Neutrophils in health and

disease : an Overview
Journal of oral and maxillofacial pathology, Volume 10, Issue 1, Jan – June 2006

Rashmi SM, Alka DK,

Ramakant SN

Presented by :
Dr.Kush Pathak
 Introduction
• Neutrophils are so named because of their neutral staining with wright stain.

• Also known as PMNs, polys or microphages.

• Are round in shape and approx. 12 – 14 µm in diameter.

• Multilobed shape of nucleus contributes in extreme cell elasticity.

• When infection occurs, chemotactic agents are generated, that result in

migration of neutrophils to the site of the infection and activation of
neutrophil defensive function.
LIFE HISTORY
• Neutrophils have limited capacity to regenerate expended
lysosomal and specific enzymes, which are rapidly depleted
by phagocytic activity.

• Cytosolic and granule components can be delivered by

apoptotic process (programmed death).
 Subcellular Structure Of Neutrophils

• Immunoelectron microscopy and subcellular fractionation are

used to understand functions of neutrophils.

• Types of neutrophils :
 Azurophilic (primary) granules.
 Specific (Secondary) granules.
 Gelatinase (tertiary) granules.
 Secretory vesicles.
• Granules have fine granular matrix bounded by a typical
membrane.

• Shape of granules varies from round to grain of rice and small

dumbles.

• Number varies from 500 – 1500/ granulocyte.

• Large amount of proteins and traces of lipids and nucleic acids

are present in granules.
• These granules are essential for post phagocytic function of PMN leucocytes and their constituents are essential in
inflammation.

• Azurophilic granules contain myeloperoxidase, defensins, lysozyme, azurocidin etc., which have antibacterial
function.

• Greenish coloration to pus is imparted by myeloperoxidase.

• Specific granules are 3 times more common in cytoplasm and release of it’s contents like collagenase,
apolactoferrin, lysozyme, histaminase, etc., may modify the infalmmatory process.
 Sex Indicated By Leukocytes
• Only one X chromosome is essential for normal activity of cell
; the other in normal XX female remains unextended and thus
is visible as a chromatin body.

• This chromatin body of neutrophil of female is a small mass,

usually adjacent to the nuclear membrane that stains deeply
with haematoxylin, cresyl violet, fuchsin gallocyanin, Feulgen
and thionine and is about 0.7 to 1.2 µm.

• They take a form of drumstick projecting from one of the

nuclear lobes.
• Confirmation of X chromosome in drumstick has been
provided by in situ hybridization.
 LEUKOCYTES IN THE DENTOGINGIVAL AREA
AND SALIVA

• Predominantly leukocytes present in a clinically healthy gingival

sulci are neutrophils.

• Gingival sulcus is the main portal for entry of leukocytes in the oral
cavity.

• These cells have phagocytic and killing capacity and thus constitute
a protective mechanism against plaque extension in the gingival
sulcus.

• pH changes in periodontal environment could alter the balance

between the host and the bacteria.
• Saliva contains all forms of leukocytes along with desquamated
epithelial cells. Among them, majority is of PMNs.

• In saliva, living PMNs are sometime known as

‘OROGRANULOCYTES’. The rate of their migration in the oral
cavity is called ‘OROGRANULOCYTIC MIGRATORY RATE’.

• Skougoard in 1994, proposed that the rate of migration is

correlated with the severity of gingival inflammation and hence is
reliable index for assessing gingivitis.
NEUTROPHIL INTERFACE WITH MICRO
ORGANISMS
• PMNs form a “leukocyte wall” interposed between the
periodontal plaque mass and junctional and sulcular
epithelium.

• This leukocyte wall may function both as a secretory and as a

digestive organ.

• Crevicular neutrophils exhibit both partial degranulation and

ingested bacteria.
 CONTROL OF PERIODONTAL BACTERIA BY
NEUTROPHILS

• Miller in 1984 suggested that, initially periodontopathic organisms

encounter plasma factors. Result of this encounter is the initiation of
inflammation as evidenced by the recovery of compliment product
from the crevice. If the compliment is not successful in controlling
the pathogen, the host defense turns to neutrophil.

• Neutrophils within the gingival crevice provide the first cellular host
mechanisms to control periodontal bacteria.
• If PMNs, are unsuccessful, monocytes are recruited which
infiltrate in the connective tissue and then by developing into
the macrophages, digest the antigen.

• Thus, neutrophils are important because they control

periodontal micro-ecology prior to the involvement of chronic
inflammatory cells.
• The ability of neutrophils to destroy microorganisms with the
intracellular enzymes can be evaluated by the nitroblue
tetrazolium reduction test (NBT).

• Normal neutrophils contain enzymes that convert colorless

NBT to dark blue granules within the cell and if these dark
blue granules are not seen, neutrophils will not destroy
bacteria.
• Studies evaluating the other aspects of neutrophil function
include the following :

 The Steroid Challenge, which assesses neutrophil reserves in

the bone marrow.

 The Epinephrine Challenge, which assesses neutrophil reserves

in the marginal pool.

 The Endotoxin Challenge, which measures the ability of

neutrophils to migrate to the peripheral tissues.
ORAL MANIFESTATIONS ASSOCIATED WITH
DECREASE IN CIRCULATING NEUTROPHILS
AND NEUTROPHIL DYSFUNCTION
• Oral ulcer, advanced periodontal disease, pericoronitis, and pulpal
infections in patients with severe neutropenia should be considered
potentially life threatening because they may lead to bacteremia
and septicemia.

• The most susceptible tissue to pathologic changes in the oral cavity

due to neutrophil dysfunction are the tissues of the periodontium.

• Presence of PMNs in connective tissue, junctional epithelium and

gingival crevice or periodontal pocket of the periodontium is a
characteristic feature of chronic periodontal disease
Conditions Neutrophil Abnormility Oral manifestation
Agranulocytes Almost complete absence of Appear as necrotizing ulcer of
granulocytes or PMNs oral mucosa, tonsils and
pharynx.
Mainly gingiva and palate are
involved.
Lesion appear as ragged
necrotic ulcer covered by a
gray or even black
membrane.
Excessive salivation is seen.

Cyclic neutropenia A periodic or cyclic diminution Severe gingivitis, stomatitis

in circulating with ulceration.
polymorphonuclear In children, repeated insult of
neutrophilic leukocytes as a an infection often leads to
result of bone marrow considerable loss of
maturation arrest. These supporting bone around the
regress spontaneously only to teeth.
recur in subsequent rhythmic
pattern.
NEUTROPHILIC ABNORMALITIES WITH ORAL
MANIFESTATIONS
• About one half of the patients with Down Syndrome suffer from chemotactic
defects in PMN.

• In down syndrome, PMNs fail to exert their normal bactericidal function.

• Leukocyte alkaline phosphatase activity (LAP) of the various granulocytic

enzymes present in the neutrophil has been studied as a diagnostic tool.

• LAP activity is reported to be altered in pregnancy, mylofibrosis, polycythemia

vera, leukemoid reaction, Cushing’s syndrom, hypophosphataemeia, familial
lymphomas, melanomas, endometrial carcinoma and chronic myeloid leukemia.
• Till date there is no explanation for the alteration of LAP
activity. The suggested hypothesis for these alteration are,

 Production of abnormal alkaline phosphatase.

 Decreased production of blood cells.

Condition Neutrophil Abnormality Oral manifestations

Leukocyte adhesion Mostly children are affected. Recurrent infections with

deficiency They have multiple defect in pyogenic bacteria including
neutrophil and mononuclear severe periodontal disease.
phagocyte adhesion
dependent functions,
including chemotaxis and
CR, mediated phagocytosis.

Hyperimmunoglobulinemia Defects in chemotaxis of Recurrent infection with

E (Job’s syndrome) (rare, PMNs. opportunistic organisms like,
complex autosomal, Staphylococcus aureus and
recessive disorder) Candida albicans.
Condition
Chediak – Higashi syndrome
(rare disease) often fatal in
early life as a result of
lymphoma like terminal
phase, hemorrhage or
infection with an autosomal
recessive mode of
inheritance.
Papillon – Lefevre syndrome Reduced PMN motility (Van
is a rare autosomal recessive Dyke 1984) and reduced
disorder.
Neutrophil Abnormality
Structural defect where in the Ulceration of the oral
transformation of
Azurophilic and specific
granules into giant bodies
called ‘megabodies’ is
characteristic. Functional
neutrophil defects include
decreased chemotaxis,
degranulation and
microbicidal activity.
PMN bactericidal activity
(Sham El et 1984) have been
reported.
Oral manifestation
mucosa, severe gingivitis,
glossitis, periodontal disease,
severe bone loss and
recurrent pyogenic infection.
Mutation in the vesicle
trafficking regulator gene
LYST.
Rapid generalised destruction
of alveolar bone
Early loss of Deciduous and
Permanent teeth
Tissues heal rapidly without
sequelae until the permanent
teeth eruption.
Recently associated with
individuals with a mutation
in cathepsin C gene.
Condition Neutrophil Abnormality Oral Manifestation

Specific granules deficiency Represents a faliure to pack Severe Periodontitis and

(SGD) is a rare disease, which is whole group of proteins (both ulceration.
probably autosomal recessive specific and Azurophilic) into
granules. Deficiency of these
components results in depressed
respiratory burst activity,
diminished ability to respond to
chemotaxis and poor
phagocytosis. Therefore
intraphagolysosomal killing is
predictably sluggish

Localized juvenile Periodontitis High lymphocyte counts and

increased number of immature
granulocytes in peripheral blood.
This may be due to 2 factors : (a)
Neutrophils intrinsically
defective. (b) Extrinsic factors in
sera of LJP patients alter
neutrophil functions.
 PULPAL INFLAMMATION

• Neutrophil is the most common type of leukocyte found in

pulpal inflammation. Although eosinophils and basophils are
also detected.

• They are major cell types in micro abscess formation and very
effective at destroying and phagocytizing bacteria or dead
cells.
 PERIAPICAL LESIONS

• Inflammatory response here is similar to any where else in the

body.
 WOUND HEALING

• Neutrophils have number of functions :

 Cell lysis.

 Phagocytosis of cells, debris, exudate, foreign particles, immune complexes and erythrocytes.

 Fibrinolysis by phagocytosis and extracellular lysis.

 Assisting in epithelial cell migration.

 Release of proteolytic enzymes such as elastase, which can
destroy the fibronectin coating of fibroblasts that enable them
to differentiate and migrate.

 An influence on blood flow and albumin extravasation in

granulation tissue.
 IMPLANTS

• Neutrophil granulocyte is the primary defender against

bacterial invasion, but it has also been implicated as an
effector cell in several inflammatory tissue destructive
diseases including periodontitis.

• Neutrophil derived enzymes in the crevicular fluid have been

used as risk markers for tissue destruction in periodontal
disease and may also prove useful as a sensitive marker of
destructive process around implants.
• Elastase, which is a serine protease derived from the primary
granule of the neutrophil is also shown to correlate with bone
reduction around implants.

• It is released extracellulary during phagocytosis and may

therefore serve as a marker of neutrophil activity.
 CRITICAL EVALUATION

• Author didn’t mention about classification of neutrophils.

• Author didn’t discuss Neutrophil Extracellular Traps (NETs).

• Lack of images in the article.

• Did not discuss about various clinical manifestations.

DISCUSSION
• Definition: Neutrophils are the most common type of white blood cells,
comprising about 50-70% of all white blood cells.

• They are Phagocytic.

• They are the first immune cells to arrive at a site of infection, through a
process known as chemotaxis.

• They are the main component of pus and are responsible for it’s white
color.
• Neutrophils can release a net of fibers called a neutrophil
extracellular trap (NET), which serves to trap and kill
microbes outside of the cell.

• Neutrophil granulocytes are generally referred to as

either neutrophils or polymorphonuclear
neutrophils (or PMNs).
• The are subdivided into segmented neutrophils (or segs) and banded
neutrophils (bands).

 Segmented Neutrophils – presence of more than one nucleus

fragment.

 Band Neutrophils – Only one nucleus fragment present.

[J.K. Mui et al. Automated classification of Blood cell Neutrophils. The

journal of Histochemistry and Cytochemistry 1977;25,7; 633 - 640]
The cytoplasm is
transparent because it’s
granules are small and
faintly pink colored.
Immature neutrophils have
a band-shaped or
horseshoe-shaped nucleus
and are known as band
cells.
This scanning
electron
micrograph
shows a
neutrophil
(yellow)
engulfing rods
of Bacillus
anthracis
(orange), the
etiological
agent of
anthrax
• It’s name is derived from it’s staining characteristics on
hematoxylin and eosin.
• Neutrophilic white blood cells stain with a neutral pink color.
• There nucleus is divided into 2 – 5 lobes.
 MEASUREMENT OF NEUTROPHILS

• They have an average diameter of 12 – 15 µm in peripheral

blood smears.

• Cell nucleus of a female neutrophil shows a small additional X

chromosome structure, known as “neutrophil drumstick”.

• The standard normal range of a neutrophil count is 2.5-7.5 x

109/L.
 LIFE SPAN
• The average lifespan of non-activated neutrophils in the circulation
is about 1 - 4 days.

• On activation, they marginate (position themselves adjacent to the

blood vessel endothelium), and undergo selectin -dependent capture
followed by integrin - dependent adhesion in most cases, after
which they migrate into tissues, where they survive for 1–2 days.

• Neutrophils themselves get phagocytosed by macrophages after

digestion of pathogens. PECAM -1 and phosphatidylserine are
involved in this process.
 CHEMOTAXIS

• Neutrophils undergo a process called chemotaxis, which

allows them to migrate toward sites of infection or
inflammation.

• Cell surface receptors allow neutrophils to detect chemical

gradients of molecules such as interlukin -8(IL-8), interferon
gamma (IFN-gamma), and C5a, which these cells use to direct
the path of their migration.
 ANTIMICROBIAL FUNCTION

• Being highly motile, neutrophils quickly reach the site of

inflammation.

• Release cytokines, which in turn amplify inflammatory reactions by

several other cell types.

• Play a key role in the front-line defense against invading pathogens.

• [http://en.wikipedia.org/wiki/Neutrophil_granulocyte#Anti-microbial
Function]
• Neutrophils have 3 strategies for directly attacking
microorganisms:

 Phagocytosis

 Release of soluble anti – microbials

 Generation of neutrophil extracellular traps

 PHAGOCTOSIS

• Neutrophils are phagocytes, capable of ingesting

microorganisms.
 Activation of
Respiratory burst
NADPH oxidase

Superoxide

Hydrogen Superoxide
Peroxide dismutases

Hypochlorous acid
(HCIO)
Neutrophil granulocyte
migrates from the blood
vessel to the matrix,
sensing proteolytic
enzymes, in order to
determine intercellular
connections (to the
improvement of its
mobility) and envelop
bacteria through
Phagocytosis.
Robbins’ Pathologic Basis of Disease
7th edition
 DEGRANULATION

• Neutrophils also release an assortment of proteins in three

types of granules by a process called degranulation.

 Specific granules - Lactoferrin and Cathelicidin(hCAP18).

 Azurophilic granules - myeloperoxidase, bactericidal/

permeability increasing protein (BPI), Defensins and Serine
protease neutrophil elastase and Cathepsin G.

 Tertiary granules – Cathepsin and gelatinase.

Neutrophil granules
 NEUTROPHIL EXTRACELLULAR TRAPS

• Activation of neutrophils causes the release of web-like

structures of DNA: which is the third mechanism for killing of
bacteria. [Brinkmann et al. Neutrophil extracellular traps kill bacteria .Science
2004 ; 303 ;March 2004; 1532 – 1535]

• These traps contain web fibers composed of chromatin and

serine proteases that trap and kill microbes extracellulary.

• It provides a high local concentration of antimicrobial

components and bind, disarm, and kill microbes independent
of phagocytic uptake.
Inactivated neutrophils Activated neutrophils
• They serve as a physical barrier that prevents further spread of
pathogens. Trapping of bacteria may be a particularly important role
for NETs in sepsis, where NETs are formed within blood vessels.
[Stephen R Clark et al. “ Platelet TLR4 activates neutrophil extracellular traps to ensnare
bacteria in septic blood”. Nature medicine 2007;13;4: 463- 469]

• They play an important role in inflammatory diseases, as NETs could

be detected in preclampsia, a pregnancy related inflammatory
disorder in which neutrophils are known to be activated. [Gupta, Ak et al.
"Neutrophil NETs: a novel contributor to preeclampsia-associated placental
hypoxia?". Semin Immunopathol 2007; 29 : 163–7]
 ROLE IN DISEASES

Neutropenia -
Low neutrophil counts are termed as neutropenia, and makes a
patient highly susceptible to infections.

This can be congenital or can even develop later in life due to

aplastic anemia and some leukemia.

Can also be a side effect of some medications, mainly

chemotherapy.
• Functional Disorders –
 They are often heriditary.

 They are disorders of phagocytosis or deficiencies in

the respiratory burst. (as in chronic granulomatous disease and
myeloperoxidase deficiency).
• Alpha 1- antitrypsin deficiency –
 Here important neutrophil enzyme elastase is not inhibited by
alpha 1- antitrypsin, leading to excessive tissue damage in the
presence of inflammation –most prominently pulmonary
emphysema.
• Familial Mediterranean fever (FMF)
 A mutation in pyrin (or marenostrin), which is expressed
mainly in neutrophil granulocytes, leads to active acute phase
response and causes attacks of fever, arthralgia, peritonitis and
amyloidosis.
• Leukocyte Adhesion Deficiency –
 Is a human genetic disease that shows autosomal recessive inheritance.

 Patients have recurrent bacterial infections, diminished pus formation,

impaired wound healing.

 Oral characteristic features include generalized periodontitis, progressive

alveolar bone loss, premature tooth loss and severe gingival inflammation.

[Thomas E Van Dyke and George A. Hoop. Neutrophil function and Oral Disease. Oral Biology and
Medicine 1990; 1, 2 ; 117 – 133]
• AIDS –
 Abnormalities in leukocyte number and functions contribute to the high incidence of
infection in patients with HIV.

 Leukopenia is a frequent occurrence in such cases.

 Oral manifestations related to neutrophil dysfunction in AIDS are oral lesions and
periodontal breakdown.

 AIDS associated periodontitis and ANUG are also characteristic feaures.

[Thomas E Van Dyke and George A. Hoop. Neutrophil function and Oral Disease. Oral Biology and Medicine 1990; 1, 2 ; 117 – 133]
 CONCLUSION

• Neutrophils are a very important line of defense against micro

organisms and a decrease in their number or any dysfunction
of these neutrophils can lead to serious abnormalities,
regardless of age. The tissue most sensitive to pathological
changes in the oral cavity is periodontium.
 References
• Guyton AS, Hall J E. Textbook of Medical Physiology, 10th edition.
Pennsylvania : Elsevier ;2000. 392 – 399

• Stephen R Clark et al. “ Platelet TLR4 activates neutrophil extracellular

traps to ensnare bacteria in septic blood”. Nature medicine 2007;13;4:
463- 469

• Gupta, Ak et al. "Neutrophil NETs: a novel contributor to preeclampsia-

associated placental hypoxia?". Semin Immunopathol 2007; 29 : 163–7
• Brinkmann et al. Neutrophil extracellular traps kill bacteria
.Science 2004 ; 303 ;March 2004; 1532 – 1535

• J.K. Mui et al. Automated classification of Blood cell

Neutrophils. The journal of Histochemistry and Cytochemistry
1977;25,7; 633 – 640

• Thomas E Van Dyke and George A. Hoop. Neutrophil

function and Oral Disease. Oral Biology and Medicine 1990;
1, 2 ; 117 - 133