Granulocytes Overview

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.

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 bronchoalveolar-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.

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.

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.