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VISHNU DENTAL COLLEGE

DEPARTMENT OF PERIODONTICS AND IMPLANTOLOGY

SEMINAR ON

NEUTROPHILS IN PERIODONTAL DISEASES

2013-2016

GUIDED BY: Dr. C.D.DWARAKANATH

PRESENTED BY: Dr. K.SAI SUDHA

Contents
Introduction
Neutrophil Morphology & its characteristics
Role of phagocytes in host defense
Neutrophils & periodontal tissues
Altered neutrophil functions & periodontitis
-Chronic periodontitis
-Localized aggressive periodontitis
Neutrophil defects classification
Periodontal

diseases

abnormalities
Neutrophil assays
Conclusion
References

associated

with

neutrophil

INTRODUCTION
Neutrophils are multinucleated granulocytic leukocytes constituting 40-70% of
total circulatory leukocytes found in blood.The name neutrophils is derived from the
property of these cells to stain neutrally with Wrights stain in peripheral smear
preparations.They function like a double edged sword that is they act as protective
proinflammatory cells,at the same time they contain certain cellular elements which
when released can potentiate tissue destruction in the host.
They are defence personnel that constitute the front of bodys battleground against
infection by foreign substances such as bacteria and viruses.In the body the
neutrophils proliferate and mature in the red bone marrow of long bones such as the
femur,radius and ulnar.They are then released into the blood stream where they
circulate for hours before entering the tissues.Once they have done so their fate is
unknown but they probably have a life span of only 24-48 hours.
About 10 billion polymorphonuclear neutrophils enter and leave the circulation on a
daily basis.
The importance of these cells in combating infections is demonstrated through the
increased susceptibility of individuals with defective neutrophil production and/or
function against bacterial infections.Neutrophils also play a critical role in combating
infections originating in the periodontal tissues in the oral cavity.This importance has
been strongly emphasized by a wide variety of research work carried out on Localized
Juvenile Periodontitis.It is therefore a vital cell in the defence mechanism of oral and
extra oral tissues and hence a detail knowledge about its development, biochemistry,
function and clinical implications of these properties is a must for every clinician

MORPHOLOGY OF NEUTROPHILS AND ITS


PRECURSORS
A peripheral smear preparation of bone marrow when observed under the microscope
shows neutrophils in various stages of development.The following are the various
stages of granulocyte maturation.
Myeloblast

Premyelocyte

Myelocyte
Metamyelocyte
Band Cell
Mature Poly Morpho Nuclear Neutrophil
- Proliferation is exhibited only in stages 1 and 2. Maturation seen in the latter stages
occurs as progressive indentation and lobulation of the nucleus.There is appearance of
intracellular granules which are of non specific type to begin with and are transformed
to the specific type as maturation progresses.The cells also exhibit acquisition of
ameboid movement through the various stages of the maturative process.
-Loss of metachromatisation is also evidenced through the process of maturation.The
most reliable method of visualisation of the azurophilic granules under light
microscope is to stain the cells for peroxidase.

- Some mature neutrophils in females have drumstick or club shaped nuclear


appendages.These contain an inactivated X chromosome which has been confirmed by
specific nucleic acid probes via insitu hybridization techniques..

COMPONENTS OF NEUTROPHILS
Contents of neutrophilic granules can be summarized as follows;
Azurophil granules

Specific granules

- Acid hydrolases

- Lysozyme

- Cathepsin B

- Apolactoferrin

- Cathepsin D

- Collagenase

- Cathepsin G

- Cobalamine binding

- Chloroacetate esterase

protein

- Elastase

- C5a cleaving enzyme

- Beta glucoronidase

- Plasminogen activator.

- Beta glucosaminidase

- Cytochrome b558

- Beta galactosidase
- L-mannosidase
- L-fucosidase
- Beta
glycerophosphatase
- Arylsulphatase
- Lysozyme
- Myeloperoxidase
- Defensins
-Bacterial Permeability
Increasing Proteins
(BPI)

Tertiary granules
- Gelatinase

PLASMA MEMBRANE AND CYTOMATRIX PROTEINS

Membrane receptors: Neutrophils have receptors for


-

Opsonins [ Fc portion of Ig G-1,G-2,C3b,C3bi]

Chemotactic factors [C5a,leukotreine B4]

Exogenous modulators of neutrophil functions


# Platelet activating factor
# Beta adrenergic agents
# Adenosine
# Interleukin-1
# Tumour necrosis factor

Associated with these are several G proteins ie guanine nucleotide


binding proteins.They mediate transudation between receptors and
agonists such as cyclic nucleotides,diacyl glycerol and inositol
polyphosphates.

Receptors for growth factors [Granulocyte Macrophage-Colony


Stimulating Factor,Granulocyte-Colony Stimulating Factor]

Insulin binding sites have also been demonstrated.

Adherence proteins:
-

CD 11 and CD-18 complexes

There are three subtypes


# LFA-1
# MAC-1
# P150, P95.
Each contains a sub unit of CD 11a,11b and 11c complexed to a beta
subunit of CD-18.

Patients lacking these complexes show increased susceptibility to


infections.

Matrix proteins:
-

Many cytoproteins are found in the neutrophils.

Actin is one of the most abundant proteins.

Tubulin and microtubule associated proteins.They confer spatial


organization on the cytoplasm and its organelles.

Motility proteins are as given in the table below.

Protein
Actin

Function
Polymerises into filaments.

Actin binding protein


L actinin
Prolifin Acumenin Gelasolin
Tropomyosin

Cross links the actin filaments into a gel.


Bundles actin filaments into sheaves.
Regulates length of actin filaments.
Stabilises actin filaments;myosin causes gel to
contract.

Membrane enzymes:
-

Amiloride sensitive Na+ - H+ antiporte which regulates cytoplasmic


and phagolysosomal enzymes.

Oubain sensitive Na+ - K+ ATPase helps maintain the membrane


potential.

Ca+2 Mg+2 ATPase which expels calcium ions from the cell thus
maintaining its cytosol concentration at 0.1 micro milliliters.

Respiratory burst oxidase responsible for the synthesis of microbial


oxidants resides in the plasma membrane.

Lipids: They are distributed between the plasma membrane and the membrane
of intracellular organelles.They contitute 5% of neutrophil wet weight.

They include:
-

Phospholipid choline

Phosphatidyl ethanolamine

Sphingomyelin

Phosphatidylserine

Phosphatidylinositol

Phosphatidic acid

Triglyceride

Glycolipid

Cholestrol

Rare phosphoinotides act as a source of inositol 1,4,5 triphosphate(a


calcium releasing mediator) and diacyl glycerol(activates protein
kinases).

Arachadionic acid is found in phospholipids.

Enzymes that help split these phospholipids such as phospholipase


A2,several types of phospholipase C and phospholipase D are found
partly in the membrane and partly in the cytosol.

Cytosolic constituents:
-

As in other cells neutrophils contain in their cytoplasm the usual housekeeping enzymes necessary for the maintenance of cellular energy
supplies.

They include glycolytic enzymes,enzymes of HMP shunt,protein and


lipid synthesizing enzymes.

Antioxidant enzymes include


# Superoxide dismutase
# Catalase
# Glutathione peroxidase
# Glutathione reductase
# Organic hydroperoxidase
# Low molecular weight antioxidants include ascorbic acid ,taurine and
tocopherol.

NADPH is a source of electrons for production of microbial oxidants.

NADPH is provided by the HMP shunt enzymes Glucose-6-Phosphate


dehydrogenase(G-6-PD) and 6-Phosphogluconate dehydrogenase.

Lipoxygenases and cycloxygenases are also found in the cytosol.

Other components include calmodulin and protein kinase C.

Why are neutrophils important?


The strategy of host defense against periodontal infection may be viewed as similar to
that used in combatting any local infection. Initially, potential periodontal pathogens
encounter plasma factors, such as complement, within the crevicular or extracellular
fluids. The result of this encounter is the initiation of inflammatory processes, as
evidenced by the recovery of complement activation products from the gingival
crevice. If complement ( antibody) is not successful in controlling the pathogen, the
host defense turns to neutrophils.Neutrophils within the gingival crevice provide the
first cellular host mechanism to control periodontal bacteria. They bring an astounding
arsenal of antimicrobial weaponry packaged within almost all of their cellular
compartments. If neutrophils are unsuccessful in controlling the pathogen (i.e.,
reducing bacterial antigen levels sufficiently), monocytes are recruited which infiltrate
the connective tissue, develop into tissue macrophage, and either digest the antigen
completely (and signal repair processes with cytokines such as transforming growth
factor ) or present partially digested antigen in association with major
histocompatibility complex (MHC)-encoded class II molecules to lymphocytes.
Perhaps to avoid systemic infection, chronic periodontal inflammation may produce
localized specific immune responses and a cytokine-orchestrated amputation of
connective tissue which we call periodontal disease. Thus, neutrophils are important
because they control the periodontal microecology prior to the involvement of chronic
inflammatory cells. In contrast, monocytes and lymphocytes dictate tissue responses to
the periodontal microecology. Given the distinct tasks accorded to the acute and
chronic immune elements, it may be proposed simplistically that either hypofunction
of neutrophils or hyperfunction of monocytes/lymphocytes may result in
increased susceptibility to periodontal diseases.

Neutrophil functions
Neutrophil response to infections can be divided into 3 Rs.
Recruitment

Response

Resolution

Recruitment
Neutrophils circulating in the blood stream are able to adhere to
endothelial cells , A process that involve a highly regulated interplay between adhesion
molecules and their ligands.
Two process occurs in this stage :
A. Adhesion process
B. Chemotaxis .
Transendothelial migration

Step 1 Rolling

Step 2 an insult to local tissue.

Step 3 signaling the endothelium

Step 4 increased rolling

Step 5 signal for rolling arrest

Step 6 strong adhesion ( arrested rolling)

Step 7 zipper phase.

Step 8 degradation of basement membrane.


The directed movement of leukocytes from blood into the local tissues is
central to inflammation. Transendothelial migration is an interaction between
leukocytes and endothelium that results in leukocytes pushing its way between
the endothelial cells to exit the blood and enter into tissues. Defects in
Transendothelial migration results in aggressive periodontitis reflecting the
importance of this process in periodontal diseases.

Step 1 : Leukocytes use the lectin (a nonenzymatic carbohydrate binding


protein) , designated L-selectin to interact with carbohydrate molecules known
as vascular addressins on the luminal surface of the endothelial cells. This brief
interaction manifests itself as a rolling of the leukocyte along the lumenal
surface of the endothelium, a process where by leukocytes essentially pauses to
inspect the endothelium.
Step 2 : A local insult triggers the release of variety of inflammatory signals
such as interleukin-1-beta, tumor necrosis factor alpha from the cells in the
tissue especially from the resident leukocytes known as mast cells.
Step 3 : Mast cells are crucial in initiating neutrophil recruitment against
bacteria and respond to anaphylotoxins such as C3a and C5a.
Step 4 : inflammatory signals and lipopolysaccharides can stimulate
endothelial cells to express P-selectin and E-selectin on their luminal
surfaces. Either of these selectins can bind carbohydrate molecules found on
the leukocyte. This appears microscopically as an increasing in number of
leukocytes attached to the luminal surface of the endothelium or increased
rolling.
Step 5 : the stimulated endothelium also releases chemokines. Chemokines
are small peptide cytokines , first recognized for their chemoattractant
activities , which play a fundamental role of selective signals for leukocytes to
exit the blood.
Step 6 : chemokines function as a signal for rolling arrest. The interaction of
a chemokines , interleukin-8 with the leukocyte receptorCXCR2 causes the
leukocyte to shed L-selectin and upregulate the integrin ,leukocyte function
associated antigen -1

( LFA-1). LFA-1 binds ICAM-2 which is expressed

constitutively by endothelium. This results in rolling arrest because the


phagocyte becomes firmly associated with the endothelium.
Step 7 : CD31 ,a transmembrane glycoprotein is a hemophilic adhesion
molecule because CD31 molecules bind to one another. The binding of CD31
on endothelium with CD31 on leukocytes guides leukocytes to the boundaries
between endothelial cells. This zipper effect has been proposed as a

mechanism of minimizing the leakage of fluid. As the endothelium unzips its


CD31 , the leukocytes rapidly zips between the endothelial cells.
Step 8 : leukocytes accumulate briefly between the basement membrane and
endothelial cell . this pause may reflect a period of secretion of proteases to
degrade the basement membrane . leukocytes possess several proteases
including urokinase plasminogen activator receptor (uPAR). The uPAR leads to
the activation of collagenase , which then may degrade the basement
membrane and enable the leukocyte to enter the connective tissue.

Step 9 : Chemotaxis . once the leukocyte enters the connective


tissue , it must be able to locate and migrate to the site of insult .
this is accomplished by chemotaxis which depends on the
leukocytes ability to sense the chemical gradient across the cell
body and migrate in the direction of the increasing concentration.
The phagocytes sense only a limited number of chemicals
chemotaxins for which it has receptors or chemotaxin receptors. The
receptors for chemotaxis belong to a family called the G-protein
coupled family.
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, Leukotrienes B4, neutrophil chemotactic factor &
platelet activating factor
FMLP is a modified amino acid present in most of bacteria but not in human
thus it serves as a tell tale sign that bacteria are present within host tissues
RESPONSE

This stage includes,

Phagocytosis

Intracellular killing

Extracellular killing

Recognition of the Invader

Complement :
The net effect of complement activation is to augment
opsonization of bacteria by antibodies, to allow some antibodies to kill
bacteria, to recruit phagocytes to the site of complement activation and to
attack the membrane of pathogens forming pores in the cell and lysis.

The six regulators of complement activation are encoded in a tight cluster of


chromosome

1. Factor H
2. Membrane cofactor protein (MCP)
3. Complement receptor 1(CR1)
4. Complement receptor 2 (CR2)
5. Decay accelerating factor (DAF)
6. C4 binding protein (C4BP)
Complement is activated through three different pathways.
They are , the classical pathway , the lectin pathway and the alternative
pathway.Although multiple pathways exist for the initial activation of complement,
all pathways ultimately result in the production of a protease called C3 convertase
that is covalently bound to the surface of the pathogen.
Interaction of C3 with the C3 convertase generates
two main effector molecules from each C3 molecule: an opsonin called C3b
that binds covalently to pathogen surfaces, and a small peptide mediator of
inflammation called C3a.

Complement receptors on neutrophils can be directly affected by complement


components.

C5a increases the expression of CR1 and CR3 on the surface of cells and
increases their adherence to vessel walls, their migration toward sites of
antigen deposition (chemotaxis) and their ability to ingest particles.

Fc receptors :
An Fc receptor is a protein found on the surface of certain cells -

including natural killer cells, macrophages, neutrophils, and mast cells - that
contribute to the protective functions of the immune system.

Their activity stimulates phagocytic or cytotoxic cells to destroy microbes, or


infected cells by antibody-mediated phagocytosis or antibody-dependent cellmediated cytotoxicity . Antigen recognition is conferred not by specificity of
the responding cells, but by the specificity of the antibody.

Binding of immunoglobulin by the Fc receptors can activate a number of


different biological processes depending on the cell type and receptor type.

These processes include phagocytosis, antibody-dependent cellular


cytotoxicity, clearance of immune complexes, degranulation and activation of
the respiratory burst and release of inflammatory mediators.

Fc receptors specific for IgG, IgE and IgA have been identified and referred to
as FcR, FcR and FcR respectively. Different Fc receptors can be found
expressed widely on monocytes, neutrophils, platelets, B cells, eosinophils,
basophils and trophoblasts.

FcR classes and distribution


In humans, three major classes of FcR have been identified:
FcRI (CD64), FcRII (CD32) and FcRIII (CD16)

FcR expression by neutrophils :

Human neutrophils constitutively express two Fc receptors:


FcRIIA and FcRIII-B. There are two genetically determined structurally
and functionally different allotypes of human FcRII-A.The homozygous
FcyRII-A-H131 allotype (Histidine type) interacts efficiently with complexed
human IgG2, whereas the homozygous FcyRIIa-R131 allotype (arginine)does
so only poorly. Both FcRII-A and FcRIII-B appear to be important for
optimum phagocytosis.

Phagocytosis occurs when the neutrophil encounters the bacteria.


Phagocytosis is greatly enhanced by coating of the bacteria with antibody or

complement.
These coating molecules (collectively called opsonins) facilitate binding and
internalization via cell surface receptors including Fc receptors (receptors for
the Fc fragment of IgG) and receptors for complement, specifically CR1 and

CR3.
As soon as the neutrophil recognizes the opsonins that are bound to the
bacterial surface , it engulfs the microorganism via invagination of the plasma
membrane, which encloses the microbe in a phagosome.
Phagosome undergoes fusion with other organelles to form
phagolysosomes which are specialized structures for killing microbes through
acidification of the contents and release of the antibacterial molecules from
neutrophil granules.

DELIVERY OF ANTIMICROBIAL SUBSTANCES


Neutrophils are terminally-differentiated cells no longer concerned with growth or
division. Their main function is to deliver antimicrobial substances to bacterial and
fungal targets. Because neutrophils are not concerned with their own survival, they are
free to use delivery modes which can be suicidal. Neutrophils deliver antimicrobial
substances by 4 different mechanisms
Delivery of Oxygen Metabolites

When phagocytes contact certain stimuli (such as opsonized bacteria or


chemoattractants), they consume dioxygen if it is available (a process called the
"respiratory burst") by transferring 1 or 2 electrons from cytosolic NADPH to the
extracellular dioxygen via the NADPH oxidase system, forming potentially toxic
Superoxide anion (02") and hy drogen peroxide (H202), respectively. The formation of
chlorinating oxidants requires the secretion of myeloperoxidase (MPO) from the
azurophil granule.

Secretion
Extracellular secretion involves the mobilization of cytoplasmic granules, fusion
between the granule and plasma (or phagosomal) membranes, and discharge of
granule contents into the external environment.
Phagocytosis
Phagocytosis is the engulfment of particles within a membrane- bound structure called
the "phagosome." Fusion between the cytoplasmic granules (lysosomes) and the
phagosome form the "phagolysosome," and represents a specialized form of secretion.
The phagolysosomal fusion process is dependent upon intracellular calcium and
requires the activity of a mixture of 3 "synexin-like" cytosolic fusogenic proteins.
Antimicrobial substances are delivered at very high concentrations by
intraphagolysosomal secretion and respiratory burst activity. In addition, phagocytosis
isolates the ingested organism within an extremely stringent environment.
Death (Cytolysis and/or Apoptosis)
Cytosolic and granule components can be delivered by apoptotic (programmed death)
or cytolytic mechanisms.Apoptosis/cytolysis may be important in controlling
mucocutaneous candidiasis.

To kill microbes neutrophils can use both oxygen-dependent and oxygenindependent means. Oxygen dependent killing depends on generation of
reactive oxygen intermediates (ROI),through the action of NADPH oxidase
system.
Superoxide production: the NADPH oxidase

Neutrophil metabolism can occur through multiple pathways.


Neutrophils in the resting phase metabolize through glycolysis.When activated,
they switch to the hexose monophosphate shunt pathway and produce large
quantities of peroxide and active oxygen intermediates.
The NADPH oxidase system is the major source of reactive
oxygen within phagocytic cells and is composed of five components; three
membrane associated and two cytosolic . Together, the components making up
the NADPH oxidase system are responsible for the reaction:
NADPH+202

202- +

H+ +

NADP+

The oxygen produced through NADPH


is quickly dismutated to hydrogen peroxide in the cellular milieu by the
reaction.The H202 thus formed is a weak oxidizing agent that can oxidize -SH
groups, inactivating some proteins, and at high concentrations is bactericidal.

Myeloperoxidase.
Myeloperoxidase is thought to be one of the major components
involved in the antibacterial activity of phagocytic cells. Forty to seventy
percent of the O2 consumed during the respiratory burst appears as HOCl in
the reaction:
H2O2+C1-

HOCl + H2O

Stimulation of oxygen-dependent agents


The components of the NADPH oxidase complex must be
translocated to the plasma membrane or membrane of the phagolysosome
before the complex can be activated. Reactive oxidant production can be
stimulated by a number of agonists including N-formyl-L-methionyl- L-leucyl1.-phenylalanine, platelet-activating factor, leukotriene B4 and opsonized
bacteria. Pretreatment of the cells with cytochalasin B or cytokines such as
granulocyte-macrophage colony-stimulating factor greatly enhances the
production of reactive oxygen metabolites. Agents such as N-formyl-Lmethionyl- L-leucyl-L-phenylalanine, C5a granulocyte macrophage
colony-stimulating factor and other neutrophil stimulants have been shown to
induce translocation of granules, resulting in upregulation of some receptors

and cytochrome b onto the cell plasma membrane. Thus, events that prime the
neutrophil significantly enhance the ability of cells to generate reactive
oxidants.
Oxygen-independent antimicrobial proteins of neutrophils.
Many of the most potent oxygen-independent antimicrobial proteins
reside within the azurophil granules and include defensins, cathepsin G,
elastase, proteinase 3, azurocidin, lysozyme and bactericidal or permeabilityincreasing protein. Also, specific granules contain several oxygen-independent
antimicrobial substances that may function extracellularly, including
lactoferrin, lysozyme and B12-binding protein (cobalophilin).

Defensins :
Defensins are small (approximately 3200- 4000 Da), cationic
amphipathic, arginine- and cysteine- rich peptides. They make up 5-7% of the
total protein and 30-50% of the azurophil granule content of human
neutrophils. Defensins are membrane-permeabilizing molecules that can kill a
variety of gram-negative bacteria, gram-positive bacteria, fungi and eukaryotic
cells. The Capnocytophaga spp. are more sensitive to the human defensins
and are killed by these peptides under both aerobic and anaerobic conditions.
Cathepsin G

,is a neutral serine

protease found in the granules of neutrophils. Cathepsin G has proteolytic and


esterolytic activities resembling that of chymotrypsin. Miyasaki & Bodeau.
have found that cathepsin G is the most potent nonoxidative antimicrobial
agent against periodontal pathogens found in the human neutrophil.
Extracellular killing
At the time of invagination of the surface membrane and
formation of phagocytic vacuoles, some of the neutrophil lysosomal enzymes
may escape into the extracellular fluid, especially if the particle is too large to
ingest. Recently neutrophils were shown to entrap microbes in the extracellular
space through release of web of fibres containing DNA , histones and granule
derived bactericidal molecules. These neutrophilic extracellular traps (NET )
may serve to limit secondary damage to surrounding tissue by preventing

random diffusion of granule contents (Brinkmann et al.2004). Vitkov et al.


(2009) showed that NETs are abundant on gingival surface and in the
gingival crevicular exudates in patients with chronic periodontitis.
RESOLUTION

Successful resolution of inflammation is a prerequisite for

restoration of healthy tissue. Because acute inflammation is characterized by


an abundance of infiltrating neutrophils , their elimination and the restoration
of normal number of tissue leukocytes, is a critical step in the progression of
tissue repair.
Neutrophils & periodontal disease
Half the leukocytes infiltrating the junctional epithelium and 90% of the leukocytes
isolated from crevicular fluid are neutrophils. Whereas neutrophils predominate the
junctional epithelium and the gingival crevice, lymphocytes and
monocytes/macrophage predominate within the subjacent connective tissue. The
concentration of neutrophils in the periodontal tissues exceeds the concentration of
neutrophils in blood. In minimally-inflamed gingivitis, 2.5 x 107 neutrophils/cm3
infiltrate the gingival connective tissue and 1.7 x 108 neutrophils/cm3 are found
in the junctional epithelium (blood levels normally range from 1 x 106 to 4 x 106
neutrophils/cm3). Rather than forming a homogenous bacteria/leukocyte mixture in
the gingival crevice, the neutrophils form a "leukocyte wall" interposed between the
periodontal plaque mass and the junctional and sulcular (or pocket) epithelium.
The "leukocyte wall" may function both as a secretory and as a digestive organ, and
crevicular neutrophils exhibit both partial degranulation and ingested bacteria.
Plaque micro organisms do not normally enter the tissues , so in order to kill them
-

Neutrophils must leave the tissues & enter gingival crevice or periodontal
pocket

PMNs 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 opsonized & unopsonized bacteria bacteria are susceptible to killing but
opsonisation increases the efficiency.

Unattached bacteria could be killed in traditional manner, but this is unusal because
-

Neutrophil function is inhibited by microbial factors such as endotoxins,


formyl peptides & by host factors ike 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

Altered phagocytic function & periodontal disease

Periodontal disease is common sequel associated with compromised


phagocytic no. / function

For some aggressive 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 PMNs have shown to be important in various


disease entities, of which periodontitis is a common sequel

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.a infection

Altered phagocyte function has been used as a model for understanding


periodontal pathology in LJP

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 decreased chemotaxis (7075%)

LJP pts appear to be healthy & have not been documented to exhibit increased
susceptibility to other infection, as would be expected in pts exhibiting
impaired neutrophil functions

The manifestation of this disease i.e. massive tissue damage & bone loss occur
in the 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

Following observations suggest that extrinsic factors in sera may alter neutrophils
function in LJP Induction of cytokines:

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
neutrophils in healthy serum

Also healthy neutrophils treated with LJP sera function similar to LJP
neutrophils

Contact neutrophils

Alter function of PMNs

Chemotaxis & chemotactic receptors adherence superoxide anion &


degranulation

Reduced migration of PMN to site of infection

Thus neutrophils exposed to cytokines exhibit all the characteristics of LJP PMNs

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 JP 1996)

ALTERED NEUTROPHIL FUNCTION & CHRONIC PERIODONTITIS

PMN mediated tissue injury

Bacteria & its products modulate PMN function

Delayed neutrophil apoptosis

A. Neutrophil mediated tissue injury was first demonstrated by Deguchi et.al


a. Oxygen radical sp produced by PMNs can attack every biologically relevant
molecule & cause damage. In addition, they also modulate various cellular
activities which are mediators in sequence of events leading to tissue injury
b. O2
c. H 2O 2

NO
vascular adhesion & activation of PAF

b. PMN degranulation releases several proteolytic enzymes that can cause host tissue
damage
i.

Crevicular fluid PMNs release upto 5 times more elastase & collagenase than
peripheral blood PMNs in patients 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 glucoronidase activity

iii.

Lactoferrin enhances PMN adhesiveness & is synergistic with its enzymes

iv.

c. Activated PMNs also release proinflammatory mediators such as


leukotriene B4 & PAF that are potent stimulators of neutrophil chemotaxis,

adhesion, oxidative burst & degranulation, thus amplifying neutrophil


mediated tissue injury
v.

They have capacity to cleave complement components via alternate pathway &
to activate kinin system reactions, which in turn perpetuate & magnify
inflammation

B. Bacteria & its products modulate PMN function


-

Bacterial products such as LPS & proteinases have 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 fibroblast by increasing


the adherence of PMN to fibroblasts (Deguchi)

Bacteria may directly interfere with neutrophil phagocytosis by modulating


complement activity

Proteolytic activity of P.gingivalis is an 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

C. Delayed neutrophil apoptosis & periodontitis

Circulating neutrophils have a short half life & onset of apoptotic process is
associated with loss of several important functions such as adhesion &
phagocytosis (Dransfield 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 , GM CSF (granulocyte macrophage


colony stimulating factor) may delay neutrophil apoptosis by increasing their
mitochondrial stability,& down regulating gene expression
(Tsiyjmoto & Shimizir 2000)

Recent studies have shown that bacterial products isolated from different
starins of P.gingivalis also delay neutrophil apoptosis in dose dependent
manner (Preshaw et al 1999)

NEUTROPHIL DYSFUNCTION
Neutrophils employment for the destruction of invading microorganisms
can be divided into six stages or function.
1. Rolling along vascular endothelium
2. Adherence to the endothelial lining
3. Migration (chemotaxis) towards the site of

infection

4. Adherence to microorganisms
5. Engulfment of bacteria ( phagocytosis)
6. Intracellular killing

Defects in any of these functions or decrease in the number of


neutrophils capable of responding to the site of infection may result in
susceptibility to infection.
These quantitative and qualitative defects may be
1. Inherited
2. Acquired
3. Dug induced

Neutropenia is clinically significant when the neutrophil count falls


below 1000 cells/ l.
Normal adult range is 1800-8000 cells/ l.
When the neutrophil count is less than 200 cells\ l, control of
endogenous microbiota will be impaired and risk for serious infection
increases.
Less than 200 cells/ l leads to in ability to produce inflammatory
response
Neutropenia can be
1. Inherited
2. Congenital or acquired due to infection
3. Malignancy
4. Certain medication
5. Autoimmune
6. Nutritional deficiencies/hematopiotic diseases
PATHOPHYSIOLOGY OF NEUTROPENIA
1. Abnormalities of bone marrow stem cell development
2. Impaired release of neutrophils from the bone marrow
3. Abnormalities in distribution of neutrophils between the
circulating and marginating pool of blood
4. Decreased survival of neutrophils in the blood
Diagnosis of neutropenia is based on clinical signs and symptoms as
well as absolute neutrophil count.
CLASSIFICATION

OF

LABORATORY DIAGNOSIS

NEUTROPHIL

DISORDERS

&

Defects in the process of margination occurs at 2 levels


First one is defect in a specific neutrophil ligand, saily lewis x protein
(CD 1 5 5 ), here loss of neutrophils ability to roll along the endothelial
lining occurs.
The defect is detected through flow cytometry using a
commercially

available

monoclonal

antibody

detected

against

the

membrane surface antigen associated with CD 1 5 3 . The disease associated


with this deficit is leukocyte adhesion deficiency type-II ( LAD-II).
Defects
CD18\

in

neutrophils surface

integrins

CD 1 8 /CD 1 1 a ,

CD 1 8 /CD 1 1 b ,

CD 1 1 c prevent the adhesion of neutrophil to the endothelium. This

prevents the migration of neutrophils to the site of infection. Since


these integrins also responsible for neutrophil adhesion to opsonised
bacteria, the neutrophil ability to phagocytize bacteria is compromised.
Defects in the CD 1 8 and CD 1 1 are identified by flow cytometry.
Deficiency of these integrins is termed as LAD-I
DEFECTS IN NEUTROPHIL CHEMOTAXIS
It can be either
1. Inherited
2. Secondary to variety of infections or medication
Any alteration in the neutrophil cytoskeleton or its ability to sense or
respond to a chemotactic gradient will interfere with the cells ability to
reach the site of infection.
Chemotactic defects can be detected in vivo by Rebuck skin window
method and invtro by Boyden chamber/ agrose technique
Rebuck skin window measures the movement neutrophils on a glass
slide placed on the superficial abrasion made on the patients skin.

Boyden chamber evaluates chemotaxis by quantifying the migration of


neutrophils from an upper chamber through a microscope filter into a
power chamber containing a chemoattractant.
Primary defects in the neutrophil defects are rare, impairment of
phagocytosis is due to deficiencies in certain immunoglobulin isotopes
and other opsonising factors. Assays of phagocytosis utilize either
radiolabelled microorganisms or inert particles that are detectable
within the cell after phagocytosis.
DEFCTS IN INTRACELLULAR KILLING
Divided into disorders affecting
1. Oxidative pathway
2. Non oxidative pathway
Two main conditions with defects in non-oxidative pathway are
1. Chidiak higashi syndrome - (diagnosed by peripheral blood
smear containing Azurophilic granules)
2.

Specific granule deficiency - (diagnosed by Wright stain


confirming the absence of secondary granules)

The

neutrophils

oxidative

killing

mechanism

involves

two

main

enzymes that in rare instances can be dysfunctional. The first one


involves defect in the NADPH oxidase, seen in chronic granulomatous
disease.
Diagnostic tests are nitro blue tetrazolium test. Here neutrophils are
stimulated and incubated with the colorless nitro blue tetrazolium, if the
oxidative burst is defective no precipitate is formed. If the NADPH
oxidase is functioning the nitro blue tetrazolium is reduced to blueblack formazin precipitate.

A more sensitive test is flow cytometry . Here hydrogen peroxide


converts dihydrorhodamine to rhodamine and this is detected by using
florescent label.
The second one is absence of Myeloperoxidase, a defect of this enzyme
lead to lack of hypochlorus acid and a delay in bacterial killing.
Diagnosed by using peroxidase staining of blood films, flow cytometry,
and direct assay of enzyme activity.
CHIDIAK HIGASHI SYNDROME
Its a rare autosomal recessive disorder affecting neutrophils, due to
mutation in the vesicle trafficking regulatory gene.
Manifests early in life as
1. Occulo cutanous albinism
2. Photophobia
3. P yogenic infections and lymph adenopathy
Oral findings include
1. Gingivitis
2. Ulcerations of both tongue and buccal mucosa
3. Early onset periodontitis leading to loss of both the dentitions.
Treatment

includes

bone

marrow

transplantation

for

correcting

neutrophil abnormalities.
Hallmark of this syndrome is presence of large Azurophilic granules in
the cytoplasm of the neutrophils. These large inclusions impair
neutrophil migration, possibly by inhibiting cell deformability. Which
causes neutrophils unable to phagocytose microbes. The patients of this
syndrome are more prone for recurrent infections in early childhood.

DNA probe technology reveals the presence of pathogenic flora like,


A.A, P.G, and P.I
Lab diagnosis is made by presence of Azurophilic granules within the
neutrophils.
CHRONIC GRANULOMATOUS DISEASE
It is an extremely rare syndrome characterized by staphylococcus,
proteus or pseudomonas species infections. Etiology is congenital
defects in the enzyme NADPH oxidase.
The defect prevents the free oxygen radicals from being produced and
neutrophils inability to kill intracellular organisms predispose patients
to recurrent bacterial infections.
Oral complications are gingivitis and periodontitis.
Treatment for this disease mainly concentrates on control of infections.
1.

Cotrimoxazole-broad spectrum activity on most of the causative


organisms

2.

Antifungal prophylaxis with itraconazole

3.

Interferon- -restores

oxidase

activity

in

neutrophils

and

monocytes
4.

Corticosteriods

low

doses

to

manage

symptomatic

complications such as colitis and hollow organ obstruction.


5.

Bone marrow transplantation is the most predictable treatment.

HYPER IMMUNOGLOBULIN E SYNDROME

Inherited as an autosomal dominant trait that affects the dentition,


skeleton, connective tissue and immune system.
Classically it has a triad of s ymptoms
1. Abscess

2. Pneumonia
3. Elevated serum IGE levels.
.
Etiology is defect in neutrophil chemotaxis
There are 3 hypothesis exists for the chemotactic defect
1. IGE

against

an

infecting

bacterium

causes

the

release

of

histamine, that may inhibit neutrophil chemotaxis.


2. Bacterial

antigens

cause

monocytes

to

secrete

chemotaxis

inhibiting mediators or IgG.


3. Mononuclear

cells

create

specific

factor,

which

inhibit

neutrophil chemotaxis.
Oral findings are
1. Ulcerations and gingivitis,
2. Over retention of primary teeth.
Treatment includes
1. Antibiotics
2. Local debridement
3. Surgical incision and drainage of infections
4. Treatment with cimitidine or ranitidine alone or in combination
with H1 receptor antagonists shows improvement.
LAZY LEUKOCYTE SYNDROME
Characterized by recurrent infections due to deficiency in neutrophil
chemotaxis and a systemic neutropenia. While the phagocytic function
of neutrophils is normal, within the bone marrow the quantity and
morphology of neutrophils leads to diminished in vivo migration of
neutrophils into the tissue and to sites of inflammation.
Oral complications are oral ulcerations, gingivitis and periodontitis.
Lab diagnosis is by absolute neutrophil count.

AGRANULOCYTOSIS
Characterized by decrease in or disappearance of granular leukocytes
including neutrophils. The decreased number of granulocytes can result
from

either

decreased

production

or

an

increased

peripheral

destruction of cells.
Decreased production of granulocytes is usually due to
1. Drugs
2. Chemicals
3. Ionizing radiation
4. Infection
5. Vitamin deficiencies
6. Bone marrow tumors
7. Bone marrow hyperplasia
Clinically patients present with necrotizing gangrenous lesions of
mucous

membrane

including

oral,

gastrointestinal

and

vaginal

membranes but without purulence.


Oral signs and symptoms are painful stomatitis, spontaneous bleeding
and necrotic tissue, periodontitis.
Lab diagnosis is by absolute neutrophil count.
LEUKOCYTE ADHESION DEFICIENCY .
LAD-I is a disorder that involves a deficiency in the membrane
integrins.
LAD-I is inherited as an autosomal recessive pattern.
CD 1 8 /CD 1 1 a binds neutrophils to endothelium via intercellular adhesion
molecules (ICAM)
CD 1 8 /CD 1 1 b

(MAC-1)

binds

ICAM

complement mediated phagocytosis.

and

complement

facilitates

The function of the CD 1 8 /CD 1 1 c is not well understood.


Deficiency of these integrins prevents neutrophil from adhering to the
vessel wall at the site of infection. Inspite of leukocytosis neutrophils
are unable to migrate into the effected tissues.
Clinical features like ulcerations and necrosis of tissue without
purulence, delayed umbilical core separation, periodontitis.
Lab diagnosis is by Rebuck skin window method or boyden chamber
flow cytometry, detects the deficiency of CD 1 8 orCD 1 1 b integrins.
LAD-II
Etziomi etal in 1992 first described it.
Clinical features are short stature, mental retardation, craniofacial
abnormalities, and recurrent infections.
The neutrophil defect in LAD-II is of saily-lewis x glycoprotoien
(CD 1 5 5 ). Which allows neutrophils to attach to selectins (CD 6 2 E ) on the
endothelial

surface.

The

neutrophils

are

unable

to

migrate

extravenously.
Oral manifestations are periodontitis and tooth loss.
Lab diagnosis is by Rebuck skin window method or Boyden chamber
flow cytometry. Detects the deficiency of saily lewis x ligand.
PAPILLON LEFEVRE SYNDROME
In 1924 Papillon and Lefevre introduced this syndrome, its an

autosomal recessive disease due to mutation in the cathepsin C gene . 2


essential features of this s yndrome are
Hyperkeratosis of palms and soles

1. Genaralised rapid destruction of the periodontal attachment


apparatus, resulting in premature loss of both the dentitions.

2. Other findings like


1. Ectopic calcification of falx ceribri and choriod plexus.
2. Increased susceptibility to infections
3. Mental retardation
4. Endocrine dessorders
5. Aggressive periodontitis
Etiology is variable deficits in chemotaxis, phagocytosis and intra
cellular killing.
Lab diagnosis is by chemotaxis and phagocytic tests.
DOWNS SYNDROME
Etiology is variable deficits in chemotaxis, phagocytosis and intra
cellular killing.
Clinical features are mental retardation increased susceptibility to
infection, cardiac malformation.
Bousw a and Van dijk examined both exogenous and endogenous
factors, which predispose affected patients to aggressive periodontitis.
Exogenous factors divided into local and secondary factors.
Local factors - oral hygiene
Secondary factors - tongue thrust, malocclusion and lack of lip seal.

Endogenous factors - defects in neutrophils leads to deficits in


chemotaxis, phagocytosis and intra cellular killing.
Halinen etal found increased levels of neutrophil collagenase (MMP 8 )
in the saliva and G.C.F of downs syndrome patients.
Lab diagnosis is by chemotactic and phagocytic tests.
MYELOPEROXIDASE DEFICIENCY
Myeloperoxidase located in the Azurophilic lysosomes of neutrophils
and monocytes. M yeloperoxidase H 2 O 2 -cl s ystem represents the most
efficient component of the oxygen dependent antimicrobial system.
Myeloperoxidase deficiency can be congenital or acquired.
Most of the individuals with Myeloperoxidase deficiency have no risk
of infection or other clinical manifestations.

CONCLUSION
Neutrophils are a critical arm of the defence against periodontitis , but bacterial
evasion of the neutrophil microbicidal machinery coupled with delayed neutrophil
apoptosis may transform the neutrophil from defender to perpetrator.
Future research is required inorder to define the neutrophil pathways involved in
promoting repair versus inducing tissue damage during periodontal tissue regeneration

References

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Neutrophil

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tissue

injury

in

periodontal

disease

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The neutrophil mechanisms of controlling periodontal bacteria


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Neutrophil function & dysfunction in periodontal disease


Current opinion in periodontology 1994

Alterations in phagocyte function & periodontal infections


Micheal Daniel & Van Dyke JP 1996

Neutrophil defects as risk factors for periodontal disease


Shapira et. At. JP- 1994

Altered neutrophil function in LJP: Intrinsic or Induced Sudha


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