Endodontic Topics 2004, 8, 88–103 Copyright r Blackwell Munksgaard

All rights reserved ENDODONTIC TOPICS 2004

1601-1538

Cell-to-cell interactions
CSONGOR KISS

The narrow anatomic periodontal space between the root surface and the alveolar bone is relatively poor in cells.
However, a broad range of noxious stimuli, most frequently root canal infection, will result in the accumulation of a
large number of inflammatory cells in the periapical region of the affected tooth/root. The encounter between
invading microorganisms and host cells as well as the interactions of resident and immigrating cells will determine
the course of the inflammatory events that may take place. This review attempts to summarize current knowledge
on how cellular interactions may contribute to dynamic equilibrium between protective, self-maintaining,
propagating, destructive and downregulating events in apical periodontitis.

Introduction Chronic apical periodontitis represents a dynamic

Apical periodontitis is an inflammatory response of the
periapical area to irritative stimuli from the root canal
space, most frequently root canal infection. Depending
on the intensity and duration of the initiating event,
periapical inflammatory lesions may develop either in
an acute or a chronic direction. The histopathological
appearance of the lesion correlates with the clinical
course of apical periodontitis (1). In acute apical
periodontitis lesions, vasodilatation, vascular conges-
tion, edema, extravasation of macrophages and neu-
trophil leukocytes involve the periapical ligament.
Lamina dura disruption and limited bone resorption
of the neighboring spongiosa can be noticed. As a
result of the first line of host defense, the spreading of
microorganisms from the infected root canal is limited.
Occasionally, virulent root canal microbes may gain
access through the apical foramen to establish a film on
the external root surface or by forming nests within the
previously healthy periapex. In response, an abundant
number of neutrophil leukocytes accumulate and the
incipient acute lesion develops into primary periapical
abscess (2). Acute exacerbation of a pre-existing
chronic lesion results in a similar histological picture,
leading to the development of secondary abscess. Both
primary abscess formation and episodes of exacerba-
tion are associated with significant bone loss and to
some apical root resorption giving space for the
development of a chronic lesion (3).
balance between exogenous irritative agents, usually the
root canal microbiota and their by-products, and host
defense mechanisms, where the latter are not able to
destroy and completely eliminate the pathogenic factors
but, by forming a histological as well as functional
barrier, effectively prevent from their further invasion
(4). The chronic lesion, termed periapical granuloma, is
made up by mononuclear and polymorphonuclear
leukocytes and fibrovascular elements. The histological
appearance of periapical granuloma represents every
momentum of the periradicular inflammatory process
from the acute incipient response to the burned out
end-stage lesion. Neighboring histological structures,
i.e. necrotic, exudative, granulomatous and fib-
rous zones penetrate into the surrounding alveolar
bone in an onion leaf-like fashion starting from
the apical foramen (Fig. 1). According to the pre-
dominant zone(s), lesions can be classified as exudative,
granulomatous, granulofibrotic and fibrotic granulo-
mas (5, 6).
In about 50% of periapical granulomas, epithelial
strands originating from the epithelial rests of Malassez
can be identified (7, 8). Coalescing epithelial strands
enclose a hollow space forming radicular cysts in about
6–55% of all chronic periapical lesions. The cavity of pocket
or bay cyst communicates with the root canal. In contrast,
the true cyst is independent of the root canal system. Even
in cysts, the lumen is almost always embedded into the
inflammatory granulation tissue (1, 2, 9).

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 Cell-to-cell interactions

Fig. 1. Schematic presentation of periapical granuloma.

Necrotic, exudative, granulomatous and fibrotic zones of
the periapical inflammatory lesion form an onion leaf-like
structure around of the tooth apex.

Apical periodontitis – dynamic chemokines (Fig. 2A). These regulatory glycoproteins

equilibrium between protective and
destructive mechanisms
The periapical inflammatory cell infiltrate in chronic apical
periodontitis consists of virtually every component of the
specific and non-specific branches of the immune system
(1, 2). Accumulating immune-inflammatory cells effec-
tively kill microorganisms preventing tissues surrounding
the tooth apex from excessive bacterial invasion. However,
anti-infective effector mechanisms are not restricted to
protect the host but also destroy normal tissue components
resulting in bone resorption and ultimately, tooth loss (3,
4, 9). The lesion has the potential to compromise general
health, as discussed by I. Marton elsewhere in this issue.
Although the periapical inflammation is fuelled by the
constant release of microorganisms and their by-products
from the root canal space, autocrine and paracrine loops of
stimulation may contribute to the promotion and main-
tenance of the lesion (3). According to some investigators,
true cysts may be considered to be autonomous lesions
once initiated as they may not respond successfully to
proper endodontic treatment (10, 1).
Initiation of the apical periodontitis
lesion – interaction between resident
host cells and root canal microbes
provide a week stimulus for attracting and activating
macrophages and polymorphonuclear leukocytes (11,
12). This low-level activity of innate immunity has been
suggested to play a key role in maintaining clinically
healthy periodontal and periapical tissues (13, 14).
Early inflammatory events in response to
root canal infection
In root canal infection, as soon as the bacterial front
penetrates the tooth apex, cell wall or soluble compo-
nents of endodontopathic bacteria may upregulate the
expression of first-line proinflammatory cytokines, such
as interleukin (IL)-1 and tumor necrosis factor (TNF)-
a, cell adhesion molecules and chemokines inducing a
robust influx of inflammatory cells into the periodontal
ligament (Fig. 2B). Indeed in human chronic periapical
granuloma, our group has observed differential in situ
distribution of IL-8, monocyte chemoattractant pro-
tein-1 (MCP-1) and Rantes (Fig. 3). IL-8 was found
primarily in the cytoplasm of the Malassez epithelial
cells, MCP-1 immunoreactivity was confined to en-
dothelial cells (15). Subsequently, a second wave of
inflammatory mediators, produced by the stimulated
resident and newly recruited cells, may amplify,
propagate and prolong the expression of cell adhesion
molecules and chemokine genes (16, 17).

Contribution of innate immunity to The role of neuropeptides in eliciting

maintain a healthy periapex periapical inflammation
Epithelial and endothelial cells within the periodontal In cases of non-infectious pulp necrosis root canal and
ligament surrounding the healthy root apex constitu- periapical inflammation can be elicited by sensory
tively express low levels of cell adhesion molecules and neuropeptides that are released from nociceptive nerve

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endings by a variety of noxious stimuli (18, 19).
Neuropeptides induce the production of proinflamma-
tory cytokines, such as TNF-a and IL-1 that propagate
protective and destructive inflammatory changes (20).
In an elegant model of periodontitis, contralateral
increase in calcitonin gene-related peptide (CGRP) and
CGRP messenger RNA (mRNA) expression accom-
panied by inflammation and bone loss was demon-
strated in rats having received intragingival injection of
a lipopolysaccharide (LPS) preparation at the first right
90
mandibular molar (21). In periapical granuloma
samples, CGRP and substance P (SP) immunoreactivity
was confined to nerve fibers in close proximity to blood
vessels in areas associated with the collection of
inflammatory cells, including TNF-a-containing mast
cells. The vascular endothelial cells stained positively for
E-selectin and intercellular adhesion molecule-1
(ICAM-1). In contrast, clinically healthy periapical
ligment contained only a few macrophages and
lymphocytes. No immunostaining for CGRP, SP,
 Cell-to-cell interactions

Fig. 3. Differential expression of chemokines in human periapical granuloma. (A) Expression of interleukin-8 as red
staining in the cytoplasm of the epithelial cells of Malassez. Alkaline phosphatase anti-alkaline phosphatase (APAAP)
reaction, ! 250 (15). (B) MCP-1-specific monoclonal antibody labels the surface of small blood vessels (red staining)
penetrating the granulomatous zone (APAAP reaction, ! 250) (15).

TNF-a, E-selectin and ICAM-1 was detected (22, 23).
Indeed stimuli mediated by sympathetic nerve fibers
were shown to increase TNF-a production in experi-
mental pulp infections and periapical lesions of
unilaterally sympathectomized rats (24). In addition
to the potential inflammatory mediator function in
sterile lesions, neuropeptides were demonstrated to co-
operate with LPS from endodontopathic bacteria (25).
The exudative phase – robust
recruitment of phagocytes regulated
by redundant signals
The initial interaction between root canal microbes and
resident host cells may set the stage for mounting a
robust and rapid protective response reaction by
attracting a large number of phagocytic cells, mono-
cyte/macrophages and neutrophil leukocytes, to the
sites of bacterial invasion, mainly to the immediate
vicinity of the apical foramen (1). A variety of partially
overlapping signals is involved in this process. The
recruitment of leukocytes requires them to transiently
attach to endothelial cells, migrate through them and
then to interact with cells and substrates in the
extravascular tissue. This cascade is governed by a set
of cell adhesion molecules, such as selectins and
integrins and the receptors for them (26). The driving
force, the chemotactic signal can be provided by
complement components of the alternative pathway
of complement activation and, more significantly, by
chemokines (27, 28). Pleiotropic (IL-1, TNF), lineage-
restricted (granulocyte macrophage-colony stimulating
factor (GM-CSF)) and lineage-specific (granulocyte
colony-stimulating factor and monocyte colony-stimu-
lating factor) cytokines activate these infiltrating
leukocytes to stimulate their effector functions that

3
Fig. 2. Schematic presentation of the evolution of the periapical inflammatory response. (A) Epithelial and endothelial
cells within the periodontal ligament surrounding the healthy root apex constitutively express low levels of cell adhesion
molecules and chemokines providing a week stimulus for attracting and activating macrophages and polymorphonuclear
leukocytes. Low-level activity of innate immunity plays a key role in maintaining clinically healthy periodontal and
periapical tissues. (B) Root canal infection results in pulp necrosis. Endodontopathic bacteria can be found within the
root canal, at the apical foramen and within the lesion per se. Bacteria and their by-products upregulate the expression of
cell adhesion molecules, chemokines and proinflammatory cytokines attracting an abundant number of neutrophil
leukocytes and monocyte-macrophages that represent the first line of defense. (C) Antigen-presenting cells (dendritic
cells and macrophages) present bacterial antigens to CD41 T-helper 1 (Th1) lymphocytes via the antigen-specific T-cell
receptor in conjunction with major histocompatibility complex Class II molecules. Primed Th1 cells produce interferon-
c and interleukin-2. The former cytokine stimulates monocyte-macrophage cells establishing a positive loop of
stimulation. The latter cytokine promotes the proliferation of further antigen-specific Th1 cells and other activated
lymphocytes resulting in granulation tissue formation. (D) In established periapical granulomas Th2 cells outnumber
Th1 cells resulting in the stimulation of B-lymphocytes and plasma cells producing immunoglobulins and in a change of
the CD4/CD8 ratio in favor of the latter cells. The cytokine milieu provided by Th2 cells and CD81 lymphocytes can
stimulate the proliferation of epithelial cells and may contribute to the healing of the lesion.

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contribute to both protective and self-destructive
events.
Induction of the exudative phase in
transgenic mice
Much of our understanding stems from the investiga-
tion of transgenic mice. In these animals, the gene for
either the ligand or the receptor participating in
leukocyte recruitment and function has been disrupted
rendering the animals deficient in functions regulated
by the given ligand–receptor interaction. Probably as a
result of the overlap in signals that attract leukocytes to
sites of infection, it has been proven that almost all
ligand–receptor pairs involved in the process can be
bypassed by proper functioning of unperturbed signal
pathways (16). For example, the dental pulp of mice
with functional deletions of receptors to IL-1, TNF, or
both was inoculated with anaerobic pathogens. Poly-
morphonuclear and mononuclear phagocyte recruit-
ment occurred to the greatest extent in double negative
mice, and to a lesser extent in IL-1 receptor (IL-1R) or
TNF receptor (TNFR) mice, and the least in wild-type
mice (29). This study confirmed that despite being
involved in the upregulation of cell adhesion molecules
and chemokines influencing the migration of mono-
cytes and neutrophil leukocytes, IL-1/IL-1R- as well as
TNF/TNFR-independent pathways for phagocyte
recruitment can be induced by endodontopathic
bacteria (30, 31).
Role of integrins and their ligands
ICAM-1, a ligand for the heterodimer integrin
molecule CD11/CD18 seems to be essential for
neutrophil recruitment induced by Gram-negative
bacterial stimuli (32). Both CD11/CD18-dependent
and -independent pathways for neutrophil emigration
have been identified suggesting that Gram-negative
bacteria induce predominantly CD11/CD18-depen-
dent emigration, whereas Gram-positive bacteria in-
duce predominantly CD11/CD18-independent
emigration (16). Additional b1 integrins, very late
antigen (VLA)-5 and VLA-6 were suggested to
contribute to LPS-induced neutrophil migration (33).
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The role of chemokines
The complex process of chemotaxis, i.e. the directed
migration of phagocytes from within the capillary bed,
through the endothelial cells, across the interstitium,
between epithelial cells, into the site of infection may
require sequential interactions with different chemo-
kines expressed by different local cells or attached to
matrix components (15, 34, 35). Chemokines of the a
(CXC) family have been shown essential to leukocyte
migration induced by bacteria or LPS (36, 37). In vitro
incubation of neutrophil leukocytes with TNF-a and
GM-CSF was demonstrated to upregulate receptors for
and responsiveness to CC chemokines, members of the
chemokine b family (38, 39).
Role of nuclear transcription factors
The overlapping stimulatory effects exerted by ligand–
receptor pairs involved in leukocyte migration and
chemotaxis may converge to the nuclear transcription
factor NF-kB regulating the expression of the genes for
many cell adhesion molecules and chemokines and
their receptors (40). Investigation of transgenic mice,
deficient in TNF and in RelA, a 65 kDa subunit of NF-
kB suggested that RelA may promote the coordinated
expression of adhesion molecules and chemokines
essential to leukocyte infiltration in response to
Gram-negative bacteria and LPS (41).
Infiltrating phagocytes represent the
dominant cell population of the exudative
phase
Once reaching at the infected periapical space, the role
of leukocytes is clearly protective. The infiltrating
nature of these cells has been firmly established.
Neutrophil leukocytes are virtually absent in the
healthy periodontal ligament therefore, they must be
attracted from the bone marrow via the circulating
blood in case of root canal and concomitant periapical
infection. Most neutrophil leukocytes have been shown
metabolically inactive or dead in human periapical
lesions indicating their short-lived effector cell nature
(42). Newly recruited macrophages clearly outnumber
resident macrophages as demonstrated by immunohis-
tochemistry, in situ hybridization and ultrastructural
analysis. Actively phagocytozing macrophages of in-
filtrating phenotype were found in great numbers just
 Cell-to-cell interactions

beneath the periapical abscess in experimentally in- Table 1. Host regulatory factors induced by endo-
duced rattine periapical lesion (43). The presence of dontopathic microorganisms and their by-products
large, cytophagocytic macrophages containing apopto-
tic neutrophil leukocytes characterized human periapi- Cell adhesion molecules and their ligands
cal lesions. Although macrophages present in the ICAM-1
human lesion actively transcribe and translate genes as
CD11/18
indicated by detecting polyadenosine RNA and ribo-
somal RNA, they exhibit only a very low level of LFA-1
proliferative activity (42). VLA-5, VLA-6

CD29, CD49
Protective and destructive functions of Chemokines and their receptors
infiltrating phagocytes
IL-8
Inhibiting leukocyte recruitment or activation either by
KC
disrupting regulatory signals mediated by IL-1, TNF,
IL-6 and their receptors or by depressing bone marrow Macrophage inflammatory protein-2

function have been shown to result in a more wide- MCP-1

spread local bacterial infiltration, even in generalized Rantes
infections (17, 29, 44). Although local tissue damage
has been connected with the release of reactive oxygen Proinflammatory cytokines, their receptors and antago-
nists
and nitrogen radicals and matrix metalloproteases
during phagocytosis, interestingly however, osteolytic GM-CSF

periapical lesions were larger in IL-1R-, TNFR- and in IL-1

IL-6-deficient transgenic mice (29, 44–46). In turn,
IL-1R
pharmacological enhancement of the number and
activity of circulating neutrophils and monocytes IL-1Ra

resulted in the reduction of periapical bone and soft IL-6

tissue lost in mice (47). These observations suggest that IFN-c
the more effective is the local control of infection, the
TNF
less tissue damage occur during periapical lesion
initiation. Reactive oxygen and nitrogen intermediates and arachi-
donic acid metabolites

Regulatory molecules derived from
endodontopathic bacteria participate
in the initiation and progression of
the periapical inflammatory process
Bacteria do not need necessarily emigrate from the
infected root canal. Among their soluble products a
number of compounds has been defined that may elicit
and sustain a vigorous inflammatory response in the
periapical region. In addition to attract phagocytes,
representing the first line of defense, directly or by
inducing the expression of proinflammatory cytokines
and cell adhesion molecules by resident host cells,
bacterial molecules can stimulate the specific immune
responses (Table 1). The appearance of lymphocytes
and plasma cells will transform the early periapical
Superoxide radical
Hydrogen peroxide
Leucotrienes
Prostaglandins
Nuclear transcription factors
NF-jB
IE2-protein
ICAM-1, intercellular adhesion molecule 1; LFA,
lymphocyte function-associated antigen; VLA, very late
antigen; IL, interleukin; KC, member of the chemokine
family; MCP, monocyte chemoattractant protein; Rantes,
member of the chemokine family; GM-CSF, granulocyte
macrophage-colony stimulating factor; IFN, interferon;
TNF, tumor necrosis factor; IE, immediate early.

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lesion into periapical granuloma. LPSs represent
probably the most potent, and best-studied bacterial
factors. In addition, some more recently characterized
microbial molecules may contribute to the pathome-
chanism of apical periodontitis.
The role of LPSs in periapical inflammation
LPS present in and released from cell walls of Gram-
negative bacteria are probably the best-characterized
components of endodontopathic bacteria to induce
cytokines and other inflammatory compounds. Most
studies have used peripheral blood monocytes and
fibroblasts as target cells. LPSs isolated from Actino-
bacillus actinomycetemcomitans, Fusobacterium nucle-
atum, Porphyromonas gingivalis and Prevotella
intermedia, for example, have been shown to induce
the production of an array of proinflammatory and
chemotactic cytokines including GM-CSF, interferon
(IFN)-g, IL-1a and -b, IL-6, IL-8, MCP-1 and TNF-a
(48). Local IL-1, IL-6 and GM-CSF production was
repeatedly detected in human dental pulp and periapi-
cal lesions (49–52). Expression of IL-6 mRNA was
confirmed in immortalized oral keratinocytes upon
challenge with LPS from endodontopathic bacteria by
RT-PCR (14). The broad concentration range of
activity and the colorful biological spectrum of LPS
depend on a number of factors (Table 2).
The availability of CD14 antigen
The availability of CD14 antigen and LPS-binding
protein are absolute requirements of LPS–receptor
binding and signal translation (48). CD14 can be
present either on the surface of the targeted cell, or as a
soluble compound, which in a complex with LPS and
LPS-binding protein stimulates the target cell.
Target tissue specificity
LPS preparations extracted from the same endodonto-
pathic bacteria were shown to induce cytokine secre-
tion in oral but not in skin-derived epithelial cells (14).
Constitutive differences
Recently, a hyperinflammatory phenotype has been
defined in humans. Peripheral blood monocytes,
derived from such subjects, produce 3- to 10-fold
94
Table 2. Factors influencing the biological activities
of lipopolysaccharides (LPS)
Availability of cell surface or soluble CD14 antigen and
LPS-binding protein
Target tissue specificity
Constitutively determined hyper/hyporesponsive pheno-
type
The source of LPS
Induction of agonistic and antagonistic cytokines and cell
adhesion molecules
greater amounts of IL-1b and TNF-a than those
obtained from normal individuals (53, 54). In murine
monocytes, an impaired ability to respond to LPS has
been attributed to a mutation in the gene that encodes
Toll-like receptor 4 (55, 56). In humans, polymorph-
isms, most often single nucleotide polymorphisms of
genes encoding for cytokines and enzymes influencing
the function of immune cells have been shown to alter
the intensity of immune reactions and may be
responsible for the hypo/hyperresponsive pattern of
monocytes and macrophages (57–59).
The source of LPS
LPS derived from A. actinomycetemcomitans, Actino-
bacillus israeli and F. nucleatum were shown to
upregulate ICAM-1, IL-8 and MHC Class II antigens
in oral epithelial cell lines, whereas P. gingivalis LPS
downregulated the former two molecules. In contrast
to the strong and moderate stimulation by F. nucle-
atum and A. israeli, respectively, LPS from Actinoba-
cillus viscosus did not increase constitutive expression of
MHC Class II molecules. The inability of certain
endodontopathic bacteria to induce MHC Class II
expression or their potential to inhibit IL-8 accumula-
tion may result in a decreased level of inflammatory
cellular infiltrate thereby contributing to bacterial
invasion (13, 14, 60).
Induction of agonistic and antagonistic
cytokines and cell adhesion molecules
F. nucleatum-derived LPS were shown to induce MHC
Class II, ICAM-1, IL-1 and IL-6 molecules and

suppressed the manifestation of co-stimulatory mole-
cules CD70 and CD80/86 in oral epithelial cells (14).
In the lack of a signal via CD80/86 molecules, T-cell
activation will be impaired despite proper antigen
presentation through MHC Class II antigens and
despite ICAM-1 binding to its receptor, lymphocyte
function-associated antigen-1 (61). Moreover, LPS
may induce simultaneous expression of IL-1 and its
antagonist IL-1Ra (48). Thus, LPS-induced differen-
tial expression of key regulatory molecules in resident
root canal and periradicular cells may result either in a
powerful initiation of the inflammatory lesion or in a
state of unresponsiveness contributing to the charac-
teristic long-term quiescent periods observed in many
chronic periapical lesions.
The role of other bacterial compounds in
periapical inflammation
There are a number of further cell components of
endodontopathic bacteria which are able to initiate
inflammation by inducing inflammatory cytokines
(48). Among these, a few compounds deserve parti-
cular interest.
Fimbrial protein extracts
Fimbrial protein extracts from P. gingivalis were
demonstrated to increase IL-1a and IL-1b mRNS
expression even in macrophages from an ‘LPS-unre-
sponsive’ mouse strain (62). In addition to IL-1a and
IL-1b, the fimbrial protein extract of this microorgan-
ism was demonstrated to stimulate the production of
further proinflammatory cytokines, TNF-a, IL-6 and
GM-CSF, with bone-resorptive activities and two
chemokines, IL-8 and KC (63, 64). The latter two
chemotactic cytokines play a role in driving neutrophil
emigration from blood and in promoting endothelial
and epithelial cell proliferation (28, 65, 66). A 12-mer
amino acid sequence between residues 69 and 80
connected with the biological activities of the native
fimbrial protein has been identified, opening a pathway
for developing targeted therapy (63).
Surface-associated material
The biological activity of easily solubilized components
of certain Gram-negative bacteria, termed surface-
associated material (SAM), may exceed that of corre-
Cell-to-cell interactions
sponding LPS preparations. SAM prepared from A.
actinomycetemcomitans, Eikenella corrodens and P.
gingivalis was shown to directly stimulate IL-6 synth-
esis of human gingival fibroblasts without affecting
transcription of IL-1 and TNF genes that induce the
expression of IL-6 by positive loops of stimulation
upon challenge with LPS (67).
Heat shock proteins
A 64 kDa protein component of A. actinomycetemco-
mitans SAM with potent osteolytic activity has been
identified as a homologue of the 60 kDa Escherichia coli
GroEL protein, member of the bacterial heat shock
protein (Hsp) family (68). Hsp64 exerted a powerful
proliferative stimulus on porcine periodontal ligament
epithelial cells in low concentrations but proved
cytotoxic for immortalized skin epithelial cells in large
concentrations (69). Bacterial Hsp’s, frequently pre-
sented as immunodominant antigens in mammals, have
a significant homology with human Hsp’s and cross-
react in vitro with anti-human Hsp antibodies (70, 71).
Antibodies to Hsp’s of endodontopathic bacteria may
cross-react with human Hsp’s exposed in an injured
root canal or periapical tissue, promoting the initial
inflammatory response (72).
An emerging role of viruses in the
periapical inflammatory process
A growing attention has been focused recently on two
members of human herpesviruses, Cytomegalovirus
(CMV) and Ebstein–Barr virus (EBV) playing an
etiopathogenic role in the development of root canal
and periapical lesions. The research group at the
University of Southern California has repeatedly
confirmed the association between CMV and EBV
infection and symptomatic periapical pathosis (73, 74).
CMV and EBV infections induce an array of inflam-
matory and chemotactic cytokines that stimulate the
expression of further activation and cell adhesion
molecules, recruit inflammatory effector cells to the
site of infection and contribute to bone resorption.
Increase in the expression of IL-1b gene is particularly
abundant as IE2 protein, one of the immediate early
(IE) CMV gene products, has been shown to provide
robust transactivation of the IL-1b gene without
requiring the involvement of the far-upstream enhancer
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of the gene (75). These effects may become especially
powerful in synergy with LPS (76).
Granulation tissue formation – fine-
tuned cellular interactions establish a
balance between protective,
destructive and reparative reactions
If the invading microorganisms cannot be completely
eliminated in course of the anti-infective reactions of
the exudative phase and the irritative effects of soluble
bacterial by-products and viruses persist, the lesion
becomes chronic and a characteristic, complex granula-
tion tissue will be established. Granulomatous inflam-
mation is a protective host response built up to limit
periapical tissue damage by localizing root canal
microbiota and the inflammatory response. The initia-
tion and formation of granulomas is largely dependent
on the interaction of antigen-presenting cells with
CD41 T-lymphocytes (77, 78). Antigen-presenting
cells activate CD41 T-lymphocytes through their
antigen-specific receptors (TCR) in conjunction with
MHC Class II molecules (79).
Interaction of antigen-presenting cells and
T-lymphocytes
The research group from the Tokyo Medical and
Dental University has performed a detailed study of
antigen-presenting cells in experimentally induced
periapical lesions in the rat (43). Early lesions,
established after 28 days of unsealed pulp exposure
were studied. In contrast to MHC Class II-negative,
actively phagocytozing macrophages accumulating in
the vicinity of periapical abscess, MHC Class II-positive
macrophages were found evenly interspersed among
other cell types within the cell-rich granulomatous
zone. The various appearances of these permanently
immigrating cells may mirror the heterogeneity the
stage of differentiation at the time of observation.
MHC Class II-positive dendritic cells (DCs) were also
noticed in great numbers, mostly located in the outer,
fibrotic border of the lesion. Both MHC Class II-
positive macrophages and DC established cell-to-cell
contacts with MHC Class II-negative lymphocytes.
These results indicate that local antigen presentation
takes place in periapical lesions with DC being able to
activate both naive and memory T-cells during primary
96
and secondary immune responses, respectively and
MHC Class II-positive macrophages eliciting a sec-
ondary immune response (43). A similar distribution
and morphology of macrophages labeled with anti-
CD14, anti-CD68 and anti-human leukocyte antigen-
DR monoclonal antibodies was observed in human
periapical lesions (Fig. 4) (80, 81). Characterizing the
isoforms of leukocyte common antigen CD45, naive
(CD45RA and CD45RC) and memory (CD45RO and
CD45RB) T-lymphocytes were found in almost equal
numbers within the granulation tissue confirming the
possibility, extrapolating from the results of the rattine
model, that both primary and secondary T-cell
responses can be initiated in human periapical lesions
(82).
T-helper cell subpopulations
According to their cytokine expression pattern, CD41
T-cells can be classified as T-helper 1 (Th1) cells that
produce and secrete IL-2 and IFN-g and Th2 cells that
produce and secrete IL-4, IL-5, IL-6, IL-10 and IL-13.
Positive loops of stimulations exist between macro-
phages and Th1 cells as well as within the Th1 and Th2
subpopulations, respectively. In contrast, Th1 and Th2
cells exert a mutually inhibitory effect on each other.
Th1-type cytokines augment cytotoxic T-cell func-
tions, stimulate the expression of proinflammatory and
bone resorptive molecules in other cell types, whereas
Th2-type cytokines participate in B-lymphocyte stimu-
lation to mount a humoral immune response and in
Fig. 4. Distribution of CD141 macrophages in the
granulomatous zone of periapical granuloma. The
antigen–antibody reaction was visualized by the avidin–
biotin–peroxidase complex method resulting in a brown
staining of CD141 macrophages, ! 250 (80).
 Cell-to-cell interactions

dampening the inflammatory reactions (83). As ex-

pected, Th1-lymphocytes prevail in early, expanding
periapical lesions (84, 85). As a result of positive loops
of stimulation, the number of Th1 cells increases
rapidly within the lesion (Fig. 2C).

The origin of T-lymphocytes in

granulomatous inflammatory lesions
The origin of these cells has been characterized
precisely in the schistosoma-induced granuloma mod-
el. In this model, granulomatous inflammatory lesions
in the livers of mice were induced by the parasite
Schistosoma mansoni. The very diverse TCR repertoire
of the individual granulomas in the liver of the same
infestated mouse indicate that most of the T-cells are
recruited to the lesions and are activated systemically.
At the same time, nucleotide sequence analyses of
individually sized products from the complementarity-
determining region 3 (CDR3) of the TCR gene from
single granuloma indicate that a fraction of T-cells
expand locally at the lesion site. Furthermore, it has
been shown, that non-schistosoma-specific T-cells
home to the granuloma if they are activated, resulting
in a change of the original Th1 vs. Th2 and CD41 vs.
CD81 balance (86). The low level of S-phase prolifer-
ating and cycling lymphocytes, assessed by in situ
hybridization using a histone probe and by Ki-67
immunostaining in contrast to their transcriptional and
translational activity support the view that a similar
scenery may be operative in the human periapical lesion
(42).
Fig. 5. Distribution of major histocompatibility complex
Class II-expressing cells in the granulomatous zone of
periapical granuloma. Small, lymphocyte-like and larger
mononuclear cells resembling macrophages display a
circumferential brown staining using the avidin–biotin–
peroxidase complex (ABC) method, ! 250 (80).
Humoral immune response in human
periapical granuloma lesions
In more advanced lesions, Th2 cells slowly outnumber
Th1 lymphocytes. Recently, bacterial Hsp’s were
shown to contribute to switching the Th1 response
towards Th2 (89). The resulting change in the cytokine
milieu is accompanied by a steady increase in the
number of B-lymphocytes and plasma cells present in
the lesion (Fig. 6) (51, 90). The functional significance
of local humoral immunity in periapical lesions is
underscored by the observation that both T/B-null
RAG-2 SCID and B-cell-deficient mice, but not T-cell-
or C5-deficient mice, have increased susceptibility to

T-lymphocyte activation markers in human

periapical granuloma lesions
Similar to the schistosoma-induced granulomas, T-
lymphocytes express activation markers, such as IL-2
receptor a-chain (CD25) and MHC Class II molecules
in expanding periapical lesions, implying their active
participation in anti-infective and proinflammatory
reactions (Fig. 5) (80, 87). This hypothesis was
confirmed recently by demonstrating that cyclosporine Fig. 6. Distribution of B-lymphocytes and plasma cells in
A treatment resulted in a dose-dependent decrease in the granulomatous zone of periapical granuloma.
lesion size accompanied by a significant reduction in Numerous plasma cells and B-lymphocytes reacting with
the mixed heavy chain (immunoglobulin (Ig)A1IgG1
the number of CD251 cells in rat molars exposed to the
IgM) anti-serum in the granulomatous zone. Reactive
oral environment when compared with non-treated cells appear in brown with the peroxidase anti-peroxidase
controls (88). method. ! 160 (114).

97
Kiss
the development of disseminating anaerobic infections
following experimental root canal infection with a
mixture of anaerobic pathogens (91).
Changing Th/suppressor ratio
In contrast to early, expanding periapical granulomas,
CD81 cytotoxic/suppressor T-lymphocytes usually out-
number CD41 helper/inducer T-cells in advanced
lesions contributing either to a more effective elimination
of infected and destructed cells or to the suppression of
the intensity of inflammatory reactions, or both (3, 78).
Possible mechanism of cyst
development
The CD41 Th2/CD81-rich nature is even more
obvious in periradicular cysts that were demonstrated
to develop late during the course of experimentally
induced periapical inflammation (92, 93). The factors
that initiate and promote the proliferation of the
epithelial rests of Malassez to form either pocket or
true radicular cysts, are not well understood. Antigen-
presenting cells and CD81 cytotoxic/suppressor T-
lymphocytes have been shown repeatedly to be
associated with pre-existing and newly formed epithe-
lium (81, 94). It has been suggested that the local
cytokine milieu provided by the neighboring macro-
phages and CD81 cells may contribute to epithelial
proliferation. Epithelial cells, indeed, have only been
shown to be in the phase of active proliferation in
human periapical lesions (42). Another group of
investigators observed that intensive immunostaining
for Hsp27 was in coincidence with the presence of local
infiltration of immune cells suggesting that Hsp’s may
contribute to epithelial proliferation (95). In contrast,
CD57 antigen-positive cells may play a role in
degeneration of the epithelial lining of late radicular
cysts as they have been shown to be present in atrophic
radicular cysts in a significantly greater cell density than
in radicular cysts with hyperplastic epithelium (96).
More recently, a role for mitogen-activated protein
(MAP) kinases has been suggested in promoting
epithelial proliferation and differentiation. Based on
the finding that two ubiquitous MAP kinases, ERK1
and ERK2 were overexpressed in differentiated and
highly proliferative epithelial cells, as compared with
those containing non-differentiated, quiescent epithe-
98
lial rests, investigators at the University of Manitoba
proposed a model, in which LPS and proinflammatory
cytokines stimulated proliferation and differentiation
of the epithelial rests of Malassez by means of MAP
kinase activation (97).
Mechanism of bone resorption in
periapical lesions
The most prominent destructive event connected with
both periapical granuloma formation and cyst develop-
ment is the resorption of alveolar bone, although the rate
of bone loss is not as high as in the exudative phase(s),
i.e. during initiation and exacerbation(s) of apical
periodontitis lesions. The effector cells of this degen-
erative process are osteoclasts. However, osteoblasts
regulate the dynamic equilibrium of bone remodeling.
The signaling is mediated by the interaction of a recently
discovered cell membrane-bound ligand–receptor pair,
members of the TNFR and ligand families (98).
Osteoblasts express receptor activator of NF-kB ligand
(RANKL) that binds to its receptor, RANK expressed on
the surface of osteoclast precursor cells. As a result of
RANKL–RANK binding, osteoclast precursors differ-
entiate into mature osteoclasts, mature osteoclasts
become activated resulting in bone resorption. Osteo-
blasts also express and secrete the soluble form of
RANKL, osteoprotegerin (OPG) which, by acting as a
decoy receptor, inhibits RANKL–RANK interaction and
thus bone resorption. Reciprocal gene expression of
RANKL and OPG has been shown to play a key role in
the coupling of osteoblast–osteoclast interaction (99).
Humoral regulatory molecules of bone resorption and
formation, such as hormones and cytokines exert their
effects largely by influencing RANKL–RANK interac-
tion directly or by changing the ratio of RANKL/OPG
reciprocal gene expression (100). Prostaglandins appear
to act even more indirectly as permissive cofactors of
bone-resorptive cytokines (101, 102).
Results from the extensively studied rat periapical
granuloma model indicate that IL-1a is involved in the
bone destruction induced by bacterial root canal
infection (103). In human periapical lesions, IL-1b is
the predominant bone-resorptive cytokine (104).
Bone-resorbing activity of radicular and periapical
CMV infection has also been connected with the ability
of the virus to stimulate IL-1 release (75, 76). Other
cytokines that have been implicated in alveolar bone

loss are TNFa and IL-6, IL-18 (76, 105). As ‘bone-
resorptive’ cytokines may alter the balance in favor of
bone formation by their ability to stimulate osteoblasts
and their precursors on the one hand, and taking the
overlapping nature of the effects induced by them, it is
probably not surprising that disrupting the function of
one of these cytokines did not result in a reduced bone
loss in IL-1R-, TNFR- and in IL-6-deficient transgenic
mice exposed to root canal infection (29, 44).
Bacterial enzymes, LPS and other cell-wall compo-
nents may be involved directly in bone degradation as
demonstrated under experimental conditions (106–
108). However, endodontopathic bacteria only rarely
establish themselves outside of the external root canal
surface in granulomatous periapical lesions (109).
Moreover, the concentration of LPS to directly elicit
bone resorption is in the range of 10 " 3 g/L, whereas as
tiny concentration of LPS as 10 " 9 g/L was demon-
strated to induce the release of endogenous bone
resorptive cytokines and prostaglandins (110, 111).
That is, the probability for an indirect involvement of
endodontopathic microorganisms in the process of
alveolar bone loss in established periapical lesions is 106
times as much as their direct role.
Healing of the periapical lesions
Having eliminated successfully the initiating root canal
infection by proper endodontic and restorative treat-
ment, repairing mechanisms exerted by the granulation
tissue may prevail over tissue destructive events. With
the exception of a not exactly characterized portion of
true radicular cysts, which may take an autonomous
character, the majority of periapical lesions improve
significantly over an extended period of time suggesting
that even radicular cysts in general are treatable by
conventional endodontics (112, 113). The lesion may
heal completely by remineralization as indicated by the
disappearance of the former radiolucent area. Finally, the
lesion may develop into a fibrotic periapical granuloma,
or periapical scar containing only a few cells, mostly
fibroblasts and lacking an inflammatory character.
Concluding remarks
Constant irritation from the root canal space will result in
an inflammatory response within the periapical area.
Noxious stimuli are most often provided by endodonto-
Cell-to-cell interactions
pathic bacteria, that, in interaction with resident host
cells, recruit large number of phagocytes, representing
the first line of host defense. Subsequent appearance of
lymphocytes and plasma cells will transform the early
periapical lesion into periapical granuloma. The char-
acteristic lesion would not develop in the absence of
permanent release of bacteria and their by-products from
the infected root canal. On the other hand, the formation
of a granulation tissue, walled off by a fibrotic capsule
towards the neighboring healthy tissues is entirely
dependent on the presence and proper function of host
cells and regulatory molecules. Inhibiting leukocyte
recruitment or activation either by disrupting regulatory
signals mediated by host-derived cell-bound or soluble
regulatory compounds or by depleting the sources of the
infiltrating cells result in a widespread local bacterial
infiltration, even in generalized infections. Thus, a
complex, fine-tuned interaction between microorgan-
isms, resident host cells and infiltrating leukocytes,
recruited by them will determine the dynamic equili-
brium between protective, destructive and reparative
events in apical periodontitis. Much of our under-
standing of this process comes from experimental models
and represents extrapolations based on investigations of
similar responses occurring in different tissues and body
parts of humans and other mammalian species. The
elucidation of the exact role of cell-to-cell interactions in
human apical periodontitis requires further research
utilizing recent advances of molecular biologic methods.
Acknowledgements
Microphotographs were taken in collaboration with Drs
Ildikó J. Márton, Balazs Dezsö, Zoltan Nemes and Antal Rot.
The help of Dr Edit Bárdi in preparing computer graphics is
kindly acknowledged. This work was supported by grants of
the National Research Development Fund (OTKA) Nos.
T038307 and T046588, and of the Health Science Council
(ETT) of the Hungarian Ministry of Health No. 225/2003.
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