A Guide to Granulocyte Markers

Granulocytes are hematopoietic cells of the innate immune system, distinguished by their lobed nuclei and the presence of cytoplasmic granules containing antimicrobial proteins. Originating from bone marrow, billions of short-lived granulocytes are produced every day to maintain a steady-state population in circulation. During infection, new granulocytes are generated to combat the microbial threat; this process is called emergency granulopoiesis.

There are four types of granulocytes: neutrophils, basophils, eosinophils, and mast cells. Neutrophils are the most common type of granulocyte, and basophils are the least. Although each type of granulocyte possesses unique cellular markers and functions, there is a surprising amount of overlap between these cell types. This guide summarizes the most commonly used markers used to identify and discriminate neutrophils, basophils, eosinophils, and mast cells in humans.

Figure: The diagram highlights the four main granulocyte types and some of their overlapping protein markers. 

Neutrophil Markers

Neutrophils are granulocytes that feature a multi-lobed nucleus of two to five lobes, with a higher number generally indicating a more mature neutrophil. These cells can account for as much as 70% of all circulating leukocytes in an adult human, playing a key role in innate immunity by phagocytosing microorganisms and degrading them with cytotoxic granule proteins.   

Early neutrophil precursors express cytoplasmic myeloperoxidase (MPO; a granule protein), ANPEP (CD13), and CD33. Upon maturation, these cells sequentially upregulate effector molecules, including ITGAM (CD11b), FCGR3A (CD16), and MME (CD10). Neutrophils express ANPEP periodically throughout maturation. Sialylated carbohydrate antigen CD65 is another indicator of maturity, as its presence emerges only after the progenitor marker CD34 disappears.

Like many immune cells, neutrophils express a wide array of cellular markers that can be used for identification and functional characterization. These include cytokine and chemokine receptors (IL5RA, CCR3, CCR5, CXCR2), pattern recognition receptors (CD14, TLR7, TLR8), adhesion molecules (ITGAM, IGTAX, CEACAM8, CD65), and enzymes (MME, ANPEP, FUT4, MPO). Other commonly expressed neutrophil receptors include CD33 and FCGR3A (CD16). MPO is an important granule protein used by neutrophils to destroy microorganisms.

These are but a few of the markers expressed by neutrophils. For a more in-depth summary, see our guide on neutrophil markers.

Eosinophil Markers

Eosinophils are granulocytes with a bilobed nucleus characterized by highly condensed chromatin. They possess acidophilic granules with electron-dense cores upon ultrastructural examination. Common granule proteins found in eosinophils include major basic protein (MBP), eosinophil peroxidase (EPO, encoded by EPX), eosinophil cationic protein (ECP, encoded by RNASE3), and eosinophil-derived neurotoxin (EDN, encoded by RNASE2). Eosinophils release these granule proteins extracellularly to combat parasites, which are too large to be phagocytosed by neutrophils. These granulocytes also feature lipid bodies, which form after eosinophil activation and participate in eicosanoid synthesis. Only 1-5% of circulating leukocytes are eosinophils.

Classically, eosinophils have been defined as CD45+CD11b+CD11c+CD13+CD15+CD33+. However, more recent investigations using larger and more complex flow cytometric panels have uncovered a surprising amount of heterogeneity among eosinophils, including substantial overlap with neutrophils. Eosinophils have been reported to express the following array of markers: immunoglobulin receptors (FCGR2A), complement receptors (CR1, CR3), cytokine receptor (IL3RA, IL2RA), adhesion molecules (ITGA4), prostaglandin receptors (PTGDR2), platelet-activating factor receptor (PTAFR), and sialic acid-binding Ig-like lectin 8 (SIGLEC8).

Like neutrophils, eosinophils express IL5RA, CCR3, TLR7, TLR8, ITGAM, ITGAX, ANPEP, CD33, CEACAM8, FUT4, and CD65. It is worth noting that although IL5RA can be expressed by neutrophils, its expression is more commonly associated with eosinophils, which depend on the receptor for maturation and survival via the cytokine IL-5. In fact, IL-5 has been shown to confer eosinophilic properties in neutrophils, revealing a potential capacity for transdifferentiation among granulocytes. Similarly, CCR3 and CCR5 are more typical for eosinophils (used to respond to eotaxin and RANTES) but have also been reported on neutrophils; and, more perplexingly, neutrophils have been shown to produce the eosinophil granule ECP.

Learn more about eosinophil markers here.

Mast Cell Markers

Unique among granulocytes due to their unlobed nucleus, mast cells differentiate and reside within tissues, associating largely with blood vessels and epithelial surfaces, where they participate in pathogen defense and, more notably, allergic responses. Human mast cells either express tryptase (TPSAB1) or mast cell-specific chymase (CMA1) as granule proteins. Mast cells also secrete histamine, a key molecule that mediates allergy.

CD117 (encoded by KIT) and FcεR1 (encoded by FCER1A) are key markers for mast cells. Other mast cell markers include immunoglobulin receptors (FCGR2A), inhibitory G-protein coupled receptors (ADRB2, ADORA2B, PTGER2), cytokine and chemokine receptors (IL3RA, IL5RA, IL9R, IL10RA, CSF2RA, CCR3, CCR5, CXCR2, CXCR4), enzymes (ENPP3, CPE), and CD33.

Learn more about mast cell markers here. 

Basophil Markers

Basophils are the rarest of granulocytes at <1% of circulating leukocytes. Despite their lobed nuclei, basophils are functionally and molecularly similar to mast cells, participating also in acute and chronic allergic responses. They express FCER1A (FcεR1), cytokine and chemokine receptors (IL3RA, IL5RA, IL2RA, CCR2, CCR3), enzymes (ENPP3), and CD33. Unlike mast cells, basophils differentiate in the bone marrow and seldom proliferate after maturation.

Genes expressed uniquely by basophils (among granulocytes, at least) include CD36, CD38, CD40LG, and CD9.

Table of Granulocyte Markers

The table below lists characteristic granulocyte proteins as described by review literature. The list includes a variety of marker types, including receptors, adhesion molecules, and enzymes. Accompanying each marker are links to relevant antibodies and ELISA kits that can be used to detect granulocytes in vitro and in vivo. The associated products are offered by a variety of manufacturers and can serve as a useful reference for granulocyte characterization.

Note: *This is a carbohydrate antigen, not a gene-encoded protein. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only). 

References

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2. Gorczyca W, Sun ZY, Cronin W, Li X, Mau S, Tugulea S. Immunophenotypic pattern of myeloid populations by flow cytometry analysis. Methods Cell Biol. 2011;103:221-266. doi:10.1016/B978-0-12-385493-3.00010-3

3. Gustafson MP, Lin Y, Maas ML, et al. A method for identification and analysis of non-overlapping myeloid immunophenotypes in humans. PLoS One. 2015;10(3):e0121546. Published 2015 Mar 23. doi:10.1371/journal.pone.0121546

4. de Ruiter K, van Staveren S, Hilvering B, et al. A field-applicable method for flow cytometric analysis of granulocyte activation: Cryopreservation of fixed granulocytes. Cytometry A. 2018;93(5):540-547. doi:10.1002/cyto.a.23354

5. Orfao A, Matarraz S, Pérez-Andrés M, et al. Immunophenotypic dissection of normal hematopoiesis. J Immunol Methods. 2019;475:112684. doi:10.1016/j.jim.2019.112684

6. Berdnikovs S. The twilight zone: plasticity and mixed ontogeny of neutrophil and eosinophil granulocyte subsets. Semin Immunopathol. 2021;43(3):337-346. doi:10.1007/s00281-021-00862-z