Lecture #6: Neutrophils

Granulocyte/macrophage progenitor

gives rise to monocytes, neutrophils, eosinophils, basophils and mast cells

Common myeloid progenitor

Originates from the pluripotent stem cell and gives rise to the granulocyte/macrophage progenitor

Pluripotent stem cell

The origin of every white blood cell; it gives rise to the common myeloid progenitor

Neutrophil location (and %)

Peripheral blood, in about 60-75%

Life-span of neutrophils

short lived, only being in circulation for about 6-12 hours

Neutrophil average diameter

10-20 micrometers

Main function of neutrophils

phagocytose and kill pathogens

Equine neutrophils

white or slightly pink cytoplasm with no visible granules; their nuclei is long, thin and knobby with clumps of condensed chromatin.

Feline neutrophils

cytoplasm that is white and lacks visible granules

Canine neutrophils

cytoplasm that contains small pink specific or secondary granules

Equine neutrophils (photo)

Feline neutrophils (photo)

Canine neutrophils (photo)

Panther neutrophils (photo)

Foal neutrophils (photo)

Shark neutrophils (photo)

Iguana neutrophils (photo)

Parrot neutrophils (photo)

Difference of heterophil to neutrophil

granules are large and stained deep orange to red

Granulopoiesis (definition)

Process in which neutrophils are developed

Granulopoiesis (location)

Occurs in the bone marrow

Order of granulopoiesis

HSC --> MPP --> LMPP --> GMP --> Myeloblast --> Promyelocyte --> Myelocyte --> Metamyelocyte --> Band Cell --> Neutrophil

Primary granules include

• Myeloperoxidase

• Lysozyme

• Elastase

• Beta-glucuronidase

• Cathepsin Beta

Secondary granules include

• Lysozyme

• Collagenase

• Matrix metalloprotease-8

Tertiary granules include

• Gelatinase (MMP-9)

• Arginase I

• Cytochrome b558

Location where neutrophils are abundant

liver, spleen, lungs and bone marrow

Extravasation

Process where the neutrophils leave the circulation and migrate through the tissue

Extravasation requirements

requires a defined sequence of interactions between adhesion molecules, while also requiring chemokines to direct the cells to the site of the insult

Emigration from the blood stream requires changes in:

• Endothelial cells

• Neutrophils

• Integrins

• Emigration

What are selectins?

Adhesion molecules of importance during extravasation of white blood cells

Types of selectins

P-selectin and E-selectin, L-selectin

Where are P-selectin and E-selectin found?

Endothelial cells of blood vessels

Where are L-selectin found?

Neutrophils (in this particular case)

Steps of extravasation

• Tethering

• Slow Rolling

• Full arrest

• Firm adhesion

• Intraluminal crawling

• Transmigration

Neutrophils antimicrobial mechanisms

1. Phagocytosis

2. Degranulation

3. NETosis

Phases of phagocytosis

• Neutrophils become activated, which occurs upon

encountering activated endothelium and exposure to

cytokines

• Encounter/adhere to target

• Ingest/engulf target

• Destroy target

What does CD mean in neutrophils

Cluster of differentiation

Most relevant receptors for neutrophils

• Opsonins

• Leukotrienes

• Complement

• Cytokines

• Attaching neutrophils to blood vessel walls

Function of opsonins

coating the microbe, making it positively charged and therefore easier to grab

What charge do microbes and neutrophils both have?

Negative

Where does ingestion of a microbe occur

Within the neutrophil, which engulfs the bacteria

How is the membrane-bound vacuole that contains the bacteria called

Phagosome

Phagolysosome

Junction of phagosome and lysosome

Process by which the microbes inside the phagolysosome are destroyed

respiratory burst and the action of the granule-associated lytic enzymes and antimicrobial peptides

Respiratory burst

consists of using reactive oxygen species, such as O2-, H2O2 and OH-, to damage membranes.

Examples of granule-associated lytic enzymes and antimicrobial peptides

• Proteolytic enzymes: degrade bacteria and tissue

• Lactoferrin: binds iron (which bacteria require)

• Defensins: bactericidal, recruit and activate other WBC

Degranulation

represents a much faster mechanism to kill pathogens; it consists of the release of granules to the extracellular space

Secretory granules used during degranulation would include

proteases, myeloperoxidase, reactive oxygen species, lysozyme and cationic proteins

Secretory granules used during degranulation are characterized by being

some of the most toxic, readily releasable factors

What are NETs

extracellular webs of microbicidal cytosolic and granule proteins assembled on a scaffold of decondensed chromatin or mitochondrial DNA

NET formation is induced by

various pathogenic triggers

Steps of the NET formation pathway (1/2)

• Nuclear delobulation and the disassembly of the nuclear envelope

• Cellular depolarization and chromatin decondensation

• Plasma membrane rupture and release of NETs

Neutrophils fate

short lived with a high rate of spontaneous apoptosis, with most of them only surviving a few days

When does apoptosis of neutrophils occur?

presence of inflammatory stimuli

Which system is in charge of removing the apoptotic neutrophils

mononuclear phagocytic system (macrophages)

The ingestion of neutrophils by the mononuclear phagocytic system (macrophages) triggers and induces:

trigger the secretion of anti-inflammatory cytokines (IL-10 and TGF-Beta) and the resolution of the inflammatory process

Benefits of neutrophils

Phagocytosis of pathogens and clearance of dead cells and debris, anti-microbial peptides, influence other cells (T cells) and aid in resolution of inflammation

Detrimental effects of neutrophils

Tissue damage via excessive MPO, NE, MMP production, oxidative burst and NETs, impaired function during co-infections and drivers of asthma exacerbation

Molecules involved in the extravasation process

Selectins, Integrins and Chemokines

Extravasation steps

Rolling, tethering, adhering and transmigration

Steps of phagocytosis

Activation, adherence, ingestion and destruction

Additional antimicrobial mechanisms of neutrophils

Degranulation, NET formation