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Neutrophil Markers

Heather Van Epps, PhD | 6th March 2025

Neutrophils are a population of granulocytic cell characterized by unique multi-lobed nuclei and abundant granules. Neutrophils are the most abundant subset of white blood cell in humans (50-70% of all leukocytes) and are best known for their pivotal role in innate immune defenses. Discovered in the late 1800s by Ehrlich and Metchnikoff, neutrophils were long thought to be terminally differentiated innate cells with a singular function: to seek out and destroy extracellular invaders by a variety of mechanisms including phagocytosis, degranulation, production of reactive oxygen species (ROS), and neutrophil extracellular trap (NET) formation.1 However, more recent studies have revealed that these cells display remarkable phenotypic and functional plasticity, with diverse roles in bone marrow, blood and tissues, both at steady-state and during stress.1-6

Neutrophils – also known as polymorphonuclear neutrophils or PMNs – have traditionally been difficult to study owing to their short half-life (mostly <1 day), post-mitotic nature, and sensitivity to manipulation. However, sophisticated genetic, genomic, and single-cell technologies—as well as the appreciation that neutrophils can survive for up to 5 days in certain contexts—have dramatically increased our understanding of neutrophil diversity and functional plasticity. There are a variety of markers that identify neutrophils at various stages of maturation and in specific contexts that allow researchers to study the phenotype and function of these cells in health and disease.

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Table of Contents

Determinants of Neutrophil Identity

Neutrophils are abundant in the circulation, comprising 50-70% of all circulating leukocytes in humans (10-20% in mice).5,7 These cells are also found in virtually all tissues of the body, where they exhibit a range of phenotypes and functions, including many that are unrelated to immune defenses. Like M1 and M2 macrophages, neutrophils can have both proinflammatory and anti-inflammatory functions, and they have important roles in homeostatic processes in tissues.

The phenotype and function of neutrophils is dictated by their state of maturation, migration status, and location in bone marrow, blood, or tissues (Table 1). Various subsets of neutrophils have been described, including low-density neutrophils (LDN), granulocytic myeloid-derived suppressor cells (g-MDSC or PMN-MDSC), and tumor-associated neutrophils (TAN)—with many studies using distinct terms to describe similar neutrophil populations. Indeed, a universally accepted neutrophil nomenclature is lacking,8 and whether the various neutrophil subsets that have been described represent durable populations or simply reflect differential states of maturation or activation dictated by environmental cues is a matter of ongoing debate and investigation.

Cell Stage Location Function Regulation
HSPCs, GMPs, Pre-neutrophils Bone marrow Granulopoiesis Growth factors (e.g. G-CSF, GM-CSF)

Cytokines, chemokines (e.g. CXCL12, IL-1β, IL-6)

Transcription factors
Immature neutrophils Response to stress (e.g. cancer, infection)
Mature neutrophils Immune defense, response to stress
Circulating neutrophils Blood Immune defense, acute inflammation Endotoxins and pathogen-associated molecular patterns (PAMPs)

Circadian-controlled transcription factors and chemokine receptors
Aged neutrophils Removal from blood, circadian immune defense, vascular protection
Tissue neutrophils Tissues Anticipation of infection, tissue-specific roles such as pulmonary transcription, regulation of bone marrow niches Tissue-derived signals

Microbiotal metabolites

Table 1: Neutrophil heterogeneity in location, function and regulation. Adapted from 5

Pan Neutrophil Markers

Several molecules are expressed by most neutrophil populations and are used in various combinations to identify mature neutrophils in blood and tissues, and these markers vary by species. Pan neutrophil markers that identify most neutrophils in the blood and tissues include:

Mouse

  • CD11b
  • Ly6G/GR1
  • CXCR2

Human

  • CD15
  • CD16
  • CD33
  • CD62L
  • CD66b

None of these markers is unique to neutrophils, however. For example, CD11b is also expressed on other myeloid cells, and CD62L is expressed on naive T cells.

IHC - Anti-CD11b Antibody [ITGAM/3340] (A249064)

Figure 1: IHC of human tonsil stained with Anti-CD11b Antibody [ITGAM/3340] (A249064).

Flow cytometry - Anti-CD15 Antibody [MEM-158] (FITC) (A85936)

Figure 2: Flow cytometry analysis of human peripheral whole blood stained with Anti-CD15 Antibody [MEM-158] (FITC) (A85936).

Neutrophil Developmental Markers

Neutrophils are continuously produced in the bone marrow from granulocyte–monocyte progenitors (GMPs) via a stepwise process called granulopoiesis (Figure 3). At steady-state, neutrophils are released into the bloodstream as mature cells; however, immature neutrophils are also released from the bone marrow in response to inflammatory signals. Neutrophil production can be augmented during states of inflammation by a process called emergency granulopoiesis.

Diagram showing development of neutrophils from multipotent progenitors.

Figure 3: Neutrophil development via granulopoiesis. Neutrophils develop in the bone marrow from hematopoietic stem cells (HSCs) via various progenitor subtypes, ultimately released into the blood as mature neutrophils. Based on transcriptional profiles, monocytes are the closest related cells to neutrophils. GMP: granulocyte/monocyte progenitor; LMPP: lymphoid-primed multipotent progenitor; MPP: multipotent progenitor.

Expression of surface markers changes during neutrophil maturation, with certain markers such as CD49d (Integrin alpha 4) expressed only on human pre-neutrophils and others (e.g., CD10) expressed only on fully mature human neutrophils. Levels of expression of cell surface proteins vary during neutrophil maturation (Table 2), and these markers differ between humans and mice (Table 3).5,6

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band cell Neutrophil
HLA-DR + - - - - -
CD34 + - - - - -
CD49d ++ ++ ++ + - -
CD15 + +++ +++ +++ +++ ++
CD33 +++ +++ ++ + + +
CD62L ++ ++ ++ ++ ++ ++
CXCR2 + + + + ++ ++
CXCR4 ++ ++ ++ ++ + +
CD18 ++ + +++ ++ ++ ++
CD66b - +++ +++ ++ ++ ++
CD24 - - ++ ++ ++ ++
CD11b - - +/++ ++ ++ ++
CD11c - - ++ ++ ++ ++
CD177 - - + + + +
CD16 - - - + ++ +++
CD87 - - - - ++ ++
CD35 - - - - ++ ++
CD10 - - - - - ++

Table 2: Expression of surface markers during granulopoiesis in humans. Expression key: (-) Not expressed (+) Low (++) Medium (+++) High. Based on data from 8

Pre-neutrophil Immature neutrophil Mature neutrophil
Mouse LIN-
CD117+
Siglec-F-
CD11b+
Ly6G+
CXCR4+		 LIN-
CD115-
Siglec-F-
CD11b+
Ly6G+
CXCR2-
CD101-		 LIN-
CD115-
Siglec-F-
CD11b+
Ly6G+
CXCR2+
CD101+
Human LIN-
CD66b+
CD15+
CD33mid
CD49dmid
CD101-		 LIN-
CD66b+
CD15+
CD33mid
CD49-
CD101mid
CD10-
CD16mid		 LIN-
CD66b+
CD15+
CD33mid
CD49-
CD101mid
CD10+
CD16hi

Table 3: Markers of different stages of neutrophil development in mouse and human. Adapted from 5

Mature neutrophils are continuously released from the bone marrow into the bloodstream, but the phenotype of mature neutrophils is not uniform. Studies of mouse and human neutrophils have identified specific subpopulations within the circulating PMN pool at steady state.6 For example, one study identified a population of pro-angiogenic neutrophils in mouse and human blood that express CD49d, vascular endothelial growth factor receptor 1 (VEGFR1) and the chemokine receptor CXCR4.9

Markers of Neutrophil Priming & Aging

Mature neutrophils in the blood can be primed by a variety of stimuli, including inflammatory cytokines, chemokines, mitochondrial contents, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs).10,11 In response to priming, neutrophils upregulate molecules involved in extravasation into tissues, including CD11b, CD63, and CD66a, with concomitant increases in functional capacity, including ROS production and phagocytosis.10

The phenotype of circulating, mature neutrophils is also regulated by a circadian process of neutrophil aging, in which aged PMNs eventually return to the bone marrow, liver or spleen for clearance by macrophages.5,12 Neutrophil aging is characterized by diurnal changes in expression of surface receptors, with levels of CXCR4 and CD11b increasing and levels of CD62L decreasing as the cells age. 5

IHC - Anti-CD63 Antibody [MX-49.129.5] (A250748)

Figure 4: IHC of human tonsil stained with Anti-CD63 Antibody [MX-49.129.5] (A250748).

Flow cytometry - Anti-CD62L Antibody [LT-TD180] (FITC) (A85808)

Figure 5: Flow cytometry analysis of human peripheral whole blood stained with Anti-CD62L Antibody [LT-TD180] (FITC) (A85808).

Tissue Neutrophil Markers

Neutrophils migrate from the bloodstream into tissues, both at steady-state and in response to infection or disease. Most circulating neutrophils express low levels of ICAM-1 and high levels of CXCR1, whereas PMN found in tissues express low levels of both molecules.6,13 Upregulation of specific adhesion molecules that are typically low in circulating neutrophils has been shown to be necessary for migration into distinct tissues; for example, L-selectin (CD62L) is required for neutrophil migration to the spleen, and ICAM-1 for migration to the liver.15

Neutrophils can also migrate in the reverse direction—from tissues into blood—and these reverse migrated PMN have been shown to have the opposite phenotype (ICAM-1highCXCR1low) to most circulating PMNs.

Neutrophils are present in most tissues of the body, including blood, liver, lung, spleen, intestine, and skin,1,2 where they acquire new phenotypic and functional properties via transcriptional and epigenetic reprogramming in response to tissue-specific signals. For example:

  • In lungs and intestine, neutrophils have been shown to differentially express genes involved in vascular growth and repair, required for homeostatic and stress-induced angiogenesis.2
  • In lymphoid tissue, a population of neutrophils has been identified that express an activated phenotype (CD11bhigh, CD62Llow, CXCR2low), major histocompatibility class II (MHCII), and costimulatory molecules (CD80, CD86), suggesting the capacity to function as antigen presenting cells to stimulate adaptive immune responses.15
  • PMNs from the colon and oral cavity of mice express particularly high levels of CD66a and CD11b.14

IHC - Anti-ICAM1 Antibody [P2A4] (A248905)

Figure 6: IHC of human lymph node stained with Anti-ICAM1 Antibody [P2A4] (A248905).

Flow cytometry - Recombinant Anti-CXCR1 Antibody [DMC470] (A318720)

Figure 7: Flow cytometry analysis of Expi293 cells transfected with human CXCR1 (blue) or with an irrelevant protein (red) stained with Recombinant Anti-CXCR1 Antibody [DMC470] (A318720).

Other Neutrophil Subsets

Tumor-Associated Neutrophils (TANs)

Tumor-associated neutrophils (TAN) were first described in mice and were initially divided into functionally distinct tumor-suppressive (N1) and pro-tumor (N2) subtypes.7,16 In humans, TANs primarily function to promote tumor growth (i.e. N2), and clinical studies show that an increased neutrophil-to-lymphocyte ratio is associated with poor clinical outcomes in many cancer types in humans;17,19 human neutrophils can also help to restrain tumor growth in specific contexts.4

Other subsets of neutrophils, including immature neutrophils and interferon-γ (IFNγ)-activated neutrophils, have also been described in the tumor microenvironment in humans and mice.1 Whether different populations of TANs exert anti-tumor or pro-tumor functions depends in part on the tumor milieu. For example, TGFβ polarizes TANs toward a pro-tumorigenic phenotype. IFNγ, by contrast, induces anti-tumor responses in neutrophils, which can activate CD8+ T cells and render tumors responsive to anti-PD-1 therapy.1 Subsets of TANs are characterized by varying expression of neutrophils surface markers, which differ in mice and humans (Table 4).

Immature neutrophils Pro-tumor neutrophils Anti-tumor neutrophils IFN-stimulated neutrophils
Mouse Ly6G+
CD11b+
CD117+
CD170lo
CD101-
CD84+
JAML		 Ly6G+
CD11b+
PD-L1+
CD170hi		 Ly6G+
CD11b+
CD170lo
CD170+ (in CRC)
CD54+
CD16+		 Ly6G+
CD11b+
IFIT1
IRF7
RSAD2
Human CD66b+
CD11b+
CD117+
CD10-
CD16int/lo
LOX1+
CD84+
JAML		 CD66b+
CD11b+
CD170hi
PD-L1		 CD66b+
CD11b+
CD101+
CD177+
CD170lo
CD54+
CD86+
HLA-DR+
CD15hi		 CD66b+
CD11b+
IFIT1
IRF7
RSAD2

Table 4: Expression of surface markers on subsets of tumor-associated neutrophils. CRC: colorectal cancer. Adapted from 1

Neutrophil Myeloid-Derived Suppressor Cells (PMN-MDSC)

Pro-tumor TANs with T-cell suppressor activity are often referred to as granulocytic myeloid-derived suppressor cells (g-MDSC or PMN-MDSC), although these cells can also be induced in response to inflammation and trauma.20 In mice, PMN-MDSC express CD11b, CD14, Ly6G and low levels of Ly6C;4,21 human PMN-MDSC markers include CD11b, CD33, CD15, LOX-1 (humans). Both mouse and human PMN-MDSC have been shown to express CD84, FATP2, and TRAIL-R25.4,22 It is important to note that there are no markers that definitively distinguish PMN-MDSC from other neutrophil populations, and the characterization of these cells relies on tests of their suppressive activity.6

Low-Density Neutrophils

Low-density neutrophils (LDNs)—so called because they reside in the low-density fraction on density gradient fractionation—are a population of neutrophils first identified in patients with infections or inflammatory diseases and in cancer (noting that PMN-MDSC were initially thought to mostly reside within the low-density fraction). The phenotypic and functional distinction between LDNs and normal-density neutrophils (NDNs) is not completely clear, although LDNs appear to include both immature and mature PMNs, with many of the latter showing evidence of activation or degranulation. LDNs can have both pro-inflammatory and anti-inflammatory functions (Figure 8).6 LDNs have been described as exhibiting higher levels of CD10, CD15, CD16b and CD66b than NDNs, though no markers have been identified yet to definitively distinguish LDNs from NDNs.23

Diagram showing similarities between T cytotoxic and T helper cells.

Figure 8: LDN populations isolated from density gradient fractionation of blood. Two main LDN populations have been reported so far: immunosuppressive and proinflammatory, sometimes referred to as polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and low-density granulocytes (LDGs), respectively. Whether NDN populations fulfilling similar functions in people with these conditions also exist is currently unclear. Adapted from 6.

Neutrophil Marker Antibodies

CD11b Antibodies
CD15 Antibodies
CD16 Antibodies
CD33 Antibodies
CD62L Antibodies
CD66b Antibodies
CD177 Antibodies
CXCR1 Antibodies
CXCR2 Antibodies
CXCR4 Antibodies
Integrin alpha 4 Antibodies
Ly6G Antibodies

References

  1. Herro, R., & Leighton Grimes, H. The diverse roles of neutrophils from protection to pathogenesis. Nature Immunol 25, 2209-2219 (2024).
  2. Ballesteros, I., et al. Co-option of Neutrophil Fates by Tissue Environments. Cell 183, 1282–1297 (2020).
  3. Burn, G. L., Foti, A., Marsman, G., Patel, D. F. & Zychlinsky, A. The neutrophil. Immunity 54, 1377–1391 (2021).
  4. Eruslanov, E. Nefedova Y., & Gabrilovich, D.I. The heterogeneity of neutrophils in cancer and its implication for therapeutic targeting. Nature Immunol 26, 17-28 (2025).
  5. Ng, L.G., Ostuni, R. & Hidalgo, A. Heterogeneity of neutrophils. Nat Rev Immunol 19, 255–265 (2019).
  6. Silvestre-Roig, C., Fridlender, Z.G., Glogauer, M., & Scapini, P. Neutrophil Diversity in Health and Disease. Trends Immunol 40, 565-583 (2019).
  7. Zhang, F. et al. Neutrophil diversity and function in health and disease. Signal Transduct Targeted Ther 9, 343 (2024).
  8. McKenna, E., et al. Neutrophils: Need for Standardized Nomenclature. Front Immunol 12, 602963 (2021).
  9. Massena, S., et al. Identification and characterization of VEGF-A–responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans. Blood 126, 2016-2026 (2015).
  10. Miralda, I., Uriarte, S. M., McLeish, K. R. Multiple Phenotypic Changes Define Neutrophil Priming. Front Cell Infect Microbiol 7, 217 (2017).
  11. Fine, N., et al. Primed PMNs in healthy muse and human circulation are first responders during acute inflammation. Blood Adv 3, 1622-1637 (2019).
  12. Paudel, S., Ghimire, L., Jin L., Jeansonne, D., & Jeyaseelan, S. Regulation of emergency granulopoiesis during infection. Front Immunol 13, 961601 (2022).
  13. Duffy, D., et al. Neutrophils transport antigen from the dermis to the bone marrow, initiating a source of memory CD8+ T cells. Immunity 37, 917-929 (2012).
  14. Chadwick, J. W., et al. Tissue-specific murine neutrophil activation states in health and inflammation. J Leukoc Biol 110, 187-195 (2021).
  15. He, W., et al. Circadian Expression of Migratory Factors Establishes Lineage-Specific Signatures that Guide the Homing of Leukocyte Subsets to Tissues. Immunity 49, 1175-1190 (2018).
  16. Fridlender, Z. G. et al. Polarization of tumor-associated neutrophil phenotype by TGF-beta: ‘N1’ versus ‘N2’ TAN. Cancer Cell 16, 183–194 (2009).
  17. Mouchli, M., Reddy, S., Gerrard, M., Boardman, L. & Rubio, M. Usefulness of neutrophil-to-lymphocyte ratio (NLR) as a prognostic predictor after treatment of hepatocellular carcinoma. Ann. Hepatol. 22, 100249 (2021).
  18. Pirozzolo, G., Gisbertz, S. S., Castoro, C., van Berge Henegouwen, M. I. & Scarpa, M. Neutrophil-to-lymphocyte ratio as prognostic marker in esophageal cancer: a systematic review and meta-analysis. J Thorac Dis 11, 3136–3145 (2019).
  19. Vartolomei, M. D. et al. Prognostic role of pretreatment neutrophil-to-lymphocyte ratio (NLR) in patients with non-muscle-invasive bladder cancer (NMIBC): A systematic review and meta-analysis. Urol Oncol 36, 389–399 (2018).
  20. Gabrilovich, D., & Nagaraj, S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 9, 162-174 (2009).
  21. Kramer, E. D., Abrams, S. I. Granulocytic Myeloid-Derived Suppressor Cells as Negative Regulators of Anticancer Immunity. Front Immunol 11, 1963 (2020).
  22. Pettinella, F., et al. Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors. Cell Rep Med 5, 101380 (2024).
  23. Rankin, A. N., Hendrix, S. V., Naik, S. K., Stallings, C. L. Exploring the Role of Low-Density Neutrophils During Mycobacterium tuberculosis Infection. Front Cell Infect Microbiol 12, 901590 (2022).
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