking. The aim of this study was to investigate age-related changes in neutrophils regarding neutrophil subpopulations, their potential of activation, DNA release and NET formation.Methods: Neutrophil subpopulations (HDNs and LDNs) were isolated from 25 healthy individuals subdivided into 3 groups (<45 years, n=8; 45-54, n=9; >54, n=8). Neutrophil characteristics, potential of activation and the ability of NET formation was investigated using flow cytometry. Externalisation of DNA was detected by a DNA release assay.Results: HDN and LDN counts did not differ between age-groups. However, with increasing age we observed a shift in neutrophil subpopulations towards a lower amount of mature LDNs, characterized by their expression of membrane receptors CD62L and CD16. Upon stimulation, neutrophils of older individuals showed significantly higher release of DNA. HDNs of younger participants increased activation markers (CD66b and CD11b) to a higher extent compared to those of older individualsConclusion: Neutrophils and their ability of activation, DNA release and NET formation change with age and this might contribute to the higher risk of infection and thrombosis at advanced age. Furthermore, these results highlight the importance and necessity of age-matching in studies that focus on neutrophil characteristics.

Background: Neutrophils are a heterogeneous population of leukocytes, which can be subdivided into high and low density neutrophils (HDNs/LDNs). They are known to fight intruders with different mechanisms, including the release of neutrophil extracellular traps (NETs), which have also been associated with thrombosis. Risk of both, thrombosis and infections, increase with age. Differences in neutrophil subpopulations and functionality have been shown in various disease states, but investigations in healthy subjects and their dependence on age are lacking. The aim of this study was to investigate age-related changes in neutrophils regarding neutrophil subpopulations, their potential of activation, DNA release and NET formation. Methods: Neutrophil subpopulations (HDNs and LDNs) were isolated from 25 healthy individuals subdivided into 3 groups (<45 years, n=8; 45-54, n=9; >54, n=8). Neutrophil characteristics, potential of activation and the ability of NET formation were investigated using flow cytometry. Externalisation of DNA was detected by a DNA release assay. Results: HDN and LDN counts did not differ between age-groups. However, with increasing age we observed a shift in neutrophil subpopulations towards a lower amount of mature LDNs, characterized by their expression of membrane receptors CD62L and CD16. Upon stimulation, neutrophils of older individuals showed significantly higher release of DNA. NET formation was associated with increasing age. HDNs of younger participants increased activation markers (CD66b and CD11b) to a higher extent compared to those of older individuals. Conclusion: Neutrophils and their ability of activation, DNA release and NET formation change with age and this might contribute to the higher risk of infection and thrombosis at advanced age. Furthermore, these results highlight the importance and necessity of age-matching in studies that focus on neutrophil characteristics.