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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)
40
9/
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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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