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 normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and 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.
Highlights
Heterogeneity is a characteristic of human neutrophil populations (1), yet whether this represents truly diverse neutrophil subsets, plasticity during the immune response or differences in maturation state is undetermined
The median number of Low density neutrophils (LDNs) identified by flow cytometry following traditional Percoll gradient of 12mls blood (N=5) was 794,000 which accounted for 2.95% of total neutrophils isolated on average (Figure 1F)
The median number of LDNs isolated by the new protocol from (N=10) healthy donors was 630,000 from 12 mls whole blood, which accounted for 3.62% of total neutrophils isolated on average (Figure 1G)
Summary
Heterogeneity is a characteristic of human neutrophil populations (1), yet whether this represents truly diverse neutrophil subsets, plasticity during the immune response or differences in maturation state is undetermined. LDNs have a similar density to peripheral blood mononuclear cells (PBMCs) and are isolated alongside these cells, in contrast to normal density neutrophils (NDNs) which segregate with other polymorphonuclear (PMN) cells during density exclusion (3, 4). LDNs have been described in pregnancy (5), autoimmune disease (6, 7), cancer (8, 9), infection (10– 13) and inflammation (14, 15), and speculated to contribute to pathophysiology. Across these studies, LDNs do not represent one discrete neutrophil sub-population, but rather a spectrum of multiple. Isolating LDN populations by density exclusion produces mixed populations of cells, and the lack of consistent LDN markers means that cell sorting techniques from whole blood are unsuitable
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