Dr Harshavardhan PatwalDepartment of Periodontics

CONTENTS1. INTRODUCTION

2. FORMATION OF BLOOD CELLS- HEMATOPOIESIS

3. INNATE & ADAPTIVE IMMUNITY

4. HUMORAL & CELL MEDIATED IMMUNITY

5. HUMORAL IMMUNE RESPONSE - PRIMARY IMMUNE RESPONSE - SECONDARY IMMUNE RESPONSE

6. ANTIBODIES

7. B LYMPHOCYTES - ACTIVATION

8. ROLE OF ANTIGEN PRESENTING CELLS

9. COSTIMULATION & ROLE OF B CELLS

10. B CELL- T CELL INTERACTION

11. AUTOIMMUNITY OF B CELLS

12. ANTIBODIES IN GCF

13. VACCINES

INTRODUCTION…

B LYMPHOCYTES: A WBC, agranulocyte derived from the

lymphoid series of pluripotent stem cells.

CHRONIC PERIODONTITIS: Defined as “ An infectious disease resulting in

inflammation within the supporting tissues of the teeth, progressive attachment loss and bone loss”

FORMATION OF BLOOD CELLS- HEMATOPOIESIS

During fetal development generation of all blood cells occurs initially in blood & islands of yolk sac &para aortic mesenchyme.

Later in liver and and spleen.

Gradually –Bone marrow (marrow of flat bones)

Puberty- sternum, vertebra, iliac bone & ribs.

All blood cells originate from a common stem cell that becomes committed to differentiate along particular lineages (i.e.. Erythroid, megakaryocytic, granulocyte, monocyte & lymphocyte)

The bone marrow is the site of all circulating blood cells in the adult, including immature lymphocytes & is the site of maturation of B cell maturation.

INNATE / NATURAL / NATIVE IMMUNITY

Resistance to infections that an individual possesses by virtue of his genetic & constitutional makeup.

The principal component of innate immunity:

Physical or chemical component- epithelia, anti microbial on epithelia surfaces

Phagocytic cells- Neutrophils, macrophages, NK Blood proteins- members of complement system inflammatory mediators Proteins called cytokines-regulate innate

immunity

ADAPTIVE/SPECIFIED/ACCQUIRED IMMUNITY

Immune response is stimulated in response to infectious agents & increase in magnitude & defensive capabilities with each successive exposure to particular microbe.

Particular characteristic- ability to remember. - respond more

vigorously. Components –T& B Lymphocytes & their products

2 TYPES Humoral immunity Cell mediated immunity

CHARACTERSTICS OF IMMUNE CELLS

CharacteristiCharacteristicscs

B cellB cell T cellT cell

Site of Site of productionproduction

Bursa Bursa equivalentequivalent

ThymusThymus

Type of Type of immunityimmunity

Humoral Humoral Cell mediatedCell mediated

Sub population Sub population Plasma cellsPlasma cells Memory B cellsMemory B cells

Helper Helper Suppressor Suppressor

Presence of IgGPresence of IgG ++ __Presence of Presence of receptors for Agreceptors for Ag

++ ++

Life span Life span Short Short Long & shortLong & shortSecretory Secretory productsproducts

Antibodies Antibodies Lymphokines Lymphokines

HUMORAL IMMUNE RESPONSE:1. Primary immune response.2. Secondary immune response.

PRIMARY IMMUNE RESPONSE The increase in Ab titre/ Ag specific T cells

resulting from exposure of a host to an Ag for the 1st time

SECONDARY IMMUNE RESPONSE Subsequent the antigen is exposed 2nd time. More rapid onset & longer in duration. Mediated by Memory B cells. They have

greater specificity Ags when compared to primary exposure.

IgG major role.

ANTIBODIES

B cell- Plasma cells produces Antibodies/ Immunoglobulins.

Ig- 20-25% (15mg/ml) of total serum protein(60-70mg/ml)

9 genetically distinct isotype of Ig: IgE IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2. When B cell exit BM they possess receptor

bearing only IgM. To form secondary isotypes B cell must enter

pathway of differentiation. In this memory pathway B cells undergo ‘isotype switching’. Eg: A.a, can be controlled by neutrophils when opsonised by IgG isotype.

STRUCTURE OF IMMUNOGLOBULIN

Vl- Varaible light chains

Vh- Variable heavy chains

Ch- Heavy chain‘C’region

Cl- Light chain constant

region

A globular protein antigen bound to antibody moleculeshows how a Ag binding site can accommodate soluble macromolecules in their native configuration.Red –heavy chains of Ab, Yellow- Light chains of Ag, Blue- Antigen

B LYMPHOCYTES

DISTRIBUTION: 30% of Lymphocytes

In peripheral blood 15-30% Lymph node 20% Bone marrow 75% Thymus 10% Tonsillar lymphocytes 50% Splenic lymphocytes 50%

ACTIVATION OF B LYMPHOCYTES 1. Synthesis of proteins Naïve B cell large lymphocyte

Proteins are cytokines: * stimulate growth & differentiating of

lymphocytes. *cytokine receptors *other protein involved in gene transcription

& cell division.

2. Cellular proliferation/ Clonal expansion:

* In response to Ag & growth factor made by Ag stimulated lymphocytes, the Ag specific lymphocytes undergo mitotic division.

* massive clonal expansion of microbe specific lymphocytes to keep pace with replicating microbes.

3. Differentiation into effector cells:

B lymphocytes differentiate into * cells actively secreting & synthesizing Ab’s- Plasma cells * Memory B cell

4. Homeostasis- Apoptosis

PLASMA CELLS

B cells are transformed into Plasma cell-Ab secreting cell.

Eccentrically placed nucleus, large blocks of chromatin located peripherally (cart wheel appearance)

Cytoplasm- large, abundant ER, well developed Golgi apparatus.

Develop at lymphoid organs- migrate to the BM.

Survive for 2-3 days Expresses only surface IgM.

MEMORY CELLS

Some of the progeny of antigens stimulated B & T lymphocytes differentiate into memory cells.

Expresses certain isotypes of membrane Ig such as IgG, IgE & IgA

Occupy lymphoid tissues throughout the body . Inactive until 2nd exposure- Ab’s produced.

ROLE OF B CELL AS ANTIGEN PRESENTING CELLS

There 3 professional Antigen presenting cells:1. Peripheral dentritic cells2. Monocyte/ macrophages3. B cells

B cell binds to soluble antigen using BCR. If Ag is bound they are ingested & processed

&parts of the antigen are presented to CD4( Helper) T cells using MHC Cl II molecule.

Th controls the proliferation of T& B Lymphocytes.

CO STIMULATION & ROLE OF B CELLS

Interaction between cells at a high level by producing costimulatory molecule.

Once APC’s (B cells) present Ag to T cell it gives out a 2nd signal.

The most impo 2nd signal is called CO STIMULATION.

Costimulation reaffirms to the Tcell that it has recognised an undesirable Ag.

In absence of co stimulation, T cells may become unresponsive /apoptotic &die.

Costimulation has 3 functions:

1. Prevents apoptosis.2. Upregulates Growth factor receptor on T

cells- therby stimulating its proliferation.3. Decreases the amount of time needed to

trigger the Tcell- Amplification

B CELL- T CELL INTERACTIONS

1. T CELL INDEPENDENT B CELL ANTIBODY RESPONSE

B cell Ag receptor (BCR) if formed partly by Ig molecule on the B cell surface which serve as highly specific Ag receptor.

Therefore B cell are capable of responding to certain Ag in the absence of T cell.

These cells do not mature (i.e. do not enter the memory pathway)

These cells maintain Ig M isotype.

2. T CELL DEPENDENT:

B cells interacts with T cells – then only can enter the memory pathway.

B cell binds to soluble antigen using BCR.

B cell presents Ag to CD4+T cells using Cl II MHC.

After Ag is presented- T cell provides an Activation signal to B cell.

T cell Activators include- transmembraneous Gp39 & Gp34 which interact with B cell receptor CD40 or OX40 respectively.

Compensatory increase in IgM- T cell Gp39 enables a B cell entry into memory pathway.

Absence of Gp39- terminal differentiation of Bcell towards Ig M ,producing plasma cells.

B cells up regulate B7-1 &B7-2 if activated by Gp39.

Co stimulatory factors (B 7-1, B7-2) enables Tcells to differentiate- proliferate & production of cytokines.

Some T cell cytokines are SWITCH FACTORS:

Th1- IL-2,IFNr Th2- IL-4,IL10 Th3- TGFß

Th1, Th2 switch on- inflammatory Ig(IgG/Ig E)

Th3 switch on- IgA (anti-inflammatory isotype)

Th0 – T cell matures to Th0,phenotype that is multifaceted.

Th1- controls altered cells,& intracellular molecule

Th2 – important in pro inflammatory responses against

extracellular antigens.

Th3 – important in anti inflammatory against extracellular

antigens.

B CELL PROGRESSION FROM -GINGIVITIS TO PERIODONTITS

Accumulating evidence, suggest that the host’s immune response to periodonpathogens may be different in those affected by adult periodontitis & those resistant to the disease, in whom it would not progress beyond gingivitis.

In 1976 Page & Schroeder classified the progression of gingival to periodontal inflammation as follows:

Clinical conditionClinical condition Histopathologic conditionHistopathologic condition

Pristine gingivaPristine gingiva Histologic perfectionHistologic perfection

Normal healthy Normal healthy gingivagingiva

Initial lesionInitial lesion

Early gingivitisEarly gingivitis Early lesion of Page & Early lesion of Page & Schroeder (few plasma cells)Schroeder (few plasma cells)

Established gingivitisEstablished gingivitis Estb lesion with no bone loss Estb lesion with no bone loss or apical migration (plasma or apical migration (plasma cell density 10-30%)cell density 10-30%)

PeriodontitisPeriodontitis Estb lesion with bone loss& Estb lesion with bone loss& apical migration from CEJ apical migration from CEJ (plasma cell density >50%)(plasma cell density >50%)

1. INTIAL LESION: predominant with PMN’s.

2. EARLY LESION: T cells ↑, B cells lymphocytes & PMN’s more than plasma cells

3. ESTABLISHED LESION: B cells↑, T cells↓ plasma cells predominant.

2 types of established lesion appear to exist: 1 remains stable & not progress for months/yrs (Lindhe et al 1975, Page et al 1975) 2ND becomes active- progressive advanced lesion

Seymour et al (1979) hypothesized that a change from T cell to B cell dominance, converted it from stability to activity involving aggressive destruction.

Page (1986) Gillette et al (1986) disagreed. Showed that B cell infiltrate mainly was associated with non progressive lesion in childhood gingivitis.

Liljenberg et al ( 1994) compared plasma cell density & found the density of plasma cell(51.3%) was very much increased in active sites as compared to inactive sites.

4. THE ADVANCED LESION: It is generally accepted that plasma cells are

predominant type in advanced lesions ( Garant & Mulvihill,1972)

GINGIVITIS

Strong innate immune response

↓ Th1 ↓ Cell mediated immunity

& Protective antibody

production ↓ STABLE LESION

PERIODONTITIS ↓ Poor innate immune

response Periodontopathic

bacteria Polyclonal B cell

activation ↓ Th2 ↓ Non protective antibody production

↓ PROGRESSIVE LESION

The fact is that stable lesion is mediated by Th1 cells & progessive lesion by Th2 cells.

The net effect of immune response to infection is regulated by a balance between T helper (Th1 & Th2 cytoines)

Th1 cytokines- IL2 &INF-r is to enhance cell mediated immunity.

Th2 cytokine IL4 is to suppress cell mediated & hence enhance the resistance associated with humoral immunity.

T lymphocytes predominant in the stable lesion, while proportions f B cells & plasma cells is increased in progressive lesion.

If innate response is poor & low levels of IL 12 is produced & a poor Th1 response occurs, it may not contain the infection.

Mast cell stimulation & subsequent production of IL 4 would encourage Th2 response, B cell activation& Ab production.

If the Ab’s are protective- clear the infection- No progression.

If it persists, continued B cell activation will produce large amounts of of IL 1 & hence tissue destruction.

It was also shown that decreased IL 10 allowed continued polyclonal B cell activation.

Also shown that CD8+T cells may participate in local response by suppressing IFNr producing cells and favouring humoral response.

CONCLUSION: A tendency for an indiviual or site to form an

extensive plasma cell infiltrate may indicate an inability to defend against peridontopathogenic bacteria & thus a predisposition to peridontitis.

B CELL & ANTIBODY REGULATION IN PERIODONTAL DISEASE

B cells and plasma cells produce Ig, which protect the host by:

1. prevention of bacterial adherence. 2. inactivation of bacterial toxins 3. acting as opsonin for phagocytosis by

neutrophils. 4. antibodies can up regulate/ down

regulate immune system.

Ab’s response to P.gingivalis & A.actinomycetemcomitans are increased in pts with periodontal disease than without . ( Kinane et al 1993, Mooney et al 1994)

Naito et al (1987) demonstrated that the serum titer to P.g was decreased in periodontitis pts following successful therapy.

Mooney et al (1995) conducted a study on specific Ab avidity to P.g & A.a in periodontitis patients before & after periodontal therapy. Found that:

-IgG levels to P.g were increased significantly

- In patients who had originally high levels of IgG & IgA to P.g had demonstrated better treatment outcomes.

- This result suggest that a pts ability to enhance his

Humoral response to suspected periodontal pathogens&

thereby improve outcome of therapy.

Seymour & Greenspan(1979) reported that majority of the lymphocytes had the phenotype of B cells and were positive for IgM & IgG.

Lindhe et al(1980) – Periodontitis lesion:31%plasma cells,5-10%lymphocytes, 5%fibroblast, 1.3%macrophages, 1.3%neutrophil. In the gingivitis lesion the ratio lymphocytes-plasma cells was 1:1,in periodontitis-1:3.

Okada et al(1983) Only few PMNs were observed. Plasma cells predominated in the central portion of the lamina propria,with the proportion positive for IgG,IgA & IgM accounting for 65.2%,11.2% & 1.3% of the total infiltrating cells.

Gillett et al(1986) : In juvenile periodontitis biopsies,>50% of the cells were plasma cells. Lesions in chronic adult periodontitis were dominated by Bcells & plasma cells.

Morinushi et al (2000 ) showed that serum anti P. gingivalis but not anti A.a antibodies were inversely correlated with gingival inflammation, suggesting an inhibition of P.gingivalis antibodies.

Gemmel et al (2001) found that a higher % of CD86+cells indicated predominance if Th2 response in both healthy/gingivitis & periodontal tissues.

Berglundh T, Donati M (2005) found that in periodontitis lesion, plasma cells most common-50% of all cells. B cells-18%. Proportion of B cells larger than T cells. The relative dominance of B cells & plasma cells may be because of imbalance between Th1 & Th2.

Auto immune reactions were also evident in periodontal lesions.

AUTO IMMUNITY OF B CELLS

Brandtzaeg & Kraus (1965) reported the presence of autoAb producing plasma cells in periodontitis lesion.

A particular group of Bcells CD5+B (B-1a)cells are found in large nos in peripheral blood of pts with autoimmune disorder.

B1 –a cells may develop into plasma cells & produce Ig of other classes other than IgM.

Afar et al (1992) & Berglundh et al (2002) showed B1-a cells in periodontitis pts were 5-6 times more than normal.

Berglundh et al (1999) in a study on local & systemic features of host response in CP before & after non surgical periodontal therapy found that the elevated no. of B1-a cells did not decline after therapy.

Aramaki et al (1998) further reported that IL 10 which is an autocrine growth factor for B1 cells was found in higher levels in gingival tissues than peripheral blood.

It was suggested that periodontal pathogens induce a hyperactive IL 10 response leading to proliferation of B1-a cells & to auto Ab production.

ANTIBODIES IN GCF:

IgG is more prevalent.

Identified about 58% B lymphocytes, 24% T lymphocytes & 18% Mononuclear phagocytes.

The ratio of T to B lymphocytes was 1:3 in GCF (reversed from normal ratio of 3:1 in peripheral blood).

In a pt with periodontal disease 1. reduction in Ab response is detrimental 2. Ab response plays a protective role ( Lamster IB, Celenti R, 1990)

VACCINES

Passive immunization of humans using monoclonal Ab’s temporarily prevents colonization of P.gingivalis.

Gemmell et al (2004) used F. nucleatum & P. gingivalis bacteria in immunisation schedule. Results showed that a vaccine candidate maybe enhanced by combination of both. Raised IgG1 and IgG2 were seen.