WBC - Granulocytes

What are the myeloid types of WBC

•Granulocytes

•Mononuclear phagocytes

List three granulocytes and two mononuclear WBCs

Granulocytes: Neutrophils, Eosinophils and Basophils

Mononuclear WBC - Lymphocytes and monocytes

What cytokines and what produces them stimulates production/maturation of neutrophils

IL-3 - T cells

GM-CSF - T cells, endothelial cells, monocytes and fibroblasts

G-CSG - Endothelial cells, placenta and monocytes

What cytokines and what produces them stimulates production/maturation of eosinophils

IL-3 - T cells

IL5 - T cells and basophils

GM-CSF - T cells, endothelial cells, monocytes and fibroblasts

What cytokines and what produces them stimulates production/maturation of basophils

IL3 - T cells

IL4 - B & T cells, eosinophils

GM- CSF T cells, endothelial cells, monocytes and fibroblasts

What are the normal ranges for Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils

	Normal [x 109/l]
Neutrophils	1.8 – 7.5
Lymphocytes	1.5 – 4.0
Monocytes	0.2 – 0.8
Eosinophils	0 – 0.4
Basophils	0.01 – 0.1

Why would neutrophils be elevated

Bacterial infection, stress, exercise, myeloproliferative diseases e.g. leukaemia

Why would lymphocytes be raised

Viral infection, lymphoproliferative diseases (e.g. lymphocytic leukaemia)

Why would Monocytes be elevated

Infection, inflammation, tissue damage, monocytic leukaemia

Why would Eosinophils count be elevated

Allergy, intestinal parasites, hypereosinophilic syndrome, eosinophilic leukaemia

Why would Basophils be elevated

Some myeloproliferative diseases (esp. Chronic granulocytic leukaemia)

What are the main roles for neutrophils, eosinophils and basophils

Neutrophils – the elimination of invading bacteria and some fungi

Eosinophils – elimination of parasitic worms (=helminths), regulation of local immune and inflammatory responses

Basophils – Immune system regulation, secretion of heparin and histamine, allergy, inflammation, parasite defence, ? tumour surveillance

Describe the structure of a neutrophil

Neutrophils:

    granules are

    neutral-staining

Describe the eosinophil structure

    granules stain with eosin (orange)

Describe a Basophils structure

granules stain intensely with methylene blue

What are the stages of neutrophil maturation

What is the granulocyte turnover

50 – 320 x 10⁹/day

  (= approx 370,000 / sec)

How long does neutrophils spend in the peripheral blood and then tissues

Peripheral blood ~ 7 hours

Tissues ~ 20 hours

What is the lifespan of an eosinophil

8-12 hours in circulation then 8 – 12 days in tissues (thymus, lower GI tract, ovary, uterus, spleen and lymph nodes)

What is the lifespan of basophils

2-3 days in circulation then die

Why is a neutrophils nucleus lobulated

To aid deformability and motility

What are the granules present in neutrophils

•Primary – discharge into phagosomes. Contain microbicidal proteins (eg Myeloperoxidase, hydrolases & lysozyme) for oxidative and non-oxidative killing. Main function is killing and digesting micro-organisms.

•Secondary – discharge into phagosomes and extracellularly. Contain e.g. hydrolases, alkaline phosphatase, lysozyme and collagenase.

•Tertiary – Contain e.g. alkaline phosphatase, gelatinase (involved in the destruction of collagen), cathepsin (a protease)

What does the neutrophils function involve

Location by chemotaxis and then phagocytosis

How does chemotaxis work

Vessel wall adhesion. Tissue damage > neutrophil adhesion to endothelial cells of vessel wall > migration into tissues. Adhesion using  adhesive membrane receptors.

Movement up a concentration gradient. Chemo-attractants bind to specific neutrophil surface receptors, e.g. complement components, esp C5a, bacterial metabolic by-products, leukotriene B4 (an inflammatory mediator, made by monocytes/macrophages). Neutrophil receptors can detect concentration changes as small as 2%.

What are the 4 steps of phagocytosis

1) Requires opsonisation (i.e. coating) of particle with e.g. IgG or IgM antibodies or complement.

Neutrophils require particle to be completely coated with opsinin for successful phagocytosis

2) Particle attaches to neutrophil via a receptor for the opsonin

3) Pseudopodia enclose particle, which is ingested > phagosome

4) Neutrophil granules fuse into a phagosome and release contents, killing invading organism.

What is a respiratory burst

Increase in O₂ consumption

Increase in glycolysis

Uprating of bactericidal processes e.g. Myeloperoxidase (MPO) activity

Increased expression of some constituents, e.g. Alkaline phosphatase

How does microbial killing usually work

What are some disorders of phagocytosis and how do they work

1) Myeloperoxidase deficiency.

    Fairly common. Partial or total. Only 20% of patients are

    immunocompromised. Oxygen free radicals (O^-) & lysozyme 

    compensate. Fungal infections are the biggest problem.

2) Respiratory burst failure – esp NADPH oxidase

  Inherited, metabolic failure of microbial killing, affects 1 in 200,000. (= Chronic Granulomatous Disease (CGD).

  Some organisms live in the phagosome > persistent infections.

  Multiple granulomas (accumulations of immune system cells).

  Non-oxidative killing partially compensates

3) Inherited defects in neutrophil adhesion / migration are very rare.

    Acquired defects, e.g. leukaemia, diabetes, renal failure, > varying degrees of susceptibility to sepsis.

 

Other neutrophil disorders

Neonate neutrophils have only 20 – 27% chemotactic activity of adults , less in premature neonates.

Neutrophil function declines with age – chemotaxis and phagocytosis significantly impaired in the elderly.

Abnormal neutrophils (e.g.hypogranular or agranular) are found in myelodysplasia (“pre-leukaemia”), common in the elderly and in leukaemia

What are some features of chronic granulocytic leukaemia

Increased and unregulated

  growth of myeloid cells in

  bone marrow.

Increased myeloid cell

 numbers in blood, many

 early (but mainly

 differentiated) forms evident.

Many cases now “cured” by

 tyrosine kinase inhibitor

 therapy eg Imatinib

(in cases of 9:22 translocation which deregulates tyrosine kinase activity)

What indicates incorrect function of neutrophils

Chronic bacterial infection

Increased susceptibility to bacterial infections

Therapy-resistant infections

Recurrent infections with nonpathogenic microorganisms

Abscesses of liver or lung

What is NBT dye reduction and whats it for

Nitroblue tetrazolium for chronic granulomatous disease

What does NBT test for

Functional test of neutrophil respiratory burst (production of active oxygen species e.g. O^-). Reduction of NBT to an insoluble blue compound by active neutrophils. Visual assessment (microscopy) of results.

Largely superceded by direct measurements of respiratory burst products using flow cytometry

How is neutrophil motility tested

Motility by assessing ability to penetrate a filter membrane or observed movement across a glass slide

How is neutrophil phagocytosis tested

•Ingestion, e.g. by observing reduction in the number of free bacteria in a bacteria + neutrophil suspension

•Killing, e.g. by observing the intra-cytoplasmic fall in numbers of phagocytosed bacteria.

Where are Eosinophils usually found

Thymus, lower GI tract, ovary, uterus, spleen and lymph nodes only in lungs in airborne allergy

What is more numerous tissue or blood eosinophils

Tissue eosinophils are several 100 x more numerous than blood eosinophils

What are eosinophils function

Functions – key mediators of allergic inflammation and elimination of parasitic worm infections by antibody-dependant cell-mediated toxicity (IgE)

What does Eosinophils cationic protein do

creates pores in the membranes of target cells allowing potential entry of other cytotoxic molecules to the cell & has anti-viral activity

What is Major Basic Protein

(toxic to parasites & epithelial cells, causes release of histamine & heparin from basophils & mast cells)

What causes Eosinophils significant local tissue damage

Eosinophils degranulation

What Interleukin does Eosinophils produce

IL-4

•Important immunoregulatory cytokine

•Affects inflammation, B-cell activation, antibody production

What is hypereosinophilic syndrome

Defined as sustained unexplained eosinophilia > 1.5 x 10⁹/l > 6 months (normal range 0-0.4)

A monoclonal population of activated T lymphocytes may be found, producing excess IL5 (so the primary problem is lymphoid in origin)

What is hypereosinophilic syndromes affect on organs

Causes organ dysfunction due to eosinophilic infiltration (heart, lungs, GI tract, spleen, skin, CNS, etc)

What does treatment of hypereosinophilic syndrome aim to do and what is the treatment

Treatment attempts to limit organ damage by control of eosinophils using steroids, hydroxyurea, chemotherapy (Imatinib) therapy.

Is hypereosinophilic syndrome a fatal disorder

Most cases are eventually fatal but 80% survival to 5 years

What are the differences between basophils and mast cells

Treatment attempts to limit organ damage by control of eosinophils using steroids, hydroxyurea, chemotherapy (Imatinib) therapy.

What activates basophils

Basophils are activated by e.g. IgE, IL-3, C5a, GM-CSF & insect venoms to release granule contents, especially histamine.

What do basophils mediate

Basophils are key mediators of immediate hypersensitivity reactions e.g. asthma, urticaria & anaphylaxis

There is also possible evidence they supress tumour growth

List what basophils secrete and the function of each secretion

•Histamine - chemotactic agent for eosinophils & is a vasodilator

•Heparin – anticoagulant

•IL-3, a basophil growth factor, regulates macrophage and granulocyte populations in inflammation.

•IL-4, attracts eosinophils and supports B-cell activation

•IL-5, basophil and eosinophil production and activation, B-cell growth and activation

•IL-13, promotes B-cell proliferation and differentiation

•TNF-α –pro-inflammatory cytokine

•GM-CSF –inflammation-associated cytokine, activates neutrophils, moderates monocyte activation and is a growth and differentiation factor for granulocytes and macrophages

What causes significant basophilia

Significant basophilia is common in Chronic Myeloid Leukaemia but otherwise rare. (Suggestions that basophil numbers correlate with plasma triglyceride levels are probably counting artefacts giving a falsely elevated count).

Basophilia to a lesser extent is found in other myeloproliferative disorders, e.g. myelofibrosis & Primary Proliferative Polycythaemia.

Why is treatment for basophil leukaemia difficult

It is a rare disorder and it’s difficult due to release of histamine and other granule contents

How are basophils tested

basophil Activation Test (BAT).

Assesses the expression of activation markers (CD63 or CD 203c) on the surface of live basophils or measures the degree of basophil degranulation.

In whole fresh blood by flow cytometry following stimulation with allergen

Usually in investigation of peanut allergy, using peanut allergen

Distinguishes between allergic and tolerant individuals

NB Performed exceptionally rarely (antigen challenge instead)

Describe a monocytes structure and contents

What are the function of monocytes

Phagocytosis

Antigen presentation (to T cells)

Cytokine production

Differentiation into macrophages and monocyte-derived dendritic cells

Critical role in both innate and adaptive immunity (phagocytic WBCs are innate only)

How can monocytes bind to endothelial cells

With different adhesive glycoproteins which facilitate adhesion

What cytokines do monocytes release

Release cytokines e.g. Pro-inflammatory IL1, IL6, IL8, G-CSF, Tumour Necrosis Factor-α (TNF-α, involved in cell signalling, apoptosis, tumour cell suppression )

What is the lifespan of monocytes

Most in circulation are short-lived (24h)

As macrophages and dendritic cells live for months or years

What causes an increased number of monocytes

in chronic infections and inflammatory conditions, e.g. tuberculosis & Crohn’s disease

What are some examples of inherited impairment of degradation of phagocytosed material

“Lipid storage diseases”, e.g. Gaucher’s disease, Niemann-Pick disease result from an accumulation of debris within macrophages

  Cause permanent cellular and tissue damage, particularly in the brain, peripheral nervous system, liver, spleen, and bone marrow.