NEUTROPHILS AND THEIR DISORDERS

READING: D Roos et al. Microbes and Infection 5 (2003) 1307-1315. OMIM-Chronic Granulomatous Disease

WHITE CELLS
TWO BROAD GROUPS: PHAGOCYTES : NEUTROPHILS, EOSINOPHILS, BASOPHILS MONOCYTES IMMUNOCYTES: LYMPHOCYTES, PLASMA CELLS

NEUTROPHIL

Granules

Nucleus

EOSINOPHIL

Cell Count
Total White Cell Count: 4-11 x 109/l Neutrophils: 2.5-7.5 x 109/l Eosinophils: 0.04-0.4 x 109/l Basophils: 0.01-0.1 x109/l Monocytes: 0.2-0.8 x 109/l Lymphocytes: 1.5-3.5 x109/l

NEUTROPHIL
Multi lobed 3-5 lobes Mature Neutrophil observed in peripheral blood Contains primary and secondary granules

Granules
Primary -Galactosidase Myeloperoxidase Esterase Lysozyme Secondary (Specific) Elastase Components of NADPH Oxidase Collagenase Lysozyme Lactoferrin Transcobalamins (TC I and TC II)

FUNCTION
PRIMARY FUNCTION - DEFENCE (1)Killing Microoraganisms (2)Phagocytosis

NADPH OXIDASE

NADPH OXIDASE
FUNCTION TO GENERATE SUPEROXIDE O2 COMPONENTS: TWO SUBUNITS a) 90 kDa B) 22 kDa These contain a cytochrome b (-245) and a haem function.

NADPH OXIDASE
OTHER COMPONENTS: A FLAVIN RAP IA Cytosolic components p47 phox (PKC dependent) P67 phox, p40 phox and rac

NADPH OXIDASE
2O2 2 O2

P22

GP 91

Plasma membrane

P4 7

p40 NADPH

p6 7

NADP

NADPH??
NADPH is derived from the hexose Monophosphate shunt Activation of NADPH oxidase involves the Consumption of a lot of oxygen. This is called the RESPIRATORY BURST as It was first observed using oxygen electrode experiments

CELL SIGNALLING
FMLP
O2 O2

PIP2 G PLC

DAG

PtdCho NADPH OXIDASE PLD 47K 67K 40K rac

PKC

Ca 2+

IP3

PKC

ACTIVATION FACTORS
1) fMet-Leu-Phe bacteria Formyl peptide derived from

At low concentrations Chemotactic Concentrations activator of NADPH Oxidase 2) Complement component C5a 3) Leukotriene B4 (LTB4)-neutrophil origin

Priming Agents
Priming increases the response of the Neutrophil following its activation. Examples: GM-CSF-sites of infection /inflammation TNF In Vitro- cytochalasin B

PRIMING AGENTS
PRE-TREAT ISOLATED NEUTROPHILS WITH GM-CSF. (Note INCREASE IN SUPEROXIDE PRODUCTION). + GM-CSF

CYT C RED

-GM-CSF

FMLP

Time

KILLING
Gram +ve O2 H2O2 MPO HOCl

Plasma membrane

NADPH OXIDASE 1 1 2 2

CYTOSOL

Granules

Granules

What Can Go Wrong?

A) Chronic Granulomatous Disease (CGD) Reduced expression of NADPH Oxidase Large subunit (90kDa) Encoded on X chromosome This accounts For 80% of CGD ( 1in 1,000,000)

OUTCOMES
Patients present in early infancy Recurring bacterial or sometimes fungal Infections. Further details on subtypes and genetics See OMIM (On-line mendelian Inheritance In man)

Lab Tests
a) Cytochrome c reduction b) Chemiluminescence c) Tetrazolium dye

Cytochrome C
O2 Cyt (Fe (III)) 550 nm Cyt c Fe (II)

Isolated Neutrophils activated with either FMet-Leu-Phe or PMA PMA Phorbol ester directly activates Protein kinase C.

Cytochrome c reduction
550 nm reduction of Cytochrome c

Normal

CGD TIME (min) FMLP

CHEMILUMINESCENCE

LUMINOL

OXIDANTS eg HOCl

FLASHES OF LIGHT

fMet-Leu-Phe

Chemiluminescence mV

Normal

CGD Time (min)

PMA

Chemiluminescence mV

Normal

CGD TIME (min)

Nitroblue Tetrazolium
Normal Blue product

CGD No reduction

Other Rare Disorders

a) Myeloperoxidase deficiency b) Chediak- Higashi Syndrome

Giant granules

Chediak-Higashi Syndrome
Autosomal recessive Giant graules in neutrophils /eosinophils Monocytes and lymphocytes Neutropenia, thrombocytopenia Partial albinism Affected children tend to die from infection Or haemorrhage

MORPHOLOGICAL DISORDERS a) Pelger Huet-autosomal dominant Bilobed shape

b) May-Hegglin-Autosomal dominant Giant platelets and inclusion of RNA in neutrophils

Alders anomaly-Deep purple granules

INFECTIOUS MONONUCLEOSIS
Infectious mononucleosis (I.M.) (Glandular fever. Symptoms: lethargy, swollen glands Lymphadenopathy, rash Rise in white cell count: ~10-20 x109/l Absolute lymphocytosis

INFECTIOUS MONONUCLEOSIS
In Blood-Presence of atypical lymphocytes

What are they?

B-Lymphocytes are infected with Epstein Barr Virus. Atypical Lymphoctes are reactive T-lymphocytes towards the infected lymphocytes. Usually peak between 7-10 days Of the illness.

SEROLOGY
Heterophile antibodies are raised in I.M. These can react with antigens from other species e.g. they can Agglutinate horse red cells

SEROLOGY
Paul Bunnel Test: I.M. antibodies can be adsorbed by Ox red cells I.M. Antibodies are NOT adsorbed by Guinea Pig kidney cells I.M. antibodies can agglutinate horse red cells

PAUL BUNNELL TEST

GPK Normal plasma I.M. + Ox red cells -

After mixing plasma with GPK and Ox red cells Add horse red cells. GPK does not adsorb ab So it can then agglutinate added horse red cells

ANTIDODIES
Heterophile Ab (IgM) Anti-viral Caspid (IgG) Anti-EBNA

3 months EBNA -anti EB nuclear antigen

6 months

Summary
Neutrophils-first line of defence NADPH OXIDASE CGD Other neutrophil disorders Lymphocytosis-benign disorder-I.M.