ROLE OF NEUTROPHILS IN

PERIODONTAL DISEASE

PRESENTED BY GUIDED BY
SHEETAL OSWAL DR C D DWARAKANATH
CONTENTS
 Introduction
 Role of phagocytes in host defense
 Neutrophil function & dysfunction
 Neutrophils & periodontal tissues
 Altered neutrophil functions &
periodontitis
-Chronic periodontitis
-Localised aggressive periodontitis
-ANUG
 Neutrophil defects-classification
 Periodontal disease associated with
neutrophil abnormalities
 Neutrophils & periodontal tissues in
systemic diseases
 Anti neutrophil therapies
-Lipoxins
-ATL
 Neutrophil assays
 Summary & conclusions
INTRODUCTION
 Why study neutrophils in periodontics???
 Is the role of neutrophils in periodontal
tissues same as in other regions???
 Are the systemic diseases with neutrophil
defects a threat to periodontal tissues???
 Role of periodontist in such conditions…..
ROLE OF PHAGOCYTIC CELLS IN HOST DEFENSE
• Neutrophils & macrophages are critical in host defense
against bacterial infections. When phagocytic cell
number of function is compromised, disease progression
& severity is markedly increased. Periodontal disease is
a common sequelae associated with altered phagocytic
response
• Neutrophils are important in periodontal disease because
they control the periodontal microecology prior to
involvement of chronic inflammatory cells. In contrast
monocytes & lymphocytes dictate tissue responses in
periodontal microecology.
• Thus it may be proposed simplicitically that either
hypofunction or altered PMN function or
hyperfunction of monocytes/ lymphocytes may result
in increased susceptibility to periodontal disease

• Also, though they are essential for host defense, these

phagocytic cells can cause some damage to healthy
tissues- bystander effect. The junctional epithelium is
particularly at risk of such damage because PMN’s
secrete their enzymes & toxins on bacteria which
adhere to it , damaging epithelial cell underneath
PMN functions can be categorized as follows:

1. Neutrophil rolling & Margination

2. Adhesion
3. Diapedesis/ transendothelial migration
4. Chemotaxis
5. Extracellular secretion, receptoe upregulation &
binding to target
6. Phagocytosis
7. Microbial killing
Functions of Neutrophils
 INFLAMMATION
INFLAMMATION
PMN’S ADHERE TO ENDOTHELIUM

L-selectins on PMN’S
Local inflammation interact with endothelium
IL-1beta & TNF Rolling
from mast cells &
leucocytes

Endothelium to express P & E selecins on

luminal surfaces
Releases
chemokines P & E selectins+ L selectins
Signal fo Rolling
arrest Increased rolling
Chemokines interact with PMN’s shed selectins
PMN receptor CxCR2 & upregulate integrins

LFA-1(Integrin B2)

Adhere to ICAM-2 on endothelium

Rolling arrest & strong adhesion

CD31(E) + CD31(L)
PMN’s locate inter endothelial junction

Zipping & unzipping DIAPEDESIS

Chemotaxis- leucocyte’s ability to sense a chemicalgradient
e its increasing concentration.
& migrate in the direction of
Chemical gradient is termed as chemotaxins for which it
has receptors called chemotactic receptors
Two types
Exogenous - Directly derived from bacteria
e.g. N-Formyl Methionyl Peptide (FMLP)
Endogenous – Those produced within the body e.g
TNF, IL-8, C5a, Leukotrines B4, neutrophil chemotactic
factor & platelet activating factor
FMLP is a modified amino acid present in most of bacteria
but not in humans-thus it serves as a tell tale sign that
bacteria are present within host tissues
 chemotaxis

Chemotactic receptors
G-coupled family
on PMN

Receptor upregulation & binding to target

IgG & iC3b Recognise pathogen

Opsonisation

Phagocytosis

Delivery of antimicrobial substances Microbial killing

Phagocytosis & Killing
 VARIOUS OPSONINS & OPSONIN RECEPTORS

Target Opsonin Receptor

Gram –ve bacteria LPS binding CD14

protein

Any cell iC3b CR3, CR4

Any cell IgG1, IgG2, IgG3 FcγRII,

Microbial killing

Neutrophils are granular leucocytes. These granules

are distinct & adapted to perform several functions.
Broadly classified into 3 categories:
Azurophilic /Primary granules
Specific/secondary granules
Secretory/ teritiary granules
Microbial killing Specific granule Microbial
s killing
Acid hydrolases -Lysozyme Gelatinase
- Cathepsin B - Apolactoferrin
- Cathepsin D - Collagenase
- Cathepsin G
- Cobalamine binding
- Chloroacetate esterase
protein
- Elastase
- Beta glucoronidase
- C5a cleaving enzyme
- Beta galactosidase - Plasminogen
- L-mannosidase activator.
- L-fucosidase - Cytochrome b558
- Beta glycerophosphatase
- Arylsulphatase
- Lysozyme
- Myeloperoxidase
- Defensins
-BPI
• Granule secretions are used as markers for
neutrophil activity.
• markers of Azurophilic granules are
myeloperoxide & glycosidase
• Markers of specific granules are lactoferrin & Vit
B12 binding protein
• Territiary granules are more readily & rapidly
secreted.Their contents are believed to play an
important role in adhesion & in replenishment of
cell surface receptors
• Deficiency of granules /its contents results in
impaired microbial killing- thus impaired host
defense
 Delivery of antimicrobial substances
• Neutrophils deliver antimicrobial substances by 4
mechanisms
-Delivery of oxygen metabolites-respiratory burst
-Extracellular secretion
-Phagocytosis- intraphagolysomal
-Cytolysis & Death

Microbial killing takes place either intraphagolysomal or

by extracellular secretion
 Mechanisms of killing bacteria

-Two mechanisms-Oxidative & Non oxidative

-The non oxidative mechanisms in general are based

on membrane disruptive antibiotic activities of
peptides, They do not require oxygen nor release
of toxic metabolites. They undergo degranulation
to release cytosolic contents & kill bacteria
-They also play a important role in preventing
bacterial colonization
-
 Non oxidative killing of periodontal bacteria
-The potential non oxidative mechanisms are known to
kill putative pathogens such as A.a & Capnocytophaga

- As highly anaerobic conditions persist in periodontal

pocket, this mechanism is of particular interest. More
than 50% of A.a is killed by this mechanism

-A.a is killed by enzymes such as lactoferrin, defensins,

& neutral serene proteases. Capnocytophaga sp is also
killed by defensins & NSP.
Cathepsin G kills most periodontal bacteria in hypoxic
a
conditions by both enzymatic (Cap sp) & non
enzymatic ( A.a) degradation.
It potentiates killing of bacteria by lysosyme, BPI, &
MPO-H2O2-Cl system-Respiratory burst
It enhances PMN phagocytosis & promote complement
mediated granulocyte chemotactic activity.
(Mechanism of controlling bacteria-Kenneth Mayasaki)
-
The oxidative mechanism of killing bacteria are mediated
by 2 entities- NADPH oxidase system & MPO system.

-The oxidative mechanisms require the presence of

oxygen & an oxidation reduction potential@>160mv

-Neutrophil stimulation results in increase oxygen

consumption by cell which leads to its activation. This
leads to release of NADPH
-
NADPH is oxidized to NADP on outer surface of PMN &
this leads to production of superoxide
2O2 +NADPH—2O2- +NADP+ +H+
2O2 +2H—O2 + H2O2
The superoxide is converted to H2O2 in the presence of
superoxide dimutase. Further in presence of H2O2.
myeloperoxide catalyses to form HOCL acid which is
lethal to most microbes
MPO-H2O2 +Cl—HOCL

HOCL further chlorinates to form chloramines (bactericidal)

 Oxidative killing of periodontal bacteria

Oxidative killing of A.a by H2O2 requires 10 times higher

conc than that anticipated in phagolysome. Further its
killing by H2O2 & oxygen is blocked by deffuroxime ,
suggesting the bactericidal activity of H2O2 against A.a
may be due to iron catalyzed reaction.

A.a is rapidly killed by MPO-H2O2-Cl system. It also

neutralizes the leucotoxin produced by A.a. It also
blocks adherence of A. viscosis & oral streptococci to
saliva coated hydroxyapatite.
OxidativeNeutrophil and
killing of A.a by H periodontal
2O2 requires tissues
10 times higher conc than that
anticipated in phagolysome. Further its killing by H2O2 & oxygen is
blocked by deffuroxime , suggesting the bactericidal activity of H2O2
against A.a may be due to iron catalyzed reaction.

A.a is rapidly killed by MPO-H2O2-Cl system. It also neutralizes the

leucotoxin produced by A.a. It also blocks adherence of A. viscosis & oral
streptococci to saliva coated hydroxyapatite.

-Plaque mo’s do not normally enter the tissues, so in order to kill them,
neutrophils must leave the tissues & enter gingival crevice or periodontal
pocket.

- PMN’s form a layer on the surface of plaque, but cannot phagocytose the
adherent bacteria which are embedded in plaque matrix. They secrete their
enzymes & kill bacteria externally without phagocytosis

-Both opsonised & unopsonised bacteria are susceptible to killing but

opsonisation increases the efficiency
-
Unattached bacteria could be killed in traditional
manner, but this is unusual because:

• Neutrophil function is inhibited by microbial

factors such as endotoxins, formyl peptides & by host
factors like degraded antibody, complement &
protease inhibitors in crevicular fluid which inhibits
phagocytosis by blocking surface receptors
• The low oxygen concentration & redox potential
in deep pockets also inhibits neutrophil function
 PMN’s produced in bone marrow

LAD-2 rolling

LAD1 Strong adhesion & diapedisis

Actin chemotaxis
Diabetes
dysfunction
phagocytosis
CGM Chediak Higashi
Myeloperoxide Microbial killing
deficiency
Degradation of mo’s
 Altered phagocytic function & Periodontal disease
-Periodontal disease is common sequelae associated
with compromised phagocytic no/ function
-For some agrressive periodontal diseases, a strong
association altered PMN function & disease has
been reported
-Neutrophil mediated tissue injury in periodontium
can cause destruction of attachment apparatus &
bone loss
-Functional abnormalities of PMN’S have shown to
be important in various disease entities, of which
periodontitis is a commom sequelae
 Altered phagocyte function & aggressive periodontitis

• Aggressive periodontitis is a clinically distinct, well

characterized form of destructive periodontitis with
circumpubertal onset, localization of bone & attachment
loss to first molars & incisors, chemotactic defects,
familial association & strong association with
A.actinomycetecomitans infection

• Altered phagocyte function has been used as a model for

understanding periodontal pathology in LJP
 Neutrophils in gingival crevice
Expression of Increased adhesion
CD11/CD18
Vascular adhesion

GP-110 Locomotion & migration

chemotaxis
Receptor for FMLP & chemotaxis
C5a & LB4 Receptor expression
Receptor
Receptor
Phagocytosis expression
expression
phagocytosis?
CYTOKINES superoxide

Microbial killing killing

killing
Altered neutrophil function –induced or intrinsic???
SudharAgarwal et al JP,96;67
The chemotactic defect is irreversible by treatment &
appears to be intrinsic to LJP neutrophils

Although patients exhibit a genetic predisposition to

LJP , the collective functional changes associated
with LJP neutrophils have as yet to be linked to
common genetic elements.

This is further complicated by following facts:

• Not all pts with clinically diagnosed LJP exhibit
e
decreased chemotaxis(70-75%)
• LJP pts appear to be healthy & have not been
documented to exhibit increased susceptibility to
other infections, as would be expected in pts
exhibiting impaired neutrophil functions.
• The manifestations of this disease i.e , massive
tissue damage & bone loss occur in presence of a
relatively low bacterial load
The inability to place these collective observations
into a clear unified hypothesis suggests that
intrinsic cellular defects may not be responsible in
altered PMN function in LJP
• The following observations suggest that extrinsic
factors in sera may alter neutrophil functions in LJP

– In response to bacterial challenge, no of

cytokines are induced by immune cells & carried
through blood .If they contact neutrophils they
alter function of neutrophils.
– LJP sera is specific, sustained & cannot be
reversed by placing LJP serum treated
neutrophils in healthy serum
– Also healthy neutrophils treated with LJP sera
function similar to LJP neutrophils
 Induction of cytokines
Contact neutrophils
TNF Alter function of PMN IL-
alter
1B
Chemotaxis & adherence Superoxide anions &
chemotactic receptors degranulation

Reduced migration of PMN to site of infection

THUS NEUTROPHILS EXPOSED TO CYTOKINES

EXHIBIT ALL THE CHARACTERISTICS OF LJP PMN’s
In conclusion, present evidence states that cytokines
produced in response to infections can alter the
functions of neutrophils. This increased level of
cytokines is a result of hyperactivation of monocytes
& it can exert significant effects both locally &
systemically

Increased cytokine production locally leads to excessive

bone loss & tissue damage in periodontium, while
systemic increase could lead to priming of
neutrophils, increased proliferation of lymphocytes &
antibodies
• An overaggressive immune response can thus
provide a basis for unified explanation for observed
altered neutrophil functions, severe tissue damage &
bone loss in periodontium, familial nature & other
immunological findings associated with
pathogenesis of this disease.
(Sudha Agarwal et.al J.P-1996)
 Altered neutrophil function & chronic periodontitis

PMN mdiated Delayed neutrophil

Tissue injury Apoptosis

Bacteria & its

products modulate
PMN function
A. Neutrophil mediated tissue injury was first
demonstrated by Deguchi. et.al

a. Oxygen radical sp produced by PMN’s can attack

every biologically relevant molecule & cause
damage. In addition, they also modulate various
cellular activities which are mediators in sequence
of events leading tot tissue injury.
O2 NO
H2O2 vascular adhesion & activation of
PAF
b. PMN degranulation releases several proteolytic
enzymes that can cause s host tissue damage
i. Crevicular fluid PMN’s release upto 5 times
more elastase & collagenese than peripheral
blood PMN’s in pts with periodontitis.They
hydrolyse several extracellular matrix proteins
& generate peptide fragments that are
chemotactic to monocytes
ii. Lamster et al has shown that these pts display
enhanced macroglobulin levels, IgM in GCF &
B-glucornidase activity
iii. Lactoferrin enhances PMN adhesiveness & is
synergistic with its enzymes.
c. Activated PMN’s also release proinflammatory
e B4 & PAF that are
mediators such as leukotrine
potent stimulators of neutrophil chemotaxis,
adhesion, oxidative burst & degranulation, thus
amplifying neutrophil mediated tissue injury.

They have capacity to cleave complement

components via alternate pathway &t o activate
kinin system reactions , which in turn perpetuate
& magnify inflammation
 Neutrophil mediated tissue injury

O2 & H2O2
LTB 4
PMN degranulation
& PAF
releases proteolytic
PAF enzymes

Tissue factor Magnify

synthesis inflammation
Reduce
catabolism of Generate
PMN’s chemotactic
substances Host tisssue damage
• B. Bacteria & its products modulate PMN function
• Bacterial products such as LPS & proteinases have
ability to modulate neutrophil response. They act
indirectly on cellular constituents of gingival tissues
activating cellular factors that induce destruction of
connective tissue & bone

• LPS produced from different bacteria varies. LPS

from A.a enhances chemotaxis via chemokinetic
effects, whereas P.gingivalis LPS inhibits
chemotaxis. (Shapira)

• Also LPS activated neutrophils led to damage of Pdl

fibroblasts by increase adherence of PMN to
fibroblasts (Deguchi)
Bacteria may directly interfere with neutrophil
phagocytosis by modulating complement activity
Proteolytic activity of P.gingivalis is a important
virulence factor.It has ability to degrade C3 & C5 in
human sera (Scheinkein) & further prevent the
accumulation of C3b on bacterial surface. This
prevents opsonic activity & interferes with
neutrophil phagocytosis.

Thus production of proteases represents a primary role

in periodontal destruction & inhibition of
phagocytosis in susceptible individual represents a
potential secondary risk factor.
Altered neutrophil
function &
chronic
periodontitis
Delayed neutrophil apoptosis & periodontitis

Circulating neutrophils have a short half life & onset of

apoptic process is associated with loss of several
important functions such as adhesion & phagocytosis
(Dransifield 1998) ,which eventually leads to their
clearance from lesion by macrophage ingestion thus
promoting the resolution of inflammation (Simon)

This constitutive tendency to undergo apoptosis prevents

neutrophils from lingering at the infection site & limits
their proinflammatory potential (Haslett)
However cytokines such as TNF-alpha & GM-CSF
may delay neutrophil apoptosis by increasing their
mitochondrial stability, reducing caspase activity
& downregulating gene expression
(Tsiyjmoto & Shimizir 2000)
Recent studies have shown that bacterial products
isolated from different strains of P. gingivalis also
delay neutrophil apoptosis in dose dependent
manner ( Preshaw et al 1999)
 Role of neutrophils in ANUG
Listgarten noted that invasion of spirochetes in ANUG
lesions is broadly grouped into 4 zones.
Bacterial zone
Neutrophil rich zone
Necrotic zone
The zone of spirochete infiltration
 Neutrophil defects

Quantitative Qualitative
Defects in adhesion-
LAD1 & LAD2
Neutropaenia
Defects in chemotaxis

Defects in phagocytosis

Defects in microbicidal
activity
 Neutropaenia
Production defects Destructive defects

Aplasia
Splenic
Infiltrative diseases sequestration

Drugs

Metabolic diseases Antineutrophil

antibodies
Infective diseases
Kostmann syndrome
Normal neutrophil count-1800-7200 cells/cumm
Neutropaenia occurs when count is > 2000 cells/cumm
moderate-> 1000cells/cumm
severe- >500 cells/cumm
Very severe- >200 cells/cumm ( inability to mount an
inflammatory response)

– Lower limit of neutrophil cell count is 1x10 9

cells/litre in whites and 1.4 x 10 8 cells /litre in
blacks.
– Any further fall can induce a serious risk of
developing recurrent infections.
– Agranulocytosis manifests as high fever,
chills,necrotising painful oral ulcers and septicemia.

– Decreased pus formation can give a misleading

picture.Eg. Lack of pneumonic consolidation is seen
in neutropenic patients.

– Chronic idiopathic neutropenia is associated with

pyoderma and otitis media in children.

– Pneumonia,lung abscesses,stomatitis,hepatic
abscesses or infection at other sites may occur.
Chronic cyclic neutropenia is characterized by
oscillatory periods of neutropenia occurring at 3
week intervals.Life threatening conditions are
uncommon.

Pseudoneutropenia
– This condition occurs because a larger
proportion of neutrophils are in the marginal
instead of circulating blood.
– Total blood neutrophil pool is normal and
infections do not occur due to this atypical
distribution of neutrophils.
• Periodontal disease is a complication of various systemic
diseases in which neutrophil function is compromised.
They are
• Chediak higashi syndrome
• Job’s syndrome
• Papillion lefreve syndrome
• Leukocyte adhesion deficiency
• Down’s syndrome
• Chronic granulomatous disease
• Specific granule defeciency
• Diabetes
DISEASE ORAL FEATURES TREATMENT

Chediak higashi Severe periodontal Fatal.

disease disease with no specific treatment
Immune def, partial advanced bone Those who survive
Oculocutaneous loss ,oral ulcers & have neurological
albinism,easy glossitis symptoms
bleeding. recurrent
infections.giant
abnormal
lysosomalgranules
DISEASE ORAL FEATURES TREATMENT

Job’s disease Coarse facies, Cause?

Staphylococcal Hypertelorism, jaw Abnormal neutrophil
Pneumonia, skin Prominence, cranial & monocyte
absceses with high synostosis & severe phagocytosis &
levels of IgE Pdl disease chemotaxis

Pappilion Lefervre Aggressive Skin lesions

syndrome periodontitis with -retinoids
Palmer & plantar rapid destruction of Periodontitis
keratosis, mental alveolar bone. -plaque control
retardation & Loss in order of
-removal of hopeless
intracranial tooth eruption.
teeth
calcification. entire dentition lost -antibiotics
Hyperhidrosis,& fine at young age
leucocyte adhesion Pyogenic infections Fatal
deficiency & periodontal
Leucocytosis, disease.
delayed wound (waldrop-1987)
heaing & less Lack CR3 , iC3B
leucocyte Receptor
mobilisation

Down’s syndrome Rapid Periodontal Supportive

Typical facial app destruction (Saxen) periodontal therapy
with epicanthic in 60 to 100% 0f & maintainence of
folds, broad nasal young adults under oral hygeine
bridge & protruding 30 yrs of age.
tongue,MR & CHD
Specific Granule
deficiency Oral ulcerations & Plaque control &
Depressed respiratory severe periodontitis antimicrobial
activity& diminished activity
ability to respond to
chemotaxis & poor
phagocytosis

Chronic granulomatous Frequent regimen of

disease
Recurrent indolent
pyogenic infections of
certain bacteria. Inability
to distroy bacteria which
gain access to C.T.
Gingivitis & oral antibiotics used
ulcers. affects periodontal
Not associated with ecology
periodontitis
Neutrophil assays

-Adhesion: commercially available monoclonal

antibody directed against membrane surface
antigens
-Phagocytosis: utilize either erect particles or
radiolabelled mo’s that are detectable within
cellafter phagocytosis following intubation.The
ingested particles are quantified to determine if
phagocytosis is impaired
-For chemotaxis
-The Rebrick skin window
-Boyden chamber
-Agarose technique

For Intracellular killing

-Chediak Higashi- Large azurophilic granules
-Specific Granule deficiency-Wright’s stain & assays for
constitute proteins

For respiratory burst activity

-Nitroblue Tetrazolium test –Formazan precipitate
-Flow cytometry- dihidrohodamine-Rhodamine
Conclusion
References
• Carranza’s clinical periodontology -8th & 10th edition
• Contemporary Periodontics- Genco & Cohen
• Periodontics- current concepts & treatment strategies
by Galgut.
• Neutrophil mediated tissue injury in periodontal
disease pathogenesis: Findings from localised
aggressive periodontitis –Van Dyke et.al.
J.P2003:74:66-75
• The neutrophil- mechanisms of controlling periodontal
bacteria—Kenneth.T.Mayasaki –J.P 91
• Neutrophil function & dysfunction in Periodontal
disease—Van Dyke & Vailkuntam
Current opinion in periodontology-1994
• Alterations in Phagocyte functions & Periodontal
infections—Michael Daniel & Van Dyke- J.P1996
• Neutrophil defects as risk factors for periodontal
diseases---Shapira et. al. J.P-1994
• Papilllion Lefervre syndrome: A review of
literature & report of 4 cases ---Hattab et al
J.P-1995
• Altered neutrophil function in LJP: Intrinsic or
Induced---Sudha Agarwal et.al J.P-96