ROLE OF NEUTROPHILS IN PERIODONTAL DISEASE

PRESENTED BY GUIDED BY SHEETAL OSWAL DR C D DWARAKANATH

CONTENTS IntroductionRole of phagocytes in host defenseNeutrophil function & dysfunctionNeutrophils & periodontal tissuesAltered neutrophil functions &

periodontitis -Chronic periodontitis -Localised aggressive periodontitis

-ANUGNeutrophil defects-classificationPeriodontal disease associated with

neutrophil abnormalities

Neutrophils & periodontal tissues in systemic diseases

Anti neutrophil therapies -Lipoxins -ATL

Neutrophil assaysSummary & conclusions

INTRODUCTIONWhy 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 & Margination2. Adhesion3. Diapedesis/ transendothelial migration4. Chemotaxis5. Extracellular secretion, receptoe upregulation &

binding to target6. Phagocytosis7. Microbial killing8. Apoptosis

Functions of Neutrophils

INFLAMMATION INFLAMMATION

PMN’S ADHERE TO ENDOTHELIUM

Rolling

Endothelium to express P & E selecins on luminal surfaces

Local inflammationIL-1beta & TNF

from mast cells & leucocytes

Increased rolling

Releases chemokines

Signal fo Rolling arrest

L-selectins on PMN’S interact with endothelium

P & E selectins+ L selectins

Chemokines interact with PMN receptor CxCR2

PMN’s shed selectins & upregulate integrins

Adhere to ICAM-2 on endothelium

LFA-1(Integrin B2)

Rolling arrest & strong adhesion

PMN’s locate inter endothelial junction

Zipping & unzipping DIAPEDESIS

CD31(E) + CD31(L)

eChemotaxis- leucocyte’s ability to sense a chemicalgradient

& migrate in the direction of its increasing concentration. 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

Receptor upregulation & binding to target

Opsonisation

Phagocytosis

Delivery of antimicrobial substances Microbial killing

Chemotactic receptors on PMN

Recognise pathogen

G-coupled family

IgG & iC3b

Phagocytosis & Killing

VARIOUS OPSONINS & OPSONIN RECEPTORS

Target Opsonin Receptor

Gram –ve bacteria LPS binding protein

CD14

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

sMicrobial killing Specific granule Microbial

killingAcid hydrolases

- Cathepsin B

- Cathepsin D

- Cathepsin G

- Chloroacetate esterase

- Elastase

- Beta glucoronidase

- Beta galactosidase

- L-mannosidase

- L-fucosidase

- Beta glycerophosphatase

- Arylsulphatase

- Lysozyme

- Myeloperoxidase

- Defensins

-BPI

-Lysozyme

- Apolactoferrin

- Collagenase

- Cobalamine binding

protein

- C5a cleaving enzyme

- Plasminogen activator.

- Cytochrome b558

Gelatinase

• 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

• 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

Delivery of antimicrobial substances

-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

-

Mechanisms of killing 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.

Non oxidative killing of periodontal bacteria

aCathepsin G kills most periodontal bacteria in hypoxic 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 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.

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.

-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

-

Neutrophil and periodontal tissues

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

rolling

Strong adhesion & diapedisis

chemotaxis

Microbial killing

phagocytosis

Degradation of mo’s

LAD-2

LAD1

Actin dysfunction

Diabetes

CGM Myeloperoxide

deficiency

Chediak Higashi

-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 phagocytic function & Periodontal disease

• 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

Altered phagocyte function & aggressive periodontitis

Neutrophils in gingival crevice

Microbial killing

Phagocytosis

chemotaxis

Vascular adhesion

Increased adhesion

chemotaxis

Receptor expressionReceptor

expressionReceptor expression

phagocytosis? superoxide

killingkilling

Expression of CD11/CD18

GP-110

Receptor for FMLP & C5a & LB4

CYTOKINES

Locomotion & migration

rAltered neutrophil function –induced or intrinsic??? Sudha Agarwal et al JP,96;67The 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:

e• Not all pts with clinically diagnosed LJP exhibit

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

alterAlter function of PMN

Chemotaxis &chemotactic receptors

adherence Superoxide anions & degranulation

Reduced migration of PMN to site of infection

THUS NEUTROPHILS EXPOSED TO CYTOKINESEXHIBIT ALL THE CHARACTERISTICS OF LJP PMN’s

Contact neutrophils

TNF IL-1B

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

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 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

sb. PMN degranulation releases several proteolytic

enzymes that can cause host tissue damagei. 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.

ec. Activated PMN’s also release proinflammatory

mediators such as leukotrine B4 & PAF that are 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

PAF

Tissue factor synthesis

Generate chemotactic substances

Reduce catabolism of

PMN’s

PMN degranulation releases proteolytic

enzymes

LTB 4 & PAF

Magnify inflammation

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)

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

Role of neutrophils in ANUG

Neutrophil defects

QualitativeQuantitative

Neutropaenia

Defects in adhesion- LAD1 & LAD2

Defects in chemotaxis

Defects in phagocytosis

Defects in microbicidal activity

Neutropaenia

Production defects Destructive defects

Aplasia

Infiltrative diseases

Drugs

Metabolic diseases

Infective diseases

Kostmann syndrome

Splenic sequestration

Antineutrophil antibodies

Normal neutrophil count-1800-7200 cells/cummNeutropaenia occurs when count is > 2000 cells/cumm

moderate-> 1000cells/cummsevere- >500 cells/cummVery 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 disease

Immune def, partial Oculocutaneous albinism,easy bleeding. recurrent infections.giant abnormal lysosomalgranules

Severe periodontal disease with advanced bone loss ,oral ulcers & glossitis

Fatal.

no specific treatment

Those who survive have neurological symptoms

DISEASE ORAL FEATURES TREATMENT

Job’s disease

Staphylococcal Pneumonia, skin absceses with high levels of IgE

Coarse facies,

Hypertelorism, jaw

Prominence, cranial synostosis & severe Pdl disease

Cause?

Abnormal neutrophil & monocyte phagocytosis & chemotaxis

Pappilion Lefervre syndrome

Palmer & plantar keratosis, mental retardation & intracranial calcification.

Hyperhidrosis,& fine body hair

Aggressive periodontitis with rapid destruction of alveolar bone.

Loss in order of tooth eruption.

entire dentition lost at young age

Skin lesions -retinoids

Periodontitis

-plaque control

-removal of hopeless teeth

-antibiotics

leucocyte adhesion deficiency

Leucocytosis, delayed wound heaing & less leucocyte mobilisation

Pyogenic infections & periodontal disease. (waldrop-1987)

Lack CR3 , iC3B Receptor

Fatal

Down’s syndrome

Typical facial app with epicanthic folds, broad nasal bridge & protruding tongue,MR & CHD

Rapid Periodontal destruction (Saxen) in 60 to 100% 0f young adults under 30 yrs of age.

Supportive periodontal therapy & maintainence of oral hygeine

Specific Granule deficiency

Depressed respiratory activity& diminished ability to respond to chemotaxis & poor phagocytosis

Oral ulcerations & severe periodontitis

Plaque control & antimicrobial activity

Chronic granulomatous disease

Recurrent indolent pyogenic infections of certain bacteria. Inability to distroy bacteria which gain access to C.T.

Gingivitis & oral ulcers.

Not associated with periodontitis

Frequent regimen of antibiotics used affects periodontal ecology

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