A Guide to Neutrophil Markers

Neutrophils, which can amount to as much as 70% of all leukocytes in the human body, represent a major component of the immune system. An estimated 10¹¹ new neutrophils are produced each day during granulopoiesis, highlighting their important role in serving as the first line of defense against invading pathogens. The conventional understanding of neutrophil functions has centered on the antimicrobial response via phagocytosis, degranulation, and the release of neutrophil extracellular traps (NETs). However, these polymorphonuclear immune cells appear to be more active and complex. Studies have shown neutrophils can produce cytokines, influence activities of other immune cells, modulate inflammation, and infiltrate tumors. In investigations involving neutrophils, it is fundamental to acknowledge that this cell type exists in heterogeneous populations, which can be classified in areas such as developmental stage, function, migration, and association with various health conditions. In this article, we focus on the common cellular markers mentioned in recent literature that have been used to describe various neutrophil subpopulations.

Markers for neutrophil maturation

The neutrophil life cycle begins with development from neutrophil precursors in the bone marrow, maturation into circulating blood neutrophils, and the eventual clearance of aged neutrophils by macrophages. Accordingly, the heterogeneity of neutrophils under various states of maturation can be observed through the differential expression of certain markers. Among these are CD15, CD11b, CD16, and CD10, which immature, differentiating neutrophils begin to express. For mature, circulating neutrophils, a phenotype of CD16^hi, CXCR2^hi, CXCR4^low, and CD62L^hi has been reported.

Aged neutrophils are often discussed as a distinct subset because their maturation also leads to changes in effector function. In addition to being morphologically smaller and containing fewer granules, aged neutrophils are reported to be effective in migrating to sites of inflammation. A general phenotype for aged cells includes CD62L^low, CXCR2^low, CXCR4^hi, CD11b^hi. and CD47^low. Other markers reported to be associated with aged neutrophils include CD11c, CD24, ICAM1, CD45, and TLR4. Further upregulation of CXCR4 marks neutrophils for senescence, leading them back to the bone marrow for turnover.

Image: Phenotypic markers can be used to characterize the various states and functions of neutrophils.

Markers for neutrophil activation

Neutrophil activation, which occurs in response to signals from sites of inflammation or infection, primes the transendothelial migration of neutrophils across the blood vessels into peripheral tissues. Leading up to this process, interactions with endothelial cell surface proteins and chemotactic molecules cause certain proteins to be exposed to the neutrophil membrane. Proteins upregulated upon activation include CD11b and CD18, corresponding to the α and β chains of the β2 integrin. The granulocyte marker CD66b, which is stored in granules, also becomes exposed upon degranulation. The presence of CD177 and PRTN3 have also been reported in some individuals. 

Markers for neutrophil migration

In addition to blood circulation, neutrophils can also migrate to various tissues under normal conditions and exhibit signature phenotypes. Among these are subsets residing in the spleen (CD62L^low, CD11b^hi, ICAM1^hi), and lymph nodes (CCR7, LFA-1, CXCR4). A specialized population of human neutrophils has been reported to reside in the marginal zone of the spleen. Termed, 'B cell helper neutrophils’ (NBH cells), this subset (CD15^int/loCD16^int/lowCD11b^hi) is believed to promote B cell proliferation and antibody production.

Antibodies Search Tool
Discover marker antibodies
from different suppliers Search

Neutrophils can return to the bloodstream after migrating to the tissue, a process known as reverse transendothelial migration. This group has a notably longer lifespan and an increased ability to produce superoxide species. Markers associated with reverse-migrated neutrophils include ICAM1, CD18, CD62L, CXCR1, CXCR2, CXCR4, and neutrophil elastase (ELANE).

Table of neutrophil markers

The table below lists human and mouse proteins used in phenotyping different populations of neutrophils as mentioned by recent literature. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for neutrophil immunophenotyping.

Note: *Some proteins are protein isoforms or multi-subunit protein complexes composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

Other neutrophil subsets

Low-density neutrophils (LDN) are a population identified in the low-density fraction from density gradient separations of inflammatory disease blood samples. However, studies have yet to fully elucidate functional differences between LDNs and normal density neutrophils. Interestingly, their numbers appear to increase with tumor growth and progression. Markers associated with LDNs include ARG1, CD15, CD33, CEACAM8 (CD66b), FCGR3A (CD16), ITGAM (CD11b), MME (CD10), and PECAM1 (CD31). 

PMN-MDSC (polymorphonuclear MDSC, also called granulocytic-MDSC) are a subset of myeloid-derived suppressor cells—so named for their ability to suppress T cell responses—that either contain or resemble neutrophils. PMN-MDSCs are a heterogeneous population that appears to contain both immature and mature neutrophils. Consistent with an immunosuppressive role, this subset is associated with worse clinical outcomes in cancer and is associated with other diseases. Proteins involved with phenotyping PMN-MDSC include ARG1, CD33, FCGR3A, and ITGAM.

Proangiogenic neutrophils (PAN) are a distinct population found to be recruited to hypoxic, non-vascularized areas and promote angiogenesis. These have been found to represent a small fraction (~3%) of total circulating neutrophils. Described phenotypically with the markers CXCR4, FLT1 (VEGFR1), and ITGA4 (CD49d), PANs have also been implicated in tumor angiogenesis. 

Markers have been described for characterizing neutrophils and their involvement in inflammation and inflammatory disease. For instance, proteins associated with the neutrophil response to inflammatory cues include CD63, CD49, CXCR2 (CD182), and the IL-17 receptor. Studies in mice have led to the classification of two subsets, PMN-1 and PMN-2, which vary in cytokine production, macrophage activation, and expression of markers, including ITGA4 (CD49d), ITGAM (CD11b), and certain toll-like receptors (TLRs). Examples of neutrophil-expressed proteins involved in inflammatory diseases include CD16, CD18, CD43, CD63 (rheumatoid arthritis); CD31, CD11c (systemic lupus erythematosus); and CD177, PRTN3 (vasculitis). 

Tumor-associated neutrophils (TAN) are a population first identified in mice, containing subsets with antitumor (N1) and tumor-promoting (N2) functions. Likewise, populations that either activate or suppress T cell activity have also been reported in humans. One such reported phenotype for TANs is CD66b+, CD15+, CD16+, CD11b+, HLA-DR−, and ARG1. Other mentioned TAN markers include CD14, CCR7, and CD86. 

References

1. Silvestre-Roig C, Hidalgo A, Soehnlein O. Neutrophil heterogeneity: implications for homeostasis and pathogenesis. Blood. 2016;127(18):2173-2181. doi:10.1182/blood-2016-01-688887
2. Lakschevitz FS, Hassanpour S, Rubin A, Fine N, Sun C, Glogauer M. Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry. Exp Cell Res. 2016;342(2):200-209. doi:10.1016/j.yexcr.2016.03.007
3. Rosales C. Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types?. Front Physiol. 2018;9:113. Published 2018 Feb 20. doi:10.3389/fphys.2018.00113
4. Deniset JF, Kubes P. Neutrophil heterogeneity: Bona fide subsets or polarization states?. J Leukoc Biol. 2018;103(5):829-838. doi:10.1002/JLB.3RI0917-361R
5. Silvestre-Roig C, Fridlender ZG, Glogauer M, Scapini P. Neutrophil Diversity in Health and Disease. Trends Immunol. 2019;40(7):565-583. doi:10.1016/j.it.2019.04.012
6. Yang P, Li Y, Xie Y, Liu Y. Different Faces for Different Places: Heterogeneity of Neutrophil Phenotype and Function. J Immunol Res. 2019;2019:8016254. Published 2019 Feb 28. doi:10.1155/2019/8016254
7. Grieshaber-Bouyer R, Nigrovic PA. Neutrophil Heterogeneity as Therapeutic Opportunity in Immune-Mediated Disease. Front Immunol. 2019;10:346. Published 2019 Mar 4. doi:10.3389/fimmu.2019.00346