Pmn’s and Its Abnormalities

8/12/2019 Pmns and Its Abnormalities

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PMNs AND ITS ABNORMALITIES


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INTRODUCTION

Periodontal diseases involve

A local infection

A host response that may result in connectivetissue alteration.

Leucocytes are critical to periodontal defense, as

most potential periodontal pathogens areknown to be resistant to the antimicrobialmechanisms of serum.


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Control of Local Infection

ACUTE INFLAMMATION

Three primary leucocytes participating in immuneresponse to periodontal diseases are

NEUTROPHILS , MONOCYTES and LYMPHOCYTES.The leucocytes infiltrate the gingiva in a temporal

and spatial order, suggesting that the strategy ofhost defense against periodontal infections is

similar to that used to combat most other localinfections.(Miyaski KT . THE NEUTROPHIL : MECHANISMS OF CONTROLLING PERIODONTAL BACTERIA. JPeriodontol 62:761, 1991 {55} ) .


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CHRONIC INFLAMMATION

It begins with the infiltration of monocytes and

lymphocytes . Unlike neutrophils , monocytesand lymphocytes primarily infiltrate

connective tissue and develop into tissue

macrophages and activated lymphocytes .


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Normal neutrophil function

Neutrophil can crawl at a rate of about 400micrometer / hour .

This may seem fast but it actually amounts to

only about 40 cell lenghts per hour . The primary role of the neutrophil is the

destruction of pathogens that threaten tissuesoutside of the blood , including the periodontium

For this reason the blood , finding targets , andkilling targets are important phagocyte functionsin the defense of the periodontium .


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Normal functions

Adherence

Chemotaxis

Phagocytosis Metabolic response

Bacteriocidal capacity

Digestion

Secretion


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Leukocyte cellular events

Leukocytes leave the vasculature routinely through

the following sequence of events:

Margination and rolling

Adhesion and transmigration Chemotaxis and activation

They are then free to participate in:

Phagocytosis and degranulation

Leukocyte-induced tissue injury


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Leucocytes activation


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Margination and Diapedesis

With increased vascular permeability, fluid leaves thevessel causing leukocytes to settle-out of the centralflow column and marginate along the endothelial

surface

Endothelial cells and leukocytes have complementarysurface adhesion molecules which briefly stick andrelease causing the leukocyte to roll along theendothelium like a tumbleweed until it eventuallycomes to a stop as mutual adhesion reaches a peak


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Margination and Rolling

Early rolling adhesion mediated by selectin

family:

E-selectin (endothelium), P-selectin (platelets,

endothelium), L-selectin (leukocytes) bind other

surface molecules (i.e.,CD34, Sialyl-Lewis X-

modified GP) that are upregulated on

endothelium by cytokines (TNF, IL-1) at injury sites


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Transmigration (diapedesis)

Occurs after firm adhesion within the systemic

venules and pulmonary capillaries via PECAM

1 (CD31)

Must then cross basement membrane

Collagenases

Integrins


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Transmigration (diapedesis)

Early in inflammatory response mostly PMNs,

but as cytokine and chemotactic signals

change with progression of inflammatory

response, alteration of endothelial celladhesion molecule expression activates other

populations of leukocytes to adhere

(monocytes, lymphocytes, etc)


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SELECTINS

Selectins, so called because they are characterized by anextracellular N-terminal domain related to sugar-bindingmammalian lectins, consist of E-selectin (CD62E, previously knownas ELAM-1), which is confined to endothelium; P-selectin (CD62P,previously called GMP140 or PADGEM), which is present in

endothelium and platelets; and L-selectin (CD62L, previously knownby many names, including LAM-1), which is expressed on mostleukocyte types.

Selectins bind, through their lectin domain, to sialylated forms ofoligosaccharides (e.g., sialylated Lewis X), which themselves arecovalently bound to various mucin-like glycoproteins (GlyCAM-1,

PSGL-1, ESL-1, and CD34). The immunoglobulin family molecules include two endothelial

adhesion molecules: ICAM-1 (intercellular adhesion molecule 1) andVCAM-1 (vascular cell adhesion molecule 1).


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INTEGRINS

Integrins are transmembrane heterodimericglycoproteins, made up of a and b chains, thatare expressed on many cell types and bind to

ligands on endothelial cells, other leukocytes,and the extracellular matrix.

The b2 integrins LFA-1 and Mac-1(CD11a/CD18 and CD11b/CD18) bind to ICAM-1, and the b1 integrins (such as VLA-4) bindVCAM-1.


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MUCIN

Mucin-like glycoproteins, such as heparan

sulfate, serve as ligands for the leukocyte

adhesion molecule called CD44. These

glycoproteins are found in the extracellular

matrix and on cell surfaces.


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Chemotaxis

Leukocytes follow chemical gradient to site of injury

(chemotaxis)

Soluble bacterial products

Complement components (C5a)

Cytokines (chemokine family e.g., IL-8)

LTB4(AA metabolite)

Chemotactic agents bind surface receptors inducing

calcium mobilization and assembly of cytoskeletal

contractile elements


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Chemotaxis and Activation

Leukocytes:

extend pseudopods with overlying surfaceadhesion molecules (integrins) that bind ECM

during chemotaxis undergo activation:

Prepare AA metabolites from phospholipids

Prepare for degranulation and release of lysosomal

enzymes (oxidative burst) Regulate leukocyte adhesion molecule affinity as

needed


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Phagocytosis:

Derived from the Greek words Eat and cell.

Phagocytosis is carried out by white blood cells:macrophages, neutrophils, and occasionally

eosinophils.

Neutrophils predominate early in infection.

Wandering macrophages: Originate from

monocytes that leave blood and enter infected

tissue, and develop into phagocytic cells.

Fixed Macrophages (Histiocytes): Located in liver,nervous system, lungs, lymph nodes, bone marrow,

and several other tissues.


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Phagocytic Cells: Macrophages (Monocytes),

Neutrophils, and Eosinophils

(Macrophages)


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Stages of Phagocytosis

1. Chemotaxis: Phagocytes are chemically attracted

to site of infection.2. Adherence: Phagocyte plasma membrane

attaches to surface of pathogen or foreign

material.

Adherence can be inhibited by capsules (S.

pneumoniae) or M protein (S. pyogenes).

Opsonization: Coating process with opsonins that

facilitates attachment. Opsoninsinclude antibodies and complement proteins.


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Phagocytes are Attracted to Site of Infection

by Chemotaxis

S f Ph i (C i d)


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Stages of Phagocytosis (Continued)

3. Ingestion: Plasma membrane of phagocytes

extends projections (pseudopods) which engulf the

microbe. Microbe is enclosed in a sac calledphagosome.

4. Digestion: Inside the cell, phagosome fuses with

lysosome to form a phagolysosome.Lysosomal enzymes kill most bacteria within 30

minutes and include:

Lysozyme: Destroys cell wall peptidoglycan

Lipases and Proteases

RNAses and DNAses

After digestion, residual bodywith undigestable

material is discharged.


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Process of Phagocytosis


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Recognition and Atachment

Opsonized by serum complement,

immunoglobulin (C3b, Fc portion of IgG)

Corresponding receptors on leukocytes (FcR,

CR1, 2, 3) leads to binding


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Contd.

Triggers an oxidative burst (next slide)

engulfment and formation of vacuole which

fuses with lysosomal granule membrane

(phagolysosome)

Granules discharge within phagolysosome and

extracellularly (degranulation)


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Oxidative burst

Reactive oxygen species formed throughoxidative burst that includes:

Increased oxygen consumption

Glycogenolysis Increased glucose oxidation

Formation of superoxide ion

2O2+ NADPH 2O2-rad + NADP+ + H+

(NADPH oxidase)

O2 + 2H+ H2O2 (dismutase)


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Reactive oxygen species

Hydrogen peroxide alone insufficient

MPO (azurophilic granules) converts hydrogenperoxide to HOCl-(in presence of Cl-), an

oxidant/antimicrobial agent Therefore, PMNs can kill by halogenation, or

lipid/protein peroxidation


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Degradation and Clean-up

Reactive end-products only active within

phagolysosome

Hydrogen peroxide broken down to water and

oxygen by catalase

Dead microorganisms degraded by lysosomal

acid hydrolases


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Leucocytes abnormalities


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Disorders of Neutrophil Numbers

Definition

NeutropeniaLess than 1500/l

NeutrophilaGreater than 7700/l

Acquired

Or

Inherited

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J. Levine


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Definition of Neutrophilia - too many

Normal ANC is 1500-7700/l Neutrophilia: abnormally high ANC

Shift to the left: d release of precursors

from the bone marrow

not necessarily associated with

neutrophilia

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NEUTROPHILIA

Often accompanied by other reactive changes, e.g. atypical lymphocytes.

Varied aetiology, e.g. excercise, emotional (anger, stress), inflammation, infection,chemicals or drugs (glucocorticoids, epinephrine, G-CSF), tumours, smoking.

Left shift results from increased granulopoiesis.

Leukaemoid reactions must be distinguished from CML.

Morphological changes include Dhle bodies (aggregates of polyribosomes), toxic

granulation (persistence of primary granules) and cytoplasmic vacuolation.


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Neutrophilia

Chronic Stimulation Excess cytokine stimulates

proliferative pool

Causes: Infection

Down's Syndrome

Pregnancy/Eclampsia

Chemotherapy recovery

Myeloproliferative disorders Marrow metastases

Acute shift frommarginating tocirculating pool measured WBC, not total

WBC

Causes: Steroid treatment Exercise Epinephrine Hypoxia Seizures Other stress

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Example: exercise induced neutrophilia

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Neutropenia: too few

Neutropenia

Definition: ANC < 1500/l

ANC 500-1000 increased risk of infection from

exposure

ANC < 500: increased risk of infection from host

organisms

African-Americans: lower normal neutrophilcounts (1000-1200)

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NEUTROPENIA(


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Acquired Causes of Neutropenia

DecreasedProduction

IncreasedDestruction

Shift toMarginating Pool

Bone marrow Peripheral

circulation

Move from the

circulating pool to

attach along the

vessel wall

Medication:

Chemotherapy

Antibiotics, etc

Autoimmune

diseases

(Rheumatoidarthritis, SLE, etc)

Severe infection

Endotoxin release

HemodialysisCardiopulmonary

bypass

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Increased Destruction

Anti-neutrophil

antibodyNeutrophil-Antibody

Complex

Uptake and

destruction of

neutrophil by the

RE system

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Shift to Marginating Pool

Circulating

Marginating

Circulating

Marginating

Severe infection / Endotoxin release

Hemodialysis

Cardiopulmonary bypass

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Evaluation of Neutropenia

If visit prompted by a fever and ANC islow, treat promptly for infection

Suspect medication: major cause of

neutropenia If no culprits, bone marrow exam for:

Malignancy

Infiltration by non-marrow cells Arrest of cell growth

Myeloproliferative disorder

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Cyclic Neutropenia

21 day cycle

autosomal dominant

fever, mouth ulcers

Treatment G-CSF

usually improves after

puberty

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Congenital Neutropenia

Maturation arrest

frequent infections,often serious

mouth sores may lose teeth or

develop severe guminfections

Increased risk ofleukemia

Tx: G-CSF, BMT

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Role of Neutrophil

Responds to chemotactic factorsreleased from damagedtissue

Rolls and attachesto the endothelial cell wall

protein and carbohydrate interactions (selectins and their ligands).

Becomes activatedby chemotactic factors

Tightly adheresthrough the integrin family of proteins.

Migratesacross the endothelial cell wall.

Phagocytizesorganisms so that they are contained within avesicle or phagosome.

Releases granule productsand reduced oxygen species (e.g.hydrogen peroxide and superoxide) to kill organisms

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Function of the Circulating Neutrophil

Chemoattractant

Attachment/rolling Activation Adhesion

Migration

Phagocytosis

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Disruption of Neutrophil Function

Steps where defects in structural components

of neutrophils results in impaired ability to

fight infection

Recruitment from the circulation

Adhesion and subsequent migration

Defective production in active oxygen metabolites

Deficiency in granules

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Defect in Attachment/Rolling

Attachment/rolling

Sialyl LewisX

Selectins

Cell surface molecules expressing Sialyl Lewis X

interact with selectin proteins on the cell

surface of endothelial cells

LAD-2 Impaired expression of sialyl LewisX -

Neutrophils do not attach and are not recruited to the site of inflammation

Chemoattractant

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Defect in Adhesion

Chemoattractant

Adhesion

Integrins on the surface of

neutrophils mediate tight adhesion

to the endothelial cell wall.

Cells then migrate.

Migration

Integrin

LAD-1 results from a defect in leukocyte integrins.Decreased to absent expression on the cell surface.

Cells can not adhere and subsequently cannot migrate.

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Clinical manifestations: LAD

53Source Undetermined (Both Images)


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Phagocytosis

Chemoattractant

Bacteria are engulfed and contained in a phagosome.

Contents of the granules are released.

Oxygen metabolites (superoxide and H2O2) kill bacteria

CGD: NADPH-Oxidase-defective

Cannot produce active oxygen species

Chediak-Higashi Syndrome: Defect in granule formation

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Chediak-Higashi Syndrome

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Chediak-Higashi Syndrome

Oculocutaneous

albinism

Photophobia

Sun sensitivity

Neuropathy

Infections, esp Staph

aureus

TX: BMT

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Chronic granulomatous disease (CGD)

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Chronic granulomatous disease: CGD

Catalase positive organisms

Staph aureus

Serratia marcescens

Burkholderia cepacia

Fungal

Skin, lungs, bones, abscesses

Granuloma formation from chronic infection

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Myeloperoxidase deficiency

One of the more common disorders

1: 4000

Decreased production of hypochlorous acid

(HOCl)

Killing takes longer than normal

Clinically silent for most people

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Diseases with Neutrophil Defects

Disease Step Molecular DefectLAD-2 Rolling Sialyl Lewis XCarbohydrate

LAD-1 AdhesionPhagocytosis

Integrinexpression

Chediak-HigashiSyndrome

MigrationDegranulation

Vacuolar sortingprotein (largegranules interferewith traversing

endothelial wall)

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Diseases with Neutrophil Defects

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LEUKOCYTE ADHESION DEFICIENCY

TYPE I :

LAD1 is an autosomal disorder (localized to

chromosome 21q22.3) characterized by the

inability of individuals to express beta2subunit (CD18) common to leukocytes

integrins LFA-1 , Mac1 and p 150/95 .

Periodontal disease is related to if one or twodefective alleles are present .

TYPE II


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TYPE II

There appears to be a selectinligand

deficiency i.e., the leukocytes do not expresssialo-Lewis x or gp150-Lewis x , referred to as

leucocyte adhesion deficiency, type II , in

which neutrophil rolling doesnot increase in

response to inflammation .

Individuals with this deficiency suffer from

recurrent bacterial infections, neutrophilia

and severe early onset bacteria .


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Localized juvenile periodontitis

Neutrophils with LJP exhibit a selective decrease inability to kill A. actinomycetamcomitans, despitenormal phagocytosis, oxygen production, and secretionof specific granule components.

Neutrophils from individuals with the classic form ofLJP are characterized by a decrease in chemotacticresponses to a variety of chemotactic factors, includingC5a, FMLP and leukotriene B4.

The neutrophil dysfunction is associated with afunctional decrease in chemotaxin receptors on thePMN surface.


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Pmn’s and Its Abnormalities