Int End J 1998

Microbiological, pathological, inflammatory,

immunological and molecular biological aspects of

periradicular disease

K . T A K A H A S H I

Department of Periodontology and Endodontology, Okayama University Dental School, Japan

Summary

Multimicrobial infection of the dental pulp triggers

inflammatory responses and ultimately causes bone

destruction in the periradicular tissues. Besides

bacteria, noxious substances such as degraded

protein components and cholesterol could also act

as antigens and elicit a host response, which can be

harmful to periradicular tissues. Histologically, a

dense infiltration of immunocompetent cells is seen

in periradicular lesions and their host reactions may

induce bone resorption. Polymorphonuclear

leucocytes (PMN) and macrophages migrate to the

periapical lesions and phagocytose pathogens as a

first line of defence. Dead PMN are quickly

phagocytosed by macrophages and this disposal

system plays a role in maintaining chronicity of the

lesions. The pathological roles of periradicular T

cells have been assessed through analysing

phenotypic markers of cell types, especially CD

antigens, but the results are still controversial.

Recently, technical developments in immunology

and molecular biology have made it possible to

investigate the pathogenesis of many diseases at

molecular level. The investigation of functional

analysis of the immune cells and their regulatory

molecules such as apoptosis-associated molecules

and adhesion molecules, will lead to a better

understanding of the pathogenesis of periradicular

lesions. The role of inflammatory mediators

including antibodies, cytokines, matrix

metalloproteinases, growth factors and arachidonic

metabolism is becoming known for these lesions.

Knowledge from these investigations improve the

understanding of the pathological mechanisms of

periradicular infections.

Keywords: chronic inflammation, host±parasite

interaction, immune response, pathological

mechanism, periapical diseases.

Introduction

Bacterial infection of the pulp may result in pulpal

destruction and ultimately elicits a host defence reaction

in periapical lesions. The pulpless tooth has a `dead space'

where there is no vascular circulation creating a suitable

environment for bacterial colonization and degradation of

protein components of body fluids.

The host defensive reaction against the irritants from

the infected root canal induces numerous inflammatory

mediators from a variety of cells. These processes operate

in the periradicular lesion to prevent the invasion of

pathogens from infected root canals over a relatively long

time and the host responses may paradoxically account

for much of the tissue damage.

Periradicular lesions are now treated successfully in

many cases compared to periodontal disease. The basic

concept of root canal treatment is based on removing the

irritants from infected root canals both mechanically and

chemically, and then obturating the root canal system to

eliminate or reduce the ingless of microorganisms. The

precise pathological mechanisms involved in the periradi-

cular lesion remain unclear. Diagnostic tools are still not

available to evaluate the progression of periapical period-

ontitis nor to predict future exacerbation of lesions. The

present review paper will summarize the major accom-

plishments of studies on periradicular lesions over the past

three decades. Furthermore, possible pathological

mechanisms of periradicular lesions and some of the

important issues remaining will be discussed.

Pathogens

Periapical infection. The major pathways of pulpal contami-

nation are exposed dentinal tubules, direct pulp exposure,

q 1998 Blackwell Science Ltd 311

International Endodontic Journal (1998) 31, 311±325

Correspondence: Dr Keiso Takahashi DDS PhD, Department of

Periodontology and Endodontology, Okayama University Dental

School, 25-1 Shikata-cho, Okayama 700, Japan.

ReviewArticle

lateral and apical foramina, and blood-transmitted bacter-

ia. It has been reported that dental extraction, periodontal

and orthodontic treatment and even brushing the teeth,

can cause bacteraemia because the oral cavity is septic

(Burket et al. 1937, Sconyers et al. 1973, Hobson & Clark

1995). Therefore, it is possible that during a bacteraemia,

circulating microorganisms could move and colonize pulp-

less teeth (anachoresis). Regardless of the infection path-

ways, it is difficult to distinguish `colonization' or

`infection' by bacteria in infected root canals and infection

modalities in periradicular lesions are still unknown.

Multibacterial infection. The important role of microbes in

pulp and periapical lesions was established by Kakehashi

et al. (1965) using an experimental rat model. Microor-

ganisms in the infected root canals may directly damage

cellular and structural components of the periapical bone

via the release of their proteolytic and noxious waste pro-

ducts (Nair et al. 1996). Bacterial byproducts also elicit an

immune response that could injure host tissues. For exam-

ple, endotoxin from Gram-negative bacteria are capable of

invoking inflammatory and immune responses (Meghji

et al. 1996). It is now generally accepted that `multibac-

terial infection' occurs in the infected root canals (Baum-

gartner 1991a, Trowbridge & Stevens 1992, Simon 1994,

Kettering & Trabinejad 1994). Many anaerobic bacteria,

such as Porphyromonas and Prevotella species, were de-

tected from infected root canals. Some anaerobic bacteria

were thought to be involved in acute periapical abscess

(Brook et al. 1981) and non-healing periradicular lesions

(Sundqvist et al. 1989). Therefore, rapid and reliable mi-

crobial identification methods have been developed to de-

tect specific microorganisms in the infected root canals.

Microbial identification. Current techniques for microbial

identification include culturing, immunological and nu-

cleic acid based methods. Culturing techniques are time-

consuming and expensive, and may fail to grow some

pathogenic organisms. In fact, depending on the culturing

methods used for bacterial identification, the types and

numbers of isolated microorganisms varied (Sundqvist

et al. 1989, Iwu et al. 1990, Barnett et al. 1990, Brook

et al. 1991, Wayman et al. 1992).

Immunological methods require specific antibodies

against targeted bacteria and then may result in false-

positive results because of a cross-reaction with non-

targeted microorganisms. Molecular biological techniques,

DNA colony lift (Cross et al. 1993) and polymerase chain

reaction (PCR) techniques are now used to detect

bacteria. However, DNA probes for some organisms have

problems with low specificity, and cloned DNA probes

may show low sensitivity (Melvin et al. 1994). PCR

methodology offers a highly sensitive and specific

detection for bacteria in biological samples and this

technique has been applied recently in periodontal

research (Riggio et al. 1996, Watanabe & Frommel

1996). However, it is possible that false positive results

may have occurred because the oral cavity is septic and

the sampling procedure would allow other bacterial con-

tamination. Therefore, much more care, such as the

complete isolation of affected teeth by using a rubber dam,

should be taken when using the PCR method to detect

targeted bacteria.

If the development of acute periapical abscess,

periapical periodontitis, refractory and treatment-resistant

periapical lesions (Nair et al. 1990, Reader et al. 1994)

could be predicted through bacterial examination, it

would prove a useful tool in the diagnosis and subsequent

treatment decisions for periradicular lesions involving

different microflora.

However, it might be difficult to obtain strong evidence

for the specific aetiological role of particular bacteria

associated with periradicular lesions because polymicrobial

infection occurs (Trowbridge & Stevens 1992). Recently,

it has been reported that bacterial DNA containing un-

methylated CpG motifs induce B cell proliferation, immu-

noglobulin and cytokine production (Krieg et al. 1995,

Klinman et al. 1996, Schwartz et al. 1997). This evidence

may indicate that any bacteria in infected root canals

which contain the unmethylated CpG motifs can elicit in-

flammatory and immune responses like mitogens in

periapical lesions. The concept of `nonspecific multibacter-

ial infection' (Takahashi et al. 1996b) also suggests that

microbial identification from infected root canals may not

be so useful for diagnosis and hence the choice of

treatment regimens for periradicular lesions at present.

Overall, the variability in the outcome of bacterial

infection is determined by differences in the virulence of

the infecting pathogens, and the effectiveness of host

response against them. Therefore, bacterial characteriza-

tion from infected root canals seems to have limited value

in assessing the risk for exacerbation of periradicular

lesions at present. More clinical investigations are required

to confirm this.

Other pathogens. It has been reported that no bacteria were

detected in cyst fluids (Nair et al. 1993, Meghji et al.

1996). Yeasts were detected from therapy-resistant peria-

pical lesions (Nair et al. 1990). Kettering & Torabinejad

(1993) have speculated the possible involvement of

viruses in infected root canals because natural killer (NK)

cells infiltrate the periapical lesions. In fact, HIV was de-

q 1998 Blackwell Science Ltd, International Endodontic Journal, 31, 311±325

312 K. Takahashi

tected in the dental pulp of patients with AIDS (Glick et al.

1989). Shinoda et al. (1986) have reported that degraded

pulpal tissue components could act as antigens. Nair et al.

(1993) speculated that cholesterol crystals would be pre-

dominant antigens in dental cysts. These results support

the hypothesis that `nonmicrobial pathogens' could, in

some cases, be crucial to the pathogenesis of periradicular

lesions.

There would be numerous different pathogens in

infected root canals and the persistence of irritation may

cause inflammatory and immunological reactions in peri-

radicular lesions. It is still not known what the immuno-

dominant pathogens in infected root canals are.

Furthermore, additional attention should be paid to the

feature of host response that operates against the irritants

in infected root canals.

Host defence processes

The histopathological feature of periapical lesions is

largely the same as that seen in other granulation tissues

that develop from the connective tissue surrounding the

damaged area. A feature common to periapical lesions, ir-

respective of the possible underlying cause, is the

persistent exudation of large number of immunocompe-

tent cells, such as polymorphonuclear leucocytes (PMN),

macrophages, lymphocytes, plasma cells, giant cells, NK

cells and mast cells (Yanagisawa 1980, Stern et al. 1981,

Nevins et al. 1985, Perrini & Fonzi 1985, Piattelli et al.

1991, Kettering & Torabinejad 1993). PMN and

macrophages are important cell types that are involved in

cell-mediated innate immunity which phagocytose

opsonized microorganisms and dead cells.

T and B cells are predominant cellular components in

human periapical lesions (Stern et al. 1982, Nilsen et al.

1984, Skang et al. 1984, Torabinojad & Kettering 1985,

Matthews & Browne 1987, Gao et al. 1988, Barkhordar

et al. 1988, Lukic et al. 1990, Tani et al. 1992a, Marton

& Kiss 1993). These cell types play a central role in

antigen-specific immune response. A number of studies

suggest that both humoral and cellular immune responses

play a role in the pathogenesis of the lesions.

The differences in the proportions of immunocompetent

cells between granulomas and cysts have been studied.

Kopp et al. (1989) have reported that helper/inducer T

cells (Th/i), suppressor/cytotoxic T cells (Ts/c),

macrophages and Ia antigen-positive cells showed a

significant increase in cysts compared to granulomas. In

contrast, Matsuo et al. (1992) showed that there were no

significant differences in the proportion of T cell subsets,

Th/i, Ts/c and immunoglobulin-positive cells. Although

agreement on the cell proportions in the two lesions has

not been reached, no marked differences have yet been

found, hence the immune responses in both lesions may

not appear to be fundamentally different.

Antigen-presenting cells. Animal experiments suggest that

Ia antigen-expressing non-lymphoid cells can be observed

and that an antigen-specific immune defence system is ac-

tive in periapical lesions (Okiji et al. 1994). Although Lan-

gerhans and dendritic cells do not function directly in

antigen elimination, these cells are capable of capturing

antigen in the periphery and migrating to lymphoid or-

gans where they present the antigen to T cells. T helper

cells recognize the association of foreign antigens and self

major histocompatibility complex and then become stimu-

lated, proliferate and release cytokines (Fig. 1). However,

the expression of Ia antigen alone on the cell surface in

vivo is not enough to prove the antigen presentation from

antigen-presenting cells (APC) to CD4 positive helper T

cells because other accessory molecules are also essential

for the event. In fact, Ia antigen-positive cells do not al-

ways show antigen-presenting ability (Geppert & Lipsky

1987, Bal et al. 1990). Furthermore, B cells and activated

T cells are also positively stained for the Ia antigens. After

all, there is only circumstantial evidence about the anti-

gen presentation and cell-to-cell interaction between APC

and helper T cells in periradicular. Therefore, further stu-

dies focusing on cell events are necessary.

T cells. T cells play a central role in cell-mediated immu-

nity. The functional analysis of periradicular T cells has

been performed immunohistochemically and an immunor-

egulatory imbalance of periradicular T cells has been sug-

gested (Trowbridge 1990). Some investigators, have

reported that Ts/c are predominant in the lesions (Kontiai-

nen et al. 1986), whilst others have reported that Th/i are

predominant (Kopp et al. 1989). The majority of T cells in

the lesions are resting (Piattelli et al. 1991). Kawashima

et al. (1996) suggested that the progression of periapical

lesions, with bone resorption, required helper T cells,

whilst suppressor T cells and plasma cells are related to

the chronicity of inflammation. These results indicate that

the immune response is involved in the development of

these lesions. In contrast, Babal et al. (1987) have re-

ported that antibody-mediated immune reactions are pre-

dominant rather than cell-mediated immunity in

periapical granulomas. In addition, an animal study has

supported T cells having a minor role in the pathogenesis

of periradicular lesions (Wallstrom et al. 1993).

Taken together, it is clear that simple evaluation of

phenotypic markers of T cell subsets does not adequately


Pathogenesis of periradicular disease 313

reflect the immune processes that may be occurring.

Therefore, the effector functions or cell activity of periradi-

cular T cells should be assessed. Immunological and

molecular biological techniques can be applied. Possible

approaches to investigate periradicular T cell functions

will now be discussed.

T cell functions can be estimated by their cytokine profiles

(Yamamura et al. 1991, Fresno et al. 1997), i.e. CD4�

helper T cells can be subdivided into T helper 1 (Th1) or T

helper 2 (Th2) cell types (Fig. 1). The humoral immune

response is prompted by Th2 cell types which produce char-

acteristic cytokines, IL-4, IL-5, IL-10 and IL-13. Th1 cells

release IL-2 and interferon-g that enhance cell-mediated

immune responses. In periodontal research, cytokine profiles

in adult periodontitis have been well investigated at both

protein and messenger RNA (mRNA) levels using immuno-

histochemistry, reverse transcription PCR and in situ hybri-

dization methodologies (Table 1). The role of periradicular T

cells can be assessed with the same methodologies.

However, cytokine profiles in periodontitis are still contro-

versial (Matsuki et al. 1992, Fujihashi et al. 1993, Yamazaki

et al. 1995, Fujihashi et al. 1996) and the cytokine profile in

inflamed tissues may be changeable because of different

antigenic challenges, treatment modalities and the stage of

the disease. Furthermore, it has been reported that different

methods for the detection and quantification of cytokines at

protein or mRNA level may give different results (Favre et al.

1997) and therefore we need to choose suitable methods for

our study aim.

Fig. 1 Th1/Th2 immune response. T helper cells recognize peptides that are bound to major histocompatibility complex (MHC) and expressed

on the cell surface of antigen-presenting cells (APC), such as macrophages and dendritic cells. The production of IL-12 by APC promotes thedevelopment of Th1 cells that activates macrophages. In contrast, Th2 cells produce IL-4, IL-5, IL-10 and IL-13, which are responsible for

antibody production. Abbreviations: Ag, antigen; B, B cell; IFN-g, interferon-g; IL, interleukin; Mf, macrophages; T, T cell; TCR, T cell receptor;

Th1, T helper 1; Th2, T helper 2.

Table 1 Target identity and detection techniques

Genome DNA sequence Dot blot

Southern blot

in situ hybridization

PCR

mRNA mRNA sequence Northern blot

in situ hybridization

RT-PCR

Protein Amino acid sequence Immunohistochemistry

and antigenicity ELISA

ELISPOT

Western blot

ELISA, enzyme-linked immunosorbent assay; ELISPOT, enzyme-linked

immunospot; PCR, polymerase chain reaction; RT-PCR, reverse

transcription±polymerase chain reaction.


314 K. Takahashi

T cell functions can also be assessed by determining the

T cell receptor (TCR) repertoire in the disease sites

(Forman et al. 1994, Caignard et al. 1994, Yamazaki et al.

1996, Hingorani et al. 1996, Mato et al. 1997). Antigen

recognition by T cells is mediated by the cell surface

receptors (Pannetier et al. 1995). The expression of the

variable region genes of the TCR alpha and beta chain

can be analysed to study the involvement of T cells in the

disease tissues. The evidence of limited repertoire usage of

T cells in disease sites suggests the clonal expansion and/

or migration of restricted antigen-specific T cells or super-

antigen activation in the inflamed sites.

By using immunohistochemical techniques, TCR

repertoire in situ can be analysed. In addition, quantita-

tive PCR and DNA sequence analysis for the third com-

plementarity-determining region of the TCR now permits

a more in-depth analysis of the repertoire of T cells

recovered from small biopsy samples (Cottrez et al.

1994). Oligoclonal expansion of local T cells in

autoimmune diseases has been suggested using RT-PCR

and single-strand conformational polymorphism method

(Hayashi et al. 1995, Struyk et al. 1995, Gulwani-

Akolkar et al. 1996). In periodontal research, Nakajima

et al. (1996) and Yamazaki et al. (1996, 1997) have

characterized the TCR repertoire usage in adult period-

ontitis and they have suggested that oligoclonal

expansion of limited repertoire of TCR-bearing T cells or

superantigen activation would occur in inflamed gingiva.

Thus, molecular techniques may be useful to understand

the functional role of periradicular T cells in the patho-

genesis of periradicular lesions.

Immunoglobulin-producing cells. There are numerous plas-

ma cells in the lesions, and immunoglobulin G (IgG)-con-

Fig. 2 Migration of leucocytes and their fate in periapical lesions. Apoptosis occurs predoninantly in PMN that are engulfed by macrophages.

This may be a disposal mechanism for dead and effete PMN in the periapical lesions. This disposal system may suppress the release of enzymesand inflammatory agents from dead PMN and thus regulate the chronicity of periapical inflammation. In contrast, lymphocytes were not

removed by apoptosis and apoptosis-suppressing molecules, bcl-2 and bcl-x could be involved in this mechanism. Abbreviations: Ag, antigen; IL,

interleukin; Mf, macrophages; T, T cell; TCR, T-cell receptor; B, B cell; PMN, Polymorphonuclear leucocytes; PL, plasma cells; MMP, matrixmetalloproteinases; FasL, Fas ligands; TNF, tumour necrosis factor.



taining plasma cells predominate with lower numbers of

IgA and a few IgM, as shown by immunofluorescence and

immunoenzymatic methods (Table 2: Toller 1971, Kuntz

et al. 1977, Morton et al. 1977, Stern 1981, Matthews &

Mason 1983, Smith et al. 1987). The distribution of im-

munoglobulin-producing cells in the periradicular lesion

suggests that the immune reaction is of systemic and ser-

um type rather than mucosal-associated. Immunological

studies for detecting immunoglobulin classes in periapical

exudates have shown similar results (Skaug 1974, Baum-

gartner 1991b, Matsuo et al. 1995). In contrast, Toller &

Holborow (1969) found more IgA- than IgG- or IgM

synthesizing plasma cells in cyst walls, which suggests

that the local immunoglobulin production changes during

cyst maturation and both systemic and mucosal immune

responses may be involved in the pathogenesis of periradi-

cular lesions.

Immunohistochemistry is an established technique for

protein detection but staining for immunoglobulin using

antibodies in inflamed tissues may be unreliable because

of the high background staining caused by non-specific

binding to other immunoglobulins within the tissues

(Ruprai et al. 1991) and serum-derived immunoglobulin

that bathes the tissues (Challacombe et al. 1978). In

addition, the detection of immunoglobulin protein in vivo

presents several technical difficulties because the protein

can be degraded by neutrophil elastase (Giannopoulou

et al. 1992), oxidant (Weiss 1989), proteolytic enzymes

(Ylipaavalniemi & Tuompo 1977) and matrix metallopro-

teinases (Teronen et al. 1995) as well as by immunoglo-

bulin-degrading proteases from bacteria (Kilian 1981).

Thus, immunolocalization of immunoglobulins at the

protein level presents many technical and interpretational

difficulties. Also, it would be better to detect the existence

of local immunoglobulin production at the gene level. In

situ hybridization is a molecular biological technique that

permits the detection of specific mRNA expression even in

small tissue samples (Table 1). This technique avoids

many of the problems alluded to above and offers the

opportunity to detect, localize and quantify the cells

producing immunoglobulins.

Human IgG and IgA consists of four and two

subclasses, respectively, IgG1, IgG2, IgG3 and IgG4, IgA1

and IgA2, and the immunoglobulin heavy chain

constant-region genes on chromosome 14 are

50-m-d-g3-g1-a1-g2-g4-e-a2±30 (Ellison et al. 1982a,b,

Flanagan et al. 1984, Huck et al. 1986). The exact

mechanisms of B cell activation, class switch, proliferation

and differentiation in vivo remain unknown (Nieuwenhuis

1981). Periapical lesions are accessible and suitable for

the molecular analysis for B cell class switch and differen-

tiation. Questions whether periapical B cells are activated,

where they proliferate and differentiate, and how they

migrate to the periapical lesions, are intriguing. We have

characterized the distribution of IgG and IgA subclass

mRNA bearing plasma cells in periapical lesions

(Takahashi et al. 1997b,c). As a result, amongst IgG

subclasses, IgG1 is predominant and suggests that protein

antigens are the major antigens in periapical lesions

(Table 3). The proportion of plasma cells that expressed

IgG2 mRNA was about 35% of the total IgG producing

cells, which is higher than the abundance of cells that

expressed IgG2 in periodontitis (23%) (Takahashi et al.

1997a,c). Therefore, lipopolysaccharide and carbohydrate

antigens may play a greater role in eliciting the immune

response in periapical lesions rather than in periodontitis.

Most IgA plasma cells are J chain negative (Takahashi

et al. 1997b). These features indicate that secretory IgA-

mediated immune defense systems appear to play little

part in the lesions. In contrast, Torres et al. (1994) have

reported the presence of secretory IgA in periapical

exudate and have suggested that secretory IgA is actively

produced in cystic lesions and that the monomeric IgA

present in granulomas is a result of transudation of serum

derived proteins into the lesions. Further research will be

necessary on this aspect.

Table 2 Immunoglobulin-producing cells in periapical lesions

Periapical status Percentages of Ig positive cells

IgG IgA IgM IgE Methods References

Cysts IgA>IgG>IgM F Toller & Hobborow 1969

Granulomas 70 14 4 10F Pulver et al. 1978

Cysts 45 45 5 5

Granulomas 69 23 2 5F Stern et al. 1981

Cysts 67 27 1 5

Granulomas 82 12 5 1 E Matthews & Mason 1983

Cysts 85 14 2 ND E Smith et al. 1987

F, fluorescence method; E, enzymmatic method; ND, not done.


316 K. Takahashi

Recently, the existence of antigen-driven differentia-

tion of B cells and the formation of germinal centres

within the lungs has been shown (Chvatchko et al.

1996). These germinal centres in non-lymphoid organs

would provide a local source of immunoglobulin-

secreting cells that may effectively contribute to the

local immune reaction. In contrast, germinal centre-like

organs are rarely seen and no local proliferation of B

lymphocytes was found in periapical lesions, whilst pro-

liferation of lymphocytes in tonsillar germinal centres

(mostly B cells) was observed (Takahashi et al. 1996a).

These results suggest that B cells in periapical tissues

may be long-living and at the resting stage (Go stage),

possessing the capability of continuously migrating

between blood and lymph nodes to participate in

immune responses in the periapical lesions.

The humoral immune response. Specific humoral immune

response against several bacteria from the infected root

canal has been investigated by the enzyme-linked immu-

nosorbent assay (ELISA) method using crude bacterial

components as antigens (Kettering et al. 1991). Mono-

clonal and oligoclonal immunoglobulins were detected in

periapical lesions (Matsumoto 1985) and periapical anti-

body-secreting cells against specific antigens of endodon-

topathic bacteria have been investigated by using

enzyme-linked immunospot (ELISPOT) assay (Ogawa et al.

1992). This evidence supports the possibility that `peria-

pical lesion relevant' plasma cells may arise via selective

homing and/or by the clonal proliferation and differen-

tiation of specific B cells in this region. The local produc-

tion of oligoclonal and monoclonal immunoglobulins in

periapical lesions (Matsumoto 1985) supports the con-

cept that the species of immunodominant antigens in in-

fected root canals are limited. PCR-based amplification of

immunoglobulin variable region genes, especially the

third complementarity-determining region, which consti-

tutes a important part of the immunoglobulin-antigen

binding site, and DNA sequence analysis may be helpful

to understand the types of unknown antigens in infected

root canals (Marks et al. 1991, Mortari et al. 1993, Raa-

phorst et al. 1996, 1997).

Investigation of immunodominant antigens may help

us to understand the pathological role of irritants in

infected root canals. Future research should focus on

detecting which antigens are immunodominant. If immu-

nodominant molecules could be found, they might be a

pathogenic factor because they are thought to be capable

of inducing an excessive antibody production in patients.

However, it may be difficult to determine the immunodo-

minant antigens from bacteria in infected root canals

because the irritants are quite different as described above

and immuneresponsiveness amongst individuals varies

widely, although the possibility that there may be

common bacteria (Sundqvist 1992) and limited immuno-

dominant antigens in infected root canals cannot be

denied.

Polyclonal B cell activation. Histopathological findings of

established periradicular lesions are similar to those

found in periodontitis in which plasma cells predominate

and this suggests that plasma cells are related to bone

loss. In addition to antigen-specific immune responses,

lymphocytes are directly activated by several microbial

products including teichoic acid, peptidoglycan and lipo-

polysaccharide (LPS). LPS of periodontal bacteria contain

several components that induce proliferation and differ-

entiation of B cells non-specifically, as say, `polyclonal B

cell activation (PBA)' (Tew et al. 1989). Antigen-specific

and non-specific lymphocyte activation may occur in

periodontal lesions. Likewise, it is possible that LPS from

anaerobic bacteria in infected root canals has PBA activ-

ity and induces proliferation and differentiation of peria-

pical B cells (Tani et al. 1992b). Recently, it has been

reported that LPS is strongly stimulatory for macro-

phages and induces these cells to release various cyto-

kines which could stimulate T cell subsets (Tough et al.

1997). Activated B cells also produce IL-1 that has a

bone-resorbing activity (Matsushima et al. 1985) and it

is possible that B cells and plasma cells in periapical le-

sions can produce inflammatory factors in addition to

antibodies that may induce bone destruction. There is

still little evidence of the role of locally produced anti-

body and other molecules produced by periapical plasma

Table 3 Ratios of immunoglobin mRNA-expressing cells

Disease sites k/l ratio IgA1/IgA (%) J chain +IgA/IgA (%) IgG sublclasses (%)

(polymeric IgA)IgG1 IgG2 IgG3 IgG4

Granulomas 1.66�0.85 75.3�11.2 1.3 (0±7.7) 57.3�7.3 34.1�5.0 4�2.8 4�2.7

Cysts 1.47�0.51 64.8�21.3 4.7 (0.3±13.6) 56�5.7 34.6�5.1 4.4�2.2 5.4�2.5

Periodontitis gingiva 2.4 65.1 ND 63 23 3 10

Reference: Takahashi et al. (1996 b, c; 1997 a, b; 1998) ND, not done.



cells. It has been reported that prostaglandin E2 is pro-

duced predominantly by plasma cells in radicular cysts,

using immunostaining and this result supports that plas-

ma cells are involved in bone resorption (Matejka et al.

1986). By contrast, it has been reported that plasma

cells are involved in tissue repair rather than the devel-

opment of periapical lesions (Akamine et al. 1994a).

Therefore, further research to analyse plasma cell-de-

rived inflammatory factors would be of interest. Molecu-

lar biological strategies for searching which mRNAs are

expressed in periapical plasma cells will prove their role

in periapical lesions more exactly.

Cell functional analysis

Cell synthesis. Previous work has shown that a large num-

ber of immunocompetent cells accumulates in the periapi-

cal lesions, although little is known about their functions.

The role of the different cell types and their interrelation-

ships has been investigated by morphological and immu-

nohistochemical studies (Matthews & Browne 1987, Tani

et al. 1992, Marton & Kiss 1993). However, morphologi-

cal observation and phenotypic analysis of cell surface

markers alone are not enough to elucidate cell function in

vivo. It is now possible to evaluate the synthetic activity of

cells using in situ hybridization for mRNA and ribosomal

RNA (rRNA) expression (Pringle et al. 1989, Danks et al.

1995, Yoshii et al. 1995). Information about the relative

concentrations of the total mRNA and rRNA would be va-

luable in assessing cellular synthetic activity and tissue

functions in vivo (Takahashi et al. 1996a). Plasma cells

showed the strongest signal for the two probes, and slight

to moderate staining could be found in the epithelium, fi-

broblasts, macrophages, endothelial cells and lymphocytes,

whereas almost all polymorphonuclear leucocytes were

negative for these probes (Takahashi et al. 1997d).

Proliferation. The size and functional activity of tissues or or-

gans depend on the balance between cell proliferation and

death. The investigation of cell proliferation in inflammatory

lesions is important from a number of standpoints; normal

cell turnover, clonal expansion of lymphocytes, tissue devel-

opment, tissue regeneration and destruction. Cell prolifera-

tion in vivo can be assessed with several markers, for

example, Ki67, PCNA and histone (S-phase) (Bacchi et al.

1993, Alison et al. 1994, Hall & Coates 1995). Lymphocytes

do not appear to proliferate greatly in periapical tissues (Ta-

kahashi et al. 1997d). These findings support the hypothesis

that specific leucocytes predominate in the periapical tissue

through selective homing or non-specific migration rather

than by local proliferation of periapical lymphocytes.

Proliferation of epithelial cells. Cyst expansion is affected by

growth of cyst epithelium and the proliferation of epithe-

lial cell rests of Malassez and is a unique reaction of peria-

pical tissues (Hill 1930, Torabinejad 1983, Li et al. 1994).

Proliferation of epithelial cells in the lesions is accompa-

nied by inflammatory cells such as PMN, lymphocytes and

plasma cells (Hill 1930). Therefore, inflammatory media-

tors from their immunocompetent cells may be involved

in the proliferation of epithelial cells. However, the mole-

cular mechanism which stimulates the epithelial cell rests

to proliferate and is responsible for their enlargement has

not been clarified.

Cytokines and bacterial endotoxin affect epithelial cell

proliferation (Meghji et al. 1996). Cyst epithelial cells may

also produce IL-1 and IL-6 to act in an autocrine manner

(Bando et al. 1993). Recently, it has been reported that

keratinocyte growth factor was produced by stromal cells

in periapical lesions and may stimulate epithelial prolifera-

tion associated with cyst formation (Gao et al. 1996).

Epithelial cells strongly express epidermal growth factor

receptor in those periapical lesions showing epithelial cell

proliferation (Lin et al. 1996). This evidence supports the

hypothesis that expansion of cysts occurs from epithelial

cell proliferation through their growth factors and the

cytokines produced by inflammatory cells.

Therapeutically, an apical cyst may resist conventional

root canal treatment. Therefore, further investigations will

be needed to understand the mechanism that stimulates

proliferation of the epithelial cell rests in periapical lesions.

Although this new evidence may not fundamentally

change how these lesions are treated, it will allow

clinicians to treat patients with more understanding and

confidence.

Cell death. Cell death also plays a major role in the organi-

zation of tissues and results from either necrosis or apopto-

sis. Necrosis follows damage to the plasma membrane,

disruption of ion gradients, rapid swelling and autolysis

(Duvall & Wyllie 1986). Russell bodies are thought to be

necrotic plasma cells that can no longer produce antibo-

dies (Simon 1994). In contrast, apoptosis is cell suicide

and exhibits characteristic signs, including cell shrinkage,

nuclear and cytoplasmic organelle condensation and DNA

fragmentation (Arends et al. 1991). Although there are

numerous inflammatory cells and necrotic tissues in peria-

pical lesions (Stern et al. 1981), little information is avail-

able about their cell death and their clearance system. It

has been investigated previously that PMN in periapical

lesions became apoptotic and are phagocytosed by macro-

phages (Takahashi et al. 1997d). Dead and/or activated

PMN release potent degradative enzymes and cytokines,


318 K. Takahashi

such as IL-1b and TNF-a (Miller et al. 1996, Takahashi

et al. 1995, Galbraith et al. 1997) and their removal sys-

tems may be crucial in preventing the release of harmful

inflammatory mediators from dead PMN and in reducing

the chronicity of inflammation (Savill et al. 1989, Jones

et al. 1993). PMN express both Fas and Fas ligands (FasL)

on their cell surface, but do not express apoptosis-inhibi-

tory molecules, such as bcl-2 and bcl-x, and this feature

may reflect their having the shortest life-span amongst

blood leucocytes (Ohta et al. 1995). In addition, PMN can

release soluble FasL and this soluble FasL could induce

apoptosis of epithelial cells that express Fas antigens (Liles

et al. 1996). Tani-Ishii et al. (1997) reported that apopto-

sis of osteoclasts is induced by anti-Fas antibody and that

TNF-a can act as an inhibitor. Such evidence supports the

possibility that TNF-a and PMN-derived soluble FasL can

regulate apoptosis of osteoclasts and then PMN play a role

in bone destruction in periradicular lesions.

Inflammatory molecules

Cytokines. The role of cytokines in the regulation of humor-

al and cellular immune responses in vivo has been thor-

oughly investigated. Recently, the existence of

proinflammatory cytokines that induce bone resorption

such as IL-1b and TNF-a in periradicular lesions has been

reported (Artese et al. 1991, Stashenko et al. 1992, Wang

& Stashenko 1993, Lim et al. 1994 Wang et al. 1997).

These cytokines are produced by several cells, such as

macrophages near to the bone resorption and osteoclasts.

(Hamachi et al. 1995, Tani-Ishii et al. 1995). It has also

been reported that osteoclasts express both type I and II

IL-1 receptors (Xu et al. 1996). This evidence supports the

paradigm that cytokines produced by immunocompetent

cells in the periapical lesions can induce bone destruction

by activating osteoclast function. Therefore, much more

attention should be paid to the action of the cytokine net-

work in the periapical lesions.

The pathological role of PMN in inflammatory sites

needs to be re-evaluated considering this new

information, suggesting that the functional activity of

PMN is broader than previously thought (Lloyd &

Oppenheim 1992). Much evidence has been demonstrated

to show that PMN are capable of generating cytokines,

such as, IL-1b, TNF-a, IL-6 and IL-8 (Cassatella 1995,

Takahashi et al. 1995). PMN may be a significant source

of IL-1 in periapical lesions and PMN-derived cytokines

may be involved in bone resorption (Miller et al. 1996). In

fact, discharge of pus has been observed from the infected

root canals diagnosed as acute periapical periodontitis. It

is therefore possible that PMN-derived cytokines besides

oxidants (Weiss 1989) and matrix metalloproteinases

(Teronen et al. 1995) play a role in the periapical inflam-

matory response.

Arachidonic acid metabolites. Prostaglandins (PGs) have

been implicated in the bone resorption of periapical lesions

(Harris et al. 1973, McNicholas et al. 1991, Wang & Sta-

shenko 1993). If PGs play a role in bone resorption of

periapical lesions, they may be released at a site accessible

to the bone. The distribution of the PGs-producing cells in

periapical lesions was analysed. Plasma cells and histiocy-

tic elements in radicular cysts and macrophages and en-

dothelial cells in dental pulp have been shown to be

positive cells for PGE2 (Matejka et al. 1986, Miyauchi et al.

1996). PGs produced from these host cells are involved in

the development of bone resorption and then might be re-

lated to clinical symptoms (Takayama et al. 1996).

Matrix metalloproteinases. Collagen is a major protein de-

stroyed during periapical periodontitis, resulting in the de-

struction of collagen fibres that attach the teeth to

surrounding bone and in the resorption of the bone itself.

The members of the family of matrix metalloproteinases

(MMPs) are key enzymes in matrix degradation. There are

three major MMPs: gelatinase, stromelysin and collagenase.

MMPs are secreted by a variety of defense and structural

cells; PMN, fibroblasts, macrophages and keratinocytes in

inflamed tissues. MMPs from neutrophils (MMP-9) and fi-

broblasts (MMP-2) are active in both the cyst wall and cyst

fluids (Teronen et al. 1995). Microorganisms produce a

variety of soluble enzymes to digest host proteins and other

molecules and thereby produce nutrients for growth.

Although microbes can produce a multiplicity of proteases,

the main protease activity in the cyst fluid is likely to be

host-cell-derived and it is therefore worth mentioning host

proteases in context with microbial proteases (Barkhordar

1987). This evidence suggests that MMPs from host cells

are involved in periapical tissue breakdown. Tissue destruc-

tion would be influenced by the imbalance of MMPs and tis-

sue inhibitors of metalloproteinases (TIMP). The production

of MMPs and TIMP by cells is regulated by many cytokines,

growth factors and hormones. Therefore, further investiga-

tion to determine the regulatory mechanism of MMPs and

TIMPs in periapical lesions is required.

Adhesion molecules. It has been reported that immunocompe-

tent cells are actively motile and capable of penetrating sev-

eral layers of epithelial lining of the cyst (Toller & Holborow

1969). Adhesion molecules are involved in leucocyte migra-

tion through epithelium and the infiltrating mechanism of

inflammatory cells to epithelial lining is feasible. The distribu-



tion of ICAM-1 and ELAM-1 expression has been reported in

radicular cysts (Bando et al. 1993). However, little informa-

tion is available as to which adhesion molecules are involved

in the process, although VLA integrins-VCAM-1 or VLA-fi-

bronectin cellular adhesion molecules maybe presumed to be

involved. Further studies will be needed to investigate cell-to-

cell and cell-to-extracellular matrix interactions through ad-

hesion molecules in periapical lesions.

Animal model (rat model). Animal experiments were per-

formed by Kakehashi et al. (1965) to elucidate the role of

the immune response against bacteria, using a pulp-expo-

sure rat model. There have been many investigations fo-

cusing on the microbial infection (Tani-Ishii et al. 1994),

the kinetics of bone resorption (Wang & Stashenko 1991,

Stashenko et al. 1992, Anan et al. 1993, Bando & Na-

gayama 1993, Yamasaki et al. 1994) and the role of im-

munocompetent cells (Stashenko & Yu 1989, Akamine

et al. 1994b, Tani et al. 1995, Kawashima et al. 1996).

However, care should be taken because there are some

species differences between human beings and rodents

and the immune systems are slightly different. For exam-

ple, IL-1a is the predominant form of IL-1 in the rat,

whilst IL-1b is predominant in humans (Wang & Stashen-

ko 1993, Stashenko et al. 1994). Unquestionably, more

precise in vivo studies are needed to investigate the role of

cytokines using clinical samples from humans and experi-

mental animal models.

This rat model also gives an opportunity to try novel

therapeutic approaches. Anti-inflammatory agents, such

as antibacterial agents, collagenase inhibitor, prostaglan-

din synthesis inhibitors and IL-1 receptor antagonists

have been tried on this model and these agents are

thought to be useful to inhibit acute inflammation and

bacterially induced bone resorption in periapical lesions

(Nobuhara et al. 1993, Anan et al. 1996).

Influence of infected root canals on systemic conditions. Anti-

genic stimulation through root canals may cause local and

systemic immune responses. Immune reactions against the

irritants in infected root canals and periapical lesions may

influence systemically (Barnes & Langeland 1966, Okada

et al. 1967, Shinoda et al. 1986, Marton & Kiss 1992). This

evidence provides a useful model for investigating the inter-

actions between chronic oral inflammation and homeostasis

of the host related to infected root canals.

Besides bacteria, potent soluble mediators, such as PGs,

cytokines and MMPs, can be produced by periapical in-

flammatory cells. It has been suggested that focal

infection in the oral cavity, such as marginal and

periapical periodontitis, may cause a bacteraemia and

ultimately exacerbate sepsis in compromised hosts; for

example, patients treated with chemotherapy following

organ transplantation. Strict infection control including

oral infection will be required in these patients and closer

cooperation between dentists and physicians is necessary.

It has been reported that certain features of rheumatoid

inflammation may occur, and free rheumatoid factor has

been detected in periapical lesions of patients with

rheumatoid arthritis (Malmstrom 1975, Malmstrom &

Natvig 1975). These reports raise the possibility that

systemic disease could have an influence on the pathological

changes of periapical lesions. Tani-Ishii et al. (1996) have

recently reported that leprosy periapical granulomas may

develop as a result of an immunological response to Myco-

bacterium leprae. Incidentally, we observed that patients with

Mycobacterium leprae, who had a dysfunction of monocyte

phagocytosis, showed a delayed healing after root canal

treatment (unpublished observation). These results suggest

that monocyte dysfunction may be a risk factor for

periapical periodontitis and that there is individual suscept-

ibility to periapical lesions depending on their host defense

and/or tissue repair ability.

Conclusions

It is apparent from the literature that immune responses

which are involved in the pathogenesis of periapical lesions

are complex and variable. There are still many questions

concerning the pathological mechanisms of the lesions. Does

host response against irritants work protectively or

harmfully? Do T cells play a minor or major role on the

pathogenesis of periapical lesions? Do periapical plasma cells

produce antigen-specific antibody, and if so, what are the

predominant antigens in infected root canals? In addition,

the processes of bone resorption and remodeling elicited by

the host-parasite interaction are still unknown for periapical

lesions as well as in other infectious diseases such as

periodontal disease. Therefore, molecular mechanisms of

bacterially induced bone destruction and bone formation,

tissue repair, growth factors for epithelial cells and the

cytokine network in periapical lesions, should be researched.

The application of microbiological, immunological and

molecular biological techniques may help us to resolve the

enigma of the pathogenesis of periapical lesions.

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