ROLE OF NEUTROPHILS IN PERIODONTAL DISEASE
PRESENTED BY SHEETAL OSWAL
GUIDED BY DR C D DWARAKANATH
CONTENTS Introduction Role of phagocytes in host defense function & dysfunction Neutrophil function Neutrophils & periodontal tissues Altered neutrophil functions & periodontitis -Chronic periodontitis -Localised aggressive periodontitis -ANUG Neutrophil defects-classification Periodonta Periodontall disease associated with neutrophil abnormalities
Neutrophils
& periodontal tissues in systemic diseases
Anti
neutrophil therapies
-Lipoxins -ATL Neutrophil Summary
assays
& 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. 2. 3. 4. 5.
Neutrophil rolling & Margination Adhesion Diapedesis/ transendothelial migration Chemotaxis Extracellular secretion, receptoe upregulation & binding to target 6. Phagocytosis 7. Microbial killing
Functions
of Neutrophils
INFLAMMATION INFLAMMATION PMN¶S ADHERE TO ENDOTHELIUM
Local inflammation IL-1beta & TNF from mast cells & leucocytes
Releases chemokines Signal fo Rolling arrest
L-selectins on PMN¶S interact with endothelium Rolling
Endothelium to express P & E selecins on luminal surfaces P & E selectins+ L selectins Increased rolling
Chemokines interact with PMN receptor CxCR2
PMN¶s shed selectins & 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 on PMN
G-coupled family
Receptor upregulation & binding to target
IgG & iC3b
Recognise pathogen Opsonisation
Phagocytosis Delivery of antimicrobial substances
Microbial killing
Phagocytosis & Killing
VARIOUS
Target
OPSONINS & OPSONIN RECEPTORS
Opsonin
Receptor
Gram ±ve bacteria LPS binding protein
CD14
Any cell
iC3b
CR3, CR4
Any cell
IgG1, IgG2, IgG3
FcRII,
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
s
Acid hydrolases
-Lysozyme
- Cathepsin B
- Apolactoferrin
- Cathepsin D
- Collagenase
- Cathepsin G - Chloroacetate esterase - Elastase - Beta glucoronidase - Beta galactosidase - L-mannosidase - L-fucosidase - Beta glycerophosphatase - Arylsulphatase - Lysozyme - Myeloperoxidase - Defensins - BPI
- Cobalamine binding protein - C5a cleaving enzyme - Plasminogen activator. -
Cytochrome b558
Microbial killing 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
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.
a a l b a cteri a in hypoxic a thepsin G kills most periodont C a tic (C a p sp) & non conditions by both enzym a tic ( A. a ) degr a d a tion. enzym a tes killing of b a cteri a by lysosyme, BPI, & It potenti a tory burst MPO-H2O2-Cl system-Respir a nces PMN ph a gocytosis & promote complement It enh a ted gr a nulocyte chemot a ctic a ctivity. medi a nism of controlling b a cteri a -Kenneth M a y a as ki) (Mech
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 +NADPH²2O2- +NADP+ +H+ 2O2 +2H²O2 + 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 +Cl²HOCL 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.
Oxidative killing of A.a by H 2O2 requires 10 times higher conc than that Neutrophil andFurther periodontal tissues anticipated in phagolysome. its killing by H 2O2 & oxygen is blocked by deffuroxime , suggesting the bactericidal activity of H 2O2 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 rolling
LAD-2 LAD1
Strong adhesion & diapedisis
Actin dysfunction
chemotaxis
Diabetes
phagocytosis CGM Myeloperoxide deficiency
Chediak Higashi Microbial killing 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 CD11/CD18
Increased adhesion Vascular adhesion Locomotion & migration
GP-110 chemotaxis
chemotaxis
Receptor for FMLP & C5a & LB4
Receptor expression Receptor Receptor
Phagocytosis
expression expression phagocytosis? superoxide
CYTOKINES
Microbial killing
killing
killing
Altered neutrophil function ±induced or intrinsic??? Sudha A garwal et al JP,96;67 The chemotactic defect is irreversible by treatment & appears to be intrinsic to LJP neutrophils
r
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(70-75%) e 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 Chemotaxis & chemotactic receptors
Alter function of PMN alter adherence
IL1B
Superoxide anions & 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
PAF
Reduce catabolism of PMN¶s
PMN degranulation releases proteolytic enzymes Tissue factor synthesis Generate chemotactic substances
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)
Role of neutrophils in ANUG 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 infiltration
Neutrophil defects Quantitative
Qualitative Defects in adhesionLAD1 & LAD2
Neutropaenia Defect Def ectss in chemot chemotaxi axiss Defects in phagocytosis Defects in microbicidal activity
Neutropaenia Production defects
Aplasia Infiltrative diseases
Destructive defects Splenic sequestration
Drugs Metabolic diseases Infective diseases Kostmann syndrome
Antineutrophil antibodies
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. T hey are
Chediak higashi syndrome Job¶s syndrome Papillion lefreve syndrome Leukocyte adhesion deficiency Down¶s syndrome Chronic granulomatous disease Specific granule defeciency Diabetes
D ISEASE
ORAL F EATURES
Chediak higashi disease
Severe periodontal disease with advanced bone loss ,oral ulcers & glossitis
Immune def, partial Oculocutaneous albinism,easy bleeding. recurrent infections.giant abnormal lysosomalgranules
M E N T TREAT
F atal.
no specific treatment T hose who survive have neurological symptoms
D ISEASE
ORAL FEATURES
M E N T TREAT
J ob¶s
Coarse facies,
Cause?
disease
Staphylococcal Pneumonia, skin absceses with high levels of IgE
Hypertelorism, jaw
Pappilion Lefervre syndrome
Aggressive periodontitis with rapid destruction of alveolar bone.
Palmer & plantar keratosis, mental retardation & intracranial calcification. Hyperhidrosis,& fine
Abnormal neutrophil Prominence, cranial & monocyte synostosis & severe phagocytosis & chemotaxis Pdl disease
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 Pyogenic infections & periodontal deficiency disease. Leucocytosis, (waldrop-1987) delayed wound
heaing & less leucocyte mobilisation
Fatal
Lack CR3 , iC3B Receptor
Rapid Periodontal Supportive destruction (Saxen) periodontal therapy Typical facial app in 60 to 100% 0f & maintainence of with epicanthic young adults under oral hygeine folds, broad nasal bridge & protruding 30 yrs of age. tongue,MR & CHD Down¶s syndrome
Specific Granule deficiency
Depressed respiratory activity& diminished ability to respond to chemotaxis & poor phagocytosis Chronic granulomatous disease Recurrent indolent pyogenic infections of certain bacteria. Inability to distroy bacteria which gain access to C.T.
Oral ulcerations & Plaque control & severe periodontitis antimicrobial activity
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
- F or chemotaxis -The Rebrick skin window -Boyden chamber -Agarose technique F or Intracellular killing -Chediak Higashi- Large azurophilic granules -Specific Granule deficiency-Wright¶s stain & assays for constitute proteins F or respiratory burst activity -Nitroblue Tetrazolium test ±Formazan precipitate -Flow cytometry- dihidrohodamine-Rhodamine
Conclusion
References Carranza¶s clinical periodontology -8 th & 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 bacteria²Kenneth.T.Mayasaki ±J.P 91
