Granulocytes and Basophils

• Granulocytes are the first line of defense upon exposure to danger.
• Granulocytes release granule contents as a response to danger sensing.
• Polymorphonuclear cells (PMNCs) are multilobulated, with segmented nuclei that are not separated.
• Granule contents directly target pathogens and danger.
• Activation of granulocytes leads to secretion of cytokines and mediators that regulate trafficking and activity of other white blood cells, including lymphocytes.
• Granulocytes are divided into neutrophils, basophils, mast cells and eosinophils.

Basophils

• Non-phagocytotic granulocytes containing large granules filled with basophilic proteins.
• Constitute about 0.5-1% of circulating leukocytes.
• Key participants in IgE-mediated reactions, releasing contents upon binding of circulating IgE.
• Infiltrate inflammatory lesions, releasing pro-inflammatory mediators such as histamine and leukotriene C4, and regulatory cytokines like IL-4 and IL-13.
• Cytokines secreted by basophils modulate the adaptive immune response.
• Infiltrate various human cancers, playing diverse roles as both promoters or inhibitors of tumorigenesis.
• Facilitate wound healing, linked to IL-4/IL-13 secretion, polarizing macrophages toward the M2 phenotype, and linked to secretion of angiogenic factors, including VEGF-A and amphiregulin.
• Vascular endothelial growth factors released by cancer cells and immune cells in the tumor microenvironment can favor basophil infiltration.
• IL-3, which is made by intra-tumoral lymphocytes, mast cells and cancer cells, is the most important growth and activating factor for human and mouse basophils. It uses the engagement of the IL-3 receptor (IL-3Ra/CD123).
• CCL3/CCL4 secreted by intra-tumoral basophils induces CD8+ T cell recruitment in the tumor microenvironment, which promotes melanoma rejection in mice.
• IL-33 affects tumorigenesis, upregulates granzyme B mRNA, and the surface expression of CD63, leading to functional and phenotypic basophil activation; furthermore, IL-33-activated basophils induce melanoma cell death in mice.
• TNF-α and granzyme B are released by mouse basophils, exerting cytotoxic effects on tumor cells; human basophils also release these under certain circumstances.
• An activated signature of CD123, CCR3, CD63, and CD203c gene expression in tumor-resident basophils is associated with improved outcomes in ovarian cancer patients.
• Topical exposure to carcinogens can cause tumor-protective IgE response through skin-infiltrating basophils.

Basophils in Cancer

• Galectin-3 is highly expressed by many types of cancer cells, indicating it indicates poor prognosis and mediates immunosuppression within the tumor microenvironment.
• Galectin-3 activates basophils to secrete IL-4/IL-13, promoting M2-like macrophages.
• IL-4-producing basophils exists in the tumors from human pancreatic cancer. Mouse models have shown that IL-4 promotes a Th2>Th1 response more conducive to tumorigenesis.
• Basophils secrete VEGF-A, which promotes angiogenesis, and induce IL-6/IL-8 secretion and promotes metastasis.
• EC-Gal-3 activates dendritic cells and monocytes, producing high levels of TNF-α/IL-6 in vitro.
• The chronic production of these cytokines and M2 cell-derived IL-10/TGF-b promotes T-cell exhaustion by up-regulating checkpoint inhibitors (e.g., PD-1).
• This interacts with tumor cell-associated markers (PD-L1) to suppress cytotoxic T cell activity.
• V-domain immunoglobulin suppressor of T-cell activation (VISTA) is another immune checkpoint receptor, regulating allergen-specific Th2-mediated immune responses.
• Dysregulated innate immune responses synergize to promote tumorigenesis.

Mast Cells

• Characterized by a large count of cytoplasmic granules containing histamine and other pharmacologically active substances.
• Play an important role in the development of allergies.

Eosinophils

• Unique blood cells with bright red granules when stained with eosin, termed "eosinophilic granulocytes," comprising >5% of circulating white blood cells.
• Play role in defense against parasitic worms, clustering invading worms and damaging the membranes through proteins released from the granules.
• Eosinophils play a role in tissue repair and regeneration, secreting IL-4, which activates muscle-resident fibrocyte-adipocyte progenitors upon muscle injury, induces proliferation of quiescent hepatocytes, and regulates liver regeneration.
• In response to IL-5, they contribute to the exacerbation of chronic allergic diseases.
• In response to amphiregulin, they contribute to tissue fibrosis of respiratory airways.
• IL-5 plays specific roles in the development and function of eosinophils, promoting their maturation in the bone marrow from GMP.
• Migration of eosinophils is regulated by IL-5.
• Eosinophils are recruited to inflamed lung through IL-5 during eosinophilic inflammation.
• Tpath2 cells, ILC2s, Mast cells, and NKT cells mainly produce IL-5 under inflammatory conditions.
• Mepolizumab, a humanized monoclonal antibody against IL-5, inhibits its biological activity.
• Benralizumab, a humanized monoclonal antibody against IL-5 receptor α, induces eosinophil apoptosis.
• Airway fibrotic response is caused by inflammatory eosinophils and is when ST2high memory Th2 cells secrete amphiregulin.
• Eosinophils activate and produce osteopontin, which is a key profibrotic inflammatory protein through amphiregulin stimulation.

Neutrophils

• Account for about 70% of leukocytes, with less than 1011 cells produced daily in the bone marrow.
• Mature neutrophils are short-lived (12-24 hours), do not divide, and contain lobulated nuclei.
• They circulate for more than 12 hours, then migrate to tissues, and are cleared by tissue-resident macrophages.
• Neutrophils reach the site of infection in response to inflammatory molecules generated by innate cells.
• They phagocyte bacteria and secrete proteins with antimicrobial effects.
• The first responders to infection and main cellular components of pus, where they accumulate and die.
• Circulating neutrophils increase depending on the infection.
• Recognize microbial pathogens and deploy functions to destroy them.
• Perform phagocytosis which is the ingestion of the microorganism into a phagocytic vacuole becoming a phagolysosome.
• Low pH and degrading enzymes destroy microorganism in the new organelle.
• Neutrophils degranulate, releasing the contents of their granules.
• Neutrophils produce the nets by producing extracellular traps (NETs) formed by DNA fibers and proteins from the granules.

Neutrophil Behavior

• Display complex behaviors compared to only pathogen-killing cells.
• Transcriptionally active and change the expression of membrane molecules, producing cytokines.
• Different cell factions depend on the tissues where they are found.
• There are existing subpopulations of neutrophils in various conditions of health and disease.
• Neutrophils and their products, including cytokines modulate the innate and adaptive immune response.
• Neutrophils and NETs promote cytokine production by macrophages, while enhancing DC recruitment and antigen presentation.
• Promote innate immune resolution and repair, regulating cytokine and growth factor production.
• Neutrophils trigger B cell expansion and antibody production, and affect T cell cytokine production and differentiation.
• In some instances they can perturbing T cell functionality.

Neutrophils in Cancer

• Exhibit diverse roles in tumor regression or enhancement of tumor expansion and metastasis.
• Produce ROS and ATP that directly attack tumor cells, along with cytokines to promote T cell antitumor properties.
• Neutrophil-derived products can also promote tumor expansion.
• Tumor cell migration, expansion, and angiogenesis can happen which inhibits cytolytic T cell function.
• Neutrophil serine proteases and NETs can pave the way for metastatic cells to escape tumor sites and spread to different tissues.

Low Density Neutrophils (LDNs)

• Neutrophil subsets are separated through density gradient centrifugation.
• Mature neutrophils (PMN) are denser, separating at the bottom of the gradient, and have classical neutrophil morphology with a lobulated nucleus and many granules.
• Peripheral blood mononuclear cells (PBMC) are separated in the upper part of the gradient.
• Low-density neutrophils, in this upper part of the gradient, comprise immature and mature neutrophils with immunosuppressive properties.
• Immature neutrophils are called granulocytic myeloid-derived suppressor cells (G-MDSC).
• LDNs were first reported in the blood of patients with autoimmune diseases and are is associated with cancer.
• Found in other pathological conditions, including sepsis, psoriasis, HIV infection, asthma, and malaria.
• LDNs are reported in natural pregnancy.
• Downregulation of T cell functions happens through the enzyme arginase which depletes, is required to ensure materno-fetal tolerance and for T cell proliferation.
• LDNs exist in the peripheral blood of pregnant women, as well as in term placentae.
• Chronic inflammation and immunosuppression appear to induce neutrophil diversity.
• LDNs from autoimmunity patients form NETs and can be responsible for sustaining chronic inflammation leading to autoimmunity.
• Elevated in peritoneal cavity from abdominal surgery of gastric cancer; these readily formed NETs selectively attached cancer cells which assisted the clustering and growth of free tumor cells disseminated in the abdomen.