BASIC IMMUNOLOGY OF PULP AND

PERIAPICAL TISSUES
Maria Tanumihardja
TEXTBOOKS:
• Ingle’s Endodontics. 6th ed, 2008. J.I.Ingle & LK Bakland.
• Endodontics. Principles and Practice. 4th ed,2009, M.Torabinejad &
R.Walton.
• Cohen’s Pathways of the pulp. 10th ed, 2011.
• Treatment of endodontic infection. Jose F.Siqueira Jr 2011.
Quintessence Publishing.
• Medical microbiology and immunology for dentistry. Nejat Düzgünes,
2016.
• Cellular and molecular immunology, 6th Ed, Saunders. Abbas AK,
Lichtman AK, Pallai S
Terminology

• Immunity→ resistance to disease, specifically infectious disease

• Immune system→ the collection of cells, tissues, and molecules that
mediate resistance to infections
• Immune response→ the coordinated reaction of these cells and
molecules to infectious microbes
• Immunology→ the study of the immune system and its responses to
invading pathogens
Host defense mechanisms
Consist of :
- Innate immunity (natural or native immunity)→ mediates the initial
protection against infections
- always present in healthy individuals
- prepare to block the entry of microbes
- eliminate microbes raidly that succeed in entering host tissues
- Adaptive immunity (specific or acquired immunity)→ protection
develops more slowly and mediates the later, more effective defense
against infections after adaptation to the presence of microbial
invaders
Host response/host resistance :
1. Innate immune response (= non specific) :
Predominant cells → odontoblast cells, PMN, macrophage,
dendrite cells, NK cells
Bacteria → PAMPs recognized by PRRs ( called TLRs)

activation of PRRs triggers numerous host responses including

opsonization, activation of complement, coagulation cascades,
phagocytosis, activation of proinflammatory signaling pathways,
and induction of apoptosis.

Macrophages also serve as APC (antigen presenting cells) by

expressing MHC class II molecules for Adaptive immunity
2. Adaptive Immunity (= acquired, specific)
Main role is T cells and B cells.
- the specifity is regulated at genetic levels in B (BCRs)
and T lymphocytes (TCRs)recognize and bind foreign
or self- antigens)
T receptor is TCRs with subpopulations : Thelper cells,
Treg, Tsup, Tcyto cells distinguished by their cell
surface markers, cytokine profiles or transcriptional
factors. Constitute for cell-mediated immunity
- B cells→ mainly produce antibodies that constitute the
host humoral immune response
INNATE IMMUNITY
• Cells of innate immunity
- Neutrophil/PMN
- 54%-63% of peripheral blood leucocytes
- short half life→ circulating ± 6 hours not recruited, apoptosis
- first defense line against mo→ acute inflammation
- defense mechanisms→ production of lysosomal enzymes, cytokines,
oxygen-derived free radicals, phagocytosis
Neutrophil/PMN

- defense proteins in 2 different granules

. Azurophilic/large granules contain lysozyme, myeloperoxidase,
defensins, acid hydrolases, neutral hydrolases (kolagenase,
elastase, cathepsin-G, and other proteinases
. Small granules contain lactoferrin, lysozyme, collagenases,
plasminogen activator, histaminases, alkaline phosphatase,
membrane-bound cytochrome b558
INNATE IMMUNITY
• Monocytes/macrophage
- circulating leucocytes, 4%-8% of blood leucocytes
- macrophages→ reside within connective tissue, long half-life in
tissues (months), principal inflammatory cells mobilized in chronic
inflammation, as APC, posses MHC-II, involved in phagocytosis
- principal produces of proinflammatory cytokines→IL-1, IL-6, TNF-α,
and IL-12
- express surface receptors (TLRs) to PAMPs (LPS, LTA, PG)
- microbial killing→ within the phagosome, oxidative burst
Figure 2-5 Maturation of mononuclear phagocytes. Mononuclear
phagocytes develop in the bone marrow, circulate in the blood as
monocytes, and are resident in all tissues of the body as macrophages.
They may differentiate into specialized forms in particular tissues. CNS,
central nervous system
INNATE IMMUNITY

• Eosinophils→only 1%-3% of circulating leucocytes

- involved in allergic and some parasitic reactions
- express some inflammatory and healing mediators
( TGF-α) and TGF-β)
Basophils→ similar life span with neutrophil & eosinophil
- involved in the mediation of allergic reactions
- share some features with connective tissue mast cells
INNATE IMMUNITY
• Mast cells
- reside in connective tissues for weeks or months
- generally dormant with little activity until stimulated by IgE (type 1
hypersensitive reaction)
- degranulation mast cells→ local (atopic) or systemic (anaphylactic)
reactions→ vasodilation and bronchoconstriction
Anaphylactic rx→ life threatening (apnea and reduced blood
pressure) mediated by release of granule contents (histamine,
arachidonic acid metabolites (PGE2, PGI1,, LTB4, LTD4), PAF, ECF-a) and
other enzymes
INNATE IMMUNITY
• Dendritic cells (DCs)
- reside in a variety of tissues incl blood, lymph, epidermis, dermis,
and secondary lymphoid organs
- also present in the dental pulp
- prevalent among the odontoblastic cell layer and around blood
vessels, periodontal ligament and periradicular lesions
- as antigen presenting cells (APC), express MHC-II
INNATE IMMUNITY
• Natural Killer Cells (NK)
- lymphocytes that do not require activation like CD4 or CD8 lymph
- member of innate immune response
- 4%-20% of circulating mononuclear cells
- produce IFN-ϒ→ capable of activating Mφ to produce cytokines
- particularly effective in killing host cells infected with viruses or
other intracellular microorganisms
INNATE IMMUNITY
• Odontoblasts
- dentin-producing cells from ectomesenchymal cells
- first line of cells encountering foreign antigens after enamel/dentin
breakdown similar to epithelial cells, part of innate immunity
- express chemokines IL-8, and antimicrobial peptides, defensins
- express TLRs→ play a significant role in the pulp innate immunity
 COMPONENTS OF INNATE IMMUNITY
CYTOKINES→secreted protein induced by mo or antigens to
trigger various cells respond of involved cells
TNF,IL-1, chemokines inflammation
INF-α,-β Resitance to viral infection
INF-γ Marophage activation
IL-12 INFγ production by NK cells and
T cells
IL-15 Proliferation of NK cells.
IL-10, TGFβ Control of inlammation.
 Functions of selected cytokines in host defence

Figure 12-1 Functions of selected cytokines in host defense. In innate immunity, cytokines produced by macrophages and NK cells mediate the early inflammatory reactions to microbes
and promote the elimination of microbes. In adaptive immunity, cytokines stimulate proliferation and differentiation of antigen-stimulated lymphocytes and activate specialized effector
cells, such as macrophages. The properties of the cytokines shown in this figure are discussed later in this chapter. APC, antigen-presenting cell.

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 ADAPTIVE IMMUNITY

• Cells of adaptive immunity→ specific receptor

molecules on T-cell antigen receptors (TCRs) and
B-cell antigen receptor (BCRs) or Igs.
T cells (cells-mediated immunity)
- found in normal pulp
- play pivotal role in adaptive immunity
- originated from bone marrow hematopoeitic stemcells
- migrate to the thymus to undergo maturation and express different
surface markers (CD44, CD3, CD4, CD8 and CD25) at various stages,
screened via positive and negative selection process
- positive selection→ mature T cells recognize foreign antigen in the
context of self MHC molecules
- 97% of T cells undergo apoptosis
T cells
- Naive T cells ( CD4+ or CD8+) circulate back and forth between the
lymphatic system and blood until they encounter foreign antigen,
presented by APCs
- Interaction between TCR and antigen peptide.MHC, between
costimulator CD28 and B7→activate signal transduction pathway →
synthesize of T-cell growth factor IL-2 and its receptor→ T cell clonal
expansion/proliferation →differentiate into armed effector T cells and
memory cells
- subpopulation of T cells→ T helper cells, T regulatory, T suppressor
and T cytotoxic cellsdistinguished by surface markers, cytokine
profiles or transcription factors
• Naive CD4 cells
Upon antigen stimulation, proliferate and differentiate into TH0, and
committed into Th1 or Th2 cells
- Monocytoid DC→ induces T helper(Th)1 responses  mainly
produce IL-2 and IFN-ϒ, activate macrophages, induce B cells to
produce opsonizing antibody
- Plasmacytoid DC→ induces Th2 responses produce IL-4, -5, -10,-13,
activate B cells to make neutralizing antibody, and various effects on
macrophages
Th1 and Th2→ modulatory effect
T cells
• Naive CD4 cells, upon antigen stimulation, proliferate and
differentiate into Th1 (IL-2, IFN-ϒ), and Th2 (IL-4, -5,-10, -13)
• Treg cells→ role in mediating immunosupression and maintenance of
tolerance
• Tsup cells→ inhibit the proliferation of Th by blocking activation of
APC and preventing upregulation of costimulatory molecules
• Tc (CD8+) cells, known as cytolitic T lymphocytes (CTLs) → kill target
cells expressing MHC-1 by perforin or cytolisin and granzymes
B cells (humoral immunity)
• Constitute the host humoral immune response
• rarely found in normal pulps
• Main role→ production of antibodies
• Also undergo negative selection
• Bone marrow stromal cells interact with progenitor B-cells→ signals
and GF (IL-7) for B-cell development
• Large quantity of antibody is secreted when B cells terminally
differentiate into plasma cells
Inflammation
A stand alone defence

Immune response
 Functions of Inflammation
1. Destroy and remove pathogens, dilute toxins
2. If destruction is not possible, to limit effects by confining the
pathogen and its products
3. Repair and replace tissue damaged by pathogen and its products
4. Can be potentially harmful
 Initiators of inflammation
• Physical agents – causing tissue necrosis
• Heat, cold, radiation
• Foreign bodies – splinters, dirt, sutures
• Chemicals – acids, alkali
• Infections
• Microbiological agents
• Bacteria, fungi, virus, protozoa
• Immune mediated
• Immune complexes (antibody bound to antigen)
• Hypersensitivity reactions
Cells and Tissues involved in Inflammation
 Causes: physiological and pathological outcomes of inflammation

Note: only inflammation induced by infection causes activation of the immune response
Inflammatory sequence
Major manifestations of Inflammation – compared to normal

1. Vascular dilation and

increased blood flow
(causing erythema –
redness and warmth)
2. Extravasation and
extravascular deposition of
plasma fluid and proteins
(edema – swelling)
3. Leukocyte emigration and
accumulation at the site of
injury
 Vascular changes and fluid leakage during acute inflammation
lead to Edema in a process called Exudation

• Transudate
• Result of hydrostatic or osmotic
imbalance
• Ultrafiltrate of plasma
• Low protein content
• Specific gravity < 1.015
• Exudate
• result of inflammation
• vascular permeability
• high protein content
• specific gravity > 1, 020
 Acute inflammation involves:
• Alteration of vascular caliber -
following very brief
vasoconstriction (seconds),
vasodilation leads to increased
blood flow and blood pooling
creating redness and warmth
• Changes of microvasculature –
increased permeability for plasma
proteins and cells creating swelling
(tumor). Fluid loss leads to
concentration of red blood cells and
slowed blood flow (statis)
• Emigration of leukocytes from
microcirculation – due to stasis and
activation leads migration towards
offending agent
Increased vascular permeability and edema: a
hallmark of acute inflammation
• Leakage is restricted to venules
of 20 – 60 µm in diameter
• caused by endothelial gaps
• usualy an immediate and
transient response (30 min)
• Gaps occur due to contraction
of e.g myosin and shortening of
the individual endothelial cell
• Loss of protein from plasma
leads to edema
• Due to reduced osmotic pressure
in vasculature
• and increased osmotic pressure in
the interstitium
• direct endothelial injury
causing necrotic cell death will
result in leakage from all
levels of microcirculation
(venules, cpillaries, and
arterioles)
• This reaction is immediate and
sustained
• Delayed prolonged leakage
begins after 2 – 12 hours and
can last several days due to
thermal, x-ray radiation or
ultraviolet radiation (sunburn)
and involves venules and
capillaries
• Leakage from new blood
vessels during tissue repair
(angiogenesis) due to
immature endothelial layer
 Phagocytosis
• Derived from the greek words ”eat and cell”. Phagocytosis is carried
out by white blood cells: macrophages, neutrophils, and occasionally
eosinophils.
• Neutrophils predominate early in infection.
• Wandering macrophages: originate from monocytes that leave blood
and enter infected tissue, and develop into phagocytic cells.
• Fixed macrophages (histiocytes): located in liver, nervous system,
lungs, lymph nodes, bone marrow, and several other tissues
 Stages of phagocytosis
1. Chemotaxis: phagocytes are chemically attracted to site of infection
2. Adherence: phagocyte plasma membrane attaches to surface of
pathogen or foreign material
• Adherence can be inhibited by capsules (S. pneumoniae) or M protein (S.
pyogenes)
• Opsonization: coating process with opsonins that facilitates attachment.
• Opsonins include antibodies and complement proteins
 Stages of phagocytosis (continued)
3. Ingestion: plasma membrane of phagocytes extends projections
(pseudopods) which engulfs the microbe. Microbe is enclosed in a
sac called phagosome.
4. Digestion: inside the cell, phagosome fuses with lysosome to form a
phagolysosome.
Lysosomal enzymes kill most bacteria within 30 minutes and include:
• Lysozyme: destroys cell wall peptidoglycan
• Lipase and proteases
• RNAses and DNAses
After digestion, residual body with indigestible material is discharged
 Neutrophils have oxidative and non-oxidative mechanisms of
killing
• NADPH oxidase system, a membrane bound enzyme complex,
reduces O2 to superoxide anion (O2-), hydrogen peroxide (H2O2), and
hydroxyl radical (OH) = oxidative burst
• H2O-MPO-halide system is thought to be the most efficient
bactericidal system (in vitro) by catalyzing the formation of bleach
(hypochlorous radical =HOCl) from H2O2 and Cl- ions which kills
bacteria by halogenation or protein and lipid peroxidation
• Bacteriocidal and cell degrading enzyme contents of lysosomal
granules (azurophil and specific granules) fuse with phagosome to
form phago-lysosome
 Oxidative burst and co-lateral damage

• The NADPH oxidase system is only active when the cytosolic subunits are
assembled with the membrane bound subunits in response to leukocyte
activation
• Inflammatory products may be released into the extracellular space
causing tissue damage and additional disease
• Release occurs transiently during “engulfing” –regurgitation during feeding
• If material is deposited on flat membranes and can not be removed (e.g
immune complexes on basement membrane = frustrated phagocytosis
• Ingestion of membranolytic material (urate cyrstals)
Immunodeficiency diseases caused by deficiencies or defects in
phagocytes (neutrophils and/or macrophages)

• Lack of neutrophil/macrophage numbers or defect of their function

can lead to life threatening infections diseases, particularly with
bacterial and fungal pathogens
• Clinically most common:
• Bone marrow suppression with decreased cell numbers (leukopenia) due to
tumour infiltrate or chemotherapy resulting in myelosuppresion (>500
neutrophils/µl is considered very severe)
• However, inherited defects of adhesion, phagolysosome and microbicidal
functions have been found
 Metabolites of Arachidonic Acid (eicosanoids)
• Membrane lipids of activated cells can be transformed into biological
active lipid mediators
• All mammalian cells except erythrocytes can produce eicosanoid
• They are autocoids = short range hormones (very short range and
half-life)
• Arachidonic acid is derived from conversion of linoleic acid
Functions of nitric oxide
(NO) in blood vessels
and macrophages.

NO is produced by two
NO synthase (NOS)
enzymes. It causes
vasodilation, and NO-
derived free radicals are
toxic to microbial and
mammalian cells
 Nitric Oxide (NO)
a pleiotropic mediator of inflammation
• NO was initially discovered as endothelium discovered as endothelium derived
relaxing factor
• NO is a soluble gas
• NO is produce by many cells including:
• Endothelial cells
• Some neurons
• Phagocytes
• Synthesized from L-arginine by nitric oxide synthase (NOS)
• Three different NOS:
• Endothelial eNOS
• Neuronal nNOS
• Inducible iNOS
• (phagocytes)
 NO modulates the inflammatory response

• NO is a potent vasodilator
• Reduces platelet aggregation
• Reduces leukocyte recruitment
• is Antimicrobial
Cytokines in
Inflammation
 Consequences of complement activation
1. Cytolisis: due to the formation of a membrane attack complex
(MAC) which produces lesions in microbial membranes.
2. Inflammation: complement components (C3a) trigger the release of
histamine, which increases vascular permeability.
3. Opsonization: complement components (C3b) bind to microbial
surface and promote phagocytosis.
4. Inactivation of complement: regulatory proteins limit damage to
host cells that may be caused by complement
Outcomes of acute inflammation: resolution, healing by fibrosis,
or chronic inflammation
 Histamine and serotonin induce vasodilation and increased
vascular permeability
Mast cell:
• richest source of histamine
• located in connective tissue
• adjacent to blood vessels
• degranulation through receptors for IgE-, IgG, histamine, bacterial
products, and anaphylatoxin C5a, physical injury, cold, heat
• release of PAF (platelet activating factor) leads to serotonin and
histamine release from activated platelets
• Mast cells are very important effector cells in hypersensitivity
reactions (anaphylactic reactions)
 Termination of acute inflammation
• Eradication of an offending agent should lead to
discontinuation of the inflammatory response
• Neutorphils have only a short life span (few hours – 1
day)
• Most mediators are very short lived and are degraded
immediately
• Anti-inflammatory cytokines (TGF-beta, and IL-10) can
inhibit the production of pro-inflammatory cytokines
(TNF)
• In arachidonic acid metabolism, lipoxin and resolvins
are generated that have anti-inflammatory activity

However, the exact mechanism by which acute inflammation

resolves remain still somewhat elusive
 Summary of Acute Inflammation
• Initial response to tissue injury
• Vascular phase
• Blood flow increased at the site of damage, increased vascular permeability
• Exudative phase
• Formation of cell-free protein rich exudate in the tissues
• Cellular phase
• Tissue infiltration by neutrophils
• Inflammatory response facilitated by cellular mediators (prostaglandins,
leukotrienes, cytokines) and plasma proteins (coagulation factors,
complement, kinins)
• Different forms of inflammation can be characterised by a particular phase
of the response (serous, haemorrhagic, fibrinous, or purulent)
 Outcome of Acute Inflammation
• Resolution – restoration of tissue to normal
• Suppuration – pus formation
• Healing – regeneration and repair
• Mediated by tissue resident and recruited macrophages
• Lipid mediators switch from pro-inflammatory prostoglandins to anti-inflammatory
lipoxins
• Lipoxins inhibit recruitment of neutorphils, promote recruitment of monocytes which remove
dead cells and initiate tissue remodelling
• Resolvins, protectins and TGF-  and growth factors produced by macrophages also important
in resolving inflammation and initiating tissue repair
• Failure of resolution of ACUTE inflammation can result in chronic
inflammation
Summary of chronic inflammation
• Continuing inflammation at the same time as healing
• Mononuclear cells predominate (includes LYMPHOCYTES, plasma
cells, and macrophages)
• Chronic inflammation can follow acute inflammation or arise de novo
• Histology – features can be non-specific or specific eg TB or syphilis
• Granulomas are aggregates of macrophages; may be accompanied by
multinucleated giant cells (can incorporate fat, foreign body type and
Langhans’ type commonly seen in TB sarcoidosis and Chron’s)
Neurogenic inflammation
• Is inflammation caused by the neuropeptides
• Neuropeptides and their receptors-→ widely distributed throughout
the body.
• During inflammation→ sprouting of afferent fibers and local increase
of inflammatory mediatorsneuropeptides release
• Pivotal neuropeptides in the induction of neurogenic inflammation:
Recruit immune cells,
- CGRP (calcitonin gene related peptide)→ vasodilation Mφ, PMNs, Limph, mast
- SP ( Substance P)→ increases vascular permeability cells
- Neuropeptides Y (NPY) and VIP (vasoactive intestinal peptide)→
inhibit inflammatory response block production of TNF-α, IL-6, IL-12
Immune response in the dental pulp & periapical lesion

• Has the same basic pattern but is affected by local factors

• Innate immune response is initiated when presented with a new
antigen, and is followed by the specific response few days later
• Immediate specific response occurs when antigen has been
recognized
• Different cells types within the dentin-pulp complex are able to
detect invading bacteria at all stages of infection
• - early disease stages→ odontoblasts
• - disease progresses→ core pulpal cellsfibroblasts, stem cells,
endothelial cells and immune cells
Those cell types express PRRs (pattern recognition receptors = TLRs) detect
bacterial components (PAMPs) activation of intracellular signalling
cascades
• All three APCs are present and active in the pulp’s response to bacteria and toxin
• Odontoblasts→- being the ideal position to detects antigen via TLRs,
produce a variety cytokines and chemokines
- the first cells to encounter antigen diffusing into the DentTub
- express different PRRs (TLR-2 of Gram +, TLR4 of Gram – mo)
The symphatetic system→ inhibits the production of proinflammatory cytokines,
T lymphocytes and other leucocytes→ produce antinociceptive molecules such as
β-endorphin and somatostatin during inflammation reduce the excitability of
pain fibers
When pulpal response succeeds : Repair and Regeneration
• Effective immune response :
- neutralizes and removes any foreign material
- allows and initiates the second part of pulp response recovery,
repair, and regeneration  relate to the severity of injury
Repair
No cells are killed→ original odontoblasts form reactionary (tertiary) dentin
Odontoblasts are killed→ stem cells (undifferentiated mesenchymal cells) form
reparative dentin
Repair of larger areas of damage→ pulp polyp
Injury and repair go on at the same time→ chronic inflammation
Immune response in the periapical lesion

• Periapical or apical lesion→ an inflammatory lesion of endodontic origin

• Periapical lesions  produced by an inflammatory response at the root
apices of teeth with non vital pulps inaccessible to the immune response
• The identified immune cells in the periapical lesion→ PMNs, mast cells,
macrophages, dendritic cells, lymphocytes, plasma cells, and osteoclasts
• In rat model: an early, active phase of lesion development→CD4+/ Th >>
Later chronic stage→CD8+ T cells >>
Apical Periodontitis
Apical Periodontitis