What are granulocytes?

Granulocytes are a heterogenous category of leukocytes, comprising neutrophils, eosinophils, basophils, and mast cells [1]. They are innate immune cells and once activated, release immunostimulatory molecules to fight-off viral and parasitic infections. Granulocytes rely on inflammatory signals to recruits them to the site of injury, infection, or allergic response to become activated and produce effector function [1]. In addition to their responses to viral and parasitic infection, granulocytes are involved in several diseases including chronic inflammation, asthma, allergies, immune regulation, autoimmunity, and cancer.

Granulocytes are characterized by the presence of secretory cytotoxic granules in their cytoplasm and are polymorphonuclear. The most abundant member of the granulocytes is the neutrophil (~60% of circulating leukocytes in human, ~20% in mice), followed by eosinophils (1–3% of circulating leukocytes, ~6% in bone marrow), while basophils are the rarest (<1% of circulating leukocytes). Mast cells are mainly tissue-resident cells.

Types of granulocytes

Associated with allergic and anaphylactic responses but also have been demonstrated to play a protective role, particularly in immunity against parasites and environmental toxins such as venoms. Like basophils, mast cells express FcεRI, which binds the Fc region of the IgE immunoglobulin secreted in response to parasitic infections and allergens [6]. Also involved in tissue repair and maintenance.

Morphology: Obscured nucleus with a dense granular cytoplasm [6].

Abundance: Low frequencies of mast cells in tissues. Long-lived tissue-resident cells found in mucosal fluid.

Granulocyte isolation and flow cytometry analysis

Granulocytes are terminally differentiated cells that can be isolated from blood, bone marrow, or tissue. 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 [1].

Neutrophils
Neutrophils are frequently isolated from whole blood by density gradient centrifugation, instead of from bone marrow.

Eosinophils
The most accessible source for eosinophils in human and mouse is blood, while the highest number is found in bone marrow. Tissue-resident eosinophils can be obtained from most tissues by digesting with tissue-appropriate proteases. Another source for eosinophils is bronchioalveolar-lavage fluid obtained from mice in allergic asthma models. Flow cytometry analyzers and sorters can be used for their characterization and isolation.

Basophils
Flow cytometry analyzers and sorters should be used for basophil characterization and isolation [7]. Murine basophils can also be cultured from bone marrow in the presence of IL-3, although the conditions of culture have been observed to give rise to phenotypic and functional differences in the resultant cells.

Mast cells
Isolating mast cells from tissues or the peritoneal cavity is labor-intensive and often suffers from poor yields [11]. The development of transformed mast cell lines has been used as models. Protocols for the differentiation of mouse mast cells from bone marrow or human mast cells from bone marrow, cord blood, or fetal liver have also been described and typically involve culture with IL-3, SCF, or both for an extended amount of time. However, these cultured cells are also not ideal surrogates for tissue-resident mast cells, as the precise microenvironments in which these cells typically differentiate is difficult to recapitulate in vitro.

Table 1. Non-exhaustive list of granulocyte cellular markers for characterizing granulocyte cells by flow cytometry.

Cell SubtypeMarkerLocalizationSpecies
Pan-granulocytesCD11bSurfaceHuman and mouse
CD13SurfaceHuman
CD15SurfaceHuman
CD16/32SurfaceMouse
CD32SurfaceHuman
CD33SurfaceHuman
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
CD15SurfaceHuman and mouse
CD17SurfaceHuman and mouse
CD24SurfaceHuman and mouse
CD35SurfaceHuman and mouse
CD43SurfaceHuman and mouse
CD66aSurfaceHuman and mouse
CD66bSurfaceHuman and mouse
CD66cSurfaceHuman
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
Mast cells
CathepsinsSecretedHuman and mouse
HistamineSecretedHuman and mouse
TNF alphaSecretedHuman and mouse
IL-4SecretedHuman and mouse
TGF betaSecretedHuman and mouse
NGFSecretedHuman and mouse
CD9SurfaceHuman and mouse
CD15SurfaceHuman and mouse
CD24SurfaceHuman and mouse
CD35SurfaceHuman and mouse
CD43SurfaceHuman and mouse
CD64SurfaceHuman and mouse
CD116SurfaceHuman and mouse
CD117 (c-kit)SurfaceKey phenotyping marker: Human and mouse
CD123SurfaceHuman and mouse
CD125SurfaceHuman and mouse
CD126SurfaceHuman and mouse
FceR1SurfaceKey phenotyping marker: Human and mouse
IL-33R (ST-2)SurfaceHuman and mouse
Basophils
IL-4SecretedHuman and mouse
IL-13SecretedHuman and mouse
HistamineSecretedHuman and mouse
CCL3 (MIP-1 alpha)SecretedHuman and mouse
CD9SurfaceHuman and mouse
CD11aSurfaceHuman and mouse
CD13SurfaceHuman and mouse
CD16SurfaceHuman
CD25SurfaceHuman and mouse
CD33SurfaceHuman and mouse
CD38SurfaceHuman and mouse
CD43SurfaceHuman and mouse
CD63SurfaceHuman and mouse
CD88 (C5a receptor)SurfaceHuman and mouse
CD123SurfaceKey phenotyping marker: Human and mouse
CD125SurfaceHuman and mouse
CD154 (CD40 ligand)SurfaceHuman and mouse
CD192 (CCR2)SurfaceHuman and mouse
CD203cSurfaceHuman
CD218 (IL-18R)SurfaceHuman and mouse
CD282 (TLR2)SurfaceHuman and mouse
CD284 (TLR4)SurfaceHuman and mouse
CD286 (TLR6)SurfaceHuman and mouse
CD294 (CRTH2)SurfaceHuman and mouse
FceR1SurfaceKey phenotyping marker
CD281 (TLR1)IntracellularHuman and mouse
CD289 (TLR9)IntracellularHuman and mouse
C/EBP alphaIntracellularHuman and mouse
GATA-2IntracellularHuman and mouse
Eosinophils
MBPsSecretedHuman and mouse
EDNSecretedHuman
EPXSecretedHuman and mouse
CD9SurfaceHuman and mouse
CD15SurfaceHuman and mouse
CD24SurfaceHuman and mouse
CD35SurfaceHuman and mouse
CD43SurfaceHuman and mouse
CD64SurfaceHuman and mouse
CD116SurfaceHuman and mouse
CD123SurfaceHuman and mouse
CD125SurfaceKey phenotyping marker: Human and mouse
CD126SurfaceHuman and mouse
CD170 (SiglecF)SurfaceKey phenotyping marker: Human and mouse
CD193 (CCR3)SurfaceKey phenotyping marker: Human and mouse
CD244SurfaceHuman and mouse
FceR1SurfaceHuman and mouse

In flow cytometry, granulocytes are easy to exclude by morphology based on FSC/SSC (Figure 1). When leukocytes are gated based on violet light scatter properties, the three main leukocyte cell populations in human blood can be distinguished–lymphocytes, monocytes, and granulocytes.

Granulocytes cytokine and chemokine profiling

Neutrophils: Activated neutrophils can secrete a variety of cytokines when either positively modulated by IFN-gamma or negatively by IL-10. Because human neutrophils typically possess several fold less total RNA than other leukocytes, on a per cell basis, they will produce significantly lower cytokine amounts than other leukocytes.

Basophil: The cytokine IL-3 serves as a potent hematopoietic growth factor involved in the differentiation from myeloid progenitor and activation of basophil function. Basophils secrete IL-4, TSLP, IL-13, and IL-25, cytokines that can promote Th2 function and contribute to immunoglobulin synthesis especially IgE production.

Eosinophils: IL-5 is a potent hematopoietic growth factor that activates eosinophils and acts synergistically with IL-3 and GM-CSF towards the development of mature eosinophils. Eotaxins 1, 2, and 3 serve as potent chemoattractants for eosinophil migration to body sites such as the lungs and intestines. Over 30 cytokines have been reported to be secreted by eosinophils with several reported to be pre-formed and stored within the eosinophil crystalloid granules.

Mast cells: Numerous secreted cytokines, chemokines, and growth factors have been identified for mouse and human mast cells and mediate allergic disease and both innate and adaptive immune response. Although the majority of these cytokines are also produced by many other cell types, their secretion by mast cells is likely dependent on the specific role of the mast cell, such as defense against parasitic infections, bacterial infections, viral infections, food allergies, and anaphylaxis, or at mucosal sites.

Multiplexed immunoassays provide a convenient and cost-effective method to profile cytokines, chemokines, and growth factors related to granulocytes. The comprehensive Immune Monitoring 65-Plex Human Panel is the largest multiplex immunoassay panel available on the market and an impactful method to monitoring granulocyte cytokine production.

Table 2: Key cytokines secreted by mast cells, neutrophils, eosinophils, and basophils.

 Mast CellsBasophilsNeutrophilsEosinophils
Cytokines, chemokines, growth factorsCCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL9, CCL10, CCL17, CXCL2, CXCL8, CXCL10, TNF, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-11, IL-13, IL-16, IL-33, G-CSF, NGF, FGF, PDGF, SCF, TGF-beta1, VEGFIL-4, IL-13, MIP-1α, TSLP, IL-25, IL-6, CCL5, CCL3, VEGF, GM-CSF, IL-3TNF-alpha, IL-4, IL-10, IFN-gamma, CCL5, IL-1alpha, IL-1beta, IL-6, CCL3, IL-8, CXCL1, CXCL8, CXCL10, CCL2, VEGF, G-CSF, FasL, TRAIL, BAFFCCL5, CCL3, IL-6, NGF, MBPs, eosinophil-derived neurotoxin (EDN), EPX, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12, IL-13, IL-16, IL-17, IL-25, IFN-gamma, TNF-alpha, CCL11, CCL13, CCL17, CXCL1, CXCL5, CXCL8, CXCL10, CXCL11, CXCL9, SCF, VEGF, TGF-beta, TGF-alpha

Table 3. Multiplex Immunoassays for immune monitoring.

SpeciesDescriptionAnalytesCat. No.
HumanImmune Monitoring 65-Plex Human PanelG-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A (CTLA-8), IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-31, LIF, M-CSF, MIF, TNF alpha, TNF beta, TSLP, BLC (CXCL13), ENA-78 (CXCL5), Eotaxin (CCL11), Eotaxin-2 (CCL24), Eotaxin-3 (CCL26), Fractalkine (CX3CL1), Gro-alpha (CXCL1), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MDC (CCL22), MIG (CXCL9), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), IP-3 alpha (CCL20), SDF-1 alpha (CXCL12), FGF-2, HGF,  MMP-1, NGF beta, SCF, VEGF-A, APRIL, BAFF, CD30, CD40L (CD154), IL-2R (CD25), TNF-RII, TRAIL (CD253), TWEAKEPX650-10065-901
MouseImmune Monitoring 48-Plex Mouse ProcartaPlex PanelBAFF, G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A (CTLA-8), IL-18, IL-19, IL-22, IL-23, IL-25 (IL-17E), IL-27, IL-28, IL-31, IL-33, LIF, M-CSF, RANKL, TNF alpha, ENA-78 (CXCL5), Eotaxin (CCL11), GRO alpha (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), MIP-2, RANTES (CCL5), Betacellulin (BTC), Leptin, VEGF-A, IL-2R, IL-7R alpha, IL-33R (ST2)EPX480-20834-901

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