Ultra-pure isolation of low density neutrophils casts doubt on their exceptionality in health and disease

Abstract

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy NDNs, aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in LDNs and normal density neutrophils (NDNs) and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4 + or CD8 + T cell proliferation or IFNγ production compared with NDNs.LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.

Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction

“…These findings were obtained when neutrophils were isolated by dextran sedimentation as well as by negative selection using magnetic beads, indicating that the differences between the neutrophil subpopulations cannot be considered an artifact due to the isolation technique used. 17 Of note, linear regression analysis revealed a strong positive correlation between the percentages of CD16 + CD66b + CD10 neg cells and circulating levels of G-CSF (Figure 4B). AMI patients with higher systemic concentrations of G-CSF have increased CD10 neg neutrophils levels, suggesting G-CSF-driven immature neutrophil release/expansion.…”

Expansion of CD10neg neutrophils and CD14+HLA-DRneg/low monocytes driving proinflammatory responses in patients with acute myocardial infarction

“…These findings were obtained when neutrophils were isolated by dextran sedimentation as well as by negative selection using magnetic beads, indicating that the differences between the neutrophil subpopulations cannot be considered an artifact due to the isolation technique used. 12 Of note, linear regression analysis revealed a strong positive correlation between the percentages of CD16 + CD66b + CD10 neg cells and circulating levels of G-CSF (Figure 4B). AMI patients with higher systemic concentrations of G-CSF have increased CD10 neg neutrophils levels, suggesting G-CSF-driven immature neutrophil release/expansion.…”

Neutrophils in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus: Same Foe Different M.O.

“…LDGs have been identified in many other disease settings, including asthma, vasculitis, multiple sclerosis and chronic kidney disease (287)(288)(289)(290). Indeed, they are even present in low numbers in healthy controls (291). Isolation of LDGs from blood is highly dependent upon the density of the isolation medium used (e.g., Ficoll, Percoll, Polymorphprep) (292) and this raises the question as to whether studies using different isolation protocols for preparation of neutrophils and LDGs from whole blood can be directly compared.…”

“…Isolation of LDGs from blood is highly dependent upon the density of the isolation medium used (e.g., Ficoll, Percoll, Polymorphprep) ( 292 ) and this raises the question as to whether studies using different isolation protocols for preparation of neutrophils and LDGs from whole blood can be directly compared. There are mixed reports on the functionality of LDGs from healthy controls, and whether they have different immunological properties (e.g., T cell suppression) to normal density neutrophils and LDGs from inflammatory disease ( 291 , 292 ). Another key question is whether LDGs represent a novel subset of neutrophils, or whether their phenotype reflects one of spectra of phenotypes that blood neutrophils may exhibit through functional plasticity.…”

“…Administration of LPS in vivo to healthy volunteers appears to support this conclusion, with an increase in immature CD16 dim band cells being observed in blood 3h after LPS challenge ( 292 ). However, it is also possible to induce a low density phenotype from normal density neutrophils in vitro by activation with agents, such as fMLP, platelet activating factor, TNFα and LPS ( 291 , 292 ), suggesting that LDGs may represent a subset of primed or activated neutrophils ( 292 ). Further work needs to be carried out to determine the true origin, phenotype and nature of LDGs and other neutrophil subsets, such as G-MDSCs ( 264 ).…”