Here we elaborate on the origin of low(er)-density neutrophils (LDNs) to better understand the variation found in literature. Supplemented with original data, we test the hypothesis that buoyant density of neutrophils is characterized by a spectrum that as a whole shifts to a lower density after activation. Both the 20% highest density (HDNs) and 20% lowest density (LDNs) neutrophils from healthy donors were isolated by Percoll of different densities. Using this method we found that LDNs were significantly better in T-cell suppression and bacterial containment than their 20% highest density counterparts. We found no statistically relevant differences in neutrophil survival or bacterial phagocytosis. Stimulation of healthy donor neutrophils with N-formylmethionyl-leucyl-phenylalanine induced LDNs co-segregating with peripheral blood mononuclear cells after Ficoll separation. These in vitro induced LDNs showed increased activation markers compared to HDNs and were comparable to the activation markers found on the LDN fraction seen in patients with chronic inflammatory conditions such as present in cancer patients. This all fits with the hypothesis that the density of neutrophils is distributed in a spectrum partially coupled to maturation. Additionally a shift in this spectrum can be induced by in vitro stimulation or by activation in disease. K E Y W O R D Sgranulocytes, LDG, neutrophil subsets INTRODUCTIONNeutrophils are main actors in the innate immune system. Until recently neutrophils were thought to belong to a relative homogeneous population of cells. However, an increasing number of studies now show heterogeneity in morphology, phenotype, function, or a combination of these factors. 1 One of the subtypes of neutrophils identified and studied is the low-density neutrophil (LDN). It was first recognized in 1986, when neutrophils co-segregated with mononuclear cells after density-gradient isolation in systemic lupus erythematosus (SLE) and rheumatoid arthritis patients. 2 Thereafter, the presence of LDNs have been shown in several, mostly chronic, diseases. 3

Neutrophils are one of the most important effector cells of the innate immune response (1). They are traditionally seen as a homogenous population of short‐lived cells mainly involved in the defence against extracellular microorganisms by phagocytosis and intracellular killing (1,2). The cells contain a large armamentarium that aids in this function and ranges from the production of reactive oxygen species by a membrane‐bound NADPH oxidase to cytotoxic proteins and peptides residing in the different granules present in the cytoplasm (3). Recently, the view of neutrophils belonging to a homogenous population of cells has been challenged, and several neutrophil phenotypes have been described that exhibit specialized functions, such as involvement in tissue repair, tumour killing and immune regulation (4). It is not clear whether these cells belong to separate parallel lineages originating from the bone marrow or that neutrophils become instructed in the distant tissues, thus changing their phenotypes. In addition, functional heterogeneity in a phenotypically homogenous population of neutrophils adds to the complexity of neutrophil phenotypes(5). This article will review the current literature describing the heterogeneity within the neutrophil compartment with respect to both phenotype and function in health and disease.

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