What are neutrophils?

Neutrophils are the dominant phagocytic granulocyte, representing up to 60% of all circulating leukocytes (~20% in mice). Neutrophils phagocytose pathogens in response to pathogen or damage associated molecular patterns (PAMPs or DAMPs). In addition, neutrophils promote the immune response by releasing granules, producing cytokines, and mediating the recruitment of other immune cells to sites of infection. On the other hand, in an exacerbated immune response, neutrophils can cause considerable damage to the host such as extensive cell death, necrosis, vascular leakage, thrombus formation, and antibody-mediated auto-immune responses.

Neutrophil development and life cycle

Neutrophils in the periphery are comparatively short-lived compared to other immune cells, 6–24 h in circulation and possibly up to 7 days in tissue during an inflammatory response. They are sourced from recently identified unipotent neutrophil precursors in the bone marrow (BM) (mouse progenitor Lin−, CD117+, Ly6A/E+, with additional markers: Siglec F-, FcεRIα-, CD16/CD32+, Ly6B+, CD162lo, CD48lo, Ly6C-, CD115-; human: CD66b+, CD117+, CD38+ CD34+/−). Neutrophils go through distinct developmental stages before getting released to circulation: promyelocytes, myelocytes, metamyelocytes, and band neutrophils (Figure 1). Neutrophils released into blood will express high levels of L-selectin (CD62L). By the end of their life cycle or once deactivated from an inflammatory event, neutrophils express CXCR4 (C-X-C chemokine receptor type 4). Increased expression of CXCR4 indicates clearance by the spleen.

Neutrophil migration into inflamed tissue

Neutrophils are in circulation until they encounter signals that initiate their trans-endothelial migration from the bloodstream to the interstitium. L-selectins and PSGL-1 mediate the rolling process along the endothelial layer. Endothelial E-selectin engages the neutrophil PSGL-1. Signaling through PSGL-1 by activated endothelium initiates β2 integrin extension, which slows down rolling, aided by the formation of membrane slings and tethers, resulting in selectin/integrin-mediated adhesion and arrest. Key chemoattractant receptors are CXCR1, as well as formyl peptide receptors 1 and 2 (FPR1, FPR2) and leukotriene B4 receptor BLT1. Note that recruitment to lung and liver seems to be selectin independent.

Neutrophil effector functions

The antimicrobial effector functions of neutrophils comprise degranulation, releasing reactive oxygen species (ROS), phagocytosis, and neutrophil extracellular traps (NET) formation. Degranulation can damage the target cell in a contact dependent manner, in which granules form pores by fusing with the membrane of the target or by exocytosis into the intracellular space. Neutrophil granules are formed in a controlled order during granulopoiesis. In addition to their cytotoxic and proteolytic effects (via e.g. MPO, cathelicidins and in human, α-defensins), their content, including surface proteins and cytokines, can also regulate migration, transmigration (metalloproteinases), phagocytosis, and NET formation. NET formation entails the expulsion of protein decorated neutrophil chromatin to trap and eliminate viruses, bacteria, fungi, and other parasites but has also been described to contribute to coagulation. Receptor activation (e.g. via TLRs) stimulates downstream events of the MEK/ERK pathway such as ROS production, neutrophil elastase release and citrullination of histone H3 that result in NET formation (Figure 2). NETs contain proteins such as elastase and potent anti-microbials as MPO, defensins, and citrullinated histone H3. While NETosis describes DNA release upon induced cell death due to prolonged stimulation, NET formation can be triggered within minutes and permit neutrophils to survive by expelling mitochondrial DNA or persist as a denucleated phagocytic cytoplast.

Neutrophils are widely accepted to possess immune modulatory functions impacting the innate and adaptive arms of the immune system (Figure 2). Neutrophils produce cytokines in response to DAMPs or PAMPs to recruit other immune cells like T cells to the site of inflammation. However, neutrophils can migrate to lymph nodes mediated by CCR7 or CXCR4 where they interact with T cells, either assuming an antigen presenting role via MHCII or MHCI, inducing regulatory T cells in pregnancy or suppress T cell activation (Arg-1, PD-L1 or CCL17 mediated). By secretion of BAFF or APRIL, neutrophils can promote B cell activation, survival, and differentiation. B-cell helper neutrophils in spleen are involved in inducing T-independent secretion of IgG and IgA by B cells.

Table 1. Characteristics of mouse and human neutrophils.

Granule contentBPI, MPO, β-glucuronidase,
lysozyme, alkaline phosphatase, and arginase-1
Defensins, BPI, MPO, β-glucuronidase, lysozyme,
alkaline phosphatase, and arginase-1
Chemokines potentially expressed and/or producedCXCL1, CXCL2/MIP-2a, CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CCL2, CCL3, CCL4, CCL5, CCL7, CCL9, CCL12, CCL17, CCL20, CCL22CXCL1, CXCL2/MIP-2a, CXCL3, CXCL4, CXCL5, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, CXCL13, CXCL16, CCL2, CCL3, CCL4, CCL17, CCL18, CCL19, CCL20
Key surface markersLy-6G (Gr-1)CD66b
FC receptor expressionDo not express FcαRI or FcγRIFcαRI; inducible expression of FcγRI

Neutrophil heterogeneity

The concept of neutrophil heterogeneity has recently raised attention as more in-depth analysis exposed distinct phenotypes that seem to be dependent on neutrophil localization and health condition. It is still unclear whether these differences identify a bona fide subpopulation or merely reflect transitional re-programming. One example is CxCR4+, VEGFR1+, and CD49d+ pro-angiogenic neutrophils, which have been described in mouse and human and are likely to be involved in extracellular matrix (ECM) remodeling.

In addition to emerging subpopulations in steady state, heterogeneity during inflammation and, more recently, cancer has long been accepted. In particular, the occurrence of low-density neutrophils (LDN) is markedly increased in inflammation or cancer. While most neutrophils in density gradient centrifugation of whole blood settle below the Ficoll layer (Normal Density Neutrophils/NDN), LDN sediment in the same fraction as mononuclear cells and comprise a mix of mature and immature neutrophils (Figure 3).  Intriguingly, the effect of these LDN seems to be pro-inflammatory under inflammatory conditions, while in cancer, they seem to exert an immune suppressive function. In mice, a similar immune suppressing population has been identified in tumor models, which seems to be TGF-b induced and was coined N2. In contrast, their counterpart N1 is pro-inflammatory and displays anti-tumorigenic function. However, no markers have been identified to adequately dissect and characterize corresponding populations in human.

Collectively, in contrast to the long held believe of neutrophils being a homogenous population, the data clearly supports considerable heterogeneity and plasticity of neutrophils whether this is due to different activation or maturation stages, environmental imprinting, or bona fide subpopulations.

Isolation of neutrophils

Granulocytes require an inflammatory signal that recruits them to the site of injury, infection, or allergic response to become activated and produce effector function. Flow cytometry can be used to determine the population of activated granulocytes. The method used for isolation of cells heavily influences the nonspecific activation of granulocytes. Human neutrophils are frequently isolated from whole blood by density gradient centrifugation (Percoll or Ficoll) due to easier accessibility of blood versus bone marrow.

For mouse research, BM offers a richer harvest compared to blood, but it has the disadvantage of being a terminal procedure. Alternatively, using eBioscience 1X RBC Lysis Buffer on murine blood on ice and spinning down the cells at 350 x g at 4ºC is an option to analyze or prepare neutrophils for downstream applications if enrichment is not required. This has the advantage of saving time in particular about their short lifespan.

There are notable differences between mouse and human immune cells and neutrophils are not an exception
(see Table 1). A useful resource to plan experiments is public databases such as https://immuneatlas.org or http://www.immgen.org.

Flow cytometry analysis of neutrophils

Under homeostatic conditions, neutrophils are most prevalent circulating in the periphery, making flow cytometry a convenient tool for their characterization.  A list of relevant immunophenotyping markers can be found in Table 2.

Table 2. Non-exclusive list for granulocyte cellular markers

Cell subtypeMarkerLocalizationSpecies
Pan-granulocytesCD11bSurfaceHuman and mouse
NeutrophilsElastaseSecretedHuman and mouse
LactoferrinSecretedHuman and mouse
IL-6SecretedHuman and mouse
IL-12SecretedHuman and mouse
TNF alphaSecretedHuman and mouse
IL-1 alpha/betaSecretedHuman and mouse
CD10SurfaceHuman and mouse
CD17SurfaceHuman and mouse
CD24SurfaceHuman and mouse
CD35SurfaceHuman and mouse
CD43SurfaceHuman and mouse
CD66aSurfaceHuman and mouse
CD66bSurfaceHuman and mouse
CD66dSurfaceHuman and mouse
CD89SurfaceHuman and mouse
CD93SurfaceHuman and mouse
CD112 (Nectin-2)SurfaceHuman and mouse
CD114 (G-CSFR)SurfaceHuman and mouse
CD116SurfaceHuman and mouse
CD157SurfaceHuman and mouse
CD177SurfaceHuman and mouse
CD181 (CXCR1)SurfaceHuman and mouse
CD282 (TLR2)SurfaceHuman and mouse
CD284 (TLR4)SurfaceHuman and mouse
CD286 (TLR6)SurfaceHuman and mouse
Ly-6G (Gr-1)SurfaceKey phenotyping marker: Mouse
Calprotectin (S100A8/A9)SurfaceHuman
CD281 (TLR1)IntracellularHuman and mouse
CD289 (TLR9)IntracellularHuman and mouse
Figure 5: Flow cytometry analysis of neutrophils using cell surface marker, Ly-6G/Ly-6C. Ly-6G/Ly-6C is a 21–25 kDa protein also known as the myeloid differentiation antigen Gr-1. Mouse splenocytes and Balb/c bone marrow cells where stained with Gr-1 (Cat. No. 48-5931-82) and 7-AAD. As expected, based on known relative expression patterns, Gr-1 clone RB6-8C5 stains cells in the bone marrow myeloid gate and not in the splenocytes gate or bone marrow lymphoid gate. Viable bone marrow cells in the lymphoid (blue histogram) and myeloid (purple histogram) gates and viable splenocytes (orange histogram) were used for analysis.

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