Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2020 May;107(5):809-818.
doi: 10.1002/JLB.5HR0120-459R. Epub 2020 Mar 14.

On the origin of low-density neutrophils

Marwan Hassani  1 , Pien Hellebrekers  1   2 , Na Chen  1 , Corneli van Aalst  1 , Suus Bongers  1   2 , Falco Hietbrink  2 , Leo Koenderman  1 , Nienke Vrisekoop  1
Affiliations
Review

On the origin of low-density neutrophils

Marwan Hassani et al. J Leukoc Biol. 2020 May.

Abstract

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-formyl-methionyl-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.

Keywords: LDG; granulocytes; neutrophil subsets.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Unstimulated neutrophils display a spectrum of densities. Neutrophils were first isolated using Ficoll density centrifugation. Thereafter isolated neutrophils were centrifuged on top of Percoll with different densities. (A) Depicted is the percentage of total neutrophils in the “PBMC layer” after density centrifugation with different densities of Percoll (n = 9). (B) The lag time in which higher density neutrophils (HDNs) and lower density neutrophils (LDNs) contain growth of GFP labeled S. Aureus (n = 7). (C) The percentage of the original population of lymphocytes that divided after stimulation in the absence of neutrophils, in the presence of all neutrophils and in the presence of either LDN or HDN of the same donor (n = 7). (D) Division index (average number of cell divisions that a cell in the original population has undergone) of lymphocytes under the same conditions as (C). (E) Proliferation index (the total number of divisions divided by the number of cells that went into division) of lymphocytes under the same conditions as (C). For all graphs median ± IQR% is shown. Data are analyzed using Friedman test without correction for multiple comparisons. (C‐E) all conditions were tested, but only statistically significant results are indicated
FIGURE 2
FIGURE 2
Density shift of neutrophils after stimulation. Depicted is the percentage of neutrophils in the PBMC fraction after stimulation of neutrophils with (A). N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF) or (B) platelet activating factor (PAF) (C‐G) Median fluorescence intensity of activation markers on unstimulated neutrophils and lower density neutrophils (LDNs) and higher density neutrophils (HDNs) after 1 nM fMLF stimulation (n = 8). (C) CD66b, (D) CD11b, (E) CD35, (F) CD62L, and (G) CD16. (H) FLUO‐4 signal for gated neutrophils is shown before and after adding 1 nM fMLF. Data are depicted as median with IQR. Data were analyzed using Friedman test without correction for multiple comparison. (A‐B) all conditions were tested, but only statistically significant results are indicated
FIGURE 3
FIGURE 3
CD16dim neutrophils (banded nucleus) are more abundant in the lower density neutrophil (LDN) fraction. (A) Depicted is the percentage of CD16dim neutrophils of healthy donors in all neutrophils, LDN and higher density neutrophils (HDN) (n = 6). (B) Presented is the percentage of the total loaded neutrophils in the PBMC (LDNs) or PMN (HDNs) layer. The dashed lines depict the normal unstimulated situation (n = 9, also see Fig. 1A), whereas the solid lines represent the neutrophils of different healthy volunteers after an in vivo LPS challenge (n = 8). (C) Percentage of CD16dim neutrophils in the LDN and HDN fraction and for all neutrophils after LPS challenge (n = 9). Data are depicted as median with IQR. Data of (A) and (C) were analyzed using Friedman test without correction for multiple comparison. Data of (B) was analyzed using a Mann‐Whitney test
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ng LG, Ostuni R, Hidalgo A. Heterogeneity of neutrophils. Nat Rev Immunol. 2019;19:255‐265. - PubMed
    1. Hacbarth E, Kajdacsy‐Balla A. Low density neutrophils in patients with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic fever. Arthritis Rheum. 1986;29:1334‐1342. - PubMed
    1. Scapini P, Marini O, Tecchio C, et al. Human neutrophils in the saga of cellular heterogeneity: insights and open questions. Immunol Rev. 2016;273:48‐60. - PubMed
    1. Pember SO, Barnes KC, Brandt SJ, et al. Density heterogeneity of neutrophilic polymorphonuclear leukocytes: gradient fractionation and relationship to chemotactic stimulation. Blood. 1983;61:1105‐1115. - PubMed
    1. Pember SO, Kinkade JM. Differences in myeloperoxidase activity from neutrophilic polymorphonuclear leukocytes of differing density: relationship to selective exocytosis of distinct forms of the enzyme. Blood. 1983;61:1116‐1124. - PubMed