neutrophils and their disorders

8/6/2019 Neutrophils and Their Disorders

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NEUTROPHILS AND THEIR

DISORDERS

READING:D Roos et al. Microbes and Infection5 (2003) 1307-1315.

OMIM-Chronic Granulomatous Disease


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WHITE CELLS

TWO BROAD GROUPS:

PHAGOCYTES :NEUTROPHILS, EOSINOPHILS, BASOPHILSMONOCYTES

IMMUNOCYTES:LYMPHOCYTES, PLASMA CELLS


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NEUTROPHIL Granules

Nucleus


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EOSINOPHIL


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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


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NEUTROPHIL

Multi lobed 3-5 lobes

Mature Neutrophil observed in peripheralblood

Contains primary and secondary granules


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Granules

Primary

-GalactosidaseMyeloperoxidaseEsteraseLysozyme

Secondary (Specific)

Elastase

Components ofNADPH OxidaseCollagenaseLysozymeLactoferrinTranscobalamins(TC I and TC II)


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FUNCTION

PRIMARY FUNCTION - DEFENCE

(1)Killing Microoraganisms(2)Phagocytosis


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NADPH OXIDASE


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NADPH OXIDASE

FUNCTION TO GENERATE SUPEROXIDE

O2

COMPONENTS:

TWO SUBUNITS a) 90 kDa B) 22 kDa

These contain a cytochrome b (-245) and ahaem function.


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NADPH OXIDASE

OTHER COMPONENTS:

A FLAVIN

RAP IA

Cytosolic components p47 phox(PKC dependent)

P67 phox, p40 phox and rac


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NADPH??

NADPH is derived from the hexoseMonophosphate shunt

Activation of NADPH oxidase involves theConsumption of a lot of oxygen.

This is called the RESPIRATORY BURST asIt was first observed using oxygen electrodeexperiments


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CELL SIGNALLING

R

GPLC

NADPH

OXIDASE

PKC47K

67K

40K rac

PIP2 DAG

PLD

PKCIP3Ca 2+

PtdCho

O2 O2

FMLP


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ACTIVATION FACTORS

1) fMet-Leu-Phe Formyl peptide derived frombacteria

At low concentrations ChemotacticConcentrations activator of NADPH Oxidase

2) Complement component C5a

3) Leukotriene B4 (LTB4)-neutrophil origin


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Priming AgentsPriming increases the response of theNeutrophil following its activation.

Examples:

GM-CSF-sites of infection /inflammation

TNF

In Vitro- cytochalasin B


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PRIMING AGENTS

PRE-TREAT ISOLATED NEUTROPHILS WITHGM-CSF. (Note INCREASE IN SUPEROXIDEPRODUCTION).

Time

CYT C

RE

D

+ GM-CSF

-GM-CSF

FMLP


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KILLING

NADPH

OXIDASE

1

12

2

MPO

O2 H2O2 HOCl

Plasma

membrane

Gram +ve

CYTOSOL Granules


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Granules


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What Can Go Wrong?

A) Chronic Granulomatous Disease (CGD)

Reduced expression of NADPH OxidaseLarge subunit (90kDa)

Encoded on X chromosome This accountsFor 80% of CGD ( 1in 1,000,000)


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OUTCOMES

Patients present in early infancy

Recurring bacterial or sometimes fungalInfections.

Further details on subtypes and genetics

See OMIM (On-line mendelian InheritanceIn man)


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Lab Tests

a) Cytochrome c reduction

b) Chemiluminescence

c) Tetrazolium dye


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Cytochrome C

Cyt (Fe (III)) Cyt c Fe (II)

O2

550 nm

Isolated Neutrophils activated with eitherFMet-Leu-Phe or PMA

PMA Phorbol ester directly activates Proteinkinase C.


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TIME (min)

550nmr

eductionof

Cytochromec

Normal

CGD

Cytochrome c reduction

FMLP


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CHEMILUMINESCENCE

LUMINOL

FLASHES

OF LIGHTOXIDANTS eg HOCl


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Time (min)

Ch

em

ilum

in

escen

cemV

Normal

CGD

fMet-Leu-Phe


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PMA

TIME (min)Ch

em

ilum

in

escen

cemV

Normal

CGD


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Nitroblue Tetrazolium

Normal

Blue product

CGDNo reduction


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Other Rare Disorders

a) Myeloperoxidase deficiency

b) Chediak- Higashi Syndrome

Giantgranules
http://eduserv.hscer.washington.edu/HuBio552/hemedist/hemecases/Intro/1628.jpg


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Chediak-Higashi Syndrome

Autosomal recessive

Giant graules in neutrophils /eosinophils

Monocytes and lymphocytes

Neutropenia, thrombocytopenia

Partial albinismAffected children tend to die from infectionOr haemorrhage


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MORPHOLOGICAL DISORDERS

a) Pelger Huet-autosomal dominantBilobed shape


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b) May-Hegglin-Autosomal dominantGiant platelets and inclusion of RNA in

neutrophils


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Alders anomaly-Deep purple granules
http://images.google.co.uk/imgres?imgurl=http://www.kalpesh.itgo.com/dohle%2520body-1.JPG&imgrefurl=http://www.kalpesh.itgo.com/PS8.htm&h=137&w=192&sz=17&tbnid=oi9RkEpJUxwJ:&tbnh=69&tbnw=97&start=6&prev=/images%3Fq%3DAlder%2527s%2Banomaly%26hl%3Den%26lr%3D%26sa%3DG


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INFECTIOUS

MONONUCLEOSIS

Infectious mononucleosis (I.M.)

(Glandular fever.

Symptoms: lethargy, swollen glandsLymphadenopathy, rash

Rise in white cell count: ~10-20 x109/l

Absolute lymphocytosis


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INFECTIOUS

MONONUCLEOSISIn Blood-Presence of atypical lymphocytes


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What are they?

B-Lymphocytes are infected with Epstein BarrVirus.

Atypical Lymphoctes are reactiveT-lymphocytes towards the infectedlymphocytes. Usually peak between 7-10 daysOf the illness.


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SEROLOGY

Heterophile antibodies are raised in I.M.

These can react with antigens fromother species e.g. they canAgglutinate horse red cells


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SEROLOGY

Paul Bunnel Test:

I.M. antibodies can be adsorbed byOx red cells

I.M. Antibodies are NOT adsorbed byGuinea Pig kidney cells

I.M. antibodies can agglutinate horse red cells


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PAUL BUNNELL TEST

Normal plasma

I.M.

GPK Ox red cells

After mixing plasma with GPK and Ox red cellsAdd horse red cells. GPK does not adsorb abSo it can then agglutinate added horse red cells

- -

+ -


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ANTIDODIES

Heterophile Ab(IgM)

3 months 6 months

Anti-viralCaspid (IgG)

Anti-EBNA

EBNA -anti EB nuclear antigen


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Summary

Neutrophils-first line of defence

NADPH OXIDASECGDOther neutrophil disordersLymphocytosis-benign disorder-I.M.

neutrophils and their disorders

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