Neutrophils comprises in humans 50-70% of all circulating leukocytes, whereas in mice they represent 10-25 of circulating leukocytes. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

Neutrophils (polymorphonuclear leukocytes “PMN) are produced in the bone marrow during hematopoiesis and released into the blood where they circulate for 7-10 h before migrating into the tissues where they have a 3 day life span. Neutrophils generally are the first to arrive at the site of inflammation and are phagocytic cells like macrophages.

Anti-Microbial Activity of Neutrophils 

Neutrophils are very adept at killing microorganisms and only two bacterial pathogens have been conclusively shown to survive and replicate within them. The short life-span of a mature neutrophil and fast turnover rate is not conducive to long-term survival of intracellular pathogens. High neutrophil turnover is due to spontaneous apoptosis coupled with subsequent removal by macrophages. Neutrophils are particularly involved in the  to bacterial/fungal infection. They are the first phagoyctes to arrive at the side of infection (usually 6-12 hours post infection). Neutrophils are capable of releasing several deleterious components that mediate the inflammatory response.

Production of ROS: Neutorphils activate the NADPH oxidase to generate large amounts of superoxide, which is a precursor of H2O2 and other forms of ROS with potent antimicrobil activity. ROS can cross the membranes of bacterial pathogens and damage their nueceic acids, prtoeins and cell membranes. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396).

Degranulation: At the site of infetion, neutrophils need to perfrom a rpaid response against microorganisms. To acheive this, they count on pre-formed effector molecuels sotres in their intracellular granules. There are four types of granules; primary or azurophil granules containing MPO, NE, Azuracidin and defensins and secondary or specific granules containing lactofferin, which sequester iron important for microbial growth. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

–defensins: The most abundant protein type in neutrophils are defensins which are used by neutrophils to kill phagocytosed pathogens.

Secretion of elastase: another proteolytic activity used by neutrophils at the site of inflammation is neutrophil elastase (NE) which has the potential to preferentially disrupt the elastic network. Neutrophils also require NE to kill some phagocytosed Gram-negative bacilli, and this activity is mediated through activation of proteases by K+ flux into the phagocytic vacuoles caused by the O2- generating system.

Cytokine release: Neutrophils, as the first line of defense of the innate immune system against invading microorganisms, display singificant antimicrobial functions, including degranulation, production of reactive oxygen species (ROS, phagocytosis and the formation of neutrophil extracellular traps (NET) (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396) These antimcirobila functions were believed to be the only prupose of neutrophils. However, in the last decade it has become clear that neutrophils display many functional responses that go beyond the simple killer of microrganisms. Neutrophils product cytokines and other infalammatory factions that regualte the entire immune system. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

Phagocytosis: is.a receptor-mediated process during which a particle larger than 0.5 um is internalized by the cell into a vacuole called the phagosome. Neutrophils recognize pthogens through PAMPs or through opsonin (antibody moecules or complement components). Neutrophil receptors for PAMPS include C-type lectins such as Dectin-2, Mincle, or DNGR-1,; savenger receptors. They also include Dectin-1, which is a recetpro for fungal beta-glucan. Other receptors such as TLRs also recognize diverse PAMPS but they do not function as phagocytic receptors. However, tLRs often collaborate with other receptors to stimulate phagocytosis. Neutrophils recognize opxonice-particles by antibody receptor (Fc receptors) or complement receptors, which induce much more eficient phagocytosis. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

Potential for CNS repair:

Both mouse and human bone marrow (BM) neutrophils, when polirized with a combination of recombinant interleukin 4 and granulocyte-colony stimulating factor (G-CSF) upregulate alternative activaiton markers and produce an array of growth factors, thereby gaining the capacity to promote neurite outgrowth. Moreover, adoptive transfer of IL-4/g-CSF polirzed BM neutrohpils into eperimental models of CNS injury triggered substantial axon regernation within the optic nerve and spinal cord. Liu, “Cytokine polarized, alternatively activated bone marrow neutrophils drive axon regeneration” Research Square).

Morphology

Mature neutrophils in circulation are about 7-10 um in diameter presenting a segmented nucleus and having a cytoplasms enriched with granules and secretory vesicles. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

Neutrophils have a characteristic nuclear morphology, express the RB-6 antigen, and lack MHC class II products.

Neutrophile migration:

To lymphoid organs: TNFalpha is important for neutrophile migration into lymphatic vessels. Tissue at the site of immunizaiton release TNFalpha whihc primed neutrophils to enter the lymphatic vessels in a CCR7-dependent manner. (Rosales “Neutrophils at the crossroads of innate and adaptive immunity”. J. Leukoc Biol 2020, 106: 377-396)

Isolation

Human neutrophils can be isolated from venous blood by the Percoll density gradient centrifugation method. Each 10 ml of whole blood can be mixed with 0.8 ml of 0.1 M EDTA (an anti-cogulant) and 25 ml of saline. The diluted blood is layered over 9 ml of Percoll at a specific density of 1.080 g/ml. After centrifugation at 400 g for 20 min at 20C, the plasma, mononuclar cell and Percoll lyers are removed. Erythrocytes are lysed by the addition of 18 ml of ice cold water for 30 s, followed by 2 ml of 10 times concentrated Pipes buffer. Cells are pelleted at 4C, the supernatant decanted and the procedure repeated. After the second hypotonic lysis, cells are washed twice with PAG buffer. ((Xia, Analytical Biochemistry 245, 93-96 (1997).

Neutrophils can be identified by labeling with CD15 (neutrophil body marker).

Neutropenia

Neutropenia is defined as a circulatory neutrophil count below 1.5 x 109/L. An absolute neutrophil count of less than 0.5 x 109/L is regarded as severe neutropenia and may be associated with life threatening infection s like pneumonia and septicaemia. A characgteristic glased mucositis occurs in the mouth, and ulceration is common.

Cause of neutropenia may be inherited as with Kostmann’s syndrome or acquired through, for example, viral infection. Chemotherpay and radiotherapy also produce neutropenia.

Antiobiotics should be given as necessary to patients with acute severe neutropenia. If the neutropenia is caused by a drug, the drug therapy should be stopped. G-CSF is also used to decrease the period of neutropenia after chemotherapy and maemopoietic transplantation.