Neutrophils : In Health And Disease 6q1923

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 ing 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

Leukocyte adhesion deficiency

Mostly children are affected. Recurrent infections with 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 E (Job’s syndrome) (rare, PMNs. complex autosomal, recessive disorder)

Oral manifestations

Recurrent infection with opportunistic organisms like, Staphylococcus aureus and Candida albicans.

Condition

Neutrophil Abnormality

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.

Structural defect where in the Ulceration of the oral transformation of mucosa, severe gingivitis, Azurophilic and specific glossitis, periodontal disease, granules into giant bodies severe bone loss and called ‘megabodies’ is recurrent pyogenic infection. characteristic. Functional Mutation in the vesicle neutrophil defects include trafficking regulator gene decreased chemotaxis, LYST. degranulation and microbicidal activity.

Papillon – Lefevre syndrome Reduced PMN motility (Van is a rare autosomal recessive Dyke 1984) and reduced disorder. PMN bactericidal activity (Sham El et 1984) have been reported.

Oral manifestation

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 therefore serve as a marker of neutrophil activity.

and may

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, inflammation.

neutrophils

quickly

reach

the

site

of

• 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 microorganisms:

3 strategies for directly attacking

 Phagocytosis  Release of soluble anti – microbials  Generation of neutrophil extracellular traps

PHAGOCTOSIS • Neutrophils are microorganisms.

phagocytes,

capable

of

ingesting

Respiratory burst

Activation of NADPH oxidase Superoxide

Hydrogen Peroxide

Hypochlorous acid (HCIO)

Superoxide dismutases

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 hypoxia?". Semin Immunopathol 2007; 29 : 163–7]

preeclampsia-associated

placental

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 preeclampsiaassociated 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